Author response:

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

Reviewer #1 (Public review):

Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the suboesophageal zone that interacts with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet-taste sensitivity and sugar-feeding behavior in male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.

Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the suboesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar-feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. found that DopR1 and Dop2R, but not DopEcR, are involved in the sexual failure-induced decrease in sugar sensitivity in these neurons. I wish the authors had further explored or discussed this discrepancy, as it is unclear how dopamine release selectively engages different receptors to modulate neuronal sensitivity in a context-dependent manner.

Our immunostaining experiments showed that three dopamine receptors, Dop1R1, Dop2R, and DopEcR were expressed in Gr5a⁺ neurons in the proboscis, which was consistent with previous findings by using RT-PCR (Inagaki et al 2012). As the reviewer pointed out, we found that Dop1R1 and Dop2R were required for courtship failure-induced suppression of sugar sensitivity, whereas Marella et al 2012 and Inagaki et al 2012 found that Dop2R and DopEcR were required for starvation-induced enhancement of sugar sensitivity. These results may suggest that different internal states (courtship failure vs. starvation) modulate the peripheral sensory system via different signaling pathways (e.g. different subsets of dopaminergic neurons; different dopamine release mechanisms; and different dopamine receptors). We have discussed these possibilities in the revised manuscript.

The data presented by Wang et al. are solid and effectively support their conclusions. However, certain aspects of their experimental design, data analysis, and interpretation warrant further review, as outlined below.

(1) The authors did not explicitly indicate the feeding status of the flies, but it appears they were not starved. However, the naive and satisfied flies in this study displayed high feeding and PER baselines, similar to those observed in starved flies in other studies. This raises the concern that sexually failed flies may have consumed additional food during the 4.5-hour conditioning period, potentially lowering their baseline hunger levels and subsequently reducing PER responses. This alternative explanation is worth considering, as an earlier study demonstrated that sexually deprived males consumed more alcohol, and both alcohol and food are known rewards for flies. To address this concern, the authors could remove food during the conditioning phase to rule out its influence on the results.

This is an important consideration. To rule out potential confound from food intake during courtship conditioning, we have now also conducted courtship conditioning in vials absent of food. In the absence of any feeding opportunity over the 4.5-hour courtship conditioning period, sexually rejected males still exhibited a robust decrease in sweet taste sensitivity compared with Naïve and Satisfied controls (Figure 1-supplement 1C). These data confirm that the suppression of PER is driven by courtship failure per se, rather than by differences in feeding during the conditioning phase.

(2) Figure 1B reveals that approximately half of the males in the Failed group did not consume sucrose yet Figure 1-S1A suggests that the total volume consumed remained unchanged. Were the flies that did not consume sucrose omitted from the dataset presented in Figure 1-S1A? If so, does this imply that only half of the male flies experience sexual failure, or that sexual failure affects only half of males while the others remain unaffected? The authors should clarify this point.

Our initial description of the experimental setup might be a bit confusing. Here is a brief clarification of our experimental design and we have further clarified the details in the revised manuscript, which should resolve the reviewer’s concerns:

After the behavioral conditioning, male flies were divided for two assays. On the one hand, we quantified PER responses of individual flies. As shown in Figure 1C, Failed males exhibited decreased sweet sensitivity (as demonstrated by the right shift of the dose-response curve). On the other hand, we sought to quantify food consumption of individual flies by using the MAFE assay (Qi et al 2005).

In the initial submission, we used 400 mM sucrose for the MAFE assay. When presented with 400 mM sucrose, approximately 100% of the flies in the Naïve and Satisfied groups, and 50% of the flies in the Failed group, extended their proboscis and started feeding, as a natural consequence of decreased sugar sensitivity (Figure 1B). We were able to quantify the actual volume of food consumed of these flies showing PER responses towards 400 mM sucrose and observed no change (Figure 1-supplement 1A, left). To avoid potential confusion, we have now repeated the MAFE assay with 800 mM sucrose, which elicited feeding in ~100% of flies among all three groups, as shown in Figure 1C. Again, we observed no change in food intake (Figure 1-supplement 1A, right).

These experiments in combination suggest that sexual failure suppresses sweet sensitivity of the Failed males. Meanwhile, as long as they still responded to a certain food stimulus and initiated feeding, the volume of food consumption remained unchanged. These results led us to focus on the modulatory effect of sexual failure on the sensory system, the main topic of this present study.

(3) The evidence linking TH-GAL4 labeled dopaminergic neurons to reduced sugar sensitivity in Gr5a+ neurons in sexually failed males could be further strengthened. Ideally, the authors would have activated TH-GAL4 neurons and observed whether this restored GCaMP responses in Gr5a+ neurons in sexually failed males. Instead, the authors performed a less direct experiment, shown in Figures 3-S1C and D. The manuscript does not describe the condition of the flies used in this experiment, but it appears that they were not sexually conditioned. I have two concerns with this experiment. First, no statistical analysis was provided to support the enhancement of sucrose responses following activation of TH-GAL4 neurons. Second, without performing this experiment in sexually failed males, the authors lack direct evidence to confirm that the dampened response of Gr5a+ neurons to sucrose results from decreased activity in TH-GAL4 neurons.

We have now quantified the effect of TH⁺ neuron activation on Gr5a⁺ neuron calcium responses. in Naïve males, dTRPA1-mediated activation of TH⁺ cells significantly enhanced sucrose-induced calcium responses (Figure 3-supplement 1C); while in Failed males, the baseline activity of Gr5a⁺ neurons was lower (Figure 3C), the same activation also produced significant (even slightly larger) effect on the calcium responses of Gr5a⁺ neurons (Figure 3-supplement 1D).

Taken together, we would argue that these experiments using both Naïve and Failed males were adequate to show a functional link between TH⁺ neurons and Gr5a⁺ neurons. Combining with the results that these neurons form active synapses (Figure 3-supplement 1B) and that the activity of TH⁺ neurons was dampened in sexually failed males (Figure 3G-I), our data support the notion that sexual failure suppresses sweet sensitivity via TH-Gr5a circuitry.

(4) The statistical methods used in this study are poorly described, making it unclear which method was used for each experiment. I suggest that the authors include a clear description of the statistical methods used for each experiment in the figure legends. Furthermore, as I have pointed out, there is a lack of statistical comparisons in Figures 3-S1C and D, a similar problem exists for Figures 6E and F.

We have added detailed information of statistical analysis in each figure legend.

(5) The experiments in Figure 5 lack specificity. The target neurons in this study are Gr5a+ neurons, which are directly involved in sugar sensing. However, the authors used the less specific Dop1R1- and Dop2R-GAL4 lines for their manipulations. Using Gr5a-GAL4 to specifically target Gr5a+ neurons would provide greater precision and ensure that the observed effects are directly attributable to the modulation of Gr5a+ neurons, rather than being influenced by potential off-target effects from other neuronal populations expressing these dopamine receptors.

We agree with the reviewer that manipulating Dop1R1 and Dop2R genes (Figure 4) and the neurons expressing them (Figure 5) might have broader impacts. For specificity, we have also tested the role of Dop1R1 and Dop2R in Gr5a⁺ neurons by RNAi experiments (Figure 6). As shown by both behavioral and calcium imaging experiments, knocking down Dop1R1 and Dop2R in Gr5a⁺ neurons both eliminated the effect of sexual failure to dampen sweet sensitivity, further confirming the role of these two receptors in Gr5a⁺ neurons.

(6) I found the results presented in Fig. 6F puzzling. The knockdown of Dop2R in Gr5a+ neurons would be expected to decrease sucrose responses in naive and satisfied flies, given the role of Dop2R in enhancing sweet sensitivity. However, the figure shows an apparent increase in responses across all three groups, which contradicts this expectation. The authors may want to provide an explanation for this unexpected result.

We agree that there might be some potential discrepancies. We have now addressed the issues by re-conducting these calcium imaging experiments again with a head-to-head comparison with the controls (Gr5a-GCaMP, +/- Dop1R1 and Dop2R RNAi).

In these new experiments, Dop1R1 or Dop2R knockdown completely prevented the suppression of Gr5a⁺ neuron responsiveness by courtship failure (Figure 6E), whereas the activities of Gr5a⁺ neurons in Naïve/Satisfied groups were not altered. These results demonstrate that Dop1R1 and Dop2R are specifically required to mediate the decrease in sweet sensitivity following courtship failure.

(7) In several instances in the manuscript, the authors described the effects of silencing dopamine signaling pathways or knocking down dopamine receptors in Gr5a neurons with phrases such as 'no longer exhibited reduced sweet sensitivity' (e.g., L269 and L288), 'prevent the reduction of sweet sensitivity' (e.g., L292), or 'this suppression was reversed' (e.g. L299). I found these descriptions misleading, as they suggest that sweet sensitivity in naive and satisfied groups remains normal while the reduction in failed flies is specifically prevented or reversed. However, this is not the case. The data indicate that these manipulations result in an overall decrease in sweet sensitivity across all groups, such that a further reduction in failed flies is not observed. I recommend revising these descriptions to accurately reflect the observed phenotypes and avoid any confusion regarding the effects of these manipulations.

We have changed the wording in the revised manuscript. In brief, we think that these manipulations have two consequences: suppressing the overall sweet sensitivity, and eliminating the effect of sexual failure on sweet sensitivity.

Reviewer #2 (Public review):

Summary:

The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.

Strengths:

The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.

Weaknesses:

The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.

We agree with the reviewer that in the current study, we did not examine the exact mechanism of how mating experience suppressed the activity of dopaminergic neurons in the SEZ. The current study mainly focused on the behavioral characterization (sexual failure suppresses sweet sensitivity) and the downstream mechanism (TH-Gr5a pathway). We think that examining the upstream modulatory mechanism may be more suitable for a separate future study.

We believe that a sustained reduction in sweet sensitivity (not limited to sucrose but extend to other sweet compounds Figure 1-supplement 1D-E) upon courtship failure suggests a generalized and sustained consequence on reward-related behaviors. Sexual failure may thus resemble a state of “primitive emotion” in fruit flies. We have further discussed this possibility in the revised manuscript.

Reviewer #3 (Public review):

Summary

In this work, the authors asked how mating experience impacts reward perception and processing. For this, they employ fruit flies as a model, with a combination of behavioral, immunostaining, and live calcium imaging approaches.

Their study allowed them to demonstrate that courtship failure decreases the fraction of flies motivated to eat sweet compounds, revealing a link between reproductive stress and reward-related behaviors. This effect is mediated by a small group of dopaminergic neurons projecting to the SEZ. After courtship failure, these dopaminergic neurons exhibit reduced activity, leading to decreased Gr5a+ neuron activity via Dop1R1 and Dop2R signaling, and leading to reduced sweet sensitivity. The authors therefore showed how mating failure influences broader behavioral outputs through suppression of the dopamine-mediated reward system and underscores the interactions between reproductive and reward pathways.

Concern

My main concern regarding this study lies in the way the authors chose to present their results. If I understood correctly, they provided evidence that mating failure induces a decrease in the fraction of flies exhibiting PER. However, they also showed that food consumption was not affected (Fig. 1, supplement), suggesting that individuals who did eat consumed more. This raises questions about the analysis and interpretation of the results. Should we consider the group as a whole, with a reduced sensitivity to sweetness, or should we focus on individuals, with each one eating more? I am also concerned about how this could influence the results obtained using live imaging approaches, as the flies being imaged might or might not have been motivated to eat during the feeding assays. I would like the authors to clarify their choice of analysis and discuss this critical point, as the interpretation of the results could potentially be the opposite of what is presented in the manuscript.

Please refer to our responses to the Public Review (Reviewer 1, Point 2) for details.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The label for the y-axis in Figure 1B should be "fraction", not "percentage".

We have revised the figure as suggested.

(2) I suggest that the authors indicate the ROIs they used to quantify the signal intensity in Figure 3E and G.

We have revised the figures as suggested.

(3) There is a typo in Figure 4A: it should be "Wilde type", not "Wide type".

We have revised the figure as suggested.

(4) The elav-GAL4/+ data in Figure 4-S1B, C, and D appears to be reused across these panels. However, the number of asterisks indicating significance in the MAT plots differs between them (three in panels B and C, and four in panel D). Is this a typo?

It is indeed a typo, and we have revised the figure accordingly.

Reviewer #2 (Recommendations for the authors):

Additional comments:

The authors should add this missing literature about dopamine and neuromodulation in courtship:

Boehm et al., 2022 (eLife) - this study shows that mating affects olfactory behavior in females.

Cazalé-Debat et al., 2024 (Nature) - Mating proximity blinds threat perception.

Gautham et al., 2024 (Nature) - A dopamine-gated learning circuit underpins reproductive state-dependent odor preference in Drosophila females.

We have added these references in the introduction section.

Has the mating behavior been quantified? How often did males copulate with mated and virgin females?

We tried to examine the copulation behavior based on our video recordings. In the “Failed” group (males paired with mated females), we observed virtually no successful copulation events at all, confirming that nearly 100% of those males experienced sexual failure. In contrast, males in the “Satisfied” group (paired with virgin females) mated on average 2-3 times during the 4.5-hour conditioning period. We have added some explanations in the manuscript.

Do the rejected males live shorter? Is the effect also visible when they are fed with normal fly food, or is it only working with sugar?

We did not directly measure the lifespan of these males. But we conducted a relevant assay (starvation resistance), in which “Failed” males died significantly faster than both Naïve and Satisfied controls, indicating a clear reduction in their ability to endure food deprivation (Figure 1-supplement 1B). Since sweet taste is a primary cue for food detection in Drosophila, and sugar makes up a large portion of their standard diet, the drop in sugar sensitivity we observed in Failed males could likewise impair their perception and consumption of regular fly food, hence their resistance to starvation.

Also, the authors mention that the reward pathway is affected, this is probably the case as sugar sensation is impaired. One interesting experiment would be (and maybe has been done?) to test rejected males in normal odor-fructose conditioning. The data would suggest that they would do worse.

We have already measured how courtship failure affected fructose sensitivity (Figure 1 supplement 1D), and we found that the reduction in fructose perception was even more profound than for sucrose. We have not yet tested whether Failed males showed deficits in odor-fructose associative conditioning. That was indeed a very interesting direction to explore. But olfactory reward learning relies on molecular and circuit mechanisms distinct from those governing taste. We therefore argue such experiments would be more suitable in a separate, follow up study.

The authors could have added another group where males are exposed to other males. It would be interesting if this is also a "stressful" context and if it would also reduce sugar preference - probably beyond the scope of this paper.

In our experiments, all flies, including those in the Naïve, Failed, and Satisfied groups, were housed in groups of 25 males per vial before the conditioning period (and the Naïve group remained in the same group housing until PER testing). This means every cohort experienced the same level of “social stress” from male-male interactions. While it would indeed be interesting to compare that to solitary housing or other male-only exposures, isolation itself imposes a different kind of stress, and disentangling these effects on sugar preference would require a separate, dedicated study beyond the scope of the present work.

Would the behavior effect also show up with experienced males? Maybe this has been tested before. Does mating rejection in formerly successful males have the same impact?

As suggested by the reviewer, we performed an additional experiment in which males that had previously mated successfully were subsequently subjected to courtship rejection. As shown in Figure 1 supplement 1F, prior successful mating did not prevent the decline in sweet sensitivity induced by subsequent mating failure, indicating that even experienced males exhibit the reduction in sugar sensitivity after rejection.

Is the same circuit present and functioning in females? Does manipulating dopamine receptors in GR5a neurons in females lead to the same phenotype? This would suggest that different internal states in males and females could lead to the same phenotype and circuit modulations.

This is indeed a very interesting suggestion. In male flies, Gr5a-specific knockdown of dopamine receptors did not alter baseline sweet sensitivity, but it selectively prevented the reduction in sugar perception that followed mating failure (Figure 6C-D), indicating that this dopaminergic pathway is engaged only in the context of courtship rejection. By extension, knocking down the same receptors in female GR5a neurons would likewise be expected to leave their basal sugar sensitivity unchanged. Moreover, because there is currently no established paradigm for inducing mating failure in female flies, we cannot yet test whether sexual rejection similarly modulates sweet taste in females, or whether it operates via the same circuit.

Reviewer #3 (Recommendations for the authors):

Suggestions to the authors:

Introduction, line 61. I suggest the authors add references in fruit flies concerning the rewarding nature of mating. For example, the paper from Zhang et al, 2016 "Dopaminergic Circuitry Underlying Mating Drive" demonstrates the role of the dopamine rewarding system in mating drive. There is a large body of literature showing the link between dopamine and mating.

We have added this literature in the introduction section.

Figure 1B and Figure Supplement 1: If I understood correctly, Figure Supplement 1A shows that the total food consumption across all tested flies remains unchanged. However, fewer flies that failed to mate consumed sucrose. I would be curious to see the results for sucrose consumption per individual fly that did eat. According to their results, individual flies that failed to mate should consume more sucrose. This would change the conclusion. The authors currently show that a group of flies that failed to mate consumed less sucrose overall, but since fewer males actually ate, those that failed to mate and did eat consumed more sucrose. The authors should distinguish between failed and satisfied flies in two groups: those that ate and those that did not.

Please see our responses to the Public Review for details (Reviewer 1, Point 2).

Figure 1C, right: For a better understanding of all the "MAT" figures, I suggest the authors start the Y axis with the unit 25 and increase it to 400. This would match better the text (line 114) saying that it was significantly elevated in the failed group. As it is, we have the impression of a decrease in the graph.

We have revised the figures accordingly.

Line 103: When suggesting a reduced likelihood of meal initiation of these males, do these males take longer to eat when they did it? In other words, is the latency to eat increased in failed males? That would be a good measure of motivational state.

We tried to analyze feeding latency in the MAFE assay by measuring the time from sucrose presentation to the first proboscis extension, but it was too short to be accurately accounted. Nevertheless, when conducting the experiments, we did not feel/observe any significant difference in the feeding latency between Failed males and Naïve or Satisfied controls.

Line 117. I don't understand which results the authors refer to when writing "an overall elevation in the threshold to initiate feeding upon appetitive cues". Please specify.

This phrase refers to the fact that for every sweet tastant we tested, including sucrose (Figure 1C), fructose and glucose (Figure 1 supplement 1D-E), the concentration-response curve in Failed males shifted to the right, and the Mean Acceptance Threshold (MAT) was significantly higher. In other words, for these different appetitive cues, mating failure raised the concentration of sugar required to trigger a proboscis extension, indicating a general elevation in the threshold to initiate feeding upon an appetitive cue.

Figure 1D. Please specify the time for the satisfied group.

For clarity, the Naïve and Satisfied groups in Figure 1D each represent pooled data from 0 to 72 hours post-treatment, as their sweet sensitivity remained stable throughout this period. Only the Failed group was shown with time-resolved data, since it was the only group exhibiting a dynamic change in sugar sensitivity over time. We have now specified this in the figure legend.

Figure 1F. The phenotype was not totally reversed in failed-re-copulated males. Could it be due to the timing between failure and re-copulation? I suggest the authors mention in the figure or in the text, the time interval between failure and re-copulation.

We’d like to clarify that the interval between the initial treatment (“Failed”) and the opportunity for re copulation was within 30 minutes. The incomplete reversal in the Failed-re-copulated group indeed raised interesting questions. One possible explanation is that mating failure reduces synaptic transmissions between the SEZ dopaminergic neurons and Gr5a⁺ sweet sensory neurons (Figure 3), and the regeneration of these transmissions takes a longer time. We have added this information to the figure legend and the Method section.

Line 227-228 and Figure 3E. The authors showed that the synaptic connections between dopaminergic neurons and Gr5a+ GRNs were significantly weakened. I am wondering about the delay between mating failure and the GFP observation. It would be informative to know this timing to interpret this decrease in synaptic connections. If the timing is relatively long, it is possible that we can observe a neuronal plasticity. However, if this timing is very short, I would not expect such synaptic plasticity.

The interval between the behavioral treatment and the GRASP-GFP experiment was approximately 20 hours. We chose this time window because it was sufficient for both GFP expression and accumulation. Therefore, the observed reduction in synaptic connections between dopaminergic neurons and Gr5a⁺ GRNs likely reflects a genuine, experience-induced structural and functional change rather than an immediate, transient effect. We have added this information to the revised manuscript for clarity in the Method section.

Line 240-243: The authors demonstrated that there is a reduction of CaLexA-mediated GFP signals in dopaminergic neurons in the SEZ after mating failure, but not a reduction in Gr5a+ GRNs. I suggest replacing "indicate" with "suggest' in line 240.

We have made the change accordingly. Meanwhile, we would like to clarify that while we observed a reduction of NFAT signal in SEZ dopaminergic neurons (Figure 3G), we did not directly test NFAT signal in Gr5a⁺ neurons. Notably, the results that the synaptic transmissions from SEZ dopaminergic neurons to Gr5a⁺ neurons were weakened (Figure 3E-F), and the reduction of NFAT signal in SEZ dopaminergic neurons (Figure 3G-I), were in line with a reduction in sweet sensitivity of Gr5a⁺ neurons upon courtship failure (Figure 3B-D).

Line 243: replace "consecutive" with "constitutive".

We have revised it accordingly.

Figure 5: I have trouble understanding the results obtained in Figure 5. Both constitutive activation and inhibition of Dop1R1 and Dop2R neurons lead to the same results, knowing that males who failed mating no longer exhibit decreased sweet sensitivity. I would have expected contrary results for both experimental conditions. I suggest the author to discuss their results.

Both activation and inhibition of Dop1R1 and Dop2R neurons eliminated the effect of courtship failure on sweet sensitivity (Figure 5). These results are in line with our hypothesis that courtship failure leads to changes in dopamine signaling and hence sweet sensitivity. If dopamine signaling via Dop1R1 and Dop2R was locked, either to a silenced or a constitutively activated state, the effect of courtship failure on sweet sensitivity was eliminated.

Nevertheless, as the reviewer pointed out, constitutive activation/inhibition should in principle lead to the opposite effect on Naïve flies. In fact, when Dop1R1⁺/Dop2R⁺ neurons were silenced in Naïve flies, PER to sucrose was significantly reduced (Figure 5C-D), confirming that these neurons normally facilitate sweet sensation. Meanwhile, while neuronal activation by NaChBac did show a trend towards enhanced PER compared to the GAL4/+ controls, it did not exhibit a difference compared to +>UAS-NaChBac controls that showed a high PER level, likely due to a potential ceiling effect. We have added the discussions to the manuscript.

Figure 7: I suggest the authors modify their figure a bit. It is not clear why in failed mating, the red arrow in "behavioral modulation" goes to the fly. The authors should find another way to show that mating failure decreased the percentage of flies that are motivated to eat sugar.

We have modified the figure as suggested.

Overall, I would suggest the authors be precautious with their conclusion. For example, line 337= "sexual failure suppressed feeding behavior". This is not what is shown by this study. Here, the study shows that mating failure decreases the fraction of flies to eat sucrose. Unless the authors demonstrate that this decrease is generalizable to other metabolites, I suggest the authors modify their conclusion.

While we primarily used sucrose as the stimulant in our experiments, we also tested responses to two other sugars: fructose and glucose (Figure 1 supplement 1D-E). In all three cases, mating failure led to a significant reduction in sweet perception, suggesting that the effect of courtship failure is not limited to a single metabolite but rather reflects a general decrease in sweet sensitivity. Meanwhile, reduced sweet sensitivity indeed led to a reduction of feeding initiation (Figure 1).

Layer 1: Signal

Layer 2: Ground

Layer 3: Power

Layer 4: Signal

Layer 5: Signal

Layer 6: Power

Layer 7: Ground

Layer 8: Signal

Layer 1: Signal

Layer 2: Ground

Layer 3: Signal

Layer 4: Signal

Layer 5: Power

Layer 6: Signal

Layer 7: Ground

Layer 8: Signal

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In their comprehensive analysis Diallo et al. deorphanise the first olfactory receptor of a nonhymenopteran eusocial insect - a termite and identified the well-established trail pheromone neocembrene as the receptor's best ligand. By using a large set of odorants the authors convincingly show that, as expected for a pheromone receptor, PsimOR14 is very narrowly tuned. While the authors first make use of an ectopic expression system, the empty neuron of Drosophila melanogaster, to characterise the receptor's responses, they next perform single sensillum recordings with different sensilla types on the termite antenna. By that, they are able to identify a sensillum that houses three neurons, of which the B neuron exhibits the narrow responses described for PsimOR14. Hence the authors do not only identify the first pheromone receptor in a termite but can even localize its expression on the antenna. The authors in addition perform a structural analysis to explain the binding properties of the receptor and its major and minor ligands (as this is beyond my expertise, I cannot judge this part of the manuscript). Finally, they compare expression patterns of ORs in different castes and find that PsimOR14 is more strongly expressed in workers than in soldier termites, which corresponds well with stronger antennal responses in the worker caste.

Strengths:

The manuscript is well-written and a pleasure to read. The figures are beautiful and clear. I actually had a hard time coming up with suggestions.

We thank the reviewer for the positive comments.

Weaknesses:

Whenever it comes to the deorphanization of a receptor and its potential role in behaviour (in the case of the manuscript it would be trail-following of the termite) one thinks immediately of knocking out the receptor to check whether it is necessary for the behaviour. However, I definitely do not want to ask for this (especially as the establishment of CRISPR Cas-9 in eusocial insects usually turns out to be a nightmare). I also do not know either, whether knockdowns via RNAi have been established in termites, but maybe the authors could consider some speculation on this in the discussion.

We agree that a functional proof of the PsimOR14 function using reverse genetics would be a valuable addition to the study to firmly establish its role in trail pheromone sensing. Nevertheless, such a functional proof is difficult to obtain. Due to the very slow ontogenetic development inherent to termites (several months from an egg to the worker stage) the CRISPR Cas-9 is not a useful technique for this taxon. By contrast, termites are quite responsive to RNAimediated silencing and RNAi has previously been used for the silencing of the ORCo co-receptor in termites resulting in impairment of the trail-following behavior (DOI: 10.1093/jee/toaa248). Likewise, our previous experiments showed a decreased ORCo transcript abundance, lower sensitivity to neocembrene and reduced neocembrene trail following upon dsPsimORCo administration to P. simplex workers, while we did not succeed in reducing the transcript abundance of PsimOR14 upon dsPsimOR14 injection. We do not report these negative results in the present manuscript so as not to dilute the main message. In parallel, we are currently developing an alternative way of dsRNA delivery using nanoparticle coating, which may improve the RNAi experiments with ORs in termites.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors performed the functional analysis of odorant receptors (ORs) of the termite Prorhinotermes simplex to identify the receptor of trail-following pheromone. The authors performed single-sensillum recording (SSR) using the transgenic Drosophila flies expressing a candidate of the pheromone receptor and revealed that PsimOR14 strongly responds to neocembrene, the major component of the pheromone. Also, the authors found that one sensillum type (S I) detects neocembrene and also performed SSR for S I in wild termite workers. Furthermore, the authors revealed the gene, transcript, and protein structures of PsimOR14, predicted the 3D model and ligand docking of PsimOR14, and demonstrated that PsimOR14 is higher expressed in workers than soldiers using RNA-seq for heads of workers and soldiers of P. simplex and that EAG response to neocembrene is higher in workers than soldiers. I consider that this study will contribute to further understanding of the molecular and evolutionary mechanisms of the chemoreception system in termites.

Strength:

The manuscript is well written. As far as I know, this study is the first study that identified a pheromone receptor in termites. The authors not only present a methodology for analyzing the function of termite pheromone receptors but also provide important insights in terms of the evolution of ligand selectivity of termite pheromone receptors.

We thank the reviewer for the overall positive evaluation of the manuscript.

Weakness:

As you can see in the "Recommendations to the Authors" section below, there are several things in this paper that are not fully explained about experimental methods. Except for this point, this paper appears to me to have no major weaknesses.

We address point by point the specific comments listed in the Recommendation to the authors chapter below.

Reviewer #3 (Public review):

Summary:

Chemical communication is essential for the organization of eusocial insect societies. It is used in various important contexts, such as foraging and recruiting colony members to food sources. While such pheromones have been chemically identified and their function demonstrated in bioassays, little is known about their perception. Excellent candidates are the odorant receptors that have been shown to be involved in pheromone perception in other insects including ants and bees but not termites. The authors investigated the function of the odorant receptor PsimOR14, which was one of four target odorant receptors based on gene sequences and phylogenetic analyses. They used the Drosophila empty neuron system to demonstrate that the receptor was narrowly tuned to the trail pheromone neocembrene. Similar responses to the odor panel and neocembrene in antennal recordings suggested that one specific antennal sensillum expresses PsimOR14. Additional protein modeling approaches characterized the properties of the ligand binding pocket in the receptor. Finally, PsimOR14 transcripts were found to be significantly higher in worker antennae compared to soldier antennae, which corresponds to the worker's higher sensitivity to neocembrene.

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size.

We thank the reviewer for these positive comments.

Weaknesses:

The manuscript would benefit from a more detailed explanation of the research advances this work provides. Stating that this is the first deorphanization of an odorant receptor in a clade is insufficient. The introduction primarily reviews termite chemical communication and deorphanization of olfactory receptors previously performed. Although this is essential background, it lacks a good integration into explaining what problem the current study solves.

We understand the comment about the lack of an intelligible cue to highlight the motivation and importance of the present study. In the current version of the manuscript the introduction has been reworked. As suggested by Reviewer 3 in the Recommendations section below, the introduction now integrates some parts of the original discussion, especially the part discussing the OR evolution and emergence of eusociality in hymenopteran social insects and in termites, while underscoring the need of data from termites to compare the commonalities and idiosyncrasies in neurophysiological (pre)adaptations potentially linked with the independent eusociality evolution in the two main social insect clades.

Selecting target ORs for deorphanization is an essential step in the approach. Unfortunately, the process of choosing these ORs has not been described. Were the authors just lucky that they found the correct OR out of the 50, or was there a specific selection process that increased the probability of success?

Indeed, we were extremely lucky. Our strategy was to first select a modest set of ORs to confirm the feasibility of the Empty Neuron Drosophila system and newly established SSR setup, while taking advantage of having a set of termite pheromones, including those previously identified in the P. simplex model, some of them de novo synthesized for this project. The selection criteria for the first set of four receptors were (i) to have full-length ORF and at least 6 unambiguously predicted transmembrane regions, and (ii) to be represented on different branches (subbranches) of the phylogenetic tree. Then it was a matter of a good luck to hit the PsimOR14 selectively responding to the genuine P. simplex trail-following pheromone main component. In the revised version, we state these selection criteria in the results section (Phylogenetic reconstruction and candidate OR selection).

The deorphanization attempts of additional P. simplex ORs are currently running.

The authors assigned antennal sensilla into five categories. Unfortunately, they did not support their categories well. It is not clear how they were able to differentiate SI and SII in their antennal recordings.

We agree that the classification of multiporous sensilla into five categories lacks robust discrimination cues. The identification of the neocembrene-responding sensillum was initially carried out by SSR measurements on individual olfactory sensilla of P. simplex workers one-by-one and the topology of each tested sensillum was recorded on optical microscope photographs taken during the SSR experiment. Subsequently, the SEM and HR-SEM were performed in which we localized the neocembrene sensillum and tried to find distinguishing characters. We admit that these are not robust. Therefore, in the revised version of the manuscript we decided to abandon the attempt of sensilla classification and only report the observations about the specific sensillum in which we consistently recorded the response to neocembrene (and geranylgeraniol). The modifications affect Fig. 4, its legend and the corresponding part of the results section (Identification of P. simplex olfactory sensillum responding to neocembrene).

The authors used a large odorant panel to determine receptor tuning. The panel included volatile polar compounds and non-volatile non-polar hydrocarbons. Usually, some heat is applied to such non-volatile odorants to increase volatility for receptor testing. It is unclear how it is possible that these non-volatile compounds can reach the tested sensilla without heat application.

The reviewer points at an important methodological error we made while designing the experiments. Indeed, the inclusion of long-chain hydrocarbons into Panel 1 without additional heat applied to the odor cartridges was inappropriate, even though the experiments were performed at 25–26 °C. We carefully considered the best solution to correct the mistake and finally decided to remove all tested ligands beyond C22 from Panel 1, i.e. altogether five compounds. These changes did not affect the remaining Panels 2-4 (containing compounds with sufficient volatility), nor did they affect the message of the manuscript on highly selective response of PsimOR14 to neocembrene (and geranylgeryniol). In consequence, Figures 2, 3 and 5 were updated, along with the supplementary tables containing the raw data on SSR measurements. In addition, the tuning curve for PsimOR14 was re-built and receptor lifetime sparseness value re-calculated (without any important change). We also exchanged squalene for limonene in the docking and molecular dynamics analysis and made new calculations.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) L 208: "than" instead of "that"

Corrected.

(2) L 527+527 strange squares (•) before dimensions

Apparently an error upon file conversion, corrected.

(3) L553 "reconstructing" instead of "reconstruct"

Corrected.

(4) Two references (Chahda et al. and Chang et al. appear too late in the alphabet.

Corrected. Thank you for spotting this mistake. Due to our mistake the author list was ordered according to the alphabet in Czech language, which ranks CH after H.

Reviewer #2 (Recommendations for the authors):

(1) L148: Why did the authors select only four ORs (PsimOR9, 14, 30, and 31) though there are 50 ORs in P. simplex? I would like you to explain why you chose them.

Our strategy was to first select a modest set of ORs to confirm the feasibility of the Empty Neuron Drosophila system and newly established SSR setup, while taking advantage of having a set of termite pheromones, including those previously identified in the P. simplex model, some of them de novo synthesized for this project. Then, it was a matter of a good luck to hit the PsimOR14 selectively responding to the genuine P. simplex trail-following pheromone main component, while the deorphanization attempts of a set of additional P. simplex ORs is currently running. In the revised version of the manuscript, we state the selection criteria for the four ORs studied in the Results section (Phylogenetic reconstruction and candidate OR selection).

(2) L149: Where is Figure 1A? Does this mean Figure 1?

Thank you for spotting this mistake. Fig. 1 is now properly labelled as Fig. 1A and 1B in the figure itself and in the legend. Also the text now either refers to either 1A or 1B.

(3) Figure 1: The authors also showed the transcription abundance of all 50 ORs of P. simplex in the right bottom of Figure 1, but there is no explanation about it in the main text.

The heatmap reporting the transcript abundances is now labelled as Fig. 1B and is referred to in the discussion section (in the original manuscript it was referred to on the same place as Fig. 1).

(4) L260-265: The authors confirmed higher expression of PsimOR14 in workers than soldiers by using RNA-seq data and stronger EAG responses of PsimOR14 to neocembrene in workers than soldiers, but I think that confirming the expression levels of PsimOR14 in workers and soldiers by RT-qPCR would strengthen the authors' argument (it is optional).

qPCR validation is a suitable complement to read count comparison of RNA Seq data, especially when the data comes from one-sample transcriptomes and/or low coverage sequencing. Yet, our RNA Seq analysis is based on sequencing of three independent biological replicates per phenotype (worker heads vs. soldier heads) with ~20 millions of reads per sample. Thus, the resulting differential gene expression analysis is a sufficient and powerful technique in terms of detection limit and dynamic range.

We admit that the replicate numbers and origin of the RNA seq data should be better specified since the Methods section only referred to the GenBank accession numbers in the original manuscript. Therefore, we added more information in the Methods section (Bioinformatics) and make clear in the Methods that this data comes from our previous research and related bioproject.

(5) L491: I think that "The synthetic processes of these fatty alcohols are ..." is better.

We replaced the sentence with “The de novo organic synthesis of these fatty alcohols is described …”

(6) L525 and 527: There are white squares between the number and the unit. Perhaps some characters have been garbled.

Apparently an error upon file conversion, corrected.

(7) L795: ORCo?

Corrected.

(8) L829-830 & Figure 4: Where is Figure 4D?

Thank you for spotting this mistake from the older version of Figure 4. The SSR traces referred to in the legend are in fact a part of Figure 5. Moreover, Figure 4 is now reworked based on the comments by Reviewer 3.

(9) L860-864: Why did the authors select the result of edgeR for the volcano plot in Figure 7 although the authors use both DESeq2 and edgeR? An explanation would be needed.

Both algorithms, DESeq2 and EdgeR, are routinely used for differential gene expression analysis. Since they differ in read count normalization method and statistical testing we decided to use both of them independently in order to reduce false positives. Because the resulting fold changes were practically identical in both algorithms (results for both analyses are listed in Supplementary table S15), we only reported in Fig. 7 the outputs for edgeR to avoid redundancies. We added in the Results section the information that both techniques listed PsimOR14 among the most upregulated in workers.

Reviewer #3 (Recommendations for the authors):

The discussion contains many descriptions that would fit better into the introduction, where they could be used to hint at the study's importance (e.g., 292-311, 381-412). The remaining parts often lack a detailed discussion of the results that integrates details from other insect studies. Although references were provided, no details were usually outlined. It would be helpful to see a stronger emphasis on what we learn from this study.

Along with rewriting the introduction, we also modified the discussion. As suggested, the lines 292-311 were rewritten and placed in the introduction. By contrast, we preferred to keep the two paragraphs 381-412 in the discussion, since both of them outline the potential future interesting targets of research on termite ORs.

As suggested, the discussion has been enriched and now includes comparative examples and relevant references about the broad/narrow selectivity of insect ORs, about the expected breadth of tuning of pheromone receptors vs. ORs detecting environmental cues, about the potential role of additional neurons housed in the neocembrene-detecting sensillum of P. simplex workers, etc. From both introduction and discussion the redundant details on the chemistry of termite communication have been removed.

This includes explanations of the advantages of the specific methodologies the authors used and how they helped solve the manuscript's problem. What does the phylogeny solve? Was it used to select the ORs tested? It would be helpful to discuss what the phylogeny shows in comparison to other well-studied OR phylogenies, like those from the social Hymenoptera.

We understand the comment. In fact, our motivation to include the phylogenetic tree of termite ORs was essentially to demonstrate (i) the orthologous nature of OR diversity with few expansions on low taxonomic levels, and (ii) to demonstrate graphically the relationship among the four selected sequences. We do not attempt here for a comprehensive phylogenetic analysis, because it would be redundant given that we recently published a large OR phylogeny which includes all sequences used in the present manuscript and analysed them in the proper context of related (cockroaches) and unrelated insect taxa (Johny et al., 2023). This paper also discusses the termite phylogenetic pattern with those observed in other Insecta. This paper is repeatedly cited on appropriate places of the present manuscript and its main observations are provided in the Introduction section. Therefore, we feel that thorough discussion on termite phylogeny would be redundant in the present paper.

The authors categorized the sensilla types. Potential problems in the categorization aside, it would be helpful to know if it is expected that you have sensilla specialized in perceiving one specific pheromone. What is known about sensilla in other insects?

We understand. In the discussion of the revised version, we develop more about the features typical/expected for a pheromone receptor and the sensillum housing this receptor together with two other olfactory sensory neurons, including examples from other insects.

As the manuscript currently stands, specialist readers with their respective background knowledge would find this study very interesting. In contrast, the general reader would probably fail to appreciate the importance of the results.

We hope that the re-organized and simplified introduction may now be more intelligible even for non-specialist readers.

(1) L35: Should "workers" be replaced with "worker antennae"?

Corrected.

(2) L62: Should "conservativeness" be replaced by "conservation"?

Replaced with “parsimony”.

(3) L129: How and why did the authors choose four candidate ORs? I could not find any information about this in the manuscript. I wondered why they did not pick the more highly expressed PsimOr20 and 26 (Figure 7).

As already replied above in the Weaknesses section, we selected for the first deorphanization attempts only a modest set of four ORs, while an additional set is currently being tested. We also explained above the inclusion criteria, i.e. (i) full-length ORF and at least 6 unambiguously predicted transmembrane regions, and (ii) presence on different branches (subbranches) of the OR phylogeny. For these reasons, we did not primarily consider the expression patterns of different ORs. As for Fig. 7, it shows differential expression between soldiers and workers, which was not the primary guideline either and the data was obtained only after having the ORs tested by SSR. Yet, even though we had data on P. simplex ORs expression (Fig. 1B), we did not presume that pheromone receptors should be among the most expressed ORs, given the richness of chemical cues detected by worker termites and unlike, e.g., male moths, where ORs for sex pheromones are intuitively highly expressed.

The strategy of OR selection is specified in the results section of the revised manuscript under “Phylogenetic reconstruction and candidate OR selection”.

(4) 198 to 200: SI, II, and III look very similar. Additional measurements rather than qualitative descriptions are required to consider them distinct sensilla. The bending of SIII could be an artifact of preparation. I do not see how the authors could distinguish between SI and SII under the optical microscope for recordings. A detailed explanation is required.

As we responded above in “Weaknesses” chapter, we admit that the sensilla classification is not intelligible. Therefore, we decided in the revised version to abandon the classification of sensilla types and only focus on the observations made on the neocembreneresponding sensillum. To recognize the specific sensillum, we used its topology on the last antennal segment. Because termite antennae are not densely populated with sensilla, it is relatively easy to distinguish individual sensilla based on their topology on the antenna, both in optical microscope and SEM photographs. The modifications affect Fig. 4, its legend and the corresponding part of the results section (Identification of P. simplex olfactory sensillum responding to neocembrene).

(5) 208: "Than" instead of "that"

Corrected.

(6) 280: I suggest replacing "demand" with "capabilities"

Corrected.

(7) 312: Why "nevertheless? It sounds as if the authors suggest that there is evidence that ORs are not important for communication. This should be reworded.

We removed “Nevertheless” from the beginning of the sentence.

(8) 321 to 323: This sentence sounds as if something is missing. I suggest rewriting it.

This sentence simply says that empty neuron Drosophila is a good tool for termite OR deorphanization and that termite ORs work well Drosophila ORCo. We reworded the sentence.

(9) 323: I suggest starting a new paragraph.

Corrected.

(10) 421: How many colonies were used for each of the analyses?

The data for this manuscript were collected from three different colonies collected in Cuba. We now describe in the Materials and Methods section which analyses were conducted with each of the colonies.

(11) 430: Did the termites originate from one or multiple colonies and did the authors sample from the Florida and Cuba population?

The data for this manuscript were collected from three different colonies collected in Cuba. We now describe in the Materials and Methods section which analyses were conducted with each of the colonies.

(12) 501: How was the termite antenna fixated? The authors refer to the Drosophila methods, but given the large antennal differences between these species, more specific information would be helpful.

Understood. We added the following information into the Methods section under “Electrophysiology”: “The grounding electrode was carefully inserted into the clypeus and the antenna was fixed on a microscope slide using a glass electrode. To avoid the antennal movement, the microscope slide was covered with double-sided tape and the three distal antennal segments were attached to the slide.”

(13)509: I want to confirm that the authors indicate that the outlet of the glass tube with the airstream and odorant is 4 cm away from the Drosophila or termite antenna. The distance seems to be very large.

Thank you for spotting this obvious mistake. The 4 cm distance applies for the distance between the opening for Pasteur pipette insertion into the delivery tube, the outlet itself is situated approx. 1 cm from the antenna. This information is now corrected.

(14) 510/527: It looks like all odor panels were equally applied onto the filter paper despite the difference in solvent (hexane and paraffin oil). How was the solvent difference addressed?

In our study we combine two types of odorant panels. First, we test on all four studied receptors a panel containing several compounds relevant for termite chemical communication including the C12 unsaturated alcohols, the diterpene neocembrene, the sesquiterpene (3R,6E)-nerolidol and other compounds. These compounds are stored in the laboratory as hexane solutions to prevent the oxidation/polymerization and it is not advisable to transfer them to another solvent. In the second step we used three additional panels of frequently occurring insect semiochemicals, which are stored as paraffin oil solutions, so as to address the breadth of PsimOR14 tuning. We are aware that the evaporation dynamics differ between the two solvents but we did not have any suitable option how to solve this problem. We believe that the use of the two solvents does not compromise the general message on the receptor specificity. For each panel, the corresponding solvent is used as a control. Similarly, the use of two different solvents for SSR can be encountered in other studies, e.g. 10.1016/j.celrep.2015.07.031.

(15) 518: delta spikes/sec works for all tables except for the wild type in Table S5. I could not figure out how the authors get to delta spikes/sec in that table.

Thank you for your sharp eye. Due to our mistake, the values of Δ spikes per second reported in Table S5 for W1118 were erroneously calculated using the formula for 0.5 sec stimulation instead of 1 sec. We corrected this mistake which does not impact the results interpretation in Table S5 and Fig. 2.

522: Did the workers and soldiers originate from different colonies or different populations?

We now clearly describe in the Material and Methods section the origin of termites for different experiments. EAG measurements were made using individuals (workers, soldiers) from one Cuban colony.

(16) Figure 6C/D: I suggest matching colors between the two figures. For example, instead of using an orange circle in C and a green coloration of the intracellular flap in D, I recommend using blue, which is not used for something else. In addition, the binding pocket could be separated better from anything else in a different color.

We agree that the color match for the intracellular flap was missing. This figure is now reworked and the colors should have a better match and the binding region is better delineated.

(17) Figure 7/Table S15: It is unclear where the transcriptome data originate and what they are based on. Are these antennal transcriptomes or head transcriptomes? Do these data come from previous data sets or data generated in this study? Figure 7 refers to heads, Table S15 to workers and soldiers, and the methods only refer to antennal extractions. This should be clarified in the text, the figure, and the table.

We admit that the replicate numbers and origin of the RNA seq data should be better specified and that the information that the RNASeq originated from samples of heads+antennae of workers and soldiers should be provided at appropriate places. Therefore, we added more information on replicates and origin of the data in the Methods section (Bioinformatics) and make clear that this data comes from our previous research and refer to the corresponding bioproject. Likewise, the Figure 7 legend and Table S15 heading have been updated.

Compte rendu détaillé : La justice face aux violences sexuelles, entre tradition punitive et voie restaurative

• Ce compte rendu explore les principaux thèmes et idées abordés lors de l'émission "Les matins de France Culture" avec Antoine Garapon, magistrat honoraire et président de la commission reconnaissance et réparation, et Aude Douinge, chargée de plaidoyer et de communication de l'association "Face à l'Inceste".

La discussion se focalise sur les limites de la justice punitive traditionnelle face aux crimes de violences sexuelles, en particulier l'inceste, et propose des alternatives telles que la justice restaurative et des évolutions législatives.

1. La nature et l'ampleur des crimes sexuels, en particulier l'inceste

• Les intervenants soulignent l'ampleur effrayante des violences sexuelles, notamment sur les enfants.

Antoine Garapon mentionne le chiffre de "160 000 enfants subissent des violences sexuelles chaque année" en France, une statistique qu'il met en perspective avec les 1600 homicides annuels, soulignant que les violences sexuelles sont "10 000 fois plus" fréquentes.

Ces crimes sont caractérisés par :

• L'identité de l'agresseur : Majoritairement des hommes, souvent majeurs. Les pères (27%), les frères (19%) et les oncles (13%) sont fréquemment cités comme agresseurs.

• Leur nature "fondatrice" et paradoxale : Antoine Garapon les décrit comme des crimes "réputés les plus graves, les plus fondateurs", mais paradoxalement "les moins condamnés, étaient même les moins dénoncés".

L'exemple des crimes sexuels commis par des prêtres est particulièrement mis en avant, car une institution qui doit annoncer le salut "sème la mort", ce qui est une contradiction totale.

• L'inimaginable et le "système du silence" : Pendant longtemps, ces crimes étaient considérés comme "au-delà du périmètre de ce qu'on était prêt à croire".

Un "système du silence" prévalait, souvent lié à un "conflit de loyauté", où la loyauté envers l'institution (comme l'Église) ou la famille était "supérieure à au crédit porté à un enfant".

L'affaire de l'Abbé Pierre est citée comme un exemple criant où "tout le monde savait" mais les autorités n'ont pas agi, abordant le crime uniquement par rapport à la loi morale, "pas un mot pour les victimes".

• La notion de "pharmakos" : La victime, appartenant au vocabulaire sacrificiel, était perçue comme "l'objet du sacrifice".

La thèse audacieuse de Dorothée Dussy, partagée par Garapon, suggère que les enfants victimes étaient en quelque sorte "le prix de l'ordre familial, de l'ordre ecclésial", participant par leur silence à l'ordre social général.

2. L'évolution de la "conscience commune" et le rôle du mouvement #MeToo

La perception de ces crimes a radicalement évolué. Reprenant la définition de Durkheim, qui définit le crime comme "ce qui choque la conscience commune", Antoine Garapon affirme qu'aujourd'hui, "ces crimes sont considérés comme étant les plus choquants dans la conscience générale. Peut-être même plus que les homicides".

• Cette évolution est attribuée à une période de "rêve d'une société postsacrificielle" et, de manière significative, au mouvement " #MeToo" qui a marqué "un grand tournant" en montrant une évolution de la sensibilité.

La société ne supporte plus que des dominés (enfants, femmes) soient l'objet de violences impunies, d'autant plus que le viol est quasi équivalent au crime en termes de répression pénale.

3. Les limites de la justice pénale traditionnelle et les souffrances des victimes

La justice pénale traditionnelle, bien qu'essentielle, montre ses limites :

• Centrée sur le coupable et l'ordre public : Elle est "très centrée sur le coupable, sur l'ordre public", plutôt que sur la victime.
• La "thérapie judiciaire" : L'expression "c'est de la thérapie judiciaire" était utilisée par certains magistrats pour déprécier l'intérêt porté aux victimes, sous-entendant que le rôle du juge n'était pas de s'occuper du rétablissement des personnes.

Cependant, Antoine Garapon soutient que "s'intéresser au rétablissement des personnes à commencer par celui de la victime, c'est de la justice".

