I have always heard of the top down conversation and really believe that we all are equal as employees whether you are a principal or a teacher. What I mean by that is that we should all have the same passion working for kids and want the same results to put kids learning first. When I talk with my mentee or my administration, I want them to know that I know they bring value to the team as a whole. This is meaningful for them and the school as they are a part of the process of what we do.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review): 

Summary: 

Taber et al report the biochemical characterization of 7 mutations in PHD2 that induce erythrocytosis. Their goal is to provide a mechanism for how these mutations cause the disease. PHD2 hydroxylates HIF1a in the presence of oxygen at two distinct proline residues (P564 and P402) in the "oxygen degradation domain" (ODD). This leads to the ubiquitylation of HIF1a by the VHL E3 ligase and its subsequent degradation. Multiple mutations have been reported in the EGLN1 gene (coding for PHD2), which are associated with pseudohypoxic diseases that include erythrocytosis. Furthermore, 3 mutations in PHD2 also cause pheochromocytoma and paraganglioma (PPGL), a neuroendocrine tumour. These mutations likely cause elevated levels of HIF1a, but their mechanisms are unclear. Here, the authors analyze mutations from 152 case reports and map them on the crystal structure. They then focus on 7 mutations, which they clone in a plasmid and transfect into PHD2-KO to monitor HIF1a transcriptional activity via a luciferase assay. All mutants show impaired activation. Some mutants also impaired stability in pulse chase turnover assays (except A228S, P317R, and F366L). In vitro purified PHD2 mutants display a minor loss in thermal stability and some propensity to aggregate. Using MST technology, they show that P317R is strongly impaired in binding to HIF1a and HIF2a, whereas other mutants are only slightly affected. Using NMR, they show that the PHD2 P317R mutation greatly reduces hydroxylation of P402 (HIF1a NODD), as well as P562 (HIF1a CODD), but to a lesser extent. Finally, BLI shows that the P317R mutation reduces affinity for CODD by 3-fold, but not NODD.  

Strengths: 

(1) Simple, easy-to-follow manuscript. Generally well-written. 

(2) Disease-relevant mutations are studied in PHD2 that provide insights into its mechanism of action. 

(3) Good, well-researched background section. 

Weaknesses: 

(1) Poor use of existing structural data on the complexes of PHD2 with HIF1a peptides and various metals and substrates. A quick survey of the impact of these mutations (as well as analysis by Chowdhury et al, 2016) on the structure and interactions between PHD2 peptides of HIF1a shows that the P317R mutation interferes with peptide binding. By contrast, F366L will affect the hydrophobic core, and A228S is on the surface, and it's not obvious how it would interfere with the stability of the protein. 

Thank you for the comment.  We have further analyzed the mutations on the available PHD2 crystal structures in complex with HIFα to discern how these substitution mutations may impact PHD2 structure and function.  This analysis has been added into the discussion.

(2) To determine aggregation and monodispersity of the PHD2 mutants using size-exclusion chromatography (SEC), equal quantities of the protein must be loaded on the column. This is not what was done. As an aside, the colors used for the SEC are very similar and nearly indistinguishable. 

Agreed. We have performed an additional experiment as suggested by the reviewer to further assess aggregation and hydrodynamic size.  The colors used in the graph were changed for clearer differentiation between samples.

(3) The interpretation of some mutants remains incomplete. For A228S, what is the explanation for its reduced activity? It is not substantially less stable than WT and does not seem to affect peptide hydroxylation. 

We agree with the reviewer that the causal mechanism for some of the tested disease-causing mutants remain unclear.  The negative findings also raise the notion, perhaps considered controversial, that there may be other substrates of PHD2 that are impacted by certain mutations, which contribute to disease pathogenesis.  A brief paragraph discussing this has been included in the discussion.

(4) The interpretation of the NMR prolyl hydroxylation is tainted by the high concentrations used here. First of all, there is a likely a typo in the method section; the final concentration of ODD is likely 0.18 mM, and not 0.18 uM (PNAS paper by the same group in 2024 reports using a final concentration of 230 uM). Here, I will assume the concentration is 180 uM. Flashman et al (JBC 2008) showed that the affinity of the NODD site (P402; around 10 uM) for PHD2 is 10-fold weaker than CODD (P564, around 1 uM). This likely explains the much faster kinetics of hydroxylation towards the latter. Now, using the MST data, let's say the P317R mutation reduces the affinity by 40-fold; the affinity becomes 400 uM for NODD (above the protein concentration) and 40 uM for CODD (below the protein concentration). Thus, CODD would still be hydroxylated by the P317R mutant, but not NODD. 

The HIF1α concentration was indeed an oversight, which will be corrected to 0.18 mM.  The study by Flashman et al.[1] showing PHD2 having a lower affinity to the NODD than CODD likely contributes to the differential hydroxylation rates via PHD2 WT.  We showed here via MST that PHD2 P317R had K[d] of 320 ± 20 uM for HIF1αCODD, which should have led to a severe enzymatic defect, even at the high concentrations used for NMR (180 uM).  However, we observed only a subtle reduction in hydroxylation efficiency in comparison to PHD2 WT.  Thus, we performed another binding method using BLI that showed a mild binding defect on CODD by PHD2 P317R, consistent with NMR data.  The perplexing result is the WT-like binding to the NODD by PHD2 P317R, which appears inconsistent with the severe defect in NODD hydroxylation via PHD2 P317R as measured via NMR.  These results suggest that there are supporting residues within the PHD2/NODD interface that help maintain binding to NODD but compromise the efficiency of NODD hydroxylation upon PHD2 P317R mutation. 

(5) The discrepancy between the MST and BLI results does not make sense, especially regarding the P317R mutant. Based on the crystal structures of PHD2 in complex with the ODD peptides, the P317R mutation should have a major impact on the affinity, which is what is reported by MST. This suggests that the MST is more likely to be valid than BLI, and the latter is subject to some kind of artefact. Furthermore, the BLI results are inconsistent with previous results showing that PHD2 has a 10-fold lower affinity for NODD compared to CODD. 

The reviewer’s structural prediction that P317R mutation should cause a major binding defect, while agreeable with our MST data, is incongruent with our NMR and the data from Chowdhury et al.[2] that showed efficient hydroxylation of CODD via PHD2 P317R.  Moreover, we have attempted to model NODD and CODD on apo PHD2 P317R structure and found that the mutation had no major impact on CODD while the mutated residue could clash with NODD, causing a shifting of peptide positioning on the protein.  However, these modeling predictions, like any in silico projections, would need experimental validation.  As mentioned in our preceding response, we also performed BLI, which showed that PHD2 P317R had a minor binding defect for CODD, consistent with the NMR results and findings by Chowdhury et al[2].  NODD binding was also measured with BLI as purified NODD peptides were not amenable for soluble-based MST assay, which showed similar K[d]’s for PHD2 WT and P317R.  Considering the absence of NODD hydroxylation via PHD2 P317R as measured by NMR and modeling on apo PHD2 P317R, we posit that P317R causes deviation of NODD from its original orientation that may not affect binding due to the other interactions from the surrounding elements but unfortunately disallows NODD from turnover.  Further study would be required to validate such notion, which we feel is beyond the scope of this manuscript.  

(6) Overall, the study provides some insights into mutants inducing erythrocytosis, but the impact is limited. Most insights are provided on the P317R mutant, but this mutant had already been characterized by Chowdhury et al (2016). Some mutants affect the stability of the protein in cells, but then no mechanism is provided for A228S or F366L, which have stabilities similar to WT, yet have impaired HIF1a activation. 

We thank the reviewer for raising these and other limitations.  We have expanded on the shortcomings of the present study but would like to underscore that the current work using the recently described NMR assay along with other biophysical analyses suggests a previously under-appreciated role of NODD hydroxylation in the normal oxygen-sensing pathway.  

Reviewer #2 (Public review): 

Summary: 

Mutations in the prolyl hydroxylase, PHD2, cause erythrocytosis and, in some cases, can result in tumorigenesis. Taber and colleagues test the structural and functional consequences of seven patientderived missense mutations in PHD2 using cell-based reporter and stability assays, and multiple biophysical assays, and find that most mutations are destabilizing. Interestingly, they discover a PHD2 mutant that can hydroxylate the C-terminal ODD, but not the N-terminal ODD, which suggests the importance of N-terminal ODD for biology. A major strength of the manuscript is the multidisciplinary approach used by the authors to characterize the functional and structural consequences of the mutations. However, the manuscript had several major weaknesses, such as an incomplete description of how the NMR was performed, a justification for using neighboring residues as a surrogate for looking at prolyl hydroxylation directly, or a reference to the clinical case studies describing the phenotypes of patient mutations. Additionally, the experimental descriptions for several experiments are missing descriptions of controls or validation, which limits their strength in supporting the claims of the authors. 

Strengths: 

(1) This manuscript is well-written and clear. 

(2) The authors use multiple assays to look at the effects of several disease-associated mutations, which support the claims. 

(3) The identification of P317R as a mutant that loses activity specifically against NODD, which could be a useful tool for further studies in cells. 

Weaknesses: 

Major: 

(1) The source data for the patient mutations (Figure 1) in PHD2 is not referenced, and it's not clear where this data came from or if it's publicly available. There is no section describing this in the methods. 

Clinical and patient information on disease-causing PHD2 mutants was compiled from various case reports and summarized in an excel sheet found in the Supplementary Information.  The case reports are cited in this excel file.  A reference to the supplementary data has been added to the Figure 1 legend and in the introduction.

(2) The NMR hydroxylation assay. 

A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B. 

As the reviewer mentioned, the assay that we developed directly measures the target proline residues.  This assay is ideal when mutations near the prolines are studied, such as A403, Y565 (He et al[3]).  In this previous work, we observed that the shifting of the target proline cross-peaks due to change in electronegativity on the pyrrolidine ring of proline in turn impacted the neighboring residues[3], which meant that the neighboring residues can be used as reporter residues for certain purposes.  In this study, we focused on investigating the mutations on PHD2 while leaving the sequence of the HIF-1α unchanged by using solely 15N-HSQC-based experiments without the need for double-labeled samples.  Nonetheless, we thank the reviewer for pointing out the confusion in the text and we have corrected and clarified our description of this assay.

B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec? 

For previous studies, we performed intercalated 15N-HSQC and 13C-CON experiments for the kinetic measurements of wild-type HIF-1α and mutants.  We observed that the shifting pattern of A403 and I566 in the 15N-HSQC spectra aligned well with the ones of P402 and P564, respectively, in the 13C-CON spectra.  Representative data has been added to Supplemental Data.

C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this? 

This is an astute observation by the reviewer.  We checked and confirmed that for all kinetic datasets, the peak intensities of the end point residue are always slightly lower than the ones of the starting.  This includes the cases for PHD2 A228S and P317R in 5B, although not as obvious as the one of PHD2 WT.  We agree with the reviewer that the sample dilution is a factor as a total volume of 16 microliters of reaction components was added to the solution to trigger the reaction after the first spectrum was acquired.  It is also likely that rate of prolyl hydroxylation becomes extremely slow with only a low amount of substrate available in the system.  Therefore, the reaction would not be 100% complete which was detected by the sensitive NMR experimentation.

(3) Data validating the CRISPR KO HEK293A cells is missing. 

We thank the reviewer for noting this oversight.  Western blots validating PHD2 KO in HEK293A cells have been added to the Supplementary Data file.

(4) The interpretation of the SEC data for the PHD2 mutants is a little problematic. Subtle alterations in the elution profiles may hint at different hydrodynamic radii, but as the samples were not loaded at equal concentrations or volumes, these data seem more anecdotal, rather than definitive. Repeating this multiple times, using matched samples, followed by comparison with standards loaded under identical buffer conditions, would significantly strengthen the conclusions one could make from the data. 

Agreed.  We have performed an additional experiment as suggested with equal volume and concentration of each PHD2 construct loaded onto the SEC column for better assessment of aggregation.  Notably, our conclusion remained unchanged.

Minor: 

(1) Justification for picking the seven residues is not clearly articulated. The authors say they picked 7 mutants with "distinct residue changes", but no further rationale is provided. 

Additional justification for the selection of the mutants has been added to the ‘Mutations across the PHD2 enzyme induce erythrocytosis’ section.  Briefly, some mutants were chosen based on their frequency in the clinical data and their presence in potential mutational hot spots.  Various mutations were noted at W334 and R371, while F366L was identified in multiple individuals.  Additionally, 9 cases of PHD2-driven disease were reported to be caused from mutations located between residues 200 to 210 while 13 cases were reported between residues 369-379, so G206C and R371H were chosen to represent potential hot spots.  To examine a potential genotype-phenotype relationship, two of the mutants responsible for neuroendocrine tumor development, A228S and H374R, were also selected.  Finally, mutations located close or on catalytic core residues (P317R, R371H, and H374R) were chosen to test for suspected defects.   

(2) A major finding of the paper is that a disease-associated mutation, P317R, can differentially affect HIF1 prolyhydroxylation, however, additional follow-up studies have not been performed to test this in cells or to validate the mutant in another method. Is it the position of the proline within the catalytic core, or the identity of the mutation that accounts for the selectivity? 

This is the very question that we are currently addressing but as a part of a follow-up study.  Indeed, one thought is that the preferential defect observed could be the result of the loss of proline, an exceptionally rigid amino acid that makes contact with the backbone twice, or the addition of a specific amino acid, namely arginine, a flexible amino acid with an added charge at this site.  Although beyond the scope of this manuscript, we will investigate whether such and other characteristics in this region of PHD2/HIF1α interface contribute to the differential hydroxylation. 

Reviewer #3 (Public review): 

Summary: 

This is an interesting and clinically relevant in vitro study by Taber et al., exploring how mutations in PHD2 contribute to erythrocytosis and/or neuroendocrine tumors. PHD2 regulates HIFα degradation through prolyl-hydroxylation, a key step in the cellular oxygen-sensing pathway. 

Using a time-resolved NMR-based assay, the authors systematically analyze seven patient-derived PHD2 mutants and demonstrate that all exhibit structural and/or catalytic defects. Strikingly, the P317R variant retains normal activity toward the C-terminal proline but fails to hydroxylate the N-terminal site. This provides the first direct evidence that N-terminal prolyl-hydroxylation is not dispensable, as previously thought. 

The findings offer valuable mechanistic insight into PHD2-driven effects and refine our understanding of HIF regulation in hypoxia-related diseases. 

Strengths: 

The manuscript has several notable strengths. By applying a novel time-resolved NMR approach, the authors directly assess hydroxylation at both HIF1α ODD sites, offering a clear functional readout. This method allows them to identify the P317R variant as uniquely defective in NODD hydroxylation, despite retaining normal activity toward CODD, thereby challenging the long-held view that the N-terminal proline is biologically dispensable. The work significantly advances our understanding of PHD2 function and its role in oxygen sensing, and might help in the future interpretation and clinical management of associated erythrocytosis. 

Weaknesses: 

(1) There is a lack of in vivo/ex vivo validation. This is actually required to confirm whether the observed defects in hydroxylation-especially the selective NODD impairment in P317R-are sufficient to drive disease phenotypes such as erythrocytosis.

We thank the reviewer for this comment, and while we agree with this statement, the objective of this study per se was to elucidate the structural and/or functional defect caused by the various diseaseassociated mutations on PHD2.  The subsequent study would be to validate whether the identified defects, in particular the selective NODD impairment, would lead to erythrocytosis in vivo.  However, we feel that such study would be beyond the scope of this manuscript.

(2) The reliance on HRE-luciferase reporter assays may not reliably reflect the PHD2 function and highlights a limitation in the assessment of downstream hypoxic signaling. 

Agreed.  All experimental assays and systems have limitations.  The HRE-luciferase assay used in the present manuscript also has limitations such as the continuous expression of exogenous PHD2 mutants driven via CMV promoter.  Thus, we performed several additional biophysical methodologies to interrogate the disease-causing PHD2 mutants.  The limitations of the luciferase assay have been expanded in the revised manuscript. 

(3) The study clearly documents the selective defect of the P317R mutant, but the structural basis for this selectivity is not addressed through high-resolution structural analysis (e.g., cryo-EM). 

We thank the reviewer for the comment.  While solving the structure of PHD2 P317R in complex with HIFα substrate is beyond the scope for this study, a structure of PHD2 P317R in complex with a clinically used inhibitor has been solved (PDB:5LAT).  In analyzing this structure and that of PHD2 WT in complex with NODD, Chowdhury et al[2] stated that P317 makes hydrophobic contacts with LXXLAP motif on HIFα and R317 is predicted to interact differently with this motif.  While this analysis does not directly elucidate the reason for the preferential NODD defect, it supports the possibility that P317R substitution may be more detrimental for enzymatic activity on NODD than CODD.  We have discussed this notion in the revised manuscript. 

(4) Given the proposed central role of HIF2α in erythrocytosis, direct assessment of HIF2α hydroxylation by the mutants would have strengthened the conclusions. 

We thank the reviewer for this comment, but we feel that such study would be beyond the scope of the present study.  We observed that the PHD2 binding patterns to HIF1α and HIF2α were similar, and we have previously assigned >95% of the amino acids in HIF1α ODD for NMR study[3]. Thus, we first focused on the elucidation of possible defects on disease-associated PHD2 mutants using HIF1α as the substrate with the supposition that an identified deregulation on HIF1α could be extended to HIF2α paralog.  However, we agree with the reviewer that future studies should examine the impact of PHD2 mutants directly on HIF2α.  

References:

(1) Flashman, E. et al. Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases. J Biol Chem 283, 3808-3815 (2008).

(2) Chowdhury, R. et al. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun 7, 12673 (2016).

(3) He, W., Gasmi-Seabrook, G.M.C., Ikura, M., Lee, J.E. & Ohh, M. Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1alpha. Proc Natl Acad Sci U S A 121, e2408104121 (2024).

Reviewer #1 (Recommendations for the authors): 

(1) To increase the impact and significance of this work, I would recommend determining the mechanism by which A228S and F366L impair PHD2. Are these mutations affecting interactions with proteins other than HIF1a? Furthermore, does the F366L mutation affect the hydroxylation rate? This should be measured. The authors should also perform a more in-depth structural analysis of these mutations and perhaps use AlphaFold to identify how these sites may be involved in other interactions. 

We thank the reviewer for the recommendations.  A paragraph discussing the quandary of A228S and F366L has been added to the discussion as well as an in-depth structural analysis of each selected mutant.  While AlphaFold is excellent at predicting protein structures overall, its capability to predict the effect of single point mutation, such as those in this study, is limited.  Therefore, it was not utilized for this paper.

(2) For the aggregation assay, I recommended injecting the same quantity of protein on the SEC. If the aggregation-prone mutants' yields were too low, then reduced amounts of the other mutants should be injected. 

Agreed.  An additional experiment was performed in which similar concentrations of each mutant protein was loaded onto the SEC column and chromatograms was normalized according to the molecular concentration.  Results from this experiment have been added to replace the previously performed aggregation assay.  Notably, the data from the revised experiment did not change the outcome or conclusion of the study.

(3) For the NMR kinetics data, the authors should discuss the impact of affinities and concentrations on the reaction rate and incorporate this analysis framework to interpret their data. 

Done.  As discussed in depth in response to Public Reviewer 1’s fourth comment, we observed only a subtle reduction in hydroxylation efficiency of HIF1aCODD by PHD2 P317R in comparison to PHD2 WT.  Upon performing BLI, we found PHD2 P317R displays only a mild binding defect on the CODD and NODD.  The WT-like binding to the NODD by PHD2 P317R appears to be inconsistent with the severe defect in NODD hydroxylation via PHD2 P317R as measured via NMR.   These results suggest that there are supporting residues within the PHD2/NODD interface that help maintain binding to NODD but compromise the efficiency of NODD hydroxylation upon PHD2 P317R mutation.

Reviewer #2 (Recommendations for the authors): 

It is unclear where the source data came from describing the patient mutations, or if it is publicly available. Several minor issues were noted with several of the figures or methods: 

(1) Figure 2C. It is not clear what data are being compared for significance. The lines don't seem to clearly distinguish this. 

Done.  The significance lines have been adjusted in the figure to better convey which data are being compared.

(2) Please incorporate the calculated biophysical constants (KD, TM, etc, average +/- std dev) from the tables into the figures or figure legends that show the data from which they are calculated.  

Done.  References to the corresponding tables have been added to the appropriate figure legends.

(3) Figure 3C, the data for F366L do not appear normalized in the same way as the other constructs. 

CD melt values for F366L were normalized in the same way as other constructs but due to noisier data acquired between 25-37°C, the top value of the sigmoidal curve is slightly higher than the other constructs (F366L: 1.066, WT: 1.007, A228S: 1.000, P317R: 1.015, R371H: 1.005). 

(4) For Figure 1B, it would be helpful to highlight the mutants characterized in the current study with a different color/symbol to help show the number of cases. 

Done.  Dots representing the selected mutants have been highlighted in red in Figure 1B.

(5) A description of the isotopic labeling of PHD2 is missing from the methods.

Due to the nature of the NMR assay, no isotopic labeling was required for PHD2.

Reviewer #3 (Recommendations for the authors): 

(1) To further strengthen the manuscript, the authors could consider exploring the relevance of their in vitro findings in a more physiological context. 

We thank the reviewer for the suggestion, and we will certainly consider furthering our investigation in a more physiological context for future studies.

(2) If technically feasible, integrating direct analyses of HIF2α regulation by the PHD2 mutants would better reflect the clinical phenotype, given the known importance of HIF2α in erythrocytosis. 

We agree that HIF2α is important in the context of erythrocytosis, but through MST we observed no difference in binding pattern between HIF1 and HIF2 and the selected PHD2 mutants.  As we had previously assigned >95% of residues for HIF1α ODD for NMR assay, we analyzed HIF1 with the supposition that any defects observed would likely apply to HIF2.  However, we agree that future studies on the impact of PHD2 mutants directly on HIF2 would be beneficial to supplement our understanding of pseudohypoxic disease.

(3) Additionally, although perhaps more suitable for future work or discussion, structural modeling or highresolution structural studies of the P317R variant could offer valuable insight into the observed NODD selectivity defect. 

We thank the reviewer for the suggestion. While solving the structure of PHD2 P317R in complex with NODD is beyond the scope of this manuscript, a crystal structure of PHD2 P317R in complex with an inhibitor has been solved and insights from this structure have been added to the discussion. 

(4) Finally, a brief clarification or discussion of the limitations of the luciferase reporter assay-especially in the context of aggregation-prone mutants-would help readers better interpret the functional data. 

We thank the reviewer for the suggestion.  The limitations of the luciferase reporter assay in regard to its inability to detect defects with aggregation-prone mutants have been elaborated on in the discussion.

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Profa. Flor Chávez.

¡Hola a tod@s!

Con el gusto de saludarles, esperando se encuentren muy bien, les pido que por favor instalen la extensión hypothes.is en su navegador (ver tablón de Classroom).

Posteriormente, en el espacio designado en , identifiquen la hipótesis del autor y la explicación o justificación que ofrece para sustentarla. Coméntenla y emitan su opinión al respecto.

También, encuentren por qué Luis Montaño afirma que la Administración es una disciplina relativamente reciente en México, con poca tradición en investigación y orientada principalmente hacia las áreas docentes.

Finalmente, respondan la siguiente pregunta: ¿Qué opina el autor acerca del liderazgo?

¡Comenzamos!

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

Taber et al report the biochemical characterization of 7 mutations in PHD2 that induce erythrocytosis.

Their goal is to provide a mechanism for how these mutations cause the disease. PHD2 hydroxylates HIF1a in the presence of oxygen at two distinct proline residues (P564 and P402) in the "oxygen degradation domain" (ODD). This leads to the ubiquitylation of HIF1a by the VHL E3 ligase and its subsequent degradation. Multiple mutations have been reported in the EGLN1 gene (coding for PHD2), which are associated with pseudohypoxic diseases that include erythrocytosis. Furthermore, 3 mutations in PHD2 also cause pheochromocytoma and paraganglioma (PPGL), a neuroendocrine tumour. These mutations likely cause elevated levels of HIF1a, but their mechanisms are unclear. Here, the authors analyze mutations from 152 case reports and map them on the crystal structure. They then focus on 7 mutations, which they clone in a plasmid and transfect into PHD2-KO to monitor HIF1a transcriptional activity via a luciferase assay. All mutants show impaired activation. Some mutants also impaired stability in pulse chase turnover assays (except A228S, P317R, and F366L). In vitro purified PHD2 mutants display a minor loss in thermal stability and some propensity to aggregate. Using MST technology, they show that P317R is strongly impaired in binding to HIF1a and HIF2a, whereas other mutants are only slightly affected. Using NMR, they show that the PHD2 P317R mutation greatly reduces hydroxylation of P402 (HIF1a NODD), as well as P562 (HIF1a CODD), but to a lesser extent. Finally, BLI shows that the P317R mutation reduces affinity for CODD by 3-fold, but not NODD.  

Strengths: 

(1) Simple, easy-to-follow manuscript. Generally well-written. 

(2) Disease-relevant mutations are studied in PHD2 that provide insights into its mechanism of action. 

(3) Good, well-researched background section. 

Weaknesses: 

(1) Poor use of existing structural data on the complexes of PHD2 with HIF1a peptides and various metals and substrates. A quick survey of the impact of these mutations (as well as analysis by Chowdhury et al, 2016) on the structure and interactions between PHD2 peptides of HIF1a shows that the P317R mutation interferes with peptide binding. By contrast, F366L will affect the hydrophobic core, and A228S is on the surface, and it's not obvious how it would interfere with the stability of the protein. 

Thank you for the comment.  We will further analyze the mutations on the available PHD2 crystal structures in complex with HIFa to discern how these substitution mutations may impact PHD2 structure and function.  

(2) To determine aggregation and monodispersity of the PHD2 mutants using size-exclusion chromatography (SEC), equal quantities of the protein must be loaded on the column. This is not what was done. As an aside, the colors used for the SEC are very similar and nearly indistinguishable. 

Agreed.  We will perform additional experiment as suggested by the reviewer to further assess aggregation and hydrodynamic size.  The colors used in the graph will be changed for a clearer differentiation between samples.

(3) The interpretation of some mutants remains incomplete. For A228S, what is the explanation for its reduced activity? It is not substantially less stable than WT and does not seem to affect peptide hydroxylation. 

We agree with the reviewer that the causal mechanism for some of the tested disease-causing mutants remain unclear.  The negative findings also raise the notion, perhaps considered controversial, that there may be other substrates of PHD2 that are impacted by certain mutations, which contribute to disease pathogenesis.  We will expand our discussion accordingly. 

(4) The interpretation of the NMR prolyl hydroxylation is tainted by the high concentrations used here. First of all, there is a likely a typo in the method section; the final concentration of ODD is likely 0.18 mM, and not 0.18 uM (PNAS paper by the same group in 2024 reports using a final concentration of 230 uM). Here, I will assume the concentration is 180 uM. Flashman et al (JBC 2008) showed that the affinity of the NODD site (P402; around 10 uM) for PHD2 is 10-fold weaker than CODD (P564, around 1 uM). This likely explains the much faster kinetics of hydroxylation towards the latter. Now, using the MST data, let's say the P317R mutation reduces the affinity by 40-fold; the affinity becomes 400 uM for NODD (above the protein concentration) and 40 uM for CODD (below the protein concentration). Thus, CODD would still be hydroxylated by the P317R mutant, but not NODD. 

The HIF1α concentration was indeed an oversight, which will be corrected to 0.18 mM.  The study by Flashman et al.[1] showing PHD2 having a lower affinity to the NODD than CODD likely contributes to the differential hydroxylation rates via PHD2 WT.  We showed here via MST that PHD2 P317R had Kd of 320 ± 20 uM for HIF1αCODD, which should have led to a severe enzymatic defect, even at the high concentrations used for NMR (180 uM).  However, we observed only a subtle reduction in hydroxylation efficiency in comparison to PHD2 WT.  Thus, we performed another binding method using BLI that showed a mild binding defect on CODD by PHD2 P317R, consistent with NMR data.  The perplexing result is the WT-like binding to the NODD by PHD2 P317R, which appears inconsistent with the severe defect in NODD hydroxylation via PHD2 P317R as measured via NMR.  These results suggest that there are supporting residues within the PHD2/NODD interface that help maintain binding to NODD but compromise the efficiency of NODD hydroxylation upon PHD2 P317R mutation. We will perform additional binding experiments to further interrogate and validate the binding affinity of PHD2 P317R to NODD and CODD.

(5) The discrepancy between the MST and BLI results does not make sense, especially regarding the P317R mutant. Based on the crystal structures of PHD2 in complex with the ODD peptides, the P317R mutation should have a major impact on the affinity, which is what is reported by MST. This suggests that the MST is more likely to be valid than BLI, and the latter is subject to some kind of artefact. Furthermore, the BLI results are inconsistent with previous results showing that PHD2 has a 10-fold lower affinity for NODD compared to CODD. 

The reviewer’s structural prediction that P317R mutation should cause a major binding defect, while agreeable with our MST data, is incongruent with our NMR and the data from Chowdhury et al.[2] that showed efficient hydroxylation of CODD via PHD2 P317R.  Moreover, we have attempted to model NODD and CODD on apo PHD2 P317R structure and found that the mutation had no major impact on CODD while the mutated residue could clash with NODD, causing a shifting of peptide positioning on the protein.  However, these modeling predictions, like any in silico projections, would need experimental validation.  As mentioned in our preceding response, we also performed BLI, which showed that PHD2 P317R had a minor binding defect for CODD, consistent with the NMR results and findings by Chowdhury et al[2].  NODD binding was also measured with BLI as purified NODD peptides were not amenable for soluble-based MST assay, which showed similar K_d’s for PHD2 WT and P317R.  Considering the absence of NODD hydroxylation via PHD2 P317R as measured by NMR and modeling on apo PHD2 P317R, we posit that P317R causes deviation of NODD from its original orientation that may not affect binding due to the other interactions from the surrounding elements but unfortunately disallows NODD from turnover.  Further study would be required to validate such notion, which we feel is beyond the scope of this manuscript.  However, we will perform additional binding experiments to further interrogate PHD2 P317R binding to NODD.   

(6) Overall, the study provides some insights into mutants inducing erythrocytosis, but the impact is limited. Most insights are provided on the P317R mutant, but this mutant had already been characterized by Chowdhury et al (2016). Some mutants affect the stability of the protein in cells, but then no mechanism is provided for A228S or F366L, which have stabilities similar to WT, yet have impaired HIF1a activation. 

We thank the reviewer for raising these and other limitations.  We will expand on the shortcomings of the present study but would like to underscore that the current work using the recently described NMR assay along with other biophysical analyses suggests a previously under-appreciated role of NODD hydroxylation in the normal oxygen-sensing pathway.  

Reviewer #2 (Public review): 

Summary: 

Mutations in the prolyl hydroxylase, PHD2, cause erythrocytosis and, in some cases, can result in tumorigenesis. Taber and colleagues test the structural and functional consequences of seven patientderived missense mutations in PHD2 using cell-based reporter and stability assays, and multiple biophysical assays, and find that most mutations are destabilizing. Interestingly, they discover a PHD2 mutant that can hydroxylate the C-terminal ODD, but not the N-terminal ODD, which suggests the importance of N-terminal ODD for biology. A major strength of the manuscript is the multidisciplinary approach used by the authors to characterize the functional and structural consequences of the mutations. However, the manuscript had several major weaknesses, such as an incomplete description of how the NMR was performed, a justification for using neighboring residues as a surrogate for looking at prolyl hydroxylation directly, or a reference to the clinical case studies describing the phenotypes of patient mutations. Additionally, the experimental descriptions for several experiments are missing descriptions of controls or validation, which limits their strength in supporting the claims of the authors. 

Strengths: 

(1) This manuscript is well-written and clear. 

(2) The authors use multiple assays to look at the effects of several disease-associated mutations, which support the claims. 

(3) The identification of P317R as a mutant that loses activity specifically against NODD, which could be a useful tool for further studies in cells. 

Weaknesses: 

Major: 

(1) The source data for the patient mutations (Figure 1) in PHD2 is not referenced, and it's not clear where this data came from or if it's publicly available. There is no section describing this in the methods.

Clinical and patient information on disease-causing PHD2 mutants was compiled from various case reports and summarized in an excel sheet found in the Supplementary Information.  The case reports are cited in this excel file.  A reference to the supplementary data will be added to the Figure 1 legend and in the introduction.

(2) The NMR hydroxylation assay. 

A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B. 

As the reviewer mentioned, the assay that we developed directly measures the target proline residues.  This assay is ideal when mutations near the prolines are studied, such as A403, Y565 (He et al[3]).  In this previous work, we observed that the shifting of the target proline cross-peaks due to change in electronegativity on the pyrrolidine ring of proline in turn impacted the neighboring residues[3], which meant that the neighboring residues can be used as reporter residues for certain purposes.  In this study, we focused on investigating the mutations on PHD2 while leaving the sequence of the HIF-1α unchanged by using solely 15N-HSQC-based experiments without the need for double-labeled samples.  Nonetheless, we thank the reviewer for pointing out the confusion in the text and we will correct and clarify our description of this assay.

B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec? 

For previous studies, we performed intercalated 15N-HSQC and 13C-CON experiments for the kinetic measurements of wild-type HIF-1α and mutants.  We observed that the shifting pattern of A403 and I566 in the 15N-HSQC spectra aligned well with the ones of P402 and P564, respectively, in the 13C-CON spectra.  Representative data will be added to Supplemental Data.

C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this? 

This is an astute observation by the reviewer.  We checked and confirmed that for all kinetic datasets, the peak intensities of the end point residue are always slightly lower than the ones of the starting.  This includes the cases for PHD2 A228S and P317R in 5B, although not as obvious as the one of PHD2 WT.  We agree with the reviewer that the sample dilution is a factor as a total volume of 16 microliters of reaction components was added to the solution to trigger the reaction after the first spectrum was acquired.  It is also likely that rate of prolyl hydroxylation becomes extremely slow with only a low amount of substrate available in the system.  Therefore, the reaction would not be 100% complete which was detected by the sensitive NMR experimentation.

(3) Data validating the CRISPR KO HEK293A cells is missing. 

We thank the reviewer for noting this oversight.  Western blots validating PHD2 KO in HEK293A cells will be added to the Supplementary Data file.

(4) The interpretation of the SEC data for the PHD2 mutants is a little problematic. Subtle alterations in the elution profiles may hint at different hydrodynamic radii, but as the samples were not loaded at equal concentrations or volumes, these data seem more anecdotal, rather than definitive. Repeating this multiple times, using matched samples, followed by comparison with standards loaded under identical buffer conditions, would significantly strengthen the conclusions one could make from the data. 

Agreed.  We will perform additional experiments as suggested with equal volume and concentration of each PHD2 construct loaded onto the SEC column for better assessment of aggregation.

Minor: 

(1) Justification for picking the seven residues is not clearly articulated. The authors say they picked 7 mutants with "distinct residue changes", but no further rationale is provided. 

Additional justification for the selection of the mutants will be added to the ‘Mutations across the PHD2 enzyme induce erythrocytosis’ section.  Briefly, some mutants were chosen based on their frequency in the clinical data and their presence in potential mutational hot spots.  Various mutations were noted at W334 and R371, while F366L was identified in multiple individuals.  Additionally, 9 cases of PHD2-driven disease were reported to be caused from mutations located between residues 200 to 210 while 13 cases were reported between residues 369-379, so G206C and R371H were chosen to represent potential hot spots.  To examine a potential genotype-phenotype relationship, two of the mutants responsible for neuroendocrine tumor development, A228S and H374R, were also selected.  Finally, mutations located close or on catalytic core residues (P317R, R371H, and H374R) were chosen to test for suspected defects.   

(2) A major finding of the paper is that a disease-associated mutation, P317R, can differentially affect HIF1 prolyhydroxylation, however, additional follow-up studies have not been performed to test this in cells or to validate the mutant in another method. Is it the position of the proline within the catalytic core, or the identity of the mutation that accounts for the selectivity? 

This is the very question that we are currently addressing but as a part of a follow-up study.  Indeed, one thought is that the preferential defect observed could be the result of the loss of proline, an exceptionally rigid amino acid that makes contact with the backbone twice, or the addition of a specific amino acid, namely arginine, a flexible amino acid with an added charge at this site.  Although beyond the scope of this manuscript, we will investigate whether such and other characteristics in this region of PHD2/HIF1α interface contribute to the differential hydroxylation. 

Reviewer #3 (Public review): 

Summary: 

This is an interesting and clinically relevant in vitro study by Taber et al., exploring how mutations in PHD2 contribute to erythrocytosis and/or neuroendocrine tumors. PHD2 regulates HIFα degradation through prolyl-hydroxylation, a key step in the cellular oxygen-sensing pathway. 

Using a time-resolved NMR-based assay, the authors systematically analyze seven patient-derived PHD2 mutants and demonstrate that all exhibit structural and/or catalytic defects. Strikingly, the P317R variant retains normal activity toward the C-terminal proline but fails to hydroxylate the N-terminal site. This provides the first direct evidence that N-terminal prolyl-hydroxylation is not dispensable, as previously thought. 

The findings offer valuable mechanistic insight into PHD2-driven effects and refine our understanding of HIF regulation in hypoxia-related diseases. 

Strengths: 

The manuscript has several notable strengths. By applying a novel time-resolved NMR approach, the authors directly assess hydroxylation at both HIF1α ODD sites, offering a clear functional readout. This method allows them to identify the P317R variant as uniquely defective in NODD hydroxylation, despite retaining normal activity toward CODD, thereby challenging the long-held view that the N-terminal proline is biologically dispensable. The work significantly advances our understanding of PHD2 function and its role in oxygen sensing, and might help in the future interpretation and clinical management of associated erythrocytosis. 

Weaknesses: 

(1) There is a lack of in vivo/ex vivo validation. This is actually required to confirm whether the observed defects in hydroxylation-especially the selective NODD impairment in P317R-are sufficient to drive disease phenotypes such as erythrocytosis. 

We thank the reviewer for this comment, and while we agree with this statement, the objective of this study per se was to elucidate the structural and/or functional defect caused by the various diseaseassociated mutations on PHD2. The subsequent study would be to validate whether the identified defects, in particular the selective NODD impairment, would lead to erythrocytosis in vivo.  However, we feel that such study would be beyond the scope of this manuscript.

(2) The reliance on HRE-luciferase reporter assays may not reliably reflect the PHD2 function and highlights a limitation in the assessment of downstream hypoxic signaling. 

Agreed.  All experimental assays and systems have limitations. The HRE-luciferase assay used in the present manuscript also has limitations such as the continuous expression of exogenous PHD2 mutants driven via CMV promoter. Thus, we performed several additional biophysical methodologies to interrogate the disease-causing PHD2 mutants. The limitations of the luciferase assay will be expanded in the revised manuscript. 

(3) The study clearly documents the selective defect of the P317R mutant, but the structural basis for this selectivity is not addressed through high-resolution structural analysis (e.g., cryo-EM). 

We thank the reviewer for the comment.  While solving the structure of PHD2 P317R in complex with HIFα substrate is beyond the scope for this study, a structure of PHD2 P317R in complex with a clinically used inhibitor has been solved (PDB:5LAT).  In analyzing this structure and that of PHD2 WT in complex with NODD, Chowdhury et al[2] stated that P317 makes hydrophobic contacts with LXXLAP motif on HIFα and R317 is predicted to interact differently with this motif. While this analysis does not directly elucidate the reason for the preferential NODD defect, it supports the possibility that P317R substitution may be more detrimental for enzymatic activity on NODD than CODD. We will discuss this notion in the revised manuscript. 

(4) Given the proposed central role of HIF2α in erythrocytosis, direct assessment of HIF2α hydroxylation by the mutants would have strengthened the conclusions. 

We thank the reviewer for this comment, but we feel that such study would be beyond the scope of the present study. We observed that the PHD2 binding patterns to HIF1α and HIF2α were similar, and we have previously assigned >95% of the amino acids in HIF1α ODD for NMR study[3]. Thus, we first focused on the elucidation of possible defects on disease-associated PHD2 mutants using HIF1α as the substrate with the supposition that an identified deregulation on HIF1α could be extended to HIF2α paralog. 

However, we agree with the reviewer that future studies should examine the impact of PHD2 mutants directly on HIF2α.  

References:

(1) Flashman, E. et al. Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases. J Biol Chem 283, 3808-3815 (2008).

(2) Chowdhury, R. et al. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun 7, 12673 (2016).

(3) He, W., Gasmi-Seabrook, G.M.C., Ikura, M., Lee, J.E. & Ohh, M. Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1alpha. Proc Natl Acad Sci U S A 121, e2408104121 (2024).

Author response:

The following is the authors’ response to the previous reviews.

Reviewer 1:

The authors frequently refer to their predictions and theory as being causal, both in the manuscript and in their response to reviewers. However, causal inference requires careful experimental design, not just statistical prediction. For example, the claim that "algorithmic differences between those with BPD and matched healthy controls" are "causal" in my opinion is not warranted by the data, as the study does not employ experimental manipulations or interventions which might predictably affect parameter values. Even if model parameters can be seen as valid proxies to latent mechanisms, this does not automatically mean that such mechanisms cause the clinical distinction between BPD and CON, they could plausibly also refer to the effects of therapy or medication. I recommend that such causal language, also implicit to expressions like "parameter influences on explicit intentional attributions", is toned down throughout the manuscript.

Thankyou for this chance to be clearer in the language. Our models and paradigm introduce a from of temporal causality, given that latent parameter distributions are directly influenced by latent parameter estimates at a previous point in time (self-uncertainty and other uncertainty directly governs social contagion). Nevertheless, we appreciate the reviewers perspective and have now toned down the language to reflect this.

Abstract:

‘Our model makes clear predictions about the mechanisms of social information generalisation concerning both joint and individual reward.’

Discussion:

‘We can simulate this by modelling a framework that incorporates priors based on both self and a strong memory impression of a notional other (Figure S3).’

‘We note a strength of this work is the use of model comparison to understand algorithmic differences between those with BPD and matched healthy controls.’

Although the authors have now much clearer outlined the stuy's aims, there still is a lack of clarity with respect to the authors' specific hypotheses. I understand that their primary predictions about disruptions to self-other generalisation processes underlying BPD are embedded in the four main models that are tested, but it is still unclear what specific hypotheses the authors had about group differences with respect to the tested models. I recommend the authors specify this in the introduction rather than refering to prior work where the same hypotheses may have been mentioned.

Thankyou for this further critique which has enabled us to more cleary refine our introduction. We have now edited our introduction to be more direct about our hypotheses, that these hypotheses are instantiated into formal models, and what our predictions were. We have also included a small section on how previous predictions from other computational assessments of BPD link to our exploratory work, and highlighted this throughout the manuscript.

‘This paper seeks to address this gap by testing explicitly how disruptions in self-other generalization processes may underpin interpersonal disruptions observed in BPD. Specifically, our hypotheses were: (i) healthy controls will demonstrate evidence for both self-insertion and social contagion, integrating self and other information during interpersonal learning; and (ii) individuals with BPD will exhibit diminished self-other integration, reflected in stronger evidence for observations that assume distinct self-other representations.

We tested these hypotheses by designing a dynamic, sequential, three-phase Social Value Orientation (Murphy & Ackerman, 2014) paradigm—the Intentions Game—that would provide behavioural signatures assessing whether BPD differed from healthy controls in these generalization processes (Figure 1A). We coupled this paradigm with a lattice of models (M1-M4) that distinguish between self-insertion and social contagion (Figure 1B), and performed model comparison:

M1. Both self-to-other (self-insertion) and other-to-self (social contagion) occur before and after learning M2. Self-to-other transfer only occurs M3. Other-to-self transfer only occurs M4. Neither transfer process, suggesting distinct self-other representations

We additionally ran exploratory analysis of parameter differences and model predictions between groups following from prior work demonstrating changes in prosociality (Hula et al., 2018), social concern (Henco et al., 2020), belief stability (Story et al., 2024a), and belief updating (Story, 2024b) in BPD to understand whether discrepancies in self-other generalisation influences observational learning. By clearly articulating our hypotheses, we aim to clarify the theoretical contribution of our findings to existing literature on social learning, BPD, and computational psychiatry.’

Caveats should also be added about the exploratory nature of the many parameter group comparisons. If there are any predictions about group differences that can be made based on prior literature, the authors should make such links clear.

Thank you for this. We have now included caveats in the text to highlight the exploratory nature of these group comparisons, and added direct links to relevant literature where able:

Introduction

‘We additionally ran exploratory analysis of parameter differences and model predictions between groups following from prior work demonstrating changes in prosociality (Hula et al., 2018), social concern (Henco et al., 2020), belief stability (Story et al., 2024a), and belief updating (Story, 2024b) in BPD to understand whether discrepancies in self-other generalisation influences observational learning. By clearly articulating our hypotheses, we aim to clarify the theoretical contribution of our findings to existing literature on social learning, BPD, and computational psychiatry.’

Model Comparison

‘We found that CON participants were best fit at the group level by M1 (Frequency = 0.59, Exceedance Probability = 0.98), whereas BPD participants were best fit by M4 (Frequency = 0.54, Exceedance Probability = 0.86; Figure 2A). This suggests CON participants are best fit by a model that fully integrates self and other when learning, whereas those with BPD are best explained as holding disintegrated and separate representations of self and other that do not transfer information back and forth.

We first explore parameters between separate fits (see Methods). Later, in order to assuage concerns about drawing inferences from different models, we examined the relationships between the relevant parameters when we forced all participants to be fit to each of the models (in a hierarchical manner, separated by group). In sum, our model comparison is supported by convergence in parameter values when comparisons are meaningful (see Supplementary Materials). We refer to both types of analysis below.’

Phase 2 analysis

‘Prior work predicts those with BPD should focus more intently on public social information, rather than private information that only concerns one party (Henco et al., 2020). In BPD participants, only new beliefs about the relative reward preferences – mutual outcomes for both player - of partners differed (see Fig 2E): new median priors were larger than median preferences in phase 1 (mean = -0.47; = -6.10, 95%HDI: -7.60, -4.60).’

‘Models of moral preference learning (Story et al., 2024) predicts that BPD vs non-BPD participants have more rigid beliefs about their partners. We found that BPD participants were equally flexible around their prior beliefs about a partner’s relative reward preferences (= -1.60, 95%HDI: -3.42, 0.23), and were less flexible around their beliefs about a partner’s absolute reward preferences (=-4.09, 95%HDI: -5.37, -2.80), versus CON (Figure 2B).’

Phase 3 analysis

‘Prior work predicts that human economic preferences are shaped by observation (Panizza, et al., 2021; Suzuki et al. 2016; Yu et al, 2021), although little-to-no work has examined whether contagion differs for relative vs. absolute preferences. Associative models predict that social contagion may be exaggerated in BPD (Ereira et al., 2018).… As a whole, humans are more susceptible to changing relative preferences more than selfish, absolute reward preferences, and this is disrupted in BPD.’

Psychometric and Intentional Attribution analysis

‘Childhood trauma, persecution, and poor mentalising in BPD are all predicted to disrupt one’s ability to change (Fonagy & Luyten, 2009).’

‘Prior work has also predicted that partner-participant preference disparity influences mental state attributions (Barnby et al., 2022; Panizza et al., 2021).’

I'm not sure I understand why the authors, after adding multiple comparison correction, now list two kinds of p-values. To me, this is misleading and precludes the point of multiple comparison corrections, I therefore recommend they report the FDR-adjusted p-values only. Likewise, if a corrected p-value is greater than 0.05 this should not be interpreted as a result.

We have now adjusted the exploratory results to include only the FDR corrected values in the text.

‘We assessed conditional psychometric associations with social contagion under the assumption of M3 for all participants. We conducted partial correlation analyses to estimate relationships conditional on all other associations and retained all that survived bootstrapping (5000 reps), permutation testing (5000 reps), and subsequent FDR correction. When not controlled for group status, RGPTSB and CTQ scores were both moderately associated with MZQ scores (RGPTSB r = 0.41, 95%CI: 0.23, 0.60, p[fdr]=0.043; CTQ r = 0.354 95%CI: 0.13, 0.56, p[fdr]=0.02). This was not affected by group correction. CTQ scores were moderately and negatively associated with shifts in individualistic reward preferences (; r = -0.25, 95%CI: -0.46, -0.04, p[fdr]=0.03). This was not affected by group correction. MZQ scores were in turn moderately and negatively associated with shifts in prosocial-competitive preferences () between phase 1 and 3 (r = -0.26, 95%CI: -0.46, -0.06, p[fdr]=0.03). This was diminished when controlled for group status (r = 0.13, 95%CI: -0.34, 0.08, p[fdr]=0.20). Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).

Prior work has predicted that partner-participant preference disparity influences mental state attributions (Barnby et al., 2022; Panizza et al., 2021). We tested parameter influences on explicit intentional attributions in Phase 2 while controlling for group status. Attributions included the degree to which they believed their partner was motived by harmful intent (HI) and self-interest (SI). According with prior work (Barnby et al., 2022), greater disparity of absolute preferences before learning was associated on a trend level with reduced attributions of SI (<= -0.23, p[fdr]=0.08), and greater disparity of relative preferences before learning exaggerated attributions of HI = 0.21, p[fdr]=0.08), but did not survive correction (Figure S4B). This is likely due to partners being significantly less individualistic and prosocial on average compared to participants (= -5.50, 95%HDI: -7.60, -3.60; = 12, 95%HDI: 9.70, 14.00); partners are recognised as less selfish and more competitive.’

Can the authors please elaborate why the algorithm proposed to be employed by BPD is more 'entropic', especially given both their self-priors and posteriors about partners' preferences tended to be more precise than the ones used by CON? As far as I understand, there's nothing in the data to suggest BPD predictions should be more uncertain. In fact, this leads me to wonder, similarly to what another reviewer has already suggested, whether BPD participants generate self-referential priors over others in the same way CON participants do, they are just less favourable (i.e., in relation to oneself, but always less prosocial) - I think there is currently no model that would incorporate this possibility? It should at least be possible to explore this by checking if there is any statistical relationship between the estimated θ_ppt^m and 〖p(θ〗_par |D^0).

Thank you for this opportunity to be clearer in our wording. We belief the reviewer is referring to this line in the discussion: ‘In either case, the algorithm underlying the computational goal for BPD participants is far higher in entropy and emphasises a less stable or reliable process of inference.’

We note in the revised Figure 2 panel E and in the results that those with BPD under M4 show insertion along absolute reward (they still expect diminished selfishness in others), but neutral priors over relative reward (around 0, suggesting expectations of neither prosocial or competitive tendencies of others). Thus, θ_ppt^m (self preference) and θ_par^m (other preference) are tightly associated for absolute, but not relative reward.

In our wording, we meant that whether under model M4 or M1, those with BPD either show a neutral prior over relative reward (M4) or a prior with large variance over relative reward (M1), showing expectations of difference between themselves and their partner. In both cases, expectation about a partner’s absolute reward preferences is diminished vs. CON participants. We have strengthened our language in the discussion to clarify this:

‘In either case, the algorithm underlying the computational goal for BPD participants is far higher in uncertainty, whether through a neutral central tendency (M4) or large variance (M1) prior over relative reward in phase 2, and emphasises a less certain and reliable expectation about others.’

To note, social contagion under M3 was highly correlated with contagion under M1 (see Fig S11). This provides some preliminary evidence that trauma impacts beliefs about individualism directly, whereas trauma and persecutory beliefs impact beliefs about prosociality through impaired trait mentalising" - I don't understand what the authors mean by this, can they please elaborate and add some explanation to the main text?

We have now clarified this in the text:

‘Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).’

I noted that at least some of the newly added references have not been added to the bibliography (e.g., Hitchcock et al. 2022).

Thankyou for noticing this omission. We have now ensured all cited works are in the reference list.

Reviewer 2:

The paper is not based on specific empirical hypotheses formulated at the outset, but, rather, it uses an exploratory approach. Indeed, the task is not chosen in order to tackle specific empirical hypotheses. This, in my view, is a limitation since the introduction reads a bit vague and it is not always clear which gaps in the literature the paper aims to fill. As a further consequence, it is not always clear how the findings speak to previous theories on the topic.’

As I wrote in the public review, however, I believe that an important limitation of this work is that it was not based on testing specific empirical hypotheses formulated at the outset, and on selecting the experimental paradigm accordingly. This is a limitation because it is not always clear which gaps in the literature the paper aims to fill. As a consequence, although it has improved substantially compared to the previous version, the introduction remains a bit vague. As a further consequence, it is not always clear how the findings speak to previous theories on the topic. Still, despite this limitation, the paper has many strengths, and I believe it is now ready for publication

Thank you for this further critique. We appreciate your appraisal that the work has improved substantially and is ready for publication. We nevertheless have opted to clarify our introduction and aprior predictions throughout the manuscript (please see response to Reviewer 1).

Reviewer 3:

Although the authors note that their approach makes "clear and transparent a priori predictions," the paper could be improved by providing a clear and consolidated statement of these predictions so that the results could be interpreted vis-a-vis any a priori hypotheses.

In line with comments from both Reviewer 1 and 2, we have clarified our introduction to make it clear what our aprior predictions and hypotheses are about our core aims and exploratory analyses (see response to Reviewer 1).

The approach of using a partial correlation network with bootstrapping (and permutation) was interesting, but the logic of the analysis was not clearly stated. In particular, there are large group (Table 1: CON vs. BPD) differences in the measures introduced into this network. As a result, it is hard to understand whether any partial correlations are driven primarily by mean differences in severity (correlations tend to be inflated in extreme groups designs due to the absence of observation in middle of scales forming each bivariate distribution). I would have found these exploratory analyses more revealing if group membership was controlled for.

Thank you for this chance to be clearer in our methods. We have now written a more direct exposition of this exploratory method:

‘Exploratory Network Analysis

To understand the individual differences of trait attributes (MZQ, RGPTSB, CTQ) with other-to-self information transfer () across the entire sample we performed a network analysis (Borsboom, 2021). Network analysis allows for conditional associations between variables to be estimated; each association is controlled for by all other associations in the network. It also allows for visual inspection of the conditional relationships to get an intuition for how variables are interrelated as a whole (see Fig S11). We implemented network analysis with the bootNet package in r using the ‘estimateNetwork’ function with partial correlations (Epskamp, Borsboom & Fried, 2018). To assess the stability of the partial correlations we further implemented bootstrap resampling with 5000 repetitions using the ‘bootnet’ function. We then additionally shuffled the data and refitted the network 5000 times to determine a p_permuted value; this indicates the probability that a conditional relationship in the original network was within the null distribution of each conditional relationship. We then performed False Discovery Rate correction on the resulting p-values. We additionally controlled for group status for all variables in a supplementary analysis (Table S4).’

We have also further corrected for group status and reported these results as a supplementary table, and also within the main text alongside the main results. We have opted to relegate Figure 4 into a supplementary figure to make the text clearer.

‘We explored conditional psychometric associations with social contagion under the assumption of M3 for all participants (where everyone is able to be influenced by their partner). We conducted partial correlation analyses to estimate relationships conditional on all other associations and retained all that survived bootstrapping (5000 reps), permutation testing (5000 reps), and subsequent FDR correction. When not controlled for group status, RGPTSB and CTQ scores were both moderately associated with MZQ scores (RGPTSB r = 0.41, 95%CI: 0.23, 0.60, p[fdr]=0.043; CTQ r = 0.354 95%CI: 0.13, 0.56, p[fdr]=0.02). This was not affected by group correction. CTQ scores were moderately and negatively associated with shifts in individualistic reward preferences (; r = -0.25, 95%CI: -0.46, -0.04, p[fdr]=0.03). This was not affected by group correction. MZQ scores were in turn moderately and negatively associated with shifts in prosocial-competitive preferences () between phase 1 and 3 (r = -0.26, 95%CI: -0.46, -0.06, p[fdr]=0.03). This was diminished when controlled for group status (r = 0.13, 95%CI: -0.34, 0.08, p[fdr]=0.20). Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).’

Discussion first para: "effected -> affected"

Thanks for spotting this. We have now changed it.

Add "s" to "participant: "Notably, despite differing strategies, those with BPD achieved similar accuracy to CON participant."

We have now changed this.

¿El TECNM capacita a sus maestros para formar profesionistas con esas características?

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

General Statements

We sincerely thank all three reviewers for their thoughtful and constructive feedback. Your comments were invaluable in improving the clarity and quality of our work.

In this study, we revisit a previously overlooked lipophilic dye, demonstrating its utility for live-cell imaging that transport in a non-vesicular pathway and label autophagy related structures. Against the backdrop of increasing attention to membrane contact sites (MCSs), bridge-like lipid transfer proteins (BLTPs), and organelle biogenesis, we aim to propose the possibility of a reversible one-way phospholipid transfer activity that really takes place in living cells.

As Reviewer #1 noted, recent cryo-EM studies (e.g., Oikawa et al.) have highlighted the importance of lipids in autophagosome formation. And there are some existed in vitro studies. However, we believe that we have to think about the consistence of simplified in vitro reconstitution and the complex real cellular environment. In addition, to our knowledge, no studies have directly tracked lipid flow dynamics over time in living cells. We believe our work contributes to this gap by combining three interesting technical approaches: (a) R18 as a lipid-tracing dye, (b) FRAP analysis on the isolation membrane, and (c) the use of Ape1 overexpression to stall autophagosome closure, enabling us to visualize reversible lipid flow in vivo. While these techniques may not appear "fancy," we hope they offer new insights that can inspire further exploration in lipid dynamics story in a real cellular environment.

We appreciate Reviewer #2's comments on our high imaging quality and Reviewer #3's recognition of our approach as an elegant way to study lipid transfer. We have revised the manuscript accordingly and included additional explanations, figure clarifications, and planned experiments to address remaining concerns.

As two key concerns were raised repeatedly by all reviewers, we would like to address them here:

1. Regarding the concern that the evidence for reversible lipid transfer from the IM to the ER is not sufficiently strong:

We are deeply grateful to Reviewer #2 for the insightful suggestion to compare the fluorescence recovery of the adjacent bleached ER to that of the ER-IM MCS, to exclude the possibility that recovery at the ER-IM MCS originates from nearby ER rather than from the IM. Following this suggestion, we performed a quantitative analysis using unbleached ER as a background. Interestingly, in every sample, the adjacent bleached ER consistently showed a significantly lower fluorescence recovery than the ER-IM MCS. We also used the IM as a background for normalization, the difference became even more pronounced, further supporting the idea that the adjacent ER could not be the source of the recovery signal at the ER-IM MCS. These findings strengthen our conclusion that phospholipid recovery at the MCS could be derived from the IM. The updated analysis and corresponding figure panels (Figure 5K, 5L, and 5M), along with the relevant text (lines 384-396), have been revised accordingly.

Regarding the concern that the evidence for R18 transfer via Atg2 as a bridge-like lipid transfer protein is not sufficiently direct:

In addition to the evidence presented in this manuscript, we have now cited our parallel study currently under revision (Sakai et al., bioRxiv 2025.05.24.655882v1), where we provide direct evidence that Atg2 indeed functions as a bridge-like lipid transfer protein, rather than a shuttle. Importantly, we also show in that study that R18 transfer requires the bridge-like structure of Atg2. This new reference has been cited in the revised manuscript, and relevant textual explanations have been added to provide further support.

We hope that the revisions and our revision plan can address the reviewers key concerns. Please find our detailed point-by-point responses below.

Response to the Reviewer ____#____1

In their study, Hao and colleagues exploited the fluorescent fatty acid R18 to follow phospholipid (PL) transfer in vivo from the endoplasmic reticulum to the IM during autophagosome formation. Although the results are interesting, especially the retrograde transport of PLs, based on the provided data, additional control experiments are needed to firmly support the conclusions.

We sincerely thank the reviewer for the positive assessment and agree that additional controls are necessary to support our conclusion. Detailed responses and corresponding revisions are provided below.

An additional point is that the authors also study the internalization of R18 into cells and found a role of lipid flippases and oxysterol binding proteins. While this information could be useful for researchers using this dye, these analyses/findings have no specific connection with the topic of the manuscript, i.e. the PL transfer during autophagosome formation. Therefore, they must be removed.

We thank the reviewer for the thoughtful comment. We understand the concern that the R18 internalization analysis may appear peripheral to the manuscript's main focus on phospholipid transfer during autophagosome formation. However, we respectfully believe that this section is critical for establishing the mechanistic basis as this study represents the first detailed in vivo application of R18 for tracing lipid dynamics. We believe it is interesting that R18 entry is not due to chemically passive diffusion or non-specific adsorption, but occurs through a biologically regulated, non-vesicular lipid transport pathway. This mechanistic context underpins the reliability of using R18 to monitor ER-to-IM lipid transport in the autophagy pathway.

To improve clarity and coherence, we have added explanatory text in the Introduction and at the start of the Results section to explicitly link the internalization assay to the subsequent autophagy-related experiments (line 94-98, 185-187). We hope this helps guide the reader through the rationale and relevance of this part of the study.

Major points:

1) In general, the quality of the microscopy images are quite poor and this make it difficult to assert some of the authors' conclusions.

We thank the reviewer for the feedback. To better address this concern, we would appreciate clarification regarding which specific images or figure panels were found to be of low quality. Overall, we believe the microscopy data presented are of sufficient resolution and clarity to support our main conclusions, as also noted by Reviewer #2 ("the high-quality images and FRAP experiments").

We acknowledge that certain phenomena-such as occasional R18 labeling of the vacuole-were not clearly explained in the original manuscript. We have now included additional clarification in the results section and mentioned this limitation in the discussion (lines 170-171, 436-438), along with a note on ongoing experiments to further investigate this point.

2) It would be important to perform some lipidomics analysis to determine in which PLs and other lipids or lipid intermediates R18 is incorporated. First, it will be important to know which the major PL species are are labelled under the conditions of the experiments done in this study. Second, the authors assume that all the R18 is exclusively incorporated into PLs and this is what they follow in their in vivo experiments. What about acyl-CoA, which has been shown to be a key player in the IM elongation (Graef lab, Cell)?

We thank the reviewer for raising this point. However, we believe this is based on a misunderstanding of the chemical nature of R18. R18 is not a free fatty acid analog and cannot be incorporated into phospholipids or acyl-CoA via metabolic pathways. Due to its chemical structure-a bulky rhodamine headgroup attached to a long alkyl chain-it cannot undergo enzymatic conjugation or incorporation into membrane lipids. This is why we did not pursue lipidomics analysis. Instead, we focused on characterizing the biological behavior of R18 through a range of live-cell assays, including temperature and ATP dependency, involvement of flippases, OSBP proteins, and Atg2, all of which support a regulated, non-vesicular lipid transport pathway. Additionally, the AF3 structural model presented in this study is consistent with this interpretation, showing no evidence of R18 forming chemical bonds with phospholipids.

3) Figure 1A and 1B. The authors conclude that Atg2 is involved in the lipid transfer since R18 does not localize to the PAS/ARS in the atg2KO cells. However, another possible explanation is that in those cells the IM is not formed and does not expand, and con sequetly R18 is present in low amounts not detectable by fluorescence microscopy. To support their conclusion, the authors must assess PAS-labelling with R18 in cells lacking another ATG gene in which Atg2 is still recruited to the PAS.

We thank the reviewer for this important suggestion. As noted, the absence of R18 at the PAS in atg2Δ cells may reflect a lack of membrane formation rather than impaired lipid transfer. However, in support of our interpretation, our previous work (Hirata E, Ohya Y, Suzuki K, 2017) has shown that R18 accumulates at PAS-like structures in delipidation mutants, where the IM fails to expand but Atg2 is still recruited (please refer to the attached revision plan for further details). This suggests that the presence of Atg2, rather than the mere existence of a mature IM, contributes to R18 localization.

To address this, we revised our statement to the more cautious: "R18 was undetectable at the PAS in atg2Δ cells," to avoid overinterpretation (lines 119-120). 4)

4) Figure 2. As written, the paragraph this figure seems to indicate that flippases are directly involved in the translocation of R18 from the PM to the ER. As correctly indicated by the authors, flippases flip PLs, not fatty acids. Moreover, there are no PL synthesizing at the PM and thus probably R18 is not flipped upon incorporation into PL. As a result, the relevance of flippase in R18 internalization is probably indirect. This must be explained clearly to avoid confusion/misunderstandings.

We thank the reviewer for this important clarification. We fully agree that flippases act on phospholipids, not fatty acids, and that R18 is not metabolically incorporated into phospholipids at the plasma membrane. However, our ongoing work (Rev. Figure 1) shows that R18 preferential labeling affinity for PS and PE in vivo (yeast phospholipid synthesis mutants), consistent with its flippase-dependent localization. Flippases are known to specifically flip PS and PE. While R18 itself is not enzymatically modified or incorporated into phospholipids, its membrane distribution may thus depend on the lipid environment and the activity of lipid-translocating proteins.

Preliminary data supporting this observation are included in the "Supplementary Figures for reviewer reference only" and are not part of the public submission.

5) A couple of manuscript has shown a (partial) role of Drs2 in autophagy. The authors must explain the discrepancy between their own results and what published, especially because they use the GFP-Atg8 processing assay, which is less sensitive than the Pho8delta60 used in the other studies.

We thank the reviewer for raising this important point. We are aware of prior reports implicating Drs2 in autophagy and in fact discussed this work directly with the authors during the course of our experiments, who kindly provided helpful suggestions. While our GFP-Atg8 processing assay did not show significant defects upon Drs2 deletion, strain background differences may explain this discrepancy. We also appreciate the suggestion to use the Pho8Δ60 assay and plan to include it in future experiments.

Additionally, authors should check whether the Atg2 and Atg18 proteins are present at the IM-ER membrane contact sites in the same rates after nutrient replenished than when cells are nitrogen-starved, since this complex would determine the lipid transfer dynamics at this membrane contact site.

We thank the reviewer for the helpful suggestion. We plan to perform additional experiments to monitor Atg18 localization during the nutrient replenishment assay.

6) Authors used a predicted Atg2 lipid-transfer mutant (Srinivasan et al, J Cel Biol, 2024), but not direct prove that this mutant is defective for this activity. As previously done for other Atg2/ATG2-related manuscripts (Osawa et al, Nat Struct Mol Biol, 2019; Valverde et al, J Cel Biol, 2019), this must be measure in vitro. Moreover, they do not show whether other known functions of Atg2 are unaffected when expressing this Atg2 mutant, e.g. formation of the IM-ER MCSs, Atg2 interaction with Atg9 and localization at the extremity of the IM...

We thank the reviewer for this concern. The lipid-transfer-deficient Atg2 mutant used here is based on the same structural rationale as in our recent parallel study (Sakai et al., bioRxiv 2025; https://www.biorxiv.org/content/10.1101/2025.05.24.655882v1, currently under revision). In that study, we addressed whether Atg2 indeed functions as a bridge-like lipid transfer protein, and also used R18 to directly demonstrate the lipid transfer defect of this Atg2 mutant in vivo.

We therefore believe that referencing this study provides mechanistic support for the use of this Atg2 mutant in the current manuscript. A citation and brief explanation have now been added to the revised text (line 315-316, 439-441). We also plan to perform the lipid transfer assay in vitro.

7) The mNG-Atg8 signal is not recovered in the fluorescent recovery assays. Based on the observation that R18 signal comes back after photobleaching, authors suggest that the supply of Atg8 is not required for IM expansion. This idea is opposite to data where the levels of Atg8 and deconjugation of lipidated Atg8 determines the size of the forming autophagosomes (e.g., Xie et al, Mol Biol Cell, 2008; Nair et al, Autophagy, 2012). Similar results have also been obtained in mammalian cells (Lazarou and Mizushima results in cell lacking components of the two ubiquitin-like conjugation systems). This discrepancy requires an explanation.

We thank the reviewer for pointing out this imprecise interpretation, and we sincerely apologize for the confusion it may have caused. We fully agree that Atg8 is essential for the expansion of the isolation membrane (IM), as supported by previous studies. In our FRAP data, mNG-Atg8 showed gradual recovery at the later timepoints, indicating that Atg8 can be replenished over time. The reason why R18 recovery appears much more rapid is likely due to the inherently fast lipid transfer activity of Atg2, the bridge-like lipid transport protein. In contrast, Atg8 signal recovery may have been delayed for two reasons: (1) slower recruitment kinetics to the IM, and (2) partial depletion of the available mNG-Atg8 protein pool due to photobleaching during the experiment.

We have revised the relevant paragraph in the manuscript (line 326-330) to clarify these points and avoid potential misinterpretation.

8) Although authors claim that there is a retrograde lipid transfer from the IM to the ER, based on the data, it quite difficult to extract these conclusions as they show a decrease in the lipid flow dynamics rather to an inversion of the lipid flow per se. Can the authors exclude that ER microdomains are formed at the ERES in contact with the IM, and consequently what they measure is a slow diffusion of R18-labeled lipid from other part of the ER to these ERES?

We appreciate the reviewer's insightful comment. Indeed, we are also considering the possibility that lipid-enriched microdomains may form in the ER and contribute to complex lipid dynamics at contact sites. However, direct visualization of such domains in cells remains technically challenging, this remains one of the important directions we aim to pursue in future studies. While our current data do not allow us to definitively state that all recovered lipids originate from the IM, our FRAP experiments provide indirect yet strong support for the possibility that at least a substantial portion of the recovered lipid signal in the ER derives from the IM. Moreover, following Reviewer 2's major point No.4, we performed a direct comparison of R18 fluorescence recovery between the photobleached ER-IM MCS region and the adjacent bleachedER region (Figure 5K and 5M). Interestingly, each sample consistently showed lower fluorescence recovery in the adjacent bleached ER near the ER-IM MCS (mean = 0.20), compared to the ER-IM MCS region (mean = 0.28). To further validate this observation, we also used the IM as a background reference for normalization. This analysis revealed a more significant difference, with the adjacent bleached ER near the ER-IM MCS showing a lower recovery (mean = 0.47) than the ER-IM MCS (mean = 0.80).

As the Reviewer2 pointed out, these results support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS is due to the signal coming from the IM, rather than from the adjacent bleached ER, which recovers more slowly and less efficiently. We have incorporated this new analysis into Figure 5, and accordingly revised the figure legend and main text (lines 384-396).

9) The retrograde PL transfer is studied in cells overexpressing Ape1, in which IM elongation is stalled. This is a non-physiological experimental setup and consequently it is unclear whether what observed applies to normal IM/autophagosomes. This event should be shown to occur in WT cells as well.

We thank the reviewer for this point. Indeed, it remains technically difficult to visualize lipid flow during normal IM expansion in vivo, as this process is rapid and transient. And to date, there are no reports directly addressing lipid flow in this process.

But the Ape1 overexpression system provides a strategic advantage by temporally extending the IM elongation phase and spatially enlarging the IM, thus offering a unique opportunity to capture membrane behavior that would otherwise be transient and difficult to resolve. Importantly, this system arrests autophagosome closure, which we leveraged to investigate the potential reversibility of phospholipid transfer in a controlled and prolonged context. Without this system, it would be exceedingly difficult for reaserchers to examine the lipid flow directionality in living cells.

Furthermore, the use of Ape1 overexpression has been widely employed in previous high-impact autophagy studies. We emphasize that our aim is to understand Atg2-mediated lipid transfer, and in this context, the Ape1 system provides a valuable and informative tool without compromising the validity of our conclusions.

10) From the images provided, it appears that R18 also labels the vacuole. The vacuole form MCSs with the IM. Can the author exclude a passage of R18 from the vacuole to the IM?

We thank the reviewer for the insightful comment. Our data suggest that R18 traffics from the plasma membrane to the ER, then to autophagy-related structures. Actually, following that, as we kown, autophagosomes will eventually reaches and fused with the vacuole. This explains the occasional weak R18 signals at the vacuole membrane, particularly in late-stage cells. We have revised the figure and clarified this point in the text to avoid oversimplification of R18 localization (lines 169-171, 426-428)

Here we also added the results of our onging work (in preparation). R18 tends to accumulate in a dot-like compartment after prolonged rapamycin treatment and incubation (Rev. Figure 2). And the vacuolar labeling of R18 correlates with the degradation status of autophagosomes, rather than reverse lipid transport from the vacuole to the IM (Rev. Figure 2). Taken together, we believe that R18 transport from the vacuole back to the IM is unlikely.

Preliminary data supporting this response are included in the "Supplementary Figures for reviewer reference only" and are not part of the public submission.

Minor points:

1) L66. One report has indicated that Vps13 may also play a role in the transfer of lipids from the ER to the IM (Graef lab, J. Cell Biol).

Thank you for pointing this out. Their excellent work also suggested that the inherent lipid transfer activity of Atg2 is required for IM expansion. We have revised the sentence (lines 67-68, 312-314) and included the appropriate citation at these two places.

2) L70. It must be indicated that IM is also called phagophore.

We have revised the sentence (line 70-71). Thank you for pointing this out.

3) L74. It is mentioned "Additionally, a hydrophobic cavity in the N-terminal region of Atg2 directly tethers Atg2 to the ER, particularly the ER exit site (ERES), which is considered a key hub for autophagosome biogenesis", but there is no experimental evidence supporting that Atg2 is involved in the tethering with the ERES.

Thank you for pointing this out. We have removed the N-terminal region part and revised the sentence accordingly (line 79-81) to avoid overstatement.

4) L90. PAS must be listed between the ARS.

We have revised the sentence (line 97-98). Thank you for pointing this out.

5) Upon deletion of ATG39 and ATG40, there is a pronounced reduction of mNG-Atg8 labelled with R18. This would suggest that these two ER-phagy receptors are required for the PL transfer from the ER to the IM, which is not the case as autophagy is mildly affected by the absence of them (e.g., Zhang et al, Autophagy, 2020).

We thank the reviewer for the important comment and agree that Atg39 and Atg40 are not required for phospholipid transfer from the ER to the IM. We have revised the text (lines 155-157). We appreciate if the reviewer could provide the DOI or PubMed ID for this paper.

6) Authors referred that "no direct evidence has been found to confirm lipid transfer at the ER-IM MCS in living cells" (lines 282-283). However, a recent paper has shown that de novo-synthesized phosphatidylcholine is incorporated from the ER to the autophagosomes and autophagic bodies (Orii et al, J Cel Biol, 2021). This reference should be mentioned in the manuscript.

Thank you for your insightful reminder. This paper beautifully demonstrated the importance of de novo-synthesized phosphatidylcholine in autophagy using electron microscopy. We have now included its citation and brief discussion in the revised manuscript (lines 74-76, 297-298). However, we respectfully note that direct observation of lipid transfer at the ER-IM MCS in living cells still remains unproven.

7) In lines 252-253, the sentence "R18 transport from the PM to the ER was partially impaired in osh1Δ osh2Δ, osh6Δ osh7Δ, and oshΔ osh4-1 cells (Figure S3). These results suggest that Osh proteins participate in transferring R18 from the PM to the ER" does not recapitulate what is observed in Fig. S3. Moreover, the Emr lab has generate a tertadeletion mutant in which the PM-ER MCSs are abolished. The authors could examine this mutant.

We thank the reviewer for this helpful comment and sincerely apologize for the lack of clarity in our original description. Our conclusion was primarily based on the partial PM accumulation of R18 observed in some osh mutant strains shown in Figure S3, which motivated us to further investigate this pathway using the OSW-1 inhibitor. We have revised the corresponding text to improve the logic and clarity of this section.

We appreciate the recommendation of the tether∆ mutant. Our preliminary tests indicate that R18 still properly labels the ER in tether∆ cells, suggesting that its localization is not due to passive diffusion at membrane contact sites, but rather involves specific transport mechanisms. As this is an initial observation, we plan to confirm the result and include it in a future revision.

Reviewer #1 (Significance (Required)):

General assistent: Strength: potential new system to monitor lipid flow Limitations: Indirect evidences and in the case of the retrograde transport of phospholipids, it could be an artefact of the employed experimental approach. Advance: Little advances because something in part already shown in vitro. No new mechanisms uncovered. Audience: Autophagy and membrane contact site fields.

We sincerely thank the reviewer for the overall evaluation. We agree that our current system offers indirect but promising evidence for lipid transfer events at ER-IM contact sites in vivo. While Atg2-mediated lipid transport has been proposed in vitro, our study adds value by (1) establishing a live-cell imaging way to monitor lipid flow in a non-vesicular transport pathway, (2) proposing a model of reversible one-way lipid transfer activity, and (3) addressing whether findings from simplified in vitro reconstitution accurately reflect the dynamics in the more complex real cellular environment.

We recognize the limitations of our current approach and plan to include additional analyses to more cautiously interpret the observed retrograde movement. Although we do not claim to identify a new mechanism, we believe our work provides an interesting framework to inspire future efforts aimed at directly probing lipid flow at membrane contact sites in vivo.

We also sincerely appreciate the reviewer's recognition of the potential value of this system for the autophagy and membrane contact site communities.

Response to the Reviewer ____#2

Non-vesicular lipid transfer plays an essential role in organelle biogenesis. Compared to vesicular lipid transfer, it is faster and more efficient to maintain proper lipid levels in organelles. In this study, Hao et al. introduced a high lipophilic dye octadecyl rhodamine B (R18), which specifically labels the ER structures and autophagy-related structures in yeast and mammalian cells. They characterised its distinct lipid entry into yeast cells via lipid flippase Neo1 and Drs2 on the plasma membrane, rather than through the endocytic pathway. They then demonstrated that R18 intracellular trafficking through plasma membrane to ER depends on "box-like" lipid transfer Osh proteins. They further looked into the "bridge-like" lipid transfer protein Atg2, using R18 as a lipid probe to track lipid transfer from ER to the isolation membrane (IM) during membrane expansion and reversible lipid transfer through IM to the ER-IM membrane contact sites (MCS) when autophagy is terminated by nutrient replenishment. The authors provide an interesting model of reversible directionality of Atg2 lipid transfer during autophagy induction and termination.

We sincerely thank the reviewer for the thoughtful and constructive summary of our work. We are grateful for the recognition of the novelty of using R18 to visualize non-vesicular lipid transfer in vivo and for highlighting the conceptual contribution of our proposed model of reversible Atg2-mediated transport during autophagy.

In response to the reviewer's valuable suggestions, we have revised key parts of the manuscript and prepared a detailed revision plan to address the specific concerns. We truly appreciate the reviewer's insights, which have been instrumental in improving the clarity of our study.

Major points:

1. Line 299-309: The FRAP assays were interesting and well performed. The authors photobleached R18 and Atg8 signal, and found R18 fluorescence recovery but not Atg8, which suggests lipid transfer occurs between ER and the IM and faster than Atg8 lipidation process during IM expansion. These results gave clear evidence that R18 can be transferred during IM expansion. The supply of Atg8 may not be not able to track within this time frame or the recovered amount of Atg8 may not be able to visualized due to the threshold limitation with confocal microcopy. This does not imply the supply of Atg8 to the IM is not required during IM expansion. This should be clarified.

We thank the reviewer for this valuable comment and fully agree that Atg8 is essential for IM expansion. We apologize for any ambiguity that may have suggested otherwise.

As pointed out, the lack of mNG-Atg8 recovery in our FRAP assay likely reflects the slower turnover of lipidated Atg8, limited observation time, and photobleaching of the existing protein pool. Notably, we observed a weak but gradual signal recovery at later time points, supporting this view. We have revised the relevant paragraph in the manuscript (line 326-330) to clarify these points and avoid potential misinterpretation.

Please clarify how the length of the IM is measured and determined in Figure 4H and Figure 5D.

We thank the reviewer for the vaulable comment. We have now clarified the method for quantifying IM length in the revised manuscript. Specifically, we modified the Statistical Analysis section of the Methods (line 642-643).

Line 336-342: The description of the results should be clarified. Based on Figure 5H, the authors observed a significant decrease in the mNG-Atg8 signal during photobleaching of the R18 signal.

We thank the reviewer for pointing out the ambiguity. We have now clarified the description in the revised manuscript. The sentence has been modified (line 360-362) as follows: "To determine whether nutrient replenishment terminates autophagy, we selectively photobleached the R18 signal and monitored the R18 (photobleached) and mNG-Atg8 (without photobleaching) signal following nutrient replenishment."

The authors photobleached ER-IM MCS and the ER region (boxed region in Figure 5J) and quantified fluorescence recovery, normalized to the IM region and an ER control. The ER control was taken from the other cell. It would be helpful to compare and analyse the fluorescence recovery of R18 in the bleached ER region near the ER-IM MCS to that in the ER-IM MCS. This would help to confirm the ER-IM MCS fluorescence recovery is due to signal coming from the IM.

We sincerely thank the reviewer for this insightful suggestion. We have now performed the suggested comparison. Interestingly, each sample consistently showed lower fluorescence recovery in the adjacent bleached ER near the ER-IM MCS (mean = 0.20), compared to the ER-IM MCS region (mean = 0.28). To further validate this observation, we also used the IM as a background reference for normalization. This analysis revealed a more significant difference, with the adjacent bleached ER near the ER-IM MCS showing a lower recovery (mean = 0.47) than the ER-IM MCS (mean = 0.80).

As the reviewer pointed out, these results support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS is due to the signal coming from the IM, rather than from the adjacent bleached ER, which recovers more slowly and less efficiently. We have incorporated this new analysis into Figure 5, and accordingly revised the figure legend and main text (lines 384-396). Again, we appreciate this constructive and helpful suggestion.

In figure 5K, the autophagic structure or IM labelled by R18 seems to be maintained when the mNG-Atg8 signal decreases or dissociates from the IM. Could the authors comment on that how they interpret the termination of the prolonged IM structure and IM shrinkage?

We thank the reviewer for this insightful observation. Based on our live-cell imaging, we speculate that following the initial dissociation of Atg8, the IM membrane undergoes a relatively slow disassembly process, potentially retracting toward the ER-IM MCS, which often localizes near ER exit sites (ERES). This suggests that IM shrinkage may proceed via Atg8-independent mechanisms. Although the precise pathway remains unclear, we occasionally observed vesiculation events during this phase, supporting the idea that membrane remodeling continues even in the absence of Atg8. In response to this comment, we have revised our manuscript to reflect these interpretations (line 494-496).

The author has shown that Atg2Δ and Atg2LT lipid transfer mutant impair R18 labelling of autophagic structures in Figure 4C. However, the evidence supporting that R18 fluorescence recovery at ER-IM MCS is mediated by reversible Atg2 lipid transfer is not direct. It would be helpful to clarify whether Atg2 stays on the enlarged autophagic membranes when the membrane has reached to its maximum length and no longer grows.

We thank the reviewer for this important suggestion. As noted in our response to Reviewer 1 (Major Point 8-2), clarifying whether Atg2/Atg18 remains at the ER-IM contact sites after IM expansion is indeed important for supporting the reversible lipid transfer model. We plan to monitor the localization of Atg18 during the nutrient replenishment assay.

Minor points:

1. Figure 2A "Dpm-GFP" is missing. The experiment replicates in Figure 2M should be indicated.

We thank the reviewer for pointing out these issues. The label for "Dpm-GFP" has been added in Figure 2A, and the number of experimental replicates for Figure 2M is now indicated in the figure legend.

Figure S2, the magenta panel should be "R18".

We thank the reviewer for catching this labeling error. We have corrected the magenta panel label in Figure S2 to "R18" in the revised version of the figure.

Line 341-342: "Figure 5H and 5J" should be "Figure 5H and 5I"

We thank the reviewer for pointing out this error. The citation has been corrected from "Figure 5H and 5J" to "Figure 5H and 5I" in the revised manuscript.

Please describe how the lipid docking model of Atg2 is generated.

We thank the reviewer for this question. We have added a description of the modeling approach in the Methods section of the revised manuscript (lines 640-646). We also added the configuration files of AlphaFold3 to the supplementary information.

Reviewer #2 (Significance (Required)):

Currently, lipid probes are emerging as powerful tools to understand membrane dynamics, integrity, and the lipid-mediated cellular functions. In this manuscript, the authors performed a detailed characterisation of octadecyl rhodamine B (R18) as a potential lipid probe, which specifically labels ER and autophagic membranes. They present high quality imaging data and performed FRAP experiments to monitor the membrane dynamics and investigate the lipid transfer directionality between the ER and autophagic structure. However, the evidence of Atg2-mediated reversible lipid transfer may not be direct and sufficient. The proposed reversible lipid transfer model is interesting and provides an explanation of lipid level regulation during autophagosome formation.

We sincerely thank the reviewer for the positive assessment of our work and for acknowledging the potential of R18 as a lipid probe, as well as the quality of our imaging and FRAP experiments. We are particularly grateful that the reviewer found the proposed model of reversible lipid transfer both interesting and relevant to the broader question of lipid regulation during autophagosome formation.

Regarding the reviewer's concern that the evidence for Atg2-mediated reversible lipid transfer may not be sufficiently direct, we agree this is a critical point. While technical limitations currently prevent direct visualization of lipid flow reversal at single-molecule resolution in vivo, we hope our revision plan strengthen the proposed model and better convey its biological relevance, while also acknowledging the current limitations and the need for further mechanistic work.

Response to the ____Reviewer #3

The authors address the question of how autophagic membrane seeds expand into autophagosomes. After nucleation, IMs expand in dependence of the bridge-like lipid transfer protein Atg2, which has been shown to tether the IM to the ER. Several studies have shown in vitro evidence for direct lipid transfer by Atg2 between tethered membranes, and previous evidence has shown that the hydrophobic groove of Atg2 implicated in lipid transfer is required for autophagosome biogenesis in vivo in yeast and mammalian cells.

In this manuscript, the authors take advantage of the dye R18, which they show accumulates mainly in the ER after a few minutes. They show specifically that the import of R18 into cells and transfer to the ER depends on the activity of flippases in the plasma membrane and OSPB-related lipid transporter. Using different sets of FRAT experiments, the authors track the fluorescence recovery of R18 in the IM, the IM-ER membrane contact site and the neighboring ER. From these experiments the authors conclude that (a) R18 is transferred to IM from the ER when IMs expand and (b) can be transferred from IMs back to the ER when autophagy is deactivated.

The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

We sincerely thank the reviewer for this thoughtful and encouraging summary. We appreciate the recognition of our approach using R18 to visualize lipid dynamics at ER-IM contact sites, and agree that in vivo quantitative data are critically needed to advance our understanding of autophagic membrane expansion.

We also fully agree with the reviewer that our current study provides indirect-but conceptually informative-support for Atg2-mediated reversible one way lipid transfer. While prior in vitro studies have demonstrated the lipid transfer capability of Atg2, our goal here was to develop a live-cell system that allows the dynamic tracking of lipid flow in vivo, and to explore the possibility of reversible transport during autophagy termination. We hope our story will offer unique insights for future studies aiming to directly probe lipid transfer mechanisms in live cells.

Regarding the reviewer's concern about the lack of direct evidence that Atg2's lipid transfer activity underlies the observed R18 dynamics, we fully acknowledge this limitation. To address this point, we would like to cite our parallel study currently under revision (Sakai et al., bioRxiv 2025.05.24.655882v1), which provides additional mechanistic evidence linking R18 dynamics to the lipid transfer function of Atg2. Further details and planned revisions are described in the responses below.

Major points:

(1) The authors use R18in FRAP experiments to follow its transfer from the ER into IMs. However, whether this transfer is mediated by Atg2 via its inherent lipid transfer activity remains indirect. The only evidence that implicates Atg2 directly is the observation that a lipid transfer deficient Atg2 variant fails to support IM expansion and autophagosome biogenesis. A similar full-length Atg2 mutant has previously been shown to block autophagosome formation in Dabrowski et al. 2023 in yeast, which the authors do not cite or discuss, suggesting the inherent lipid transfer activity of Atg2 is required for IM expansion. However, aside from this experiment, the mechanisms underlying R18 transfer remain unclear and, while they likely depend on or are at least partially mediated by Atg2, they may involve alternative mechanisms including vesicle transport or continuous membrane contacts. Moreover, for the assays with stalled or dissolving IM, it is essential for the authors to test whether Atg2 is still associated with these IMs. It is quite possible that Atg2 dissociates from maximally expanded or dissolving IMs, which would make their interpretation of the data very unlikely. Thus, it will be critical to provide consistent evidence that lipid transfer from the IM to the ER is mediated by Atg2. Ideally, the authors would label IM with BFP-Atg8, R18, and Atg2-GFP and perform their in vivo analysis.

We sincerely thank the reviewer for the critical comments and valuable suggestions. To further support the link between R18 transfer and Atg2, we would like to highlight two complementary findings. As noted in our response to Reviewer 1 (Major Point 3), R18 can still label the PAS even when Atg2 is recruited but IM expansion is impaired, suggesting that R18 trafficking occurs in an Atg2-dependent manner. In addition, in our parallel study (bioRxiv, 2025.05.24.655882v1), we demonstrated that Atg2 acts as a bridge-like lipid transfer protein. Notably, when we mutated the bridge-forming region of Atg2, R18 transport to the IM was also disrupted.

We greatly appreciate the reviewer's reminder regarding the study by Dabrowski et al., 2023, which we have now cited and discussed in the revised manuscript (lines 66-68, 312-314). Their findings that the inherent lipid transfer activity of Atg2 is required for autophagosome formation in vivo strongly reinforce our model.

Regarding the possibility of vesicle transport, we consider this contribution minimal based on R18's preferential labeling of continuous membranes and its divergence from FM4-64 staining. As for the role of continuous membrane contacts, as also mentioned in our response to Reviewer 1, our preliminary tests indicate that R18 still properly labels the ER in tether∆ cells, suggesting that its localization is not due to passive diffusion at membrane contact sites, but rather involves specific transport mechanisms. As this is an initial observation, we plan to confirm the result and include it in a future revision.

We also thank the reviewer for the suggestion to monitor Atg2 localization at the dissolving IM. As similarly pointed out by two other reviewers, we plan to track Atg18 during the nutrient replenishment assay.

Finally, we appreciate the idea of triple-labeling with BFP-Atg8, R18, and Atg2-GFP. While our preliminary attempts encountered technical difficulties such as abnormal BFP-Atg8 localization and severe bleaching during long-term imaging in yeast, we plan to optimize this approach in future experiments.

(2) Given the ER forms contact sites with many organelles using bridge-like lipid transfer proteins, how do the authors explain the preferential accumulation of R18 in ARS and not in for example PM (Fmp27), mitochondria, endosomes or vacuole (Vps13)? Why should R18 specifically transferred by Atg2 and not or to a much lower rate by Fmp27 or Vps13?

We sincerely thank the reviewer for raising this insightful question. Indeed, we have carefully considered this point. Our data indicate that R18 labeling of autophagy-related structures (ARS) depends on Atg2, as demonstrated in the present manuscript and supported by our parallel study currently under revision (bioRxiv, 2025.05.24.655882v1).

We speculate that the preferential accumulation of R18 in ARS may arise from structural and contextual differences among bridge-like LTPs, such as Atg2, Vps13, and Fmp27. Although all are capable of mediating lipid transfer, these proteins differ in their membrane tethering modes, cargo specificity, and spatial regulation. For example, Atg2 localizes specifically to ER-IM contact sites during autophagosome formation, where membrane expansion requires rapid lipid supply. In contrast, Vps13 and Fmp27 may function at more stable or less dynamic contacts, where lipid turnover or probe accessibility is more limited. We have added a brief discussion of this point in the revised manuscript to reflect this important consideration (lines 439-444).

(3) Does R18 label autophagic bodies after they are formed. Could the authors add R18 after autophagic bodies have formed in atg15 or pep4 cells?

We thank the reviewer for this excellent suggestion. To address whether R18 can label autophagic bodies post-formation, we plan to perform additional experiments by adding R18 after autophagic bodies have accumulated in atg15Δ or pep4Δ cells. This will help clarify whether R18 incorporates into pre-formed autophagic bodies or requires earlier membrane dynamics for its labeling.

(4) Since Neo1- or OSBP-defective cells do not transfer R18 from the PM to the ER or other membranes, the authors should include these strains as controls for ER-dependent R18 transfer to ARSs.

We thank the reviewer for this insightful suggestion. To further validate the ER-dependency of R18 transfer to autophagy-related structures, we plan to include Neo1- and OSBP-deficient strains as additional controls.

Comments:

The authors neglect to mention or discuss important recent literature directly related to their study:

Schutter et al., Cell (2020); Orii et al., JCB (2021); Polyansky et al., EMBOJ (2022); Dabrowski et al., JCB (2023); Shatz et al., Dev Cell (2024)

We sincerely thank the reviewer for pointing out these important and highly relevant studies. We apologize for our oversight in not citing them earlier. Each of these works has provided valuable insights that are directly related to and have greatly informed our current study. We have now cited and discussed these references in appropriate sections of the revised manuscript.

Figure 1A and B: The authors need to describe how these cells were stained with R18 in the figure legend or text to help the reader to understand how these experiments were performed. Figure legends need to indicate at which time point after rapamycin treatment cells were analyzed.

Thank you for the helpful suggestion. We have now added the corresponding information to the figure legends to clarify the staining procedure and time points.

The authors need to clarify whether mNG-Atg8 colocalization with R18 was included for dot- and ring-like structures for WT cells as shown separately in 1A but not in 1B.

Thank you for the comment. The quantification in Figure 1B includes both dot- and ring-like structures of mNG-Atg8 colocalized with R18 in WT cells, as shown in Figure 1A. We have now clarified this point in the revised figure legend.

Figure 1C: The figure legend needs to describe the conditions cells were treated with and when cells were analyzed after rapamycin treatment (presumably).

Thank you for the helpful suggestion. We have now added the corresponding information to the figure legends.

Figure 1C: The authors should combine atg15 and pep4 deletions with atg2 or atg7 as controls in which autophagic bodies are not formed.

Thank you for the valuable suggestion. We plan to perform these experiments that combine atg15 and pep4 deletions with atg2 or atg7 as controls.

Figure 1E and F: R18 stains more than just the ER in the cells shown. In addition to atg39 and atg40, authors should include atg11 to inhibit all forms of selective autophagy.

Thank you very much for the insightful comment. We agree and plan to include the atg11Δ mutant to inhibit all forms of selective autophagy.

Figure S2A and B: The figures are mislabeled. Instead of FM4-64 it should say R18. In addition to the ER, in several images it is obvious to see R18 staining the vacuole membrane (for example Figure 2A 30 degrees) and others. Thus, the strong thresholding in S2 may give the reader an oversimplified view on R18 localization. This needs to be corrected.

Thank you very much for pointing this out. We have corrected the labeling error in Figure S2A and B. Regarding the observation that R18 occasionally labels the vacuole membrane, we agree with the reviewer's comment. Based on our data, we believe that this signal likely reflects autophagosomes that have reached and fused with the vacuole, as expected in the later stages of autophagy. We have clarified this point in the text to avoid oversimplification of R18 localization (lines 169-171, 426-428).

Figure 1G and H: In 1G, there are number of R18-stained patches not co-labeled by GFP-ER. What are these patches and which organelles to they represent? In 1H, given the tight association of the ER (omegasome) with forming IMs, it is difficult to discern whether R18 labels surrounding ER membrane or the IM itself. This needs to be more closely analyzed. The authors need to quantify these data similar to the yeast data.

Thank you for the suggestion. We plan to perform additional quantification and colocalization analysis to clarify the identity of R18-positive signals in 1G and 1H.

Figure 4A-C: A full-length PLT-deficient variant of Atg2 has been analyzed by Dabrowski et al, JCB 2023 in vivo. This work needs to be cited and discussed. The analysis needs to include punctate Atg8 structures for WT cells to exclude effects due to expansion defects.

Thank you for the suggestion. We have now cited and discussed the work by Dabrowski et al., JCB 2023 in the revised manuscript (lines 67-68, 312-314). In addition, we have included an analysis of punctate Atg8 structures in WT cells to address the concern regarding potential expansion defects.

Figure 4F-H: To measure the size changes in IMs, the authors would need to perform these experiments without bleaching the mNG-Atg8 signals.

We apologize for the lack of clarity. The method for measuring IM size has now been added to the revised manuscript. In Figure 4, we note that mNG-Atg8 fluorescence actually shows a slow recovery over time. This limited recovery likely reflects both the slower turnover of Atg8 and the fact that the pre-existing Atg8 pool at the IM was partially photobleached. We have now revised the main text to clarify this point and included additional explanation (line 326-330).

Figure 5C: The authors need to indicate the bleached areas in the mNG-Atg8 image for easier orientation. It looks to me that the area that the authors mark as IM-ER MCS is really the IM in proximity to the ER. Thus, if lipid transfer to the IM has ceased, I would not expect recovery here. If the IM-ER MCS area includes IM and the ER to similar extent, I would expect exactly what the authors show: IM does not recover while ER quickly recovers. On average, we would observe reduced recovery as shown in 5D.

Thank you for the helpful suggestion, and we apologize for the oversight during figure preparation. We have now clearly indicated the bleached areas in the merged image in Figure 5C for better orientation. Additionally, we have carefully re-examined the defined ER-IM MCS region and confirm that the quantified area indeed corresponds to the contact site between the ER and the IM. And double checked the measurements shown in the figure remain correct.

Figure 5L: Since mNG-Atg8 signal homogenously disappears from the IM, it is meaningless to measure size. How do the authors measure the size of something they cannot detect?

Thank you for pointing this out. We agree with the reviewer's comment and have removed the panel from the revised version accordingly.

Figure 5K: The authors need to show the whole bleached area overtime for the reader to be able to see where the recovered R18 signal might be coming from. Currently, it is impossible to discern whether the signal comes from the IM or from slow recovery from neighboring ER.

We appreciate this insightful comment. To address the concern and following the suggestion from Reviewer 2 (Major Point No.4), we have now revised the figure to include an additional measurement of fluorescence recovery in the adjacent bleached ER (Figure 5K and 5M) (lines 384-396). These results further support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS originates from the IM, rather than from the adjacent bleached ER, which shows slower and less efficient recovery.

We have also added time-lapse videos to the supplementary information due to space limitations in the main figure.

Reviewer #3 (Significance (Required)):

The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

We sincerely thank the reviewer for this encouraging and thoughtful comment. We appreciate the recognition that our live-cell approach using a lipophilic dye provides a valuable framework to visualize lipid dynamics during autophagosome biogenesis. As the reviewer pointed out, quantitative in vivo evidence is critically needed in this field, and we hope our study contributes meaningfully toward that goal.

We also fully acknowledge the limitation. While our current data offer indirect evidence for Atg2-mediated lipid transfer, we would like to support this by our revision plan and also our parallel study (bioRxiv, 2025.05.24.655882v1) that shows Atg2 is indeed a bridge-like LTP and R18 transfer is lost in the bridge-structure defective strain. Together, we hope these can suggest that the lipid transfer activity of Atg2 underlies the observed R18 dynamics in vivo.

Author response:

The following is the authors’ response to the previous reviews

General Response to Reviewers:

We thank the Reviewers for their comments, which continue to substantially improve the quality and clarity of the manuscript, and therefore help us to strengthen its message while acknowledging alternative explanations.

All three reviewers raised the concern that we have not proven that Rab3A is acting on a presynaptic mechanism to increase mEPSC amplitude after TTX treatment of mouse cortical cultures.  The reviewers’ main point is that we have not shown a lack of upregulation of postsynaptic receptors in mouse cortical cultures. We want to stress that we agree that postsynaptic receptors are upregulated after activity block in neuronal cultures.  However, the reviewers are not acknowledging that we have previously presented strong evidence at the mammalian NMJ that there is no increase in AChR after activity blockade, and therefore the requirement for Rab3A in the homeostatic increase in quantal amplitude points to a presynaptic contribution. We agree that we should restrict our firmest conclusions to the data in the current study, but in the Discussion we are proposing interpretations. We have added the following new text:

“The impetus for our current study was two previous studies in which we examined homeostatic regulation of quantal amplitude at the NMJ.  An advantage of studying the NMJ is that synaptic ACh receptors are easily identified with fluorescently labeled alpha-bungarotoxin, which allows for very accurate quantification of postsynaptic receptor density. We were able to detect a known change due to mixing 2 colors of alpha-BTX to within 1% (Wang et al., 2005).  Using this model synapse, we showed that there was no increase in synaptic AChRs after TTX treatment, whereas miniature endplate current increased 35% (Wang et al., 2005). We further showed that the presynaptic protein Rab3A was necessary for full upregulation of mEPC amplitude (Wang et al., 2011). These data strongly suggested Rab3A contributed to homeostatic upregulation of quantal amplitude via a presynaptic mechanism.  With the current study showing that Rab3A is required for the homeostatic increase in mEPSC amplitude in cortical cultures, one interpretation is that in both situations, Rab3A is required for an increase in the presynaptic quantum.”

The point we are making is that the current manuscript is an extension of that work and interpretation of our findings regarding the variability of upregulation of postsynaptic receptors in our mouse cortical cultures further supports the idea that there is a Rab3Adependent presynaptic contribution to homeostatic increases in quantal amplitude.

Public Reviews:

Reviewer #1 (Public review):

Koesters and colleagues investigated the role of the small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cortical cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed no significant changes in GluA2 puncta size, intensity, and integral after TTX treatment in control and Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which neuronal Rab3A is required for homeostatic scaling of synaptic transmission, potentially through GluA2-independent mechanisms.

The major finding - impaired homeostatic up-scaling after TTX treatment in Rab3A KO and Rab3 earlybird mutant neurons - is supported by data of high quality. However, the paper falls short of providing any evidence or direction regarding potential mechanisms. The data on GluA2 modulation after TTX incubation are likely statistically underpowered, and do not allow drawing solid conclusions, such as GluA2-independent mechanisms of up-scaling.

The study should be of interest to the field because it implicates a presynaptic molecule in homeostatic scaling, which is generally thought to involve postsynaptic neurotransmitter receptor modulation. However, it remains unclear how Rab3A participates in homeostatic plasticity.

Major (remaining) point:

(1) Direct quantitative comparison between electrophysiology and GluA2 imaging data is complicated by many factors, such as different signal-to-noise ratios. Hence, comparing the variability of the increase in mini amplitude vs. GluA2 fluorescence area is not valid. Thus, I recommend removing the sentence "We found that the increase in postsynaptic AMPAR levels was more variable than that of mEPSC amplitudes, suggesting other factors may contribute to the homeostatic increase in synaptic strength." from the abstract.

We have not removed the statement, but altered it to soften the conclusion. It now reads, “We found that the increase in postsynaptic AMPAR levels in wild type cultures was more variable than that of mEPSC amplitudes, which might be explained by a presynaptic contribution, but we cannot rule out variability in the measurement.”.

Similarly, the data do not directly support the conclusion of GluA2-independent mechanisms of homeostatic scaling. Statements like "We conclude that these data support the idea that there is another contributor to the TTX- induced increase in quantal size." should be thus revised or removed.

This particular statement is in the previous response to reviewers only, we deleted the sentence that starts, “The simplest explanation Rab3A regulates a presynaptic contributor….”. and “Imaging of immunofluorescence more variable…”. We deleted “ our data suggest….consistently leads to an increase in mEPSC amplitude and sometimes leads to….” We added “…the lack of a robust increase in receptor levels leaves open the possibility that there is a presynaptic contributor to quantal size in mouse cortical cultures. However, the variability could arise from technical factors associated with the immunofluorescence method, and the mechanism of Rab3A-dependent plasticity could be presynaptic for the NMJ and postsynaptic for cortical neurons.”

Reviewer #2 (Public review):

I thank the authors for their efforts in the revision. In general, I believe the main conclusion that Rab3A is required for TTX-induced homeostatic synaptic plasticity is wellsupported by the data presented, and this is an important addition to the repertoire of molecular players involved in homeostatic compensations. I also acknowledge that the authors are more cautious in making conclusions based on the current evidence, and the structure and logic have been much improved.

The only major concern I have still falls on the interpretation of the mismatch between GluA2 cluster size and mEPSC amplitude. The authors argue that they are only trying to say that changes in the cluster size are more variable than those in the mEPSC amplitude, and they provide multiple explanations for this mismatch. It seems incongruous to state that the simplest explanation is a presynaptic factor when you have all these alternative factors that very likely have contributed to the results. Further, the authors speculate in the discussion that Rab3A does not regulate postsynaptic GluA2 but instead regulates a presynaptic contributor. Do the authors mean that, in their model, the mEPSC amplitude increases can be attributed to two factors- postsynaptic GluA2 regulation and a presynaptic contribution (which is regulated by Rab3A)? If so, and Rab3A does not affect GluA2 whatsoever, shouldn't we see GluA2 increase even in the absence of Rab3A? The data in Table 1 seems to indicate otherwise.

The main body of this comment is addressed in the General Response to Reviewers. In addition, we deleted text “current data, coupled with our previous findings at the mouse neuromuscular junction, support the idea that there are additional sources contributing to the homeostatic increase in quantal size.” We added new text, so the sentence now reads: “Increased receptors likely contribute to increases in mESPC amplitudes in mouse cortical cultures, but because we do not have a significant increase in GluA2 receptors in our experiments, it is impossible to conclude that the increase is lacking in cultures from Rab3A^-/- neurons.”

I also question the way the data are presented in Figure 5. The authors first compare 3 cultures and then 5 cultures altogether, if these experiments are all aimed to answer the same research question, then they should be pooled together. Interestingly, the additional two cultures both show increases in GluA2 clusters, which makes the decrease in culture #3 even more perplexing, for which the authors comment in line 261 that this is due to other factors. Shouldn't this be an indicator that something unusual has happened in this culture?

Data in this figure is sufficient to support that GluA2 increases are variable across cultures, which hardly adds anything new to the paper or to the field. 

A major goal of performing the immunofluorescence measurements in the same cultures for which we had electrophysiological results was to address the common impression that the homeostatic effect itself is highly variable, as the reviewer notes in the comment “…GluA2 increases are variable across cultures…” Presumably, if GluA2 increases are the mechanism of the mEPSC amplitude increases, then variable GluA2 increases should correlate with variable mEPSC amplitude increases, but that is not what we observed. We are left with the explanation that the immunofluorescence method itself is very variable. We have added the point to the Discussion, which reads, “the variability could arise from technical factors associated with the immunofluorescence method, and the mechanism of Rab3A-dependent homeostatic plasticity could be presynaptic for the NMJ and postsynaptic for cortical neurons.”

Finally, the implication of “Shouldn’t this be an indicator that something unusual has happened in this culture?” if it is not due to culture to culture variability in the homeostatic response itself, is that there was a technical problem with accurately measuring receptor levels. We have no reason to suspect anything was amiss in this set of coverslips (the values for controls and for TTX-treated were not outside the range of values in other experiments). In any of the coverslips, there may be variability in the amount of primary anti-GluA2 antibody, as this was added directly to the culture rather than prepared as a diluted solution and added to all the coverslips. But to remove this one experiment because it did not give the expected result is to allow bias to direct our data selection.

The authors further cite a study with comparable sample sizes, which shows a similar mismatch based on p values (Xu and Pozzo-Miller 2007), yet the effect sizes in this study actually match quite well (both ~160%). P values cannot be used to show whether two effects match, but effect sizes can. Therefore, the statement in lines 411-413 "... consistently leads to an increase in mEPSC amplitudes, and sometimes leads to an increase in synaptic GluA2 receptor cluster size" is not very convincing, and can hardly be used to support "the idea that there are additional sources contributing to the homeostatic increase in quantal size.”

We have the same situation; our effect sizes match (19.7% increase for mEPSC amplitude; 18.1% increase for GluA2 receptor cluster size, see Table 1), but in our case, the p value for receptors does not reach statistical significance. Our point here is that there is published evidence that the variability in receptor measurements is greater than the variability in electrophysiological measurements. But we have softened this point, removing the sentences containing “…consistently leads and sometimes...” and “……additional sources contributing…”.

I would suggest simply showing mEPSC and immunostaining data from all cultures in this experiment as additional evidence for homeostatic synaptic plasticity in WT cultures, and leave out the argument for "mismatch". The presynaptic location of Rab3A is sufficient to speculate a presynaptic regulation of this form of homeostatic compensation.

We have removed all uses of the word “mismatch,” but feel the presentation of the 3 matched experiments, 23-24 cells (Figure 5A, D), and the additional 2 experiments for a total of 5 cultures, 48-49 cells (Figure 5C, F), is important in order to demonstrate that the lack of statistically significant receptor response is due neither to a variable homeostatic response in the mEPSC amplitudes, nor to a small number of cultures.

Minor concerns:

(1) Line 214, I see the authors cite literature to argue that GluA2 can form homomers and can conduct currents. While GluA2 subunits edited at the Q/R site (they are in nature) can form homomers with very low efficiency in exogenous systems such as HEK293 cells (as done in the cited studies), it's unlikely for this to happen in neurons (they can hardly traffic to synapses if possible at all).

We were unable to identify a key reference that characterized GluA2 homomers vs. heteromers in native cortical neurons, but we have rewritten the section in the manuscript to acknowledge the low conductance of homomers:

“…to assess whether GluA2 receptor expression, which will identify GluA2 homomers and GluA2 heteromers (the former unlikely to contribute to mEPSCs given their low conductance relative to heteromers (Swanson et al., 1997; Mansour et al., 2001)…”

(2) Lines 221-222, the authors may have misinterpreted the results in Turrigiano 1998. This study does not show that the increase in receptors is most dramatic in the apical dendrite, in fact, this is the only region they have tested. The results in Figures 3b-c show that the effect size is independent of the distance from soma.

Figure 3 in Turrigiano et al., shows that the increase in glutamate responsiveness is higher at the cell body than along the primary dendrite. We have revised our description to indicate that an increase in responsiveness on the primary dendrite has been demonstrated in Turrigiano et al. 1998.

“We focused on the primary dendrite of pyramidal neurons as a way to reduce variability that might arise from being at widely ranging distances from the cell body, or, from inadvertently sampling dendritic regions arising from inhibitory neurons. In addition, it has been shown that there is a clear increase in response to glutamate in this region (Turrigiano et al., 1998).”

“…synaptic receptors on the primary dendrite, where a clear increase in sensitivity to exogenously applied glutamate was demonstrated (see Figure 3 in (Turrigiano et al., 1998)).

(3) Lines 309-310 (and other places mentioning TNFa), the addition of TNFa to this experiment seems out of place. The authors have not performed any experiment to validate the presence/absence of TNFa in their system (citing only 1 study from another lab is insufficient). Although it's convincing that glia Rab3A is not required for homeostatic plasticity here, the data does not suggest Rab3A's role (or the lack of) for TNFa in this process.

We have modified the paragraph in the Discussion that addresses the glial results, to describe more clearly the data that supported an astrocytic TNF-alpha mechanism: “TNF-alpha accumulates after activity blockade, and directly applied to neuronal cultures, can cause an increase in GluA1 receptors, providing a potential mechanism by which activity blockade leads to the homeostatic upregulation of postsynaptic receptors (Beattie et al., 2002; Stellwagen et al., 2005; Stellwagen and Malenka, 2006).”

We have also acknowledged that we cannot rule out TNF-alpha coming from neurons in the cortical cultures: “…suggesting the possibility that neuronal Rab3A can act via a non-TNF-alpha mechanism to contribute to homeostatic regulation of quantal amplitude, although we have not ruled out a neuronal Rab3A-mediated TNF-alpha pathway in cortical cultures.”

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate that Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength is already elevated. In this context, it is unclear if the plasticity is absent, already induced by this mutation, or just occluded by a ceiling effect due to the synapses already being strengthened. Occlusion may also occur in the mixed cultures when Rab3A is missing from neurons but not astrocytes. The authors do appropriately discuss these options. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between changes in synaptic strength and AMPA receptor trafficking during HSP, and conclude that trafficking may not be solely responsible for the changes in synaptic strength during HSP.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms, including whether Rab3A is active pre-synaptically to regulate quantal amplitude.

As Rab3A is primarily known as a pre-synaptic molecule, this possibility is intriguing. However, it is based on the partial dissociation of AMPAR trafficking and synaptic response and lacks strong support. On average, they saw a similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant due to higher variability. It is difficult to determine if this is due to biology or methodology - the imaging method involves assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed, which would be expected to be more variable than mEPSC recordings averaged across several hundred events. However, when they reduce the mEPSC number of events to similar numbers as the imaging, the mESPC amplitudes are still less variable than the imaging data. The reason for this remains unclear. The pool of sampled synapses is still different between the methods and recent data has shown that synapses have variable responses during HSP. Further, there could be variability in the subunit composition of newly inserted AMPARs, and only assessing GluA2 could mask this (see below). It is intriguing that pre-synaptic changes might contribute to HSP, especially given the likely localization of Rab3A. But it remains difficult to distinguish if the apparent difference in imaging and electrophysiology is a methodological issue rather than a biological one. Stronger data, especially positive data on changes in release, will be necessary to conclude that pre-synaptic factors are required for HSP, beyond the established changes in post-synaptic receptor trafficking.

Regarding the concern that the lack of increase in receptors is due to a technical issue, please see General Response to Reviewers, above. We have also softened our conclusions throughout, acknowledging we cannot rule out a technical issue.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a strong frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. But the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. The decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the conclusions about the GluA2 imaging as compared to the mEPSC amplitude data.

The key finding in Figure 3 is that NASPM did not eliminate the statistically significant increase in mEPSC amplitude after TTX treatment (Fig 3A).  Whether or not NASPM sensitive receptors contribute to mESPC amplitude is a separate question (Fig 3B). We are open to the possibility that NASPM reduces mEPSC amplitude in both control and TTX treated cells (p = 0.08 for both), but that does not change our conclusion that NASPM has no effect on the TTX-induced increase in mEPSC amplitude. The mechanism underlying the decrease in mEPSC frequency following NASPM is interesting, but does not alter our conclusions regarding the role of Rab3A in homeostatic synaptic plasticity of mEPSC amplitude. In addition, the Reviewer does not acknowledge the Supplemental Figure #1, which shows a similar lack of correspondence between homeostatic increases in mEPSC amplitude and GluA1 receptors in two cultures where matched data were obtained. Therefore, we do not think our lack of a robust increase in receptors can be explained by our failing to look at the relevant receptor.

To understand the role of Rab3A in HSP will require addressing two main issues:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. More concrete support for the authors' suggestion of a pre-synaptic site of control would be helpful.

We agree that definitive evidence for a presynaptic role of Rab3A in homeostatic plasticity of mEPSC amplitudes in mouse cortical cultures requires demonstrating that loss of Rab3A in postsynaptic neurons does not disrupt the plasticity, whereas loss in presynaptic neurons does. Without these data, we can only speculate that the Rab3A-dependence of homeostatic plasticity of quantal size in cortical neurons may be similar to that of the neuromuscular junction, where it cannot be receptors. We have added to the Discussion that the mechanism of Rab3A regulation of homeostatic plasticity of quantal amplitude could different between cortical neurons and the neuromuscular junction (lines 448-450 in markup,). Establishing a way to co-culture Rab3A-/- and Rab3A+/+ neurons in ratios that would allow us to record from a Rab3A-/- neuron that has mainly Rab3A+/+ inputs (or vice versa) is not impossible, but requires either transfection or transgenic expression with markers that identify the relevant genotype, and will be the subject of future experiments.

(2): Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs or a decrease in GABA release (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at those synapses.

We agree with the Reviewer, that it is important to determine the generality of Rab3A function in homeostatic plasticity. Establishing the homeostatic effect on mIPSCs and then examining them in Rab3A-/- cultures is a large undertaking and will be the subject of future experiments.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Minor (remaining) points:

(1) The figure referenced in the first response to the reviewers (Figure 5G) does not exist.

We meant Figure 5F, which has been corrected in the current response.

(2) I recommend showing the data without binning (despite some overlap).

The box plot in Origin will not allow not binning, but we can make the bin size so small that for all intents and purposes, there is close to 1 sample in each bin. When we do this, the majority of data are overlapped in a straight vertical line. Previously described concerns were regarding the gaps in the data, but it should be noted that these are cell means and we are not depicting the distributions of mEPSC amplitudes within a recording or across multiple recordings.

(3) Please auto-scale all axes from 0 (e.g., Fig 1E, F).

We have rescaled all mEPSC amplitude axes in box plots to go from 0 (Figures 1, 2 and 6).

(4) Typo in Figure legend 3: "NASPM (20 um)" => uM

Fixed.

Reviewer #2 (Recommendations for the authors):

(1) Line 140, frequencies are reported in Hz while other places are in sec-1, while these are essentially the same, they should be kept consistent in writing.

All mEPSC frequencies have been changed to sec^-1, except we have left “Hz” for repetitive stimulation and filtering.

(2) Paragraph starting from line 163 (as well as other places where multiple groups are compared, such as the occlusion discussion), the authors assessed whether there was a change in baseline between WT and mutant group by doing pairwise tests, this is not the right test. A two-way ANOVA, or at least a multivariant test would be more appropriate.

We have performed a two-way ANOVA, with genotype as one factor, and treatment as the other factor. The p values in Figures 1 and 2 have been revised to reflect p values from the post-hoc Tukey test on the specific interactions (for each particular genotype, TTX vs CON effects). The difference in the two WT strains, untreated, was not significant in the Post-Hoc Tukey test, and we have revised the text. The difference between the untreated WT from the Rab3A+/Ebd colony and the untreated Rab3AEbd/Ebd mutant was still significant in the Post-Hoc Tukey test, and this has replaced the Kruskal-Wallis test. The two-way ANOVA was also applied to the neuron-glia experiments and p values in Figure 6 adjusted accordingly.

(3) Relevant to the second point under minor concerns, I suggest this sentence be removed, as reducing variability and avoiding inhibitory projects are reasons good enough to restrict the analysis to the apical dendrites.

We have revised the description of the Turrigiano et al., 1998 finding from their Figure 3 and feel it still strengthens the justification for choosing to analyze only synapses on the apical dendrite.

Reviewer #3 (Recommendations for the authors):

Minor points:

The comments on lines 256-7 could seem misleading - the NASPM results wouldn't rule out contribution of those other subunits, only non-GluA2 containing combinations of those subunits. I would suggest revising this statement. Also, NASPM does likely have an effect, just not one that changes much with TTX treatment.

At new line 213 (markup) we have added the modifier “homomeric” to clarify our point that the lack of NASPM effect on the increase in mEPSC amplitude after TTX indicates that the increase is not due to more homomeric Ca²⁺-permeable receptors. We have always stated that NASPM reduces mEPSC amplitude, but it is in both control and treated cultures.

Strong conclusions based on a single culture (lines 314-5) seem unwarranted.

We have softened this statement with a “suggesting that” substituted for the previous “Therefore,” but stand by our point that the mEPSC amplitude data support a homeostatic effect of TTX in Culture #3, so the lack of increase in GluA2 cluster size needs an explanation other than variability in the homeostatic effect itself.

Saying (line 554) something is 'the only remaining possibility' also seems unwarranted.

We have softened this statement to read, “A remaining possibility…”.

Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC (2002) Control of synaptic strength by glial TNFalpha. Science 295:2282-2285.

Mansour M, Nagarajan N, Nehring RB, Clements JD, Rosenmund C (2001) Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement. Neuron 32:841-853. Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF-alpha. Nature 440:1054-1059.

Stellwagen D, Beattie EC, Seo JY, Malenka RC (2005) Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci 25:3219-3228.

Swanson GT, Kamboj SK, Cull-Candy SG (1997) Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition. J Neurosci 17:5869.

Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB (1998) Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391:892-896.

Wang X, Wang Q, Yang S, Bucan M, Rich MM, Engisch KL (2011) Impaired activity-dependent plasticity of quantal amplitude at the neuromuscular junction of Rab3A deletion and Rab3A earlybird mutant mice. J Neurosci 31:3580-3588.

Wang X, Li Y, Engisch KL, Nakanishi ST, Dodson SE, Miller GW, Cope TC, Pinter MJ, Rich MM (2005) Activity-dependent presynaptic regulation of quantal size at the mammalian neuromuscular junction in vivo. J Neurosci 25:343-351.

This may be a note about parallel process, but I think there is something to be said here about being reflective about our teaching and learning more about how we act and think in classroom settings, and why. We should be encouraging students to be reflective in their learning, while also stepping into that opportunity ourselves.

holy mackerel THIS. 14th amendment clearly incorporates the constitutional interpret that the requirement MUST be unanimous. Lousiana needs to stop writing fanfiction.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Recommendations for the authors):

Major comments

(1) The section on page 20 describing the proteomic analysis of EVs is poorly written and confusing, with a lot of data in the supplement. It is not clear what the proteomics data actually means.

We appreciate your feedback on the clarity of the proteomic analysis section. We have rewritten the section on page 20 with more detained information to provide a clearer explanation of the proteomics data and its biological significance. Additionally, we have incorporated a comparative analysis of the EV and total cell lysate proteomes (Fig. 8E, Supplementary Fig. S7A, Supplementary Tables 3 and 4) for supplemental data interpretation.

(2) The order of the data could be improved.

We appreciate your feedback regarding the data organization. We have reorganized the order and position of some data in a more structured and coherent manner, as suggested by the reviewers.

- Reorganization of the qPCR data (previously Fig. 1C) as Fig. 3A

- Removal of the data on the growth analysis on raffinose media (previously Fig. 7H).

-Reorganization of the spotting data of the double mutant (previously Fig 3B) to Supplementary Fig. S3B

- Reorganization of the subcellular localization data (previously Fig 3E) to Supplementary Fig. S4A

(3) The discussion is repetitive with the introduction and merely summarizes the results and speculates on the mechanism of how the absence of UGGT, leading to ERQC defects, results in defective EV biogenesis/cargo loading in C. neoformans.

We removed several repetitive sentences in the discussion and provided additional information on proteome analysis.

Other questions and comments

(1) Instead of comprehensively analyzing EVs from the UGG1 mutant, a more informative approach to better understanding how defects in N-linked glycosylation impact secretion, would be to do a proteomic analysis on the total secretions (including beta glucanase-treated cells to release classically secreted proteins from the cell wall) and EVs.

We agree that a comprehensive proteomic analysis of total secretions and classically secreted proteins would provide deeper insights into how defects in N-glycosylation impact secretion in C. neoformans. To address this concern, we performed an additional set of proteomic analyses, the proteome profiles of total cell lysates and the secretome of C. neoformans cultivated in SD broth and presented the results as Supplementary Table S5 and Supplementary Fig. S7B. These additional analyses provide further insights into the impact of UGG1 deletion on both conventional and unconventional secretion pathways, supporting a more pronounced effect of the UGG1 defect on EV-mediated trafficking. The discussion has been updated accordingly (Page 22, lines 509-514).

(2) The melanization defect in Ugg1 mutant is not strong. Could the reduction be due to partially compromised Ugg1 mutant growth at 30{degree sign}C as indicated in the spot tests. Were photos of the spot dilution assays taken at 1 and 2 days to investigate slower growth? Or alternatively were growth curves taken in a liquid culture?

For accuracy of melanin synthesis defect, in addition to analysis on L-DOPA plates, we had assessed melanin production in liquid L-DOPA medium following a 3-day incubation, and the melanin production in liquid media was normalized by cell density (OD₆₀₀). The data on normalized melanin production is now included as Fig. 4B in the revised manuscript. The defective laccase activity in the _ugg1_Δ mutant (Fig. 7C) further corroborates our melanization assay results, which is additionally mentioned in the text (Page 18, lines 393-395).

(3) Is it accurate to say that some virulence factors (i.e. melanin, capsule and phosphatases) are predominantly trafficked through EV's in C. neoformans? Have studies been done to determine the proportion of virulence factors trafficked via EV's versus traditional secretion?

We thank you for the thoughtful comments. Some virulence factors, such as urease, melanin and capsule polysaccharides, lack a signal peptide required for targeting for the conventional ER/Golgi secretion pathway. It is generally assumed that the trafficking of these factors in C. neoformans is predominantly mediated by non-conventional secretion via EVs. Additionally, even some virulence factors with signal peptides, such as laccase and phosphatases, are also transported via EVs besides the conventional secretion. The quantitative analysis to compare the proportion of virulence factors secretion via EVs versus the conventional pathway has not been yet reported, despite that genetic evidence suggests that conventional secretion also plays a significant role in the export of capsule polysaccharides. Thus, we were also careful not to highlight EV as the main route of virulence factors in the manuscript.

(4) There is insufficient background in the introduction linking what is known about the ERQC process to secretion in general. The topic changes from the ERQC process to fungal virulence factor, with a primary focus on non-classical (EV-based) secretion. Classical secretion should also be discussed without assuming that non classical (EV) secretion is the major pathway contributing to fungal virulence.

We appreciate your insightful comments highlighting the need for more background on the ERQC process and its relationship with secretion. To address the reviewer’s concerns, we have added sentences to describe the key roles of ERQC in conventional protein secretion in the Introduction (Page 5, lines 102-106).

(5) Figure 1A. What does the blue filled circle with the red outline signify? Fig 1 A legend is not well explained. A summary using material provided in the intro/discussion should be included to briefly explain the process and the differences between fungal species. Please also be aware that the intro starts describing the human ERQC process and then switches to what happens in S. cerevisiae.

We have revised Figure 1A by removing the red circle and updated the figure legend in the revised manuscript to include more detailed information about the ERQC differences across higher eukaryotes and fungal species.

(6) Figure 2A. There are no units on the Y-axis. Presumably, the scale is the same for all 3 strains.

Thank you for your comments. The Y-axis is the same for all three strains and, as in Fig. 2C, and represents the relative fluorescence intensity obtained from the HPLC analysis. We added the units on the Y-axis in Fig. 2A.

(7) If Mnl1 and 2 have proposed roles in proteasomal degradation, wouldn't they be expected to have ER retention signals, like Ugg1?

We appreciate your valuable insights regarding the absence of ER retention signals in Mnl1 and Mnl2. Previous studies have shown that Saccharomyces cerevisiae Mnl1/Htm1 does not possess canonical KDEL/HDEL-like ER retention signals. Instead, its retention in the ER lumen is facilitated through its interaction with protein disulfide isomerase Pdi1, which contains an HDEL sequence (Gauss et al. 2011). Thus, it is expected that non-canonical retention mechanisms—such as interactions with other ER proteins—could contribute to the retention of Mnl1 and Mnl2 within the ER. We added this information to the revised manuscript (Page 8, lines 154-159).

(8) Figure 1 C qPCR showing change in mRNA in response to ER stress should not be grouped in this figure. It could be standalone or discussed when the spot dilution assays are performed. Anyway, spots tests are more convincing of a role in stress response than qPCR as the ugg1 mutant is sensitive to tunicamycin, DTT and cell wall stressing agents.

As suggested by the reviewer, we have reorganized the qPCR data as a part of Figure 3 (Figure 3A) in the revised manuscript.

(9) It is odd that mns1/101 mutants are not sensitive to ER and CW stress given their proposed differing location/function in the pathway (Figure 1) determined from the N-linked profiling. Any explanation? Could there be redundancy?

We appreciate the reviewer’s observation regarding the lack of ER and CW stress sensitivity in the mns1_Δ and _mns101_Δ mutants, despite their proposed roles in _N-glycan processing. We had previously reported that the C. neoformans alg3_Δ mutant, lacking a critical enzyme responsible for the synthesis of Dol-PP-Man₆GlcNAc₂ in the _N-glycosylation pathway, exhibited clearly impaired N-glycan elongation, but showed no detectable growth defects even under stress conditions in vitro. However, alg3_Δ is avirulent in _in vivo pathogenicity (Thak et al., 2020). Similarly, the mns1_Δ_101_Δ double mutant shows glycan-processing defects that do not compromise cellular fitness under stress conditions but result in attenuated virulence in animal models. These findings suggest that some glycosylation-related defects may impact more severely _in vivo pathogenicity rather than in vitro stress sensitivity.

(10) Although the Silver-stained gels of the ugg1 mutant are not particularly informative, why weren't they (and Con A blots) performed for the other mutants?

The overall decrease of hypermannosylated glycans observed in the ugg1_Δ mutant allowed us to detect clear alterations in protein glycosylation patterns in the lectin blot using _Galanthus nivalis agglutinin, which recognizes terminal α1,2-, α1,3-, and α1,6-linked mannose residues. In contrast, the limited changes of a few glycan species in other mutants, including mns1_Δ, _mns101_Δ, and _mns1_Δ_101_Δ, are relatively subtle to be detected in the lectin blot, due to only minor differences in the average lengths of their _N-glycans compared to the WT. Therefore, we presented the lectin blotting data only for the _ugg1_Δ mutant.

(11) If there is ER stress under normal conditions in the Ugg1 mutant then technically this mutant should be growing more slowly under normal conditions. This is difficult to predict in a spot dilution assay where growth is only visualized at day three when any growth defect may have been corrected. The slower growth rather than the reduced secretion of GXM specifically is therefore more likely to be responsible for the reduced virulence.

We appreciate the reviewer’s insightful comment regarding the interplay between ER stress, growth defects, and virulence attenuation in the ugg1_Δ mutant. While retarded growth in _C. neoformans is often associated with reduced virulence, there are a few exceptions. For instance, disruptions in cell cycle progression in C. neoformans have been reported to result in larger capsule sizes, which rather enhance in vivo virulence when analyzed in Galleria mellonella infection models (García-Rodas et al., 2014). This highlights that growth defect alone is not sufficient for virulence attenuation. In the case of the _ugg1_Δ mutant, we speculate that the almost complete loss of virulence is attributed not only to its growth retardation but also to its impaired secretion of key virulence factors, including the polysaccharide capsule.

(12) The rationale for using leucine analogue 5',5',5'-trifluoroleucine (TFL), in a growth assay (Fig. 3C) to determine whether the defective ugg1Δ phenotypes are induced by ER stress caused by misfolded protein accumulation is not explained.

The leucine analogue 5',5',5'-trifluoroleucine (TFL) can be incorporated into newly synthesized proteins, disrupting normal folding and thus leading to the generation of misfolded proteins (Trotter et al., 2002; Cowie et al., 1959). In the context of a defective ERQC pathway, these misfolded proteins cannot be adequately repaired, resulting in their accumulation and triggering ER stress. Excessive ER stress may ultimately inhibit cell growth in the presence of TFL. This explanation has been incorporated into the revised manuscript (Page 11, lines 236–241).

(13) I would argue that only the Ugg1 and double Mns mutant were defective in virulence. For the single mutants, it looks like no difference was found relative to WT. The longer median survival of these mutants (if significant) is most likely due to poor infection technique.

We agree with the reviewer’s opinion that the mns1_Δ and _mns101_Δ single mutants have no significant difference in _in vivo virulence compared to the WT strain, unlike the _mns1_Δ_101_Δ double mutant which showed significant attenuated virulence. We had previously addressed that in the manuscript (Page 13, lines 267-269).

(14) The authors conclude that the ugg1Δ strain specifically is impaired in extracellular secretion of capsular polysaccharides but is this via classical (SAV1) secretion or EVs?

In addition to EV-mediated transport, capsular polysaccharide secretion can occur via the Sav1 (Sec4p)-mediated classical secretion pathway. However, our proteome data of total cell lysates indicated that the protein levels of Sav1 were comparable between the WT and _ugg1_Δ strains, suggesting that Sav1p function itself might not be impaired. Given that the _ugg1_Δ mutant exhibits altered vesicular structures (Supplementary Fig. S6) and loss of microvesicles (Fig. 8A), we speculate that a defect might occur at a post-Sav1p step, such as vesicle fusion with the plasma membrane, likely contributing to the complete defect in secretion of capsular polysaccharides in the _ugg1_Δ strain, in which EV biogenesis and defective cargo loading are severely impaired, producing EVs that lack capsular polysaccharides (Figure 8F). However, further studies should be carried out to define the contribution of SAV1 to the secretion of capsular polysaccharides in in the _ugg1_Δ strain.

(15) The rationale for doing 7 H is very confusing.

The experiment assessing raffinose utilization as a carbon source was inspired by the previous work of Garcia-Rivera et al., reporting that the _cap59_Δ mutant is unable to utilize raffinose due to a defect in the secretion of raffinose-hydrolyzing enzymes. As another way to investigate potential defects in the conventional secretion pathway, we investigated the growth of the _ugg1_Δ mutant in the presence of raffinose. Due to our extensive data length, we have decided to remove this complementary data from the manuscript.

(16) It is speculated in the discussion that ER stress impacts lipid/sterol synthesis and that LDs (lipid droplets?) aid the UPR and ERAD in degrading misfolded proteins during ER stress in S. cerevisiae. The authors mention that they observed a drastic increase in LDs in the ugg1Δ mutant. Where is this data? Even with the data, this is all speculation. The authors also speculate that increased numbers of vacuoles in ugg1 (where is the data?) could be the cause of the altered vesicular structures observed in the mutants, which may indicate abnormal lipid homeostasis caused by the ERQC defects, which could, in turn, affect EV biogenesis. Again, this is speculative.

The data on lipid droplets (LDs) and vacuole staining are presented in Supplementary Figure S6, showing a drastic increase in LDs and an increased in vacuolar size in the _ugg1_Δ mutant compared to the wild-type strain, especially in capsule-inducing conditions. In addition to such changes in vesicular structures, our preliminary data on sphingolipids and sterol analysis in the surface lipid fraction of the _ugg1_Δ mutant led us to propose the hypothesis that ERQC defects may impact lipid metabolism, which in turn could influence EV biogenesis and membrane properties. It is expected that these findings would provide a strong foundation for future studies exploring the link between ERQC, lipid homeostasis, and EV biogenesis. We have revised our speculation on the association of abnormal lipid homeostasis, caused by ERQC, with EV biogenesis more appropriately by adding the information on our preliminary data of lipid profiles and mentioning that the _ugg1_Δ mutant lacks microvesicles, which are derived from the plasma membrane (Page 24, lines 554-559).

Reviewer #2 (Recommendations for the authors):

(1) My suggestions for the authors are the same as those presented in the public review: (1) reducing the text in certain sections of the paper to improve readability for the audience, and (2) reconsidering the figures to reduce the amount of information in each one, moving some of the content to the supplementary material.

We thank the reviewer for their constructive suggestions regarding the organization and readability of the manuscript. As suggested, we addressed your concerns as follows:

(1) Reducing the text in the Introduction, Results, and Discussion sections by removing repetitive statements and simplifying complex descriptions where possible.

(2) Changing the presentation of figures: we have also reorganized the presentation of some data by moving non-essential data to the supplementary material. The updated figures and supplementary materials have been clearly referenced in the text to guide readers.

(3) Reorganization of materials and methods: some parts of methods were moved to Supplementary Information

(4) Removal of Figure 7H and the sentences describing the result

More detailed explanations on the reduction and reorganization are also described in the response to the major comments (2) and (3) made by Reviewer #1.

(2) Figure 3, for example, shows no difference in fungal growth under different cultivation conditions. This information is valuable but could be mentioned in the text, with the image provided as supplementary material, focusing the figure only on images that show significant growth differences among the strains. I suggest a similar approach for other figures so that the authors can include only the most relevant results in the main body of the article and move some figures to the supplementary materials.

For Fig. 3, the spotting data of the double mutant (previously Fig. 3B) is now presented in the supplementary information (Supplementary Fig. S3B). Additionally, the subcellular localization data (previously Fig 3E) was also moved to the supplementary material (Supplementary Fig. S4A).

Reviewer #3 (Recommendations for the authors):

(1) Line 43 "EV-mediated transport of virulence bags" doesn't make sense. EVs have been described as "virulence bags" (and are in this work later in the introduction) but this should here be "transport of virulence factors" or "compounds associated with virulence" but only if you have confirmed that the "cargo" is consistent with this- which is not evident in the abstract.

Thank you for your insightful comment. We have revised this to "EV-mediated transport of virulence factors" in line with your suggestion.

(2) Line 49 "secretory pathway" - is there not more than one secretion pathway?

Thank you for pointing this out. The term "secretory pathway" has been updated to "secretory pathways" to acknowledge the presence of both conventional and unconventional secretion mechanisms.

(3) Line 53 "recognizes folding defects, repairs them, and ensures the translocation of irreparable misfolded proteins" should be "recognizes folding defects and repairs them or ensures the translocation of irreparable misfolded proteins.

Thank you for pointing this out. We have revised the sentence as you suggested.

(4) Lines 88-90 ALG needs to be written out the first time - Asn-linked glycans. Also, consider adding that ALG genes are present in most eukaryotes as it is unclear what you are comparing C. neoformans to.

Thank you for your helpful comment. We have revised the text to write out "ALG" as "Asn-linked glycosylation" and added the sentence “ALG genes are evolutionary conserved in most eukaryotes” in the revised manuscript (Page 4, line 84).

(5) Line 99 Cryptococcus has already been abbreviated to C. so don't write it out again.

We have corrected "Cryptococcus" to “C.” throughout the manuscript after its first mention.

(6) Line 152- tunicamycin and DTT are not described yet, which may make it challenging for some readers to understand what these drugs are doing/why they were used. What is on lines 156 and 157 for these drugs should go up with the first mention of these drugs.

Thank you for your helpful suggestion. We have revised the manuscript to include the descriptions and purpose of using tunicamycin (TM) and dithiothreitol (DTT) immediately following their first mention, as recommended (Page 10, lines 208-210).

(7) The text for Figure 1 C is inaccurate. High temperature also induced KAR2, as noted above, but inaccurately stated in line 160. There is no comment on the significant UGG1 increase with tunicamycin or that KAR2 was highest in this condition.

Thank you for your thoughtful comment. We have better clarified the significant increase of UGG1 expression following tunicamycin treatment and KAR2 induction upon heat stress in the revised manuscript (Page 10, lines 216-217). Please note that Fig. 1C was revised and is now referred to as Fig. 3A.

(8) Figure 2B is not well explored/explained. There appears to be more protein in the mutant, including of higher weight in the intracellular compartment. It is difficult to ascertain if there is more too in the secretion phase with this gel. The methods do not specifically describe the concentration of protein added - just volume. Is what we are seeing a loading issue vs real differences?

Thank you for your insightful comments regarding Figure 2B. We added information on amounts of protein (30 µg per lane) in the legend of Figure 2B.

The main purpose of Fig. 2B is to examine the altered glycosylation pattern of ERQC by detecting glycoproteins using the Galanthus nivalis agglutinin, which specifically bind terminal α1,2-, α1,3-, and α1,6-linked mannose residues. The result of lectin blotting indicated that glycoproteins are more abundantly detected in the secretion fraction compared to in the soluble intracellular fraction, consistent with the general notion that more than 50% of secretory proteins are glycoproteins. Also, the more abundant proteins with decreased molecular weight in the secretion fraction of ugg1_Δ mutant supported the _N-glycan profiles with decreased hypermannosylation in _ugg1_Δ mutant. We added the purpose and more detailed interpretation on Figure 2B in the revised manuscript (Page 9, lines 174-179).

(9) Line 242 "melanin pigment" is redundant as melanin is a pigment.

We thank the reviewer for pointing out the redundancy in the phrase. We revised the text to simply state "melanin".

(10) Line 250 drops "completely" especially as the mutant did colonize the lungs of mice.

To avoid any possible misleading, we removed the term "completely" in the revised manuscript.

(11) Line 275- need to reference 18B7 as it is first introduced here.

We added the reference on the antibody 18B7 in the revised manuscript.

(12) Line 308- there are specific techniques to measure GXM size that could validate or refute the statement on "incomplete" polysaccharides. For example, DOI:10.1128/EC.00268-09.

We appreciated the valuable suggestion on specific techniques to measure GXM size, which will be one of key experiments in our future study. In the revised manuscript we cited the suggested reference to indicate the need for validation of our statement (Page 14, lines 316-318).

(13) Line 496 "mammals" - why is this used when the study is on a fungus, not a mammal? The structure of the first 2 paragraphs can be clearer to focus more on fungal biology.

We have compared both mammals and fungi to emphasize that the ERQC system is conserved among eukaryotes but diverged with a few species-specific features. This comparison is relevant in the context of understanding the evolutionary unique features of ERQC pathways in C. neoformans. We modified the first 2 paragraphs to clarify the main issue of our present study (Page 21, lines 472-483).

(14) Line 525- the ugg mutant was not avirulent as CFU was present and histopathology in the supplementary figures shows the tissue with ugg1 deletion was not normal (although the images are not especially easy to review). Yes, the mutant did not kill under your test conditions, but it was not avirulent (incapable of causing disease). Significantly attenuated or other descriptors should be utilized. Line 548 is also thus incorrect "complete loss of virulence").

We appreciate the reviewer’s concern regarding the description of the _ugg1_Δ mutant as avirulent. We agree that the use of merely “avirulent" may not fully capture the observed phenotypes in the CFU and histopathological data, since we cannot exclude the possibility that the _ugg1_Δ mutant retains the ability to establish an infection. Thus, we have revised the text by describing the _ugg1_Δ mutant as "almost avirulent".

(15) Line 597- the study by Fukuoka used kidney cells. It is misleading to not clearly state that this finding of ER stress was NOT done in fungi as the way it is presented makes it read as if this work was performed in C. neoformans. This should be clarified. This should also be double-checked and clarified for other statements, such as the reference to Harada in line 606, as this study used melanoma cells. These cell types are very different from cryptococcus- though I absolutely concur that lessons can be learned from comparative assessments.

We thank the reviewer for pointing out the need to clarify the experimental context of the cited studies. We explicitly stated the host cell types used in the referenced studies by Fukuoka et al. and by Harada et al., respectively, in the revised manuscript (Page 25, lines 560 and 568).

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

In a heroic effort, Ozanna Burnicka-Turek et al. have made and investigated conduction system-specific Tbx3-Tbx5 deficient mice and investigated their cardiac phenotype. Perhaps according to expectations, given the body of literature on the function of the two T-box transcription factors in the heart/conduction system, the cardiomyocytes of the ventricular conduction system seemed to convert to "ordinary" ventricular working myocytes. As a consequence, loss of VCS-specific conduction system propagation was observed in the compound KO mice, associated with PR and QRS prolongation and elevated susceptibility to ventricular tachycardia.

Strengths:

Great genetic model. Phenotypic consequences at the organ and organismal levels are well investigated. The requirement of both Tbx3 and Tbx5 for maintaining VCS cell state has been demonstrated.

We thank Reviewer #1 for acknowledging the effort involved in generating and characterizing the Tbx3/Tbx5 double conditional knockout mouse model and for highlighting the significance of this work in elucidating the role of these transcription factors in maintaining the functional and transcriptional identity of the ventricular conduction system. 

Weaknesses:

The actual cell state of the Tbx3/Tbx5 deficient conducting cells was not investigated in detail, and therefore, these cells could well only partially convert to working cardiomyocytes, and may, in reality, acquire a unique state.

We agree with Reviewer #1 that the Tbx3/Tbx5 double mutant ventricular conduction myocardial cells may only partially convert to working cardiomyocytes or may acquire a unique state.  The transcriptional state of the double mutant VCS cells was investigated by bulk profiling of key genes associated with specific conduction and non-conduction cardiac regions, including fast conduction, slow conduction, or working myocardium. Neither the bulk transcriptional approaches nor the optical mapping approaches we employed capture single-cell data; in both cases, the data represents aggregated signals from multiple cells (1, 2). Single cell approaches for transcriptional profiling and cellular electrophysiology would clarify this concern and are appropriate for future studies. 

(1) O’Shea C, Nashitha Kabri S, Holmes AP, Lei M, Fabritz L, Rajpoot K, Pavlovic D (2020) Cardiac optical mapping – State-of-the-art and future challenges. The International Journal of Biochemistry & Cell Biology 126:105804. doi: 10.1016/j.biocel.2020.105804. (2) Efimov IR, Nikolski VP, and Salama G (2004) Optical Imaging of the Heart. Circulation Research 95:21-33. doi: 10.1161/01.RES.0000130529.18016.35.

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive the adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First, the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

We appreciate Reviewer #2’s detailed summary of the study’s aims, methodologies, and findings, as well as their thoughtful suggestions for further analysis. We are grateful for their recognition of our genetic model’s novelty and robustness.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function.

We agree with this reviewer’s assessment of the assertions in our manuscript.  The novel combined Tbx5/Tbx3 double mutant model does not further explore the TBX5/TBX3 ratio concept, which we previously examined in detail (1). Instead, as the Reviewer notes, this manuscript focuses on testing a model that the coordinated activity of Tbx3 and Tbx5 defines specialized ventricular conduction identity. 

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model.

(2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line.

(3) Well-powered and convincing assessments of mortality and physiological phenotypes. (4) Isolation of genetically modified VCS cells using flow.

We thank Reviewer #2 for acknowledging the listed strengths of our study.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates the expression of VCS genes, which seem like distinct roles in VCS patterning. However, the authors move between different descriptions of the functional relationship and epistatic relationship between these factors, including terms like "cooperative", "coordinated", and "distinct" at various points. In a similar vein, sometimes terms like "reversion" are used to describe how VCS cells change after Tbx3/Tbx5 conditional knockout, and other times "transcriptional shift" and at other times "reprogramming". But these are all different concepts. The lack of a clear and consistent terminology for describing the phenomena observed makes the overarching claims of the manuscript more difficult to evaluate.

We discriminate prior work on the “long-standing and well-supported model’ supported by investigation of the role of Tbx5 and Tbx3 independently from this work examining the coordinated role of Tbx5 and Tbx3. Prior work demonstrated that Tbx3 represses working myocardial genes and Tbx5 activates expression of VCS genes, consistent with the reviewer’s suggestion of their distinct roles in VCS patterning. However, the current study uniquely evaluates the combined role of Tbx3 and Tbx5 in distinguishing specialized conduction identify from working myocardium, for the first time. 

We appreciate Reviewer #2’s feedback regarding the need for consistent terminology when describing the impact of the double Tbx3 and Tbx5 mutant. We will edit the manuscript to replace terms like “reversion” with “transcriptional shift” or “transformation” when describing the observed phenotype, and we will use “coordination” to describe the combined role of Tbx5 and Tbx3 in maintaining VCS-specific identity.

(2) A more direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from a single Tbx5 KO is presented for comparison to the double KO.

We thank Reviewer #2 for the suggestions that a direct comparison between Tbx5 single conditional knockout and Tbx3/Tbx5 double conditional knockout models may help isolate the specific contribution of Tbx3 deletion in addition to Tbx5 deletion. 

Previous studies have assessed the effect of single Tbx5 CKO in the VCS of murine hearts (1, 3, 5). Arnolds et al. demonstrated that the removal of Tbx5 from the adult ventricular conduction system results in VCS slowing, including prolonged PR and QRS intervals, prolongation of the His duration and His-ventricular (HV) interval (3).

Furthermore, Burnicka-Turek et al. demonstrated that the single conditional knockout of Tbx5 in the adult VCS caused a shift toward a pacemaker cell state, with ectopic beats and inappropriate automaticity (1). Whole-cell patch clamping of VCS-specific Tbx5 deficient cells revealed action potentials characterized by a slower upstroke (phase 0), prolonged plateau (phase 2), delayed repolarization (phase 3), and enhanced phase 4 depolarization - features characteristic of nodal action potentials rather than typical VCS action potentials (3). These observations were interpreted as uncovering nodal potential of the VCS in the absence of Tbx5. Based on the role of Tbx3 in CCS specification (2), we hypothesized that the nodal state of the VCS uncovered in the absence of Tbx5 was enabled by maintained Tbx3 expression. This motivated us to generate the double Tbx5

/ Tbx3 knockout model to examine the state of the VCS in the absence of both T-box TFs. In the current study, we demonstrate that the VCS-specific deletion of Tbx3 and Tbx5 results in the loss of fast electrical impulse propagation in the VCS, similar to that observed in the single Tbx5 mutant. However, unlike the Tbx5 single mutant, the Tbx3/Tbx5 double deletion does not cause a gain of pacemaker cell state in the VCS. Instead, the physiological data suggests a transition toward non-conduction working myocardial physiology. This conclusion is supported by the presence of only a single upstroke in the optical action potential (OAP) recorded from the His bundle region and VCS cells in Tbx3/Tbx5 double conditional knockout mice. The electrical properties of VCS cells in the double knockout are functionally indistinguishable from those of ventricular working myocardial cells. As a result, ventricular impulse propagation is significantly slowed, resembling activation through exogenous pacing rather than the rapid conduction typically associated with the VCS. We will edit the text of the manuscript to more carefully distinguish the observations between these models, as suggested.

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

(2) Mohan RA, Bosada FM, van Weerd JH, van Duijvenboden K, Wang J, Mommersteeg MTM, Hooijkaas IB, Wakker V, de Gier-de Vries C, Coronel R, Boink GJJ, Bakkers J, Barnett P, Boukens BJ, Christoffels VM (2020) T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system. Proc Natl Acad Sci U S A. 117:18617-18626. doi: 10.1073/pnas.1919379117.

(3) Arnolds DE, Liu F, Fahrenbach JP, Kim GH, Schillinger KJ, Smemo S, McNally EM, Nobrega MA, Patel VV, Moskowitz IP (2012) TBX5 drives Scn5a expression to regulate cardiac conduction system function. The Journal of Clinical Investigation 122:2509–2518. doi: 10.1172/JCI62617.

(4) Frank DU, Carter KL, Thomas KR, Burr RM, Bakker ML, Coetzee WA, Tristani-Firouzi M, Bamshad MJ, Christoffels VM, Moon AM (2012) Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis. Proc Natl Acad Sci U S A. 109:E154-63. doi: 10.1073/pnas.1115165109.

(5) Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE, Seidman JG (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116. doi: 10.1242/dev.01265. PMID: 15289437.

(3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.

And

the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment).

We appreciate Reviewer #2’s suggestion to expand the gene expression analysis in Tbx3/Tbx5-deficient VCS cells by including other specific genes and comparisons with “native”/actual working ventricular myocardial cells and broadening the gene panel. In this study, we evaluated core cardiac conduction system markers, revealing a loss of conduction system-specific gene expression in the double mutant VCS. Furthermore, we evaluated key working myocardial markers normally excluded from the conduction system, Gja1 and Smpx, revealing a shift towards a working myocardial state in the double mutant VCS (Figure 4). We agree that a more comprehensive analysis, such as transcriptome-wide approaches, would offer greater clarity on the extent and specificity of the observed shift from conduction to non-conduction identity. These approaches are appropriate directions for future studies.

(4) From the optical mapping data, it is difficult to distinguish between the presence of (a) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (b) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

And

In addition, the optical mapping dataset is incomplete and has alternative interpretations that are not excluded or thoroughly discussed.

We agree with Reviewer #2 that the resolution of the optical mapping experiment may be insufficient to precisely localize the conduction block due to the limited signal strength from the VCS. It is possible that the region defined as the His Bundle also includes portions of the right bundle branch. Our control mice show VCS OAP upstrokes consistent with those reported by Tamaddon et al. (2000) using Di-4-ANEPPS (1). We appreciate the Reviewer’s attention to alternative interpretations, and we will incorporate these caveats into the manuscript text. 

(1) Tamaddon HS, Vaidya D, Simon AM, Paul DL, Jalife J, Morley GE (2000) Highresolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system. Circulation Research 87:929-36. doi: 10.1161/01.res.87.10.929. 

Impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS but do not, as presented, constitute a decisive advance.

And

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented fit well within the existing paradigm of established functions of Tbx3 and Tbx5 in the VCS and in that sense do not decisively advance the field. Moreover, the authors' claims about the implications of the data are not always strongly supported by the data presented and do not fully explore alternative possibilities.

We appreciate Reviewer # 2’s acknowledgment of the elegance and novelty of the mouse model we generated. However, we respectfully disagree with their assessment that this work merely corroborates existing models without providing a decisive advance. Previous studies have investigated single Tbx5 or Tbx3 gene knockouts in-depth and established the T-box ratio model for distinguishing fast VCS from slow nodal conduction identity (1) that the reviewer alludes to in earlier comments. In contrast, this study aimed to explore a different model, that the combined effects of Tbx5 and Tbx3 distinguish adult VCS identity from non-conduction working myocardium. The coordinated Tbx3 and Tbx5 role in conduction system identify remained untested due to the lack of a mouse model that allowed their simultaneous removal. The very model the reviewer recognizes as “novel and elegantly constructed” has allowed the examination of the coordinated role of Tbx5 and Tbx3 for the first time. While we acknowledge the opportunity for additional depth of investigation of this model in future studies, the data we present provides consistent experimental support for the coordinated requirement of both Tbx5 and Tbx3 for ventricular cardiac conduction system identity. 

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterized the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of T_bx5_ and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinKcreERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterizing VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analyzed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

We appreciate the Reviewer’s comments regarding the originality and utility of our model and the strengths of our methodological approach. The Reviewer’s appreciation of the molecular and morphological analyses as well as their constructive feedback is highly valuable.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

We appreciate the reviewer’s concern regarding the morphology of the cardiac conduction system in the Tbx3/Tbx5 double conditional knockout model. We did not observe any structural abnormalities, as the Reviewer notes. We agree with their suggestion for using Genetic Inducible Fate Mapping to mark cardiac conduction cells expressing MinKCre. In fact, we utilized this approach to isolate VCS cells for transcriptional profiling. Specifically, we combined the tamoxifen-inducible MinKCreERT allele with the Cre-dependent R26Eyfp reporter allele to label MinKCre-expressing cells in both control VCS and VCS-specific double Tbx3/Tbx5 knockouts. EYFP-positive cells were isolated for transcriptional studies, ensuring that our analysis exclusively targeted conduction system-lineage marked cells. The ability to isolate MinKCre-marked cells from both controls and Tbx5/Tbx3 double mutants indicates that VCS cells persisted in the double knockout. Nonetheless, the suggestion for in-vivo marking by Genetic Inducible

Fate Mapping and morphologic analysis is a valuable recommendation for future studies. 

Reviewer #1 (Recommendations for the authors):

In a heroic effort, Ozanna Burnicka-Turek et al. have made and investigated conduction system-specific Tbx3-Tbx5 deficient mice and investigated their cardiac phenotype. Perhaps according to expectations, given the body of literature on the function of the two T-box transcription factors in the heart/conduction system, the cardiomyocytes of the ventricular conduction system seemed to convert to "ordinary" ventricular working myocytes. As a consequence, loss of VCS-specific conduction system propagation was observed in the compound KO mice, associated with PR and QRS prolongation and elevated susceptibility to ventricular tachycardia.

Previous work suggested the prediction that VCS-specific genetic ablation of both the TBX3 and TBX5 would transform fast-conducting adult VCS into cells resembling working myocardium, eliminating specialized CCS fate. The current study suggests that this prediction is at least to some extent accurate.

We appreciate Reviewer #1’s summary and recognition of our study. As the review notes, the simultaneous deletion of Tbx3 and Tbx5 in the mature ventricular conduction system (VCS) suggests a conversion of VCS to "ordinary" ventricular working myocytes. To our knowledge, this represents a novel observation and experimental model that uniquely captures the combined roles of these essential T-box transcription factors. We believe that this model offers a valuable platform for further investigation into the transcriptional mechanisms underlying conduction system specialization.

(1) The huge effort made to generate the DKO model contrasts with the limited efforts made to study the mechanism. Conditional deficiency of Tbx3 and Tbx5 creates an artificial situation that is useful for addressing fundamental mechanistic questions. The authors provide a rather superficial analysis of the changes in the VCS upon deletion of these two critically important factors and do not provide really novel insights into their requirement/function in the VCS gene regulatory network and epigenetic state. So to what extent do VCS cardiomyocytes (CMs) from Tbx3/5 DKO mice resemble "simple" working myocardium? To what extent do these cells acquire the working myocardial (epigenetic) state, do these cells have an epigenetic memory of the Tbx3/Tbx5+ history, is the enhancer usage between the modified VCS CMs and the working CMs similar or not, etc.? The assumption that the authors' data indicate that the DKO VCS CMs simply acquire a ventricular working "fate" is unlikely. Following this reasoning, the reverse experiment to induce Tbx3 and Tbx5 expression in working CMs would result in complete conversion to VCS CMs, which is also unlikely.

To answer such questions, transcriptomic and epigenetic state analysis, electrophysiologic analysis (e.g. patch-clamp), cell/subcellular level analysis, etc. would be required, as well as a comparison of the changed state of the DKO VCS CMs to that of working CMs.

This initial study focused on generating the Tbx3:Tbx5 double-conditional knockout model and characterizing the resulting physiological and molecular changes within the VCS. We analyzed transcriptomic markers of fast conduction (VCS), slow conduction (nodal), and non-conduction (working myocardium). Additionally, we applied optical mapping to evaluate the physiological consequences of the double knockout, which allowed a calculated AP of the VCS to be generated. We agree that a more in-depth mechanistic investigation of the VCS transformation upon Tbx3/Tbx5 deletion by transcriptomic or cellular electrophysiology could provide a deeper understanding of the precise transcriptional/epigenetic state of the VCS in the double knockout and clarify whether there is a partial or complete conversion of VCS cells to a simple working myocardial phenotype. The suggestions by the reviewer will be considered for future studies.

(2) Tbx3 stimulates BMP-TGFb signaling (e.g. positive loop between Tbx3-Bmp2), which in turn stimulates EMT and modulates the behavior of endocardial and mesenchymal cells. Did the authors investigate the impact of Tbx3/5 DKO on non-CM cells in and around the VCS? (see also comment 1). The insulation of the AVB for example could be a Tbx3/5 non cell autonomous target.

We appreciate the Reviewer’s suggestion to examine the impact of Tbx3/Tbx5 deletion on non-CM cells surrounding the VCS. While this is an intriguing avenue for future exploration, it falls outside the scope of the current study, which focused on the cardiomyocyte-specific roles of Tbx3 and Tbx5 in maintaining adult VCS identity.

(3) The MinK-Cre line used (from the Moskowitz lab) also recombines in the AVN (Arnolds et al 2011). The authors do not mention changes in the AVN, and systematically call the line VCS specific (which refers to the AVB, BB, PVCS I assume). This could also impact the PR interval. Please address.

The MinK-Cre line recombines in the atrioventricular bundle (AVB) and bundle branches (BB). It recombines in cardiomyocytes adjacent to the atrioventricular node (AVN). We previously interpreted these cells as the penetrating portion of the His bundle into the AVN. This line does not recombine in the vast majority, if any, physiologic nodal cells. We also assessed nodal conduction parameters by invasive electrophysiologic (EP) studies. Our data showed that non-VCS parameters, including sinus node recovery time, AV node recovery time, and atrial and ventricular effective refractory periods, remained within normal ranges in Tbx3:Tbx5-deficient mice (please see Figure 2I). These findings indicate that AVN function is preserved in the VCS-specific double knockout, reinforcing the specificity of the observed conduction defects to the ventricular conduction system.

(4) Did the authors also investigate the electrophysiological changes in the (EGFP+) DKO VCS CMs? Would these resemble the properties of ventricular working CMs, or would they still show some VCS properties? (see also comment 1).

We performed electrophysiologic analysis of the double knockout by optical mapping. Optical mapping provides tissue-level resolution, capturing the functional behavior of clusters of thousands of cells simultaneously, rather than individual cells. While this technique does not achieve single-cell resolution, it allows for a comprehensive assessment of electrophysiological changes across the VCS region. Single cell electrophysiology is a good idea for future studies. 

(5) Throughout the manuscript, the authors use "patterning" and "fate", which are applicable to development and differentiation, not to the situation where a gene is removed from fully differentiated cells in an adult organism resulting in a change of these cells. Perhaps more appropriate are "state" change and the requirement for "homeostasis/maintenance" of state.

We appreciate the Reviewer’s concern regarding the terminology used to describe changes in VCS cell identity. To ensure precision and uniformity, we replaced terms such as “fate” and “patterning” with “state” or “maintenance” to reflect the shift in cellular characteristics in a fully differentiated adult tissue context. 

Minor:

(1) Please provide all data points in bar graphs.

We have incorporated individual data points into the bar graphs as suggested, ensuring enhanced transparency and clarity in the data presentation.

“(2) Formally, gene expression levels between samples are not normally distributed. The Welch t-test used here assumes a normal distribution. Therefore, nonparametric tests should be used.

We appreciate Reviewer #1’s consideration of the appropriate statistical approach to the qPCR data and clarify our statistical approach here. Normality within each experimental group was assessed using the Shapiro-Wilk test. Between-group comparisons were conducted using Welch t-test, and multiple comparisons were corrected using the Benjamini & Hochberg method to control the false discovery rate (FDR) (71). If a significant difference was detected between two groups (t-test FDR < 0.05) but normality was rejected in any of the compared groups (Shapiro-Wilk P < 0.05), a non-parametric Wilcoxon rank-sum test was used for verification. A significant group-mean difference was confirmed at one-tailed Wilcoxon P≤0.05 (detailed in Supplementary Data Set I). Furthermore, we have updated the qRT-PCR information in each figure and their respective legends as follows. Statistical analysis was performed using R version 4.2.0. We have included a new Supplementary Data Set I, detailing the statistical analysis of qRT-PCR data. Additionally, we have revised the Methods/Statistics section to detail the applied statistical analysis. 

(3) Some of the panels of figures are tiny and cannot be evaluated. For example, in Figure 1B the actual data (expression of Tbx3/5) is impossible to see.

We appreciate the Reviewer’s observation and have revised the figures to improve visual clarity and ensure that the presented data are easily interpretable by readers.

Reviewer #2 (Recommendations for the authors):

Additional Experiments, Data, Analysis:

(1) Comparisons between both single knockouts and double knockouts at the phenotypic level are needed. In some instances, the data is shown (e.g., mortality and EKG) but direct statistical comparison is not performed. In other instances (optical mapping and gene expression), data with single knockouts are not shown. If combined VCS Tbx3/Tbx5 deletion does not change the phenotype of the VCS Tbx5 single deletion, this should be explicitly stated and discussed.

We appreciate Reviewer #2’s suggestion to compare the phenotypic outcomes of the Tbx3 and Tbx5 single conditional knockout models with those observed in Tbx3/Tbx5 double conditional knockout model. We have expanded the discussion section of our manuscript to incorporate a more detailed comparison between the double Tbx3/Tbx5 model and the single Tbx5 and Tbx3 models [1-5], highlighting the distinct phenotypic outcomes of the single and double knockouts.

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

(2) Mohan RA, Bosada FM, van Weerd JH, van Duijvenboden K, Wang J, Mommersteeg MTM, Hooijkaas IB, Wakker V, de Gier-de Vries C, Coronel R, Boink GJJ, Bakkers J, Barnett P, Boukens BJ, Christoffels VM (2020) T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system. Proc Natl Acad Sci U S A. 117:18617-18626. doi: 10.1073/pnas.1919379117.

(3) Arnolds DE, Liu F, Fahrenbach JP, Kim GH, Schillinger KJ, Smemo S, McNally EM, Nobrega MA, Patel VV, Moskowitz IP (2012) TBX5 drives Scn5a expression to regulate cardiac conduction system function. The Journal of Clinical Investigation 122:2509–2518. doi: 10.1172/JCI62617.

(4) Frank DU, Carter KL, Thomas KR, Burr RM, Bakker ML, Coetzee WA, Tristani-Firouzi M, Bamshad MJ, Christoffels VM, Moon AM (2012) Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis. Proc Natl Acad Sci U S A. 109:E154-63. doi: 10.1073/pnas.1115165109. [5] Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE, Seidman JG (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116. doi: 10.1242/dev.01265.

(2) Genome-wide expression analysis including working myocardium would provide stronger evidence for interconversion of cell states. Ideally, this would include single knockouts.

We agree that a genome-wide expression analysis, including a direct comparison with working myocardium, would provide more comprehensive insights into cell state transitions in Tbx3:Tbx5-deficient VCS cells. Additionally, incorporating single knockout models into such analyses would further clarify the distinct and cooperative contributions of Tbx3 and Tbx5 to maintaining VCS identity. This is a good suggestion for future studies.

(3) This may not be essential to support the authors' claims, but the addition of epigenetic data from single and double KO VCS using ATAC-seq (which can be performed with relatively small numbers of cells) could provide stronger evidence for cell state changes of the kind hypothesized by the authors.

We agree that epigenetic data such as ATAC-seq would complement transcriptional analyses and provide insight into chromatin states that underlie the observed cellular reprogramming. This is a good suggestion for follow-up studies to further characterize the molecular state of Tbx3:Tbx5-deficient VCS cells.

(4) Additional clarification of the optical mapping experiments to exclude alternative interpretations like focal right bundle branch block and to include single knockouts for comparison - if the Tbx5 single KO looks the same as the double KO that would be very important to know and would directly affect interpretation of the experiment.

Right septal optical mapping preparation involved removing the right ventricular free wall to directly image the right ventricular septum, which contains the VCS. In a healthy mouse, there are two peak components of the optical action potential upstroke, the first peak due to the activation of the VCS and the second due to the activation of the ventricular cardiomyocytes. Importantly, in Tbx3:Tbx5 double-conditional knockout mice, the first peak was absent, rather than delayed, indicating loss of fast conduction through the VCS. This absence suggests a shift in VCS cells toward a ventricular working myocardial phenotype, rather than a regional conduction block or delayed propagation through a structurally intact VCS.

Previous studies from our group have extensively characterized the effect of single Tbx5 knockout on the VCS in murine hearts [1, 2, 3]. Arnolds et al. demonstrated that VCSspecific Tbx5-deficiency results in significant slowing of VCS conduction, evidenced by prolonged PR and QRS intervals, along with lengthening of the atrio-Hisian interval, His duration, and Hisioventricular interval [1]. Although both single Tbx5 knockout and Tbx3:Tbx5 double knockout mice exhibit slowing of ventricular conduction system, our optical mapping studies reveal distinct differences in their electrophysiological phenotypes. Burnicka-Turek et al. showed that the single knockout of Tbx5 in the VCS leads to a shift toward a pacemaker cell state, evidenced by ectopic beats originating in the ventricles and inappropriate automaticity [3]. During spontaneous beats, electrical impulses were retrogradely activated, propagating from the ventricles to the atria [3]. Whole-cell patch clamping recordings confirmed that Tbx5-deficient VCS cells displayed action potentials resembling pacemaker cells, characterized by slower upstroke (phase 0), prolonged plateau (phase 2), delayed repolarization (phase 3), and enhanced phase 4 depolarization [3]. In contrast, our current study on VCS-specific Tbx3:Tbx5 double knockout demonstrates a loss of the VCS-specific fast conduction propagation. Optical mapping demonstrated the absence of the initial upstroke corresponding to VCS activation in the His bundle region, indicating a shift in the VCS cells toward a ventricular working myocardium state. This loss of fast conduction properties highlights a fundamental distinction between single and double knockouts, suggesting that both Tbx3 and Tbx5 are required to maintain VCS identity and function.

(1) D. E. Arnolds et al., “TBX5 drives Scn5a expression to regulate cardiac conduction system function,” J. Clin. Invest., vol. 122, no. 7, pp. 2509–2518, Jul. 2012, doi: 10.1172/JCI62617.

(2) Moskowitz, I.P., Pizard, A., Patel, V.V., Bruneau, B.G., Kim, J.B., Kupershmidt, S., Roden, D., Berul, C.I., Seidman, C.E., Seidman, J.G. (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131(16):4107-4116. 

(3) Burnicka-Turek, O., Broman, M.T., Steimle, J.D., Boukens, B.J., Peterenko, N.B, Ikegami, K., Nadadur, R.D., Qiao, Y., Arnolds, D.E., Yang, X.H., Patel, V.V., Nobrega, M.A., Efimov, I.R., Moskowitz, I.P. (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circ Res. 127(3):e94-e106. 

Methods:

(1) Additional methods on FACS are required. The methods section references a paper from 2004 (reference 67) that describes the flow sorting of embryonic cardiomyocytes. However, flow cytometric isolation of intact adult cardiomyocytes, which the authors describe in the present work, is a distinct technique and generally requires special equipment. These need to be described in more detail to be fully replicable.

We thank Reviewer #2 for highlighting the need to provide additional details regarding our flow cytometric isolation of adult VCS cardiomyocytes. While we referenced earlier methods, we agree that isolating adult cardiomyocytes requires specialized approaches. Therefore, we revised the Methods section to include a detailed description of the equipment, procedures, and adaptations specific to isolating intact adult VCS cells to ensure full replicability.

Minor Corrections:

(1) Figure 1D. Please add a statistical test for mortality between the double conditional KO and the Tbx5 conditional KO.

We have revised Figure 1D to include the statistical test comparing mortality between the Tbx3:Tbx5 double conditional knockout and the Tbx5 conditional knockout cohorts.

(2) Figure 2A, 2I, 3A: Please include all individual data points not just a bar graph with error bars.

We have added all individual data points to the bar graphs as recommended, enhancing the transparency and clarity of the data presentation.

(3) Figure 2A: Please consider separate graphs for PR and QRS with appropriately scaled Y-axis so differences are easier to see.

We appreciate Reviewer #2’s suggestion and fully agree with it. As a result, we have revised Figure 2A to include separate graphs for PR and QRS intervals, each with appropriately scaled Y-axes. This adjustment enhanced both the readability and the clarity of the observed differences.

(4) Figure 3 G-K: The figure would be easier to interpret for the reader if genotypes were shown in the figure not just in the legend.

We agree with Reviewer #2’s suggestion and have revised Figure 3 accordingly by adding genotype labels directly to the histological sections in Panels G-K. This update improves clarity, making the data easier for readers to interpret without needing to refer to the figure legend.

(5) Figure 4A, C: Are vertical axes mislabeled? They say, "CON VCS and TBX5OE VCS". Please double-check axis labels and data on the graph.

We appreciate the Reviewer bringing the mislabeling of the vertical axis in Figure 4 to our attention. We have corrected the labeling errors and ensured consistency between the graph and the underlying data.

(6) Legend to Supplementary Figure 6. Says "Tbx3:Tbx3" instead of "Tbx3:Tbx5".

We thank Reviewer #2 for pointing out the typo. It has been corrected to: “Supplementary Figure 6. Tbx3:Tbx5 double-conditional knockout mice exhibit QRS prolongation”.

(7) Discussion. The authors write, "In Tbx3:Tbx5 double VCS knockout, we observed repression of fast VCS markers and also repression of Pan-CCS markers transcribed throughout the entire CCS." The term 'repression' has a specific connotation with transcription regulators that is likely not intended in this context so perhaps 'reduced expression' would be better here?

We agree with Reviewer #2 and have replaced “repression” with “reduced expression” throughout the text (look below for references).

“In the Tbx3:Tbx5 double VCS knockout, we observed a reduction in the expression of both fast VCS markers and Pan-CCS markers transcribed throughout the entire CCS.”

(8) Discussion, the authors write, "This study combined with prior literature (1, 7, 11, 15, 26, 53, 54) indicates that the presence of both Tbx3 and Tbx5 is necessary for the specification of the adult VCS (Figure 7)." Since this work presents data from an adult conditional deletion, it's not clear how it informs our understanding of the specification, which occurs during development. Perhaps "maintenance of VCS fate" would be more appropriate here?

We agree with Reviewer #2 that the term “maintenance of VCS fate” is more appropriate in the context of our study. Accordingly, we have updated the text to reflect this terminology.

Reviewer #3 (Recommendations for the authors):

(1) Figure 2B: It is hard to see the IF images. What is the cardiac structure studied? Maybe a dashed line and a label to define the region and the structure represented will help. As the authors have described that the crosses used contain a reporter allele (R26-EYFP), a clearer way to show these results would be to include images of the linage traced cells with the reporter, not only to identify the CCS structure analyzed, but also to demonstrate that the deletion is specific to the MinK-creERT expression in the CCS.

We appreciate the Reviewer’s suggestion to improve the clarity of Figure 2B by delineating the cardiac structures analyzed. In response, we have added dashed lines and labels to highlight the regions of interest within the IF images. Unfortunately, we were unable to capture high-quality EYFP fluorescence images for these sections. However, to address this concern, we microdissected the region shown in the IF images and performed FACS to isolate EYFP-positive cells from this specific area. These sorted cells were subsequently used for qPCR analysis, which confirmed the presence of Tbx3 and Tbx5 in control samples and the successful deletion of both genes in the doubleconditional knockout samples (Figure 2C, middle panel). We believe this approach provides robust evidence for the specificity of the MinK-CreERT expression in the CCS and the efficiency of gene deletion in the targeted region.

(2) 3G-K: The authors describe the absence of morphological defects in the tissue sections of adult hearts from the different genotypes analyzed. Although this reviewer agrees that there seem to be no major defects in the general cardiac morphology of these animals, the higher magnification images suggest some tissue differences at the level of the AVN especially in the double HET, double HOMO, and the Tbx3 HOMO. Is that due to the section plane used? If so, more appropriate and comparable sections must be provided. Again, as the crosses used by the authors contain a reporter allele (R26-EYFP), it is required that the authors show that the CCS cells, where deletions are induced, are still present in equivalent areas in the mutants and that they remain in similar numbers only failing to maintain their specification into CCS due to Tbx3 and Tbx5 loss of function.

This analysis will reinforce the authors' claims on the role of Tbx5/Tbx3 in this process.

We thank the reviewer for their thorough assessment and thoughtful feedback on our histological analysis. The higher magnification images in Figure 3G-K do not specifically present the AVN. These sections primarily represent areas of the ventricular conduction system (VCS), particularly the His bundle and bundle branches, rather than the AVN itself. We do not believe that the observed morphological differences are related to AVN tissue, and there were no functional deficits attributable to the AVN in the double knockout. Furthermore, the Mink-Cre allele used in this study does not recombine in the ANV proper.   We agree that confirming the presence of CCS cells in equivalent regions across different genotypes is crucial. Our approach using FACS-based isolation of EYFP-positive cells from the VCS, followed by qPCR analysis, provides evidence that these cells remain present in double conditional knockouts, although they fail to maintain their specialized gene expression profile. This reinforces our conclusion that Tbx3 and Tbx5 are essential for maintaining the molecular identity of CCS cells, rather than their physical presence.

(3) Figure 4: The authors performed molecular analysis by qPCR and WB in Tbx5/Tbx3 double mutants to demonstrate that CCS cells lose the expression of CCS genes and express working myocardium genes. Could this be further demonstrated by ISH, HCR, or IF together with lineage tracing to provide evidence that these changes are located where the CCS tissues are in the control embryos? Analysis of 2 or 3 of these markers of each type on tissue sections would be enough.

We thank the Reviewer for their insightful suggestion regarding additional validation of our molecular findings through ISH, HCR, or IF combined with lineage tracing. However, we would like to clarify that the molecular analyses we performed by qPCR and WB were conducted on EYFP-positive cells that were specifically isolated from the ventricular conduction system (VCS) region of both control and double conditional knockout (dCKO) mice. These EYFP-positive cells were obtained through fluorescence-activated cell sorting (FACS), ensuring that our analyses were confined to the targeted VCS population. Alternate approaches are appropriate for future studies to investigate the precise genomic and molecular nature of the transformation observed in the double knockout.

(4) Discussion: in the discussion section the authors conclude that the combined role of Tbx5/Tbx3 is critical for the specification of the adult VCS. However, as the Tbx5/Tbx3 loss of function conditions are only induced in adult animals 6 weeks old, would it be more appropriate that their function is the maintenance of the VCS cell fate and that if not present these cells return to the working myocardium fate? If the authors believe that these genes are involved in the induction of VCS specification in adults, then they need to demonstrate that, before the loss of function induction at 6 weeks, these cells are not yet specified as adult VCS.

We appreciate the Reviewer’s clarification regarding terminology. We agree that our study focuses on adult-specific conditional deletion and thus reflects the maintenance, rather than the specification, of VCS cell fate. Accordingly, we have revised the text to explicitly state that Tbx3 and Tbx5 are critical for maintaining VCS identity in adult mice, and that their loss leads to a shift toward a working myocardial fate.

Minor:

(1) There is no consistency in the way the quantitative data is shown in graphs. There are some graphs showing only bars, other dot plots, and other a combination of both. The authors must homogenise the representation of quantitative data showing the different data points in dot plots and not in bar graphs.

We have standardized the quantitative data presentation across all figures, by including individual data points in bar graphs, ensuring enhanced transparency and clarity.

(2) Figure 3: The labels defining the genotypes corresponding to the different histological sections of adult hearts (Panels G-K) are missing. Panels J and K are not referenced in the text.

We thank Reviewer #3 for highlighting these omissions. We have added the genotype labels to the histological sections in Panels G-K of Figure 3 to ensure clarity. Furthermore, we have now referenced Panels J and K in the results and in the supplementary material (please look below for references).

“Histological examination of all four-chambers demonstrated no discernible differences between VCS-specific Tbx3:Tbx5 double-knockout (Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+; MinK^CreERT2/+) and control (Tbx3^+/+;Tbx5^+/+;R26^EYFP/+; MinK^CreERT2/+) mice, nor between . the double-knockout (Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+; MinK^CreERT2/+) and single-knockout models for either Tbx3 (Tbx3^fl/fl;Tbx5^+/+;R26^EYFP/+; MinK^CreERT2/+) or Tbx5 (Tbx3^+/+;Tbx5^fl/fl;R26^EYFP/+; MinK^CreERT2/+).Ventricular muscle appeared normal without hypertrophy or myofibrillar disarray and no fibrosis was present (Figure 3G, 3I, 3J, and 3K, respectively).”

“Additionally, we confirmed the absence of histological and structural abnormalities in these mice, aligning with previous findings (Figures 3A, 3F versus 3B, and 3K versus 3G, respectively)(1, 11).”

(3) Typo: Supplementary Figure 6. Tbx3:Tbx3 double-conditional knockout: it should say Tbx5:Tbx3 double-conditional knockout.

We thank Reviewer #3 for pointing out the typo. It has been corrected to: “Supplementary Figure 6. Tbx3:Tbx5 double-conditional knockout mice exhibit QRS prolongation”.

Author response:

The following is the authors’ response to the original reviews

Response to the Editors’ Comments

Thankyou for this summary of the reviews and recommendations for corrections. We respond to each in turn, and have documented each correction with specific examples contained within our response to reviewers below.

‘They all recommend to clarify the link between hypotheses and analyses, ground them more clearly in, and conduct critical comparisons with existing literature, and address a potential multiple comparison problem.’

We have restructured our introduction to include the relevant literature outlined by the reviewers, and to be more clearly ground the goals of our model and broader analysis. We have additionally corrected for multiple comparisons within our exploratory associative analyses. We have additionaly sign posted exploratory tests more clearly.

‘Furthermore, R1 also recommends to include a formal external validation of how the model parameters relate to participant behaviour, to correct an unjustified claim of causality between childhood adversity and separation of self, and to clarify role of therapy received by patients.’

We have now tempered our language in the abstract which unintentionally implied causality in the associative analysis between childhood trauma and other-to-self generalisation. To note, in the sense that our models provide causal explanations for behaviour across all three phases of the task, we argue that our model comparison provides some causal evidence for algorithmic biases within the BPD phenotype. We have included further details of the exclusion and inclusion criteria of the BPD participants within the methods.

R2 specifically recommends to clarify, in the introduction, the specific aim of the paper, what is known already, and the approach to addressing it.’

We have more thoroughly outlined the current state of the art concerning behavioural and computational approaches to self insertion and social contagion, in health and within BPD. We have linked these more clearly to the aims of the work.

‘R2 also makes various additional recommendations regarding clarification of missing information about model comparison, fit statistics and group comparison of parameters from different models.’

Our model comparison approach and algorithm are outlined within the original paper for Hierarchical Bayesian Model comparison (Piray et al., 2019). We have outlined the concepts of this approach in the methods. We have now additionally improved clarity by placing descriptions of this approach more obviously in the results, and added points of greater detail in the methods, such as which statistics for comparison we extracted on the group and individual level.

In addition, in response to the need for greater comparison of parameters from different models, we have also hierarchically force-fitted the full suite of models (M1-M4) to all participants. We report all group differences from each model individually – assuming their explanation of the data - in Table S2. We have also demonstrated strong associations between parameters of equivalent meaning from different models to support our claims in Fig S11. Finally, we show minimal distortion to parameter estimates in between-group analysis when models are either fitted hierarchically to the entire population, or group wise (Figure S10).

‘R3 additionally recommends to clarify the clinical and cognitive process relevance of the experiment, and to consider the importance of the Phase 2 findings.’

We have now included greater reference to the assumptions in the social value orientation paradigm we use in the introduction. We have also responded to the specific point about the shift in central tendencies in phase 2 from the BPD group, noting that, while BPD participants do indeed get more relatively competitive vs. CON participants, they remain strikingly neutral with respect to the overall statespace. Importantly, model M4 does not preclude more competitive distributions existing.

‘Critically, they also share a concern about analyzing parameter estimates fit separately to two groups, when the best-fitting model is not shared. They propose to resolve this by considering a model that can encompass the full dynamics of the entire sample.’

We have hierarchically force-fitted the full suite of models (M1-M4) to all participants to allow for comparison between parameters within each model assumption. We report all group differences from each model individually – assuming their explanation of the data - in Table S2 and Table S3. We have also demonstrated strong associations between parameters of equivalent meaning from different models to support our claims in Fig S11. We also show minimal distortion to parameter estimates in between-group analysis when models are either fitted hierarchically to the entire population, or group wise (Figure S10).

Within model M1 and M2, the parameters quantify the degree to which participants believe their partner to be different from themselves. Under M1 and M2 model assumptions, BPD participants have meaningfully larger versus CON (Fig S10), which supports the notion that a new central tendency may be more parsimonious in phase 2 (as in the case of the optimal model for BPD, M4). We also show strong correlations across models between under M1 and M2, and the shift in central tendenices of beliefs between phase 1 and 2 under M3 and M4. This supports our primary comparison, and shows that even under non-dominant model assumptions, parameters demonstrate that BPD participants expect their partner’s relative reward preferences to be vastly different from themselves versus CON.

‘A final important point concerns the psychometric individual difference analyses which seem to be conducted on the full sample without considering the group structure.’

We have now more clearly focused our psychometric analysis. We control for multiple comparisons, and compare parameters across the same model (M3) when assessing the relationship between paranoia, trauma, trait mentalising, and social contagion. We have relegated all other exploratory analyses to the supplementary material and noted where p values survive correction using False Discovery Rate.

Reviewer 1:

‘The manuscript's primary weakness relates to the number of comparisons conducted and a lack of clarity in how those comparisons relate to the authors' hypotheses. The authors specify a primary prediction about disruption to information generalization in social decision making & learning processes, and it is clear from the text how their 4 main models are supposed to test this hypothesis. With regards to any further analyses however (such as the correlations between multiple clinical scales and eight different model parameters, but also individual parameter comparisons between groups), this is less clear. I recommend the authors clearly link each test to a hypothesis by specifying, for each analysis, what their specific expectations for conducted comparisons are, so a reader can assess whether the results are/aren't in line with predictions. The number of conducted tests relating to a specific hypothesis also determines whether multiple comparison corrections are warranted or not. If comparisons are exploratory in nature, this should be explicitly stated.’

We have now corrected for multiple comparisons when examining the relationship between psychometric findings and parameters, using partial correlations and bootstrapping for robustness. These latter analyses were indeed not preregistered, and so we have more clearly signposted that these tests were exploratory. We chose to focus on the influence of psychometrics of interest on social contagion under model M3 given that this model explained a reasonable minority of behaviour in each group. We have now fully edited this section in the main text in response, and relegated all other correlations to the supplementary materials.

‘Furthermore, the authors present some measures for external validation of the models, including comparison between reaction times and belief shifts, and correlations between model predicted accuracy and behavioural accuracy/total scores. However it would be great to see some more formal external validation of how the model parameters relate to participant behaviour, e.g., the correlation between the number of pro-social choices and ß-values, or the correlation between the change in absolute number of pro-social choices and the change in ß. From comparing the behavioural and computational results it looks like they would correlate highly, but it would be nice to see this formally confirmed.’

We have included this further examination within the Generative Accuracy and Recovery section:

‘We also assessed the relationship (Pearson rs) between modelled participant preference parameters in phase 1 and actual choice behaviour: was negatively correlated with prosocial versus competitive choices (r=-0.77, p<0.001) and individualistic versus competitive choices (r=-0.59, p<0.001); was positively correlated with individualistic versus competitive choices (r=0.53, p<0.001) and negatively correlated with prosocial versus individualistic choices (r=-0.69, p<0.001).’

‘The statement in the abstract that 'Overall, the findings provide a clear explanation of how self-other generalisation constrains and assists learning, how childhood adversity disrupts this through separation of internalised beliefs' makes an unjustified claim of causality between childhood adversity and separation of self - and other beliefs, although the authors only present correlations. I recommend this should be rephrased to reflect the correlational nature of the results.’

Sorry – this was unfortunate wording: we did not intend to imply causation with our second clause in the sentence mentioned. We have amended the language to make it clear this relationship is associative:

‘Overall, the findings provide a clear explanation of how self-other generalisation constrains and assists learning, how childhood adversity is associated with separation of internalised beliefs, and makes clear causal predictions about the mechanisms of social information generalisation under uncertainty.’

‘Currently, from the discussion the findings seem relevant in explaining certain aberrant social learning and -decision making processes in BPD. However, I would like to see a more thorough discussion about the practical relevance of their findings in light of their observation of comparable prediction accuracy between the two groups.’

We have included a new paragraph in the discussion to address this:

‘Notably, despite differing strategies, those with BPD achieved similar accuracy to CON participants in predicting their partners. All participants were more concerned with relative versus absolute reward; only those with BPD changed their strategy based on this focus. Practically this difference in BPD is captured either through disintegrated priors with a new median (M4) or very noisy, but integrated priors over partners (M1) if we assume M1 can account for the full population. In either case, the algorithm underlying the computational goal for BPD participants is far higher in entropy and emphasises a less stable or reliable process of inference. In future work, it would be important to assess this mechanism alongside momentary assessments of mood to understand whether more entropic learning processes contribute to distressing mood fluctuation.’

‘Relatedly, the authors mention that a primary focus of mentalization based therapy for BPD is 'restoring a stable sense of self' and 'differentiating the self from the other'. These goals are very reminiscent of the findings of the current study that individuals with BPD show lower uncertainty over their own and relative reward preferences, and that they are less susceptible to social contagion. Could the observed group differences therefore be a result of therapy rather than adverse early life experiences?’

This is something that we wish to explore in further work. While verbal and model descriptions appear parsimonious, this is not straight forward. As we see, clinical observation and phenomenological dynamics may not necessarily match in an intuitive way to parameters of interest. It may be that compartmentalisation of self and other – as we see in BPD participants within our data – may counter-intuitively express as a less stable self. The evolutionary mechanisms that make social insertion and contagion enduring may also be the same that foster trust and learning.

‘Regarding partner similarity: It was unclear to me why the authors chose partners that were 50% similar when it would be at least equally interesting to investigate self-insertion and social contagion with those that are more than 50% different to ourselves? Do the authors have any assumptions or even data that shows the results still hold for situations with lower than 50% similarity?’

While our task algorithm had a high probability to match individuals who were approximately 50% different with respect to their observed behaviour, there was variation either side of this value. The value of 50% median difference was chosen for two reasons: 1. We wanted to ensure participants had to learn about their partner to some degree relative to their own preferences and 2. we did not want to induce extreme over or under familiarity given the (now replicated) relationship between participant-partner similarity and intentional attributions (see below). Nevertheless, we did have some variation around the 50% median. Figure 3A in the top left panel demonstrates this fluctuation in participant-partner similarity and the figure legend further described this distribution (mean = 49%, sd = 12%). In future work we want to more closely manipulate the median similarity between participants and partners to understand how this facilitates or inhibits learning and generalisation.

There is some analysis of the relationship between degrees of similiarity and behaviour. In the third paragraph of page 15 we report the influence of participant-partner similarity on reaction times. In prior work (Barnby et al., 2022; Cognition) we had shown that similarity was associated with reduced attributions of harm about a partner, irrespective of their true parameters (e.g. whether they were prosocial/competitive). We replicate this previous finding with a double dissociation illustrated in Figure 4, showing that greater discrepancies in participant-partner prosociality increases explicit harmful intent attributions (but not self-interest), and discrepancies in participant-partner individualism reduces explicit self-interest attributions (but not harmful intent). We have made these clearer in our results structure, and included FDR correction values for multiple comparisons.

The methods section is rather dense and at least I found it difficult to keep track of the many different findings. I recommend the authors reduce the density by moving some of the secondary analyses in the supplementary materials, or alternatively, to provide an overall summary of all presented findings at the end of the Results section.

We have now moved several of our exploratory findings into the supplementary materials, noteably the analysis of participant-partner similarity on reaction times (Fig S9), as well as the uncorrected correlation between parameters (Fig S7).

Fig 2C) and Discussion p. 21: What do the authors mean by 'more sensitive updates'? more sensitive to what?

We have now edited the wording to specify ‘more belief updating’ rather than ‘sensitive’ to be clearer in our language.

P14 bottom: please specify what is meant by axial differences.

We have changed this to ‘preference type’ rather than using the term ‘axial’.

It may be helpful to have Supplementary Figure 1 in the main text.

Thank you for this suggestion. Given the volume of information in the main text we hope that it is acceptable for Figure S1 to remain in the supplementary materials.

Figure 3D bottom panel: what is the difference between left and right plots? Should one of them be alpha not beta?

The left and right plots are of the change in standard deviation (left) and central tendency (right) of participant preference change between phase 1 and 3. This is currently noted in the figure legend, but we had added some text to be clearer that this is over prosocial-competitive beliefs specifically. We chose to use this belief as an example given the centrality of prosocial-comeptitive beliefs in the learning process in Figure 2. We also noticed a small labelling error in the bottom panels of 3D which should have noted that each plot was either with respect to the precision or mean-shift in beliefs during phase 3.

‘The relationship between uncertainty over the self and uncertainty over the other with respect to the change in the precision (left) and median-shift (right) in phase 3 prosocial-competitive beliefs .’

Supplementary Figure 4: The prior presented does not look neutral to me, but rather right-leaning, so competitive, and therefore does indeed look like it was influenced by the self-model? If I am mistaken please could the authors explain why.

This example distribution is taken from a single BPD participant. In this case, indeed, the prior is somewhat right-shifted. However, on a group level, priors over the partner were closely centred around 0 (see reported statistics in paragraph 2 under the heading ‘Phase 2 – BPD Participants Use Disintegrated and Neutral Priors). However, we understand how this may come across as misleading. For clarity we have expanded upon Figure S4 to include the phase 1 and prior phase 2 distributions for the entire BPD population for both prosocial and individualistic beliefs. This further demonstrates that those with BPD held surprisingly neutral beliefs over the expectations about their partners’ prosociality, but had minor shifts between their own individualistic preferences and the expected individualistic preferences of their partners. This is also visible in Figure S2.

Reviewer 2:

‘There are two major weaknesses. First, the paper lacks focus and clarity. The introduction is rather vague and, after reading it, I remained confused about the paper's aims. Rather than relying on specific predictions, the analysis is exploratory. This implies that it is hard to keep track, and to understand the significance, of the many findings that are reported.’

Thank you for this opportunity to be clearer in our framing of the paper. While the model makes specific causal predictions with respect to behavioural dynamics conditional on algorithmic differences, our other analyses were indeed exploratory. We did not preregister this work but now given the intriguing findings we intent to preregister our future analyses.

We have made our introduction clearer with respect to the aims of the paper:

‘Our present work sought to achieve two primary goals: 1. Extend prior causal computational theories to formalise the interrelation between self-insertion and social contagion within an economic paradigm, the Intentions Game and 2., Test how a diagnosis of BPD may relate to deficits in these forms of generalisation. We propose a computational theory with testable predictions to begin addressing this question. To foreshadow our results, we found that healthy participants employ a mixed process of self-insertion and contagion to predict and align with the beliefs of their partners. In contrast, individuals with BPD exhibit distinct, disintegrated representations of self and other, despite showing similar average accuracy in their learning about partners. Our model and data suggest that the previously observed computational characteristics in BPD, such as reduced self-anchoring during ambiguous learning and a relative impermeability of the self, arise from the failure of information about others to transfer to and inform the self. By integrating separate computational findings, we provide a foundational model and a concise, dynamic paradigm to investigate uncertainty, generalization, and regulation in social interactions.’

‘Second, although the computational approach employed is clever and sophisticated, there is important information missing about model comparison which ultimately makes some of the results hard to assess from the perspective of the reader.’

Our model comparison employed what is state of the art random-effects Bayesian model comparison (Piray et al., 2019; PLOS Comp. Biol.). It initially fits each individual to each model using Laplace approximation, and subsequently ‘races’ each model against each other on the group level and individual level through hierarchical constraints and random-effect considerations. We included this in the methods but have now expanded on the descrpition we used to compare models:

In the results -

‘All computational models were fitted using a Hierarchical Bayesian Inference (HBI) algorithm which allows hierarchical parameter estimation while assuming random effects for group and individual model responsibility (Piray et al., 2019; see Methods for more information). We report individual and group-level model responsibility, in addition to protected exceedance probabilities between-groups to assess model dominance.’

We added to our existing description in the methods –

‘All computational models were fitted using a Hierarchical Bayesian Inference (HBI) algorithm which allows hierarchical parameter estimation while assuming random effects for group and individual model responsibility (Piray et al., 2019). During fitting we added a small noise floor to distributions (2.22e^-16) before normalisation for numerical stability. Parameters were estimated using the HBI in untransformed space drawing from broad priors (μM\=0, σ²_M = 6.5; where M\={M1, M2, M3, M4}). This process was run independently for each group. Parameters were transformed into model-relevant space for analysis. All models and hierarchical fitting was implemented in Matlab (Version R2022B). All other analyses were conducted in R (version 4.3.3; arm64 build) running on Mac OS (Ventura 13.0). We extracted individual and group level responsibilities, as well as the protected exceedance probability to assess model dominance per group.’

(1) P3, third paragraph: please define self-insertion

We have now more clearly defined this in the prior paragraph when introducing concepts.

‘To reduce uncertainty about others, theories of the relational self (Anderson & Chen, 2002) suggest that people have availble to them an extensive and well-grounded representation of themselves, leading to a readily accessible initial belief (Allport, 1924; Kreuger & Clement, 1994) that can be projected or integrated when learning about others (self-insertion).’

(2) Introduction: the specific aim of the paper should be clarified - at the moment, it is rather vague. The authors write: "However, critical questions remain: How do humans adjudicate between self-insertion and contagion during interaction to manage interpersonal generalization? Does the uncertainty in self-other beliefs affect their generalizability? How can disruptions in interpersonal exchange during sensitive developmental periods (e.g., childhood maltreatment) inform models of psychiatric disorders?". Which of these questions is the focus of the paper? And how does the paper aim at addressing it?

(3) Relatedly, from the introduction it is not clear whether the goal is to develop a theory of self-insertion and social contagion and test it empirically, or whether it is to study these processes in BPD, or both (or something else). Clarifying which specific question(s) is addressed is important (also clarifying what we already know about that specific question, and how the paper aims at elucidating that specific question).

We have now included our specific aims of the paper. We note this in the above response to the reviwers general comments.

(4) "Computational models have probed social processes in BPD, linking the BPD phenotype to a potential over-reliance on social versus internal cues (Henco et al., 2020), 'splitting' of social latent states that encode beliefs about others (Story et al., 2023), negative appraisal of interpersonal experiences with heightened self-blame (Mancinelli et al., 2024), inaccurate inferences about others' irritability (Hula et al., 2018), and reduced belief adaptation in social learning contexts (Siegel et al., 2020). Previous studies have typically overlooked how self and other are represented in tandem, prompting further investigation into why any of these BPD phenotypes manifest." Not clear what the link between the first and second sentence is. Does it mean that previous computational models have focused exclusively on how other people are represented in BPD, and not on how the self is represented? Please spell this out.

Thank you for the opportunity to be clearer in our language. We have now spelled out our point more precisely, and included some extra relevant literature helpfully pointed out by another reviewer.

‘Computational models have probed social processes in BPD, although almost exclusively during observational learning. The BPD phenotype has been associated with a potential over-reliance on social versus internal cues (Henco et al., 2020), ‘splitting’ of social latent states that encode beliefs about others (Story et al., 2023), negative appraisal of interpersonal experiences with heightened self-blame (Mancinelli et al., 2024), inaccurate inferences about others’ irritability (Hula et al., 2018), and reduced belief adaptation in social learning contexts (Siegel et al., 2020). Associative models have also been adapted to characterize  ‘leaky’ self-other reinforcement learning (Ereira et al., 2018), finding that those with BPD overgeneralize (leak updates) about themselves to others (Story et al., 2024). Altogether, there is currently a gap in the direct causal link between insertion, contagion, and learning (in)stability.’

(5) P5, first paragraph. The description of the task used in phase 1 should be more detailed. The essential information for understanding the task is missing.

We have updated this section to point toward Figure 1 and the Methods where the details of the task are more clearly outlined. We hope that it is acceptable not to explain the full task at this point for brevity and to not interrupt the flow of the results.

“Detailed descriptions of the task can be found in the methods section and Figure 1.’

(6) P5, second paragraph: briefly state how the Psychometric data were acquired (e.g., self-report).

We have now clarified this in the text.

‘All participants also self-reported their trait paranoia, childhood trauma, trust beliefs, and trait mentalizing (see methods).’

(7) "For example, a participant could make prosocial (self=5; other=5) versus individualistic (self=10; other=5) choices, or prosocial (self=10; other=10) versus competitive (self=10; other=5) choices". Not sure what criteria are used for distinguishing between individualistic and competitive - they look the same?

Sorry. This paragraph was not clear that the issue is that the interpretation of the choice depends on both members of the pair of options. Here, in one pair {(self=5,other=5) vs (self=10,other=5)}, it is highly pro-social for the self to choose (5,5), sacrificing 5 points for the sake of equality. In the second pair {(self=10,other=10) vs (self=10,other=5)}, it is highly competitive to choose (10,5), denying the other 5 points at no benefit to the self. We have clarified this:

‘We analyzed the ‘types’ of choices participants made in each phase (Supplementary Table 1). The interpretation of a participant’s choice depends on both values in a choice. For example, a participant could make prosocial (self=5; other=5) versus individualistic (self=10; other=5) choices, or prosocial (self=10; other=10) versus competitive (self=10; other=5) choices. There were 12 of each pair in phases 1 and 3 (individualistic vs. prosocial; prosocial vs. competitive; individualistic vs. competitive).’  

(8) "In phase 1, both CON and BPD participants made prosocial choices over competitive choices with similar frequency (CON=9.67[3.62]; BPD=9.60[3.57])" please report t-test - the same applies also various times below.

We have now included the t test statistics with each instance.

‘In phase 3, both CON and BPD participants continued to make equally frequent prosocial versus competitive choices (CON=9.15[3.91]; BPD=9.38[3.31]; t=-0.54, p=0.59); CON participants continued to make significantly less prosocial versus individualistic choices (CON=2.03[3.45]; BPD=3.78 [4.16]; t=2.31, p=0.02). Both groups chose equally frequent individualistic versus competitive choices (CON=10.91[2.40]; BPD=10.18[2.72]; t=-0.49, p=0.62).’

(9) P 9: "Models M2 and M3 allow for either self-insertion or social contagion to occur independently" what's the difference between M2 and M3?

Model M2 hypothesises that participants use their own self representation as priors when learning about the other in phase 2, but are not influenced by their partner. M3 hypothesises that participants form an uncoupled prior (no self-insertion) about their partner in phase 2, and their choices in phase 3 are influenced by observing their partner in phase 2 (social contagion). In Figure 1 we illustrate the difference between M2 and M3. In Table 1 we specifically report the parameterisation differences between M2 and M3. We have also now included a correlational analysis of parameters between models to demonstrate the relationship between model parameters of equivalent value between models (Fig S11). We have also force fitted all models (M1-M4) to the data independently and reported group differences within each (see Table S2 and Table S3).

(10) P 9, last paragraph: I did not understand the description of the Beta model.

The beta model is outlined in detail in Table 1. We have also clarified the description of the beta model on page 9:

‘The ‘Beta model’ is equivalent to M1 in its causal architecture (both self-insertion and social contagion are hypothesized to occur) but differs in richness: it accommodates the possibility that participants might only consider a single dimension of relative reward allocation, which is typically emphasized in previous studies (e.g., Hula et al., 2018).’

(11) P 9: I wonder whether one could think about more intuitive labels for the models, rather than M1, M2 etc.. This is just a suggestion, as I am not sure a short label would be feasible here.

Thank you for this suggestion. We apologise that it is not very intitutive. The problem is that given the various terms we use to explain the different processes of generalisation that might occur between self and other, and given that each model is a different combination of each, we felt that numbering them was a lesser evil. We hope that the reader will be able to reference both Figure 1 and Table 1 to get a good feel for how the models and their causal implications differ.

(12) Model comparison: the information about what was done for model comparison is scant, and little about fit statistics is reported. At the moment, it is hard for a reader to assess the results of the model comparison analysis.

Model comparison and fitting was conducted using simultaneous hierarchical fitting and random-effects comparison. This is employed through the HBI package (Piray et al., 2019) where the assumptions and fitting proceedures are outlined in great detail. In short, our comparison allows for individual and group-level hierarchical fitting and comparison. This overcomes the issue of interdependence between and within model fitting within a population, which is often estimated separately.

We have outlined this in the methods, although appreciate we do not touch upon it until the reader reaches that point. We have added a clarification statement on page 9 to rectify this:

‘All computational models were fitted using a Hierarchical Bayesian Inference (HBI) algorithm which allows hierarchical parameter estimation while assuming random effects for group and individual model responsibility (Piray et al., 2019; see Methods for more information). We report individual and group-level model responsibility, in addition to protected exceedance probabilities between-groups to assess model dominance.’

(13) P 14, first paragraph: "BPD participants were also more certain about both types of preference" what are the two types of preferences?

The two types of preferences are relative (prosocial-competitive) and absolute (individualistic) reward utility. These are expressed as b and a respectively. We have expanded the sentence in question to make this clearer:

‘BPD participants were also more certain about both self-preferences for absolute and relative reward ( = -0.89, 95%HDI: -1.01, -0.75; = -0.32, 95%HDI: -0.60, -0.04) versus CON participants (Figure 2B).’

(14) "Parameter Associations with Reported Trauma, Paranoia, and Attributed Intent" the results reported here are intriguing, but not fully convincing as there is the problem of multiple comparisons. The combinations between parameters and scales are rather numerous. I suggest to correct for multiple comparisons and to flag only the findings that survive correction.

We have now corrected this and controlled for multiple comparisons through partial correlation analysis, bootstrapping assessment for robustness, permutation testing, and False Detection Rate correction. We only report those that survive bootstrapping and permutation testing, reporting both corrected (p[fdr]) and uncorrected (p) significance.

(15) Results page 14 and page 15. The authors compare the various parameters between groups. I would assume that these parameters come from M1 for controls and from M4 for BDP? Please clarify if this is indeed the case. If it is the case, I am not sure this is appropriate. To my knowledge, it is appropriate to compare parameters between groups only if the same model is fit to both groups. If two different models are fit to each group, then the parameters are not comparable, as the parameter have, so to speak, different "meaning" in two models. Now, I want to stress that my knowledge on this matter may be limited, and that the authors' approach may be sound. However, to be reassured that the approach is indeed sound, I would appreciate a clarification on this point and a reference to relevant sources about this approach.

This is an important point. First, we confirmed all our main conclusions about parameter differences using the maximal model M1 to fit all the participants. We added Supplementary Table 2 to report the outcome of this analysis. Second, we did the same for parameters across all models M1-M4, fitting each to participants without comparison. This is particularly relevant for M3, since at least a minority of participants of both groups were best explained by this model. We report these analyses in Fig S11:

Since the M4 is nested within M1, we argue that this comparison is still meaningful, and note explanations in the text for why the effects noted between groups may occur given the differences in their causal meaning, for example in the results under phase 2 analyses:

‘Belief updating in phase 2 was less flexible in BPD participants. Median change in beliefs (from priors to posteriors) about a partner’s preferences was lower versus. CON ( = -5.53, 95%HDI: -7.20, -4.00; = -10.02, 95%HDI: -12.81, -7.30). Posterior beliefs about partner were more precise in BPD versus CON ( = -0.94, 95%HDI: -1.50, -0.45;  = -0.70, 95%HDI: -1.20, -0.25).  This is unsurprising given the disintegrated priors of the BPD group in M4, meaning they need to ‘travel less’ in state space. Nevertheless, even under assumptions of M1 and M2 for both groups, BPD showed smaller posteriors median changes versus CON in phase 2 (see Table T2). These results converge to suggest those with BPD form rigid posterior beliefs.’

(16) "We built and tested a theory of interpersonal generalization in a population of matched participants" this sentence seems to be unwarranted, as there is no theory in the paper (actually, as it is now, the paper looks rather exploratory)

We thank the reviewer for their perspective. Formal models can be used as a theoretical statement on the casual algorithmic process underlying decision making and choice behaviour; the development of formal models are an essential theoretical tool for precision and falsification (Haslbeck et al., 2022). In this sense, we have built several competing formal theories that test, using casual architectures, whether the latent distribution(s) that generate one’s choices generalise into one’s predictions about another person, and simultaneously whether one’s latent distribution(s) that represent beliefs about another person are used to inform future choices.

Reviewer 3:

‘My broad question about the experiment (in terms of its clinical and cognitive process relevance): Does the task encourage competition or give participants a reason to take advantage of others? I don't think it does, so it would be useful to clarify the normative account for prosociality in the introduction (e.g., some of Robin Dunbar's work).’

We agree that our paradigm does not encourage competition. We use a reward structure that makes it contingent on participants to overcome a particular threshold before earning rewards, but there is no competitive element to this, in that points earned or not earned by partners have no bearing on the outcomes for the participant. This is important given the consideration of recursive properties that arise through mixed-motive games; we wanted to focus purely on observational learning in phase 2, and repercussion-free choices made by participants in phase 1 and 3, meaning the choices participants, and decisions of a partner, are theoretically in line with self-preferences irrespective of the judgement of others. We have included a clearer statement of the structure of this type of task, and more clearly cited the origin for its structure (Murphy & Ackerman, 2011):

‘Our present work sought to achieve two primary goals. 1. Extend prior causal computational theories to formalise and test the interrelation between self-insertion and social contagion on learning and behaviour to better probe interpersonal generalisation in health, and 2., Test whether previous computational findings of social learning changes in BPD can be explained by infractions to self-other generalisation. We accomplish these goals by using a dynamic, sequential social value economic paradigm, the Intentions Game, building upon a Social Value Orientation Framework (Murphy & Ackerman, 2011) that assumes motivational variation in joint reward allocation.’

Given the introductions structure as it stands, we felt providing another paragraph on the normative assumptions of such a game was outside the scope of this article.

‘The finding that individuals with BPD do not engage in self-other generalization on this task of social intentions is novel and potentially clinically relevant. The authors find that BPD participants' tendency to be prosocial when splitting points with a partner does not transfer into their expectations of how a partner will treat them in a task where they are the passive recipient of points chosen by the partner. In the discussion, the authors reasonably focus on model differences between groups (Bayesian model comparison), yet I thought this finding -- BPD participants not assuming prosocial tendencies in phase 2 while CON participant did -- merited greater attention. Although the BPD group was close to 0 on the \beta prior in Phase 2, their difference from CON is still in the direction of being more mistrustful (or at least not assuming prosociality). This may line up with broader clinical literature on mistrustfulness and attributions of malevolence in the BPD literature (e.g., a 1992 paper by Nigg et al. in Journal of Abnormal Psychology). My broad point is to consider further the Phase 2 findings in terms of the clinical interpretation of the shift in \beta relative to controls.’

This is an important point, that we contextualize within the parameterisation of our utility model. While the shift toward 0 in the BPD participants is indeed more competitive, as the reviewer notes, it is surprisingly centred closely around 0, with only a slight bias to be prosocial (mean = -0.47;  = -6.10, 95%HDI: -7.60, -4.60). Charitably we might argue that BPD participants are expecting more competitive preferences from their partner. However even so, given their variance around their priors in phase 2, they are uncertain or unconfident about this. We take a more conservative approach in the paper and say that given the tight proximity to 0 and the variance of their group priors, they are likely to be ‘hedging their bets’ on whether their partner is going to be prosocial or competitive. While the movement from phase 1 to 2 is indeed in the competitive direction it still lands in neutral territory. Model M4 does not preclude central tendancies at the start of Phase 2 being more in the competitive direction.

‘First, the authors note that they have "proposed a theory with testable predictions" (p. 4 but also elsewhere) but they do not state any clear predictions in the introduction, nor do they consider what sort of patterns will be observed in the BPD group in view of extant clinical and computational literature. Rather, the paper seems to be somewhat exploratory, largely looking at group differences (BPD vs. CON) on all of the shared computational parameters and additional indices such as belief updating and reaction times. Given this, I would suggest that the authors make stronger connections between extant research on intention representation in BPD and their framework (model and paradigm). In particular, the authors do not address related findings from Ereira (2020) and Story (2024) finding that in a false belief task that BPD participants *overgeneralize* from self to other. A critical comparison of this work to the present study, including an examination of the two tasks differ in the processes they measure, is important.’

Thank you for this opportunity to include more of the important work that has preceded the present manuscript. Prior work has tended to focus on either descriptive explanations of self-other generalisation (e.g. through the use of RW type models) or has focused on observational learning instability in absence of a causal model from where initial self-other beliefs may arise. While the prior work cited by the reviewer [Ereira (2020; Nat. Comms.) and Story (2024; Trans. Psych.)] does examine the inter-trial updating between self-other, it does not integrate a self model into a self’s belief about an other prior to observation. Rather, it focuses almost exclusively on prediction error ‘leakage’ generated during learning about individual reward (i.e. one sided reward). These findings are important, but lie in a slightly different domain. They also do not cut against ours, and in fact, we argue in the discussion that the sort of learning instability described above and splitting (as we cite from Story ea. 2024; Psych. Rev.) may result from a lack of self anchoring typical of CON participants. Nevertheless we agree these works provide an important premise to contrast and set the groundwork for our present analysis and have included them in the framing of our introduction, as well as contrasting them to our data in the discussion.

In the introduction:

‘The BPD phenotype has been associated with a potential over-reliance on social versus internal cues (Henco et al., 2020), ‘splitting’ of social latent states that encode beliefs about others (Story et al., 2023), negative appraisal of interpersonal experiences with heightened self-blame (Mancinelli et al., 2024), inaccurate inferences about others’ irritability (Hula et al., 2018), and reduced belief adaptation in social learning contexts (Siegel et al., 2020). Associative models have also been adapted to characterize  ‘leaky’ self-other reinforcement learning (Ereira et al., 2018), finding that those with BPD overgeneralize (leak updates) about themselves to others (Story et al., 2024). Altogether, there is currently a gap in the direct causal link between insertion, contagion, and learning (in)stability.’

In the discussion:

‘Disruptions in self-to-other generalization provide an explanation for previous computational findings related to task-based mentalizing in BPD. Studies tracking observational mentalizing reveal that individuals with BPD, compared to those without, place greater emphasis on social over internal reward cues when learning (Henco et al., 2020; Fineberg et al., 2018). Those with BPD have been shown to exhibit reduced belief adaptation (Siegel et al., 2020) along with ‘splitting’ of latent social representations (Story et al., 2024a). BPD is also shown to be associated with overgeneralisation in self-to-other belief updates about individual outcomes when using a one-sided reward structure (where participant responses had no bearing on outcomes for the partner; Story et al., 2024b). Our analyses show that those with BPD are equal to controls in their generalisation of absolute reward (outcomes that only affect one player) but disintegrate beliefs about relative reward (outcomes that affect both players) through adoption of a new, neutral belief. We interpret this together in two ways: 1. There is a strong concern about social relativity when those with BPD form beliefs about others, 2. The absence of constrained self-insertion about relative outcomes may predispose to brittle or ‘split’ beliefs. In other words, those with BPD assume ambiguity about the social relativity preferences of another (i.e. how prosocial or punitive) and are quicker to settle on an explanation to resolve this. Although self-insertion may be counter-intuitive to rational belief formation, it has important implications for sustaining adaptive, trusting social bonds via information moderation.’

In addition, perhaps it is fairer to note more explicitly the exploratory nature of this work. Although the analyses are thorough, many of them are not argued for a priori (e.g., rate of belief updating in Figure 2C) and the reader amasses many individual findings that need to by synthesized.’

We have now noted the primary goals of our work in the introduction, and have included caveats about the exploratory nature of our analyses. We would note that our model is in effect a causal combination of prior work cited within the introduction (Barnby et al., 2022; Moutoussis et al., 2016). This renders our computational models in effect a causal theory to test, although we agree that our dissection of the results are exploratory. We have more clearly signposted this:

‘Our present work sought to achieve two primary goals. 1. Extend prior causal computational theories to formalise and test the interrelation between self-insertion and social contagion on learning and behaviour to better probe interpersonal generalisation in health, and 2., Test whether previous computational findings of social learning changes in BPD can be explained by infractions to self-other generalisation. We accomplish these goals by using a dynamic, sequential economic paradigm, the Intentions Game, building upon a Social Value Orientation Framework (Murphy & Ackerman, 2011) that assumes innate motivational variation in joint reward allocation.‘

‘Second, in the discussion, the authors are too quick to generalize to broad clinical phenomena in BPD that are not directly connected to the task at hand. For example, on p. 22: "Those with a diagnosis of BPD also show reduced permeability in generalising from other to self. While prior research has predominantly focused on how those with BPD use information to form impressions, it has not typically examined whether these impressions affect the self." Here, it's not self-representation per se (typically, identity or one's view of oneself), but instead cooperation and prosocial tendencies in an economic context. It is important to clarify what clinical phenomena may be closely related to the task and which are more distal and perhaps should not be approached here.’

Thank you for this important point. We agree that social value orientation, and particularly in this economically-assessed form, is but one aspect of the self, and we did not test any others. A version of the social contagion phenomena is also present in other aspects of the self in intertemporal (Moutoussis et al., 2016), economic (Suzuki et al., 2016) and moral preferences (Yu et al., 2021). It would be most interesting to attempt to correlate the degrees of insertion and contagion across the different tasks.

We take seriously the wider concern that behaviour in our tasks based on economic preferences may not have clinical validity. This issue is central in the whole field of computational psychiatry, much of which is based on generalizing from tasks like ours, and discussing correlations with psychometric measures. We hope that it is acceptable to leave such discussions to the many reviews on computational psychiatry (Montague et al., 2012; Hitchcock et al., 2022; Huys et al., 2016). Here, we have just put a caveat in the dicussion:

‘Finally, a limitation may be that behaviour in tasks based on economic preferences may not have clinical validity. This issue is central to the field of computational psychiatry, much of which is based on generalising from tasks like that within this paper and discussing correlations with psychometric measures. Extrapolating  economic tasks into the real world has been the topic of discussion for the many reviews on computational psychiatry (e.g. Montague et al., 2012; Hitchcock et al., 2022; Huys et al., 2016). We note a strength of this work is the use of model comparison to understand causal algorithmic differences between those with BPD and matched healthy controls. Nevertheless, we wish to further pursue how latent characteristics captured in our models may directly relate to real-world affective change.’

‘On a more technical level, I had two primary concerns. First, although the authors consider alternative models within a hierarchical Bayesian framework, some challenges arise when one analyzes parameter estimates fit separately to two groups, particularly when the best-fitting model is not shared. In particular, although the authors conduct a model confusion analysis, they do not as far I could tell (and apologies if I missed it) demonstrate that the dynamics of one model are nested within the other. Given that M4 has free parameters governing the expectations on the absolute and relative reward preferences in Phase 2, is it necessarily the case that the shared parameters between M1 and M4 can be interpreted on the same scale? Relatedly, group-specific model fitting has virtues when believes there to be two distinct populations, but there is also a risk of overfitting potentially irrelevant sample characteristics when parameters are fit group by group.

To resolve these issues, I saw one straightforward solution (though in modeling, my experience is that what seems straightforward on first glance may not be so upon further investigation). M1 assumes that participants' own preferences (posterior central tendency) in Phase 1 directly transfer to priors in Phase 2, but presumably the degree of transfer could vary somewhat without meriting an entirely new model (i.e., the authors currently place this question in terms of model selection, not within-model parameter variation). I would suggest that the authors consider a model parameterization fit to the full dataset (both groups) that contains free parameters capturing the *deviations* in the priors relative to the preceding phase's posterior. That is, the free parameters $\bar{\alpha}_{par}^m$ and $\bar{\beta}_{par}^m$ govern the central tendency of the Phase 2 prior parameter distributions directly, but could be reparametrized as deviations from Phase 1 $\theta^m_{ppt}$ parameters in an additive form. This allows for a single model to be fit all participants that encompasses the dynamics of interest such that between-group parameter comparisons are not biased by the strong assumptions imposed by M1 (that phase 1 preferences and phase 2 observations directly transfer to priors). In the case of controls, we would expect these deviation parameters to be centred on 0 insofar as the current M1 fit them best, whereas for BPD participants should have significant deviations from earlier-phase posteriors (e.g., the shift in \beta toward prior neutrality in phase 2 compared to one's own prosociality in phase 1). I think it's still valid for the authors to argue for stronger model constraints for Bayesian model comparison, as they do now, but inferences regarding parameter estimates should ideally be based on a model that can encompass the full dynamics of the entire sample, with simpler dynamics (like posterior -> prior transfer) being captured by near-zero parameter estimates.’

Thank you for the chance to be clearer in our modelling. In particular, the suggestion to include a model that can be fit to all participants with the equivalent of the likes of partial social insertion, to check if the results stand, can actually be accomplished through our existing models.  That is, the parameter that governs the flexibility over beliefs in phase 2 under models M1 (dominant for CON participant) and M2 parameterises the degree to which participants think their partner may be different from themselves. Thus, forcibly fitting M1 and M2 hierarchically to all participants, and then separately to BPD and CON participants, can quantify the issue raised: if BPD participants indeed distinguish partners as vastly different from themselves enough to warent a new central tendency, should be quantitively higher in BPD vs CON participants under M1 and M2.

We therefore tested this, reporting the distributional differences between for BPD and CON participants under M1, both when fitted together as a population and as separate groups. As is higher for BPD participants under both conditions for M1 and M2 it supports our claim and will add more context for the comparison - may be large enough in BPD that a new central tendency to anchor beliefs is a more parsimonious explanation.

We cross checked this result by assessing the discrepancy between the participant’s and assumed partner’s central tendencies for both prosocial and individualistic preferences via best-fitting model M4 for the BPD group. We thereby examined whether belief disintegration is uniform across preferences (relative vs abolsute reward) or whether one tendency was shifted dramatically more than another.  We found that beliefs over prosocial-competitive preferences were dramatically shifted, whereas those over individualistic preferences were not.

We have added the following to the main text results to explain this:

Model Comparison:

‘We found that CON participants were best fit at the group level by M1 (Frequency = 0.59, Protected Exceedance Probability = 0.98), whereas BPD participants were best fit by M4 (Frequency = 0.54, Protected Exceedance Probability = 0.86; Figure 2A). We first analyse the results of these separate fits. Later, in order to assuage concerns about drawing inferences from different models, we examined the relationships between the relevant parameters when we forced all participants to be fit to each of the models (in a hierarchical manner, separated by group). In sum, our model comparison is supported by convergence in parameter values when comparisons are meaningful. We refer to both types of analysis below.’

Phase 1:

‘These differences were replicated when considering parameters between groups when we fit all participants to the same models (M1-M4; see Table S2).’

Phase 2:

‘To check that these conclusions about self-insertion did not depend on the different models, we found that only under M1 and M2 were consistently larger in BPD versus CON. This supports the notion that new central tendencies for BPD participants in phase 2 were required, driven by expectations about a partner’s relative reward. (see Fig S10 & Table S2). and parameters under assumptions of M1 and M2 were strongly correlated with median change in belief between phase 1 and 2 under M3 and M4, suggesting convergence in outcome (Fig S11).’

‘Furthermore, even under assumptions of M1-M4 for both groups, BPD showed smaller posterior median changes versus CON in phase 2 (see Table T2). These results converge to suggest those with BPD form rigid posterior beliefs.’

‘Assessing this same relationship under M1- and M2-only assumptions reveals a replication of this group effect for absolute reward, but the effect is reversed for relative reward (see Table S3). This accords with the context of each model, where under M1 and M2, BPD participants had larger phase 2 prior flexibility over relative reward (leading to larger initial surprise), which was better accounted for by a new central tendency under M4 during model comparison. When comparing both groups under M1-M4 informational surprise over absolute reward was consistently restricted in BPD (Table S3), suggesting a diminished weight of this preference when forming beliefs about an other.’

Phase 3

‘In the dominant model for the BPD group—M4—participants are not influenced in their phase 3 choices following exposure to their partner in phase 2. To further confirm this we also analysed absolute change in median participant beliefs between phase 1 and 3 under the assumption that M1 and M3 was the dominant model for both groups (that allow for contagion to occur). This analysis aligns with our primary model comparison using M1 for CON and M4 for BPD  (Figure 2C). CON participants altered their median beliefs between phase 1 and 3 more than BPD participants (M1: linear estimate = 0.67, 95%CI: 0.16, 1.19; t = 2.57, p = 0.011; M3: linear estimate = 1.75, 95%CI: 0.73, 2.79; t = 3.36, p < 0.001). Relative reward was overall more susceptible to contagion versus absolute reward (M1: linear estimate = 1.40, 95%CI: 0.88, 1.92; t = 5.34, p<0.001; M3: linear estimate = 2.60, 95%CI: 1.57, 3.63; t = 4.98, p < 0.001). There was an interaction between group and belief type under M3 but not M1 (M3: linear estimate = 2.13, 95%CI: 0.09, 4.18, t = 2.06, p=0.041). There was only a main effect of belief type on precision under M3 (linear estimate = 0.47, 95%CI: 0.07, 0.87, t = 2.34, p = 0.02); relative reward preferences became more precise across the board. Derived model estimates of preference change between phase 1 and 3 strongly correlated between M1 and M3 along both belief types (see Table S2 and Fig S11).’