• Difficulté d'accès à la plainte et amnésie traumatique : Les victimes souffrent d'un "empêchement d'être" et d'une "impossibilité même d'accéder à la plainte, même d'accéder à son propre souvenir".

L'"amnésie traumatique" peut durer des années, empêchant même la conscience des faits.

• Le fardeau de la preuve : Il est "très difficile de savoir ce qui s'est passé dans un collège, dans un dortoir d'un collège, dans un confessionnal, dans une famille il y a 30 ou 40 ans".

Les aveux de l'auteur restent souvent la preuve maîtresse.

• Impact dévastateur sur les victimes : Une agression sexuelle peut "détruire" une victime, et savoir que son agresseur est "couvert de gloire", "un saint homme", révolte encore plus.
• La reproduction des violences : Les auteurs de violences incestueuses ou sexuelles ont souvent eux-mêmes été abusés (au minimum la moitié des cas), créant un "engrenage" et un "climat incestuel" dans certaines familles.
• Santé mentale et espérance de vie : Aude Douinge souligne que l'inceste est "profondément traumatisant" et se cumule en moyenne avec "trois ou quatre autres traumatismes dans l'enfance".

Plus le nombre de traumatismes est élevé, plus les conséquences à l'âge adulte sont graves.

Une personne ayant subi deux traumatismes majeurs dans l'enfance a "20 ans d'espérance de vie de moins que la population générale".

Plus de la moitié des victimes d'inceste font ou ont fait une tentative de suicide.

4. La justice restaurative : une alternative centrée sur la victime

Antoine Garapon promeut la justice restaurative comme une "alternative" ou un complément à la justice pénale :

• Centrée sur la victime : Son but est de "rétablir, de réhabiliter la victime" et de lui "restituer sa parole, lui restituer une parole propre et pas une parole toujours déléguée ou substituée comme dans le procès ordinaire".
• Nomination et reconnaissance : Elle vise à ce qu'il y ait une "nomination, c'est-à-dire qu'on nomme les choses. Oui, c'était une reconnaissance. Oui, c'est bien. Le premier des besoins des victimes, c'est que la société reconnaisse". Il s'agit d'une "validation sociale de ce qui s'est passé".
• Objectif de "restituer à une victime l'énergie de vivre" : La justice restaurative est "beaucoup plus dynamique" et vise à libérer la victime de la solitude paralysante.
• Importance de la parole : Elle ne se caractérise pas par la "mise en suspicion systématique de la parole" de la victime, contrairement au processus pénal.
• Non-obligatoire : Aude Douinge insiste sur le fait que la justice restaurative "ne peut être obligatoire", car "on ne peut obliger les victimes au pardon".

5. Les évolutions législatives et les défis de la prescription

Les intervenants abordent les débats actuels autour de la prescription des crimes sexuels :

• L'imprescriptibilité : L'association "Face à l'Inceste" milite pour l'"imprescriptibilité pour les crimes d'inceste et la protection immédiate des enfants". Actuellement, le délai de prescription est de 30 ans après les 18 ans de la victime, soit jusqu'à 48 ans.
• Distinction pénal/civil : Le gouvernement réfléchit à une imprescriptibilité pour la justice civile, permettant des réparations financières, mais à charge pour la victime d'apporter des preuves. Les intervenants estiment que cela ne "prend pas le problème de face" en raison des difficultés de preuve et du risque d'aggraver la souffrance de la victime par un non-lieu.
• La procédure pénale est fondamentale : Aude Douinge souligne que la "réponse pénale reste extrêmement importante et elle doit pouvoir être offerte aux victimes puisqu'il faut rappeler que la prescription, c'est aussi le droit à l'oubli pour l'agresseur".

Elle ajoute que "le sentiment d'intranquillité qui habite la victime lui est à vie" et qu'il devrait "venir hanter l'agresseur".

• Départ de la prescription à la "consolidation" : Une solution juridique proposée serait de faire partir le délai de prescription de la date de "consolidation", c'est-à-dire le moment où le traumatisme est estimé ne plus évoluer, plutôt que de la date des faits. Cependant, la blessure psychique est fluctuante.
• L'abus de bien social comme exemple : L'exemple de l'abus de bien social, imprescriptible à partir de la découverte du délit, est donné comme modèle pour les crimes sexuels.

6. Le rôle des associations et les besoins des victimes

L'association "Face à l'Inceste", créée il y a 25 ans par une victime, Isabelle Aubry, joue un rôle crucial :

• Visibilisation de l'inceste : Leurs sondages ont révélé que "trois enfants par classe ont subi l'inceste" et que cela touche "un Français sur 10, 7,4 millions de Français".
• Combats législatifs : Ils ont milité pour la réintégration du crime d'inceste au code pénal en 2016 et la notion de "solidarité".
• Besoins des victimes : Au-delà de la réponse pénale, les victimes réclament "un soutien psychologique et un soutien indéniablement financier". La prise en charge psychologique est souvent peu soutenue et l'arrêt des thérapies est souvent dû à des raisons financières. Un formulaire pour le remboursement à 100% des soins pour les victimes d'inceste par la sécurité sociale existe mais est "trop peu connu".
• Reconnaissance et réparation : Les victimes ont besoin d'abord et avant tout de "cette reconnaissance et que la société légitime ce qu'elles ont vécu et viennent leur dire oui, ce qui vous est arrivé et a existé et on va le reconnaître".

7. Vers une "autre justice" et la "politisation de l'intime"

Antoine Garapon plaide pour une "autre justice", plus "accomplie", qui intègre différentes facettes :

• Réarticulation des justices : Il appelle à une "réarticulation entre la justice civile, la justice restaurative et la justice pénale".
• "Politisation de l'intime" : Le défi est de savoir "comment les pouvoirs publics vont pouvoir s'emparer de relations intimes intelligemment pour mettre fin à cette ce très très grand nombre, ce trop grand nombre de violences sexuelles".
• Respect des désirs de la victime : Il est crucial de "respecter les désirs de la victime", qu'il s'agisse d'une demande de punition, d'une demande protectrice pour se dégager et vivre dans l'anonymat.
• Les droits de l'auteur : Tout en se concentrant sur la victime, il est rappelé que "l'auteur aussi a des droits" et bénéficie de la présomption d'innocence.

En conclusion, la discussion met en lumière la nécessité d'une approche plus globale et empathique face aux violences sexuelles, qui ne se limite pas à la seule punition de l'agresseur mais qui inclut une reconnaissance profonde de la souffrance des victimes, un soutien adapté, et des mécanismes de réparation qui favorisent leur reconstruction et leur capacité à vivre.

Synthèse : Le Consentement au Cœur du Débat en France

• Ce document explore la notion de consentement, en soulignant son émergence comme un concept central dans le débat public français, notamment sous l'impulsion de mouvements féministes et de procès emblématiques.

Il met en lumière la complexité de cette notion, les défis liés à sa compréhension et son application, ainsi que les efforts déployés pour l'intégrer pleinement dans la loi et les mentalités.

1. Le Consentement : Une Notion Émergente et Centralisée

• Le mouvement féministe et des affaires judiciaires retentissantes ont placé le consentement au premier plan des préoccupations sociétales.

Le procès des viols de Mazan, avec la condamnation de Dominique Pélico pour avoir drogué et violé sa femme pendant dix ans, a été un catalyseur majeur.

Une des personnes interrogées souligne la simplicité apparente mais la profondeur de la notion :

"Quand une fille dit non, j'ai l'impression quand même que souvent ça sous-entend que c'est c'est non. Non.

D'accord. Ah ouais. Ah faut bien c'est pas si simple. Faut bien choper le truc hein. Oui ou non ? Deux petits mots de trois lettres. Mais qui change absolument tout."

Cette prise de conscience a conduit à des appels à inscrire le consentement dans la loi, exigeant que les agresseurs présumés prouvent avoir obtenu un accord explicite avant tout acte sexuel.

Le slogan "Jamais sans mon consentement" est devenu un cri de ralliement dans les cortèges féministes.

2. La Compréhension du Consentement : Défis et Manques

• Malgré son importance croissante, la compréhension du consentement reste un défi, en particulier chez les jeunes.

Pauline, victime de viol à 14 ans par son premier petit ami, témoigne de la difficulté à identifier le viol et à en parler, d'autant plus en l'absence d'éducation sexuelle adéquate : "Je savais pas ce que c'était les rapports.

Donc pour moi c'était un peu la norme entre guillemets... je savais pas trop comment en parler et après j'ai mis du temps avant de d'accepter aussi le terme viol parce que c'est un mot quand même très fort."

Elle évoque aussi l'influence de la pornographie, qui "ne parle pas du tout" du consentement à cet âge.

Les témoignages révèlent que le "non" n'est pas toujours respecté, et que la peur peut paralyser les victimes, comme Elodie qui a été agressée sexuellement à 17 ans : "J'étais tellement peur que c'est comme si j'étais paralysée. J'arrivais pas à crier. J'étais vraiment tétanisée."

3. L'Éducation et la Prévention : Des Outils Essentiels

Face à ces lacunes, des interventions en milieu scolaire se multiplient. Une gendarme intervient dans un collège pour expliquer le consentement aux élèves de 3ème.

Elle définit l'agression sexuelle comme "le fait de toucher les parties intimes sans consentement, sans son autorisation."

Elle insiste sur la clarté du "oui" ou du "non", verbal ou par des gestes, et surtout, sur le fait qu'en l'absence de réponse, il faut considérer que c'est un "non".

L'importance de parler "sans cacher les mots" est soulignée par la gendarme, car "on a beau dire non du plus plus fort qu'on peut, si l'autre en face n'entend pas, il fera quand même ce qu'il a envie de faire qui est illégal."

Ces interventions sont jugées cruciales, car la discussion sur le consentement est "très peu abordée aussi bien par les parents à la maison qui peuvent être embarrassés... et même les établissements scolaires sont parfois dépourvus de moyens."

4. La Réalité des Violences Sexuelles : Souvent le Fait de Proches

Un point crucial est la démystification de l'image de l'agresseur. Contrairement à l'imaginaire collectif, un violeur n'est pas toujours un inconnu armé : "Dans 90 % des cas, l'agresseur connaît sa victime.

Dans la moitié des cas, c'est son partenaire ou un ex amoureux." De plus, les femmes sont majoritairement les victimes, avec 91% des auteurs de violences sexuelles étant des hommes.

5. La Complexité Juridique et la Subjectivité du Consentement

• Les affaires de viol sont souvent complexes, mêlant souffrances et ressentiments. L'avocat Robin Binsard souligne que la "question de la preuve est toujours au centre des débats" et que la "vérité est parfois plurielle".

Un accusé, qui nie les viols dont il est accusé malgré la condamnation à 7 ans de prison, exprime cette ambiguïté : "La notion de consentement est pour moi acquise...

À aucun moment, ell m'ont elles m'ont dit non clairement." Il ajoute avoir dit à une victime "C'est comme un viol, ce n'en est pas un," illustrant la "limite très fine" de la compréhension.

• La magistrate Genola Jolicose récuse la notion de "parole contre parole", affirmant que le rôle de la cour est de "contextualiser, de comprendre que ça n'est pas simplement une situation qui nous est décrite mais en réalité un système.

Tout ça est adossé à la culture du viol, au patriarcat, à la domination des femmes et c'est ça qui change tout."

6. L'Inscription Légale du Consentement : L'Exemple International

Le débat sur l'inscription du consentement dans la loi française s'inspire de législations étrangères :

• Suède (2018) : Nécessité d'un consentement verbal ou physique.
• Espagne (2022) : Un rapport sexuel sans consentement explicite est un viol ("solo sí es sí").
• Canada : Premier pays à définir le consentement pénalement comme donné "librement et avec enthousiasme, continu, précis, requis pour chaque activité et éclairé". Éléonore Noël, chercheuse en sciences sociales au Canada, explique que cela change tout car l'enjeu principal n'est plus la violence ou la contrainte, mais l'absence de consentement.

7. Changer l'Imaginaire Collectif pour une Culture du Consentement

• Pour lutter contre la "culture du viol" et promouvoir une "culture de consentement", il est essentiel de "développer un imaginaire positif autour du consentement".

Les films et les médias sont critiqués pour leurs représentations stéréotypées où l'insistance masculine est glorifiée et le "non" féminin est souvent interprété comme un "oui" latent.

Des initiatives, comme l'association Sex et Consentement, proposent des supports (cartes postales, préservatifs) avec des messages explicites pour normaliser la demande de consentement.

Les jeunes interrogés y voient un moyen de "nous forcer à réfléchir et à demander à l'autre aussi si elle est d'accord oui ou non."

En conclusion, l'émission souligne une transformation profonde des mentalités et du cadre légal autour du consentement en France, tirant des leçons des expériences individuelles et des législations internationales pour mieux protéger les victimes et éduquer les nouvelles générations.

Reviewer #1 (Public review):

Summary:

In this study, Bonnifet et al. profile the presence of L1 ORF1p in the mouse and human brain and report that ORF1p is expressed in the human and mouse brain specifically in neurons at steady state and that there is an age-dependent increase in expression. This is a timely report as two recent papers have extensively documented the presence of full-length L1 transcripts in the mouse and human brain (PMID: 38773348 & PMID: 37910626). Thus, the finding that L1 ORF1p is consistently expressed in the brain is important to document and will be of value to the field.

Strengths:

Several parts of this manuscript appear to be well done and include the necessary controls. In particular, the documentation of neuron-specific expression of ORF1p in the mouse brain is an interesting finding with nice documentation. This will be very useful information for the field.

Weaknesses:

Several parts of the manuscript appear to be more preliminary and need further experiments to validate their claims. In particular, the data suggesting expression of L1 ORF1p in the human brain and the data suggesting increased expression in the aged brain need further validation. Detailed comments:

(1) The expression of ORF1p in the human brain shown in Fig. 1j is puzzling. Why are there two strong bands in the WB? How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? While the authors discuss that others have found double bands when examining human ORF1p, there are also several labs that report only one band. This discrepancy in the field should at least be discussed and the uncertainties with their findings should be acknowledged.

(2) The data showing a reduction in ORF1p expression in the aged mouse brain is an interesting observation, but the effect magnitude of effect is very limited and somewhat difficult to interpret. This finding should be supported by orthogonal methods to strengthen this conclusion. For example, by WB and by RNA-seq (to verify that the increase in protein is due to an increase in transcription).

(3) The transcriptomic data using human postmortem tissue presented in Figure 4 and Figure 5 are not convincing. Quantification of transposon expression on short read sequencing has important limitations. Longer reads and complementary approaches are needed to study the expression of evolutionarily young L1s (see PMID: 38773348 & PMID: 37910626 for examples of the current state of the art). As presented, the human RNA data is inconclusive due to the short read length and small sample size. The value of including an inconclusive analysis in the manuscript is difficult to understand. With this data set, the authors cannot investigate age-related changes in L1 expression in human neurons.

(4) In line with these comments, the title should be changed to better reflect the findings in the manuscript. A title that does not mention "L1 increase with aging" would be better.

Compte-rendu détaillé : Éducation Populaire et Liens avec l'École

Source : Extraits de "France culture être et savoir Tuerie dans un lycée de Nantes L'éducation populaire, quelles relations avec l'école 11192-28.04.2025-ITEMA_24116950-2025C14993S0118-NET_MFC_1282E914-3484-4849-8E09-1FE806076BE5-21.mp3"

Introduction : Un événement tragique comme point de départ et la nécessité de l'éducation populaire

L'émission s'ouvre sur le rappel d'une attaque au couteau survenue le 24 avril dans un lycée privé de Nantes, Notre Dame de toutes aide, où un élève de seconde a poignardé quatre camarades, causant la mort d'une lycéenne.

L'agresseur, Justin P., 16 ans, était inconnu des services de police et ses camarades le décrivent comme "un jeune homme perturbé".

Suite à cet événement, la sociologue Nathalie Paton, spécialiste des school shootings aux États-Unis, intervient pour commenter les réactions politiques, notamment la proposition du Premier ministre d'installer des portiques de sécurité.

Elle juge cette mesure "démesurée et presque légèrement délirante" dans le contexte français, soulignant que les school shootings sont un phénomène isolé en France, contrairement aux États-Unis où ils sont quotidiens et où de telles mesures n'ont pas prouvé leur efficacité, pouvant même générer un sentiment d'insécurité.

L'analyse des motivations de l'agresseur tend vers une "belle psychose" et un "délire", comme en témoigne un manifeste mêlant des références disparates (Hitler, Écoid).

Nathalie Paton souligne l'importance d'une approche psychiatrique pour comprendre cet acte, soulignant que le jeune homme était "clairement très mal, très délirant" et que son acte a été un "passage à l'acte" débordant d'une "grande angoisse".

Thème central : Le sous-financement de la pédopsychiatrie et de la médecine scolaire

Le cas de Nantes met en lumière les graves lacunes de la prise en charge de la santé mentale des jeunes en France. Nathalie Paton insiste sur l'état "extrêmement préoccupant" de la pédopsychiatrie française et le "délaiement" de la médecine scolaire.

Elle s'interroge sur l'absence de repérage et de prise en charge préalable de l'agresseur : "Qu'est-ce qui fait que il n'avait pas été pris en charge avant ?

Ça ça paraît difficile de penser que ça allait déborder pour la première fois ce jour-là."

Elle dénonce le manque de psychologues scolaires et le fait que la psychiatrie soit considérée comme une "médecine pauvre" par les politiques publiques, manquant cruellement de "politiques et d'investissements".

Cette première partie de l'émission sert de tremplin pour aborder le rôle crucial de l'éducation populaire dans la construction du lien social et la prévention, en complément de l'école.

L'Éducation Populaire : Histoire, Valeurs et Fonctions L'émission explore ensuite en détail le monde de l'éducation populaire, souvent invisible mais pourtant essentiel pour deux tiers des enfants et adolescents français (périscolaire, centres de loisirs, colonies de vacances, activités sportives et artistiques).

1. Fondements et mission historique : Former le citoyen éclairé

• Hélène Lacassagne, présidente de la Ligue de l'enseignement (créée en 1866 par Jean Macé), souligne la vocation profondément politique et républicaine de l'éducation populaire : "Les fondements même sont des fondements tout à fait républicains.

Il s'agit de faire en sorte que le vote populaire soit pas ne soit pas détourné parce que parce que ce vote populaire ne serait pas éclairé."

L'objectif est de "favoriser la création d'une école d'une école publique laïque" et de "former les citoyens pour que la démocratie s'exerce vraiment dans la République."

La Ligue agit "un mouvement complémentaire de l'école publique et elle agit y compris au sein de l'école publique."

2. Une éducation "au côté ou à côté de l'école" : Complémentarité et différences

L'éducation populaire se positionne en complément de l'école, mais avec des approches différentes. Wahid Ben Hamed, directeur du centre de formation des CEMÉA Île-de-France, insiste sur la nature des métiers de l'éducation populaire : "C'est des métiers du lien social.

C'est des métiers de la cohésion sociale." Il met en avant la dimension collective de l'apprentissage : "On apprend ensemble on apprend lorsqu'on se met autour d'objets communs."

Distinction fondamentale : L'absence de jugement et de compétition

Une différence majeure avec l'école est l'absence de jugement et d'évaluation. Laurent Bess, maître de conférence en histoire contemporaine, explique que "les animateurs par principe refusent de juger que ce soit les pratiques ou les réalisations des enfants alors que bah l'enseignant, il dit ce qui est vrai, ce qui est faux, ce qui est juste, ce qui est bon."

Cette approche favorise une "volonté de conserver la cohérence du groupe" en "abolissant ce jugement qui crée effectivement des différences entre les enfants."

Wahid Ben Hamed renchérit en affirmant : "C'est pas un concours, c'est jamais c'est ce qui différencie par exemple de la profession d'enseignant."

Pour lui, l'enjeu est de "réinterroger les représentations du groupe" pour "favoriser l'émancipation".

Il cite l'exemple du sport où l'on peut "imaginer autre chose" que le simple fait de gagner ou de perdre.

3. L'évolution de l'éducation populaire : Des cours du soir aux loisirs émancipateurs

Laurent Bess retrace l'histoire de l'éducation populaire, situant son "âge d'or" entre l'entre-deux-guerres et les années 1970.

Si au 19ème siècle, elle était davantage centrée sur des modèles scolaires (cours du soir), elle se transforme dans l'entre-deux-guerres autour de la "démocratisation des loisirs", visant à permettre aux enfants des milieux populaires d'accéder à de nouvelles pratiques (artistiques, sportives, plein air).

Des instituteurs ont d'abord encadré ces activités via les "œuvres laïques", avant d'être progressivement remplacés par des professionnels, les "animateurs socioculturels".

Aujourd'hui, l'accent est mis sur "l'aspect non scolaire de l'éducation populaire sur la reconnaissance des individus l'accent mis sur des relations qui se veulent horizontales des pratiques qui se veulent ludiques qui visent à former toujours."

Bien que l'ambition de former le citoyen demeure, le contenu politique est "moins mise en avant".

4. Le rôle crucial du "vivre ensemble" et de la "transformation sociale"

Patricia Ménard, directrice du périscolaire pour l'école du Four au sein de la Fondation Léo Lagrange (fondée en 1936), insiste sur les valeurs de son institution : "le vivre ensemble, la découverte et l'épanouissement de l'enfant et la mixité culturelle."

Elle définit le "vivre ensemble" comme "partager, c'est être ensemble, essayer de comprendre les autres, c'est vivre ensemble en tant que citoyen aussi sur un dans le loisir au sein de l'école, d'avoir les mêmes règles de l'école et du loisir, c'est être un enfant parmi toute une collectivité et être à plusieurs pour être bien en fait."

• Mohamed Magassa, coordinateur au centre de ressources documentaires des CEMÉA Île-de-France et président de l'association Reconnectus, met en avant l'importance de "rendre acteurs" les jeunes, de les "accompagner sur ces actions" pour qu'ils "s'approprient l'émancipation".

Il souligne que l'éducation populaire vise la "transformation sociale", en "essayant d'ouvrir une porte et de s'approprier en fait ce qu'on lui propose."

Défis et Perspectives de l'Éducation Populaire

1. La précarité des financements et ses conséquences

La question du financement est jugée "cruciale" par Hélène Lacassagne. Mohamed Magassa explique que son association dépend "systématiquement" de "subventions" et "d'appels à projet".

Hélène Lacassagne déplore que les appels à projet et les marchés publics se soient "substitués à la subvention", ce qui pose un "une vraie difficulté parce que le diagnostic n'est plus porté par l'association."

Elle regrette que cela mette en danger la "capacité d'innovation" des associations, autrefois moteurs de dispositifs comme les bibliobus.

• Elle critique également le fait que le système actuel "met en concurrence les associations là où naturellement quand on travaille sur un territoire... le travail de l'éducation populaire, c'est de mettre en réseau, c'est de construire du projet sur un diagnostic partagé avec d'autres associations, d'autres acteurs."

Ce modèle, qui exige du temps, est menacé par des politiques publiques qui ne "rencontrent pas les personnes pour lesquelles elle a été inventée", car le "dernier kilomètre, c'est le premier" pour les acteurs de terrain.

2. Le défi de l'attractivité des métiers et de l'innovation pédagogique

Les métiers de l'éducation populaire sont "pas très bien payés".

La motivation des professionnels comme Cyriel, une animatrice Léo Lagrange qui a créé l'atelier "raconte-toi", réside dans le sens de leur travail : "Je n'ai pas l'impression d'aller au travail.

En fait tous les jours, on a une situation différente et moi je trouve que c'est une chance de pouvoir leur transmettre des valeurs et les écouter."

Wahid Ben Hamed insiste sur "l'innovation pédagogique" au sein des centres de formation des CEMÉA, qui accueillent de nombreux jeunes ayant "une méfiance et une réticence au fait d'apprendre" suite à un "échec" ressenti vis-à-vis de l'Éducation Nationale.

L'approche des CEMÉA est non-verticale : "on part du principe que les gens qui sont ici et les apprenants ont des choses à nous apprendre nous à formateur en tant que formateur. Ils ont des des choses à apprendre au groupe qui est là."

L'exemple de la "Newton Room" au collège Jean-Mermoz d'Angers, un atelier scientifique scandinave, illustre cette volonté d'innover pour rendre les mathématiques "concrètes" et offrir des outils de qualité.

Ce type de partenariat vise à valoriser l'école publique et à lui donner une "étiquette" pour "exister sur des des établissements qui ont pignon sur rue" (privés).

3. Accueillir tous les publics et déconstruire les sujets sensibles

• L'éducation populaire s'adresse à l'ensemble de la population, y compris les "milieux populaires" qui ressentent un fort "sentiment de relégation".

Hélène Lacassagne souligne la nécessité d'une approche qui ne soit pas seulement "prestataire" mais qui permette de "recréer une relation, de remettre les de faire vraiment éducation populaire, c'est-à-dire de mettre les personnes en situation, de porter l'action, d'être non pas dans une relation de de consommation d'une action, mais d'être associé au diagnostic, au faire et à l'évaluation de la chose de façon à ce que les personnes se sentent reconnu en égale dignité avec les autres citoyennes, les autres citoyens."

Mohamed Magassa explique comment son association Reconnectus aborde les "questions vives" avec les jeunes.

Ces derniers "ramènent en fait les sujets qu'ils avaient entendu à l'école pour les déconstruire avec nous", abordant par exemple la discrimination avant la laïcité.

Leur propre expérience de la discrimination leur permet de mieux accompagner les jeunes : "le sujet de la laïcité s'impose à travers la discrimination."

Conclusion

• L'émission met en lumière la fragilité de la pédopsychiatrie et de la médecine scolaire en France, des lacunes qui peuvent avoir des conséquences dramatiques comme le cas de Nantes.

Face à cela, l'éducation populaire apparaît comme un pilier essentiel, bien que souvent sous-estimé et sous-financé.

Son rôle complémentaire de l'école, axé sur le lien social, l'émancipation individuelle et collective, et l'absence de jugement, en fait un acteur clé pour répondre aux besoins des jeunes et des familles.

Cependant, la pérennité et la capacité d'innovation de l'éducation populaire sont menacées par les modes de financement actuels, qui entravent la co-construction de projets adaptés aux réalités du terrain et au "premier kilomètre" des citoyens.

Le plaidoyer des intervenants est clair : reconnaître et soutenir davantage ce secteur pour qu'il puisse continuer à former des citoyens éclairés et à renforcer le tissu social.

Author response:

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

Reviewer 1:

Comment 1: Indirect Estimates of White Matter Connections: While dMRI is a valuable tool, it inherently provides indirect and inferred information about neural pathways. The accuracy and specificity of tractography can be influenced by various factors, including fiber crossing, partial volume effects, and algorithmic assumptions. A potential limitation in the accuracy of indirect estimates might affect the precision of spatial extent measurements, introducing uncertainty in the interpretation of cortico-thalamic connectivity patterns. Addressing the methodological limitations associated with indirect estimates and considering complementary approaches could strengthen the overall robustness of the findings.

We appreciate the reviewer’s comment and agree tractography is an indirect estimate and subject to limitations. Regarding this manuscript, the key question is not whether the anatomical tracts are without false positives or negatives, and in fact we argue that this question is outside the scope of this manuscript and has been addressed in several previous studies (e.g. Thomas et al. 2015, Schilling et al., 2020, Grisot et al. 2021, and many others). Instead, the key question for this manuscript is whether the focality of termination patterns within the thalamus is systematically biased in a way that the observation of a hierarchy effect is artifactual. The many supplementary analyses in this manuscript do help address this question and increase our confidence that the indirect nature of tractography does not systematically bias the EDpc1 measure such that association areas only appear to have more diffuse connectivity patterns relative to sensorimotor areas.

Comment 2: An over-arching theme of my review is that, each time I found myself wondering about a detail, a null, or a reference, I had only to read the next sentence or paragraph to find my concern handled in a clear and concise fashion. This is, in my opinion, the mark of work of the highest order. I congratulate the authors on their excellent work, which I believe will be impactful and well-received.

I have no notes that I feel can help improve what is already an impeccable piece of work.

We thank the reviewer for the kind comment.

Reviewer #2:

Comment 1: Structural thalamocortical connectivity was estimated from diffusion imaging data obtained from the HCP dataset. Consequently, the robustness and accuracy of the results depend on the suitability of this data for such a purpose. Conducting tractography on the cortical-thalamic system is recognized as a challenging endeavor for several reasons. First, diffusion directions lose their clearly defined principal orientations once they reach the deep thalamic nuclei, rendering the tracking of structures on the medial side, such as the medial dorsal (MD) and pulvinar nuclei difficult. Somewhat concerning is those are regions that authors found to show diffuse connectivity patterns. Second, the thalamic radiata diverge into several directions, and routes to the lateral surface often lack the clarity necessary for successful tracking. It is unclear if all cortical regions have similar levels of accuracy, and some of the lateral associative regions might have less accurate tracking, making them appear to be more diffuse, biasing the results.

As mentioned in the weakness section, it is crucial to address the need for better validation or the inclusion of control analyses to ensure that the results are not systematically biased due to known issues, such as the difficulty in tracking the medial thalamus and the potential for higher false positives when tracking the lateral frontal cortex.

We thank that reviewer for bringing up an important point. To determine if some areas of the thalamus were more difficult to track and, in turn, biased the EDpc1 measure we added an additional supplemental figure (S31). In this figure, shown below, we calculate the total SC of all ipsilateral cortical areas to each thalamic voxel. We show that, indeed, medial thalamic voxels have a lower total streamline count to ipsilateral cortex, and we see reduced total streamline counts to lateral thalamic areas and the very posterior end of the thalamus. We determined if some cortical areas preferentially projected to parts of the thalamus with lower ipsilateral total SC (i.e. by calculating the overlap between SC and total cortical SC for each thalamic voxel) and found only a weak relationship with our measure. Furthermore, we regressed each voxel’s mean ipsilateral cortical SC from streamline count matrix. We found that the EDpc1 measure didn’t significantly change after the regression.

Additionally, we note that this analysis assumes that all thalamic voxels should have equal strength of connectivity (i.e., total SC) to the ipsilateral cortex and that such a measure is a proxy for “accuracy.” While both of these assumptions may not be entirely valid, this figure does demonstrate that potential reductions in tracking from the medial thalamus does not significantly affect the EDpc1 measure.

Comment 2: While the methodology employed by the authors appears to be state-of-the-art, there exists uncertainty regarding its appropriateness for validation, given the well-documented issues of false positives and false negatives in probabilistic diffusion tractography, as discussed by Thomas et al. 2014 PNAS. Although replicating the results in both humans and non-human primates strengthens the study, a more compelling validation approach would involve demonstrating the method's ability to accurately trace known tracts from established tracing studies or, even better, employing phantom track data. Many of the control analyses the authors presented, such as track density, do not speak to accuracy.

In addition to or response to Reviewer 1 Comment 1, we would like to add the following:

We agree with the reviewer that tractography methods have known limitations. We would also like to point out that several studies have already performed the studies suggested by the reviewer. Many studies have compared tracts reconstructed from diffusion data using tractography methods to tracer-derived connections (eg. Thomas et al., 2014, as mentioned by the reviewer; Donahue et al., 2016, J Neurosci; Dauguet et al., 2007 NeuroImage; Gao et al., 2013 PloS One; van den Heuvel et al., 2015, Hum Brain Map; Azadbakht et al., 2015 Cereb Cortex; Ambrosen et al., 2020 NeuroIamge). Notably, studies comparing tractography and tracer-derived white matter tracts in the same animal (e.g. Grisot et al., 2021; Gao et al., 2013 PloS One) have demonstrated that tractography errors may be inflated in studies comparing tractography and tracer-derived connections in different animals.

Additionally, others have employed phantoms to assess the validity of tractography methods (e.g. Drobnjak et al., 2021). For the purposes of this manuscript, phantom data would not be an adequate control because phantom data would likely not capture the biological complexities of tracking subcortical white matter tracts and identifying projections within subcortical grey matter.

While a comparison of our tractography-derived ED measure to ED calculated on terminations from tracer studies within the thalamus from several somatomotor and associative regions in macaques would provide additional confidence for our results, such a control is certainly outside the scope of this study. Additionally, such a study would not provide a ground truth comparison for the human data. Even if this hypothetical experiment was performed, a negative finding would not refute our results, as any differences could be attributed to evolutionary differences. Unfortunately, there exists no ground truth to compare human white matter connectivity patterns to, which is why we stress-tested our results in as many ways as possible. These stress tests revealed that our main findings are very robust.

Specifically, as the key validity question of our study was whether there was a confound that systematically biased the ED measure as to make the hierarchy effect artifactual, the control analyses we performed to determine if track density, cortical geometry, bundle integrity, etc in fact do speak the robustness of the results. Regarding the track density analyses we argue that these control analyses do speaks to accuracy. The reviewer mentioned above that some cortical areas may be biased because their anatomical tracts may be more difficult to reconstruct using tractography. The mean streamline count is meant to reflect the density of a fiber bundle, but corticothalamic tracts that are more difficult to track will, by nature, have fewer streamline counts. So, the mean streamline not only reflects the density of a fiber bundle but also how easily that tract is to reconstruct. Therefore, if it was the case that cortical areas with more difficult to reconstruct white matter tracts to the thalamus are also more diffuse, then we should observe a strong positive correlation between the ED measure and the mean streamline count, which we tested directly and found only a weak correlation (Fig. S11). This is true for tracking to the entire thalamus, and the additional supplemental Figure S31 shows that reduced tracking to specific parts of the thalamus (e.g. the medial portion) also does not strongly relate to the ED measure. So, tracts that are more difficult to reconstruct may also be more diffuse, but this seems to add only a little noise and does not account for the strong relationship between the ED measure and T1w/T2w and RSFCpc1 measures the reflect the cortical hierarchy.

Comment 3: If tracking the medial thalamus is indeed less accurate, characterized by higher false positives and false negatives, it could potentially lead to increased variability among individual subjects. In cases where results are averaged across subjects, as the authors have apparently done, this could inadvertently contribute to the emergence of the "diffuse" motif, as described in the context of the associative cortex. This presents a critical issue that requires a more thorough control analysis and validation process to ensure that the main results are not artifacts resulting from limitations in tractography.

Additionally, conducting a control analysis to demonstrate that individual variability in tracking endpoints within the thalamus, when averaged across subjects, does not artificially generate a more diffuse connectivity pattern, is essential.

We thank the reviewer for bringing up this point, and the reviewer is correct that a simple group average of streamline counts across that thalamus could make some thalamic patterns appear more diffuse if those patterns vary slightly in location across people. The simplest way to address this concern is to show that diffuse patterns are present in individual subjects. Fig. 2 panels B, C, H, and I are all subject-level figures, which show that we can replicate the group level findings in Fig. 2 panels F, G. Specifically, Fig 2. Panels H and I show that the effect of association areas exhibiting more diffuse connectivity patterns within the thalamus relative to sensorimotor areas is generalizable across subjects.

To the reviewer’s point, the other way that averaged streamline counts could make focal connections seem diffuse is by averaging within cortical areas (e.g. to test the possibility that association areas may have highly variability focal patterns, and when averaged within the cortical area it makes these focal patterns appear more diffuse). To test this, we show that we can replicate the hierarchy effect at the vertex level, by calculating the extent of connectivity patterns for every cortical vertex and correlated vertex-level EDpc1 values to vertex-level T1w/T2w and RSFC_pc1 values (Fig S20).

Hopefully the data shown in Fig. 2 (replication at the individual level) and Fig. S20 (replication at the vertex level) ameliorate the reviewer’s concerns that averaging highly variable focal connectivity patterns within the thalamus (either across people or across vertices) does not artifactually produce diffuse thalamic connectivity patterns for associative cortical areas.

Comment 4: Because the authors included data from all thresholds, it seems likely that false positive tracks were included in the results. The methodology described seems to unavoidably include anatomically implausible pathways in the spatial extent analyses.

The thresholding approach taken in the manuscript aimed to control for inter-areal differences in anatomical connection strength that could confound the ED estimates. Here I am not quite clear why inter-areal differences in anatomical connection strength have to be controlled. A global threshold applied on all thalamic voxels might kill some connections that are weak but do exist. Those weak pathways are less likely to survive at high thresholds. In the meantime, the mean ED is weighted, with more conservative thresholds having higher weights. That being said, isn't it possible that more robust pathways might contribute more to the mean ED than weaker pathways?

This is a good point from the reviewer, and we appreciate them bringing up these points about our thresholding rationale. We would like to clarify two points: why it was appropriate for our question to threshold thalamic voxels for each cortical area separately and why we iteratively thresholded thalamic voxels.

Regarding thalamic connectivity differences between cortical areas: a global threshold would indeed exclude weak, but potentially true, connections. This was part of our rationale for thresholding thalamic voxels for each cortical area separately. Too conservative of a global threshold would exclude all thalamic voxels for some cortical areas and too liberal of a threshold would include many potentially false positive connections for other cortical areas. Our method of thresholding each cortical area’s thalamic voxels separately ensured that we were sampling thalamic voxels in an equitable manner across cortical areas. We updated the text to clarify this:

Methods section, pg. 11, section Framework to quantify the extent of thalamic connectivity patterns via Euclidean distance (ED)

“We used Euclidean distance (ED) to quantify the extent of each cortical area's thalamic connectivity patters. Probabilistic tractography data require thresholding before the ED calculation. To avoid the selection of an arbitrary threshold (Sotiropoulos et al., 2019, Zhang et al., 2022), we calculated ED for a range of thresholds (Figure 1a). Our thresholding framework uses a tractography-derived connectivity matrix as input. We iteratively excluded voxels with lower streamline counts for each cortical parcel such that the same number of voxels was included at each threshold. At each threshold, ED was calculated between the top x\% of thalamic voxels with the highest streamline counts. This produced a matrix of ED values (360 cortical parcels by 100 thresholds). This matrix was used as input into a PCA to derive a single loading for each cortical parcel. While alternative thresholding approaches have been proposed, this framework optimizes the examination of spatial patterns by proportionally thresholding the data, enabling equitable sampling of each cortical parcel's streamline counts within the thalamus.

This approach controlled for inter-areal differences in anatomical connection strength that could confound the ED estimates. In contrast, a global threshold, which is applied to all cortical areas, may exclude all thalamic streamline counts for some cortical areas that are more difficult to reconstruct, thus making it impossible to calculate ED for that cortical area, as there are no surviving thalamic voxels from which to calculate ED. This would be especially problematic for white matter tracts are more difficult to reconstruct (e.g. the auditory radiation), and cortical areas connected to the thalamus by those white matter tracts would have a disproportionate number of thalamic voxels excluded when using a global threshold.”

Regarding thalamic connectivity differences across the thalamus for a given cortical area, the thresholding method we use does include anatomically implausible connections in the ED calculation because we sample voxels iteratively, and as more and more thalamic voxels are included in the ED analysis the likelihood that they reflect spurious connections increases. This approach made the most sense to us, because there is no way to identify a threshold that only includes true positive connections. And since this method does not exist, we sampled all thresholds and leveraged the behavior of the ED metric across thresholds to quantify the spread of a connectivity pattern. As the reviewer points out, since the measure is effectively “weighted,” more “robust” or anatomically plausible pathways should contribute more to the EDpc1 rather than weaker pathways. This is exactly the balanced approach we aimed for: a measure that is driven by connections that have the highest likelihood of being a true positive but does not rely on an arbitrary threshold.

We did also replicate our main findings after thresholding and binarizing the data for separate thresholds, which show that our main effect was strongest only when thalamic voxels with the highest streamline counts (which are assumed to have a lower chance of being false positives) are included in the ED calculation (Fig. S5). This more traditional method of thresholding also supported our results, and increases our overall confidence that associative cortical areas have more diffuse connectivity patterns within the thalamus relative to somatomotor areas.

Comment 5: In the introduction, there is a bit of ambiguity that needs clarification. The overall goal of the study appears to be the examination of anatomical connectivity from the cortex to the thalamus, specifically whether a cortical region projects to a single thalamic subregion or multiple thalamic subregions. However, certain parts of the introduction also suggest an exploration of the concept of thalamic integration, which typically means a single thalamic region integrating input from multiple cortical regions (converging input). These two patterns, many cortical regions to one thalamic region versus one cortical region to many different thalamic regions, represent distinct and fundamentally different concepts that should be clarified in the manuscript.

We thank the reviewer for pointing out this ambiguity and have edited the introduction to clarify this point:

Our argument for a potential mechanism for integration is the following: because corticothalamic connectivity is topographically organized, if a cortical area has a more diffuse anatomical projection across the thalamus that means its connections overlap with more cortical areas. To the reviewer’s point, our argument is simply that one cortical area targeting multiple thalamic nuclei inherently suggests that such a cortical area has overlapping connectivity patterns with many other cortical areas in the same thalamic subregion. We have updated the introduction to clarify this further.

Intro, pg 1.

“Studies of cortical-thalamic connectivity date back to the early 19th century, yet we still lack a comprehensive understanding of how these connections are organized (see 13 and 14 for review). The traditional view of the thalamus is based on its histologically-defined nuclear structure (6). This view was originally supported by evidence that cortical areas project to individual thalamic nuclei, suggesting that the thalamus primarily relays information (15). However, several studies have demonstrated that cortical connectivity within the thalamus is topographically organized and follows a smooth gradient across the thalamus (16–21). Additionally, some cortical areas exhibit extensive connections within the thalamus, which target multiple thalamic nuclei (22? ). These extensive connections may enable information integration within the thalamus through overlapping termination patterns from different cortical areas, a key mechanism for higher-order associative thalamic computations (23– 25). However, our knowledge of how thalamic connectivity patterns vary across cortical areas, especially in humans, remains incomplete. Characterizing cortical variation in thalamic connectivity patterns may offer insights into the functional roles of distinct cortico-thalamic loops (6, 7).”

Discussion, pg 9. Section: The spatial properties of thalamic connectivity pat- terns provide insight into the role of the thalamus in shaping brain-wide information flow.

“In this study, we demonstrate that association cortical areas exhibit diffuse anatomical connections within the thalamus. This may enable these cortical areas to integrate information from distributed areas across the cortex, a critical mechanism supporting higher-order neural computations. Specifically, because thalamocortical connectivity is organized topographically, a cortical area that projects to a larger set of thalamic subregions has the potential to communicate with many other cortical areas. We observed that anterior cingulate cortical areas had some of the most diffuse thalamic connections. This observation aligns with findings from Phillips et al. that area 24 exhibited the most diffuse anatomical terminations across the mediodorsal nucleus of the thalamus relative to other prefrontal cortical area…”

Reviewer 3:

Comment 1: Potential weaknesses of the study are that it seems to largely integrate aspects of the thalamus that have been already described before. The differentiation between sensory and association systems across thalamic subregions is something that has been described before (see: Oldham and Ball, 2023; Zheng et al., 2023; Yang et al., 2020 Mueller, 2020; Behrens, 2003).

It is true that previous studies have shown that corticothalamic systems vary between sensory and associative cortical areas. Furthermore, there is much evidence that indicates that the sensory-association hierarchy is a major principle of brain organization in general. However, how and why these circuits are different is still not fully known, both across the whole brain and in corticothalamic circuits specifically.

Our study is the first to compare patterns of anatomical connectivity within the thalamus and determine if cortical areas vary in the extent of those patterns. So our main finding isn't that sensory and association cortical areas show differences in thalamic connectivity, it is that they specifically show differences in their pattern of connectivity within the thalamus. This provides a unique insight into how sensory and associative systems differ in their thalamic connectivity in primates.

Additionally, we show evidence that provides some insight into why these differences may exist. Although we cannot provide causal evidence, our data suggest that differences in patterns of anatomical connectivity within the thalamus were related to how different cortical areas process information via the thalamus, which aligns with speculations from Phillips et al 2021.

So our main finding isn't that sensory and association cortical areas show differences in thalamic connectivity, is it that they specifically show differences in their pattern of connectivity within the thalamus and these differences may help us understand how these cortical areas process information and, in turn, how they may support different types of computations, both of which are major goals in neuroscience. To better clarify this in the manuscript, we made the following changes:

Discussion, Paragraph 1, pg 8:

“This study contributes to the rich body of literature investigating the organization of cortico-thalamic systems in human and non-human primates. Prior research has shown that features of thalamocortical connectivity differ between sensory and association systems, and our work advances this understanding by demonstrating that these systems also differ in the pattern and spatial extent of their anatomical connections within the thalamus. Using dMRI-derived tractography across species, we show that these connectivity patterns vary systematically along the cortical hierarchy in both humans and macaques. These findings are critical for establishing the anatomical architecture of how information flows within distinct cortico-thalamic systems. Specifically, we identify reproducible tractography motifs that correspond to sensorimotor and association circuits, which were consistent across individuals and generalize across species. Collectively, this study offers convergent evidence that the spatial pattern of anatomical connections within the thalamus differs between sensory and association cortical areas, which may support distinct computations across cortico-thalamic systems.”

Comment 2: (1) Why not formally test the association between humans and macaques by bringing the brains to the same space?

We thank the reviewer for this query. We were primarily interested in using the macaque data as a validation of the human data, because it was acquired at a much higher resolution, there are no motion confounds, and it provides a bridge with the tract tracing literature in macaques. We are currently studying interspecies differences in patterns of thalamic connectivity, as well as extensions of our approach into structure-function coupling, and we believe these topics warrant their own paper.

Comment 3: (2) Possibly flesh out the differences between this study and other studies with related approaches a bit further.

We updated the discussion section to better clarify the differences in this study from previous research. See response to Reviewer 3 Comment 1 for text changes.

Comment 4: (3) The current title entails 'cortical hierarchy' but would 'differentiation between sensory and association regions' not be more correct? Or at least a reflection on how cortical hierarchy can be perceived?

We treat these phrases as synonymous terms. Our definition of cortical hierarchy is a smooth transition in features between sensory and motor areas to higher-order associative areas. The use of cortical hierarchy is meant to reflect that our measure continuously varies across the cortex. We updated the manuscript to make this clearer:

Abstract, pg 1.

“Additionally, we leveraged resting-state functional MRI, cortical myelin, and human neural gene expression data to test if the extent of anatomical connections within the thalamus varied along the cortical hierarchy, from sensory and motor to multimodal associative cortical areas.”

Comment 5: (4) For the core-matrix map, there is a marked left-right differences and also there are only two donors in the right hemisphere, possibly note this as a limitation?

We thank the reviewer for this observation. We updated Fig. S28 Panel D to show that the correspondence between EDpc1 and the Core-Matrix (CPc) cortical maps holds when the correlation was done for left and right cortex, separately.

Document d'information détaillé sur les études de genre :

Ce document d'information examine les principales thématiques et les idées ou faits les plus importants concernant les études de genre, en s'appuyant sur les extraits de l'émission "France Culture Questions du soir : le débat Études de genre : pourquoi tant de polémiques".

1. La nature controversée des études de genre :

• Division de l'opinion : Les études de genre suscitent des réactions diverses.

Certains les perçoivent comme une "remise en cause des repères", tandis que d'autres les considèrent comme un "outil utile pour penser les inégalités". * Controverses politiques et médiatiques : Aux États-Unis, des recherches ont été "freinées voire arrêtées sous l'administration Trump".

En France, des "polémiques régulières alimentent la méfiance, même dans les sphères ministérielles".

Le collectif "La Manif pour tous" s'oppose à l'intrusion du "gender à l'école", affirmant que cela "favoriserait l'indifférenciation entre les sexes et la théorie du genre", et que l'idéologie du genre à l'école "signifie propager l'idée aux enfants qu'ils peuvent changer d'identité sexuelle".

• Menace perçue sur les repères anthropologiques : Pour les opposants, les études de genre menacent les "repères viscéraux auxquels nous sommes attachés en terme d'anthropologie, c'est-à-dire qu'est-ce que l'homme, qu'est-ce que la femme, de quoi a besoin un enfant".

2. Qu'est-ce que les études de genre ?

• Un champ d'étude multidisciplinaire : Éric Fassin, sociologue, décrit les études de genre comme un "champ d'étude" mobilisant "des disciplines différentes qui sont mobilisées.

Ça va des sciences sociales, à la philosophie, mais aussi à la biologie ou à toutes sortes de disciplines."

• Pluralité des théories : Il n'existe pas une "théorie du genre" monolithique, mais "des théories qui peuvent s'opposer". Sylviane Agacinski, philosophe, confirme qu'il s'agit d'une "caricature", d'une "simplification" de parler d'une idéologie monolithique, car "il y a plusieurs théories, c'est-à-dire il y a aussi plusieurs usages du mot genre."

• Un concept central : le "genre" comme "sexe social" : Le concept de genre a été "approprié par le féminisme à partir des années 70" et s'est transformé. Il signifiait initialement le "sexe social", comme l'a utilisé Ann Oakley.

Cette notion est cruciale pour comprendre que "quand on parle des femmes, on parle toujours à mon avis simultanément des femmes telles qu'elles sont dans telle ou telle société, dans telle ou telle culture.

C'est-à-dire que en tant que sexe [...] elles sont toujours socialisées, de même que le masculin est toujours socialisé."

• Origine dans les mouvements sociaux : Ce champ d'étude est né de "mouvements sociaux et en particulier du féminisme mais aussi des mouvements sociaux liés aux minorités sexuelles en général." Cela souligne le lien entre "le savoir et la politique".

3. Le débat sur la biologie et le sexe :

• Critique du "biologisme" : Le reproche courant est que les études de genre nieraient l'importance de la biologie. Cependant, Éric Fassin explique que ce qui est critiqué n'est pas la biologie en tant que fait, mais le "biologisme", c'est-à-dire "l'idée que nous serions tout entier posé par cette définition."

• La perspective d'Anne Fausto-Sterling : Cette biologiste féministe utilise le concept de genre pour "déconstruire l'idée même de notre rapport à la biologie".

Elle remet en question la dualité homme/femme, soulignant une "variété bien plus grande que le simple sexe mâle et femelle" et la possibilité de penser le sexe à "différents niveaux : chromosomal, hormonal, formation des organes génitaux, gonades, et développement humain".

Elle propose que la discipline biologique propose "des manières d'organiser le réel" mais que cela "ne veut pas dire que c'est le réel".

• Catégorisation et hiérarchie : Éric Fassin insiste sur le fait que "catégoriser, c'est-à-dire organiser le monde selon des catégories, c'est pas simplement décrire de manière neutre, c'est toujours déjà organiser des hiérarchies."

• Le point de vue de Sylviane Agacinski sur la reproduction et le sexe : Agacinski rejette l'approche de Fausto-Sterling comme un "biologisme réductionnisme". Pour elle, "la définition du sexe se donne par la fécondité, par la reproduction".

Elle considère que la distinction mâle/femelle est "universelle" et que les personnes intersexes, bien qu'humaines, sont des "exceptions" qui "confirment la règle".

• Le sexe comme fait politique et d'état civil : Éric Fassin soutient que le sexe n'est pas "juste une donnée biologique, c'est un fait politique", citant la possibilité de "changer de sexe selon certaines conditions qui sont variables selon les pays et selon les époques".

Il utilise l'exemple de Donald Trump qui veut "restaurer le sexe biologique", montrant que "c'est un fantasme la biologie" dans ce cas.

La controverse sur "l'homme enceinte" découle de l'abandon de la stérilisation pour le changement de sexe, montrant que "c'est l'État, c'est la politique qui détermine le sexe." Sylviane Agacinski conteste l'idée que l'on puisse "changer de sexe" facilement, affirmant que les réalités physiologiques persistent.

4. Les études de genre face à l'individualisme et aux normes sociales :

• Critique de l'individualisme : Éric Marty suggère que les études de genre, avec leur aspiration à la "gender fluidité" et au "genderless", pourraient être en "parfaite harmonie avec le discours néolibéral" et le masque d'un "ordre social" ou une "idéologie".
• Réponse des études de genre : Éric Fassin rejette cette critique comme un "contresens". Le féminisme et les études de genre ne visent pas à la disparition des normes, mais à questionner le fait que "ces normes, elles sont historiques et politiques, autrement dit, elles sont susceptibles de changer".
• Renégociation des normes : Pour Fassin, il ne s'agit pas d'une "disparition des normes" mais d'une "renégociation des normes, les repenser, imaginer d'autres normes".

Les violences sexuelles en sont un exemple, où il y a eu "une prise de conscience que il y a des normes démocratiques, c'est-à-dire de respecter la liberté, c'est-à-dire la capacité de consentir et l'égalité".

• Asymétrie des sexes et violence : Sylviane Agacinski insiste sur l'asymétrie de force physique entre hommes et femmes, qui explique selon elle pourquoi les femmes "souffrent de violence sexuelle".

5. Pourquoi les études de genre cristallisent-elles tant de polémiques ?

• Touche à l'intimité et aux peurs : Éric Fassin explique que la controverse vient du fait que "ça touche à notre intimité et mobiliser l'intimité, les peurs sur l'intimité et sur les changements de l'ordre amoureux, de l'ordre sexuel et bien c'est politiquement efficace".

• Un langage politique pour les rapports de pouvoir : Il souligne une deuxième partie de la définition des études de genre, telle que donnée par Joan Scott : "une manière de signifier les rapports de pouvoir".

Cela signifie que le genre "ne parle pas seulement des hommes et des femmes", mais aussi d'"immigration, de laïcité, d'islam, d'identité nationale, etc."

C'est un "langage politique pour mobiliser des troupes" et jouer sur des "questions raciales, sur des questions économiques". * Instrumentalisation politique : Sylviane Agacinski reconnaît une "instrumentalisation" et une "utilisation politique". Elle évoque des "violences activistes" qui peuvent se mêler à la "réflexion et la théorie", ce qu'elle déplore.

• Lien entre féminisme et politique : Éric Fassin insiste sur le caractère "politique" de toutes ces questions, soulignant que "les féministes ne parlent pas d'une seule voix" et s'affrontent parce que ce sont des "enjeux démocratiques".

Il alerte sur le fait que des leaders comme Trump, Milei, Orban et Poutine "défendent l'idée que l'ordre sexuel et bien ça ne doit pas bouger", ce qui a des "effets sur des gens bien réels et pas simplement sur des minorités sexuelles mais aussi sur des femmes."

• En résumé, les études de genre sont un champ académique diversifié qui questionne les constructions sociales et politiques des catégories de sexe et de genre.

Elles sont l'objet de vifs débats, souvent politisés, concernant la nature du sexe, la relativité des normes sociales et leur rôle dans la compréhension et la contestation des rapports de pouvoir.

Note de synthèse : Les différences cognitives entre les sexes

Source : Extraits de la conférence "Les différences cognitives entre les sexes : lesquelles ? pourquoi ? comment ?" animée par Franck Ramus.

Cette conférence aborde la question complexe des différences cognitives entre les sexes, en s'appuyant sur des données scientifiques pour démystifier les idées reçues et explorer les diverses causes possibles (biologiques, environnementales, sociales).

Thèmes principaux et idées clés :

1. Existence de différences cognitives moyennes et leur nature :

• Il existe des différences cognitives moyennes entre les sexes en langage, mathématiques, mémoire et attention.
• Ces différences sont souvent commentées, exagérées ou niées, mais la science cherche à comprendre leur origine et leur ampleur.
• Citation : "oui il existe des différences cognitives moyennes entre les sexes en langage en mathématiques en mémoire en attention mais ces différences sont-elles biologiques ?

quel rôle a joué notre évolution dans cette différenciation ? sont-elles construites par l'éducation ? sont-elles universelles ou liées à une époque et une culture et un environnement ?

et surtout que peut-on vraiment prouver aujourd'hui quand on interroge la réalité de ces écarts ?"

2. Le cas des performances en mathématiques chez les filles et les garçons :

• Les rapports officiels (ADEP, inspection générale) montrent que les filles sont minoritaires dans les options scientifiques au lycée et dans les filières scientifiques de l'enseignement supérieur, une situation stable depuis 20 ans après une progression.

• Des évaluations nationales récentes ont révélé que les garçons sont en moyenne plus performants en mathématiques dès le milieu du CP.

• Cet écart est spécifique aux mathématiques, car globalement, les filles ont de meilleurs résultats scolaires dans d'autres matières (français, langues, etc.).

• Ce phénomène n'est pas franco-français et s'observe dans la plupart des pays de l'OCDE (évaluations PISA), à quelques exceptions près comme la Finlande où il n'y a pas de différence ou elle est inversée.

• Citation : "dès le dès le milieu du CP il se passe quelque chose au cours du CP qui fait que tout d'un coup les filles commencent à perdre du terrain sur les garçons dans l'apprentissage des mathématiques et c'est assez spécifique aux mathématiques parce que finalement c'est dans un contexte où globalement les filles ont des meilleurs résultats scolaires que les garçons".

3. Analyse des causes potentielles des différences : un modèle multifactoriel :

• Le rapport de l'inspection générale écarte l'origine biologique des différences en mathématiques, privilégiant les causes psychologiques et sociologiques, notamment la menace du stéréotype.

Franck Ramus propose un modèle causal hypothétique incluant :

• Facteurs internes à l'individu : préférences, motivations, capacités cognitives.
• Facteurs environnementaux : influences familiales et sociales (socialisation de genre, stéréotypes, modèles).
• Facteurs biologiques précoces : génome (chromosomes sexuels), hormones sexuelles (testostérone).
• Facteurs contextuels : peuvent biaiser la mesure des performances (ex: menace du stéréotype, compétitivité).

4. Analyse critique des facteurs sociaux :

• Menace du stéréotype :Des expériences (ex: tâche de Rey-Osterrieth présentée comme test de dessin vs. géométrie) montrent que le fales it de mentionner les mathématiques peut faire baisser la performance des filles.
• Cependant, les méta-analyses et études à grande échelle récentes remettent en question sa réplicabilité et suggèrent que son effet est très faible (taille d'effet proche de 0,07 après correction des biais de publication).
• Son rôle dans les situations d'évaluation réelles n'est pas clairement établi.
• Socialisation de genre (activités genrées et organisation genrée de la famille) :Une étude sur la cohorte ELF (4000 enfants suivis de la naissance au CP) n'a trouvé aucun effet des "activités genrées" (choix de jeux, etc.) sur les performances en mathématiques.
• En revanche, l'organisation genrée de la famille (répartition stéréotypique des tâches parentales) a un petit effet sur les performances en mathématiques des filles au CP (explique environ 7% de la différence garçons-filles).

Les filles issues de familles plus stéréotypiques ont de moins bons scores.

• Influence des enseignants :Les caractéristiques des enseignants (sexe, formation) peuvent moduler les performances des filles. Un écart plus important est observé chez les filles ayant un instituteur à formation scientifique.
• Des études montrent que plus les enseignants ont des stéréotypes de genre (explicites ou implicites), plus les scores des filles en maths diminuent, sans effet sur les garçons.
• Contexte de l'évaluation (compétitivité, pression temporelle) :Les filles ont tendance à moins bien performer dans les contextes d'évaluation compétitifs ou chronométrés.
• Les garçons préfèrent davantage les tâches compétitives que les filles.
• Cependant, ce n'est pas spécifique aux mathématiques et ne semble pas entièrement expliquer les écarts observés dans les évaluations nationales.

5. La perspective évolutionnaire et les différences à travers les espèces :

• Il existe de nombreuses différences entre les sexes observables dans toutes les espèces, souvent liées à la reproduction.
• La reproduction sexuée anisogame (gamètes de tailles différentes) entraîne des stratégies reproductives différentes :
• Investissement parental : plus important pour les femelles (grossesse, allaitement).
• Potentiel reproductif : plus élevé chez les mâles (un homme peut potentiellement avoir beaucoup plus de descendants qu'une femme).
• Compétition intrasexuelle : plus forte chez les mâles, menant à la sélection de traits (taille, force, agressivité).
• Dimorphisme sexuel : différences de forme/taille (ex: mâles plus grands pour la lutte).
• Ces prédictions de la théorie de l'évolution sont vérifiées chez l'humain (critères de choix du partenaire, agressivité, violence).
• Citation : "Toutes ces prédictions de la théorie de l'évolution elles sont vérifiées non seulement dans plein d'espèces mais aussi chez l'être humain et chez la plupart des mammifères donc de ce point de vue on n'a pas l'air exceptionnel du tout".
• L'hypothèse d'une origine 100% socioconstructiviste des différences humaines est jugée "peu plausible" car elle impliquerait une annulation de l'héritage évolutif et une émergence "par hasard" de différences qui correspondent exactement aux prédictions évolutionnaires.

6. Lien entre la cognition, l'évolution et les mathématiques :

• Les mathématiques ne sont pas une capacité cognitive primaire sélectionnée, mais un objet culturel complexe.

• Capacités visuo-spatiales (rotation mentale 3D) : Les hommes sont en moyenne meilleurs.

Des études chez les bébés (3-16 mois) et des "expériences de la nature" (filles avec hyperplasie congénitale des surrénales) suggèrent une origine biologique précoce, potentiellement liée à la testostérone prénatale/postnatale.

• Citation : "c'est un indice possible que pour une capacité cognitive bien spécifique il pourrait y avoir un avantage au garçon qui soit observé dès la naissance et que peut-être il est là parce que il a été sélectionné".

• Cependant, le lien entre ces capacités spécifiques et la performance globale en mathématiques est complexe et non linéaire, car les mathématiques sollicitent de nombreuses fonctions cognitives (mémoire, raisonnement, etc.) où les avantages sont répartis entre les sexes.

• Les tests de compétences mathématiques précoces chez les bébés (dès 6 mois) ne montrent pas de différences entre garçons et filles.

7. Différences de préférences et de variabilité :

• Préférences :Les hommes ont un intérêt plus fort pour les objets et les systèmes matériels ("systématisation").
• Les femmes ont un intérêt plus fort pour les personnes et les relations sociales ("empathisation").
• Ces préférences sont observées dès le plus jeune âge (choix de jouets) avec des tailles d'effet très importantes.
• Des indices suggèrent un rôle des facteurs biologiques précoces (filles avec hyperplasie, nouveau-nés).
• Ces préférences peuvent entraîner un engagement différent dans les activités et, à terme, des différences de compétences.
• Variabilité :Les garçons/hommes présentent une plus grande variabilité dans leurs scores en mathématiques et dans la plupart des traits cognitifs et cérébraux.
• Citation : "toutes les barres qui sont au-dessus de la ligne zéro ici et ben elles disent que les garçons ont des scores de math plus variables que les filles".

• Cette plus grande variabilité masculine est expliquée par la plus grande variabilité reproductive des mâles (potentiel de descendants extrêmes), ce qui favorise la sélection de phénotypes extrêmes (très bons ou très mauvais).

• Conséquence : Même avec une moyenne égale, une plus grande variabilité masculine signifie qu'il y aura plus d'hommes aux extrêmes (à la fois les meilleurs et les moins bons), ce qui impacte les filières sélectives (ingénierie, sciences).

Conclusion générale :

• Les différences entre les sexes sont variées en ampleur et en niveaux de preuve.
• Les différences liées à la reproduction et au comportement social sont importantes et bien étayées par des preuves biologiques précoces.
• Les différences dans des capacités cognitives spécifiques sont plus petites et leurs preuves d'origine biologique précoce sont plus faibles (ex: rotation mentale 3D).
• Il n'y a pas de différence significative d'intelligence générale entre les sexes.

Concernant les différences en mathématiques :

• Les différences cognitives innées spécifiques aux mathématiques ne sont pas clairement prouvées.
• Les différences de préférence semblent être un facteur explicatif plus robuste, car elles sont précoces et peuvent influencer l'engagement et le développement des compétences.
• La socialisation de genre (notamment l'organisation genrée de la famille et les stéréotypes des enseignants) a un effet causal prouvé, bien que modeste.
• La menace du stéréotype a un effet très faible et sa réalité est débattue.
• La plus grande variabilité masculine peut expliquer la surreprésentation des garçons aux extrêmes (donc dans les filières d'excellence) même si la moyenne est similaire.

En somme, l'explication des différences en mathématiques est multifactorielle, impliquant des interactions complexes entre facteurs biologiques, environnementaux et sociaux, sans qu'aucune cause unique ne puisse tout expliquer.

La focalisation sur les mathématiques est justifiée par leur lien avec les carrières rémunératrices, mais une attention similaire devrait être portée aux difficultés des garçons en français/langage.

Synthèse des Thèmes et Idées Principales : La Grande Solitude des Adolescents et le Manque de Liens Sociaux

L'émission "France culture être et savoir" aborde la problématique croissante de la solitude chez les adolescents, un phénomène qui dépasse même les questions de santé mentale, et ses conséquences sur la violence et le bien-être général des jeunes.

Les intervenantes, Louia Bris (coordinatrice jeunesse), Laurence Touroude (spécialiste des sciences de l'éducation), Sophie Vénétitay (secrétaire générale du SNES FSU) et Marie-Rose Morau (pédopsychiatre), explorent les causes de cette solitude et les pistes pour recréer du lien social.

1. La Solitude Existentielle et Banale des Adolescents

Le thème central de l'émission est la "solitude immense et banale de nos enfants" (Introduction de l'émission).

Marie-Rose Morau souligne que les adolescents d'aujourd'hui se sentent "isolé", ce qui est "la chose la plus grave et la plus importante qui arrive à nos adolescents aujourd'hui".

Cette solitude est si prégnante que même une jeune stagiaire de seconde, Agathe, témoigne de classes où "on est tous restés inconnus les uns des autres", où "on se parlait pas tant que ça" et où elle ne connaissait pas "les prénoms des gens de ma classe".

Ce manque de lien est perçu comme une "solitude existentielle" par les adolescents eux-mêmes, qui, une fois hospitalisés, apprécient les échanges mais craignent que "quand je vais sortir, ma solitude va recommencer."

2. Le Manque de Liens Sociaux et la Difficulté à "Vivre Ensemble"

Plusieurs facteurs sont identifiés comme contribuant à ce manque de lien social :

• L'individualisation de la réussite scolaire et les réformes éducatives : Marie-Rose Morau critique la "stratégie même à l'intérieur des classes avec des options, avec des" spécialités qui, bien que liées à la "réforme du bac", encouragent les élèves à "réussir individuellement".

Cela conduit à une méfiance entre eux car "ils se connaissent pas bien qui justement qui a pas ces liens de générosité de et de fraternité". L'école, qui devrait être un lieu collectif, a selon elle "renoncé à être ce lieu collectif".

• L'impact des écrans et des réseaux sociaux : Louia Bris observe que "de plus en plus, les parents vont plutôt nous dire en fait, il préfère rester à la maison, il préfère être devant ses écrans". Agathe, la stagiaire, l'explique directement : "Les jeunes passent de nos jours beaucoup plus de temps sur les réseaux sociaux qu'en personne avec les gens parce que bah ils peuvent communiquer, ils peuvent s'envoyer des messages, des vocaux comme si c'était la vie réelle." Ce comportement entraîne un repli sur soi où des élèves "s'enferment, ils ont leur casques, leur téléphone, ils parlent à personne".

• La disparition des espaces collectifs : Les intervenantes soulignent le manque de lieux où les adolescents peuvent se rassembler et construire ensemble. Marie-Rose Morau déplore l'absence de "lieux où ces adolescents construisent finalement contre nous, j'allais dire contre la génération d'avant qui a encore le pouvoir, construisent ses propres valeurs". Sophie Vénétitay ajoute que "la démocratie lycéenne, la démocratie collégienne, ça fait partie de tous ces espaces qui aujourd'hui nous manquent pour créer du collectif et créer du vivre ensemble aujourd'hui." Elle cite également la disparition progressive des "clubs, les associations, la maison des lycéens".

• L'incapacité à décoder les émotions et interagir : Louia Bris, travaillant en centre social, constate une "incapacité à des moments à créer du lien avec les autres" et une difficulté à comprendre "ce que l'autre ressent".

Ce constat est partagé par les écoles, centres de loisirs et clubs de sport locaux, tous d'accord sur le fait que les jeunes "ne savent vraiment plus communiquer".

3. Les Conséquences du Manque de Lien : Violence, Souffrance Psychologique et Isolement du Personnel

Le manque de liens a des répercussions graves :

• Augmentation de la violence : L'émission s'ouvre sur les récents faits divers violents impliquant des adolescents, soulignant une "violente sidérante" et un "manque d'empathie" chez certains jeunes. Marie-Rose Morau relie ces violences à la souffrance des adolescents : "certains vont réagir en se faisant du mal à eux-mêmes, d'autres vont réagir en faisant du mal aux autres."
• Souffrance psychologique et diagnostics précoces sans prise en charge : Marie-Rose Morau met en garde contre les politiques de "repérage ultra précoce" des fragilités, affirmant que "on peut pas prévenir [les passages à l'acte] sauf par des actions de société". Elle dénonce un système où des "diagnostics très précoces" sont faits (par exemple de troubles du neurodéveloppement), mais où les interventions sont retardées de "1 an, 2 ans, 3 ans" ou inexistantes, les familles étant "laissé à elle-même".
• Fragilité du cadre institutionnel et isolement des adultes : Laurence Touroude met en lumière les dysfonctionnements du cadre institutionnel scolaire, notamment le "manque de circulation de l'information", l'"inconsistance du cadre" (règles fluctuantes), et le manque de soutien de la hiérarchie envers les Assistants d'Éducation (AED). Sophie Vénétitay souligne que les professionnels de l'éducation sont "par définition, par nature, par essence isolé et seul", gérant des situations complexes sans formation adéquate et sans le temps nécessaire pour un travail collectif.

4. Pistes et Solutions : Recréer du Lien et du Collectif Malgré la gravité de la situation, des solutions sont esquissées :

• Priorité à la construction de liens et de communautés : Marie-Rose Morau insiste sur la nécessité d'"aider ces adolescents tous à être en relation, à se sentir membre d'une communauté ici, la communauté scolaire mais aussi aussi autour de l'école et dans la famille".
• Renforcer le rôle des professionnels de l'éducation et les soutenir : Les AED sont un personnel "hyper important" qui "peut avoir un lien très différent avec les élèves" car ils sont des "adultes auxquels on peut parler". Il est crucial qu'ils soient "soutenus" et non "discrédité" par leur hiérarchie, et qu'ils bénéficient de formations sur "l'accompagnement et sur le lien".
• Accorder du temps et des espaces pour le collectif : Sophie Vénétitay déplore le manque de "temps et des espaces où on pourrait se retrouver pour faire du collectif". Agathe suggère des "temps d'échange", des "lieux d'échange" comme "une salle avec des canapés où tu peux te regrouper entre jeunes, parler et on n'est pas forcément sur nos téléphones".
• Développer l'éducation populaire et l'autonomie : Les centres sociaux, comme celui de Louia Bris, offrent un "accompagnement global" en créant un cadre "un tout petit peu plus souple" et en incitant les jeunes à l'autonomie, par exemple en les accompagnant à des sorties culturelles pour leur donner "les codes de juste le truc de se dire 'On y va, on voit qu'on peut ça prend 20 minutes en transport, que du coup c'est accessible, que finalement personne ne demande rien au musée.'" Ces lieux démontrent qu'il est possible de "créer des liens" et que "nos lieux fonctionnent". Louia Bris évoque le succès de son centre qui a accueilli "160 jeunes de toute la France", prouvant que "on peut le faire et que les centres sociaux ont trouvé peut-être un bout de réponse qui pourrait être dupliqué dans d'autres structures."
• Remettre en question la compétition et l'individualisme : Le dialogue doit s'élargir pour interroger "comment est-ce qu'on fait société dans l'école et en dehors ?" La compétition scolaire, la valorisation de l'individuel au détriment du collectif, et même l'idée que "le collectif était dangereux" pendant le Covid, sont des freins à la création de lien.

En conclusion, l'émission met en lumière une crise profonde du lien social chez les jeunes, exacerbée par les évolutions sociétales et éducatives.

Face à cette "angoisse de cette solitude" (Laurence Touroude), il est impératif de repenser collectivement les cadres et les espaces qui permettent la construction du "vivre ensemble" et le partage de la parole.

Reviewer #3 (Public review):

Summary:

This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. The experimental design and results are robust, the manuscript's discussion has been satisfactorily updated. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.

Strengths:

The manuscript asks an important question in plant-microbe interaction studies with interesting findings.

Overall, the experiments are detailed, thorough and very well-designed. The findings appear to be robust.

The authors provide results that are not overinterpreted and are instead measured and logical.

Weaknesses:

No major weaknesses.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.

Strengths:

The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.

Weaknesses:

No serious weaknesses, although the manuscript could be improved slightly from technical and communication standpoints.

Reviewer #2 (Public Review):

Summary:

The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in Nfixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.

Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.

Strengths:

This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.

The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.

Weaknesses:

The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe.

Reviewer #3 (Public Review):

Summary:

This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.

Strengths:

The manuscript asks an important question in plant-microbe interaction studies with interesting findings.

Overall, the experiments are detailed, thorough, and very well-designed. The findings appear to be robust.

The authors provide results that are not overinterpreted and are instead measured and logical.

Weaknesses:

No major weaknesses. However, a well-thought-out discussion integrating all the findings and interpreting them is lacking; in its current form, the discussion lacks 'boldness'. The primary question of the study - how plants differentiate between pathogens and symbionts - is not discussed in light of the findings. The concluding remark, "Taken together, our results indicate that successful development of the root nodule symbiosis requires cross-talk between NF-triggered symbiotic signaling and plant immune signaling mediated by RIN4," though accurate, fails to capture the novelty or significance of the findings, and left me wondering how this adds to what is already known. A clear conclusion, for eg, the phosphorylation of RIN4 isoforms by SYMRK at S143 modulates immune responses during symbiotic interactions with rhizobia, or similar, is needed.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

I have no major criticism of the work, although it could be improved by addressing the following minor points:

(1) Page 8, Figure 2 legend. Consider changing "proper symbiosis formation" to "normal nodulation" or something that better reflects control of nodule development/number.

We thank you for the suggestion, the legend was changed to “...required for normal nodule formation” (see Page 10, revised manuscript)

(2) Page 9. Cut "newly" from the first sentence of paragraph 2, as S143 phosphorylation was identified previously.

Thank you for the suggestion, we removed “newly” from the sentence.

(3) Page 10, Figure 3. Panels B showing green-fluorescent nodules are unnecessary given the quantitative data presented in the accompanying panel A. This goes for similar supplemental figures later.

We appreciate the comment; regarding Figure 3 (complementing rin4b mutant, we updated the figures according to the other reviewer’s comment) and Suppl Figure 6 (OE phenotype of phospho-mimic/negative mutants), we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See Page 10, revised manuscript)

(4) Consider swapping Figure 3 for Supplemental Figure S7, which I think shows more clearly the importance of RIN4 phosphorylation in nodulation.

We appreciate the comment and have swapped the figures according to the reviewer’s suggestion. Legend, figure description, and manuscript text have been updated accordingly. (See page 12 and 38, revised manuscript)

(5) Page 10. Replace "it will be referred to S143..." with "we refer to S143 instead of ....".

We replaced it according to the comment.

(6) Page 11, delete "While" from "While no interactions could be observed...".

We deleted it according to the suggestion.

(7) Page 33, Fig S5. How many biological replicates were performed to produce the data presented in panel C and what do the error bar and asterisk indicate? Check that this information is provided in all figures that show errors and statistical significance.

Thank you for the remark. The experiment was repeated three times, and this note was added to the figure description. All the other figure legends with error bar(s) were checked whether replicates are indicated accordingly.

(8) Page 37, Fig S11, panel B. Are averages of data from the 2 biological and 3 technical replicates shown? Add error bars and tests of significant difference.

Averages of a total of 6 replicates (from 2 biological replicates, each run in triplicates) are shown. We thank the reviewer for pointing out the missing error bars and statistical test, we have updated the figure accordingly.

(9) Fig S12. Why are panels A, C, E, and G presented? The other panels seem to show the same data more clearly- showing the linear relationship between peak area ratio and protein concentration.

We have taken the reviewer’s comment into consideration and revised the figure, removing the calibration curves and showing only four panels. The figure legend has been corrected accordingly. (Please see page 43, revised masnuscript). The original figure (unlike other revised figures) had to be deleted from the revised manuscript,as it caused technical issues when converting the document into pdf.

Reviewer #2 (Recommendations For The Authors):

Some small suggestions:

(1) It's good to include a protein schematic for RIN4 in Figure 1.

We appreciate the reviewer’s suggestion and we have drawn a protein schematic and added it to Figure 1. The figure legend was updated accordingly.

(2) There appears to be incorrect labeling in Figure 2c; please double-check and make the necessary corrections.

With respect, we do not understand the comment about incorrect labeling. Would the reviewer please help us out and give more explanation? In Figure 2C, RIN4a and RIN4b expression was checked in transgenic roots expressing either EV (empty vector) or different silencing constructs targeting RIN4a/b.

Reviewer #3 (Recommendations For The Authors):

I enjoyed the level of detail and precision in experimental design.

A discussion point could be - What does it mean that nodule number but not fixation is affected? Is RIN4 only involved in the entry stage of infection but not in nodules during N-fixation?

Current/Our data suggest that RIN4 does indeed appear to be involved in infection. This hypothesis is supported by the findings that RIN4a/b was found phosphorylated in root hairs but not in root (or it was not detected in the root). The interaction with the early signaling RLKs also suggests that RIN4 is likely involved in the early stage of symbiosis formation.

How would the authors explain their observation "However, the motif is retained in non-nodulating Fabales (such as C. canadensis, N. schottii; SI Appendix, Figure S2) and Rosales species as well." What does this imply about the role in symbiosis that the authors propose?

We appreciate the reviewer’s question. The motif seems to be retained, however, it might be not only the motif but also the protein structure that in case of nodulating plants might be different. We have not investigated the structure of RIN4, how it would look based on certain features/upon interaction with another protein and/or post-translational modification(s). Griesman et al, (2018) showed the absence of certain genes within Fabales in non-nodulating species, we can speculate that these absent genes can’t interact with RIN4 in those species, therefore the lack of downstream signaling could be possible (in spite of the retained motif in non-nodulating species). At this point, there is not enough data or knowledge to further speculate.

qPCR analysis of symbiotic pathway genes showed that both NIN-dependent and NIN-independent branches of the symbiosis signaling pathway were negatively affected in the rin4b mutant. Please derive a conclusion from this.

We appreciate the comment, it also prompted us to correct the following sentence; original: “Since NIN is responsible for induction of NF-YA and ERN1 transcription factors, their reduced expression in rin4b plants was not unexpected (Fig. 5). “As ERN1 expression is independent of NIN (Kawaharada et al, 2017). The following sentences were also deleted as it represented a repetition of a statement above these sentences: “Soybean NF-YA1 homolog responded significantly to rhizobial treatment in rin4b plants, whereas NF-YA3 induction did not show significant induction (Fig. 5).“

We added the following conclusion/hypothesis: “Based on the results of the expression data presented above, it seems that both NIN-dependent and NINindependent branches of the symbiotic signaling pathways are affected in the rin4b mutant background. This indicates that the role of RIN4 protein in the symbiotic pathway can be placed upstream of CYCLOPS, as the CYCLOPS transcription activating complex is responsible (directly or indirectly) for the activation of all TFs tested in our expression analysis (Singh et al, 2014/47, 48).” (Please see Page 16, revised manuscript)

The authors are highly encouraged to write a thoughtful discussion that would accompany the detailed experimental work performed in this manuscript.

We appreciate the comment, and we did some work on the discussion part of the document. (Please see Pages 17-19, revised manuscript)

Some minor suggestions for overall readability are below.

What about immune signaling genes? Given that authors hypothesize that "Absence of AtRIN4 leads to increased PTI responses and, therefore, it might be that GmRIN4b absence also causes enhanced PTI which might have contributed to significantly fewer nodules." Could check marker immune signaling gene expression FLS2 and others.

We appreciate the reviewer’s comment, and while we believe those are very interesting questions/suggestions, answering them is out of the scope of the current manuscript. Partially because it has been shown that several defenseresponsive genes that were described in leaf immune responses could not be confirmed to respond in a similar manner in root (Chuberre et al., 2018). It was also shown that plant immune responses are compartmentalized and specialized in roots (Chuberre et al., 2018). If we were looking at immune-responsive genes, the signal might be diluted because of its specialized and compartmentalized nature. Another reason why these questions cannot be answered as a part of the current manuscript is because finding a suitable immune responsive gene would require rigorous experiments (not only in root, but also in root hair (over a timecourse) which would be a ground work for a separate study (root hair isolation is not a trivial experiment, it requires at least 250-300 seedlings per treatment/per time-point).

Regarding FLS2, it is known in Arabidopsis that its expression is tissue-specific within the root, and it seems that FLS2 expression is restricted to the root vasculature (Wyrsch et al, 2015). In our manuscript, we showed that RIN4a/b is highly expressed in root hairs, as well as RIN4 phosphorylation was detectable in root hair but not in the root; therefore, we do not see the reason to investigate FLS2 expression.

"in our hands only ERN1a could be amplified. One possible explanation for this observation is that primers were designed based on Williams 82 reference genome, while our rin4b mutant was generated in the Bert cultivar background." Is the sequence between the two cultivars and the primers that bind to ERN1b in both cultivars so different? If not, this explanation is not very convincing.

At the time of performing the experiment the genomic sequence of the Bert cultivar (used for generating rin4b edited lines) was not publicly available. In accordance with the reviewer’s comment, we removed the explanation, as it does not seem to be relevant. (See page 16, revised manuscript)

The figures are clear and there is a logical flow. The images of fluorescing nodules in Figure 2,3 panels with nodules are not informative or unbiased .

We appreciate the comment, as for Figure 3 (complementing rin4b mutant), we updated the figures according to the other reviewer’s comment and Suppl. Figure 6 (OE phenotype of phospho-mimic/negative mutants) we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See pages 10, 12 and 38, revised manuscript)

What does the exercise in isolation of rin4 mutants in lotus tell us? Is it worth including?

Isolation of the Ljrin4 mutant suggests that RIN4 carries such an importance that the mutant version of it is lethal for the plant (as in Arabidospis, where most of the evidence regarding the role of RIN4 has been described), and an additional piece of evidence that RIN4 is similarly crucial across most land plant species.

Sentence ambiguous. "Co-expression of RIN4a and b with SymRKßΔMLD and NFR1α _resulted in YFP fluorescence detected by Confocal Laser Scanning Microscopy (SI Appendix, Figure S8) suggesting that RIN4a and b proteins closely associate with both RLKs." Were all 4 expressed together?

Thank you for the remark. Not all 4 proteins were co-expressed together. We adjusted the sentence as follows: “Co-expression of RIN4a/ and b with SymRKßΔMLD as well as and NFR1α resulted in YFP fluorescence…” I hope it is phrased in a clearer way. (See page 13, revised manuscript)

Minor spelling errors throughout.. Costume-made (custom made?)

Thank you for noticing. According to the Cambridge online dictionary, it is written with a hyphen, therefore, we added a hyphen and corrected the manuscript accordingly.

CRISPR-cas9 or CRISPR/Cas9? Keep it consistent throughout. CRISPR-cas9 is the latest consensus.

We corrected it to “CRISPR-Cas9” throughout the manuscript.

References are missing for several 'obvious statements' but please include them to reach a broader audience. For example the first 5 sentences of the introduction. Also, statements such as 'Root hairs are the primary entry point for rhizobial infection in most legumes.'.

Thank you for the comment. To make it clearer, we also added reference #1, after the third sentence of the introduction, as well as we added an additional review as reference. This additional review was also cited as the source for the sentence “Root hairs are the primary…” (Please see page 2, revised manuscript)

Can you provide a percent value? Silencing of RIN4a and RIN4b resulted in significantly reduced nodule numbers on soybean transgenic roots in comparison to transgenic roots carrying the empty vector control. Also, this wording suggests it was a double K.D. but from the images, it appears they were individually silenced.

We appreciate the reviewer's comment. We observed a 50-70% reduction in the number of nodules. We adjusted the text according to the reviewer's remark. (See page 9, revised manuscript)

Now add AI into the mix. There will be even more detection software.

Reviewer #2 (Public review):

Oracová et al. present data supporting a role for SIMC1/SLF2 in silencing plasmid DNA via the SMC5/6 complex. Their findings are of interest, and they provide further mechanistic detail of how the SMC5/6 complex is recruited to disparate DNA elements. In essence, the present report builds on the author's previous paper in eLife in 2022 (PMID: 36373674, "The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers") by showing the role of SIMC1/SLF2 in localisation of the SMC5/6 complex to plasmid DNA, and the distinct requirements as compared to recruitment to DNA damage foci. Although the findings of the manuscript are of interest, we are not yet convinced that the new data presented here represents a compelling new body of work and would better fit the format of a "research advance" article. In their previous paper, Oracová et al. show that the recruitment of SMC5/6 to SV40 replication centres is dependent on SIMC1, and specifically, that it is dependent on SIMC1 residues adjacent to neighbouring SLF2.

Other comments

(1) The mutations chosen in Figure 1 are quite extensive - 5 amino acids per mutant. In addition, they are in many cases 'opposite' changes, e.g., positive charge to negative charge. Is the effect lost if single mutations to an alanine are made?

(2) In Figure 2c, it isn't clear from the data shown that the 'SLF2-only' mutations in SMC6 result in a substantial reduction in SIMC1/SLF2 binding.

(3) In the GFP reporter assays (e.g. Figure 3), median fluorescence is reported - was there any observed difference in the percentage of cells that are GFP positive?

(4) The potential role of the large T antigen as an SMC5/6 evasion factor is intriguing. However, given the role of the large T antigen as a transcriptional activator, caution is required when interpreting enhanced GFP fluorescence. Antagonism of the SMC5/6 complex in this context might be further supported by ChIP experiments in the presence or absence of large T. Can large T functionally substitute for HBx or HIV-Vpr?

(5) In Figure 5c, the apparent molecular weight of large T and SMC6 appears to change following transfection of GFP-SMC5 - is there a reason for this?

Reviewer #3 (Public review):

Summary:

This study by the Boddy and Otomo laboratories further characterizes the roles of SMC5/6 loader proteins and related factors in SMC5/6-mediated repression of extrachromosomal circular DNA. The work shows that mutations engineered at an AlphaFold-predicted protein-protein interface formed between the loader SLF2/SIMC1 and SMC6 (similar to the interface in the yeast counterparts observed by cryo-EM) prevent co-IP of the respective proteins. The mutations in SLF2 also hinder plasmid DNA silencing when expressed in SLF2-/- cell lines, suggesting that this interface is needed for silencing. SIMC1 is dispensable for recruitment of SMC5/6 to sites of DNA damage, while SLF1 is required, thus separating the functions of the two loader complexes. Preventing SUMOylation (with a chemical inhibitor) increases transcription from plasmids but does not in SLF2-deleted cell lines, indicating the SMC5/6 silences plasmids in a SUMOylation dependent manner. Expression of LT is sufficient for increased expression, and again, not additive or synergistic with SIMC1 or SLF2 deletion, indicating that LT prevents silencing by directly inhibiting 5/6. In contrast, PML bodies appear dispensable for plasmid silencing.

Strengths:

The manuscript defines the requirements for plasmid silencing by SMC5/6 (an interaction of Smc6 with the loader complex SLF2/SIMC1, SUMOylation activity) and shows that SLF1 and PML bodies are dispensable for silencing. Furthermore, the authors show that LT can overcome silencing, likely by directly binding to (but not degrading) SMC5/6.

Weaknesses:

(1) Many of the findings were expected based on recent publications.

(2) While the data are consistent with SIMC1 playing the main function in plasmid silencing, it is possible that SLF1 contributes to silencing, especially in the absence of SIMC1. This would potentially explain the discrepancy with the data reported in ref. 50. SLF2 deletion has a stronger effect on expression than SIMC1 deletion in many but not all experiments reported in this manuscript. A double mutant/deletion experiments would be useful to explore this possibility.

(3) SLF2 is part of both types of loaders, while SLF1 and SIMC1 are specific to their respective loaders. Did the authors observe differences in phenotypes (growth, sensitivities to DNA damage) when comparing the mutant cell lines or their construction? This should be stated in the manuscript.

(4) It would be desirable to have control reporter constructs located on the chromosome for several experiments, including the SUMOylation inhibition (Figures 5A and 5-S2) and LT expression (Figure 5D) to exclude more general effects on gene expression.

(5) Figure 5A: There appears to be an increase in GFP in the SLF2-/- cells with SUMOi? Is this a significant increase?

(6) The expression level of SFL2 mut1 should be tested (Figure 3B).

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Syed et al. investigate the circuit underpinnings for leg grooming in the fruit fly. They identify two populations of local interneurons in the right front leg neuromere of ventral nerve cord, i.e. 62 13A neurons and 64 13B neurons. Hierarchical clustering analysis identifies 10 morphological classes for both populations. Connectome analysis reveals their circuit interactions: these GABAergic interneurons provide synaptic inhibition either between the two subpopulations, i.e., 13B onto 13A, or among each other, i.e., 13As onto other 13As, and/or onto leg motoneurons, i.e., 13As and 13Bs onto leg motoneurons. Interestingly, 13A interneurons fall into two categories, with one providing inhibition onto a broad group of motoneurons, being called "generalists", while others project to a few motoneurons only, being called "specialists". Optogenetic activation and silencing of both subsets strongly affect leg grooming. As well as activating or silencing subpopulations, i.e., 3 to 6 elements of the 13A and 13B groups, has marked effects on leg grooming, including frequency and joint positions, and even interrupting leg grooming. The authors present a computational model with the four circuit motifs found, i.e., feed-forward inhibition, disinhibition, reciprocal inhibition, and redundant inhibition. This model can reproduce relevant aspects of the grooming behavior.

Strengths:

The authors succeeded in providing evidence for neural circuits interacting by means of synaptic inhibition to play an important role in the generation of a fast rhythmic insect motor behavior, i.e., grooming. Two populations of local interneurons in the fruit fly VNC comprise four inhibitory circuit motifs of neural action and interaction: feed-forward inhibition, disinhibition, reciprocal inhibition, and redundant inhibition. Connectome analysis identifies the similarities and differences between individual members of the two interneuron populations. Modulating the activity of small subsets of these interneuron populations markedly affects the generation of the motor behavior, thereby exemplifying their important role in generating grooming.

We thank the reviewer for their thoughtful and constructive evaluation of our work. We are encouraged by their recognition of the major contributions of our study, including the identification of multiple inhibitory circuit motifs and their contribution to organizing rhythmic leg grooming behavior. We also appreciate the reviewer’s comments highlighting our use of connectomics, targeted manipulations, and modeling to reveal how distinct subsets of inhibitory interneurons contribute to motor behavior.

Weaknesses:

Effects of modulating activity in the interneuron populations by means of optogenetics were conducted in the so-called closed-loop condition. This does not allow for differentiation between direct and secondary effects of the experimental modification in neural activity, as feedforward and feedback effects cannot be disentangled. To do so, open loop experiments, e.g., in deafferented conditions, would be important. Given that many members of the two populations of interneurons do not show one, but two or more circuit motifs, it remains to be disentangled which role the individual circuit motif plays in the generation of the motor behavior in intact animals.

We appreciate the reviewer’s point regarding the role of sensory feedback in our experimental design. We agree that reafferent (sensory) input from ongoing movements could contribute to the behavioral outcomes of our optogenetic manipulations. However, our aim was not to isolate central versus peripheral contributions, but rather to assess the role of 13A/B neurons within the intact, operational sensorimotor system during natural grooming behavior.

These inhibitory neurons form recurrent loops, synapse onto motor neurons, and receive proprioceptive input—placing them in a position to both shape central motor output and process sensory feedback. As such, manipulating their activity engages both central control and sensory consequences.

The finding that silencing 13A neurons in dusted flies disrupts rhythmic leg coordination highlights their role in organizing grooming movements. Prior studies (e.g., Ravbar et al., 2021) show that grooming rhythms persist when sensory input is reduced, indicating a central origin, while sensory feedback refines timing, coordination, and long-timescale stability. We concluded that rhythmicity arises centrally but is shaped and stabilized by mechanosensory or proprioceptive feedback. Our current results are consistent with this view and support a model in which inhibitory premotor neurons participate in a closed-loop control architecture that generates and tunes rhythmic output.

While we agree that fully removing sensory feedback and parsing distinct roles for neurons that participate in multiple circuit motifs would be desirable, we do not see a plausible experimental path to accomplish this - we would welcome suggestions!

We considered the method used by Mendes and Mann (eLife 2023) to assess sensory feedback to walking, 5-40-GAL4, DacRE-flp, UAS->stop>TNT + 13A/B-spGAL4 X UAS-csChrimson. This would require converting one targeting system to LexA and presents significant technical challenges. More importantly, we believe the core interpretation issue would remain: broadly silencing proprioceptors would produce pleiotropic effects and impair baseline coordination, making it difficult to distinguish whether observed changes reflect disrupted rhythm generation or secondary consequences of impaired sensory input.

We will clarify in the revised manuscript that our behavioral experiments were performed in freely moving flies under closed-loop conditions. We thank the reviewer for highlighting these important considerations and will revise the manuscript to better communicate the scope and interpretation of our findings.

Reviewer #2 (Public review):

Summary:

This manuscript by Syed et al. presents a detailed investigation of inhibitory interneurons, specifically from the 13A and 13B hemilineages, which contribute to the generation of rhythmic leg movements underlying grooming behavior in Drosophila. After performing a detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits, the authors build on this anatomical framework by performing optogenetic perturbation experiments to functionally test predictions derived from the connectome. Finally, they integrate these findings into a computational model that links anatomical connectivity with behavior, offering a systems-level view of how inhibitory circuits may contribute to grooming pattern generation.

Strengths:

(1) Performing an extensive and detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits.

(2) Making sense of the largely uncharacterized 13A/13B nerve cord circuitry by combining connectomics and optogenetics is very impressive and will lay the foundation for future experiments in this field.

(3) Testing the predictions from experiments using a simplified and elegant model.

We thank the reviewer for their thoughtful and encouraging evaluation of our work. We are especially grateful for their recognition of our detailed connectome analysis and its contribution to understanding the organization of premotor inhibitory circuits. We appreciate the reviewer’s comments highlighting the integration of connectomics with optogenetic perturbations to functionally interrogate the 13A and 13B circuits, as well as their recognition of our modeling approach as a valuable framework for linking circuit architecture to behavior.

Weaknesses:

(1) In Figure 4, while the authors report statistically significant shifts in both proximal inter-leg distance and movement frequency across conditions, the distributions largely overlap, and only in Panel K (13B silencing) is there a noticeable deviation from the expected 7-8 Hz grooming frequency. Could the authors clarify whether these changes truly reflect disruption of the grooming rhythm?

We are re-analyzing the whole dataset in the light of the reviews (specifically, we are now applying LMM to these statistics). For the panels in question (H-J), there is indeed a large overlap between the frequency distributions, but the box plots show median and quartiles, which partially overlap. (In the current analysis, as it stands, differences in means were small yet significant.) However, there is a noticeable (not yet quantified) difference in variability between the frequencies (the experimental group being the more variable one). If the activations/deactivations of 13A/B circuits disrupt the rhythm, we would indeed expect the frequencies to become more variable. So, in the revised version we will quantify the differences in both the means and the variabilities, and establish whether either shows significance after applying the LMM.

More importantly, all this data would make the most sense if it were performed in undusted flies (with controls) as is done in the next figure.

In our assay conditions, undusted flies groom infrequently. We used undusted flies for some optogenetic activation experiments, where the neuron activation triggers behavior initiation, but we chose to analyze the effect of silencing inhibitory neurons in dusted flies because dust reliably activates mechanosensory neurons and elicits robust grooming behavior, enabling us to assess how manipulation of 13A/B neurons alters grooming rhythmicity and leg coordination.

(2) In Figure 4-Figure Supplement 1, the inclusion of walking assays in dusted flies is problematic, as these flies are already strongly biased toward grooming behavior and rarely walk. To assess how 13A neuron activation influences walking, such experiments should be conducted in undusted flies under baseline locomotor conditions.

We agree that there are better ways to assay potential contributions of 13A/13B neurons to walking. We intended to focus on how normal activity in these inhibitory neurons affects coordination during grooming, and we included walking because we observed it in our optogenetic experiments and because it also involves rhythmic leg movements. The walking data is reported in a supplementary figure because we think this merits further study with assays designed to quantify walking specifically. We will make these goals clearer in the revised manuscript and we are happy to share our reagents with other research groups more equipped to analyze walking differences.

(3) For broader lines targeting six or more 13A neurons, the authors provide specific predictions about expected behavioral effects-e.g., that activation should bias the limb toward flexion and silencing should bias toward extension based on connectivity to motor neurons. Yet, when using the more restricted line labeling only two 13A neurons (Figure 4 - Figure Supplement 2), no such prediction is made. The authors report disrupted grooming but do not specify whether the disruption is expected to bias the movement toward flexion or extension, nor do they discuss the muscle target. This is a missed opportunity to apply the same level of mechanistic reasoning that was used for broader manipulations.

While we know which two neurons are labeled based on confocal expression, assigning their exact identity in the EM datasets has been challenging. One of these neurons appears absent from our 13A reconstructions of the right T1 neuropil in FANC, although we did locate it in MANC. However, its annotation in MANC has undergone multiple revisions, making confident assignment difficult at this time. Since we can’t be sure which motor neurons and muscles are most directly connected, we did not want to predict this line’s effect on leg movements.

(4) Regarding Figure 5: The 70ms on/off stimulation with a slow opsin seems problematic. CsChrimson off kinetics are slow and unlikely to cause actual activity changes in the desired neurons with the temporal precision the authors are suggesting they get. Regardless, it is amazing that the authors get the behavior! It would still be important for the authors to mention the optogenetics caveat, and potentially supplement the data with stimulation at different frequencies, or using faster opsins like ChrimsonR.

We were also surprised - and intrigued - by the behavioral consequences of activating these inhibitory neurons with CsChrimson. We tried several different activation paradigms: pulsed from 8Hz to 500Hz and with various on/off intervals. Because several of these different stimulation protocols resulted in grooming, and with different rhythmic frequencies, we think the phenotypes are a specific property of the neural circuits we have activated, rather than the kinetics of CsChrimson itself.

We will include the data from other frequencies in a new Supplementary Figure, we will discuss the caveats CsChrimson’s slow off-kinetics present to precise temporal control of neural activity, and we will try ChrimsonR in future experiments.

Overall, I think the strengths outweigh the weaknesses, and I consider this a timely and comprehensive addition to the field.

Thank you!

Reviewer #3 (Public review):

Summary:

The authors set out to determine how GABAergic inhibitory premotor circuits contribute to the rhythmic alternation of leg flexion and extension during Drosophila grooming. To do this, they first mapped the ~120 13A and 13B hemilineage inhibitory neurons in the prothoracic segment of the VNC and clustered them by morphology and synaptic partners. They then tested the contribution of these cells to flexion and extension using optogenetic activation and inhibition and kinematic analyses of limb joints. Finally, they produced a computational model representing an abstract version of the circuit to determine how the connectivity identified in EM might relate to functional output. The study, in its current form, makes an important but overclaimed contribution to the literature due to a mismatch between the claims in the paper and the data presented.