‘My second concern pertains to the psychometric individual difference analyses. These were not clearly justified in the introduction, though I agree that they could offer potentially meaningful insight into which scales may be most related to model parameters of interest. So, perhaps these should be earmarked as exploratory and/or more clearly argued for. Crucially, however, these analyses appear to have been conducted on the full sample without considering the group structure. Indeed, many of the scales on which there are sizable group differences are also those that show correlations with psychometric scales. So, in essence, it is unclear whether most of these analyses are simply recapitulating the between-group tests reported earlier in the paper or offer additional insights. I think it's hard to have one's cake and eat it, too, in this regard and would suggest the authors review Preacher et al. 2005, Psychological Methods for additional detail. One solution might be to always include group as a binary covariate in the symptom dimension-parameter analyses, essentially partialing the correlations for group status. I remain skeptical regarding whether there is additional signal in these analyses, but such controls could convince the reader. Nevertheless, without such adjustments, I would caution against any transdiagnostic interpretations such as this one in the Highlights: "Higher reported childhood trauma, paranoia, and poorer trait mentalizing all diminish other-to-self information transfer irrespective of diagnosis." Since many of these analyses relate to scales on which the groups differ, the transdiagnostic relevance remains to be demonstrated.’

We have restructured the psychometric section to ensure transparency and clarity in our analysis. Namely, in response to these comments and those of the other reviewers, we have opted to remove the parameter analyses that aimed to cross-correlate psychometric scores with latent parameters from different models: as the reviewer points out, we do not have parity between dominant models for each group to warrant this, and fitting the same model to both groups artificially makes the parameters qualitatively different. Instead we have opted to focus on social contagion, or rather restrictions on , between phases 1 and 3 explained by M3. This provides us with an opportunity to examine social contagion on the whole population level isolated from self-insertion biases. We performed bootstrapping (1000 reps) and permutation testing (1000 reps) to assess the stability and significance of each edge in the partial correlation network, and then applied FDR correction (p[fdr]), thus controlling for multiple comparisons. We note that while we focused on M3 to isolate the effect across the population, social contagion across both relative and absolute reward under M3 strongly correlated with social contagion under M1 (see Fig S11).

‘We explored whether social contagion may be restricted as a result of trauma, paranoia, and less effective trait mentalizing under the assumption of M3 for all participants (where everyone is able to be influenced by their partner). To note, social contagion under M3 was highly correlated with contagion under M1 (see Fig S11). We conducted partial correlation analysis to estimate relationships conditional on all other associations and retained all that survived bootstrapping (1000 reps), permutation testing (1000 reps), and subsequent FDR correction. Persecution and CTQ scores were both moderately associated with MZQ scores (RGPTSB r = 0.41, 95%CI: 0.23, 0.60, p = 0.004, p[fdr]=0.043; CTQ r = 0.354 95%CI: 0.13, 0.56, p=0.019, p[fdr]=0.02). MZQ scores were in turn moderately and negatively associated with shifts in prosocial-competitive preferences () between phase 1 and 3 (r = -0.26, 95%CI: -0.46, -0.06, p=0.026, p[fdr]=0.043). CTQ scores were also directly and negatively associated with shifts in individualistic preferences (; r = -0.24, 95%CI: -0.44, -0.13, p=0.052, p[fdr]=0.065). This provides some preliminary evidence that trauma impacts beliefs about individualism directly, whereas trauma and persecutory beliefs impact beliefs about prosociality through impaired mentalising (Figure 4A).’

(1) As far as I could tell, the authors didn't provide an explanation of this finding on page 5: "However, CON participants made significantly fewer prosocial choices when individualistic choices were available" While one shouldn't be forced to interpret every finding, the paper is already in that direction and I found this finding to be potentially relevant to the BPD-control comparison.

Thank you for this observation. This sentance reports the fact that CON participants were effectively more selfish than BPD participants. This is captured by the lower value of reported in Figure 2, and suggests that CON participants were more focused on absolute value – acting in a more ‘economically rational’ manner – versus BPD participants. This fits in with our fourth paragraph of the discussion where we discuss prior work that demonstrates a heightened social focus in those with BPD. Indeed, the finding the reviewer highlights further emphasises the point that those with BPD are much more sensitive, and motived to choose, options concerning relative reward than are CON participants. The text in the discussion reads:

‘We also observe this in self-generated participant choice behaviour, where CON participants were more concerned over absolute reward versus their BPD counterparts, suggesting a heighted focus on relative vs. absolute reward in those with BPD.’

(2) The adaptive algorithm for adjusting partner behavior in Phase 2 was clever and effective. Did the authors conduct a manipulation check to demonstrate that the matching resulted in approximately 50% difference between one's behavior in Phase 1 and the partner in Phase 2? Perhaps Supplementary Figure suffices, but I wondered about a simpler metric.

Thanks for this point. We highlight this in Figure 3B and within the same figure legend although appreciate the panel is quite small and may be missed.  We have now highlighted this manipulation check more clearly in behavioural analysis section of the main text:

‘Server matching between participant and partner in phase 2 was successful, with participants being approximately 50% different to their partners with respect to the choices each would have made on each trial in phase 2 (mean similarity=0.49, SD=0.12).’

(3) The resolution of point-range plots in Figure 4 was grainy. Perhaps it's not so in the separate figure file, but I'd suggest checking.

Apologies. We have now updated and reorganised the figure to improve clarity.

(4) p. 21: Suggest changing to "different" as opposed to "opposite" since the strategies are not truly opposing: "but employed opposite strategies."

We have amended this.

(5) p. 21: I found this sentence unclear, particularly the idea of "similar updating regime." I'd suggest clarifying: "In phase 2, CON participants exhibited greater belief sensitivity to new information during observational learning, eventually adopting a similar updating regime to those with BPD."

We have clarified this statement:

‘In observational learning in phase 2, CON participants initially updated their beliefs in response to new information more quickly than those with BPD, but eventually converged to a similar rate of updating.’

(6) p. 23: The content regarding psychosis seemed out of place, particularly as the concluding remark. I'd suggest keeping the focus on the clinical population under investigation. If you'd like to mention the paradigm's relevance to psychosis (which I think could be omitted), perhaps include this as a future direction when describing the paradigm's strengths above.

We agree the paragraph is somewhat speculative. We have omitted it in aid of keeping the messaging succinct and to the point.

(7) p. 24: Was BPD diagnosis assess using unstructured clinical interview? Although psychosis was exclusionary, what about recent manic or hypomanic episodes or Bipolar diagnosis? A bit more detail about BPD sample ascertainment would be useful, including any instruments used to make a diagnosis and information about whether you measured inter-rater agreement.

Participants diagnosed with BPD were recruited from specialist personality disorder services across various London NHS mental health trusts. The diagnosis of BPD was established by trained assessors at the clinical services and confirmed using the Structured Clinical Interview for DSM-IV (SCID-II) (First et al., 1997). Individuals with a history of psychotic episodes, severe learning disability or neurological illness/trauma were excluded. We have now included this extra detail within our methods in the paper:

‘The majority of BPD participants were recruited through referrals by psychiatrists, psychotherapists, and trainee clinical psychologists within personality disorder services across 9 NHS Foundation Trusts in the London, and 3 NHS Foundation Trusts across England (Devon, Merseyside, Cambridgeshire). Four BPD participants were also recruited by self-referral through the UCLH website, where the study was advertised. To be included in the study, all participants needed to have, or meet criteria for, a primary diagnosis of BPD (or emotionally-unstable personality disorder or complex emotional needs) based on a professional clinical assessment conducted by the referring NHS trust (for self-referrals, the presence of a recent diagnosis was ascertained through thorough discussion with the participant, whereby two of the four also provided clinical notes). The patient participants also had to be under the care of the referring trust or have a general practitioner whose details they were willing to provide. Individuals with psychotic or mood disorders, recent acute psychotic episodes, severe learning disability, or current or past neurological disorders were not eligible for participation and were therefore not referred by the clinical trusts.‘

Prueba

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Vision is a highly active process. Humans move their eyes 3-4 times per second to sample information with high visual acuity from our environment, and where eye movements are directed is critical to our understanding of active vision. Here, the authors propose that the cost of making a saccade contributes critically to saccade selection (i.e., whether and where to move the eyes). The authors build on their own recent work that the effort (as measured by pupil size) that comes with planning and generating an eye movement varies with saccade direction. To do this, the authors first measured pupil size for different saccade directions for each participant. They then correlated the variations in pupil size obtained in the mapping task with the saccade decision in a free-choice task. The authors observed a striking correlation: pupil size in the mapping task predicted the decision of where to move the eyes in the free choice task. In this study, the authors provide a number of additional insightful analyses (e.g., based on saccade curvature, and saccade latency) and experiments that further support their claim that the decision to move the eyes is influenced by the effort to move the eyes in a particular direction. One experiment showed that the same influence of assumed saccade costs on saccade selection is observed during visual search in natural scenes. Moreover, increasing the cognitive load by adding an auditory counting task reduced the number of saccades, and in particular reduced the costly saccades. In sum, these experiments form a nice package that convincingly establishes the association between pupil size and saccade selection.

We thank the reviewer for highlighting the novelty and cogency of our findings.

In my opinion, the causal structure underlying the observed results is not so clear. While the relationship between pupil size and saccade selection is compelling, it is not clear that saccade-related effort (i.e., the cost of a saccade) really drives saccade selection. Given the correlational nature of this relationship, there are other alternatives that could explain the finding. For example, saccade latency and the variance in landing positions also vary across saccade directions. This can be interpreted for instance that there are variations in oculomotor noise across saccade directions, and maybe the oculomotor system seeks to minimize that noise in a free-choice task. In fact, given such a correlational result, many other alternative mechanisms are possible. While I think the authors' approach of systematically exploring what we can learn about saccade selection using pupil size is interesting, it would be important to know what exactly pupil size can add that was not previously known by simply analyzing saccade latency. For example, saccade latency anisotropies across saccade directions are well known, and the authors also show here that saccade costs are related to saccade latency. An important question would be to compare how pupil size and saccade latency uniquely contribute to saccade selection. That is, the authors could apply the exact same logic to their analysis by first determining how saccade latencies (or variations in saccade landing positions; see Greenwood et al., 2017 PNAS) vary across saccade directions and how this saccade latency map explains saccade selection in subsequent tasks. Is it more advantageous to use one or the other saccade metric, and how well does a saccade latency map correlate with a pupil size map?

We thank the reviewer for the detailed comment. 1) The reviewer first points out the correlational nature of many of our results. Thereafter, 2), the reviewer asks whether saccade latencies and landing precision also predict saccade selection, and could be these potential predictors be considered alternative explanations to the idea of effort driving saccade selection? Moreover, what can pupil size add to what can be learned from saccade latency?

In brief, although we report a combination of correlational and causal findings, we do not know of a more parsimonious explanation for our findings than “effort drives saccade selection”. Moreover, we demonstrate that oculomotor noise cannot be construed as an alternative explanation for our findings.

(1) Correlational nature of many findings.

We acknowledge that many of our findings are predominantly correlational in nature. In our first tasks, we correlated pupil size during saccade planning to saccade preferences in a subsequent task. Although the link between across tasks was correlational, the observed relationship clearly followed our previously specified directed hypothesis. Moreover, experiments 1 and 2 of the visual search data replicated and extended this relationship. We also directly manipulated cognitive demand in the second visual search experiment. In line with the hypothesis that effort affects saccade selection, participants executed less saccades overall when performing a (primary) auditory dual task, and even cut the costly saccades most – which actually constitutes causal evidence for our hypothesis. A minimal oculomotor noise account would not directly predict a reduction in saccade rate under higher cognitive demand. To summarize, we have a combination of correlational and causal findings, although mediators cannot be ruled out fully for the latter. That said, we do not know of a more fitting and parsimonious explanation for our findings than effort predicting saccade selection (see following points for saccade latencies). We now address causality in the discussion for transparency and point more explicitly to the second visual search experiment for causal evidence.

“We report a combination of correlational and causal findings. Despite the correlational nature of some of our results, they consistently support the hypothesis that saccade costs predicts saccade selection [which we predicted previously, 33]. Causal evidence was provided by the dual-task experiment as saccade frequencies - and especially costly saccades were reduced under additional cognitive demand. Only a cost account predicts 1) a link between pupil size and saccade preferences, 2) a cardinal saccade bias, 3) reduced saccade frequency under additional cognitive demand, and 4) disproportional cutting of especially those directions associated with more pupil dilation. Together, our findings converge upon the conclusion that effort drives saccade selection.”

(2) Do anisotropies in saccade latencies constitute an alternative explanation?

First of all, we would like to to first stress that differences in saccade latencies are indeed thought to reflect oculomotor effort (Shadmehr et al., 2019; TINS). For example, saccades with larger amplitudes and saccades where distractors need to be ignored are associated with longer latencies. Therefore, even if saccade latencies would predict saccade selection, this would not contrast the idea that effort drives saccade selection. Instead, this would provide convergent evidence for our main novel conclusion: effort drives saccade selection. There are several reasons why pupil size can be used as a more general marker of effort (see responses to R2), but ultimately, our conclusions do not hinge on the employed measure of effort per se. As stressed above in 1), we see no equally parsimonious explanation besides the cost account. Moreover, we predicted this relationship in our previous publication before running the currently reported experiments and analyses (Koevoet et al., 2023). That said, we are open to discuss further alternative options and would be looking forward to test these accounts in future work against each other – we are welcoming the reviewers’ (but also the reader’s) suggestions.

We now discuss this in the manuscript as follows:

“We here measured cost as the degree of effort-linked pupil dilation. In addition to pupil size, other markers may also indicate saccade costs. For example, saccade latency has been proposed to index oculomotor effort [100], whereby saccades with longer latencies are associated with more oculomotor effort. This makes saccade latency a possible complementary marker of saccade costs (also see Supplemen- tary Materials). Although relatively sluggish, pupil size is a valuable measure of attentional costs for (at least) two reasons. First, pupil size is a highly established as marker of effort, and is sensitive to effort more broadly than only in the context of saccades [36–45, 48]. Pupil size therefore allows to capture not only the costs of saccades, but also of covert attentional shifts [33], or shifts with other effectors such as head or arm movements [54, 101]. Second, as we have demonstrated, pupil size can measure saccade costs even when searching in natural scenes (Figure 4). During natural viewing, it is difficult to disentangle fixation duration from saccade latencies, complicating the use of saccade latency as a measure of saccade cost.

Together, pupil size, saccade latency, and potential other markers of saccade cost could fulfill complementary roles in studying the role of cost in saccade selection.”

Second, we followed the reviewer’s recommendation in testing whether other oculomotor metrics would predict saccade selection. To this end, we conducted a linear regression across directions. We calculated pupil size, saccade latencies, landing precision and peak velocities maps from the saccade planning task. We then used AICbased backward model selection to determine the ‘best’ model model to determine which factor would predict saccade selection best. The best model included pupil size, latency and landing precision as predictors (Wilkinson notation: saccade preferences ~ pupil size + saccade latency + landing precision). Pupil size (b \=-42.853, t \= 4.791, p < .001) and saccade latency (b \=-.377, t \= 2.106, p \= .043; see Author response image 1) predicted saccade preferences significantly. In contrast, landing precision did not reach significance (b \= 23.631, t \= 1.675, p \= .104). This analysis shows that although saccade latency also predicts saccade preferences, pupil size remains a robust predictor of saccade selection. These findings demonstrate that minimizing oculomotor noise cannot fully explain the pattern of results.

Author response image 1.

The relationship between saccade latency (from the saccade planning task) and saccade preferences averaged across participants. Individual points reflect directions and shading represents bootstrapped 95% confidence intervals.

We have added this argument into the manuscript, and discuss the analysis in the discussion. Details of the analysis have been added to the Supporting Information for transparency and further detail.

“A control analysis ruled out that the correlation between pupil size and saccade preferences was driven by other oculomotor metrics such as saccade latency and landing precision (see Supporting Information).”

“To ascertain whether pupil size or other oculomotor metrics predict saccade preferences, we conducted a multiple regression analysis. We calculated average pupil size, saccade latency, landing precision and peak velocity maps across all 36 directions. The model, determined using AIC-based backward selection, included pupil size, latency and landing precision as predictors (Wilkinson notation: saccade preferences  pupil size + saccade latency + landing precision). The analysis re- vealed that pupil size (β = -42.853, t = 4.791, p < .001) and saccade latency (β = -.377, t = 2.106, p = .043) predicted saccade preferences. Landing precision did not reach significance (β = 23.631, t = 1.675, p = .104). Together, this demonstrates that although other oculomotor metrics such as saccade latency contribute to saccade selection, pupil size remains a robust marker of saccade selection.”

In addition to eye-movement-related anisotropies across the visual field, there are of course many studies reporting visual field anisotropies (see Himmelberg, Winawer & Carrasco, 2023, Trends in Neuroscience for a review). It would be interesting to understand how the authors think about visual field anisotropies in the context of their own study. Do they think that their results are (in)dependent on such visual field variations (see Greenwood et al., 2017, PNAS; Ohl, Kroell, & Rolfs, 2024, JEP:Gen for a similar discussion)?

We agree that established visual field anisotropies are fascinating to be discussed in context of our own results. At the reviewer’s suggestion, we now expanded this discussion.

The observed anisotropies in terms of saccade costs are likely related to established anisotropies in perception and early visual cortex. However, the exact way that these anisotropies may be linked remains elusive (i.e. what is cause, what is effect, are links causal?), and more research is necessary to understand how these are related.

“The observed differences in saccade costs across directions could be linked to established anisotropies in perception [80–86], attention [87–92], saccade charac- teristics [87, 88, 92, 93], and (early) visual cortex [94–98] [also see 99]. For example, downward saccades are more costly than upward saccades, which mimics a similar asymmetry in early visual areas wherein the upper visual field is relatively under- represented [94–98]; similarly stronger presaccadic benefits are found for down- compared with upward saccades [87, 88]. Moreover, upward saccades are more pre- cise than downward saccades [93]. Future work should elucidate where saccade cost or the aforementioned anisotropies originate from and how they are related - something that pupil size alone cannot address.”

We also added that the finding that more precise saccades are coupled with worse performance in a crowding task might be attributed to the increased effort associated with more precise saccades (Greenwood et al., 2017).

“Adaptive resource allocation from, and to the oculomotor system parsimoniously explains a number of empirical observations. For example, higher cognitive demand is accompanied by smooth pursuits deviating more from to-be tracked targets [137], reduced (micro)saccade frequencies [Figure 4; 63, 64, 138, 139], and slower peak saccade velocities [140–142]. Relatedly, more precise saccades are accompanied with worse performance in a crowding task [93].”

Finally, the authors conclude that their results "suggests that the eye-movement system and other cognitive operations consume similar resources that are flexibly allocated among each other as cognitive demand changes. The authors should speculate what these similar resources could mean? What are the specific operations of the auditory task that overlap in terms of resources with the eye movement system?

We agree that the nature of joint resources is an interesting question. Our previous discussion was likely too simplistic here (see also responses to R3). We here specifically refer to the cognitive resources that one can flexibly distribute between tasks.

Our data do not directly speak to the question of what the shared resources between the auditory and oculomotor tasks are. Nevertheless, both tasks charge working memory as saccade targets are mandatorily encoded into working memory prior to saccade onset (Van der Stigchel & Hollingworth, 2018), and the counting task clearly engages working memory. This may indicate some domain-generality between visual and auditory working memory during natural viewing (see Nozari & Martin, 2024 for a recent review), but this remains speculative. Another possibility is that not the working memory encoding associated with saccades per se, but that the execution of overt motor actions itself also requires cognitive processing as suggested by Beatty (1982): “the organization of an overt motor act places additional demands on informationprocessing resources that are reflected in the task-evoked pupillary response”.

We have added upon this in more detail in the results and discussion sections.

“Besides the costs of increased neural activity when exerting more effort, effort should be considered costly for a second reason: Cognitive resources are limited. Therefore, any unnecessary resource expenditure reduces cognitive and behavioral flexibility [22, 31, 36, 116]. As a result, the brain needs to distribute resources between cognitive operations and the oculomotor system. We found evidence for the idea that such resource distribution is adaptive to the general level of cognitive demand and available resources: Increasing cognitive demand through an additional pri- mary auditory dual task led to a lower saccade frequency, and especially costly sac- cades were cut. In this case, it is important to consider that the auditory task was the primary task, which should cause participants to distribute resources from the ocu- lomotor system to the counting task. In other situations, more resources could be distributed to the oculomotor system instead, for example to discover new sources of reward [22, 136]. Adaptive resource allocation from, and to the oculomotor system parsimoniously explains a number of empirical observations. For example, higher cognitive demand is accompanied by smooth pursuits deviating more from to-be tracked targets [137], reduced (micro)saccade frequencies [Figure 4; 63, 64, 138, 139], and slower peak saccade velocities [140–142]. Relatedly, more precise saccades are accompanied with worse performance in a crowding task [93]. Furthermore, it has been proposed that saccade costs are weighed against other cognitive operations such as using working memory [33, 143–146]. How would the resources between the oculomotor system and cognitive tasks (like the auditory counting task) be related? One possibility is that both consume from limited working memory resources [147, 148]. Saccades are thought to encode target objects in a mandatory fashion into (vi- sual) working memory [79], and the counting task requires participants to keep track of the auditory stream and maintain count of the instructed digit in working mem- ory. However, the exact nature of which resources overlap between tasks remain open for future investigation [also see 149]. Together, we propose that cognitive re- sources are flexibly (dis)allocated to and from the oculomotor system based on the current demands to establish an optimal balance between performance and cost minimization.”

Reviewer #2 (Public Review):

The authors attempt to establish presaccadic pupil size as an index of 'saccade effort' and propose this index as one new predictor of saccade target selection. They only partially achieved their aim: When choosing between two saccade directions, the less costly direction, according to preceding pupil size, is preferred. However, the claim that with increased cognitive demand participants would especially cut costly directions is not supported by the data. I would have expected to see a negative correlation between saccade effort and saccade direction 'change' under increased load. Yet participants mostly cut upwards saccades, but not other directions that, according to pupil size, are equally or even more costly (e.g. oblique saccades).

Strengths:

The paper is well-written, easy to understand, and nicely illustrated.

The sample size seems appropriate, and the data were collected and analyzed using solid and validated methodology.

Overall, I find the topic of investigating factors that drive saccade choices highly interesting and relevant.

We thank the reviewer for pointing out the strengths of our paper.

Weaknesses:

The authors obtain pupil size and saccade preference measures in two separate tasks. Relating these two measures is problematic because the computations that underly saccade preparation differ. In Experiment 1, the saccade is cued centrally, and has to be delayed until a "go-signal" is presented; In Experiment 2, an immediate saccade is executed to an exogenously cued peripheral target. The 'costs' in Experiment 1 (computing the saccade target location from a central cue; withholding the saccade) do not relate to Experiment 2. It is unfortunate, that measuring presaccadic pupil size directly in the comparatively more 'natural' Experiment 2 (where saccades did not have to be artificially withheld) does not seem to be possible. This questions the practical application of pupil size as an index of saccade effort

This is an important point raised by the reviewer and we agree that a discussion on these points improves the manuscript. We reply in two parts: 1) Although the underlying computations during saccade preparation might differ, and are therefore unlikely to be fully similar (we agree), we can still predict saccade selection between (Saccade planning to Saccade preference) and within tasks (Visual search). 2) Pupil size is a sluggish physiological signal, but this is outweighed by the advantages of using pupil size as a general marker of effort, also in the context of visual selection compared with saccade latencies.

(1) Are delayed saccades (cost task) and the much faster saccades (preference task) linked?

As the reviewer notes the underlying ‘type’ of oculomotor program may differ between voluntarily delayed-saccades and those in the saccade preference task. There are, however, also considerable overlaps between the oculomotor programs as the directions and amplitudes are identical. Moreover, the different types of saccades have considerable overlap in their underlying neural circuitry. Nevertheless, the underlying oculomotor programs likely still differ in some regard. Even despite these differences, we were able to measure differences across directions in both tasks, and costs and preferences were negatively and highly correlated between tasks. The finding itself therefore indicates that the costs of saccades measured during the saccade planning task generalize to those in the saccade preference task. Note also that we predicted this finding and idea already in a previous publication before starting the present study (Koevoet et al., 2023).

We now address this interesting point in the discussion as follows:

“We observed that aOordable saccades were preferred over costly ones. This is especially remarkable given that the delayed saccades in the planning task likely differ in their oculomotor program from the immediate saccades in the preference task in some regard.”

(2) Is pupil size a sensible measure of saccade effort?

As the reviewer points out, the pupillary signal is indeed relatively sluggish and therefore relatively slow and more artifical tasks are preferred to quantify saccade costs. This does not preclude pupil size from being applied in more natural settings, as we demonstrate in the search experiments – but a lot of care has to be taken to control for many possible confounding factors and many trials will be needed.

That said, as saccade latencies may also capture differences in oculomotor effort (Shadmehr et al., 2019) they are a possible alternative option to assess effort in some oculomotor tasks (see below on why saccade latencies do not provide evidence for an alternative to effort driving saccade selection, but converging evidence). Whilst we do maintain that pupil size is an established and versatile physiological marker of effort, saccade latencies provide converging evidence for our conclusion that effort drives saccade selection.

As for the saccade preference task, we are not able to analyze the data in a similar manner as in the visual search task for two reasons. First, the number of saccades is much lower than in the natural search experiments. Second, in the saccade preference task, there were always two possible saccade targets. Therefore, even if we were able to isolate an effort signal, this signal could index a multitude of factors such as deciding between two possible saccade targets. Even simple binary decisions go hand in hand with reliable pupil dilations as they require effort (e.g. de Gee et al., 2014).

There are three major reasons why pupil size is a more versatile marker of saccade costs than saccade latencies (although as mentioned, latencies may constitute another valuable tool to study oculomotor effort). First, pupil size is able to quantify the cost of attentional shifts more generally, including covert attention as well as other effector systems such as head and hand movements. This circumvents the issue of different latencies of different effector systems and also allows to study attentional processes that are not associated with overt motor movements. Second, saccade latencies are difficult to interpret in natural viewing data, as fixation duration and saccade latencies are inherently confounded by one another. This makes it very difficult to separate oculomotor processes and the extraction of perceptual information from a fixated target. Thus, pupil size is a versatile marker of attentional costs in a variety of settings, and can measure costs that saccade latencies cannot (i.e. covert attention). Lastly, pupil size is highly established as a marker of effort which has been demonstrated across wide range of cognitive tasks and therefore not bound to eye movements alone (Bumke, 1911; Koevoet et al., 2024; Laeng et al., 2012; Loewenfeld, 1958; Mathôt, 2018; Robison & Unsworth, 2019; Sirois & Brisson, 2014; Strauch et al., 2022; van der Wel & van Steenbergen, 2018).

We now discuss this as follows:

“We here measured cost as the degree of effort-linked pupil dilation. In addition to pupil size, other markers may also indicate saccade costs. For example, saccade latency has been proposed to index oculomotor effort [100], whereby saccades with longer latencies are associated with more oculomotor effort. This makes saccade latency a possible complementary marker of saccade costs (also see Supplemen- tary Materials). Although relatively sluggish, pupil size is a valuable measure of attentional costs for (at least) two reasons. First, pupil size is a highly established as marker of effort, and is sensitive to effort more broadly than only in the context of saccades [36–45, 48]. Pupil size therefore allows to capture not only the costs of saccades, but also of covert attentional shifts [33], or shifts with other effectors such as head or arm movements [54, 101]. Second, as we have demonstrated, pupil size can measure saccade costs even when searching in natural scenes (Figure 4). During natural viewing, it is difficult to disentangle fixation duration from saccade latencies, complicating the use of saccade latency as a measure of saccade cost. Together, pupil size, saccade latency, and potential other markers of saccade cost could fulfill complementary roles in studying the role of cost in saccade selection.”

The authors claim that the observed direction-specific 'saccade costs' obtained in Experiment 1 "were not mediated by differences in saccade properties, such as duration, amplitude, peak velocity, and landing precision (Figure 1e,f)". Saccade latency, however, was not taken into account here but is discussed for Experiment 2.

The final model that was used to test for the observed anisotropies in pupil size across directions indeed did not include saccade latencies as a predictor. However, we did consider saccade latencies as a potential predictor originally. As we performed AICbased backward model selection, however, this predictor was removed due to the marginal predictive contribution of saccade latency beyond other predictors explaining pupil size.

For completeness, we here report the outcome of a linear mixed-effects that does include saccade latency as a predictor. Here, saccade latencies did not predict pupil size (b \= 1.859e-03, t \= .138, p \= .889). The asymmetry effects remained qualitatively unchanged: preparing oblique compared with cardinal saccades resulted in a larger pupil size (b \= 7.635, t \= 3.969, p < .001), and preparing downward compared with upward saccades also led to a larger pupil size (b \= 3.344, t \= 3.334, p \= .003).

The apparent similarity of saccade latencies and pupil size, however, is striking. Previous work shows shorter latencies for cardinal than oblique saccades, and shorter latencies for horizontal and upward saccades than downward saccades - directly reflecting the pupil sizes obtained in Experiment 1 as well as in the authors' previous study (Koevoet et al., 2023, PsychScience).

As the reviewer notes, there are substantial asymmetries across the visual field in saccade latencies. These assymetries in saccade latency could also predict saccade preferences. We will reply to this in three points: 1) even if saccade latency is a predictor of saccade preferences, this would not constitute as an alternative explanation to the conclusion of effort driving saccade selection, 2) saccade latencies show an up-down asymmetry but oblique-cardinal effects in latency may not be generalizable across saccade tasks, 3) pupil size remains a robust predictor of saccade preferences even when saccade latencies are considered as a predictor of saccade preferences.

(1) We want to first stress that saccade latencies are thought to reflect oculomotor effort (Shadmehr et al., 2019). For example, saccades with larger amplitudes and saccades where distractors need to be ignored are associated with longer latencies. Therefore, even if saccade latencies predict saccade selection, this would not contrast the idea that effort drives saccade selection. Instead, this would provide convergent evidence for our main conclusion – effort predicting saccade selection (rather than pupil size predicting saccade selection per se).

“We here measured cost as the degree of effort-linked pupil dilation. In addition to pupil size, other markers may also indicate saccade costs. For example, saccade latency has been proposed to index oculomotor effort [100], whereby saccades with longer latencies are associated with more oculomotor effort. This makes saccade latency a possible complementary marker of saccade costs (also see Supplemen- tary Materials). Although relatively sluggish, pupil size is a valuable measure of attentional costs for (at least) two reasons. First, pupil size is a highly established as marker of effort, and is sensitive to effort more broadly than only in the context of saccades [36–45, 48]. Pupil size therefore allows to capture not only the costs of saccades, but also of covert attentional shifts [33], or shifts with other effectors such as head or arm movements [54, 101]. Second, as we have demonstrated, pupil size can measure saccade costs even when searching in natural scenes (Figure 4). During natural viewing, it is difficult to disentangle fixation duration from saccade latencies, complicating the use of saccade latency as a measure of saccade cost. Together, pupil size, saccade latency, and potential other markers of saccade cost could fulfill complementary roles in studying the role of cost in saccade selection.”

(2) We first tested anisotropies in saccade latency in the saccade planning task (Wilkinson notation: latency ~ obliqueness + updownness + leftrightness + saccade duration + saccade amplitude + saccade velocity + landing error + (1+obliqueness + updownness|participant)). We found upward latencies to be shorter than downward saccade latencies (b \= -.535, t \= 3.421, p \= .003). In addition, oblique saccades showed shorter latencies than cardinal saccades (b \= -1.083, t \= 3.096, p \= .002) – the opposite of what previous work has demonstrated.

We then also tested these latency anisotropies in another dataset wherein participants (n \= 20) saccaded toward a single peripheral target as fast as possible (Koevoet et al., submitted; same amplitude and eccentricity as in the present manuscript). There we did not find a difference in saccade latency between cardinal and oblique targets, but we did observe shorter latencies for up- compared with downward saccades. We are therefore not sure in which situations oblique saccades do, or do not differ from cardinal saccades in terms of latency, and even in which direction the effect occurs.

In contrast, we have now demonstrated a larger pupil size prior to oblique compared with cardinal saccades in two experiments. This indicates that pupil size may be a more reliable and generalizable marker of saccade costs than saccade latency. However, this remains to be investigated further.

(3) To gain further insights into which oculomotor metrics would predict saccade selection, we conducted a linear regression across directions. We created pupil size, saccade latencies, landing precision and peak velocities maps from the saccade planning task. We then used AIC-based model selection to determine the ‘best’ model to determine which factor would predict saccade selection best. The selected model included pupil size, latency and landing precision as predictors (Wilkinson notation: saccade preferences ~ pupil size + saccade latency + landing precision). Pupil size (b \=-42.853, t \= 4.791, p < .001) and saccade latency (b \=-.377, t \= 2.106, p \= .043) predicted saccade preferences significantly. In contrast, landing precision did not reach significance (b \= 23.631, t \= 1.675, p \= .104). This analysis shows that although saccade latency predicts saccade preferences, pupil size remains a robust predictor of saccade selection.

“To ascertain whether pupil size or other oculomotor metrics predict saccade preferences, we conducted a multiple regression analysis. We calculated average pupil size, saccade latency, landing precision and peak velocity maps across all 36 directions. The model, determined using AIC-based backward selection, included pupil size, latency and landing precision as predictors (Wilkinson notation: saccade preferences  pupil size + saccade latency + landing precision). The analysis re- vealed that pupil size (β = -42.853, t = 4.791, p < .001) and saccade latency (β = -.377, t = 2.106, p = .043) predicted saccade preferences. Landing precision did not reach significance (β = 23.631, t = 1.675, p = .104). Together, this demonstrates that although other oculomotor metrics such as saccade latency contribute to saccade selection, pupil size remains a robust marker of saccade selection.”

The authors state that "from a costs-perspective, it should be eOicient to not only adjust the number of saccades (non-specific), but also by cutting especially expensive directions the most (specific)". However, saccade targets should be selected based on the maximum expected information gain. If cognitive load increases (due to an additional task) an effective strategy seems to be to perform less - but still meaningful - saccades. How would it help natural orienting to selectively cut saccades in certain (effortful) directions? Choosing saccade targets based on comfort, over information gain, would result in overall more saccades to be made - which is non-optimal, also from a cost perspective.

We thank the reviewer for this comment. Although we do not fully agree, the logic is quite close to our rationale and it is worth adding a point of discussion here. A vital part of the current interpretation is the instruction given to participants. In our second natural visual search task, participants were performing a dual task, where the auditory task was the primary task, whilst the search task was secondary. Therefore, participants are likely to adjust their resources to optimize performance on the primary task – at the expense of the secondary task. Therefore, less resources are made available and used to searching in the dual than in the single task, because these resources are needed for the auditory task. Cutting expensive directions does not help search in terms of search performance, but it does reduce the cost of search, so that more resources are available for the prioritized auditory task. Also note that the search task was rather difficult – participants did it, but it was tough (see the original description of the dataset for more details), which provides another reason to go full in on the auditory task at expense of the visual task. This, however, opens up a nice point of discussion: If one would emphasize the importance of search (maybe with punishment or reward), we would indeed expect participants to perform whichever eye movements are getting them to their goal fastest – thus reducing the relative influence of costs on saccade behavior. This remains to be tested however - we are working on this and are looking forward to discussing such findings in the future.

Together, we propose that there is a trade-off between distributing resources either towards cognitive tasks or the oculomotor system (also see Ballard et al., 1995; Van der Stigchel, 2020). How these resources are distributed depends highly on the current task demands (also see Sahakian et al., 2023). This allows for adaptive behavior in a wide range of contexts.

We now added these considerations to the manuscript as follows (also see our previous replies):

“Do cognitive operations and eye movements consume from a similar pool of resources [44]? If so, increasing cognitive demand for non-oculomotor processes should result in decreasing available resources for the oculomotor system. In line with this idea, previous work indeed shows altered eye-movement behavior un- der effort as induced by dual tasks, for example by making less saccades under increased cognitive demand [62–64]. We therefore investigated whether less sac- cades were made as soon as participants had to count the occurrence of a specific digit in the auditory number stream in comparison to ignoring the stream (in Exp. 2; Figure 4a). Participants were instructed to prioritize the auditory digit-counting task over finding the visual search target. Therefore, resources should be shifted from the oculomotor system to the primary auditory counting task. The additional cognitive demand of the dual task indeed led to a decreased saccade frequency (t(24) = 7.224, p < .001, Cohen’s d = 1.445; Figure 4h).”

I would have expected to see a negative correlation between saccade effort and saccade direction 'change' under increased load. Yet participants mostly cut upwards saccades, but not other directions that, according to pupil size, are equally or even more costly (e.g. oblique saccades).

The reviewer’s point is taken from the initial comment, which we will address here. First, we’d like to point out that is it not established that saccade costs in different directions are always the same. Instead, it is possible that saccade costs could be different in natural viewing compared with our delayed-saccade task. Therefore, we used pupil size during natural viewing for the search experiments. Second, the reviewer correctly notes that oblique saccades are hardly cut when under additional cognitive demand. However, participants already hardly execute oblique saccades when not confronted with the additional auditory task (Figure 4b, d), making it difficult to reduce those further (i.e. floor effect). Participants chose to cut vertical saccades, possibly because these are more costly than horizontal saccades.

We incorporated these point in our manuscript as follows:

“To test this, we analyzed data from two existing datasets [63] wherein participants (total n = 41) searched for small targets (’Z’ or ’H’) in natural scenes (Figure 4a; [64]). Again, we tested whether pupil size prior to saccades negatively linked with saccade preferences across directions. Because saccade costs and preferences across directions could differ for different situations (i.e. natural viewing vs. saccade preference task), but should always be negatively linked, we established both cost and preferences independently in each dataset.”

“We calculated a saccade-adjustment map (Figure 4g) by subtracting the saccade preference map in the single task (Figure 4f) from the dual task map (Fig- ure 4d). Participants seemingly cut vertical saccades in particular, and made more saccades to the top right direction. This pattern may have emerged as vertical saccades are more costly than horizontal saccades (also see Figure 1d). Oblique saccades may not have been cut because there were very little oblique saccades in the single condition to begin with (Figure 4d), making it difficult to observe a further reduction of such saccades under additional cognitive demand (i.e. a floor effect).”

Overall, I am not sure what practical relevance the relation between pupil size (measured in a separate experiment) and saccade decisions has for eye movement research/vision science. Pupil size does not seem to be a straightforward measure of saccade effort. Saccade latency, instead, can be easily extracted in any eye movement experiment (no need to conduct a separate, delayed saccade task to measure pupil dilation), and seems to be an equally good index.

There are two points here.

(1) What is the practical relevance of a link between effort and saccade selection for eyemovement research and vision science?

We see plenty – think of changing eye movement patterns under effort (be it smooth pursuits, saccade rates, distributions of gaze positions to images etc.) which have substantial implications for human factors research, but also neuropsychology. With a cost account, one may predict (rather than just observe) how eye movement changes as soon as resources are reduced/ non-visual demand increases. With a cost account, we can explain such effects (e.g. lower saccade rates under effort, cardinal bias, perhaps also central bias) parsimoniously that cannot be explained by what is so far referred to as the three core drivers of eye movement behavior (saliency, selection history, goals, e.g., Awh et al., 2012). Conversely, one must wonder why eye-movement research/vision science simply accepts/dismisses these phenomena as such, without seeking overarching explanations.

(2) What is the usefulness of using pupil size to measure effort?

We hope that our replies to the comments above illustrate why pupil size is a sensible, robust and versatile marker of attentional costs. We briefly summarize our most important points here.

- Pupil size is an established measure of effort irrespective of context, as demonstrated by hundreds of original works (e.g. working memory load, multiple object tracking, individual differences in cognitive ability). This allows pupil size to be a versatile marker of the effort, and therefore costs, of non-saccadic attentional shifts such as covert attention or those realized by other effector systems (i.e. head or hand movements).

- Our new analysis indicates that pupil size remains a strong and robust predictor of saccade preference, even when considering saccade latency.

- Pupil size allows to study saccade costs in natural viewing. In contrast, saccade latencies are difficult to assess in natural viewing as fixation durations and saccade latencies are intrinsically linked and very difficult to disentangle.

- Note however, that we think that it is interesting and useful so study effects of effort/cost on eye movement behavior. Whichever index is used to do so, we see plenty potential in this line of research, this paper is a starting point to do so.

Reviewer #3 (Public Review):

This manuscript extends previous research by this group by relating variation in pupil size to the endpoints of saccades produced by human participants under various conditions including trial-based choices between pairs of spots and search for small items in natural scenes. Based on the premise that pupil size is a reliable proxy of "effort", the authors conclude that less costly saccade targets are preferred. Finding that this preference was influenced by the performance of a non-visual, attentiondemanding task, the authors conclude that a common source of effort animates gaze behavior and other cognitive tasks.

Strengths:

Strengths of the manuscript include the novelty of the approach, the clarity of the findings, and the community interest in the problem.

We thank the reviewer for pointing out the strengths of our paper.

Weaknesses:

Enthusiasm for this manuscript is reduced by the following weaknesses:

(1) A relationship between pupil size and saccade production seems clear based on the authors' previous and current work. What is at issue is the interpretation. The authors test one, preferred hypothesis, and the narrative of the manuscript treats the hypothesis that pupil size is a proxy of effort as beyond dispute or question. The stated elements of their argument seem to go like this:

PROPOSITION 1: Pupil size varies systematically across task conditions, being larger when tasks are more demanding.

PROPOSITION 2: Pupil size is related to the locus coeruleus.

PROPOSITION 3: The locus coeruleus NE system modulates neural activity and interactions.

CONCLUSION: Therefore, pupil size indexes the resource demand or "effort" associated with task conditions.

How the conclusion follows from the propositions is not self-evident. Proposition 3, in particular, fails to establish the link that is supposed to lead to the conclusion.

We inadvertently laid out this rationale as described above, and we thank the reviewer for pointing out this initial suboptimal structure of argumentation. The notion that the link between pupil size and effort is established in the literature because of its neural underpinnings is inaccurate. Instead, the tight link between effort and pupil size is established based on covariations of pupil diameter and cognition across a wide variety of tasks and domains. In line with this, we now introduce this tight link predominantly based on the relationships between pupil size and cognition instead of focusing on putative neural correlates of this relationship.

As reviewed previously (Beatty, 1982; Bumke, 1911; Kahneman, 1973; Kahneman & Beatty, 1966; Koevoet et al., 2024; Laeng et al., 2012; Mathôt, 2018; Sirois & Brisson, 2014; Strauch et al., 2022; van der Wel & van Steenbergen, 2018), any increase in effort is consistently associated with an increase in pupil size. For instance, the pupil dilates when increasing load in working memory or multiple object tracking tasks, and such pupillary effects robustly explain individual differences in cognitive ability and fluctuations in performance across trials (Alnæs et al., 2014; Koevoet et al., 2024; Robison & Brewer, 2020; Robison & Unsworth, 2019; Unsworth & Miller, 2021). This extends to the planning of movements as pupil dilations are observed prior to the execution of (eye) movements (Koevoet et al., 2023; Richer & Beatty, 1985). The link between pupil size and effort has thus been firmly established for a long time, irrespective of the neural correlates of these effort-linked pupil size changes.

We again thank the reviewer for spotting this logical mistake, and now revised the paragraph where we introduce pupil size as an established marker of effort as follows:

“We recently demonstrated that the effort of saccade planning can be measured with pupil size, which allows for a physiological quantification of saccade costs as long as low-level visual factors are controlled for [33]. Pupil size is an established marker of effort [36–44]. For instance, loading more in working memory or tracking more objects results in stronger pupil dilation [44–52]. Pupil size not only reflects cognitive (or mental) effort but also the effort of planning and executing movements [37, 53, 54]. We leveraged this to demonstrate that saccade costs can be captured with pupil size, and are higher for oblique compared with cardinal directions [33]. Here, we addressed whether saccade costs predict where to saccade.”

We now mention the neural correlates of pupil size only in the discussion. Where we took care to also mention roles for other neurotransmitter systems:

“Throughout this paper, we have used cost in the limited context of saccades.

However, cost-based decision-making may be a more general property of the brain [31, 36, 114–116]. Every action, be it physical or cognitive, is associated with an in- trinsic cost, and pupil size is likely a general marker of this [44]. Note, however, that pupil dilation does not always reflect cost, as the pupil dilates in response to many sensory and cognitive factors which should be controlled for, or at least considered, when interpreting pupillometric data [e.g., see 39, 40, 42, 117]. Effort-linked pupil dilations are thought to be, at least in part, driven by activity in the brainstem locus coeruleus (LC) [40, 118–120] [but other neurotransmitters also affect pupil size, e.g. 121, 122]. Activity in LC with its widespread connections throughout the brain [120, 123–127] is considered to be crucial for the communication within and between neu- ral populations and modulates global neural gain [128–132]. Neural firing is costly [22, 133], and therefore LC activity and pupil size are (neuro)physiologically plausible markers of cost [40]. Tentative evidence even suggests that continued exertion of effort (accompanied by altered pupil dilation) is linked to the accumulation of glutamate in the lateral prefrontal cortex [134], which may be a metabolic marker of cost [also see 116, 134, 135]. “

(2) The authors test one, preferred hypothesis and do not consider plausible alternatives. Is "cost" the only conceivable hypothesis? The hypothesis is framed in very narrow terms. For example, the cholinergic and dopamine systems that have been featured in other researchers' consideration of pupil size modulation are missing here. Thus, because the authors do not rule out plausible alternative hypotheses, the logical structure of this manuscript can be criticized as committing the fallacy of aOirming the consequent.

As we have noted in the response to the reviewer’s first point, we did not motivate our use of pupil size as an index of effort clearly enough. For the current purpose, the neural correlates of pupil size are less relevant than the cognitive correlates (see previous point). We reiterate that the neuromodulatory underpinnings of the observed pupil size effects (which indeed possibly include effects of the cholinergic, dopaminergic and serotonergic systems), while interesting for the discussion on the neural origin of effects, are not crucial to our conclusion. We hope the new rationale (without focusing too much on the (irrelevant) exact neural underpinnings) convinces the reviewer and reader.

Our changes to the manuscript are shown in our reply to the previous comment.

The reviewer notes that other plausible alternative hypotheses could explain the currently reported results. However, we did not find a more parsimonuous explanation for our data than ‘Effort Drives Saccade Selection’. Effort explains why participants prefer saccading toward specific directions in (1) highly controlled and (2) more natural settings. Note that we also predicted this effect previously (Koevoet et al., 2023). Moreover, this account explains (3) why participants make less saccades under additional cognitive demand, and (4) why especially costly saccades are reduced under additional cognitive demand. We are very open to the reviewer presenting other possible interpretations of our data so these can be discussed to be put to test in future work.

(3) The authors cite particular publications in support of the claim that saccade selection is influenced by an assessment of effort. Given the extensive work by others on this general topic, the skeptic could regard the theoretical perspective of this manuscript as too impoverished. Their work may be enhanced by consideration of other work on this general topic, e.g, (i) Shenhav A, Botvinick MM, Cohen JD. (2013) The expected value of control: an integrative theory of anterior cingulate cortex function. Neuron. 2013 Jul 24;79(2):217-40. (ii) Müller T, Husain M, Apps MAJ. (2022) Preferences for seeking effort or reward information bias the willingness to work. Sci Rep. 2022 Nov 14;12(1):19486. (iii) Bustamante LA, Oshinowo T, Lee JR, Tong E, Burton AR, Shenhav A, Cohen JD, Daw ND. (2023) Effort Foraging Task reveals a positive correlation between individual differences in the cost of cognitive and physical effort in humans. Proc Natl Acad Sci U S A. 2023 Dec 12;120(50):e2221510120.

We thank the reviewer for pointing us toward this literature. These papers are indeed relevant for our manuscript, and we have now incorporated them. Specifically, we now discuss how the costs of effort are weighed in relation to possible rewards during decision-making. We have also incorporated work that has investigated how the biomechanical costs of arm movements contribute to action selection.

“Our findings are in line with established effort-based models that assume costs to be weighed against rewards during decision-making [102–107]. In such studies, reward and cognitive/physical effort are often parametrically manipulated to as- sess how much effort participants are willing to exert to acquire a given (monetary) reward [e.g. 108, 109]. Whereas this line of work manipulated the extrinsic costs and/or rewards of decision options (e.g. perceptual consequences of saccades [110, 111] or consequences associated with decision options), we here focus on the intrin- sic costs of the movement itself (in terms of cognitive and physical effort). Relatedly, the intrinsic costs of arm movements are also considered during decision-making: biomechanically aOordable movements are generally preferred over more costly ones [26–28]. We here extend these findings in two important ways. First, until now, the intrinsic costs of saccades and other movements have been inferred from gaze behavior itself or by using computational modelling [23, 25–28, 34, 35, 112]. In con- trast, we directly measured cost physiologically using pupil size. Secondly, we show that physiologically measured saccade costs predict where saccades are directed in a controlled binary preference task, and even during natural viewing. Our findings could unite state-of-the-art computational models [e.g. 23, 25, 34, 35, 113] with physiological data, to directly test the role of saccade costs and ultimately further our understanding of saccade selection.”

(4) What is the source of cost in saccade production? What is the currency of that cost? The authors state (page 13), "... oblique saccades require more complex oculomotor programs than horizontal eye movements because more neuronal populations in the superior colliculus (SC) and frontal eye fields (FEF) [76-79], and more muscles are necessary to plan and execute the saccade [76, 80, 81]." This statement raises questions and concerns. First, the basis of the claim that more neurons in FEF and SC are needed for oblique versus cardinal saccades is not established in any of the publications cited. Second, the authors may be referring to the fact that oblique saccades require coordination between pontine and midbrain circuits. This must be clarified. Second, the cost is unlikely to originate in extraocular muscle fatigue because the muscle fibers are so different from skeletal muscles, being fundamentally less fatigable. Third, if net muscle contraction is the cost, then why are upward saccades, which require the eyelid, not more expensive than downward? Thus, just how some saccades are more effortful than others is not clear.

Unfortunately, our current data do not allow for the specification of what the source is of differences in saccade production, nor what the currency is. We want to explicitly state that while pupil size is a sensitive measure of saccade costs, pupil size cannot directly inform what underlying mechanisms are causing differences in saccade costs across conditions (e.g. directions). Nevertheless, we do speculate about these issues because they are important to consider. We thank the reviewer for pointing out the shortcomings in our initial speculations.

Broadly, we agree with the reviewer that a neural source of differences in costs between different types of saccades is more likely than a purely muscular account (also see Koevoet et al., 2023). Furthermore, we think that the observed differences in saccade costs for oblique vs. cardinal and up vs. down could be due to different underlying mechanisms. While we caution against overinterpreting single directions, tentative evidence for this may also be drawn by the different time course of effects for up/down versus cardinal/oblique, Figure 1c.

Below we speculate about why some specific saccade directions may be more costly than others:

Why would oblique saccades be more costly than cardinal saccades? We thank the reviewer for pointing out that oblique saccades additionally require coordination between pontine and midbrain circuits (Curthoys et al., 1984; King & Fuchs, 1979; Sparks, 2002). This point warrants more revised discussion compared to our initial version. We have incorporated this as follows:

“The complexity of an oculomotor program is arguably shaped by its neural underpinnings. For example, oblique but not cardinal saccades require communication between pontine and midbrain circuits [73–75]. Such differences in neural complexity may underlie the additional costs of oblique compared with cardinal saccades. Besides saccade direction, other properties of the ensuing saccade such as its speed, distance, curvature, and accuracy may contribute to a saccade’s total cost [22, 33, 53, 76, 77] but this remains to be investigated directly.”

Why would downward saccades be more costly than upward saccades? As the reviewer points out: from a net muscular contraction account of cost, one would expect the opposite pattern due to the movement of the eyelid. Instead, we speculate that our findings may be associated with the well-established anisotropy in early visual cortex along the vertical meridian. Specifically, the upper vertical meridian is represented at substantially less detail than the lower vertical meridian (Himmelberg et al., 2023; Silva et al., 2018). Prior to a saccade, attention is deployed towards the intended saccadic endpoint (Deubel & Schneider, 1996; Kowler et al., 1995). Attention tunes neurons to preferentially process the attended location over non-attended locations. Due to the fact that the lower visual field is represented at higher detail than the upper visual field, attention may tune neuronal responses differently when preparing up- compared with downward saccades (Hanning et al., 2024; Himmelberg et al., 2023). Thus, it may be more costly to prepare down- compared with upward saccades. This proposition, however, does not account for the lower costs associated horizontal compared with up- and downward saccades as the horizontal meridian is represented at a higher acuity than the vertical merdian. This makes it unlikely that this explains the pattern of results completely. Again, at this point we can only speculate why costs differ, yet we demonstrate that these differences in cost are decisive for oculomotor behavior. We now explicitly state the speculative nature of these ideas that would all need to be tested directly.

We have updated our discussion of this issue as follows:

“The observed differences in saccade costs across directions could be linked to established anisotropies in perception [80–86], attention [87–92], saccade charac- teristics [87, 88, 92, 93], and (early) visual cortex [94–98] [also see 99]. For example, downward saccades are more costly than upward saccades, which mimics a similar asymmetry in early visual areas wherein the upper visual field is relatively under- represented [94–98]; similarly stronger presaccadic benefits are found for down- compared with upward saccades [87, 88]. Moreover, upward saccades are more pre- cise than downward saccades [93]. Future work should elucidate where saccade cost or the aforementioned anisotropies originate from and how they are related - something that pupil size alone cannot address.”

(5) The authors do not consider observations about variation in pupil size that seem to be incompatible with the preferred hypothesis. For example, at least two studies have described systematically larger pupil dilation associated with faster relative to accurate performance in manual and saccade tasks (e.g., Naber M, Murphy P. Pupillometric investigation into the speed-accuracy trade-off in a visuo-motor aiming task. Psychophysiology. 2020 Mar;57(3):e13499; Reppert TR, Heitz RP, Schall JD. Neural mechanisms for executive control of speed-accuracy trade-off. Cell Rep. 2023 Nov 28;42(11):113422). Is the fast relative to the accurate option necessarily more costly?

We thank the reviewer for this interesting point that we will answer in two ways. First, we discuss the main point: the link between pupil size, effort, and cost. Second, we discuss the findings described specifically in these two papers and how we interpret these from a pupillometric account.

First, one may generally ask whether 1) any effort results in pupil dilation, 2) whether any effort is costly, and 3) whether this means that pupil dilation always reflects effort and cost respectively. Indeed, it has been argued repeatedly, prominently, and independently (e.g., Bumke, 1911; Mathôt, 2018) that any change in effort (no matter the specific origin) is associated with an evoked pupil dilation. Effort, in turn, is consistently and widely experienced as aversive, both across tasks and cultures (David et al., 2024). Effort minimization may therefore be seen as an universal law of human cognition and behavior with effort as a to-be minimized cost (Shadmehr et al., 2019; Hull 1943, Tsai 1932). However, this does not imply that any pupil dilation necessarily reflects effort or that, as a consequence thereof, any pupil dilation is always signaling cost. For instance, the pupil dark response, the pupil far response and changes in baseline pupil size are not associated with effort. Baseline and task-evoked pupil dilation responses have to be interpreted differently (see below), moreover, the pupil also changes (and dilates) due to other factors (see Strauch et al., 2022; Mathôt, 2018, Bumke 1911, Loewenfeld, 1999 for reviews).

Second, as for Naber & Murphy (2020) & Reppert at al. (2023) specifically: Both Reppert et al. (2023) and Naber & Murphy (2020) indeed demonstrate a larger baseline pupil size when participants made faster, less accurate responses. However, baseline pupil size is not an index of effort per-se, but task-evoked pupil dilation responses are (as studied in the present manuscript) (Strauch et al., 2022). For work on differences between baseline pupil diameter and task-evoked pupil responses, and their respective links with exploration and exploitation please see Jepma & Nieuwenhuis (2011). Indeed, the link between effort and larger pupil size holds for task evoked responses, but not baseline pupil size per se (also see Koevoet et al., 2023).

Still, Naber (third author of the current paper) & Murphy (2020) also demonstrated larger task-evoked pupil dilation responses when participants were instructed to make faster, less accurate responses compared with making accurate and relatively slow responses. However, this difference in task-evoked response gains significance only after the onset of the movement itself, and peaks substantially later than response offset. Whilst pupil dilation may be sluggish, it isn’t extremely sluggish either. As feedback to the performance of the participant was displayed 1.25s after performing the movement and clicking (taking about 630ms), we deem it possible that this effect may in part result from appraising the feedback to the participant rather than the speed of the response itself (in fact, Naber and Murphy also discuss this option). In addition to not measuring saccades but mouse movements, it is therefore possible that the observed evoked pupil effects in Naber & Murphy (2020) are not purely linked to motor preparation and execution per se. Therefore, future work that aims to investigate the costs of movements should isolate the effects of feedback and other potential factors that may drive changes in pupil size. This will help clarify whether fast or more accurate movements could be linked to the underlying costs of the movements.

Relatedly, we do not find evidence that pupil size during saccade planning predicts the onset latency of the ensuing saccade (please refer to our second response to Reviewer 2 for a detailed discussion).

Together, we therefore do not see the results from Reppert et al. (2023) and Naber & Murphy (2020) to be at odds with our interpretation of evoked pupil size reflecting effort and cost in the context of planning saccades.

We think that these are considerations important to the reader, which is why we now added them to the discussion as follows:

“Throughout this paper, we have used cost in the limited context of saccades.

However, cost-based decision-making may be a more general property of the brain [31, 36, 114–116]. Every action, be it physical or cognitive, is associated with an in- trinsic cost, and pupil size is likely a general marker of this [44]. Note, however, that pupil dilation does not always reflect cost, as the pupil dilates in response to many sensory and cognitive factors which should be controlled for, or at least considered, when interpreting pupillometric data [e.g., see 39, 40, 42, 117].”

(6) The authors draw conclusions based on trends across participants, but they should be more transparent about variation that contradicts these trends. In Figures 3 and 4 we see many participants producing behavior unlike most others. Who are they? Why do they look so different? Is it just noise, or do different participants adopt different policies?

We disagree with the transparency point of the reviewer. Note that we deviated from the norm here by being more transparent than common: we added individual data points and relationships rather than showing pooled effects across participants with error bars alone (see Figures 2c, 3b,c, 4c,e,f).

Moreover, our effects are consistent and stable across participants and are highly significant. To illustrate, for the classification analysis based on cost (Figure 2E) 16/20 participants showed an effect. As for the natural viewing experiments (total > 250,000 fixations), we also find that a majority of participants show the observed effects: Experiment 1: 15/16 participants; Experiment 2: 16/25 participants; Experiment 2 – adjustment: 22/25 participants.

We fully agree that it’s interesting to understand where interindividual variation may originate from. We currently have too little data to allow robust analyses across individuals and zooming in on individual differences in cost maps, preference maps, or potential personalized strategies of saccade selection. That said, future work could study this further. We would recommend to hereby reduce the number of directions to gain more pupil size data per direction and therefore cleaner signals that may be more informative on the individual level. With such stronger signals, studying (differences in) links on an individual level may be feasible and would be interesting to consider – and will be a future direction in our own work too. Nonetheless, we again stress that the reported effects are robust and consistent across participants, and that interindividual differences are therefore not extensive. Moreover, our results from four experiments consistently support our conclusion that effort drives saccade selection.

Recommendations for the authors:  

Reviewer #1 (Recommendations For The Authors):

- Based on the public review, I would recommend that the authors carefully review and correct the manuscript with regard to the causal conclusions. The study is largely correlational (i.e. the pupil was only observed, not manipulated) and therefore does not allow causal conclusions to be drawn about the relationship between pupil size and saccade selection. These causal conclusions become even more confusing when pupil size is equated with effort and saccade cost. As a consequence, an actual correlation between pupil size and saccade selection has led to the title that effort drives saccade selection. It would also be helpful for the reader to summarize in an additional section of the discussion what they consider to be a causal or correlational link based on their results.

We agree with the reviewer, and we have indeed included more explicitly which findings are correlational and which causal in detail now. As outlined before we do not see a more parimanious explanation for our findings than our title, but we fully agree that the paper benefits from making the correlational/causal nature of evidence for this idea explicitly transparent.

“We report a combination of correlational and causal findings. Despite the correlational nature of some of our results, they consistently support the hypothesis that saccade costs predicts saccade selection [which we predicted previously, 33]. Causal evidence was provided by the dual-task experiment as saccade frequencies - and especially costly saccades were reduced under additional cognitive demand. Only a cost account predicts 1) a link between pupil size and saccade preferences, 2) a cardinal saccade bias, 3) reduced saccade frequency under additional cognitive demand, and 4) disproportional cutting of especially those directions associated with more pupil dilation. Together, our findings converge upon the conclusion that effort drives saccade selection.”

- Can the authors please elaborate in more detail on how they transformed the predictors of their linear mixed model for the visualization in Figure 1f? It is difficult to see how the coeOicients in the table and the figure match.

We used the ‘effectsize’ package to provide effect sizes of for each predictor of the linear mixed-effects model (https://cran.r-project.org/web/packages/effectsize/index.html). We report absolute effect sizes to make it visually easier to compare different predictors. These details have now been included in the Methods section to be more transparent about how these effect sizes were computed.

“Absolute effect sizes (i.e. r) and their corresponding 95% confidence intervals for the linear mixed-effects models were calculated using t and df values with the ’effectsize’ package (v0.8.8) in R.”

- Could the authors please explain in more detail why they think that a trial-by-trial analysis in the free choice task adds something new to their conclusions? In fact, a trialby-trial analysis somehow suggests that the pupil size data would enter the analysis at a single trial level. If I understand correctly, the pupil size data come from their initial mapping task. So there is only one mean pupil size for a given participant and direction that goes into their analysis to predict free choice in a single trial. If this is the case, I don't see the point of doing this additional analysis given the results shown in Figure 2c.

The reviewer understands correctly that pupil size data is taken from the initial mapping task. We then used these mean values to predict which saccade target would be selected on a trial-by-trial basis. While showing the same conceptual result as the correlation analysis, we opted to include this analysis to show the robustness of the results across individuals. Therefore we have chosen to keep the analysis in the manuscript but now write more clearly that this shows the same conceptual finding as the correlation analysis.

“As another test of the robustness of the effect, we analyzed whether saccade costs predicted saccade selection on a trial-by-trial basis. To this end, we first determined the more aOordable option for each trial using the established saccade cost map (Figure 1d). We predicted that participants would select the more aOordable option. Complementing the above analyses, the more aOordable option was chosen above chance level across participants (M = 56.64%, 95%-CI = [52.75%-60.52%], one-sample t-test against 50%: t(19) = 3.26, p = .004, Cohen’s d = .729; Figure 2e). Together, these analyses established that saccade costs robustly predict saccade preferences.”

Reviewer #2 (Recommendations For The Authors):

The authors report that "Whenever the difference in pupil size between the two options was larger, saccades curved away more from the non-selected option (β = .004, SE = .001, t = 4.448, p < .001; Figure 3b), and their latencies slowed (β = .050, SE = .013, t = 4.323, p < .001; Figure 3c)". I suspect this effect might not be driven by the difference but by a correlation between pupil size and latency.

The authors correlate differences in pupil size (Exp1) with saccade latencies (Exp2), I recommend correlating pupil size with the latency directly, in either task. This would show if it is actually the difference between choices or simply the pupil size of the respective individual option that is linked to latency/effort. Same for curvature.

The reviewer raises a good point. Please see the previous analyses concerning the possible correlations between pupil size and saccade latency, and how they jointly predict saccade selection.

Our data show that saccade curvature and latencies are linked with the difference in pupil size between the selected and non-selected options. Are these effects driven by a difference in pupil size or by the pupil size associated with the chosen option?

To assess this, we conducted two linear mixed-effects models. We predicted saccade curvature and latency using pupil size (from the planning task) of the selected and nonselected options while controlling for the chosen direction (Wilkinson notation: saccade curvature/latency ~ selected pupil size + non-selected pupil size + obliqueness + vertical + horizontal + (1+ selected pupil size + non-selected pupil size|participant). We found that saccades curved away more from costlier the non-selected targets (β \=1.534, t \= 8.151, p < .001), and saccades curved away from the non-selected target less when the selected target was cheaper (β \=-2.571, t \= -6.602, p < .001). As the costs of the selected and non-selected show opposite effects on saccade curvature, this indicates that the difference between the two options drives oculomotor conflict.

As for saccade latencies, we found saccade onsets to slow when the cost of the selected target was higher (b \= .068, t \= 2.844, p \= .004). In contrast, saccade latencies were not significantly affected by the cost of the non-selected target (β \= -.018, t \= 1.457, p \= .145), although numerically the effect was in the opposite direction. This shows that latencies were primarily driven by the cost of the selected target but a difference account cannot be fully ruled out.

Together, these analyses demonstrate that the difference in costs between two alternatives reliably affects oculomotor conflict as indicated by the curvature analysis. However, saccade latencies are predominantly affected by the cost of the selected target – even when controlling for the obliqueness, updownness and leftrightness of the ensuing saccade. We have added these analyses here for completeness, but because the findings seem inconclusive for saccade latency we have chosen to not include these analyses in the current paper. We are open to including these analyses in the supplementary materials if the reviewer and/or editor would like us to, but have chosen not to do so due to conciseness and to keep the paper focused.

I was wondering why the authors haven't analyzed the pupil size in Experiment 2. If the pupil size can be assessed during a free viewing task (Experiment 3), shouldn't it be possible to also evaluate it in the saccade choice task?

We did not analyze the pupil size data from the saccade preference task for two reasons. First, the number of saccades is much lower than in the natural search experiments (~14.000 vs. ~250.000). Second, in the saccade preference task, there were always two possible saccade targets. Therefore, even if we were able to isolate an effort signal, this signal could index a multitude of factors such as deciding between two possible saccade targets (de Gee et al., 2014), and has the possibility of two oculomotor programs being realized instead of only a single one (Van der Stigchel, 2010).

Discussion: "due to stronger presaccadic benefits for upward compared with downward saccades [93,94]". I think this should be the other way around.

We thank the reviewer for pointing this out. We have corrected our mistake in the revised manuscript.

Saccade latencies differ around the visual field; to account for that, results / pupil size should be (additionally) evaluated relative to saccade onset (rather than cue offset). It is interesting that latencies were not accounted for here (Exp1), since they are considered for Exp2 (where they correlate with a pupil size difference). I suspect that latencies not only correlate with the difference in pupil size, but directly with pupil size itself.

We agree with the reviewer that locking the pupil size signal to saccade onset instead of cue offset may be informative. We included an analysis in the supporting information that investigates this (see Figure S1). The results of the analysis were conceptually identical.

The reviewer writes that latencies were not accounted for in Experiment 1. Although saccade latency was not included in the final model reported in the paper, it was considered during AIC-based backward model selection. As saccade latency did not predict meaningful variance in pupil size, it was ultimately not included in the analysis as a predictor. For completeness, we here report the outcome of a linear mixed-effects that does include saccade latency as a predictor. Here, saccade latencies did not predict pupil size (β \= 1.859e-03, t \= .138, p \= .889). The assymetry effects remained qualitatively unchanged: preparing oblique compared with cardinal saccades resulted in a larger pupil size (β \= 7.635, t \= 3.969, p < .001), and preparing downward compared with upward saccades also led to a larger pupil size (β \= 3.344, t \= 3.334, p \= .003).

In addition, we have included a new analysis in the supporting information that directly addresses this issue. We will reiterate the main results here:

“To ascertain whether pupil size or other oculomotor metrics predict saccade preferences, we conducted a multiple regression analysis. We calculated average pupil size, saccade latency, landing precision and peak velocity maps across all 36 directions. The model, determined using AIC-based backward selection, included pupil size, latency and landing precision as predictors (Wilkinson notation: saccade preferences  pupil size + saccade latency + landing precision). The analysis re- vealed that pupil size (β = -42.853, t = 4.791, p < .001) and saccade latency (β = -.377, t = 2.106, p = .043) predicted saccade preferences. Landing precision did not reach significance (β = 23.631, t = 1.675, p = .104). Together, this demonstrates that although other oculomotor metrics such as saccade latency contribute to saccade selection, pupil size remains a robust marker of saccade selection.”

We have also added this point in our discussion:

“We here measured cost as the degree of effort-linked pupil dilation. In addition to pupil size, other markers may also indicate saccade costs. For example, saccade latency has been proposed to index oculomotor effort [100], whereby saccades with longer latencies are associated with more oculomotor effort. This makes saccade latency a possible complementary marker of saccade costs (also see Supplemen- tary Materials). Although relatively sluggish, pupil size is a valuable measure of attentional costs for (at least) two reasons. First, pupil size is a highly established as marker of effort, and is sensitive to effort more broadly than only in the context of saccades [36–45, 48]. Pupil size therefore allows to capture not only the costs of saccades, but also of covert attentional shifts [33], or shifts with other effectors such as head or arm movements [54, 101]. Second, as we have demonstrated, pupil size can measure saccade costs even when searching in natural scenes (Figure 4). During natural viewing, it is difficult to disentangle fixation duration from saccade latencies, complicating the use of saccade latency as a measure of saccade cost. Together, pupil size, saccade latency, and potential other markers of saccade cost could fulfill complementary roles in studying the role of cost in saccade selection.”

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Author response:

The following is the authors’ response to the current reviews.

We thank Reviewers for highlighting the strengths of our work along with suggestions for future directions.

We agree with the Reviewers that RPS26 depletion may impact not only RAN translation initiation and codon selection (as showed in the experiments in Figure 4G), but also other mechanisms, such as speed of PIC scanning, as we stated in the discussion. Although, we did provide the data showing that mRNA of exogenous FMR1-GFP does not change upon RPS26 depletion (Figure 3B&C), hence observed effect most likely stems from translation regulation. In addition, an experiment with ASO-ACG treatment (Figure 4G) suggests that near cognate start codon selection or speed of PIC scanning may be a part of the regulation of RAN translation sensitive to RPS26 depletion. In addition, our latest unpublished results (Niewiadomska D. et al., in revision), indicate that FMRpolyG in fusion with GFP is fairly stable, in particular, while derived from long repeats (>90xCGG), suggesting that the protein stability is not at play in RPS26-dependent regulation.

We would like to stress that in order to avoid bias in result interpretation and to mimic the natural situation, the majority of experiments concerning levels of FMRpolyG were performed in cell models with stable expression of ACG-initiated FMRpolyG. Currently, we do not possess a cell model with stable expression of AUG-initiated FMRpolyG, and the experiments based on transient transfection system would not necessarily be comparable to the results obtained in stable expression system. However, we believe that the experiment presented in Figure 2B serves as a good control for overall translation level upon RPS26 depletion indicating that RPS26 insufficiency does not affect global translation and the observed regulation is specific to some mRNAs including the one encoding FMRpolyG frame. We also show that the level of ca. 80% of identified canonical proteins, including FMRP, did not change upon RPS26 silencing (SILAC-MS, Figure 4A). Indeed, we did not explore the ribosome composition upon RPS26 and TSR2 depletion, although, most likely the pool of functional ribosomes in the cell is sufficient enough to support the basal translation level (SUnSET assays, Figure 2B & 5C). However, we cannot exclude possibility that for some mRNAs, including one encoding for FMRpolyG, the observed effect can be partially caused by lowering the number of fully active ribosomes, especially in experiments with transient transfection experiments where transgene expression is hundreds times higher than for average native mRNA.

Finally, we agree with the Reviewer that in vitro translation assay would provide the evidence of direct effect of RPS26 on FMRpolyG level, however, we did not manage to overcome technical difficulties in obtaining cellular lysate devoid of RPS26 from vendor companies.

The following is the authors’ response to the original reviews.

General Comments

We thank Reviewers for the critical comments and experimental suggestions. We considered most of the advices in the revised version of the manuscript, which allowed for a more balanced interpretation of the results presented, and further supported major statement of the manuscript that insufficiency of the RPS26 and RPS25 plays a role in modulating the efficiency of noncanonical RAN translation from FMR1 mRNA, which results in the production of toxic polyglycine protein (FMRpolyG). Firstly, performing new experiments, we showed that silencing of the RPS26 and its chaperone protein TSR2, which regulates loading/exchange of RPS26 in maturing small ribosome subunit, did not elicit global translation inhibition. Secondly, we demonstrated that in contrary to RPS26 and RPS25 depletion, silencing the RPS6 protein, a core component of 40S subunit, did not affect FMRpolyG production, further supporting the specific effect of RPS26 and RPS25 on RAN translation regulation of mutant FMR1 mRNA. We also observed that depletion of RPS26, RPS25 and RPS6 had significant negative effect on cells proliferation which is in line with previously published results indicating that insufficiencies of ribosomal proteins negatively affect cell growth. Moreover, we showed that FMRpolyG production is significantly affected by RPS26 depletion while initiated at ACG, but not other near cognate start codons. Importantly, translation of FMRP initiated at canonical AUG codon of the same mRNA upstream the CGGexp was not affected by RPS26 silencing, similarly to vast majority of the human proteome. This implies that RAN translation of FMR1 mRNA mediated by RPS26 insufficiency is likely to be dependent on start codon selection/fidelity. In essence, we provide a series of evidences indicating that cellular amount of 40S ribosomal proteins RPS26 and RPS25 is important factor of CGGrelated RAN translation regulation. Finally, we also decided to tone down our claims. Now, we state that the RPS26/25/TSR2 insufficiency or depletion, affects RAN translation, rather than composition of 40S ribosomal subunit per se influences RAN translation. We have addressed all specific concerns below and made changes to the new version of manuscript.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Tutak et al use a combination of pulldowns, analyzed by mass spectrometry, reporter assays, and fluorescence experiments to decipher the mechanism of protein translation in fragile X-related diseases. The topic is interesting and important.

Although a role for Rps26-deficient ribosomes in toxic protein translation is plausible based on already available data, the authors' data are not carefully controlled and thus do not support the conclusions of the paper.

We sincerely appreciate your rigorous, insightful, and constructive feedback throughout the revision process. We believe your guidance has been instrumental in significantly enhancing the quality of our research. Below, we have addressed your comments pointby-point.

Strengths:

The topic is interesting and important.

Weaknesses:

In particular, there is very little data to support the notion that Rps26-deficient ribosomes are even produced under the circumstances. And no data that indicate that they are involved in the RAN translation. Essential controls (for ribosome numbers) are lacking, no information is presented on the viability of the cells (Rps26 is an essential protein), and the differences in protein levels could well arise from block in protein synthesis, and cell division coupled to differential stability of the proteins.

We agree that data presented in the first version of the manuscript did not directly address the following processes: ribosome content, global translation rate and cell viability upon RPS26 depletion. Therefore we addressed some of the issues in the revised version of the manuscript. In particular, we showed that RPS26 and TSR2 knock down did not inhibit global translation (new Figure 2B & 4C), hence we concluded that the changes of FMRpolyG level did not arise from general translational shut down. On the other hand, RPS26, RPS25 and RPS6 depletion negatively affected cells proliferation (new Figure 2A,5D,6C), which is in line with a number of previously published researches (e.g. Cheng et al, 2019; Havkin-Solomon et al, 2023). However, the rate of proliferation abnormalities is limited. We agree that observed effects on RAN translation from mutant FMR1 mRNA may stem from the combination of altered protein synthesis, conditions of the cells but also cis-acting factors of mRNA sequence/structure. In new experiments we showed that single nucleotide substitution of ACG by other near cognate start codons change sensitivity of RAN translation to insufficiency of RPS26 (new Figure 4F). Also the inhibitory effect of antisense oligonucleotide binding to the region of 5’UTR containing ACG initiation codon (ASO_ACG) is different in cells differing in amount of RPS26 (new Figure 4G).

We also agree that our data only partially supports the role of RPS26-defficient ribosomes in RAN translation. Therefore, we have toned down our claims. Now, we state that the RPS26/25/TSR2 insufficiency or depletion affects RAN translation. We also changed the title of the manuscript to: “Insufficiency of 40S ribosomal proteins, RPS26 and RPS25, negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions” (Previously it was: “Ribosomal composition affects the noncanonical translation and toxicity of polyglycine-containing proteins in fragile X-associated conditions”.

Specific points:

(1) Analysis of the mass spec data in Supplemental Table S3 indicates that for many of the proteins that are differentially enriched in one sample, a single peptide is identified. So the difference is between 1 peptide and 0. I don't understand how one can do a statistical analysis on that, or how it would give out anything of significance. I certainly do not think it is significant. This is exacerbated by the fact that the contaminants in the assay (keratins) are many, many-fold more abundant, and so are proteins that are known to be mitochondrial or nuclear, and therefore likely not actual targets (e.g. MCCC1, PC, NPM1; this includes many proteins "of significance" in Table S1, including Rrp1B, NAF1, Top1, TCEPB, DHX16, etc...).

The data in Table S6/Figure 3A suffer from the same problem.

I am not convinced that the mass spec data is reliable.

We thank Reviewer for the comment concerning MS data; however, we believe that it may stem from misunderstanding of the data presented in Table S3 and S6. Both tables represent the output from MaxQuant analysis (so-called ProteinGroup) of MS .raw files, without any filtering. As stated in the Material&Methods, we applied default parameters suggested by MaxQuant developers to analyze MS data, these include identification of proteins based on at least 1 unique peptide, and thus some of the proteins with only 1 unique peptide are shown in Tables S1 and S3. Reviewer is also right that in this output table common contaminants, such as keratins are included. However, these identifications are denoted as “CON_”, and are further filtered out during statistical analysis in Perseus software. During the statistical analysis we first filtered out irrelevant protein groups identifications, such as contaminants, or only identified by site modifications.

We have changed the names of Supplementary Table files, giving more detailed description. We hope this will help to avoid misunderstanding for broader public. Secondly, when comparing the data presented in Table S3 and volcano plot presented in Figure 1B, one can notice that indeed the majority of identified proteins are not statistically significant (grey points), thus not selected for further stratification. Lack of significance of these proteins may be partially due to poor MS identification, however, they are not included in the following parts of the manuscript. Further, we selected only eight proteins (out of over 150) for stratification by orthogonal techniques, thus we argue that this step validates the biological relevance of chosen candidate RAN-translation modifiers. One should also keep in mind that pull down samples analyzed by MS often yield lower intensity and identification rates, when comparing to whole cell analysis, as a result of lower protein input or stringent washes used during sample preparation.

Regarding the data presented in Table S6 (SILAC data), we argue that these data are of very good quality. More than 2,000 proteins were identified in a 125min gradient, with over 80% of proteins that were identified with at least 2 unique peptides. Each of three biological replicates was analyzed three times (technical replicates), giving total of 9 high resolution MS runs. Together, we strongly believe that this data is of high confidence.

(2) The mass-spec data however claims to identify Rps26 as a factor binding the toxic RNA specifically. The rest of the paper seeks to develop a story of how Rps26-deficient ribosomes play a role in the translation of this RNA. I do not consider that this makes sense.

Indeed, we identified RPS26 as a protein that co-precipitated with FMR1 containing expanded CGG repeats (Supplementary Figure 1G) and found that depletion of RPS26 hindered RAN translation of FMRpolyG, suggesting that RPS26 positively affects RAN translation. However, we did not state that RPS26 directly interacts with toxic RNA. In order to confirm the specificity of RAN translation regulation by RPS26 insufficiency, we tested whether depletion of other 40S ribosomal protein, RPS6, affects FMRpolyG synthesis. Our experiments showed that there was no any significant effect on RAN translation efficiency post RPS6 silencing (new Figure 5C). Importantly, we showed that RPS26 depletion did not inhibit global translation (new Figure 2B). In addition, mutagenesis of near-cognate start codon (new Figure 4F) and ASO_ACG treatment (new Figure 4G) provided the evidences that modulation of FMRpolyG biosynthesis by RPS26 level may depend on start codon selection. In essence, our data suggest that RPS26 depletion specifically affects synthesis of FMRpolyG, but not FMRP derived from the same FMR1 mRNA with CGGexp. However, we do not claim that the observed effect is the consequence of a direct interaction between RPS26 and 5’UTR of FMR1 mRNA. Downregulation of FMRpolyG biosynthesis could be an outcome of the alteration of ribosomal assembly, decrease of efficiency and fidelity of PIC scanning/initiation or impeded elongation or a combination of all these processes. In the manuscript we presented the results of experiments which tested many of these possibilities.

(3) Rps26 is an essential gene, I am sure the same is true for DHX15. What happens to cell viability? Protein synthesis? The yeast experiments were carefully carried out under experiments where Rps26 was reduced, not fully depleted to give small growth defects.

We agree with the Reviewer that RPS26 and DHX15 are essential proteins, similarly to all RNA binding proteins, and caution should be taken during experimental design. To address this, we titrated different concentrations of siRPS26, and found that administration of 5 nM siRPS26, which just partially silenced RPS26, decreased FMRpolyG by around 50% (new Figure 1D). This impact was even greater with 15 nM siRPS26, as we observed around 80% decrease of FMRpolyG.

Havkin-Solomon et al. (2023), showed that proliferation rate is decreased in cells with mutated C-terminus of RPS26, which is required for contacting mRNA. In accordance with this study, we showed that cells with knocked down RPS26 proliferate less efficiently (new Figure 2A), but depletion of RPS26 did not impact the global translation (new Figure 2B). In addition, our SILAC-MS data indicates that ~80% of proteins with determined expression level were not affected by RPS26 insufficiency, and ~20% of the proteins turned out to be sensitive to RPS26 decrease. Although, these data do not take into account the protein stability.

(4) Knockdown efficiency for all tested genes must be shown to evaluate knockdown efficiency.

The current version of the manuscript contains representative western blots with validation of knock-down efficiency (for example in Figure 3B, C, E, Figure 6A) and we included knock-down validations where applicable (Figures 1D, 2B, 4G and 5C).

(5) The data in Figure 1E have just one mock control, but two cell types (control si and Rps26 depletion).

Mock control corresponds to the cells treated with lipofectamine reagent and was included in the study to determine the “background” signal from cells treated with delivery agent and reagents used to measure the apoptosis process. These cells were neither expressing FMRpolyG, nor siRNAs. Luminescence signals were normalized to the values obtained from mock control. We added more details describing this assay in the Figure 1 legend.

(6) The authors' data indicate that the effects are not specific to Rps26 but indeed also observed upon Rps25 knockdown. This suggests strongly that the effects are from reduced ribosome content or blocked protein synthesis. Additional controls should deplete a core RP to ascertain this conclusion.

We agree that observed effects may stem from reduced ribosome content, however, we argue that this is the only possibility and explanation. Previously, it was shown that RPS25 regulates G4C2-related RAN translation, but knock out of RPS25 does not affect global translation (Yamada S, 2019, Nat. Neuroscience). Similarly, we showed that KD of RPS26 or TSR2 did not reduce significantly global translation rate (SUnSET assay; new Figure 2B and 5C, respectively).

Moreover, in a new version of manuscript we included a control experiment, where we silenced core ribosomal protein (RPS6) and found that RPS6 depletion did not affect RAN translation from mutant FMR1 mRNA (new Figure 5C), thus strengthening our conclusion about specific RAN translation regulation by the level of RPS26 and RPS25.

Finally, our observation aligns well with current knowledge about how deficiency of different ribosomal proteins alters translation of some classes of mRNAs (Luan Y, 2022, Nucleic Acids Res; Cheng Z, 2019, Mol Cell). It was shown that depletion of RPS26 affects translation rate of different mRNAs compared to depletion of other proteins of small ribosomal subunit.

(7) Supplemental Figure S3 demonstrates that the depletion of S26 does not affect the selection of the start codon context. Any other claim must be deleted. All the 5'-UTR logos are essentially identical, indicating that "picking" happens by abundance (background).

Supplementary Figure 3D represents results indicating that the mutation in -4 position (from G to A) did not affect the RAN translation regardless of RPS26 presence or depletion. However, this result does not imply that RPS26 does not affect the selection of start codon of sequence- or RNA structure-context. We verified this particular -4 position, as it was suggested previously as important RPS26-sensitive site in yeasts (Ferretti M, 2017, Nat Struct Mol Biol). We agree with Reviewer that all 5’UTR logos presented in our paper did not show statistical significance for neither tested position for human mRNAs. On the contrary, we observed that regulation sensitive to RPS26 level depends on the selection of start codon of RAN translation, in particular ACG initiation (new Figure 4F&G). RPS26 depletion affected ACG-initiated but not GTG- or CTG-initiated RAN translation.

In the previous version of the manuscript, we wrote that we did not identify any specific motifs or enrichment within analyzed transcripts in comparison to the background. On the other hand, we found that the GC-content among analyzed transcripts is higher within 5’UTRs and in close proximity to ATG in coding sequences (Figure 4D), what suggests the importance of RNA stable structures in this region. In addition, we showed that mRNAs encoding proteins responding to RPS26 depletion have shorter than average 5’UTRs (new Figure 4E).

(8) Mechanism is lacking entirely. There are many ways in which ribosomes could have mRNA-specific effects. The authors tried to find an effect from the Kozak sequence, unsuccessfully (however, they also did not do the experiment correctly, as they failed to recognize that the Kozak sequence differs between yeast, where it is A-rich, and mammalian cells, where it is GGCGCC). Collisions could be another mechanism.

Indeed, collisions as well as other mechanisms such as skewed start codon fidelity may have an effect on efficiency of FMRpolyG biosynthesis. In the current version of the manuscript, we show that RPS26 amount-sensitive regulation seems to be start codonselection dependent (new Figure 4F&G).

Reviewer #2 (Public Review):

Summary:

Translation of CGG repeats leads to the accumulation of poly G, which is associated with neurological disorders. This is a valuable paper in which the authors sought out proteins that modulate RAN translation. They determined which proteins in Hela cells bound to CGG repeats and affected levels of polyG encoded in the 5'UTR of the FMR1 mRNA. They then showed that siRNA depletion of ribosomal protein RPS26 results in less production of FMR1polyG than in control. There are data supporting the claim that RPS26 depletion modulates RAN translation in this RNA, although for some results, the Western results are not strong. The data to support increased aggregation by polyG expression upon S26 KD are incomplete.

We thank the Reviewer for critical comments and suggestions. We sincerely appreciate your rigorous, insightful, and constructive feedback throughout the revision process.

Below each specific point, we addressed the mentioned issues.

Strengths:

The authors have proteomics data that show the enrichment of a set of proteins on FMR1 RNA but not a related RNA.

We thank Reviewer for appreciation of provided MS-screening results, which identified proteins enriched on FMR1 RNA with expanded CGG repeats.

Weaknesses:

- It is insinuated that RPS26 binds the RNA to enhance CGG-containing protein expression. However, RPS26 reduction was also shown previously to affect ribosome levels, and reduced ribosome levels can result in ribosomes translating very different RNA pools.

In previous version of the manuscript we did not state that RPS26 binds directly to RNA with expanded CGG repeats and we did not show the experiment indicating direct interaction between studied RNA and RPS26. What we showed is that RPS26 was enriched on FMR1 RNA MS samples, however, we did not verify whether it is direct or indirect interaction. We also tried to test hypothesis that lack of RPS26 in PIC complex may affect efficiency of RAN translation initiation via specific, previously described in yeast Kozak context (Ferretti M, 2017, Nat Struct Mol Biol). As we described this hypothesis was negatively validated. However, we showed that other features of 5’UTR sequences (e.g. higher GC-content or shorter leader sequence) are potentially important for translation efficiency in cells with depleted RPS26.

Indeed, RPS26 is involved in 40S maturation steps (Plassart L, 2021, eLife) and its insufficiency or mutations or blocking its inclusion to 40S ribosome may result in incomplete 40S maturation, which subsequently might negatively affect translation per se. However, we did not observe global translation inhibition after RPS26 depletion or depletion of TSR2, the chaperon involved in incorporation/exchange RPS26 to small ribosomal subunit (new Figure 2B and 5C). In addition, our SILAC-MS data indicates that majority of studied proteins (including FMRP, the main product of FMR1 gene) were not affected by RPS26 depletion which can be carefully extrapolated to global translation. In revised manuscript we also showed that relatively low silencing of RPS26 also decreased FMRpolyG production in model cells (new Figure 1D).

We agree that reduced ribosome levels can result in different efficiency of translation of different RNA pools. We enhance this statement in revised manuscript. However, we also showed that the same mRNA containing different near cognate start codons (single/two nucleotide substitution) specific to RAN translation, or targeting this codon with antisense oligonucleotides resulted in altered sensitivity of FMR1 mRNA translation to RPS26 depletion (new Figure 4F).

- A significant claim is that RPS26 KD alleviates the effects of FMRpolyG expression, but those data aren't presented well.

We thank the Reviewer for this comment. In the new version of the manuscript, we have added new microscopic images and improved the explanation of Figure 1E. We have also completed the interpretation of Figure 1F in the main text, figure image as well as figure legend, and we hope that these changes will ameliorate understanding of our data.

Recommendations For The Authors:

- A significant claim is that RPS26 KD alleviates the effects of FMR polyG expression, but those data aren't presented well:

Figure 1D (supporting data in S2) and 2D - the authors need to show representative images of a control that has aggregation and indicate aggregates being counted on an image. The legend states that there are no aggregates, but the quantification of aggregates/nucleus is ~1, suggesting there are at least 1 per cell. It is preferred to show at least a representative of what is quantified in the main figure instead of a bar graph.

The representative images of control and siRPS26-treated cells are now shown in revised version of Figure 1E. Additionally, we completed the Figure legend concerning this part, as well as extended description of the experiment in Materials&Methods section.

Figure 1E - it is unclear what luminescence signal is being measured. Is this a dye for an apoptotic marker? More information is needed in the legend.

This information was added to the legend of modified Figure 1F (previously 1E) as suggested.

- Some of the Western blots are not very convincing. Better evidence for the changes in bar graphs would improve how convincing the data are:

Fig 2B. The western for FMR95G in the first model is not very convincing. The difference by eye for the second siRNA seems to give a larger effect than the first for 95G construct but they appear almost the same on the graph. More supporting information for the quantification is needed.

We provided better explanation for WB quantification in M&M section in the manuscript. Alos, we provided additional blot demonstrating independent biological replicate of the mentioned experiment in supplementary materials (Supplementary Figure S2E).

Figure 4A, the blots for RPS26 and FMR95G are not convincing. They are quite smeary compared to all of the others shown for these proteins in other figures. Could a different replicate be shown?

We provided additional blot demonstrating the effect on transiently expressed FMRpolyG affected by depletion of TSR2 in COS7 cell line (Supplementary Figure S4A).

Figure 5A and 5B blots are not ideal. Could a different replicate be shown? Or show multiple replicates in the supplemental figure?

We provided additional blots from the same experiment, although data is not statistically significant, most likely due to low quality of normalization factor, which is Vinculin (Supplementary Figure S5A). Nevertheless, the level of FMRpolyG is decreased by ~70% after RPS25 silencing in SH-SY5Y cells.

Figure 2C. Please use the same y axes for all four Westerns in B and C. One would like to compare 95 and 15 repeats, but it is difficult when the y axes are different.

Thank you for this comment. The y axis was adjusted as suggested by the Reviewer.

Figure 3D-The text suggests a significant difference between positive and negative responders that is not clear in the figure.

In the main body of the manuscript we state that: “We did not observe any significant differences in the frequency of individual nucleotide positions in the 20-nucleotide vicinity of the start codon relative to the expected distribution in the BG”, which is in line with the graph showed in Figure 4D (previously 3D).

Reviewer #3 (Public Review):

Tutak et al provide interesting data showing that RPS26 and relevant proteins such as TSR2 and RPS25 affect RAN translation from CGG repeat RNA in fragile X-associated conditions. They identified RPS26 as a potential regulator of RAN translation by RNAtagging system and mass spectrometry-based screening for proteins binding to CGG repeat RNA and confirmed its regulatory effects on RAN translation by siRNA-based knockdown experiments in multiple cellular disease models and patient-derived fibroblasts. Quantitative mass spectrometry analysis found that the expressions of some ribosomal proteins are sensitive to RPS26 depletion while approximately 80% of proteins including FMRP were not influenced. Since the roles of ribosomal proteins in RAN translation regulation have not been fully examined, this study provides novel insights into this research field. However, some data presented in this manuscript are limited and preliminary, and their conclusions are not fully supported.

(1) While the authors emphasized the importance of ribosomal composition for RAN translation regulation in the title and the article body, the association between RAN translation and ribosomal composition is apparently not evaluated in this work. They found that specific ribosomal proteins (RPS26 and RPS25) can have regulatory effects on RAN translation (Figures 1C, 2B, 2C, 2E, 4A, 5A, and 5B), and that the expression levels of some ribosomal proteins can be changed by RPS26 knockdown (Figure 3B, however, the change of the ribosome compositions involved in the actual translation has not been elucidated). Therefore, their conclusive statement, that is, "ribosome composition affects RAN translation" is not fully supported by the presented data and is misleading.

We thank the Reviewer for critical comments and suggestions. We agree that the initial title and some statements in the text were misleading and the presented data did not fully support the aforementioned statement regarding ribosomal composition affecting FMRpolyG synthesis. Therefore, in the revised version of the manuscript we included a control experiment indicating that depletion of another core 40S ribosomal protein (RPS6) did not impact the FMRpolyG synthesis (new Figure 5C), which supports our hypothesis that RPS26 and RPS25 are specific CGG-related RAN translation modifiers. To precisely deliver a main message of our work, we changed the title that will indicate the specific effect of RPS26 and RPS25 insufficiency on RAN translation of FMRpolyG. Proposed title: “Insufficiency of 40S ribosomal proteins, RPS26 and RPS25 negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions”. We also changed all statements regarding “ribosomal composition” in main text of the new version of manuscript.

(2) The study provides insufficient data on the mechanisms of how RPS26 regulates RAN translation. Although authors speculate that RPS26 may affect initiation codon fidelity and regulate RAN translation in a CGG repeat sequence-independent manner (Page 9 and Page 11), what they really have shown is just identification of this protein by the screening for proteins binding to CGG repeat RNA (Figure 1A, 1B), and effects of this protein on CGG repeat-RAN translation. It is essential to clarify whether the regulatory effect of RPS26 on RAN translation is dependent on CGG repeat sequence or near-cognate initiation codons like ACG and GUG in the 5' upstream sequence of the repeat. It would be better to validate the effects of RPS26 on translation from control constructs, such as one composed of the 5' upstream sequence of FMR1 with no CGG repeat, and one with an ATG substitution in the 5' upstream sequence of FMR1 instead of near-cognate initiation codons.

We agree that the data presented in the manuscript implies that insufficiency of RPS26 plays a pivotal role in the regulation of CGG-related RAN translation and in the revised version of the manuscript we included a series of experiments indicating that ACG codon selection seems to be an important part of RPS26 level-dependent regulation of polyglycine production (new Figure 4F&G; see point 3 below for more details). Importantly, in the luciferase assay showed on Figure 4F we used the AUG-initiated firefly luciferase reporter as normalization control.

Moreover, to verify if FMRpolyG response to RPS26 deficiency depends on the type of reporter used, we repeated many experiments using FMRpolyG fused with different tags. The luciferase-based assays were in line with experiments conducted on constructs with GFP tag (new Figure 1D), thus strengthening our previous data. Moreover, in the series of experiments, we show that FMRP synthesis which is initiated from ATG codon located in FMR1 exon 1, was not affected by RPS26 depletion (Figure 3E & 4C), even though its translation occurs on the same mRNA as FMRpolyG. This indicates a specific RPS26 regulation of polyglycine frame initiated from ACG near cognate codon.

(3) The regulatory effects of RPS26 and other molecules on RAN translation have all been investigated as effects on the expression levels of FMRpolyG-GFP proteins in cellular models expressing CGG repeat sequences Figures 1C, 2B, 2C, 2E, 4A, 5A, and 5B). In these cellular experiments, there are multiple confounding factors affecting the expression levels of FMRpolyG-GFP proteins other than RAN translation, including template RNA expression, template RNA distribution, and FMRpolyG-GFP protein degradation. Although authors evaluated the effect on the expression levels of template CGG repeat RNA, it would be better to confirm the effect of these regulators on RAN translation by other experiments such as in vitro translation assay that can directly evaluate RAN translation.

We agree that there are multiple factors affecting final levels of FMRpolyG-GFP proteins including aforementioned processes. We evaluated the level of FMR1 mRNA, which turned out not to be decreased upon RPS26 depletion (Figure 3B&C), therefore, we assumed that what we observed, was the regulation on translation level, especially that RPS26 is a ribosomal protein contacting mRNA in E-site. We believe that direct assays such as in vitro translation may be beneficial, however, depletion of RPS26 from cellular lysate provided by the vendor seems technically challenging, if not completely impossible. Instead, we focused on sequence/structure specific regulation of RAN translation with the emphasis on start-codon initiation selection. It resulted in generating the valuable results pointing out the RPS26 role in start codon fidelity (Figure 4F&G). These new results showed that translation from mRNAs differing just in single or two nucleotide substitution in near cognate start codon (ACG to GUG or ACG to CUG), although results in exactly the same protein, is differently sensitive to RPS26 silencing (new Figure 4F). Similar differences were observed for translation efficiency from the same mRNA targeted or not with antisense oligonucleotide complementary to the region of RAN translation initiation codon (new Figure 4G). These results also suggest that stability of FMRpolyG is not affected in cells with decreased level of RPS26.

(4) While the authors state that RPS26 modulated the FMRpolyG-mediated toxicity, they presented limited data on apoptotic markers, not cellular viability (Figure 1E), not fully supporting this conclusion. Since previous work showed that FMRpolyG protein reduces cellular viability (Hoem G, 2019,Front Genet), additional evaluations for cellular viability would strengthen this conclusion.

We thank the Reviewer for this suggestion. We addressed the apoptotic process in order to determine the effect of RPS26 depletion on RAN translation related toxicity (Figure 1F). In revised version of the manuscript, we also added the evaluation on how cells proliferation was affected by RPS26, RPS25, RPS6 and TSR2 depletion. Our data indicate that TSR2 silencing slightly impacted the cellular fitness (new Figure 5D), whereas insufficiencies of RPS26, RPS25 and RPS6 had a much stronger negative effect on proliferation (new Figure 2A, 5D, 6C), which is in line with previous data (Cheng Z 2019, Mol Cell; Luan Y, 2022, Nucleic Acids Res). The difference in proliferation rate after treatment with siRPS26 makes proper interpretation of cellular viability assessment very difficult.

Recommendations For The Authors:

(1) It would be nice to validate the effects of overexpression of RPS26 and other regulators on RAN translation, not limited to knockdown experiments, to support the conclusion.

We did not performed such experiments because we believed that RPS26 overexpression may have no or marginal effect on translation or RAN translation. It is likely impossible to efficiently incorporate overexpressed RPS26 into 40S subunits, because the concentration of all ribosomal proteins in the cells is very high.

(2) It would be better to explain how authors selected 8 proteins for siRNA-based validation (Figure 1C, 1D, S1D) from 32 proteins enriched in CGG repeat RNA in the first screening.

We selected those candidates based on their functions connected to translation, structured RNA unwinding or mRNA processing. For example, we tested few RNA helicases because of their known function in RAN translation regulation described by other researchers. This explanation was added to the revised version of the manuscript.

(3) Original image data showing nuclear FMRpolyG-GFP aggregates should be presented in Figure 1D.

The representative images of control and siRPS26-treated cells are now shown in modified version of Figure 1E and described with more details in the legend.

(4) Image data in Figure 2A and 2D have poor signal/noise ratio and the resolution should be improved. In addition, aggregates should be clearly indicated in Figure 2D in an appropriate manner.

The stable S-FMR95xG cellular model is characterized by very low expression of RANtranslated FMR95xG, therefore, it is challenging to obtain microscopic images of better quality with higher GFP signal. In the L-99xCGG model expression of transgene is higher. Therefore, we provided new image in the new version of Figure 3D (former 2D). Moreover, we showed aggregates on the image obtained using confocal microscopy (new Supplementary Figure 2D).

(5) The detailed information on patient-derived fibroblast (age and sex of the patient, the number of CGG repeats, etc.) in Figure 2F needed to be presented.

This information was added to the figure legend (Figure 3F; previously 2F) and in the Material and Methods section as suggested.

(6) It would be better to normalize RNA expression levels of FMR1 and FMR1-GFP by the housekeeping gene in Figure S2C, like other RT-qPCR experimental data such as Figure 2B.

Normalization of FMR1-GFP to GAPDH is now shown in modified version of Figure S2C (right graph) as requested by the Reviewer.

(7) It would be better to add information on molecular weight on all Western blotting data.

(8) Marks corresponding to molecular weight ladder were added to all images.

Full blots, including protein ladders were deposited in Zenodo repository, under doi: 10.5281/zenodo.13860370

References

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Plassart L, Shayan R, Montellese C, Rinaldi D, Larburu N, Pichereaux C, Froment C, Lebaron S, O’donohue MF, Kutay U, et al (2021) The final step of 40s ribosomal subunit maturation is controlled by a dual key lock. Elife 10

En ese sentido son interesantes las prácticas de investigación y publicación reproducibles popularizadas desde el Norte Global, pero practicadas acá incluso antes (de su popularización norteña). También sistemas como Smalltalk en los 70's que en la figura de la imagen incluían todo lo necesario para que un sistema se ejecutara (ventaja que he experimentado en primera persona al poder acceder aún hoy a mis simulaciones de la tesis de maestría presentada en 2007, dejadas en el mismo estado en que las dejé durante la sustentación) y que inspiraron luego sistemas como Docker (aunque como es habitual con toda la complejidad incidental que no incluye Smalltalk). Hoy en día sistemas reproducibles y declarativos como Nix parecen brindar una garantía respecto a la reproducibilidad y acceso futuro de creaciones digitales.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and proinvasive phenotype.

In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with highgrade DCIS cells in patient tissue samples.

Specifically in invasive MCF10DCIS.com cells, they showed that overcrowding or overconfluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting to the fact that there are a high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PMlocalized TRPV4 channels.

In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.

Strengths:

The study is elegantly designed and the findings are novel. Their findings on this mechanotransduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility, and invasiveness will have a great impact in the cancer field and are potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. The authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, and pharmacological means, and showed a good correlation between different phenomena.

Weaknesses:

A major emphasis in the study is on pharmacological means to relate TRPV4 channel function to the phenotype. I believe the use of genetic means would greatly enhance the impact and provide compelling proof for the involvement of TRPV4 channels in the associated phenotype.

In this regard, I wonder if siRNA-mediated knockdown of TRPV4 in over-confluent cells (or knockout) would lead to an increase in cell volume and normalize the intracellular calcium levels back to normal, thus ultimately leading to a decrease in cell invasiveness.

We greatly appreciate the positive feedback regarding the design of our study and the novelty of our findings. We also acknowledge the valuable suggestion to complement our pharmacological approaches with genetic manipulation of TRPV4.

In response to the comment regarding siRNA-mediated knockdown or knockout of TRPV4, we fully agree that this would further substantiate our findings. In the revised manuscript, we implemented shRNA targeting TRPV4 to investigate its functional effects on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes, assessed through singlecell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript, and detailed descriptions of these findings are included below.

Using the shRNA approach that resulted in ~50% reduction of TRPV4 expression

(Supplementary Figure 6A and 6B show TRPV4 expression levels via IF and immunoblots, respectively), we examined the effect of reduced TRPV4 on intracellular calcium levels in MCF10DCIS.com cells under normal density (ND) and stress conditions (confluent; Con and hyperosmotic; PEG) using Fluo-4 AM imaging (Fig. 4S-X). We found that shRNA TRPV4 slightly decreased calcium levels in ND cells, likely due to fewer active calcium channels at the plasma membrane resulting from lower TRPV4 expression (as shown in the summary plot in Fig. 4W). With fewer active calcium channels, cells treated with shRNA TRPV4 exhibited less reduction in intracellular calcium levels under cell crowding conditions compared to control cells. Additionally, hyperosmotic stress using PEG 300 induced smaller calcium spikes in shRNA cells compared to the significant spike observed in control cells. This reduced calcium response to Con and hyperosmotic stress in shRNA cells was reflected in the decreased cell volume reduction by PEG 300 shown in Fig. 4Y. Consequently, shRNA-mediated TRPV4 reduction impaired cell volume plasticity in MCF10DCIS.com cells and abolished the pro-invasive mechanotransduction capability involving cell volume reduction, as evidenced by no increase in cell motility (both cell diffusivity and directionality) under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive

mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.

Reviewer #2 (Public review):

Summary:

The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.

Strengths:

The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.

Weaknesses:

However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.

We are deeply grateful for the positive assessment of our study and its contribution to advancing our understanding of mechanical signaling in cancer progression. We also greatly appreciate the suggestion to incorporate genetic ablation experiments to further validate the role of TRPV4 in cell crowding responses.

As noted in our response to Reviewer #1, we employed an shRNA approach to investigate the functional effects of TRPV4 knockdown on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes. We assessed these effects using Fluo-4 AM calcium assay, single-cell volume measurements, and single-cell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript and are described in detail in our response to Reviewer #1's "weaknesses" comment.

Reducing TRPV4 expression levels by shRNA diminished mechanosensing intracellular calcium changes under cell crowding and hyperosmotic conditions using PEG 300 treatment. Furthermore, a significantly reduced cell volume plasticity was observed under hyperosmotic conditions in shRNA treated cells compared to control cells (Fig. 4S-X). This diminished mechanosensing capability abolished the pro-invasive mechanotransduction effect, as assessed by single cell motility under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.

Reviewer #1 (Recommendations for the authors):

The way the results or discussion section is written. It was a little confusing for me to relate to some phenomena. For example, it is not clear how TRPV4 inhibition (due to overcrowding) leads to a decrease in intercellular calcium levels, especially when TRPV4 channels were intercellular (not on the PM) to begin with (in normal density (ND) conditions). Along the same lines, how GSK219 causes a dip in calcium levels in ND cells when TRPV4 channels are primarily intercellular (Figure 4E). If most of the TRPV4 channels that are translocated to the PM in response to cell crowding are in an inactive state, how do they confer enhanced cell volume plasticity relative to non-invasive cell lines?

Thank you very much for raising this important point. We fully agree with your concern and have significantly revised the manuscript to clarify this aspect. Specifically, we have emphasized that a modest level of TRPV4 channels are constitutively active at the plasma membrane in normal density (ND) cells. This is now discussed in detail in the context of Fig. 4:

Page 14: “Considering these factors, we hypothesized that cell crowding might inhibit calcium-permeant ion channels that are constitutively active at the plasma membrane, including TRPV4, which would then lower intracellular calcium levels and subsequently reduce cell volume via osmotic water movement.”

Page 16-17: “… However, the temporal profile of Fluo-4 intensity in Fig. 4E, which corresponds to the time points marked in Fig. 4D (t₁: baseline and t₂: dip), clearly shows the dip at t₂, indicated by ΔCa (the vertical dashed line between the dip and baseline). This modest Fluo-4 dip at t₂ represents the inhibition of activity by GSK219 on a small population of constitutively active TRPV4 channels at the plasma membrane under ND conditions.

In Con cells, 1 nM GSK219 caused a smaller dip in Fluo-4 intensity compared to the one observed in ND cells, with no subsequent changes. This is likely due to fewer constitutively active TRPV4 at the plasma membrane in Con cells than in ND cells. …These findings suggest that a substantial portion of TRPV4 channels relocated to the plasma membrane under cell crowding was inactive, and some constitutively active TRPV4 channels already present in the membrane became inactive as a result of cell crowding.”

'Internalization' might be a better word than 'uptake' in the following line in the results section

"...activating TRPV4 under cell crowding conditions triggered channel uptake, indicating that TRPV4 trafficking depended on the channel's activation status."

Thank you very much for this suggestion. As recommended, we replaced ‘uptake’ with internalization’ on page 18: 

“However, in Con cells, where a large number of inactive TRPV4 channels are likely located at the plasma membrane, GSK101 treatment notably reduced plasma membrane-associated TRPV4 in a dose-dependent manner through internalization (Fig. 4O, 4Q), consistent with previous findings65. These data suggest that plasma membrane TRPV4 levels were largely

regulated by the channel activity status. Specifically, channel activation led to the internalization of TRPV4, while channel inhibition promoted the relocation of TRPV4 to the plasma membrane.”

1. Out of curiosity:

2. Is there any information on what the intercellular TRPV4 channels are doing in the cytosol and in the nucleus? Is there any role of intercellular calcium stores in the proposed pathway?

We greatly appreciate this insightful question. Although we were unable to find studies specifically exploring the roles of cytosolic TRPV4, a recent study (Reference 74) identified a role for nuclear TRPV4 in regulating calcium within the nucleus. We speculate that when TRPV4 activity is severely impaired, such as with additional TRPV4 inhibition under cell crowding conditions, some TRPV4 channels may be redirected to the nucleus. This redistribution could help maintain nuclear calcium homeostasis.

This discussion is included on page 18 of the manuscript:

“These findings suggest that further TRPV4 inhibition under crowding conditions triggers a distinct trafficking alteration. Recent studies have implicated nuclear TRPV4 in regulating nuclear Ca2+ homeostasis and Ca2+-regulated transcription74. In light of this study and our findings, TRPV4 may relocate to the nucleus as a compensatory mechanism to maintain nuclear calcium regulation. This relocation could reflect an adaptive response to preserve calcium-dependent transcriptional programs or other nuclear processes essential for cell survival under mechanical stress.”

One recommendation is to add some explanation or some minor details for the convenience of the reader. For example:

At normal or lower confluence, cells show an acute large dip in intercellular calcium when an inhibitor is applied implying that there are a few TRPV4 channels on the PM and they are constitutively active.

Thank you very much for highlighting this important point and for the helpful suggestion to improve clarity. We have significantly revised the text associated with Fig. 4 to ensure this point is clear. Specifically, we have added the following explanation on page 16:

"This modest Fluo-4 dip at t2 represents the inhibition of activity by GSK219 on a small population of constitutively active TRPV4 channels at the plasma membrane under ND conditions."

Reviewer #2 (Recommendations for the authors):

(1) Figure 1. The authors frequently change the medium to prevent acidification in overconfluent cultures. A cell viability assay should be performed to ensure that the over-confluent cells remain healthy and viable during the experiments. There are commercial kits that can be easily used to quantify the number of viable cells and the extent of cell toxicity. The number of viable cells would provide a more reliable basis for comparison between normal density and overconfluent conditions.

Thank you very much for raising this important point. We have consistently observed that cell crowding does not induce significant cell death in MCF10DCIS.com cells. To address your recommendation, we performed a viability assay using propidium iodide (PI) to selectively stain dead cells and WGA-488 to stain all live cells. Cell death was quantified under normal density (ND) conditions and at 1, 3, 5, 7, and 10 days post-confluence.

Our results indicate that cells remain similarly viable post-confluence, with minimal cell death

(~1.5%) compared to ND cells (~0.75%). These findings are summarized in Supplementary Figure 2, demonstrating that over-confluent cultures remain healthy and viable during the experiments.

(2) Figure 2. To determine whether the reduction in cell volume is reversible, over-confluent cells can be further diluted back to normal density. Additionally, the reversibility of TRPV4 channel trafficking to the plasma membrane should be assessed under these conditions in IF experiments and cell surface biotinylation.