Strengths:

The authors have identified an interesting question and use a strong set of complementary tools to address it:

(1) They analysed serial‐section TEM data to obtain reconstructions of every 13A and 13B neuron in the prothoracic segment. They manually proofread over 60 13A neurons and 64 13B neurons, then used automated synapse detection to build detailed connectivity maps and cluster neurons into functional motifs.

(2) They used optogenetic tools with a range of genetic driver lines in freely behaving flies to test the contribution of subsets of 13A and 13B neurons.

(3) They used a connectome-constrained computational model to determine how the mapped connectivity relates to the rhythmic output of the behavior.

We appreciate the reviewer’s thorough and constructive feedback on our work. We are encouraged by their recognition of the complementary approaches used in our study.

Weaknesses:

The manuscript aims to reveal an instructive, rhythm-generating role for premotor inhibition in coordinating the multi-joint leg synergies underlying grooming. It makes a valuable contribution, but currently, the main claims in the paper are not well-supported by the presented evidence.

Major points

(1) Starting with the title of this manuscript, "Inhibitory circuits generate rhythms for leg movements during Drosophila grooming", the authors raise the expectation that they will show that the 13A and 13B hemilineages produce rhythmic output that underlies grooming. This manuscript does not show that. For instance, to test how they drive the rhythmic leg movements that underlie grooming requires the authors to test whether these neurons produce the rhythmic output underlying behavior in the absence of rhythmic input. Because the optogenetic pulses used for stimulation were rhythmic, the authors cannot make this point, and the modelling uses a "black box" excitatory network, the output of which might be rhythmic (this is not shown). Therefore, the evidence (behavioral entrainment; perturbation effects; computational model) is all indirect, meaning that the paper's claim that "inhibitory circuits generate rhythms" rests on inferred sufficiency. A direct recording (e.g., calcium imaging or patch-clamp) from 13A/13B during grooming - outside the scope of the study - would be needed to show intrinsic rhythmogenesis. The conclusions drawn from the data should therefore be tempered. Moreover, the "black box" needs to be opened. What output does it produce? How exactly is it connected to the 13A-13B circuit?

We will modify the title to better reflect our strongest conclusions: “Inhibitory circuits coordinate rhythmic leg movements during Drosophila grooming”

Our optogenetic activation was delivered in a patterned (70 ms on/off) fashion that entrains rhythmic movements but does not rule out the possibility that the rhythm is imposed externally. In the manuscript, we state that we used pulsed light to mimic a flexion-extension cycle and note that this approach tests whether inhibition is sufficient to drive rhythmic leg movements when temporally patterned. While this does not prove that 13A/13B neurons are intrinsic rhythm generators, it does demonstrate that activating subsets of inhibitory neurons is sufficient to elicit alternating leg movements resembling natural grooming and walking.

Our goal with the model was to demonstrate that it is possible to produce rhythmic outputs with this 13A/B circuit, based on the connectome. The “black box” is a small recurrent neural network (RNN) consisting of 40 neurons in its hidden layer. The inputs are the “dust” levels from the environment (the green pixels in Figure 6I), the “proprioceptive” inputs (“efference copy” from motor neurons), and the amount of dust accumulated on both legs. The outputs (all positive) connect to the 13A neurons, the 13B neurons, and to the motor neurons. We refer to it as the “black box” because we make no claims about the actual excitatory inputs to these circuits. Its function is to provide input, needed to run the network, that reflects the distribution of “dust” in the environment as well as the information about the position of the legs.

The output of the “black box” component of the model might be rhythmic. In fact, in most instances of the model implementation this is indeed the case. However, as mentioned in the current version of the manuscript: “But the 13A circuitry can still produce rhythmic behavior even without those external sensory inputs (or when set to a constant value), although the legs become less coordinated.” Indeed, when we refine the model (with the evolutionary training) without the “black box” (using a constant input of 0.1) the behavior is still rhythmic and sustained. Therefore, the rhythmic activity and behavior can emerge from the premotor circuitry itself without a rhythmic input.

The context in which the 13A and 13B hemilineages sit also needs to be explained. What do we know about the other inputs to the motorneurons studied? What excitatory circuits are there?

We agree that there are many more excitatory and inhibitory, direct and indirect, connections to motor neurons that will also affect leg movements for grooming and walking. Our goal was to demonstrate what is possible from a constrained circuit of inhibitory neurons that we mapped in detail, and we hope to add additional components to better replicate the biological circuit as behavioral and biomechanical data is obtained by us and others. We will add this clarification of the limits of the scope to the Discussion.

Furthermore, the introduction ignores many decades of work in other species on the role of inhibitory cell types in motor systems. There is some mention of this in the discussion, but even previous work in Drosophila larvae is not mentioned, nor crustacean STG, nor any other cell types previously studied. This manuscript makes a valuable contribution, but it is not the first to study inhibition in motor systems, and this should be made clear to the reader.

We thank the reviewer for this important reminder and we will expand our discussion of the relevant history and context in our revision. Previous work on the contribution of inhibitory neurons to invertebrate motor control certainly influenced our research and we should acknowledge this better.

(2) The experimental evidence is not always presented convincingly, at times lacking data, quantification, explanation, appropriate rationales, or sufficient interpretation.

We are committed to improving the clarity, rationale, and completeness of our experimental descriptions. We will revisit the statistical tests applied throughout the manuscript and expand the Methods.

(3) The statistics used are unlike any I remember having seen, essentially one big t-test followed by correction for multiple comparisons. I wonder whether this approach is optimal for these nested, high‐dimensional behavioral data. For instance, the authors do not report any formal test of normality. This might be an issue given the often skewed distributions of kinematic variables that are reported. Moreover, each fly contributes many video segments, and each segment results in multiple measurements. By treating every segment as an independent observation, the non‐independence of measurements within the same animal is ignored. I think a linear mixed‐effects model (LMM) or generalized linear mixed model (GLMM) might be more appropriate.

We thank the reviewer for raising this important point regarding the statistical treatment of our segmented behavioral data. Our initial analysis used independent t-tests with Bonferroni correction across behavioral classes and features, which allowed us to identify broad effects. However, we acknowledge that this approach does not account for the nested structure of the data. To address this, we will re-analyze key comparisons using linear mixed-effects models (LMMs) as suggested by the reviewer. This approach will allow us to more appropriately model within-fly variability and test the robustness of our conclusions. We will update the manuscript based on the outcomes of these analyses.

(4) The manuscript mentions that legs are used for walking as well as grooming. While this is welcome, the authors then do not discuss the implications of this in sufficient detail. For instance, how should we interpret that pulsed stimulation of a subset of 13A neurons produces grooming and walking behaviours? How does neural control of grooming interact with that of walking?

We do not know how the inhibitory neurons we investigated will affect walking or how circuits for control of grooming and walking might compete. We speculate that overlapping pre-motor circuits may participate in walking and grooming because both behaviors have extension flexion cycles at similar frequencies, but we do not have hard experimental data to support. This would be an interesting area for future research. Here, we focused on the consequences of activating specific 13A/B neurons during grooming because they were identified through a behavioral screen for grooming disruptions, and we had developed high-resolution assays and familiarity with the normal movements in this behavior. We will clarify this rationale in the revised discussion.

(5) The manuscript needs to be proofread and edited as there are inconsistencies in labelling in figures, phrasing errors, missing citations of figures in the text, or citations that are not in the correct order, and referencing errors (examples: 81 and 83 are identical; 94 is missing in text).

We will carefully proofread the manuscript to fix all figure labeling, citation order, and referencing errors.

Reviewer #2 (Public review):

Summary:

This manuscript by Syed et al. presents a detailed investigation of inhibitory interneurons, specifically from the 13A and 13B hemilineages, which contribute to the generation of rhythmic leg movements underlying grooming behavior in Drosophila. After performing a detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits, the authors build on this anatomical framework by performing optogenetic perturbation experiments to functionally test predictions derived from the connectome. Finally, they integrate these findings into a computational model that links anatomical connectivity with behavior, offering a systems-level view of how inhibitory circuits may contribute to grooming pattern generation.

Strengths:

(1) Performing an extensive and detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits.

(2) Making sense of the largely uncharacterized 13A/13B nerve cord circuitry by combining connectomics and optogenetics is very impressive and will lay the foundation for future experiments in this field.

(3) Testing the predictions from experiments using a simplified and elegant model.

Weaknesses:

(1) In Figure 4, while the authors report statistically significant shifts in both proximal inter-leg distance and movement frequency across conditions, the distributions largely overlap, and only in Panel K (13B silencing) is there a noticeable deviation from the expected 7-8 Hz grooming frequency. Could the authors clarify whether these changes truly reflect disruption of the grooming rhythm? More importantly, all this data would make the most sense if it were performed in undusted flies (with controls) as is done in the next figure.

(2) In Figure 4-Figure Supplement 1, the inclusion of walking assays in dusted flies is problematic, as these flies are already strongly biased toward grooming behavior and rarely walk. To assess how 13A neuron activation influences walking, such experiments should be conducted in undusted flies under baseline locomotor conditions.

(3) For broader lines targeting six or more 13A neurons, the authors provide specific predictions about expected behavioral effects-e.g., that activation should bias the limb toward flexion and silencing should bias toward extension based on connectivity to motor neurons. Yet, when using the more restricted line labeling only two 13A neurons (Figure 4 - Figure Supplement 2), no such prediction is made. The authors report disrupted grooming but do not specify whether the disruption is expected to bias the movement toward flexion or extension, nor do they discuss the muscle target. This is a missed opportunity to apply the same level of mechanistic reasoning that was used for broader manipulations.

(4) Regarding Figure 5: The 70ms on/off stimulation with a slow opsin seems problematic. CsChrimson off kinetics are slow and unlikely to cause actual activity changes in the desired neurons with the temporal precision the authors are suggesting they get. Regardless, it is amazing that the authors get the behavior! It would still be important for the authors to mention the optogenetics caveat, and potentially supplement the data with stimulation at different frequencies, or using faster opsins like ChrimsonR.

Overall, I think the strengths outweigh the weaknesses, and I consider this a timely and comprehensive addition to the field.

Reviewer #3 (Public review):

Summary:

The authors set out to determine how GABAergic inhibitory premotor circuits contribute to the rhythmic alternation of leg flexion and extension during Drosophila grooming. To do this, they first mapped the ~120 13A and 13B hemilineage inhibitory neurons in the prothoracic segment of the VNC and clustered them by morphology and synaptic partners. They then tested the contribution of these cells to flexion and extension using optogenetic activation and inhibition and kinematic analyses of limb joints. Finally, they produced a computational model representing an abstract version of the circuit to determine how the connectivity identified in EM might relate to functional output. The study, in its current form, makes an important but overclaimed contribution to the literature due to a mismatch between the claims in the paper and the data presented.

Strengths:

The authors have identified an interesting question and use a strong set of complementary tools to address it:

(1) They analysed serial‐section TEM data to obtain reconstructions of every 13A and 13B neuron in the prothoracic segment. They manually proofread over 60 13A neurons and 64 13B neurons, then used automated synapse detection to build detailed connectivity maps and cluster neurons into functional motifs.

(2) They used optogenetic tools with a range of genetic driver lines in freely behaving flies to test the contribution of subsets of 13A and 13B neurons.

(3) They used a connectome-constrained computational model to determine how the mapped connectivity relates to the rhythmic output of the behavior.

Weaknesses:

The manuscript aims to reveal an instructive, rhythm-generating role for premotor inhibition in coordinating the multi-joint leg synergies underlying grooming. It makes a valuable contribution, but currently, the main claims in the paper are not well-supported by the presented evidence.

Major points

(1) Starting with the title of this manuscript, "Inhibitory circuits generate rhythms for leg movements during Drosophila grooming", the authors raise the expectation that they will show that the 13A and 13B hemilineages produce rhythmic output that underlies grooming. This manuscript does not show that. For instance, to test how they drive the rhythmic leg movements that underlie grooming requires the authors to test whether these neurons produce the rhythmic output underlying behavior in the absence of rhythmic input. Because the optogenetic pulses used for stimulation were rhythmic, the authors cannot make this point, and the modelling uses a "black box" excitatory network, the output of which might be rhythmic (this is not shown). Therefore, the evidence (behavioral entrainment; perturbation effects; computational model) is all indirect, meaning that the paper's claim that "inhibitory circuits generate rhythms" rests on inferred sufficiency. A direct recording (e.g., calcium imaging or patch-clamp) from 13A/13B during grooming - outside the scope of the study - would be needed to show intrinsic rhythmogenesis. The conclusions drawn from the data should therefore be tempered. Moreover, the "black box" needs to be opened. What output does it produce? How exactly is it connected to the 13A-13B circuit? The context in which the 13A and 13B hemilineages sit also needs to be explained. What do we know about the other inputs to the motorneurons studied? What excitatory circuits are there? Furthermore, the introduction ignores many decades of work in other species on the role of inhibitory cell types in motor systems. There is some mention of this in the discussion, but even previous work in Drosophila larvae is not mentioned, nor crustacean STG, nor any other cell types previously studied. This manuscript makes a valuable contribution, but it is not the first to study inhibition in motor systems, and this should be made clear to the reader.

(2) The experimental evidence is not always presented convincingly, at times lacking data, quantification, explanation, appropriate rationales, or sufficient interpretation.

(3) The statistics used are unlike any I remember having seen, essentially one big t-test followed by correction for multiple comparisons. I wonder whether this approach is optimal for these nested, high‐dimensional behavioral data. For instance, the authors do not report any formal test of normality. This might be an issue given the often skewed distributions of kinematic variables that are reported. Moreover, each fly contributes many video segments, and each segment results in multiple measurements. By treating every segment as an independent observation, the non‐independence of measurements within the same animal is ignored. I think a linear mixed‐effects model (LMM) or generalized linear mixed model (GLMM) might be more appropriate.

(4) The manuscript mentions that legs are used for walking as well as grooming. While this is welcome, the authors then do not discuss the implications of this in sufficient detail. For instance, how should we interpret that pulsed stimulation of a subset of 13A neurons produces grooming and walking behaviours? How does neural control of grooming interact with that of walking?

(5) The manuscript needs to be proofread and edited as there are inconsistencies in labelling in figures, phrasing errors, missing citations of figures in the text, or citations that are not in the correct order, and referencing errors (examples: 81 and 83 are identical; 94 is missing in text).

Reviewer #2 (Public review):

Summary:

This study examines whether the localization of endocytic proteins to presynaptic periactive zones depends on synaptic activity or active zone scaffolds. Using a combination of genetic and pharmacological perturbations in Drosophila and mouse neurons, the authors show that proteins such as Dynamin, Amphiphysin, AP-180, and others are still recruited to periactive zones even when evoked release or active zone architecture is disrupted. While the results are mostly negative, the study is methodologically solid and contributes to a more nuanced understanding of synaptic vesicle recycling machinery.

Strengths:

(1) The experimental design is careful and systematic, covering both fly and mammalian systems.

(2) The use of advanced genetic models (e.g., Liprin-α quadruple knockout mice) is a notable strength.

(3) High-resolution imaging (STED, Airyscan) is well used to assess spatial localization.

(4) The findings clarify that certain core assumptions - such as strict activity dependence of endocytic recruitment - may not hold universally.

Weaknesses:

(1) The study would benefit from a clearer positive control to demonstrate activity-dependent recruitment (e.g., Endophilin).

(2) The reliance on Tetanus toxin in the Drosophila NMJ experiments in my eyes is a limitation, as it does not block all presynaptic fusion events; this should be discussed more directly.

(3) The potential role of Dynamin in organizing other periactive zone proteins is not addressed and could be an important next step.

(4) Some small changes in protein levels upon silencing are reported; their biological meaning (e.g., compensation vs. variability) is not fully clarified.

(5) While alternative organizing mechanisms (actin, lipids, adhesion molecules) are mentioned, a more forward-looking discussion of how to test these models would be helpful.

(6) The authors should consider including, or at least discussing, a well-established activity-dependent endocytic protein (e.g., Endophilin) as a positive control to help contextualize the negative findings.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors endeavor to capture the dynamics of emotion-related brain networks. They employ slice-based fMRI combined with ICA on fMRI time series recorded while participants viewed a short movie clip. This approach allowed them to track the time course of four non-noise independent components at an effective 2s temporal resolution at the BOLD level. Notably, the authors report a temporal sequence from input to meaning, followed by response, and finally default mode networks, with significant overlap between stages. The use of ICA offers a data-driven method to identify large-scale networks involved in dynamic emotion processing. Overall, this paradigm and analytical strategy mark an important step forward in shifting affective neuroscience toward investigating temporal dynamics rather than relying solely on static network assessments

Strengths:

(1) One of the main advantages highlighted is the improved temporal resolution offered by slice-based fMRI. However, the manuscript does not clearly explain how this method achieves a higher effective resolution, especially since the results still show a 2s temporal resolution, comparable to conventional methods. Clarification on this point would help readers understand the true benefit of the approach.

(2) While combining ICA with task fMRI is an innovative approach to study the spatiotemporal dynamics of emotion processing, task fMRI typically relies on modeling the hemodynamic response (e.g., using FIR or IR models) to mitigate noise and collinearity across adjacent trials. The current analysis uses unmodeled BOLD time series, which might risk suffering from these issues.

(3) The study's claims about emotion dynamics are derived from fMRI data, which are inherently affected by the hemodynamic delay. This delay means that the observed time courses may differ substantially from those obtained through electrophysiology or MEG studies. A discussion on how these fMRI-derived dynamics relate to - or complement - is critical for the field to understand the emotion dynamics.

(4) Although using ICA to differentiate emotion elements is a convenient approach to tell a story, it may also be misleading. For instance, the observed delayed onset and peak latency of the 'response network' might imply that emotional responses occur much later than other stages, which contradicts many established emotion theories. Given the involvement of large-scale brain regions in this network, the underlying reasons for this delay could be very complex.

Concerns and suggestions:

However, I have several concerns regarding the specific presentation of temporal dynamics in the current manuscript and offer the following suggestions.

(1) One selling point of this work regarding the advantages of testing temporal dynamics is the application of slice-based fMRI, which, in theory, should improve the temporal resolution of the fMRI time course. Improving fMRI temporal resolution is critical for a research project on this topic. The authors present a detailed schematic figure (Figure 2) to help readers understand it. However, I have difficulty understanding the benefits of this method in terms of temporal resolution.

a) In Figure 2A, if we examine a specific voxel in slice 2, the slice acquisitions occur at 0.7s, 2.7s, and 4.7s, which implies a temporal resolution of 2s rather than 0.7s. I am unclear on how the temporal resolution could be 0.7s for this specific voxel. I would prefer that the authors clarify this point further, as it would benefit readers who are not familiar with this technology.

b) Even with the claim of an increased temporal resolution (0.7s), the actual data (Figure 3) still appears to have a 2s resolution. I wonder what specific benefit slice-based fMRI brings in terms of testing temporal dynamics, aside from correcting the temporal distortions that conventional fMRI exhibits.

(2) In task-fMRI, the hemodynamic response is usually estimated using a specific model (e.g., FIR, IR model; see Lindquist et al., 2009). These models are effective at reducing noise and collinearity across adjacent trials. The current method appears to be conducted on unmodeled BOLD time series.

a) I am wondering how the authors avoid the issues that are typically addressed by these HRF modeling approaches. For example, if we examine the baseline period (say, -4 to 0s relative to stimulus onset), the activation of most networks does not remain around zero, which could be due to delayed influences from the previous trial. This suggests that the current time course may not be completely accurate.

b) A related question: if the authors take the spatial map of a certain network and apply a modeling approach to estimate a time series within that network, would the results be similar to the current ICA time series?

(3) Human emotion should be inherently fast to ensure survival, as shown in many electrophysiology and MEG studies. For example, the dynamics of a fearful face can occur within 100ms in subcortical regions (Méndez-Bértolo et al., 2016), and general valence and arousal effects can occur as early as 200ms (e.g., Grootswagers et al., 2020; Bo et al., 2022). In contrast, the time-to-peak or onset timing in the BOLD time series spans a much larger time range due to the hemodynamic delay. fMRI findings indeed add spatial precision to our understanding of the temporal dynamics of emotion, but could the authors comment on how the current temporal dynamics supplement those electrophysiology studies that operate on much finer temporal scales?

(4) The response network shows activation as late as 15 to 20s, which is surprising. Could the authors discuss further why it takes so long for participants to generate an emotional response in the brain?

(5) Related to 4. In many theories, the emotion processing stages-including perception, valuation, and response-are usually considered iterative processes (e.g., Gross, 2015), especially in real-world scenarios. The advantage of the current paradigm is that it incorporates more dynamic elements of emotional stimuli and is closer to reality. Therefore, one might expect some degree of dynamic fluctuation within the tested brain networks to reflect those potential iterative processes (input, meaning, response). However, we still do not observe much brain dynamics in the data. In Figure 5, after the initial onset, most network activations remain sustained for an extended period of time. Does this suggest that emotion processing is less dynamic in the brain than we thought, or could it be related to limitations in temporal resolution? It could also be that the dynamics of each individual trial differ, and averaging them eliminates these variations. I would like to hear the authors' comments on this topic.

(6) The activation of the default mode network (DMN), although relatively late, is very interesting. Generally, one would expect a deactivation of this network during ongoing external stimulation. Could this suggest that participants are mind-wandering during the later portion of the task?

Reviewer #2 (Public review):

In this manuscript, Timblin et al provide a model where exogenous CoA is taken up by macrophages and utilized to support transcriptional events associated with activation. They provide a series of important findings, and for the most part, the data are clear and convincing. However, additional clarity on a few points would be helpful.

First of all, the contention that endogenous TLR ligands from the bone marrow cultures are driving the tonic signaling that makes exogenous CoA beneficial in unstimulated cells seems counter to the well-described anergic state of myeloid cells derived from TLR-null mice. This reviewer's understanding was that myeloid cells in MyD88 nulls or similar are developmentally anergic due to the lack of TLR stimulation in vivo. The data here (Figure 5G, etc) show these cells have much lower TLR responses, but the authors attribute it to loss of response to endogenous ligands during the cultures rather than in vivo. Testing some of the phenotypes ex vivo, etc, might make this argument more compelling and rule out that this is an effect in vivo.

Second, the data suggesting that CoA enhances anti-microbial activity via itaconate production needs additional context and/or clarification. Interactions between itaconate and CoA have been demonstrated. Itaconate exposure can deplete the CoA pool as it is converted into Itaconyl-CoA. The Irg-/- cells should not have reduced CoA due to the lack of the need to activate itaconate for metabolism. Has this been addressed by the authors? I believe that low levels of itaconate production have been shown in "resting" bone marrow cultures. The data show a full log of more bugs in the macs that lack Irg, confirming that endogenous itaconate is at work. In addition, itaconate, which is made very quickly and is likely there in considerable amounts in 4 hrs, is known to affect transcription via action on TET2. Perhaps this explains some of the connections to CoA?

Lastly, the idea that Acetyl-CoA phenocopies CoA suggests that CoA is the effector is interesting but could be supported more. Did the authors do the "unlabeling" experiment with Acetyl-CoA to confirm that it is readily converted to the CoA pool?

Do the ACLY inhibitors have the expected effects on the ChIP seq data?

Reviewer #2 (Public review):

The authors aimed to investigate whether gamma synchrony serves a functional role in figure-ground perception. They specifically sought to test whether the stimulus-dependence of gamma synchrony, often considered a limitation, actually facilitates perceptual grouping. Using the theory of weakly coupled oscillators (TWCO), they developed a framework wherein synchronization depends on both frequency detuning (related to contrast heterogeneity) and coupling strength (related to proximity between visual elements). Through psychophysical experiments with texture discrimination tasks and computational modeling, they tested whether human performance follows patterns predicted by TWCO and whether perceptual learning enhances synchrony-based grouping.

Strengths:

(1) The theoretical framework connecting TWCO to visual perception is innovative and well-articulated, providing a potential mechanistic explanation for how gamma synchrony might contribute to both feature binding and separation.

(2) The methodology combines psychophysical measurements with computational modeling, with a solid quantitative agreement between model predictions and human performance.

(3) In particular, the demonstration that coupling strengths can be modified through experience is remarkable and suggests gamma synchrony could be an adaptable mechanism that improves with visual learning.

(4) The cross-validation approach, wherein model parameters derived from macaque neurophysiology successfully predict human performance, strengthens the biological plausibility of the framework.

Weaknesses:

(1) The highly controlled stimuli are far removed from natural scenes, raising questions about generalisability. But, of course, control (almost) excludes ecological validity. The study does not address the challenges of natural vision or leverage the rich statistical structure afforded by natural scenes.

(2) The experimental design appears primarily confirmatory rather than attempting to challenge the TWCO framework or test boundary conditions where it might fail.

(3) Alternative explanations for the observed behavioral effects are not thoroughly explored. While the model provides a good fit to the data, this does not conclusively prove that gamma synchrony is the actual mechanism underlying the observed effects.

(4) Direct neurophysiological evidence linking the observed behavioral effects to gamma synchrony in humans is absent, creating a gap between the model and the neural mechanism.

Achievement of Aims and Support for Conclusions:

The authors largely achieved their primary aim of demonstrating that human figure-ground perception follows patterns predicted by TWCO principles. Their psychophysical results reveal a behavioral "Arnold tongue" that matches the synchronization patterns predicted by their model, and their learning experiment shows that perceptual improvements correlate with predicted increases in synchrony.

The evidence supports their conclusion that gamma synchrony could serve as a viable neural grouping mechanism for figure-ground segregation. However, the conclusion that "stimulus-dependence of gamma synchrony is adaptable to the statistics of visual experiences" is only partially supported, as the study uses highly controlled artificial stimuli rather than naturalistic visual statistics, or shows a sensitivity to the structure of experience.

Likely Impact and Utility:

This work offers a fresh perspective on the functional role of gamma oscillations in visual perception. The integration of TWCO with perceptual learning provides a novel theoretical framework that could influence future research on neural synchrony.

The computational model, with parameters derived from neurophysiological data, offers a useful tool for predicting perceptual performance based on synchronization principles. This approach might be extended to study other perceptual phenomena and could inspire designs for artificial vision systems.

The learning component of the study may have a particular impact, as it suggests a mechanism by which perceptual expertise develops through modified coupling between neural assemblies. This could influence thinking about perceptual learning more broadly, but also raises questions about the underlying mechanism that the paper does not address.

Additional Context:

Historically, the functional significance of gamma oscillations has been debated, with early theories of temporal binding giving way to skepticism based on gamma's stimulus-dependence. This study reframes this debate by suggesting that stimulus-dependence is exactly what makes gamma useful for perceptual grouping.

The successful combination of computational neuroscience and psychophysics is a significant strength of this study.

The field would benefit from future work extending (if possible) these findings to more naturalistic stimuli and directly measuring neural activity during perceptual tasks. Additionally, studies comparing predictions from synchrony-based models against alternative mechanisms would help establish the specificity of the proposed framework.

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:29][^2^][2]:

La vidéo présente une discussion approfondie sur la santé mentale au travail avec Samah Karaki. Elle aborde l'importance de traiter la santé mentale au travail de la même manière que dans d'autres domaines de la vie, en soulignant que le cerveau ne fait pas de distinction entre les différents environnements.

Points forts: + [00:00:15][^3^][3] Santé mentale au travail * L'importance de prendre soin de sa santé mentale * Pas de différence entre le travail et la famille pour le cerveau * L'influence de toutes les expériences vécues sur la santé mentale + [00:01:17][^4^][4] Le rôle des 'happiness officers' * Questionnement sur leur efficacité * Les besoins humains fondamentaux doivent être satisfaits * La nécessité de créer un environnement de travail sain + [00:05:01][^5^][5] La séparation entre travail et vie personnelle * Discussion sur une série télévisée dystopique * Impossibilité de séparer complètement les deux domaines * L'impact de la charge mentale des deux environnements + [00:06:02][^6^][6] Les micro-traumatismes quotidiens * Comparaison avec le stress chronique chez les animaux * L'importance de la prévention avant le soin * Alignement des environnements avec les besoins humains + [00:07:12][^7^][7] Les besoins humains fondamentaux * Discussion sur l'évolution des besoins humains * L'importance de l'autonomie, la reconnaissance et la certitude * La nécessité d'un environnement de travail qui ne menace pas ces besoins + [00:11:00][^8^][8] La productivité et le bonheur au travail * La productivité comme objectif implicite ou explicite * La stabilité émotionnelle améliore la performance * La différence entre motivations intrinsèques et extrinsèques Résumé de la vidéo [00:24:33][^1^][1] - [00:47:23][^2^][2]:

La vidéo aborde l'importance de trouver du sens dans son travail, indépendamment du poste ou de la mission. Elle souligne la nécessité d'une transparence sur les objectifs et l'utilité de chaque rôle au sein d'une entreprise. La discussion porte également sur la gestion de l'incertitude et l'importance de la reconnaissance et du respect des compétences individuelles pour prévenir le burn-out et le bore-out.

Points forts: + [00:24:33][^3^][3] Trouver du sens au travail * Importance de la connexion avec la mission de l'entreprise * Nécessité de comprendre l'utilité de son travail * Exemple de la NASA pour illustrer le sens au travail + [00:35:05][^4^][4] Le bore-out et l'épuisement professionnel * Risque d'épuisement lorsque les compétences ne sont pas sollicitées * Concept de "flow" et équilibre entre compétences et défis * Impact du bore-out sur la santé mentale et le suicide professionnel + [00:43:46][^5^][5] Gérer l'incertitude et promouvoir la diversité * Approches pour faire face à l'incertitude dans le monde professionnel * Importance de la transparence et de la tolérance à l'erreur * Valeur des profils atypiques et de la diversité des perspectives Résumé de la vidéo 00:47:25 - 00:54:35: La vidéo traite de l'importance de la diversité des perspectives et de l'écoute équitable dans le milieu professionnel pour aborder la complexité croissante du monde. Elle souligne la nécessité d'inclure des profils atypiques et de valoriser chaque voix, indépendamment du genre ou de l'apparence physique, pour apprendre les uns des autres et mieux gérer l'incertitude.

Points forts: + [00:47:25][^1^][1] Diversité des perspectives * La diversité est cruciale pour comprendre le monde complexe * Les profils atypiques apportent des vues uniques * Chaque trajectoire singulière a de la valeur + [00:48:37][^2^][2] Écoute équitable * Il est important que tous les profils se sentent légitimes à s'exprimer * L'écoute doit être sincère et non discriminatoire * L'apparence physique ne devrait pas influencer l'attention accordée + [00:50:22][^3^][3] Importance du collectif * Face à l'incertitude, la collaboration est essentielle * La discrimination positive est nécessaire mais insuffisante sans écoute active * Chaque personne a quelque chose à enseigner aux autres + [00:52:17][^4^][4] Conseils pour les leaders * Reconnaître que personne n'a toutes les réponses * Valoriser l'intelligence collective et l'expertise diverse * Encourager la formulation des bonnes questions par tous

Exercise to Consider: Set a timer for 1 minute. Then, write down any words that come to mind, that you believe describes yourself.. Once the timer ends, review the list of words, and then respond to the following reflective questions: 1. How many of the words you listed are subjective vs. objective? 2. How many of the words listed are things that you have said about yourself vs what others have said about you? 3. How many of the words listed are what you want to be vs. who you are? 4. Are there any words that you intentionally did not write down? If so, why? 5. How does the way you responded to the above questions reveal about your self-concept?

Briefing : Révolutionner la productivité des associations grâce au No-Code et à l'IA

Introduction

Ce briefing récapitule les points clés abordés lors du webinaire organisé par Solidatech, en partenariat avec Contournement et Nocode Forgood.

L'objectif principal de cette session était de démontrer comment les outils "no-code" et l'intelligence artificielle (IA) peuvent permettre aux associations de "gagner des dizaines d’heures par mois" et de renforcer leur impact numérique.

1. Solidatech : Renforcer l'impact numérique des associations

• Solidatech est une organisation française fondée en 2008, dédiée à l'accompagnement des associations dans leur transformation numérique.

Composée d'une douzaine de personnes, elle opère depuis Paris et les Deux-Sèvres, où se situe sa coopérative d'insertion, les Ateliers du Bocage (mouvement Emmaüs), spécialisée dans le réemploi de matériel bureautique. Solidatech est également le satellite français du réseau international TechSoup.

Public cible :

Plus de 42 000 associations inscrites gratuitement. Divers statuts juridiques : associations (locales ou plus grandes), fondations, fonds de dotation, bibliothèques publiques. Toutes tailles d'organisations, avec ou sans employés (y compris celles composées uniquement de bénévoles).

Piliers d'accompagnement :

• Accès à des outils et matériels à tarifs réduits : Offre de logiciels et matériels reconditionnés (ordinateurs, écrans, smartphones, etc.) avec des remises allant de -30% à -90%, voire des gratuités. Exemples de partenaires : Cisco, Dell.
• Développement des usages numériques :Centre de ressources gratuit.
• Équipe support basée dans les Deux-Sèvres pour le conseil et le choix des licences.
• Outil de diagnostic numérique pour évaluer la maturité numérique.
• Étude nationale annuelle sur la place du numérique dans le milieu associatif.
• Accompagnement et formation :Newsletters régulières.
• Partenariat Prestatek (annuaire de prestataires de services).
• Webinaires thématiques variés.
• Formations certifiées Qualiopi sur des sujets comme la conformité RGPD, la communication digitale, la recherche de financement, la gestion de dons, et l'utilisation d'outils (Microsoft 365, Canva).
• Impact : Solidatech aide les associations à réaliser des "économies monétaires [et] en temps", à gagner en maturité numérique et à se professionnaliser.

2. Le No-Code et l'IA : Définitions et promesses

Erwan Kezzard, cofondateur de Contournement, a introduit les concepts de no-code et d'IA générative comme des leviers majeurs pour optimiser le temps. Il souligne que "le temps... c'est une ressource extrêmement importante, notamment quand on travaille soit aux sources contraintes".

Définition du No-Code :

• "Le nocode comme son anxie son son les les l'exprime et l'exige ce sont des outils qui permettent de réaliser visuellement intuitivement des projets numériques sans forcément avoir de compétences informatiques en code."

• Permet de créer des sites web, des petites applications, d'automatiser des tâches, de créer des solutions internes, etc., de manière visuelle et intuitive.

• Exemple : Excel n'est pas du no-code ; le no-code permet de "créer ses propres outils".

Définition de l'IA Générative :

Il s'agit des IA accessibles comme "les Chat GPT, Mistral et autres qui sont euh bah des technologies auxquelles on peut assez rapidement demander des choses demander de retraiter du contenu demander de faire des recherches et elle nous répond".

Potentiel et Bénéfices :

• L'objectif est de "gagner des dizaines d'heures par mois" en évitant les "manipulations répétitives, des tâches non informatisées ou des tâches informatisées mal optimisées".
• Principal usage : l'optimisation de la productivité, c'est-à-dire "travailler mieux pour faire autant ou faire moins". Cela concerne l'optimisation des "ops" (opérations quotidiennes) en administratif, RH, gestion de projet, etc.
• Exemple de gain de temps : "si cinq fois par jour je passe 5 minutes à faire une tâche à la fin de l'année j'aurais passé 6 jours plein 6 jours de travail à ne faire que ça".

3. Les Trois Briques Fondamentales du No-Code Erwan Kezzard a illustré les capacités du no-code à travers trois piliers principaux, souvent combinés :

Bases de données visuelles :

Outil clé : Airtable (alternative française : TimeTonic).

• Fonctionnalité : Ressemble à Excel mais est une "base de données", où chaque ligne est une fiche. Les colonnes ont des types de données spécifiques (liens, sélecteurs, pièces jointes, dates).
• Avantages : Création rapide de vues filtrées et segmentées ("vues" pour stagiaires, commerciaux, DG), gestion des accès, formulaires d'entrée de données (créé en "moins de 25 secondes").
• Concept de "relations" : Possibilité de lier des entrées entre différentes tables (ex: lier des prospects à des entreprises), ce qui résout de nombreux problèmes de ressaisie et de cohérence des données. Permet de naviguer entre les données comme sur une application.
• Permet de construire des "CRM que je me fais moi-même" et des "intranets".

Automatisation et interconnexion :

• Outil clé : Zapier (alternatives : Make, N8N - open source mais plus technique).
• Fonctionnalité : Connecte différents outils pour automatiser des processus.
• Exemple : "à chaque fois que dans Airtable il y a une nouvelle entrée dans la table entreprise alors automatiquement va dans le chat GPT demande-lui 'Tu es un expert en politique RSE...' puis prends cette convers enfin trouve la ligne dans Air Table et mets à jour cette ligne avec l'info directement ici".
• Permet d'automatiser des notifications (Teams, Slack), des envois de mails personnalisés, la création de documents (PDF), etc.
• L'IA "fait qu'un seul boulot" (poser la question), le no-code "fait le boulot" des tuyaux d'interconnexion.

Interfaces (Sites web, applications mobiles/web) :

• Outil clé : Glide (pour applications mobiles), Software (pour applications web / intranets).
• Fonctionnalité : Permet de créer des applications mobiles ou web à partir d'une base de données existante (ex: Airtable).
• Avantages : Ne nécessite aucune installation ni hébergement, permet de modifier l'apparence et les fonctionnalités intuitivement. "je peux modifier cette application mobile changer l'apparence changer l'info qui apparaît où et cetera".

4. Philosophie et Positionnement de Contournement & Nocode Forgood

Contournement :

• Métier : "former les équipes et les individus au nos code et Alia pour leur permettre de travailler plus efficacement".
• Ne vise pas à lancer la "prochaine start-up à la mode" mais à "gagner du temps", "digitaliser" et "fluidifier ses processus".
• Offre de formations en présentiel, téléprésentiel, et e-learning (plateforme avec abonnement à coût accessible, réductions prochainement à 50-100€/mois).

Accompagne aussi des publics éloignés de l'emploi.

• Vision du no-code : une "compétence complémentaire" valorisable sur le marché de l'emploi ("je suis chargé de communication... je me forme quelques jours au nocode je sais digitaliser automatiser dans mon métier et ça m'apporte quelque chose").
• Met en garde contre le "miroir aux alouettes" et le "charlatanisme" liés au no-code comme "nouveau métier".

Nocode France :

• "La communauté la plus active au monde dans le Nocode qui est française".
• Composée de 15 000 personnes qui "s'entraident bénévolement", offrant conseils et orientations.

Nocode Forgood :

• Mission : "donner accès aux outils no code les plus démocratiques du numérique pour rendre la vie plus simple aux assos et leur permettre de démultiplier leur impact".
• Fait découvrir le no-code (masterclass) et surtout met en relation des associations avec des "nocodeurs et des nocodeuses engagés".
• Approche "MVP" (Minimum Viable Product) : "commencer petit", "réaliser le plus vite possible un morceau qui fonctionne et après de l'adapter". L'objectif est d'aider les associations à "faire leur skateboard" (amorce), puis de les accompagner.
• Projets : les nocodeurs peuvent travailler bénévolement (avec contrepartie de formations Contournement) ou à "tarif solidaire".

5. Exemples concrets de succès

• Les Francofolies : "15 personnes un seul informaticien". En deux jours de formation Airtable, ils ont gagné "plusieurs dizaines d'heures par semaine" notamment sur le reporting carbone.

Ils ont aussi fait appel à une experte Ania pour des projets plus complexes, mais ont aussi décidé de ne pas "nocoder" certains processus peu chronophages.

• La MedNum : Coopérative qui gère son sociétariat, ses projets et son organisme de formation avec Airtable (base de données) et Make (automatisation).

Gagne "énormément de temps". Utilise aussi Notion pour la documentation interne et les ressources textuelles.

• Wildlife Impact Network (via Nocode Forgood) : Création d'un site avec Softer et d'une galerie de projets finançables avec Airtable en deux jours.
• Naestan (via Nocode Forgood) : Création d'un outil de pilotage et de reporting interne pour une association d'aide aux jeunes Afghans, réalisé avec CODA.
• Nocode Forgood (interne) : Automatisation de la génération de brouillons de posts LinkedIn à partir de retours d'expérience d'associations, via Make et l'IA.

6. Bonnes pratiques et avertissements

• Cartographier avant de se lancer : "Une bonne pratique c'est de cartographier avant de se lancer".
• Ne pas tout no-coder à outrance : "pas besoin de tout nous coder les meilleurs outils ça peut être de trouver des outils spécialisés". Si un processus fonctionne bien, ne pas le modifier.
• Outils modernes et interconnectables : Privilégier les outils qui peuvent se connecter entre eux (vérifier la compatibilité Zapier ou Make). Exemple : Assoconnect est intégrable avec Zapier et Make.
• Collaboration avec l'IT et les juristes : "appuyez-vous toujours sur l'IT sur le juridique sur les décisionnaires ne faites pas du shadow IT dans votre coin sur le nocode s'il y a des gens qui doivent être décisionnaires avec vous ça peut exposer à des risques de données mal géré et cetera de sécurité".
• Formation : Même si le no-code est accessible, un minimum de formation est nécessaire. "Au bout d'une journée ou de 2 à 5 jours de formation les gens peuvent commencer à faire des choses".
• Appui sur des experts externes : Recommandé pour éviter les erreurs (ex: données publiques par erreur) et structurer des projets plus complexes.
• Coût : "Un outil no code qui se respecte est payant déjà un dans un outil de code qui se respecte est fremium". Les tarifs commencent souvent entre 15 et 30€/mois. Il faut prévoir "entre 50 et 100 € de budget mensuel" pour faire beaucoup de choses. C'est un investissement rapidement amorti.
• RGPD et stockage des données :L'hébergement aux États-Unis n'est pas intrinsèquement non-RGPD. De nombreux outils américains sont "RGPD compliant".
• Il est crucial de consulter un juriste pour les données sensibles.
• "Le RGPD rappelons que c'est un process où vous vous devez faire toute une démarche de nous par exemple contournement on a tout un registre où on dit où sont stocké quelle donné et on fait gaffe régulièrement à supprimer les données qui ont plus de 3 ans".
• Les outils no-code payants ne "vendent" généralement pas vos données, leur modèle économique étant basé sur l'abonnement. Le risque principal est lié aux exigences gouvernementales (Cloud Act, Patriot Act).
• Migration de bases de données : Simple via l'import CSV dans Airtable (ou TimeTonic, Notion). Possibilité de synchroniser des bases existantes (ex: Excel) avec Airtable via Zapier/Make.
• Différence Notion vs. Airtable : Notion est "plus orienté je prends des notes", gestion de "contenu riche", "espace collaboratif tout en un" (wiki, documentation interne). Airtable est centré sur la "donnée" et sa structuration.

7. Outils de productivité IA complémentaires

Whisper Flow : Outil de dictée vocale permettant de "dicter et ne plus taper quasiment au clavier". Reconnaissance précise de la syntaxe et de la ponctuation. Dict AI : Application mobile française et souveraine pour "prendre en note les réunions automatiquement" et générer des comptes-rendus.

Conclusion

Le no-code et l'IA représentent une opportunité significative pour les associations de toutes tailles d'améliorer leur efficacité opérationnelle et de se professionnaliser.

Des organisations comme Solidatech, Contournement et Nocode Forgood jouent un rôle essentiel dans la démocratisation de ces technologies, en offrant des ressources, des formations et un accompagnement adapté, tout en soulignant l'importance de l'éthique, de la sécurité des données et d'une approche pragmatique dans leur adoption.

Briefing : Réussir son projet numérique associatif

Ce document synthétise les points clés du webinaire "[Webinaire] De l'idée à la réalisation : comment réussir votre projet numérique associatif ?", animé par Gautier Jeanzac de Pastec et une représentante de Solidatech.

Il vise à fournir une méthodologie claire et des conseils pratiques pour les associations souhaitant entreprendre un projet numérique.

I. Solidatech : Un partenaire pour la transformation numérique des associations

Solidatech est présenté comme un programme de solidarité numérique créé en 2008, porté par les Ateliers du Bocage (mouvement Emmaüs). Son objectif est de "renforcer l'impact des associations par le numérique" et "renforcer votre impact à travers une meilleure utilisation du numérique".

1. Bénéficiaires et Éligibilité :

• Principalement les associations loi 1901, mais aussi les fondations RUP, fonds de dotation, et bibliothèques publiques.
• L'inscription est gratuite et ouverte "quel que soit votre secteur d'activité, que aussi vous vous soyez bah voilà vous fonctionnez entièrement avec que des bénévoles ou au contraire des des centaines de salariés".
• Plus de 42 000 associations sont déjà inscrites.

2. Modes d'action pour accompagner les associations :

• Faciliter l'accès au numérique :Logiciels à tarifs réduits (Microsoft, Adobe, Zoom, ainsi que des solutions françaises comme Assoctoc, Spirit, Net Explorer, Insia).
• Matériel informatique reconditionné (par les Ateliers du Bocage) et neuf (grâce à des partenaires comme Cisco et Dell).
• Accompagnement au développement des usages du numérique :Centre de ressources et outil d'autodiagnostic.
• Formations (Solidatech est un organisme de formation certifié Qualiopi), webinaires thématiques mensuels.
• Newsletters.

Prestations de conseil sur mesure.

Coproduction et diffusion de savoirs :Réalisation d'une étude nationale sur la place du numérique dans le projet associatif tous les 3 ans (5e édition lancée mi-avril 2025, dernière en 2022).

II. Pastec : Expertise en conseil et développement informatique pour les projets à impact

• Gautier Jeanzac représente Pastec, une société coopérative qui est une "agence de conseil et développement en informatique" spécialisée dans l'accompagnement de "projets à impact social et ou environnemental", travaillant "beaucoup avec des associations".

1. Expertises Métiers de Pastec :

• Conseil et gestion de projet / Direction technique partagée : Aide à la conception (architecture, planning) et au pilotage du développement produit.
• Développement de produits numériques : Basé sur un cahier des charges, en "code traditionnel" ou "no code".
• Numérique responsable : Conception de services "éco-conçus", "accessibles" et "respectueux du RGPD" (Règlement Général sur la Protection des Données).

2. Rôle du Chef de Produit (Gautier Jeanzac) :

Point de convergence entre la vision du client, les attentes des utilisateurs et les possibilités techniques. Aide à piloter la vie du produit en intégrant ces différentes perspectives.

III. La formalisation du besoin : Clé de voûte du projet numérique

La transformation numérique est définie comme "l'idée d'intégrer des technologies numériques dans l'ensemble des activités de l'association".

1. Freins et Opportunités de la transformation numérique :

• Freins majeurs dans les associations : "le coût, le temps et les compétences".
• Opportunités : "améliorer l'efficacité opérationnelle, moderniser des services pour les bénéficiaires, renforcer l'impact des associations".
• Exemple : Numérisation de processus administratifs chronophages (ex: suivi des bénévoles via fichiers Excel) pour gagner du temps et "passer ce temps-là du coup on peut le passer sur ces métiers".

2. Du besoin métier au produit numérique :

• Identifier un besoin métier : Souvent issu d'un "point de douleur" (ex: "processus d'adhésion très complexe", "beaucoup de saisies manuelles").
• Question clé : "où est-ce qu'on dépense trop d'énergie et de temps ?"
• Rédiger un cahier des charges : Document "de synthèse en fait de tous ces besoins", permettant de "prendre du recul avant de vous lancer dans la dans le développement".
• C'est "le document qui vous permet d'expliquer et d'expliciter le ou les objectifs à atteindre de votre service ou de votre outil numérique".
• Sert de référence pour le développement, que ce soit avec un prestataire ou en interne.

3. Structure recommandée d'un cahier des charges (6 parties) :

• Contexte : Qui est l'association, son histoire, ses objectifs, l'origine du besoin numérique (passé, présent, futur du projet).
• Lexique : Définir les termes et acronymes propres à l'association pour une compréhension externe.
• Technique : Préciser les éléments techniques existants (langage, hébergement, développeur précédent, contraintes spécifiques, migration des données, contrat de maintenance, RGPD).
• Règles d'utilisation (User Stories) : Décrire les interactions des utilisateurs avec le service sous forme de "petites histoires". Ex: "En tant que bénévole de l'association, je souhaite pouvoir me connecter à un espace membre qui m'indique depuis combien de temps je suis bénévole". Inclure le résumé, les détails et les critères d'acceptation.
• UX (User Experience) et UI (User Interface) :UX : "la capacité à concevoir ce qu'on un parcours utilisateur" (comment l'utilisateur interagit avec le logiciel et ses différentes étapes).
• UI : "l'interface utilisateur" (ce que l'utilisateur va voir, l'aspect visuel, les maquettes, les wireframes - schémas d'écran).
• Annexes : Tout document complémentaire pertinent.

4. Exemple de CRM pour une association d'entrepreneuriat :

Le cahier des charges a permis de suivre les interactions avec les parties prenantes. Le besoin de CRM est venu d'un "audit qui a fait remonter le besoin". Illustration concrète des user stories et des contraintes techniques (intégration dans un SI global, respect RGPD).

IV. La construction du produit : Intégrer les utilisateurs et itérer

La phase de construction insiste sur l'importance d' "intégrer en fait les équipes les bénévoles vos utilisateurs et vos utilisatrices dans la réflexion de ce à quoi va ressembler votre produit".

1. Mener des actions terrain (Interviews Utilisateurs) :

• Objectif : Comprendre les points de douleur et les besoins des utilisateurs (ce qu'ils veulent, ce qu'ils pensent de l'existant, comment améliorer).
• Cinq grandes étapes :Préparation efficace : Définir les informations à recueillir et le "fil rouge" de l'entretien.
• Début d'entretien : Poser le cadre, mais surtout insister sur le fait qu'il n'y a "pas de bonnes réponses" pour encourager l'honnêteté.
• Pendant l'entretien : Parler "20%" et écouter "80% du temps". Privilégier les "questions ouvertes".
• Après l'entretien : Traduire les besoins en fonctionnalités.
• Priorisation : Développer en priorité ce qui "coûte le moins de temps et qui apporte le plus pour mes utilisateurs" (les "victoires rapides").

2. Méthodologie Agile et "Petits Pas" :

• Mettre l'utilisateur au cœur de la réflexion.
• Commencer par un "plus petit lot possible de fonctionnalité à développer" (un "skateboard" avant la "voiture").
• Processus itératif : Proposer de nouvelles fonctionnalités, les utilisateurs les utilisent, remontent de nouveaux besoins, et le cycle recommence.
• Exemple : Formulaire d'adhésion simple (nom, email) puis ajout progressif de champs (adresse, région) basés sur les retours des équipes.
• Avantages de l'approche itérative : Développer uniquement les fonctionnalités demandées, éviter le budget inutile, créer des outils adaptés aux besoins.
• Observation d'usage : Observer physiquement les utilisateurs pour comprendre leurs interactions et ajuster le produit.

V. Le développement : Code traditionnel vs No Code Deux grandes familles de développement sont présentées :

1. Code traditionnel :

Avantages : Peu ou pas de limites, possibilité de "tout faire" pour répondre à des besoins très précis. Inconvénients : Plus long et donc plus cher (coût basé sur le temps de développement), "complicité technique" qui rend la compréhension difficile pour les non-initiés (nécessite un bon partenaire pour "vulgariser les points techniques").

2. No Code :

• Définition : Outils pour créer des applications ou sites sans coder, comme des "Legos" (assemblage de blocs).
• Avantages : "Rapidement mettre une solution en place", itérer vite, coût "un petit peu moins cher".
• Inconvénients : "Moins personnalisable", nécessite une grande vigilance sur "l'écoconception, l'accessibilité et parfois du RGPD", outils propriétaires (la solution ne vous appartient pas).

3. Exemples d'outils No Code :

• CRM : Monday, Airtable (attention au RGPD car hébergement aux USA, lié au Patriot Act), Grist (alternative RGPD, hébergeable où l'on veut).
• Formulaires : Type Form, Tally (version hébergée en Europe, plus RGPD).
• Sites vitrines : Wix, Webflow, Bubble (Canva peut être une alternative si l'association est à l'aise avec l'outil, car il existe des versions premium gratuites pour les associations).
• Applications web : Bubble, XAR.
• Automatisation : Make, Zapper.
• Wireframes : wireframe.cc (gratuit, simple), Canva, Paint, PowerPoint. Pour des maquettes plus complexes : Figma, suite Adobe (Adobe Express est gratuit en version premium pour les associations).

VI. Travailler avec une agence et derniers conseils

Le développement d'outils numériques, qu'il soit en code ou no code, "demande du temps et des compétences techniques".

Si une association n'a pas le temps et préfère passer par une agence, plusieurs conseils sont donnés :

• Équipe interne dédiée au pilotage : Avec "un pouvoir décisionnel" pour faciliter les évolutions.
• Approche collaborative et construite : Intégrer toutes les parties prenantes, surtout les utilisateurs finaux.
• Ne pas hésiter à demander et exiger la vulgarisation : S'assurer de comprendre ce qui se passe techniquement pour pouvoir reprendre la main si besoin.
• Adopter la stratégie des petits pas : Éviter de développer des fonctionnalités inutiles et optimiser le budget.
• Être adaptable : Trouver le juste milieu entre la vision initiale et les retours des utilisateurs.
• Garder du temps pour se former : Comprendre les aspects techniques du produit.

Concernant l'utilisation de l'IA pour le développement :

Réponse nuancée : "J'en sais rien". * Vigilance : Dépend des outils et modèles utilisés. * Prudence : Ne pas déployer des choses non maîtrisées pour éviter les erreurs ou des problèmes ingérables.

• Ce briefing offre une feuille de route complète, de l'identification du besoin à la concrétisation du projet, en soulignant l'importance de l'écoute des utilisateurs et d'une approche itérative et adaptable.

Note d'information : L'engouement des jeunes pour les soins de la peau (Skincare) et ses implications

• Ce document de synthèse examine la tendance croissante des jeunes, en particulier la "génération Alpha" (née après 2010), à adopter des routines de soins de la peau complexes, souvent inspirées par les médias sociaux.

Il met en lumière les motivations de cette tendance, les risques potentiels pour la santé et les mesures prises ou recommandées par les professionnels de la santé et l'industrie.

Thèmes Principaux :

• L'adoption précoce des routines de soins par les enfants et préadolescents : Une tendance omniprésente, alimentée par les réseaux sociaux et l'influence des pairs.

• Les risques pour la santé dermatologique et hormonale : L'utilisation de produits non adaptés aux peaux jeunes peut entraîner irritations, allergies, et des effets synergiques potentiellement dangereux à long terme.

• L'impact de l'industrie cosmétique et des influenceurs : Les marques ciblent spécifiquement ce nouveau public, créant des produits attrayants mais parfois inappropriés.

• La réaction des professionnels et les tentatives de régulation : Dermatologues, cosmétologues, et pharmaciens expriment leur inquiétude et appellent à une meilleure information et à des restrictions.

Synthèse Détaillée :

1. L'engouement généralisé pour le "Skincare" chez les jeunes

• Une routine complexe dès le plus jeune âge : Charlotte, 12 ans, décrit une routine typique de "skincare" incluant nettoyage, lotion, masques et crèmes hydratantes. Elle affirme que "90% [des filles de sa classe] en font [du skincare], il y en a même qui en font beaucoup plus que moi qui mettent énormément de produits".

• Influence des pairs et des réseaux sociaux : Charlotte a découvert cette tendance par ses amis et "en regardant internet aussi il y avait plein de gens qui commençaient à parler de ça". TikTok est devenu "un réseau où l'on expose sa skinc routine sa collection de crème et de lotion". La génération Alpha "copie leurs aîné influenceuses leur style leur vocabulaire".

• Perception du "skincare" comme un jeu : À 10-12 ans, "prendre soin de sa peau c'est devenu un jeu qu'on expose".

• Obsession pour la prévention du vieillissement : Des enfants "vantaient de la crème anti-ride persuadé que plus on commence tôt moins on aura de ride". Une fillette de 7 ans s'inquiétait de son "stock d'acide hyaluronique" qui disparaissait.

• Stratégies d'acquisition : Pour gérer le "sacré budget" des produits, les jeunes utilisent des "techniques avec mes amis" pour obtenir des échantillons gratuits dans des grands magasins comme Globus et Manor, se faisant connaître comme "très connu" par les vendeuses.

2. Les dangers et préoccupations des professionnels

• Produits inadaptés aux peaux jeunes : La dermatologue pédiatre constate que "ce qu'elles utilisent ne sont pas des produits adaptés à leur peau qu'en plus d'habitude elles n'ont pas besoin de crème ni de quoi que ce soit à cet âge là".

• Risques d'irritations et d'allergies : Des amies de Charlotte ont déjà eu "plein de boutons ou des rougeurs" suite à l'utilisation de produits. La dermatologue s'inquiète de savoir "est-ce qu'il va y avoir des irritations est-ce qu'il va y avoir des allergies est-ce que ça peut être plus grave".

• Effet "cocktail" et perturbations hormonales : L'utilisation de multiples produits crée un "effet de cocktail" ou "synergique" où "chacun va influencer l'autre", ce qui "semble qu'à long terme ce soit plus dangereux qu'on l'avait penser au départ". Il y a une inquiétude quant à la "perturber [des] hormones à long terme".

• Ingrédients problématiques pour les enfants :Rétinol : "Le rétinol par exemple peut irriter les peaux d'enfant". Un masque testé contenait du rétinol et un dérivé se transformant en "acide rétinoïque qui est interdit dans le domaine cosmétique".

• Pellings à pH très acide : Peuvent provoquer "des picotements", "des rougeurs", et même "des brûlures".

• Perturbateurs endocriniens : Certains masques en contiennent, comme le produit Skin Republic.

• Extraits éclaircissants : Utilisés pour l'éclaircissement, alors que les jeunes n'ont pas de problèmes de tâches cutanées ("on va pas aller essayer d'éclaircir des tâches qui n'existent pas").

• Molécules toxiques ou à effet "Botox végétal" : Le spilantol (extrait d'acmella) est "vraiment à éviter chez l'enfant" en raison de son "profil toxicologique qui n'est pas forcément très rassurant".

• Ingrédients œstrogéniques ou corticomimétiques : Un masque "le pire" contenait de l'origan à caractère œstrogénique et des extraits à effet corticomimétique.

• Allergènes interdits : Un masque liquide contenait un allergène parfumé ("hydroxy isoexyl 3 cycloexène carboxaldéide") désormais "interdit par la réglementation européenne et donc en Suisse aussi".

• Masques peel-off : Contiennent des "matières plastiques" pour obtenir l'effet pelable, en plus de générer "beaucoup de déchets" avec les emballages à usage unique.

3. Le rôle de l'industrie cosmétique

• Ciblage des jeunes : L'industrie cosmétique voit cette génération comme "le nouveau public cible". De "nouvelles marques qui ressemblent à des bonbons naissent chaque mois".

• Marketing trompeur : Les jeunes sont "finalement victimes des publicités puisque l'industrie cherche à agrandir son cercle de clientèle donc il faut toujours consommer plus".

• Manque d'information : Les très jeunes filles connaissent "un rayon sur les crèmes leurs effets leurs indications mais qui n'ont pas conscience que certains contenus ne sont pas adaptés".

• Absence de mention d'âge recommandé : "Légalement les fabricants n'en ont pas l'obligation", mais ils "réalisent maintenant que leurs produits ne sont pas toujours utilisés par le public qui était visé".

4. Réactions et recommandations

• Restrictions en Suède : La principale chaîne de pharmacies en Suède a décidé de "ne plus vendre certains produits au moins de 15 ans des antirides notamment", suite à l'augmentation des demandes de "produits skinc avancés" par des enfants de 8 à 10 ans. La réaction du public a été "très très nombreuses... toutes positives".

• Appel à la prudence en Suisse : L'association des cosmétiques et détergents en Suisse a publié un communiqué précisant que "les produits cosmétiques anti-âge n'ont pas vocation à être utilisés sur des pots d'enfants" et que "les parents doivent veiller à ce que des produits cosmétiques adaptés au besoins de la peau des enfants et des adolescents soient utilisés".

• Sensibilisation des vendeuses : Des tests réalisés par Inès et Lia (10 et 11 ans) ont montré que les vendeuses dans des enseignes comme Douglas, Kiko et Marionnaud sont désormais sensibilisées, déconseillant l'achat de produits inadaptés en raison de l'âge ("trop forte et que ça pouvait nous brûler la peau") et les mettant en garde contre "ce qu'on avait vu sur Youtube".

• Vente libre problématique : Cependant, ces mêmes produits peuvent être achetés en "se servant directement sur l'étalage sans passer par le conseil d'une vendeuse", ce qui n'est "rien d'illégal".

• Nécessité de ralentir la création de marques : La professeur de cosmétologie estime qu'il faut "arrêter... qu'il y ait moins de marque mais de meilleure qualité".

• Recommandation d'éviter le "layering" : La superposition de cosmétiques ("layering") est "aberrant" même pour l'adulte car cela crée un "effet occlusif" et "accumulation qui n'est pas bon pour la peau", et "encore moins pour l'enfant".

• Interdiction des ingrédients dangereux : Une analyse en laboratoire a révélé que 10 des 15 masques testés contenaient des "ingrédients problématiques pour des peaux jeunes" et ne devraient "pas être utilisé par des enfants ou des adolescentes", avec un cas de produit "illégal" car contenant un allergène interdit par la réglementation européenne.

En conclusion, l'engouement des jeunes pour le "skincare" est une tendance préoccupante en raison des risques potentiels pour leur santé dermatologique et hormonale, liés à l'utilisation de produits inadaptés et à des pratiques excessives.

L'industrie est invitée à davantage de responsabilité, et les parents sont appelés à une vigilance accrue, tandis que des mesures restrictives, comme celles prises en Suède, pourraient être envisagées plus largement.

Synthèse Détaillée : Comprendre et Combattre les Préjugés

Ce document explore en profondeur la nature des préjugés, des stéréotypes et de la discrimination, leurs origines, leurs manifestations subtiles et leurs impacts.

Il propose également des stratégies concrètes pour les identifier, les mesurer et les réduire, soulignant l'importance de la conscience de soi et de l'éducation.

1. Distinction des Concepts : Stéréotypes, Préjugés et Discrimination

Le document établit une distinction claire entre trois concepts souvent confondus :

• Stéréotype : Défini comme "une croyance, une opinion, où l’on plaque des caractéristiques à tout un groupe social".

Ces croyances peuvent être personnelles ou, plus souvent, "des croyances partagées qu'on a apprises sans s’en rendre compte, via nos proches, nos environnements sociaux, les médias, etc."

Les stéréotypes peuvent être "positifs" (ex: "les garçons sont généralement doués en maths") ou "négatifs" (ex: "les filles seraient moins douées en maths").

Il est crucial de noter que les stéréotypes ne sont pas seulement descriptifs mais aussi prescriptifs, créant des attentes.

• Préjugé : Représente "l’émotion, le sentiment, les attitudes négatives qu’on peut avoir vis-à-vis d’un groupe social", souvent associées à des stéréotypes.

Les préjugés sont renforcés dans des contextes où l'on se sent menacé par un groupe, que ce sentiment soit fondé ou non.

Une personne confrontée à une réalité qui contredit ses stéréotypes peut générer une "réaction négative : un préjugé."

• Discrimination : Désigne des "comportements spécifiques où l’on traite différemment les individus selon leur groupe social perçu".

Cela peut se manifester par des "remarques déplacées, ou encore ignorer ou désavantager volontairement quelqu’un."

• Il est souligné que ces concepts ne sont pas toujours interconnectés de manière linéaire :

"On peut avoir connaissance des stéréotypes, même les plus négatifs, sans que cela génère en nous des préjugés.

On peut aussi avoir des préjugés, sans que cela aboutisse à des comportements discriminants."

La conscience de ses propres préjugés peut même permettre de s'ajuster pour éviter les comportements discriminants.

2. Le Masque des Préjugés et les Stratégies de Dissimulation

Les préjugés sont souvent dissimulés ou exprimés de manière détournée, car "nos préjugés avancent masqués."

• Humour de dénigrement : Une stratégie courante est l'utilisation de l'humour, où "une remarque sexiste, suivie d'un « Oh mais ça va, c'est une blague ! »" sert de "couverture" pour "rendre socialement plus acceptable l’expression de préjugés, tout en pouvant se défendre qu’il s’agit là de préjugés."

• Rhétorique pseudodémocratique : Dès les années 40, Adorno et ses collègues ont étudié cette stratégie où les personnes "préfèrent atténuer l’expression de leur préjugé en utilisant le conditionnel, ou des conjonctions comme mais."

L'exemple classique est "Je ne suis pas raciste, mais..." ou "Ce n'est pas que j'ai des préjugés, mais...". Ces discours à forts préjugés sont "déguisé[s] sous l’apparence d’ouverture et de tolérance."

• Déni et sous-estimation : Paradoxalement, "on peut avoir tendance à sous-estimer ses propres préjugés, à croire qu’on n’en a pas, et paradoxalement, c'est ce qui peut alimenter des stéréotypes et conduire à des discriminations."

Les personnes ayant des préjugés se répètent, à elles-mêmes et aux autres, "ne pas en avoir, comme s’il suffisait de dire Je ne suis absolument pas raciste. Pour annuler le reste du propos."

3. Préjugés Implicites et Leurs Origines

Le document introduit la notion de préjugés implicites : "Oui. Et on parle dès lors de préjugés implicites."

• Nature des préjugés implicites : Contrairement aux préjugés explicites (conscients), les préjugés implicites sont "inaccessibles, automatiques, omniprésents, et influencent d’une manière unique nos jugements et comportements."

Ils agissent "comme des tâches en arrière plan" de notre conscience, influençant nos interactions sans que nous nous en rendions compte.

"Autrement dit, on peut sincèrement penser ne pas avoir de préjugés et pourtant en avoir en toile de fond."

• Sources des préjugés : Qu'ils soient implicites ou non, les stéréotypes et préjugés sont "construits, appris très tôt dès notre enfance auprès de nos parents, de notre famille, de nos environnements sociaux, à partir de certains évènements vécus, ou encore à travers les médias de masse."

L'exemple donné est celui de la télévision dans les années 80, où "plus les gens regardaient la télévision, plus leurs préjugés racistes et sexistes se retrouvaient fortifiés" à force de voir des "représentations stéréotypées."

4. Les Microagressions : Manifestations Subtiles de la Discrimination

Le document met en lumière les "microagressions" comme des "comportements plus subtils et bien plus présents au quotidien."

Ces comportements, bien que peu visibles, ont des conséquences préjudiciables lorsqu'ils sont répétés :

• Exemples : "faire des blagues sexistes, avoir tendance à couper la parole et à la monopoliser, mépriser l’identité d’une personne, par exemple en la mégenrant, ne pas tenir compte de l’avis d’une personne en raison de sa couleur de peau, de son genre, orientation sexuelle, handicap, etc."

• Impact : Elles entraînent une "diminution de l’estime personnelle, dépression, anxiété, sentiment d’impuissance, culpabilisation" chez les victimes, et contribuent à "maintenir et renforcer les inégalités dans la société."

5. Mesurer les Préjugés : Les Méthodes des Psychologues

Les psychologues utilisent diverses méthodes pour étudier les préjugés, notamment :

• Questionnaires d'auto-évaluation : Simples, mais sujets au "biais de désirabilité sociale", où les gens modifient leurs réponses pour être bien perçus. L'anonymat peut atténuer ce biais.

• Observation comportementale : Des situations mises en scène permettent d'observer les comportements des gens sans qu'ils sachent qu'ils sont testés.

L'exemple donné est celui de l'aide apportée à des personnes hétérosexuelles ou homosexuelles, montrant un "pourcentage plus élevé d’aide dans la condition “hétérosexuel”."

• Mesures physiologiques : Fréquence cardiaque, activité électrique du cerveau (IRM fonctionnelle).

Il est mentionné que "lorsqu’on présente à des personnes blanches sous IRM fonctionnelle des photos d’hommes noirs, ils auront une réponse émotionnelle négative, ce qui n’est pas le cas devant la photo d’un homme blanc," interprétée comme une "perception de menace."

• Tests d'association implicites (IAT) : Mesurent le temps de réaction entre des catégories de groupes sociaux et des mots à valence positive ou négative, révélant des "associations automatiques, des attitudes implicites."

• La combinaison de plusieurs méthodes permet de "capturer nos préjugés à plusieurs niveaux, des plus explicites aux plus implicites."

6. Les Motivations Derrière la Dissimulation des Préjugés

Le conflit intérieur entre le désir d'exprimer des émotions et le maintien de valeurs contradictoires explique pourquoi les préjugés sont cachés. Deux types de motivations sont identifiés :

• Motivations internes : Liées aux "valeurs personnelles, telles que l’altruisme, la tolérance ou l’égalité."

Ceux qui sont animés par ces motivations "lutter[ont] contre eux [leurs préjugés]" et "travailler[ont] sur moi-même, en diminuant mes propres préjugés de manière autonome." Ils ont effectivement de plus faibles préjugés.

• Motivations externes : Dictées par "des motifs extérieurs, sociaux, normatifs," comme la "crainte par exemple d’être socialement mal perçus" (biais de désirabilité sociale).

Ces personnes "tenter[ont] de résoudre ce conflit en évitant d’exprimer mes préjugés sans vraiment travailler à les diminuer."

Leurs préjugés sont généralement plus forts et ils les déguisent par "des blagues dégradantes, des rhétoriques pseudo-démocratiques ou diverses justifications tel que des arguments naturalistes : « ce sont les faits », « c’est la biologie », « c’est la nature »."

7. L'Impact des Normes Sociales et de la Rhétorique Politique

Le document aborde la manière dont les normes sociales influencent l'expression des préjugés :

• Changement des normes : Si les normes sociales deviennent plus permissives envers l'expression des préjugés, "les personnes qui jusqu’ici cachaient leurs préjugés risquent de les exprimer davantage, parfois avec force, comme un barrage qui vient de céder."

• L'effet d'encouragement ("effet Trump") : La rhétorique de figures politiques, comme celle de Donald Trump en 2016, "participe à banaliser l’expression de préjugés, à les rendre comme socialement plus acceptable."

Cela déplace les normes sociales, autorisant "les personnes qui jusqu’ici réprimaient leurs préjugés selon des motivations externes" à "exprimer le fond de leur pensée, parfois avec grande violence."

8. L'Héritage des Stéréotypes et Préjugés : L'Exemple de "Couleur Café"

Le document utilise la chanson "Couleur Café" de Serge Gainsbourg pour illustrer comment les stéréotypes implicites et les préjugés coloniaux peuvent être ancrés dans la culture populaire :

• Interprétation contrastée : Alors que pour beaucoup, c'est une chanson qui "célèbre la diversité," pour les personnes de couleur, elle "réduit [notre identité] à une couleur et à un produit colonial, le café."

• Clichés coloniaux : La chanson réactive "tout un imaginaire et des préjugés coloniaux," associant "femmes noires, produits coloniaux et sexualité."

Le texte explique l'origine de l'image de la femme métisse "dansante, au service de l’homme blanc," créée par la "propagande coloniale" pour attirer de jeunes colons.

• Persistance et déconstruction : Aujourd'hui, les femmes noires et métisses "essayent de se débarrasser de ce cliché," ce qui est difficile car "les gens pensent que c’est un cliché positif."

Le document appelle non pas à la censure, mais à "écouter les paroles et essayer de comprendre l’histoire des symboles et des préjugés," utilisant la chanson comme un "outil parfait pour analyser les stéréotypes et faire de la pédagogie antiraciste."

9. Stratégies pour Réduire et "Hacker" les Préjugés

Malgré leur omniprésence, il est affirmé que les préjugés "peuvent être diminués, voire hackés, y compris quand ils sont implicites."

• Auto-régulation et reconnaissance : "Apprendre à reconnaître ses préjugés, à ne pas les nier, sans se juger sévèrement," en voyant cela comme une "occasion de les travailler pour s’en débarrasser."

Avec l'entraînement, cela peut devenir automatique.

• Aider autrui : En évitant les jugements sévères, "poser des questions, sur ses émotions, l’amener à prendre conscience par lui-même que certaines attitudes et comportements peuvent être problématiques."

• Développer des valeurs : Cultiver des "valeurs d’égalité et de tolérance de manière autonome," issues de motivations internes.

• Prise de perspective et empathie : "Imaginer le monde du point de vue d’une autre personne, se mettre à sa place," par l'écoute de témoignages ou la fiction.

• Éviter le déni des inégalités : Refuser la croyance que "les préjugés et les discrimination n’auraient plus vraiment cours," car cela "favorise les préjugés" en rendant aveugle à leur gravité et aux microagressions. Préférer une "approche multi culturelle qui valorise les différences."

• Environnements diversifiés et inclusifs : Favoriser dès l'enfance des "environnements sociaux diversifiés et inclusifs" (théorie du contact intergroupe).

• Représentations positives dans les médias : Promouvoir une meilleure image des divers groupes sociaux.

• Éviter les cadres compétitifs : Limiter les situations qui renforcent le clivage "nous contre eux."

• Programmes de sensibilisation et de formation : Dans les écoles et sphères professionnelles, "sensibiliser à la diversité, en expliquant ce que sont les stéréotypes, les préjugés, et les moyens de lutter contre," et "exposer des récits contre-stéréotypés, de proposer des prises de perspectives."

• En résumé, le document offre un aperçu complet de la complexité des préjugés, allant de leur définition conceptuelle à leurs manifestations les plus subtiles, et propose des voies claires pour leur réduction individuelle et sociétale.

Well laid out/easy to follow.

Synthèse et Analyse Approfondie des Cancers Professionnels et de leur Invisibilité en France

Ce document de synthèse explore les multiples facettes de l'invisibilité des cancers professionnels en France, s'appuyant sur les travaux du Giscope (Groupe d'Intérêt Scientifique de recherche sur les cancers professionnels) en Seine-Saint-Denis, notamment les recherches d'Anne Marchand, sociologue et historienne, et les commentaires de Nathalie Bajos.

Il met en lumière les mécanismes institutionnels, scientifiques, sociaux et culturels qui contribuent à cette invisibilité, malgré une prévalence significative et des conséquences humaines et sociales dramatiques.

1. Le Cloisonnement Historique et Institutionnel entre Santé au Travail et Santé Publique

• Un thème central est le cloisonnement historique et persistant entre l'espace du travail et l'espace de vie en matière de santé. Ce cloisonnement, analysé par l'historien Thomas Lerou, a conduit à "l'effacement progressif du corps ouvrier dans les préoccupations sanitaires et politiques" dès les 18e et 19e siècles. Il a créé une séparation artificielle entre l'hygiène industrielle et l'hygiène publique, cette dernière devenant "l'hygiène d'une partie seulement du public ignorant ce qui se déroule dans l'espace de travail".

Cette dichotomie a des conséquences majeures :

• Approche fragmentée de la santé : Elle empêche de "penser la santé des individus et des populations dans leur globalité" et "laisse dans l'ombre de nombreux facteur d'inégalité sociale".
• Campagnes de prévention inadaptées : Les campagnes de prévention contre le cancer sont "exclusivement centrées (...) sur la modification des comportements dits individuels", ignorant le rôle des conditions de travail et la responsabilité de l'État et des employeurs. Cela conduit à une approche qui "pointe la responsabilité des seuls individus" tout en laissant dans l'ombre les "cancérogènes présents dans le monde du travail".

• Angle mort de la recherche en santé publique : Le travail est souvent "un angle mort des approches en santé", comme si les lieux de travail n'étaient pas aussi des lieux de vie où l'on passe une grande partie de son temps.

• L'Épidémie Cachée : La Sous-Estimation et la Sous-Déclaration des Cancers Professionnels

• Les sources révèlent une sous-estimation et une sous-déclaration massives des cancers d'origine professionnelle, contrastant avec l'augmentation constante de l'incidence du cancer en France (doublée depuis les années 1990).

• Disparité Chiffrée : En 2023, seules 1452 reconnaissances de cancers professionnels ont été enregistrées, majoritairement liées à l'amiante. Or, les estimations épidémiologiques consensuelles indiquent que "4 à 8 % des nouveaux cas de cancer seraient d'origine professionnelle", soit "jusqu'à 34 644 cas par an". Cette énorme divergence crée un "phénomène un peu circulaire : moins il y a de cancer professionnel reconnus moins les personnes atteintes de cancer seront en mesure de penser le lien entre leur travail et leur maladie moins elles le déclareront en maladie professionnelle".

• Exposition Généralisée : L'étude Sumi révèle que "11 % des salariés en moyenne des secteurs publics et privés (...) sont exposés à au moins un cancérogène dans leur activité habituelle de travail". Ces expositions sont fortement inégalitaires, touchant particulièrement les ouvriers qualifiés de l'industrie automobile (90% exposés), les intérimaires, et les jeunes de moins de 25 ans.
• Polyexposition : La "poliexposition", c'est-à-dire l'exposition simultanée ou successive à différents cancérogènes, "démultiplie le risque de contracter un cancer". Un exemple frappant est celui d'un homme exposé à 17 cancérogènes identifiés au cours de son parcours professionnel.

• Longue Latence : Le caractère différé des effets des cancérogènes (20 à 50 ans après l'exposition) rend le lien causal difficile à établir pour les victimes et le corps médical. De plus, il est "impossible scientifiquement et médicalement de distinguer un facteur sur l'autre dans sa survenue" (ex: amiante vs tabac pour le cancer du poumon).

• Les Mécanismes d'Invisibilisation des Cancers Professionnels

• Plusieurs facteurs, imbriqués et complexes, contribuent à cette invisibilité :

3.1. Les Données Officielles et la Prévalence de l'Amiante

• Loupe déformante : Les chiffres de reconnaissance de l'Assurance Maladie sont le "premier facteur de cette invisibilité sociale", donnant l'impression que les cancers professionnels sont rares et majoritairement liés à l'amiante. L'amiante est "l'arbre qui cache la forêt des autres cancérogènes".
• Maladies "signatures" et droits spécifiques : L'existence de maladies "signature" (mésothéliome) et de droits spécifiques (retraite anticipée, FIVA) pour les victimes de l'amiante a paradoxalement renforcé cette perception limitée des cancers professionnels.

3.2. Le Cadre Juridique et Administratif : Les Tableaux de Maladies Professionnelles

• Objet de négociation et de rapport de force : Les tableaux de maladies professionnelles, créés par le Code de la Sécurité Sociale, sont le "résultat de négociation entre représentant de syndicat de salariés et représentant de syndicat d'employeur". Chaque terme choisi est le fruit de "rapports de force", ouvrant ou fermant les conditions de reconnaissance.
• Restrictions et obsolescence : Ces tableaux sont des "objets mouvants du droit" mais leur contenu est souvent "très en deçà des connaissances scientifiques". L'exemple du cancer de la vessie lié aux amines aromatiques, dont le "titre" nécessite "un bac + 12 en chimie pour arriver à relier son travail à ce cancer", illustre la complexité et l'inadéquation.
• Cancer du sein : un exemple d'invisibilité levée : L'absence de tableau pour le cancer du sein a longtemps masqué son origine professionnelle, le cantonnant à une "certaine fatalité". Les efforts de syndicats et de recherches ont permis de "rendre visible le facteur professionnel dans cette épidémie", montrant l'impact potentiel de l'inscription dans un tableau.
• La "maladie négociée" : La maladie professionnelle n'est pas une catégorie médicale mais "une catégorie juridico-politique", une "maladie négociée", ce qui la rend distincte de la causalité médicale.

3.3. L'Ignorance des Expositions et le Sentiment de Protection des Salariés

• Manque d'information : La plupart des personnes touchées "ignoraient avoir été exposées à des substances cancérogènes". Cette ignorance peut venir de la méconnaissance des dangers de substances (comme l'amiante dans les années 80) ou de leur présence insidieuse et imperceptible (rayonnements ionisants, produits chimiques sans odeur ni effet immédiat).
• Fausse impression de sécurité : Les salariés ont le "sentiment d'avoir été protégés" car ils imaginent que "sauf situation accidentelle tout est maîtrisé dans l'entreprise" ou que les substances dangereuses seraient interdites.
• Dispositifs trompeurs :Valeurs Limites d'Exposition Professionnelle (VLEP) : Les VLEP sont le "fruit de compromis sociaux" et ne signifient pas l'absence de risque, car "la plupart des cancérogènes sont sans effet de seuil".
• Surveillance Médicale Renforcée (SMR) : La SMR, bien que réservée aux salariés exposés, "ne protège en rien" mais peut créer l'illusion de protection ("Il pensait qu'on le protégeait en fait on l'endormait").
• Équipements de Protection Individuelle (EPI) : Les EPI sont souvent inefficaces ou utilisés pour d'autres raisons (protection du produit), brouillant la perception du risque (ex: gants en salle blanche).

3.4. Le Manque d'Intérêt pour la Déclaration et l'Indemnisation Insuffisante

• L'horizon indemnitaire : Le "montant proposé au mieux (...) ne peut dépasser le montant mensuel des derniers salaires", ce qui est souvent "pas assez pour devenir moteur d'engagement". L'indemnisation est "forfaitaire" et très éloignée de ce qu'une victime obtiendrait devant un tribunal.
• Dispositifs concurrents : Le dispositif d'invalidité est perçu comme "plus facile et plus rémunérateur", orientant les victimes loin de la reconnaissance en maladie professionnelle. Cette stratégie "contribue largement à rendre invisible les effets du travail sur la santé et donc les cancers professionnels" et "socialise le coût de ces maladies à l'ensemble de la collectivité" au lieu qu'il soit financé par les employeurs.
• Signification de l'argent : L'argent de l'indemnisation revêt différentes significations culturelles. L'ignorance du principe "pollueur-payeur" fait que certains ne veulent pas "coûter davantage à la Sécu", ou ressentent de la "honte" à "assimiler cette démarche à une demande d'aide sociale". Pour les veuves, l'argent peut "brûler les doigts", générant une stigmatisation sociale.

3.5. Le Rôle Déterminant et les Lacunes du Corps Médical

• Formation insuffisante : Les médecins sont "très peu formés sur ce volet très spécifique du droit de la sécurité sociale" (environ "une dizaine d'heures sur leurs dizaines années d'études").
• Difficulté à établir le lien : Formés à la causalité médicale, ils "appréhendent avec beaucoup de circonspection cette catégorie médico-administrative" et sont nombreux à refuser de rédiger un certificat médical pour des patients fumeurs, ignorant ou refusant d'admettre la présomption d'origine professionnelle.
• Crainte du conflit : Le certificat médical initial (CMI) est un "espace de conflit" et peut entraîner des convocations devant le Conseil de l'Ordre à la demande d'employeurs. La tâche de "certifier" une origine professionnelle les éloigne de leur cœur de métier, le soin.
• Manque d'interrogatoire : Dans l'ensemble, les médecins "interrogent très peu les activités exercées et encore moins les conditions de travail" de leurs patients.

3.6. Les Inégalités d'Accès à la Reconnaissance et les Transformations du Travail

• Charge de la preuve : La présomption d'origine professionnelle des tableaux est limitée, et le salarié doit souvent "apporter des preuves de la maladie", "des preuves de l'emploi" (certificats de travail, fiches de paye) et surtout "des preuves de l'activité habituelle de travail de l'activité exposante jusqu'à 40 ans en amont de la survenue de la maladie".
• Fragilité des parcours : Cette capacité à prouver les activités exposantes est "très inégalement distribuée". Elle est plus facile pour les salariés avec une "stabilité professionnelle" ou qui peuvent compter sur un "réseau syndical ou de retraités dynamiques" (mineurs, dockers).
• Travail morcelé et sous-traitance : La situation est "bien plus dur pour des salariés isolés", ceux "qui ont connu des parcours très morcelés" (jusqu'à 35-40 employeurs), et surtout pour les "salariés des entreprises sous-traitantes", qui sont à la fois "parmi les plus exposés et les moins reconnus". La sous-traitance, légalisée dans les années 70, est devenue un moyen de "contourner leurs obligations" et d'"externaliser des activités qui étaient les plus pénibles et les plus exposantes", renforçant l'invisibilité.
• Intérimaires et travailleurs migrants : Les intérimaires, dont les documents ne disent "absolument rien du site sur lequel ils ont travaillé", et les "migrants travailleurs agricoles saisonniers", souvent "affectés au traitement chimique là où les risques toxiques sont les plus importants mais dont la maladie si elle survient ne sera pas visible en France ni reliée au travail", sont particulièrement vulnérables.

3.7. Le Manque de Traçabilité Institutionnelle

• Volatilité réglementaire : La "valse des réglementations" empêche la mise en place d'un dispositif stable garantissant une "traçabilité rigoureuse dans le temps des expositions cancérogènes" sur de longues périodes (20, 30, 40 ans).

• Le Caractère Structurel de l'Invisibilité et l'Enjeu de Justice Sociale

• L'analyse de la genèse de la catégorie "cancer professionnel" révèle une "certaine récurrence dans les obstacles à la construction de la connaissance". Dès le début du 20e siècle, malgré une identification précoce des cancers liés à des industries spécifiques (houille, colorants, rayons X), les mêmes constats d'échec de déclaration et de reconnaissance se répètent. Les affiches de 1938 exhortant les médecins à déclarer les maladies professionnelles témoignent de cette problématique ancienne.

• Absence de données fiables : Les données sur le cancer sont "incomplètes", avec des registres qui ne couvrent "moins d'un quart de la population en France", et qui présentent des biais (population plus rurale, plus âgée, plus favorisée, moins de personnes d'origine étrangère). Les zones les plus polluées (sites Seveso) sont souvent non couvertes. La proposition de loi pour créer un registre national des cancers est une étape "indispensable".

• Fabrication de "non-problèmes" : L'invisibilité des cancers professionnels s'inscrit dans une dynamique de "fabrique des non-problèmes ou comment éviter que la politique s'emmêle".
• Question de justice sociale : En définitive, cette invisibilité pose la "question de la valeur différentielle des vies" et constitue une "question de justice sociale", comme le souligne Nathalie Bajos.

En conclusion, la lutte contre les cancers professionnels exige bien plus que des campagnes de prévention individuelles.

Elle nécessite une réforme profonde des mécanismes de reconnaissance, une formation accrue du corps médical, une meilleure traçabilité des expositions, une indemnisation plus juste, et surtout, un changement de paradigme qui intègre pleinement la santé au travail dans la santé publique, reconnaissant le lieu de travail comme un lieu de vie essentiel.

Note de synthèse : La sous-reconnaissance des maladies professionnelles en France : un problème de santé publique et de justice sociale

Ce briefing documente l'ampleur et les causes de la sous-déclaration et de la sous-reconnaissance des maladies professionnelles en France. Il s'appuie sur une analyse processuelle et met en lumière les multiples obstacles rencontrés par les victimes, ainsi que les lacunes du système juridique et médical actuel.

1. Ampleur et enjeu de la sous-reconnaissance

Les maladies professionnelles (MP) représentent un problème de santé publique majeur et invisible en France, exacerbant les inégalités sociales de santé.

• Chiffres alarmants : En 2022, on estime à plusieurs centaines de milliers le nombre de maladies professionnelles non déclarées. Concernant spécifiquement les cancers professionnels, la fourchette est estimée entre 67 000 et 99 000 cas, dont seulement 4 000 sont déclarés.
• Coût financier et social : La sous-déclaration transfère le coût de ces maladies de la branche « Accidents du Travail et Maladies Professionnelles » (AT/MP), financée par les cotisations patronales, vers la branche « Maladie » du régime général, désavantageant financièrement les victimes qui ne bénéficient pas des indemnisations plus favorables (prise en charge des frais de santé, indemnités journalières, rentes viagères). Au-delà de l'enjeu financier, il existe un enjeu symbolique fort pour les victimes.
• Inégalités sociales : L'exposition aux risques professionnels est fortement inégalitaire. Les ouvriers sont nettement plus exposés aux contraintes physiques intenses et aux cancérogènes que les cadres ou les professions intermédiaires, soulignant un lien direct entre position sociale et risques pour la santé au travail.

2. Cadre juridique et ses limites

• Le droit à la santé et à la sécurité au travail repose sur deux principes : la responsabilité de l'employeur et la réparation des atteintes à la santé. La loi de 1898 (étendue en 1919 aux MP) a instauré un système d'assurance forfaitaire, supprimant la nécessité de prouver la faute de l'employeur, mais en contrepartie, le droit à poursuivre pénalement l'employeur a été perdu.

• Les tableaux de maladies professionnelles : Créés pour identifier les pathologies éligibles à la reconnaissance et à l'indemnisation, ils sont aujourd'hui considérés comme obsolètes et incomplets. Leur création est le fruit de négociations entre l'État, les syndicats de salariés et d'employeurs, où les considérations économiques et les arguments patronaux ont historiquement pesé lourd.

• Critiques de l'ANSES (2024) : Les tableaux sont jugés "incomplets", "loins" de la réalité des expositions et "hétérogènes". Les diagnostics d'exclusion méconnaissent le principe de présomption d'origine.
• Ralentissement de l'actualisation : Seulement 2 créations et 5 révisions de tableaux sont intervenues au régime général entre 2010 et 2018. Ce ralentissement est dû à la divergence des intérêts patronaux (financeurs uniques de la branche AT/MP), l'absence de consensus sur la présomption d'imputabilité, et le caractère plurifactoriel des pathologies.

3. Le processus de reconnaissance : une "course d'obstacles"

• La reconnaissance d'une maladie professionnelle est un processus complexe, jalonné d'obstacles à chaque étape :
• Exposition au risque : Les contraintes et expositions physiques varient considérablement selon le sexe et la position sociale. Par exemple, les ouvriers sont majoritairement plus exposés aux risques cancérogènes. Le cas de l'amiante est emblématique : classé cancérogène en 1977, son usage n'a été interdit qu'en 1997, et il reste présent dans de nombreux matériaux, exposant encore des travailleurs.
• Reconnaissance par le salarié de l'origine professionnelle :
• Méconnaissance des maladies : De nombreuses MP restent mal connues, notamment les troubles psychiques qui représentent un "angle mort très important".
• Difficulté à établir le lien travail-maladie : Il est souvent "pas évident pour un individu de faire le lien entre sa maladie et son travail, de considérer que le travail puisse être pathogène". Cette démarche est source de "tension normative".
• Déni du danger : Dans certains métiers, des salariés développent une "attitude de déni du danger pour tenter d'y faire face", comme le montre le travail de Christophe Dejours sur les ouvriers du bâtiment ou l'exemple des coiffeuses.
• Méconnaissance des droits : Même en ayant conscience du lien travail-maladie, de nombreux salariés ignorent les droits qui leur sont ouverts.
• Absence de perception d'intérêt : Certains ne perçoivent pas d'intérêt matériel, financier, ni même symbolique à la déclaration, surtout en cas de traitements lourds où "les démarches de réparation sont vues comme secondaires par rapport aux enjeux de soins, voire inutiles ou même néfastes".
• Peur des représailles : La crainte de perdre son emploi ou de subir des représailles de l'employeur ou des collègues est un frein majeur.
• Déclaration de la maladie professionnelle :
• Responsabilité de la victime : Contrairement aux accidents du travail, c'est la victime elle-même (ou ses ayants droit) qui doit effectuer la demande, nécessitant un certificat médical initial.
• Complexité administrative et juridique : La procédure est "compliquée" et "complexe", décourageant de nombreux potentiels bénéficiaires.
• Manque d'information et de soutien des médecins : La formation des professionnels de santé sur les MP est insuffisante (environ 10h en 2e cycle des études médicales, contre une moyenne européenne de 25h). Certains médecins du travail sont réticents à accompagner les démarches, parfois par méconnaissance des avantages financiers pour le salarié ou par crainte de l'impact négatif sur l'emploi du patient.
• Obstruction des employeurs : Les employeurs n'ont "évidemment pas d'intérêt financier à ce qu'il y ait des maladies professionnelles déclarées". Ils peuvent exercer des pressions, proposer des ruptures conventionnelles en échange de la renonciation, ou contester le lien avec l'accident/la maladie.
• Reconnaissance administrative et médico-légale :
• Obstruction des caisses primaires d'assurance maladie (CPAM) : Des "obstructions importantes" sont documentées, se manifestant par une "très forte hétérogénéité entre les caisses dans les délais de traitement des dossiers".
• Caractère différé des maladies : Le "temps de latence" entre l'exposition et l'apparition des symptômes (plusieurs années, voire décennies pour certains cancers) rend difficile la traçabilité des expositions passées.
• Ressources inégales face à la contestation : Les "grandes entreprises qui ont les moyens de recourir à des cabinets d'avocats spécialisés" peuvent manipuler les données et "mettre en scène l'ignorance pour contester la parole des travailleurs et limiter le coût de la reconnaissance". La stratégie de certains avocats est de "repérer un bon cas" pour créer une jurisprudence favorable.

4. Perspectives et recommandations

• La situation est marquée par une "stabilité" voire une "inertie" des logiques sociales et institutionnelles qui construisent la non-reconnaissance. Des "petites mesures" ne suffiront pas ; une "modification de la loi" est nécessaire.
• Manque de connaissance et biais dans la recherche :
• Analyses genrées : Il y a un "manque de connaissance" et une "absence particulière" des analyses genrées en santé publique sur les MP, malgré leur pertinence. Les études épidémiologiques sur les cancers professionnels ont historiquement porté majoritairement sur les hommes, sous-estimant les expositions et les cancers chez les femmes (ex: agentes de nettoyage).
• Populations vulnérables : Les travailleurs étrangers ou d'origine étrangère, et les personnes en situation de précarité, sont confrontés à des obstacles particuliers et sont "pas du tout assez étudiées". Une approche intersectionnelle est nécessaire pour articuler les rapports sociaux de domination (classe, genre, race).
• Facteurs de risque et multiexpositions : La recherche doit continuer à identifier les facteurs de risque, notamment pour les maladies plurifactorielles (expositions professionnelles et environnementales simultanées). L'idée qu'on puisse isoler une cause unique est une "illusion épidémiologique". La question est de savoir si une exposition professionnelle favorise ou aggrave la maladie, indépendamment d'autres causes (ex: tabac, environnement).
• Nécessité de modifier la loi et le système de reconnaissance :
• Formation des professionnels de santé : Renforcer la formation des médecins sur les MP, même si cela ne suffit pas sans mesures structurelles.
• Traçabilité et information : Améliorer la traçabilité des expositions et l'information des salariés sur leurs droits.
• Contrôle et sanctions : Renforcer l'arsenal de contrôle et de sanction des entreprises qui procèdent à des déclarations incomplètes.
• Actualisation et refonte des tableaux : Actualiser les tableaux de maladies professionnelles, voire en modifier l'objectivation des pathologies en se basant sur les examens recommandés par les sociétés savantes ou la Haute Autorité de Santé.
• Refonte globale du système : La loi elle-même est interrogée pour sa capacité à produire la non-déclaration. Une refonte du système de reconnaissance est indispensable pour tenir compte :
• De l'évolution des modes de management.
• De la plus grande discontinuité des carrières professionnelles.
• De l'augmentation des produits dangereux et de la reconnaissance de leur dangerosité.
• Des multiexpositions.
• De la "question fondamentale de la reconnaissance des troubles psychiques qui ne font toujours pas l'objet d'un tableau de maladie professionnelle".
• De l'exclusion de certains travailleurs.
• En conclusion, la reconnaissance des maladies professionnelles est un "enjeu majeur de santé publique" qui contribue aux inégalités de santé. C'est un enjeu "politique", "scientifique" et "éthique" qui nécessite des changements profonds pour sortir de l'inertie actuelle. Les expériences de terrain, comme celle du GISCOPE, peuvent dessiner des pistes concrètes pour une transformation fondamentale.

Compte rendu détaillé : La soumission chimique – Comprendre, Identifier et Lutter

Ce document de briefing est basé sur la conférence de Juliette Descœurs, praticien hospitalier et biologiste au laboratoire de toxicologie de la Péronie, et experte près la cour d'appel de Montpellier, axée sur le phénomène de la "soumission chimique".

Il vise à synthétiser les concepts clés, les substances impliquées, les méthodes d'analyse et les stratégies de prévention.

1. Définition et Distinction de la Soumission Chimique

Juliette Descœurs débute par une définition précise de la soumission chimique : « l'administration volontaire de substances psycho-actives à l'insu de la victime ou sous la menace à des fins à la fois soit délictuelles pour des vols des signatures de documents par exemple ou à des fins criminelles pour faire des agressions sexuelles des viols de la pédophilie ou des affaires que l'on retrouve dans des maisons de retraite ou même intrafamiliales ».

Elle établit une distinction cruciale avec la "vulnérabilité chimique". Cette dernière désigne la « consommation volontaire par une personne de substance psychoactive qui conduirait à un état de vulnérabilité », où les agressions sont majoritairement perpétrées sur des victimes ayant consommé de l'alcool et/ou du cannabis.

• Un point important soulevé est l'utilisation des substances comme « une stratégie pour instaurer une genre d'emprise par addiction à des psychotropes provoqués et alimentés par l'exploiteur ».

Cette emprise est renforcée par le fait que « l'obtention de ces substances est réalisée par l'exploiteur lui-même », transformant l'emprise chimique en un moyen de « contraindre des personnes en situation de vulnérabilité à commettre des délits des crimes ou même à se mettre en danger ».

2. Le "Produit Idéal" pour la Soumission Chimique

Pour un agresseur, le produit idéal de soumission chimique présente plusieurs caractéristiques :

Facilité d'obtention. Goût agréable ou sans goût. Forme liquide ou soluble, facilement dissoluble dans un milieu aqueux. Invisible. Actif à faible dose et à action rapide. Deux critères principaux sont recherchés pour classer ces substances :

Vitesse d'élimination : « rapide ».

Mécanisme d'action : « sédation, un effet amnésiant, une stimulation sexuelle, une action myorelaxante pour pouvoir faire un peu ce qu'on veut de la victime, une diminution de ces des réactions de défense ».

3. Substances Utilisées dans la Soumission Chimique

Les substances sont classées en deux catégories principales :

3.1. Substances Non Médicamenteuses :

• GHB (Gamma-Hydroxybutyrate) : Surnommé "liquide extasie" ou "drogue du violeur".

• Forme : Poudre blanche soluble, liquide inodore et incolore.

• Utilisation : Principalement dans les milieux festifs à des fins illégales en raison de son « effet amnésiant et inducteur de sommeil ». Souvent consommé avec de l'alcool.

• Effets : Forte sensation de chaleur et d'ivresse (comparable à l'alcool) à faibles doses, puis quiétude, légère euphorie, désinhibition. À fortes doses : vertiges, perte de coordination, nausées, vomissements, coma, dépression respiratoire.

• Furtivité : Qualifié de "furtif" car il disparaît rapidement du sang (jusqu'à 5-6h) et des urines (jusqu'à 10-12h). Sa particularité est sa double production : physiologique (taux basal de 2 à 3 mg/L) et in vitro (formation dans les échantillons mal conservés), rendant l'analyse complexe et nécessitant de prendre en compte la conservation, le seuil physiologique et les délais de prélèvement.