Thank you for this suggestion. We reseeded the previously overcrowded (OC) cells at normal density and observed that their TRPV4 distribution predominantly returned to being intracellular, with only modest plasma membrane localization, as shown by line analysis (Supplementary Figure 10A-C, page 13). Furthermore, their invasiveness decreased to levels comparable to the original normal density (ND) cells (Supplementary Figure 3C and 3E, page 6). These results demonstrate the reversibility of TRPV4 trafficking changes and the increase in invasiveness under mechanical stress.

Page 6. "The enhanced invasiveness of MCF10DCIS.com cells under cell crowding was largely reversible. When OC cells were reseeded at normal density for invasion assays, their invasive cell fraction decreased to approximately 15%, slightly lower (p = 0.012) than the initial value of around 24% (Suppl. Fig. 3C, 3E)."

Page 13. “We investigated whether TRPV4 relocation to the plasma membrane induced by cell crowding is reversible, as suggested by its impact on invasiveness (Suppl. Fig. 3E). To test this, previously OC MCF10DCIS.com cells were reseeded under ND conditions. We then assessed TRPV4 localization via immunofluorescence (IF) imaging to determine if most channels returned to the cytoplasm and could be relocated to the plasma membrane under mechanical stress, such as hyperosmotic conditions. Consistent with their initial ND state, reseeded ND MCF10DCIS.com cells displayed intracellular TRPV4 distribution (Suppl. Fig. 10A). Upon exposure to hyperosmotic stress (74.4 mOsm/Kg PEG300), TRPV4 was again relocated to the plasma membrane (Suppl. Fig. 10B). These findings, quantified through line analysis (Suppl. Fig. 10C), demonstrate that the mechanosensing response of MCF10DCIS.com cells is reversible.”

(3) Figure 3B. A control using intracellular proteins such as GAPDH or Tubulin is missing. Including this control would help exclude the possibility of cell rupture or compromised cell membranes in crowded environments, which is very common in a cell crowding environment.

Thank you very much for pointing this out. The control lanes (GAPDH) were already included in the full gel results shown in Supplementary Figure 5. For the immunoprecipitation and immunoblotting of surface-biotinylated cell lysates, we did not expect to detect GAPDH; however, some GAPDH signals were still observed. As shown for MCF10DCIS.com cells, less GAPDH was detected under OC conditions, but the immunoprecipitated samples displayed significantly higher levels of TRPV4 on the cell surface compared to ND cells (Supplementary Figure 5A). For the whole cell lysates, TRPV4 protein levels were comparable across different cell lines based on the immunoblot results, with consistent GAPDH signals serving as a loading control (Supplementary Figure 5B).

(4) Figure 4. To convincingly demonstrate TRPV4 relocation to the plasma membrane, IF should be performed under non-permeable conditions (i.e., without detergents like saponin). This approach ensures that only plasma membrane proteins are accessible to antibodies, reducing intracellular background. The same approach should be applied to Piezo1 and TfR.

Thank you for this suggestion. We observed that under non-permeable conditions, primary antibodies could still access intracellular proteins. To address this issue, we employed extracellular-binding TRPV4 antibodies to selectively detect TRPV4 relocation to the plasma membrane under hyperosmotic conditions (74.4 mOsm/kg PEG 300) in live MCF10DCIS.com cells, as shown in Supplementary Figure 9. These results clearly demonstrate the plasma membrane relocation of TRPV4 under hyperosmotic conditions, distinguishing it from control conditions. Unfortunately, we were unable to identify high-affinity extracellular-binding antibodies for Piezo1 and TfR. Nevertheless, our findings strongly support the mechanosensing plasma membrane relocation of TRPV4.

Essential Weakness:

Throughout the study, only TRPV4 inhibitors and activators were used to show that TRPV4 relocation is associated with intracellular calcium concentration and cell size changes. It is crucial to use TRPV4 KO or KD cells to confirm that the observed effects are specific to TRPV4 and not due to off-target effects on other proteins. Additionally, fusing a plasma membrane targeting sequence to TRPV4 to make a constitutive plasma membrane-localized construct could demonstrate the opposite effect.

Thank you very much for this important comment. As noted in our response to Reviewer #1, we employed an shRNA approach to investigate the functional effects of TRPV4 knockdown on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes. We assessed these effects using Fluo-4 AM calcium assay, single-cell volume measurements, and single-cell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript and are described in detail in our response to Reviewer #1's "weaknesses" comment.

Reducing TRPV4 expression levels by shRNA diminished mechanosensing intracellular calcium changes under cell crowding and hyperosmotic conditions using PEG 300 treatment. Furthermore, a significantly reduced cell volume plasticity was observed under hyperosmotic conditions in shRNA treated cells compared to control cells (Fig. 4S-X). This diminished mechanosensing capability abolished the pro-invasive mechanotransduction effect, as assessed by single cell motility under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.

Minor Points:

The introduction section is poorly written; many results currently included in the introduction would be more appropriately placed in the discussion section. The long redundant introduction makes the article hard to read through.

Thank you very much for pointing this out. In the revised introduction, we have significantly reduced references to the results, streamlining the section to make it more concise and focused. This adjustment ensures the introduction is clearer and avoids redundancy, improving the readability of the manuscript.

interesting- so the central plaza was kind of a trade hub, something to which access could be gained. They go on to write that traders would have needed to pass through the gates to the main clan area (which would have required sponsorship, as mentioned in one of the historical sources) in order to reach the trade area, which also offered a kind of spiritual protection (but to outsiders, posed spiritual/physical risks)

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

"Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

Strengths:

The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials according to the experts' consensus-recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on the rigor, the EEG methods is beyond my expertise as a reviewer.

Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.

The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.

The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

Weaknesses:

Though the methods employed in the paper are generally strong, there are certain aspects that are not clearly described in the Materials & Methods section, such as a description of the statistical analyses used for hypothesis testing.

Thank you for pointing this out. A description of the statistical models used in the analyses of EEG biomarkers has been added to the Materials and Methods:

“First, exponent and offset values were averaged across all electrodes and analyzed using a 2x2 repeated measures ANOVA with group (dyslexic, control) as a between-subjects factor and condition (resting state, language task) as a within-subjects factor. Age was included in the analyses as a covariate due to the correlation between variables. Next, exponent and offset values were averaged across electrodes corresponding to the left (F7, FT7, FC5) and right inferior frontal gyrus (F8, FT8, FC6), and to the left (T7, TP7, TP9) and right superior temporal sulcus (T8, TP8, TP10). The electrodes were selected based on the analyses outlined by Giacometti and colleagues (2014) and Scrivener and Reader (2022). For these analyses, a 2x2x2x2 repeated measures ANOVA with age as a covariate was conducted with group (dyslexic, control) as a between-subjects factor and condition (resting state, language task), hemisphere (left, right), and region (frontal, temporal) as within-subjects factors. Results for the alpha and beta bands were calculated for the same clusters of frontal and temporal electrodes and analyzed with a similar 2x2x2x2 repeated measures ANOVA; however, for these analyses, age was not included as a covariate due to a lack of significant correlations.”

We also expanded the description of the statistical models used in the analyses of MRS biomarkers:

“To analyze the metabolite results, separate univariate ANCOVAs were conducted for Glu, GABA+, Glu/GABA+ ratio and Glu/GABA+ imbalance measures with group (control, dyslexic) as a between-subjects factor and voxel gray matter volume (GMV) as a covariate. Additionally, for the Glu analysis, age was included as a covariate due to a correlation between variables. Both frequentist and Bayesian statistics were calculated. Glu/GABA+ imbalance measure was calculated as the square root of the absolute residual value of a linear relationship between Glu and GABA+ (McKeon et al., 2024).”

With regard to metabolite quantification, it is unclear why the authors chose to analyze and report metabolite values in terms of creatine ratios rather than quantification based on a water reference given that the MRS acquisition appears to support using a water reference.

We have decided to use the ratio of Glu and GABA to total creatine (tCr), as this is still a common practice in MRS studies at 7T (e.g., Nandi et al., 2022; Smith et al., 2021). This approach normalizes the signal, reducing the impact of intensity variations across different regions and tissue compositions. Additionally, total creatine concentration is considered relatively stable across different brain regions, which is particularly important in our study, where a functional localizer was used to establish the left STS region individually. Our decision was further influenced by previous studies on dyslexia (Del Tufo et al., 2018; Pugh et al., 2014) which have reported creatine ratios and included GM volume as a covariate in their models, thus providing comparability. It is now indicated in the Results:

“For comparability with previous studies in dyslexia (Del Tufo et al., 2018; Pugh et al., 2014) we report Glu and GABA as a ratio to total creatine (tCr).”

and in the Method sections:

“Glu and GABA+ concentrations were expressed as a ratio to total-creatine (tCr; Creatine + Phosphocreatine) following previous MRS studies in dyslexia (Del Tufo et al., 2018; Pugh et al., 2014).

We did not estimate absolute concentrations using water signals as a reference, as this would require accounting for water relaxation times, which may vary across our age range. Nevertheless, our dataset has been made publicly available for future researchers to calculate and compare absolute values.

Del Tufo, S. N., Frost, S. J., Hoeft, F., Cutting, L. E., Molfese, P. J., Mason, G. F., Rothman, D. L., Fulbright, R. K., & Pugh, K. R. (2018). Neurochemistry Predicts Convergence of Written and Spoken Language: A Proton Magnetic Resonance Spectroscopy Study of Cross-Modal Language Integration. Frontiers in Psychology, 9, 1507. https://doi.org/10.3389/fpsyg.2018.01507

Nandi, T., Puonti, O., Clarke, W. T., Nettekoven, C., Barron, H. C., Kolasinski, J., Hanayik, T., Hinson, E. L., Berrington, A., Bachtiar, V., Johnstone, A., Winkler, A. M., Thielscher, A., Johansen-Berg, H., & Stagg, C. J. (2022). tDCS induced GABA change is associated with the simulated electric field in M1, an effect mediated by grey matter volume in the MRS voxel. Brain Stimulation, 15(5), 1153–1162. https://doi.org/10.1016/j.brs.2022.07.049

Pugh, K. R., Frost, S. J., Rothman, D. L., Hoeft, F., Del Tufo, S. N., Mason, G. F., Molfese, P. J., Mencl, W. E., Grigorenko, E. L., Landi, N., Preston, J. L., Jacobsen, L., Seidenberg, M. S., & Fulbright, R. K. (2014). Glutamate and choline levels predict individual differences in reading ability in emergent readers. Journal of Neuroscience, 34(11), 4082–4089. https://doi.org/10.1523/JNEUROSCI.3907-13.2014

Smith, G. S., Oeltzschner, G., Gould, N. F., Leoutsakos, J. S., Nassery, N., Joo, J. H., Kraut, M. A., Edden, R. A. E., Barker, P. B., Wijtenburg, S. A., Rowland, L. M., & Workman, C. I. (2021). Neurotransmitters and Neurometabolites in Late-Life Depression: A Preliminary Magnetic Resonance Spectroscopy Study at 7T. Journal of Affective Disorders, 279, 417–425. https://doi.org/10.1016/j.jad.2020.10.011

GABA is typically quantified using J-editing sequences as lower field strengths (~3T), and there is some evidence that the GABA signal can be reliably measured at 7T without editing, however, the authors should discuss potential limitations, such as reliability of Glu and GABA measurements with short-TE semi-laser at 7T.

In addition, MRS measurements of GABA are known to be influenced by macromolecules, and GABA is often denoted as GABA+ to indicate that other compounds contribute to the measured signal, especially at a short TE and in the absence of symmetric spectral editing.

A general discussion of the strengths and limitations of unedited Glu and GABA quantification at 7T is warranted given the interest of this work to researchers who may not be experts in MRS.

While we agree with the Reviewer that at 3T, it is recommended to use J-edited MRS to measure GABA (Mullins et al., 2014), the better spectral resolution at 7T allows for more reliable results for both metabolites using moderate echo-time, non-edited MRS (Finkelman et al., 2022). In this study, we used a short echo time (TE), which is optimal for Glu but not ideal for GABA, as it interferes with other signals. We are grateful to the Reviewer for suggesting the addition of a short paragraph to the Discussion, describing the practicalities of 3T and 7T MRS and changing the abbreviation to GABA+ to inform readers of possible macromolecule contamination:

“We chose ultra-high-field MRS to improve data quality (Özütemiz et al., 2023), as the increased sensitivity and spectral resolution at 7T allows for better separation of overlapping metabolites compared to lower field strengths. Additionally, 7T provides a higher signal-to-noise ratio (SNR), improving the reliability of metabolite measurements and enabling the detection of small changes in Glu and GABA concentrations. Despite these theoretical advantages, several practical obstacles should be considered, such as susceptibility artifacts and inhomogeneities at higher field strengths that can impact data quality. Interestingly, actual methodological comparisons (Pradhan et al., 2015; Terpstra et al., 2016) show only a slight practical advantage of 7T single-voxel MRS compared to optimized 3T acquisition. For example, fitting quality yielded reduced estimates of variance in concentration of Glu in 7T (CRLB) and slightly improved reproducibility levels for Glu and GABA (at both fields below 5%). Choosing the appropriate MRS sequence involves a trade-off between the accuracy of Glu and GABA measurements, as different sequences are recommended for each metabolite. J-edited MRS is recommended for measuring GABA, particularly with 3T scanners (Mullins et al., 2014). However, at 7T, more reliable results can be obtained using moderate echo-time, non-edited MRS (Finkelman et al., 2022). We have opted for a short-echo-time sequence, which is optimal for measuring Glu. However, this approach results in macromolecule contamination of the GABA signal (referred to as GABA+).”

Finkelman, T., Furman-Haran, E., Paz, R., & Tal, A. (2022). Quantifying the excitatory-inhibitory balance: A comparison of SemiLASER and MEGA-SemiLASER for simultaneously measuring GABA and glutamate at 7T. NeuroImage, 247, 118810. https://doi.org/10.1016/j.neuroimage.2021.118810

Mullins, P. G., McGonigle, D. J., O'Gorman, R. L., Puts, N. A., Vidyasagar, R., Evans, C. J., Cardiff Symposium on MRS of GABA, & Edden, R. A. (2014). Current practice in the use of MEGA-PRESS spectroscopy for the detection of GABA. NeuroImage, 86, 43–52. https://doi.org/10.1016/j.neuroimage.2012.12.004

Özütemiz, C., White, M., Elvendahl, W., Eryaman, Y., Marjańska, M., Metzger, G. J., Patriat, R., Kulesa, J., Harel, N., Watanabe, Y., Grant, A., Genovese, G., & Cayci, Z. (2023). Use of a Commercial 7-T MRI Scanner for Clinical Brain Imaging: Indications, Protocols, Challenges, and Solutions-A Single-Center Experience. AJR. American Journal of Roentgenology, 221(6), 788–804. https://doi.org/10.2214/AJR.23.29342

Pradhan, S., Bonekamp, S., Gillen, J. S., Rowland, L. M., Wijtenburg, S. A., Edden, R. A., & Barker, P. B. (2015). Comparison of single voxel brain MRS AT 3T and 7T using 32-channel head coils. Magnetic Resonance Imaging, 33(8), 1013–1018. https://doi.org/10.1016/j.mri.2015.06.003

Terpstra, M., Cheong, I., Lyu, T., Deelchand, D. K., Emir, U. E., Bednařík, P., Eberly, L. E., & Öz, G. (2016). Test-retest reproducibility of neurochemical profiles with short-echo, single-voxel MR spectroscopy at 3T and 7T. Magnetic Resonance in Medicine, 76(4), 1083–1091. https://doi.org/10.1002/mrm.26022

Further, the single MRS voxel location is a limitation of the study as neurochemistry can vary regionally within individuals, and the putative excitatory/inhibitory imbalance in dyslexia may appear in regions outside the left temporal cortex (e.g., network-wide or in frontal regions involved in top-down executive processes). While the functional localization of the MRS voxel is a novelty and a potential advantage, it is unclear whether voxel placement based on left-lateralized reading-related neural activity may bias the experiment to be more sensitive to small, activity-related fluctuations in neurotransmitters in the CON group vs. the DYS group who may have developed an altered, compensatory reading strategy.

We agree that including only one region of interest for the MRS measurements is a potential limitation of our study, and we have now added this information to the Discussion:

“Moreover, since the MRS data was collected only from the left STS, it is plausible that other areas might be associated with differences in Glu or GABA concentrations in dyslexia.”

However, differences in Glu and GABA concentrations in this region were directly predicted by the neural noise hypothesis of dyslexia. We acknowledge that this information was missing in the previous version of the manuscript. It is now included in the Results:

“Moreover, the neural noise hypothesis of dyslexia identifies perisylvian areas as being affected by increased glutamatergic signaling, and directly predicts associations between Glu and GABA levels in the superior temporal regions and phonological skills (Hancock et al., 2017).”

as well as in the Discussion:

“Nevertheless, the neural noise hypothesis predicted increased glutamatergic signaling in perisylvian regions, specifically in the left superior temporal cortex (Hancock et al., 2017).”

Figure 1 contains a lot of information, and it may be helpful to split it into 2 figures (EEG vs. MRS) so that the plots could be made larger and the reader could more easily digest the information.

(a) I would also recommend displaying separate metabolite fit plots for each group, since the current presentation in panel F makes it appear that the MRS data is examined by testing differences between groups across the full spectrum (where the lines diverge), which really isn't the case.

(b) The GABA peak is not visible in the spectrum, and Glutamate and GABA both have multiple peaks that should be shown on the spectrum. This may be best achieved by displaying the individual metabolite sub-spectra below the full spectrum

Thank you for these suggestions. We have split the information into two Figures following the Reviewer’s recommendations.

It is not clear why the 3T structural images were used for segmentation and calculation of tissue fraction if 7T structural images were also acquired (which would presumably have higher resolution).

Generally, T1-weighted images from the 7T scanner exhibit more artifacts than those from the 3T scanner due to higher magnetic field inhomogeneity. These artifacts are especially pronounced in regions near air-tissue interfaces, such as the temporal lobes. Therefore, we chose the 3T structural images for segmentation and tissue fraction calculations and clarified this in the Method section:

“Voxel segmentation was performed on structural images from a 3T scanner, coregistered to 7T structural images in SPM12, as the latter exhibited excessive artifacts and intensity bias in the temporal regions”.

The basis set includes a large number of metabolites (27), including many low-concentration metabolites/compounds (e.g., bHG, bHB, Citrate, Threonine, ethanol) that are typically only included in studies targeting specific metabolites in disease/pathology. Please justify the inclusion of this maximal set of metabolites in the basis set, given that the inclusion of overlapping low-concentration metabolites may influence metabolite measurements of interest (https://doi.org/10.1002/mrm.10246).

There is still no consensus in the MR community on which metabolites should be included in the model of human cerebral 1H-MR spectra. Typically, only major contributors such as NAA, Cr, Cho, Lac, mI, and possibly Glx are evaluated. Some studies also include additional metabolites like Ace, Ala, Asp, GABA, Glc, Gly, sI, NAAG, and Tau. In this study, as in a few others, further metabolites such as PCh, GPC, PCr, GSH, PE, and Thr were introduced and this approach seems suitable for high-field spectra (Hofmann et al., 2002).

Hofmann, L., Slotboom, J., Jung, B., Maloca, P., Boesch, C., & Kreis, R. (2002). Quantitative 1H-magnetic resonance spectroscopy of human brain: Influence of composition and parameterization of the basis set in linear combination model-fitting. Magnetic Resonance in Medicine, 48(3), 440–453. https://doi.org/10.1002/mrm.10246

Please provide a figure indicating the localization of the MRS voxel for a sample subject.

A figure indicating the localization of the MRS voxel for a sample subject was added to the MRS checklist.

It would be helpful to include Table S1 in the main article.

Table S1 from the Supplementary Material has now been added to the main manuscript as Table 1 in the Results section.

Please report descriptive statistics for EEG and MRS measures in Table S1.

We have added a new Table S1 in the Supplementary Material, providing descriptive statistics for EEG and MRS E/I balance measures, presented separately for the dyslexic and control groups.

I recommend avoiding using the terms "direct" and "indirect" to contrast MRS and EEG measures of E/I balance. Both of these measures are imperfect and it is misleading to say that MRS is a "direct" measure of neurotransmitters. There is also ambiguity in what is meant by "direct": in contrast to EEG, MRS does not measure neural activity and does not provide high-resolution temporal information, so in a sense, it is less direct.

Thank you for this suggestion. We have replaced the terms 'direct' and 'indirect' biomarkers with 'MRS' and 'EEG' biomarkers throughout the text.

There are many cases throughout the results in which Bayes and frequentist stats seem to contradict each other in terms of significance and what should be included in the models, especially with regard to the interaction effects (the Bayes factors appear to favor non-significant interactions). I think this is worth considering and describing to offer more clarity for the readers.

We agree that a discussion of the divergent results between Bayesian and frequentist models was missing in the previous version of the manuscript. To provide greater clarity for the readers, we have conducted follow-up Bayesian t-tests in every case where the results indicated the inclusion of non-significant interactions with the effect of group in the model. These additional analyses have been performed for the exponent, offset, as well as for beta bandwidth in the Supplementary Material. We have also added a paragraph addressing these discrepancies in the Discussion:

“Remarkably, in some models, results from Bayesian and frequentist statistics yielded divergent conclusions regarding the inclusion of non-significant effects. This was observed in more complex ANOVA models, whereas no such discrepancies appeared in t-tests or correlations. Given reports of high variability in Bayesian ANOVA estimates across repeated runs of the same analysis (Pfister, 2021), these results should be interpreted with caution. Therefore, following the recommendation to simplify complex models into Bayesian t-tests for more reliable estimates (Pfister, 2021), we conducted follow-up Bayesian t-tests in every case that favored the inclusion of non-significant interactions with the group factor. These analyses provided further evidence for the lack of differences between the dyslexic and control groups. Another source of discrepancy between the two methods may stem from the inclusion of interactions between covariates and within-subject effects in frequentist ANOVA, which were not included in Bayesian ANOVA to adhere to the recommendation for simpler Bayesian models (Pfister, 2021).”

Pfister, R. (2021). Variability of Bayes factor estimates in Bayesian analysis of variance. The Quantitative Methods for Psychology, 17(1), 40-45. doi:10.20982/tqmp.17.1.p040

It would be helpful to indicate whether participants in the DYS group had a history of reading intervention/remediation. In addition to showing that the DYS group performed lower than the CON group on reading assessments as a whole and given their age, was the performance on the reading assessments at an individual level considered for inclusion in the study? (i.e., were participants' persistent poor reading abilities confirmed with the research assessments?)

We were unable to assess individual reading skills due to the lack of standardized diagnostic norms for adult dyslexia in Poland. Therefore, participants in the dyslexic group were recruited based on a previous clinical diagnosis of dyslexia, and reading and reading-related tasks were used for group-level comparisons only. This information has been added to the Methods section:

“Since there are no standardized diagnostic norms for dyslexia in adults in Poland, individuals were assigned to the dyslexic group based on a past diagnosis of dyslexia.”

Unfortunately, we did not collect information about participants' history of reading intervention or remediation. In this context, we acknowledge that including a sample of adult participants is a potential limitation of our study, however, this was already mentioned in the Discussion.

Regarding the fMRI task, please indicate whether the participants whose threshold and/or contrast was changed for localization were from the DYS or CON group.

This information is now added to the Method section:

“For 6 participants (DYS n = 2, CON n = 4), the threshold was lowered to p < .05 uncorrected, while for another 6 participants (DYS n = 3, CON n = 3) the contrast from the auditory run was changed to auditory words versus fixation cross due to a lack of activation for other contrasts.”

Reviewer #2 (Public Review):

Summary:

This study utilized two complementary techniques (EEG and 7T MRI/MRS) to directly test a theory of dyslexia: the neural noise hypothesis. The authors report finding no evidence to support an excitatory/inhibitory balance, as quantified by beta in EEG and Glutamate/GABA ratio in MRS. This is important work and speaks to one potential mechanism by which increased neural noise may occur in dyslexia.

Strengths:

This is a well-conceived study with in-depth analyses and publicly available data for independent review. The authors provide transparency with their statistics and display the raw data points along with the averages in figures for review and interpretation. The data suggest that an E/I balance issue may not underlie deficits in dyslexia and is a meaningful and needed test of a possible mechanism for increased neural noise.

Weaknesses:

The researchers did not include a visual print task in the EEG task, which limits analysis of reading-specific regions such as the visual word form area, which is a commonly hypoactivated region in dyslexia. This region is a common one of interest in dyslexia, yet the researchers measured the I/E balance in only one region of interest, specific to the language network.

We agree with the Reviewer that including different tasks for the EEG biomarkers assessment would be valuable. However, this limitation was already addressed in the Discussion:

“Importantly, our study focused on adolescents and young adults, and the EEG recordings were conducted during rest and a spoken language task. These factors may limit the generalizability of our results. Future research should include younger populations and incorporate a broader array of tasks, such as reading and phonological processing, to provide a more comprehensive evaluation of the E/I balance hypothesis.”

Further, this work does not consider prior studies reporting neural inconsistency; a potential consequence of increased neural noise, which has been reported in several studies and linked with candidate-dyslexia gene variants (e.g., Centanni et al., 2018, 2022; Hornickel & Kraus, 2013; Neef et al., 2017). While E/I imbalance may not be a cause of increased neural noise, other potential mechanisms remain and should be discussed.

Thank you for referring us to other works reporting neural variability in dyslexia. We agree that a broader context regarding sources of reduced neural synchronization, beyond E/I imbalance, was missing in the previous version of the manuscript. We have now included these references in the Discussion:

“Furthermore, although our results do not support the idea of E/I balance alterations as a source of neural noise in dyslexia, they do not preclude other mechanisms leading to less synchronous neural firing posited by the hypothesis. In this context, there is evidence showing increased trial-to-trial inconsistency of neural responses in individuals with dyslexia (Centanni et al., 2022) or poor readers (Hornickel and Kraus, 2013) and its associations with specific dyslexia risk genes (Centanni et al., 2018; Neef et al., 2017). At the same time, the observed trial-to-trial inconsistency was either present only in a subset of participants (Centanni et al., 2018), limited to some experimental conditions (Centanni et al., 2022), or specific brain regions – e.g., brainstem in Hornickel and Kraus (2013), left auditory cortex in Centanni et al. (2018), or left supramarginal gyrus in Centanni et al. (2022).”

A better description of the exponent and offset components is needed at the beginning of the results, given that the methods are presented in detail at the end. I also do not see a clear description of these components in the methods.

A description of the aperiodic components is now included in the Results:

“In the initial step of the analysis, we analyzed the aperiodic (exponent and offset) components of the EEG spectrum. The exponent reflects the steepness of the EEG power spectrum, with a higher exponent indicating a steeper signal; while the offset represents a uniform shift in power across frequencies, with a higher offset indicating greater power across the entire EEG spectrum (Donoghue et al., 2020).”

as well as in the Materials and Methods:

“Two broadband aperiodic parameters were extracted: the exponent, which quantifies the steepness of the EEG power spectrum, and the offset, which indicates signal’s power across the entire frequency spectrum.”

Reviewer #3 (Public Review):

Summary:

This study by Glica and colleagues utilized EEG (i.e., Beta power, Gamma power, and aperiodic activity) and 7T MRS (i.e., MRS IE ratio, IE balance) to reevaluate the neural noise hypothesis in Dyslexia. Supported by Bayesian statistics, their results show solid 'no evidence' of EI balance differences between groups, challenging the neural noise hypothesis. The work will be of broad interest to neuroscientists, and educational and clinical psychologists.

Strengths:

Combining EEG and 7T MRS, this study utilized both the indirect (i.e., Beta power, Gamma power, and aperiodic activity) and direct (i.e., MRS IE ratio, IE balance) measures to reevaluate the neural noise hypothesis in Dyslexia.

Weaknesses:

The authors may need to provide more data to assess the quality of the MRS data.

We have addressed the following specific recommendations of the Reviewer providing more data about the quality of the MRS data.

The authors may need to explain how the number of subjects is determined in the MRS section.

We have clarified the MRS sample description in the Results section:

“Due to financial and logistical constraints, 59 out of the 120 recruited subjects, selected progressively as the study unfolded, were examined with MRS. Subjects were matched by age and sex between the dyslexic and control groups. Due to technical issues and to prevent delays and discomfort for the participants, we collected 54 complete sessions. Additionally, four datasets were excluded based on our quality control criteria, and three GABA+ estimates exceeded the selected CRLB threshold. Ultimately, we report 50 estimates for Glu (21 participants with dyslexia) and 47 for GABA+ and Glu/GABA+ ratios (20 participants with dyslexia).”

Is there a reason why theta and gamma peaks were not observed in the majority of participants? What are the possible reasons that likely caused the discrepancy between this study and previously reported relevant studies?

We have now added a discussion about the absence of oscillatory peaks in the theta and gamma bands to the Discussion section:

“We could not perform analyses for the gamma oscillations since in the majority of participants the gamma peak was not detected above the aperiodic component. Due to the 1/f properties of the EEG spectrum, both aperiodic and periodic components should be disentangled to analyze ‘true’ gamma oscillations; however, this approach is not typically recognized in electrophysiology research (Hudson and Jones, 2022). Indeed, previous studies that analyzed gamma activity in dyslexia (Babiloni et al., 2012; Lasnick et al., 2023; Rufener and Zaehle, 2021) did not separate the background aperiodic activity. For the same reason, we could not analyze results for the theta band, which often does not meet the criteria for an oscillatory component manifested as a peak in the power spectrum (Klimesch, 1999). Moreover, results from a study investigating developmental changes in both periodic and aperiodic components suggest that theta oscillations in older participants are mostly observed in frontal midline electrodes (Cellier et al., 2021), which were not analyzed in the current study.”

Hudson, M. R., & Jones, N. C. (2022). Deciphering the code: Identifying true gamma neural oscillations. Experimental Neurology, 357, 114205. https://doi.org/10.1016/j.expneurol.2022.114205

Klimesch, W. (1999). EEG alpha and theta oscillations reflect cognitive and memory performance: A review and analysis. Brain Research Reviews, 29(2-3), 169-195. https://doi.org/10.1016/S0165-0173(98)00056-3

Based on Figure 1F, the quality of the MRS data may be contaminated by the lipid signal, especially for the DYS group. To better evaluate the MRS data, especially the GABA measurements, the authors need to show:

(a) the placement of the MRS voxel on the anatomical images;

Averaged MRS voxel placement was already presented in Figure 1 (now Figure 2) in the manuscript. Now, we have also added exemplary single-subject images to the MRS checklist in the Supplement.

(b) Glu and GABA model functions

We have now provided more meaningful Glu and GABA indications in Figure 2.

(c) CRLB for GABA

We have added respective estimates to the Supplement:

%CRLB of Glu: mean 2.96, SD = 0.79

%CRLB of GABA: mean 10.59, SD = 2.76

%CRLB of NAA: 1.76 SD = 0.46

Further, the authors added voxel's gray matter volume as a covariate when performing separate ANCOVAs. The authors may need to use alpha correction or 1-fCSF correction to corroborate these results.

We chose to use the ratio of Glu and GABA to total creatine (tCr), as this remains a common practice in MRS studies at 7T (e.g., Nandi et al., 2022; Smith et al., 2021). This decision was also influenced by previous dyslexia studies (Del Tufo et al., 2018; Pugh et al., 2014) and is now clarified in the Results and Methods sections.

Regarding alpha correction, a recent paper (García-Pérez et al., 2023) recommends: 'In general, avoid corrections for multiple testing if statistical claims are to be made for each individual test, in the absence of an omnibus null hypothesis.' Since we report null findings, further alpha correction would not significantly impact the results.

García-Pérez, M. A. (2023). Use and misuse of corrections for multiple testing. Methods in Psychology, 8, 100120. https://doi.org/10.1016/j.metip.2023.100120

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors follow up on their published observation that providing a lower glucose parental nutrition (PN) reduces sepsis from a common pathogen [Staphylococcus epidermitis (SE)] in preterm piglets. Here they found that a higher dose of glucose could thread the needle and get the protective effects of low glucose without incurring significant hypoglycemia. They then investigate whether the change in low glucose PN impacts metabolism to confer this benefit. The finding that lower glucose reduces sepsis is important as sepsis is a major cause of morbidity and mortality in preterm infants, and adjusting PN composition is a feasible intervention.

Strengths:

(1) They address a highly significant problem of neonatal sepsis in preterm infants using a preterm piglet model.

(2) They have compelling data in this paper (and in a previous publication, ref 27) that low glucose PN confers a survival advantage. A downside of the low glucose PN is hypoglycemia which they mitigate in this paper by using a slightly high amount of glucose in the PN.

(3) The experiment where they change PN from high to low glucose after infection is very important to determine if this approach might be used clinically. Unfortunately, this did not show an ability to reduce sepsis risk with this approach. Perhaps this is due to the much lower mortality in the high glucose group (~20% vs 87% in the first figure).

(4) They produce an impressive multiomics data set from this model of preterm piglet sepsis which is likely to provide additional insights into the pathogenesis of preterm neonatal sepsis.

Weaknesses:

(1) The high glucose control gives very high blood glucose levels (Figure 1C). Is this the best control for typical PN and glucose control in preterm neonates? Is the finding that low glucose is protective or high glucose is a risk factor for sepsis?

This work is a follow-up from our previous work where we explored different PN glucose regimens. Taken together our experiments heavily imply that glucose provision is associated to severity in a seemingly linear manner. In the clinical setting, there is no fixed glucose provision, but guidelines specify ranges that are acceptable. However, these guidelines do not take possible infections into account and are designed to optimize growth outcomes. Increased provision of glucose to preterm neonates may therefore increase their infection risk, but parenteral glucose cannot be entirely avoided as it would lead to hypoglycaemia and associated brain damage. In the present paper the reduced glucose PN reflects the lowest end of the recommended PN glucose intake. More work is needed to figure out the best glucose provision to infected preterm newborns, balancing positive and negative factors.

(2) In Figure 1B, preterm piglets provided the high glucose PN have 13% survival while preterm piglets on the same nutrition in Figure 6B have ~80% survival. Were the conditions indeed the same? If so, this indicates a large amount of variation in the outcome of this model from experiment to experiment.

In the follow-up experiment outlined in Figure 6 we reduced the follow-up time to 12 hours in an effort to minimize the suffering of the animals. We did this because we could detect relevant differences in the immune response between High and low glucose infected pigs as 12 hours. If we had extended the follow-up experiment to 22 hours we would likely have seen a much increased mortality.

(3) Piglets on the low glucose PN had consistently lower density of SE (~1 log) across all time points. This may be due to changes in immune response leading to better clearance or it could be due to slower growth in a lower glucose environment.

We agree with this assessment and have adjusted our result section to reflect this.

(4) Many differences in the different omics (transcriptomics, metabolomics, proteomics) were identified in the SE-LOW vs SE-HIGH comparison. Since the bacterial load is very different between these conditions, could the changes be due to bacterial load rather than metabolic reprogramming from the low glucose PN?

We analyzed the relationship between bacterial burdens and mortality and found that it did not correlate within each of the treatment groups. We have now added this data to the results section as supplemental and report this fact in the section called “Reduced glucose supply increases hepatic OXPHOS and gluconeogenesis and attenuates inflammatory pathways”. This finding inspired us to further explore the relationship between bacterial burdens and infection responses in our model which has resulted in our recent preprint: Wu et at. Regulation of host metabolism and defense strategies to survive neonatal infection. BioRxiv 2024.02.23.581534; doi: https://doi.org/10.1101/2024.02.23.581534

Reviewer #2 (Public Review):

Summary:

The authors demonstrate that a low parenteral glucose regimen can lead to improved bacterial clearance and survival from Staph epi sepsis in newborn pigs without inducing hypoglycemia, as compared to a high glucose regimen. Using RNA-seq, metabolomic, and proteomic data, the authors conclude that this is primarily mediated by altered hepatic metabolism.

Strengths:

Well-defined controls for every time point, with multiple time points and biological replicates. The authors used different experimental strategies to arrive at the same conclusion, which lends credibility to their findings. The authors have published the negative findings associated with their study, including the inability to reverse sepsis-related mortality after switching from SE-high to SE-low at 3h or 6h and after administration of hIAIP.

Weaknesses:

(1) The authors mention, and it is well-known, that Staph epi is primarily involved in late-onset sepsis. The model of S. epi sepsis used in this study clearly replicates early-onset sepsis, but S. epi is extremely rare in this time period. How do the authors justify the clinical relevance of this model?

The distinction between early and late onset sepsis makes sense clinically because they are likely to be caused by different organisms and therefore require different empirical antibiotic regimes. Early onset sepsis is caused by organisms transferred perinatally often following chorioamnionitis or uro-gential maternal infections (Strep. agalacticae/E. coli) whereas Late onset sepsis is likely caused by organisms from indwelling catheters or mucosal surfaces, most often coagulase negative staphylococci. Timing of an infection after birth of course plays a role, but the virulence factors of the pathogen probably plays a large role in shaping the immune response. Therefore, even though the infection in our model is initiated on the first day after birth, the organism that we use, Staph epidermidids, makes it a better model for pathogenesis of late onset sepsis. However, it is also important to acknowledge that the pathophysiology of “sepsis” may be similar despite timing and pathogen and depends on the degree of immune activation and downstream effects on organs.

(2) The authors find that the neutrophil subset of the leukocyte population is diminished significantly in the SE-low and SE-high populations. However, they conclude on page 10 that "modulations of hepatic, but not circulating immune cell metabolism, by reduced glucose supply..." and this is possible because the authors have looked at the entire leukocyte transcriptome. I am curious about why the authors did not sequence the neutrophil-specific transcriptome.

We collected the whole blood transcript during the experiments, which reflect the transcription profile of all the circulating leucocytes. Since we did not do single cell RNA sequencing during the experiment there is no possibility of isolating the neutrophil transcriptome at this time. Your point however is valid and we will reconsider incorporating single cell transcriptomics in future experiments.

(3) The authors use high (30g/k/d) and low (7.2g/k/d) glucose regimens. These translate into a GIR of 21 and 5 mg/k/min respectively. A normal GIR for a preterm infant is usually 5-8, and sometimes up to 10. Do the authors have a "safe GIR" or a threshold they think we cannot cross? Maybe a point where the metabolism switch takes place? They do not comment on this, especially as GIR and glucose levels are continuous variables and not categorical.

Our reduced glucose PN was chosen as it corresponded with the low end of recommended guidelines for PN glucose intake. There likely is not a “safe GIR” as the clinical responses to glucose intake during infections do not seem binary but increase with glucose intake. It is also important to remember that the reduced glucose intervention still resulted in significant morbidity and a 25% mortality within 22 hours. There is therefore still vast room for improvement, but even though further reduction in PN glucose would probably provide further protection it would entail dangerous hypoglycaemia (as described in our previous paper). The findings in this current paper has prompted us to explore several strategies to replace parenteral glucose with alternative macronutrients. Thus, the optimal PN for infected newborns would probably differ from standard PN in all macronutrients and will require much more pre- and clinical research.

(4) In Figures 2B and C the authors show that SE-high and SE-low animals have differences in the oxphos, TCA, and glycolytic pathways. The authors themselves comment in the Supplementary Table S1B, E-F that these same metabolic pathways are also different in the Con-Low and Con-high animals, it is just the inflammatory pathways that are not different in the non-infected animals. How can they then justify that it is these metabolic pathways specifically which lead to altered inflammatory pathways, and not just the presence of infection along with some other unfound mechanism?

It is to be expected that the inflammatory pathways do not differ between the Con-Low and Con-High groups as there is no infection to induce these pathways. The identified metabolic pathways that differ between SE-High and SE-Low animals seem to us the best explanation of the differences in clinical phenotype.

(5) The authors mention in Figure 1F that SE-low animals had lower bacterial burdens than SE-high animals, but then go on to infer that the inflammatory cytokine differences are attributed to a rewiring of the immune response. However, they have not normalized the cytokine levels to the bacterial loads, as the differences in the cytokines might be attributed purely to a difference in bacterial proliferation/clearing.

Please see our response to reviewer #1

(6) The authors mention that switching from SE-high to SE-low at 3 or 6 h time points does not reduce mortality. Have the authors considered the reverse? Does hyperglycemia after euglycemia initially, worsen mortality? That would really conclude that there is some metabolic reprogramming happening at the very onset of sepsis and it is a lost battle after that.

A very good point that we have not explored yet, we have added this consideration to the discussion and slightly amended our conclusions of this follow-up experiment.

Reviewer #3 (Public Review):

Summary:

Baek and colleagues present important follow-up work on the role of serum glucose in the management of neonatal sepsis. The authors previously showed high glucose administration exacerbated neonatal sepsis, while strict glucose control improved outcomes but caused hypoglycemia. In the current report they examined the effect of a more tailored glucose management approach on outcomes and examined hepatic gene expression, plasma metabolome/proteome, blood transcriptome, as well as the the therapeutic impact of hIAIP. The authors leverage multiple powerful approaches to provide robust descriptive accounts of the physiologic changes that occur with this model of sepsis in these various conditions. Strengths:

(1) Use of preterm piglet model.

(2) Robust, multi-pronged approach to address both hepatic and systemic implications of sepsis and glucose management.

(3) Trial of therapeutic intervention - glucose management (Figure 6), hIAIP (Figure 7).

Weaknesses:

(1) The translational role of the model is in question. CONS is rarely if ever a cause of EOS in preterm neonates. The model. uses preterm pigs exposed at 2 hours of age. This model most likely replicates EOS.

Please see our response to Reviewer #2

(2) Throughout the manuscript it is difficult to tell from which animals the data are derived. Given the ~90% mortality in the experimental CONS group, and 25% mortality in the intervention group, how are the data from animals "at euthanasia" considered? Meaning - are data from survivors and those euthanized grouped together? This should be clarified as biologically these may be very different populations (ie, natural survivor vs death).

This is a very valid point. For all endpoints that are analyzed “at euthanasia” the age of the animal will vary. Some will have been euthanized early due to clinical deterioration and some will have survived all the way to the end of the experiment. This needs to be kept in mind when interpreting the results. We have further highlighted this point in the discussion and made it clear to the reader at what time-point each analysis was performed.

(3) With limited time points (at euthanasia ) for hepatic transcriptomics (Figure 2), plasma metabolite (Figure 3) blood transcriptome (Figure 4), and plasma proteome (Figure 5) it is difficult to make conclusions regarding mechanisms preceding euthanasia. Per methods, animals were euthanized with acidosis or clinical decompensation. Are the reported findings demonstrative of end-organ failure and deterioration leading to death, or reflective of events prior?

Yes, all organ specific endpoints are snapshots of the state of the animals at the time of euthanasia, pooling together animals that succumbed to sepsis and those that survived to 22 hours post infection. These results therefore reflect the end-state of the infection we cannot be sure when the differences between groups manifested themselves. However, given the stark differences in plasma lactate at 12 hours post infection it is likely that changes to metabolism occurred before most of animals succumbed to sepsis.

We agree this is a weakness in our model, but we have since published a pre-print where we have further explored how metabolic adaptations shape the fate of similarly infected preterm pigs: BioRxiv 2024.02.23.581534; doi: https://doi.org/10.1101/2024.02.23.581534

(4) Data are descriptive without corresponding "omics" from interventions (glucose management and/or hIAIP) or at least targeted assessment of key differences.

We only did in-depth analysis of the glucose intervention as this showed the most promising clinical effects that warranted further in-depth investigation. It is possible that further insights could be gained from in-depth analysis of the other interventions but given that there were no obvious clinical befits we refrained from that.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

I am intrigued that mortality was not correlated to bacterial burden. Please provide the "data not shown" as this would help the reader understand better whether the difference in bacterial burden is driving the phenotypes and findings of the low glucose group.

We have added this data to supplementary figure 1.  

Reviewer #2 (Recommendations For The Authors):

(1) I would urge the authors to consider a neutrophil-specific transcriptomic analysis. I understand that this would add significantly to the resubmission process. If the authors wish to include that as a future direction instead, they need to specifically mention the limitations of whole blood transcriptomics and how different immune cell types react differently to bacterial antigens.

We agree with your considerations but we cannot include that data using the whole blood method applied in the experiment. We have added your consideration to the discussions.

(2) I urge the authors to remove any impression that this is a model of late-onset sepsis, which is implied from the introduction, lines 3 and 4.

Our intention was not to directly suggest that our model is a perfect reflection of late-onset sepsis but rather to highlight the relevance of using a pathogen commonly associated with LOS. We believe our model primarily captures the effects of intense pro-inflammatory immune activation, which may have parallels with various forms of sepsis, including LOS.

Reviewer #3 (Recommendations For The Authors):

Drawing on the robust nature of your "omics", identify key measures and test whether they are altered earlier in the development of clinical sepsis. Test whether these are altered by the intervention.

A very valid point, at the moment it is not possible for us to explore this within the confines of these experiments. But, building upon these findings and the ones in our recent preprint we are confident that shifts in hepatic ratio of Oxidative phosphorylation and gluconeogenesis vs glycolysis shape the immune response to infections in neonates. In our upcoming experiments we are planning to incorporate plasma metabolomics at earlier timepoints to monitor when shifts in metabolism occur. However, given the heterogeneity of pigs, as opposed to inbred rodent models, sacrificing animals at fixed timepoints to gauge their organ function will be hard to interpret as it is impossible to know what the end state of the particular animal would have been. Therefore longitudinal sampling of liver tissue, during the course of infection would be challenging.

staging talk- talk regarding performance itself

banter = consistent team realignments

4 types of communication not for the front stage?

control over perceptions i control over contact made!!

takes very little to signal this meaning- susceptible to misinterpretation

explicó cómo mejorar las relaciones personales (5:23-26, 38-42; 7:1-5, 12), cómo mantener la pureza moral (5:27-32) y cómo llevar una vida con sentido (6:19-24; 7:24-27). Pero no solo dijo cómo conseguir sabiduría, sino que lo demostró dando explicaciones, razones y pruebas.

Lo que sí heredó de sus padres humanos fue el derecho a ocupar el trono de David, pues ellos eran descendientes de este rey (Hechos 13:22, 23). Cuando Jesús se bautizó, en el año 29 de nuestra era, Jehová lo ungió con espíritu santo y dijo: “Este es mi Hijo amado” (Mateo 3:16, 17). ¡Al fin había llegado la Descendencia! (Gálatas 3:16).

moleculas del sistema inmune en tortugas: Lisozymas con actividad antibacteriana y otrs moleculas son catelicidinas con actividad antifungica yactividad antibacteriana, incluso mas potente que farmacos ampicilina y bencilpenicilina. En el cocodrilo siames se aislo una pequeña proteina cationica 2008). A small cationic protein was isolated from the Siamese crocodile (Crocodylus siamensis), which demonstrated antibacterial activity against S. typhi, E. coli, S. aureus, Staphylococcus epidermidis, K. pneumoniae, P. aeruginosa and Vibrio chorelae (Preecharram et al., 2008). These antimicrobial peptides offer potent protection for reptiles against infection as well as provide exciting opportunities in the search for new clinical or agricultural antibiotics.

Cómo una señal que Jehová estaba contento con su obra. ellos estaban advertidos y recordaban las palabras de Jesús acerca de que Satanás se enojaría, y los atacaría. Juan 15:20

Los amos de la Joven perdieron una fuente de ingresos, así que tomaron venganza, y empezaron hablar mal de Pablo y Silas, y los arrastraron ante jueces que tenían prejuicios, así que mediante acusaciones falsas, los encerraron en un calabozo muy oscuro y con cepo en los pies. Hechos 16:19-24

El que esté de pie cuide de no caer. El pueblo que Dios bendijo, el pueblo escogido. - Terminó adorando a otros dioses - cometieron inmoralidad sexual que les costó la vida a Miles de ellos - pusieron a prueba dudando de Dios - y se quejaron.

Estos son ejemplos de cómo podemos perder nuestra relación con Jehová si no nos mantenemos alerta 1 Corintios 10:6-12

Esforzarnos cómo si buscáramos tesoros. Implica, - estar atento a que necesitamos su consejo - tener un corazón humilde para aceptar su guía y llegar a amarla - pedir con fuerza y determinación que necesitamos entendimiento Entonces si llegaremos a temer a Dios que es el principio de la sabiduría. Proverbios 2:1-6

Aunque lo amaba muchísimo y su poder es infinito, decidió no intervenir. ¿Por qué? Por amor. Jesús dijo: “Dios amó tanto al mundo que entregó a su Hijo unigénito para que nadie que demuestre tener fe en él sea destruido, sino que tenga vida eterna” (Juan 3:16).

Para liberarnos del pecado y la muerte, Jehová estuvo dispuesto a sufrir un dolor inimaginable permitiendo que su Hijo fuera ejecutado

porque las trataba con empatía y compasión (Luc. 15:1, 2).

Además, las recompensaba por mostrar que tenían fe en él cómo con Zaqueo (Luc. 19:1-10).

Jesús reflejó a la perfección la misericordia de su Padre (Juan 14:9).

Con sus palabras y con sus acciones, nos mostró que Jehová nos ama y es muy compasivo, y quiere ayudarnos a vencer al pecado. (Luc. 5:27, 28).

Jesús prestaba especial atención a quienes se sentían muy mal porque eran pecadores, y los invitaba a seguirlo.

Por eso tomaba la iniciativa de acercarse a hombres y mujeres que tenían la reputación de ser pecadores. De hecho, él mismo puso este ejemplo: “Los que están sanos no necesitan un médico, pero los enfermos sí”. Y añadió: “No vine a llamar a justos, sino a pecadores” (Mat. 9:12, 13).

Lo demostró con hechos, perdonado los pecados de la mujer pecadora que le lavó sus pues con sus lágrimas, o cuando le enseño importantes verdades a la samaritana. Y cuando resucitó muertos con el poder que Dios le dió (Luc. 7:37-50). (Juan 4:7, 17-19, 26). (Mat. 11:5).

“¡Miren, el Cordero de Dios que quita el pecado del mundo!” Juan 1:29

según la Ley, casi todas las cosas se purifican con sangre Hebreos 9:22

porque no es posible que la sangre de toros y de cabras elimine los Hebreos 10:1-4

Hebreos 10:12. Pero este hombre ofreció un solo sacrificio para siempre por los pecados y se sentó a la derecha de Dios,+

Génesis 3:15 Dios habló de una “descendencia” que sería muy importante porque con el tiempo aplastaría a Satanás y arreglaría todos los problemas que él causó en el jardín de Edén (1 Juan 3:8).

No siempre se refiere a lo mismo - es lo que hacemos que Dios no quiere que hagamos - es lo que no hacemos que Dios quiere que hagamos. - y es la imperfección heredado de Adán y Eva, que conduce a la muerte. (Rom. 5:12).

Aunque es un enemigo terrible : estamos totalmente convencidos de que podemos derrotar al pecado con la ayuda de Jehová.

Author response:

The following is the authors’ response to the original reviews.

The detailed, thorough critique provided by the three reviewers is very much appreciated. We believe the manuscript is greatly improved by the changes we have made based on those reviews. The major changes are described below, followed by a point by point response.

Major Changes:

(1) We revised our model (old Fig. 10; new Fig. 9) to keep the explanation focused on the data shown in the current study. Specifically, references to GTP/GDP states of Rab3A and changes in the presynaptic quantum have been removed and the mechanisms depicted are confined to pre- or post-synaptic Rab3A participating in either controlling release of a trophic factor that regulates surface GluA2 receptors (pre- or postsynaptic) or directly affecting fusion of GluA2-receptor containing vesicles (postsynaptic).

(2) We replaced all cumulative density function plots and ratio plots, based on multiple quantile samples per cell, with box plots of cell means. This affects new Figures 1, 2, 3, 5, 6, 7 and 8. All references to “scaling,” “divergent scaling,” or “uniform scaling,” have been removed. New p values for comparison of means are provided above every box plot in Figures 1, 2, 3, 5, 6, 7 and 8. The number of cultures is provided in the figure legends.

(3) We have added frequency to Figures 1, 2 and 8. Frequency values overall are more variable, and the effect of activity blockade less robust, than for mEPSC amplitudes. We have added text indicating that the increase in frequency after activity blockade was significant in neurons from cultures prepared from WT in the Rab3A+/- colony but not cultures prepared from KO mice (Results, lines 143 to 147, new Fig. 1G. H). The TTX-induced increase in frequency was significant in the NASPM experiments before NASPM, but not after NASPM (Results, lines 231 to 233, new Fig. 3, also cultures from WT in Rab3A+/- colony). The homeostatic plasticity effect on frequency did not reach significance in WT on WT glia cultures or

WT on KO glia cultures, possibly due to the variability of frequency, combined with smaller sample sizes (Results, lines 400 to 403, new Fig. 8). In the cultures prepared from WT mice in the Rab3A+/Ebd colony, there was a trend towards higher frequency after TTX that did not reach statistical significance, and in cultures prepared from mutant mice, the p value was large, suggesting disruption of the effect, which appears to be due to an increase in frequency in untreated cultures, similar to the behavior of mEPSC amplitudes in neurons from mutant mice (Results, lines 161-167). In sum, the effect of activity on frequency requires Rab3A and Ca2+-permeable receptors, and is mimicked by the presence of the Rab3A Earlybird mutant. We have also added a discussion of these results (Discussion, lines 427-435). 

(4) In the revised manuscript we have added analysis of VGLUT1 levels for the same synaptic sites that we previously analyzed GluA2 levels, and these data are described in Results, lines 344 to 371, and appear in new Table 2. In contrast to previous studies, we did not find any evidence for an increase in VGLUT1 levels after activity blockade. We reviewed those studies to determine whether there might be differences in the experimental details that could explain the lack of effect we observed. In (De Gois et al., 2005), the authors measured mRNA and performed western blots to show increases in VGLUT1 after TTX treatment in older rat cortical cultures (DIV 19). The study performs immunofluorescence imaging of VGLUT1 but only after bicuculline treatment (it decreases), not after TTX treatment. In (Wilson et al.,

2005), the hippocampal cultures are treated with AP5, not TTX, and the VGLUT1 levels in immunofluorescence images are reported relative to synapsin I. That the type of activity blockade matters is illustrated by the failure of Wilson and colleagues to observe a consistent increase in VGLUT1/Synapsin ratio in cultures treated with AMPA receptor blockade (NBQX; supplementary information). These points have been added to the Discussion, lines 436 to 447.)

Reviewer #1:

(1) (model…is not supported by the data), (2) (The analysis of mEPSC data using quantile sampling…), (3) (…statistical analysis of CDFs suffers from n-inflation…), (4) (How does recording noise and the mEPSC amplitude threshold affect “divergent scaling?”) (5) (…justification for the line fits of the ratio data…), (7) (A comparison of p-values between conditions….) and (10) (Was VGLUT intensity altered in the stainings presented in the manuscript?)

The major changes we made, described above, address Reviewer #1’s points. The remaining points are addressed below.

(6) TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data. 

The p values based on CDF comparisons were problematic, but the point we were making is that they were much larger for amplitudes measured in cultures prepared from Rab3A-/- mice (Fig. 1, p = 0.04) compared to those from cultures prepared from Rab3A+/+ mice (Fig. 1, p = 4.6 * 10-4). Now that we are comparing means, there are no significant TTX-induced effects on mEPSC amplitudes for Rab3A-/- data. However, acknowledging that some increase after activity blockade remains, we describe homeostatic plasticity as being impaired or not significant, rather than abolished, by loss of Rab3A, (Abstract, lines 37 to 39; Results, lines 141 to 143; Discussion, lines 415 to 418).

(8) There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3AEbd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+ and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?

The Reviewer is concerned that the increase in mEPSC amplitude in the presence of the Rab3A point mutant may be through a ‘non-Rab3A’ mechanism (a concern raised by the lack of such effect in cultures from the Rab3A-/- mice), and secondly, that the already large mEPSC cannot be further increased by the homeostatic plasticity mechanism. It must always be considered that a mutant with an altered genetic sequence may bind to novel partners, causing activities that would not be either facilitated or inhibited by the original molecule. We have added this caveat to Results, lines 180 to 186 We added that a number of other manipulations, implicating individual molecules in the homeostatic mechanism, have caused an increase in mEPSC amplitude at baseline, potentially nonspecifically occluding the ability of activity blockade to induce a further increase (Results lines 186 to 189). Still, it is a strong coincidence that the novel activity of the mutant Rab3A would affect mEPSC amplitude, the same characteristic that is affected by activity blockade in a Rab3A dependent manner, a point which we added to Results, lines 189 to 191.

(9) Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs -15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.

Our results, even with the modest sample size of 11 cells, are clear: NASPM does not disrupt the effect of TTX treatment on mEPSC amplitude (new Fig. 3A). It also looks like there is a greater magnitude effect of NAPSM on frequency in TTX-treated cells; we note this, but point out that nevertheless, these mEPSCs are not contributing to the increase in mEPSC amplitude (Results, lines 238-241). 

(11) The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?

We had reported that GluA2 area showed the most prominent increase following activity blockade, with intensity changing very little. When we examined the integral, it closely matched the change in area. We have added the values for integral to new Fig. 5 D, H; new Fig. 6 A-C; new Fig. 7 A-C and new Table 1 (for GluA2) and new Table 2 (for VGLUT1). These results are described in the text in the following places: Results, lines 289-292; 298-299; 311-319; 328-324). For VGLUT1, both area and intensity changed modestly, and the integral appeared to be a combination of the two, being higher in magnitude and resulting in smaller p values than either area or intensity (Results, lines 344-348; 353-359; new Table 2).

(12) The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and the smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold? What was the rationale for a threshold of 400 a.u. or 450 a.u.? How does this threshold compare to the mEPSC threshold of 3 pA.

This concern is partially addressed by no longer comparing the rank ordered mEPSC amplitudes with the rank ordered GluA2 receptor characteristics. We had used multiple thresholds in the event that an experiment was not analyzable with the chosen threshold (this in fact happened for VGLUT1, see end of this paragraph). We created box plots of the mean GluA2 receptor cluster size, intensity and integral, for experiments in which we used all three thresholds, to determine if the effect of activity blockade was different depending on which threshold was applied, and found that there was no obvious difference in the results (Author response image 1). Nevertheless, since there is no need to use a different threshold for any of the 6 experiments (3 WT and 3KO), for new Figures 5, 6 and 7 we used the same threshold for all data, 450; described in Methods, lines 746 to 749. For VGLUT1 levels, it was necessary to use a different threshold for Rab3A+/+ Culture #1 (400), but a threshold of 200 for the other five experiments (Methods, lines 751-757). The VGLUT1 immunofluorescent sites in Culture #1 had higher levels overall, and the low threshold caused the entire AOI to be counted as the synapse, which clearly included background levels outside of the synaptic site. Conversely, to use a threshold of 400 on the other experiments meant that the synaptic site found by the automated measurement tool was much smaller that what was visible by eye. In our judgement it would have been meaningless to adhere to a single threshold for VGLUT1 data.

Author response image 1.

Using different thresholds does not substantially alter GluA2 receptor cluster size data. A) Rab3A+/+ Culture #1, size data for three different thresholds, depicted above each graph. B) Rab3A+/+ Culture #2, size data for three different thresholds, depicted above each graph. Note scale bar in A is different from B, to highlight differences for different thresholds. (Culture #3 was only analyzed with 450 threshold).

The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size.

(ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 1820%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)? Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors.

Our comparison between mEPSC amplitude and GluA2 receptor cluster characteristics has been reexamined in the revised version using means rather than rank-ordered data in rank-order plots or ratio plots. Importantly, all of these methods revealed that in one out of three WT cultures (Culture #3) GluA2 receptor cluster size (old Fig. 8, old Table 1; new Fig. 6, new Table 1), intensity and integral (new Fig. 6, new Table 1) values decreased following activity blockade while in the same culture, mEPSC amplitudes increased. It is based on this lack of correspondence that we conclude that increases in mEPSC amplitude are not fully explained by increases in GluA2 receptors, and suggest there may be other contributors. These points are made in the Abstract (lines 108-110); Results (lines 319 to 326; 330337; 341-343) and the Discussion (lines 472 to 474). To our knowledge, there are not any reports that quantitatively compare receptor levels (area, intensity or integrals) to mEPSC amplitudes in the same cultures. We examined the comparisons very closely for 5 studies that used TTX to block activity and examined receptor levels using confocal imaging at identified synapses (Hou et al., 2008; Ibata et al., 2008; Jakawich et al., 2010a; Xu and Pozzo-Miller, 2017; Dubes et al., 2022). We were specifically looking for whether the receptor data were more variable than the mEPSC amplitude data, as we found. However, for 4 of the studies, sample sizes were very different so that we cannot simply compare the p values. Below is a table of the comparisons.

Author response table 1.

In Xu 2017 the sample sizes are close enough that we feel comfortable concluding that the receptor data were slightly more variable (p < 0.05) than mEPSC data (p<0.01) but recognize that it is speculative to say our finding has been confirmed. A discussion of these articles is in Discussion, lines 456-474.

(iv) It is not entirely clear if their imaging experiments will sample from all synapses. Other AMPAR subtypes than GluA2 could contribute, as could kainite or NMDA receptors.

While our imaging data only examined GluA2, we used the application of NASPM to demonstrate Ca2+permeable receptors did not contribute quantitatively to the increase in mEPSC amplitude following TTX treatment. Since GluA3 and GluA4 are also Ca2+-permeable, the findings in new Figure 3 (old Fig. 4) likely rule out these receptors as well.  There are also reports that Kainate receptors are Ca2+-permeable and blocked by NASPM (Koike et al., 1997; Sun et al., 2009), suggesting the NASPM experiment also rules out the contribution of Kainate receptors. Finally, given our recording conditions, which included normal magnesium levels in the extracellular solution as well as TTX to block action-potential evoked synaptic transmission, NMDA receptors would not be available to contribute currents to our recordings due to block by magnesium ions at resting Vm. These points have been added to the Methods section, lines 617 to 677 (NMDA); 687-694 (Ca2+-permeable AMPA receptors and Kainate receptors).

Furthermore, the statement “complete lack of correspondence of TTX/CON ratios” is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/-, the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2, particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.

We are no longer presenting ratio plots in the revised manuscript, so we do not base our conclusion that mEPSC amplitude data is not always corresponding to GluA2 receptor data on the difference in behavior of TTX/CON ratio values, but only on the difference in direction of the TTX effect in one out of three cultures. We agree with the reviewer that the ratio plots are much more sensitive to differences between control and treated values than the rank order plot, and we feel these differences are important, for example, there is still a homeostatic increase in the Rab3A-/- cultures, and the effect is still divergent rather than uniform. But the comparison of ratio data will be presented elsewhere.

(13) Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?

While this result is most obvious in CDF plots, we still observe a trend towards smaller mEPSC amplitudes after TTX treatment in two of three individual cultures prepared from Rab3A-/- mice when comparing means (new Fig. 7, Table 1) which did not reach statistical significance for the pooled data (new Fig. 5, new Table 1). There was not any evidence of this decrease in the larger data set (new Fig. 1) nor for Rab3A-/- neurons on Rab3A+/+ glia (new Fig. 8). Given that this effect is not consistent, we did not comment on it in the revised manuscript. It may be that there is a non-Rab3A-dependent mechanism that results in a decrease in mEPSC amplitude after activity blockade, which normally pulls down the magnitude of the activity-dependent increase typically observed. But studying this second component would be difficult given its magnitude and inconsistent presentation.

Reviewer #1 (Recommendations For the Authors):

(1) Abstract, last sentence: The conclusion of the present manuscript should be primarily based on the results presented. At present, it is mainly based on a previous publication by the authors.

We have revised the last sentence to reflect actual findings of the current study (Abstract, lines 47 to 49).

(2) Line 55: “neurodevelopmental”

This phrase has been removed.

(3) Line 56: “AMPAergic” should be replaced by AMPAR-mediated

This sentence was removed when all references to “scaling” were removed; no other instances of “AMPAergic” are present.

(4) Figure 9: The use of BioRender should be disclosed in the Figure Legend.

We used BioRender in new Figures 3, 7 and 8, and now acknowledge BioRender in those figure legends.

(5) Figure legends and results: The number of cultures should be indicated for each comparison.

Number of cultures has been added to the figure legends.

(6) Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.

Agreed, therefore we have removed CDFs and the KS test comparison p values. All comparisons in the revised manuscript are for cell means.

(7) Line 623ff: The argument referring to NMJ data is weak, given that different types of receptors are involved.

We still think it is valid to point out that Rab3A is required for the increase in mEPC at the NMJ but that ACh receptors do not increase (Discussion, lines 522 to 525). We are not saying that postsynaptic receptors do not contribute in cortical cultures, only that there could be another Rab3A-dependent mechanism that also affects mEPSC amplitude.

(8) Plotting data points outside of the ranges should be avoided (e.g., Fig. 2Giii, 7F).

These two figures are no longer present in the revised manuscript. In revising figures, we made sure no other plots have data points outside of the ranges.

(9) The rationale for investigating Rab3AEbd/Ebd remains elusive and should be described.

A rationale for investigating Rab3AEbd/Ebd is that if the results are similar to the KO, it strengthens the evidence for Rab3A being involved in homeostatic synaptic plasticity. In addition, since its phenotype of early awakening was stronger than that demonstrated in Rab3A KO mice (Kapfhamer et al., 2002), it was possible we would see a more robust effect. These points have been added to the Results, lines 118 to 126.

(10) Figures 3 and 4, as well as Figure 5 and 6 could be merged.

In the revised version, Figure 3 has been eliminated since its main point was a difference in scaling behavior. Figure 4 has been expanded to include a model of how NASPM could reduce frequency (new Fig. 3.) Images of the pyramidal cell body have been added to Figure 5 (new Fig. 4), and Figure 6 has been completely revised and now includes pooled data for both Rab3A+/+ and Rab3A-/- cultures, for mEPSC amplitude, GluA2 receptor cluster size, intensity and integral.

(11) Figure 5: The legend refers to MAP2, but this is not indicated in the figure.

MAP2 has now been added to the labels for each image and described in the figure legend (new Fig. 4).

Reviewer #2:

Technical concerns:

(1) The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006). It is important to ensure there is enough spiking activity before doing any activity manipulation. Similarly it is also unknown whether spiking activity is normal in Rab3AKO/Ebd neurons.

In the studies cited by the reviewer, NMDA currents were detected under experimental conditions in which magnesium was removed. In our recordings, we have normal magnesium (1.3 mM) and also TTX, which prevents the necessary depolarization to allow inward current through NMDA receptors. This point has been added to our Methods, lines 674 to 677. We acknowledge we do not know the level of spiking in cultures prepared from Rab3A+/+, Rab3A-/- or Rab3A_Ebd/Ebd_ mice. Given the similar mEPSC amplitude for untreated cultures from WT and KO studies, we think it unlikely that activity was low in the latter, but it remains a possibility for untreated cultures from Rab3A_Ebd/Ebd_ mice, where mEPSC amplitude was increased. These points are added to the Methods, lines 615 to 622.

(2) Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.

We no longer include any cumulative distributions or ratio plot analysis in the revised version. We have added the following text to Methods, lines 703 to 720:

“MiniAnalysis selects many false positives with the automated feature when a small threshold amplitude value is employed, due to random fluctuations in noise, so manual re-evaluation of the automated process is necessary to eliminate false positives. If the threshold value is set high, there are few false positives but small amplitude events that visually are clearly mEPSCs are missed, and manual re-evaluation is necessary to add back false negatives or the population ends up biased towards large mEPSC amplitudes. As soon as there is a manual step, bias is introduced. Interestingly, a manual reevaluation step was applied in a recent study that describes their process as ‘unbiased (Wu et al., 2020). In sum, we do not believe it is currently possible to perform a completely unbiased detection process. A fully manual detection process means that the same criterion (“does this look like an mEPSC?”) is applied to all events, not just the false positives, or the false negatives, which prevents the bias from being primarily at one end or the other of the range of mEPSC amplitudes. It is important to note that when performing the MiniAnalysis process, the researcher did not know whether a record was from an untreated cell or a TTX-treated cell.”

(3) Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapse on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.

We identified neurons with a pyramidal shape and a prominent primary dendrite at 60x magnification without the zoom feature. This should have been made clear in the description of imaging. We have added an image of the two selected cells to our figure of dendrites (old Fig. 5, new Fig. 4), and described this process in the Methods, lines 736 to 739, and Results, lines 246 to 253. Given the morphology of the neurons selected it is highly unlikely that the dendrites we analyzed came from interneurons.

Conceptual Concerns

The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The authors claim that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study’s contribution to the field is very limited.

We have demonstrated that loss of Rab3A and expression of a Rab3A point mutant disrupt homeostatic plasticity of mEPSC amplitudes, and that in the absence of Rab3A, the increase in GluA2 receptors at synaptic sites is abolished. Further, we show that this effect cannot be through release of a factor, like TNFα, from astrocytes. In the new version, we add the finding that VGLUT1 is not increased after activity blockade, ruling out this presynaptic factor as a contributor to homeostatic increases in mEPSC amplitude. We show for the first time by examining mEPSC amplitudes and GluA2 receptors in the same cultures that the increases in GluA2 receptors are not as consistent as the increases in mEPSC amplitude, suggesting the possibility of another contributor to homeostatic increases in mEPSC amplitude. We first proposed this idea in our previous study of Rab3A-dependent homeostatic increases in mEPC amplitudes at the mouse neuromuscular junction. In sum, we dispute that there is only one novel finding and that we have no insights into mechanism. We acknowledge that we have no direct evidence for regulation from the presynaptic side, and have removed this claim from the revised manuscript. We have retained the Discussion of potential mechanisms affecting the presynaptic quantum and evidence that Rab3A is implicated in these mechanisms (vesicle size, fusion pore kinetics; Discussion, lines 537 to 563). One way to directly show that the amount of transmitter released for an mEPSC has been modified after activity blockade is to demonstrate that a fast off-rate antagonist has become less effective at inhibiting mEPSCs (because the increased glutamate released out competes it; see (Liu et al., 1999) and (Wilson et al., 2005) for example experiments). This set of experiments is underway but will take more time than originally expected, because we are finding surprisingly large decreases in frequency, possibly the result of mEPSCs with very low glutamate concentration that are completely inhibited by the dose used. Once mEPSCs are lost, it is difficult to compare the mEPSC amplitude before and after application of the antagonist. Therefore we intend to include this experiment in a future report, once we determine the reason for the frequency reduction, or, can find a dose where this does not occur.

(1) Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019). Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.

Within the analyses in the revised manuscript, which are now based only on comparison of cell/dendrite means, we find a very good match in the magnitude of increase for the pooled data of mEPSC amplitudes and GluA2 receptor cluster sizes (+19.7% and +20.0% respectively; new Table 1). However, when looking at individual cultures, we had one of three WT cultures in which mEPSC amplitude increased 17.2% but GluA2 cluster size decreased 9.5%. This result suggests that while activity blockade does lead to an increase in GluA2 receptors after activity blockade, the effect is more variable than that for mEPSC amplitude. We went back to published studies to see if this has been previously observed, but found that it was difficult to compare because the sample sizes were different for the two characteristics (see Author response table 1). We included these particular 5 studies because they use the same treatment (TTX), examine receptors using imaging of identified synaptic sites, and record mEPSCs in their cultures (although the authors do not indicate that imaging and recordings are done simultaneously on the same cultures.) Only one of the studies listed by the Reviewer is in our group (Ibata et al., 2008). The study by (Tan et al., 2015) uses western blots to measure receptors; the study by (Silva et al., 2019) blocks activity using a combination of AMPA and NMDA receptor blockers; the study by (Pozo et al., 2012) correlates mEPSC amplitude changes with imaging but not in response to activity blockade, instead for changing the expression of GluA2. While it may seem like splitting hairs to reject studies that use other treatment protocols, there is ample evidence that the mechanisms of homeostatic plasticity depend on how activity was altered, see the following studies for several examples of this (Sutton et al., 2006; Soden and Chen, 2010; Fong et al., 2015). A discussion of the 5 articles we selected is in the revised manuscript, Discussion, lines 456 to 474. In sum, we provide evidence that activity blockade is associated with an overall increase in GluA2 receptors; what we propose is that this increase, being more variable, does not fully explain the increase in mEPSC amplitude. However, we acknowledge that the disparity could be explained by the differences in limitations of the two methods (Discussion, lines 469-472).

(2) The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010b; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.

We have added the information that Rab3A could control BDNF from the postsynaptic cell and included the two references provided by the reviewer, Discussion, lines 517 to 518. We have added new evidence, recently published, that the Rab3 family has been shown to regulate targeting of EGF receptors to rafts (among other plasma membrane molecules), with Rab3A itself clearly present in nonneuronal cells (Diaz-Rohrer et al., 2023) (added to Discussion, lines 509 to 515).

(3) The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the NASPM wash-in experiments clearly show that the GluA1 homomer contributions have not changed before and after TTX treatment.

We have revised this section in the Discussion, lines 534 to 536, to clarify that any change due to GluA1 homomers should have been detectable by a greater ability of NASPM to reverse the TTX-induced increase.

Reviewer #2 (Recommendations for the Authors):

For authors to have more convincing arguments in general, they will need to clarify/improve certain details in their data collection by addressing the above technical concerns. Additionally, the authors should design experiments to test whether Rab3A regulates scaling from pre- or post-synaptic site. For example, they could sparsely knock out Rab3A in WT neurons to test the postsynaptic possibility. On the other hand, their argument for a presynaptic role would be much more compelling if they could show whether there are clear functional changes such as in vesicle sizes and release probability in the presynaptic terminal of Rab3AKO neurons.

An important next step is to identify whether Rab3A is acting pre- or post-synaptically (Discussion, lines 572 to 573), but these experiments will be undertaken in the future. It would not add much to simply show vesicle size is altered in the KO (and we do not necessarily expect this since mEPSC amplitude is normal in the KO). It will be very difficult to establish that vesicle size is changing with activity blockade and that this change is prevented in the Rab3A KO, because we are looking for a ~25% increase in vesicle volume, which would correspond to a ~7.5% increase in diameter. Finally, we do not believe demonstrating changes in release probability tell us anything about a presynaptic role for Rab3A in regulating the size of the presynaptic quantum.

Reviewer #3 (Public Review)

Weaknesses: However, the rather strong conclusions on the dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. The key issue is the GluA2 immunostaining in comparison with the mEPSC recordings. Their imaging method involves only assessing puncta clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, judging from the sample micrographs (Fig. 5). To my knowledge, this is a new and unvalidated approach that could represent a particular subset of synapses not representative of the synapses contributing to the mEPSC change (they are also sampling different neurons for the two measurements; an additional unknown detail is how far from the cell body were the analyzed dendrites for immunostaining.) While the authors acknowledge that a sampling issue could explain the data, they still use this data to draw strong conclusions about the lack of AMPAR trafficking contribution to the mEPSC amplitude change. This apparent difference may be a methodological issue rather than a biological one, and at this point it is impossible to differentiate these. It will unfortunately be difficult to validate their approach. Perhaps if they were to drive NMDAdependent LTD or chemLTP, and show alignment of the imaging and ephys, that would help. More helpful would be recordings and imaging from the same neurons but this is challenging. Sampling from identified synapses would of course be ideal, perhaps from 2P uncaging combined with SEP-labeled AMPARs, but this is more challenging still. But without data to validate the method, it seems unwarranted to make such strong conclusions such as that AMPAR trafficking does not underlie the increase in mEPSC amplitude, given the previous data supporting such a model.

In the new version, we soften our conclusion regarding the mismatch between GluA2 receptor levels and mEPSC amplitudes, now only stating that receptors may not be the sole contributor to the TTX effect on mEPSC amplitude (Discussion, lines 472 to 474). With our analysis in the new version focusing on comparisons of cell means, the GluA2 receptor cluster size and the mEPSC amplitude data match well in magnitude for the data pooled across the 3 matched cultures (20.0% and 19.7%, respectively, see new Table 1). However, in one of the three cultures the direction of change for GluA2 receptors is opposite that of mEPSC amplitudes (Table 1, Culture #3, -9.5% vs +17.2%, respectively).

It is unlikely that the lack of matching of homeostatic plasticity in one culture, but very good matching in two other cultures, can be explained by an unvalidated focus on puncta associated with MAP2 positive dendrites. We chose to restrict analysis of synaptic GluA2 receptors to the primary dendrite in order to reduce variability, reasoning that we are always measuring synapses for an excitatory pyramidal neuron, synapses that are relatively close to the cell body, on the consistently identifiable primary dendrite. We measured how far this was for the two cells depicted in old Figure 5 (new Fig. 4). Because we always used the 5X zoom window which is a set length, and positioned it within ~10 microns of the cell body, these cells give a ball park estimate for the usual distances. For the untreated cell, the average distance from the cell body was 38.5 ± 2.8 µm; for the TTX-treated cell, it was 42.4 ± 3.2 µm (p = 0.35, KruskalWallis test). We have added these values to the Results, lines 270 to 274.

We did not mean to propose that AMPA receptor levels do not contribute at all to mEPSC amplitude, and we acknowledge there are clear cases where the two characteristics change in parallel (for example, in the study cited by Reviewer #2, (Pozo et al., 2012), increases in GluA2 receptors due to exogenous expression are closely matched by increases in mEPSC amplitudes.) What our matched culture experiments demonstrate is that in the case of TTX treatment, both GluA2 receptors and mEPSC amplitudes increase on average, but sometimes mEPSC amplitudes can increase in the absence of an increase in GluA2 receptors (Culture #3, Rab3A+/+ cultures), and sometimes mEPSC amplitudes do not increase even though GluA2 receptor levels do increase (Culture #3, Rab3A-/- cultures). Therefore, it would not add anything to our argument to examine receptors and mEPSCs in NMDA-dependent LTP, a different plasticity paradigm in which changes in receptors and mEPSCs may more closely align. It has been demonstrated that mEPSCs of widely varying amplitude can be recorded from a single synaptic site (Liu and Tsien, 1995), so we would need to measure a large sample of individual synapse recordings to detect a modest shift in average values due to activity blockade. In addition, it would be essential to express fluorescent AMPA receptors in order to correlate receptor levels in the same cells we record from (or at the same synapses). And yet, even after these heroics, one is still left with the issue that the two methods, electrophysiology and fluorescent imaging, have distinct limitations and sources of variability that may obscure any true quantitative correlation.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is quite unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. It is also unclear why the authors argue this proves that NASPM was at an effective concentration (lines 399-400). Further, the amplitude data show a strong trend towards smaller amplitude. The p value for both control and TTX neurons was 0.08 – it is very difficult to argue that there is no effect. And the decrease is larger in the TTX neurons. Considering the strong claims for a presynaptic locus and the use of this data to justify only looking at GluA2 by immunostaining, these data do not offer much support of the conclusions. Between the sampling issues and perhaps looking at the wrong GluA subunit, it seems premature to argue that trafficking is not a contributor to the mEPSC amplitude change, especially given the substantial support for that hypothesis. Further, even if trafficking is not the major contributor, there could be shifts in conductance (perhaps due to regulation of auxiliary subunits) that does not necessitate a pre-synaptic locus. While the authors are free to hypothesize such a mechanism, it would be prudent to acknowledge other options and explanations.

We have created a model cartoon to explain how NASPM could reduce mEPSC frequency (new Fig. 3D). mEPSCs that arise from a synaptic site that has only Ca2+-permeable AMPA receptors will be completely blocked by NASPM, if the NASPM concentration is maximal. The reason we conclude that we have sufficient NASPM reaching the cells is that the frequency is decreased, as expected if there are synaptic sites with only Ca2+-permeable AMPA receptors. We previously were not clear that there is an effect of NASPM on mEPSC amplitude, although it did not reach statistical significance (new Fig. 3B). Where there is no effect is on the TTX-induced increase in mEPSC amplitude, which remains after the acute NASPM application (new Fig. 3A). We have revised the description of these findings in Results, lines 220 to 241. In reviewing the literature further, we could find no previous studies demonstrating an increase in conductance in GluA2 or Ca2+-impermeable receptors, only in GluA1 homomers. In other words, any conductance change would have been due to a change in GluA1 homomers, and should have been visible as a disruption of the homeostatic plasticity by NASPM application. We have added text to Results, lines 211 to 217; 236-241; Discussion, lines 420 to 422; 526-536 and Methods, lines 685 to 695 regarding this point.

The frequency data are missing from the paper, with the exception of the NASPM dataset. The mEPSC frequencies should be reported for all experiments, particularly given that Rab3A is generally viewed as a pre-synaptic protein regulating release. Also, in the NASPM experiments, the average frequency is much higher in the TTX treated cultures. Is this statistically above control values?

This comment is addressed by the major change #3, above.

Unaddressed issues that would greatly increase the impact of the paper:

(1) Is Rab3A activity pre-synaptically, post-synaptically or both. The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where is it acting (pre or post) would aid substantially in understanding its role (and particularly the hypothesized and somewhat novel idea that the amount of glutamate released per vesicle is altered in HSP). They could use sparse knockdown of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

This is similar to the request of Reviewer #2, Recommendations to the Authors. An important next step is to identify whether Rab3A is working pre- or postsynaptically. However, it is possible that it is acting pre-synaptically to anterogradely regulate trafficking of AMPAR, as we have depicted in our model, new Fig. 9. To demonstrate that the presynaptic quantum is being altered, we would need to show that vesicle size is increased, or the amount of transmitter being released during an mEPSC is increased after activity blockade. To that end, we are currently performing experiments using a fast off-rate antagonist. As described above in response to Reviewer #2’s Conceptual Concerns, we find dramatic decreases in frequency not explained by the 30-60% inhibition observed for the largest amplitude mEPSCs, which suggests the possibility that small mEPSCs are more sensitive than large mEPSCs and therefore may have less transmitter. Due to these complexities and the delay while we test other antagonists to see if the effect is specific to fast-off rate antagonists, we are not including these results here.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs and/or a decrease of GABA-packaging in vesicles (ie the opposite of whatever is happening at excitatory synapses.). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling, an effect only at excitatory synapses would argue for a more specific role just at these synapses.

It will be important to determine if homeostatic synaptic plasticity at inhibitory synapses on excitatory neurons is sensitive to Rab3A deletion, especially in light of the fact that unlike many of the other molecules implicated in homeostatic increases in mEPSCS, Rab3A is not a molecule known to be selective for glutamate receptor trafficking (in contrast to Arc/Arg3.1 or GRIP1, for example). Such a study would warrant its own publication.

Reviewer #3 (Recommendations for the Authors):

There are a number of minor points or suggestions for the authors:

Is RIM1 part of this pathway (or expected to be)? Some discussion of this would be nice.

RIM, Rab3-interacting molecule, has been implicated at the drosophila neuromuscular junction in a presynaptic form of homeostatic synaptic plasticity in which evoked release is increased after block of postsynaptic receptors (Muller et al., 2012), a plasticity that also requires Rab3-GAP (Muller et al., 2011). To our knowledge there is no evidence that RIM is involved in the homeostatic plasticity of mEPSC amplitude after activity blockade by TTX. The Rim1a KO does not have a change in mEPSC amplitude relative to WT (Calakos et al., 2004), but that is not unexpected given the normal mEPSC amplitude in neurons from cultures prepared from Rab3A-/- mice in the current study. It would be interesting to look at homeostatic plasticity in cortical cultures prepared from Rim1a or other RIM deletion mice, but we have not added these points to the revised manuscript since there are a number of directions one could go in attempting to define the molecular pathway and we feel it is more important to discuss the potential location of action and physiological mechanisms.

Is the Earlybird mutation a GOF? More information about this mutation would help.

We have added a description of how the Earlybird mutation was identified, in a screen for rest:activity mutants (Results, lines 118 to 123). Rab3A Earlybird mice have a shortened circadian period, shifting their wake cycle earlier and earlier. When Rab3A deletion mice were tested in the same activity raster plot measurements, the shift was smaller than that for the Earlybird mutant, suggesting the possibility that it is a dominant negative mutation.

The high K used in the NASPM experiments seems a bit unusual. Have the authors done high K/no drug controls to see if this affects the synapses in any way?

We used the high K based on previous studies that indicated the blocking effect of the Ca2+-permeable receptor blockers was use dependent (Herlitze et al., 1993; Iino et al., 1996; Koike et al., 1997). We reasoned that a modest depolarization would increase the frequency of AMPA receptor mEPSCs and allow access of the NASPM.  We have added this point to the Methods, lines 695 to 708. 

The NASPM experiments do not show that GluA1 does not contribute (line 401), only that GluA1 homomers are not contributing (much – see above). GluA1/A2 heteromers are quite likely involved. Also, the SEM is missing from the WT pre/post NASPM data.

Imaging of GluA2-positive sites will not distinguish between GluA2 homomers and GluA2-GluA1 heteromers, so we have added this clarification to Results, lines 242 to 246. We have remade the NASPM pre-post line plots so that the mean values and error bars are more visible (new Fig. 3B, C).

It seems odd to speculate based on non-significant findings (line 650-1), with lower significance (p = 0.11) than findings being dismissed in the paper (NASPM on mEPSC amplitude; p = 0.08).

We did not mean to dismiss the effect of NASPM on mEPSC amplitude (new Fig. 3B), rather, we dismiss the effect of NASPM on the homeostatic increase in mEPSC amplitude caused by TTX treatment (new Fig. 3A). We have emphasized this distinction in Results, lines 223 to 225, and Discussion, lines 420 to 422, as well as adding that the stronger effect of NASPM on frequency after TTX treatment suggests an activity-dependent increase in the number of synapses expressing only Ca2+ permeable homomers (Results, lines 236 to 241; Discussion, lines 431 to 435).

Fig. 4 could be labeled better (to make it clear that B is amplitude and C is freq from the same cells).

Fig. 4 has been revised—now the amplitude and frequency plots from the same condition (new Fig. 3, B, C; CON or TTX) are in a vertical line and the figure legend states that the frequency data are from the same cells as in Fig. 3A.

The raw amplitude data seems a bit hidden in the inset panels – I would suggest these data are at least as important as the cumulative distributions in the main panel. Maybe re-organizing the figures would help.

We have removed all cumulative distributions, rank order plots, and ratio plots. The box plots are now full size in new Figures 1, 2, 5, 6, 7 and 8.

I’m not sure I would argue in the paper that 12 cells a day is a limiting issue for experiments. It doesn’t add anything and doesn’t seem like that high a barrier. It is fine to just say it is difficult and therefore there is a limited amount of data meeting the criteria.

We have removed the comment regarding difficulty.

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Los reyes que Jehová consideraba fieles lo amaban con todo su corazón. Nota La Biblia a menudo utiliza la palabra corazón para describir todo lo que una persona es por dentro, lo que incluye sus deseos, pensamientos, inclinaciones, actitudes, habilidades, motivos y metas. Ejemplos, - Jehosafat “buscó a Jehová con todo su corazón” (2 Crón. 22:9). - Josías: “Antes de él no hubo ningún rey como él, que volviera a Jehová con todo su corazón” (2 Rey. 23:25). - Salomón, “no sirvió a Jehová su Dios con un corazón completo” (1 Rey. 11:4). - Abiyam, “No sirvió a Jehová su Dios con un corazón completo” (1 EhempliRey. 15:3).

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this paper, Steinemann et al. characterized the nature of stochastic signals underlying the trial-averaged responses observed in the lateral intraparietal cortex (LIP) of non-human primates (NHPs), while these performed the widely used random dot direction discrimination task. Ramp-up dynamics in the trial averaged LIP responses were reported in numerous papers before. However, the temporal dynamics of these signals at the single-trial level have been subject to debate. Using large-scale neuronal recordings with Neuropixels in NHPs, allows the authors to settle this debate rather compellingly. They show that drift-diffusion-like computations account well for the observed dynamics in LIP.

Strengths:

This work uses innovative technical approaches (Neuropixel recordings in behaving macaque monkeys). The authors tackle a vexing question that requires measurements of simultaneous neuronal population activity and hence leverage this advanced recording technique in a convincing way

They use different population decoding strategies to help interpret the results.

They also compare how decoders relying on the data-driven approach using dimensionality reduction of the full neural population space compare to decoders relying on more traditional ways to categorize neurons that are based on hypotheses about their function. Intriguingly, although the functionally identified neurons are a modest fraction of the population, decoders that only rely on this fraction achieve comparable decoding performance to those relying on the full population. Moreover, decoding weights for the full population did not allow the authors to reliably identify the functionally identified subpopulation.

Weaknesses:

No major weaknesses beyond a few, largely clarification issues, detailed below.

We thank Reviewer 1 (R1) for this summary. The revised manuscript incorporates R1’s suggestions, as detailed below.

Reviewer #2 (Public Review):

Steinemann, Stine, and their co-authors studied the noisy accumulation of sensory evidence during perceptual decision-making using Neuropixels recordings in awake, behaving monkeys. Previous work has largely focused on describing the neural underpinnings through which sensory evidence accumulates to inform decisions, a process which on average resembles the systematic drift of a scalar decision variable toward an evidence threshold. The additional order of magnitude in recording throughput permitted by the methodology adopted in this work offers two opportunities to extend this understanding. First, larger-scale recordings allow for the study of relationships between the population activity state and behavior without averaging across trials. The authors’ observation here of covariation between the trial-to-trial fluctuations of activity and behavior (choice, reaction time) constitutes interesting new evidence for the claim that neural populations in LIP encode the behaviorally-relevant internal decision variable. Second, using Neuropixels allows the authors to sample LIP neurons with more diverse response properties (e.g. spatial RF location, motion direction selectivity), making the important question of how decision-related computations are structured in LIP amenable to study. For these reasons, the dataset collected in this study is unique and potentially quite valuable.

However, the analyses at present do not convincingly support two of the manuscript’s key claims: (1) that ”sophisticated analyses of the full neuronal state space” and ”a simple average of Tconin neurons’ yield roughly equivalent representations of the decision variable; and (2) that direction-selective units in LIP provide the samples of instantaneous evidence that these Tconin neurons integrate. Supporting claim (1) would require results from sophisticated population analyses leveraging the full neuronal state space; however, the current analyses instead focus almost exclusively on 1D projections of the data. Supporting claim (2) convincingly would require larger samples of units overlapping the motion stimulus, as well as additional control analyses.

We thank the reviewer (R2) for their careful reading of our paper and the many useful suggestions.

As detailed below, the revised manuscript incorporates new control analyses, improved quantification, and statistical rigor, which now provide compelling support for key claim #1. We do not regard claim #2 as a key claim of the paper. It is an intriguing finding with solid support, worthy of dissemination and further investigation. We have clarified the writing on this matter.

Specific shortcomings are addressed in further detail below:

(1) The key analysis-correlation between trial-by-trial activity fluctuations and behavior, presented in Figure 5 is opaque, and would be more convincing with negative controls. To strengthen the claim that the relationship between fluctuations in (a projection of) activity and fluctuations in behavior is significant/meaningful, some evidence should be brought that this relationship is specific - e.g. do all projections of activity give rise to this relationship (or not), or what level of leverage is achieved with respect to choice/RT when the trial-by-trial correspondence with activity is broken by shuffling.

We do not understand why R2 finds the analysis opaque, but we are grateful for the lucid recommendations. The relationships between fluctuations in neural activity and behavior are indeed “specific” in the sense that R2 uses this term. In addition to the shuffle control, which destroys both relationships (Reviewer Figure 1), we performed additional control analyses that preserve the correspondence of neural signals and behavior on the same trial. We generated random coding directions (CDs) by establishing weight vectors that were either chosen from a standard normal distribution or by permuting the weights assigned to PC-1 in each session. The latter is the more conservative measure. Projections of the neural responses onto these random coding directions render 𝑆rand(𝑡). Specifically, the degree of leverage is effectively zero or greatly reduced. These analyses are summarized in a new Supplementary Figure S10. The bottom row of Figure S10 also addresses the question, “What degree of leverage and mediation would be expected for a theoretical decision variable?” This is accomplished by simulating decision variables using the drift-diffusion model fits in Figure 1c. The simulation is consistent with the leverage and (incomplete) mediation observed for the populations of Tcon neurons. For details see Methods, Simulated decision variables and Leverage of single-trial activity on behavior.

(2) The choice to perform most analysis on 1D projections of population activity is not wholly appropriate for this unique type of dataset, limiting the novelty of the findings, and the interpretation of similarity between results across choices of projection appears circular:

We disagree with the characterization of our argument as circular, but R2 raises several important points that will probably occur to other careful readers. We address them as subpoints 2.1–2.4, below. Importantly, we are neither claiming nor assuming that the LIP population activity is one-dimensional. We have revised the paper to avoid giving this impression. We are also not claiming that the average of Tin neurons (or the 1D projections) explains all features of the LIP population, nor would we expect it to, given the diversity of response fields across the population. Our objective is to identify the specific dimension within population activity that captures the decision variable (DV), which has been characterized successfully as a one-dimensional stochastic process—that is, a scalar function of time. We have endeavored to clarify our thinking on this point in the revised manuscript (e.g., lines 97–98, 103–104).

(2.1) The bulk of the analyses (Figure 2, Figure 3, part of Figure 4, Figure 5, Figure 6) operate on one of several 1D projections of simultaneously recorded activity. Unless the embedding dimension of these datasets really does not exceed 1 (dimensionality using e.g. participation ratio in each session is not quantified), it is likely that these projections elide meaningful features of LIP population activity.

We now report the participation ratio (4.4 ± 0.4, mean ± s.e. across sessions), and we state that the first 3 PCs explain 67.1±3.1% of the variance of time- and coherence-dependent signals used for the PCA. We agree that the 1D projections may elide meaningful features of LIP population activity. Indeed, we make this point through our analysis of the Min neurons. We do not claim that the 1D projections explain all of the meaningful features of LIP population activity. They do, however, reveal the decision variable, which is our main focus. These 1D signals contain features that correlate with events in the superior colliculus, summarized in Stine et al. (2023), attesting to their biological relevance.

(2.2) Further, the observed similarity of results across these 1D projections may not be meaningful/interpretable. First, the rationale behind deriving Sramp was based on the ramping historically observed in Tin neurons during this task, so should be expected to resemble Tin.

The Reviewer is correct that we would expect 𝑆ramp to resemble the ramping observed in Tin neurons. We refer to this approach as hypothesis-driven. It captures the drift component of drift-diffusion. It is true that the Tcon neurons exhibit such ramps in their trial average firing rates, but this does not guarantee in

that the single-trial population firing rates would manifest as drift-diffusion. Indeed Latimer et al. (2015) concluded that the ramp-like averages comprise stepping from a low to a high firing rate on each trial at a random time. Therefore, while R2 is right to characterize the similarity of Tcon to the ramp direction in in trial-averaged activity as unsurprising, their similarity on single trials is not guaranteed.

(2.3) Second, Tin comprises the largest fraction of the neuron groups sampled during most sessions, so SPC1 should resemble Tin too. The finding that decision variables derived from the whole population’s activity reduce essentially to the average of Tin neurons is thus at least in part ’baked in’ to the approach used for deriving the decision variables.

This is incorrect. The Tcon in neurons constitute only 14.5% of the population, on average, across the sessions (see Table 1). This misunderstanding might contribute to R2’s concern about the importance of these neurons in shaping PC1. It is not simply because they are over-represented. Also, addressing R2’s concern about circularity, we would like to remind R2 that the selection of Tin neurons was based only on their spatial selectivity in the delayed saccade task. We do not see how it could be baked-in/guaranteed that a simple average of these neurons (i.e. zero degrees of freedom) yields dynamics and behavioral correlations that match those produced by dimensionality-reduction techniques that (𝑖) have degrees of freedom equal to the number of neurons and (𝑖𝑖) are blind to the neurons’ spatial selectivity. We have additionally modified what is now Supplementary Figure S13 (old Supplementary Figure S8), which portrays the mean accuracy of choice decoders trained on the neural activity of all neurons, only Tin neurons, all but the Tin neurons, and all but Tin and Min neurons, respectively. Figure S13 now highlights how much more readily choice can be decoded from the small population of Tin neurons than the remainder of the population.

(2.4) The analysis presented in Figure S6 looks like an attempt to demonstrate that this isn’t the case, but is opaque. Are the magnitudes of weights assigned to units in Tin larger than in the other groups of units with preselected response properties? What is their mean weighting magnitude, in comparison with the mean weight magnitude assigned to other groups? What is the null level of correspondence observed between weight magnitude and assignment to Tin (e.g. a negative control, where the identities of units are scrambled)?

The revised Figure S6—what is now Figure S9—displays more clearly that the weights assigned to Tcon and Tips neurons (purple & yellow, respectively) are larger in magnitude than those assigned in in to other neurons (gray). Author response table 1 shows a more detailed breakdown of the groups. Note that the length of the vector of weights is one. We are unsure what R2 means by “the null level of correspondence.” Perhaps it helps to know that the mean weight of the “other neurons” is close to zero for all four coding directions. However, it is the overlap of the weights and the relative abundance of non-Tin neurons that is more germane to the point we are making. To wit, knowing the weight (or percentile) of a neuron is a poor predictor that it belongs to the Tin category. This point is most clearly supported by the logistic regression (Fig. S9, bottom row). In other words, the large group of non-Tin neurons contribute substantially to all four coding directions examined in Figure S9. Thus, the similarity between Tin neurons and PC1 is not simply due to an over-representation of Tin neurons as suggested in item 2.3.

Author response table 1.

Mean weights assigned to neuron classes in four coding directions.

(3) The principal components analysis normalization procedure is unclear, and potentially incorrect and misleading: Why use the chosen normalization window (±25ms around 100ms after motion stimulus onset) for standardizing activity for PCA, rather than the typical choice of mean/standard deviation of activity in the full data window? This choice would specifically squash responses for units with a strong visual response, which distorts the covariance matrix, and thus the principal components that result. This kind of departure from the standard procedure should be clearly justified: what do the principal components look like when a standard procedure is used, and why was this insufficient/incorrect/unsuitable for this setting?

We used the early window because it is a robust measure of overall excitability, but we now use a more conventional window that spans the main epoch of our analyses, 200–600 ms after motion onset. This method yields results qualitatively similar to the original method. We are persuaded that this is the more sensible choice. We thank R2 for raising this concern.

(4) Analysis conclusions would generally be stronger with estimates of variability and control analyses: This applies broadly to Figures 2-6.

We have added estimates of variability and control analyses where appropriate.

Figure 2 shows examples of single-trial signals. The variability is addressed in Figure 3a and the new Supplementary Figure S5.

Figure 3 now contains error bars derived by bootstrapping (see Methods, Variance and autocorrelation of smoothed diffusion signals). We have also added Supplementary Figure S5, which substantiates the sublinearity claim using simulations.

Figure 4 (i) We now indicate the s.e.m. of decoding accuracy (across sessions) by the shading in Figure 4a. (ii) The black symbols in new Supplementary Figure S8 show the mean±s.e.m. for all pairwise comparisons shown in Figure 4d & e. (iii) Supplementary Figure S8 also summarizes two control analyses that deploy random coding directions (CDs) in neuronal state space. The upper row of Fig S9 compares the observed cosine similarity (CoSim)—between the CD identified by the graph title and the other four CDs labeled along the abscissa—with values obtained with 1000 random CDs established by random permutations of the weight assignments. The brown symbols are the mean±sdev of the CoSim (N=1000). The error bars are smaller than the symbols. We use the cumulative distribution of CoSim under permutation to estimate p-values (p<0.001 for all comparisons). We used a similar approach to estimate the distribution of the analogous correlation statistics between signals rendered by random directions in state space (Figure S8, lower row). For additional details, please see Methods, Similarity of single-trial signals.

Figure 5: The rigor of all claims associated with this figure is adduced from two control analyses and a simulation. The first control breaks the trial-by-trial correspondence between neural signals and behavior (Reviewer Figure 1). The second control shows that neural activity does not have substantial leverage on behavior when projected onto random directions in state space (Supplementary Figure S10, top). Simulations of decision variables using parameters derived from the fits to the behavioral data (Figure 1) support a degree of leverage and mediation comparable to the values observed for 𝑆Tincon (Supplementary Figure S10, bottom). For additional details, please see Methods (Leverage of single-trial activity on behavior) and the reply to item 1, above.

Figure 6: Panels c&d show estimates of variability across neurons and experimental sessions, respectively. The reported p-value is based on a permutation test (see Methods, Correlations between Min and Tconin ). The correlations shown in panel e (heatmap) are derived from pooled data across sessions. The reported p-value is based on a permutation test (see Methods, Correlations between Min and Tconin ).

Reviewer #3 (Public Review):

Summary:

The paper investigates which aspects of neural activity in LIP of the macaque give rise to individual decisions

(specificity of choice and reaction times) in single trials, by recording simultaneously from hundreds of neurons. Using a variety of dimensionality reduction and decoding techniques, they demonstrate that a population-based drift-diffusion signal, which relies on a small subset of neurons that overlap choice targets, is responsible for the choice and reaction time variability. Analysis of direction-selective neurons in LIP and their correlation with decision-related neurons (T con in [Tconin ] neurons ) suggests that evidence integration occurs within area LIP.

Strengths:

This is an important and interesting paper, which resolves conflicting hypotheses regarding the mechanisms that underlie decision-making in single trials. This is made possible by exploiting novel technology (Primatepixels recordings), in conjunction with state-of-the-art analyses and well-established dynamic random dot motion discrimination tasks.

General recommendations:

(1) Please tone down causal language. You presentcompelling correlativeevidencefor the idea thatLIP population activity encodes the drift-diffusion DV. We feel that claims beyond that (e.g., ”Single-trial drift-diffusion signals control the choice and decision time”) would require direct interventions, and are only partially supported by the current evidence. Further examples are provided in point 1) of Reviewer 1 below.

We have adopted the recommendation to “tone down the causal language.” Throughout the manuscript, we strive to avoid conveying the false impression that the present findings provide causal support for the decision mechanism. However, other causal studies of LIP support causality in the random dot motion task (Hanks et al., 2006; Jeurissen et al., 2022). It is therefore justifiable to use terms that imply causality in statements intended to convey hypotheses about mechanism. We agree that we should not give the false impression that the present support for said mechanism is adduced from causal perturbations in this study, as there were none.

(2) Please provide a commonly used, data-driven quantification of the dimensionality of the population activity – for example, using participation ratio or the number of PCs explaining 90 % of the variance. This will help readers evaluate the conclusions about the dimensionality of the data.

Principal component analysis reveals a participation ratio of 4.4 ± 0.4 (mean ±s.e., across sessions), and the first 3 PCs explain 67.1 ± 3.1 percent of the variance. The dimensionality of the data is low, but greater than one. We state this in Methods (Principal Component Analysis) and in Results (Single-trial drift-diffusion signals approximate the decision variable, lines 200–201).

(3) Please justify the normalization procedure used for PCA: Why use the chosen normalization window (±25ms around 100ms after motion stimulus onset) for standardizing activity for PCA, rather than the more common quantification of mean/standard deviation across the full data window? What do the first principal components look like when the latter procedure is used?

We now use a more conventional window that spans the main epoch of our analyses, 200–600 ms after motion onset. This method yields results qualitatively similar to the original method. We are persuaded that this is the more sensible choice.

(4) Please provide estimates of variability for variance and autocorrelation in Fig. 3 (e.g., through bootstrapping). Further, simulations could substantiate the claim about the expected sub-linearity at later time points (Fig. 3a) due to the upper stopping bound and limited firing rate range.

We thank the reviewers for these helpful recommendations. The revised Fig. 3 now contains error bars derived by bootstrapping (see Methods, Variance and autocorrelation of smoothed diffusion signals). We have also added Supplementary Figure S5, which substantiates the sub-linearity claim using simulations.

(5) Please add controls and estimates of variability for decoding across sessions in Fig. 4: what are the levels of within-trial correlation/cosine similarity for random coding directions? What is the variability in the estimates of values shown in a/d/e?

We have addressed each of these items. (1) Figure 4a now shows the s.e.m. of decoding accuracy (across sessions). (2) Regarding the variability of estimates shown in Figure 4d & e, the standard errors are displayed in the new supplementary Figure S8. It makes sense to show them there because there is no natural way to represent error on the heat maps in Figure 4, and Figure S8 concerns the comparison of the values in Figure 4d&e to values derived from random coding directions. (3) Random coding directions lead to values of cosine similarity and within-trial correlation that do not differ significantly from zero. We show this in several ways, summarized in our reply to Public Review item 4. Additional details are in the revised manuscript (Methods, Similarity of single-trial signals) and the new Supplementary Figure S8.

(6) Please perform additional analysis to strengthen the claim from Fig. 6, that Min represents the integrand and not the integral. The analysis in Fig. 6d could be repeated with the integral (cumulative sum) of the single-trial Min signals. Does this yield an increase in leverage over time?

The short answer is, yes in part. Reviewer Figure 2a provides support for leverage of the integral on choice, and this leverage, like 𝑆Tincon (t), increases as a function of time. The effect is present in all seven sessions that have both Mleftin and Mrightin neurons (all 𝑝 < 1𝑒 − 10). However, as shown in panel b, the same integral fails to demonstrate more than a hint of leverage on RT. All correlations are barely negative, and the magnitude does not increase as a function of time. We suspect—but cannot prove—that this failure arises because of limited power and the expected weak effect. Recall that the mediation analysis of RT is restricted to longer trials. Moreover, the correlation between the Min difference and the Tin signal is less than 0.1 (heatmap, Fig. 6e), implying that the Min difference explains less than 1% of the variance of 𝑆Tin(𝑡). We considered including Reviewer Figure 2 in the paper, but we feel it would be disingenuous (cherry-picking) to report only the positive outcome of the leverage on choice. If the editors feel strongly about it, we would be open to including it, but leaving these analyses out of the revised manuscript seems more consistent with our effort to deëmphasize this finding. In the future, we plan to record simultaneously from populations MT and LIP neurons (Min and Tin, of course) and optimize Min neuron yield by placing the RDM stimulus in the periphery.

(7) Please describe the complete procedure for determining spatially-selective activity. E.g.: What response epoch was used, what was the spatial layout of the response targets, were responses to all ipsi- vs contralateral targets pooled, what was the spatial distribution of response fields relative to the choice targets across the population?

We thank the reviewers for pointing out this oversight. We now explain this procedure in the Methods (lines 629–644):

Neurons were classified post hoc as Tin by visual-inspection of spatial heatmaps of neural activity acquired in the delayed saccade task. We inspected activity in the visual, delay, and perisaccadic epochs of the task. The distribution of target locations was guided by the spatial selectivity of simultaneously recorded neurons in the superior colliculus (see Stine 2023 for details). Briefly, after identifying the location of the SC response fields, we randomly presented saccade targets within this location and seven other, equally spaced locations at the same eccentricity. In monkey J we also included 1–3 additional eccentricities, spanning 5–16 degrees. Neurons were classified as Tin if they displayed a clear, spatially-selective response in at least one epoch to one of the two locations occupied by the choice targets in the main task. Neurons that switched their spatial selectivity in different epochs were not classified as Tin. The classification was conducted before the analyses of activity in the motion discrimination task. The procedure was meant to mimic those used in earlier single-neuron studies of LIP (e.g., Roitman & Shadlen 2002) in which the location of the choice targets was determined online by the qualitative spatial selectivity of the neuron under study. The Tcon neurons in the in present study were highly selective for either the contralateral or ipislateral choice target used in the RDM task (AUC = 0.89±0.01; 𝑝 < 0.05 for 97% of neurons, Wilcoxon rank sum test). Given the sparse sampling of saccade target locations, we are unable to supply a quantitative estimate of the center and spatial extent of the RFs.

(8) Please clarify if a neuron could be classified as both Tin and Min. Or were these categories mutually exclusive?

These categories are mutually exclusive. If a neuron has spatially-selective persistent activity, as defined by the method described above, it is classified as a Tin neuron and not as an Min neuron even if it also shows motion-selective activity during passive motion viewing. We now specify this in the Methods (lines 831–832).

Reviewer #1 (Recommendations For The Authors):

𝑅∗1.1a Causal language (Line 23-24): “population activity represents […] drift” and “we provide direct support for the hypothesis that drift-diffusion signal is the quantity responsible for the variability in choice and RT” reads at first sight as if the authors claim that they present evidence for a causal effect of LIP activity on choice. The authors areotherwisenuanced and carefultopointout thattheir evidence is correlational. What seems to be meant is that the population activity/drift-diffusion signal ”approximates the DV that gives rise to the choices […]” (cf. line 399). I would recommend using such alternative phrasing to avoid confusion (and the typically strong reactions by readers against misleading causal statements).

We have adopted the reviewer’s recommendation and have modified the text throughout to reduce causal language. See our response to General Recommendation 1.

𝑅∗1.1b Relatedly, any discussion about the possibility of LIP being causally involved in evidence integration (e.g. lines 429-445 [Au: now 462–478]) should also comment on the possibility of a distributed representation of the decision variable given that neural correlates of the DV have been reported in several areas including PFC, caudate and FEF.

We believe this is possible. However, we hope to avoid discussions about causality given that it is not a focus of the paper. Although it is somewhat tangential, we have shown elsewhere that LIP is causal in the sense that causal manipulations affect behavior, but it is also true that causality does not imply necessity, and similarly, lack of necessity does not imply “only correlation.” Regarding distributed representations, it is worth keeping in mind the cautionary counter-example furnished by the SC study (Stine et al., 2023). The firing rates measured by averaging over trials are similar in SC and LIP; both manifest as coherence and direction-dependent ramps, leading to the suggestion that they form a distributed representation of the decision variable. With single-trial resolution, we now know that LIP and SC exhibit distinct dynamics—drift-diffusion and bursting, respectively. It remains to be seen if single-trial resolution achievable by simultaneous Neuropixels recordings from prefrontal areas and LIP reveal shared or distinct dynamics.

𝑅∗1.2 How was the spatially selective activity determined? The classification of Tin neurons is critical to this study - how was their spatial selectivity determined? Please describe this in similar detail as the description of direction selectivity on lines 681-690 [Au: now 824–832]. E.g.: what response epoch was used, what was the spatial layout of the response targets, were responses to all ipsi- vs contralateral targets pooled, and what was the spatial distribution of response fields relative to the choice targets across the population?

We now explain the selection procedure in Methods (lines 629–644). Please see our reply to General Recommendation 7, above.

𝑅∗1.3 Could a neuron be classified as both Tin and Min, or were these categories mutually exclusive? Please clarify. (This goes beyond the scope of the current study: but did the authors find evidence for topographic organization or clustering of these categories of neurons?)

These categories are mutually exclusive. Please see our response to General Recommendation 8, above.

𝑅∗1.4 Contrary to the statement on line 121, the trial averages in Fig. 2a, 2b show coherence dependency at the time of the saccade in saccade-aligned traces for the coding strategies, except for STin (fig. 2c). Is this a result of the choice for t1 (= 0.1s)? (The authors may want to change their statement on line 121.) Relatedly, do the population responses for the two coding strategies Sramp and SPC1 depend on the epoch used to derive weights for individual neurons?

We have revised the description to accommodate R2’s observation. 𝑆ramp retains weak coherence-dependence before saccades towards the choice target contralateral to the recording site. This was true in four of the eight sessions. For 𝑆PC1, there is no longer a coherence dependency for the Tin choices, owing to the change in normalization method (see revised Figure 2b).