• Alcool : Souvent le "numéro 1" des substances retrouvées, agissant comme un "alter ego du GHB" avec des effets euphorisants, désinhibants, stimulants et amnésiques. Les cas suspects de GHB se révèlent souvent être des intoxications alcooliques sévères (3 à 4 g/L).

• Extasie (MDMA) : Mentionnée en lien avec l'affaire du sénateur Joël Guerriot. Présente une forte prévalence dans ce type de situations.

• Effet : Antactogène, c'est-à-dire qu'il « altère le consentement de la victime » et provoque une « amnésie antérograde ». Bien que stimulant, il est un bon candidat pour la soumission chimique car il agit comme « un adjuvant de l'humeur par effet euphorisant et en abolissant la méfiance », rendant la victime « participative ».

• Catinones de synthèse, cannabis, cocaïne.

• Hallucinogènes :Scopolamine : Action sédative, provoque hallucinations, amnésie et pertes de conscience.

• Ayahuasca (dérivé de la diméthyltriptamine) : Souvent associée aux rituels chamaniques, la littérature montre que les victimes se retrouvent souvent dans un « état second avec une impossibilité de s'opposer à des agressions sexuelles ».

3.2. Substances Médicamenteuses :

• Benzodiazépines et "Z-drugs" (hypnotiques apparentés) : (Bromazépam, diazépam, alprazolam, zolpidem, zopiclone). Mentionnées en écho au procès de Mazan (affaire Gisèle Pélico). Des langues bleues (additif du clonazépam) ont été observées dans des vidéos.

• Propriétés : Anxiolytiques, anticonvulsivantes, sédatives, myorelaxantes et surtout « amnésiantes très recherchées par les agresseurs ». L'« amnésie antérograde et lacunaire » est particulièrement retrouvée avec les benzodiazépines hypnotiques sédatives à demi-vie courte, permettant aux agresseurs de faire accomplir des actes dont la victime ne gardera aucun souvenir.

• Antihistaminiques (H1) : (Atarax, désloratadine, Aérius).

• Effets : Sédation, somnolence, étourdissement, ralentissement des réflexes.

• Neuroleptiques : Souvent détournés de leur usage, retrouvés dans des cas de maisons de retraite où des personnes âgées sont en état léthargique après administration à leur insu.

4. Statistiques et Prévalence (Enquête CEIP-A 2022)

En 2022, sur 1229 signalements suspects, 97 cas de soumission chimique ont été jugés vraisemblables. Les victimes étaient « essentiellement des femmes et dans des milieux festifs ».

La majorité des substances impliquées étaient médicamenteuses, mais une part non négligeable de substances non médicamenteuses était également présente.

Top 1 non médicamenteuses : MDMA (extasie), cocaïne, cannabis. Top 1 médicamenteuses : Benzodiazépines (bromazépam, zopiclone, hydroxyzine) et tramadol.

5. Prise en Charge en Laboratoire (CHU La Péronie)

Le laboratoire du CHU La Péronie a mis en place un protocole avec les urgences, l'Institut de Médecine Légale et la pharmacologie pour la prise en charge des cas suspects de soumission chimique.

• Circuits de prélèvement :

• Conservation : Demande de 4 tubes minimum (hépariné, fluoré, EDTA, urine).

• Clinique : Demande de tubes supplémentaires si nécessaire pour des analyses complémentaires.

• Gestion des échantillons : Détruits après 3 ans sans saisie de justice ; traités en cas de réquisition judiciaire.

• Formulaire de réception des prélèvements : Essentiel pour compiler un maximum d'informations rapidement.

• Informations clés : Date/heure supposée des faits, date/heure des prélèvements, prise en charge thérapeutique, traitement habituel de la victime, consommation éventuelle de stupéfiants.

• Signes non cliniques : Agression physique (signes de violence), préjudice matériel (perte de carte bancaire, chéquier), découverte de substances sur les lieux.

• Signes cliniques (neuropsychiques) :Durée de la phase léthargique et qualité du réveil : Différents selon les molécules (ex: GHB : sédation courte, réveil rapide et complet ; benzodiazépines : somnolence prolongée, réveils difficiles).

• Troubles de la vigilance, propos désorganisés, troubles du comportement, hallucinations.

• Amnésie : Classiquement « antérograde » (empêche l'enregistrement de nouveaux souvenirs) et « lacunaire » (absence de souvenir d'un moment de la vie).

• Autres symptômes : Vomissements (GHB, absents avec antihistaminiques H1), sécheresse buccale marquée (effets anticholinergiques des antihistaminiques).

• Types de prélèvements pour l'analyse :

• Sang : Permet de remonter à la quantité prise, mais fenêtre de détection courte (quelques heures).

• Urine : Fenêtre de détection plus large grâce à la présence de métabolites.

• Cheveux : « Stabilité exceptionnelle » car matrice biologique non biodégradable.

• Principe : 1 cm de cheveux correspond à environ un mois de condition de vie.

• Avantages : Constitue une « sauvegarde des molécules » avec lesquelles le sujet a été en contact, documente un usage répété ou une exposition unique. Réalisé en zone de confidentialité et conservé à température ambiante.

6. Moyens de Lutte et de Prévention

• Mission gouvernementale sur la soumission chimique (rapport du 12 mai 2025) : Juliette Descœurs met l'accent sur la recommandation 43 : « un soutien à la recherche scientifique en galénique et en toxicologie qui permettra d'entraver la lutte contre le détournement criminel de médicaments ».

• L'Agence du Médicament demande aux laboratoires titulaires d'une AMM de « modifier l'aspect visuel de tels médicaments en ajoutant par exemple des colorants (...) un goût, une odeur facilement identifiable et peut-être une texture aussi inhabituelle (faire des grumeaux à la surface) », pour permettre aux victimes de repérer un éventuel détournement.

• Mouvement #metoo et visibilisation : Les agressions sexistes et sexuelles facilitées par l'absorption non consentie de substances psychoactives ont été mises en lumière dans le cadre d'événements festifs (#balancetonbar) et de la sphère privée (affaires Sandrine Josu et Gisèle Pécelico).

• Campagne de sensibilisation (mai 2023) : Lancée par le CRAFS (Société Francophone des Sciences Pharmaceutiques Officinales), l'Ordre National des Pharmaciens, et l'association #Mandorpa, avec le soutien du Secrétaire d'État à l'égalité entre les femmes et les hommes. Le CRAFS est la « plateforme référente de santé publique qui informe sur les substances actuellement de soumission chimique ».

• Ce briefing souligne l'importance d'une approche multidisciplinaire pour comprendre, identifier et combattre la soumission chimique, impliquant à la fois la recherche scientifique, l'adaptation des médicaments, la sensibilisation du public et une prise en charge médico-légale rigoureuse.

Compte-rendu détaillé du Séminaire International sur le Travail Collaboratif à l’École

Ce compte-rendu explore les thèmes principaux et les idées essentielles abordées lors du séminaire international sur le travail collaboratif à l’école, organisé par France Éducation Internationale (FEI).

Il met en lumière la philosophie de la co-construction, l'importance des initiatives de terrain, les défis de la généralisation des bonnes pratiques et le rôle crucial de l'inclusion et de la diversité dans l'éducation.

1. France Éducation Internationale : Un Opérateur de Coopération Éducative axé sur la Co-construction

France Éducation Internationale (FEI), anciennement CIEP, se positionne comme l'opérateur clé du Ministère de l'Éducation Nationale et de la Jeunesse pour la coopération éducative mondiale.

Sa philosophie centrale est la "co-construction", qui rejette l'exportation unilatérale du savoir-faire français au profit d'une collaboration adaptée aux contextes locaux.

• Rôle et Mission de FEI : FEI englobe diverses activités, de l'aide au développement à la mobilité (assistants de langue), en passant par la reconnaissance des diplômes et la recherche pédagogique.
• Approche Comparatiste : Le séminaire lui-même illustre cette approche, comme le souligne le représentant de FEI : « le séminaire d'aujourd'hui a été conçu dans cet esprit avec une approche volontairement comparatiste qui est celle de notre revue internationale d'éducation de Sèvres ». Cette méthode permet d'apprendre des expériences diversifiées, avec des études de cas en France (Creuse, Aube), en Inde, au Royaume-Uni et au Mexique.
• Héritage et Vision : L'ambition de FEI, héritée de la vision de Gustave Monod lors de la création du Centre International d'Études Pédagogiques en 1945, reste inchangée : « établir et maintenir des liens à l'international et bénéficier de ces échanges pour alimenter les travaux de recherche en vue de méthode plus active d'enseignement ». L'idée fondatrice est que « on travaille avec les autres on aide les autres mais les autres nous instruisent ».

2. Le Conseil National de la Refondation (CNR) – Volet Éducatif : "Notre école, faisons-la ensemble"

Le lancement du volet éducatif du CNR, baptisé "Notre école, faisons-la ensemble", représente une approche "révolutionnaire" pour le système éducatif français, historiquement très centralisé.

• Confiance aux Acteurs de Terrain : Plutôt que des injonctions verticales, la démarche repose sur la confiance accordée aux équipes pédagogiques locales. Le ministre souligne : « non par je ne sais quel injonction verticale venue du ministère mais par autant de dynamiques collective locale autour de l'école d'initiative et d'innovation portée par les équipes pédagogiques de confiance donnée aux acteurs de terrain ».
• Dynamique Collective Locale : Plus de 17 000 écoles et établissements (près d'un tiers du total) se sont engagés dans des concertations locales impliquant toutes les parties prenantes (enseignants, élèves, parents, collectivités). Cela a déjà abouti au dépôt de plus de 5 500 projets pédagogiques.
• Soutien Financier et Accompagnement : Ces projets bénéficient d'un soutien financier via le fonds d'innovation pédagogique, permettant de lever les freins liés au transport et au financement, notamment dans les zones rurales isolées. Comme l'indique l'une des présentatrices : « grâce au fond cette barrière peut être levée et là la démarche de notre école faisons là ensemble est une véritable aide à la réussite des élèves et à la lutte contre les inégalités ».

3. Études de Cas Françaises : Illustrations du Travail Collaboratif

Deux projets français, issus du CNR, sont présentés comme des exemples concrets de travail collaboratif :

• Collège de Parsac (Creuse) : Améliorer la lecture et l'orthographe
• Objectifs : Améliorer la lecture à haute voix et la fluence, ainsi que l'orthographe et l'expression écrite, de la maternelle à la troisième.
• Démarche : Né d'un diagnostic local basé sur les résultats des tests nationaux, le projet a impliqué des concertations avec les enseignants, les élèves, les parents et les collectivités locales.
• Actions : Incluent des ateliers d'écriture avec le groupe "Les Goguettes", la lecture d'albums par les élèves de 6ème aux maternelles ("Les Grands chez les Petits"), des nuits de la lecture, la rédaction d'un journal inter-degrés et un projet d'observation d'abeilles solitaires. L'élève Apolline témoigne : « J'aime lire des livres aux enfants de l'école ».
• Impact : Création d'un continuum pédagogique et interdisciplinaire, renforcement des liens entre le collège et les écoles, et une dynamique de confiance accordée au terrain.
• École Primaire de Mézières-lès-Briennes (Aube) : Parcours linguistique en allemand
• Objectif : Mettre en place un parcours linguistique cohérent en allemand dès l'école maternelle, visant le niveau A1+ à la fin du cycle 3.
• Contexte : Fortement motivé par le faible niveau en langues vivantes (notamment révélé par les tests Eva Langues) et un jumelage actif avec une ville allemande.
• Acteurs Impliqués : Enseignantes aux compétences en allemand, professeur agrégé d'allemand du collège, comité de jumelage, collectivités locales, parents d'élèves.
• Actions : Sensibilisation précoce à la diversité linguistique (dispositif Élysée), enseignement bilingue (dispositif Émile), correspondance avec une Kita allemande, mobilités d'élèves et d'enseignants, et à terme, une heure d'histoire en allemand au collège.
• Philosophie : « une approche méthodologique innovante qui va bien au-delà de l'enseignement des langues vivantes visant la citoyenneté la curiosité des élèves l'engagement de tous partenaire et acteurs l'interculturalité et la motivation ».

4. Perspectives Internationales sur le Travail Collaboratif

Les études de cas du Mexique et de l'Inde soulignent la portée universelle et l'adaptabilité du travail collaboratif.

• Mexique (Carlos Ornellas) : Le travail collaboratif est une réponse essentielle dans des contextes difficiles, marqués par la violence sociale et les inégalités. Des initiatives simples, émanant du terrain, permettent aux enfants de sortir de situations de violence et de misère. Comme le souligne M. Ornellas, cela met en évidence que « les initiatives viennent du terrain et c'est que là que c'est là que se construisent la véritable expertise ».
• Inde (Salini Borka) : Une initiative dans un État indien, mobilisant un ensemble d'acteurs éducatifs autour d'un syndicat, a permis d'inventer de nouvelles méthodes. Cela prouve qu'« un collectif qui connaît le terrain et beaucoup plus performant que toute idée qui peuvent qui peut qui peut surnager sans avoir cette connaissance fine du terrain et des ajustements nécessaires pour pour une meilleure éducation ».

5. Caractéristiques Clés du Travail Collaboratif et Conditions de Succès

Plusieurs intervenants ont identifié des principes fondamentaux pour la réussite et la généralisation du travail collaboratif :

• Intelligence Collective et Engagement : Le travail collaboratif repose sur la mobilisation de « l'intelligence collective », mutualisant les compétences des élèves, des équipes pédagogiques, et de l'environnement extérieur. Il nécessite un « engagement basé sur une motivation » (M. Ndoy).
• Dialogue Inclusif basé sur l'Évaluation : Le dialogue doit être « inclusif » et fondé sur « l'évaluation des résultats de l'apprentissage », permettant d'identifier des problèmes concrets à résoudre.
• Changement Culturel et Renouvellement des Pratiques : Le travail collaboratif bouleverse l'organisation pédagogique traditionnelle et conduit à un changement culturel chez les enseignants, les incitant à considérer que « tout enfant est éducable » (M. Ndoy) et à s'intéresser aux apprenants en difficulté.
• La Question de la "Réforme" vs. "Initiative" : Le professeur Antonio Nóvoa remet en question la pertinence du terme "réforme" au profit d'"initiative", de "partage" et de "collaboration".

Il met en garde contre les projets qui restent « une exception à la au quotidien scolaire » et ne touchent pas le « corps le centre du travail scolaire ». Il insiste sur la nécessité d'une nouvelle organisation de l'école (emploi du temps, organisation des classes, curriculums) pour que le travail soit au cœur du processus. * Optimisme et "Agir Ensemble" : La rectrice Carole Drucker Godard met en avant l'« optimisme » et l'« envie » générés par ces projets. Elle souligne que l'« agir ensemble est fédéré par des valeurs partagées » et que l'effort doit être partagé. * Les Facteurs de Réussite de la Rectrice :Communication : Essentielle pour expliquer les bienfaits des pratiques innovantes et « transmettre l'envie ». * Auto-évaluation et Rétroaction : Pour ajuster et améliorer continuellement les projets. * Formation des Enseignants : Développer la culture du travail collaboratif, notamment via des formations continues comme les "constellations". * Accompagnement des Projets : Par les chefs d'établissement et les services académiques. * Incitation à la Dynamique de Projet : Être attentif aux appels à projets pertinents. * Lien avec la Recherche : Impliquer des chercheurs pour enrichir la réflexion et l'action. * Reproductibilité vs. Reproduction (M. Ndoy) : Pour la généralisation, il ne s'agit pas de reproduire une expérience telle quelle, mais de comprendre et recréer les « facteurs qui ont conduit à la réussite » dans de nouveaux contextes. L'« analyse diagnostic » est primordiale. * Créer les Conditions de la Collaboration (M. Nóvoa) : La collaboration n'est pas automatique. Elle nécessite des « espaces ouverts », une autre « organisation des temps de l'école » et des « environnements éducatifs » qui favorisent la diversité et le travail conjoint. « Il n'y a pas d'inclusion sans diversité ». * La "Publication de l'Âme" (M. Nóvoa citant Steiner) : La communication ne se limite pas au journalisme ; il s'agit de "publier les innovations", de les partager, pour créer une "action commune".

En conclusion, le séminaire a souligné que le travail collaboratif à l'école est bien plus qu'une simple méthode pédagogique.

Il représente une transformation profonde des pratiques, des cultures et des organisations scolaires, enracinée dans la confiance envers les acteurs de terrain, la mutualisation des intelligences et un engagement commun pour la réussite et l'épanouissement de tous les élèves.

Bien que des défis subsistent, notamment la systématisation des changements, l'optimisme quant à la capacité des acteurs à innover est palpable.

Author response:

Public Review

Joint Public Review:

This manuscript presents an algorithm for identifying network topologies that exhibit a desired qualitative behaviour, with a particular focus on oscillations. The approach is first demonstrated on 3-node networks, where results can be validated through exhaustive search, and then extended to 5-node networks, where the search space becomes intractable. Network topologies are represented as directed graphs, and their dynamical behaviour is classified using stochastic simulations based on the Gillespie algorithm. To efficiently explore the large design space, the authors employ reinforcement learning via Monte Carlo Tree Search (MCTS), framing circuit design as a sequential decision-making process.

This work meaningfully extends the range of systems that can be explored in silico to uncover non-linear dynamics and represents a valuable methodological advance for the fields of systems and synthetic biology.

Strengths

The evidence presented is strong and compelling. The authors validate their results for 3-node networks through exhaustive search, and the findings for 5-node networks are consistent with previously reported motifs, lending credibility to the approach. The use of reinforcement learning to navigate the vast space of possible topologies is both original and effective, and represents a novel contribution to the field. The algorithm demonstrates convincing efficiency, and the ability to identify robust oscillatory topologies is particularly valuable. Expanding the scale of systems that can be systematically explored in silico marks a significant advance for the study of complex gene regulatory networks.

Weaknesses

The principal weakness of the manuscript lies in the interpretation of biological robustness. The authors identify network topologies that sustain oscillatory behaviour despite perturbations to the system or parameters. However, in many cases, this persistence is due to the presence of partially redundant oscillatory motifs within the network. While this observation is interesting and of clear value for circuit design, framing it as evidence of evolutionary robustness may be misleading. The "mutant" systems frequently exhibit altered oscillatory properties, such as changes in frequency or amplitude. From a functional cellular perspective, mere oscillation is insufficient - preservation of specific oscillation characteristics is often essential. This is particularly true in systems like circadian clocks, where misalignment with environmental cycles can have deleterious effects. Robustness, from an evolutionary standpoint, should therefore be framed as the capacity to maintain the functional phenotype, not merely the qualitative behaviour.

A secondary limitation is that, despite the methodological advances, the scale of the systems explored remains modest. While moving from 3- to 5-node systems is non-trivial, five elements still represent a relatively small network. It is somewhat surprising that the algorithm does not scale further, particularly when considering the performance of MCTS in other domains - for instance, modern chess engines routinely explore far larger decision trees. A discussion on current performance bottlenecks and potential avenues for improving scalability would be valuable.

Finally, it is worth noting that the emergence of oscillations in a model often depends not only on the topology but also critically on parameter choices and the nature of the nonlinearities. The use of Hill functions and high Hill coefficients is a common strategy to induce oscillatory dynamics. Thus, the reported results should be interpreted within the context of the modelling assumptions and parameter regimes employed in the simulations.

We thank the reviewers for their careful consideration of our work and for the interesting feedback and scientific discussion. We are working on a revised text based on their recommendations, which will include some of the discussion below.

This work meaningfully extends the range of systems that can be explored in silico to uncover non-linear dynamics and represents a valuable methodological advance for the fields of systems and synthetic biology.

We thank the reviewers for their positive assessment of our work’s impact!

The use of reinforcement learning to navigate the vast space of possible topologies is both original and effective, and represents a novel contribution to the field. The algorithm demonstrates convincing efficiency, and the ability to identify robust oscillatory topologies is particularly valuable. Expanding the scale of systems that can be systematically explored in silico marks a significant advance for the study of complex gene regulatory networks.

We appreciate these kind comments about our work’s merits. We are excited to share our reinforcement learning (RL) based method with the fields of systems and synthetic biology, and we consider it a valuable tool for the systematic analysis and design of larger-scale regulatory networks!

The principal weakness of the manuscript lies in the interpretation of biological robustness. The authors identify network topologies that sustain oscillatory behaviour despite perturbations to the system or parameters… [However, these] "mutant" systems frequently exhibit altered oscillatory properties, such as changes in frequency or amplitude. From a functional cellular perspective, mere oscillation is insufficient - preservation of specific oscillation characteristics is often essential. This is particularly true in systems like circadian clocks, where misalignment with environmental cycles can have deleterious effects. Robustness, from an evolutionary standpoint, should therefore be framed as the capacity to maintain the functional phenotype, not merely the qualitative behaviour.

We thank the reviewers for their attention to this point. In the large-scale circuit search, summarized in Figures 4A and 4B, we ran a search for 5-component oscillators that can spontaneously oscillate even when subjected to the deletion of a random gene. Some of the best performing circuits under these conditions exhibited a design feature we call “motif multiplexing,” in which multiple smaller motifs are interleaved in a way that makes oscillation possible under many different mutational scenarios. Interestingly, despite not selecting for preservation of frequency, the 3Ai+3Rep circuit (a 5-gene circuit highlighted in Figure 5) anecdotally appears to have a natural frequency that is robust to partial gene knockdowns, although not to complete gene deletions. As shown in Figure 5C, this circuit has a natural frequency of 6 cycles/hr (with one particular parameterization), and it can sustain a knockdown of any of its 5 genes to 50% of the wild-type transcription rate without altering the natural frequency by more than 20%.

However, we agree that there are salient differences between this training scenario and natural evolution. The revised text will clarify that these differences limit what conclusions can be drawn about biological evolution by analogy. As the reviewers point out, we use the presence of spontaneous oscillations (with or without the deletion) as a measure of fitness, regardless of frequency, so as to screen for designs with promising behavior. Also, the deletion mutations introduced during training likely represent larger perturbations to the system than a typical mutation encountered during genome replication (for example, a point mutation in a response element leading to a moderate change in binding affinity). Finally, we do not introduce any entrainment. Real circadian oscillators are aligned to a 24-hour period (“entrained”) by environmental inputs such as light and temperature. For this reason, natural circadian clocks may have natural frequencies that are slightly shorter or longer than 24 hours, although a close proximity to the 24-hour period does seem to be an important selective factor [1].

...despite the methodological advances, the scale of the systems explored remains modest. While moving from 3- to 5-node systems is non-trivial, five elements still represent a relatively small network. It is somewhat surprising that the algorithm does not scale further, particularly when considering the performance of MCTS in other domains - for instance, modern chess engines routinely explore far larger decision trees. A discussion on current performance bottlenecks and potential avenues for improving scalability would be valuable.

We thank the reviewers for their attention to this point. The main limitation we encountered to exploring circuits with more than 5 nodes in this work was the poor computational scaling of the Gillespie stochastic simulation algorithm, rather than a limitation of MCTS itself. While the average runtime of a 3-node circuit simulation was roughly 7 seconds, this number increased to 18-20 seconds with 5-node circuits. For this reason, we limited the search to topologies with ≤15 interaction arrows (15 sec/simulation). In general, the simulation time was proportional to the square of the number of transcription factors (TFs). We will revise the text to include the reason for stopping at 5 nodes, which is significant for understanding CircuiTree’s scaling properties.

With regards to scaling, an important advantage of CircuiTree is its ability to generate useful candidate designs after exploring only a portion of the search space. Like exhaustive search, given enough time, MCTS will comprehensively explore the search space and find all possible solutions. However, for large search spaces, RL-based agents are generally given a finite number of simulations (or time) to learn as much as possible.

Across machine learning (ML) applications [2] and particularly with RL models [3], this training time tends to obey a power law with respect to the underlying complexity of the problem. Thus we can use the complexity of the 3-node and 5-node searches to infer the current scaling limits of CircuiTree. The first oscillator topology was discovered after 2,280 simulations for the 3-node search, and in the 5-node search, the first oscillator using 5 nodes appeared at ~8e5 simulations, resulting in a power law of Y ~ 84.4 X^0.333. Thus, useful candidate designs may be found for 6-node and 7-node searches after 4.5e7 and 5.26e9 simulations, respectively, even though these spaces contain 1.5e17 and 2.5e23 topologies, respectively. Thus, running a 7-node search with the current implementation of CircuiTree would require resources close to the current boundaries of computation, requiring roughly 1.8 million CPU-hours, or 2 weeks on 5,000 CPUs, assuming a 1-second simulation. These points will be incorporated into both the results and discussion sections in our revised text.

However, we are optimistic about CircuiTree’s potential to scale to much larger circuits with modifications to its algorithm. CircuiTree uses the original (so-called “vanilla”) implementation of MCTS, which has not been used in professional game-playing AIs in over a decade. Contemporary RL-based game-playing engines leverage deep neural networks to dramatically reduce the training time, using value networks to identify game-winning positions and policy networks to find game-winning moves. AlphaZero, developed by Google DeepMind to learn games by self-play and without domain knowledge, outperformed all other chess AIs after 44 million training games, much smaller than the 10^43 possible chess states [4]. Similarly, the game of go has 10¹⁷⁰ possible states, but AlphaZero outperformed other AIs after only 140 million games [4]. Large circuits live in similarly large search spaces; for example, 19-node and 20-node circuits represent spaces of 10¹⁷² and 10¹⁹⁰ possible topologies. The revised text will include this discussion and identify value and policy networks, as well as more scalable simulation paradigms such as ODEs and neural ODEs, as our future directions for improving CircuiTree’s scalability.

Finally, our revised discussion will note some important differences between game-playing and biological circuit design. Unlike deterministic games like chess, the final value of a circuit topology is determined stochastically, by running a simulation whose fitness depends on the parameter set and initial conditions. Thus, state-for-state, it is possible that training an agent for circuit design may inherently require more simulations to achieve the same level of certainty compared to classical games. Additionally, while we often possess a priori knowledge about a game such as its overall difficulty or certain known strategies, we lack this frame of reference when searching for circuit designs. Thus, it remains challenging to know if and when a large space of designs has been “satisfactorily” or “comprehensively” searched, since the answer depends on data that are unknown, namely the quantity, quality, and location of solutions residing in the search space.

Not accounting for redundancy due to structural symmetries

Finally, it is worth noting that the emergence of oscillations in a model often depends not only on the topology but also critically on parameter choices and the nature of the nonlinearities. The use of Hill functions and high Hill coefficients is a common strategy to induce oscillatory dynamics. Thus, the reported results should be interpreted within the context of the modelling assumptions and parameter regimes employed in the simulations.

In our dynamical modeling of transcription factor (TF) networks, we do not rely on continuum assumptions about promoter occupancy such as Hill functions. Rather, we model each reaction - transcription, translation, TF binding/unbinding, and degradation - explicitly, and individual molecules appear and disappear via stochastic birth and death events. Many natural TFs are homodimers that bind cooperatively to regulate transcription; similarly, we assume that pairs of TFs bind more stably to their response element than individual TFs. Thus, our model has similar cooperativity to a Hill function, and it can be shown that in the continuum limit, the effective Hill coefficient is always ≤2. Our revision will clarify this aspect of the modeling and include a derivation of this property. Currently, the parameter values used in the figures are shown in Table 2. In the revised text, these will be displayed in the body of the text as well for clarity.

Bibliography (1) Spoelstra, K., Wikelski, M., Daan, S., Loudon, A. S. I., & Hau, M. (2015). Natural selection against a circadian clock gene mutation in mice. PNAS, 113(3), 686–691. https://doi.org/https://doi.org/10.1073/pnas.1516442113<br /> (2) Neumann, O., & Gros, C. (2023). Scaling Laws for a Multi-Agent Reinforcement Learning Model. The Eleventh International Conference on Learning Representations. Retrieved from https://openreview.net/forum?id=ZrEbzL9eQ3W (3) Jones, A. L. (2021). Scaling Scaling Laws with Board Games. arXiv [Cs.LG]. Retrieved from http://arxiv.org/abs/2104.03113 (4) Silver, D., Hubert, T., Schrittwieser, J., Antonoglou, I., Lai, M., Guez, A., Lanctot, M., Sifre, L., Kumaran, D., Graepel, T., Lillicrap, T., Simonyan, K., & Hassabis, D. (2018). A general reinforcement learning algorithm that Masters Chess, Shogi, and go through self-play. Science, 362(6419), 1140–1144. https://doi.org/10.1126/science.aar6404

Author response:

Reviewer #1 (Public Review):

Summary: 

BMP signaling is, arguably, best known for its role in the dorsoventral patterning, but not in nematodes, where it regulates body size. In their paper, Vora et al. analyze ChIP-Seq and RNA-Seq data to identify direct transcriptional targets of SMA-3 (Smad) and SMA-9 (Schnurri) and understand the respective roles of SMA-3 and SMA-9 in the nematode model Caenorhabditis elegans. The authors use publicly available SMA-3 and SMA-9 ChIP-Seq data, own RNA-Seq data from SMA-3 and SMA-9 mutants, and bioinformatic analyses to identify the genes directly controlled by these two transcription factors (TFs) and find approximately 350 such targets for each. They show that all SMA-3-controlled targets are positively controlled by SMA-3 binding, while SMA-9-controlled targets can be either up or downregulated by SMA-9. 129 direct targets were shared by SMA-3 and SMA-9, and, curiously, the expression of 15 of them was activated by SMA-3 but repressed by SMA-9. Since genes responsible for cuticle collagen production were eminent among the SMA-3 targets, the authors focused on trying to understand the body size defect known to be elicited by the modulation of BMP signaling. Vora et al. provide compelling evidence that this defect is likely to be due to problems with the BMP signaling-dependent collagen secretion necessary for cuticle formation. 

We thank the reviewer for this supportive summary. We would like to clarify the status of the publicly available ChIP-seq data. We generated the GFP tagged SMA-3 and SMA‑9 strains and submitted them to be entered into the queue for ChIP-seq processing by the modENCODE (later modERN) consortium. Due to the nature of the consortium’s funding, the data were required to be released publicly upon completion. Nevertheless, we have provided the first comprehensive analysis of these datasets.

Strengths: 

Vora et al. provide a valuable analysis of ChIP-Seq and RNA-Seq datasets, which will be very useful for the community. They also shed light on the mechanism of the BMP-dependent body size control by identifying SMA-3 target genes regulating cuticle collagen synthesis and by showing that downregulation of these genes affects body size in C. elegans. 

Weaknesses: 

(1) Although the analysis of the SMA-3 and SMA-9 ChIP-Seq and RNA-Seq data is extremely useful, the goal "to untangle the roles of Smad and Schnurri transcription factors in the developing C. elegans larva", has not been reached. While the role of SMA-3 as a transcriptional activator appears to be quite straightforward, the function of SMA-9 in the BMP signaling remains obscure. The authors write that in SMA-9 mutants, body size is affected, but they do not show any data on the mechanism of this effect. 

We thank the reviewer for directing our attention to the lack of clarity about SMA-9’s function. We will revise the text to highlight what this study and others demonstrate about SMA-9’s role in body size. We also plan to analyze additional target genes to deepen our model for how SMA-3 and SMA-9 interact functionally to produce a given transcriptional response.

(2) The authors clearly show that both TFs can bind independently of each other, however, by using distances between SMA-3 and SMA-9 ChIP peaks, they claim that when the peaks are close these two TFs act as complexes. In the absence of proof that SMA-3 and SMA-9 physically interact (e.g. that they co-immunoprecipitate - as they do in Drosophila), this is an unfounded claim, which should either be experimentally substantiated or toned down. 

A physical interaction between Smads and Schnurri has been amply demonstrated in other systems. The limitation in the previous work is that only a small number of target genes was analyzed. Our goal in this study was to determine how widespread this interaction is on a genomic scale.  Our analyses demonstrate for the first time that a Schnurri transcription factor has significant numbers of both Smad-dependent and Smad-independent target genes. We will revise the text to clarify this point.

(3) The second part of the paper (the collagen story) is very loosely connected to the first part. dpy-11 encodes an enzyme important for cuticle development, and it is a differentially expressed direct target of SMA-3. dpy-11 can be bound by SMA-9, but it is not affected by this binding according to RNA-Seq. Thus, technically, this part of the paper does not require any information about SMA-9. However, this can likely be improved by addressing the function of the 15 genes, with the opposing mode of regulation by SMA-3 and SMA-9. 

We appreciate this suggestion and will clarify how SMA-9 and its target genes contribute to collagen organization and body size regulation.

(4) The Discussion does not add much to the paper - it simply repeats the results in a more streamlined fashion. 

We thank the reviewer for this suggestion. We will add more context to the Discussion.

Reviewer #2 (Public Review): 

In the present study, Vora et al. elucidated the transcription factors downstream of the BMP pathway components Smad and Schnurri in C. elegans and their effects on body size. Using a combination of a broad range of techniques, they compiled a comprehensive list of genome-wide downstream targets of the Smads SMA-3 and SMA-9. They found that both proteins have an overlapping spectrum of transcriptional target sites they control, but also unique ones. Thereby, they also identified genes involved in one-carbon metabolism or the endoplasmic reticulum (ER) secretory pathway. In an elaborate effort, the authors set out to characterize the effects of numerous of these targets on the regulation of body size in vivo as the BMP pathway is involved in this process. Using the reporter ROL-6::wrmScarlet, they further revealed that not only collagen production, as previously shown, but also collagen secretion into the cuticle is controlled by SMA-3 and SMA-9. The data presented by Vora et al. provide in-depth insight into the means by which the BMP pathway regulates body size, thus offering a whole new set of downstream mechanisms that are potentially interesting to a broad field of researchers. 

The paper is mostly well-researched, and the conclusions are comprehensive and supported by the data presented. However, certain aspects need clarification and potentially extended data. 

(1) The BMP pathway is active during development and growth. Thus, it is logical that the data shown in the study by Vora et al. is based on L2 worms. However, it raises the question of if and how the pattern of transcriptional targets of SMA-3 and SMA-9 changes with age or in the male tail, where the BMP pathway also has been shown to play a role. Is there any data to shed light on this matter or are there any speculations or hypotheses? 

We agree that these are intriguing questions and we are interested in the roles of transcriptional targets at other developmental stages and in other physiological functions, but these analyses are beyond the scope of the current study.

(2) As it was shown that SMA-3 and SMA-9 potentially act in a complex to regulate the transcription of several genes, it would be interesting to know whether the two interact with each other or if the cooperation is more indirect. 

A physical interaction between Smads and Schnurri has been amply demonstrated in other systems. Our goal in this study was not to validate this physical interaction, but to analyze functional interactions on a genome-wide scale.

(3) It would help the understanding of the data even more if the authors could specifically state if there were collagens among the genes regulated by SMA-3 and SMA-9 and which. 

We thank the reviewer for this suggestion and will add the requested information in the text.

(4) The data on the role of SMA-3 and SMA-9 in the regulation of the secretion of collagens from the hypodermis is highly intriguing. The authors use ROL-6 as a reporter for the secretion of collagens. Is ROL-6 a target of SMA-9 or SMA-3? Even if this is not the case, the data would gain even more strength if a comparable quantification of the cuticular levels of ROL-6 were shown in Figure 6, and potentially a ratio of cuticular versus hypodermal levels. By that, the levels of secretion versus production can be better appreciated. 

rol-6 has been identified as a transcriptional target of this pathway. The level of ROL-6 protein, however, is not changed in sma-3 and sma-9 mutants, indicating that there is post-transcriptional compensation. We will include these data in the revised manuscript.

(5) It is known that the BMP pathway controls several processes besides body size. The discussion would benefit from a broader overview of how the identified genes could contribute to body size. The focus of the study is on collagen production and secretion, but it would be interesting to have some insights into whether and how other identified proteins could play a role or whether they are likely to not be involved here (such as the ones normally associated with lipid metabolism, etc.). 

We will add this information to the Discussion.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Work by Brosseau et. al. combines NMR, biochemical assays, and MD simulations to characterize the influence of the C-terminal tail of EmrE, a model multi-drug efflux pump, on proton leak. The authors compare the WT pump to a C-terminal tail deletion, delta_107, finding that the mutant has increased proton leak in proteoliposome assays, shifted pH dependence with a new titratable residue, faster-alternating access at high pH values, and reduced growth, consistent with proton leak of the PMF.

Strengths:

The work combines thorough experimental analysis of structural, dynamic, and electrochemical properties of the mutant relative to WT proteins. The computational work is well aligned in vision and analysis. Although all questions are not answered, the authors lay out a logical exploration of the possible explanations.

Weaknesses:

There are a few analyses that are missing and important data left out. For example, the relative rate of drug efflux of the mutant should be reported to justify the focus on proton leak. Additionally, the correlation between structural interactions should be directly analyzed and the mutant PMF also analyzed to justify the claims based on hydration alone. Some aspects of the increased dynamics at high pH due to a potential salt bridge are not clear.

Reviewer #2 (Public review):

Summary:

This manuscript explores the role of the C-terminal tail of EmrE in controlling uncoupled proton flux. Leakage occurs in the wild-type transporter under certain conditions but is amplified in the C-terminal truncation mutant D107. The authors use an impressive combination of growth assays, transport assays, NMR on WT and mutants with and without key substrates, classical MD, and reactive MD to address this problem. Overall, I think that the claims are well supported by the data, but I am most concerned about the reproducibility of the MD data, initial structures used for simulations, and the stochasticity of the water wire formation. These can all be addressed in a revision with more simulations as I point out below. I want to point out that the discussion was very nicely written, and I enjoyed reading the summary of the data and the connection to other studies very much.

Strengths:

The Henzler-Wildman lab is at the forefront of using quantitative experiments to probe the peculiarities in transporter biophysics, and the MD work from the Voth lab complements the experiments quite well. The sheer number of different types of experimental and computational approaches performed here is impressive.

Weaknesses:

The primary weaknesses are related to the reproducibility of the MD results with regard to the formation of water wires in the WT and truncation mutant. This could be resolved with simulations starting from structures built using very different loops and C-terminal tails.

The water wire gates identified in the MD should be tested experimentally with site-directed mutagenesis to determine if those residues do impact leak.

We appreciate the reviewers thoughtful consideration of our manuscript, and their recognition of the variety of experimental and computational approaches we have brought to bear in probing the very challenging question of uncoupled proton leak through EmrE.

We did record SSME measurements with MeTPP+, a small molecule substrate at two different protein:lipid ratios. These experiments report the rate of net flux when both proton-coupled substrate antiport and substrate-gated proton leak are possible. We will add this data to the revision, including data acquired with different lipid:protein ratio that confirms we are detecting transport rather than binding. In brief, this data shows that the net flux is highly dependent on both proton concentration (pH) and drug-substrate concentration, as predicted by our mechanistic model. This demonstrates that both types of transport contribute to net flux when small molecule substrates are present.

In the absence of drug-substrate, proton leak is the only possible transport pathway. The pyranine assay directly assesses proton leak under these conditions and unambiguously shows faster proton entry into proteoliposomes through the ∆107-EmrE mutant than through WT EmrE, with the rate of proton entry into ∆107-EmrE proteoliposomes matching the rate of proton entry achieved by the protonophore CCCP. We have revised the text to more clearly emphasize how this directly measures proton leak independently of any other type of transport activity. The SSME experiments with a proton gradient only (no small molecule substrate present) provide additional data on shorter timescales that is consistent with the pyranine data. The consistency of the data across multiple LPRs and comparison of transport to proton leak in the SSME assays further strengthens the importance of the C-terminal tail in determining the rate of flux.

None of the current structural models have good resolution (crystallography, EM) or sufficient restraints (NMR) to define the loop and tail conformations sufficiently for comparison with this work. We are in the process of refining an experimental structure of EmrE with better resolution of the loop and tail regions implicated in proton-entry and leak. Direct assessment of structural interactions via mutagenesis is complicated because of the antiparallel homodimer structure of EmrE. Any point mutation necessarily affects both subunits of the dimer, and mutations designed to probe the hydrophobic gate on the more open face of the transporter also have the potential to disrupt closure on the opposite face, particularly in the absence of sufficient resolution in the available structures. Thus, mutagenesis to test specific predicted structural features is deferred until our structure is complete so that we can appropriately interpret the results.

In our simulation setup, the MD results can be considered representative and meaningful for two reasons. First, the C-terminal tail, not present in the prior structure and thus modeled by us, is only 4 residues long. We will show in the revision and detailed response that the system will lose memory of its previous conformation very quickly, such that velocity initialization alone is enough for a diverse starting point. Second, our simulation is more like simulated annealing, starting from a high free energy state to show that, given such random initialization, the tail conformation we get in the end is consistent with what we reported. It is also difficult to sample back-and-forth tail motion within a realistic MD timescale. Therefore, it can be unconclusive to causally infer the allosteric motions with unbiased MD of the wildtype alone. The best viable way is to look at the equilibrium statistics of the most stable states between WT- and ∆107-EmrE and compare the differences.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The work is well done and well presented. In my opinion, the authors must address the following questions.

(1) It is unclear to a non-SSME-expert, why the net charge translocated in delta_107 is larger than in WT. For such small pH gradients (0.5-1pH unit), it seems that only a few protons would leave the liposome before the internal pH is adjusted to be the same as the external. This number can be estimated given the size of the liposomes. What is it? Once the pH gradient is dissipated, no more net proton transport should be observed. So, why would more protons flow out of the mutant relative to WT?

We appreciate the complexity of both the system and assay and have made revisions to both the main text and SI to address these points more clearly. While we can estimate liposomes size, we cannot easily quantify the number of liposomes on the sensor surface so cannot calculate the amount of charge movement as suggested by the reviewer. We have revised Fig. 3.2 and added additional data at low and high pH with different lipid to protein ratios to distinguish pre-steady state (proton release from the protein) and steady state processes (transport). An extended Fig. 3.2 caption and revised discussion in the main text clarify these points.

We have also revised SI figure 3.2 to include an example of transport driven by an infinite drug gradient. Drug-proton antiport results in net charge build-up in the liposome since two protons will be driven out for every +1 drug transported in. This also creates a pH gradient is created (higher proton concentration outside). The negative inside potential inhibits further antiport of drug. However, both the negative-inside potential and proton gradient will drives protons back into the liposome if there is a leak pathway available. This is clearly visible with a reversal of current negative (antiport) to positive (proton backflow), and the magnitude of this back flow is larger for ∆107-EmrE which lacks the regulatory elements provided by the C-terminal tail. We have amended the main text and SI to include this discussion.

(2) Given the estimated rate of transport, size of liposomes, and pH gradient, how quickly would the SSME liposomes reach pH balance?

Since SSME measurements are due to capacitive coupling and will represent the net charge movement, including pre-steady state contributions, the current values will be incredibly sensitive to individual rates of alternating access, proton and drug on- and off-rates. Time to pH balance would, therefore, differ based on the construct, LPR, absolute pH or drug concentrations as well as the magnitude of the given gradients. For this reason, we necessarily use integrated currents (transported charge over time) when comparing mutants as it reflects kinetic differences inherent to the mutant without over-processing the data, for example, by normalizing to peak currents which would over emphasize certain properties that will differ across mutants. This process allows for qualitative comparisons by subjecting mutants to the same pH and substrate gradients when the same density of transporter construct is present, and care is given to not overstate the importance of the actual quantities of charges that are moving as they will be highly context dependent. This is clearly seen in Fig 3.2 where the current is not zero and the net transported charge is still changing at the end of 1 second. We have amended SI figure 3.2 and the main text to include this discussion.

(3) Given that H110 and E14 would deprotonate when the external pH is elevated above 7 and that these protons would be released to external bulk, the external bulk pH would decrease twice as much for WT compared to delta107. This would decrease the pH gradient for WT relative to the mutant. Can these effects be quantified and accounted for? Would this ostensibly decrease the amount of charge that transfers into the liposomes for WT? How would this impact the current interpretation that the two systems are driven by the same gradient?

The reviewer is correct that there will be differences in deprotonation of WT and ∆107 and the amount of proton release will also change with pH. We have amended Figure 3.2 to clarify this difference and its significance. For the proton gradient only conditions in Figure 3, each set of liposomes were equilibrated to the starting pH by repeated washings and incubation before measurement occurred. For example, for the pH 6.5 inside, pH 7 outside condition, both the inside and outside pH were equilibrated at 6.5, and both E14 residues will be predominantly protonated in WT and ∆107, and H110 will be predominantly protonated in WT-EmrE. Upon application of the external pH 7 solution, protons will be released from the E14 of either construct, with additional proton being released from H110 for WT-EmrE causing a large pre-steady state negative contribution to the signal (Fig. 3.2A). Under this pH condition, we the peak current correlates with the LPR, as this release of protons will depend on density of the transporter. However, we also see that the longer-time decay of the signal correlates with the construct (WT or ∆107) and is relatively independent of LPR, consistent with a transport process rather than a rapid pre-steady state release of protons. Therefore, when we look at the actual transported charge over time, despite the higher contribution of proton release to the WT-EmrE signal, the significant increase in uncoupled proton transport for the C-terminal deletion mutant dominates the signal.

As a contrast, we apply this same analysis to the pH 8 inside, pH 8.5 outside condition where both sets of transports will be deprotonated from the start (Fig. 3.2B). Now the peak currents, decay rates, and transported charge over time are all consistent for a given construct (WT or ∆107). The two LPRs for an individual construct match within error, as the differences in overall charge movement and transported charge over time are independent of pre-steady-state proton release from the transporter at high pH.

(4) A related question, how does the protonation of H110 influence the potential rate of proton transport between the two systems? Does the proton on H110 transfer to E14?

The protonation of H110 will only influence the rate of transport of WT-EmrE as its protonation is required for formation of the hydrogen bonding network that coordinates gating. However, protonation of both E14s will influence the rate of proton transport of both systems as protonation state affects the rate of alternating access which is necessary for proton turnover. This is another reason we use the transported charge over time metric to compare mutants as it allows for a common metric for mutants with altered rates which are present in the same density and under the same gradient conditions. We do not have any evidence to support transfer of proton from H110 to E14, but there is also no evidence to exclude this possibility. We do not discuss this in the manuscript because it would be entirely speculative.

(5) Is the pKa in the simulations (Figure 6B) consistent with the experiment?

We calculated the pKa from this WT PMF and got a pKa of 7.1, which is in close proximity of the experimental value of 6.8

(6) Why isn't the PMF for delta_107 compared to WT to corroborate the prediction that hydration sufficiently alters both the rate and pKa of E14?

We appreciate the reviewer’s suggestion and agree that a direct comparison would be valuable. However, several factors limit the interpretability of such an analysis in this context:

(a) Our data indicate that the primary difference in free energy barriers between WT and Δ107 lies in the hydration step rather than proton transport itself. To fully resolve this, a 2D PMF calculation via 2D umbrella sampling would be required which can be very expensive. Solely looking at the proton transport side of this PMF will not give much difference.

(b) Given this, the aim for us to calculate this PMF is to support our conjecture that the bottleneck for such transport is the hydrophobic gate.

(7) The authors suggest that A61 rotation 'controls the water wire formation' by measuring the distribution of water connectivity (water-water distances via logS) and average distances between A61 and I68/I67. Delta_107 has a larger inter-residue distance (Figure 6A) more probable small log S closer waters connecting E14 and two residues near the top of the protein (Figure 5A). However, it strikes me that looking at average distances and the distribution of log S is not the best way to do this. Why not quantify the correlation between log S and A61 orientation and/or A61-I68/I71 distances as well as their correlation to the proposed tail interactions (D84-R106 interactions) to directly verify the correlation (and suggest causation) of these interactions on the hydration in this region. Additionally, plotting the RMSD or probability of waters below I68 and I171 as a function of A61-I68 distances and/or numbers over time would support the log S analysis.

The reviewer requested that we provide direct correlation analyses between A61 orientation, residue distances (A61-I68/I71), and water connectivity (logS) to better support the claim about water wire formation, rather than relying solely on average distances and distributions.

We appreciate the reviewer’s suggestion to strengthen our analysis with direct correlations. However, due to the slow kinetics of hydration/dehydration events, unbiased simulation timescales do not permit sufficient sampling of multiple transitions to perform statistically robust dynamic correlation analyses. Instead, our approach focuses on equilibrium statistics, which reveal the dominant conformational states of WT- and Δ107-EmrE and provide meaningful insights into shifts in hydration patterns.

(8) It looks like the D84-R106 salt bridge controls this A61-I68 opening. Could this also be quantifiably correlated?

As discussed in response to the previous question, the unbiased simulation timescales do not permit sufficient sampling of multiple transitions to perform statistically robust dynamic correlation analyses.

(9) The NMR results show that alternating access increases in frequency from ~4/s for WT at low and high pH to ~17/s for delta_107 only at high pH. They then go on to analyze potential titration changes in the delta_107 mutant, finding two residues with approximate pKa values of 5.6 and 7.1. The former is assigned to E14, consistent with WT. But the latter is suggested to be either D84, which salt bridges to R106, or the C-terminal carboxylate. If it is D84, why would deprotonation, which would be essential to form the salt bridge, increase the rate of alternating access relative to WT?

We note that the faster alternating access rate was observed for TPP+-bound ∆107-EmrE, not the transporter in the absence of substrate. In the absence of substrate the relatively broad lines preclude quantitative determination of the alternating access rate by NMR making it difficult to judge the validity of the reviewers reasoning. Identification of which residue (D84 or H110) corresponds to the shifted pKa is ultimately of little consequence as this mutant does not reflect the native conditions of the transporter. It is far more important to acknowledge that both R106 and D84 are sensitive to this deprotonation as it indicates these residues are close in space and provides experimental support for the existence of the salt bridge identified in the MD simulations, as discussed in the manuscript.

(10) In a more general sense, can the authors speculate why an efflux pump would evolve this type of secondary gate that can be thrown off by tight binding in the allosteric site such as that demonstrated by Harmane? What potential advantage is there to having a tail-regulated gate?

This was likely a necessity to allow for better coupling as these transporters evolved to be more promiscuous. The C-terminal tail is absent in tightly coupled family members such as Gdx who are specific for a single substrate and have a better-defined transport stoichiometry. We have included this discussion in the main text and are currently investigating this phenomenon further. Those experiments are beyond the scope of the current manuscript.

(11) It is hard to visualize the PT reaction coordinate. Is the e_PT unit vector defined for each window separately based on the initial steered MD pathway? If so, how reliant is the PT pathway on this initial approximate path? Also, how does this position for each window change if/when E14 rotates? This could be checked by plotting the x,y,z distributions for each window and quantifying the overlap between windows in cartesian space. These clouds of distributions could also be plotted in the protein following alignment so the reader can visualize the reaction coordinate. Does the CEC localization ever stray to different, disconnected regions of cartesian phase space that are hidden by the reaction coordinate definition?

The unit vector e_PT is the same across all windows based on unbiased MD. Therefore, the reaction coordinate (a scalar) is the vector from the starting point to the CEC, projected on this unit vector. E14 rotation does not significantly change the window definition a lot unless the CEC is very close to E14, where we found this to be a better CV. For detailed discussions about this CV, especially a comparison between a curvilinear CV, please see J. Am. Chem. Soc. 2018, 140, 48, 16535–16543 “Simulations of the Proton Transport” and its SI Figure S1.In the Supplementary Information, we added figure 6.1 to show the average X, Y, Z coordinates of each umbrella window.

(12) Lastly, perhaps I missed it, but it's unclear if the rate of substrate efflux is also increased in the delta_107 mutant. If this is also increased, then the overall rate of exchange is faster, including proton leak. This would be important to distinguish since the focus now is entirely on proton leaks. I.e., is it only leak or is it overall efflux and leak?

We have amended SI figure 3.2 to include a gradient condition where an infinite drug gradient is created across the liposome. The infinite gradient allows for rapid transport of drug into the liposomes until charge build-up opposes further transport. This peak is at the same time for both LPRs of WT- and ∆107-EmrE suggesting the rate of substrate transport is similar. Differences in the peak heights across LPRs can be attributed to competition between drug and proton for the primary binding site such that more proton will be released for the higher density constructs as described above. This process does also create a proton gradient as drug moving in is coupled to two protons moving out so as charge build-up inhibits further drug movement, the building proton gradient will also begin to drive proton back in which is another example of uncoupled leak. Here, again we see that this back-flow of protons or leak is of greater magnitude for ∆107-EmrE proteoliposomes that for those with WT-EmrE. We have included this discussion in the SI and main text.

Minor

(1) Introduction - the authors describe EmrE as a model system for studying the molecular mechanism of proton-coupled transport. This is a rather broad categorization that could include a wide range of phenomena distal from drug transport across membranes or through efflux pumps. I suggest further specifying to not overgeneralize.

We revised to note the context of multidrug efflux.

Reviewer #2 (Recommendations for the authors):

Simulations. The initial water wire analysis is based on 4 different 1 ms simulations presented in Figure 5. The 3 WT replicates show similar results for the tail-blocking water wire formation, but the details of the system build and loop/C-terminal tail placement are not clear. It does appear that a single C-terminal tail model was created for all WT replicates. Was there also modeling for any parts of the truncation mutant? Regardless, since these initial placements and uncertainties in the structures may impact the results and subsequent water wire formation, I would like a discussion of how these starting structures impacted the formation or not of wires. I think that another WT replicate should be run starting from a completely new build that places the tail in a different (but hopefully reasonable location). This could be built with any number of tools to generate reasonable starting structures. It's critical to ensure that multiple independent simulations across different initial builds show the same water wire behavior so that we know the results are robust and insensitive to the starting structure and stochastic variation.

We thank Reviewer 2 for their suggestion regarding the discussion of the initial structure. In our simulations, the C-terminal tail was initially modeled in an extended conformation (solvent-exposed) to mimic its disordered state prior to folding. This approach resembles an annealing process, where the system evolves from a higher free-energy state toward equilibrium. Notably, across all three replicas, we observed consistent folding of the tail onto the protein surface, supporting the robustness of this conformational preference.

For the Δ107 truncation mutant, minimal modeling was required, as most experimental structures resolve residues up to S105 or R106. To rigorously assess the influence of the starting configuration, we analyzed the tail’s dynamics using backbone dihedral angle auto- and cross-correlation functions (new Supplementary Figures 10.1 and 10.2). These analyses reveal rapid decay of correlations—consistent with the tail’s short length (5 residues) and high flexibility—indicating that the system "forgets" its initial configuration well within the simulation timescale. Thus, we conclude that our sampling is sufficient to capture equilibrium behavior, independent of the starting structure.

What does the size of the barrier in the PMF (Figure 6B) imply about the rate of proton transfer/leak and can the pKa shift of the acidic residue be estimated with this energy value compared to bulk?

We noticed this point aligns with a related concern raised by Reviewer 1. For a detailed discussion please refer to Point 5 in our response to Reviewer 1.

Experimental validation. The hypotheses generated by this work would be better buttressed if there were some mutation work at the hydrophobic gate (61, 68, 71) to support it. I realize that this may be hard, but it would significantly improve the quality.

Due to the small size of the transporter, any mutagenesis of EmrE should necessarily be accompanied by functional characterization to fully assess the effects of the mutation on rate-limiting steps. We have revised the manuscript to add a discussion of the challenges with analyzing simple point mutants and citing what is known from prior scanning mutagenesis studies of EmrE.

Reviewer #1 (Public review):

Summary:

This study addresses the roles of polyunsaturated fatty acids (PUFAs) in animal physiology and membrane function. A C. elegans strain carrying the fat-2(wa17) mutation possesses a very limited ability to synthesize PUFAs and there is no dietary input because the E. coli diet consumed by lab grown C. elegans does not contain any PUFAs. The fat-2 mutant strain was characterized to confirm that the worms grow slowly, have rigid membranes, and have a constitutive mitochondrial stress response. The authors showed that chemical treatments or mutations known to increase membrane fluidity did not rescue growth defects. A thorough genetic screen was performed to identify genetic changes to compensate for the lack of PUFAs. The newly isolated suppressor mutations that compensated for FAT-2 growth defects included intergenic suppressors in the fat-2 gene, as well as constitutive mutations in the hypoxia sensing pathway components EGL-9 and HIF-1, and loss of function mutations in ftn-2, a gene encoding the iron storage protein ferritin. Taken together, these mutations lead to the model that increased intracellular iron, an essential cofactor for fatty acid desaturases, allows the minimally functional FAT-2(wa17) enzyme to be more active, resulting in increased desaturation and increased PUFA synthesis.

Strengths:

(1) This study provides new information further characterizing fat-2 mutants. The authors measured increased rigidity of membranes compared to wild type worms, however this rigidity is not able to be rescued with other fluidity treatments such as detergent or mutants. Rescue was only achieved with polyunsaturated fatty acid supplementation.

(2) A very thorough genetic suppressor screen was performed. In addition to some internal fat-2 compensatory mutations, the only changes in pathways identified that are capable of compensating for deficient PUFA synthesis was the hypoxia pathway and the iron storage protein ferritin. Suppressor mutations included an egl-9 mutation that constitutively activates HIF-1, and Gain of function mutations in hif-1 that are dominant. This increased activity of HIF conferred by specific egl-9 and hif-1 mutations lead to decreased expression of ftn-2. Indeed, loss of ftn-2 leads to higher intracellular iron. The increased iron apparently makes the FAT-2 fatty acid desaturase enzyme more active, allowing for the production of more PUFAs.

(3) The mutations isolated in the suppressor screen show that the only mutations able to compensate for lack of PUFAs were ones that increased PUFA synthesis by the defective FAT-2 desaturase, thus demonstrating the essential need for PUFAs that cannot be overcome by changes in other pathways. This is a very novel study, taking advantage of genetic analysis of C. elegans, and it confirms the observations in humans that certain essential PUFAs are required for growth and development.

(4) Overall, the paper is well written, and the experiments were carried out carefully and thoroughly. The conclusions are well supported by the results.

Weaknesses:

Overall, there are not many weaknesses. The main one I noticed is that the lipidomic analysis shown in Figs 3C, 7C, S1 and S3. While these data are an essential part of the analysis and provide strong evidence for the conclusions of the study, it is unfortunate that the methods used did not enable the distinction between two 18:1 isomers. These two isomers of 18:1 are important in C. elegans biology, because one is a substrate for FAT-2 (18:1n-9, oleic acid) and the other is not (18:1n-7, cis vaccenic acid). Although rarer in mammals, cis-vaccenic acid is the most abundant fatty acid in C. elegans and is likely the most important structural MUFA. The measurement of these two isomers is not essential for the conclusions of the study.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors present a novel CRISPR/Cas9-based genetic tool for the dopamine receptor dop1R2. Based on the known function of the receptor in learning and memory, they tested the efficacy of the genetic tool by knocking out the receptor specifically in mushroom body neurons. The data suggest that dop1R2 is necessary for longer-lasting memories through its action on ⍺/ß and ⍺'/ß' neurons but is dispensable for short-term memory and thus in ɣ neurons. The experiments impressively demonstrate the value of such a genetic tool and illustrate the specific function of the receptor in subpopulations of KCs for longer-term memories. The data presented in this manuscript are significant.

Reviewer #2 (Public Review):

Summary:

This manuscript examines the role of the dopamine receptor, Dop1R2, in memory formation. This receptor has complex roles in supporting different stages of memory, and the neural mechanisms for these functions are poorly understood. The authors are able to localize Dop1R2 function to the vertical lobes of the mushroom body, revealing a role in later (presumably middle-term) aversive and appetitive memory. In general, the experimental design is rigorous, and statistics are appropriately applied. While the manuscript provides a useful tool, it would be strengthened further by additional mechanistic studies that build on the rich literature examining the roles of dopamine signaling in memory formation. The claim that Dop1R2 is involved in memory formation is strongly supported by the data presented, and this manuscript adds to a growing literature revealing that dopamine is a critical regulator of olfactory memory. However, the manuscript does not necessarily extend much beyond our understanding of Dop1R2 in memory formation, and future work will be needed to fully characterize this reagent and define the role of Dop1R2 in memory.

Strengths:

(1) The FRT lines generated provide a novel tool for temporal and spatially precise manipulation of Dop1R2 function. This tool will be valuable to study the role of Dop1R2 in memory and other behaviors potentially regulated by this gene.

(2) Given the highly conserved role of Dop1R2 in memory and other processes, these findings have a high potential to translate to vertebrate species.

Weaknesses:

(1) The authors state Dop1R2 associates with two different G-proteins. It would be useful to know which one is mediating the loss of aversive and appetitive memory in Dop1R2 knockout flies.

We thank you for the insightful comment. We agree that it would be very useful to know which G-proteins are transmitting Dop1R2 signaling. To that extent, we examined single-cell transcriptomics data to check the level of co-expression of Dop1R2 with G-proteins that are of interest to us. (Figure 1 S1)

Lines 312-325

“Some RNA binding proteins and Immediate early genes help maintain identities of Mushroom body cells and are regulators of local transcription and translation (de Queiroz et al., 2025; Raun et al., 2025). So, the availability of different G-proteins may change in different lobes and during different phases of memory. The G-protein via which GPCRs signal, may depend on the pool of available G-proteins in the cell/sub-cellular region (Hermans, 2003)., Therefore, Dop1R2 may signal via different G-proteins in different compartments of the Mushroom body and also different compartments of the neuron. We looked at Gαo and Gαq as they are known to have roles in learning and forgetting (Ferris et al., 2006; Himmelreich et al., 2017). We found that Dop1R2 co-expresses more frequently with Gαo than with Gαq (Figure 1 S1). While there is evidence for Dop1R2 to act via Gαq (Himmelreich et al., 2017). It is difficult to determine whether this interaction is exclusive, or if Dop1R2 can also be coupled to other G-proteins. It will be interesting to determine the breadth of G-proteins that are involved in Dop1R2 signaling.”

(2) It would be interesting to examine 24hr aversive memory, in addition to 24hr appetitive memory.

This is indeed an important point and we agree that it will complete the assessment of temporally distinct memory traces. We therefore performed the Aversive LTM experiments and include them in the results.

Lines 208-228

“24h memory is impaired by loss of Dop1R2

Next, we wanted to see if later memory forms are also affected. One cycle of reward training is sufficient to create LTM (Krashes & Waddell, 2008), while for aversive memory, 5-6 cycles of electroshock-trainings are required to obtain robust long-term memory scores (Tully et al., 1994). So, we looked at both, 24h aversive and appetitive memory. For aversive LTM, the flies were tested on the Y-Maze apparatus as described in (Mohandasan et al., (2022).

Flipping out Dop1R2 in the whole MB causes a reduced 24h memory performance (Figure 4A, E). No phenotype was observed when Ddop1R2 was flipped out in the γ-lobe (Figure 4B, F). However, similar to 2h memory, loss of Ddop1R2 in the α/β-lobes (Figure 4C, G) or the α’/β’-lobes (Figure 4D, H) causes a reduction in memory performance. Thus, Dop1R2 seems to be involved in aversive and appetitive LTM in the α/β-lobes and the α’/β’-lobes.

Previous studies have shown mutation in the Dop1R2 receptor leads to improvement in LTM when a single shock training paradigm is used (Berry et al., 2012). As we found that it disrupts LTM, we wanted to verify if the absence of Dop1R2 outside the MB is what leads to an improvement in memory. To that extent, we tested panneuronal flip-out of Dop1R2 flies for 6hr and 24hr memory upon single shock using the elav-Gal4 driver. We found that it did not improve memory at both time points (Figure 4 S1). Confirming that flipping out Dop1R2 panneuronally does not improve LTM (Figure 4 S1C) and highlighting its irrelevance in memory outside the MB.”

(3) The manuscript would be strengthened by added functional analysis. What are the DANs that signal through Dop1R. How do these knockouts impact MBONs?

We thank you for this question. We indeed agree that it is a highly relevand and open question, how distinct DANs signal via distinct Dopamine receptors. Our work here uniquely focusses on Dop1R2 within the MB. We aim to investigate other DopRs and the connection between DANs in the future using similar approaches.

(4) Also in Figure 2, the lobe-specific knockouts might be moved to supplemental since there is no effect. Instead, consider moving the control sensory tests into the main figure.

We thank you for this suggestion and understand that in Figure 2 no significant difference is seen. However, we have emphasized in the text that the results from the supplementary figures are just to confirm that the modifications made at the Dop1R2 locus did not alter its normal function.

Lines 156-162

“We wanted to see if flipping out Dop1R2 in the MB affects memory acquisition and STM by using classical olfactory conditioning. In short, a group of flies is presented with an odor coupled to an electric shock (aversive) or sugar (appetitive) followed by a second odor without stimulus. For assessing their memory, flies can freely choose between the odors either directly after training (STM) or at a later timepoint.

To ensure that the introduced genetic changes to the Dop1R2 locus do not interfere with behavior we first checked the sensory responses of that line”

(5) Can the single-cell atlas data be used to narrow down the cell types in the vertical lobes that express Dop1R2? Is it all or just a subset?

This is indeed an interesting question, and we thank you for mentioning it. To address this as best as we could, we analyzed the single cell transcriptomic data from (Davie et al., 2018) and presented it in Figure 1 S1.

Reviewer #3 (Public Review):

Summary:

Kaldun et al. investigated the role of Dopamine Receptor Dop1R2 in different types and stages of olfactory associative memory in Drosophila melanogaster. Dop1R2 is a type 1 Dopamine receptor that can act both through Gs-cAMP and Gq-ERCa2+ pathways. The authors first developed a very useful tool, where tissue-specific knock-out mutants can be generated, using Crispr/Cas9 technology in combination with the powerful Gal4/UAS gene-expression toolkit, very common in fruit flies.

They direct the K.O. mutation to intrinsic neurons of the main associative memory centre fly brain-the mushroom body (MB). There are three main types of MB-neurons, or Kenyon cells, according to their axonal projections: a/b; a'/b', and g neurons.

Kaldun et al. found that flies lacking dop1R2 all over the MB displayed impaired appetitive middle-term (2h) and long-term (24h) memory, whereas appetitive short-term memory remained intact. Knocking-out dop1R2 in the three MB neuron subtypes also impaired middle-term, but not short-term, aversive memory.

These memory defects were recapitulated when the loss of the dop1R2 gene was restricted to either a/b or a'/b', but not when the loss of the gene was restricted to g neurons, showcasing a compartmentalized role of Dop1R2 in specific neuronal subtypes of the main memory centre of the fly brain for the expression of middle and long-term memories.

Strengths:

(1) The conclusions of this paper are very well supported by the data, and the authors systematically addressed the requirement of a very interesting type of dopamine receptor in both appetitive and aversive memories. These findings are important for the fields of learning and memory and dopaminergic neuromodulation among others. The evidence in the literature so far was generated in different labs, each using different tools (mutants, RNAi knockdowns driven in different developmental stages...), different time points (short, middle, and long-term memory), different types of memories (Anesthesia resistant, which is a type of protein synthesis independent consolidated memory; anesthesia sensitive, which is a type of protein synthesis-dependent consolidated memory; aversive memory; appetitive memory...) and different behavioral paradigms. A study like this one allows for direct comparison of the results, and generalized observations.

(2) Additionally, Kaldun and collaborators addressed the requirement of different types of Kenyon cells, that have been classically involved in different memory stages: g KCs for memory acquisition and a/b or a'/b' for later memory phases. This systematical approach has not been performed before.

(3) Importantly, the authors of this paper produced a tool to generate tissue-specific knock-out mutants of dop1R2. Although this is not the first time that the requirement of this gene in different memory phases has been studied, the tools used here represent the most sophisticated genetic approach to induce a loss of function phenotypes exclusively in MB neurons.

Weaknesses:

(1) Although the paper does have important strengths, the main weakness of this work is that the advancement in the field could be considered incremental: the main findings of the manuscript had been reported before by several groups, using tissue-specific conditional knockdowns through interference RNAi. The requirement of Dop1R2 in MB for middle-term and long-term memories has been shown both for appetitive (Musso et al 2015, Sun et al 2020) and aversive associations (Plaçais et al 2017).

Thank you for this comment. We believe that the main takeaway from the paper is the elegant tool we developed, to study the role of Dop1R2 in fruit flies by effectively flipping it out spatio-temporally. Additionally, we studied its role in all types of olfactory associative memory to establish it as a robust tool that can be used for further research in place of RNAi knockouts which are shown to be less efficient in insects as mentioned in the texts in line 394-398.

“The genetic tool we generated here to study the role of the Dop1R2 dopamine receptor in cells of interest, is not only a good substitute for RNAi knockouts, which are known to be less efficient in insects (Joga et al., 2016), but also provides versatile possibilities as it can be used in combination with the powerful genetic tools of Drosophila.”

(2) The approach used here to genetically modify memory neurons is not temporally restricted. Considering the role of dopamine in the correct development of the nervous system, one must consider the possible effects that this manipulation can have in the establishment of memory circuits. However, previous studies addressing this question restricted the manipulation of Dop1R2 expression to adulthood, leading to the same findings than the ones reported in this paper for both aversive and appetitive memories, which solidifies the findings of this paper.

We thank you for this comment and we agree that it would be important to show a temporally restricted effect of Dop1R2 knockout. To assess this and rule out potential developmental defects we decided to restrict the knockout to the post-eclosion stage and to include these results.

Lines 230-250

“Developmental defects are ruled out in a temporally restricted Dop1R2 conditional knockout.

To exclude developmental defects in the MB caused by flip-out of Dop1R2, we stained fly brains with a FasII antibody. Compared to genetic controls, flies lacking Dop1R2 in the mushroom body had unaltered lobes (Figure 4 S2C).

Regardless, we wanted to control for developmental defects leading to memory loss in flip-out flies. So, we generated a Gal80ts-containing line, enabling the temporal control of Dop1R2 knockout in the entire mushroom body (MB). Given that the half-life of the receptor remains unknown, we assessed both aversive short-term memory (STM) and long-term memory (LTM) to determine whether post-eclosion ablation of Dop1R2 in the MB produced differences compared to our previously tested line, in which Dop1R2 was constitutively knocked out from fertilization. To achieve this, flies were maintained at 18°C until eclosion and subsequently shifted to 30°C for five to seven days. On the fifth day, training was conducted, followed by memory testing. Our results indicate that aversive STM was not significantly impaired in Dop1R2-deficient MBs compared to control flies (Figure 4 S3), consistent with our previous findings (Figure 2). However, aversive LTM was significantly impaired relative to control lines (Figure 4 S3), which also aligned with prior observations. These findings strongly indicate that memory loss caused by Dop1R2 flip-out is not due to developmental defects.”

(3) The authors state that they aim to resolve disparities of findings in the field regarding the specific role of Dop1R2 in memory, offering a potent tool to generate mutants and addressing systematically their effects on different types of memory. Their results support the role of this receptor in the expression of long-term memories, however in the experiments performed here do not address temporal resolution of the genetic manipulations that could bring light into the mechanisms of action of Dop1R2 in memory. Several hypotheses have been proposed, from stabilization of memory, effects on forgetting, or integration of sequences of events (sensory experiences and dopamine release).

We thank you for this comment. We agree that it would be interesting to dissect the memory stages by knocking out the receptor selectively in some of them (encoding, consolidation, retrieval). However, our tool irreversibly flips out Dop1R2 preventing us from investigating the receptor’s role in retrieval. Our results show that the receptor is dispensable for STM formation (Figure 2, Figure 4 Supplement 3), suggesting that it is not involved in encoding new information. On the other hand, it is instead involved in consolidation and/or retrieval of long-term and middle-term memories (Figure 3, Figure 4, Figure 5B).

Overall, the authors generated a very useful tool to study dopamine neuromodulation in any given circuit when used in combination with the powerful genetic toolkit available in Drosophila. The reports in this paper confirmed a previously described role of Dop1R2 in the expression of aversive and appetitive LTM and mapped these effects to two specific types of memory neurons in the fly brain, previously implicated in the expression and consolidation of long-term associative memories.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) On the first view, the results shown here are different from studies published earlier, while in the same line with others (e.g. Sun et al, for appetitive 24h memories). For example, Berry et al showed that the loss of dop1R2 impairs immediate memory, while memory scores are enhanced 3h, 6h, and 24h after training. Further, they showed data that shock avoidance, at least for higher shock intensities, is reduced in mutant (damb) flies. All in all, this favors how important it is to improve the genetic tools for tissue-specific manipulation. Despite the authors nicely discussing their data with respect to the previous studies, I wondered whether it would be suitable to use the new tool and knock out dop1R2 panneuronally to see whether the obtained data match the results published by Berry et al.. Further, as stated in line 105ff: "As these studies used different learning assays - aversive and appetitive respectively as well as different methods, it is unclear if Dop1R2 has different functions for the different reinforcement stimulus" I wondered why the authors tested aversive and appetitive learning for STM and 2h memory, but only appetitive memory for 24h.

Thank you for this comment. To that extent, as mentioned above in response to reviewer #2, we included in the results the aversive LTM experiment (Figure 4). Moreover, we performed experiments along the line of Berry et al. using our tool as shown in Figure 4 S1. Our results support that Dop1R2 is required for LTM, rather than to promote forgetting.

(2) Line 165ff: I can´t find any of the supplementary data mentioned here. Please add the corresponding figures.

Thank you for pointing this out. In that line we don’t refer to any supplementary data, but to the Figure 1F, showing the absence of the HA-tag in our MB knock-out line. We have clarified this in the text (lines 151-153)

(3) I can't imagine that the scale bar in Figure 1D-F is correct. I would also like to suggest to show a more detailed analysis of the expression pattern. For example, both anterior and posterior views would be appropriate, perhaps including the VNC. This would allow the expression pattern obtained with this novel tool to be better compared with previously published results. Also, in relation to my comment above (1), it may help to understand the functional differences with previous studies, especially as the authors themselves state that the receptor is "mainly" expressed in the mushroom body (line 99). It would be interesting to see where else it is expressed (if so). This would also be interesting for the panneuronal knockdown experiment suggested under (1). If the receptor is indeed expressed outside the mushroom body, this may explain the differences to Berry et al.

Thank you for noting this, there was indeed a mistake in the scale bar which we now fixed. Since with our HA-tag immunostaining we could not detect any noticeable signal outside of the MB, we decided to analyze previously existing single cell transcriptomics data that showed expression of the receptor in 7.99% of cells in the VNC and in 13.8% of cells outside the MB (lines 98-100) confirming its sparse expression in the nervous system. The lack of detection of these cells is likely due to the sparse and low expression of the protein. The HA-tag allows to detect the endogenous level of the locus (it is possible that a Gal4/UAS amplification of the signal might allow to detect these cells).

Regarding the panneuronal knockout, we decided to try to replicate the experiment shown in Berry et al. in Figure 4 S1 and found that Dop1R2 is required for LTM.

(4) Related to learning data shown in Figures 2-4, the authors should show statistical differences between all groups obtained in the ANOVA + PostHoc tests. Currently, only an asterisk is placed above the experimental group, which does not adequately reflect the statistical differences between the groups. In addition, I would like to suggest adding statistical tests to the chance level as it may be interesting to know whether, for example, scores of knockout flies in 3C and 3D are different from the chance level.

Many thanks for this correction, we agree with the fact that the way significance scores were shown was not informative enough. We fixed the point by now showing significance between all the control groups and the experimental ones. We also inserted the chance level results in the figure legends.

(5) Unfortunately, the manuscript has some typing errors, so I would like to ask the authors to check the manuscript again carefully.

Some Examples:

Line 31: the the

Line 56: G-Protein

Line 64: c-AMP

Line 68: Dopamine

Line 70: G-Protein (It alternates between G-protein and G-Protein)

Line 76: References are formatted incorrectly

Line 126: Ha-Tag (It alternates between Ha and HA)

Line 248: missing space before the bracket...is often found

Thank you for noticing these errors, we have now corrected the spelling throughout the manuscript.

(6) In the figures the axes are labelled Preference Index (Pref"I"). In the methods, however, the calculation formula is defined as "PREF".

We thank you for drawing attention to this. To avoid confusion, we changed the definition in the methods section so that it could be clear and coherent (“Memory tests” paragraph in the methods section).

“PREF = ((N_arm1 - N_arm2) 100) / N_total the two preference indices were calculated from the two reciprocal experiments. The average of these two PREFs gives a learning index (LI). LI = (PREF₁ + PREF₂) / 2.

In case of all Long-term Aversive memory experiments, Y-Maze protocol was adapted to test flies 24 hours post training. Testing using the Y-Maze was done following the protocol as described in (Mohandasan et al., 2022) where flies were loaded at the bottom of 20-minutes odorized 3D-printed Y-Mazes from where they would climb up to a choice point and choose between the two odors. The learning index was then calculated after counting the flies in each odorized vial as follows: LI = ((N_CS- - N_CS+) 100) / N_total. Where NCS- and NCS+ are the number of flies that were found trapped in the untrained and trained odor tube respectively.

Reviewer #2 (Recommendations For The Authors):

(1) In Figures 2 and 3, the legends running two different subfigures is confusing. Would be helpful to find a different way to present.

Thank you for your suggestion. We modified how we present legends, placing them vertically so that it is clearer.

(2) Use additional drivers to verify middle and long-term memory phenotypes.

We agree that it would be interesting to see the role of Dop1R2 in other neurons. To that extent, we looked at long term aversive memory in flies where the receptor was panneuronaly flipped out, and did not find evidence that suggested involvement of Dop1R2 in memory processes outside the MB. (Figure 4 S1)

(3) Additional discussion of genetic background for fly lines would be helpful.

Thank you for your advice. We have mentioned the genetic background of flies in the key resources table of the methods sections. Additionally, we also included further explanation on how the lines were created and their genetic background (see “Fly Husbandry” paragraph in the methods section).

“UAS-flp;;Dop1R2 cko flies and Gal4;Dop1R2^cko flies were crossed back with ;;Dop^cko flies to obtain appropriate genetic controls which were heterozygous for UAS and Gal4 but not Dop1R2^cko.”

Reviewer #3 (Recommendations For The Authors):

Line 109 states that to resolve the problem a tool is developed to knock down Dop1R2 in s spatial and temporal specific manner- while I agree that this is within the potential of the tool, there is no temporal control of the flipase action in this study; at least I cannot find references to the use of target/gene switch to control stages of development or different memory phases. However the version available for download is missing supplementary information, so I did not have access to supplementary figures and tables.

Thank you for the comment, as mentioned before it would be great to be able to dissect the memory phases. We show in lines 232 – 250 and Figure 4 S3 that the temporally restricted flip-out to the post-eclosion life stage gave us coherent results with the previous findings, ruling out potential developmental defects.

In relation to my comment on the possible developmental effects of the loss of the gene, Figure 1F could showcase an underdeveloped g lobe when looking at the lobe profiles. I understand this is not within the scope of the figure, but maybe a different z projection can be provided to confirm there are no obvious anatomical alterations due to the loss of the receptor.

We understand the doubt about the correct development of the MB and we thank you for your insightful comment. To that extent we decided to perform a FasII immunostaining that could show us the MB in the different lines (Figure 4 S2) and it appears that there are no notable differences in the lobes development in our knockout line.

It seems that the obvious missing piece of the puzzle would be to address the effects of knocking out Dop1R2 in aversive LTM. The idea of systematically addressing different types of memory at different time points and in different KCs is the most attractive aspect of this study beyond the technical sophistication, and it feels that the aim of the study is not delivered without that component.

We agree and we thank you for the clarification. As mentioned above in response to Reviewer #2, we decided to test aversive LTM as described in lines –208-228, Figure 4, Figure 4 S1.

Some statements of the discussion seem too vague, and I think could benefit from editing:

Line 284 "however other receptors could use Gq and mediate forgetting"- does this refer to other dopamine receptors? Other neuromodulators? Examples?

Thank you for pointing this out. We Agree and therefore decided to omit this line.

Line 289 "using a space training protocol and a Dop1R2 line" - this refers to RNAi lines, but it should be stated clearly.

That is correct, we thank you for bringing attention to this and clarified it in the manuscript.

–Lines 329-330

“Interestingly, using a spaced training protocol and a Dop1R2 RNAi knockout line another study showed impaired LTM (Placais et al., 2017).”

The paragraph starting in line 305 could be re-written to improve clarity and flow. Some statements seem disconnected and require specific citations. For example "In aversive memory formation, loss of Dop1R2 could lead to enhanced or impaired memory, depending on the activated signaling pathways and the internal state of the animal...". This is not accurate. Berry et al 2012 report enhanced LTM performance in dop1R2 mutants whereas Plaçais et al 2017 report LTM defects in Dop1R2 knock-downs, but these different findings do not seem to rely on different internal states or signaling pathways. Maybe further elaboration can help the reader understand this speculation.

We agree and we thank you for this advice. We decided to add additional details and citations to validate our speculation

Lines 350-353

“In aversive memory formation, loss of Dop1R2 could lead to enhanced or impaired memory, depending on the activated signaling pathways. The signaling pathway that is activated further depends on the available pool of secondary messengers in the cell (Hermans, 2003) which may be regulated by the internal state of the animal.”

"...for reward memory formation, loss of Dop1R2 seems to impair memory", this seems redundant at this point, as it has been discussed in detail, however, citations should be provided in any case (Musso 2015, Sun 2020)

Thank you for noting this. We recognize the redundancy and decided to exclude the line.

Finally, it would be useful to additionally refer to the anatomical terminology when introducing neuron names; for example MBON MVP2 (MBON-g1pedc>a/b), etc.

Thank you for this suggestion. We understand the importance of anatomical terminologies for the neurons. Therefore, we included them when we introduce neurons in the paper.

We thank you for your observations. We recognize their value, so we have made appropriate changes in the discussion to sound less vague and more comprehensive.

Author response:

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

Reviewer #1 (Public review):

Summary:

Using highly specific antibody reagents for biological research is of prime importance. In the past few years, novel approaches have been proposed to gain easier access to such reagents. This manuscript describes an important step forward toward the rapid and widespread isolation of antibody reagents. Via the refinement and improvement of previous approaches, the Perrimon lab describes a novel phage-displayed synthetic library for nanobody isolation. They used the library to isolate nanobodies targeting Drosophila secreted proteins. They used these nanobodies in immunostainings and immunoblottings, as well as in tissue immunostainings and live cell assays (by tethering the antigens on the cell surface).

Since the library is made freely available, it will contribute to gaining access to better research reagents for non-profit use, an important step towards the democratisation of science.

Strengths:

(1) New design for a phage-displayed library of high content.

(2) Isolation of valuble novel tools.

(3) Detailed description of the methods such that they can be used by many other labs.

We are grateful for these supportive comments.

Weaknesses:

My comments largely concentrate on the representation of the data in the different Figures.

We have made adjustments according to the reviewer’s recommendations.

Reviewer #2 (Public review):

Summary:

In this study, the authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. The authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, the authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.

Strengths:

The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their application for immunostaining and immunoblotting experiments. Data are convincing and the manuscript is well-written, clear, and easy to read.

We thank the reviewer for these supportive comments.

Weaknesses:

On the eight Drosophila secreted proteins selected to screen for nanobodies, the authors failed to identify nanobodies for three of them. While the authors mentioned potential improvements of the protocol in the discussion, none of them have been tested in this manuscript.

We prepared all eight antigens by single-step IgG purification (see Materials and Methods) without additional biophysical quality control (e.g., size-exclusion chromatography). Consequently, we cannot definitively determine whether the three “no-binder” cases resulted from the aggregation or misfolding of the antigens, versus gaps in our naive library’s sequence space. While approaches such as additional purification steps or affinity maturation of weak binders would likely rescue these difficult targets, comprehensive pipeline optimization is beyond the scope of establishing and validating the phage-displayed nanobody platform. We have clarified this limitation and suggested these strategies in third paragraph of the Discussion.

The same comment applies to the experiments using membrane-tethered forms of the antigens to test the affinity of nanobodies identified by ELISA. Many nanobodies fail to recognize the antigens. While authors suggested a low affinity of these nanobodies for their antigens, this hypothesis has not been tested in the manuscript.

We observed that several nanobodies with strong ELISA signals showed reduced binding to membrane-displayed antigens. This discrepancy may result from low affinity of the nanobodies or differences in post-translational modifications (e.g., glycosylation) and antigen context between secreted IgG-fusion proteins (used for panning/ELISA) and GPI- or mCD8-anchored proteins. In an ongoing work, we have performed affinity maturation of the nanobodies and successfully increased the affinity toward the target antigen. These results will be reported separately.

Improving the protocol at each step for nanobody selection would greatly increase the success rate for the discovery of nanobodies with high affinity.

We fully agree that systematic optimization—from antigen preparation (e.g., additional purification steps) through screening conditions (e.g., buffer composition, additional affinity-maturation steps)—could substantially increase the success rate and nanobody affinity. These represent important directions for future work.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Figure 3. The merge of two GFP channels does not make much sense. Can the authors not use artificial colours? And show the panels at higher resolution, such that a viewer can really see and judge what they are seeing? The same comments apply to all Supplementary Figures.

We appreciate the reviewer’s comment. In the revised Figure 3, we have replaced the cyan/green overlay with red/green overlay and used enlarged pictures so that GFP-positive cells and corresponding nanobody staining are clearly visible. We applied the same layout to all relevant Supplementary Figures.

(2) Figure 4. Also, in this Figure, it is not really possible to see what the authors say one should see. The resolution should be higher, and arrows or arrowheads should point to important structures.

We appreciate the reviewer’s comment. In the revised Figure 4A, we have added arrows to point to the immunostaining signal in cells with smaller nuclei and added inset panels to show a closer view of representative NbMip-4G staining.

Reviewer #2 (Recommendations for the authors):

(1) Images are sometimes quite small and difficult to interpret. For example, Figures S2C-D.

We thank the reviewer for this suggestion. In the revised figures, we have replaced the cyan/green overlay with red/green overlay and used enlarged pictures that clearly show GFP-positive cells alongside their corresponding nanobody staining.

(2) Supplemental figures are not always cited in the text.

Thank you for the comment. To eliminate this misunderstanding, we have updated the Nesfatin1 nanobody screen data as Supplementary Figure 1 and Mip nanobody screen data as Supplementary Figure 2. We have made the corresponding changes in the Results section.

Reviewer #1 (Public review):

Summary:

Participants in this study completed three visits. In the first, participants received experimental thermal stimulations which were calibrated to elicit three specific pain responses (30, 50, 70) on a 0-100 visual analogue scale (VAS). Experimental pressure stimulations were also calibrated at an intensity to the same three pain intensity responses. In the subsequent two visits, participants completed another pre-calibration check (Visit 2 of 3 only). Then, prior to the exercise NALOXONE or a SALINE placebo-control was administered intravenously. Participants then completed 1 of 4 blocks of HIGH (100%) or LOW (55%) intensity cycling which was tailored according to a functional threshold power (FTP) test completed in Visit 1. After each block of cycling lasting 10 minutes, participants entered an MRI scanner and were stimulated with the same thermal and pressure stimulations that corresponded to 30, 50, and 70 pain intensity ratings from the calibration stage. Therefore, this study ultimately sought to investigate whether aerobic exercise does indeed incur a hypoalgesia effect. More specifically, researchers tested the validity of the proposed endogenous pain modulation mechanism. Further investigation into whether the intensity of exercise had an effect on pain and the neurological activation of pain-related brain centres were also explored. Results show that in the experimental visits (Visit 2 and 3), when participants exercised at two distinct intensities as intended. Power output, heart rate, and perceived effort ratings were higher during the HIGH versus LOW intensity cycling. In particular. HIGH intensity exercise was perceived as "hard" / ~15 on the Borg (1974, 1998) scale, whereas LOW intensity exercise was perceived as "very light" / ~9 on the same scale.

The fMRI data from Figure 1 indicates that the anterior insula, dorsal posterior insula and middle cingulate cortex show pronounced activation as stimulation intensity and subsequent pain responses increased, thus linking these brain regions with pain intensity and corroborating what many studies have shown before.

Results also showed that participants rated a higher pain intensity in the NALOXONE condition at all three stimulation intensities compared to the SALINE condition. Therefore, the expected effect of NALOXONE in this study seemed to occur whereby opioid receptors were "blocked" and thus resulted in higher pain ratings compared to a SALINE condition where opioid receptors were "not blocked". When accounting for participant sex, NALOXONE had negligible effects at lower experimental nociceptive stimulations for females compared to males who showed a hyperalgesia effect to NALOXONE at all stimulation intensities (peak effect at 50 VAS). Females did show a hyperalgesia effect at stimulation intensities corresponding to 50 and 70 VAS pain ratings. The fMRI data showed that the periaqueductal gray (PAG) showed increased activation in the NALOXONE versus SALINE condition at higher thermal stimulation intensities. The PAG is well-linked to endogenous pain modulation.

When assessing the effects of NALOXONE and SALINE after exercise, results showed no significant differences in subsequent pain intensity ratings.

When assessing the effect of aerobic exercise intensity on subsequent pain intensity ratings, authors suggested that aerobic exercise in the form of a continuous cycling exercise tailored to an individual's FTP is not effective at eliciting an exercise-induced hypoalgesia response -irrespective of exercise intensity. This is because results showed that pain responses did not differ significantly between HIGH and LOW intensity exercise with (NALOXONE) and without (SALINE) an opioid antagonist. Therefore, authors have also questioned the mechanisms (endogenous opioids) behind this effect.

Strengths:

Altogether, the paper is great piece of work that has provided some truly useful insight into the neurological and perceptual mechanisms associated with pain and exercise-induced modulation of pain. The authors have gone to great lengths to delve into their research question(s) and their methodological approach is relatively sound. The study has incorporated effective pseudo-randomisation and conducted a rigorous set of statistical analysis to account for as many confounds as possible. I will particularly credit the authors on their analysis which explores the impact of sex and female participants' stage of menses on the study outcomes. It would be particularly interesting for future work to pursue some of these lines of research which investigate the differences in the endogenous opioid mechanism between sexes and the added interaction of stage of menses or training status - all of which the authors point out in their discussion.

There are certainly many other areas that this article contributes to the literature due to the depth of methods the research team have used. For example, the authors provide much insight into: the impact of exercise intensity on the exercise-induced hypoalgesia effect; the impact of sex on the endogenous opioid modulation mechanism; and the impact of exercise intensity on the neurological indices associated with endogenous pain modulation and pain processing. All of which, the researchers should be credited for due to the time and effort they have spent completing this study. Indeed, their in-depth analysis of many of these areas provides ample support for the claims they make in relation to these specific questions. As such, I consider their evidence concerning the fMRI data to be very convincing (and interesting).

Weaknesses:

Although the authors have their own view of their results, I, however, do still maintain a slightly different take on what the post-exercise pain ratings seem to show and its implications for judging whether an exercise-induced hypoalgesia effect is present or not and whether this is related to the opioid system.

For example, my basic assumptions relate to data which appears to show that there is an exercise-induced hypoalgesia effect as average pain ratings are ~30% lower than pre-calibrated/resting pain ratings within the SALINE condition at the same temperature of stimulation. Then, it appears there is evidence for the endogenous opioid mechanism as the NALOXONE condition demonstrates a minimal hypoalgesia effect after exercise. I.e., NALOXONE indeed blocked the opioid receptors, and such inhibition prevented the endogenous opioid system from taking effect.

However, through a comprehensive revision of their work, the authors have addressed many areas that myself and my fellow reviewer have questioned and provided a comprehensive set of responses and edits about this. So while I may have some opposing views on the mechanisms at play, I believe that each reader can decide and interpret the data for themselves which has been presented well by the authors.

Reviewer #1 (Public review):

Summary:

An interesting manuscript from the Carrington lab is presented investigating the behavior of single vs double GPI-anchored nutrient receptors in bloodstream form (BSF) T. brucei. These include the transferrin receptor (TfR), the HpHb receptor (HpHbR), and the factor H receptor (FHR). The central question is why these critical proteins are not targeted by host-acquired immunity. It has generally been thought that they are sequestered in the flagellar pocket (FP), where they are subject to rapid endocytosis - any Ab:receptor complexes would be rapidly removed from the cell surface. This manuscript challenges that assumption by showing that these receptors can be found all over the outer cell body and flagella surfaces, if one looks in an appropriate manner (rapid direct fixation in culture media).

The main part of the manuscript focuses on TfR, typically a GPI1 heterodimer of very similar E6 (GPI anchored) and E7 (truncated, no GPI) subunits. These are expressed coordinately from 15 telomeric expression sites (BES), of which only one can be transcribed at a time. The authors identify a native E6:E7 pair in BES7 in which E7 is not truncated and therefore forms a GPI2 heterodimer. By in situ genetic manipulation, they generate two different sets of GPI1:GPI2 TfR combinations expressed from two different BESs (BES1 and BES7). Comparative analyses of these receptors form the bulk of the data.

The main findings are:

(1) Both GPI1 and GPI2 TfR can be found on the cell body/flagellar surface. (2) Both are functional for Tf binding and uptake. (3) GPI2 TfR is expressed at ~1.5x relative to GPI1 TfR. (4) Ultimate TfR expression level (protein) is dependent on the BES from which it is expressed.

Most of these results are quite reasonably explained in light of the hydrodynamic flow model of the Engstler lab and the GPI valence model of the Bangs lab. Additional experiments, again by rapid fixation, with HpHbR and FHR, show that these GPI1 receptors can also be seen on the cell surface, in contrast to published localizations.

It is quite interesting that the authors have identified a native GPI2 TfR. However, essentially all of the data with GPI2 TfR are confirmatory for the prior, more detailed studies of Tiengwe et al. (2017). That said, the suggestion that GPI2 was the ancestral state makes good evolutionary sense, and begs the question of why trypanosomes prefer GPI1 TfR in 14 of 15 ESs (i.e., what is the selection pressure?).

Strengths and weaknesses:

(1) BES7 TfR subunit genes (BES7_Tb427v10): There are actually three (in order 5'-3'): E7gpi, E6.1 and E6.2. E6.1 and E6.2 have a single nucleotide difference. This raises the issue of coordinate expression. If overall levels of E6 (2 genes) are not down-regulated to match E7 (1 gene), this will result in a 2x excess of E6 subunits. The most likely fate of these is the formation of non-functional GPI2 homodimers on the cell surface, as shown in Tiengwe et al. (2017), which will contribute to the elevated TfR expression seen in BES7.

(2) Surface binding studies: This is the most puzzling aspect of the entire manuscript. That surface GPI2 TfR should be functional for Tf binding and uptake is not surprising, as this has already been shown by Tiengwe et al. (2017), but the methodology for this assay raises important questions. First, labeled Tf is added at 500 nM to live cells in complete media containing 2.5 uM unlabeled Tf - a 5x excess. It is difficult to see how significant binding of labeled TfR could occur in as little as 15 seconds under these conditions. Second, Tiengwe et al. (2017) found that trypanosomes taken directly from culture could not bind labeled Tf in direct surface labeling experiments. To achieve binding, it was necessary to first culture cells in serum-free media for a sufficient time to allow new unligated TfR to be synthesized and transported to the surface. This result suggests that essentially all surface TfR is normally ligated and unavailable to the added probe. Third, the authors have themselves argued previously, based on binding affinities, that all surface-exposed TfR is likely ligated in a natural setting (DOI: 10.1002/bies.202400053). Could the observed binding actually be non-specific due to the high levels of fixative used?

(3) Variable TfR expression in different BESs: It appears that native TfR is expressed at higher levels from BES7 compared to BES1, and even more so when compared to BES3. This raises the possibility that the anti-TfR used in these experiments has differential reactivity with the three sets of TfRs. The authors discount this possibility due to the overall high sequence similarities of E6s and E7s from the various ESs. However, their own analyses show that the BES1, BES3, and BES7 TfRs are relatively distal to each other in the phylogenetic trees, and this Reviewer strongly suspects that the apparent difference in expression is due to differential reactivity with the anti-TfR used in this work. In the grand scheme, this is a minor issue that does not impact the other major conclusions concerning TfR localization and function, nor the behavior of HpHbR and FHR. However, the authors make very strong conclusions about the role of BESs in TfR expression levels, even claiming that it is the 'dominant determinant' (line 189).

(4) Surface immuno-localization of receptors: These experiments are compelling and useful to the field. To explain the difference with essentially all prior studies, the authors suggest that typical fixation procedures allow for clearance of receptor:ligand complexes by hydrodynamic flow due to extended manipulation prior to fixation (washing steps). Despite the fact that these protocols typically involve ice-cold physiological buffers that minimize membrane mobility, this is a reasonable possibility. Have the authors challenged their hypothesis by testing more typical protocols themselves? Other contributing factors that could play a role are the use of deconvolution, which tends to minimize weak signals, and also the fact that investigators tend to discount weak surface signals as background relative to stronger internal signals.

(5) Shedding: A central aspect of the GPI valence model (Schwartz et al., 2005, Tiengwe et al., 2017) is that GPI1 reporters that reach the cell body surface are shed into the media because a single dimyristoylglycerol-containing GPI anchor does not stably associate with biological membranes. As the authors point out, this is a major factor contributing to higher steady-state levels of cell-associated GPI2 TfR relative to GPI1 TfR. Those studies also found that the size/complexity of the attached protein correlated inversely with shedding, suggesting exit from the flagellar pocket as a restricting factor in cell body surface localization. The amount of newly synthesized TfR shed into the media was ~5%, indicating that very little actually exits the FP to the outer surface. In this regard, is it possible to know the overall ratio of cell surface:FP:endosomal localized receptors? Could these data not be 'harvested' from the 3D structural illumination imaging?

Reviewer #1 (Public review):

Summary:

In the manuscript by Winke et al, the authors present evidence that fear-induced analgesia is mediated by somatostatin projection cells from the vlPAG to the RVM. This study uses a mouse model of fear-induced analgesia, and incorporates optogenetic circuit manipulation with behaviour and electrophysiology to gain a meaningful insight into a novel circuit involved in fear-induced analgesia.

Strengths:

(1) This is a well-constructed study with appropriate controls and analyses.

(2) Alternative interpretations of the data are systematically considered and eliminated via rational experiments. The authors are commended for a nice piece of experimental work.

(3) The vlPAG is a known region of pain modulation, and this study adds valuable insight to the circuit involved in fear-associated analgesia.

Weaknesses:

(1) Only male mice are included in this study.

(2) Animals are excluded from analyses based on clearly defined criteria, but it is not clear how many mice were excluded from each group.

(3) The authors implement a pain sensitivity assay that involves a hot plate with progressively increasing temperature. The time to nociceptive responses is reported. Without reporting the actual temperature at which the mice respond, it makes it difficult to compare nociceptive responses to previously published work (which typically use a defined and static hotplate temperature).

(4) The authors present evidence that inhibition of SST vlPAG cells enhances spinal nociceptive electrophysiological responses, but the corresponding pain sensitivity is not altered (Figure 2, CS- condition). The reason for the discrepancy between electrophysiological and behavioural responses is not clear.