We also corrected an error in the Methods section. Specifically, the ramp ends at 𝑡1 \= 0.05 s before the time of the saccade, not 𝑡1 \= 0.1 s. While we no longer emphasize the similarity of traces aligned to saccade, it is reasonable to find issue with the observation that they retain a dependency on coherence (𝑆ramp only) because, according to theory, traces associated with Tin choices should reach a common positive threshold at decision termination. That said, for the Ramp direction there may be a reason to expect this discrepancy from theory. The deterministic part of drift-diffusion includes an urgency signal that confers positive convexity to the deterministic drift. This accelerating nonlinearity is not captured by the ramp, and it is more prominent at longer decision times, thus low coherences. We do not share this interpretation in the revised manuscript, in part because retention of coherence dependency is present in only half the sessions (see Reviewer Figure 3) The correction to the definition of 𝑡1 also provides an opportunity to address R2’s final question (“Relatedly,…?”). For 𝑆ramp this particular variation in 𝑡1 does not affect 𝑆ramp, and 𝑆PC1 no longer retains coherence dependency for Tin choices. Note that our choice of 𝑡0 and 𝑡1 is based on the empirical observation that the ramping activity in response averages of Tin neurons typically begins 200 ms after motion onset and ends 50–100 ms before initiation of the saccadic choice. The starting time (𝑡0) is also supported by the observation that the decoding accuracy of a choice-decoder begins to diverge from chance at this time (Figure 4a).

𝑅∗1.5 It is intriguing that Sramp and SPC1 show dynamics that look so similar (fig. 2a, 2b). How do the weights assigned to each neuron in both strategies compare across the population?

The weights assigned to each neuron are very similar across the two strategies as indicated by a cosine similarity (0.65 ± 0.04, mean ±s.e.m. across sessions).

𝑅∗1.6 Tin neurons, which show dynamics closely resembling different coding directions (fig. 2) and the decoders do not have weights that can distinguish them from the rest of the population in each of these analyses (fig. S7). Is it fair to interpret these findings as evidence for broad decision-related co-variability in the recorded neural population in LIP?

Yes, our results are consistent with this interpretation. However, it is worth reiterating that decoding performance drops considerably when Tin neurons are not included (see Supplementary Figure S13). Thus, this broad decision-related co-variability is present but weak.

𝑅∗1.7 It is intriguing that the decoding weights of the different decoders did not allow the authors to reliably identify Tin neurons. Could this be, in part, due to the low dimensionality of the population activity and task that the animals are presumably overtrained on? Or do the authors expect this finding to hold up if the population activity and task were higher dimensional?

Great question! We can only speculate, but it seems possible that a more complex, “higher dimensional” task could make it easier to identify Tin neurons. For example, a task with four choices instead of two may decrease correlations among groups of neurons with different response fields. We have added this caveat to the discussion (lines 459-–461). One minor semantic objection: The animal has learned to perform a highly contrived task at low signal-to-noise. The animal is well-trained, not over-trained.

𝑅∗1.8 Lines 135-137 [Au: now 141–142]: The similarity in the single trial traces from different coding strategies (fig. 2a-2c, left) is not as evident to me as the authors suggest. It might be worthwhile computing the correlation coefficients between individual traces for each pair of strategies and reporting the mean correlation to support the author’s point.

We report the mean correlation between single-trial signals generated by the chosen dimensionality reduction methods in Figure 4e. We show the variability in this measure in Supplementary Figure S8. We have also adjusted the opacity of the single-trial traces in Figure 2, left.

𝑅∗1.9 Minor/typos:

-line 74: consider additionally citing Hyafil et al. 2023.

-line 588: ”that were strongly correlated”?

-line 615: ”were the actual drift-diffusion process were...”.

-line 717: ”a causal influence” -> ”no causal influence”.

Fig. 6: panel labels e vs d are swapped between the figure and caption.

Fig. 3c: labels r1,3 & r2,3 are flipped.

We have addressed all of these items. Thank you.

Reviewer #2 (Recommendations For The Authors):

𝑅∗2.1 (Figure 2) Determine whether restricting the analysis to 1D projections of the data is a suitable approach given the actual dimensionality of the datasets being analyzed:

- Should show some quantification of the dimensionality of the recorded activity; could do this by quantifying the dimensionality of population activity in each session, e.g. with participation ratio or related measures (like # PCs to explain some high proportion of the variance, e.g. 90 %). If much of the variation is not described in 1 dimension, then the paper would benefit from some discussion/analysis of the signals that occupy the other dimensions.

We now report the participation ratio (4.4 ± 0.4, mean ±s.e. across sessions), and we state that the first 3 PCs explain 67.1 ± 3.1% of the variance of the time- and coherence-dependent signals used for the PCA (mean ±s.e). We agree that the 1D projections may elide meaningful features of LIP population activity. Indeed, we make this point through our analysis of the Min neurons. To reiterate our response above, we do not claim that the 1D projections explain all of the meaningful features of LIP population activity. They do, however, reveal the decision variable, which is our main focus. These 1D signals contain features that correlate with events in the superior colliculus, summarized in Stine et al. (2023), attesting to their biological relevance.

The Reviewer is correct that our approach presupposes a linear embedding of the 1D decision variable inthepopulationactivity. Inotherwords, anonlinearrepresentationofthe1Ddecisionvariableinpopulation activity could have an embedding dimensionality greater than 1, and there may well be a non-linear method that reveals this representation. To test this possibility, we decoded choice on each trial from population activity using (1) a linear decoder (logistic classifier) or (2) a multi-layer neural network, which can exploit non-linearities. We found that, for each session, the two decoders performed similarly: the neural network outperforms the logistic decoder (barely) in just one session. The analysis suggests that the assumption of linear embedding of the decision variable is justified. We hope this analysis convinces the reviewer that “sophisticated analyses of the full neuronal state space” and “a simple average of [Tcon ] neurons” do in indeed yield roughly equivalent representations of the decision variable. We have included the results of this analysis in Supplementary Figure S12. See also item 2 of the Public response.

𝑅∗2.2 (Figure 3) Add estimates of variability for variance and autocorrelation through time from single-trial signals:

–   E.g. by bootstrapping. Would be helpful for making rigorous the discussion of when the deviation from the theory is outside what would be expected by chance, even if it doesn’t change the specific conclusions here.

–   If possible, it would help (by simulations, or maybe an added reference if it exists) to substantiate the claim about the expected sub-linearity at later time-points (Figure 3a) due to the upper stopping bound and limited firing rate range.

We thank the reviewer for this helpful comment. The revised Fig. 3 now contains error bars derived by bootstrapping (see Methods, §Variance and autocorrelation of smoothed diffusion signals). We have also added Supplementary Figure S5, which substantiates the sub-linearity claim using simulations.

𝑅∗2.3 (Figure 4) Add controls and estimates of variability for decoding across sessions:

–   As a baseline - what is the level of within-trial correlation/cosine similarity when random coding directions are used?

–   What is the variability in the estimates of values shown in a/d/e?

We have addressed each of these items. (1) Figure 4a now shows the s.e.m. of decoding accuracy (across sessions). (2) Regarding the variability of estimates shown in Figure 4d & e, the standard errors are displayed in the new Supplementary Figure S8. It makes sense to show them there because (i) there is no natural way to represent error on the heat maps in Figure 4, and (ii) S8 concerns the comparison of the values in Figure 4d & e to values derived from random coding directions. (3) Random coding directions lead to values of cosine similarity and within-trial correlation that do not differ significantly from zero. We show this in several ways, summarized in our reply to Public Review item 4. Additional details are in the revised manuscript (Methods: Similarity of single-trial signals) and the new Supplementary Figure S8. We also provide this information in response to Recommendation 5, above.

𝑅∗2.4 (Figure 5) Add negative controls and significance tests to support claims about trends in leverage:

–   What is the level of increase in leverage attained from random 1D projections of the data, or other projections where the prior would be no leverage?

–   What is the range of leverage values fit for a simulated signal with a ground-truth of no trend?

We have added two control analyses. In addition to a shuffle control, which destroys the relationship (Review Figure 1) we performed additional analyses that preserve the correspondence of neural signals and behavior on the same trial. We generated random coding directions (CDs) by establishing weight-vectors that were either chosen from a Normal distribution or by permuting the weights assigned to PC-1 in each session. The latter is the more conservative measure. Projections of the neural responses onto these random coding directions render 𝑆rand(𝑡). Specifically, the degree of leverage is effectively zero or very much reduced. These analyses are summarized in a new Supplementary Figure S10. The distributions of our test statistics (e.g., leverage on choice and RT) under the variants of the null hypothesis also support traditional metrics of statistical significance. Figure S10 (bottom row) also provides an approximate answer to the question: What degree of leverage and mediation would be expected for a theoretical decision variable? Briefly, we simulated 60,000 trials using the race model that best fits the behavioral data of monkey M. For any noise-free representation of a Markovian integration process, the leverage of an early sample of the DV on behavior would be mediated completely by later activity as the latter sample—up to the time of commitment—subsumes all variability captured by the earlier sample. We, therefore, generated 𝑆sim(𝑡) by first subsampling the simulated data to match the trial numbers of each session. To evaluate a DV approximated from the activity of 𝑁 Tconin neurons per session rather than the true DV represented by the entire population, we generated 𝑁 noisy instantiations of the signal for each of the subsampled, simulated trials. The noisy decision variable, 𝑆sim (t) is the mean activity of these 𝑁 noise-corrupted signals. The simulation is consistent with the leverage and incomplete mediation observed for the populations of Tcon neurons. For in additional details, see Methods, §Leverage of single-trial activity on behavior) and Supplementary Figure S10, caption. See also our response to item 1 of the Public Response.

𝑅∗2.5 The analysis is performed across several signed coherence levels, with data detrended for each signed coherence and choice to enable comparison of fluctuations relative to the relevant baseline; are results similar for the different coherences?

The results are qualitatively similar for individual coherences. There is less power, of course, because there are fewer trials. The analyses cannot be performed for coherences ≥ 12.8% because there are not enough trials that satisfy the inclusion criteria (presence of left and right choice trials with RT ≤ 670 ms). Nonetheless, leverage on choice and RT is statistically significant for 27 of the 30 combinations of motion strengths < 12.8% × three signals (𝑆ramp, 𝑆PC1 and 𝑆Tin) × behavioral measures (RT and choice) (RT: all 𝑝 < 0.008, Fisher-z; choice: all 𝑝 < 0.05, t-test ). The three exceptions are trials with 6.4% coherence rightward motion, which do not correlate significantly with RT on leftward choice trials. Reviewer Figure 4 shows the results of the leverage and mediation analyses, using only the 0% coherence trials.

𝑅∗2.6 (Figure 6) Additional analysis to strengthen the claim that Min represents the integrand and not the integral:

a. Repeating the analysis in Figure 6d with the integral (cumulative sum) of the single-trial Min signals and instead observing a significant increase in leverage over time would be strong evidence for this interpretation. If you again see no increase, then it suggests that the activity of these units (while direction selective) may not be strongly yoked to behavior. This scenario (no increasing leverage of the integral of Min on behavior through time) also raises an intriguing alternative possibility: that the noise driving the ’diffusion’ of drift-diffusion here may originate in the integrating circuit, rather than just reflecting the complete integration of noise in the stream of evidence itself.

b. Repeating the analysis in Figure 6d with the projection of the M subspace onto its own first PC (e.g. take the union of units {Mrightin, Mleftin} [our ], do PCA just on those units’ single

trial activities, identify the first PC, and project those activities on that dimension to obtain SPC1-M.

c. Ameliorating the sample-size limitation by relaxing the criteria for inclusion in Min - performing the same analyses shown, but including all units with visual RFs overlapping the motion stimulus, irrespective of their direction selectivity.

a. Reviewer Figure 2a provides support for leverage of the integral on choice, and this leverage, like , increases as a function of time. The effect is present in all seven sessions that have both and neurons (all 𝑝 < 1𝑒 − 10). However, as shown in panel b, the same integral fails

to demonstrate more than a hint of leverage on RT (all correlations are negative) and the magnitude does not vary as a function of time. We suspect—but cannot prove—that this failure arises because of limited power and the expected weak effect. Recall that the mediation analysis of RT is restricted to longer trials and that the correlation between the Min difference and the signal is less than 0.1 over the heatmap in Fig. 6e, implying that the Min difference explains less than 1% of the variance of 𝑆Tin(𝑡). We considered including Reviewer Figure 2 in the paper, but we feel it would be disingenuous (cherrypicking) to report only the positive outcome of the leverage on choice. If the editors feel strongly about it, we would be open to including it, but leaving these analyses out of the revised manuscript seems more consistent with our effort to deëmphasize this finding. In the future, we plan to record simultaneously from populations MT and LIP neurons (Min and Tin, of course) and optimize Min neuron yield by placing the RDM stimulus in the periphery. We also provide this information in response to Recommendation (6) above.

b.  We tried the R’s suggestion to apply PCA to the union of Min neurons , , fully expecting PC1 to comprise weights of opposite sign for the right and left preferring neurons, but that is not what we observed. Instead, the direction selectivity is distributed over at least two PCs. We think this is a reflection of the prominence of other signals, such as the strong visual response and normalization signals (see Shushruth et al., 2018). In the spirit of the R’s suggestion, we also established an “evidence coding direction” using a regression strategy similar to the Ramp CD applied to the union of Min neurons. The strategy produced a coding direction with opposite signed weights dominating the right and left subsets. The projection of the neural data on this evidence CD yields a signal similar to the difference variable used in Fig. 6e (i.e., signals that are approximately constant firing rates vs time and scale as a function of signed coherence). These unintegrated signals exhibit weak leverage on choice and RT, consistent with Figure 6d. However, the integrated signal has leverage on choice but not RT, similar to the integral of the difference signal in Reviewer Figure 2.

c.   We do not understand the motivation for this analysis. We could apply PCA or dPCA (or the regression approach, described above) to the population of units with RFs that overlap the motion stimulus, but it is hard to see how this would test the hypothesis that direction-selective neurons similar to those in area MT supply the momentary evidence. As mentioned, we have very few Min neurons (as few as two in session 3). Future experiments that place the motion stimulus in the periphery would likely increase the yield of Min neurons and would be better suited to study this question. As such, we do not see the integrand-like responses of Min neurons as a major claim of the paper. Instead, we view it as an intriguing observation that deserves follow-up in future experiments, including simultaneous recordings from populations of MT and LIP neurons (Min and Tin, of course). We have softened the language considerably to make it clear that future work will be needed to make strong claims about the nature of Min neurons.

𝑅∗2.7 Other questions: Figure 2c is described as showing the average firing rate of units in Tconin on single trials, but must also incorporate some baseline subtraction (as the shown traces dip into negative firing rates). Whatbaselineissubtracted? Aretheseresidualsignals, asdescribedforlaterfigures, orisadifferent method used? (Presumably, a similar procedure is used also for Figure 2a/b, given that all single-trial traces begin at 0.). Is the baseline subtraction justified? If the dataset really does reflect the decision variable with single-trial resolution, eliminating the baseline subtraction when visualizing single-trial activity might actually help to make the point clearer: trials which (for any reason) begin with a higher projection on the particular direction that furnishes the DV would be predicted to reach the decision bound, at any fixed coherence, more quickly than trials with a smaller projection onto this direction.

We thank the reviewer for this comment. For each trial, the mean activity between 175 ms and 225 ms after motion onset was subtracted when generating the single-trial traces. The baseline subtraction was only applied for visualization to better portray the diffusion component in the signal. Unless otherwise indicated, all analyses are computed on non-baseline corrected data. We now describe in the caption of Figure 2 that “For visualization, single-trial traces were baseline corrected by subtracting the activity in a 50 ms window around 200 ms.” Examples of the raw traces used for all follow-up analyses are displayed in Reviewer Figure 6.

Reviewer #3 (Recommendations For The Authors):

I only have a few comments to make the paper more accessible:

𝑅∗3.1 I struggle to understand how the linear fitting from -1 to 1 was done. More detail about how the single cell single-trial activity was generated to possibly go from -1 to 1 or do I completely misunderstand the approach? I assume the data standardization does that job?

We have rephrased and added clarifying detail to the section describing the derivation of the ramp signal in the Methods (Ramp direction).

We applied linear regression to generate a signal that best approximates a linear ramp, on each trial, 𝑖, that terminates with a saccade to the choice-target contralateral to the hemisphere of the LIP recordings. The ramps are defined in the epoch spanning the decision time: each ramp begins at 𝑓𝑖(𝑡0) = −1, where 𝑡0 \= 0.2 s after motion onset, and ends at 𝑓𝑖(𝑡1) = 1, where 𝑡1 \= 𝑡sac − 0.05 s (i.e., 50 ms before saccade initiation). The ramps are sampled every 25 ms and concatenated using all eligible trials to construct a long saw-tooth function (see Supplementary Figure S2). The regression solves for the weights assigned to each neuron such that the weighted sum of the activity of all neurons best approximates the saw-tooth. We constructed a time series of standardized neural activity, sampled identically to the saw-tooth. The spike times from each neuron are represented as delta functions (rasters) and convolved with a non-causal 25 ms boxcar filter. The mean and standard deviation of all sampled values of activity were used to standardize the activity for each neuron (i.e., Z-transform). The coefficients derived by the regression establish the vector of weights that define 𝑆ramp. The algorithm ensures that the population signal 𝑆ramp(𝑡), but not necessarily individual neurons, have amplitudes ranging from approximately −1 to 1.

𝑅∗3.2 It is difficult to understand how the urgency signal is derived, to then generate fig S4.

The urgency signal is estimated by averaging 𝑆𝑥(𝑡) at each time point relative to motion onset, using only the 0% coherence trials. We have clarified this in the caption of Supplementary Figure S4.

Author response image 1.

Shuffle control for Fig. 5. Breaking the within-trial correspondence between neural signal, 𝑆(𝑡), and choice suppresses leverage to near zero.

Author response image 2.

Leverage of the integrated difference signal on choice and RT. Traces are the average leverage across seven sessions. Same conventions as in Figure 5.

Author response image 3.

Trial-averaged 𝑆ramp activity during individual sessions. Same as Figure 2b for individual sessions for Monkey M (left) and Monkey J (right). The figure is intended to illustrate the consistency and heterogeneity of the averaged signals. For example, the saccade-aligned averages lose their association with motion strength before left (contra) choices in sessions 1, 2, 5, and 6 but retain the association in sessions 3, 4, 7, and 8.

Author response image 4.

Drift-diffusion signals have measurable leverage on choice and RT even when only 0%-coherence trials are included in the analysis.

Author response image 5.

Raw single-trial activity for three types of population averages. Representative single-trial activity during the first 300 ms of evidence accumulation using two motion strengths: 0% and 25.6% coherence toward the left (contralateral) choice target. Unlike in Figure 2 in the paper, single-trial traces are not baseline corrected by subtracting the activity in a 50 ms window around 200 ms. We highlight a number of trials with thick traces and these are the same trials in each of the rows.

Escuchar, consolar , fortalecer y orar (1 Tesalonicenses 5:14). Escúchenlos con empatía (Santiago 1:19). (Proverbios 12:25).

(1 Pedro 1:22; 5:6, 7).

(Santiago 5:14, 15)

deben tener presente el ejemplo de justicia de Jehová al dar consejos bíblicos cuando es necesario. Y aunque no estan buscando defectos pueden percibir si algún miembro de la congregación puede dar “un paso en falso sin darse cuenta”.

Como los ancianos saben que Jehová no es cruel ni duro al aplicar la justicia, seguirán este consejo: “Traten de corregir al hombre con espíritu apacible” (Gálatas 6:1).

Aunque tengan que ser muy directos y advertirlo de las consecuencias, tendrán presente que es una ovejita de Jehová (Lucas 15:7).b Cuando un consejo se da por amor y con amor, es más probable que la persona recapacite y acepte la corrección.

Que cómo los príncipes de Jehová, imiten la justicia de Jesús

“¡Mira! Un rey reinará con rectitud, y príncipes gobernarán con justicia” (Isaías 32:1).

Lamentablemente es muy común en las familias ver mucho maltrato y es bastante triste. Muchos esposos, esposas y padres se portan como jueces inflexibles con su familia. No paran de decirles cosas crueles e hirientes, y hasta los golpean.

Pero entre los siervos de Dios no debe haber palabras crueles, sarcasmo ni ningún tipo de maltrato (Efesios 4:29, 31; 5:33; 6:4). Y los mandatos de Jesús de dejar de juzgar y dejar de condenar también son para la familia.

Recordemos que practicar la justicia implica tratar a los demás como nos trata Jehová. En vez de ser áspero y duro con quienes lo aman, él “es muy cariñoso y misericordioso” (Santiago 5:11).

Este párrafo es muy importante

Primero, no nos corresponde el discípulo Santiago nos recuerda que “solo hay un Legislador y Juez”: Jehová. Luego hace una pregunta que nos hace reflexionar: “¿Quién eres tú para juzgar a tu prójimo?” (Santiago 4:12; Romanos 14:1-4).

Segumdo somos imperfectos, es fácil juzgar a otros de forma injusta. Muchos factores - los prejuicios, - el resentimiento, - los celos - sentimientos de superioridad pueden distorsionar nuestra forma de ver a los demás.

Tercero no podemos leer los corazones de nadie ni saber todos los detalles de su situación personal.

Cuarto no contamos con toda la informacion, y eso puede inducirnos a sacar concludiines equivocadas. En especial si nos apresuramos.

en conclusión sólo Jehová puede juzgar con justicia y nosotros no podemos juzgar a los demás y si nuestra imperfección ve fallas en ellos , podemos imitar a Jehová , concentrandonos mejor en sus virtudes

porqué Jehová nos pone un ejemplo perfecto al tratarnos con misericordia y bondad Salmo 130:3).

y si somos agradecidos , debemos tratar a los demás del mismo modo (Salmo

Porque con la predicación se advierte a las personas, acerca de su juicio, y mediante ésa obra no se podrá decir que Jehová es injusto a no darle oportunidad a los que se comportan mal Marcos 13:10; Mateo 24:3). (Mateo 24:14, 21, 22). (Jonás 3:1-10).

¿Y qué es lo que Jehová espera de ti? ¡Solo que practiques la justicia, ames la lealtad y andes con modestia junto a tu Dios!” (Miqueas 6:8)

Estamos en deuda con Jehová porque él nos rescató del pecado y la muerte y debido la fe que tenemos en el sacrificio de su hijo Jesucristo, se perdona nuestros pecados y podemos tener vida eterna 1 Juan 1:7; 4:9

• porque pueden debilitar nuestra relación con Jehová
• y también pueden ser el primer paso que nos lleve a cometer pecados más graves

La realidad es que todos tenemos nuestros puntos débiles o tentaciones. Quizá sea comer o beber con exceso, el entrenamiento, el dinero, o quizá la inmoralidad sexual. El párrafo también menciona, espíritu de independencia, el temor al hombre o el mal carácter. “cada uno es probado al ser atraído y seducido por su propio deseo” (Sant. 1:14).

Como vimos nuestra imperfección puede llevarnos a dar pasos que terminen en pecado.

Pero estar ocupados dando pasos que agraden a Jehová, son la mejor protección

Participar en las reuniones y predicamos, se fortalece nuestra amistad con Dios, nos prepara cómo mejores maestro y estimulamos a nuestros hermanos (Mat. 28:19, 20; Heb. 10:24, 25).

Si estamos solos Leer y estudiar la Palabra de Dios y meditar en ella nos ayudará a alejarnos de lo malo y al mismo tiempo fortalecerá nuestro amor por la ley de Jehová (Jos. 1:8; Sal. 1:2, 3; 119:97, 101).

Y recordar que Jesús nos aconsejó orar constantemente para resistir las tentaciones (Mat. 26:41). Y nosotros sabemos que Jehová bendice ésas oraciones y al mismo tiempo lo hacen Feliz

Seguir un buen programa de actividades espirituales nos ayuda a rechazar las tentaciones

Porque podemos atraer lo que pensamos, en el sentido que. Con el tiempo se fortalece el deseo de cumplir esos pensamientos.

Por éso debemos resistir y evitar esos pensamientos cuando surjan. Para evitar que crezcan hasta convertirse en deseos intensos, y difíciles de resistir y que pueden llevarnos a cometer un pecado grave (Filip. 4:8; Col. 3:2; Sant. 1:13-15).

Dijo: “He hecho un pacto con mis ojos” (lea Job 31:1). Respetar ese pacto lo ayudaría a no cometer nunca adulterio. Nosotros también podemos tomar en nuestro interior la firme decisión de no hacer nada que pudiera llevarnos a caer en una tentación.

Debió haber evitado pasar por ése lugar, donde sabía que estaba esa mujer, con la que de repente cometió inmoralidad, pero la realidad es que ése acto fue el resultado de muchos pasos anteriores que se pudieron haber evitado. Proverbios 7

Que tenemos puntos débiles in duda, nos conviene ser honrados con nosotros mismos y reconocer los campos en los que no somos fuertes (2 Cor. 13:5).

Porque estaban desprevenidos. Jesús mismo les había advertido que se mantuvieran despiertos. Pero se durmieron y la situación los agarró tan de sorpresa que terminaron haciendo exactamente lo que habían dicho que nunca harían: abandonar a Jesús (Mat. 26:56).

“EL ESPÍRITU está dispuesto, pero la carne es débil” (Mat. 26:41b).a

Jesús tiene claro que somos imperfectos y aunque tengamos buenas intenciones, podemos caer en el error de pensar o actuar con exceso de confianza, cómo decir que a nosotros o a mi, nada nada me apartará de hacer siempre lo correcto. Porque amo a Jehová y Jesús.

Jeremías 18:1-11 Jehova en su misericordia los trata cómo el alfarero que lo intenta una y otra vez hasta que su artesanía queda como el espera. El es igual el da oportunidad para que los que obran con maldad, puedan cambiar, si se dejan moldear por su palabra.

check for pinkness, grey skin, guppy breathing, color of the palms and dilation in the eyes.

Reference to the issue presented at the beginning of this page, how are people supposed to judge aboutness/classify things without familiarizing themselves with the topic/basic knowledge

• Les mandaba a los padres y a las madres que pasaran bastante tiempo con sus hijos y les enseñaran de Jehová (Deuteronomio 6:6, 7).
• Prohibía el incesto y lo castigaba con la muerte (Levítico, capítulo 18).
• prohibía el adulterio, que acaba con tantas familias y hace que pierdan su seguridad y dignidad.
• protegía a las viudas y los huérfanos, y castigaba severamente a quienes los maltrataban (Éxodo 20:14; 22:22-24).

Si alguien se endeudaba, el acreedor no podía entrar en la casa de esa persona para quedarse con algún objeto como garantía del pago. - debía esperarlo afuera y dejar que el deudor le trajera el objeto que serviría de garantía. - Así se respetaban los derechos de quienes vivían en esa casa. - Si el objeto era el manto del deudor, tenía que devolvérselo antes de la noche, pues quizá lo necesitara para cubrirse (Deuteronomio 24:10-14).

porque dios es justo, no trata a nadie con parcialidad, y enseña s sus siervos que sean como el

• Actitud correcta, Sin duda, saber que se ofrece por verdadero interés en nosotros, y no por frustración o por egoísmo.
• El segundo factor es basarse en la Palabra de Dios (2 Timoteo 3:16). Todo lo que digamos debe estar fundamentado en la Biblia, sea que leamos directamente de ella o no. Con cuidado de no imponer opiniones personales. Ni manipular textos bíblicos para respaldar nuestras ideas.
• El tercer factor es ofrecer la recomendación con bondad, respetando la dignidad de cada uno, así será más fácil aceptarla (Colosenses 4:6).

(Santiago 5:14, 15).

confirmarle que la congregación lo necesita y aprecia (1 Corintios 12:12-26).

leerle algún pasaje bíblico que le recuerde cuánto se interesa Jehová por él (Salmo 34:18;

Mateo 10:29-31).

Si nos tomamos el tiempo necesario para confortarlo con alguna “buena palabra” que salga de nuestro corazón, se sentirá más amado y valorado (Proverbios 12:25).

.h2

Preguntas para meditar

• ¿Cuándo fue la última vez que felicité a alguien por su labor? (1 Corintios 11:2; Revelación 2:1-3.)

• ¿Demuestro respeto a los demás pidiendo las cosas con cortesía y dándoles las gracias? (Génesis 13:14; Juan 11:41.)

• ¿Hablo casi siempre de mí mismo, o me intereso también por las ideas y sentimientos de los demás? (Filipenses 2:3, 4; Santiago 1:19.)

• ¿Para qué empleo la información que tengo sobre las vidas ajenas? ¿Para edificar, o para derrumbar? (Proverbios 15:1, 2.)

• ¿Qué actitud demuestro hacia el lenguaje soez, y qué revela esa actitud sobre lo que tengo en el corazón? (Lucas 6:45; Santiago 3:10, 11.)

sweet but usually the brother's can't do much

Reviewer #2 (Public Review):

Summary:

The authors demonstrate that a low parenteral glucose regimen can lead to improved bacterial clearance and survival from Staph epi sepsis in newborn pigs without inducing hypoglycemia, as compared to a high glucose regimen. Using RNA-seq, metabolomic, and proteomic data, the authors conclude that this is primarily mediated by altered hepatic metabolism.

Strengths:

Well-defined controls for every time point, with multiple time points and biological replicates.

The authors used different experimental strategies to arrive at the same conclusion, which lends credibility to their findings.

The authors have published the negative findings associated with their study, including the inability to reverse sepsis-related mortality after switching from SE-high to SE-low at 3h or 6h and after administration of hIAIP.

Weaknesses:

(1) The authors mention, and it is well-known, that Staph epi is primarily involved in late-onset sepsis. The model of S. epi sepsis used in this study clearly replicates early-onset sepsis, but S. epi is extremely rare in this time period. How do the authors justify the clinical relevance of this model?

(2) The authors find that the neutrophil subset of the leukocyte population is diminished significantly in the SE-low and SE-high populations. However, they conclude on page 10 that "modulations of hepatic, but not circulating immune cell metabolism, by reduced glucose supply..." and this is possible because the authors have looked at the entire leukocyte transcriptome. I am curious about why the authors did not sequence the neutrophil-specific transcriptome.

(3) The authors use high (30g/k/d) and low (7.2g/k/d) glucose regimens. These translate into a GIR of 21 and 5 mg/k/min respectively. A normal GIR for a preterm infant is usually 5-8, and sometimes up to 10. Do the authors have a "safe GIR" or a threshold they think we cannot cross? Maybe a point where the metabolism switch takes place? They do not comment on this, especially as GIR and glucose levels are continuous variables and not categorical.

(4) In Figures 2B and C the authors show that SE-high and SE-low animals have differences in the oxphos, TCA, and glycolytic pathways. The authors themselves comment in the Supplementary Table S1B, E-F that these same metabolic pathways are also different in the Con-Low and Con-high animals, it is just the inflammatory pathways that are not different in the non-infected animals. How can they then justify that it is these metabolic pathways specifically which lead to altered inflammatory pathways, and not just the presence of infection along with some other unfound mechanism?

(5) The authors mention in Figure 1F that SE-low animals had lower bacterial burdens than SE-high animals, but then go on to infer that the inflammatory cytokine differences are attributed to a rewiring of the immune response. However, they have not normalized the cytokine levels to the bacterial loads, as the differences in the cytokines might be attributed purely to a difference in bacterial proliferation/clearing.

(6) The authors mention that switching from SE-high to SE-low at 3 or 6 h time points does not reduce mortality. Have the authors considered the reverse? Does hyperglycemia after euglycemia initially, worsen mortality? That would really conclude that there is some metabolic reprogramming happening at the very onset of sepsis and it is a lost battle after that.

Author response:

The following is the authors’ response to the current reviews.

We thank the reviewers and editor for their careful review of our work. We believe the resulting manuscript is much stronger. We agree with the comments made by Reviewer #2 regarding additional histology and neuronal data analysis, which will be presented in subsequent work.

The following is the authors’ response to the original reviews.

Reviewer 1 (Public Weaknesses):

It was not always clear what the lesion size was. This information is important for future applica- tions, for example, in the visual cortex, where neurons are organized in retinotopy patterns.

We thank the reviewer for this feedback. While there is some variation in lesion volume for a given parameter set, we have added more details of the volumes of lesions created in our testing (Fig. 4 and Fig. 5).

It would be helpful if the author could add some discussion about whether and how this method could be used in other types of array/multi-contact electrodes, such as passive neuropixels, S- probes, and so on. In addition, though an op-amp was used in the design, it would still be helpful if the author could provide a recommended range for the impedance of the electrodes.

We thank the reviewer for this suggestion. We have both added a demonstration of use in a differ- ent multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

“We demonstrated that our electrolytic lesioning technique works with a linear multicontact probe by testing with a U-Probe in ex vivo rabbit cortex. There are no particular limitations that would prevent our specific electrolytic lesioning technique and device from working with any passive multielectrode probe. The main requirements for use are that the probe has two electrodes that can directly (via whatever necessary adapters) connect to the lesioning device, such that arbitrary current can be passed into them as the anode and cathode. This would limit use of probes, like Neuropixels, where the on-chip acquisition and digitization circuitry generally precludes direct connection to electrodes [1], [2]. The impedance of the multielectrode probe should not be an issue, due to the use of an op amp. We showed use  with a Utah array (20-800 kΩ) and a U-Probe (1-1.5 MΩ). The specific op amp used here has a voltage range of ± 450 V, which assuming a desired output of 150 µA of current would limit electrode impedance to 6 MΩ. Though a different op amp could easily be used to accommodate a higher electrode impedance, it is unlikely that this would be necessary, since most electrodes have impedances between 100 kΩ to 1 MΩ [3].”

Reviewer 2 (Public Weaknesses):

In many of the figures, it is not clear what is shown and the analysis techniques are not well described.

We thank the reviewer for this feedback. We hope that our edits to both the figures and the text have improved clarity for readers.

The flexibility of lesioning/termination location is limited to the implantation site of the multielec- trode array, and thus less flexible compared to some of the other termination methods outlined in Appendix 2.

We thank the reviewer for this point. You are right that the lesioning location is limited to the multielectrode array’s implantation site, while other methods in Appendix 2 do not require prox- imity of the lesion location and the electrophysiology recording site. However, we believe that the closeness of the lesioning location to the microelectrode array is a strength - guaranteeing record- ings from the perilesional area - even with the small negative of reduced flexibility. Multielectrode arrays can be implanted in many areas of cortex. If one wanted to study distal effects of a lesion, additional electrophysiology probes could be implanted to record from those areas. We have noted this on Page 3, Line 117.

“While the link between the lesion location and the multielectrode location technically con- strains the lesion to an area of cortex in which a multielectrode array could be implanted, we see the connection as a positive, because it ensures recording some neuroelectrophysiology from the perilesional area in which recovery is hypothesized to occur (see Appendix 1Data Availabilityappendix.41).”

Although the extent of the damage created through the Utah array will vary based on anatomical structures, it is unclear what is the range of lesion volumes that can be created with this method, given a parameter set. It was also mentioned that they performed a non-exhaustive parameter search for the applied current amplitude and duration (Table S1/S2) to generate the most suitable lesion size but did not present the resulting lesion sizes from these parameter sets listed. Moreover, there’s a lack of histological data suggesting that the lesion size is precise and repeatable given the same current duration/amplitude, at the same location.

We thank the reviewer for this thoughtful feedback. We have added figures (Figs. 4 and 5), where we show the relationship between estimated lesion volume and the current amplitude and duration parameters. These figures include more data from the tests in Supplementary File 1 and Supplementary File 2. While there is some variation in lesion volume for a given current amplitude and duration, there is still a clear relationship between the parameters and lesion volume.

It is unclear what type of behavioral deficits can result from an electrolytic lesion this size and type (∼3 mm in diameter) in rhesus macaques, as the extent of the neuronal loss within the damaged parenchyma can be different from past lesioning studies.

While we appreciate the reviewer’s interest in the behavioral deficits associated with our lesions in rhesus macaques, reporting these falls beyond the scope of this manuscript. Future work will explore the behavioral deficits associated with these lesions

The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study.

We apologize for not being more explicit about the parameters used for the lesion in Monkey F. We have added this in Results on Page 5, Line 209 and in Methods on Page 19, Line 586.

“After this validation and refinement, one proof-of-concept lesion (150 µA direct current passed through adjacent electrodes for 45 seconds) was performed in an in vivo sedated rhe- sus macaque (Monkey F) in order to validate the safety of the procedure.”

“This lesion was created by applying 150 µA of direct current to two adjacent electrodes in the microelectrode array for 45 seconds.”

We also clarified the parameters used for the other lesions in Monkeys H and U in Results on Page 7, Line 233 and in Methods on Page 19, Line 586.

“In all of the fourteen lesions across two awake-behaving rhesus macaques (150 µA direct current passed through adjacent electrodes for 30 or 45 seconds (30s for Monkey U and 45s for Monkey H, except lesion H200120 which was for 50 seconds)), the current source worked as expected, providing a constant current throughout the duration of the procedure.”

“In these lesions, 150 µA of direct current was applied to two adjacent electrodes in the mi- croelectrode array for 30 or 45 seconds (30s for Monkey U, 45s for Monkey H), except in lesion H200120 where current was applied for 50 seconds.”

Monkey F was euthanized shortly after the lesion, so we now mention this on Page 19, Line 583.

“Based on this, and a lack of physiological signs of pain from the anaesthetized pig studies, a lesion was performed on a sedated rhesus macaque who was subsequently euthanized due to unrelated health complications (Monkey F; 16 year-old adult, male rhesus macaque) in order to further verify safety before use in awake-behaving rhesus.”

Because Monkey F was sedated and then euthanized shortly after, there is no behavioral data. As the lesion in sedated Monkey F was used to validate the safety of the procedure, any further data and analysis fall beyond the scope of this manuscript.

As an inactivation method, the electrophysiology recording in Figure 5 only showed a change in pairwise comparisons of clustered action potential waveforms at each electrode (%match) but not a direct measure of neuronal pre and post-lesioning. More evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning. Some exam- ples of this can be showing the number of spike clusters identified each day, as well as analyzing local field potential and multi-unit activity.

The reviewer has pointed out some short comings of the original analysis, which we believe have since been addressed with the revised analysis. LFP and spiking activity are functional measures that are more ambiguous in terms of loss and are also the subject of another manuscript currently under revision.

The advantages over recently developed lesioning techniques are not clear and are not discussed.

We thank the reviewer for noting this. We have added a section, also responding to their later request for us to compare our work to Khateeb et al. 2022, by adding a section to the Discussion on Page 16, Line 434.

“Perhaps the most unique advantage of our technique in comparison with other existing inactivation methods lies in Design Consideration #1: stable electrophysiology pre- and post-inactivation (Appendix 1Data Availabilityappendix.41). While several methods exist that allow for localization and size control of the inactivation (Design Consideration #2) and cross compatibility across regions and species (Design Consideration #3), few have achieved compatibility with stable electrophysiology. For example, some studies record electrophysiology only after the creation of the lesion, preventing comparison with baseline neuronal activity [4]. One recent study, Khateeb, et al., 2022, developed an inactivation method that is effectively combined with stable electrophysiology by creating photothrombotic lesions through a chronic cranial window integrated with an electrocorticography (ECoG) array [5], which may be appropriate for applications where local field potential (LFP) recording is sufficient. This approach has trade-offs with regards to the three design considerations presented in Appendix 1Data Availabilityappendix.41.

While Khateeb, et al., present a toolbox with integrated, stable electrophysiology from an ECoG array pre- and post- inactivation (Design Consideration #1), it demonstrated recordings from an ECoG array with limited spatial resolution. While a higher density ECoG array that would provide higher spatial resolution could be used, increasing the density of opaque electrodes might occlude optical penetration and constrain photothrombotic lesions. Further, ECoG arrays are limited to recording LFP, not electrophysiology at single neuron resolution, potentially missing meaningful changes in the neuronal population activity after lesioning. Khateeb, et al., demonstrated localization and control the size of inactivation (Design Consideration #2). In this manuscript, we have shown that the amount and duration of direct current are significant determinants of lesion size and shape, while with photothrombotic lesions, light intensity and aperture diameter are the significantly relevant parameters. One potential advantage of photothrombotic approaches is the use of optical tools to monitor anatomical and physiological changes after lesioning through the cranial window, though the research utility of this monitoring remains to be demonstrated.

Although the method presented by Khateeb, et al., shows some cross-compatibility (Design Consideration #3), it has greater limitations in comparison with the method presented here. For example, while Khateeb, et al., notes that the approach could be adapted for use in smaller organisms, no modification is needed for use in other species with this work’s approach–so long as a multielectrode probe is implantable. In this manuscript we demon- strate electrolytic lesioning spanning two multielectrode probes across rabbits, pigs, sheep, and rhesus macaques, and our same device could be easily used with other smaller species, like rats, in which multielectrode probes have been successfully implanted [6]. Further, the approach in Khateeb, et al., is limited to superficial brain structures, due to the need for opti- cal accessibility. As noted, fiber optics could allow access to deeper structures, which would bring associated additional tissue damage, but deeper structure lesioning was not demon- strated. In contrast, the approach presented here can be used in any region of cortex in which a multielectrode probe can be implanted, which, depending on the probe used, does not limit it to surface structures. For example, we demonstrated use of our lesioning tech- nique with a linear U-probe (Fig. 8figure.caption.25), which could be used to reach deeper layers of cortex or specific deep cortical structures. In both techniques, the location of the lesion is tied to the location of the electrophysiology (for Khateeb et al., wherever the cra- nial window and ECoG array are; for this technique, wherever the multielectrode probe has been implanted), which ensures that the electrophysiology will include recordings from the perilesional area. Neither work addresses the potential of their technique to induce chronic post-lesion behavioral effects, which is a key goal for future work.”

There is a lack of quantitative histological analysis of the change in neuronal morphology and loss.

We appreciate the reviewer’s desire for a quantitative histological analysis, however this falls out- side of the scope of this manuscript. We are not attempting to make strong claims about the number of neurons lost through lesioning or thoroughly characterize morphological changes in the neurons. The histology is intended to show that lesioning did lead to a loss of neurons, but the precise num- ber of neurons lost is neither in scope nor is likely to be highly conserved across lesions.

There is a lack of histology data across animals and on the reliability of their lesioning techniques across animals and experiments.

We thank the reviewer for this point. As stated above, we have now added Fig. 4 and Fig. 5, which includes volume estimates based on the histology from more of our ex vivo and in vivo testing across animals.

There is a lack of data on changes in cortical layers and structures across the lesioning and non- lesioning electrodes.

We acknowledge that the histology does not have the level of detail that is expected from many modern studies. However, the goal here was dramatically different: we sought to calibrate a novel lesion device, ensure it’s safe use in large mammals (specifically, non-human primates) and pro- vide estimates of the lesion size to compare with the literature. The extent of histology that could be performed and the tools available to us prevent such an in depth analysis. We can say based on shank length of the Utah arrays used and known anatomy that we have affected layer 2/3 and maybe a bit of layer 4.

Reviewer 1 (Recommendations For The Authors):

Figure 5b. It would be helpful if the author could plot the delta match separately for the lesion elec- trodes, near neighbor electrodes, and far neighbors. This would help understand the lesion effect, specifically whether the effect is selective (e.g., more potent for the lesion and adjacent electrodes.)

The fact that neuron loss is not particularly selective can already be seen in the spike waveform plots, arranged spatially on the array. Plenty of clear change is observed far from the lesion elec- trodes (marked with black dots) as well as nearby. We have made mention of this localized non- specificity in the main text and have ensured to remphasize in the figure legened. While a nice suggestion, we currently don’t feel this result rises to the level of a figure given it is not highly specific spatially.

Reviewer 2 (Recommendations For The Authors):

Overall the quality of the paper, the figures and the analysis used could be significantly improved. There is a lack of scientific rigor in the presentation of figures and analysis techniques. It is not clear what the authors are trying to communicate through the figures and their choice of figures to show is confusing (see below).

We thank the reviewer for their pointed critiques and believe we have addressed their concerns with many changes to the text, a revamped waveforms analysis, and both the expansion and addition of results.

The neurophysiology data shown doesn’t suggest neuronal loss, it only shows change which needs strong control data to show it is due to a lesion.

As detailed below, we have presented a revised analysis that provides this control. While the reviewer is right to point out we can distinguish actual neuron loss from neuron silencing, we be- lieve the new analysis rigorously indicates new rates of sample turnover beyond those expected from healthy state.

The histology figure should be replaced with a high-quality representation without folds.

We understand the reviewer’s suggestion. While ideally we would have many histology slices from each lesion, due to cost, we were only able to collect one histology slice per lesion. The folds were introduced by the company that performed the H&E staining, and we unfortunately cannot remove the folds. Therefore, despite the folds, this is the best and only image from this lesion. We hope that the markings on the figure and the comment in the caption is sufficient to explain to readers that the folds are not a result of the lesion but instead a result of the histology process.

The authors suggest that this lesioning method will be compatible with any available multielec- trode probe theoretically. Since all testing was done with a Utah array, it will be helpful to add an explanation about potential constraints that will make a given array compatible with this method.

We thank the reviewer for this suggestion. As stated above, we have both added a demonstration of use in a different multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

The authors should cite and discuss previous studies using electrolytic lesioning in awake-behaving animals to study the causal connection between the brain and behavior. (One example study: Morissette MC, Boye SM. Electrolytic lesions of the habenula attenuate brain stimulation reward. Behavioural brain research. 2008 Feb 11;187(1):17-26.)

We thank the reviewers for this suggestion. We have added a mention of existing electrolytic le- sioning studies on Page 2, Line 88.

“Prior termination studies mostly measure behavioral output, with no simultaneous measures of neuronal activity during the behavior, impairing their ability to provide insight into the causal connection between the brain and behavior [7]–[11], or with no baseline (i.e., pre- lesion) measures of neuronal activity [4].”

The authors should compare their technique with other recent lesioning studies in primates (e.g. Khateeb et al, 2022)

We again thank the reviewer for this point. Specifically not mentioning Khateeb et al. 2022 was a submission error on our part; we cited the paper in Appendix 2 in the version uploaded to the eLife submission portal, but we had uploaded the version prior to citing it to bioRxiv. We have combined addressing this with addressing a previous comment, as mentioned above, with a section in the Discussion on Page 16, Line 434.

In Appendix 2, the authors suggest that a major limitation of optogenetics and chemogenetic in- activation methods is the lack of rhesus-compatible constructs. However, several viral constructs have successful implementation in rhesus monkeys so far (e.g. Galvan A, Stauffer WR, Acker L, El-Shamayleh Y, Inoue KI, Ohayon S, Schmid MC. Nonhuman primate optogenetics: recent advances and future directions. Journal of Neuroscience. 2017 Nov 8;37(45):10894-903; Tremblay et al, Neuron 2020)

We thank the reviewer for pointing us to these papers. We have added a more thorough description of what we meant by lack of rhesus-compatible constructs in that Appendix.

“However, other challenges exist with using optogenetics as an inactivation method in nonhu- man primates, including difficulty reliably affecting behavior [12]. While several constructs for rhesus macaques have been developed [13], [14], reports of successfully inducing be- havioral effects have a small effect size and are less numerous than might be expected [12], and several null results have been published [15]–[17]. Other remaining challenges include the need to develop a head-mounted, battery powered light delivery system for multi-day delivery of light and difficulty integrating illumination with simultaneous chronic neuro- electrophysiology.”

For Figure 5b, only pairwise comparison results from monkey U (L11-14) are shown. It is unclear why such results from monkey H were shown in Figure 5a but not in 5b.

We thank the reviewer for pointing out this unconventional one monkey result. As described in the original submission, we previously omitted Monkey H from the analysis in Figure 5b (now Figure 7) since some of the lesions were closely spaced together, preventing well defined pre- and post- lesion rates of turnover. Never-the-less we have included Monkey H in all the revised analysis and believe even the less cleanly separated data shows useful indications of neuron loss or silencing evoked by the lesion.

Behavioral data (during a motor task) from the awake behaving monkeys (U and H) would greatly strengthen the claim that this lesioning method is capable of creating a behavioral effect and can be adopted to study the relationship between neural function and behavior outcomes.

While we are grateful for the reviewer’s interest in the application of our lesioning technique to studies involving behavior, a behavioral analysis of the effects of our electrolytic lesions falls be- yond the scope of this Tools and Resources manuscript. We would also like to point out that we do not claim that we have achieved a behavioral deficit in this manuscript.

Figure 2 would benefit from an illustration of the Utah array placement and the location of the sites used for lesioning. The authors can either overlay the illustrations on the current ex-vivo and histology images or create a separate schematic to demonstrate that for the readers. Also, Figure 2B needs to be replaced with one without the folds to avoid confusion for the readers.

We have added Figure 2 - figure supplement 1, which shows both the location within the Utah array of the two electrodes used to create the lesions as well as the relative size of the surface area of the lesion and the array. Unfortunately, as the lesion was created under the array, the exact location of the array relative to the lesion is unknown.

As mentioned above, Figure 2B is the only histological image from that lesion. We hope that the markings in the image as well as the caption sufficiently explain that the folds are unrelated to the lesion itself.

Figure 3, the conical region is not well delineated. Data across animals and lesion volume with respect to different parameters should be included.

We have included a supplemental figure, Figure 3 - figure supplement 1, where we have used a dashed white line to clearly indicate the area of damaged parenchyma, in case it was not clear in Figure 3a. We have also added volume estimates from lesions across animals and different param- eters. The ex vivo estimates are shown in Figure 4 and the in vivo estimates are shown in Figure 5.

Figure 4: it is not clear what is being communicated, and where the voltage traces are from.

We thank the reviewer for noting this confusion. We have added some lines in the text to explain what the voltage traces show, both in the caption to Fig. 6 and in the text on Page 7, Line 238.

“Traces only capture the values while the lesioning device was turned on (45 seconds for most lesions and 50 seconds for lesion H200120). A) Voltage traces. Discontinuity at the beginning of the traces indicates transient voltages that were too rapid to be captured by the voltmeter, lasting between 0.13 and 0.33 s. The fluctuating voltages, especially the rapid in- crease in voltage at the beginning of lesioning, emphasize the importance of using a current source to deliver consistent amounts of current into the brain.”

“The voltage across the microelectrode array fluctuated much more than the current did, em- phasizing that we made the correct choice in using a current source to ensure delivery of consistent amounts of current into the brain (Fig. 6figure.caption.19).”

Figure 5: why did the authors choose to use matching units as a measure of the lesion? It is surprising that there are still units on the location that the authors claim to be a lesion. To clarify that it would be helpful to show the location of the lesion in Figure 4a. Also, what can we conclude about the lesion induction when we see units on the lesion electrode? The change in unit match shows that there is a change in the network (although the authors need to show control for that so we know those changes don’t happen due to natural dynamics). It is not clear what is the time duration for pre-pre and post-post (i.e. minutes, seconds, hours). Do these comparisons come from the same time frame or are they coming from two fragments of time for both pre and post- conditions?

Aside from post-mortem histology and tissue assays, there is no good way to confirm neuron loss with chronically implanted electrode arrays in nonhuman primates. Waveforms were chosen as they are the one readily isolated physical measure of the system we are injuring. Although functional measures of activity could indicate neuron loss (topic of following papers), there are many conceivable changes in firing rate patterns that could manifest spuriously as loss, making the estimation of loss even more ambiguous and challenging this way.

We believe the new Figure 7 will make the procedure much more clear, while also providing the control requested by the reviewer, illustrating that new statistical categories of altered waveforms emerge during a lesion, beyond those associated with typical changes in waveform composition within multi-unit recordings seen during recording sample turnover fom healthy animals. We further note that by confining this analysis to four day spans at most, we have limited the impact of daily sample turnover described in the literature (Gallego, 2020).

The time duration for pre-session versus pre-session (pre-post and post-post), is some multiple of the approximate 24 hours between each daily recording session. Therefore, since restricting our- selves to four days separation, between 24 and 96 hours. Spikes are sampled from successful trial periods (so on the order of seconds, compiled into minutes across the whole recording session). Although already described in the main text, these points have been reemphasized in the figure legend.

CNO (line 931) needs to be explained.

We thank the reviewer for this point. We have defined CNO and its relevance in Appendix 2.

“Additionally, chronic inactivation over days may be logistically challenging, as the half life of clozapine N-oxide (CNO, a ligand used to activate DREADD receptors) is on the order of hours.”

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Referee #2

Evidence, reproducibility and clarity

Summary:

Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously ex-pressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crys-tallography, cryo-EM, chromatography, and mass photometry, they reveal that unlike other dNKs, Gi dNK forms a tetramer. They characterize important regions required for tetramerization and pos-tulate that this tetramerization evolved to provide Gi dNK with a heightened affinity for deoxy-adenosine.

Major comments and questions:

• The claims in this manuscript are well-supported, and I found no major issues with experi-mental methods.
• The authors provide a structure of tetrameric dNK and suggest that this tetramer leads to the increased affinity to substrate compared to non-giardia dNKs. They also show through mu-tations that removing the novel dimerization regions decreases substrate affinity by 100-fold. However, I was left unclear about why the tetramer would lead to such high affinity for substrate compared to two dimers. This is especially notable, since the authors state that there are no signs of cooperativity, which is a common way that oligomerization may lead to heightened affinity. If the authors have no current evidence explaining this, they can con-sider adding a short amount of discussion speculating on the mechanism and future direc-tions of study.

Minor comments and questions:

• The authors state that dATP acts as a mixed inhibitor and not a simple competitive inhibitor, and that previous studies have shown that this is because the dNTP competes in two loca-tions (line 163). Is it also possible that competitive inhibition + allosteric regulation could be causing this behavior instead?
• In the introduction (line 93), non-TK1-like dNKs are described as "not structurally related to TK1-like". This left me unclear, are they still interrelated among themselves?
• I was left confused by lines 106-116 in the introduction, where the specificities of dNKs in giardia are discussed. This is touched upon again in the discussion, but it was not clear here that there are several deoxyribonucleotides unaccounted for.
• When describing enzyme assays (Line 145), the authors say there is no salt dependence, but there looks to be MgCl2 always included in the assays (presumably for the ATP).
• I was confused by the y-axis of Fig 2. How is enzyme activity lower when dAdo is added? I think I read "enzyme activity" as total substrate depleted, when it is actually referring ex-clusively to the given non-dAdo substrate in each column.
• Lines 239 - 255 and Figure 3 were a little unclear to me. Specifically, I was having trouble following in the text which dimer is in the ASU, which is symmetry related, and matching those terms with which are canonical and non-canonical.
• The authors suggest that in the experiment shown in Figure S9 (Line 285), low activity may be caused by minor impurities. I'm not sure why impurities would lower activity sig-nificantly. Could there be other differences in experimental conditions that are at play in-stead?
• (Optional) From looking at the crystallography stats, I think the authors can potentially push the resolution more. At higher resolutions, Rmerge may become high, but depending on the data collection strategy, Eiger detectors can lead to high Rmerge just out of sheer data redundancy. Cc 1/2 can be a more useful metric in these contexts.
• For Figure S8, the Polder map feature in Phenix are another option for showing ligand oc-cupancy in an unbiased way. Did the authors try this?
• It's disappointing that the tetramers show so much preferred orientation in the cryo-EM. With that said, while the nominal resolution is 4.8 Å, I think that with the streakiness the EM structure looks worse resolution than that.

Referees cross-commenting

Overall, I agree with Reviewer #1's evaluation, and don't have any further suggestions or thoughts at this time.

Significance

Medical relevance: G. intestinalis is a parasite that causes 190 million cases of giardiasis per year. While treatable, there is evidence that giardia are developing a resistance to the main treatment at the moment, metronidazole. Thus, the authors provide a compelling case for the medical relevance of their investigation of Gi dNK for further pharmaceutical development. They provide further evi-dence for this by showing that several deoxyadenosine analogs bind the dNK and inhibit giardia growth. This work represents a very useful first step into a potential avenue for medical develop-ment. It's important to note that clinical studies are not within the purview of this research. Howev-er, in the discussion, the authors provide several comments on the promise of this avenue for future research.

Conceptual, technical, and mechanistic relevance: Through biochemical and structural study, the authors provide a compelling framework to understand an enzyme that is very important to the unique lifestyle of giardia parasites. From an evolutionary standpoint, the authors provide insight into how giardia can survive even without major components of de novo DNA synthesis.

The authors principally use well-established tools and techniques of the enzymology field. but do so to characterize a unique and previously uncharacterized enzyme system. This enzyme proves to be notable not just for its medical significance, but because it is unique among its family (non-TK1-like deoxynucleotide kinases) in its strong affinity for substrate and tetrameric quater-nary structure. One relatively novel technique used in the study is mass photometry, which is a relatively new and exciting way to characterize native proteins at very low concentrations. Using this technique helps the authors overcome a common criticism of structural studies in which the high concentrations or crowding conditions of techniques like crystallography and cryo-EM may be inducing non-physiological oligomers.

In summary, this work represents a meaningful addition to the protein structure-function literature. While it will principally be of interest to basic/fundamental researchers who study the mechanistic detail of protein function and evolution, it also provides a foundation for future transla-tional work and antiparasitic drug design.

Reviewer's background: I received my PhD in chemistry studying the structure and function of another enzyme key to DNA metabolism (except in giardia), ribonucleotide reductase. My back-ground is in structural biology and biochemistry. I do not have sufficient expertise to comment on studies performed on G. intestinalis growth and susceptibility to deoxyadenosine analogs.

Author Response

The following is the authors’ response to the original reviews.

Public review

Reviewer 1

Zhang et al. tackle the important topic of primate-specific structural features of the brain and the link with functional specialization. The authors explore and compare gyral peaks of the human and macaque cortex through non-invasive neuroimagery, using convincing techniques that have been previously validated elsewhere. They show that nearly 60% of the macaque peaks are shared with humans, and use a multi-modal parcellation scheme to describe the spatial distribution of shared and unique gyral peaks in both species.

We thank the reviewer for his/her summary and affirmation of our work.

The claim is made that shared peaks are mainly located in lower-order cortical areas whereas unique peaks are located in higher-order regions, however, no systematic comparison is made. The authors then show that shared peaks are more consistently found across individuals than unique peaks, and show a positive but small and non-significant correlation between cross-individual counts of the shared peaks of the human and the macaque i.e. the authors show a non-significant trend for shared peaks that are more consistently found across humans to be those that are also more found across macaques.

Answer: We appreciate the reviewer for raising questions about our work. In order to provide a more systematic comparison for the conclusion that ‘shared peaks are mainly located in lowerorder cortical areas whereas unique peaks are located in higher-order regions’, we have conducted two additional experiments. Following the reviewers’ suggestions, we conducted a statistical analysis of the ratio of shared and unique peaks within different brain networks (as depicted in Figure 2 (b)), and also presented the specific distribution quantities of the two types of peaks in both low- and high-order brain networks (as detailed in the corresponding Table 1). Through these three experiments, we have obtained a more systematic and comprehensive conclusion that ‘shared peaks are more distributed in lower-order networks, while unique peaks are more in higher-order networks’.

In order to identify if unique and shared peaks could be identified based on the structural features of the cortical regions containing them, the authors compared them with t-tests. A correction for multiple comparisons should be applied and t-values reported. Graph-theoretical measures were applied to functional connectivity datasets (resting-state fMRI) and compared between unique and shared peak regions for each species separately. Again the absence of multiple comparison correction and t-values make the results hard to interpret. The same comment applies to the analysis reporting that shared peaks are surrounded by a larger number of brain regions than unique peaks. Finally, the potentially extremely interesting results about differential human gene expression of shared and unique peaks regions are not systematically reported e.g. the 28 genes identified are not listed and the selection procedure of 7 genes is not fully reported.

Answer: We appreciate the reviewer for their suggestions about the statistical analysis in our manuscript. Firstly, we applied False Discovery Rate (FDR) correction to all experiments involving multiple comparisons throughout the entire manuscript, and the corrected t-values are reported (Table 2-5 and A5-A6). Additionally, in response to the reviewers’ guidance regarding the gene analysis section, we provided a list of 28 genes (Table A7) selected by lasso, along with the t-values obtained from Welch’s t-test for the expression of the two type of peaks. The functions corresponding to the seven genes with final t-values below 0.05 are reported in Table 6.

The paper is well written and the methods used for data processing are very compelling i.e. the peak cluster extraction pipeline and cross-species registration. However, the analysis and especially the reporting of statistics, as they stand now, constitutes the main weakness of the paper. Some aspects of the statistical analysis need to be clarified.

Reviewer 2

The authors compared the cortical folding of human brains with folding in macaque monkey brains to reveal shared and unique locations of gyral peaks. The shared gyral peaks were located in cortical regions that are functionally similar and less changed in humans from those in macaques, while the locations of unique peaks in humans are in regions that have changed or expanded functions. These findings are important in that they suggest where human brains have changed more than macaque brains in their subsequent evolution from a common ancestor. The massive analysis of comparative results provides evidence of where humans and macaques are similar or different in cortical markers, as well as noting some of the variations within each of the two primates.

Answer: Gratitude to the reviewer for his/her summary and appreciation of our cross-species work.

Strengths:

The study includes massive detail.

Weaknesses:

The manuscript is too long and there is not enough focus on the main points.

Answer: We appreciate the reviewer for pointing out the shortcomings in our manuscript. Firstly, considering the manuscript is too long, we have chosen to retain only the core experiments and relevant analyses in the main text. Relatively minor conclusions have been moved to the supplementary information, such as original Table 1 is now moved to the Supplementary Information as Table A1 (locations of all shared clusters). Additionally, some non-essential expressions in the original manuscript have been removed.

Our experiments primarily revealed the existence of partially shared cortical landmarks, known as gyral peaks, in both humans and macaques. We found that these shared and unique peaks are mainly distributed across low- and high-order brain networks. To emphasize this main point, we added two experiments on top of the existing ones to provide a more systematic explanation of this conclusion. We conducted a statistical analysis of the ratio of shared and unique peaks within different brain networks (as depicted in Figure 2 (b)), and also presented the specific distribution quantities of the two types of peaks in both low- and high-order brain networks (as detailed in the corresponding Table 1). By combining the results of these two experiments with the original manuscript’s statistical findings on the proportions of the two type of peaks in different brain networks, the conclusion that ‘shared and unique peaks are predominantly located in low-order and high-order brain networks’ becomes more prominent.

A brief listing of previous views on why fissures form and what factors are important would be helpful.

Answer: In response to this suggestion from the reviewer, we have incorporated some previous views on why fissures form and what factors are important into the ‘Introduction’ section.

‘Cortical folds are important features of primate brains. The primary driver of cortical folding is the differential growth between cortical and subcortical layers. During the gyrification process in the cortex, areas with high-density stiff axonal fiber bundles towards gyri. The brain’s folding pattern formed through a series of complex processes. The folding patterns in the brain, formed through a series of complex processes, are found to play a crucial role in various cognitive and behavioral processes, including perception, action, and cognition (Fornito et al. 2004; Cachia et al. 2018; Yang et al. 2019; Whittle et al. 2009).’

Reviewer 1 (Recommendations For The Authors):

(1) Figure 3b shows a non-significant trend for shared peaks that are more consistently found across humans to be those that are also more found across macaques. In the discussion, lines 218-219, the fact that the correlation is not significant should be reported more clearly.

Answers: We thank the reviewer for this question. We revised the Line 218-219 (now Line 257-259) as follows: ‘2. Consistency: The inter-individual consistency of shared peaks within each species was greater than that of unique peaks. The consistency of shared peaks in the human and macaque brains exhibits a positive correlation (non-significant though).’

(2) It is not fully clear how much shared peaks are mostly distributed in the higher-order cortex, especially in the macaque. It is reported in the results lines 132-133 that ‘In the macaque brain, shared peak cluster centers most distributed in the V2, DMN, and CON (Figure.2 (d)), while unique peak cluster centers most distributed in the DMN, Language (Lan), and Dorsal-attention (DAN)’ but not further discussed. Please develop this point in the discussion. Further, the results presented in Figures 2 and A1 are actually quite different and this shall be better described in the results. Given that shared and unique peaks can be found in the same region, this analysis would gain importance by applying a comparison test for the selection of regions where the most shared or unique peaks are found. The sentence lines 306-308 should be accordingly revised.

It is hard to understand what the 0-3% corresponds to in Figures 2 and A1?

Please also correct in both legends and in the text the labeling of panels and add in the legends a brief description of panel (c). In the legend of Figure 2, ‘shared peaks’ in the second sentence shall be replaced by ‘unique peaks’.

Answers: We thank the reviewer for these questions and suggestions. Our responses to them are itemized as follows:

A1: In general, to clarify the distribution of shared and unique peaks in the high-order and loworder networks, we divided 12 brain networks in Cole-Anticevic atlas into the low-order networks (visual 1 (V1), visual 2 (V2), auditory (Aud), somatomotor (SMN), posterior multimodal (PMN), ventral multimodal (VMN), and orbito-affective networks (OAN)) and higher-order networks (include cingulo-opercular (CON), dorsal attention (DAN), language (Lan), frontoparietal (FPN), default mode network (DMN)) based on previous research (Golesorkhi et al. 2022; Ito, Hearne, and Cole 2020). On this lower/higher -order division, we reported the number of shared and unique peaks in both species in Author response table 1. It is found that, whether in humans or macaques, shared peaks are more distributed in lower-order networks, while unique peaks are more in higher-order networks. This observation is particularly pronounced in humans.

Author response table 1.

The number of shared and unique peaks in lower- and higher-order brain networks of the two species. Lower-order networks include visual 1 (V1), visual 2 (V2), auditory (Aud), somatomotor (SMN), posterior multimodal (PMN), ventral multimodal (VMN), and orbito-affective networks (OAN), higher-order networks include cingulo-opercular (CON), dorsal attention (DAN), language (Lan), frontoparietal (FPN), default-mode network (DMN).

In the main text, Figure 2 (referring to Author response figure 1 later in the text.) illustrates the proportions of shared and unique peaks across 12 brain networks in both species. In each pie chart, we have specifically highlighted the top three ranked brain regions. Although the pie chart also generally supports the above results, two brain networks deserve further discussion. They are DMN and CON, two higher-order networks that have higher ranks in terms of shared peak count (the second-ranked and the third-ranked on macaque shared peaks; the fourth-ranked and the fifth-ranked on human shared peaks).

The cingulo-opercular network (CON) is a brain network associated with action, goal, arousal, and pain. However, a study found three newly discovered areas of the primary motor cortex that exhibit strong functional connectivity with the CON region, forming a novel network known as the somato-cognitive action network (SCAN) (Gordon et al. 2023). The SCAN integrates body control (motor and autonomic) and action planning, consistent with the findings that aspects of higher-level executive control might derive from movement coordination (Llinás 2002; Gordon et al. 2023). CON may be shared in the form of the SCAN network across these two species. This could explain in part the results in Author response figure 1 that shared peaks are more on CONs.

Author response image 1.

Pie chart shows the count of shared and unique peaks across different brain networks for both human and macaque. Right panel shows the Cole-Anticevic (CA) networks (Ji et al. 2019) on human surface as a reference.

Default-mode network (DMN) is a ensemble of brain regions that are active in passive tasks, including the anterior and posterior cingulate cortex, medial and lateral parietal cortex, and medial prefrontal cortex (Buckner, Andrews-Hanna, and Schacter 2008). Although DMN is considered a higher-order brain network, numerous studies have provided evidence of its homologous presence in both humans and macaques. Many existing studies have confirmed the similarity between the DMN regions in humans and macaques from various perspectives, including cytoarchitectonic (Parvizi et al. 2006; Buckner, Andrews-Hanna, and Schacter 2008; Caminiti et al. 2010) and anatomical tracing (Vincent et al. 2007). These studies all support the notion that some elements of the DMN may be conserved across primate species (Mantini et al. 2011). In general, the partial sharing of DMN between humans and macaques may be attributed to the higher occurrence of shared peaks within the DMN.

These results have been added to Table 2 along with corresponding text and discussion section.

A2: The difference between the results of Figure 2 and Figure A1 (now Figure A2) is whether the peak count is normalized by cortical area, which hugely varies across networks. For example, among the 12 brain networks, the three networks with the largest surface areas are the DMN, SMN and CON, and the three networks with the smallest area are OAN, PMN and VMN. The area difference between networks can be as large as 18-fold. Therefore, it is not difficult to find that, although the DMN ranks high in both shared and unique peak counts during statistical analysis (Figure 2 (a)), it is relatively small in Figure A2 after area normalization. In contrast, VMN ranks lower in peak count statistics but exhibits a substantial proportion after area normalization (For example, 38% of macaque shared peaks are distributed in the VMN region, but there are actually only four peaks). However, the two pie charts deliver the same message that there are more shared peaks in lower-order networks, while unique peaks are more in higher-order networks (except for macaques, where shared peaks are also distributed significantly in DMN and CON).

Following the suggestion from the reviewer, we adopted a new approach to present the ratio between shared peak count and unique peak count for each network (see Author response figure 2), such that the networks where the most shared or unique peaks are found can be easily highlighted. To mitigate potential imbalances in proportions caused by differences in the absolute numbers of each category (shared or unique) of peak, the proportions of peaks within their respective categories were utilized in the calculations. In Author response figure 2, the pink and green color bins represent ratios of shared and unique peaks, respectively. The dark blue dashed line represents the 50% reference line. In general, from left to right in the figure, the ratio of shared peaks decreases gradually while the ratio of unique peaks increases, suggesting that shared peaks are more (>0.5, above the dashed line) on lower-order networks (orange font), while unique peaks are generally more on higher-order networks (blue font). In specific, in human brains, the networks with a higher abundance of shared peaks are Aud, VMN, V1, SMN, and V2; whereas in macaques, they are CON, VMN, V1, V2, FPN, and SMN. Again, in the human brains, the disparity between shared and unique peaks tends to be more significant (further away from the reference line), for both lower-order and higher-order networks, respectively. In contrast, in the macaque brains, the disparity between shared and unique peaks is less significant (closer to the reference line). The ratio of shared and unique peaks is around 0.5 for 6 out of all 10 networks (including both lower and higher-order ones).

Author response image 2.

The ratio of shared and unique peaks in each brain network in the Cole-Anticevic (CA) atlas. The pink and green color bins represent ratios of shared and unique peaks, respectively. The dark blue dashed line represents the 50% reference line. For each brain region, the sum of the ratios of shared and unique peaks is equal to 1.

Based on these analyses, the sentence lines 306-308 (now Line 368-370) has been revised as follows: ‘In the human brain, the more shared peaks (about 65%) are located in lower-order brain regions, while unique peaks are mainly (about 74%) located in higher-order regions. However, this trend is relatively less pronounced in the macaque brain.’

These results have been added to Figure 2 (b) along with corresponding text and discussion section.

A3: In response to the third suggestion from the reviewer, we have clearly labeled the brain region names corresponding to 0% to 3% in Figure 2 (now Figure 2 (a)) and Figure A1 (now Figure A2).

Author response image 3.

Pie chart shows the count of shared and unique peaks across different brain networks for both human and macaque. Right panel shows the Cole-Anticevic (CA) networks (Ji et al. 2019) on human surface as a reference.

A4: Finally, we would like to express our gratitude to the reviewer for pointing out our mistakes.

We have made improvements to Figure 2 and revised the figure captions accordingly.

(3) The conclusions regarding the spatial relationship between peaks and functional regions shall be revised (Lines 187-188, 228-229, and 329-330). In the macaque, the results are opposite in the two atlases used. Further, in the human, it is not clear how multiple comparison corrections will impact statistics and some atlases show opposite results, although conclusions hold true in the majority of human atlases.

Answers: We thank the reviewer very much for this suggestion. We have added the results of the Cole-Anticevic atlas for macaques in the main text, which also has the observation that shared>unique (Author response table 2, corresponds to Table 5 in main text), namely, there are more diverse brain regions around shared peaks than around unique peaks. Therefore, out of the commonly used three macaque atlases, two (Markov91 and Cole-Anticevic) conform to this observation, while BA05 does not. We utilized false discovery rate (FDR) correction for multiple comparisons, and the corrected p-values are reported in Tables (in the revised main text and are shown below). Results on atlas with multiple resolutions are reported in Author response table 4) (Table A6 in the Supplementary Information). The observation that more diverse brain regions around shared peaks than around unique peaks, holds for human atlases in Author response table 3) (Table 4 in main text), where the atlas resolutions ranges from 7 parcels to 300 parcels, demonstrating the robustness of the conclusion. It is noted that the observation is not consistent on atlases with relatively lower resolutions (e.g., BA05 for macaque, n=30 and Yeo2011 for human, n=7) or, in particular, higher resolutions (e.g., Schaefer-500, and Vosdewael-400, n>300). This inconsistency could be reasonable since the resolution of the parcellation itself will largely determines the chance of a cortical region appear in a peak’s neighborhood, if the parcellation is too coarse or too fine. For example, if n=1 (the entire cortex is the only one region) or n=30k (each vertex is a region), each peak will has the same number of neighboring regions for these two extreme cases (one brain region for each peak for n=1; around 30 vertices for each peak for n=30k).

In conclusion, we observed that there are more diverse brain regions around shared peaks than around unique peaks for multiple brain atlases with a median parcellation resolution. These results have been added to Tables 4, 5, and A6 along with corresponding text and discussion section.

Author response table 2.

The mean values (±SD) of brain regions that appeared within a 3-ring neighborhood for shared and unique peaks in 3 common macaque atlases. For both Markov91 and Cole-Anticevic atlas, the shared peaks has more variety of functional regions around it than the unique peaks. But for the altas BA05, the conclusion was reversed. The bold font represent the larger values between the shared peak and unique peaks. All p<0.001, after false discovery rate (FDR) corrected.

(4) For Tables 2-4, A4, and Figure 3a, please indicate in all the legends if values correspond to Mean plus minus Standard Deviation, report t-value, and n in the legend or in the text.

Answers: We thank the reviewer very much for this suggestion. We added the ‘mean (±SD)’ in the notes of Tables 2-4, A4 (now A6), and Figure 3 (a). All the t and n values of t-test are reported in tables or in the main text.

(5) Please create a statistical section in the Methods to describe more precisely the tests used e.g. for t-tests, if datasets follow a normal distribution with unknown variance. In the case of multiple comparisons like in e.g. Table 2-4, A4, please report what multiple comparisons correction was used to adjust the significance level.

Author response table 3.

The mean values (±SD) of brain regions that appeared within a 3-ring neighborhood for shared and unique peaks in 10 common human atlases. All the shared peaks in the table have a greater number of neighboring brain regions compared to the unique peaks. All p<0.001, false discovery rate (FDR) corrected.

Author response table 4.

The mean values (±SD) of brain regions where shared and unique peaks appeared within a 3-ring neighborhood in 21 common human atlases. The p-values were corrected by FDR.

Answers: Thanks for the reviewer’s suggestion, we added a ‘Statistic Analysis’ section in the ‘Materials and Methods’ part:

‘All variables used in the two-samples t-test follow a normal distribution check and all p-values were corrected for multiple comparisons using the false discovery rate (FDR) method. Moreover, in order to identify differently expressed genes between shared and unique peaks, we employed the Welch’s t-test, given the unequal sample sizes for shared and unique peaks. For all tests, a p-value <0.05 was considered significant (FDR corrected).’

For the experiments of multiple comparisons such as Table 2-4, A4 (now A6), etc., we have added explanations in the main text, multiple comparisons correction has been corrected by false discovery rate (FDR), p-value<0.05 is considered significant.

(6) It would be of great interest to provide the full list of the 28 genes that significantly contributed to the classification of shared and unique peaks. Please provide a description of the Welch’s t-test results. From the 7 genes selected, only two are discussed. Could the authors please describe briefly the function of the other genes? Although we understand that they are not associated with neuronal activity and brain function.

Answers: We thank the reviewer for these suggestions. We have provided a complete list of 28 genes selected by LASSO in the Author response table 5. Additionally, Welch’s t-test was employed to calculate p-values for the expression differences of each gene in shared and unique peak clusters, and the results are also reported in the Author response table 5.

Author response table 5.

The 28 genes selected by LASSO and their corresponding p-values from Welch’s t-test.

Seven genes showed significant differential expression between shared and unique peaks in Welch’s t-test. These genes were PECAM1, TLR1, SNAP29, DHRS4, BHMT2, PLBD1, KCNH5. Brief descriptions of their functions are listed in Author response table 6. All gene function descriptions were derived from the NCBI website (https://www.ncbi.nlm.nih.gov/).

These results have been added to Tables 6 and A7 along with corresponding text.

(6) For comparison, could the authors provide a supplementary figure of shared peak clusters like in Figure 1b but displayed on the surface of the macaque brain template?

Answers: We thank the reviewer very much for this suggestion and we have incorporated a display of shared peak clusters on the macaque brain template surface (Author response figure 4, corresponds to Figure A1 of Supplementary Information.)

(7) Could the author develop or rephrase the sentence lines 69-72 which remains unclear?

Answers: We appreciate the reviewer’s feedback and have revised this sentence to ensure clarity. The sentences from line 69 to 72 have been revised to ‘In the study of macaques, it has been observed that the peak consistently present across individuals is located on more curved gyri (S. Zhang, Chavoshnejad, et al. 2022). Similar conclusions have been drawn in human brain research (S. Zhang, T. Zhang, et al. 2023).’ Now, this sentence corresponds to lines 74-77 in the main text.

(8) Line 99: please indicate which section.

Author response table 6.

Seven genes were selected using LASSO that showed significant differential expression in shared and unique peaks.

Answers: We thank the reviewer very much for this suggestion and we revised this sentence to ‘The definition of peaks and the method for extracting peak clusters within each species are described in the Materials and Methods section’.

(9) In Figure 3b, please report R2 and p-value. A semi-log might be more appropriate given the overdispersion of Human Peak Counts.

Answers: We thank the reviewer very much for this suggestion. Linear regression analysis was conducted on the average counts of all corresponding shared peak clusters of human and macaque. The horizontal and vertical axes of the Author response figure 5 (b) represent the average count of shared peaks in the macaque and human brains, respectively. The Pearson correlation coefficient (PCC) of the interspecies consistency of the left and right brain is 0.20 and 0.26 (p>0.05 for both), respectively. The result of linear regression shows that there is a positive correlation in the inter-individual consistency of shared peaks between macaque and human brains, but it is not statistically significant (with R2 for the left and right brain are 0.07 and 0.01, respectively).

Author response image 4.

Shared peak clusters of macaque, shows on macaque brain template.

The goodness of fit (R2), pearson correlation coefficient (PCC), and their respective p-values were indicated in Author response figure 5 (b). To avoid overdispersion, the peak count of the human brain is displayed in a semi-log format.

The updated Figure and results are presented in Figure 3 of the main text.

(10) Line 177: please indicate where in the Supplementary Information.

Answers: Thank you for the reviewer’s reminder. We have incorporated the results of the human brain structural connectivity matrix into Table A5 in the Supplementary Information and provided corresponding indications in the main text.

(11) Line 226: please correct ‘(except for betweeness [and efficiency] of the’.

Answers: We thank the reviewer very much for this suggestion and we added ‘and efficiency’ in original Line 173 and 226 (now Line 206 and 267) after ‘betweeness’.

(12) The gene expression dataset used is from the Allen Human Brain Atlas (AHBA). Reference to Hawrylycz et al., 2012 Nature. 2012 Sep 20;489(7416):391-399. doi: 10.1038/nature11405 shall be made and abbreviation defined at first use in the text.

Answers: We added the full name ‘Allen Human Brain Atlas’ when AHBA is first mentioned, along with the reference suggested by the reviewer.

Author response image 5.

(a) Mean peak count (±SD) covered by shared and unique peak clusters in two species. ***indicates p<0.001. The t-values for the t-tests in humans and macaques are 4.74 and 2.67, respectively. (b) Linear regression results of the consistency of peak clusters shared between macaque and human brains. The pink and blue colors represent the left and right hemispheres, respectively. The results of the linear regression are depicted in the figure. While there was a positive correlation observed in the consistency of gyral peaks between macaque and human, the obtained p-value for the fitted results exceeded the significance threshold of 0.05.

(13) Line 17: remove ‘are’.

Answers: We thank the reviewer very much for this suggestion and we removed ‘are’ in Line 17 (now Line 18).

(14) Line 201: remove ‘is used’.

Answers: We thank the reviewer very much for this suggestion and we removed ‘is used’ in Line 201 (now Line 237).

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Caminiti, Roberto et al. (2010). “Understanding the parietal lobe syndrome from a neurophysiological and evolutionary perspective”. In: European Journal of Neuroscience 31.12, pp. 2320–2340.

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Golesorkhi, Mehrshad et al. (2022). “From temporal to spatial topography: hierarchy of neural dynamics in higher-and lower-order networks shapes their complexity”. In: Cerebral Cortex 32.24, pp. 5637–5653.

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Zhang, Songyao, Poorya Chavoshnejad, et al. (2022). “Gyral peaks: Novel gyral landmarks in developing macaque brains”. In: Human Brain Mapping 43.15, pp. 4540–4555.

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Author Response

We thank both the editors and the Reviewers for their thoughtful comments and recommendations, that will certainly help us improve the manuscript. Below we address in a brief format some of the comments made, and then outline the changes to the manuscript that we plan to implement in the revision.

We see three interrelated issues in the comments of the Reviewers:

• the length and complexity of the manuscript;

• the link to previously proposed formalisms;

• the impact of adopting the proposed information-theoretic framework.

With regard to all of these issues, we would first like to highlight that the overall goal of our effort was to integrate con tributions to understanding the mechanisms underlying cognitive control across multiple different disciplines, using the information theoretic framework as a common formalism, while respecting and building on prior efforts as much as possible. Accordingly, we sought to be as explicit as possible about how we bridge from prior work using information theory, as well as neural networks and dynamical systems theory, which contributed to length of the original manuscript. While we continue to consider this an important goal, we will do our best to shorten and clarify the main exposition by reorganizing the manuscript as suggested by Reviewer #1 (i.e., in a way that is similar to what we did in our previous Nature Physics paper on multitasking). Specifically, we will move a substantially greater amount of the bridging material to the Supple mentary Information (SI), including the detailed discussion of the Stroop task, and the description of the link to Koechlin & Summerfield’s [L1] information theory formalism. We will also now include an outline of the full model at the beginning of the manuscript, that includes control and learning, and then more succinctly describe simplifications that focus on specific issues and applications in the remainder of the document.

Along similar lines, we will revise and harmonize our presentation of the formalism and notations, to make these more consistent, clearer and more concise throughout the document. Again, some of the inconsistencies in notation arose from our initial description of previous work, and in particular that of Koechlin & Summerfield[L1] that was an important inspiration for our work but that used slightly different notations. An important motivation for our introduction of new notation was that their formulation focused on the performance of a single task at a time, whereas a primary goal of our work was to extend the information theoretic treatment to simultaneous performance of multiple tasks. That is, in focusing on single tasks, Koechlin & Summerfield could refer to a task simply as a direct association between stimuli and responses, whereas we required a way of being able to refer to sets of tasks performed at once (”multitasks”), which in turn required specification of internal pathways. Moreover, they do not provide a mechanism to compute the conditional information Q(a|s) of a response/action s conditioned to a stimulus s does not provide a way to compute it explicitly. Our formalism instead provides a way to explicitly unpack this expression in terms of the efficacies –automatic (Eq. 5) or controlled (Eq. 15)– which can also account for the competition between different stimuli {s1, s2, . . . sn}. It also describes explicitly the competition between multiple tasks (Eq. 18, and Eq. 25 for multiple layers), because different ways of processing schemes for the same combinations of stimuli/responses can incur different levels of internal dependencies and thus require different control strategies.

To mitigate any confusion over terminology we will, as noted above, move a detailed discussion of Koechlin & Summer- field’s formulation, and how it maps to the one we present, to the SI, while taking care to introduce ours clearly at the beginning of the main document, and use it consistently throughout the remainder of the document. We will also make an important distinction – between informational and cognitive costs – more clearly, that we did not do adequately in the original manuscript.

Finally, to more clearly and concretely convey what we consider to be the most important contributions, we will restrict the number of examples we present to ones that relate most directly to the central points (e.g., the effect and limits of control in the presence of interference, and the differences in control strategy under limited temporal horizons). Accompanying our revision, we will also provide a full point-by-point response to the comments and questions raised by the Reviewers. We summarize some the key points we will address below.

PRELIMINARY REPLY TO THE REPORT OF REVIEWER #1

We want to thank the Reviewer for the time and effort put into reviewing our paper and constructive feedback that was provided. We also thank the Reviewer for recognizing the need for a clear computational account of how ”control” manages conflicts by scheduling tasks to be executed in parallel versus serially, and for the positive evaluation on our “efforts of the authors to give these intuitions a more concrete computational grounding.”. As noted in the general reply above, we regret the lack of clarity in several parts of the manuscript and in our introduction and use of the formalism. We consider the following to be the main points to be addressed:

• the role of task graphs and their mapping to standard neural architectures

• the description of entropy and related information-theoretic concepts;

• confusing choice of symbols in our notation between stimuli/responses and serialization/reconfiguration costs;

• missing definition of response time;

Regarding the first part point, we acknowledge that the network architectures we focus on do not draw direct inspiration from conventional machine learning models. Instead, our approach is rooted in the longstanding tradition of using (often simpler, but also more readily interpretable) neural network models to address human cognitive function and how this may be implemented in the brain [L2]; and, in particular, the mechanisms underlying cognitive control (e.g., [L3, L4]). In this context, we emphasize that, for analytical clarity, we deliberately abstract away from many biological details, in an effort to identify those principles of function that are most relevant to cognitive function. Nevertheless, our network architecture is inspired by two concepts that are central to neurobiological mechanisms of control: inhibition and gain modulation. Specifi- cally, we incorporate mutual inhibition among neural processing units, a feature represented by the parameter β. This aspect of our model is consistent with biologically inspired frameworks of neural processing, such as those discussed by Munakata et al. (2011)[L5], reflecting the competitive dynamics observed in neural circuits. Moreover, we introduce the parameter ν to represent a strictly modulatory form of control, akin to the role of neuromodulators in the brain. This modulatory control adjusts the sensitivity of a node to differences among its inputs (e.g., Servan-Schreiber, Printz, & Cohen, (1990)[L6]; Aston-Jones & Cohen (2005)[L7]). Finally, as the Reviewer notes, additional hidden layers can improve expressivity in neural networks, enabling the efficient implementation of more complex tasks, and are a universal feature of biological and artificial neural systems. We thus examined multitasking capability under the assumption that multiple hidden layers are present in a network; irrespective of whether they are needed to implement the corresponding tasks.

Regarding the second point, as noted above, we believe that the confusion arose from our review of the work by Koechlin & Summerfield. In their formalism, in which an action a is chosen (from a set of potential actions) with probability p(a), the cost of choosing that action is − log p(a). This is usually referred to as the information content or, alternatively, the localized entropy [L8]. As the Reviewer correctly observed, the canonical (Shannon) entropy is actually the expectation lEa[− log p(a)] over the localized entropies of a set of actions. In summarizing their formulation, we misleadingly stated that ”they used standard Shannon entropy formalism as a measure of the information required to select the action a.” We will now correct this to state: “[..] they used local entropy (− log p(a)) as a measure of the information required to select the action a, that can be treated as the cost of choosing that action.” We follow this formulation in our own, referring to informational cost as Ψ, and generalizing this to include cases in which more than one action may be chosen to perform at a time.

Regarding the third point, the confusion is due to our use of the letters S and R for both the stimulus and response units (in Sec. II.B) and then serialization and reconstruction costs (in eqs 31-33). We will fix this by renaming the serialization and reconstruction costs more explicitly as S er and Rec.

Finally, we realized we never explicitly stated the expression of the response time we used, but only pointed to it in the literature. In the manuscript we used the expression given in Eq. 53 of [L9], which provides response times as function of the error rates ER and the number of options .

PRELIMINARY REPLY TO THE REPORT OF REVIEWER #2

We want to thank the Reviewer for recognizing our effort to ”rigorously synthesize ideas about multi-tasking within an information-theoretic framework” and its potential. We also thank the Reviewer for the careful comments.

To our best understanding, and similarly to Reviewer #1, the main comments of the Reviewer are on:

• the length and density of the paper;

• the presentation of the Koechlin & Summerfield’s formalism, and the mismatch/lack of clarity of ours in certain points;

• the added value of the information theoretic formalism.

Regarding the first two points, which are common to Reviewer #1, we plan to move a significant part of the manuscript to the Supplementary Information, both to improve readability and make the manuscript shorter, as well as to provide one consistent and cleaner formalism (in particular with regards to the typos and errors highlighted by the Reviewer). In par- ticular, with respect to the comment on Eq. 4-5-6, we will clarify that the probability p[ fi j] is the probability that a certain input dimension (i in this case) is selected by on node j to produce its response (averaged over the individual inputs in each input dimension). We will also take care to make sure that the definition and domain of the various probabilities and probability distributions we use are clearly delineated (e.g. where the costs computed for tasks and task pathways come from).

Regarding the third point, we hope that our work offers value in at least two ways: i) it helps bring unity to ideas and descriptions about the capacity constraints associated with cognitive control that have previously been articulated in different forms (viz., neural networks, dynamical systems, and statistical mechanical accounts); and ii) doing so within an information theoretic framework not only lends rigor and precision to the formulation, but also allows us to cast the allocation of control in normative form – that is, as an optimization problem in which the agent seeks to minimize costs while maximizing gains. While we do not address specific empirical phenomena or datasets in the present treatment, we have done our best to provide examples showing that: a) our information theoretic formulation aligns with treatments using other formalisms that have been used to address empirical phenomena (e.g., with neural network models of the Stroop task); and b) our formulation can be used as a framework for providing a normative approach to widely studied empirical phenomena (e.g., the transition from control-dependent to automatic processing during skill acquisition) that, to date, have been addressed largely from a descriptive perspective; and that it can provide a formally rigorous approach to addressing such phenomena.

[L1] E. Koechlin and C. Summerfield, Trends in cognitive sciences 11, 229 (2007).

[L2] J. L. McClelland, D. E. Rumelhart, P. R. Group, et al., Explorations in the Microstructure of Cognition 2, 216 (1986).

[L3] J. D. Cohen, K. Dunbar, and J. L. McClelland, Psychological Review 97, 332 (1990).

[L4] E. K. Miller and J. D. Cohen, Annual review of neuroscience 24, 167 (2001).

[L5] Y. Munakata, S. A. Herd, C. H. Chatham, B. E. Depue, M. T. Banich, and R. C. O’Reilly, Trends in cognitive sciences 15, 453 (2011).

[L6] D. Servan-Schreiber, H. Printz, and J. D. Cohen, Science 249, 892 (1990).

[L7] G. Aston-Jones and J. D. Cohen, Annu. Rev. Neurosci. 28, 403 (2005).

[L8] T. F. Varley, Plos one 19, e0297128 (2024).

[L9] T. McMillen and P. Holmes, Journal of Mathematical Psychology 50, 30 (2006).

[00'00'09] Ces travaux s'intéressent à la place et au rôle. [00'00'14] De l'école et des structures socio-éducatives dans la construction des iniques des inégalités sociales et scolaires. [00'00'21] Il étudie les processus de scolarisation en lien avec les processus de socialisation et ça, ça nous intéresse particulièrement dans l'équipe vissée. [00'00'33] Donc articulations, deux de ces processus sur les territoires. [00'00'38] Les plus sensibles. [00'00'41] Ces interrogations de recherche. Alors je vais en donner quelques-unes et puis il développera. Comment les professionnels gérent-ils les désordres dans des institutions éducatives normées? [00'00'53] Comment sont décennies? comment sont désignés, comment sont nommés ces enfants et ces jeunes en difficulté? [00'00'59] Quelles sont les politiques publiques mises en place pour les prévenir ou les gérer? qu'en est-il d'une prise en charge spécialisée ou d'une protection éducative? va prolonger le débat de ce matin. [00'01'09] Que nous apprennent aussi les évaluations des politiques et des programmes de prévention sur la violence à l'école, le décrochage scolaire ou la délinquance juvénile. [00'01'20] Benjamin moignard a coordonné une recherche qui s'intitulait lutter contre le décrochage scolaire par la prévention de la violence entre pairs et la justice restaurative en milieu scolaire. [00'01'32] Il codirige actuellement: [00'01'35] Une recherche à heiner avec eric debarbieux sur la prévention des violences à l'école. [00'01'42] Voilà. [00'01'46] Merci. [00'01'47] Cette présentation. [00'01'49] Merci beaucoup de l'invitation, genet. [00'01'52] C'est très agréable de pouvoir. [00'01'55] Venir vous présenter mes résultats de ces travaux et puis de d'intervenir dans le cadre d'une journée d'étude en plus qui qui s'inscrit dans la durée, parce que y'a pas beaucoup d'universités qui qui réussissent. [00'02'06] Comme ça avoir une septième édition. [00'02'09] De ces, de ces journées d'études. [00'02'12] Je suis très heureux de pouvoir y contribuer. [00'02'14] Contribuer cette année. [00'02'16] J'ai profité de l'occasion pour présenter un projet de recherche. [00'02'22] Pas de recherche de coloc. [00'02'24] Pas encore. [00'02'25] Officialisé, qui va être officialisé d'ici une dizaine de jours. [00'02'30] C'est un colloque, le seize, dix, sept, dix, huit novembre deux mille seize, qui se tiendra à l'université paris-est créteil. [00'02'36] Autour d'eux des nouvelles problématiques éducatives, avec un enjeu qui est celui de penser des questions. [00'02'43] Qui sont souvent prises sous l'angle des milieux professionnels. [00'02'49] Travail social d'un côté comme de l'autre. [00'02'52] L'un des enjeux de ce colloque, c'est penser ces questions ensemble et sur les trois journées. Il y aura une journée qui sera aussi consacrée à restituer des expérimentations de terrain, des projets de terrain autour de ces nouvelles problématiques éducatives. [00'03'07] Avec quatre langues dans ce colloque: français, l'anglais, le portugais espagnol. [00'03'12] Et des des intervenants du terrain, qui viendront aussi de ne pas mal d'endroits du monde. Donc, je me permets de me faire cette. Cette information peut intéresser certains d'entre vous. [00'03'30] Alors sur la ma présentation pour cet après-midi manger. [00'03'35] J'ai l'avantage intervenir en deuxième sept jours. [00'03'39] Agréable parce qu'en plus, j'avais une power point avant. Donc je savais qu'il y avait certaines choses sur laquelle je n'avais pas besoin de revenir. Donc, je vais pas reprendre un certain nombre de choses que tu as déjà présenté. [00'03'52] Qui ont été présentés ici ce matin sur les questions de définition notionnels. J'en parlerai un petit peu, mais mais assez rapidement. [00'04'01] Je vais m'appuyer pour pour mettre au travail avec vous cette question de d'une urgence, sur une recherche que j'ai menée autour des exclusions temporaires au collège. [00'04'13] En région parisienne. [00'04'15] En particulier, et en prenant c'est cette recherche comme un révélateur de la manière avec laquelle on on peut s'intéresser à ce que j'appelle la recomposition des espaces scolaires et éducatifs, comment les acteurs éducatifs, au sens large du terme, non pas seulement d'un point de vue, [00'04'35] Scolaire, mais bien aussi beaucoup d'autres acteurs éducatifs. Il y en a dans la salle. [00'04'39] Quand s'opère. Je pense à des dispositifs municipaux, para municipaux, je pense aux au développement, dans les conseils généraux, les conseils régionaux, d'un certain nombre de formes d'encadrement de la jeunesse qui n'existaient pas il y a dix ou quinze ans et qui marque une recomposition forte des espaces scolaires éducatifs et qui interrogent peut-être de manière [00'04'59] Un peu spécifique, mais certains aspects. [00'05'03] Des situations, des situations d'urgence. [00'05'08] Je vous présenterai cette salle intervention. Il y aura quatre grandes grandes parties. [00'05'15] D'abord poser et prendre le temps. [00'05'18] Une dizaine de minutes de, de poser le contexte et punir les entrées théoriques qui sont les miennes. [00'05'24] Pour voir un peu d'où je parle et puis mettre aussi éventuellement en discussion certains postulats que je vais poser d'entrée. Certains peuvent être un peu. [00'05'34] Choquant. J'ai passé un terme, en tout cas interpellé, donc n'hésitez pas à m'interpeller ensuite sur c'est sur ces postulats. [00'05'42] Mais c'est important de ne de situer de delahousse pas. [00'05'46] Deuxième partie de l'intervention. [00'05'50] Qui portera plus particulièrement sur le fait de rendre compte de ce que c'est que ces exclusions temporaires et de leurs mesures. Il y a très peu de mesures sur ces, ce type de sanction, qui est une sanction relativement dure dans l'arsenal. [00'06'05] Diy répressif à disposition des équipes. [00'06'08] Non que je prendrai le temps de présenter de manière assez détaillée. [00'06'13] Ces résultats. Là, une troisième partie n'en est sonné, dans laquelle je m'intéresserai plutôt au dispositif. [00'06'19] Qui prennent en charge les élèves qui sont temporairement exclus, et là, on touche bien à cette question de la recomposition des espaces scolaires éducatifs. Ces dispositifs, ce sont des dispositifs qui sont portés par des pairs qui sont portés. [00'06'34] Par des associations de prévention spécialisée qui sont portées par beaucoup de structures de l'intervention sociale et du travail social. [00'06'41] En dehors de de l'école et qui pose un certain nombre de questions sur les formes de prise en charge et sur la manière avec laquelle, peut-être, un certain nombre de situations d'urgence- j'y reviendrai- peuvent être masqués malgré. [00'06'54] Une volonté de prise en charge. [00'06'57] Ça nous amène à la quatrième. [00'07'00] Quatrième partie, qui viendra un peu ramasser les différents éléments qu'on aura pu voir avant pour les re positionner autour des des enjeux qu'on discute ensemble aujourd'hui. [00'07'15] Donc première partie. [00'07'18] Autour du, du contexte et de quelques précisions. [00'07'23] Épistémologiques qui permettront de dire un peu de delahousse par. [00'07'28] Premier élément. [00'07'30] Important, celui de que je considère, dans ses travaux avec d'autres, qu'on est dans une période de forte recomposition des espaces scolaires et éducatifs. [00'07'40] Dans les démocraties modernes et que c'est très important pour penser les questions éducatives aujourd'hui, pas seulement les pensées à l'aune des structures du vingtième siècle. Je le dis un peu vite comme ça, mais il y a un enjeu très fort à considérer que les frontières de la prise en charge des enfants [00'07'57] Et des jeunes. [00'07'58] Sont en train de bouger de manière relativement, relativement forte. [00'08'03] Que les territoires institutionnels de l'école, du travail social, de l'intervention sociale, des structures socio-éducatives, socioculturelles sont en train plutôt de s'entremêler que seulement de se distinguer, ce qui avait été un des marqueurs des formes de prise en charge éducative du vingtième siècle. [00'08'23] On voit que c'est ces institutions où ces structures deviennent de plus en plus perméables les unes aux autres, et que c'est un marqueur qui est partagé du point de vue international. Ce n'est pas simplement quelque chose qu'on observe en france. [00'08'38] C'est quelque chose qu'on qu'on observe de manière relativement cohérente dans la plupart des démocraties contemporaines. [00'08'45] Ce set, recomposition des espaces scolaires, éducatifs, elle va de pair- et ça, par contre, c'est un marqueur français très singulier- avec l'affirmation de l'échelon local dans les politiques éducatives. [00'08'58] Qui est relativement une inflammation relativement récente en france, beaucoup plus ancienne dans d'autres pays, beaucoup de pays anglo-saxons en particulier. En france, on considère que c'est depuis une dizaine ou quinzaine d'années, tout au plus on a une accélération. [00'09'12] Deux dispositifs locaux qui sont mis en place, développer, déployer. Avec un changement d'échelle, un changement d'ampleur, ça veut dire quoi? très concrètement? ça veut dire que on n'est plus seulement. Il y a toujours eu des dispositifs locaux, il y a toujours eu une action locale. [00'09'27] Éducative, mais elle n'a pas toujours été structurée comme elle l'est depuis une dizaine d'années. Je vais donner quelques exemples, par exemple sur les collectivités locales et territoriales, les conseils généraux, les conseils régionaux, dans leurs obligations administratives et légales. [00'09'43] Jusqu'à présent, il n'y a pas d'obligation à prendre en charge les aspects pédagogiques ou des problématiques éducatives. [00'09'52] Lié à l'école, par exemple, de manière forte. Pourtant, il y a beaucoup de dispositifs qui se démultiplient, qui se mettent en place depuis une dizaine, une quinzaine d'années, y compris en lien avec l'école, mais pas par des acteurs scolaires. Il y a des politiques locales qui sont développées et qui était inimaginable il y a encore quinze ans. [00'10'12] Une bonne et simple raison si je prends le registre scolaire, mais on pourrait en prendre d'autres sur le le registre scolaire en particulier, il était inimaginable d'avoir des travailleurs sociaux qui puisse s'intéresser à la prendre en charge, la question du décrochage scolaire, par exemple, de manière avec une ampleur. [00'10'31] Relativement forte. C'est le cas aujourd'hui et c'est quelque chose de relativement récent et il faut bien le positionner comme tel. [00'10'40] Deuxième élément de cette recomposition des espaces scolaires et éducatifs: le fait qu'on assiste à une mobilisation d'acteurs pluriels est, là aussi, qui s'accélère avec le temps. [00'10'50] On a des professionnels de l'éducation, on a des professionnels de l'intervention sociale et du travail social, mais on observe le fait que de plus en plus de professionnels du champ éducatif interviennent dans l'école. [00'11'04] De plus en plus aussi d'acteurs scolaires changent de métier. [00'11'08] Évolue pas beaucoup vont vers les métiers du travail social. [00'11'13] Mais en tout cas, on voit bien qu'il y a une une, une mobilité. [00'11'17] Qui est relativement récente et qui témoigne de cette recomposition des espaces scolaires et éducatifs. [00'11'25] Dernier élément sur ce premier point: deux, deux contexte, le fait que on assiste, [00'11'31] Selon les contextes nationaux. [00'11'33] À des recompositions différenciées. [00'11'36] Selon les pays. Je m'explique entre des pays où les systèmes éducatifs, les systèmes d'encadrement de la jeunesse sont plutôt anciens, liés à des révolutions industrielles, ne trouve pas dans dans tout le détail. [00'11'49] Historique. Mais, en gros, dans les pays qui ont massifier leurs systèmes d'enseignement dans les années soixante, dix, quatre, vingt, cette recomposition est plus marquée que dans d'autres pays, les pays dits émergents- je prends des raccourcis de vocabulaire parce qu'on pourrait discuter ces termes-là- mais qui sont des pays très puissants du point de vue économe. [00'12'09] Mike. Je pense à des pays d'amérique latine, par exemple, comme le brésil ou l'argentine, pour ne citer qu'eux, qui sont des pays où cette recomposition est encore très relative, parce que les institutions éducatives sont moins fortes que dans les pays à tradition démocratique plus anciennes. On voit que c'est souvent là-dessus que se se joue les choses. [00'12'28] Et on a un certain nombre de travaux qui attestent bien de ça. Je pense aux travaux d'un carré et paillé, par exemple, qui s'intéresse à la transformation des systèmes éducatifs dans les pays du sud et qui montre que même si on a des tendances fortes à la recomposition des espaces scolaires éducatifs, elle est distincte entre des pays européens et occidentaux en général. [00'12'48] Dans lesquels les institutions se rapprochent les unes des autres de manière de plus en plus forte et des pays en voie de développement dans lesquels les institutions sont plutôt dans une phase d'affirmation de structuration. Mais on peut imaginer, sans avoir une lecture seulement chronologique, mais on peut imaginer malgré tout que finalement, c'est recompose. [00'13'08] Mission soient liées à des mouvements historiques qu'on pourra retrouver, retrouver ailleurs. [00'13'14] Donc ça, c'est un premier élément. [00'13'16] De contexte épistémologique qui me semble important de de rappeler, sur lequel je reviendrai de manière plus illustrative. Là, c'est vraiment. [00'13'24] Pardon, posez les les les grandes notions et les grands axes théoriques. [00'13'31] Desquels je je pars. [00'13'33] Deuxième élément, il y en aura trois en tout. Le deuxième, c'est le fait de considérer, dans mes travaux, [00'13'41] Ce qu'on a appelé les hampes, les nouvelles problématiques éducatives. [00'13'45] Qui sont des qu'on appellera des objets de mobilisation partagée, qui peuvent recouper des situations d'urgence. Ces nouvelles problématiques éducatives, c'est quoi? pour être très concret, c'est par exemple la violence à l'école, par exemple le décrochage scolaire, par exemple les questions de genre. C'est par exemple: [00'14'03] Discrimination, etc. Etc. Des problématiques qui sont qui ont émergé dans l'espace scolaire d'abord, mais dans l'espace éducatif plus largement ensuite, de manière relativement récente. [00'14'17] Souvent ces nouvelles problématiques éducatives. Elles sont travaillées y compris scientifiquement. [00'14'22] Les unes indépendamment des autres. On a des spécialistes de la violence à l'école, on a des spécialistes du décrochage scolaire, on a des spécialistes des discriminations sexuées, racialisées, etc. Et il nous semble- et c'est un travail qu'on a fait avec stéphanie ruby, qui a un intérêt à essayer de penser ensemble ces nouvelles problématiques éducatives- [00'14'43] Et de les penser ensemble avec quatre grandes entrées pour pour les interroger. D'abord, ces nouvelles problématiques éducatives. Elles perturbent ou questionnent le fonctionnement ordinaire de l'école ou remettent en cause ses fonctions sociales élémentaires. C'est des thématiques- je les ai données- qui font que, qui alimentent cette idée d'institutions en crise déco. [00'15'02] En crise. [00'15'04] Qui est en crise toujours depuis en gros qu'elle et massifier. [00'15'09] À un moment donné. C'est aussi interroger cette ce constat. La deuxième élément: elle interroge l'articulation des, des, des [00'15'20] Missions traditionnelles de l'école autour de l'instruction, de la transmission des savoirs et de la construction des apprentissages avec des formes éducatives plus large. C'est quoi? très concrètement? ça renvoie toujours à cet éternel débat que vous connaissez bien, entre écoles comme espace d'instruction ou école comme espace d'éducation, et on pourrait [00'15'40] Repositionner la question sur d'autres espaces éducatifs. On assiste à une forme de scolarisation du ce, du social- c'est des termes qu'utilise chokri belaïd- et on voit de plus en plus de structures socio-éducatives. Non, ce n'est absolument pas le métier que de faire de l'école, finalement, proposer aussi des formes d'intervention très scolaire. [00'16'00] Ou même des thématiques d'intervention qui sont totalement liées à des enjeux scolaires. Par ailleurs, [00'16'06] Troisième élément, ces nouvelles problématiques éducatives. Elles ne peuvent être considérées que comme tel, qu'à la condition qu'elles soient le résultat d'une forme de promotion, de désignation et de publicisation. C'est le terme qu'utilise ces failles, en particulier dans le débat public. [00'16'24] Et qui les rendre, qui les rendent lisibles et intelligibles. Je prendrai un exemple pour essayer d'illustrer ça: l'exemple de la violence à l'école. [00'16'33] Que la violence à l'école aujourd'hui. Moi, je travaille beaucoup cette question-là depuis depuis longtemps maintenant. [00'16'38] Quand je dis, quand on me dit sur quoi vous travaillez en général, je m'amuse pas à répondre: la recomposition des espaces scolaires éducatives, mais parce que cela les joindre. [00'16'47] Jeudi: la violence à l'école. [00'16'49] Et non quand on est la violence à l'école. On a dû avoir du boulot. C'est violences à l'école. Il y a beaucoup de travail. [00'16'57] C'est vrai qu'on ne manque pas de boulot. [00'17'00] Sauf que si on considère ça de manière un peu distancé, [00'17'05] On est là typiquement dans un, un objet sociologique, mais qui est objet aussi un objet médiatique très fort. [00'17'13] Avec, finalement, un décalage extrêmement important entre la réalité sociale de ce qu'est la violence à l'école aujourd'hui. [00'17'20] Et son traitement médiatique, public, politique, qui en fait un problème énorme, alors que, très objectivement, on a des difficultés, on a des, des, des [00'17'30] Ce que dit anne barrère: on est dans un nouvel âge de l'organisation scolaire, avec une multiplication d'incidents, avec tout un tas de choses. Mais si je prends par contre la question des violences les plus durs, les plus les plus spectaculaires, jamais l'école n'a été aussi sûre qu'elle ne l'est aujourd'hui, y compris quand elle était réservée à une petite élite qui était pourtant très, très mobilisée. [00'17'51] Sur. Il y a des travaux historiques très intéressant là-dessus- qui raconte par exemple la manière avec laquelle, chaque année, dans le collège, [00'17'59] Le collège henri quatre à paris, il y avait des émeutes. [00'18'03] Lycéenne qui finissait par des morts, avec des surveillants et des surveillants généraux, je passais des heures dans la salle. [00'18'11] Des surveillants généraux qui étaient pendus. [00'18'13] Dans la cour des établissements fassent pas tout à fait. Vous imaginez la chose aujourd'hui coincée, c'est alors. Ça ne veut pas dire qu'il y a pas de violence à l'école, ça veut simplement dire que c'est typiquement le le le le type d'objets. [00'18'26] Qui peut être considéré comme un objet, qui est devenu un problème public qui fait l'objet d'une attention. [00'18'33] Médiatique particulière d'une attention politique particulière d'une attention scientifique. [00'18'38] Particulière et spécifique. Donc, ça ne veut pas dire qu'il n'y a pas d'objet, ça veut simplement dire qu'on recompose et qu'on reconsidère l'objet aussi comme un objet et un problème public. En d'autres termes, on va en considérer un certain nombre comme [00'18'54] Comme pertinent. Ça veut pas dire que les nouvelles problématiques éducatives suffisent à dire toute la réalité des difficultés ou des problèmes. [00'19'02] Que rencontre l'école ou les structures socio-éducatives. C'est simplement celles qui vont être l'objet d'une attention publique, médiatique, politique, scientifique, spécifique. [00'19'13] Dernier élément. Donc, bon, ça, je l'ai déjà dit. Elles sont désignées au travers d'objets comme l'échec scolaire, la violence à l'école, le décrochage de discriminations ethniques, racialisés, etc. [00'19'22] Et donc, c'est autour de ces problématiques éducatives que s'organisent les alliances éducatives dans des espaces scolaires et éducatifs recomposés. Je m'explique: c'est bien souvent sur ces thèmes-là- pas seulement sur ces thèmes là, mais beaucoup sur ces thèmes-là- que l'on va voir se rencontrer des professionnalité qui, jusqu'à présent, étaient plutôt séparées. [00'19'42] L'exemple des peureux est assez significatif par rapport à ça. Mes pères, eux, vont travailler ces questions de l'échec scolaire. C'est question du décrochage scolaire et d'autres questions encore. [00'19'52] Des structures jeunesse, des associations de proximité vont travailler ces questions et ces objets-là. Et donc, on voit bien, et il ne s'agit pas de dire que ce n'est pas pertinent de les travailler, mais il s'agit de de penser la manière avec laquelle, finalement, on en vient à voir des lignes qui bougent sur les espaces institutionnels. [00'20'13] Des institutions les unes par rapport aux autres, les territoires des prises en charge de la jeunesse, en particulier, sont en train de bouger fortement par rapport à ces, à ces éléments-là, bien entendu, il faut pas non plus réduire à ses seules questions- là, je ne traite pas là- des aspects socioculturels, culturels, qui [00'20'33] Norme est, mais ce n'est pas mon objet, dans le cadre d'objets qui sont plutôt ceux de la marge, de la déviance du problème, pour nous dire de manière très, très générique: [00'20'44] Alors on observe cette, cette évolution très forte, très forte, dennis. [00'20'48] Dernier élément: [00'20'50] De contexte, quelque chose là aussi, qui est qui, qui me semble. [00'20'54] Vraiment très, très important. [00'20'56] Le fait qu'on observe, dans cette recomposition des espaces scolaires, éducatifs, l'émergence, l'accélération. [00'21'03] De la présence des fameux dispositif. [00'21'07] Je suis sûr que, comme on a une salle composée en partie de deux personnes mobilisées sur le terrain éducatifs, vous avez tous la connaissance de multiples, voire d'une multitude de dispositifs spécifiques d'intervention. [00'21'22] Qui permettent la prise en charge d'un certain nombre de problèmes de questions. [00'21'27] Assez fort. [00'21'30] Ces dispositifs. C'est quelque chose qui est très issu du monde du travail social, de l'intervention sociale, qui était assez peu marquée dans le secteur de de l'école. [00'21'40] Et en france en particulier. C'est moins vrai dans d'autres pays, mais en france, c'est un marqueur important. On est dans un système. [00'21'47] D'école qui était très centralisée, très descendante, l'éducation nationale, très peu de place à aux espaces locaux. On sait que depuis deux mille cinq, il y a beaucoup de choses qu'on très fortement et évolué par rapport à ça. Je rentre pas dans le détail- et on a vu l'émergence de beaucoup de dispositifs dans l'école, autour de l'école. [00'22'07] Que barère dans une, dans un article de deux mille treize. [00'22'12] Elle propose trois, trois marqueurs pour pour définir ces dispositifs et je je pense qu'ils sont vraiment très, très, très opérant sur les questions qui nous, qui nous intéresse aujourd'hui. Mendy. [00'22'24] Les dispositifs émergent autour d'eux. D'abord, d'un rapport singulier de la structure à son territoire. Un dispositif, ce n'est pas un dispositif national. Un dispositif, c'est quelque chose qui répond à un besoin particulier, à un besoin local spécifique et particulier. Ce n'est pas une moi qui va dire ce qu'est un dispositif. D'accord, on est sur quelque. [00'22'44] Chose de plus immédiat, de plus lié à l'échelon local. Donc, ça recoupe ce que je présentais tout à l'heure sur la mobilisation de de l'échelon local. [00'22'54] Deuxième élément: on est sur un rapport singulier. [00'23'00] J'ai oublié de mettre mon chronomètre en marche. [00'23'02] Un rapport. [00'23'04] Singulier au public visé. Je voulais beaucoup insister là-dessus. [00'23'08] Entre traitements ciblés et universaliste. Ça veut dire quoi? ça veut dire que on est dans un moment aussi de l'action sociale, de l'intervention sociale, de l'action éducative, ou la désignation des publics n'est plus une désignation universelle comme elle a longtemps marqué. [00'23'26] Les politiques éducatives, qu'elles soient locales, nationales, voire internationales, au travers des des grandes wenger. [00'23'33] Ou d'institutions comme l'onu, dans la prise en charge des enfants et des jeunes, et c'est très vrai pour les enfants et les jeunes, même si c'est aussi vrai pour d'autres types de de population. [00'23'44] Qu'est-ce que ça veut dire très, très concrètement? ça veut dire, par exemple, que on en est sur des systèmes de désignation qui ne considèrent pu des niveaux et des bassins, par exemple des bassins territoriaux de manière générale, qui diront: mais tous les enfants et les jeunes de ce territoire ont droit à une prise en charge spécifique, parce qu [00'24'03] C'est un territoire ségréguées ou en difficulté. Ça existe encore, bien sûr, mais en termes de tendances qu'on voit de plus en plus arriver, c'est plutôt la désignation de ces territoires spécifiques dans lesquels, effectivement, il y a une concentration de populations dites en difficulté. Mais encore faut-il que lesdites populations, c [00'24'23] Sois prête. [00'24'25] Soit. [00'24'28] En mesure de mériter la prise en charge dont elles peuvent faire l'objet. [00'24'32] Évidemment, c'est moins vrai pour des cas extrêmement difficiles, extrêmement lourds, tels que les présenter bernadette ce matin. Mais de manière plus générique, je vais vous donner un exemple: l'éducation prioritaire en france. [00'24'43] Il y a un certain nombre de travaux qui montrent sa trame. Ça a très bien, c'est une tendance assez générale. Depuis, en gros, les années deux mille, on est passé d'une logique de zone- les ep- à une logique de plus en plus ciblée d'abord sur les établissements et puis ensuite sur les individus. Quelque chose qu'avait bien illustré ce mouvement là, mais qui est toujours d'une certaine actualité en deux mille six peu. [00'25'04] Que vous vous souvenez, on avait créé les internats d'excellence. [00'25'07] Les internats d'excellence, c'était dire: on va plus aider tous les enfants de zep à mieux réussir. [00'25'13] On renverse la manière avec laquelle on considère ça. On dit: on va permettre aux élèves les plus méritants. [00'25'20] De réussir et d'avoir accès aux meilleures écoles et donc de créer quelques internats d'excellence- il y en a une dizaine sur le territoire- qui doivent permettre d'accueillir ces élèves méritants et de leur donner. [00'25'35] Plus, le fameux, fameux slogan: donner plus à ceux qui ont ceux qui ont moins. Sauf qu'en termes de proportion, on passe de politique dans les années quatre, vingt quatre, vingt dix, qui donnent plus, même si on peut discuter ce plus, mais ce n'est pas l'objet d'aujourd'hui, mais on pourrait vraiment discuter juste un chiffre quand même, parce que ça [00'25'54] Préoccupe toujours. En deux mille dix, il y avait eu un en deux mille douze, un rapport de la de la cour des comptes qui montrait que l'etat investissait plus dans n'importe quel collège parisien que dans un collège. [00'26'10] Deux rep fin de de zep à l'époque de de rares. [00'26'14] Donc de l'éducation prioritaire de l'académie de créteil. [00'26'17] Et pourtant, c'était eux qui étaient censés être prioritaires. Mais quand on regarde au final, [00'26'21] Les dotations et l'inégalité de dotation entre les académies faisait que les des académies les plus en difficulté. C'est le cas de créteil, de versailles, de strasbourg, de marseille. [00'26'30] En particulier, était nettement moins doté d'autres académiquement. C'est une parenthèse. [00'26'35] Ce qu'il faut retenir là, sur cette question du rapport singulier pour au public visé, c'est cette, ce glissement vers le fait qu'on soit de plus en plus sur des publics dits méritants, sur des publics qui doivent faire la preuve de leurs capacités. [00'26'50] A être pris en charge. [00'26'52] Isabelle astier, le monde très bien dans un ouvrage que je vous recommande parce que c'est un des rares ouvrages qui qui travaille à la fois la question sociale et la question éducative. [00'27'00] Qui s'appelle les nouvelles règles du social, qui est un ouvrage qui est paru en deux mille sept et qui renvoie très, très bien à ça. Comment finalement, sur les populations les plus en difficulté? [00'27'11] On renvoie quand même une part de la responsabilité de la prise en charge à cette population. Il faut quand même, même si elles sont en difficulté, qu'elles se montre en capacité de d'être d'être prises en charge. Et les dispositifs sont des dispositifs qui visent des publics particuliers, pas seulement sur le seul aspect. [00'27'31] Deux du mérite, mais aussi sur le fait que ils visent des populations spécifiques, par exemple les élèves dits en décrochage, par exemple les élèves identifiés comme à risque pour tout un tas de sur tout un tas de de deux registres. [00'27'47] Donc, c'est pas des dispositifs qui s'adressent à tous ces dispositifs qui s'adressent à quelques-uns parmi une population. [00'27'53] Plus large. [00'27'55] Troisième élément: [00'27'56] De ces dispositifs, le fait que il pose un rapport singulier à l'organisation et à ses temporalités. C'est quelque chose de très important. Là aussi, c'est pas des politiques publiques lancées. [00'28'07] Avec un grand barouf, huit ministres présents, etc. On est pas sur ses registres là. On est sur des dispositifs qui renvoient au pouvoir des acteurs locaux d'agir. [00'28'17] Et avec cet impératif de l'action est souvent de l'action dans l'urgence, au sens où on a identifié un problème sur un terrain, sur un territoire, on doit pouvoir mettre en place une réponse politique, institutionnelle, éducative, sur le champ qui nous, qui nous intéresse ici, et on va mettre en place un dispositif, donc un dispositif de réussite. [00'28'37] A l'école, un dispositif de prise en charge du décrochage, un dispositif d'accès à la culture des jeunes, un dispositif de, etc. Etc. Qui vient traduire une politique. [00'28'47] Local, en sus de politique nationale éventuellement, mais un marqueur très fort sur le fait que on a constaté, on a diagnostiqué des besoins spécifiques sur un territoire qui soit en général pas si spécifique que ça, mais les acteurs locaux considèrent que inné, ce sont des des des des choses particulières qui nécessitent une intervention et la force. [00'29'07] Dispositif, c'est qu'elle le permet. [00'29'09] Sont des. Finalement, si on prend une image, la politique publique, c'est une armée qu'il faut organiser, réglementer, etc. Le dispositif, c'est un petit commando qui va très, très vite à mettre en place, c'est des cellules, c'est des choses dont on voit vraiment, il y a, il y a un basculement à partir des années deux mille. [00'29'30] Une accélération, depuis deux mille dix, à la démultiplication de ces types et de ses formes d'intervention. La [00'29'42] Donc l'enjeu- je rappelle un reste- c'était vraiment de situer quelques grands axes. [00'29'47] Théorique et épistémologique. Alors c'est toujours difficile à deux heures et demie. [00'29'51] Après le repas: [00'29'52] Mais si je le mets à la fin, ça ne fait pas, parce que ça m'obligerait à revenir systématiquement sur sur plein de choses. Mais on s'approche. [00'30'02] Deux choses un peu plus. [00'30'05] Un peu plus significative du point de vue du deux deux des résultats de recherche. [00'30'12] Avec donc l'après, je vous présente ce travail sur les exclusions temporaires, que j'utilise ici comme un révélateur des tensions à l'école et qui permet de réinterroger un certain nombre de choses autour du caractère d'urgence. Je vous ai dit, je viendrai à l'urgence plutôt en fin d'intervention pour pour ramasser et remettre tout ça en perspective. [00'30'34] Point, juste pour qu'on parte tous à peu près sur la laine. [00'30'40] Les mêmes considérations sur ce que c'est que ces exclusions temporaires. [00'30'44] Donc dont je vais vous parler après. Les exclusions temporaires sur les textes prévoient que ce sont des mesures qui doivent être exceptionnelles. [00'30'52] C'est ce que prévoient les textes. Il y a eu plusieurs réformes. Deux de ces textes [00'30'57] Qui sont liés au code de l'éducation, qui sont liées à ce qu'on appelle [00'31'00] D d? c'est pas le code. J'ai perdu les mesures disciplinaires dans le second degré. [00'31'06] Avec des choses extrêmement balisé. Je vais pas rentrer dans le détail de tout, c'est pas l'enjeu, mais simplement dire que ces exclusions temporaires, elles sont limitées à huit jours maximum. C'était trois semaines avant deux mille cinq. Après deux mille cinq, on est passé à huit jours maximum. [00'31'22] C'est des exclusions qui sont à la seule sous la responsabilité des chefs d'établissement. [00'31'28] Légalement, c'est qu'il y a que le chef d'établissement qui peut promulguer des exclusions temporaires. [00'31'33] La réalité est dans la vraie vie, mais beaucoup de de ces pays en particulier, [00'31'38] Qui ont une délégation de fait de la part du chef d'établissement pour promulguer ces exclusions temporaires. [00'31'46] Elles sont parmi les sanctions les plus dures à la disposition des personnels scolaires. [00'31'52] Après les exclusions temporaires, c'est l'exclusion définitive. [00'31'55] Là une précision: je vais parler seulement d'exclusion temporaire externalisée, c'est-à-dire dans les chiffres que je vais vous présenter. [00'32'03] Le texte législatif prévoit que une exclusion temporaire [00'32'07] Peut être faite à l'intérieur d'un établissement non prise en charge et continuité éducative dans un établissement pendant le temps de l'exclusion. Il y a l'obligation légale pour les acteurs scolaires de s'assurer de la continuité scolaire et éducative du parcours des élèves pendant le temps de leur exclusion, donc en d'autres termes, pendant les trois jours gamin est pas à l'école ou les quatre jours où les cinq jours [00'32'28] Les six jours, les huit jours, il est censé non seulement avoir du travail, mais en plus pouvoir rattraper le travail. [00'32'35] Qui est fait pendant le temps de son absence. En d'autres termes, ce que pensent les textes, c'est l'idée que il n'y a pas de double peine: le fait d'être à la fois exclu du temps scolaire et, en plus, d'être largué. [00'32'46] À son retour. [00'32'48] Absolument pas le cas. Qu'on soit très, très clair par rapport à ça. Il y a très peu d'établissements, il y en a quelques-uns, qui mettent en place cette continuité éducative, mais il y en a extrêmement peu. Moi, je parlerai là des exclusions temporaires et externalisées, et pas des exclusions temporaires internalisée. Il y en a quelques-unes, mais là encore, c'est très, très marginal, même si on voit que [00'33'08] Ça commence à venir. [00'33'10] Pourquoi on a travaillé cette question des exclusions temporaires, parce que il y a une corrélation très forte, que monte déjà beaucoup de travaux sur la la question, entre les exclusions scolaires et puis les phénomènes de décrochage, voire de de rupture scolaire. On a des liens, y compris de causalité, qu'on ne trouve pas si fréquemment que ça en matière d'éducation, qui sont [00'33'30] Extrêmement fort. [00'33'31] Sur le l'usage et le registre de l'exclusion temporaire. Il y avait ce premier argument là. L'autre argument, c'était con j'ai mené des des travaux sur d'autres questions dans les établissements scolaires et on constatait qu'il y avait un volume d'élèves exclus extrêmement significatif. [00'33'48] Avec parfois des tiers de classe entière, avec des élèves exclus temporairement. Et quand on a essayé d'avoir les chiffres sur les académies de créteil et de versailles, sur lesquels on travaillait, on a réussi à avoir aucun chiffre. Et plus que de ne pas avoir de chiffres, on a constaté qu'il y avait, de la part de l'institut, [00'34'08] Eussions scolaires. Un certain embarras sur ce sujet-là. [00'34'12] Quand on disait: mais combien est un gamin temporairement exclu? là, on nous répondait presque toujours. Je vais vous donner les chiffres des conseils de discipline. [00'34'21] Non, ce n'est pas. La question n'était pas tout à fait. Celle à la question, c'était: combien d'exclusion temporaire? les conseils de discipline, c'est quelque chose de complètement différent. Là, on a l'obligation légale d'avoir ces signes, des signalements et des remontés au rectorat, pas sur les exclusions temporaires. Et on a constaté que non seulement, il y avait une sorte d'invisibilité de ces rues, [00'34'41] Montée, mais que, même s'il n'y avait pas de chiffres officiels, il y avait une, une, une préoccupation très forte des acteurs éducatifs. [00'34'52] Et des acteurs institutionnels sur le fait que [00'34'56] Cette proportion d'actions temporaire était très forte, mais sans avoir de chiffres. Donc il n'en fallait pas plus pour qu'on s'y intéresse. [00'35'03] Ça faisait pas très, très plaisir et, en plus, on n'avait pas vraiment de de chiffres. Donc, on a essayé de travailler sur cette question-là. [00'35'13] Juste en quelques mots. [00'35'17] Je ne vais pas m'appesantir. Mais là aussi, c'est l'enjeu pour moi de vous présenter ces éléments-là. C'est en rester à une présentation de résultats. [00'35'27] Brut et surtout que il y a pas mal de de tableaux de chiffres, etc. Ça peut paraître un peu un peu. [00'35'33] Lointain ou froid. [00'35'36] On a travaillé ces questions sur plusieurs années, avec une première phase exploratoire dans trois départements. [00'35'42] Quatre vingt treize, soixante dix sept, quatre vingt onze. [00'35'45] Et on a pu travailler- c'est cette question- que parce qu'on avait des relais dans les établissements. [00'35'53] Scolaire en particulier. [00'35'55] Quand on faisait des demandes officielles de remontée des exclusions temporaires, on n'arrivait pas du tout. [00'36'01] Donc, je vais être très transparent là aussi. [00'36'03] On a réussi parce que moi, j'étais, j'intervenais dans le master deux. [00'36'09] Maif. C'est donc au fur et à mesure des années, mais j'ai un réseau de sur l'académie de créteil qui est extrêmement fort et extrêmement précieux. [00'36'18] Je leur fais une spéciale dédicace à la caméra, s'ils me regarde après au moment du montage, tout ça. [00'36'24] Parce que sans eux. [00'36'26] Y avait pas de remontée de ces données. [00'36'29] Il y avait quelques profs, il y avait aussi des chefs d'établissements croisés dans des des réseaux syndicaux ou militants par ailleurs, mais c'était quand même beaucoup les cèpes et beaucoup d'essais sur notre échantillon. Je vous présenterai en détail après qui n'ont pas remonté ces informations là officiellement. [00'36'46] Dire qu'on a accepté la mise en place d'un protocole de recension interne à la vie scolaire, qui nous ont fait remonter parce que les chefs d'établissement ne souhaitaient pas que ce soit remonté et trouvé que c'était une donnée trop sensible pour prendre le risque de dévoiler le volume de ces exclusions temporaires. Ce n'était pas systématiquement le cas. Il y a quand même eu un certain nombre d'établissements et [00'37'06] Prix. On a fait des restitutions en établissement sur ses ses relevés. [00'37'12] Mais très majoritairement quand même, c'était des données plutôt données en. [00'37'18] En vis-à-vis. [00'37'22] Au départ aussi, je vous dis tout comme ça: [00'37'24] On est à l'aise. On avait imaginé, vous savez, le vrai fantasme de sociologue. On avait une grille de deux mesures, de ces exclusions temporaires, avec tout un tas de détails sur les motifs leur le prof. [00'37'40] Presque le temps qu'il faisait enfin un truc. On avait près d'une quinzaine de variables, et puis, au bout d'un mois, l'essai en question, nous ont rappelé, nous ont dit: vous êtes gentil, mais fait-on un peu de choses à faire? donc, là, c'est pas possible de le renseigner comme ça, c'est trop détaillé. Et c'est comme ça qu'on en est arrivé à détailler seulement quatre variables: le relevé du nombre d'exclusions prononcée par semaine. [00'37'59] La durée de chaque exclusion, le sexe de l'élève exclu et sa classe. Il y a un truc qu'on aurait voulu garder, mais qu'on n'a pas pu garder, c'est le prof. [00'38'09] Et on ne l'a pas gardé pour des raisons, y compris éthiques, parce que on avait des remontées. On a commencé à coder le nom des profs pour, parce que si on a une remontée nominative, on rentre dans des circuits ou on ne peut pas se contenter d'être sous le manteau. En gros, [00'38'24] Mais le codage des profs, ça rajoutait vraiment beaucoup de contraintes pour laisser les collègues qui faisaient le boulot sur le terrain de recension de se rappeler que mes primes, c'était m grecque et x, c'était m machin. Donc, on apprend: n'a pas fait ça, et c'est une vraie limite. Mais j'en dirai quelques mots après. [00'38'44] On a eu. [00'38'46] Des renseignements mensuels sur sur deux années. [00'38'51] Par quatre vingt pour cent de ses quinze pour cent personnel de direction, quatre pour cent d'enseignants et, à chaque fois, évidemment, la garantie de l'anonymat des établissements et des personnels qui ont, qui ont participé à cette enquête. C'est quelque chose, et c'est un travail qui est toujours en cours dans certains établissements, mais on a on a arrêté dans un très grand nombre d'établissements, puisque là on est arrivé. [00'39'12] A travailler dans soixante seize établissements. Donc, les chiffres que je vais vous présenter sont tirés d'une enquête sur soixante seize établissements. [00'39'19] J'ai, j'ai. Les tableaux je vous présente sont très simplifiés. Ça n'a pas pris plein de précautions statistiques sur les écarts de moyenne, sur des choses comme ça. [00'39'28] Pour les rendre intelligibles. [00'39'30] Ensuite, vous voyez aussi qu'on a des mois où on avait moins d'établissements qui répondaient et d'autres, moi, on n'avait plus d'établissements qui répondaient. Donc qui, c'est des chiffres. [00'39'41] Qui sont produits avec des limites, mais quand même sur des échantillons qui sont qui nous semblent particulièrement. [00'39'50] Significatif, à défaut d'être représentatif. [00'39'54] En tout, ces soixante seize établissements, ça représente trente huit min. Six cent soixante quinze élèves, et vous voyez aussi que c'est pas un échantillon représentatif. [00'40'02] Deux, deux, aucune académie, puisque quatre vingt onze à l'académie de versailles, de quatre, vingt treize et soixante dix sept l'académie de créteil. C'est volontairement qu'on a fait ça, qu'on a mis des établissements d'autres académies, pour pas qu'on dise après: c'est un échantillon de l'académie de créteil. [00'40'17] Y a beaucoup d'établissements de la scène s'ennuient parce que c'est là qu'on a beaucoup de réseau. Donc, c'est pour ça que je dis aussi: c'est pas représentatif ni de la seine-saint-denis, ni de l'académie de créteil, c'est juste un grand nombre d'établissements. On a été voir un petit peu ce qui se passait et ce qui se faisait avec certaines limites. Il y a des établissements de l'éducation prioritaire, mais il y a pas que des [00'40'37] Établissement de l'éducation prioritaire. Vous voyez, et sur l'ensemble des élèves, on a treize pour de filles et quatre. Vingt sept pour cent garçons ont été temporairement exclus sur ses relevés. Je reviendrai aussi éventuellement par la suite. [00'40'53] Ça va. [00'40'56] Maintenant on arrive au résultat. [00'41'04] D'après vous sur un établissement moyen? [00'41'09] Combien on a, on va dire, un établissement de six cents élèves. [00'41'14] Combien on a d'exclusion. [00'41'17] Par moi, ou plutôt combien on a d'élèves exclus par mois. [00'41'20] Une proportion d'après vous. [00'41'25] Vous diriez quoi: [00'41'27] On prend un mois. Alors, je prends pas le mois de janvier, je prends pas le mois de décembre. [00'41'32] Ce que ces adeptes des gens de l'education nationale de la rue m'attendais, ça dépend septembre, janvier, et ils auront raison. [00'41'39] Donc, moi, je suis, voila, je suis sur la moyenne sur un mois, sans enjeu, sans conseil de classe, sans tout ça. [00'41'48] Un mois ordinaire, vous diriez quoi? [00'41'53] Non, le nombre d'élèves exclus. Nash ne parle pas du nombre de journées d'exclusion. Ça, c'est ma deuxième question. [00'41'59] La première, c'est combien d'élèves sont concernés dans un établissement de six cents élèves en gros? [00'42'05] Par mois sur l'exclusion temporaire d'après vous. [00'42'11] Quatre. [00'42'17] Design. [00'42'22] Je vais quinze à droite. [00'42'28] Par mois, par mois, par mois. [00'42'32] Vingt cinq attention. [00'42'36] Six cent, jeudi six cents. [00'42'38] Six, sans moi ordinaire. On va prendre le moins de le mois de mars, un mois calme. [00'42'44] Mois de mars. Six cents élèves, établissement ordinaire, il n'est pas classé, je ne sais pas quoi, et c'est une normalité. [00'42'55] Donc on est entre quatre et vingt cinq. [00'43'01] Alors, comme tu me poses des questions compliquées, [00'43'06] Je vais en prendre une autre. C'est combien de journée d'exclusion? [00'43'11] Comme ça t'as trois jours. Un combien ça représente. [00'43'16] Voyez si on prend quatre élèves. [00'43'19] Été exclu trois jours en moyenne. Ça fait trois fois quatre quoi. [00'43'24] Combien, d'après vous, est-ce que vous avez une idée de la proportion? [00'43'34] C'est plus dur. [00'43'41] Combien. [00'43'44] Deux jours en moyenne d'exclusion. [00'43'48] Mais alors au final, donc, si on dit, il y a en gros et à vingt, vingt, trois journées, [00'43'54] Dans deux mois. [00'43'58] C'est quoi? c'est une cinquantaine, une centaine. [00'44'00] Vingt cinq n. [00'44'04] Trente. [00'44'06] On se met d'accord sur trente. [00'44'10] Non, je sens qu'il y a pas un enthousiasme. Alors il y a trente. Et à quoi d'autre? [00'44'16] Combien tu dis. [00'44'17] Vingt cinq. [00'44'19] Tu pinaille. [00'44'24] C'est comme le jeu en train de me dire. En fait, ça fait très. [00'44'28] Dans le le truc quand je regardais quand j'étais petit. [00'44'34] Une famille en or. [00'44'35] Un un petit peu les exclusions. [00'44'40] Vingt cinq trente. [00'44'43] Soixante quinze. [00'44'46] Ok. [00'44'49] Très bien, ça rigole pas. [00'44'54] Alors ça, ça vous présente les résultats. [00'44'58] De cette enquête par moi. [00'45'01] Vous voyez sur cette colonne, là, vous avez le nombre d'élèves exclus. [00'45'07] Ce que ça représente sur chacun des mois, sur les soixante seize établissements. [00'45'11] Ok, là vous avez le nombre d'élèves exclus en moyenne par mois et par établissement. [00'45'18] Donc ça veut dire qu'on est à quarante cinq élèves exclus temporairement. [00'45'23] En septembre, en moyenne par mois et par établissement. Si un élève a été exclu deux fois, par contre, il compte deux fois. [00'45'31] Ça peut, ça peut arriver, parce qu'en septembre, comme ça fais pas ça fait. Bref. [00'45'36] En par jours ouvrés dans l'établissement. Ça nous fait chaque jour une moyenne de deux virgule. Cinquante-quatre élèves l'un, par exemple, qui sont exclus temporairement. Mais on est en octobre, on a une forte, forte augmentation à sept, vingt neuf. [00'45'53] Et là, vous avez la précision du nombre de jours ouvrés selon les mois sur cette année-là. [00'45'58] Ok. [00'45'59] Tout ce que je vais vous présenter, la fin, pas tous, mais cet aspect exclusion, vous le retrouvez de manière détaillée dans un, dans un article de deux lits, j'y s. [00'46'09] Qui s'appelle les exclusions temporaires, une routine punitive, et il est en libre accès en ligne. Je vous donnerai les coordonnées à la fin, si ça vous intéresse, pour aller voir deux de plus près. [00'46'19] Donc, vous voyez que sur nos, sur nos chiffres là, ce que vous évoquez, ont été entre quatre et vingt cinq élèves, dont on voit qu'on est vraiment très au-dessus. [00'46'30] Sur une moyenne d'établissement. [00'46'35] Pareil sur le nombre d'élèves exclus ou bien le nombre de journées de l'exclusion. Une précaution cependant: là on a un effet, on a une moyenne. [00'46'45] Du coup, ça veut dire quoi? ça veut dire que on a en fait une volatilité. [00'46'49] Des. [00'46'50] Des deux. [00'46'53] Du nombre d'exclusions. [00'46'54] Qui est assez significatif, et des établissements qui excluent vraiment beaucoup, et des exclus des établissements qui excluent vraiment peu. [00'47'01] Mais la moyenne, évidemment, va écraser ces différences. Là donc, je reviendrai sur ces différences. Mais malgré tout, avec des chiffres comme ça, ça veut dire qu'on a près de la moitié, voire les trois quarts de l'échantillon qui sont sur des exclusions forte, voire très forte. [00'47'20] Il y a deux chiffres sur lesquels je voudrais attirer votre attention. [00'47'26] Il y a d'abord- vous voyez que on est pas sur quelque chose de régulier. [00'47'30] S'avère que, selon les mois, on a une différenciation du nombre moyen d'élèves exclus. [00'47'37] Vous voyez que le mois de septembre, on est à deux cinquante quatre. On a octobre, à sept vingt neuf. [00'47'42] Ça augmente, la ça re diminue, augmente, sa re diminue, etc. [00'47'46] Comment vous vous comprenez ce chiffre, la sept vingt neuf. [00'47'52] Pourquoi- et on l'a sur deux ans, et on l'a vérifié sur deux ans- on a une explosion en octobre. [00'47'57] Quelles sont vos hypothèses pour ça? [00'48'02] Ouais, mais là t'es, en octobre. [00'48'16] Ouais, donc, de fait, c'est effectivement y a ça, il y a des élèves qui sont: [00'48'23] Qui sont ainsi touchés. Mais qui sont ces élèves? [00'48'27] Si tu. [00'48'30] Ouais, mais ça te fait quand même beaucoup d'élèves mal orientés lin. [00'48'38] Ça, c'est toutes classes confondues. [00'48'40] Ouais. [00'48'48] Ouais. [00'48'53] C'est. [00'48'57] Ouais, c'est, ça peut être avant les vacances, mais mais sur les autres, moi aussi, il y a, tu vois, sur les autres mois, il y a aussi les vacances de pâques, les vacances de. [00'49'09] D'après vous, si on prend en sixième, cinquième, quatrième, troisième, c'est plutôt lesquels? [00'49'15] En octobre. Je parle en octobre. [00'49'19] Le quatrième, troisième. [00'49'41] Hum, hum et bienveillant à l'égard des sixièmes. [00'49'57] En fait, c'est les élèves de sixième et cinquième qui sont massivement exclus sur le mois d'octobre et c'est ça qui explique l'explosion par rapport aux autres mois. [00'50'06] Si on regarde les chiffres, vous voyez, là on a quarante six pour cent des élèves exclus. [00'50'11] Qui sont des élèves de sixième en septembre. Vingt quatre pour cent qui sont des élèves de cinquième. Quarante sept pour cent. [00'50'18] En octobre et trente deux pour cent. [00'50'21] Sur les élèves de cinquième et vous voyez qu'après les répartitions, elles sont beaucoup moins marquées. [00'50'26] Là, c'est vraiment septembre-octobre. C'est très fort si je le présente autrement. [00'50'32] Si je réunis sixième, cinquième, quatrième, troisième, vous le voyez très nettement. [00'50'36] Parce qu'on a un début d'année qui est très marqué par des exclusions temporaires vers les petits, les sixièmes et cinquièmes. [00'50'43] Et après, en revanche, on a une augmentation sur les mois de janvier, février et mars, un grand classique pour mes bahuts et dans les établissements. Sur la deuxième partie de l'année, on a les grands contient. [00'50'55] À utiliser contient moins que au début de l'année et on voit là les le rapport finalement s'inverser. Mais c'est c'est ça qui est très marquant sur ce, sur ces chiffres, là, c'est le fait qu'on a une concentration des exclusions temporaires sur des élèves très jeunes et qui vont- nous, c'est notre analyse aussi- qui vont. [00'51'15] Dans le sens d'une affirmation d'un cadre scolaire en rupture avec le cadre scolaire précédent. [00'51'20] Une affirmation aussi du pouvoir des adultes sur les élèves. [00'51'25] Qui marque un niveau d'exclusion et de recours à l'exclusion très fort, malgré le fait que ce soit une mesure exceptionnelle. [00'51'33] Un censé être une mesure exceptionnelle. [00'51'36] Et vous allez voir pourquoi ça va rejoindre une interrogation sur le caractère d'urgence et que ça peut être quelque chose d'assez révélateur d'effets pervers d'un certain nombre de choses. [00'51'53] Ça va sur ça. S'il y a des questions sur ce tableau et ses premières données, [00'52'02] Vous êtes dégouté, en fait. [00'52'07] Julie. [00'52'12] Parce que les grands sont plus là, en fait. [00'52'15] Donc là, la les qu'à noël. Du coup, l'écart proportionnel fait que les, même si c'est pas trop en nombre, c'est proportionnellement, ça retouche plus les petits que les grands, parce que les grands sont, et en particulier les grands, les plus en rupture ou les plus en opposition à l'école, sont plus là. [00'52'31] Nous, l'hypothèse qu'on fait et qu'on formule, et vous allez voir qu'on l'a retrouvé après sur d'autres, d'autres aspects de ce travail. [00'52'38] C'est que, finalement, l'exclusion temporaire est devenue une sanction ordinaire. [00'52'42] Elle a plus du tout de caractère exceptionnel. [00'52'44] C'est une sanction qui est très peu coûteuse dans les établissements. Une heure de colle, c'est plus contraignant qu'une exclusion temporaire. De fait, une heure de colle, il faut l'encadrer pour accueillir le gamin, faut le gérer. Une exclusion temporaire, il y en a pas, il y en a pas besoin. C'est une sanction, nous disent les chefs d'établissements, dont c'est la responsabilité de la donner ou pas. [00'53'04] Qui est perçu comme la seule sanction vraiment significative par beaucoup d'équipes éducatives. Là encore, je parle de manière un peu générique. Il y a évidemment des équipes ou c'est pas le cas, mais de manière générale, on voit qu'il y a une sensibilité très forte à cette question de l'ordre scolaire, de la déviance à l'ordre scolaire qui appelle une manifestation. [00'53'24] Au nom de la solidarité de l'équipe de direction, donc très souvent les équipes pédagogiques. [00'53'30] Considère qu'elle passe d'abord par une exclusion temporaire. Pas systématiquement, mais c'est vraiment un des marqueurs qui permet d'expliquer cette explosion du nombre d'exclusion temporaire. [00'54'04] Regardez, je vais vous montrer quelque chose. [00'54'13] Là. Quand j'arrive là dans un mois, j'ai fini. [00'54'17] Et j'ai mis bonus. [00'54'20] Si la question distinction fille garçon est posée. [00'54'26] Donc. [00'54'29] Je vous dirai ça en bonus. [00'54'49] Non sur les motifs, mais on peut déjà dévoilé sage, mais je vais revenir. Mais la grande difficulté, c'est qu'on [00'54'56] Il y a deux aspects. Le premier aspect, qui est sur le fait que cette sanction soit devenue d'un usage ordinaire. Si elle est devenue d'un usage ordinaire, il y a deux hypothèses: soit c'est une guerre permanente dans les salles de classe et donc ça va dans le sens aussi d'une forme de spectacularisation de la violence à l'école. [00'55'15] De tout ça, nous, ce qu'elle ne sait pas, ce qu'on observait du point de vue ethnographique dans les classes, dans les établissements. [00'55'22] C'est un travail que j'ai fait aussi avec deux doctorantes, julienne garnier et myriam wolinski, qui ont fait beaucoup de terrain dans ces établissements. [00'55'31] Et c'est pas du tout ce qu'on ce qu'on ce qu'on a constaté. Y a des difficultés, il y a des difficultés, il y a des incidents liés à tout ça, mais les motifs, très souvent, sont des motifs qui renvoie plutôt au danger de la mise en scène de l'enseignant ou de l'enseignante face à sa classe que à un événement qui est [00'55'51] Advenu et qui nécessitent l'intervention d'une sanction de ce type-là. Je m'explique ce qu'on nous renvoie beaucoup et quand j'ai des entretiens avec des enseignants et des enseignantes, c'est: je peux pas laisser passer ça parce que c'est risqué par rapport aux autres exemples. [00'56'09] Un gamin qui lève les yeux au ciel. [00'56'11] On a trouvé beaucoup de billets sur insolence, puisque les motifs après [00'56'15] On pourrait revenir à nos traces. [00'56'18] Sur les fiches d'incidents, etc. Ce n'est pas toujours très, très, très étayé. Quoi, ces insolences? insolence: trois jours d'exclusion demandée. Insolences répétées. [00'56'27] Et quand on creuse, on a parfois attention. Parfois, il y a des fois, c'est pas ça, mais on a trouvé beaucoup, beaucoup de fois, des, des, des des choses de ce type là, c'est-à-dire à lever les yeux au ciel. [00'56'40] Ah. [00'56'41] À siffloter à des choses qui sont plutôt du registre de la bande. Un début d'opposition, mais vraiment très, très lointaine. [00'56'50] Et ce besoin de dire tout de suite stop, avec cette crainte du débordement très, très forte chez les enseignants qu'on a interrogés, pas chez tous. Il y en a un certain nombre qui même sont très préoccupés par la proportion et l'usage ordinaire de ces sanctions, mais beaucoup d'autres- pas forcément des jeunes enseignants d'ailleurs- beaucoup d'autres qui disent de toute manière, [00'57'11] On est dans un rapport de deux du maître à l'élève, qui fait que je ne peux pas laisser passer ce genre de comportement est le seul. La seule manière de ne pas la laisser passer, c'est l'exclusion temporaire. Les heures de colle, ça sert à rien, et puis en plus, sinon il faut que je revienne le mercredi. On a des noms, qu'on a des marqueurs, comme ça, donc les, les motifs, on en a beaucoup qui sont finalement sur les registres. [00'57'32] Très secondaire et qui correspondent pas à ce que préconise le code de l'éducation, mais ça témoigne en revanche d'une crispation très forte et c'est pour ça qu'on le prend vraiment, nous, comme un révélateur. [00'57'43] Deux. Deux d'un certain nombre d'enjeux autour de l'école, d'une crispation très forte entre les enseignants et les élèves, mais surtout des attendus en termes de comportement. [00'57'54] Des jeunes et une tolérance à la sociabilité juvénile beaucoup moins forte qu'elle n'a pu être. [00'58'06] Si on prend ces chiffres là et qu'on les projette sur un département comme la seine-saint-denis- même si on pourrait les projeter sur d'autres départements- [00'58'13] Cette projection là, elle vous montre combien il y a d'élèves chaque jour en moyenne dehors, selon les mois, pour exclusion temporaire. [00'58'22] Sur un département comme la seine-saint-denis, a cent vingt établissements. On est à cent soixante sept élèves en septembre. Quatre cent quatre, vingt neuf, trois cent douze, etc. Etc. Tous les jours. [00'58'34] C'est ce qui fait qu'on a appelé ça. [00'58'37] J'ai intitulé un article que vous trouverez ainsi en ligne, s'il vous intéresse. [00'58'41] Le collège fantôme. [00'58'43] On assure un certain nombre de mois, l'équivalent de l'effectif d'un établissement. [00'58'49] Des élèves temporairement exclus qui sont dehors. [00'58'52] Qui sont dans la rue. [00'58'53] Dans un département comme la seine-saint-denis. [00'58'56] Le. Le gros écueil, et je j'attire votre attention là-dessus, c'est qu'on pourrait dire: oui, mais ça, c'est la seine-saint-denis. [00'59'03] Donc. [00'59'05] C'est pas pareil ailleurs. [00'59'06] Sauf qu'on a eu l'occasion de travailler avec le, la médiatrice de l'éducation nationale, qui, dans son dernier rapport, a fait remonter des préoccupations et une augmentation très forte des interpellations par les familles. [00'59'22] Au médiateur de l'éducation nationale, au service règle taureau de parents qui conteste? [00'59'28] L'usage des exclusions temporaires en seine-saint-denis pas beaucoup, beaucoup dans d'autres départements. [00'59'35] Et on fait l'hypothèse sur la base d'échanges qu'on a avec d'autres collègues, même s'il n'y a pas d'autres recherches ont été menées. [00'59'41] Sur des échantillons aussi large. [00'59'43] Qui a un usage aujourd'hui en france très fort, de l'exclusion temporaire est extrêmement fort et qui est un marqueur de différenciation avec d'autres pays. [00'59'55] Très comparable: ou l'exclusion temporaire est soit limitée, soit interdite, et ça, c'est un marqueur d'ailleurs très intéressant avec des pays dans lesquels les institutions scolaires est en train de s'affirmer. [01'00'06] Qui ne comprennent même pas qu'on autorise le fait d'interdire à des élèves d'aller à l'école, quoi qu'il ait fait. [01'00'13] Je travaille beaucoup sur le brésil et la comparaison avec le entre la france et le brésil, on a des situations autrement plus compliquées. [01'00'21] Au brésil que ce qu'on peut vivre en france, même s'il y en a qui sont compliqués, il n'y a pas de souci là-dessus. [01'00'26] L'exclusion temporaire est interdite. [01'00'28] Vous devez trouver une solution en interne, d'accueil. Et là, vous voyez qu'on est bien sûr aussi- et vous allez voir comment on glisse- vers ces enjeux de recomposition des espaces scolaires et éducatifs et de mobilisation de nouveaux acteurs de l'école dont on va considérer que ils sont indispensables pour faire vivre. [01'00'49] Des réponses institutionnelles. [01'00'51] De prise en charge. [01'00'53] De ces publics dits défavorisés ou qui ne correspondent pas à l'image que l'on a. [01'01'01] Élève ordinaire. [01'01'09] Du coup. [01'01'10] Vous voyez que dans ces [01'01'14] Par rapport à cette problématique. Là, on a beaucoup d'élèves. [01'01'17] Qui sont temporairement exclus, avec très peu de dispositifs mis en place par l'éducation nationale pour les prendre en charge. [01'01'25] Et on a vu émerger de nombreux dispositifs de prise en charge des élèves temporairement exclus, portés par des acteurs non scolaires. [01'01'35] J'insiste là-dessus. [01'01'37] Porté par des perreux, portés par des centres de prêt spécialisés, portés par des structures d'animation ou d'éducation populaire, portée par des maisons de quartier, des musées, etc. Etc. [01'01'54] Et le registre de l'urgence a été très, très utilisé pour justifier de la mise en place de ces dispositifs. En gros, on constatait qu'il y avait la présence de jeunes. [01'02'06] Parfois en difficulté, voire en grande difficulté, dans la rue, et qu'il est nécessaire de les prendre en charge. La question n'a pas été de dire comment l'école arrête d'exclure. [01'02'17] La question a été de dire comment les travailleurs sociaux et institutions socio-éducatives- des institutions socio-éducatives- prennent en charge ces éléments. [01'02'25] C'est un glissement, moi, qui me semble très important à avoir en tête. [01'02'30] Il n'y a pas et il y a très peu de réflexion finalement sur le fait que ces élèves soient à la rue. [01'02'34] La réflexion, c'est comment on encadre ses élèves alors qu'ils devraient être à l'école, ce qui devrait être le cheminement dans dans le cadre légal ordinaire. Ça devrait être ça. [01'02'45] Donc, à mon sens, c'est encore un marqueur de plus de cette recomposition des espaces scolaires et éducatifs, des centres de prévention spécialisée s'autorise aujourd'hui. [01'02'54] À prendre part à la résolution d'un problème scolaire. [01'02'57] C'était sans doute culturellement inenvisageable il y a dix ou quinze ans. [01'03'01] Culturellement. [01'03'02] Sur le registre d'éducateur ne met pas notre boulot. L'école fait n'importe quoi on devrait. Je ne dis pas que l'école fait bien ou pas bien. Je ne me situe pas sur ce, ce, ce positionnement là, mais culturellement, [01'03'14] On observe bien ce glissement là qui est un marqueur fort des nouvelles formes de prise en charge sur le caractère. [01'03'22] De l'urgence à répondre à cette contrainte là. [01'03'26] Un entretien avec un, un coordinateur, un responsable de collectivités territoriales qui a mis en place un dispositif de ce type-là. Après, il faut le dire aussi, il y avait une grosse demande des collèges. [01'03'40] Il ne voulait pas entendre parler de garder ces jeunes. Mais ces jeunes étaient là dans la mise en place du dispositif, dans ce qui a poussé à se mobiliser, c'est bien aussi la difficulté, dans certaines villes, à gérer ces jeunes. On se retrouve avec des coups de fil des élus locaux qui avaient des grosses difficultés à gérer les abords des établissements ou certains espaces publics. [01'03'56] Car on s'est rendu compte que si une partie de ces jeunes avaient clairement décroché de l'école, d'autres avaient été mis à la porte- a été exclu temporairement. [01'04'01] Le problème, c'est que, sans encadrement, ce public devenait difficile à gérer dans certaines communes en plus, avec des nombre de jeunes concernés très significatifs. Donc, je dirais que la mise en place du dispositif s'inscrit clairement dans ce besoin d'encadrement d'une partie de la jeunesse sur le territoire. Nous, on peut pas se substituer à l'école. On peut regretter certaines situations, mais on ne peut pas remplacer l'école et d'ailleurs, je pense que ce n'est pas facile. Alors, notre solution: [01'04'22] À nous, collectivités, ces dispositifs. [01'04'24] Voyez, on revient sur ce que j'ai évoqué en introduction. [01'04'28] Cette organisation spécifique qui répond à un besoin particulier. [01'04'35] Du coup. [01'04'36] Juste un point. [01'04'38] Là ce que je vais vous présenter sur les dispositifs. Ça a été mis en place dans le cas d'une recherche sur les formes d'encadrement des collégiens exclus et au travers d'une évaluation d'un dispositif départemental d'accueil des collégiens exclus en seine-saint-denis. Là aussi, vous trouverez le rapport, s'il vous intéresse. [01'04'55] De cette évaluation. [01'04'58] En ligne. [01'05'00] Juste pour situer la recherche. Il y avait un volet qualitatif avec des entretiens auprès d'élèves, auprès de responsables de dispositifs, auprès de principaux de collèges de sep, etc. Des observations in situ également. Un volet quantitatif avec des questionnaires, ont été administrés à la fois. [01'05'19] Sur des échantillons académiques et à la fois sur des échantillons d'élèves. [01'05'24] Qui sont passés par le le dispositif. [01'05'27] Travail. Qu'on a fait donc avec juliette garnier? j'ai myriam waf, qui est stéphanie ruby. [01'05'37] Ce qu'on observe sur ces dispositifs. [01'05'40] C'est qu'on a. [01'05'41] Et ça, c'est un marqueur important, j'insiste: on n'est plus à l'aune d'un dispositif sur une seule commune ou dans un seul établissement, on est sur une organisation où ces types de dispositifs qui deviennent très importants- celui dont on parle là y concerne vingt sept villes- il y a vingt neuf dispositifs locaux qui sont mis en place. [01'06'02] Ça touche soixante treize des cent vingt collèges de la seine-saint-denis. [01'06'06] Avec comme constat le fait que c'est nécessaire de lutter contre le décrochage au travers de la mise en place de ces dispositifs et que l'encadrement éducatif des élèves perturbateurs nécessite l'intervention de spécialistes. [01'06'20] Fais que. Mais les élèves qui sont temporairement exclus, le diagnostic qui est fait par les collectivités locales, c'est que si ils sont exclus temporairement, c'est que ce sont des élèves très difficiles. [01'06'31] Ce qui est assez logique dans une lecture. [01'06'34] Diners et traditionnel. Vous voyez que c'est soixante huit pour cent de dispositifs ce sont des services municipaux qui les portent. Trente deux pour cent ce sont des associations. [01'06'43] Ce qui est important de noter aussi, c'est que ce sont jamais d'une ville à l'autre les mêmes dispositifs. [01'06'48] Il y a le même public, il y a le même financement, mais il y a des ajustements selon les dispositifs des lieux. [01'06'58] Dans la manière avec laquelle est organisé l'accueil, via des dispositifs qui prennent les enfants que une semaine, il y a des dispositifs qui prennent seulement trois jours. Il y a des dispositifs qui font beaucoup d'activités culturelles, il y en a qui n'en font aucune, qu'il y a des dispositifs qui sont, avec des psychologues, beaucoup. Quatre vingt pour cent des dispositifs font [01'07'17] L'avenir des psychologues. [01'07'18] D'autres ne le font pas avant. Voilà, vous avez donc un cadre générique. [01'07'23] Autour de ces dispositifs. [01'07'25] Mais une déclinaison locale spécifique et particulière, qui va répondre au caractère d'urgence local de manière spécifique. Et ça, c'est un marqueur très fort, c'est qu'à chaque fois, c'est: oui, mais nous, ici, athènes, dans ta ville, c'est pas comme dans la ville de juste à côté. Nous, ce qu'on fait dans le dispositif, c'est plutôt ça, et la ville à côté de nous, ce qu'on fait, c'est c'est plutôt comme ça. [01'07'45] C'est. Ne s'agit pas de dire que c'est pas justifié. Il s'agit de constater qu'on a une mobilisation de registres de justification qui s'ancre sur une légitimité de lecture, de diagnostic local très spécifique et pas du tout sur l'application d'une politique nationale, comme on a marqué longtemps les politiques éducatives locales. [01'08'12] Une des des choses qu'on a constaté très rapidement sur ces dispositifs. [01'08'17] C'est que, devant l'ampleur des exclusions temporaires, [01'08'23] On s'est interrogé sur qui est la cible qui est finalement touchée par ces dispositifs. [01'08'29] Le conseil général. Lui, sa démarche était de dire zéro pourcent de collégiens exclus à rue. [01'08'34] On doit pouvoir prendre en charge tous les élèves temporairement exclus. [01'08'38] Sauf que la proportion d'élèves temporairement exclus est-elle? [01'08'43] Qu'il faudrait multiplier par dix, voire par quinze, la capacité d'accueil des dispositifs existants pour permettre l'accueil systématique de tous les élèves. [01'08'53] Sur les vingt sept villes dont il est question, il y en a trois qui réussissent à accueillir systématiquement tous les élèves. [01'09'01] Parce que y a un accord avec les collèges, le secteur, qui fait que tous les élèves temporairement exclus sont accueillis dans le dispositif, tous. [01'09'10] Mais dans tous les autres établissements, dans toutes les autres villes, on ne voit. Et trois sur vingt sept, c'est très minoritaire. Non, toutes les autres, c'est très majoritaire. [01'09'19] Les élèves sont orientés ou non dans le dispositif. [01'09'24] Il y en a qui vont, y en a qui vont pas, et c'est l'établissement scolaire qui fait le choix d'orienter ses élèves vers le dispositif, avec toujours le besoin d'un accord de la famille. [01'09'35] Et de l'élève. [01'09'38] C'est clair, la source, ces dispositifs, comment ils sont. [01'09'45] Et juste, c'est des dispositifs qui sont en dehors de l'école. [01'09'49] Dans des espaces soient liés aux associations ou structures. [01'09'53] Soit dédié pour quelques-uns dans l'établissement, mais on a vraiment pas beaucoup. [01'10'02] Ce qu'on nous a dit très vite, avant qu'on vérifie ça avec des une enquête un peu plus approfondie, c'est que quelque chose qui revenait beaucoup. Les porteurs de dispositifs étaient très étonnés du public. [01'10'13] Mon disait: ce n'était pas du tout le public auquel je m'attendais. [01'10'16] Par exemple, monsieur vanneau nous dit: on n'en voit pas tous les élèves au pair, c'est assez peu. [01'10'22] Pour dire clairement: on ne voit pas les cas désespérés dont on sait très bien qu'ils feront rien que ça les intéressera pas. Ou les récidivistes, multirécidivistes, pareil, on les envoie pas non plus. [01'10'31] Alors que d'autres qui sont un peu plus sur la tangente, on les prend en quelque sorte en amont. [01'10'36] Donc, ce sont les élèves qui n'ont pas beaucoup de sanctions- dernière euh, pas vraiment non- et qui sont dans la coopération avec les référents du collège, dont on sait que quand on va lui proposer, il va faire un peu la tête, mais on sait qu'on va réussir à le convaincre. [01'10'49] Et ça, c'est quelque chose de très fort. C'est là qu'on s'est rendu compte que, finalement, les élèves, il y avait des dispositifs qui étaient mis en place, créer. [01'10'58] Les élèves qui étaient orientés. [01'11'00] Orienté dans ces dispositifs étaient massivement pas les élèves les plus en difficulté. [01'11'06] De décrochage, voire de glissement vers des activités déviantes et délinquantes, parce que c'est ça qui avait motivé en plus les collectivités à mettre en place. [01'11'16] Ces dispositifs. Là, c'était un enjeu de prévention de la délinquance. Il faut beaucoup se méfier de ces rapports de cause à effet: décrochage, délinquance. Mais là, malgré tout, sans rentrer dans ces considérations-là, on voit, et ce qu'on a constaté, c'est que il y avait une orientation d'hélène. [01'11'33] Qui était plutôt ceux d'élèves dont on dira qu'ils sont scolairement bien doté. [01'11'38] Qui reste, des élèves qui ont eu un incident avec un enseignant, etc. Mais pas des élèves en forte rupture. [01'11'46] Pas des élèves en situation d'urgence. [01'11'59] Un responsable associatif nous nous dit comme ça nous dit ainsi. Mais en revanche, je suis très surpris parce que ce n'est pas le public des jeunes en difficulté que l'on reçoit ici. Il y en a quelques-uns, mais franchement, par rapport à d'autres, c'est rien, ils ont fait une bêtise. Il y en a certains qui ont des problèmes relationnels avec des profs ou avec un assistant d'éducation. [01'12'14] Non, on ne peut pas dire qu'il y ait de grosses difficultés, même sincèrement. Des fois, nous intervenons, nous nous disent: mais ça va en fait. [01'12'22] Enfin, il nous disait ça au début, parce que maintenant ils savent que c'est pas des terreurs non plus. Mais c'est vrai aussi qu'on a été un peu surpris. Moi en tout cas, je sais que c'est pas l'image que je me faisais- un élève exclu. [01'12'37] On a essayé de regarder ça dans le détail sur une mesure de l'appréciation du climat scolaire par ses élèves pris en charge dans le dispositif. [01'12'47] Pour vous expliquer la lecture de ce tableau. On est parti de questionnaires, les questionnaires debarbieux qui mesurent le climat scolaire. Climat scolaire, c'est. [01'12'59] Les niveaux relationnels, l'appréciation relationnelle. [01'13'03] Des élèves envers les profs, envers la vie scolaire, le sentiment de justice, le sentiment d'injustice, sentiment de sécurité, etc. [01'13'11] Théoriquement, lorsqu'on a des élèves en forte rupture scolaire, ils ont une perception du climat scolaire très dégradé. [01'13'19] Ok, les travaux qui portent là-dessus, soixante miniers étant ceux de debarbieux. Cette playa montre ça: les élèves en forte rupture, voire un fort risque de décrochage, sont les élèves qui ont une perception très négative du climat scolaire. [01'13'32] Inversement, les autres ont une vision, les, les élèves les mieux dotés ont une vision plus positive du climat scolaire. [01'13'39] Là vous voyez que si on va, [01'13'42] Ça, ça vous donne les résultats d'un questionnaire. [01'13'46] Plus c'est écrit là, plus le score est proche de quatre, plus la perception du climat scolaire est négative. [01'13'52] Ok. [01'13'53] Là vous avez l'échantillon. [01'13'56] Expérimental ici donc, c'est-à-dire l'échantillon. [01'14'00] Des élèves qui sont passés dans le dispositif. [01'14'03] Là vous avez un échantillon endémique de l'académie de créteil. [01'14'08] Théoriquement, on devrait avoir beaucoup d'élèves de l'échantillon expérimental ici. [01'14'14] Cette ligne là, elle marche, en gros, la laisser les élèves pour une version, une vision positive du climat, à des degrés divers. Voyez qu'on a classé en trois, en trois catégories, mais en gros, là, c'est très, très positif. C'est des gamins qui répondent: ouais, c'est super, l'école, c'est génial, j'adore. [01'14'31] Là, les quarante pourcents, c'est ce qu'ils disent. C'est très bien l'école, mais [01'14'38] Là, c'est des gamins qui disent: ça va. [01'14'40] Ok, sur ces trois catégories, on a à peu près là-dessus. Là, c'est ça va pas trop. [01'14'46] Ça va pas du tout. C'est n'importe quoi de façon l'école, tous pourris, tout ça. [01'14'51] Donc théoriquement, si on était sur des profils d'élèves. [01'14'55] En forte opposition, en forte rupture. [01'14'59] En décrochage, etc. On devrait avoir nettement plus de proportion d'élèves ici qu'on en a. Vous voyez qu'il y a une proximité très forte entre les deux échantillons, ce qui nous a amenés à dire que, finalement, les élèves qui étaient pris en charge dans le dispositif était représentatif des élèves de l'académie, pas du tout d'élèves en situation. [01'15'18] De risque ou d'urgence tels que la cible avait été pensé par les pouvoirs publics pour justifier de la mise en place de ces dispositifs. [01'15'27] Voyez le l'écart. [01'15'35] Non, ça veut dire qu'ils sont pas pris en charge dans les dispositifs d'exclusion. [01'15'41] Ils sont exclus, mais restent dans la rue. [01'15'44] Malgré le fait qu'il y ait des dispositifs de proposée qui visent à les prendre en charge. [01'15'51] Parce que c'est spécifiquement cette cible là, les deux partenaires, c'est la pj, c'est des des intervenants qui sont beaucoup plus tournés, c'est la protection de l'enfance. [01'16'01] Beaucoup plus tournée vers ses angela que ceux des élèves ordinaires. Et du coup, je redis, il y a quelques dispositifs, ou c'est pas le cas- ceux qui réussissent à toucher ces élèves là, c'est ceux où il y a une systématisation de l'orientation. [01'16'15] Des dispositifs verts. [01'16'18] Des élèves exclus vers les dispositifs parce qu'ils y passent tous. [01'16'22] Mais ça n'est possible que dans des établissements qui excluent relativement peu. [01'16'26] Dans des établissements qui excluent beaucoup. C'est pas possible, parce que le flux est-elle. [01'16'30] Que on n'y arrive pas et quand le flux est important, et on n'envoie pas les élèves les plus en difficulté, on envoie ceux qui sont le moins. [01'16'38] En. [01'16'39] Nos positions à l'école, avec voilà, avec un motif qui a été relativement. Il y en a plusieurs, j'en ai évoqué quelques-uns, mais il y en a un autre que je n'ai pas évoqué. [01'16'47] Qui est que les professionnels considèrent que c'est pas en une semaine qu'on va pouvoir résoudre son problème. C'est pas faux. [01'16'54] Mais. [01'16'56] La difficulté, c'est que du coup, les dispositifs et les intervenants. [01'17'01] Encadre des élèves qui sont pas ceux pour qui ça pourrait être un levier vers l'orientation, vers d'autres dispositifs, d'autres formes de prise en charge, etc. Etc. [01'17'16] Deuxième problème qu'on a, sa question, qu'on pose aux fins de cette recherche là et qui interroge plus particulièrement les porteurs de dispositifs: [01'17'27] C'est le fait que les dispositifs en viennent finalement à à prendre en charge plutôt une forme d'externalisation. [01'17'36] De la prise en charge des élèves. [01'17'38] Un travail véritablement partenariale avec l'institution scolaire. [01'17'43] On a, par exemple, un responsable de dispositif- et c'est quelque chose qu'on a beaucoup, beaucoup entendu- qui nous dit: le gros problème, c'est que les établissements, ils savent nous trouver, mais ils ne s'impliquent dans rien de leur côté. On signe la convention, il amène l'élève avec le peut, on fait le point rapidement par rapport à l'incident et ensuite, ciao fait la fête avec lui, par exemple, sur la continuité scolaire. Il nous bassine là-haut, il fait allusion à sa direction avec la con. [01'18'03] Continuité scolaire, mais on n'a rien. On demande à chaque fois si les profs ont donné quelque chose. On n'a jamais rien en trois ans, une fois. Une fois, il y a un prof qui a filmé un truc, le sait peu. A chaque fois, il est désolé, le pauvre. C'est même devenu une blague. Je lui dis: quand on en arrive? quand on arrive à ça, comme d'habitude, il me répond: comme d'hab. [01'18'19] Ça. Ça renvoie à cette question de ce que j'évoquais tout à l'heure de la continuité scolaire et de la tendance. [01'18'26] A cette recomposition des espaces scolaires et éducatifs et à une offre nouvelle mais qui, si sur le papier, elle est très positionnée sur des aspects partenariaux, d'organisations pluriprofessionnelles, etc. Etc. Est très difficile à faire vivre sur le terrain sur un registre de coopération qui soit réel avec. [01'18'45] Une institution scolaire qui n'est pas dans cette culture là- même si évidemment, un certain nombre d'établissements ne le font pas du tout de cette manière-là- que l'âge d'un point de vue générique, sur un grand nombre d'établissements. Mais on a aussi des exemples d'établissements qui travaillent très en lien avec, par exemple, des eux et qui sont sur une logique partenaire. [01'19'06] De long terme, mais c'est pas la tendance générale. La tendance générale, c'est plutôt celle d'une externalisation de la prise en charge de ces élèves. [01'19'15] Et ce n'est même pas une externalisation de la prise en charge des plus difficiles. On pourrait se dire si c'était difficile, mais c'est même pas les plus difficiles, mais par contre, ça témoigne bien d'une difficulté dans la construction d'un rapport de partenariat à gérer et prendre en charge, designer, gérer et prendre en charge des élèves dont on pourrait compter. [01'19'35] Sidéré que parce qu'ils sont en situation d'urgence pour une raison ou pour une autre liée à un événement scolaire et parfois à d'autres événements, [01'19'43] Puisse être accompagné. Là ce qu'on constate- et c'est pour ça que j'ai utilisé le fait que on avait finalement des dispositifs qui pouvaient masquer- [01'19'51] Des situations d'urgence, le sentiment collectif sur ces bassins, là, c'est, il y a une prise en charge qui est proposée. [01'19'58] Une: on sait qu'il y a un problème sur les exclusions, mais il y a une prise en charge qui est proposée pour les élèves en difficulté, sauf que nous, les élèves les plus en difficulté, je redis, à l'exception de quelques bassins- [01'20'10] Ne sont pas pris en charge. [01'20'17] Du coup les familles aussi. Sur cette question-là, on voit que ça alimente une forme de, de de rupture. Le terme est peut-être fort, mais de entendu, d'autres ont théorisé rail you beauty, etc. Ont théorisé aux shakes cette question du malentendu scolaire, et là, on est exactement dans le type de chose qui peut l'alimenter. Un exemple d'entretien avec une. [01'20'37] Mère de celio, douze ans, qui a été exclu pour la deuxième fois aussi. Je dois bien reconnaître que cela m'inquiète beaucoup, et son père aussi d'ailleurs. Deux exclusions en trois mois, mais il va aller jusqu'où comme ça. Alors, on lui a dit, on lui a dit: de toute manière, il est privé de tout jusqu'aux prochaines vacances: plus de consoles, plus de foot, plus de sorties exclus. C'est grave quand même être exclu. [01'20'56] Et je lui demande: vous pensez que c'est grave? [01'20'58] Je pense que c'est très grave. Bon, après aussi, c'est vrai que j'ai l'impression que dans ce collège, il donne facilement une exclusion. Moi, je crois qu'à mon époque, c'était plus pour des trucs graves une exclusion, que maintenant, je crois que c'est un peu plus utilisé. Mais moi, je me dis: ils savent ce qu'ils font, ils savent où ils vont. Moi, je vois mon fils, si vous l'écoutez, il n'a rien fait et c'est injuste. Moi, je lui dis: de toute manière, [01'21'19] Raison et t'as joué, t'as perdu. Mais bon, maintenant il faudrait que ça se calme, parce que s'il me fait une troisième exclusion, alors là, mais même si les motifs sont trop, pas trop grave, je crois. [01'21'29] Moi, je pense qu'une exclusion, c'est grave. Vous voulez bien me dire ce que c'était, les motifs? oui, oui, je veux bien. Je ne suis pas fier, mais ça va à la première. Ça faisait deux fois qu'il avait oublié son carnet, alors il a pris une journée. La deuxième, c'était pour insolence avec le prof de maths. Il a levé les yeux au ciel. Il a pris trois jours. [01'21'46] Donc, c'est voilà ça. Ça renvoie bien à ce qu'on évoquait tout à l'heure sur le l'utilisation et les usages des registres punitifs plutôt que des registres éducatifs au sein d'une institution scolaire. [01'21'58] Hé. [01'22'00] Avec un jour. J'ai pris cet extrait là. Il y en a beaucoup d'autres où les parents comprennent pas, en fait. [01'22'06] Comment ça se fait que leur, leurs enfants, leur fils ou leur fille est exclu temporairement pour ça. [01'22'13] Mais malgré tout, se retrouve face à cette injonction à soutenir la décision de l'école pour ne pas paraître en décalage avec des familles. [01'22'25] Moins proches scolairement, on dira des moins dotés scolairement, font cet effort là également, mais en imaginant du coup que leur et leurs enfants sont vraiment dans une attitude extrêmement défiante à l'égard de l'institution scolaire. Et donc, ça provoque des inquiétudes et c'est ce que nous disent beaucoup les porteurs de dispositifs. Il ne l'est pas avec les élèves qu'on a des soucis. [01'22'45] C'est avec les parents qui vivent très mal le fait que leurs gamins soient pris en charge dans une structure socio-éducative, etc. Etc. Alors que c'est pas pour le coup un public qui est en besoin ou en demande forte de ce type d'encadrement. [01'23'03] Très souvent, les dispositifs sont appelés à la rescousse par les établissements sur le registre de. Vous avez une meilleure relation. [01'23'11] À la, à la famille que nous, et donc, c'est cet aspect supplétif, il est très demandé sur le registre de la proximité avec la famille. [01'23'20] Reste que on retrouve, dans un certain nombre des entretiens qu'on a pu faire, cette même difficulté. Mon cas a déjà été montré plein de fois. Je pense aux travaux de teint, je pense aux travaux. [01'23'31] De gamme, je pense, beaucoup de travaux sur ces questions-là. Qui monte cette difficulté a avec les populations. [01'23'39] Les les moins dotés. [01'23'42] Culturellement, sur des proximités avec les travailleurs sociaux. [01'23'45] Qui alimentent les les parfois les malentendus. Aussi j'ai mis celui-ci. [01'23'51] On aurait pu en mettre d'autres- mme m, qui est la maman d'amadou, quatorze ans. Deuxième exclusion: [01'23'56] Moi, je pensais que c'était bien pour lui, mais au début je ne voulais pas qu'il y aille aussi. Mais ça faisait un peu peur au début, en fait. [01'24'02] Et je demande: mais qu'est-ce ce qui faisait peur? vous pouvez me dire un peu? [01'24'06] C'était en fait, c'était en fait. J'avais mal, mal compris. En fait, comme il travaille avec une cellule là, au début je croyais que c'était une cellule comme en prison. [01'24'14] Alors je refusais. Et puis l'éducateur m'a expliqué. Il a rigolé aussi. Il s'est un peu foutu de ma gueule, mais c'était gentil. [01'24'20] Ah bon, mais pourquoi? [01'24'22] La rigolé, en fait à cause du nom, parce qu'il travaille à une cellule. Lui et moi, je croyais que c'était une cellule de prison pour les enfants, mais il m'a dit que c'était le même nom, mais que ce n'était pas du tout une prison, que c'était pour qu amadou ne soit pas dehors, qu'il soit pris en charge par des éducateurs. Alors j'ai compris et j'ai dit: d'accord, et après, c'était bien, car en fait, il n'allait pas dehors. Le père de cette ville se présente sous l'intitulé cellule. [01'24'42] Superbe. [01'24'44] Et voilà après, sur les familles. On a beaucoup, de nous deux. [01'24'49] Sur les selles qu'on a interrogé de cet acte, soit, un côté, une incompréhension sur pourquoi est-il là, ici. Là, s'il est là, c'est que c'est très grave et c'est un vrai problème, soit ça, typiquement, c'est dans une, dans un pays heureux, qui prend en charge systématiquement les gamins, et c'est plutôt des gamins qui sont déjà plutôt en rupture avec l'école qui y sont accueillis. [01'25'10] Et on a juste ce qu'on observe déjà par ailleurs sur les difficultés relationnelles ordinaires, entre grands guillemets, entre travailleur social et population. [01'25'21] Puis marginalisés. Rien de plus que ce qu'on observe par ailleurs. [01'25'27] Dernier élément: les élèves. [01'25'29] J'ai mis un extrait aussi de d'entretien d'élèves. [01'25'33] Pour savoir un peu comment les les les élèves parlent du dispositif. [01'25'38] Il y a deux catégories. Il y a ceux qui considèrent qu'ils y sont, mais qu'ils ont rien à y faire. Ils sont nombreux, mais ils sont polis. [01'25'44] Ils font le truc et ils disent à la fin: non, mais c'était bien, c'était bien, c'était sympa. [01'25'50] Mais qui sont pas dans dans des niveaux de défiance à l'école. Fort après. On en a d'autres qui sont ceux qui sont plutôt dans des situations de de de rupture, plus plus, plus fortes, voire parfois très fortes. Ceux qui sont pris en charge systématiquement ou les quelques-uns qui, malgré tout, sont pris en charge. [01'26'10] Quand même qui sont pris en charge. [01'26'13] Qui sont des éléments en grande difficulté. [01'26'16] Ce qui nous a beaucoup marqué, c'est le fait que ces élèves-là considérer que la vraie punition, ce n'était pas tant d'être dans le dispositif ou deux, mais d'être privés d'école. Effectivement, le marqueur symbolique était très, très fort et il le formule assez bien. [01'26'32] J'ai mis deux extraits. [01'26'33] Abdoulaye qui nous dit: tu réfléchis bien quand t'es exclu avec l'éducateur et tout, tu vois, qui es-tu que t'aurais pu? t'aurais pu réagir autrement? [01'26'42] Mais je pense que ça arrange pas. Ça n'arrange pas la situation, pour pas te mentir. Je pense que tu vois, pour moi, c'est un site encore plus à faire de la merde, c'est sûr. [01'26'51] Mais pourquoi ça incite encore plus à faire de mien? [01'26'53] Parce que ça fout la haine. Après, quand tu reviens en cours, tu vois les gens, les gens, ils travaillent, et toi t'es en retard. Je comprends rien à ce que dit le prof. Après, tu peux pas suivre. [01'27'01] Comment tu fais pour suivre, bien tranquille, bien posé, quand tu comprends pas. Après, tu vas parler. Après, je ne sais pas, tu vas faire de la merde après quand exclu, c'est pire. Et du coup, t'es revenu au collège, tu voulais te calmer, mais même c'est pas facile. Tu vas parler, tu vas énerver, et voilà, tu vois là, tu vas encore plus être exclue. [01'27'18] Un autre qui nous dit: [01'27'20] Donc l'enquêtrice lui demande: ça sert à quoi pour vous de venir ici? ça sert à nous casser les couilles par nous? [01'27'26] C'est pas grave, on parle comme tu veux. Moi je pense que ça nous donne une leçon quand même. Cuisson deux, il y a deux élèves. [01'27'32] Ça nous donne une leçon. Quand même, tu dis ça parce que c'est la première fois que t'es exclu. [01'27'36] Si ça sert à nous apprendre des trucs sur comment il faut se tenir, pas se laisser emporter et tout. [01'27'42] Moi, je pense que pour apprendre des trucs, c'est mieux d'être en cours. [01'27'46] Et puis, c'est pas ça qui va nous faire comprendre, ça va pas nous faire comprendre. [01'27'50] Comment il faut se tenir en cours, et tout ça. Là, on sait déjà. En plus, ils croient quoi. On est éduqués, on sait déjà. [01'27'57] Ouais, c'est vrai. Après les profs, ils disent: t'es mal élevé. C'est normal qu'on s'énerve. Moi, je n'accepte pas qu'on dise que mes parents, ils font mal leur travail. [01'28'05] Et on aurait pu, voilà, avoir encore d'autres, d'autres extraits d'entretiens. On retrouve vraiment très souvent ces marques là, c'est-à-dire sur le premier. Le fait de dire finalement, le retour dans l'établissement est plus dur. [01'28'17] Que. [01'28'19] Qu'avant, parce que il y a un an, parce que on n'arrive pas à raccrocher fin. C'est ce que déjà très, très bien montré dans les, dans les travaux sur le décrochage, cette difficulté a raccroché. Je pense aux travaux de du l'azalée, en particulier sur ces questions-là. [01'28'34] Et le deuxième aspect, plutôt sur le fait qu'il y a une remise en cause. [01'28'39] De la la, la lecture institutionnelle des familles sur l'importance de l'école, sur le fait de comment on doit se tenir à l'école, sur une entreprise de normalisation autour de ce qu'est l'école. [01'28'51] Loin d'être partagé par les, par les élèves. [01'28'57] Pour conclure. [01'28'59] Sept points. [01'29'00] Le premier point, le fait que cette routine punitive [01'29'04] Fait qu'on est passé d'une mesure qui est pensée comme une mesure d'urgence à une mesure ordinaire. Une mesure d'urgence où elle est censée être exceptionnelle, où elle est censée répondre à un caractère. C'est indiqué dans les textes. [01'29'18] D'urgence en août, entre autres choses. [01'29'21] Et qu'on voit bien, avec les chiffres que je vous ai proposé, qu'on n'est pas du tout sur le l'exceptionnel, l'extraordinaire, comme disait bernadette ce matin, mais bien sûr l'ordinaire. [01'29'31] Deuxième aspect: [01'29'33] Des élèves en difficulté, à la difficulté de travailler avec les élèves. [01'29'37] Nous, nous sommes que ça, c'est un point important qui interroge vraiment l'école dans sa capacité à penser. [01'29'44] La question, j'ai, j'y reviendrai, mais la question de l'éduc habilité des élèves, c'est-à-dire, quelle tolérance finalement aux socialisations et aux sociabilités juvéniles ordinaire en milieu scolaire? l'école est effectivement pas la rue, l'école est effectivement. [01'29'58] Une institution est un espace spécifique marqué par une forme scolaire fortement. [01'30'02] Instituée. Mais pour autant, on voit bien que l'enjeu d'une école démocratique pose aussi la question de la cohabitation de la norme scolaire, des normes scolaires, avec les normes juvéniles. Là, ce qu'on observe, nous, sur nos terrains, c'est une crispation très forte sur ces registres de tolérance à l'écart, à la norme, et qui pose la question de l'entreprise d'éduquer. [01'30'22] Unité? est-ce qu'elle est encore posée en ces termes-là ou pas, dans un certain nombre d'établissements? [01'30'29] Le fait d'interroger la la continuité éducative. Ce qu'on a observé, c'est que les élèves qui sont les plus en rupture, les plus en difficulté, sont ceux qui sont le plus exclues, mais, en plus, qui sont le moins prise en charge du point de vue socio-éducatif. Après, par ailleurs, donc, on en vient à ce paradoxe fort, ce sont les [01'30'49] Les élèves qui ont le plus besoin d'école, qui, finalement, du point de vue du registre des apprentissages, en bénéficient le moins. Je ne suis pas rentré dans le détail d'autres travaux qu'on mène. Et puis des collègues, comme arnaud dubois, par exemple, soulignant lemoine à limoges. Arnaud dubois, versailles. [01'31'03] Travail sur les exclusions de classe. Travail sur: [01'31'08] Les, les temps d'exclusion dans l'établissement, sur des choses comme ça, et on voit qu'il y a beaucoup d'élèves qui, de plus en plus, ont une trajectoire scolaire marquée. [01'31'19] Par un manque d'école, pas sur le fait qu'ils n'y vont plus, mais dans l'école elle-même. On a plus, finalement, de de prise en charge à hauteur de ce qui pourrait être attendu pour ses élèves. [01'31'32] Quatrième élément: le fait qu'on a des alliances éducatives. [01'31'37] Qui pose la question? [01'31'39] De l'autonomie- deux de ces dispositifs- et du risque d'externalisation par l'école. [01'31'45] D'une partie de ses missions et de la prise en charge des élèves. [01'31'48] Moi, je vais être très, très clair par rapport à ça. Je pense qu'il y a un vrai danger à ce qu'il y a une dérive d'une prise de position, y compris avec des enjeux. [01'31'57] On peut tout à fait entendre de parfois financier ou de développement des structures. [01'32'05] Sur des problématiques éducatives et scolaires qui font que, l'école étant en difficulté, faites appel à ces structures là, mais dans une logique d'externalisation plus que de partenariat. Et il me semble que ça, c'est vraiment un marqueur fort de la recomposition des espaces scolaires et éducatifs qui pose et qui porte atteinte, à un moment donné, au projet. [01'32'25] Démocratique de l'école, des travailleurs sociaux et des intervenants sociaux ne peuvent pas faire à la place de l'école ce que l'école devrait faire. Ça ne veut pas dire qu'ils ne peuvent pas travailler sur des enjeux scolaires ou autres avec l'école, mais en revanche, on sait que sur le registre des apprentissages, sur le registre des savoirs, sur le registre la reconnaissance institute, [01'32'45] Lionel dans les trajectoires de vie. L'école reste dans les sociétés démocratiques. C'est le modèle pour l'instant. Ça sera peut-être un autre plus tard, mais en tout cas, c'est quand même ça, aujourd'hui, l'école, le principal levier qui est censé assurer d'une forme de mobilité et d'émancipation. Parler, parler, savoir. Donc, je vois que ça, ça pose vraiment des vraies questions. [01'33'04] Et que du coup. [01'33'05] Il faut réussir à dépasser. [01'33'08] Des dispositifs qu'on a appelé avec stéphanie rubi, les dispositifs how to the league stellaire, qui s'auto alimente, qui sont légitimes par eux-mêmes et pour eux-mêmes, qui prennent en charge un public qui n'est pas celui pour lequel ils sont conçus. Ils sont pensés et organisés. On sait très bien qu'il y a toujours une marge. Il y a aussi pas mal de travaux là-dessus en politique publique embarque. [01'33'28] Séculier de sociologie politique. [01'33'31] Qui montre bien qu'on ne peut pas être sur des modes de ciblage, à moins d'être dans des formes de big browser norme avec des fichiers, avec plein de choses. [01'33'39] Rib. [01'33'41] On ne peut pas avoir toujours la la, la cible qui est visée parfaitement, mais entre le toujours et le presque jamais, il y a il y a quand même une petite marge qui est peut-être importante. [01'33'53] Surtout quand on parle des élèves les plus en les plus en difficulté. [01'33'58] Une attention à avoir sur le cac. [01'34'00] Le fait que on a douze pour cent de notre échantillon. [01'34'04] Qui sont les établissements qui excluent peu. [01'34'07] De façon pérenne et stable. [01'34'10] Avec trois à quatre élèves exclus temporairement par mois. [01'34'14] On voyait qu'on est très loin de ce qu'on avait tout à l'heure. Quand même, douze pour cent de l'échantillon, c'est pas beaucoup. [01'34'19] Mais c'est quand même significatif. Et dans ceux-là les élèves. [01'34'24] En situation de d'urgence, eux, sont pris en charge dans les dispositifs starck. Les dispositifs ont la place de prendre en charge ces élèves. Là, on est plus dans une gestion de flux qui interdit la prise en charge des élèves en situation d'urgence, on est dans une gestion éducative des élèves en grande difficulté. C'est très différent et nous, c'est vraiment quelque chose. [01'34'44] Bataille comporte, sur ces terrains, là, pour essayer de, de, de. [01'34'50] D'attirer l'attention sur le fait qu'on doit pouvoir avoir une réflexion collective sur ces enjeux. Là. [01'34'56] Évidemment, un certain nombre de limites sur cette recherche. D'abord, le fait qu'elle est, elle pose le la focale sur un territoire. [01'35'03] Particuliers. [01'35'04] Ce serait intéressant de comparer avec des espaces plus mixtes, moins ségréguées. Là, on est quand même beaucoup sur des espaces ségréguées. Après, je fais l'hypothèse que les différences serait pas si énorme que ça. On a fait sur des échantillons plus petits, au travers des enquêtes de climat scolaire et de victimation, des études, là, sur un échantillon par exemple, des détails. [01'35'25] En rep, on a près de quatorze pour cent des élèves qui des classes avoir été temporairement exclus dans l'année. [01'35'31] Sur l'année l'année dernière. Quatorze pour cent, ça fait quand même beaucoup, de beaucoup d'élèves. [01'35'38] On a peu. Aussi, on n'a pas travaillé la question de l'articulation avec les exclusions de ponctuelles de classe qui ne sont pas comptabilisées ici. On sait qu'en place, et seulement les exclusions temporaires. [01'35'51] On a pas non plus- je l'ai évoqué rapidement tout à l'heure- travailler sur les caractéristiques sociologiques des enseignants, qui excluent ou qui n'exclut pas, parce que là, on a observé aussi une forte différenciation à l'intérieur des établissements. On a des dés, on a peu d'établissements où c'est très homogène sur qui exclue. Il y a un effet là, établissement qui masque le. [01'36'10] Fais que des étapes des enseignants excluent beaucoup, demandent beaucoup d'exclusion parce que c'est pas eux qui excluent un seul chef d'établissement. [01'36'16] Et d'autres qui n'en demandent quasiment jamais. [01'36'19] Ça, c'est aussi quelque chose qui serait intéressant de travailler. [01'36'25] Et enfin, voilà le travail sur les critères en usage d'élection- ou de mon élection- au dispositif. C'est quelque chose qu'on a un peu travaillé, mais qu'on est en train de d'approfondir de manière plus plus fine. [01'36'39] Ça, c'est des éléments bibliographiques autour de ce que j'ai, ce que j'ai présenté là, vous trouverez sur ce lien. Alors c'est un peu long, mais sinon, via google, vous trouverez. [01'36'51] Tous ces articles, la fin, ça c'est un livre qui est à paraître en deux mille seize. Mais ces deux articles et d'autres, vous les trouverez en libre accès ici. [01'37'00] Et voilà. [01'37'03] J'étais là parce, puisque je n'étais pas très longtemps, [01'37'08] Au fil des années. [01'37'58] Dans le droit chemin, si je puis dire. Est-ce qu'il y a d'autres raisons qui sont données par l'établissement et est-ce que toi, tu en vois d'autres? comment est-ce qu'on peut approfondir ce jour-là? [01'38'09] Par rapport à ça. [01'38'12] Moi, il me semble qu'il y a un enjeu essentiel- c'est ce qu'on a observé dans le changement sur certains dispositifs, certains qui n'accueille quasiment aucun gamin vraiment en difficulté et qui l'ont fait l'année suivante- c'est la gestion de flux. [01'38'26] C'est assez terrible, mais le fait que quand t'es dans un établissement où t'as en gros dix gamins par jour, exclus [01'38'31] Il y a quasiment pas de travail éducatif, voire aucun travail éducatif, sur cette question de l'accompagnement et de l'orientation vers le dispositif. [01'38'41] Pour autant, le dispositif est très important parce qu'il nous permet- je dis nous- [01'38'45] Dans un établissement, d'avoir une réponse institutionnelle à une difficulté locale qui est reconnue, s'avère qu'on a jamais eu. De quand on a fait des restitutions dans certains établissements ont donné des chiffres, on a toujours eu de la surprise. [01'38'59] Mais on n'a jamais eu ni de la négation, ni du contentement, aucun établissement. Alibaba, oui, on exclut vingt élèves par jour. Et alors, c'est quoi le problème? jamais on n'a pas eu, on a. On a toujours ressenti une équipe très mal à l'aise, voire très en difficulté par rapport à ça, mais du coup, quand on en a dix, par [01'39'18] Jour à orienter n'en remet pas en cause le le dispositif. On est tout à fait dans le registre méritocratique que j'évoquais tout à l'heure. Il y a ceux qui méritent d'aller dans le dispositif et d'avoir une prise en charge, et à ceux qui ne méritent pas cette prise en charge, et quand on met dos à dos ce qu'ils méritent, c'est-à-dire qui sont le moins, [01'39'38] Éloigné de la norme scolaire et ce qui ne mériterait pas parce qu'ils feraient pas assez d'efforts. [01'39'43] On est sur ce registre là, mais beaucoup sous la pression du flux. [01'39'46] Dans les étamines, dans les dispositifs où ils accueillent les élèves les plus en difficulté, mais pas que cela. Il y a des fois des élèves qui sont pas non plus en très grande difficulté, mais qui ont été exclus. On est, il y a beaucoup plus de distance par rapport à, à ce registre de la légitimité à être orientés ou pas dans le dispositif, parce que l'exclusion devient un marqueur. [01'40'05] Et c'est salles angela, c'est que, finalement, le marqueur est déplacé. [01'40'09] Pour les structures socio-éducatives, leurs marqueurs, c'est l'exclusion. [01'40'13] Et c'est ça la grande surprise de ce qu'il disait. Mais on pensait qu'un élève exclu un élève. [01'40'17] Vraiment, il avait été loin quoi avant d'être exclu et de se rendre compte qu'en fait, non. [01'40'22] Et c'est pour ça que sur cette, ce travail-là, il y a notre sens, il articule plusieurs dimensions. Ce n'est possible que parce que il y a recomposition des espaces scolaires, éducatifs, que parce que les travailleurs sociaux se mettent sur ce marché- entre grands guillemets- de la lutte contre le décrochage scolaire, que parce que il y a des flux. [01'40'42] Important. Que voilà, enfin. On voit les choses s'articuler après, au fur et à mesure, mais cette question de la responsabilité renvoyée aux élèves, elle est très forte. [01'40'53] Merci beaucoup. [01'40'55] Ça va, ça va, ça fonctionne, je pense. [01'40'57] Donc, je voulais dire que, en fait, j'étais très intéressé sur cette question de d'un dispositif qui cible en fait des jeunes qui sont dans une situation un peu moyenne, qui sont pas les plus en difficulté, parce qu'elle rejoint beaucoup d'autres écrits. [01'41'17] Sur des interventions sociales. [01'41'21] Qui montre que je pense en particulier à la thèse de linda la vitry, qui a eu un prix du monde l'année dernière. [01'41'29] Sur le travail des conseillers à pôle emploi. [01'41'32] Et qui montre aussi que, en fait, [01'41'37] Il est pour des raisons de gestion de flux. [01'41'40] Mais aussi pour des raisons de comment dirais-je? [01'41'46] Deux: dévaluation du travail des déconseillés. [01'41'51] Finalement, ils ont comme cœur de cible des des personnes qui ne sont pas des les les chômeurs de longue durée et les plus en difficulté, mais que cette politique d'activation dont ils parlent, dont ils parlent beaucoup, s'adressent à des gens qui sont susceptibles de [01'42'11] Revenir dans le monde du travail quand même. [01'42'13] Plus rapidement que d'autres. [01'42'15] Et c'est aussi ce qu'on voit dans les travaux, par exemple, de jean-philippe melchior. [01'42'21] Sur les. [01'42'24] L'évaluation du travail des des travailleurs sociaux. Chez hum, je me souviens bien ça part, ça parlait de travailleurs sociaux des carpates, mais je ne suis plus très sûr. En tout cas, ça parlait du travail social aussi dans un autre domaine d'intervention, qui montre que pour des questions d'évaluation de leur travail, [01'42'44] Il règle les problèmes qui vont être réglés les plus vite. [01'42'48] Le plus rapidement, donc, de fait, leur. Leur travail ne se centre pas sur les personnes qui auraient le plus besoin de leur intérieur intervention. Donc, ma sœur, ma remarque, c'est: c'est peut-être justement que [01'43'04] Finalement, quels sont les mécanismes? [01'43'09] Qui font que ce sont ces personnes-là qui sont le plus ciblés, parce que peut-être que c'est pas seulement pour des questions de [01'43'20] Peut-être que ça rejoint des questions de gestion de flux et que ce que, comme tu le disais, et que finalement, il y a et fin. [01'43'28] C'est assez frappant, ce cette centration sur des, sur la situation moyenne. [01'43'33] Après. La difficulté pour les travailleurs sociaux, c'est qu'ils ont pas la main sur le public qu'ils accueillent. [01'43'40] C'est là, c'est pas eux qui, en fonction de. [01'43'44] Exemple d'un impératif de résultat. [01'43'47] Nous, quand on a fait l'évaluation, la grande préoccupation, c'était alors: est-ce que ça marche? quel pourcentage d'élèves qui, quand tu sors du dispositif, n'est plus exclu? [01'43'56] Donc la première chose à dire, évidemment, c'est: un dispositif d'une semaine peut pas suffire à expliquer le fait qu'un gamin soit exclu ou pas après, mais c'est le fantasme très fort et compréhensible. [01'44'08] Mais de ne des financeurs et des dés. [01'44'13] Les administrations qui mettent en place ce dispositif là, mais les travailleurs sociaux. [01'44'18] C'est les épées ou les chefs d'établissement qui leur envoient les gamins. N'oublions pas la main sur l'horizon, pas la prise sur qui vient et le fait de ne choisir ou non. [01'44'29] De favoriser tel ou tel. [01'44'32] Le public a même été, ce que ce que j'essaie d'illustrer, la surprise pour beaucoup, deux d'entre eux à dire: non, c'était, voire même parfois, on a observé des situations vraiment cocasses, c'est-à-dire sur, par exemple, le rapport à la norme scolaire, des ateliers sur citoyenneté. [01'44'49] Citoyenneté- je ne sais plus comment ça plait, mais quelque chose ça ne s'appelait pas comme ça- mais en gros, des citoyens, des énormes scolaires. [01'44'56] Gamin. Ma voilà ce que l'école profilé, comme ces conflits, comme ça. [01'45'01] Et. Mais les intervenants avaient préparé quelque chose pour des gamins dont ils imaginaient que je n'étais pas trop au fait de de ses aspects normatifs là. Et puis si? [01'45'13] Des gamins un proche. [01'45'15] Pas comme un élève, un élève droite. [01'45'17] Mais un prof viendra dire ça parce que c'est un adulte. [01'45'20] Incorporez meilleur, très, très forte des relations de deux, de domination, voire de pouvoir, entre profs et élèves qui étaient, qui étaient très, très marqués. Ça tombait très à plat ces ateliers. [01'45'31] Mais avec ce ce, cette difficulté pour retrait exprimé de ne pas même pas pouvoir avoir ce choix là, finalement, et c'est là que se pose la question de la sous-traitance ou du partenariat. [01'45'42] Comme comment. [01'45'44] Sur un rapport partenarial, tu dois pouvoir composer et travailler sur quel est le type de gamin qu'on va prendre en charge et pourquoi ceux-là et pas les autres. Là, on l'a peu vu. Ça existe un peu, mais on l'a peu vu. [01'45'59] Oui, j'aurais des réflexions par rapport à tout ce que vous avez dit et par rapport à ce qu'on a dit ce matin. [01'46'06] Notamment. [01'46'07] Comment dire par rapport à l'exclusion en tant que telle? [01'46'12] Par rapport au constat que vous en faites, par rapport au motif d'exclusion au jour d'aujourd'hui. [01'46'17] J'ai le sentiment que les modalités d'exclusion telle qu'elle était prévue, [01'46'22] Dans l'échelle des sanctions punitives au niveau des collèges. [01'46'26] N'est plus appliquée. [01'46'28] Tenant, c'est-à-dire que on a perdu de vue le, le cadre. [01'46'32] Générateur de cette exclusion et que la banalisation de cette mesure vient aussi du fait que la mise en place des dispositifs supplétifs viennent traiter les conséquences de l'exclusion et ce qui est visible de l'exclusion, et non pas la question de fond, la question de [01'46'53] Faux, c'est plus par rapport à la, je dirais à la légalité et à l'origine de la légalité de cette mesure punitive. Est-ce que les collèges et les chefs d'établissement qui prennent au jour d'aujourd'hui cette décision? [01'47'07] Prenne dans le cadre, je dirais, légale et formalisé. [01'47'11] Qui était prévu à l'origine. [01'47'15] La deuxième. Ces question, c'est aussi. [01'47'17] A, qui appartiennent les enfants. [01'47'20] Par rapport à la responsabilité. L'école est obligatoire jusqu'à seize ans et les enfants qui sont à l'école sont sous la responsabilité. [01'47'28] Du directeur d'école et de l'éducation nationale. [01'47'31] Quand ils sont exclus? quelle est la responsabilité lorsqu'ils ne soit pas pris dans une structure éducative? puisque, si j'ai bien compris, [01'47'41] C'est une action volontaire. [01'47'43] De l'élève qui accepte d'aller dans le, le pays heureux. Il n'est pas obligé d'y aller, puisqu'il a le choix de l'élève qui va y aller. Et il y a le choix, l'acceptation de l'élève. Dans quelle mesure on lui laisse un choix à partir du moment où l'école est obligatoire? est-ce que ce n'est pas la solution de facilité? [01'48'02] De dire: [01'48'04] On se dégage pendant qu'on vient dire, pendant deux, trois jours, on est plus responsable de lui et s'il arrive quelque chose, l'assurance scolaire va-t-elle le couvrir? quel est le débat- je dirais juridique, légal- en termes de responsabilité, de prise en charge par rapport à cette, à cet enfant qui n'a pas l'autonomie? [01'48'25] A la fois civile et décisionnelle pour palier à son comportement. [01'48'32] Donc, c'est toutes ces questions-là qui sont paradoxales, en fait. [01'48'37] Sur sur l'aspect. Le premier aspect, l'aspect légal. [01'48'43] Le cadre légal depuis, en gros, depuis deux mille cinq. Ça s'amorce en deux mille, mais depuis deux mille cinq, il a vraiment changé en matière de procédure disciplinaire, dans les ordres, dans nos établissements du second degré en particulier, en valorisant beaucoup, quelles que soient les gouvernements d'ailleurs, [01'49'02] Le registre éducatif. [01'49'04] En insistant sur la notion de sanction éducative, en insistant sur [01'49'09] Des aspects de prise en charge de tout ça, mais ce qu'on observe, c'est qu'il y a un hiatus très fort entre les textes et l'esprit des textes. [01'49'19] Et la réalité des pratiques. [01'49'21] Sur le, sur le terrain. [01'49'25] Du coup sur ces pratiques, et c'est ce fort niveau de l'usage des sanctions de manière générale, parce que les versions temporaires, mais sur les enquêtes climat que j'évoquais, c'est plus de cinquante pour cent des élèves qui sont qui sont collés dans l'année. [01'49'40] Dans certains établissements ça monte à quatre vingt. [01'49'43] Il y a même des établissements où ces quatre, vingt, seize pour cent des élèves sont collés dans l'année. Donc, le registre de la sanction éducative, il est. [01'49'53] Loin. [01'49'54] Il est très, très loin. Mais il témoigne aussi de plusieurs choses. D'abord, d'une vraie souffrance des équipes enseignantes- et je pense qu'il faut pas les prendre de haut et il ne faut pas déconsidérer ça- c'est-à-dire que ça témoigne de difficultés ressenties par les premiers acteurs concernés, et donc c'est préoccupant. [01'50'12] On peut pas juste dire que que on a une dérive autoritaire de de l'école. Il y a quelque chose là qui est de l'ordre. [01'50'22] De la souffrance enseignante des dit françoise fantôme qu'il faut pouvoir travailler. [01'50'28] Et entendre. Deuxième élément: on a une difficulté des chefs d'établissement. Ça, ça témoigne de beaucoup de choses en termes de difficultés de management pour les chefs d'établissement. [01'50'39] Elle a écrit des nouveaux manager de la république, en parlant des chefs d'établissement est clairement beaucoup non. On dit l'exclusion temporaire, c'est une variable. [01'50'49] Importante d'achat de la paix sociale au sein de l'établissement. C'est un marqueur symbolique extrêmement fort. [01'50'56] Pour les équipes. [01'50'57] Ça témoigne d'une solidarité. [01'50'59] Très forte envers les vraies difficultés- entre guillemets- éprouvée par les, par les équipes, et beaucoup de chefs nous ont dit: on sait que ça, ça a rien à voir, on sait que c'est pire avec certains élèves, mais au moins, [01'51'13] Ça permet de d'être un peu libéré sur des enjeux. [01'51'20] Locaux. [01'51'22] Et internes. [01'51'24] Après: est-ce que l'exclusion temporaire est légal ou pas? [01'51'28] Elle est légale à partir du moment où elle déborde pas des cadres de d'une exclusion de plus de huit jours, etc. Sur la continuité éducative, la loi prévoit que le chef d'établissement doit s'assurer de la continuité éducative et scolaire. [01'51'42] Mais. [01'51'44] S'il ne le fait pas, ça le regarde lui et il est peu mis en cause par son institution par rapport à ça. Mais c'est là-dessus quand même que la médiatrice de l'éducation nationale alerte dans son rapport en disant: là y a un lien, un problème. [01'51'59] Et on voit bien que l'institution scolaire est pas très à l'aise avec cette question des exclusions temporaires parce que, effectivement, c'est pas cohérent avec l'esprit de la loi. Mais le décalage avec le terrain est-elle? [01'52'14] Que donc? on peut imaginer deux choses: soit la loi rattrape le terrain, et c'est demander à chaque fois qu'il y a une réforme sur les procédures. [01'52'22] Là-dessus. [01'52'23] Des syndicats demandent à ce que soit supprimée la valence éducative. [01'52'28] Sur, par exemple, l'exclusion temporaire, la continuité scolaire et l'obligation d'encadrement éducatif des élèves, en disant de façon: ça ne se fait pas. Donc, plutôt que de faire comme si ça se faisait, ça se fait pas pour l'instant, ça, ça n'a pas été, ou alors c'est essayer de faire vivre d'autres types de mesures qui commencent à se développer, qui existe dans l'arsenal législatif. [01'52'48] Les mesures de responsabilisation. [01'52'50] Les exclusions, inclusions que moi j'appelle les suspensions, comme les appelle les québécois, qui me semble plus juste. [01'52'57] Ou alors des mesures plus radicales qui existent dans un certain nombre de pays: la suède, l'allemagne, l'espagne. [01'53'03] Brésil, le portugal, l'interdiction des exclusions temporaires. [01'53'07] Mais ça décompose ça. Si vous voulez animer un, une conférence avec des équipes de proches, [01'53'15] Et que ce soit dynamique. Vous commencez par ça. [01'53'19] Moi je le fais, c'est-à-dire: [01'53'22] Poser cette question-là supprimant les exclusions temporaires. Et ça renvoie tout de suite à plusieurs aspects: l'aspect institutionnel, le registre punitif de l'institution, puisque foucault a déjà montré, ça a été montré et remontré par rapport à ça, le poids symbolique, l'exclusion du corps de l'élève, pour le coup. Voilà, c'est, c'est très, très sensé. [01'53'42] Cible extrêmement sensible. [01'53'44] Mais ça reste dans le cadre de la loi, avec après des des libertés qui sont prises par les chefs d'établissement. [01'53'53] Peut-être, je vais répondre à la question. De même, sur la question des filles. [01'54'02] Bonus. [01'54'11] Donc les filles, en fait, on a. C'est quelque chose qui nous avait. [01'54'16] Interpellé sur nos premières enquêtes ethnographiques sur les dispositifs. Je caricature un peu, mais ce qu'on a plus beaucoup de temps, mais en gros, on avait des, des garçons qui étaient assez calmes. [01'54'27] Assez stéréotypés sur des élèves moyens au comportement assez ordinaire. [01'54'33] Et défis. [01'54'35] Qui était remontée, mais bien comme il faut et qui correspond beaucoup plus aux profils d'élèves dites déviantes que les garçons. [01'54'43] On a fait un dispositif de dispositifs, trois dispositifs. On s'est rendu compte que ce n'était pas systématique, mais que, très souvent, les filles qui étaient orientées dans les dispositifs étaient sur des registres normatifs beaucoup plus durs que ceux des garçons. [01'54'57] De fait, ce qu'on a constaté. [01'54'59] D'abord la. La deuxième chose qui nous a mis la puce à l'oreille, c'est que, selon les années, [01'55'04] On avait, je l'ai dit tout à l'heure, en gros, treize pour cent des exclusions. C'est pour les filles, mais dans les dispositifs, on avait vingt à vingt cinq pour cent de filles. [01'55'12] Accueil, théoriquement, s'il n'y avait pas de sexuation. [01'55'16] De l'orientation dans le dispositif. [01'55'18] On devrait avoir treize pour cent de filles dans les dispositifs net tv. Vingt à vingt cinq pour cent. [01'55'24] Donc, on a essayé de comprendre pourquoi. [01'55'27] On a regardé des chiffres. Si vous voyez, j'ai repris juste quelques variables sur, par exemple, l'appréciation des élèves dans les questionnaires qu'on avait fait passer sur la sanction: est-ce que la sanction que t'as reçu allez juste, ou bien il n'est pas juste? [01'55'41] Vous voyez qu'elle est juste pour cinquante sept pour cent des garçons, pas juste pour quarante cinq pour cent. [01'55'47] Et juste pour quarante cinq pour cent défis. [01'55'50] Un juste pour vingt trois pour cent des garçons. [01'55'53] Et un juste pour trente et un pour cent des filles. Donc, les filles c'était plus plus dur que les, que les garçons. [01'55'59] Il y a plein de variables qu'on avait comme ça, ou, en fait, les filles répondaient toujours sur les essais, sur les indices de climat scolaire. [01'56'05] La perception des filles était beaucoup plus négative, sur celles qui étaient dans le dispositif, que celle des garçons. [01'56'11] On a regardé aussi. Vous voyez, là, vous avez les pourcentages d'élèves. [01'56'17] Par niveau de classe. [01'56'21] Et vous voyez la queue au niveau des défis et des garçons. On a des filles qui arrivent plus [01'56'28] Tardivement que les garçons et on avait des filles plus grandes en moyenne que les garçons qui étaient accueillies dans les dispositifs. Quand on creuse un peu tout ça, et au bout de deux ans et demi d'enquête, on a eu finalement des registres. [01'56'45] Normatif de la part des des établissements et de la part des travailleurs sociaux. [01'56'50] Très fort du point de vue de la sexuation, de l'orientation dans ces dispositifs, sur un registre qui est que les filles étaient récupérables et que les garçons étaient perdus. [01'56'59] C'est, et on a intitulé un article qui n'est pas encore publié comme ça: [01'57'04] C'est cette idée que les filles qui étaient orientées dans ces dispositifs. [01'57'10] Là aussi, je parle de manière générique. Ils n'avancent pas toutes les filles, mais as un grand nombre des filles qui étaient orientées dans ces dispositifs. [01'57'19] L'était pas en sixième, pas en cinquième, mais plutôt en quatrième, voire en troisième, avec cette idée que [01'57'28] Vraiment, on les envoyait quand, dans l'école, on n'y arrivait plus. [01'57'32] Mais que, comme elle était fille, [01'57'36] Ce n'était pas normal qu'elle se comporte comme ça. [01'57'39] Et que donc, comme ce n'était pas normal, c'était sans doute- elle devait avoir des difficultés familiales. [01'57'45] Et que, comme du coup ce n'était pas leur faute, on rentrait pas dans le registre que j'évoquais tout à l'heure. [01'57'50] De responsabilisation, de la prise en charge qu'on faisait peser sur les garçons qui, de façon, était dur, mais parce qu'il était dur. [01'57'57] Pas parce qu'il avait des problèmes familiaux ou des voilà. [01'58'01] Donc, on a vraiment une sexuation de l'orientation dans les dispositifs qui est très marqué par rapport à ça, parce que non seulement on a beaucoup de filles, [01'58'08] Proportionnellement, devront avoir treize pour cent. On a parfois dix pour cent de plus. [01'58'13] Mais en plus, les filles qui y vont sont des figues, sont pas du tout sur le même profil que le garçon et si on re découpe nos questionnaires de tout à l'heure, comprend que les garçons, on enlève les filles sur l'échantillon expérimental. [01'58'26] L'échantillon du coup expérimental des élèves qui sont passés dans le dispositif. L'appréciation climat est nettement plus positive que celle de la moyenne académique. [01'58'35] Mais. [01'58'36] Du coup. [01'58'40] Et puis on a défi. Voilà ça, c'est un extrait d'entretien. [01'58'43] On a des filles qui sont beaucoup sur des des exclus, sur des questions de problèmes de groupes de bande. [01'58'51] D'affrontements entre groupes juvénile manière très, très forte. C'est très identifié. [01'58'56] Et il y avait un un, un responsable de dispositif qui nous le dit, mais en fait, qui s'en était pas rendu compte. C'est quelque chose qui n'est jamais verbalisé. [01'59'05] Et c'est très important ça, jamais un dispositif ou un acteur de dispositif nous a dit: nous, on reçoit plus de filles que ce qu'on devrait recevoir. [01'59'12] Et les filles sont. [01'59'14] Bizarrement pas comme les garçons. Il y a toujours un peu de distance par rapport à ça. Là, m kalla, quatre vingt dix, quatre vingt quinze pour cent des élèves que l'on accueille viennent pour des choses. Il y a des violences verbales, physiques, insolence et filles-garçons. C'est la même chose, c'est pareil. [01'59'27] Des actes individuels ou collectifs dont c'est la même chose. On a beaucoup de filles qui viennent de certains collèges et qui se battent énormément. [01'59'33] Et puis, en fin de rencontre, il reprend précisément les données informatisées. À propos: défi, accueil et commente abassi. Dix des vingt filles qui sont venues proviennent en fait d'un même établissement, dont une est revenue deux fois, quatre sont de quatrième, de troisième, quatre de cinquième. C'est bien le même réseau d'interconnaissance. Elles se connaissent. Oui, elles n'ont pas été orientés à la suite d'une action collective, excepté. [01'59'53] Élève, les sept autres se connaissent et un phénomène de groupe très important pour ses fils a beaucoup de bagarres, parfois très violentes. Elles sont suivies par un éduc. Il y a un pic entre en cinquième, quatrième du même collège, puis on voit aussi beaucoup de garçons plus dur et on a plus de problèmes avec ces élèves-là. Les filles sont violentes et pour les bagarres, tout est filmé automatiquement et mis sur anti-poux cave. [02'00'11] C'est un site. [02'00'13] Cave qui pue ligne ou sur des où voilà qui balance des, des, des vidéos de ce type-là. Il nous demande de travailler en amont, de développer en amont un travail éducatif avant, l'exclusion, avec des actions collectives ou individuelles. [02'00'31] Et donc on a avoué, en gros: [02'00'35] On a trois éléments, l'un sur les éléments de sexuation. Le premier, c'est beaucoup de choses sur les ailes, les garçons de sixième, cinquième, [02'00'42] Pour les cadrer, les garçons sont plus sanctionnés que les filles. Sur sixième, cinquième et plus exclusif, les filles, en sixième, cinquième, le registre des filles pubères. Qui faut remo l'arri re moraliser avec les sanctions pour les, pour les sauver, ça c'est vraiment très, très, très, très utilisé. [02'01'02] Et puis l'appréhension, déficit scénariste des élèves et de leurs familles, mais surtout des filles, patent des garçons. [02'01'09] Ce registre de si les filles se comportent comme ça, si elles se battent, qu'elles sont dans des bandes. [02'01'14] C'est qu'elles ont un problème familial. [02'01'17] Le garçon sait que c'est un garçon dur de quartier populaire inédit. [02'01'21] La fille. Elle est perdue. [02'01'23] Donc, effectivement, il y a un marqueur fort là-dessus et donc un marqueur aussi sur la sexuation de la prise en charge. [02'01'36] Moi, je voulais poser une question sur l'amont de tout ça. [02'01'40] Parce que finalement, [02'01'43] Les enseignants, les chefs d'établissement ont énormément recours aux exclusions temporaires. [02'01'49] Alors peut-être qu'ils sont en souffrance, j'entends bien, peut-être aussi qu'ils sont démunis. [02'01'55] Du point de vue pédagogique, et donc, à ce moment-là, on en revient peut-être à des questions de deux formations. [02'02'02] Ou, en tout cas, ils ont l'air démunis sur la mise en œuvre de conditions éducatives, finalement, qui font sens pour les gamins. Donc, on est vraiment sur le paris de l'est. L'éducatibilité, quoi? enfin, je crois qu'il y a quelque chose qui est assez. [02'02'18] C'est troublant. [02'02'20] Et puis on retrouve ça: [02'02'23] Voici ce que si on avait eu un échange de mails là-dessus. Mais on retrouve ça dans: [02'02'31] Dans ce qu'écrit eric pereira, par exemple, sur le projet d'éducation, il renvoie un devoir d'hospitalité, un devoir, une préséance de l'accueil, du créera un devoir de sollicitude et un devoir de confiance. Donc, qu'est-ce qui se joue? voilà où sont ses devoirs, là, dans le paris des ducs habilités, des enfants. [02'02'50] En amont de tout ça. [02'02'52] Avant finalement d'en arriver à ce ou être complètement démunis et pas savoir quoi faire, et pour se retrouver face à des des- voilà des décisions qui ne sont pas forcément cohérent, mais juste. [02'03'06] Et puis arrivé aussi à cette situation de souffrance, parce que ça, c'est vraiment un signe de souffrance du monde enseignant, enfin moi, [02'03'13] Mais on est en plein dans le nom, dans ce paradoxe d'avoir des institutions éducatives. [02'03'21] Très institué, au sens où il énumère et: [02'03'25] Forte. [02'03'28] Mais qui sont aussi des institutions éducatives et scolaires, là en l'occurrence. [02'03'33] Dans lequel les registres entre inscription as instructions, pardon et éducation. [02'03'39] Berne. [02'03'41] En vigueur au fur et à mesure de la démocratisation et la massification des systèmes. [02'03'46] Et ça, c'est quelque chose que la la comparaison internationale, sur lequel la comparaison internationale est vraiment intéressante. [02'03'52] On voit que, dans les systèmes éducatifs, massifier, démocratiser depuis un certain temps, avec des niveaux d'accès aux diplômes et de ce qu'on appelle des sociétés des diplômes, sert de de fait une obligation à la diplomation pour. [02'04'07] Justifier la mobilité des trajectoires sociales. [02'04'12] Un ancrage. [02'04'14] Qui est de plus en plus difficile à établir sur le registre du cp habilité au profit de celui de l'instruction et de la diplomation, comme s'il y avait une forme d'antagonisme entre le fait d'éduquer ou d'instruire en france, qui est très fort parce que, historiquement, en plus, il est constitutif quand même du second degré. [02'04'33] Parce que ce dont on parle là, c'est du collège, qui est construit sur des références disciplinaires. [02'04'38] Des références disciplinaires qui sont issues d'une histoire et d'une tradition. [02'04'42] Très forte qui fait peu de cas des enjeux d'éducation. Voir qu'il est qu'il l'est. [02'04'47] Qui renonce fin? qui c'est un? [02'04'50] Lorsqu'on qu'on rejette très fortement, là, on sait que le modèle démocratique d'une école oblige. [02'04'56] À travailler. Cette question n'éduque habilité. [02'04'58] Et du registre de l'éduc habilité. Donc, [02'05'03] Il y a ça comme premier point. Le deuxième point, c'est les nouvelles problématiques éducatives. On voit aussi qu'elle qu'elle rentre dans les inquiétudes. Elles sont prises comme des inquiétudes par les nouveaux professionnels de l'éducation. Je pense aux travaux d'une vingtaine greyhound, perrier et joineau sur les nouveaux enseignants. On voit que ces préoccupations sur la conduite de classe, [02'05'23] Sur les questions de genre à l'école, sur tout ça. Ça inquiète beaucoup et donc, c'est très visible et ça alimente sans doute aussi des inquiétudes fortes qui ne sont pas traduites sur le plan du registre éducatif, qui nécessite forcément de l'instabilité. Elles sont traduites, selon beaucoup, sur le registre de la délégation à la société ou à des travailleurs sociaux, dont ça. [02'05'43] Doit être ou dont c'est censé être le métier, mais pas comme un retour au cœur du métier d'enseignant et donc du registre de les éducation et déductibilité. Mais c'est moi, je pense, que là-dessus, il y a, il y a vraiment, et c'est pour ça que ces enjeux autour de la recomposition des espaces scolaires et éducatifs, [02'06'00] De les repositionner comme des marqueurs de l'est de ce qu'est l'école démocratique aujourd'hui. C'est vraiment important et que la comparaison internationale est très révélatrice par rapport à ça. [02'06'10] Je prends l'exemple, vois. [02'06'12] Redonne celui-là, mais du brésil, il y a dix ans. [02'06'16] Le brésil, on était sur un registre très paternaliste. [02'06'22] Avec un enjeu. [02'06'24] D'encadrement dans l'école publique était celui que les élèves aillent à l'école. [02'06'27] Et qu'on les entoure, et qu'on les aime, et que tout ça. [02'06'31] En douze ans, treize ans, on a complètement changé nos registres. [02'06'36] En donnant une priorité aux apprentissages, pas au fait d'aller à l'école, mais d'être scolarisés et d'avoir des diplômes. [02'06'42] Et de rhodes, rattraper les autres puissances dans la compétition internationale sur ce que c'est que des compétences à obtenir. [02'06'49] Et on voit apparaître des problèmes et des préoccupations sur la violence à l'école, le décrochage scolaire, etc. C'est pas que ça existait pas avant, c'est que ça devient un problème public qui pénètre aussi les préoccupations des enseignants et dont la réponse apportée n'est pas celle du registre éducatif, elle est souvent celle du registre disciplinaire qui, à mon sens, est inadaptée. [02'07'10] Ah ça. Donc, ça veut dire aussi que, à un moment donné, et sans tomber dans un écueil qui opposerait une pédagogie d'un côté, les disciplinaires de l'autre, qui est aussi ridicule que plein de choses. [02'07'23] La question du modèle démocratique de l'école. [02'07'26] Ne peut pas ne pas penser. Le registre éducatif, aujourd'hui, dans la formation des enseignants, c'est optionnel. [02'07'34] C'est pas pas quelque chose qui est systématiquement mis en place, cette question-là. [02'07'40] Et on est ce que je suis là-dessus, mais on est le seul pays du monde. [02'07'45] Et pourtant, je suis très attaché au cep en tant que personne. [02'07'49] Et de ressources, et d'étudiants, etc. Mais on est quand même le seul pays du monde qui a délégué à un corps particulier dans le second degré. [02'07'58] La mission d'éducation. [02'08'01] Ça n'existe nulle part ailleurs. [02'08'03] Donc les profs. Il y a une tendance est à une organisation quand même institutionnelle, qui pousse pas à ce que les registres éducatifs concernent tout le monde, puisqu'on a des spécialistes même en interne de l'éducation. [02'08'16] Et je pense que c'est un vrai à la fois. Ça témoigne de beaucoup de choses, mais c'est aussi un levier à [02'08'22] Interrogée: [02'08'31] Par rapport aux douze pour cent des établissements où il y a moins de d'exclusion. Est-ce que vous avez constaté? [02'08'39] Un fonctionnement différent? est-ce que le dispositif interne à l'établissement? [02'08'46] Vous direz quelque chose de de nouveau. [02'08'49] Par rapport aux autres établissements. [02'08'52] Alors on n'a pas fait d'enquête systématique au sens. [02'08'57] Performatif du terme. [02'08'59] Il faudrait plus de travail, plus fin, pour vraiment répondre précisément à la question. Donc, ce que je vais vous donner, c'est plutôt de l'ordre du sentiment. [02'09'08] Que du l'ordre du résultat de recherche. [02'09'11] Mon sentiment, c'est que, sur ces douze pourcents d'établissement, y a toujours une volonté très forte de la direction de limiter le nombre d'actions temporaires. [02'09'18] On n'a pas un seul établissement où on n'a pas un chef ou une cheffe d'établissement engagé sur cette question-là et, du coup, engagé sur le registre éducatif. C'est très, très clair. Ça, c'est pas du tout des des chefs d'établissement manager au sens de ce que décrit anne barrère, c'est des chefs d'établissement. [02'09'36] Plutôt à l'ancienne, plutôt sur des registres militants, par ailleurs parfois militant d'éducation populaire. [02'09'41] Qui défendent une certaine idée et qui se cognent parfois à une partie de l'équipe- mais une partie seulement- et qui sont très soutenues par une autre partie de l'équipe. [02'09'51] C'est des chefs d'établissements qui sont là depuis longtemps et ce sont des équipes où il y a pas un turnover de soixante dix pour cent par an. [02'09'56] Ça, c'est systématique aussi. [02'09'58] On sait que c'est des équipes relativement stabilisées dans nos académies. Ça veut dire quoi? ça veut dire vingt trente pour cent de turnover par an, mais pas soixante dix, parce qu'on a en moyenne de plus de soixante pour cent. [02'10'11] Donc c'est, c'est énorme. [02'10'14] Ça, c'est le deuxième. Deuxième élément. [02'10'19] Ce sont presque toujours des établissements où il y a des exclusions. [02'10'23] Externalisez nous, il y a aussi des exclusions internalisée. Donc, ça veut dire concrètement que l'exclusion externalisée [02'10'30] Rajouter une couche en fait. [02'10'32] Préalable excité, exclu temporairement. C'est que vraiment. [02'10'36] Il y a eu un, un acte fort. On ne trouve pas d'exemple dans ces établissements, là où l'exclusion temporaire c'est parce que t'as levé les yeux au ciel ou que tu as été absent, son adhésion à dix pourcent des gamins exclus temporairement qu'ils le sont pour absentéisme. [02'10'52] Dix pour cent. [02'10'54] C'est un homme essayé, mais c'est très révélateur. Je crois que c'est ça qui est. [02'11'00] Donc on a on assets ces aspects là. [02'11'03] Après. [02'11'06] Surtout fin. Le marqueur, c'est- ça renvoie à ce qu'on se disait- c'est que des établissements travaillent beaucoup la question du climat scolaire, même s'ils ne disent pas comme ça. [02'11'15] Mais qui travaille la question de la bienveillance envers les élèves qui travaillent la question. [02'11'21] De comment on arrive à stabiliser une équipe dans une académie où c'est pas stable. Mais on y arrive en travaillant sur des projets, en étant soutenus parfois par les municipalités sur des des projets. [02'11'31] De classe. [02'11'32] Transplanter une femme ne s'appelle pas comme ça, mais de classe. [02'11'36] Le terme m'échappe. [02'11'39] Classes de neige, classes vertes, classes- voilà, il les appelle pas comme ça, mais bon, vous voyez l'idée. Classe de découverte, etc. [02'11'47] Et des établissements qui travaillent beaucoup avec les partenaires locaux. [02'11'54] Père, quand tu en as service, éducation, des vies, etc. Mais vraiment en partenariat et qu'on construit des partenariats souvent de longue durée, parfois pas avec des mairies, ça peut être des associations de proximité, mais où il y a des retours et des allers-retours. [02'12'10] Assez, assez fort pour que les ressources soient mutualisées et que il y ait vraiment des appuis là-dessus. [02'12'16] Donc, c'est pas ce que je vous dis. Là, c'est vraiment de l'ordre du jeu, redire du sentiment. Il y a peut-être des choses qui jouent sur la taille des établissements, sur l'environnement, sursis, sur ça, mais ça n'a pas du tout testé, ça, c'est par contre systématiquement. On a les chefs d'établissements très mobilisés là-dessus. Ça veut pas dire que d'autres, qui n'ont pas ces résultats, ne le sont pas aussi. [02'12'36] On a des chefs d'établissements qui voudraient bien arriver à ça, mais qui n'y arrivent pas et qui nous disent: je ne peux pour l'instant. Je ne peux pas parce que si je fais ça, je mets le feu à mon établissement et je ne veux pas mettre le feu à mon établissement. Donc, on, on est sur ces difficultés là, et des équipes aussi qui nous disent: dans certains établissements, on a fait les restitutions et on dit: chez vous, c'est cent quarante. [02'12'56] Cinq élèves ont été exclus en octobre. [02'12'58] Et qui disent: c'est pas possible, et parfois ça nous arrivait d'avoir des et c'est pas notre rôle, et donc il faut qu'on soit très vigilant quand on travaille sur ça. [02'13'08] À constater des clivages d'équipe très fort entre les tenants d'une vision plutôt instructions, plutôt dur. [02'13'16] De l'encadrement et des tenants d'une vision, on dira, plus éducative, plus bienveillante, avec des tensions très, très forte au sein des, au sein des équipes. [02'13'51] Si je veux bien. C'est une expérience qui a lieu dans un collège qui s'appelle guillaume de normandie. [02'13'57] Quartier de la guérinière à caen, où ils ont essayé, à l'interne du collège, de mettre en place quelque chose avant que la situation n'explose. Non, quand un élève est susceptible d'être exclu, il peut fréquenter un endroit qui s'appelle respire. Alors, je pensais que c'était respire, euh. En fait, c'est pas du tout ça, c'est air, euh, plus long. [02'14'17] Un espace vert. [02'14'19] J'ai une collègue qui est allée chercher entre midi et deux, ce que voulait dire tous pire. [02'14'23] Et ce serait en fait. C'est en fait un lieu qui évoque le, le fait d'avoir eu une chance supplémentaire. Enfin, voilà, il se trouve dans ce lieu dont deux personnes de l'établissement que l'élève peut choisir. [02'14'36] Qui peuvent être l'infirmière. Un enseignant implique un personnel de service fin. C'est vraiment des personnes de tous les niveaux de du du college. [02'14'44] Ou il vient pour exprimer ses difficultés. Pourquoi il en est là? qu'est ce qui est difficile. Voilà, et elle disait que c'était d'abord, c'était très bien accepté par les élèves. [02'14'55] Qui viennent, qui déversent, en fait un flot de choses. [02'14'59] Un lieu d'accueil de la parole avant tout, et que les élèves, une fois, qui y sont allés une fois redemande, a fréquenté cela parce que c'est pour eux en fait un loup. Ils viennent déposer leurs difficultés. [02'15'10] Qui évite que ça, ça, ça dégénère. Est-ce que c'est un peu ça? [02'15'17] C'est ça. C'est-à-dire, l'idée, c'est de ne pas venir sur les exclusions temporaires, de ne pas aller jusque-là, non de trouver des solutions. [02'15'27] Avant. [02'15'28] Donc là, c'est un dispositif qui est tentait, mais [02'15'32] Il y a aussi si, malgré tout pouvoir à l'exclusion temporaire, elle est internalisée via une prise en charge sous une réflexion éducative. [02'15'42] A l'interne et une prise en charge collective. [02'15'46] Ne repose pas seulement sur un individu. Ça repose sur sur une partie de un groupe ou une équipe. [02'15'52] C'est là-dessus que on est du coup dans une approche très différenciée. [02'15'57] Et les enseignants sont pas moins. [02'16'00] Impacter. [02'16'02] Que notre profession, sur une tendance très générale qu'on observe, sur ce qu'on a déjà dit ce matin et tout à l'heure. [02'16'10] Reste le fait de pas être sur un registre de responsabilisation des élèves ou des jeunes ou des enfants ou des adultes sur le fait qu'il y ait ou non droit à un accompagnement spécifique dans des conditions. [02'16'22] Spécifique aussi ce type de dispositif. C'est ça à faire, c'est non, je sais pas si l'élève, il a le droit ou pas de bénéficier de ça, et il y va parce que ça fait partie de quelque chose qui est plutôt- même si le terme est galvaudé, mais bienveillant de manière générale. J'imagine que si j'allais faire des relevés dans cet établissement des écrits. [02'16'43] Temporaire, je ne serai pas à cent cinquante par par mois sur sur octobre. [02'16'48] Non [02'16'51] Bite, c'est qu'en fait ça stigmatise certains profs, parce que il y a des élèves qui viennent se plaindre de relations avec certains profs et ils ne savent pas quoi faire de ça. Un cercueil ayant fait un corporatisme qui fait que on peut pas aller. [02'17'06] C'est difficile d'aller mettre en difficulté un collègue. Voilà alors que c'est des choses qui sont vraiment très bien identifiées et qui remontre. [02'17'13] Ouais, c'est ça recoupe, nous le constat qu'on a fait sur. Alors, après, il y a les cas particuliers, il y a des profs qui [02'17'22] Voilà qui est exclu. [02'17'25] Qui demande énormément. Ceci dit, quand il en demande vraiment trop, les chefs d'établissement régulent. Aussi sévère que il y a quand même. [02'17'33] Souvent dans les équipes, c'est plutôt des groupes de d'enseignants. [02'17'38] Qui font appel à ce type de mesures, mais qui renvoient à des registres éducatifs et pédagogiques. [02'17'45] Singulier après moi. Il me semble qu'effectivement une des voix, et c'est ce qu'on en termes de recommandations, tout en restant très prudent là-dessus- [02'17'53] Mais quand même. [02'17'55] Le fait qu'on ait beaucoup de pays qui, de plus en plus, aillent vers une interdiction. [02'18'00] Des exclusions temporaires et mise en place de suspension. [02'18'04] Internaliser. Ça doit quand même nous préoccuper. [02'18'08] On sait que le le rapport entre on ne peut pas d'un côté. [02'18'13] Déployer des tas de dispositifs de lutte contre le décrochage, puis de l'autre, [02'18'18] Savoir qu'on fabrique parfaitement du décrochage pour un certain nombre d'élèves au travers de ces mesures qui sont parfaitement légales même si, dans l'esprit de la loi, elles sont pas appliquées comme l'esprit de la loi le dit. Mais on est vraiment quand même là-dessus, sur un paradoxe fort. Et dernier point par rapport à ce que vous dites: [02'18'36] Je pense aussi qu'il faut bien qu'on bien distinguer le fait de quelques cas d'élèves très difficiles. [02'18'43] Qui pose des idées, des contraintes et parfois qui laisse des établissements démunis. [02'18'48] Qu'il n'y arrive plus. [02'18'50] Avec certains gamins, mais on a vraiment pas beaucoup finalement. [02'18'54] Et là, l'un des constats que l'on a fait sur nos terrasses et que la tendance était de formaliser des dispositifs pour cela. [02'19'03] De les appliquer à tous et qu'au final, cela n'était même plus du tout concerné. [02'19'09] Et que il y avait de fait une proportionnalité à avoir selon les cadres et selon la manière avec laquelle les élèves, en l'occurrence, vivaient. [02'19'20] Vivez à l'école, mais que les cas qu'on avait vraiment de très grandes souffrances d'élèves qui nécessitaient des prises en charge, y compris psychologique parfois, qui, de fait, sont plus du seul registre des équipes éducatives traditionnelles, étaient malgré tout. [02'19'35] Très marginale, même si elle existe. [02'19'38] Surtout pas glisser vers comment on construit l'ordinaire en fonction des cas les plus durs, mais plutôt comment on construit une ordinaire en fonction de tous, même si on se donne des moyens autres pour les cas les plus difficiles qu'on a à traiter. [02'20'17] La conclusion. [02'20'20] T'es dur à faire la gueule. [02'20'22] Ça. [02'20'28] Non, mais après, c'est juste vous dire que c'est une femme. [02'20'32] Travaux, qui qui continue ses travaux, qu'on fait localement un peu de bruit. Femmes, mobiliser aussi le, la direction académique, le conseil généraux. [02'20'43] Stc. [02'20'44] Ce qu'il y a quand même était réjouissant, c'est que, même si nos résultats étaient pas toujours très enthousiasmant- ce que je disais rapidement et peut-être trop rapidement tout à l'heure- [02'20'52] Il y a jamais eu de. [02'20'55] De contentement à ces résultats, nous à nous. Au début, quand on les a présentés, y compris dans des établissements, on craignait ça, qu'on lise bas. [02'21'02] Et alors. [02'21'04] Ouais, on exclut beaucoup. [02'21'06] Ça fait. Il y a des dispositifs qui sont prévus pour d'autres types d'élèves. C'est pas eux qui vont comme ça. On fait ce qu'on peut avec ce qu'on a. [02'21'13] Et on n'a jamais eu ça sert, qu'on a eu vraiment une forme de de remise en cause- le terme est peut-être fort- mais en tout cas de d'essayer de travailler à comment on peut réussir à réduire ces, ces exclusions, en les prenant comme un symptôme chez nous. C'est toujours là-dessus qu'on insiste aussi. [02'21'30] Les exclusions temporaires en tant que tel, on s'en fout, on s'en fiche un peu, se passe pas la question, n'est-ce pas? voilà, c'est une sanction parmi plein d'autres. C'est plutôt le révélateur que c'est et comment ça peut devenir pour des équipes des espaces de mobilisation sur des enjeux essentiels, quand on travaille avec des équipes et quand on travaille en plus des équipes. [02'21'51] Air et des acteurs éducatifs qui sont autour de l'école sur ce type de question. Là, ça pose les fondamentaux. On revient vraiment sur la question de déductibilité, la question de la bienveillance, la question de l'encadrement éducatif, la question des socialisations juvéniles, la question de la confrontation des normes scolaires éducatives adultes aux normes [02'22'11] Juvénile et enfantine. On revient sur tout ça et que, à un moment donné, pour travailler sur des milieux complexes, il faut trouver des leviers. [02'22'20] Pour parler de cette complexité et ce levier là, dans ces espaces là, c'est devenu un levier parmi d'autres pour essayer de travailler ensemble mieux et en posant vraiment les fondamentaux sur ce que c'est que l'acte d'éduquer aujourd'hui.

[00'00'07] Alors je vais, je propose qu'on commence. [00'00'10] Sans tarder- on s'est donné à environ deux heures- pour non pas épuisé sujet, mais on y votive. [00'00'17] Tout à teneur et tous les enjeux, dont bienvenue à toutes et tous, ravis de vous retrouver. [00'00'22] Ce soir pour le second volet de notre colloque deux mille vingt trois, consacré au bien-être des élèves. [00'00'27] La première partie, organisée il y a un mois environ, a rassemblé deux cents participants autour des interventions de carbet, chef du bureau de de la santé et de l'action sociale de la direction générale de l'enseignement scolaire- la fameuse dgesco au ministère de l'education nationale, et eric delamarre, défenseur des droits défenseur des enfants adjoint. [00'00'48] Du guéridon, défenseur des droits des enfants et droit tout court. Pas ce premier temps d'échange nous a permis d'explorer les enjeux de la santé mentale des enfants et des jeunes dans le contexte de crises multiples que notre société subit. [00'01'02] Aujourd'hui est donc le rôle majeur que l'école doit jouer pour prévenir et y repérer ces mots, parce que nous croyons. [00'01'09] Fortement été, foncièrement, l'école de la première chance, avec si peu aussi pour nous, quelque chose d'extrêmement important d'avoir une école forte y est présent, puissante pour pouvoir répondre aux besoins des enfants et des jeunes. Des constats ont été posés, mais également des pistes pour améliorer la prévention, le dépistage et la prise en charge des enfants qui en avaient besoin. [00'01'29] Ce webinaire et, si vous souhaitez, visible en replay sur notre site internet. Et c'est peu pour un sou, point et éphémère. [00'01'36] Donc, aujourd'hui, seconde partie de ce colloque qui va nous permettre de zoomer sur la place des parents à l'école. Une place importante parce que c'est une question fondamentale et évidemment, chers élèves, si peu puisqu'elle met, de fait, la question de la co-éducation au centre des échanges et ainsi, le rôle et la place des parents dans l'école, sujet inépuisable et jaunâtre. [00'01'56] Ulis. [00'01'57] Comme le de l'unesco et il revendique le projet éducatif de la cible. L'école étanche est essentielle pour faire société. [00'02'05] Elle est essentielle également pour l'éducation et peut contribuer et contribuent à changer la vie. Elle me permet aussi d'éradiquer, comme le dit l'unesco, la pauvreté et elle participe pleinement à la construction de l'epr de la paix. Ils ont une place tout à fait centrale pour faire société. Alors, école et familles doivent avancer la main dans la main afin d'assurer le bien-être de l'homme. [00'02'24] Enfant. Cela veut dire que l'école doit veiller à tisser un lien étroit et respectueux avec les parents et, bien sûr, vice-versa. C'est un dialogue animé. Y a un équilibre sensible tout à fait particulière à construire. [00'02'39] Tout en respectant les prérogatives de chacun, bien entendu, et les droits de chacun également. Nous vivons, et l'actualité nous montrera tous les jours, dans une société en pension, en proie, non, à de nombreux doutes. [00'02'52] Donc, dans ce contexte, cet équilibre est parfois à l'image de société compliquée. [00'02'58] Difficile à trouver. [00'03'00] Enfant. [00'03'01] Famille, école porn, pensons un trépied fondateur, et nous allons donc parler ce soir d'un écosystème où tout le monde doit trouver sa place. [00'03'09] Tu as dit cela, pardon, doit se construire. [00'03'13] Très régulièrement, parfois même sur reconstruire chaque jour. [00'03'17] Plus un enfant, un adolescent se sentira entouré. [00'03'21] Toute nue à l'école par ses parents, six enseignants parlent, toutes les personnes et tous les adultes qui fréquentent l'établissement scolaire pullulent seront en situation d'apprendre sereinement. Il sera ainsi dans les meilleures dispositions possibles pour issue, pour réussir son parcours scolaire et construire son projet de vie. Et on rebouche avec la question de l'unesco et du projet éducatif de l'évier peu. [00'03'42] Que j'évoquais il y a quelques instants. [00'03'44] La qualité de la relation parents- école influe très directement sur la qualité du climat scolaire d'un établissement et c'est aujourd'hui en fait une évidence. [00'03'53] A partir de là, consolider les liens entre enfants, parents, écoles, apporte un cadre sécurisant. [00'04'00] C'est rassurant pour l'enfant dans ses apprentissages du bouquet quelques instants. [00'04'03] L'or pour cela. [00'04'05] Des textes fondateurs au code de l'éducation et la loi d'orientation. De mille neuf cent quatre-vingt-neuf, le décret d'appliquer le décret d'août deux mille six, reconnaissent et définissent la place des parents d'élèves dans la communauté éducative. [00'04'18] Faire vivre la co-éducation, c'est un partage de l'acte éducatif et, pour les parents et l'institution scolaire, agir autour des enfants, chacun dans son rôle, chacun dans ses compétences. [00'04'29] Et attire donc le d'emblée votre attention sur un glissement sémantique auquel nous assistons depuis quelques années. [00'04'36] Le glissement du terme de parents qu'aux éducateurs, comme je l'évoquais, au regard du texte et de la culture qui s'est construite dans ce pays depuis maintenant quelques décennies, a à un vocable de parents partenaires de l'éducation et de l'école. [00'04'51] Et on le réaffirme ici, comme nous le faisions à chaque fois que cela est nécessaire. Un partenaire, sa sœur, choisit: [00'04'57] Et on peut même s'en séparer quand on n'en veut plus. [00'05'00] Alors qu'à l'inverse, un parent est un co éducateur qui ne se choisit pas, il sera toujours là, il est constitutif même de l'enfant et de l'élève. [00'05'09] Donc il est inconscient, incontournable, qui sera forcément toujours légitime. [00'05'14] Donc, c'est un terme sur lequel on tient dans elijah, à remettre quelques, quelques bornes pour éviter ce glissement de prédicateurs à partenaires et en affirmant un terme qui est tout à fait important et essentiel pour la vie: peut, si le terme deco éducateur. [00'05'29] Donc à partir de là. La relation parents- école est donc au cœur des questions qui animent le vivre ensemble. [00'05'35] Et cela participe de la façon chaque parent, chaque enfant vit les valeurs de la république. Et cela renvoie même à la notion de contrat social, puisqu'on évoquait que l'école, c'était une façon faire société. Ça construisait pleinement la société et au travers de la relation que l'on a à l'école, en tant que enfant, en tant que parents, en tant qu'enseignant, [00'05'56] Se joue aussi la confiance que l'on peut avoir dans les institutions de la république. [00'06'00] Donc, là y a un point extrêmement d'actualité et très sensible à évoquer, mais je pense que ce sera au cœur des débats, des échanges, ce soit la thématique de ce colloque. Est donc au cœur des questions qui nient notre pays. Ils posent même la crise démocratique. [00'06'14] Elle traverse en ce moment la crise sociale et crise économique et la crise démocratique. [00'06'20] Tous ces enjeux nécessitent en finir avec les discours simplistes ou certains parents serait considéré comme démissionnaire et absolument irrémédiablement en dehors de l'école. [00'06'29] Et d'autres seraient tout à fait experts. Il est tout à fait adaptés et rompu aux exercices du système éducatif. [00'06'37] Il est donc indispensable de donner à chaque parent les moyens de comprendre et de s'approprier le fonctionnement du système vif éducatif, afin qu'il puisse assumer son rôle en toute connaissance de cause. À cela, la fier peut participe, comme une association, comme fédération reconnue d'utilité publique, au travers de la coopération, de l'accompagnement. [00'06'57] D'entraide, de l'information, de l'information, et c'est d'ailleurs l'exorciste auquel on va se livrer, tout son sens que ce soit. C'est ce qui fonde notre bénévolat, nos engagements. [00'07'06] Il est urgent de créer les conditions d'un véritable accueil des parents, l'école. [00'07'10] Et nous aurons gagné le jour où tous les enfants trouveront normal de voir leurs parents. [00'07'15] Et leurs enseignants: échanger, dialoguer, se rencontrer, de façon simple, évidente, sans avoir à se dire à mes parents sont en train de discuter avec le prof, ou avec maître, ou l'enseignant, et ce soir, je risque fort de me prendre la foudre. Donc là, son mari va créer de cette habitude, cette chose, tout à fait non. [00'07'35] Mal d'un dialogue régulier, fréquent et apaisé entre parents et enseignants, on aura gagné. [00'07'41] Et évidemment, plus les enfants voient les parents dans l'école, plus, comment je l'évoquais, le climat scolaire s'en ressent. Alors, j'aime brosser très rapidement les enjeux et on va voir avec nos deux intervenants ce soir. [00'07'54] Ce qu'il estime important de de pointer, bien entendu, toutes les pistes de travail, toutes les pistes à explorer pour pouvoir rendre cette coéducation active, efficace et pleine et entière au quotidien. Et pour cela, on a plaisir d'accueillir deux intervenants: oléoduc, qui est agrégé d'histoire- je vais revenir- et jérémy fontana, qui va nous rejoindre dans quelques minutes. [00'08'14] Alors en quelques mots. Jean-luc duc est donc agrégé d'histoire, spécialiste enfance, en sciences de l'éducation, et a notamment été directeur adjoint du fem de créteil, où il a mis en place des formations sur les relations, justement, parents- enseignants, qui a publié de nombreux ouvrages que vous trouverez ainsi facilement, librairies et articles sur cette question, fut autorité et il a une expertise tout à fait pointue sur le fond. [00'08'34] Fonctionnement du système éducatif. [00'08'36] L'assurance. Individus qui ont évoqué il y a quelques instants, tous les deux que l'on sait bien l'évier peut y a eu déjà eu l'occasion de s'exprimer nombreuses reprises, d'intervenir lors du colloque de journées thématiques organisées par notre fédération, dont c'est avec plaisir que on va, seront l'accueil ce soir. Bienvenue parmi nous, et il aura l'occasion de revenir sur l'importance d'ouvrir l'école. C'est pas ici qu'on dit: [00'08'57] Contraire aux familles et tout battu, tout particulièrement, pardon, aux familles plus élevés éloignés de l'école. [00'09'04] Et on accueillera, à partir de dix-neuf heures environ, jérémy fontana e, qui est un enseignant en sciences économiques et sociales et, depuis dix ans, mis au point une méthode pédagogique basée sur un suivi au plus près des élèves et un dialogue tout à fait constant avec les familles. Les résultats, notamment en termes de réussite au baccalauréat, ont été spectaculaires et donc il y revient. [00'09'24] Drap. Il a publié un ouvrage qui s'appelle l'école de la réconciliation. [00'09'28] Qui est paru en deux mille vingt deux, qui a fait beaucoup, beaucoup d'écho et pour lesquels vous trouverez beaucoup d'articles ou d'émissions de radio sur le sujet ou de passage notamment la télé ou au quotidien. [00'09'40] Et ils vont témoigner ce soir de son expérience au lycée. [00'09'44] Dans un lycée de drancy où il a pu grandement améliorer la réussite et la confiance en soi, en soi de ses élèves, ni- on l'évoquait tout à l'heure- l'importance de cette confiance et de cette sérénité. [00'09'55] L'école. C'est maintenant cette méthode- pardon les vêtements- reprise par de nombreux enseignants, et aussi de dire: fait école. Je crois même savoir- mon nom charmant parle avec lui- qu'un film est en préparation. [00'10'07] Réconcilier les élèves avec l'école nécessite d'abord de réconcilier les familles avec l'institution. [00'10'14] Le rôle de la famille est donc fondamental. Les on va voir ensemble comment il fait pour nouer ce lien entre l'équipe de julien confiance entre l'équipe éducative, et également comment il permet à ses élèves de reprendre confiance en eux là où sont peut-être un sentiment d'échec. Monsieur ou du bonsoir, et vous avez la parole. [00'10'35] Peter, je vous remercie et mon intervention, être récent, vous en donnera pas dans la prolongation de ce que vous disiez. J'ai été très heureux du rappel, effectivement, qu'il n'y a pas de parents démissionnaires, il y a. [00'10'54] Des parents désemparés par rapport à une école qui se complexifie et pour moi, c'est un enjeu primordial, effectivement, de permettre à tous les parents, et notamment ceux qui sont éloignés de l'école. [00'11'14] De maîtriser. [00'11'17] Notre système éducatif. [00'11'19] Je voudrais dire également que la notion de bien-être [00'11'25] L'enjeu de diminuer la souffrance. [00'11'30] À l'école et pour moi aujourd'hui. [00'11'34] Un enjeu décisif. [00'11'37] Nous sommes dans une période de tension. On a des élèves stressés. [00'11'44] Angoissé. [00'11'46] Parce que je crois qu'un des éléments, c'est que en ce vingt-et-unième siècle, [00'11'53] L'école doit redéfinir son sens et ses missions pour éviter effectivement qu'elle soit un moment de souffrance. Pourquoi je dis redéfinir son sens et ses missions? je vais prendre un seul exemple: l'élève qui [00'12'13] Va rentrer à l'école maternelle en deux mille vingt-trois. [00'12'17] Il sera. Prenons la retraite à soixante. Deux ans encore au travail. [00'12'24] Autour des années deux mille quatre-vingts. [00'12'28] Or, nous ne savons pas effectivement quelles vont être les enjeux des compétences nécessaires à partir de deux mille quarante-deux mille quarante-cinq. Y a des mutations extrêmement rapides dans les compétences. Donc, [00'12'48] De manière primordiale la grande mutation de l'école qu'un certain nombre de gens ont saisi, mais qui plus que jamais est vrai, c'est que le diplôme n'est plus une finalité, mais c'est un point de départ par rapport à la faute. [00'13'08] Formation tout au long de la vie. [00'13'12] Pourquoi je dissèque germain en relation avec la souffrance à l'école, c'est que si on a fait des améliorations sur le décrochage scolaire immédiat, [00'13'25] Où, en france, nous avons diminué de moitié. [00'13'29] Nous avons un accroissement de ce que j'appelle, moi, le décrochage scolaire à terme, c'est-à-dire que des jeunes qui ont été en souffrance dans l'école sont dans le refus de plan de reconversion de la formation continue et que de ce [00'13'49] Point de vue là, quand on voit les chiffres de la formation continue en entreprise, ce sont les plus diplômés qui ont été dans une situation favorable à l'école qui l'accepte, et pas les autres. Donc, pour moi plus que jamais, [00'14'09] Combattre la souffrance à l'école, développer le bien-être, c'est un enjeu de société si on veut effectivement réussir la formation tout au long de la vie et réussir cet enjeu de la confiance, de la diminution du stress. [00'14'29] Et de l'angoisse. Ça signifie un travail sur d'autres formes de pédagogie, voire d'évaluation, et pour moi, c'est un chantier extrêmement important. [00'14'42] Et ça signifie aussi: [00'14'45] Une présence plus importante de toutes les familles à l'école, et notamment celles qui sont éloignées de l'école. Et de ce point de vue là, trois réflexions. [00'15'01] La première, c'est que moi, j'ai souhaité. J'ai pas toujours eu gain de cause. [00'15'09] Pas sur ce thème là que dans les programmes scolaires ddm, c'est dès le primaire et au collège, la place des parents soit évoquée dans l'école, pour que celle-ci soit normale par rapport aux jeunes. [00'15'27] Votre président. [00'15'29] Rappelaient effectivement que le jeune, quand il voit ses parents, c'est un élément de stress, voire d'angoisse. Sur mes caisses qui se sont dit: il faut mettre les élèves, si vous me le permettez, dans le coup en leur expliquant que c'est normal que leurs parents, membres de la communauté et du [00'15'49] Créative, soit présent dans l'école. Ça doit faire partie effectivement de la formation à la citoyenneté des jeunes. Nos jeunes, dans le cadre de la citoyenneté, sont de futurs parents et il faut qu'ils sachent effectivement le. [00'16'09] Le rôle, la finalité des parents d'élèves par rapport à l'école. Deuxième piste, et qui est pour moi très importante, par rapport au bien-être à l'école, c'est que lejeune, et c'est normal, il va, pendant sa scolarité, avoir des barrières. [00'16'29] Affranchir, baisser ses barrières. C'est le travail en commun des enseignants. [00'16'38] Et des parents, et il faut une cohérence du soutien à l'élève. Pour moi, la coéducation, c'est effectivement la définition de la cohérence menée par rapport à la réussite des jeunes, par rapport aux apprentis. [00'16'58] Sage entre les parents et l'école. [00'17'02] Il y a une notion que, moi, j'ai essayé d'introduire dans un certain nombre de réflexions, c'est de dire que c'est pas toujours effectivement épanouissant à l'école de faire tel ou tel exercice. Mr brown, comparaison sportive. Si je veux réussir dans [00'17'22] Un sport collectif, il faut faire effectivement des tours et des tours de stade à des moments. Si je veux réussir en natation, il faut faire des longueurs et des longueurs. Pour moi, enseignants et parents, c'est des coups entraîneurs pour la réussite de l'élève. [00'17'42] Et si y a pas qu'aux entraînements, on risque d'y avoir effectivement problème. Donc, c'est extrêmement important que les parents s'approprient ce qui va être l'émission, le sens. [00'18'02] De l'école, de l'année scolaire, que va faire leurs enfants. C'est pourquoi je pense important de dire aux enseignants de présenter ce que j'ai appelé, à chaque rentrée, le plan de vol de l'année, c'est-à-dire d'où je pars. [00'18'23] Où je vais arriver. [00'18'24] Par rapport à ma classe en primaire, par rapport à ma discipline. [00'18'29] En college. Comment je vais m'y prendre? comment je vais mener les évaluations? [00'18'36] En faisant effectivement que l'évaluation soit autre chose que la notation par ski. Évaluer un élève, ce n'est pas seulement noter, c'est aussi mener un diagnostic pour pouvoir donner des réponses. Et l'enjeu de [00'18'57] Inscrire dans la formation des enseignants l'évaluation diagnostique, la culture du diagnostic. [00'19'04] Est un enjeu. [00'19'06] Extrêmement important, qui devrait d'ailleurs irriguer l'ensemble du système éducatif. Ça veut dire, de ce point de vue-là, redonner tout son sens au projet d'établissement qui est le projet commun de la cause. [00'19'26] Communauté éducative et pour moi, si vous me permettez cette notion de communauté éducative, qui fait effectivement qu'on découpa le jeune en tranches de saucisson selon qu'il est en classe, qu'il est dans tel ou tel lieu, qu'il est dans telle activité, est extrêmement important. [00'19'47] Et puisque on a rappelé qu'il m'est arrivé aussi d'écrire concernant la laïcité, je pense que, en termes de citoyenneté, une communauté éducative vivant avec toutes les composantes de ce qui fait un établissement, tous les mots. [00'20'06] Métiers de ski fait un établissement, pas seulement les enseignants et l'administration, mais aussi les personnels ouvriers. Service de cantine. [00'20'18] Des élèves, les élèves, cette communauté, vie éducative vivante, c'est le meilleur rempart contre le communautarisme. Communauté éducative contre communautarisme, c'est un vrai enjeu. C'est pour ça que je ne comprends pas pourquoi, effectivement, y a pas. [00'20'38] Pas cette volonté dans le combat qui est affirmé de faire que les parents soient parties prenantes, y compris- je viens de le voir et j'y suis pour- de la rédaction du projet d'établissement. Dieu pour moi et l'expression, adaptée au terrain de ce que [00'20'59] La communauté éducative pense le mieux possible pour atteindre un certain nombre d'éléments. [00'21'07] Donc, c'est vrai que ça bouge, ça bouge lentement. Nous avons des problèmes culturels qui s'opposent dans le cadre d'un certain nombre. [00'21'22] Deux secteurs sur la place des familles. Pourtant, on le voit bien à l'étranger, dans les pays nordiques, en allemagne. [00'21'36] Au québec ou ailleurs, des parents qui sont reconnus comme parties prenantes de l'école. C'est un plus par rapport à la réussite des jeunes. Ici aussi, on ne le dit pas assez, un plus par rapport, effectivement. [00'21'54] Ou à laquelle, à condition d'enseignement des parents. Et donc, de ce point de vue-là, ce qu'on avait fait à l'iufm de créteil, et qui s'était arrêté lors de la mastérisation, c'était de faire que la formation au travail avec les familles [00'22'15] Soit un axe obligatoire de formations, parce qu'il nous semblait important, pour les conditions d'enseignement des jeunes, qu'ils se sentent aussi soutenus par les familles comme par les enseignants. [00'22'32] Et l'enjeu, effectivement, du bien-être à l'école, que je dirais l'enjeu de la souffrance. C'est un enjeu extrêmement important parce que, dans une société de tension, [00'22'48] Où on peut avoir des élèves stressés, des élèves angoissés, ou je ne suis pas sûr que les modalités [00'22'57] Du nouveau baccalauréat ne soient pas des éléments de stress, c'est faire que l'école ne soit pas un moment d'épanouissement permettant lejeune d'être dans une situation d'apprendre à apprendre et, comme je le disais, au dos. [00'23'16] Débute cette intervention dans un monde en mutation. [00'23'21] Ou le souci, effectivement, d'être dans une [00'23'27] Volonté de formation tout au long de la vie. Ça signifie que l'école doit s'interroger sur les moyens de faire que ça soit un lieu d'épanouissement qui puisse rejaillir sur l'ensemble du rapport du jeune au sav. [00'23'47] Voir y a là un chantier extrêmement important, parce que c'est le chantier du futur. Ça signifie aussi qu'on considère les parents comme des membres à part entière de la communauté éducative et qu'on comprenne bien que la [00'24'07] Coéducation, c'est respecter la place de chacun, mais de voir que, dans le soutien aux élèves, que dans le processus, effectivement, d'encouragement de l'élève, c'est important que famille et école [00'24'28] Marche ensemble. Le jeune tyran a trois ans à l'école, il va sortir à dix-huit ans. Pendant ses quinze ans, il pourra avoir des moments d'incertitude, de difficultés. Il faut qui sentent, la communauté éducative, les parents, dans une cohérence. [00'24'48] De soutien. C'est un vrai enjeu, effectivement, de société et je trouve très intéressant que, sur ce thème du bien-être live, cbd. [00'25'01] Et eu la volonté de faire un webinaire. Voilà ce que je voulais dire en introduction, mais je suis prêt à répondre et à développer. [00'25'12] Nombre de points. [00'25'16] Merci beaucoup pour ce propos introductif alors, y a quelques petites heures, voire des réactions dans le tchat tout court. [00'25'22] Un. [00'25'23] Ne la première que qu'on vous dit vous poser ce que l'on voit fleurir les, les unités nigériennes, les environnements numériques de travail pour les élèves, dans la relation principalement maintenant. [00'25'37] Collège, lycée. Mais on voit, effectivement, des dispositifs numériques se sont développés. [00'25'43] En alimentaire. [00'25'44] Je voudrais avoir votre regard sur ces outils. [00'25'48] Ainsi, son facteur d'inclusion des parents dans l'école, ou facteur d'exclusion de des parents dans l'école, dit comment on peut prendre à bras-le-corps ces outils-là dans la relation parents- école. Effectivement, parce qu'un portail, des fois, on reste devant et on n'ouvre pas la porte. [00'26'05] C'est une bonne question. [00'26'09] Je crois qu'il faut pas une vision magique par rapport aux au t, il faut aussi faire une analyse de ce que sont les familles autour de l'école, parce que on a quand même au niveau de la fracture numérique- et vous citiez tout à l'heure que vous [00'26'28] Vous aviez l'adjoint claire hédouin. Je l'entendais hier à la télévision dans une émission, la vingt heures expliqué, qui avait un quart de la population, qui étaient éloignés du numérique, ce qui posait question par rapport à la ds. [00'26'48] Matériel, isolation d'un certain nombre de services publics. Donc, de ce point de vue là, si je mets en place un t, i faut que je voie avec des associations partenaires, voir l'établissement lui-même, pour assurer effectivement. [00'27'08] Le fait que tous les parents soient sensibilisés au numérique. [00'27'14] Deuxième élément, c'est peut-être mon âge, mais je pense qu'à des moments, par rapport à l'enfant, par rapport au diagnostic qu'on va mener, le face à face est important, y compris, si vous me permettez, pour reprendre une expression, pour qu'il se transmet. [00'27'34] Formons côte à côte à côté de l'élève. Donc, je ne crois pas que le numérique puisse remplacer. [00'27'43] Tout les contacts, parce qu'il y aura des moments. [00'27'48] Ou il faudra mieux expliciter qu'effectivement la et la famille se sentent partie prenante. Et quand je dis la famille et on voit bien dans notre société, c'est que nous avons pendant tout départ parler lepage. [00'28'08] Parents. [00'28'09] Je dis toujours quand j'interviens dès parents, y a diversité de structures familiales et n'ayons pas présent à l'esprit qui aurait des parents idéal. Il n'existe pas. Y a des parents, y a des enjeux spécifiques par rapport, dans un certain cas, l'absence du père, [00'28'29] Par rapport à d'autres à telle situation. Je pense que, de ce point de vue-là, justement, des parents qui participent l'ensemble du projet d'établissement sont les mieux à même que le projet d'établissement travaille sur un territoire. Et dernier point, moins. [00'28'49] Je trouve totalement anormal tant ce que j'appelle la cohérence éducative, c'est dire le fait qu'on traite ensemble jeunes dans l'école, dans les structures socio-culturelles que les parents sont et les organisations représentatives, départ soit absent tout le travail autour des cités du [00'29'09] Créative autour des territoires éducatifs ruraux. [00'29'13] J'ai tenu le discours que je suis en train de vous dire à l'invitation d'un forum dans les yvelines, des cités éducatives, par que ça me semble effectivement quelque chose qui dénie une partie du rôle éducative et qui peut fragiliser les enfants. Donc, sur votre question d'une hum. [00'29'33] Eric, oui, mais il faut bien analyser. Ça peut être un outil d'immédiateté d'information. [00'29'43] Mais l'éducation, c'est pas toujours l'immédiateté, c'est du temps, et ça, ça ne peut pas éliminer des conversations en face à face. [00'29'58] Pour l'anecdote. On connaît les établissements où les élèves disent à leurs parents que pronote et payant pour être tout à fait certain qu'inutile ne change pas l'appli. [00'30'07] Pis pour eux, pour le petit clin d'oeil. Une autre question qui a été posée dans le chatte: [00'30'14] On a vu, notamment avec laredo me, yann, moix, arriver et se poser, dans les nouveaux mots, qui, qui, qui sont utilisés dans notre, dans notre jargon commun, la notion d'alliance éducative. [00'30'29] Quel est votre sentiment sur la notion de l'alliance éducative? [00'30'35] Moi, j'aime bien le mode coéducation, parce que co, ça veut dire qu'on a des éléments en commun, et c'est important. [00'30'46] Il y a le métier de l'enseignant, la transmission, y a le rôle des parents éducateurs de leurs enfants. Il y a tout une zone en commun qu'il faut gérer. Qui fait effectivement ca nécessite que cohérence, et le terme deco, et là le terme [00'31'06] D'alliance compte de pays sally y par de phénomènes différents, et on est ensemble. Moi, ce que je préfère dans la coéducation centaine guêpes- à la mode en france, c'est le terme de construire des compromis avec des familles qui sera un désaccord sur telle ou telle chose? et moi, [00'31'27] Moi, quand j'étais enseignant et il y avait des parents avec lequel, sur les modalités d'évaluation, notamment à ses notes. [00'31'37] Tel type de travail, vas y fallait, non pas contraindre ceux que trop souvent fait, mais essayer de convaincre. Pour construire un compromis où les parents sachent où on va, pourquoi on va. Et ça faisait effectivement qu'à la limite, on revoyait l'année d'après. [00'31'57] Ses parents, plus investis dans l'école, de pont à coéducation et culture du compromis avec les familles. Et ce que je vois trop souvent, y compris ce que nous disent les médiateurs et médiatrices académiques de l'éducation nationale, c'est que cette notion du compromis [00'32'18] Pour faire avancer l'élève et la société était un élément de la citoyenneté. Chacun apporte un peu du sien à avancent dans le sens, il est en diminution. Henry lai, au cœur d'un projet social. [00'32'35] Justement tout à l'heure, j'évoquais le, le glissement de la notion de communicateur, communauté éducative- là, on a parlé de la notion d'alliance éducative que vous avez eu sont très futile au profit de la coéducation. Comment l'implanter au début du du du webinaire? la notion de partenaire, est-ce que vous l'avez aussi vu apparaître? est-ce que vous l'avez constaté? [00'32'54] Où est-ce qu'on est du sol? la si peu à s'étonner de ce glissement sémantique qui pousse un peu partout. [00'33'00] Je l'ai vu et j'ai eu la même critique que vous par rapport à la journée là sur les cités éducatives. J'ai vu les parents d'élèves apparaître à côté des associations et d'autres, dans les partenaires, ont pour moi, effectivement. [00'33'18] Je vais reprendre: leurs partenaires ont choisi: les parents y sont effectivement les éducateurs de leurs enfants et, justement, ce positionnement tend à déresponsabiliser les parents. Et aujourd'hui, en un discours sur la responsabilité des parents de paix, [00'33'38] Personnes qui veulent, d'une certaine manière ou à travers un certain nombre d'écrits, les faire sortir des instances de l'école. Pour moi, justement, responsabiliser, c'est donner des pouvoirs, des possibilités d'être présent. [00'33'59] Et on voit bien lisse. [00'34'01] Moi j'ai fait. [00'34'03] Ça date, mais en deux mille un pour le conseil de l'europe, et à l'époque, il y avait la fcb qu'était partenaire une étude sur les relations entre les parents et l'école dans les divers pays européens, qui est d'ailleurs paru. [00'34'21] En synthèse dans la revue deux du centre international d'études pédagogiques de sèvres. Et on voit bien, là où des parents, des responsabilités, ça ne déresponsabilise pas les enseignants, mais ça fait effectivement, tient une discussion où chacun ça. [00'34'41] Propri les enjeux de l'autre est dans une période de mutation. J'essayais d'expliquer où né. Là où les programmes changent, ou les modalités pédagogiques peuvent changer ou, comme je le disais, c'est un phénomène de fond culturel. Notre diplôme n'est plus une fin. [00'35'01] Finalité, mais un point de départ. Pour reprendre une expression j'ai entendu en finlande, par rapport aux enjeux, effectivement, de skai, l'école de demain, donnons leur place aux parents pour qui participent avec nous. À y a une chose que je dis souvent, c'est: [00'35'21] Quand on fait un bon repas. [00'35'24] On peut goûter le repas, même s'il est excellent, mais si on veut le reproduire, il faut qu'on connaisse tous les ingrédients qu'on a mis dedans. Pour moi, ça ne me gêne pas que les parents connaissent les ingrédients qui font effectivement ce qu'est la transmission du savoir scolaire. [00'35'43] Ça les rendra effectivement plus efficaces pour être deux contre des coups entraîneurs. [00'35'49] À un jugement, une discussion mucha et tient à ce que vous avez face à vous des parents très investis dans le dans l'école. [00'35'56] Il se considère à part entière comme dico educateur, qu'il revendique chaque matin, chaque jour. [00'36'02] Plus près du terrain. [00'36'04] On a du mal. [00'36'07] On a réussi à entraîner avec nous d'autres parents pour faire du mal. [00'36'12] On arrive plus ou moins bien. [00'36'14] Notamment, y avait rendez-vous immanquables, comme le conseil d'école, le conseil de classe, lorsqu'on arrive à investir les parents à nos côtés au sein du conseil d'administration, par exemple. [00'36'24] Il est aujourd'hui. [00'36'27] Mais nous aussi les premiers agents. [00'36'29] De ce premier coach. Cet entraînement est long quand on se pose la question de savoir si vous avez quelques éléments à nous communiquer pour faciliter l'implication des parents dans l'école, puisque c'est aussi ça qu'on vient chercher ce soir vu. Écoutez-moi, j'ai une expérience avec un nouveau président. [00'36'47] Qui était à l'époque dans le quatre-vingt-treize. [00'36'51] Il se trouve qu'un élu d'une ville de seine-saint-denis m'avait entendu. [00'36'56] Il était en tant que parents. [00'36'59] Développer une formation sur parents enseignants et il l'a fait en invitant votre président et moi-même. Une réunion pour toutes les familles de la ville. C'était à la courneuve, sur l'entrée en sixième. [00'37'15] Comment le faire. Il y avait une salle bourrée, comme sans doute silex et pierre avait été la seule, ou s'il avait été une conférence, qui était là, et le discours a pu être tenu sur les enjeux du passage à en sixième, ce qui était là, et [00'37'35] Et puis, bien évidemment, rodrigo arenas a développé le rôle des fédérations de parents d'élèves dans un discours. Écoutez donc. Je pense que solliciter des élus pour des moments décisifs, où c'est la mairie qui va inviter les chercheurs et les parents, nous avez dit, on est [00'37'56] T'es très heureux que ça soit quelqu'un qui soit ni un directeur d'école ni un chef d'établissement. C'est quelqu'un- je mets des guillemets parce que je ne le suis pas- mais qui était neutre par rapport à l'analyse de l'école et pas jugé partie. Et bien, ce type de chose qui peut être suscite, [00'38'16] Qu'est, qui existe sûrement ailleurs. C'est à mon avis quelque chose d'important et souvent, les élus de demande ce qui peut faire. Mais moi, j'ai un souvenir de cette réunion, de la pertinence des questions qui étaient posées, du fait, effectivement, qu'on a pu expliquer à ses parents qui avaient [00'38'36] Pas de délégués parents par classe en primaire, mais qui en avait au collège, donc de leur donner toute une série d'éléments que, peut-être, on ne l'aurait pas donné à l'entrée. Voilà une initiative. Je crois que les parents, plus que jamais, sont en demande de comprendre l'école. [00'38'56] Si je regarde dans d'autres pays. [00'38'59] Nous sommes le seul pays en france où il y a des émissions grand public sur les chaînes publiques, sur la cuisine, la santé, les chiens, les chats. [00'39'13] Le jardinage. En france, il y a aucune émission grand public sur l'école, à l'inverse de ce qui existe dans des pays où, sur le modèle de l'ancienne émission de six messes et carrère d'encausse, on étudie quelle langue choisir. Pourquoi on a changé tel programme. [00'39'34] Hum, qu'est-ce que ça signifie? l'orientation professionnelle? j'en passe. Il y a des tas de sujets. En france, l'école reste un délit d'initié. Donc, le fait qu'on ait ces réunions, ouvert pour casser ce délit d'initié, c'est un enjeu important et une des propositions des fédérations de parents. [00'39'54] Grand pourrait être qu'enfin on ait une émission grand public. Quand je vois ce qui se fait ailleurs d'ailleurs, ça peut être amusant et pas stressant, mais pour faire que l'école soit l'affaire de tous, et là y a un souci effectivement d'information, j'oserais dire de vulgarisation. [00'40'14] Notre école délit d'initié de plus en plus complexe. [00'40'19] Euh, y a une question. Je vois une question extrêmement riche. Savez, dans l'ouvrage qu'on avait fait au moment de nos modules parents enseignants, en faisant d'ailleurs [00'40'34] Le mémoire professionnel que devait faire chaque enseignant, soit autour de la relation parents saignant, et on avait publié dans un petit ouvrage les meilleurs mémoires et ce que nous avait dit des témoignages des enseignants. C'est la première peur des parents, c'était [00'40'54] Celle de représailles s'il venait poser des questions par rapport à l'école. Donc, je crois que c'est un discours extrêmement important de la part des chefs d'établissement, des enseignants, de dire que une discussion entre adultes, qu'on est là, n'a pas à avoir de retour. [00'41'15] Par rapport à l'enfant. Mais je suis heureux qu'on souligne ça puisque à l'époque- et ça date, c'était deux mille sept, c'était la première angoisse des parents. C'est six. J'interviens: est-ce qu'on n'aura pas des représailles par rapport à mon enfant de la part de l'enseignant? [00'41'35] La petite question: est-ce qu'à votre avis aujourd'hui, au regard du cursus de formations et de la masterisation des les enseignants? [00'41'44] La question de la relation parents- enseignants. [00'41'48] Suffisamment intégré et cultivé. [00'41'52] Puisque vous donnez un des précurseurs. Donc je me permets de de vous poser la question. [00'41'59] Parce qu'on pourra varier un stéréotype et n'hésitez pas à déconstruire sir ci-joint supporter de d'enseignants qui sont plutôt en réussite avec l'école depuis toujours, qui n'ont pas fait. [00'42'10] A eu du chagrin nicholson. [00'42'12] Ni des élèves, ni ne ressemblent pas à ce qu'ils ont été comme élève keita, le vrai enjeu. D'où l'importance de travailler sur la relation. Alors, moi, je vais être clair. [00'42'24] Depuis la mastérisation? c'est non. Ça veut dire que, au mieux, ils ont une conférence. [00'42'33] Et malgré ma retraite, effectivement, je continue à être sollicité par des cv pour faire une conférence sur les enjeux des relations par enseignant, ce que j'accepte toujours en étonnant qu'il n'ait pas d'autre, même si pierre périer fait les. [00'42'53] Tiennent du côté de l'ouest, mais je dirais, ils n'ont pas de formations, ils ont de l'information, ce qui est complètement démunis par rapport à ça. Par exemple, qu'est-ce que c'est mener un entretien avec des parents? qu'est-ce que c'est effectivement? [00'43'13] La communication avec des parents, réfléchir sur les différents types de parents pour faire que on ne considère pas que sont des bons parents, que les parents connivence. Ça, aujourd'hui, c'est absent de la formation. Sissi aide. [00'43'33] Xii, j'ai vu à dijon très bien. [00'43'36] Ah, ils ont des choses, mais aujourd'hui, dans le cahier des charges, [00'43'42] Dès. [00'43'44] J'y ai fait des zines spé, ce n'est pas effectivement une priorité. Or, pour moi, c'est un vrai enjeu, parce que vous avez raison, vu ce que sont les concours de recrutement, vu ce qu'est le profil qui viennent, [00'44'04] C'est plus seulement, je dirais, des catégories sociales, parce qu'on voit une mutation des catégories. [00'44'12] Sociale au niveau du recrutement, notamment prof d'école, mais c'est le fait que cités et des élèves qui ont été en réussite à l'école et non en souffrance est donc pour moi. Ils vont avoir d'énormes difficultés par rapport à des élèves en souffrance, j'allais dire y compris. [00'44'32] Et je le vois bien sur l'académie de créteil, où je continue à suivre un certain nombre de choses. S'ils viennent de milieux défavorisés et qu'ils ont réussi à adhérer à l'école, ils vont vouloir reproduire ce schéma par rapport à des élèves qui n'ont peut être pas lu. [00'44'52] Les mêmes situations, exactement que donc, un vrai enjeu de formation des personnels à la diversité des parents d'élèves. C'est pour ça que je reprenais: c'est pas les parents d'élèves, c'est des parents d'élèves dont, de ce point de vue-là, ça bouge. [00'45'10] Je connais pas les collègues de dijon, je travaille beaucoup avec pierre perrier à rennes, et tout le travail qu'il a fait, tous les documents qu'on a, y compris utiliser avec athée d'écart monde, sur la manière de s'adresser à divers publics. Donc, de ce côté-là, il y a des choses qui bougent. Cela étant, [00'45'30] Bouger, c'est une chose. Avoir une impulsion ministérielle, c'est important pour les enseignants. [00'45'41] Et de ce point de vue-là, pour le moment, en termes d'impulsion, de mise en avant de projets avec les familles, ben, on reste plutôt sur sa faim, même si, aujourd'hui, ce que vous évoquez par rapport au bien-être et à la souffrance, [00'46'01] En fait qu'un certain nombre de structures. [00'46'05] Bouche sur la question, je pense par rapport aux sollicitations. J'ai eu des des caf départementale où j'ai eu un certain nombre de sollicitations et où je leur dis toujours: c'est très bien que vous fassiez ça, inviter les fédérations de parents d'élèves également des hum. [00'46'26] Également, c'est-à-dire qu'à aujourd'hui, on voit bien, il y a des parents, je le disais d'entrée, désemparée par rapport à une école qui ne ressemble pas à celle qu'il l'étaient, par rapport. [00'46'39] Un certain nombre. [00'46'42] A dijon. Le module est conçu en font un fort, en conformation avec les fédérations de parents d'élèves. Vous ecoutez, je vais y mulhouse, manga d'exception, julie joindre. Mais c'est une chose qui est importante, y compris pour pouvoir travailler. Nous le travaillons dans ces éléments. [00'47'01] Là avec, effectivement, la réalité de l'entretien, parce que, vous me permettez l'expression, ont un peu l'impression que quand on va avoir le concours, capet sous serp. [00'47'15] C'est un peu comme l'évangile de la pentecôte, où les langues de feu permettaient aux apôtres de parler toutes les langues. Là, les enseignants vont savoir menés tous les types d'entretien. Rond s'aperçoit que c'est une professionnalité de l'entretien et que, justement, la co-intervention avec des parents [00'47'35] La permet de travailler la nature de l'entretien et diminue les tensions. [00'47'41] Très bien, a dit. Merci beaucoup pour ce complément. On a une question du comment rassurer sur la place de chacun dans la co-éducation, lorsqu'on a effectivement dit des enjeux, peut-être du territoire, alors qu'ils n'ont pas dû l'être. Si on parle bien coéducation et hideuse de la communauté éducative, alors je n'ai pas dit dans mon on peut. Maintenant, on allait dire que mon avis. [00'48'01] Pense à rajouter de cible, mais y a un proverbe africain qui dit qu'il faut tout un village pour élever un enfant. [00'48'08] On l'utilise beaucoup ces derniers temps, donc je vais veiller à ne pas lui dire mon introduction. Mais le le sens du propriétaire, c'est: comment peut-on rassurer chacun sur sa place? [00'48'17] Mais vous avez raison, je ne l'ai pas utilisé, même si je l'avais utilisé dans la conférence sur les cités éducatives dans les yvelines pour, effectivement, [00'48'27] A impliqué. [00'48'29] Les parents. Je crois que c'est très clair. Il s'agit pas transformer les pas. On a là quelque chose qui a complètement [00'48'39] Un désarçonné- et les enseignants, les parents, qu'est la période du covid. [00'48'44] Où on a été dans une certaine situation, n'ait eu l'impression qu'on demandait aux parents de faire le métier enseignant. Et je pense que pèse dans ces éléments-là cette période des deux ans du covid. Donc, il faut être clair chaque. [00'49'04] Ca a son métier et légitime dans ses fonctions. L'enseignant a été légitimé par un concours ou par un recrutement par un corps d'inspection. Le parent est légitime, donc chacun. Je le disais. [00'49'24] Ouais. [00'49'26] A cinq. Pourtant, y a terrain- comme c'est pour ça que j'aime bien le terme de coéducation- chez, justement, le terrain commun qui est amenée parce que tout ne se fait pas à l'école, tout ne s'apprend pas. Terrain commun. [00'49'45] Ou jour-nuit. Y a un certain nombre de jeunes qui sont plus de temps devant les réseaux sociaux- si je prends sur un an- que dans une salle de classe. Voilà un terrain commun à échanger- parents- enseignants- sur ces questions, parce que le [00'50'06] Au moins. [00'50'08] On l'aborde, ça risque et on le voit, développement du complotisme développant d'autres choses, comme je le vois passer. Bien évidemment, les parents peuvent être des ressources et sur cette question-là, on peut organiser un forum en faisant intervenir des [00'50'28] Parents qui sont là. Mais c'est un vrai enjeu, ce terrain, comme si on le désert, y a déséquilibre si tous les parents. [00'50'39] Réplique: pas inquiète les parents, connivence. Ce déséquilibre va défavoriser un certain nombre d'élèves. Donc, c'est important. Par rapport à un élément, vous disiez comment faire venir les parents qui peuvent être impressionnés par l'enseignant, le chef d'établissement. [00'50'57] Je le disais tout à l'heure, la communauté éducative, ces vingt métiers dans une école, des a e, s, h, mentionne un homme suffisant: assistants d'éducation, personnels, ouvriers de santé, sociale, de services. Pensons, dans la relation avec les familles, à faire de. [00'51'17] C'est personnel, des ponts. [00'51'20] Avec les familles, souvent, à l'inverse de l'enseignant yvon, vont habiter dans les mêmes yeux que les parents. Souvent, ils vont effectivement les connaître. Servons nous aussi, dans les écoles comme dans les collèges et lycées, de ces personnels qui sont partie prenante. [00'51'40] De la communauté éducative et, au fond, ça montrera aux parents que l'école c'est vraiment une micro-société parce a des tas d'autres choses, et moi, je conseille aux enseignants. [00'51'51] Notamment, quatrième et troisième, quand il commence l'éducation à l'orientation, de le commencer par une enquête dans l'établissement sur les diplômes qu'ont tous les personnels, de la secrétaire au chef de cuisine, et tout pour montrer effectivement qu'une société, c'est un tout. Et [00'52'12] Casser cette séparation de l'intellectuel et du manuel, si on n'avait pas assez professions dans l'établissement, ne poserait pas, et mon expérience a montré, et les projets que j'ai pu suivre, que si on fait que ces personnels se sentent partie. [00'52'32] Prenante du projet de l'école. Ce sont des ponts par rapport aux familles souvent les plus défavorisées. Et pensons aussi à une chose: [00'52'42] A s'en servir de ces personnels s'il y a des problèmes de traduction avec des familles maîtrisant mal le français. [00'52'50] Comme m'a dit un jour une femme de service, c'était une paire de gifles pour moi que l'établissement ait payé un traducteur alors que, vous le savez bien, je maîtrise le français. Je parle la même langue que la personne que vous avez reçu. Si on avait pensé à ça, on l'intègre, ça faisait. [00'53'10] Un pont avec, effectivement, le parcours formidable, un tranquille question avant que l'on passe à un. Je voudrais pas gêner ces groupes. Alors, et pas de soucis, on est, on est bien, bien, bien en étant. [00'53'24] Une question dont le tchate évoquait les les, l'impact du confinement, la crise sanitaire. Je l'ai évoqué là. Est-ce que vous pourriez revenir aujourd'hui pour savoir si les traces de deux de cet isolement sont toujours perceptibles, si ça empêche des parents aujourd'hui de repousser la porte d'entrée de l'état? [00'53'45] Glissement scolaire du portail ou de la porte du collège et lycée. [00'53'49] Nous voilà au mit point et on nous signalons: chatte nue, belles ressource d'attiser quart monde sur la place des parents. Mais je crois que vous l'avez évoqué, indien, qui me est amer, c'est la pire période, chiot. [00'54'01] Sur les gains des ressources, sur comment faire venir les parents l'école, mais est-ce que je reviens sur la question qui gardien aujourd'hui? [00'54'09] Est-ce qu'on a pansé les plaies du covid et du confinement sur la relation parents- école? et après, j'en ai deux autres à vous poser la pâte. Alors je serai très rapide. Pas totalement. Y a des parents qui se sont sentis isolés, méprisés par une école qui les a laissé tout seul, je pense. [00'54'29] Que, de ce point de vue-là, ça sera lentement et on n'a pas pris la mesure, effectivement, du choc que ça a pu ressentir. Même chose, des enseignants ou des parents ont fait l'école, après l'école, et qu'ils n'étaient pas d'accord avec eux pour telle et telle raison. Les pédagogies, [00'54'49] Situés se sont crispés par rapport aux familles. Là encore, on n'a pas soldé totalement à cette période dite de la continuité pédagogique. [00'55'01] Qui n'a vraiment pas suffisamment fait l'objet de formations, qui n'a pas suffisamment effectivement été soldée. Je pense que on en a encore pour un ou deux ans dans les traces. [00'55'15] Par rapport à sa grande famille. Jeudi. Traces, parce que elles ont été importantes, notamment par rapport aux parents qui avaient des enfants au moment de l'apprentissage de la lecture et qui se sont sentis un peu isolés. Ça donnait d'ailleurs le fit. [00'55'35] Financement de toute une série. [00'55'39] De gens qui nous ont relancé des manuels des années cinquante, voir. [00'55'44] Antérieurement. Donc là il y a eu un vrai problème sur une gestion insuffisante de ce que signifiait le confinement et la commune, la continuité éducative. [00'55'58] Est-ce qu'il ne faudrait pas, là aussi, à l'échelle des établissements? [00'56'02] Instaurer un droit à la déconnexion. [00'56'06] Au sens où l'esprit peut considérer que ces unités qui, s'ils sont mal maîtrisés par les familles ou par les enseignants, [00'56'14] Génère des notifications. [00'56'16] Vie. [00'56'17] Didi digne d'une des devoirs ou des communications à dix heures ou à des moments des fois un peu compliqué, pour les familles comme pour les enseignants. [00'56'26] Est-ce qu'il ne faudrait pas travailler dans le règlement intérieur de chaque établissement? [00'56'31] Elle a un code de bonne conduite et de bonnes pratiques autour de ces unités. [00'56'36] Parce que moi bien apparaître. [00'56'39] Des familles qui voient contact des enseignants des moments totalement inapproprié. [00'56'44] Un ou des enseignants qui publient des fois des devoirs ou des consignes à des moments pas toujours très adaptés et qui viennent perturber la vie, la famille, le week-end ou au dernier moment. [00'56'55] Parce que ça peut arriver à un proche, n'a pas anticipé un devoir, ou à la fin. Mais en tout cas, quand ça arrive le dimanche matin, pour le lundi matin, mardi matin, dans la famille, ça crée un émoi certain quand on s'apprête à partir quelque part où on pensait pouvoir voir un dimanche tranquille. Bon voilà, est-ce que du côté des filles qui participent, du coup, climat scolaire à part entière. Mais tant qu'ils sont, [00'57'15] J'arrive en paysage. Ce n'est pas quelque chose à réfléchir? et après, j'aurai une dernière question. Porte alors sur cette question. Je vais être très rapide. Oui, y a des choses à réfléchir. [00'57'24] Il se trouve que, entre autres casquettes, [00'57'28] J'essaye du de président du comité d'éthique et déontologie pour l'académie de créteil et qu'au cours de la séance d'hier, on s'est dit que, par rapport au problème d e n t, [00'57'42] Ouch à des politiques très différentes selon des établissements. Il fallait sans doute qu'on écrive une recommandation pour les écoles, les collèges et lycées sur le code de bonne conduite, le droit à la déconnexion, et donc, on va se mettre au travail et consulter sur cette question dans le cadre du comité. [00'58'02] D'éthique et d'odontologie de l'académie de créteil. Voyez, vous êtes au cœur. [00'58'07] Eh bien, euh, d'une autre réflexion, et je pense que seront auditionnés les fédérations de parents d'élèves. [00'58'14] Nous voilà. Euh, je pense qu'ils se feront un plaisir d'être contributeurs et auditionné pour alimenter vos réflexions en ce domaine. A, en tout cas, dans les prochaines semaines, la fibule va pousser. [00'58'25] Une faveur de ce droit à la déconnection, parce que ibrahim, mais nous le leterme, si vous voulez, ça sera une partie. C'est vraiment un code de bonne conduite. Pas, on voit tout et n'importe quoi. Des choses très laxistes dans des établissements, des choses très restrictives. Donc, de ce point de vue là, le ministère ne faisant pas, on s'est dit: [00'58'45] Décidez, on va travailler avec le clemi et on va travailler à consulter pour essayer, pour la rentrée, d'avoir ce texte. L'as la décision d'hier soir. [00'58'58] On a écouté, les grands esprits se rencontrent. J'en profite, pour faire une parenthèse, pour envoyer l'ensemble des personnes qui sont connectées ce soir à la dernière revue du parent, qui est disponible sur la plateforme, notamment caféine, ou, bien entendu, sur le site internet de la fibule, puisqu'on est une interview croisée de sociologues et de spécialistes en droit bien, en usage des écrans et [00'59'18] S'interroge sur cette question des unités à l'école et du lien parent- enfant, de la question de l'autonomie de l'enfant dans l'usage de ces outils et de la relation que ça peut générer dans les familles aussi. Donc, [00'59'30] Chose intéressante à regarder dans la dernière revue des parents à ce sujet. Dernière question pour vous, monsieur duc, et jérémy va pouvoir prendre la parole. [00'59'39] Dans la relation parents- enfants. [00'59'42] Bouzid lucky demi-mot, et on vous rejoint amplement là-dessus, sur le stress induit par la réforme du musicien. [00'59'50] Qui produit des effets dans une anxiété et un calendrier scolaire, par calendrier de travail, absolument fou chez les élèves et chez les enseignants. [01'00'00] Mais le poids de cette réforme du lien étroit qui est maintenant nourrie avec parcoursup, fait que on a des parents qui deviennent des négociateurs en note et qui mettent une sorte de pression. [01'00'13] Au vu des enjeux qu'il y a avec parcoursup et un jour sur les lili suite à des études supérieures, on en est là aussi dans quelque chose qui se dégrade probablement. [01'00'24] Au regard de l'enjeu de la note de l'évaluation nous, quel est votre regard là-dessus? en quoi ça joue sur le climat parents, enfants et élèves? parce que au lycée, qui sont pleinement mon lucia, autant de stress chez les élèves. [01'00'38] Chez les meilleurs, comme toujours les amis, les élèves en difficulté. D'ailleurs, c'est pas son. On voit bien que la question du stress, lire à tous les étages. Donc, quel est votre regard là-dessus? [01'00'50] Et n'hésitez pas à réagir. Et après je parviens à me mélanger. J'ai très rapide. Vous avez parfaitement raison. [01'00'58] L'analyse qu'on peut en faire et que j'en ai fait par rapport à divers contacts. C'est d'abord chez des parents, et souvent des parents de milieu. [01'01'11] Plus favorisés. [01'01'14] Que la moyenne, qui sont dans le cadre de la négociation parce que ils ont une stratégie sur leurs enfants. Deuxième élément: quand je regarde ce qui s'est passé à peu près dans toutes les régions, la surprise, sur ces éléments de négociation, de ces crispations sexe a quasiment plus touche. [01'01'33] Chez des établissements privés sous contrat que des établissements publics où les parents disaient: on paye, donc qu'on a des droits. Et de ce point de vue là, il y a eu beaucoup de réactions de deux syndicats de l'enseignement privé par rapport à ça. J'ai même eu des demandes d'interviews. [01'01'53] Selon sur qu'est-ce que c'est que la relation parent enseignant. J'ai pas le temps de le faire, mais ah, je dirais, de ce point de vue là, on a bien vu qu'un type de parents aujourd'hui- robert bat lyon appelait ça les consommateurs d'école- qui ont aujourd'hui une stratégie pour leurs enfants et qui attend. [01'02'14] De l'école qu'elle se plaque dans cette stratégie, y compris en marchant sur d'autres. C'est une vraie question. C'est effectivement, je dirais, un effet totalement pervers de la réforme actuelle du baccalauréat. Je pense qu'il. [01'02'34] La vraie chantier pour effectivement faire que, parcoursup, les spécialités s'assoient, pas une course. Effectivement, obtenir telle ou telle chose, mec, ça s'inscrit vraiment dans le projet de vie de l'élève. Et quand je disais que mon inquiétude, c'est [01'02'54] Dès que la souffrance, le stress mis par ses élèves face qui soit dans un refus de l'accès au savoir et qu'on ait une situation canton leur proposera dans dix ans des plans de reconversion nécessaires par rapport aux compétences. Ils en ont tellement soupé de. [01'03'14] Tel ou tel type d'approche dans l'école, c'est qu'ils ne veulent pas le faire. Pour moi, c'est gros d'un décrochage scolaire à terre de gens qui ont longtemps souffert à l'école qu'ils ne voudront pas y aller. Donc, là, il y a un chantier à revoir sur le baccalauréat d'évidence. Alors, merci beaucoup, monsieur ducasse. [01'03'34] Sur la la réforme du lycée. On est en plein dedans. [01'03'38] Ouah, effectivement, on participe au meeting national de de nous sur la de suivi de cette réforme. Nous pouvons, on ne doit pas être avare de retour. [01'03'49] Sauf qu'on observe en ce moment même à boston. [01'03'52] Spécialité sur la pression due aux élèves. [01'03'55] Compris avec l'absence d'élèves, maintenant qu'ils ont joué la spécialité, avons eu affaire. Moi, la rocky termine. C'est pour ça que je dis que ça met en cause leur rapport aux savoirs et aux apprentissages des élèves. Ce type est effectivement de dévaluation, notation. Alors je je me permettrai, mais sans être trop long, épargner du temps. [01'04'16] Sur jérémy. C'est pas faute d'avoir prévenu à tous les étages sur les dangers. [01'04'21] De cette escapade. Désaccord avec jérémy, qu'on voit là, sur les conséquences. On craignait en tout cas, ronse, rapporteur nous d'une note, droit à l'erreur. [01'04'29] Pouvant faire l'objet d'un effacement. [01'04'32] Quand on constate que lydia. [01'04'36] Un accident, parcours de façon à faire baisser la pression sur la course, à la note et à la moyenne pour pouvoir parler. Arrive en parcours pendant, parcoursup, tel qu'on l'entend, et y faire baisser notamment la pression, tous les étages. La question du calendrier qu'on évoque après. [01'04'51] Après les les épreuves de ce pays, l'absence des élèves, mais ce n'est pas l'objet de ce surnom. Tout cas, on est au cœur de la relation parents, école et enseignants, parce que là, sur le lycée, la pression était extrêmement forte. [01'05'03] Merci beaucoup pour votre intervention, monsieur odieux. Il y a bientôt. [01'05'06] Reste avec nous, je vous en prie. [01'05'08] On passe la parole à jérémy. [01'05'10] Son adieu, que j'ai présenté tout à l'heure. [01'05'14] Comme étant quelqu'un qui avait travaillé d'arrache-pied sur une méthode. [01'05'19] De façon à redonner confiance à ses élèves et à tisser un lien tout à fait particulier avec les parents, comme étant des coûts et chercheurs à part entière, est l'un des partenaires majeurs. Donc, hum, jérémy, on vous passe la parole pour pas que l'inquisition votre temps. Vous avez tout le temps qu'il vous faut pour intervenir et on prendra les questions. [01'05'39] Dans le château, juste après, juste après votre intervention. [01'05'44] C'est à vous. [01'05'45] Merci, vous m'entendez bien parfaitement. [01'05'48] Merci beaucoup, excusez-moi de vous avoir rejoint plutôt moi. J'étais en classe. [01'05'53] Je suis au lycée. [01'05'55] Et je finis les cours à dix-huit heures. [01'05'58] J'ai un peu tardé pour quitter le lycée. Du coup, j'y suis resté afin de de pouvoir faire cette intervention, non pas dans la rue. [01'06'05] Jusqu'à aulnay-sous-bois, où j'habite, mais mais ici même. [01'06'09] Et du coup, suis navré d'avoir manqué les débuts. Excusez-moi. [01'06'13] Le le. Le projet réconciliation qui a été évoqué consiste en effet à mettre ce que l'on appelle l'alliance entre les parents et les professeurs ou, au centre de tout. [01'06'24] Il est souvent présenté sur ce projet ou cette méthode. [01'06'28] Comme quelque chose qui a comme but de faire réussir tous les élèves ou qui a comme but de de d'intégrer les parents, en quelque sorte. [01'06'36] Alors qu'en fait la réalité, c'est que ce projet pédagogique, cette méthode qu'on a élaboré, moi d'abord seul, puis avec un collègue, au bout de quelques années, on a commencé à travailler dessus il y a dix ans maintenant, en deux mille douze. [01'06'47] A drancy ici même. [01'06'49] En fait, l'origine du projet, c'était un sentiment d'impuissance que nous, on avait en tant qu'enseignant, puisque, évidemment, au lycée delacroix, à drancy, les élèves ont des caractéristiques particulières. On est dans un département, celui de la famille, qui est le plus pauvre de la métropole. Évidemment, les élèves, en raison de deux de leurs origines sociales et en raison du [01'07'09] Caractère injuste du système scolaire, comme vous savez, je crois. [01'07'13] Ne sont pas aidés du coup aveux. Ils sont non pas en terrain hostile, mais sur un terrain qui n'est pas favorable. [01'07'18] L'école française est difficile avec les enfants de catégories populaires. [01'07'23] Donc, bien sûr, qui a ses caractéristiques particulières. En est: [01'07'26] Drancy ses parents. [01'07'28] Michel delacroix drancy n'est pas un lycée tout à fait comme les autres, mais dans le même temps, [01'07'32] Les élèves que nous avons en face de nous, en seconde générale et technologique, en première et terminale générale, ce sont d'abord et avant tout des ados et donc, du coup, ils font beaucoup de petites bêtises. [01'07'43] Ils ont beaucoup la flemme, il manque beaucoup de confiance en eux, ils font très peu de de choses que nous, les professeurs, leur demandant de faire. Ils ne s'investissent pas assez et ne prennent pas assez au sérieux notre parole d'enseignant. Bref, il y a dix ans, notre, notre point de départ de la méthode, ayant un sentiment d'impuissance parce que on avait face à nous des classes d'élèves qui ne sont pas méchants, mais qui sont pas [01'08'04] Foncièrement de bonne volonté. [01'08'07] J'imagine qu'en tant que parent d'élève, on ayant des enfants ou des ados à la maison pourrait témoigner du fait que, parfois, ils peuvent manquer de de de d'enthousiasme au sujet des efforts à fournir vis-à-vis de l'école et donc, évidemment, vous pouvez imaginer ce que ce que ça veut dire quand il y en a vingt cinq, trente ou trente cinq. [01'08'25] En face de nos enseignants et donc, en fait, on n'y arrivait pas. [01'08'28] Et donc on a commencé à à faire appel aux parents d'élèves. [01'08'33] Dans ce qui ressemble à de la coéducation, en effet, mais moi, j'ai l'impression que notre projet. [01'08'38] Diffère peut-être un petit peu de la coéducation, dans la mesure ou non, on considère que on ne peut pas y arriver sans les parents. Donc, on ne veut pas mettre les parents ou les familles sur un piédestal, mais on considère que, de par l'influence, de par l'autorité que les parents ont sur leurs enfants, [01'08'52] Qui est dix fois supérieure à la nôtre. [01'08'54] Du coup, nous ne pouvons pas arriver à mettre les élèves pleinement au travail, ne vont pas faire en sorte que les élèves ne soient plus absents, qu'il n'est plus de bagdad et qu'il est bien tous leurs matériels, qu'ils fassent bien leur révision, pas juste la veille au soir, mais deux, trois jours avant, etc. On peut pas arriver à faire tout cela, à obtenir tout cela comme résultat, sans le soutien des parents et donc [01'09'15] Finalement. [01'09'17] Lorsque les années de d'expérimentation, d'élaboration de la méthode nous a permis de deux mètres. [01'09'23] Un jour, c'est une sorte de protocole, de façon à ce que les parents des élèves soient systématiquement et tous à nos côtés, c'est-à-dire que nous allons les chercher très tôt. En fait, c'est une démarche qui est très proactive, si je puis dire parler. [01'09'38] Tout à l'heure de de pronote de l'environnement institutionnel qui existe. [01'09'43] En fait, et y a pascal qui est qui a érigé, au moment même où je parle au sujet de l'ump. [01'09'50] Le npd dysfonctionne complètement chez nous. C'est un désastre. Un pronote ne fonctionne pas bien du tout à environ quinze pour cent des parents d'élèves se connectent ici à apprenants et lorsqu'il se connecte, ils ont du mal parfois. [01'10'03] Parce que pronote est un outil qui n'est pas du tout intuitif ou qui parle bien foutu du tout. [01'10'08] Donc, en fait, on on casse un petit peu le rapport institutionnel que souvent, nous, les professeurs, avons avec les parents, que les parents ont souvent avec nous, le professeurs. [01'10'18] Et l'idée d'aller chercher les parents personnellement. [01'10'21] En se présentant, en leur parlant de manière gentil. [01'10'24] Leur proposant de faire alliance, en leur disant qu'on a besoin d'eux. Donc, c'est à la fois très valorisant pour les parents, mais c'est aussi, en quelque sorte, responsabiliser. Enfin, non pas qu'il y ait besoin de la responsabilité, excusez-moi, mais c'est simplement que les parents ont parfois le sentiment particulier. Ici, il y a une forte foi dans l'école comme ascenseur social que l'école est capable de faire connaître une autre. [01'10'45] Tensions sociales à leurs enfants et que c'est le rôle des professeurs de deux de cire. [01'10'49] Deux si employer les parents, d'élèves, disais-je. [01'10'54] Elles ont souvent une forte foi dans l'école comme égalé, comme dans ascenseur social et sauf que nous, nous leur disons que nous sommes compétents, qu'ils peuvent compter sur nous. C'est l'objet de notre premier message, mais en même temps notre premier appel téléphonique individuel, ce qui ne se fait pas du tout ici à drancy, et d'une manière générale, je crois, les parents qui sont contactés individuellement par les, par les professeurs. [01'11'14] Au tout début de l'année scolaire, ça serra. C'est ce que nous nous faisons et qui, du coup, mêler les parents dans un état d'esprit très favorable. Ils ont l'impression que [01'11'22] Souvent, ils ont l'impression que nous sommes très investis, ce qui est pas tout à fait le cas. On n'est pas plus investi que les collègues, mais simplement, ce temps en plus comprend en début d'année permet de créer une relation humaine. Finalement, c'est en ça que on casse un petit peu. [01'11'36] Pardon, le verbe casser n'est pas bon. [01'11'39] Hum, on ne dépend pas de l'environnement institutionnel, pronote et lointain, qui ne fonctionne pas bien et qui sont institutionnels et, du coup, l'institutionnalisation des choses comporte une part de déshumanisation, je le crois. Là, l'idée est de leur parler directement, individuellement, avec la voix, de les rassurer sur le fait que nous sommes compétents et nous allons faire reus. [01'11'59] Sir leurs enfants, mais nous leur disons que nous avons besoin d'eux, donc on les responsabilise. Intrigue, mais dans la mesure où on leur dit que nous sommes capables de faire de grandes et belles choses avec leurs enfants. [01'12'11] Et qu'ils peuvent compter sur nous. Mais en même temps, nous avons besoin qu'il fasse une partie du travail qui souvent, hé hé, hé hé. [01'12'19] Elle est implicite, elle n'est pas, elle n'est pas formulée de manière claire par les enseignants. [01'12'24] Il y a beaucoup de travaux en sociologie qui montrent que nous, les professeurs, on a tendance à attendre de la part des familles en a un comportement de parents d'élèves- idéal pour nous enseignants- mais sans exactement expliquer aux parents comment faire ce travail-là. Donc, très concrètement, par exemple, vous voyez, moi, demain, dans ma petite matière en sciences économiques et sociales, du parent, je [01'12'44] Je vois votre enfant chaque semaine et chaque semaine une évaluation. Moi, je dis aux enfants que il faut réviser plusieurs fois dans la semaine, trois fois de près. Ça prend cinq minutes, dont soixante secondes. En terminale, évidemment, la charge de travail plus lourde. Mais en second, je leur dis: réviser trois fois cinq minutes votre cours dans la semaine. [01'13'00] Sauf que moi, je ne les vois qu'une fois par semaine, donc je peux pas leur rappeler, ni donc j'ai besoin que les parents [01'13'05] Deux jours après, trois jours, après, quatre jours en paix. [01'13'08] Sans attendre la veille de l'évaluation, rappelle, tu te souviens, et tu qui s'aiment, avec m fontanel et une évaluation, et donc, n'oublie pas de passer cinq minutes, pas grand-chose, mais fais le, sinon tu vas oublier. Et donc les parents, avec des consignes claires qui viennent de de nous, de manière individuelle, si je puis dire. [01'13'24] Font un, un effort en transmettant ces consignes holà, et donc ils s'impliquent davantage ou de manière peut-être plus pratique, concrète que qu'auparavant, ce qui favorise du coup le l'investissement des élèves qui vont faire ces efforts là et qui, du coup, progressent et derrière un cercle vertueux s'enclenche, c'est-à-dire que les élèves en début d'année nous redoutent beaucoup. [01'13'45] Mon collègue et moi, parce que ils ont peur de ces professeurs qui sont en contact avec leurs parents. On envoie des petits comptes compte-rendu chaque semaine aux familles pour les tenir au courant. On s'engage à ce que la relation soit régulière. Elle est à la fois précoce, comme vous l'avez compris, et surtout elle est très régulière. [01'14'00] Puisque on envoie des petits semaines chaque semaine, qui sont assez courts et assez superficiels, mais qui visent à la fois à tenir notre promesse en tant que enseignant et, dans le même temps, à [01'14'10] A à faire en sorte que les parents restent mobilisés et continuent de faire ce qu'ils n'ont. En tant que proviseur, nous nous arrange, c'est-à-dire: [01'14'18] Passez un deuxième, deuxième couche, repasser le message du nom d'un patient à la maison qu'on a passé à l'école. C'est-à-dire: n'oublie pas d'une part, ne révise pas juste la veille, n'oublie pas, tes affaires ne traînent pas trop dans la récréation, parce que elle arrive à la fin de la récré, il faut monter en classe pour être en retard, etc. Donc, le fait que les parents passent une deuxième couche nous assure du sérieux des élèves. [01'14'39] Mais comme les parents ont quinze mille choses à gérer, comme vous le savez, ils ont tendance à oublier ces consignes la sont que nous nous le rappelons chaque semaine. D'une certaine manière. Donc, c'est le coeur de la méthode consiste à créer cette relation précoce, régulière, comme je vous disais, qui, assez rapidement, fait place à la confiance, est une chose qui existe assez tôt, comme vous savez, hélas, dans dans, dans l'école pubs. [01'14'59] Blick que dans l'école privée. Je crois aussi. [01'15'02] Et en fait, cette confiance là, cette alliance la fête. Les élèves assez rapidement se mettent au travail et très rapidement. [01'15'12] Progressent et réussissent, ce qui permet à la fois de supprimer la frustration chez les élèves, chez les enseignants et chez les pains. Et ce qui est formidable, c'est que de cette méthode-là que, à l'origine, moi j'utilisais pour ma petite matière, puis mon collègue, et moi, avec mon collègue, que mathématiquement, que les élèves, petit à petit, se sont mis à appliquer. [01'15'32] Dans d'autres matières, ainsi que les élèves sont pas bêtes rampantes quand ils révisent plus que la veille du contrôle. [01'15'38] Bah, ça marche. On suède, ça marche en maths et donc bas. De même, il l'applique en histoire-géo, dans les autres langues, dans les autres matières, comme les langues vivantes, la philosophie, par exemple. [01'15'48] Respecter un physique, etc. Les élèves, du coup, petit à petit, généralisent leurs efforts et mal [01'15'56] Élargir cette alliance avec d'autres. [01'15'59] D'autres professeurs de l'équipe pédagogique qui nous transmettent les informations, qu'on remonte aux familles. Et donc, laisse la messe qui, à l'origine, n'était qu'au sujet de ma matière, la matière de mon collègue et moi de deux mathias. [01'16'11] S'applique à toutes les matières, donc, où la plupart des matières, en fonction aussi de certains professeurs qui ne veulent pas forcément remonter les informations famille, on respecte absolument, mais disons que les élèves se mettent au travail pleinement, arrête de gâcher leur potentiel, et donc, ça change beaucoup de choses avant pour eux, pour les professeurs, et puis pour les parents. Et je finirai là avant de répondre volontiers. [01'16'31] Beau à vos questions. [01'16'35] Les le la, la joie que nous on a ici à drancy, depuis cinq ans, y a pas un seul élève qui ne décroche le covid a eu peu d'impact sur nous. Cette phrase semble très, très peu probables, mais [01'16'49] On n'a pas du tout été, ou très peu été, affectés par le covid, puisque les élèves étaient tous en ligne en cours. [01'16'55] Il n'y avait pas beaucoup de professeurs qui ont été frustrés du fait que [01'17'01] Le covid parfois, ou le contexte très compliqué servait d'excuse aux élèves pour ne pas assister aux cours en ligne ou ne pas faire les efforts nécessaires. Nous élève grâce au soutien- les parents étaient tous en ligne, faisait tous les efforts. Il y a une véritable continuité pédagogique pour le couple, alors que ça ressemble quand même un slogan débile du ministère de l'education nationale. Pour nous, on a eu la chance de devises. [01'17'21] Donc, c'est vraiment formidable et ça fait cinq ans qu'il y a plus de décrochage dans nos classes. C'est extraordinaire, avec notamment une grande réussite au baccalauréat, mais pas que parce que maintenant la méthode je l'utilise moins pour toutes mes classes. Entrée en seconde. [01'17'33] Et ce qui est formidable, c'est que les résultats assez formidable et impressionnant que que les classes ont obtenu à drancy, ici. [01'17'42] Nous ont poussé, mon collègue et moi, à faire connaître la méthode de façon à ce que d'autres professeurs qui le souhaiteraient, qui auraient envie, [01'17'49] Du coup. Qui voudrait essayer de créer cette alliance avec les parents pour ne plus subir et pour ne plus souffrir du sentiment d'impuissance qui était le nôtre, nous-mêmes, y a dix ans? [01'17'59] De deux façons alors: présenter ces outils-là et de de les encourager à se lancer. Et ça fait un an et demi maintenant que c'est le cas, grâce à la médiatisation en grasset: un livre qui est sorti, un film documentaire qui sort dans quelques mois au cinéma, en septembre. [01'18'14] Qui montre une année à drancy. [01'18'16] Et en fait, [01'18'18] L'année dernière, il y avait une dizaine de professeurs qui appliquait la méthode et cette année, il y a une centaine de professeurs. [01'18'26] Un peu partout en france, à la fois des professeurs du lycée général, technologique, professionnel, de college. [01'18'31] Professeur des écoles maternelles. [01'18'34] Et élémentaire, enfin la fois maternelle et primaire. Pardon, [01'18'39] Y a des professeurs qui sont en banlieue, en quartier populaire, comme nous, en zone rurale, en zone péri-urbaine, en centre-ville aussi, c'est très intéressant. Y a en fait l'une des forces de la méthode du projet vient de de son, son adaptabilité, si je puis dire. C'est que chaque professeur, en fonction de son propre contexte, ses propres élèves. [01'18'56] De sa propre trajectoire personnelle au site, son propre caractère, de sa propre matière, s'approprie de façon très subjective cette méthode-là qui consiste finalement, pour le résumer, à créer cette alliance avec les parents, comme vous l'avez compris, je pense. [01'19'09] Et ce qui est formidable, c'est que les collègues vivent tous ce sentiment de libération, de ne plus avoir l'impression de de de d'être seuls face aux élèves et donc de subir, ou d'être seul face aux élèves et donc d'espérer que leur bonne volonté sera suffisante, ce qui est rarement le cas. Et donc, avec, avec cette alliance là, les collègues, [01'19'29] Qui soit en école primaire, en collège ou en lycée, ont l'impression de redécouvrir leur métier. Donc, c'est formidable, indispensable, qu'on appelle le projet réconciliation avec un s, parce que c'est à la fois réconciliation entre les parents, les profs, entre les élèves et le travail, puis entre les profils et leur métier lui-même, si je puis dire. [01'19'46] Alors. Merci beaucoup pour ce propos. Il y a il y a de nombreuses réactions qui vont globalement dans le même sens ici. Vous pourriez nous repréciser comment fonctionne le lien avec les parents? on a bien entendu l'appel qui est fait. [01'20'02] Un un les enseignants, l'envoi des insultes qui fait régulièrement, mais cela veut dire que un parent reçoit, séduit, dix estimés dans la semaine de chaque enseignant, un par discipline. [01'20'15] L'appel jusqu'à huit appels différents ou spécial. Comment vous cour du lycée et comment monde ou gérer le côté intrusif ou pas? comment est-ce que c'est perçu côté parents? [01'20'25] Oui. [01'20'27] Alors ne, comme je disais, on nous pendant des années. On est des seuls à faire ça. [01'20'33] L'année dernière, on était une dizaine et cette année, on a une centaine, dont il n'existe pas en france aujourd'hui, de parents d'élèves recevant plusieurs coups de téléphone, puisque les professeurs qui appliquent cette méthode sont sont isolés. On est très peu nombreux. Je pense que le projet va beaucoup grandir et je je vais m'y attacher personnellement pour l'aider. [01'20'52] Dissout les vingt prochaines années. Je pense qu'on sera quelques milliers dans quelques années, mais pour l'instant, ce n'est pas du tout le cas. Et donc, ce qui se passe aujourd'hui, c'est que, du point de vue d'un apparent, il reçoit le coup de téléphone d'un ou d'une prof qui lui propose de le tenir au courant tout au long de l'année, chaque semaine, avec des sa messe, au sujet de de sa matière. [01'21'11] Un professeur du projet qu'il nous faut en tant que prof principal dont, comme je le disais et comme nous ont influencé, il récupère les informations au sein de l'équipe pédagogique, il envoie un essai collectif, [01'21'20] Hé, hé. [01'21'23] Et c'est tout, c'est-à-dire que les parents d'élèves reçoivent à ces messes par semaine et les réactions des parents sont extraordinairement positives parce que, dans les quartiers populaires en particulier, beaucoup de parents ont l'impression de ne pas être légitime. [01'21'36] L'école, comme vous le savez, n'est pas l'endroit le plus accueillant pour les parents et en particulier pour les parents qui n'ont pas forcément fait de longues études, comme c'est beaucoup le cas en un quartier populaire où beaucoup de parents qui n'ont pas fait leurs études. [01'21'51] Souvent courtes, en france, dans un milieu, dans un pays étranger, et donc pour eux, l'école, le rapport à, l'écrit, toutes les choses qui vont de soi pour les professeurs, pour l'école, pour l'institution. [01'22'02] Ou pour une association comme la fcb, ne va pas de soi pour plein de parents, comme vous le savez, et donc, le fait de deux de leur tendre la main, de les valoriser et de leur dire qu'on a besoin d'eux et, en même temps, de leur assurer que nous allons bien faire notre travail et qu'on s'engage à les tenir au courant du coup, provoquerait des réactions très, très positives. [01'22'22] Chez les deux tiers des parents et puis parfois un peu dubitative, chez un indépendant qui dit: mais c'est quoi ce truc, un peu bizarre. [01'22'31] C'est la première fois qu'on propose un truc comme ça, en effet, et donc la nouveauté surprend toujours et donc, après nous, on on la manière dont on parle aux parents de manière très gentille et très douce, au fait que les parents souvent nous suivent et surtout, très vite les résultats. [01'22'47] Son sont très convaincants que les enfants [01'22'50] Les enfants se mettent au boulot, progresse. [01'22'53] Parle davantage de l'école à la maison. [01'22'56] Les parents recevoir, reçoivent les notes, les appréciations qui sont de plus en plus positives. Donc, les parents, à la fin de l'année, nous disent tous: mais c'est superbe, très câlin. Et je me permets de finir cette réponse en en réagissant à une réaction je viens de voir passer. [01'23'10] Il faut que je réponde à la question du temps, mais sur la généralisation, sophie duval. [01'23'15] C'est souvent une remarque, en effet, qui est gentille mais qui est dangereuse. Il faut absolument pas que notre manière de faire soit généralisée, parce que, tout d'abord, elle n'est pas généralisable, c'est-à-dire que, comme vous le sentez, il y a beaucoup dans la méthode qui dépend de l'état d'esprit ou de du tempérament, si je puis dire, des enseignants qui se lancent faveur. [01'23'35] Part de folie, je crois à une part d'optimisme. [01'23'38] Pour aller chercher les parents et penser que il est possible de créer cette alliance là. [01'23'43] Autrement dit, pour le dire de manière familière, si demain à bingham macron. [01'23'48] Qui ont cherché à en savoir plus, mais que nous avons repoussé gentiment. Et si, demain, il il généralise cette méthode, ce sera un échec cuisant, puisque les professeurs, comme vous le savez, n'aiment pas qu'on leur impose des choses. La liberté pédagogique est fondamentale et, surtout, la méthode n'a n'a de de sens et ne peut être efficace. [01'24'08] Que si elle est incarnée ou que si elle est appliquée de façon volontaire, donc demain, ainsi, sans l'oblige, ce sera pas du tout efficace. Et puis ça, ça, même si je vous remercie pour le compliment, même duval. Mais simplement, c'est aussi sous-entendre que il n'existe qu'une manière de faire. Ce qui est sûr, c'est que [01'24'28] Cette manière de faire réussit. [01'24'30] Mais il faut se garder, si vous le voulez bien, de deux. [01'24'35] Deux, de penser que, du coup, il n'y a que celle-ci. Je pense qu'il y a tout à fait, sur la base du volontariat, on est d'accord, de très belles choses à faire, et c'est ce que moi, je fais de manière individuelle. Je vais chercher les collègues, un parrain, pour qu'on soit une petite armée dans dans quelque temps. [01'24'48] Un, deux, trois questions complément. Nous, on a largement le temps. Nous devons nous sous obligé du vôtre. [01'24'54] Bar-le-duc, du coup, dans cette relation, le redire, réanimer peut être aussi, si je peux m'exprimer ainsi, [01'25'01] Au niveau de votre lycée? quelle est la place du sérieux ou du? oui, j'ai vu passer l'information. Alors, c'est une très bonne question, parce que c'est vrai que la relation avec les parents est souvent dévolue ou perçue comme quelque chose relevant du domaine réservé des cbd. Eux, c'est eux qui, comme vous le savez, je crois, ou je me permets de delà de rappeler- [01'25'19] Est un métier qui n'existe qu'en france. [01'25'22] Mais disons que dans notre lycée, en raison du décrochage, [01'25'26] Très important et des difficultés considérables qui existent ici. [01'25'30] Origines sociales. Surtout, laisser peu joint un travail de de de pompiers. [01'25'37] Ou de policier, je ne sais pas trop comment le dire. Ce qui est sûr, c'est que dans les deux cas, ce soit. [01'25'42] Policiers, aux pompiers qui réagissent quand il y a des rapports pour après les élèves, pour qu'ils arrêtent de sécher les cours ou de s'absenter de manière parler ou de manière structurelle. [01'25'51] Les, les professeurs, les cibles par dance croulent sous le travail et ont souvent l'impression de de d'être là trop tard. [01'25'59] Ou de debat faire vraiment. [01'26'02] Un travail tel que il il l'espère au début, comme nous enseignant aussi d'ailleurs. Et donc, en fait, souvent, les civils sont soulagés que nous prenions en charge le travail avec les familles, parce que ça leur enlève un peu de leur poids. [01'26'17] De la charge de travail qui est considérable. Et surtout, ça leur donne un autre statut aux yeux de la classe. C'est-à-dire que ici au moins, en tant que professeur, je, je, je viens. Thermalisme, le travail de flic, pardon de fliquer les absences, etc. Que font souvent les sbires. Du coup, les citoyens n'ont pas non plus besoin de le faire et donc, du coup, les élus [01'26'36] Eve redoute a habituellement laissé peu, parce qu'il ne perçoit comme des gendarmes chinois, en tout cas. [01'26'43] Là, maintenant, c'est plutôt moi qu'ils redoutent et donc ils vont voir différemment et c'est eux qui peuvent jouer un rôle du coup qui est nouveau et qui est très enrichissant intellectuellement pour eux, parce que son rôle d'adulte extérieur à l'équipe enseignante et qui, en même temps, [01'26'56] Cadre fait partie de la communauté éducative et donc, du coup, ils vont recevoir les élèves de manière individuelle pour le les encourager, pour les les accompagner, plutôt que d'être davantage dans le fonçage de sourcils que nous on con, qu'on utilise, si je puis dire. Nos relations avec si peu sont très bonnes parce que [01'27'17] D'accord, ça leur donne du souffle. [01'27'19] Merci beaucoup pour ces précisions, pour votre question et pour la moitié finesse, outre le fait d'avoir attribué l'opérateur, [01'27'26] Est-ce que vous utilisez votre iphone, onze perso, et comment vous gérez votre droit à la déconnection et celui des familles? [01'27'34] Oui, on en ingère un à la fin de l'intervention de deux, de jean-louis a dit que le droit à la connexion est fondamental, en fait, [01'27'44] On est. On est cent cinquante-trois aujourd'hui. Donc y en a beaucoup qui utilisent leur téléphone perso, comme c'est mon cas. [01'27'51] D'autres qui utilisent une deuxième carte sim ou un autre téléphone portable ou le téléphone du lycée. Il y en a qui passent par le mail ou par le n? t lorsque les familles sont connectées. Donc, vraiment, encore une fois, la méthode. [01'28'04] Et elle l'est réapproprié et utilisé de manière très différente en fonction des collègues. [01'28'09] Mais du coup, moi je le fais avec mon numéro perso. J'ai vu passer la question du temps. Je peux, si vous m'avez posé, excusez-moi. [01'28'16] Hum et voulut: tant que ça vous prend. [01'28'19] Deuxièmement, le temps. Je ne fais que ça prend deux, trois heures en début d'année pour appeler les parents. Ça, ça prend du temps. [01'28'26] Ce combat. Cinq minutes au téléphone, avec chacun un mot. [01'28'29] Mais ensuite, ça prend une demi-heure par semaine l'envoi de sms, parce que avec classe par classe ou tout confondant par classe, et les collègues qui se lancent dans le, dans la méthode, ne le font qu'avec une classe, parce que le travail avec les familles, nous-mêmes, on a fait que avec une seule classe pendant cinq ans. Aujourd'hui, je le fais pour l'ensemble de mes classes, mais pas avant pas. [01'28'48] Dix ans. C'est la sixième année que j'ai généralisé mes classes, parce que travailler avec les familles de manière régulière est quelque chose qui est tellement gueule. [01'28'56] On est tellement pas préparée. Non enseignant n'a jamais dit que les parents pouvaient être une aide. [01'29'00] Et puis, on l'imagine tout simplement pas, et puis on a déjà la tête sous l'eau, pour plein de raisons, comme vous savez, je pense. [01'29'07] Que, du coup, apprivoiser cette relation régulière, forte, avec les parents prend du temps. Donc, tous les collègues qui se lancent, ne le font qu'avec une classe et ça prend une demi-heure par an, par semaine, pas plus. [01'29'18] Grâce à des petites techniques que je ne dévoilerai pas ici, mais que je dévoile aux collectivités, selon si elle est professeur parmi les parents d'élèves ici présente et écrivez-moi avec grand plaisir. [01'29'27] Mon adresse académique. [01'29'29] Mais, mais en fait la question du temps de travail. [01'29'34] À laquelle on pense spontanément est en fait une non question. Manche entourée à drancy de collègues qui sont dévoués, qui sont investis, parfois plus que moi. Moi, j'ai un emploi du temps sur trois jours, ces trois jours qui sont intenses, mais je ne viens que trois jours par semaine au lycée. [01'29'49] Je ne retouche plus à mes cours, je n'en touche plus à mes évaluations depuis plusieurs années. S'entourer de collègues qui emportent des copies pendant les vacances, qui refont leurs cours pendant les pendant les vacances, le week-end. [01'29'59] Qui organise des, des sorties, des des projets, qui font intervenir des intervenants externes et etc. [01'30'08] Et qui en fait pour beaucoup, comme sont dans une relation encore. [01'30'14] Solitaire face aux élèves, parce que c'est comme ça qu'on nous a formés et c'est comme ça quatre, vingt dix neuf pour cent d'entre nous. [01'30'20] En faisant ce, ce, ce métier-là, [01'30'23] Du coup. Oui, il passe beaucoup de temps à refaire leurs cours, en espérant que la qualité intrinsèque du cours permettent d'amener à eux les élèves. [01'30'32] Je je ne consacre absolument pas ce temps-là ni à préparer ses cours de manière aussi. [01'30'37] Appliquer avec mes collègues et, dans le même temps, j'utilise de manière très pragmatique les cuisses et m comme outil d'évaluation, ce qui me prend très peu de temps à corriger. Donc, c'est: voyez, si la question n'est pas mon travail plus, c'est plutôt: comment est-ce qu'on? [01'30'52] Qu'est-ce qu'on choisit? comment ont choisi sa crémation? [01'30'55] Et donc, au final, on travaille pas plus que les collègues, ou on travaille différemment, on contourne l'obstacle, on va chercher les parents, parce que face aux élèves, on n'y arrive pas. [01'31'04] Il part quand on fait davantage. Papa. Les parents ont fait davantage travailler, les parents, si je puis dire, et et les élèves travaillent davantage, ce qui fait que nous, derrière, au final, le travail est très facilité à la fin de l'année scolaire. On travaille pas puisque que les collègues et surtout, par contre, on ne souffre plus en-dessous, on ne subit plus, et donc c'est formidable. [01'31'22] Petite question. [01'31'24] Également: est-ce que après cinq à six années de recul, et donc d'un bilan, [01'31'29] Plus ou moins collectif et à géométrie variable selon les classes ou dans l'établissement, vous avez constaté un effet. [01'31'37] Positif, neutre ou négatif sur le conseil de classe, son rôle, l'investissement des parents dans le conseil de classe. [01'31'45] Ils ont également au sein du conseil d'administration? est-ce que ça a eu un effet sur la place dans l'établissement des parents au sein des instances qui sont prévues par les textes? considèrent la laïcité peu présent, en particulier avec ces peuples, même si ce n'est pas la seule, ce sont, comme vous savez. [01'32'01] Elle est présente à drancy, mais [01'32'06] Le, le, le. L'école est si lointaine des familles ici. [01'32'11] Que c'est un travail. [01'32'13] Qui? qui est, qui est noble? [01'32'15] Hey, qui est courageux et et moi, j'ai des, j'ai des bonnes relations avec eux, avec les, avec les représentants de la fcb. [01'32'24] Notamment, mais, mais, mais. Quatre-vingts pour cent des familles ne sont pas en contact avec avec les associations ou avec les représentants. [01'32'33] Quatre, vingt quinze pour cent ne savent pas quelles sont les les enjeux du conseil d'administration, par exemple, donc, encore une fois, en quartiers populaires, là où l'école est perçue comme une institution dans laquelle elle et les parents ne se sentent pas forcément les bienvenus. [01'32'47] Je ne dis pas que c'est ce que l'école fait, je dis juste que les parents se sentent éloignés et donc, même avec la méthode qui est la nôtre, qui fait que les parents sont très investis, s'applique de manière extraordinaire et ailée et les résultats qui sont moins impressionnants ici sont absolument autant dû à notre engagement que ça. [01'33'07] Celui des élèves, des familles. [01'33'08] Bon pour. Pour autant, cet engagement et cet investissement considérable des parents ne se fait que dans le cadre de la relation individuelle que nous avons créé ensemble et donc l'institution. [01'33'21] L'institution détruit, lorsque le distingue, détruira. Pardon, mais je veux dire que que que il n'y a pas de de de conséquence d'un point de vue de l'établissement ou d'un point de vue de de de l'institution. On est dans un gros lycée et je ne suis pas moi-même au au conseil d'administration, parce que j'ai déjà [01'33'40] A la tête sous l'eau, si je puis dire, avec mes élèves et et donc moi-même, je m'implique assez peu dans l'institution et je, comme vous le sentez, je redoute quelque peu l'institution. Bon, donc, pas d'effet va dire sur la dynamique collective au sein de l'école, de sur l'amnésie faron, d'accord, ok. [01'33'57] Et puis, excusez-moi, comme on est dans un très, très grand lycée, il y a deux mille cinq cents élèves issus de son professeur, on est deux à appliquer cette méthode. Donc, notre, notre, notre expérimentation aussi fontaine, incorporez le souhaitez, n'est qu'une expérimentation parmi tant d'autres. Et donc le lait. [01'34'14] Nous ne sommes pas grand-chose pour l'instant. Alors là, vous me faites nîmes accueil ma curiosité: comment est-ce que vous gérez la distorsion- je l'appli comme ça- qui peut y avoir entre des professeurs qui, comme vous, tissus ces passerelles régulières avec les familles? on les utilise, élèves, harris, aimé, par mail, etc. Et les enseignants qui ne le [01'34'34] Font pas par choix pédagogiques ou parce que ils ont investi autrement. Faites refondre leurs cours comme vous évoquez. Il n'y a pas de jugement, mais cela a un effet. [01'34'45] Vendre une distorsion, préfère vers une plus pervers. [01'34'48] Absolument. [01'34'50] Évidemment, je vous disais, les deux tiers des parents sont enthousiastes dès le premier jour. Le dernier tiers finit par l'être au bout de quelques semaines, à quelques mois. [01'34'58] Et donc, il est évident que les parents ont beaucoup, beaucoup d'affection pour nous. Je dis nous à la fois, mon collègue et moi. Et c'est ce que vivent les collègues un peu partout en france qui reprennent la méthode. [01'35'08] Le lien tissé est extraordinairement fort et beaucoup de parents sont soulagés de deux deux d'avoir cette relation. Il avait l'impression parfois que, que, que, que l'école était lointaine. [01'35'21] Y'a aucune famille pour répondre à une question du mois passé à melody. [01'35'25] Y a aucune famille qui dit non. Puisque qu'on appelle les parents, on leur dit: est-ce que vous êtes d'accord pour qu'on vous tienne au courant de l'évolution de votre enfant? [01'35'33] Favoriser la réussite de celui-ci. Donc, du coup. [01'35'37] Le, le, les les. [01'35'41] Les parents ne disent tous oui, et c'est pareil pour les collègues du du. En formulant les choses d'une certaine manière, il n'y a pas de soucis. [01'35'48] Et après. C'est vrai que parfois les parents [01'35'51] Nous nous écrivons, nous disent: ah ah, ah, bah, c'est dommage qu'il n'y ait pas d'autres professeurs qui passa. [01'35'55] Bah, c'est. C'est dommage que que y'a plus de profs qui reprennent cette méthode-là. Et, comme nous, on est très attachés à la liberté pédagogique, même si je vois qu'il y en a qui râlent dans les commentaires sur le fait que la liberté pédagogique empêche de généraliser. Encore une fois, et la généralisation n'est pas souhaitable. Si vous avez du respect pour ce que nous faisons ou si vous trouvez ça intéressant, je vous prie. [01'36'15] Guide de mesurer vos jugements. [01'36'18] Parce que, encore une fois, généralisé, ou appelé à la généralisation, ou avoir l'impression que on rase les murs en salle des profs, comme je viens de voir passer à david dumont, c'est se tromper sur la réalité de la profession. [01'36'33] Non entouré de collègues qui sont, encore une fois, aussi investis et et qui sont aussi motivés et, si je puis dire, ou dévoué que nous. C'est juste qu'ils n'utilisent pas les mêmes outils. [01'36'44] Et que, évidemment, d'un point de vue extérieur, on peut avoir envie de, de, de de plaquer cette méthode. Mais mais non, on pense que c'est pas souhaitable, parce que quand, quand, comme notre but est de faire école et de développer ce moment-là, pour nous, la seule manière de faire c'est par l'attrait et parle là par là, là-bas. [01'37'03] One volonté et donc il faut surtout pas blesser les collègues du coup. Pour répondre à votre question, [01'37'08] On demande aux familles de de faire attention parce que parfois les parents- y compris remise de bulletin n [01'37'13] Parfois n'arrive pas à se retenir de dire un récit: bien, et c'est pratique pour aller faire ça en maths aussi ou en histoire-géo. [01'37'19] Et donc en fait: [01'37'22] Les, les parents d'élèves. On leur demande de retenir ces jugements là, et puis, non, s'excuse auprès des collègues- aussi payer cet impact là, parce que, encore une fois, [01'37'32] Y a pas de y a pas de recette miracle. Il y a plein de façons différentes de faire réussir les élèves et donc, on veut surtout pas l'imposer aux colis. Alors justement, jérémy, question pour les lily et lily, les enseignants comme vous qui font le choix de cette méthode de surveillance lire: est-ce que c'est venu influer sur les appréciations du bulletin trimestriel aussi? [01'37'52] Ou pas, le lait, les nôtres, les vôtres. [01'37'55] Est-ce que vous, du coup, parce que comme il y a sur ce lien, on va dire hebdomadaire, ou du moins très fripon, est une famille, bref, où il quitte les lèvres ou le conseil de casque joue potentiellement sur votre matière un rôle différent et l'appréciation qui est mise dans le bulletin trimestriel, est-ce que ça fait évoluer le contenu ou est-ce que? [01'38'16] Tel qu'il est: mon mois, cinq ans, et pour vous- moi je parle pour vous, en fait, il eut le le, les choses se déroulent de manière. [01'38'24] Assez simple finalement et c'est presque inquiétant, dit comme ça les élèves en début d'année. [01'38'29] Redoute un petit peu et se mettent au travail et réussissent tous. [01'38'33] Donc moi, j'ai pas un élève qui ne réussit pas aussi. [01'38'37] Inquiétante, wolfgang, semble cette, cette affirmation. Moi, j'ai pas un bulletin d'élèves. J'ai deux cents élèves de seconde chaque année. J'ai trente élèves de terminale. Ça fait cinq ans qu'il y a plus d'échecs. C'est dire que y a pas une appréciation qui n'est pas positive, y'a pas. [01'38'51] Un élève qui, à la fin d'un semestre en semestre, ici. [01'38'54] Dis: c'est trop dur à suez ou j'y arrive pas, y compris des élèves qui travaillent, les difficultés qui sont extraordinaires, y compris les élèves qui sont virés en cours d'année, les conseils de discipline et tout le tintouin, y compris des élèves. [01'39'07] Dont on a l'impression parfois que ils ne sont pas adaptés pour le système scolaire. [01'39'11] Les élèves réussissent tous vraiment, et donc, après moi, je je passe un plaisir pour moi de d'enseigner, parce que comme les élèves font de super efforts, font des supers progrès, le conseil de classe et est à la rédaction des bulletins, même s'il est chronophage. [01'39'27] Le sampson plaisir, parce que le bilan est systématiquement positif. [01'39'32] Et donc ça, ça pas eu trop d'impact, aura cinq ans. Ça m'a rendu plus optimiste, je pense, parce que les élèves réussissent systématiquement et donc les appréciations ne reflètent absolument, je pense, que le travail avec les familles- je n'aime pas beaucoup parler musicale. [01'39'45] Besoin de vous convaincre là-dessus. Le fait d'être en lien régulier avec les familles m'oblige à une certaine rigueur, m'oblige à une certaine. [01'39'54] Modestie aussi sur mes moyens, à une certaine heure. [01'39'59] Exigences, et donc les parents renforcent mon autorité et il me [01'40'04] Et il me renforce beaucoup. [01'40'06] Par le fait que je leur ai promis que ça allait bien se passer et, d'une certaine manière, [01'40'12] J'oubliais de me débrouiller derrière pour que ça se passe bien, que que tous les élèves réussissent. Je crois beaucoup dans la capacité des professeurs à réinventer leur cours, à réinventer leur manière de faire, et c'est pour ça aussi que, même si je comprends beaucoup de commentaires qui défilent et qui, de manière consciente ou non, le dénigrent- une partie de mes collègues, [01'40'29] Une partie des enseignants. Moi, je, j'ai absolument confiance dans la capacité tous les profs de français à se réinventer pour faire mieux. [01'40'37] Mais je pense que là, la première des conditions, comme face à des élèves, c'est de les respecter et même peut-être de les encourager ou de les choyer. [01'40'45] Justement, euh, par rapport à tout ça, commence le votre hiérarchie, le chef d'établissement, corps, d'inspection, à regarder votre méthode. [01'40'54] Vice-président, encourager celui de freiner, même si vous avez un devoir de réserve. [01'40'59] Derrière lequel vous pouvez, vont-ils vous retrancher si vous souhaitez, mais comment c'est perçu? [01'41'04] Comment va la situe. Ce sont ces expérimentations qui, maintenant, fut généralisée, y fait école que du bien moins celui-ci se diffuse en se généralisant et qu'il en fait. [01'41'16] La direction nous a accompagné quand on a commencé. [01'41'21] Comme elle accompagne tous les expérimentations. [01'41'25] L'orientation pédagogique dans notre lycée. [01'41'28] Comme je disais, notre projet n'en est qu'un parmi les trente ou cinquante qui existent ici. [01'41'33] Et donc la direction fait un travail de suivi. [01'41'36] Comme je vous disais, il y a deux cents professeurs et une direction à quatre ou cinq têtes. Y ont beaucoup, beaucoup de choses à gérer et donc, monsieur, ou les choses que, de manière lointaine, nous [01'41'46] N'a jamais demandé le feu vert au chef. On a utilisé notre liberté pédagogique, on allait chercher les familles et chaque année, des miracles viennent en classe. De cette manière-là, le le, la direction, de manière pragmatique, a vu que ça marchait et nous soutient. [01'42'01] Nous a permis de continuer, mon collègue et moi, en tant que binôme professeurs principaux, tous les deux. [01'42'07] Mais après, si vous voulez, les personnels de direction sont quand même assez loin de notre réalité d'enseignants inquiets face aux élèves, terres, cours évalués, etc. Accompagnés de petits. Donc, on l'aura jamais vraiment demandé l'heure mon feu vert. On n'a jamais vraiment obtenu, et encore moins pour l'institution vis-à-vis de laquelle quand [01'42'27] Vous l'avez compris, j'ai pas beaucoup de de des stimuli de tes sentiments positifs. [01'42'34] Moi, je me suis fait inspecté deux fois avant. On dit plus inspecter ma poche, les éléments. En fin d'année, ils disent bravo, merci, c'est génial. Et surtout, ils ont beaucoup grandi. Les élèves adorent. C'est les premiers et les premiers représentants de premier porte-parole de la méthode. [01'42'51] Mais en fait, le, le l'institution, comme vous le savez, a une gestion des ressources humaines qui est catastrophique dans l'éducation nationale. Donc, moi, je fais fils de de l'institution. J g j'ai débattu il y a six mois dans une émission sur france deux, avec à peine gars. [01'43'09] A qui je ne demande rien, si ce n'est de de d'augmenter, mes camarades et moi. [01'43'13] Qui. [01'43'15] Va être fait de manière ridicule, a-t-on appris aujourd'hui ma femme. Voilà le but de cesser ce discours pénible sur les profs, qui sont des feignasses, etc. [01'43'22] Mais, mais, sinon lui-même m'a demandé: est-ce que, est-ce que les escaliers? [01'43'29] Est-ce que le chef d'établissement vous soutiens? je dis oui, mais voilà. Est-ce que le benzène vous soutient? non, mais je pense pas. Qui c'est qui? on est que le recteur de soutien. Non, donc, je pense que l'institution a un rôle à jouer, mais on dit beaucoup aux collègues: lancez-vous. Et l'esprit de la méthode est aussi de deux, deux m. [01'43'49] Brouillé avec ce qu'on a dit julien. [01'43'51] Alors petite question, est-ce? [01'43'53] Peut-être la dernière ou l'une des dernières reconstruites. [01'43'57] Est-ce que vous vous sentez? [01'44'01] Ouais, je joue sur le mot de de se sentir seul. Vous évoquez face aux élèves à aura un contexte d'enseignement. Est-ce qu'avec cette méthode, il vous là, vous l'avez dépouillé? extrémité, vous vous sentez moins seul dans votre relation aux élèves. Est-ce que c'est iso par rapport à avant vingt? [01'44'18] Comment je l'assume dans les pratiques pédagogiques pour l'acide nue: sarah, sarah, samus, impuissances, pas avoir évolué. C'est le cœur de la méthode, si vous voulez. [01'44'27] Aujourd'hui, j'avais, j'avais, j'ai fait un contrôle avec des élèves de seconde a et quatre classes de seconde. Aujourd'hui, j'avais cent élèves. [01'44'37] Il n'en manquait pas un. [01'44'40] Non pas par la grâce du saint-esprit, ou alors on parlait de tout à l'heure de jean-louis duc la, une fédération laïque. [01'44'46] Sien. [01'44'48] Je n'ai pas entendu. Je disais: nous sommes une fédération laïque, donc yverdon est donc réduite. Que vous ne croyez pas publiquement en disant à la grâce du saint-esprit. En tout cas, moi j'y crois à titre personnel, mais c'est pas ça qui fait que tous les élèves étaient là. [01'45'02] Les élèves étaient tout cela parce que les parents savaient qu'aujourd'hui les contrôles et que ça fait trois à quatre jours qu'ils disent aux élèves: attention, n'oublie pas de réviser quinze minutes à plus cent ans en suède, la porte contre là où il avait quinze minutes, trente minutes maximum. Je les charge pas trop en travail. Mais donc du coup, les parents savaient qu'aujourd'hui et contrôle, ça fait une semaine qu'ils en parlent aux élèves. [01'45'22] Les épreuves de spécialité sont passées en terminale générale. [01'45'27] Ça fait un mois maintenant. Comme vous savez, il ne manque pas un seul élève de terminale à mes cours. Hier, on a fait cours- il était tout cela- lundi aussi. [01'45'37] Demain nous serons tout cela. [01'45'39] Les parents d'élèves disent aux enfants: il faut aller en cours même si tes copains n'y vont pas. Donc, en fait, assez justement parce que je ne suis plus seul qu'un jeu, que je ne souffre plus absolument, et comme je vous le disais de manière brève tout à l'heure, le fait de ne plus être seul m'oblige aussi à une forme de réussite à aider. C'était explicité dans les commentaires d'une passé tout à l'heure, excusez-moi. [01'45'59] Commentaire auquel je réponds. Pas le fait de muscler, mais mais en effet, j'ai une sorte d'obligation de résultat qui est très désagréable parce que nous, en tant qu'enseignants, on n'a pas d'obligation de résultat. Je pense que c'est une bonne chose, dans l'absolu même. [01'46'10] Cette obligation là me sort de ma zone de confort autant que nous sortons les élèves de leur zone de confort et que nous sortons les parents aussi de leur zone de confort, parce que, avec des, des consignes ou des informations qui sont précises et claires, [01'46'23] Nous mettons un petit peu la pression aussi sur les parents d'élèves qui, parfois, ayant plein de choses à gérer, [01'46'28] Ils ne veulent pas forcément jouer complètement le rôle que nous nommerons. Qui joue. Donc, l'idée est de se sortir collectivement de la zone de confort. [01'46'35] Et ici, encore une fois, la ville arrête avec mon auto promo. S'il y a des déchets, pas s'il y a des enseignants parmi les professeurs, forcément. Il y a quelques-uns qui sont morts, il fait donc. Il y en a qui sont intéressés, mêlant à titre enseignant. Écrivez-moi avec plaisir. J'ai écrit mon, mon adresse mail dans le, dans le, dans le dans la chatte. [01'46'54] Jérémy pour enfant teigneux, arobase, assez créteil pour faire. Mais il y a plein de questions auxquelles j'ai pas répondu. Je vous prie de m'excuser. Là, une question qui est qui est revenu à plusieurs reprises et que je n'ai pas posé. Si suspentes, donnons-leur la dernière. Votre conclure. Comment faites-vous avec les familles qui ont des difficultés avec le français, pour le comprendre, lui parler la langue n'est pas. [01'47'14] Un obstacle. [01'47'16] En fait, si vous voulez, dans toutes les langues du monde, huit sur dix, on comprend ce que ça veut dire. Et donc, c'est ça qui est formidable, avec la méthode, pour les collègues qui se lancent. [01'47'25] Et elle casse complètement le fatalisme qui est très répandu à la fois chez les élèves, chez les parents aussi. [01'47'32] Puis chez nous en tant qu'enseignant. [01'47'34] Parce que quand les élèves se mettent à produire des efforts considérables et à faire des progrès considérables, [01'47'40] Ça change complètement notre vision de ce qui est possible, de ce qui est impossible ou de ce qui relève de difficultés ou d'obstacles. La langue n'est absolument pas un obstacle. Donc, il arrive chez les parents tamouls en particulier, des parents chinois ou des parents turcs. Eux, c'est les les communautés où y a le un peu moins francophones que dans les communautés. [01'48'00] Slave maghrébines, ou d'origine maghrébine ou d'origine d'afrique noire, qui maîtrisent bien plus la langue française. [01'48'09] Et qui sont parfois en france depuis plus d'une génération. Donc, lorsque les parents maîtrisent peu la langue, on on on demande à parler au grand frère, grande sœur ou petit frère ou cousin, aux collègues voisins, on se débrouille. Mais une fois que les parents ont compris que nous étions des profs gentils, [01'48'29] Il nous aime bien et du coup, il parle de nous à leurs enfants. Du coup les enfants font plus d'efforts et derrière, le cercle vertueux s'enclenche. [01'48'37] Merci beaucoup pour cette intervention, jérémy. On en arrive là. Le mail intimidante tchat stéphanie c, vous pouvez le le retrouver. [01'48'46] On combat le republier, si besoin était. En tout cas, merci beaucoup. On arrive au terme de ce webinar colloque. [01'48'52] Quittez donc moi, mon lieu centré sur les relations. [01'48'56] Parents, école, et on y ajouter dès l'introduction: [01'49'00] Qu'est-ce que j'ai pu faire? et puis, avec ce propos, monsieur duc, et le vôtre, la relation. [01'49'05] École, parents et enfants, puisqu'on voit bien que on est là sur un équilibre de deux à trois pieds qui qu'il faut construire. Vous êtes une méthode. Vous avez propose une méthode plus pratique pour l'alimenter et l'alimenter au quotidien, la rendre la plus nourrissante possible, et non que les bouses interventions avec monsieur le duc se sont parfaitement complétées. [01'49'25] Effectivement pas parents démissionnaires, mais souvent des parents désemparés. Nous rentrons, je n'arrive à leur donner les outils. A, effectivement, ils s'en emparent, ils arrivent, vous en êtes. [01'49'35] L'un des témoignages vivants, la capacité investir les des portes qui s'ouvrent et avec eux également, tout le travail que nous pouvons faire en tant que fédération parents d'élèves, via les conseillers locaux dans les établissements, et vous l'avez rappelé. Merci, elle est extrêmement utile. [01'49'51] L'importance de nourrir cette relation aujourd'hui, puisque dans le rapport à la formation et l'évaluation, on prépare aussi le la capacité pour les personnes en situation de travail d'une formation continue par la suite sur moi, ni à accepter ou à refuser ce dispositif de formation continue de reconversion, puisque ils ruinent timothy. [01'50'10] Richesse. J'étais parfaitement. [01'50'13] D'illustration. Si les métiers de demain n'existent pas encore pour nombre d'entre eux, dont il faudra effectivement que nous soyons et que nos enfants sont particulièrement en capacité de s'adapter à cette nouvelle donne du métier qui évolue, qui équivalent et évoluer. [01'50'28] L'importance également du projet d'établissement dont on ne cesse de dire qu'elle est au coeur. [01'50'33] De la réflexion et d'animation de la communauté éducative au sens large, mais dont on voit bien les difficultés que l'on a à faire vivre. Ou aujourd'hui, le une épice de denis par jean-louis leduc, est travailler en tant que fédération parents d'élèves sur les moments charnières clés de la scolarité, au niveau de la sixième niveau, plutôt de l'entrée au lycée. Quand vit-on la carapace? [01'50'53] Cité pour les fédérations et les parents d'élèves à nouer. [01'50'56] Des liens particuliers sur ces moments-clés communes, comme parcoursup d'ailleurs, mais bon, même si t'arrives en fin de parcours, les soins à donner les clés de compréhension, et donc, tony, des liens y a à rendre, fécond ces sites, chimie, ces temps d'échanges et donc de contact entre l'école, les enfants, tir y puis les parents, et [01'51'17] Voilà où on a noté tous ces éléments-là pour vous, diront aussi, est désigné comme tenu par aucun devoir de réserve, vous dire toutes les inquiétudes qu'on a sur la rentrée. Deux mille vingt trois, puisque, pour pouvoir accomplir tous ces, tous ces liens, tous ces temps d'échanges, dans toute cette animation d'une relation étroite, parents, enfants, écoles- [01'51'38] Il faut des enseignants présents, il faut des adultes investis et en capacité thermicien dans les établissements. [01'51'44] On va vous publier dans quelques instants, si vous n'avez pas encore signé, une pétition pour protéger l'école publique qui réunit dans elijah plus de soixante-dix mille signatures. On attend un mois. Un mois et demi plus tard a un très beau score pour, pour notre fédération. Et on ne compte pas s'en arrêter là, bien au contraire. On estime, on espère pouvoir. [01'52'04] Pour toucher les cent dix mille et aller au sénat ou à l'assemblée avec cette pétition. [01'52'10] Aidez-nous, et aidez-nous, pardon à à, à, à rendre publique, à diffuser le plus largement possible autour de vous cette pétition, parce que, avec la suppression de mille cinq cents postes à la rentrée prochaine, ça pose des questions très claires sur la capacité à avoir des adultes face aux élèves, dans de bonnes conditions de scolarité, faire parler du climat scolaire, parler du bien-être à l'école. [01'52'30] Paul parler. [01'52'31] De la capacité à être bien lancé dans sa scolarité. Ça touchait évidemment en premier lieu la question du moyen y a, de l'ambition qu'on l'on a pour l'école et pour ses enseignants, pour ses enfants et pour ses parents. Donc, n'hésitez pas, s'il vous plait, à diffuser cette pétition le plus largement possible. On a besoin de vous. [01'52'48] En tout cas, merci beaucoup pour la qualité des échanges avec nos deux intervenants ce soir là, la multitude des questions qu'on a essayé de relayer le plus largement possible. [01'52'58] Auprès de jean-louis duc et de jérémy fontana, eux. [01'53'03] Bien sûr ce webinaire et il sera visible dans quelques jours sur notre site internet comme la première partie. [01'53'09] On vous donne rendez-vous bientôt. Et la question du climat scolaire. [01'53'13] On va continuer à travailler avec si peu, puisque c'est le thème central de notre congrès de vierzon, qui se déroulera mi-juin. Donc, on va continuer de creuser ce sillon, notamment avec l'académie d'orléans qui a proposé un certain nombre d'outils très concrets et qui a beaucoup travaillé sur cette notion de climat scolaire et qui va même conduire à une généralisation. [01'53'33] Tion d'outils et de dispositifs. Mais ça, on en parlera au congrès de vierzon. [01'53'37] Qui nous accueillera. En tout cas, merci beaucoup. [01'53'40] On ne lâche pas ce qu'on fait, parce qu'on voit bien qu'aujourd'hui, dans le contexte de crises multiples que vivent nos enfants et que l'on vit nous-mêmes la question, l'école centrale, c'est un projet de société et donc on va continuer à s'y investir. Merci beaucoup, en tout cas, pour le les interventions des uns et des autres. On vous dit à très bientôt. Et puis, n'hésitez pas à nous alimenter. [01'54'00] Faire part de vos questionnements pour qu'on puisse s'en faire l'écho et continuer à faire des propositions concrètes pour améliorer la scolarisation des enfants et des adolescents et, surtout, à promouvoir et à défendre l'école publique. Merci beaucoup et à bientôt. [01'54'18] Oh.

Author Response

Responses to public reviews

Reviewer 1

We thank the reviewer for the valuable and constructive comments and are pleased that the re-viewer finds our study timely and our behavioral results clear.

1) The RSA basically asks on the lowest level, whether neural activation patterns (as measured by EEG) are more similar between linked events compared to non-linked events. At least this is the first question that should be asked. However, on page 11 the authors state: "We ex-amined insight-induced effects on neural representations for linked events [...]". Hence, the critical analysis reported in the manuscript fully ignores the non-linked events and their neu-ral activation patterns. However, the non-linked events are a critical control. If the reported effects do not differ between linked and non-linked events, there is no way to claim that the effects are due to experimental manipulation - neither imagination nor observation. Hence, instead of immediately reporting on group differences (sham vs. control) in a two-way in-teraction (pre vs. post X imagination vs. observation), the authors should check (and re-port) first, whether the critical experimental manipulation had any effect on the similarity of neural activation patterns in the first place.

We completely agree that the non-link items are a critical control. Therefore, we had reported not only the results for linked but also for non-linked events on page 15, lines 336-350. We clarified this important point now on page 12 lines 283-286:

“Subsequently, we examined insight-induced effects on neural representations for linked (vs. non-linked) events by comparing the change from pre- to post-insight (post-pre) and the difference between imagination and observation (imagination - observation) between cTBS and sham groups using an independent cluster-based permutation t-test.”

Moreover, to directly compare linked and non-linked events we performed a four-way in-teraction including link vs. non-link. This analysis yielded a significant four-way interaction, showing that the interaction of time (pre vs. post), mode of insight (imagination vs. obser-vation) and cTBS differed for linked vs. non-linked items. We then report the follow-up analyses, separately for linked and non-linked events. Please see pages 12-13, lines 287-294:

“First, we included the within-subject factors time (pre vs. post), mode of insight (imagina-tion vs. observation) and link (vs. non-link) by calculating the difference waves. Subse-quently we conducted a cluster-based permutation test comparing the cTBS and the sham groups. This analysis yielded a four-way interaction within a negative cluster in a fronto-temporal region (electrode: FT7; p = 0.007, ci-range = 0.00, SD = 0.00). This result indicates that the impact of cTBS over the angular gyrus on the neural pattern reconfiguration follow-ing imagination- vs. observation-based insight may differ between linked and non-linked events. For linked events, this analysis yielded a […]”

2) Overall, the focus on the targeted three-way interaction is poorly motivated. Also, a func-tional interpretation is largely missing.

In order to better explain our motivation for the three-way interaction, we em-phasized in the introduction the importance of disentangling potential differences due to the mode of insight, given the known role of the angular gyrus in imagination on pages 4-5, lines 107-115:

“Considering this involvement of the angular gyrus in imaginative processes, we expected that the effect of cTBS on the change in representational similarity from pre- to post-insight will differ based on the mode of insight – whether this insight was gained via imagination or observation. Specifically, we expected a more pronounced impairment in the neural recon-figurations when insight is gained via imagination, as this function may depend more on an-gular gyrus recruitment than insight gained via observation. Additionally, we expected cTBS to the left angular gyrus to interfere with the increase in neural similarity for linked events and with the decrease of neural similarity for non-linked event.”

As discussed on page 21 (starting from line 478; see also the intro on page 4), we expected that the angular gyrus would be particularly implicated in imagination-based insight, given its known role in imagination (e.g.: Thakral et al., 2017). Moreover, given the angular gyrus’s strong connectivity with other regions, the results observed may not be driven by this re-gion alone but also by interconnected regions, such as the hippocampus. We clarified these important points at the very end of the discussion on pages 23-24, lines 543-560:

“Furthermore, the differential impact of cTBS to the angular gyrus on neural reconfigura-tions between events linked via imagination and those linked via observation may be at-tributed to its crucial role in imaginative processes (Ramanan et al., 2018; Thakral et al., 2017). Another intriguing aspect to consider is that the stimulated site was situated in the more ventral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Uddin et al., 2010). This stronger connectivity between the ventral angular gyrus and the hippocampus may shed light on the greater impact of cTBS to the angular gyrus on im-agination-based insight. Given the angular gyrus’s robust connectivity with other brain re-gions, including the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also origi-nate from these interconnected regions. This notion may bear particular importance given the required accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the an-gular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gy-rus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014).”

3) "Interestingly, we observed a different pattern of insight-related representational pattern changes for non-linked events." It is not sufficient to demonstrate that a given effect is pre-sent in one condition (linked events) but not the other (non-linked events). To claim that there are actually different patterns, the authors would need to compare the critical condi-tions directly (Nieuwenhuis et al., 2011).

We completely agree and now compared the two conditions directly. Specifical-ly, we now report the significant four-way interaction, including the factor link vs. non-link, before delving into separate analyses for linked and non-linked events on pages 12-13, lines 287-294:

“First, we included the within-subject factors time (pre vs. post), mode of insight (imagina-tion vs. observation) and link (vs. non-link) by calculating the difference waves. Subse-quently we conducted a cluster-based permutation test comparing the cTBS and the sham groups. This analysis yielded a four-way interaction within a negative cluster in a fronto-temporal region (electrode: FT7; p = 0.007, ci-range = 0.00, SD = 0.00). This result indicates that the impact of cTBS over the angular gyrus on the neural pattern reconfiguration follow-ing imagination- vs. observation-based insight may differ between linked and non-linked events. For linked events, this analysis yielded a […]”

4) "This analysis yielded a negative cluster (p = 0.032, ci-range = 0.00, SD = 0.00) in the parieto-temporal region (electrodes: T7, Tp7, P7; Fig. 3B)." (p. 11). The authors report results with specificity for certain topographical locations. However, this is in stark contrast to the fact that the authors derived time X time RSA maps.

We did derive time × time similarity maps for each electrode within each partic-ipant, which allowed us to find a cluster consisting of specific electrodes. We apologize for not making this aspect clear enough and have, therefore, modified the respective part of our methods section on page 38, lines 951-952:

“In total, this analysis produced eight Representational Dissimilarity Matrices (RDMs) for each electrode and each participant.”

5) "These theta power values were then combined to create representational feature vectors, which consisted of the power values for four frequencies (4-7 Hz) × 41 time points (0-2 sec-onds) × 64 electrodes. We then calculated Pearson's correlations to compare the power pat-terns across theta frequency between the time points of linked events (A with B), as well as between the time points of non-linked events (A with X) for the pre- and the post-phase separately, separately for stories linked via imagination and via observation. To ensure un-biased results, we took precautions not to correlate the same combination of stories twice, which prevented potential inflation of the data. To facilitate statistical comparisons, we ap-plied a Fisher z-transform to the Pearson's rho values at each time point. This yielded a global measure of similarity on each electrode site. We, thus, obtained time × time similarity maps for the linked events (A and B) and the non-linked events (A and X) in the pre- and post-phases, separately for the insight gained through imagination and observation." (p. 34+35).

If RSA values were calculated at each time point and electrode, the Pearson correlations would have been computed effectively between four samples only, which is by far not enough to derive reliable estimates (Schönbrodt & Perugini, 2013). The problem is aggra-vated by the fact that due to the time and frequency smoothing inherent in the time-frequency decomposition of the EEG data, nearby power values across neighboring theta frequencies are highly similar to start with. (e.g., Schönauer et al., 2017; Sommer et al., 2022).

Alternative approaches would be to run the correlations across time for each electrode (re-sulting in the elimination of the time dimension) or to run the correlations at each time point across electrodes (resulting in the elimination of topographic specificity).

At least, the authors should show raw RSA maps for linked and non-linked events in the pre- and post-phases separately for the insight gained through imagination and observa-tion in each group, to allow for assessing the suitability of the input data (in the supple-ments?) before progressing to reporting the results of three-way interactions.

Although we do see the reviewer’s point, we think that an RSA specific to the theta range yielding electrode specific time × time similarity maps must be run this way, otherwise, as you pointed out, one or the other dimension is compromised. Running an RSA across time for each electrode will lead to computing a similarity measure between the events without information on when these stimuli become more or less similar, thereby ig-noring the temporal dynamics crucial to EEG data and not taking advantage of the high temporal resolution. Conversely, conducting an RSA across electrodes might result in an overall similarity measure per participant, disregarding the spatial distribution and potential variations among electrodes. Although EEG has limited spatial resolution, different elec-trodes can capture differences that may aid in understanding neural processing. However, as suggested by the reviewer, we included the raw RSA maps for linked and non-linked events separately for pre- and post-phases, imagination and observation and link and non-link in the supplement and refer to these data in the results section on pages 12-13, lines 293-295:

“For linked events, this analysis yielded a negative cluster (p = 0.032, ci-range = 0.00, SD = 0.00) in the parieto-temporal region (electrodes: T7, Tp7, P7; Fig. 3B; Figure 3 – Figure sup-plement 1).”

And on page 15, lines 339-341:

“This analysis yielded a positive cluster (p = 0.035, ci-range = 0.00, SD = 0.00) in a fronto-temporal region (electrode: FT7; Fig. 3C; Figure 3 – Figure supplement 2).”

Reviewer 2

We thank the reviewer for the very helpful and constructive comments and appreciate that the reviewer finds our study relevant to all areas of cognitive research.

1) While the observed memory reconfiguration/changes are attributed to the angular gyrus in this study, it remains unclear whether these effects are solely a result of the AG's role in re-configuration processes or to what extent the hippocampus might also mediate these memory effects (e.g., Tambini et al., 2018; Hermiller et al., 2019).

We agree that, in addition to the critical role of the angular gyrus, there may be an involvement of the hippocampus. We point now explicitly to the modulatory capacities of angular gyrus stimulation on the hippocampus. Please see page 4, lines 81-88:

“One promising candidate that may contribute to insight-driven memory reconfiguration is the angular gyrus. The angular gyrus has extensive structural and functional connections to many other brain regions (Petit et al., 2023), including the hippocampus (Coughlan et al., 2023; Uddin et al., 2010). Accordingly, previous studies have shown that stimulation of the angular gyrus resulted in altered hippocampal activity (Thakral et al., 2020; Wang et al., 2014). Furthermore, the angular gyrus has been implicated in a myriad of cognitive func-tions, including mental arithmetic, visuospatial processing, inhibitory control, and theory-of-mind (Cattaneo et al., 2009; Grabner et al., 2009; Lewis et al., 2019; Schurz et al., 2014).”

We further added a new paragraph to the discussion pointing at the possibility that not solely the angular gyrus but another brain region, such as the hippocampus, may have me-diated the changes observed in our study on pages 23-24, lines 546-562:

“Another intriguing aspect to consider is that the stimulated site was situated in the more ventral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Ud-din et al., 2010). This stronger connectivity between the ventral angular gyrus and the hip-pocampus may shed light on the greater impact of cTBS to the angular gyrus on imagination-based insight. Given the angular gyrus’s robust connectivity with other brain regions, includ-ing the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also originate from these interconnected regions. This notion may bear particular importance given the re-quired accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the angular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gyrus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus.”

2) Another weakness in this manuscript is the use of different groups of participants for the key TMS intervention, along with underspecified or incomplete hypotheses/predictions.

In our view, the chosen between-subjects design is to be preferred over a crossover design for several reasons. First, our choice aimed to eliminate potential se-quence effects that may have adversely affected performance in the narrative-insight task (NIT). Second, this approach ensured consistency in expectations regarding the story links while also mitigating potential differences induced by fatigue. Additionally, we accounted for the potential advantage of a within-subject design – the stimulation of the same brain – by utilizing neuro-navigated TMS for targeting the stimulation coordinate. Finally, it is im-portant to note that we measured the event representations pre- and post-insight and that also the mode of insight was manipulated within-subject. Thus, our design did include a within-subject component and we are convinced that the chosen paradigm balances the different strengths and weaknesses of within-subject and between-subjects designs in the best possible manner. We specified our rationale for choosing a between-subjects ap-proach in the introduction on page 5, lines 122-126:

“We intentionally adopted a mixed design, combining both between-subjects and within-subject methodologies. The between-subjects approach was chosen to minimize the risk of carry-over effects and sequence biases. Simultaneously, we capitalized on the advantages of a within-subject design by altering the pre- to post-insight comparison and the mode of insight (imagination vs. observation) within each participant.”

Moreover, to provide a comprehensive portrayal of the two groups, we incorporated de-scriptions concerning trait and state variables alongside age and motor thresholds and in-cluded t-test comparisons between these variables on page 7, lines 157-160:

“Notably, the groups did not differ on levels of subjective chronic stress (TICS), state and trait anxiety (STAI-S, STAI-T), depressive mood (BDI), imaginative capacities (FFIS), person-ality dimensions (BFI), age, and motor thresholds (for descriptive statistics see Table 1; all p > 0.053).”

And further included age and motor thresholds as control variables in Table 1 on page 18, lines 402-404:

“Overall, levels of subjective chronic stress, anxiety, and depressive mood were relatively low and not different between groups. The groups did further not differ in terms of per-sonality traits, imagination capacity, age or motor thresholds (all p > 0.053; see Table 1).”

For greater precision in outlining our hypotheses, we specified these at the end of the in-troduction on pages 4-55, lines 107-118:

“Considering this involvement of the angular gyrus in imaginative processes, we expected that the effect of cTBS on the change in representational similarity from pre- to post-insight will differ based on the mode of insight – whether this insight was gained via imagination or observation. Specifically, we expected a more pronounced impairment in the neural recon-figurations when insight is gained via imagination, as this function may depend more on an-gular gyrus recruitment than insight gained via observation. Additionally, we expected cTBS to the left angular gyrus to interfere with the increase in neural similarity for linked events and with the decrease of neural similarity for non-linked events. We further predicted that cTBS to the left angular gyrus would reduce the impact of (imagination-based) insight into the link of initially unrelated events on memory performance during free recall, given its higher variability compared to other memory measures.”

3) Furthermore, in some instances, the types of analyses used do not appear to be suitable for addressing the questions posed by the current study, and there is limited explanation pro-vided for the choice of analyses and questionnaires.

We addressed this concern by inserting a new section “control variables” in the methods explaining our rationale for employing the different questionnaires as control var-iables on pages 40-41, lines 1003-1019:

“Control variables In order to ensure that the observed effects were solely attributable to the TMS manipula-tion and not influenced by other factors, we comprehensively evaluated several trait and state variables. To account for potential variations in anxiety levels that could impact our re-sults, we specifically measured state and trait anxiety using STAI-S and STAI-T (Laux et al., 1981), thus minimizing the potential confounding effects of anxiety on our findings (Char-pentier et al., 2021). Additionally, we evaluated participants’ chronic stress levels using the TICS (Schulz & Schlotz, 1999) to exclude any group variations that might explain the effect on memory, cosidering the well-established impact of stress on memory (Sandi & Pinelo-Nava, 2007; Schwabe et al., 2012). Moreover, we assessed participants’ depressive symp-toms employing the BDI (Hautzinger et al., 2006), to guarantee group comparability on this clinical measure. We further assessed fundamental personality dimensions using the BFI-2 (Danner et al., 2016) to exclude any potential group discrepancies that could account for dif-ferences observed. Lastly, we assessed participants’ imaginative capacities using the FFIS (Zabelina & Condon, 2019), to ensure uniformity across groups regarding this central varia-ble, considering the significant role of imagination in relation to the cTBS-targeted angular gyrus (Thakral et al., 2017).”

We further specified why we chose to analyze our behavioral data using LMMs on page 34, lines 849-85:

“For our behavioral analyses we opted to employ linear-mixed models (LMM), given their high robustness regarding the underlying distribution and high sensitivity to individual varia-tion (Pinheiro & Bates, 2000; Schielzeth et al., 2020).”

Moreover, we added an explanation on why we opted for the RSA approach in the meth-ods section on page 37, lines 920-923:

“This method is ideally suited to measure neural representation changes and was specifical-ly chosen as it has been previously identified as the preferred approach for quantifying in-sight-induced neural changes (Grob et al., 2023b; Milivojevic et al., 2015).”

To clarify on the rationale behind our coherence analysis, we incorporated an explanatory sentence in the methods section on page 39, lines 966-967:

“Due to the robust connectivity between the angular gyrus and other brain regions (Petit et al., 2023; Seghier, 2013), we proceeded with a connectivity analysis as a next step.”

Reviewer 3

We thank the reviewer for the constructive and very helpful comments. We are pleased that the reviewer considered our experimental design to be strong and our behavioral results to be striking.

1) My major criticism relates to the main claim of the paper regarding causality between the angular gyrus and the authors' behavior of interest. Specifically, I am not convinced by the evidence that the effects of stimulation noted in the paper are attributable specifically to the angular gyrus, and not other regions/networks.

While our results showed specific changes after cTBS over the angular gyrus, demonstrating a causal involvement of the angular gyrus in these effects, we completely agree that this does not rule out an involvement of additional areas. In particular, there is evidence suggesting that cTBS over parietal regions, such as the angular gyrus, could poten-tially influence hippocampal functioning. We address this issue now in a new paragraph that we have added to the discussion, on pages 23-24, lines 546-564:

“Another intriguing aspect to consider is that the stimulated site was situated in the more ventral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Ud-din et al., 2010). This stronger connectivity between the ventral angular gyrus and the hip-pocampus may shed light on the greater impact of cTBS to the angular gyrus on imagination-based insight. Given the angular gyrus’s robust connectivity with other brain regions, includ-ing the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also originate from these interconnected regions. This notion may bear particular importance given the re-quired accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the angular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gyrus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus. Expanding upon this idea, it is conceivable that targeting a more dorsal segment of the angular gyrus might exert a stronger influence on observation-based linking – an aspect that warrants future in-vestigations.”

Responses to reviewer recommendations

Reviewer 1

1) On page 26, the authors write: "[...] different video events (A, B, and X) were recalled from day one [...]". I may have missed this point, but I had the impression that the task was con-ducted within one day.

Indeed, this study was conducted within a single day. We rephrased the respec-tive statement accordingly. Please see page 7, lines 149-153:

“To test this hypothesis and the causal role of the angular gyrus in insight-related memory reconfigurations, we combined the life-like video-based narrative-insight task (NIT) with representational similarity analysis of EEG data and (double-blind) neuro-navigated TMS over the left angular gyrus in a comprehensive investigation within a single day.”

We further included this information in the methods section on page 27, lines 634-635:

“In total, the experiment took about 4.5 hours per participant and was completed within a single day. ”

Reviewer 2

1) There is a substantial disconnection between the introduction and the methods/results sec-tion. One reason is that there is not sufficient detail regarding the hypotheses/predictions and the specific types of analyses chosen to test these hypotheses/predictions. Additionally, it is not explained what comparisons and outcomes would be informative/expected. This should be made clear. Second and related to the above, the rationale for conducting certain types of analyses (correlation, coherence, see below) sometimes is not specified.

To address this concern, we elaborated on our hypotheses incorporating specif-ic predictions for the free recall, given its higher variability than the other memory measures, and for imagination vs. observation at the end of the introduction on pages 4-5, lines 107-122:

“Considering this involvement of the angular gyrus in imaginative processes, we expected that the effect of cTBS on the change in representational similarity from pre- to post-insight will differ based on the mode of insight – whether this insight was gained via imagination or observation. Specifically, we expected a more pronounced impairment in the neural recon-figurations when insight is gained via imagination, as this function may depend more on an-gular gyrus recruitment than insight gained via observation. Additionally, we expected cTBS to the left angular gyrus to interfere with the increase in neural similarity for linked events and with the decrease of neural similarity for non-linked events. We further predicted that cTBS to the left angular gyrus would reduce the impact of (imagination-based) insight into the link of initially unrelated events on memory performance during free recall, given its higher variability compared to other memory measures. Considering the high connectivity profile of the angular gyrus within the brain (Seghier, 2013), we conducted an EEG connec-tivity analysis building upon prior findings concerning alterations in neural reconfigurations. To establish a link between neural and behavioral findings, we chose a correlational ap-proach to relate observations from these two domains.”

Moreover, we made our rationale for the employed analyses more explicit and specified why we chose to analyze our behavioral data using LMMs on page 34, lines 849-851:

“For our behavioral analyses we opted to employ linear-mixed models (LMM), given their high robustness regarding the underlying distribution and high sensitivity to individual varia-tion (Pinheiro & Bates, 2000; Schielzeth et al., 2020).”

Moreover, we added an explanation on why we opted for the RSA approach in the meth-ods section on page 37, lines 920-923:

“This method is ideally suited to measure neural representation changes and was specifical-ly chosen as it has been previously identified as the preferred approach for quantifying in-sight-induced neural changes (Grob et al., 2023b; Milivojevic et al., 2015).”

To clarify on the rationale behind our coherence analysis, we incorporated an explanatory sentence in the methods section on page 39, lines 966-967:

“Due to the robust connectivity between the angular gyrus and other brain regions (Petit et al., 2023; Seghier, 2013), we proceeded with a connectivity analysis as a next step.”

2) The authors suggest that besides Branzi et al. (2021), this is one of the first studies showing that memory update is linked to the AG. I suggest having a look at work from Tambini, Nee, & D'Esposito, 2018, JoCN, and other papers from Joel Voss' group that target a similar re-gion of AG/Inferior parietal cortex. Many studies, using multiple TMS protocols, have now shown this brain region is causally involved in episodic and associative memory encoding.

As mentioned above, further consideration of this literature is important as it delves into the region's hippocampal connectivity (and other network properties), and how that mediates the memory effects. Indeed because of the nature of the methods employed in this study, we do not know if the memory-related behavioural effects are due to TMS-changes induced at the AG's versus the hippocampal' s level, or both. How do the current findings square with the existing TMS effects from this region? Can the connectivity profile of the target re-gion highlighted by previous studies provide further insight into how the current behaviour-al effect arises? Some comments on this could be added to the discussion.

We completely agree that the other studies showing enhanced associative memory after TMS to parietal regions need to be addressed. Therefore, we updated the discussion on page 20, lines 449-453:

“Interestingly, recent work has additionally indicated that targeting parietal regions with TMS led to alterations in hippocampal functional connectivity, thereby enhancing associa-tive memory (Nilakantan et al., 2017; Tambini et al., 2018; Wang et al., 2014), potentially shedding light on the underlying mechanisms involved.”

Moreover, we included a section specifically addressing the possibility that the effects ob-served may pertain to having modulated other regions via the targeted region and updated the discussion on pages 23-24, lines 543-562:

“Furthermore, the differential impact of cTBS to the angular gyrus on neural reconfigura-tions between events linked via imagination and those linked via observation may be at-tributed to its crucial role in imaginative processes (Ramanan et al., 2018; Thakral et al., 2017). Another intriguing aspect to consider is that the stimulated site was situated in the more ventral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Uddin et al., 2010). This stronger connectivity between the ventral angular gyrus and the hippocampus may shed light on the greater impact of cTBS to the angular gyrus on im-agination-based insight. Given the angular gyrus’s robust connectivity with other brain re-gions, including the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also origi-nate from these interconnected regions. This notion may bear particular importance given the required accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the an-gular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gy-rus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus.”

3) Another comment I have regards the results observed for the observation vs imagination insight conditions. The authors mention that the 'changes in representational similarity for the observation condition should be interpreted with caution, as these seemingly opposite changes appeared to be at least in part driven by group differences already in the pre-phase before participants gained insight.' I wonder what these group differences are and whether the authors have any hypothesis about what factors determined them.

We could only speculate about the basis of the observed pre-insight phase dif-ferences. However, we provide now the raw RSA data as supplemental material to make the pattern of the (raw) RSA findings in the pre- and post-insight phases more transparent. We refer the interested reader to this material on pages 12-13, lines 293 to 295:

“For linked events, this analysis yielded a negative cluster (p = 0.032, ci-range = 0.00, SD = 0.00) in the parieto-temporal region (electrodes: T7, Tp7, P7; Fig. 3B; Figure 3 – Figure sup-plement 1).”

And on page 15, lines 339-341:

“This analysis yielded a positive cluster (p = 0.035, ci-range = 0.00, SD = 0.00) in a fronto-temporal region (electrode: FT7; Fig. 3C; Figure 3 – Figure supplement 2).”

Furthermore, the age of participants is not reported separately for the two groups (cTBS to AG vs Sham), I think. This should be reported including a t-test showing that the two groups have the same age.

We agree and report now explicitly that groups did not significantly differ in rel-evant control variables including age. Please see page 7, lines 157-160:

“Notably, the groups did not differ on levels of subjective chronic stress (TICS), state and trait anxiety (STAI-S, STAI-T), depressive mood (BDI), imaginative capacities (FFIS), person-ality dimensions (BFI), age, and motor thresholds (for descriptive statistics see Table 1; all p > 0.053).”

And further included age and motor thresholds as control variables in Table 1 on page 18, lines 402-412:

“Overall, levels of subjective chronic stress, anxiety, and depressive mood were relatively low and not different between groups. The groups did further not differ in terms of per-sonality traits, imagination capacity, age or motor thresholds (all p > 0.053; see Table 1).”

The fact this study is not a within-subject design makes difficult the interpretation of the results and this should be recognised as an important limitation of the study.

As outlined above, a within-subject design would in our view come with several disadvantages, such as significant sequence/carry-over effects. Moreover, the neural rep-resentation change was measured in a pre-post design, enabling us to measure the insight-driven neural reconfiguration at the individual level.

We clarify our rationale for the between-subjects factor TMS in the introduction on page 5, lines 122-126:

“We intentionally adopted a mixed design, combining both between-subjects and within-subject methodologies. The between-subjects approach was chosen to minimize the risk of carry-over effects and sequence biases. Simultaneously, we capitalized on the advantages of a within-subject design by altering the pre- to post-insight comparison and the mode of insight (imagination vs. observation) within each participant.”

Furthermore, we included our rationale for choosing a between-subjects approach for the crucial TMS manipulation in the methods section on page 25, lines 601-604:

“We implemented a mixed-design including the within-subject factors link (linked vs. non-linked events), session (pre- vs. post-link), and mode (imagination vs. observation) as well as the between-subjects factor group (cTBS to the angular gyrus vs. sham) to mitigate the risk of carry-over effects and sequence biases of the crucial cTBS manipulation.”

4) The angular gyrus is a heterogeneous region with multiple graded subregions. The one tar-geted in the present study is the ventral AG which has strong connections with the episodic-hippocampal memory system. I was wondering if this might explain why the AG TMS ef-fects on representational changes have been observed for events linked via imagination but not direct observation. Perhaps the stimulation of a more 'visual' AG subregion (see Hum-phreys et al., 2020, Cerebral Cortex) would have resulted in a different (opposite) pattern of results. It would be good to add some comments on this in the discussion.

We appreciate this interesting perspective offered regarding the potential out-comes of our study, particularly in relation to the activation of a more ventral sub region of the angular gyrus. We incorporated this idea into our discussion, alongside considerations regarding the potential effects of a more dorsal angular gyrus stimulation on observation-based linking. However, caution is warranted recognizing the inherent limitations posed by the precision of TMS manipulations, which is further underscored by our electric field simu-lations, utilizing a 10 mm radius. We included this section in the discussion on pages 23-24, lines 546-569:

“Another intriguing aspect to consider is that the stimulated site was situated in the more ventral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Ud-din et al., 2010). This stronger connectivity between the ventral angular gyrus and the hip-pocampus may shed light on the greater impact of cTBS to the angular gyrus on imagina-tion-based insight. Given the angular gyrus’s robust connectivity with other brain regions, including the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also originate from these interconnected regions. This notion may bear particular importance given the re-quired accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the angular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gyrus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus. Expanding upon this idea, it is conceivable that targeting a more dorsal segment of the angular gyrus might exert a stronger influence on observation-based linking – an aspect that warrants future in-vestigations. Yet, while acknowledging the functional heterogeneity within the angular gy-rus (Humphreys et al., 2020), pinpointing specific sub regions via TMS remains challenging due to its limited focal precision at the millimeter level (Deng et al., 2013; Thielscher & Kammer, 2004), as reinforced by our electric field simulations utilizing a 10 mm radius. Hence, drawing definitive conclusions regarding distinct angular gyrus sub regions requires future research employing rigorous checks to assess the focality of their stimulation.”

5) Regarding the methods section, I have the following specific queries. It is unclear what is the purpose of the coherence and correlation analyses (pages 35, 36). Could the authors pro-vide further clarification on this? These analyses seem not to be mentioned anywhere in the introduction. This should be clarified briefly in the introduction and then in the methods sec-tion. The same for the questionnaires (anxiety, stress, etc): It is unclear the reason for col-lecting this type of data. This should be clarified in the introduction as well.

We agree, and have updated the introduction as follows on page 5, lines 118-122:

“Considering the high connectivity profile of the angular gyrus within the brain (Seghier, 2013), we conducted an EEG connectivity analysis building upon findings from the RSA anal-yses concerning alterations in neural reconfigurations. To establish a link between neural and behavioral findings, we chose a correlational approach to relate observations from these two domains.”

We additionally provided an explanation for including these questionnaires in the introduc-tion on page 5, lines 126-129:

“To control for any group differences beyond the TMS manipulation, we gathered various control variables through questionnaires, including trait- and state-anxiety, depressive symptoms, chronic stress levels, personality dimensions, and imaginative capacities.”

Moreover, we elaborated on the underlying rationale guiding our chosen analytical ap-proaches. Therefore, we specified why we chose to analyze our behavioral data using LMMs on page 34, lines 849-851:

“For our behavioral analyses we opted to employ linear-mixed models (LMM), given their high robustness regarding the underlying distribution and high sensitivity to individual varia-tion (Pinheiro & Bates, 2000; Schielzeth et al., 2020).”

Furthermore, we added an explanation on why we opted for the RSA approach in the methods section on page 37, lines 920-923:

“This method is ideally suited to measure neural representation changes and was specifical-ly chosen as it has been previously identified as the preferred approach for quantifying in-sight-induced neural changes (Grob et al., 2023b; Milivojevic et al., 2015).”

To clarify on the rationale behind our coherence analysis, we incorporated an explanatory sentence in the methods section on page 39, lines 966-967:

“Due to the robust connectivity between the angular gyrus and other brain regions (Petit et al., 2023; Seghier, 2013), we proceeded with a connectivity analysis as a next step.”

6) The preregistration webpage is in German. This is not ideal as it means that the information is available only to German speakers.

This webpage can easily be switched to English by changing the settings in the top right corner:

To address this issue, we included a description of how to set the webpage to English in the methods section on page 25, lines 581-582:

“For translation to English, please adjust the page settings located in the top right corner.”

7) Page 18. 'NIT' and 'MAT' - avoid abbreviations when possible.

We included the full name for the narrative-insight task (NIT) on page 7, line 151, line 153, and line 165, page 8 lines 177-178 and line 187, page 19 on line 427, page 26 on line 615, line 629 and line 632, page 27, line 653, page 30, lines 730-731, page 31, line 754, page 35, line 870, line 873, and page 36 and line 885.

We further included the full name for the multi-arrangements task (MAT) on page 19, lines 428-429.

8) Line 21....we further observed DECREASED...should be replaced with INCREASED, if I am not wrong.

We checked the sentence again and it looks correct to us, since it describes the change for observation-based insight, not imagination-based insight. We clarified that this finding pertains to observation-based linking by modifying the sentence on page 23, lines 525-528, as follows:

“Following cTBS to the angular gyrus, we further observed decreased pattern similarity for non-linked events in the observation-based condition, resembling the pattern change ob-served in the sham group for linked events, which may highlight the role of the angular gy-rus in representational separation during observation-based linking”

Reviewer 3

1) The major claim of the paper is that the angular gyrus is causally involved in insight-driven memory reconfiguration. To the authors' credit, they localized stimulation to the angular gyrus using an anatomical scan, the strength of the estimated electromagnetic field in the angular gyrus correlated with their behavioral results, and there were also brain-behavior correlations involving sensors located in the parietal lobe. However, the minimum evidence needed to claim causality is 1) evidence of a behavioral change (which the authors found) and 2) evidence of target engagement in the angular gyrus. It is also important to show brain-behavior correlations between target engagement and behavior. Although the au-thors stimulated the angular gyrus, that does not mean that rTMS specifically affected this region or that the behavioral results can be attributed to rTMS effects on the angular gyrus. As the authors point out, the angular gyrus has dense connections with other regions such as the hippocampus. In fact, several studies have shown that angular gyrus (or near AG) stimulation affects the hippocampal network (Wang et al., 2014, Science; Freedberg et al. 2019, eNeuro; Thakral et al., 2020, PNAS). EEG also has a poor spatial resolution, so even though the results were attributable to parieto-temporal sensors, this is not sufficient evi-dence to claim that the angular gyrus was modulated. Source localization would be re-quired to reconstruct the signal specifically from the AG. Thus, with the manuscript written as is, the authors can claim that "cTBS to the angular gyrus modulates insight-driven memory reconfiguration," but the current claim is not sufficiently substantiated.

While acknowledging the potential role of the angular gyrus in driving the ob-served changes, we recognize that the available evidence may not be sufficient. Conse-quently, we have introduced several modifications within our manuscript to address this concern.

In the revised Introduction, we now explicitly address the possibility of a stimulation of the hippocampus via the angular gyrus on page 4, lines 84-85:

“Accordingly, previous studies have shown that stimulation of the angular gyrus resulted in altered hippocampal activity (Thakral et al., 2020; Wang et al., 2014).”

Additionally, we included relevant evidence demonstrating previous instances of targeted stimulation of the angular gyrus, which led to alterations in hippocampal connectivity and associative memory. These insights have been included in the discussion on page 20, lines 449-453:

“Interestingly, recent work has additionally indicated that targeting parietal regions with TMS led to alterations in hippocampal functional connectivity, thereby enhancing associa-tive memory (Nilakantan et al., 2017; Tambini et al., 2018; Wang et al., 2014), potentially shedding light on the underlying mechanisms involved.”

Next, we have integrated crucial modifications essential for establishing a conclusive infer-ence of causality in our study. Moreover, we now explore the potential mediation of the effects observed from angular gyrus stimulation through other brain regions, like the hip-pocampus. In addition, we have highlighted prior work where such stimulation coincided with alterations in associative memory. For the updated discussion section, please see pag-es 23-24, lines 538-562:

“Although our study provided evidence suggesting a causal role of the angular gyrus in in-sight-driven memory reconfigurations – highlighted by behavioral changes after cTBS to the angular gyrus, neural changes in left parietal regions, and relevant brain-behavior associa-tions – it is important to acknowledge the limitations imposed by the spatial resolution of EEG. Consequently, the precise source of the observed signal changes in the parietal re-gions remains uncertain, potentially tempering the definitive nature of these findings. Fur-thermore, the differential impact of cTBS to the angular gyrus on neural reconfigurations between events linked via imagination and those linked via observation may be attributed to its crucial role in imaginative processes (Ramanan et al., 2018; Thakral et al., 2017). An-other intriguing aspect to consider is that the stimulated site was situated in the more ven-tral portion of the angular gyrus, recognized for its stronger connectivity to the episodic hippocampal memory system in contrast to its more dorsal counterpart (Seghier, 2013; Ud-din et al., 2010). This stronger connectivity between the ventral angular gyrus and the hip-pocampus may shed light on the greater impact of cTBS to the angular gyrus on imagina-tion-based insight. Given the angular gyrus’s robust connectivity with other brain regions, including the hippocampus (Seghier, 2013), it is plausible that the observed changes might not solely stem from alterations within the angular gyrus itself, but could also originate from these interconnected regions. This notion may bear particular importance given the re-quired accessibility to the hippocampus during imaginative processes (Benoit & Schacter, 2015; Grob et al., 2023a; Zeidman & Maguire, 2016). Interactions between the angular gyrus and the hippocampus may give rise to rich memory representations (Ramanan et al., 2018). In line with this, recent studies have demonstrated that cTBS to the angular gyrus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus.”

We further replaced terms that imply inhibition of the angular gyrus with a more operation-ally descriptive phrase:

“cTBS to the angular gyrus”

2) The authors frequently claim that cTBS is "inhibitory stimulation" and that inhibition of the angular gyrus caused their effects. There is a common misconception within the cognitive neuroscience literature that stimulation is either "inhibitory" or "excitatory," but there is no such thing as either. The effects of rTMS are dependent on many physiological, state, and trait-specific variables and the location of stimulation. For example, while cTBS does repro-ducibly inhibit behavior supported by the motor cortex (Wilkinson et al., 2010, Cortex; Rosenthal et al., 2009, J Neurosci), cTBS of the posterior parietal cortex reproducibly en-hances hippocampal network functional connectivity and episodic memory (Hermiller et al., 2019, Hippocampus; Hermiller et al., 2020, J Neurosci). The authors reference the Huang et al. (2005) paper as evidence of its inhibitory effects but work in this paper is not sufficient to broadly categorize cTBS as inhibitory. First, Huang et al. stimulated the motor cortex and measured the effects on corticospinal excitability, which is significantly different from what the current authors are measuring. Furthermore, this oft-cited study only included 9 sub-jects. Other studies have found that the effects of theta-burst are significantly more varia-ble when more subjects are used. For example, intermittent theta-burst, which is assumed to be excitatory based on the Huang paper, was found to produce unreliable excitatory ef-fects when more subjects were examined (Lopez-Alonso, 2014, Brain Stimulation). Thus, the a priori assumption that stimulation would be inhibitory is weak and cTBS should not be dis-cussed as "inhibitory."

We agree and included now a statement in the methods section that explicitly states that cTBS effects may be region-specific on page 33, lines 817-819:

“Nonetheless, the effects of cTBS appear to vary based on the targeted region, with cTBS to parietal regions demonstrating the capability to enhance hippocampal connectivity (Hermiller et al., 2019, 2020).”

We further substituted all terminology suggestive of an inhibitory effect with the phrase:

“cTBS to the angular gyrus”.

However, it is important to note, that while other studies (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014) found increased hippocampal connectivity after rTMS to a parie-tal region as well as enhanced associative memory, we observed impaired memory for the linked events. We included this clarification in the discussion on page 24, lines 558-562:

“In line with this, recent studies have demonstrated that cTBS to the angular gyrus resulted in enhanced hippocampal connectivity and improved associative memory (Hermiller et al., 2019; Tambini et al., 2018; Wang et al., 2014). However, it should be noted that our study detected impaired associative memory following cTBS to the angular gyrus.”

3) The hypothesis at the end of the introduction did not strike me as entirely clear. From this hypothesis, it seems that the authors are just comparing the differences in memory and re-configuration during imagination-based insight links. However, the authors also include ob-servation-based links and a non-linking condition, which seem ancillary to the main hy-pothesis. Thus, I am confused about why these extra factors were included and exactly what statistical results would confirm the authors' hypothesis.

We agree, and have clarified our hypotheses on pages 4-5, lines 107-115:

“Considering this involvement of the angular gyrus in imaginative processes, we expected that the effect of cTBS on the change in representational similarity from pre- to post-insight will differ based on the mode of insight – whether this insight was gained via imagination or observation. Specifically, we expected a more pronounced impairment in the neural recon-figurations when insight is gained via imagination, as this function may depend more on an-gular gyrus recruitment than insight gained via observation. Additionally, we expected cTBS to the left angular gyrus to reduce the increase in neural similarity for linked events and in-crease of neural dissimilarity for non-linked events.”

4) Many of the distributions throughout the paper do not look normal. Was normality checked? Are non-parametric stats warranted?

We evaluated and reported the normality assumption in our behavioral anal-yses. Despite the non-normal distribution of our data, we chose to utilize linear-mixed models due to their robust performance even in case of deviations from normal distribu-tions. This update in our methods section can be found on page 36, lines 890-896:

“After outlier correction, we identified non-normality in our data using a Shapiro-Wilk test (narrative-insight task: W = 0.92, p < 0.001; multi-arrangements task: W = 0.94, p < 0.001; forced-choice recognition: W = 0.50, p < 0.001; free recall details: W = 0.85, p < 0.001; free recall naming of linking events: W = 0.94, p < 0.001). However, we mitigated this by employ-ing linear-mixed models (LMMs), recognized for their robustness even with non-normally distributed data (Schielzeth et al., 2020).”

We recalculated the correlational analysis between the RSA data and the behavioral recall of linking events by using the Spearman method on page 13, lines 306-308:

“Furthermore, to address a deviation from the normality assumption, the correlational analysis was repeated using the Spearman method, which indicated an even stronger cor-relation (r(59) = 0.32, p = 0.012).”

We further recalculated the correlation between the change in coherence for linked events and the recall of details for events linked via imagination on page 16, lines 376-378:

“Please note that for addressing a deviation from the normality assumption, the correla-tional analysis was repeated using the Spearman method, which yielded a significant corre-lation of similar strength (r(59) = 0.31, p = 0.015).”

Our EEG analyses , including RSA and coherence analyses, utilized a cluster-based permuta-tion test (Fieldtrip; Oostenveld et al., 2011). These tests do not assume a normal distribu-tion by utilizing empirical sampling for statistical inference. This approach ensures robust-ness without constraints imposed by specific distributional assumptions. Subsequent t-tests, stemming from significant clusters identified in the initial non-parametric analyses, were extensions of the robust non-parametric approach and did not require additional normality testing.

5) Can the authors include more detail about the sham coil? Was it subthreshold? Did the EMF cross the skull?

The sham coil, also obtained from MAG & More GmbH, München, Germany, provided a similar sensory experience; however, the company did not specify any field strength (n.a.) as this coil was purposefully designed to prevent the induction of an elec-tromagnetic field (EMF) capable of penetrating the skull, thereby ensuring it had no impact on the brain. We clarified on this point in the methods section on pages 31-32, lines 772-778:

“Two identically looking but different 70 mm figure-of-eight-shaped coils were used de-pending on the TMS condition: The PMD70-pCool coil (MAG & More GmbH, München, Germany) with a 2T maximum field strength was used for cTBS, while the PMD70-pCool-SHAM coil (MAG & More GmbH, München, Germany), with minimal magnetic field strength, was employed for sham, providing a similar sensory experience, with stimulation pulses being scattered over the scalp and not penetrating the skull.”

6) There are differences between exclusion criteria in pre-registration and report. For example, BMI is an exclusion factor in the report, but not in the pre-registration. Can the authors provide a reason for this deviation?

This discrepancy is due to (partial) participant recruitment from previous fMRI studies conducted in our lab that involved a stress induction protocol (as a structural MRI image was needed for the ‘neuronavigated’ TMS). Owing to the distinct cortisol stress reac-tivity observed in individuals with varying body mass indices (BMIs), participants with a BMI below 19 or above 26 kg/m² were excluded from these studies. To maintain consistency within our sample, only participants meeting these criteria were included. We elaborated on this point in the methods section on page 25, lines 586-592:

“Participants were screened using a standardized interview for exclusion criteria that com-prised a history of neurological and psychiatric disease, medication use and substance abuse, cardiovascular, thyroid, or renal disease, evidence of COVID-19 infection or expo-sure, and any contraindications to MRI examination or TMS. Additionally, participants with a body mass index (BMI) below 19 or above 26 kg/m² were excluded. This decision stemmed from recruiting some participants from prior studies that incorporated stress induction pro-tocols, which imposed this specific criterion (Herhaus & Petrowski, 2018; Schmalbach et al., 2020).”

7) Were impedances monitored and minimized during EEG?

Yes, they were monitored. We clarified this point in the methods section on page 34, lines 845-847:

“We maintained impedances within a range of ± 20 μV using the common mode sense (CMS) and driven right leg (DRL) electrodes, serving as active reference and ground, re-spectively”

8) I think there may be a typo related to the Thakral coordinates. I believe Thakral used MNI coordinates -48,-64, 30, whereas the authors stated they used -48,-67,30. Is this a mistake?

Upon reevaluation of our study coordinates, we identified a slight deviation in our stimulation coordinates compared to those reported by Thakral et al. (2017; +3mm on the y-axis). This variance resulted from the required MNI to Talairach (TAL) transformations necessary for utilizing the neuronavigation software Powermag View! (MAG & More GmbH, München, Germany). Notably, this deviation was consistent across all participants in our study. While TMS is more precise than tDCS, its focality is not as fine-grained down to the millimeter level. Despite this, our electric field simulations, adopting a 10mm radius, ef-fectively encompassed the original coordinates specified by Thakral et al. (2017). This radius ensured coverage over the intended target area, mitigating the impact of this minor devia-tion on the overall study outcomes. We updated the methods section accordingly on page 33, lines 800-806:

“Based on the individual T1 MR images, we created 3D reconstructions of the participants' heads, allowing us to precisely locate the left angular gyrus coordinate (MNI: -48, -67, 30), initially derived from previous work (Thakral et al., 2017), for TMS stimulation. Despite a mi-nor deviation in coordinates due to necessary MNI to Talairach transformations for soft-ware compatibility (Powermag View! by MAG & More GmbH, München, Germany), our methodology ensured precise localization of the angular gyrus target area.”

9) How was the tail of the coil positioned during stimulation? Was it individualized so that the lobes of the coil are perpendicular to the nearest gyrus, as is commonly done?

The coil handle always pointed upwards to maintain optimal positioning with the coil holder. We followed the positioning procedure in the neuronavigation software Powermag View!, which did not indicate any positioning of the coil handle but specified the position and angle of the coil itself. To incorporate this aspect, we updated the legend of figure 2 on page 11, lines 260-261:

“Please note that in the study, the coil handle was oriented upwards; however, in this illus-tration, it has been intentionally depicted as pointing downwards for better visibility pur-poses.”

We further updated the method section on page 33, lines 723-824:

“The coil was positioned tangentially on the head and mechanically fixed in a coil holder, with its handle pointing upwards to maintain its position”

I believe that most student feel this way. They want to be able to tell themselves what to do and make their own rules, so that way whenever they break their rules they're able to take full responsibility for it instead of taking responsibilities for the rules that the teacher provided for them.

Children learn morals through socialization

Obsidian for Academic Publishing - A Walkthrough with Jason Yuh (4)

Al interior de su nota de capítulo, Jason hace resúmenes (algo sumamente importante) y divide en headers cada subcapítulo, junto con el número de la página.

Dentro de cada header, están en guiones (bullets) el número de la página seguido de un breve resumen o nota rápida – apuntes de lo que le interesa, un poco à la Luhmann.

En el número de página utiliza la "f", que en castellano sería la "s", de following o subsecuente.

En cada número(s) de página(s), Jason dice que se ha entrenado para atrapar el argumento principal o la premisa de la sección, para hacerlo en niveles de párrafos, y que cuando a veces no es disciplinado, lo hace al nivel de la sección, o al menos al nivel del capítulo.

Jason también utiliza apuntes al estilo: "skimmed mostly from 36-46". Para dejar en claro la huella de lo que leyó, pero también de lo que no leyó.

Tiene una nota "global" llamada "Abstracts", donde están todos los resúmenes de todos los artículos que ha leído.

Porque Jason hace abstracts de cada artículo que lee. Si hay uno ya hecho, lo copia y pega... Pero escribe siempre su propio resumen, en sus propias palabras, para retener mejor lo que leyó.

Su principal enfoque al momento de hacer un abstract:

• Cuáles son la conclusiones principales.
• Cuáles son las premisas principales que apoyan la conclusión.
• Cómo el argumento se "intersect" con las preguntas de investigación.

Sobre el tercer punto, es importante que abajo del abstract siempre pone un code-block (o cualquier cosa que sea visual funciona, que cambie el color y llame la atención), donde escribe cómo se relaciona el abstract con su investigación.

Jason dice que seguramente olvidará el artículo en pocas horas... Pero que el code-block le ayuda a recordar por qué ese artículo es importante para él y para su investigación.

Utiliza también la opción de "embedding" para poner los abstracts + code-blocks de cada capítulo en su nota principal del libro.

¿Cómo, en fin, Jason hace sus abstracts? Pregunta Anthony.

Jason intenta escribirlo sin mirar a las notas, solo de la memoria, para resumir todo lo que acaba de leer en un solo párrafo. Y después de escribir, revisa, sección por sección, para ver si se olvidó de algo o si escribió mal algo. Edita su abstract, elimina, añade y modifica cosas...

le caratteristiche principali sono l'uso delle forme più basilari dei verbi in questo caso all'infinito, negazione espressa solamente con il pas dopo il verbo, annullamento delle forme di genere e numeriche, uso abbondante del verbo gagner e dell'espressione y a. là usato largamente come dimostrativo, con soppressione di a e de. La pronuncia cambia da tribù a tribù.

Author Response

Thank you for your thorough critique and thoughtful suggestions for improving our manuscript, "Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.” The reviewers’ detailed comments suggest that showing multiple types of graphs to demonstrate the presence of divergent scaling of mEPSC amplitudes in cultures from Rab3A wild type, and its disruption in cultures from Rab3A knockout mice, had the unintended consequence of obscuring the major results of our study. Furthermore, our proposal that the difference in characteristics of scaling of GluA2 receptor expression compared to that of mEPSC amplitudes, based on the ratio plots, indicated that a mechanism other than postsynaptic receptors likely contributes to the homeostatic increase in mEPSC amplitude was not convincing to the reviewers. Reviewers 2 and 3 point out these results might be explained by differences in the limitations and artifacts of the two very distinct techniques, electrophysiology and fluorescence imaging. In the revision we will acknowledge that a greater variability in the signal, or, more issues with signal over noise, might be present in imaging experiments compared to electrophysiology. This could explain the lack of identical effects on GluA2 receptors compared to mEPSC amplitudes in the matched experiments, but we maintain it is also possible that a greater variability in GluA2 responses is biologically meaningful. Further, an issue with the accuracy of imaging experiments to report the true receptor effects would also call into question the conclusion that receptors always increase after activity blockade. Finally, the graphs illustrating the detailed characteristics of scaling with rank order and ratio plots required pooling multiple samples per cell, which precludes application of standard statistical methods to determine whether effects or differences reach statistical significance. Therefore, we will remove the cumulative distribution functions, rank order plots, and ratio plots, and show only analyses that involve a single sample per cell. This major change will simplify and clarify the main findings, that homeostatic plasticity of both mEPSC amplitude and GluA2 receptor expression in mouse cortical cultures involves the synaptic vesicle protein Rab3A operating in neurons rather than astrocytes. We will focus our comparison between mEPSC amplitudes and receptors in the same cultures to differences between the magnitude of effects on the mean or median, and will make clear that overall, our data can be explained by two possibilities: 1) the presynaptic vesicle protein is acting via regulation of postsynaptic receptors alone, or, it is regulating both postsynaptic receptors and another contributor to mEPSC amplitude, possibly amount of transmitter released by a single vesicle. Either way, it is very surprising that this presynaptic protein is involved in postsynaptic changes, so our results represent a novel contribution to the field of homeostatic plasticity. In sum, the changes we propose should go a long way towards addressing the majority of the reviewers’ major critiques.

A related issue raised by the reviewers was that the model describing potential presynaptic mechanisms of Rab3A in homeostatic plasticity was not supported by direct evidence (Figure 10). We meant the model to introduce the possibility of a presynaptic contribution to mEPSC amplitude and to stimulate future research, but clearly did not communicate its speculative nature, neither in the Figure legend nor in our discussion of potential mechanisms. In the revision, we will restrict the model to the direct findings in this study. Additionally, we will state where appropriate, that while previous findings at the mouse NMJ are consistent with a presynaptic role for Rab3A (Wang et al., 2011), in the current study there is no direct evidence for this idea in cortical cultures other than the quantitative differences in the fold increases in mEPSC amplitudes and GluA2 receptors which were assayed in the same cultures.

We will submit a revised version addressing each of the reviewer’s concerns and suggestions as described above and below; these major modifications will greatly improve the readability of the manuscript and clarify the main results.

Reviewer #1

Koesters and colleagues investigated the role of the presynaptic small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed an increase in GluA2 puncta size and intensity in wild type, but not Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which presynaptic Rab3A is required for homeostatic scaling of synaptic transmission through GluA2-dependent and independent mechanisms.

While the title of the manuscript is mostly supported by data of solid quality, many conclusions, as well as the final model, cannot be derived from the results presented. Importantly, the results do not indicate that Rab3A modulates quantal size on both sides of the synapse. Moreover, several analysis approaches seem inappropriate.

The following points should be addressed:

1) The model shown in Figure 10 is not supported by the data. The authors neither provide evidence for two different functional states of Rab3A being involved in mEPSC amplitude modulation, nor for a change in glutamate content of vesicles. Furthermore, the data do not fully support the conclusion of a presynaptic role for Rab3A in homeostatic scaling.

We will revise the model, removing presynaptic mechanisms for Rab3A and restricting it to the direct findings in this study.

2) The analysis of mEPSC data using quantile sampling followed by ratio calculation is not meaningful under the tested experimental conditions because of the following reasons:

(i) The analysis implicitly assumes that all events have been detected. The prominent mEPSC frequency increase after TTX suggests that this is not the case, i.e., many (small) mEPSCs are likely missed under control conditions.

We explicitly addressed the potential contribution of missed mEPSCs that are below threshold in (Hanes et al., 2020). We found that even simulating a threshold of 7 pA, applied to data artificially modified by uniformly multiplying the control data set, did not generate a ratio plot with the increasing ratio over 75% of the data that we observe in the experimental data. Overall, the findings from simulating a threshold and a uniform multiplicative factor illustrate that the threshold issue does not cause major changes to the data. Furthermore, in cultures from Rab3A+/+ mice from the Rab3AEbd/+ colony, the mEPSC amplitudes were significantly smaller than those recorded in cultures from Rab3A+/+ mice from the Rab3A+/- colony (lines 327-329, 11 pa vs 13 pA), indicating that if there were smaller mEPSCs occurring in the Rab3A+/+ data set, we would have detected them. Although for these reasons we feel it is unlikely our ratio plot analysis is invalid, to clarify the result that homeostatic plasticity of mEPSC amplitude requires functioning Rab3A, we will remove the ratio plots.

(ii) The analysis is used to conclude how events of a certain size are altered by TTX treatment. However, this analysis compares the smallest mEPSCs of the TTX condition with the smallest control mEPSCs, but this is not a pre-post experimental design. Variation between cells and between coverslips will markedly affect the results and lead to misleading interpretations.

The rank order plot is a well-established plot to examine the mathematical transformation caused by homeostatic plasticity, first used in (Turrigiano et al., 1998). We included it here to facilitate comparison of our findings with previous results. We introduced the ratio plot in (Hanes et al., 2020), finding it shows more clearly differences occurring in the range of small mEPSC values. The reviewer is correct in that we are assuming the smallest mEPSCs before treatment should be matched with the smallest mEPSCs after treatment. It is almost impossible to do a pre-post experimental design for mEPSCs. Even when applying a treatment, for example acute perfusion with a receptor antagonist, to a single cell and recording mEPSCs before and after the treatment, it is not a true pre-post design at the level of mEPSC amplitudes, which come from many different inputs. The power of the method is that different characteristic mathematical transformations for different experimental conditions (e.g., genotype or activity protocol) support the idea that those conditions either involve different mechanisms or have altered the mechanism. Such differences might be missed by only comparing means or medians. However, we found no evidence that loss of Rab3A or expression of the Rab3A Earlybird mutant altered the mathematical transformation due to homeostatic plasticity, other than to reduce its magnitude across all amplitudes. Therefore, including these complex analyses is not adding anything to the finding that Rab3A plays a role in homeostatic plasticity of mEPSC amplitudes and they will be removed in the revision.

(iii) The ratio (TTX/control) vs. control plots seem to suffer from a division by small value artifact (see Figure 6F).

The reviewer is referring to findings on the ratio plot for receptor cluster area. Because the large ratios for the smallest control areas occur in the cultures prepared from wild type mice, and to a much lower extent in cultures prepared from Rab3A knockout mice, we think there is a biologically relevant increase in the TTX/CON ratio, since an artifact due to division by small values should be present in both data sets. However, we cannot rule out that the differences in ratio plot behavior between receptors and mEPSC amplitudes result from the different limitations in detection of receptor clusters vs. the limits of detection of mEPSCs, so we will remove the ratio plots and focus on comparison of means or medians.

Correspondingly, ratio-analysis differs considerably for different control conditions (Fig. 1Giii, Fig. 2Giii, Fig. 6C, Fig. 9A).

The reviewer is correct to point out that the ratio plot shows differences across control conditions (note, these differences are not obvious with the more standard rank order plot). The magnitude of the 50th percentile ratio differs across control conditions, and behaviors of the largest mEPSCs also differ, with some ratios going down at the highest control amplitudes (1Giii, 6C), and others continuing to increase with increasing control amplitude (2Giii, 9A). They all share the divergent increasing ratio from smallest mEPSC amplitude to around the 20 pA level. We attribute the differences in magnitude to the differences in experimental conditions: 1Giii is Rab3A+/+ from the +/+ colony; 1Giii is Rab3A+/+ from the Ebd/+ colony; 6C is a set of Rab3A+/+ cultures assayed several years after the set in 1Giii; 9A is a different culture condition altogether, with neurons being plated onto an already formed bed of astrocytes. Effects on the largest mEPSCs are likely attributable to the small number and high variability of amplitudes in this range. Since the variability in the very sensitive ratio plot have taken away from the main findings of homeostatic plasticity being disrupted in the absence of functioning Rab3A in neurons, we will remove the rank-order and the ratio plots from the manuscript.

3) As noted by the authors in a previous publication (Hanes et al. 2020), statistical analysis of CDFs suffers from ninflation. In addition, the quantile sampling method chosen violates an important assumption of the K-S test. Indeed, pvalues for these comparisons are typically several orders of magnitude smaller. Given that the statistical N most likely corresponds to the number of cultures (see, e.g., https://doi.org/10.1371/journal.pbio.2005282), CDF comparisons are not informative and should thus not be used to draw conclusions from the data. The plots can be informative, though.

As the reviewer acknowledges, we were very careful in (Hanes et al., 2020) to state that the p values could not be used to determine significance in the KS test of cumulative distributions for pooled data because the KS test assumes a single sample per cell. We also suggested in that study that the p values could be used in a comparative way for looking at data sets with similar (inflated) n values to say something about bigger or smaller differences. We failed to reiterate those caveats here. In reviewing the article “What is N” by (Lazic et al., 2018) (which we very much appreciate being shown by the reviewer), we agree that in the current study where we are attempting to show how the effect of homeostatic plasticity is or is not altered by loss of Rab3A function, it is imperative that we be able to make conclusions about statistical significance. The pooling approach is essential for having some sense of the mEPSC amplitude distributions, but that is not necessary for looking at the effect of Rab3A. Therefore, we will remove all analyses that involve pooling of multiple mEPSC amplitudes per cell.

4) How does recoding noise and the mEPSC amplitude threshold affect "divergent scaling"?

We addressed this in our 2020 paper (Hanes et al., 2020) where we showed that the experimental homeostatic increase in mEPSC amplitude cannot be simulated with uniform, multiplicative synaptic scaling whether we included or excluded distortion caused by a detection threshold.

5) What is the justification for the line fits of the ratio data/how was the fit range chosen?

We are assuming the reviewer is referring to the line fits for the rank-order data. If so, the fit range is the entire range of the data. This issue will be addressed by the removal of the rank-order plots from the manuscript.

6) TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data.

Based on the p-values for comparison of means with a Kruskal-Wallis test, we would argue that TTX application does not show a significant increase in mEPSC amplitude in Rab3A-/- neurons (Figure 1 p-value = .318; Figure 9 p-value = .125) when comparing to untreated control mEPSC amplitude means. It is only when we use the KS test and the inflated n’s that we get a barely significant results, p = 0.042. Based on the Lazic article (Lazic et al., 2018), we would now conclude that we cannot use the KS p value in that analysis. We have tried to be clear that the effect of TTX application on mEPSC amplitude in Rab3A-/- neurons is not completely abolished, but rather is dramatically reduced, which we acknowledge in the manuscript (line 279). This issue will be addressed by removal of CDFs from the manuscript.

7) Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.

Although we feel that comparison of magnitude of effects can be stated in a qualitative way for similar sized pooled data sets with larger or smaller p-values, we agree that statistical significance has no meaning. This issue will be addressed by removing the CDF plots from the manuscript.

8) There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3Aebd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+, and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?

We have acknowledged in the manuscript that one explanation for a failure to exhibit homeostatic plasticity in the cultures from Rab3A Earlybird mutant mice is that the already large basal amplitude occludes any further increase (line 366). In the revision we will make sure the occlusion possibility is highlighted, but we will also discuss other proteins that have been implicated in homeostatic plasticity that have caused an increase in mEPSC amplitude and/or AMPA receptors at baseline, for example, Arc/Arg3.1 KO (Shepherd et al., 2006; Beique et al., 2011); Homer KO (Hu et al., 2010) and inhibition of mir-186-5p (Silva et al., 2019).

9) Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs. 15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.

We will acknowledge that Ca2+-permeable AMPARs could be contributing to the frequency increase following activity blockade and will also include analyses of frequency throughout the manuscript.

10) The authors discuss previous papers showing changes in VGLUT1 intensity. Was VGLUT intensity altered in the stainings presented in the manuscript?

We will perform analyses VGLUT1 intensity and include them in the manuscript.

11) The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?

The changes in GluA2 integrals look exactly like the changes in cluster size and were not included to simplify the results. But with the removal of the CDFs, rank order, and ratio plots, we can easily include integral measurements. What we did not observe was an additive effect with intensity and size such that the effects on integral were of greater magnitude or statistical significance than either alone. We will include the integral plots in the revised manuscript.

12) The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold?

What was the rationale for a threshold of 400 a.u. or 450 a.u.?

We have acquired AOIs of receptor clusters at multiple threshold levels, and can examine whether the results are altered when using a low, medium or high threshold level.

How does this threshold compare to the mEPSC threshold of 3 pA?

The issue with values being below threshold in untreated cultures has been the concern in interpreting effects on mEPSC amplitudes, specifically, whether this mismatch contributes to divergent scaling. A problem of values being below a toohighly set threshold in the control and becoming detectable after the homeostatic plasticity produces a lower ratio than expected, because now there are values in the treated condition that were not present in the control condition. Instead, for GluA2 receptor cluster size, we observed higher TTX/CON ratios at the low end of the data set. So, based on this, the thresholds chosen for imaging are not having the same effect, if that is what is being asked. This issue will be addressed by removing ratio plots.

The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size. (ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 18-20%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)?

We agree that our data show GluA2 receptors increase as based on cluster size, and did not mean to imply otherwise. Our conclusion that there is another contributor to mEPSC amplitude other than receptors is based on two main findings, 1) that the ratio plots for mEPSC amplitudes and receptor cluster size have distinctively different behaviors, and 2) that there are differences in either magnitude or direction of the TTX effect across 6 matched cultures, 3 from WT animals and 3 from TTX animals (see more explanation of this point below, in response to Reviewer 3). To our knowledge, no one has reported homeostatic plasticity effects on a culture by culture basis, and no one has compared imaging results and physiological results for the same cultures. We will remove the ratio plots and the conclusions based on the differences in behavior for mEPSC amplitudes and receptor cluster size. We will acknowledge in the revision that the differences in magnitude and direction across the 6 matched cultures could be due to the differences in limitations and artifacts of imaging fluorescent antibody staining vs. the limitations and artifacts of detecting mEPSCs electrophysiologically. However, we will continue to state that our results could also be due to the possibility that mEPSC amplitude is not changing in lockstep with receptor levels in every situation. To support this proposal, we will discuss those articles that include both measurements, and point out where mEPSC amplitude measurements and receptor levels match and where they do not.

Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors.

We will add the caveat that antibody labeling efficiency can vary between coverslips. Although we prepared single solutions that were applied to all coverslips in an experiment, this was not possible for the primary antibody to GluA2, which was added to live cultures in individual wells.(iv) It is not entirely clear if their imaging experiments will sample from all synapses. We will add to Materials and Methods that we sample from all the synapses that could be detected by the researcher on the primary dendrite of the pyramidal cell.

Other AMPAR subtypes than GluA2 could contribute, as could kainate or NMDA receptors.

This is true, other AMPARs (GluA3 and/or GluA4) could be contributing, but we only looked at the receptors well established to be contributing to homeostatic plasticity (GluA1 and GluA2). We will acknowledge the possible contribution of other AMPARs in the revised manuscript.

Furthermore, the statement "complete lack of correspondence of TTX/CON ratios" is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/- , the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2), particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.

We will remove the ratio plots from the manuscript and the arguments about differences between GluA2 receptors and mEPSC amplitudes that were based on them. However, we maintain that we have demonstrated a lack of consistent effect for GluA2 receptors and mEPSCs in the matched culture experiments. Yes, the readout of homeostatic plasticity in ratio plots for mEPSCs in the Rab3AKO reach over 1.1 in Figure 1, and as high a 1.2 in the cultures where Rab3AKO neurons were plated on Rab3AWT glia (Figure 9). Our point is that if we had measured GluA2 receptor responses to TTX in those same experiments, the ratios should have been above 1. However, in the experiments in which we measured both mEPSCs and GluA2 receptors, the ratios do not match. In culture #1, the ratio for mEPSCs was at 1 for more than 50% of the data, but for GluA2 receptors, was below 1 for more than 50% of the data. In culture #3, the ratio for mEPSCs was below 1 for more than 50% of the data, but for GluA2 receptors was close to 1.2 for 50% of the data. Only for culture #2 do the ratios appear to match. In the revised manuscript, the evidence that GluA2 receptors and mEPSCs are not changing in parallel will be based on the behavior of means or medians in untreated vs TTXtreated cultures, rather than ratio plots. It could be argued that we need a greater number of matched experiments to make conclusions, but the whole point of a matched experiment is that it should always show the same result—we are no longer dealing with the variability in the homeostatic plasticity itself. We will add a statement that the only three explanations left for the failure of mEPSC amplitudes and GluA2 receptors to change in parallel are 1) a true mismatch, 2) a sampling issue, or 3) technical artifacts that occur in one culture and not another.

13) Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?

Figure 7A depicts the pooled data that are shown separately for 3 cultures in Figure 8. We observed mEPSC amplitudes being smaller after TTX treatment in some range of the data for all three Rab3AKO cultures, suggesting that this may be a biological result rather than random variation around no change (which would be a ratio of 1). However, this effect is not significant at the level of means, nor in the KS test (which has the issue of inflated n in any case), so we did not highlight this point. This issue will be addressed by the removal of the CDF plots from the manuscript.

Reviewer #2

Technical concerns:

1) The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006).

The (Watt et al., 2000) study recorded mEPSCs in 0 Mg2+ (Figure 1). The (Sutton et al., 2006) study also shows an average mEPSC waveform (Figure 1D) that was recorded from in 0 Mg2+. Our extracellular recording solution contains Mg2+ (1.3 mM) so we likely are not observing NMDA-mediated currents because they are blocked with Mg2+ when strong depolarizations are prevented with TTX in the recording solution. We will add the idea that the NMDA currents are blocked by Mg2+ to Material and Methods.

It is important to ensure there is enough spiking activity before doing any activity manipulation.

We agree that it would be best if network spiking activity were monitored alongside mEPSC recordings, for example by culturing on multi-electrode arrays. Data from these measurements might explain culture to culture variability in homeostatic responses. To our knowledge, most other studies investigating homeostatic plasticity do not monitor network spiking activity in the same cultures that assay mEPSC amplitudes. This is something that the field should move towards. We will add the caveat that activity was not directly measured to the manuscript.

Similarly, it is also unknown whether spiking activity is normal in Rab3A KO/Ebd neurons.

Since we did not measure spiking activity, we cannot address whether the disruption in homeostatic plasticity in cultures prepared from Rab3A KO and Rab3AEbd/Ebd mutant mice is due to an alteration in network activity. If activity were already low in cultures prepared from these genetically altered mice, we would expect mEPSC amplitudes to be increased, compared to those measured in cultures from WT animals. That is not the case in cultures from Rab3A KO mice, so it is unlikely that network activity is reduced. However, mEPSC amplitudes are increased in Rab3AEbd/Ebd cultures, leaving open this possibility. It would have to be a defect unique to neurons in culture, since the Rab3AEbd/Ebd mouse appears normal in every way, suggesting action potential activity is occurring in the brains of these animals in vivo. We will add the possibility that activity is altered in the cultures from Rab3AKO and Rab3AEbd/Ebd to the manuscript.

2) Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.

MiniAnalysis (a standard program used for mEPSC event detection and analysis) selects many false positives with the automated feature (due to the very small sizes of events that are close to the noise level) so manual re-evaluation of the automated process is necessary to eliminate false positives. As soon as there is a manual step, bias is introduced. Interestingly, a manual reevaluation step was applied in a recent study that describes their process as ‘unbiased” (Wu et al., 2020). The alternative is to apply a very large threshold, reducing or eliminating false positives. However, this has the effect of biasing the data towards large events. In sum, we do not believe it is currently possible to perform a completely unbiased detection process. We feel that it is important to include as many small events as possible to reduce the problem of having events in the TTX experimental group that were not matched by events in the control experimental group, for the rank order and ratio plots, so setting the threshold low and manually detecting events accomplishes this. We will add to the Materials and Methods section that the person selecting events did not have information on whether the record was from an untreated or a TTX-treated cell at the time of selection. All of these issues, the potential for skewing the CDFs, and bias potentially interfering in the true rank order and ratio relationships, are addressed by removal of the CDFs, ratio and rank-order plots from the manuscript.

3) Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapses on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.

MAP2, in addition to labeling dendrites, also labels the cell body, and we used the cell structure revealed by MAP2 staining to select pyramidal-shaped neurons. The selection of the primary dendrite of a pyramidal neuron was stated in lines 239-240 in Materials and Methods and lines 1094 in the figure legend, but we had not explicitly stated how we knew it was a pyramidal neuron. We will include a low power picture of each of the selected pyramidal neurons in the revision.

Conceptual concerns:

The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The author claims that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study's contribution to the field is very limited.

We acknowledge that a presynaptic mechanism is involved in the regulation of homeostatic plasticity by Rab3A is not supported by direct evidence in cortical cultures in this study. But we disagree that the study’s contribution is very limited.

The revised manuscript will emphasize that there are only two possible mechanisms by which Rab3A is acting in homeostatic plasticity. Either this presynaptic vesicle protein is regulating postsynaptic receptors (an extremely surprising result for which we do have direct evidence), or, it is regulating quantal size from both sides of the synapse (supported by direct evidence from our previous study at the mouse neuromuscular junction in vivo, where receptors are not being upregulated during homeostatic plasticity, and, by indirect evidence in the current study, that receptors and mEPSCs are not being identically regulated in the same cultures). Furthermore, the first idea that follows from the effect of Rab3A on receptors is that it would be regulating release of factors from astrocytes, since this is a mechanism that has been shown to be involved in homeostatic plasticity, and we clearly disprove this hypothesis.

1) Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019).

We agree with the reviewer that many studies show an increase in receptors and mEPSC amplitudes after activity blockade. This is why we were very surprised in our initial experiments to find that there was not a consistent robust increase in receptors in our cultures. At that point we were only imaging, and we assumed that it was homeostatic plasticity that was not always robust. We decided it was essential to measure mEPSC amplitudes and image receptors in the same cultures. We expected to observe larger and smaller effects on mEPSC amplitudes from culture to culture that were paralleled by larger and smaller effects on receptors, but this is not what happened. We have gone back to the literature to look more closely at whether variability across cultures has ever been shown for mEPSC amplitudes, receptors, or both. In a survey of 14 studies, none report results culture by culture. To our knowledge, we are the first to report this variability in the receptor response, and the lack of correlation between mEPSC amplitudes and receptor responses, in the same cultures. That said, for the 4 examples provided by the reviewer, only 1 reports evidence relevant to our study that receptors and mEPSC amplitudes ‘occur side by side,’ which is the (Ibata et al., 2008) study. Here, 24 hr of TTX treatment of rat cortical cultures causes synaptically localized GluA2 receptors in confocal imaging, and mEPSC amplitudes, to both increase to around 130%. The (Pozo et al., 2012) study is not a study of activity blockade but of the effects of overexpressing beta-integrins in rat hippocampal cultures, and this causes both GluA2 receptors and mEPSC amplitudes to increase, but the GluA2 level is not restricted to synaptic sites, and, is expressed as the surface fraction (surface receptor/total receptor—total receptor being surface intensity plus internalized intensity) which increases from 0.5 to 0.55, or to 110%, while mEPSC amplitude increases to ~180%. The (Tan et al., 2015) study only provides Western blot data to show an increase of receptors to 125% in mouse cortical cultures in response to 48 hr TTX, with mEPSC amplitudes increased to ~140%, but the Western blot technique measures synaptic and nonsynaptic receptors on excitatory and inhibitory neurons, as well as receptors on astrocytes. Finally, in (Silva et al., 2019), the culture conditions for the imaging data and the mEPSC amplitude data are markedly different, with ‘low-density’ Banker cultures being used for the former, and ‘high-density’ cultures used for the latter, and the protocol to induce activity blockade is different from ours (noncompetitive AMPA and NMDA blockers); synaptic GluA2 receptors are increased to ~280% and mEPSC amplitudes to ~170%. In the revision we will carefully summarize the previous evidence for receptors and mEPSC amplitude responses to activity blockade. Since it is known that different protocols trigger different molecular mechanisms, for example, TTX + APV triggers a homeostatic plasticity that can be completely reversed by acute application of blockers of Ca-permeable receptors, whereas TTX alone triggers a plasticity that is insensitive to these blockers (Sutton et al., 2006), Figure 4E; (Soden and Chen, 2010); Figure 4A), we will keep our discussion restricted to studies using TTX alone for at least 24 hr. We will acknowledge that our finding that GluA2 receptors and mEPSC amplitudes are not varying in lockstep from culture to culture suggests there is another contributor to mEPSC amplitude, but that we cannot rule out it is due to a greater variability in signal, or more issues with signal over noise, in imaging experiments compared to electrophysiology experiments.

Studies surveyed about reporting results by culture:

(Ju et al., 2004; Stellwagen et al., 2005; Shepherd et al., 2006; Sutton et al., 2006; Cingolani and Goda, 2008; Hou et al., 2008; Ibata et al., 2008; Chang et al., 2010; Hu et al., 2010; Jakawich et al., 2010; Beique et al., 2011; Tatavarty et al., 2013; Diering et al., 2014; Sanderson et al., 2018)

Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.

We agree with the reviewer that there is no way to compare absolute levels from one type of experimental technique to another, but whatever differences in technical issues there are for the two techniques, they should cause systemic errors and should not contribute to the differences between experiments. Most of the issues with imaging come down to variability in the intensity of fluorescence from experiment to experiment, since the antibody solutions are made anew each time, as is the fixation solution. In addition, the confocal microscope function can vary over time and give brighter or dimmer images. But those kinds of artifacts are addressed by using the same solutions on control and TTX-treated coverslips, and imaging control and TTX-treated coverslips in the same single 2-3 hour imaging session, so that whatever issues there are, they cannot contribute to the TTX effect itself. Therefore when we compare the TTX effect (TTX measurements compared to untreated measurements) from culture to culture and find that in one WT culture there was no increase in receptors but there was in mEPSC amplitude, it is difficult to explain how a limitation specific to the antibody imaging technique could produce such a result. Similarly, when we get the opposite result, that in one KO culture, receptors increased but mEPSC amplitudes did not, it is unclear how limitations in signal detection would produce such a result in one culture but not another. The one exception to this is that the primary GluA2 antibody has to be added individually to each coverslip before returning the dishes to the incubator in order to avoid the disruption to live cells that a complete removal of media would have had. The only remaining ‘artifact’ that could explain the results would be a greater variability in the imaging experiments due to limitations in the signal or the signal to noise ratio. In the revision we will report additional characteristics of imaging experiments, such as average intensity for each coverslip, and for each experiment, to address whether variability in fluorescence levels could explain the variability in TTX effects we observe. We will include the possibility that the mismatches in GluA2 receptors and mEPSCs could be caused by greater variability in the imaging experiments.

2) The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.

In the revision, the model will focus on the direct findings of the manuscript and tone down the speculation about BDNF signaling, but in the Discussion we will add the possibility that a Rab3A-BDNF interaction could occur either presynaptically or postsynaptically. Interestingly, these articles suggest the postsynaptic BDNF is affecting presynaptic function, namely mEPSC frequency. It is conceivable it could presynaptically affect the vesicle’s release of transmitter.

3) The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the Naspm wash-in experiments clearly show that GluA1 homomer contributions have not changed before and after TTX treatment.

Our apologies to the reviewer that we were not clear on this point. In lines 396 to 400 we were describing the significant effects that NASPM had on mEPSC frequency on both untreated and TTX-treated cells, despite having only modest, and not quite significant effects on mEPSC amplitude. We conclude from these results that there are synaptic sites that have only GluA1 homomers, and the mEPSCs from these sites are blocked 100% by NASPM. There may be an increase in such GluA1-only synapses after activity blockade, but nevertheless, these events do not contribute to the amplitude increase. So we did not mean to suggest that there is a shift from Glua2 containing to GluA1 containing receptors that leads to the amplitude increase and fully agree with the reviewer that the GluA1 homomer contributions to amplitude have not changed before and after TTX. We will clarify the difference between the contribution of GluA1 homomers to amplitude and frequency in the revised manuscript.

Reviewer #3

Summary: The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength seems already elevated. In this context, it is unclear if the plasticity is absent or just occluded by a ceiling effect due the synapses already being strengthened. The authors do appropriately discuss both options. There are also differences in genetic background between the Rab3A KO and Earlybird mutants that could also impact the results, which are also noted. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between synaptic strength during HSP and AMPA receptor trafficking, and conclude that trafficking is largely not responsible for the changes in synaptic strength.

Strengths: This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms.

Weaknesses: However, the rather strong conclusions on the dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. The key issue is the GluA2 immunostaining in comparison with the mESPC recordings. Their imaging method involves only assessing puncta clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, judging from the sample micrographs (Fig 5). To my knowledge, this is a new and unvalidated approach that could represent a particular subset of synapses not representative of the synapses contributing to the mEPSC change. (they are also sampling different neurons for the two measurements; an additional unknown detail is how far from the cell body were the analyzed dendrites for immunostaining. While the authors acknowledge that a sampling issue could explain the data, they still use this data to draw strong conclusions about the lack of AMPAR trafficking contribution to the mEPSC amplitude change. This apparent difference may be a methodological issue rather than a biological one, and at this point it is impossible to differentiate these. It will unfortunately be difficult to validate their approach. Perhaps if they were to drive NMDA-dependent LTD or chemLTP, and show alignment of the imaging and ephys, that would help. More helpful would be recordings and imaging from the same neurons but this is challenging. Sampling from identified synapses would of course be ideal, perhaps from 2P uncaging combined with SEP-labeled AMPARs, but this is more challenging still. But without data to validate the method, it seems unwarranted to make such strong conclusions such as that AMPAR trafficking does not underlie the increase in mEPSC amplitude, given the previous data supporting such a model.

We chose the primary dendrite to ensure we were not assaying dendrites from inhibitory neurons or on axons, but we will add in the revision that it is a limitation of our methods that we are not sampling all the synapses for each neuron. The majority of previous studies that establish that receptors are increased side by side with mEPSCs did not measure receptors and mEPSCs in the same cells, nor even in the same cultures. There is a recent study which employs dual recordings, transfection of GluA2 and VGlut1 constructs, and infusion of dyes to highlight cell morphology (Letellier et al., 2019), so in principle an experiment could be done in which synaptic GluA2 sites are imaged in a cell in which the mEPSCs are also measured. It would be difficult to make these measurements in the same cells before and after TTX treatment, since there is a high likelihood of damaging the cell upon electrode withdrawal and with the imaging process itself. In theory, only a few such experiments would be necessary to establish whether receptors and mEPSC amplitudes are varying in lockstep, and we will consider this for a future study. As stated in response to conceptual concern #1 in Reviewer 2’s comments, we will review the literature on previous studies’ demonstrations of increases in receptors and mEPSC amplitudes following activity blockade in more detail, including how the synaptic sites to be imaged were chosen, to address whether our selection of sites touching the primary dendrite is unvalidated.

A sample from 3 articles:

(Ibata et al., 2008), only information is that ‘distal dendrites’ were examined. The authors do not use a dendritic label. (Jakawich et al., 2010), ‘neurons with pyramidal-like morphology were selected for imaging,’ and ‘principal dendrite of each neuron was linearized’—but how these were identified is not clear, since MAP2 or other cellular labels are not described.

(Silva et al., 2019), ‘dendrites with similar thickness and appearance were randomly selected using MAP2 staining,’ which suggests synaptic sites with GluA2 and VGLUT1 were selected on the basis of being close to or touching the MAP2 positive dendrite, although this is not stated explicitly.

We can perform length measurements on the dendrites imaged and report this information in the revision, but the primary dendrite is the closest dendrite to the cell body.

We have addressed the potential contribution of technical artifacts arising from the two distinct methods of measurement, imaging and electrophysiology, in our response to conceptual concern #1 of Reviewer 2.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is quite unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. It is also unclear why the authors argue this proves that the NASPM was at an effective concentration (lines 399-400).

We observed a clear effect of NASPM reducing mEPSC frequency. We will state more clearly that we infer from the loss of mEPSCs after NASPM that such mEPSCs were from synaptic sites that had only GluA1 homomers, and acknowledge that this is an interpretation. We will also clarify that if our inference is correct, it would indicate that the dose of NASPM we used was 100% effective at blocking GluA1 homomers. The alternative explanation would be a presynaptic effect of NASPM, which has never been reported, to our knowledge.

Further, the amplitude data show a strong trend towards smaller amplitude. The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. And the decrease is larger in the TTX neurons. Considering the strong claims for a pre-synaptic and the use of this data to justify only looking at GluA2 by immunostaining, these data do not offer much support of the conclusions. Between the sampling issues and perhaps looking at the wrong GluA subunit, it seems premature to argue that trafficking is not a contributor to the mEPSC amplitude change, especially given the substantial support for that hypothesis. Further, even if trafficking is not the major contributor, there could be shifts in conductance (perhaps due to regulation of auxiliary subunits) that does not necessitate a pre-synaptic locus. While the authors are free to hypothesize such a mechanism, it would be prudent to acknowledge other options and explanations.

We did not mean to suggest that there is no effect of NASPM on mEPSC amplitude. We will clarify that our data indicate that there is no effect of NASPM on the TTX effect on mEPSC amplitude. We agree with the reviewer that the effect of NASPM on frequency is of larger magnitude after TTX treatment, although the p value is larger than that for untreated cells, likely due to greater variability. We interpret this to mean that TTX treatment increases the proportion of synapses that have only GluA1 homomers. Nevertheless, the increase in GluA1 homomer sites does not appear to contribute to the overall increase in amplitude following TTX treatment, and we wanted to find the mechanism of the amplitude increase. That is why we focused on GluA2 receptors. We will acknowledge the limitation of basing our conclusions on only GluA2 receptors in the revision, as well as the possibility that there is a change in conductance. As stated in our response to Reviewer 2, we do not mean to state that GluA2 receptors do not go up after activity blockade, we find that this is the case. We are proposing an additional mechanism contributing to mEPSC amplitude to explain the different responses for GluA2 receptors vs. mEPSC amplitudes in some of the 6 matched experiments (3 WT and 3 KO).

The frequency data are missing from the paper, with the exception of the NASPM dataset. The mEPSC frequencies should be reported for all experiments, particularly given that Rab3A is generally viewed as a pre-synaptic protein regulating release. Also, in the NASPM experiments, the average frequency is much higher in the TTX treated cultures. Is this statistically above control values?

We will report frequency measurements for all experiments shown. Following TTX treatment, frequency variability increases enormously, with cells having as high as > 10 mEPSCs per second, and other TTX-treated cells with frequencies as low as < 1 mEPSC per second, so the TTX effect on frequency, and whether this effect is present or not in Rab3A KO and Rab3AEbd/Ebd is not completely clear, which is why we did not include those results previously.

Unaddressed issues that would greatly increase the impact of the paper:

1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role (and particularly the hypothesized and somewhat novel idea that the amount of glutamate released per vesicle is altered in HSP). They could use sparse knock-down of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

We agree with the reviewer that this is the most important question to answer next. The approach suggested by the reviewer would be to record from Rab3A KO neurons in a culture where the majority of its inputs are Rab3A positive. If the TTX effect is absent from these cells, it would strongly indicate that postsynaptic Rab3A is required for homeostatic plasticity. There are not currently transgenic mice expressing GFP forms of Rab3A, so we would have to create one, or, transiently transfect Rab3A-GFP into Rab3AKO neurons. Given that under our experimental conditions, we require a very high density of neurons to observe the increase in mEPSC amplitude, it would be difficult to get the ratio of Rab3A-expressing neurons high enough using transfection to be sure that a given postsynaptic cell lacking Rab3A had a normal number of Rab3A-positive inputs and almost no Rab3A-negative inputs. It may be that the opposite experiment is more doable—an isolated Rab3A-positive neuron in a sea of Rab3A-negative neurons, which could be accomplished with a very low transfection efficiency. Another approach would be to use the fast off rate antagonist gamma-DGG, which is more effective against low glutamate concentrations than high glutamate concentrations (see (Liu et al., 1999; Wu et al., 2007). If gamma-DGG were less effective at reducing mEPSC amplitude in TTX-treated cells, compared to untreated cells, it would support the hypothesis that activity blockade leads to an increase in the amount of transmitter per vesicle fusion event. Further, if the change in gamma-DGG sensitivity after activity blockade were disrupted in cultures from Rab3A KO cells, it would support a presynaptic role for Rab3A in homeostatic plasticity of mEPSC amplitude. We have begun these experiments but are finding the surprising result that within a single recording, small mEPSCs and large mEPSCs appear to be differentially sensitive to gamma-DGG. To confirm that this is a biological characteristic, rather than an issue with the detection threshold, we will be repeating our experiments with a slow off rate antagonist that has same effect regardless of transmitter concentration. The complexity of these results precludes including them in the current manuscript.

2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs and/or a decrease of GABA-packaging in vesicles (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at these synapses.

The next question, after it is determined where Rab3A is acting, is whether it is required for other forms of homeostatic plasticity. This includes plasticity of GABA mIPSCs on pyramidal neurons, but also mEPSCs on inhibitory neurons, and, the downscaling of mEPSCs (and upscaling of mIPSCs) when activity is increased, by bicuculline for example. We will add a statement about future experiments examining other forms of plasticity to the discussion, and include examples where a molecular mechanism has mediated multiple forms, and those that have been shown to be very specific.

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Reviewer #1 (Public Review):

Koesters and colleagues investigated the role of the presynaptic small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed an increase in GluA2 puncta size and intensity in wild type, but not Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which presynaptic Rab3A is required for homeostatic scaling of synaptic transmission through GluA2-dependent and independent mechanisms.

While the title of the manuscript is mostly supported by data of solid quality, many conclusions, as well as the final model, cannot be derived from the results presented. Importantly, the results do not indicate that Rab3A modulates quantal size on both sides of the synapse. Moreover, several analysis approaches seem inappropriate.

The following points should be addressed:

1. The model shown in Figure 10 is not supported by the data. The authors neither provide evidence for two different functional states of Rab3A being involved in mEPSC amplitude modulation, nor for a change in glutamate content of vesicles. Furthermore, the data do not fully support the conclusion of a presynaptic role for Rab3A in homeostatic scaling.<br /> 2. The analysis of mEPSC data using quantile sampling followed by ratio calculation is not meaningful under the tested experimental conditions because of the following reasons: (i) The analysis implicitly assumes that all events have been detected. The prominent mEPSC frequency increase after TTX suggests that this is not the case, i.e., many (small) mEPSCs are likely missed under control conditions. (ii) The analysis is used to conclude how events of a certain size are altered by TTX treatment. However, this analysis compares the smallest mEPSCs of the TTX condition with the smallest control mEPSCs, but this is not a pre-post experimental design. Variation between cells and between coverslips will markedly affect the results and lead to misleading interpretations. (iii) The ratio (TTX/control) vs. control plots seem to suffer from a division by small value artifact (see Figure 6F). Correspondingly, ratio-analysis differs considerably for different control conditions (Fig. 1Giii, Fig. 2Giii, Fig. 6C, Fig. 9A).<br /> 3. As noted by the authors in a previous publication (Hanes et al. 2020), statistical analysis of CDFs suffers from n-inflation. In addition, the quantile sampling method chosen violates an important assumption of the K-S test. Indeed, p-values for these comparisons are typically several orders of magnitude smaller. Given that the statistical N most likely corresponds to the number of cultures (see, e.g., https://doi.org/10.1371/journal.pbio.2005282), CDF comparisons are not informative and should thus not be used to draw conclusions from the data. The plots can be informative, though.<br /> 4. How does recoding noise and the mEPSC amplitude threshold affect "divergent scaling"?<br /> 5. What is the justification for the line fits of the ratio data/how was the fit range chosen?<br /> 6. TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data.<br /> 7. Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.<br /> 8. There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3Aebd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+, and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?<br /> 9. Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs. -15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.<br /> 10. The authors discuss previous papers showing changes in VGLUT1 intensity. Was VGLUT intensity altered in the stainings presented in the manuscript?<br /> 11. The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?<br /> 12. The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold? What was the rationale for a threshold of 400 a.u. or 450 a.u.? How does this threshold compare to the mEPSC threshold of 3 pA? The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size. (ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 18-20%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)? Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors. (iv) It is not entirely clear if their imaging experiments will sample from all synapses. Other AMPAR subtypes than GluA2 could contribute, as could kainate or NMDA receptors.<br /> Furthermore, the statement "complete lack of correspondence of TTX/CON ratios" is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/- , the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2), particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.<br /> 13. Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?

s pacientes más jóvenes tienen más probabilidades de presentar el síndrome de Goodpasture completo, con hemoptisis, caída repentina de la hemoglobina, fiebre, disnea y hematuria, y los pacientes de mayor edad tienen más probabilidades de presentar glomerulonefritis aislada