Reviewer #3 (Public review):

Summary:

Conditioned analgesia refers to the ability of a learned fear cue to suppress pain-related behavior and neural activity. Understudied, the authors developed a novel conditioned analgesia procedure in which a cue that had been paired or unpaired with shock was played while a hot plate increased temperature. Compared to several control conditions, the authors found increased latency to a nociceptive response (paw licking). The authors identified somatostatin neurons in the periaqueductal gray as a likely mediator of the behavior. They then showed that: (1) stimulating vlPAG-SST neurons blocked nociceptive response latency increases to the CS+, (2) stimulating vlPAG-SST neurons suppressed fear retrieval freezing, (3) stimulating vs. inhibiting vlPAG-SST neurons drove opposing modulation of c-fibers and Aδ-fibers, (4) direct-projecting vlPAG SST neurons modulate freezing while RVM-projecting vlPAG SST neurons modulate conditioned analgesia.

Strengths:

These experiments have many strengths. The behavioral assay is chief among them. The assay is robust and controls for confounding factors to reveal a repeatable effect of a shock-paired cue to delay nociceptive responding. The optogenetic experiments provide the correct level of temporal precision, given the authors' time-specific interest in cued responding. Combining neuronal manipulations with spinal recordings is particularly innovative, especially in the context of more behavioral neuroscience-based assays. All-in-all, I found this to be an exceptionally strong set of experiments.

Weaknesses:

No obvious weaknesses were identified by this Reviewer.

Document de Synthèse : "Genre et torts épistémiques" - Camille Zimmerman Introduction : Présentation de Camille Zimmerman et de ses recherches

Camille Zimmerman est une doctorante dont les recherches se situent à l'intersection de la philosophie et des arts de la scène, co-dirigée par Amandine Catala (Université du Québec à Montréal - UQAM, Chaire de recherche du Canada sur l'injustice et l'agentivité épistémique) et Grégory Schiller (UGA, Laboratoire Litt&Arts, co-directeur du Performance Lab).

Son travail vise à développer une "pédagogie des corps" pour mieux comprendre l'aspect incarné de l'agentivité épistémique, un concept initialement élaboré par Miranda Fricker en 2007 dans son ouvrage Epistemic Injustice: Power and the Ethics of Knowing.

La question centrale qui l'anime est de savoir "en quoi et comment notre capacité à utiliser, générer et communiquer du savoir sollicite-t-elle notre corps, nos sensibilités, nos gestes, nos actions, et pas seulement, comme le défend la tradition dominante en philosophie, notre raison ?"

Plus encore, elle s'interroge sur ce que cette incarnation de l'agentivité épistémique nous apprend sur "les mécanismes tacites de l'oppression, notamment de genre, c'est-à-dire sur leur fonctionnement comme sur les stratégies que nous pouvons développer pour résister à ces mécanismes."

Son objectif est de donner aux corps un rôle émancipateur dans l'échange des savoirs, malgré ou grâce à leur vulnérabilité.

Ses travaux s'inscrivent dans l'épistémologie sociale, reconnaissant que la connaissance n'est pas produite de manière isolée mais à travers des relations sociales où des "torts" peuvent se manifester.

1. Biais Sexistes, Stéréotypes et Menaces du Stéréotype dans le Partage du Savoir

Camille Zimmerman commence par clarifier les distinctions entre stéréotypes, préjugés et biais, soulignant leur impact sur la perception de soi et des autres dans le partage du savoir.

Stéréotypes, Préjugés et Biais :

• Un stéréotype est un raccourci cognitif qui simplifie le traitement de l'information quotidienne. Il peut être utile mais devient problématique lorsqu'il est "trop réducteur" et "négatif", menant à des préjugés.

• Les préjugés sont des jugements ou des croyances négatives envers des groupes sociaux.

• Les biais sont des associations néfastes qui accentuent les préjugés et affectent la perception des personnes.

• Menace du Stéréotype : Introduit par Steele et Aronson (1995) pour comprendre l'échec scolaire des minorités ethniques, ce concept décrit comment, en tant que membre d'un groupe marginalisé, la conscience des stéréotypes négatifs à son égard génère un climat de stress ou d'anxiété.

Cette anxiété peut paradoxalement "rejouer" et confirmer le stéréotype (ex: "les femmes sont trop émotionnelles", "pas bonnes en maths").

La menace du stéréotype "impacte vraiment la manière dont on va nous-même agir dans la situation et rejouer et confirmer en fait les stéréotypes négatifs à notre égard."

Application au Genre et à la Philosophie :

• Une sous-représentation des femmes en philosophie (peu de philosophes femmes citées dans le canon, peu d'enseignantes) renforce la menace du stéréotype. Les étudiantes manquent de modèles et peuvent internaliser l'idée qu'elles "ne sont pas faites pour ça", ce qui accroît la peur de mal performer.

• Les biais sexistes se manifestent par des réflexions associant les femmes à l'émotion (les rendant "pas crédibles"), par une attention portée à leur apparence plutôt qu'à leur propos, ou par une réduction de leur espace de parole, exacerbant le stress et la pression lors de l'expression.

• Stratégies de Déconstruction : Pour contrer ces biais, il est proposé d'augmenter la représentation des femmes en philosophie (inviter des conférencières, afficher des portraits), et de diversifier le canon philosophique.

L'intervenante cite des initiatives comme le groupe "Philo Situé" (anciennement "Philo-Sophia") et le Symposium de philosophie féministe au Québec, qui visent à créer des espaces de reconnaissance professionnelle pour les femmes en philosophie.

2. Les Injustices Épistémiques : Cadre Théorique et Manifestations

Les recherches de Camille Zimmerman s'appuient sur le concept d'injustice épistémique, issu de l'épistémologie sociale, qui étudie les "torts" commis dans les relations de partage du savoir.

• Définition et Origines : L'injustice épistémique concerne les torts éthiques commis à l'égard de notre "agentivité épistémique", c'est-à-dire notre droit à développer notre capacité à utiliser, communiquer et transmettre du savoir.

Ces torts minent la confiance en soi. Le concept a été formellement introduit par Miranda Fricker en 2007, mais ses racines se trouvent dans les théories du Black Feminism et du standpoint theory (années 60-70), soulignant d'emblée une dimension intersectionnelle des oppressions (genre, race, classe sociale).

Les Deux Formes Majeures d'Injustice Épistémique (selon Fricker) :

• L'Injustice Testimoniale : Concerne la crédibilité du témoignage. Lorsqu'une personne appartenant à un groupe social non dominant (basé sur le genre, la race, la classe, les capacités, l'orientation sexuelle, etc.) partage du savoir, on va "arbitrairement [lui] donner ou pas du crédit".

Ce n'est pas lié à la véracité du propos, mais à l'identité de la personne.

C'est une situation "systématique" et non accidentelle, qui "mine [la] confiance" de la personne.

L'accent est mis sur le "contexte social" plutôt que sur un coupable individuel.

• L'Injustice Herméneutique : Caractérisée par un "déficit d'intelligibilité" ou un "manque d'intelligibilité".

Elle se produit lorsque les termes ou les concepts utilisés par une personne appartenant à un groupe non dominant ne sont pas compris ou reconnus par le groupe dominant, qui impose ses propres "ressources herméneutiques" (langage, concepts).

Cela conduit à une "marginalisation" et une "exclusion" du partage du savoir.

L'exemple emblématique est celui du harcèlement sexuel avant que le terme n'existe, où l'absence de mot pour nommer une expérience vécue empêche sa reconnaissance juridique et sociale (exemple de Suzanne Brer).

L'exploitation herméneutique est "un vrai enjeu politique" car elle maintient les groupes marginalisés "en marge".

3. La Conception Pluraliste de l'Agentivité Épistémique et la Place de l'Affect

La critique de la définition traditionnelle (logocentrique et rationnelle) de l'agentivité épistémique est au cœur des recherches de Camille Zimmerman et de sa directrice, Amandine Catala.

• Critique du Logocentrisme : La définition de Fricker, basée sur la rationalité, réduit l'intelligence et exclut ceux qui ne s'expriment pas verbalement ou rationnellement (ex: personnes en situation de handicap, neurodiverses).

Cela pose problème car si une personne n'est pas considérée comme un agent épistémique, elle ne peut pas défendre le fait qu'elle subit des injustices épistémiques. * Les Cinq Caractéristiques de l'Agentivité Épistémique Pluraliste (selon Alexis Shotwell) : Cette nouvelle approche élargit la définition de la connaissance au-delà du "savoir propositionnel" (basé sur le langage et la vérifiabilité logique) pour inclure d'autres formes de savoir cruciales pour comprendre l'expérience humaine, notamment les expériences d'oppression.

• Savoir Propositionnel : Formulation verbale, vérifiable (le "know that" en anglais).
• Savoir-faire : Connaissance pratique acquise par l'expérience corporelle (ex: faire du vélo), irréductible à un manuel.
• Savoir Incarné : Intégration des codes sociaux et adaptation corporelle aux normes (ex: posture, ton de voix, performance du genre). Non verbal, implicite, et pourtant crucial pour la crédibilité sociale.
• Savoir Tacite : Ce qui n'a pas besoin d'être explicité par le langage (sens commun, stéréotypes), permettant les raccourcis dans l'interaction.
• Savoir Affectif : La manière dont on vit, éprouve, ressent les choses. "Disent aussi quelque chose par rapport à mon identité." C'est une "forme de compréhension des choses, des sens."
• L'Affect comme Ressource Épistémique : La conception pluraliste réhabilite l'affect, souvent dévalorisé et associé aux femmes, comme une source de connaissance.
• Dans la Marginalisation Herméneutique : Le vécu corporel, émotionnel et sensible, même sans mots, "nous révèle une importance par rapport à ce qui se passe." Il peut catalyser la recherche de termes ou de reconnaissance (ex: Saraha Ahmed et la violence subie à vélo, dont le corps garde la mémoire).
• Dans la Menace du Stéréotype : Le "climat de stress", l'anxiété et la fatigue ne sont pas neutres et affectent la performance épistémique. Reconnaître l'impact de l'environnement (ex: salles de conférence avec portraits d'hommes blancs) permet de comprendre comment l'affect mine l'agentivité.
• L'Exploitation Épistémique (Nora Berenstein) : Ce concept, mis en lumière par des chercheur·es racisé·es, montre comment les groupes privilégiés, face aux explications des marginalisé·es sur leur oppression, "discréditent les ressources épistémiques qu'ils apportent et les force[nt] à produire un travail cognitif et émotionnel supplémentaire." La personne opprimée est contrainte de s'expliquer, malgré l'effort physique et émotionnel que cela représente, car son expérience n'est pas reconnue si elle n'est pas "adéquatement expliquée".

L'ignorance active du privilégié est ainsi reproduite.

Ce phénomène met clairement en évidence l'importance du corps et de l'affect dans les injustices épistémiques.

4. Pédagogies de l'Inconfort et Vulnérabilité Épistémique

Pour faire face à ces injustices et valoriser l'affect, Camille Zimmerman propose d'explorer les pédagogies de l'inconfort.

• Bell Hooks et l'Intégrité en Éducation : Inspirée par Paulo Freire, Bell Hooks soutient que la salle de classe n'est pas un espace neutre politiquement.

Les rapports de pouvoir et les identités sociales influencent l'expérience de l'enseignement et de l'apprentissage.

Elle promeut l'enseignement avec "intégrité", en intégrant le corps et l'esprit, cherchant le "désir d'apprendre" des étudiant·es et la connexion avec leurs vécus.

L'enseignant·e doit également considérer son "état émotionnel" et la place de l'affect dans son propre enseignement.

• Megan Boler et le Sentiment d'Appartenance : Boler, spécialiste des pédagogies féministes, insiste sur l'importance de créer un "sentiment d'appartenance" en classe.

Elle reconnaît que l'espace n'est pas intrinsèquement sécuritaire pour tou·tes et qu'il peut devenir insécuritaire. Il s'agit de travailler à partir du ressenti des étudiant·es pour améliorer l'espace et discuter ouvertement des rapports de pouvoir.

Boler explore également comment les normes sociales (liées au genre, par exemple) influencent l'expression des émotions et peuvent desservir les individus dans certains contextes (ex: l'injonction à "combattre son idée" en philosophie).

• La Vulnérabilité Épistémique (Erin Gilson) : Ce concept est proposé comme une "vertu" en opposition à l'ignorance active.

Il s'agit d'accepter d'être vulnérable, de reconnaître que nos propres croyances ne sont pas les seules valides, et que l'écoute des croyances d'une personne issue d'une identité sociale différente peut "déconstruire ma propre croyance."

Cette démarche, bien que "déstabilisante", est essentielle pour une "ouverture des esprits" et pour ne pas "résister à ne pas comprendre".

Elle permet de reconnaître l'importance des affects dans la capacité à utiliser, partager et générer du savoir.

Conclusion Générale

La présentation de Camille Zimmerman propose une exploration approfondie des injustices épistémiques, en particulier celles liées au genre, en insistant sur le rôle central de l'incarnation et des affects.

Le cheminement s'articule comme suit :

• Identification des Problèmes : Les biais sexistes et la menace du stéréotype influencent négativement le partage du savoir.
• Cadre Théorique : Les injustices épistémiques (testimoniale et herméneutique) décrivent les torts subis, souvent invisibles et liés à l'identité sociale.
• Refonte Conceptuelle : La conception pluraliste de l'agentivité épistémique, intégrant le savoir incarné, tacite et affectif, déconstruit les limites du logocentrisme et valorise les affects comme sources de connaissance.
• Stratégies Pédagogiques : Les pédagogies de l'inconfort (Bell Hooks, Megan Boler) et le développement de la vulnérabilité épistémique (Erin Gilson) sont des approches concrètes pour transformer les espaces d'apprentissage et de partage du savoir, en reconnaissant et en travaillant avec le vécu émotionnel et corporel.
• L'objectif final est de créer des environnements où l'inconfort (lorsqu'il est géré constructivement) peut devenir "un indicateur dans ma propre éducation à accepter la croyance de l'autre ou à rentrer dans une collaboration plutôt que de maintenir ma propre croyance", ouvrant ainsi la voie à une compréhension plus riche et équitable du savoir.

Briefing sur la Cohérence Cardiaque : Gestion du Stress et Au-delà

Ce document de briefing vise à synthétiser les concepts clés, les mécanismes physiologiques, les bénéfices et les applications pratiques de la cohérence cardiaque, tels que présentés par Caroline Sévoz-Couche, neuropharmacologue et chercheuse à l'Inserm, spécialiste du nerf vague.

1. Qu'est-ce que la Cohérence Cardiaque ?

La cohérence cardiaque est une technique de respiration contrôlée qui vise à harmoniser les variations de la fréquence cardiaque avec la fréquence respiratoire.

Bien que popularisée en Occident plus récemment, ses principes sont connus et pratiqués depuis des millénaires dans les civilisations orientales, notamment à travers le Pranayama du yoga.

• Définition et Historique : C'est une technique simple et rapide, souvent associée à une origine américaine récente. Cependant, la régulation de la fréquence respiratoire est connue depuis des milliers d'années dans les civilisations orientales.

"L'expire produit la stabilité du mental", une phrase écrite il y a près de 2000 ans, souligne l'importance de l'expiration contrôlée.

En Occident, elle est apparue au 18ème siècle et a été popularisée en France par le Dr. David Servan-Schreiber.

• Non une Simple Relaxation : Il est crucial de comprendre que la cohérence cardiaque n'est pas une simple relaxation menant à l'endormissement. Au contraire, elle prépare l'esprit aux actes d'attention, augmentant la concentration et la rapidité de prise de décision.

"Ce n'est pas une relaxation où on s'endort... c'est-à-dire qu'en fait quand on va faire la cohérence cardiaque on va être beaucoup plus sensible à tout ce qui va se passer autour à l'environnement on va être beaucoup plus concentré on va avoir des prises de décisions beaucoup plus rapide".

Elle peut être pratiquée avant une compétition sportive ou une tâche nécessitant une grande vigilance.

2. Le Mécanisme Physiologique Clé : La Variabilité Cardiaque et la Fréquence de Résonance

Le cœur ne bat pas à une fréquence parfaitement régulière ; il présente une variabilité constante, essentielle pour s'adapter aux stimuli internes et externes.

La cohérence cardiaque vise à optimiser cette variabilité.

• Variabilité Cardiaque (VFC) : Un cœur en bonne santé présente des oscillations rapides de sa fréquence. Une faible variabilité est souvent associée à une pathologie. Lors d'une respiration spontanée (environ 15 cycles/minute), ces oscillations sont de faible amplitude.

• Optimisation à 6 cycles/minute : La pratique de la cohérence cardiaque à 6 respirations par minute (soit un cycle respiratoire de 10 secondes) maximise l'amplitude des oscillations cardiaques.

Cette fréquence est la plus efficace car elle entre en résonance avec les oscillations automatiques naturelles du corps, qui sont également de 6 par minute.

"Ces amplitudes sont maximales avec une fréquence respiratoire spécifique de 6 par minute".

• Analogie de la Balançoire : Le principe est similaire à celui d'une balançoire : si l'on pousse la balançoire à sa fréquence naturelle, l'amplitude de son mouvement augmente considérablement. De même, en synchronisant la respiration avec les fréquences oscillatoires naturelles du corps, on amplifie la variabilité cardiaque.

• Cohérence des Oscillations, pas des Fréquences : Il est important de noter que ce ne sont pas les fréquences cardiaques (ex: 60-80 battements/minute) et respiratoires (6 cycles/minute) qui sont mises en cohérence, mais le nombre d'oscillations cardiaques par minute induites par la respiration et celles produites automatiquement par le corps, qui se synchronisent à 6 par minute.

3. Aspects Techniques de la Respiration en Cohérence Cardiaque

Pour maximiser les bénéfices, certaines pratiques respiratoires sont recommandées :

• Ratio Inspiration/Expiration : Le ratio idéal est de 5 secondes d'inspiration et 5 secondes d'expiration pour un cycle de 10 secondes (6 cycles/minute).
• Un léger allongement de l'expiration (ex: 4 sec inspiration / 6 sec expiration) est acceptable et conserve une bonne amplitude.
• Un allongement excessif de l'expiration (ex: 3 sec inspiration / 7 sec expiration) ou une inspiration plus longue que l'expiration réduit significativement l'amplitude des oscillations et peut être contre-productif ("on perd la cohérence").
• Volume Thoracique : Il est crucial d'augmenter le volume thoracique pour maintenir une bonne ventilation. "Il faut penser à avoir une grande amplitude thoracique quand on fait la cohérence cardiaque".
• Respiration Nasale : L'inspiration par le nez est fortement recommandée car elle active davantage les ondes cérébrales bénéfiques (ondes thêta) et apporte d'autres avantages physiologiques (filtration de l'air, humidification, meilleure oxygénation, réduction des ronflements).

"Si vous voulez avoir des effets au niveau central il faut penser à l'inspiration il faut respirer par le nez". L'expiration peut se faire par la bouche ou le nez.

4. Le Rôle Central du Nerf Vague

La cohérence cardiaque stimule de manière maximale le nerf vague, un nerf cranien crucial pour le système nerveux parasympathique.

• Le Nerf Vague : Un "Vagabond" : Nommé d'après le latin "vagare" (errer, vagabonder), le nerf vague innerve de nombreux organes depuis le cerveau (cœur, poumons, tube digestif, etc.).
• Effets sur les Organes Périphériques : Sa stimulation a des effets positifs variés :
• Cardiaques : Amélioration de la variabilité cardiaque.
• Digestifs : Amélioration des sensations de goût, augmentation de la sécrétion des sucs gastriques, amélioration de la motilité gastro-intestinale.
• Anti-inflammatoires : Libération de neurotransmetteurs qui réduisent l'inflammation systémique et organique (ex: poumons chez les asthmatiques).
• Boucle Vago-vagale et Effets Cérébraux : Seulement 20% des fibres du nerf vague descendent du cerveau vers les organes, tandis que 80% remontent. Cette boucle vagale permet une influence bidirectionnelle. La stimulation du nerf vague par la cohérence cardiaque active des structures cérébrales clés :
• Cortex insulaire et cingulaire : Impliqués dans l'intéroception (perception des signaux internes du corps) et la prise de décision.
• Amygdale : Régulation du stress.
• Hippocampe : Mémoire et cognition.
• Modulation du Système Limbique : Cette activation cérébrale conduit à une amélioration de :
• La concentration, la mémoire de travail et l'apprentissage.
• La prise de décision et le contrôle de l'impulsivité (réduction des comportements impulsifs liés à l'alimentation, l'alcool, la drogue, augmentant le temps d'abstinence).
• Le seuil de la douleur (augmentation).
• La gestion du stress et la diminution des symptômes anxieux et dépressifs.

5. Bénéfices et Applications Prouvées Scientifiquement

De nombreuses études scientifiques, incluant des groupes placebo, ont démontré l'efficacité de la cohérence cardiaque.

• Réduction Immédiate du Stress et de l'Anxiété : Une session de 5 minutes chez des volontaires sains (musiciens avant un concert, sportifs avant une compétition) réduit significativement l'anxiété et le niveau de stress.

Effets à Long Terme et Persistance :

• Basketballeurs : 20 minutes/jour pendant 10 jours ont montré une diminution de l'anxiété qui persiste pendant au moins un mois après l'arrêt de la pratique, contrairement aux groupes contrôle.
• Vétérans atteints de SSPT : 20 minutes/jour pendant 4 semaines ont significativement diminué la perception du stress, de la douleur et des émotions négatives.
• Patients déprimés résistants aux traitements : Sessions hebdomadaires de 20 minutes pendant 10 semaines, combinées à 20 minutes quotidiennes non supervisées, ont entraîné une diminution "très importante" de la dépression et de l'anxiété, visible dès 4 semaines.
• Pression Artérielle : Une étude a montré une diminution de la pression artérielle qui persistait 6 mois après l'arrêt de la pratique chez ceux qui l'avaient faite sur le long terme.
• Adaptation à l'Âge et aux Pathologies :
• Bien que l'amplitude des oscillations puisse être légèrement moindre après 40 ans ou chez des patients atteints de diabète, la cohérence cardiaque augmente toujours cette amplitude par rapport à la respiration spontanée, offrant des bénéfices à tous.

"Quel que soit l'âge quel que soit la condition il tout le monde peut faire la cohérence cardiaque et avoir un effet bénéfique".

• Gestion de la Douleur : Des sessions courtes (5 minutes) avant un événement douloureux (ex: prise de sang) peuvent réduire la sensation de douleur.
• Sommeil : Améliore le temps d'endormissement (latence d'endormissement). Il est plus efficace de la pratiquer juste avant de se coucher.

6. Protocoles de Pratique Recommandés

La durée et la fréquence de la pratique varient en fonction des objectifs :

• "Traitement Ponctuel" / Avant un Événement : 5 minutes (5 secondes inspiration, 5 secondes expiration, inspiration nasale).
• "Traitement de Fond" Léger (Concentration, Mémoire, Stress ponctuel) : 10 minutes par jour pendant au moins 2-3 semaines.
• "Traitement de Fond" Important (Anxiété, Dépression, Douleur Chronique) : 20 minutes par jour pendant 4 à 8 semaines et au-delà.
• Il est préférable de faire des sessions de 10 minutes plutôt que de multiples sessions de 5 minutes pour un effet plus rémanent.
• Les effets peuvent persister au moins un mois après l'arrêt pour une pratique prolongée.

7. Outils et Aides à la Pratique

De nombreuses ressources facilitent la pratique de la cohérence cardiaque :

• Vidéos et Applications :
• Sites web : vimeo.com, onedeepbreath.
• Chaînes YouTube avec animations visuelles (ex: celle de Caroline Sévoz-Couche avec nuages ou flèches).
• Applications mobiles (iOS/Android) : "Cardia", "Breathing App", "Breath+ (permet de visualiser la fréquence cardiaque en direct et la calque sur la respiration idéale)".
• Montres Connectées : Certaines montres proposent des fonctionnalités de cohérence cardiaque.
• Pratique sans Outil : Il est possible de la pratiquer n'importe où en comptant mentalement 5 secondes d'inspiration nasale et 5 secondes d'expiration.

8. Questions et Remarques Importantes

• Différence avec d'autres Techniques de Respiration : La respiration carrée (inspiration-pause-expiration-pause) peut être utilisée pour d'autres objectifs (relaxation), mais elle n'est pas optimale pour la cohérence cardiaque car les pauses interrompent la "cohérence" des oscillations et réduisent l'amplitude.

Pour la cohérence cardiaque, l'absence de pause ou de très courtes pauses (0,5 sec) est préférable. * Complémentarité avec d'autres Thérapies : La cohérence cardiaque est reconnue par certains professionnels de la santé, notamment en psychiatrie et psychologie positive, comme un outil efficace et complémentaire à d'autres techniques (hypnose clinique, EMDR), car elle active des structures cérébrales similaires. * Contre-indications ou Effets Indésirables : Très rares. La seule prudence concerne les personnes sujettes aux malaises vagaux très prononcés. Certains moments de vie (ex: 3ème trimestre de grossesse) peuvent rendre la pratique plus difficile en raison de contraintes physiques. * Cohérence Cardiaque et Effort Sportif : Non indiquée pendant un effort sportif intense, car celui-ci active le système nerveux sympathique, qui s'oppose à l'action du nerf vague (parasympathique). * L'objectif et la concentration : Il ne faut pas trop se concentrer sur un objectif pendant la pratique elle-même, car cela peut augmenter la partie sympathique et réduire les effets bénéfiques. L'important est de se concentrer sur la technique respiratoire. * Processus d'Habituation à l'Anxiété : Bien que ce ne soit pas directement un processus d'habituation, la stimulation vagale par la cohérence cardiaque peut aider le cerveau des patients déprimés (et potentiellement anxieux) à mieux accepter les nouvelles informations et à sortir des ruminations, en réactivant les systèmes inhibiteurs de l'impulsivité.

En conclusion, la cohérence cardiaque est une pratique basée sur des mécanismes physiologiques solides, facilement accessible et aux bénéfices avérés sur la gestion du stress, l'anxiété, la dépression, la douleur, la concentration, la mémoire et le sommeil, quel que soit l'âge ou la condition de santé.

Sa simplicité et son efficacité en font un outil précieux pour le bien-être général.

What really resonates with my students is the addition of the following to each goal: Goal 1: "I'm ok" Goal 2: "You're ok" Goal 3: "That's not ok" Goal 4: Let's do something about it"

Synthèse de la Conférence : "A la recherche du temps perdu, les enfants face aux écrans" - Grégoire Borst

Cette conférence de Grégoire Borst, professeur de psychologie du développement et chercheur en neurosciences cognitives, vise à démystifier et nuancer le débat public autour de l'impact des écrans sur les enfants et adolescents, en s'appuyant sur des données scientifiques.

Il met en lumière les idées fausses véhiculées et propose une approche plus complexe et contextuelle de la problématique, notamment à travers le prisme du rapport "À la recherche du temps perdu" auquel il a contribué.

1. Démystification des Idées Reçues sur les Écrans

Grégoire Borst commence par interroger l'audience sur des affirmations courantes concernant les écrans, pour ensuite les déconstruire systématiquement :

• Baisse de l'intelligence des nouvelles générations : "il y a aucune de ces informations qui n'est vraie qui est vraie en tout cas pas posé comme cela il y a nécessité d'avoir d'introduire un peu plus de complexité làdessus".
• Troubles neurodéveloppementaux (troubles des apprentissages, TSA, TDAH) : Les écrans ne peuvent pas en être la cause. "par définition les troubles du neurodéveloppement ne peuvent pas avoir comme cause l'exposition aux écrans". Il insiste sur la fausse information concernant "l'autisme virtuel", qui est une "fake news" nuisible, surtout dans un pays ayant déjà une prise en charge déficitaire des TSA.
• Symptômes dépressifs des adolescents : "il y a aucune donnée qui suggère ça la dépression c'est multifactoriel et les les réseaux sociaux en tant que tel ça peut pas être la cause de la dépression".
• Dépendance/Addiction : La communauté médicale n'a pas reconnu d'addiction aux jeux vidéo, aux réseaux sociaux ou à l'usage des écrans en général, "pour l'instant en tout cas en l'état actuel de nos connaissances la communauté médicale a décidé que il n'y avait pas d'addiction ni au jeux vidéos ni aux réseaux sociaux ni à un ensemble d'usages qui peut être fait des des écrans".

2. Statistiques Clés sur l'Équipement et le Temps d'Écran

Borst souligne l'importance de s'appuyer sur des données fiables et met en garde contre les sondages peu rigoureux.

• Difficulté d'évaluation : Il est intrinsèquement difficile d'évaluer le temps d'écran réel des enfants et adolescents.
• Taux d'exposition : Augmente avec l'âge. La dernière cohorte consolidée pour l'ensemble des enfants et adolescents date de 2017, donc les chiffres actuels sont probablement plus élevés.
• Équipement personnel (hors smartphone) :7-12 ans : environ 1,6 écran personnel (console, etc.).
• 13-19 ans : 2,9 écrans personnels.
• Smartphones :Seulement 35% des 7-12 ans ont un smartphone (contre une idée répandue de 90% ou 100% au collège).
• L'âge moyen d'acquisition pour ceux qui en possèdent un est de 9 ans et 8 mois.
• Il est crucial de comprendre que ce chiffre s'applique à une sous-population spécifique, pas à la moyenne générale.
• Ordinateurs personnels :19% des 7-12 ans.
• 60% des 13-19 ans.
• Consoles de jeu :58% des 7-12 ans.
• 63% des 13-19 ans.
• Utilisation des smartphones (15-17 ans) : Sur 4h43 d'utilisation déclarée par jour, 2h43 sont consacrées à des activités liées à l'école. Cela "permet de relativiser considérablement je dirais la variable temps d'écran c'est évidemment ça dépend de ce que vous y faites".
• Accès aux réseaux sociaux :63% des 7-10 ans déclarent être inscrits sur les réseaux sociaux (contre 49% déclaré par les parents), alors que l'âge légal est 13 ans.
• 91% des 11-14 ans ont accès aux réseaux sociaux.

3. La Complexité des Effets des Écrans : Corrélation vs. Causalité

Grégoire Borst insiste sur la distinction fondamentale entre corrélation et causalité, illustrant son propos avec l'exemple humoristique de la consommation de margarine et des taux de divorce.

• Piège de l'association : De nombreuses études montrent des associations entre le temps d'écran et des effets sur le développement, mais cela ne prouve pas un lien de causalité direct. "on a beaucoup plus de mal à mettre en évidence des liens de causalité".
• Directionnalité : Une association peut signifier que le temps d'écran affecte le développement, ou que des vulnérabilités préexistantes (liées au développement cognitif et socio-émotionnel) conduisent à une plus grande consommation d'écrans. Seules les études longitudinales peuvent commencer à établir une directionnalité.
• Variables tierces : L'importance cruciale de contrôler les variables comme le "milieu social d'origine des familles". "Toute étude pour faire simple prochaine étude que vous regardez sur cette questionl si dans le modèle statistique on a va pas contrôler pour le milieu social d'origine vous prenez l'étude vous l'achetez à la poubelle elle veut rien dire en soi".

4. Effets Spécifiques Documentés et Nuances

• Développement langagier :
• Une étude française (coHorte Eden) montre qu'il n'y a pas d'effet du temps global d'exposition à 2 ans sur les compétences langagières à 5-6 ans.
• Cependant, "quand on a la télé allumée pendant les repas on a des effets négatifs sur le développement langengagé de l'enfant à 5 ou 6 ans".
• Méta-analyses : Confirment un effet négatif du temps d'écran sur le développement langagier (taille d'effet faible, 16% d'écart-type). Le "bruit de fond" de la télévision a un effet négatif similaire.
• Co-visionnage et programmes ludo-éducatifs : Des effets positifs sont observés. "le simple covisionnage avec un enfant d'un contenu audiovisuel a un effet positif sur le développement Lang langagé de l'enfant qui est de la même taille que les effets négatifs observés pour le temps d'exposition global".
• Qualité du contenu : L'enjeu est de sortir de la "question du temps d'écran et qu'on rentre dans quelque chose de beaucoup plus complexe qui est la question de la qualité du contenu et des interactions qui peuvent exister des interactions sociales autour du contenu audiovisuel". Éviter les contenus pauvres en langage oral (ex: Télétubbies).
• Sommeil : C'est un domaine où "toutes les études sont concordantes globalement" sur l'impact négatif des écrans.
• Adolescents : impact négatif si utilisé dans l'heure précédant le coucher.
• Jeunes enfants : impact à tout moment de la journée.
• Impact en cascade : Le sommeil étant crucial pour le développement cérébral et l'apprentissage, des perturbations peuvent affecter d'autres fonctions cognitives comme le langage.
• Sensibilisation parentale : 49% des parents d'enfants de moins de 11 ans ignorent l'impact des écrans sur le sommeil de leurs enfants.
• Facteurs multiples : Le déficit de sommeil des adolescents n'est pas uniquement dû aux écrans ; la structure du système éducatif (heures de cours tôt le matin) y contribue également.
• Sédentarité : 33% des enfants de moins de 3 ans ne pratiquent aucune activité physique. La sédentarité est liée à l'obésité et aux risques de maladies cardiovasculaires. "C'est un vrai enjeu de santé publique".
• Réseaux Sociaux et Bien-être Adolescent :
• Il existe un lien statistique entre le temps passé sur les réseaux sociaux et le bien-être, mais la taille de l'effet est très faible (0,4% du bien-être).
• Il existe des facteurs de vulnérabilité individuels où l'usage peut avoir des effets négatifs.
• Effets positifs : Une étude (2016) suggère que le temps passé sur les réseaux sociaux à 10 ans peut avoir un effet positif sur le développement de l'empathie affective et cognitive.

"les réseaux sociaux ça rend pas les adolescents moins empathiqu de façon générale ça peut même avoir des effets positifs y compris sur des domaines qui relèvent des compétences psychosociales ou socioémotionnelles".

5. La Place du Numérique à l'École

Faible équipement en France : Le taux d'équipement numérique des élèves français est "très très faible" comparé à d'autres pays comme la Suède. "on est très en retard sur le numérique éducatif".

Effets positifs : La mise à disposition d'équipements mobiles individuels (tablettes) à l'école peut avoir "des effets positifs y compris sur les apprentissages scolaires fondamentaux que ce soit en 5e ouou en 4e on a des effets positifs sur les compétences mathématiques sur la compréhension de l'oral et sur la compréhension de l'écrit".

6. Recommandations et Conclusion

Le rapport de la commission sur les écrans (disponible sur le site de l'Élysée) propose un ensemble de recommandations, mettant l'accent sur la complexité des enjeux et la nécessité d'une action publique nuancée et informée par la science.

La conférence souligne l'importance d'un discours mesuré, basé sur des preuves solides, pour éviter les alarmismes infondés et orienter efficacement les politiques publiques.

L'accent doit être mis sur la qualité des interactions et des contenus, ainsi que sur l'environnement global de développement de l'enfant (sommeil, activité physique, milieu social), plutôt que sur une simple restriction du temps d'écran.

Note de synthèse : Comprendre l'Adolescence à travers le Prisme des Neurosciences et de la Psychologie du Développement (Mathieu Cassotti)

Cette synthèse est basée sur les extraits de la conférence "C'est pas moi, c'est mon cerveau" de Mathieu Cassotti, Professeur en psychologie du développement à l'Université Paris Cité.

L'objectif principal de cette intervention est de déconstruire les perceptions souvent négatives et biaisées de l'adolescence pour proposer une compréhension plus nuancée, fondée sur les spécificités du développement cérébral et les interactions avec l'environnement social.

1. La Perception Biaisée de l'Adolescence

Traditionnellement, l'adolescence est perçue sous l'angle de la prise de risque, des difficultés et de la vulnérabilité. Les adultes ont du mal à comprendre le fonctionnement des adolescents, et cette incompréhension est réciproque.

Les adolescents, malgré les cours de biologie, ont peu d'informations sur leur propre cerveau et ses spécificités. La psychologie a souvent abordé l'adolescence sous l'angle des psychopathologies.

Cassotti et son collègue Grégoire Bord ont cherché à changer cette perspective, arguant que notre perception est souvent biaisée par un "biais de génération" : "on a tendance à considérer que les générations d'après les nôtres sont d'après la nôtre est toujours moins bien que la nôtre les générations d'après sont toujours moins bien ah bah de mon temps on était meilleur".

Ce biais nous pousse à ne pas suffisamment explorer les aspects positifs de cette période. L'engagement des adolescents pour des causes comme la justice sociale ou le climat est souvent minimisé ou dévalorisé par les adultes.

Le livre "C'est pas moi, c'est mon cerveau" vise à vulgariser le fonctionnement du cerveau adolescent pour les adolescents eux-mêmes, afin qu'ils "comprennent un petit peu mieux leur fonctionnement pour qu'ils puissent à partir de cette compréhension euh soit euh faire en sorte de changer un certain nombre de comportements soit au contraire de se sentir légitime de pouvoir essayer de de faire quelque chose et d'essayer de le changer".

2. Le Cerveau Adolescent : Une Période de Développement et de Plasticité

Le cerveau adolescent, bien que similaire en forme à celui de l'adulte, est encore en plein développement, une période qui peut s'étendre "jusqu'à 25 ans".

Cette immaturité n'est pas uniquement biologique mais est fortement influencée par l'environnement.

L'adolescence est une "fenêtre de plasticité particulière" avec des changements tardifs et d'importantes différences interindividuelles (par exemple, le début de la puberté).

Une découverte clé des 20 dernières années en neurosciences, notamment grâce aux travaux de Bétio Ky, est la maturation progressive et hétérogène du cerveau :

Les régions impliquées dans la réactivité émotionnelle (système limbique) maturent beaucoup plus rapidement. Les régions impliquées dans la régulation et le contrôle (cortex préfrontal) maturent plus tardivement.

Ce décalage explique en partie la spécificité de l'adolescence : une "maturation fonctionnelle des systèmes de la réactivité émotionnelle mais pas encore c'est du contrôle".

Cela conduit à une "hypersensibilité émotionnelle" observée dans de nombreuses recherches, notamment face aux stimuli positifs comme les visages exprimant la joie, qui activent fortement le réseau de la récompense.

Cependant, cette immaturité n'est pas une "incapacité" mais plutôt une période "en cours d'apprentissage". L'environnement joue un rôle crucial dans le développement de l'autorégulation émotionnelle.

L'apprentissage de la verbalisation, de l'identification et de la gestion des émotions est essentiel et "très peu enseigné de façon explicite aux adolescents".

3. Émotions Complexes et Prise de Décision

L'hypersensibilité émotionnelle des adolescents n'est pas uniforme pour toutes les émotions.

Si elle est bien établie pour la joie et la peur, les émotions sociales comme la honte, la culpabilité ou la jalousie, ainsi que le regret et le soulagement, sont plus complexes.

Le Regret : Les adolescents ressentent moins de regret que les adultes et l'anticipent moins.

Le regret, défini comme la différence entre le résultat obtenu et ce qui aurait pu être obtenu avec un autre choix, est soutenu par le cortex préfrontal, qui est encore immature. Cela peut expliquer une moindre capacité à apprendre de leurs erreurs dans des situations où le regret est un signal d'apprentissage clé.

Prise de Risque : Concernant la prise de décision et de risque, les adolescents sont "aussi bons que les adultes" à partir de 15 ans lorsqu'ils disposent de toutes les informations nécessaires.

Cependant, leur capacité à apprendre des feedbacks de l'environnement (positifs ou négatifs) est plus difficile que chez les adultes, en particulier lorsque l'information sur le risque n'est pas explicite et doit être extraite de l'expérience.

"ce n'est pas uniquement une question de contrôle c'est aussi parce que ils sont pas des preneurs de risque complètement fous c'est c'est aussi parfois une capacité à apprendre de des feedback qu'il vont avoir de leur environnement positif et négatif et donc là c'est plus difficile que chez les adultes".

4. Influence Sociale et Conformisme

Contrairement à l'idée répandue, les adolescents ne sont pas "davantage des moutons que nous le sommes nous en tant qu'adultes".

Les adultes sont également très sensibles au conformisme social, même dans des situations où la bonne réponse est évidente.

Les études montrent que le conformisme social est maximal chez les enfants et "minimal à l'adolescence à l'âge de 17 ans".

Cependant, cette tendance s'inverse dans les situations d'incertitude et surtout en contexte social, où les adolescents peuvent montrer une "hypersensibilité au contexte social", particulièrement dans les situations de prise de risque.

En présence de leurs pairs, ils sont enclins à prendre plus de risques.

L'imagerie cérébrale révèle que cette influence sociale n'affecte pas le contrôle mais stimule le réseau de la récompense. Deux interprétations sont proposées :

• Le contexte social stimule la sensibilité aux récompenses immédiates.

• La "représentation qu'ils ont de la norme sociale de ce qui est valorisé par leur père" inclut la prise de risque, menant à une récompense sociale. Cette seconde interprétation est privilégiée par Cassotti, d'autant plus que l'effet est plus prononcé chez les garçons que chez les filles, reflétant des normes sociales différentes.

• Lorsque l'observateur change (par exemple, la mère au lieu des pairs), l'activation du striatum ventral (récompense) diminue et les régions de contrôle s'activent, réduisant la prise de risque.

Cela souligne l'importance des "normes sociales et l'impact en vérité de ces normes sociales sur les adolescents sont sont différentes".

Il est crucial de travailler sur le conformisme social avec les adolescents, non pour le supprimer (ce qui est impossible), mais pour les aider à "comprendre cette dynamique avoir une vraie représentation explicite du fait qu'il existe ce conformisme social et détecter dans certaines situations où il faut pas se conformer".

La peur de l'exclusion est un moteur puissant du conformisme, et il est important d'aider les adolescents à relativiser ce "coût affectif".

5. Écouter et Soutenir les Adolescents : La Créativité comme Moteur

Cassotti insiste sur l'importance d'écouter davantage les adolescents et de ne pas se contenter de leur dicter des solutions.

Il propose un "changement de paradigme" où les adolescents deviennent "acteurs de la façon de résoudre les problèmes", en particulier pour des défis complexes comme la transition écologique, pour lesquels les adultes n'ont pas toujours les solutions.

Le laboratoire de Cassotti s'engage dans la co-conception de recherche avec les adolescents, leur fournissant des "outils pour penser, les outils pour réfléchir par eux-mêmes et proposer les solutions par eux-mêmes".

La Réactivité Émotionnelle comme Moteur d'Engagement : L'hypersensibilité émotionnelle des adolescents aux injustices sociales peut être un puissant moteur d'exploration et d'engagement.

Cette réactivité émotionnelle est non seulement plus forte mais "dure plus longtemps dans le temps" que chez les adultes, qui ont tendance à diluer leurs émotions, à utiliser des mécanismes de coping pour réguler les affects négatifs, ou à ne pas savoir quoi faire.

Cette "spécificité de l'adolescence qu'on peut documenter d'un point de vue neuro" peut être utilisée comme un élément de stimulation pour "une volonté d'agir et de changer les choses".

Créativité : Les adultes ont souvent des "blocages cognitifs" qui les empêchent de proposer des solutions créatives, retombant au niveau d'enfants de CM1/CM2 pour des problèmes complexes.

Le cerveau a tendance à rechercher des solutions par analogie, ce qui mène souvent à des réponses convenues et peu originales.

Par exemple, pour le problème de l'œuf lâché de 10m, la plupart des solutions se regroupent en trois catégories : ralentir la chute, amortir la chute, ou protéger l'œuf.

Il est essentiel de "sortir de ce cadre là" en explorant de nouvelles connaissances (par exemple, les propriétés naturelles de l'œuf, jouer sur le problème lui-même).

Même les idées "farfelues" peuvent être précieuses car elles "déclenchent comme activation de connaissance pour pouvoir ensuite aller explorer des solutions nouvelles".

Il y a un "vrai enjeu à travailler avec les adolescents pour les aider et les soutenir dans leur démarche plutôt que pour les enfermer".

En somme, Mathieu Cassotti invite à reconsidérer l'adolescence non pas comme une période de problèmes à gérer, mais comme une phase de développement unique avec des spécificités neurologiques et psychologiques qui, si elles sont comprises et soutenues, peuvent devenir de puissants atouts pour l'innovation, l'engagement social et la résolution de problèmes complexes.

Note de synthèse : "Le Gaslighting, outil de pensée et arme d’émancipation" - Hélène Frappat

• Cette note de synthèse s'appuie sur une retranscription d'une conférence donnée par Hélène Frappat, philosophe, traductrice et écrivaine, autour de son essai "Le Gaslighting ou l'art de faire taire les femmes" (2023).

L'intervention explore la notion de "gaslighting" non seulement comme concept psychologique, mais aussi comme outil philosophique et politique pour comprendre les mécanismes de manipulation, de déshumanisation et de destruction du langage.

Thèmes principaux et idées clés :

1. Le Gaslighting : Un concept philosophique et politique issu du cinéma

• Origine du terme : Le terme "gaslighting" provient directement du film éponyme de George Cukor (1944), lui-même une adaptation d'une pièce de théâtre. Le film met en scène la manipulation d'une femme par son mari qui la convainc qu'elle est folle, notamment en abaissant l'intensité des lampes à gaz (d'où le nom) et en niant la réalité de ses perceptions.
• Fonctionnement du gaslighting : Il s'agit d'une destruction du langage humain et du sens commun, une "déliaison" du langage. Le but est de "tuer l'autre dans le langage", de le faire douter de sa propre perception du réel, de le rendre fou ou docile. Frappat souligne que le gaslighting n'est pas tant une question d'intentionnalité psychologique que de mécanismes de pouvoir.
• Dimension politique du gaslighting : Hélène Frappat étend le concept au-delà des relations interpersonnelles pour l'appliquer à la sphère politique. Elle cite des exemples contemporains comme le macronisme ("bienveillance" détournée de son sens) ou le négationnisme ("qui se souvient des Arméniens ?"), où des mots sont volés, des faits niés, et des réalités alternatives imposées. Elle affirme que le négationnisme est une forme de gaslighting.

2. Le cinéma comme outil de pensée et de révélation

• Le cinéma comme "thatrum" : Reprenant l'étymologie du mot "théâtre" (le lieu où l'on regarde), Frappat insiste sur le cinéma comme "lieu du regard", de la "convergence des regards", et donc comme lieu de la pensée et du désir. Elle critique l'utilisation réductrice du cinéma comme simple illustration d'idées.
• "Les films nous regardent" : Le cinéma n'est pas passif ; il nous confronte, nous interroge et révèle des enjeux très contemporains. Elle analyse le film Gaslight de Cukor comme une réflexion sur le mariage traditionnel comme "maison de l'horreur" et un "manuel de déshumanisation" de la femme.
• Révolutionner le regard : Le film Basic Instinct de Paul Verhoeven est présenté comme un contrepoint à Vertigo d'Hitchcock. Si dans Vertigo, l'héroïne est "regardée" (et tuée), dans Basic Instinct, l'héroïne "regarde" (et tue), incarnant l'émancipation par le regard et la voix. La "chatte" de Sharon Stone devient un "regard qui nous regarde", un lieu de subjectivité qui tue, une subversion de la prédation du regard masculin.

3. Le lien entre violence conjugale et violence politique : La voix de la femme niée et réappropriée

• La voix comme enjeu de pouvoir : L'étranglement de la voix est un thème central. Le film Gaslight montre comment la voix d'Ingrid Bergman est progressivement éteinte, du contenu de ses phrases à la tonalité de son son. Ce mécanisme d'étouffement de la voix féminine est historique.
• La généalogie de la voix féminine répugnante : S'appuyant sur les travaux d'Anne Carson (The Gender of Sound), Frappat retrace la longue histoire de la dénégation de la voix féminine dans la philosophie grecque, notamment chez Aristote. Celui-ci, par une "pseudo-analogie" absurde entre la machine à tisser et les testicules, lie la voix grave (masculine) à la suprématie politique et la voix aiguë (féminine, homosexuelle, des "calamites") à l'absence d'autorité. La voix de la femme est perçue comme un "flux" lié au corps (utérus, menstruations, larmes), donc impur et répugnant, devant être verrouillé.
• Le silence comme "cosmos" de la femme : Citant Sophocle ("le silence est le cosmos de la femme"), Frappat souligne que le silence a longtemps été la "loi" imposée aux femmes.
• L'acte de "pleurer à la place de" : L'écrivaine se voit comme celle qui "pleure à la place de" (référence à Something's Got to Give de Cukor), non pas pour une écriture larmoyante, mais pour incarner un travail d'empathie, de se mettre à la place de l'autre, dévoyé par des discours politiques comme celui de Macron sur la "bienveillance".

4. La méthode d'écriture : La "nonfiction" et l'analogie comme mode de pensée

• La forme comme méthode : Pour Hélène Frappat, la méthode d'écriture est intrinsèquement liée à la forme. Elle se reconnaît dans le genre de la "nonfiction" (Rebecca Solnit, Anne Carson), qui transgresse les frontières des genres littéraires et mélange roman, essai, traduction, et critique cinématographique.
• La démarche analogique : Sa pensée est profondément analogique, cherchant à "faire le lien" entre des domaines, des époques et des œuvres apparemment disparates (philosophie de Kant, tragédie grecque, films hollywoodiens, littérature enfantine comme Fantomette). Elle rejette les hiérarchies bourgeoises du "bon goût" et la chronologie linéaire.
• L'écriture comme acte de résistance : L'écriture, en particulier le fait de "dire la vérité" et de "témoigner" (comme le journal dans 1984 d'Orwell), est une arme contre le gaslighting. La "suspension consentie de la croyance" (Coleridge), permise par l'ironie, est une manière de ne plus consentir aux récits qui nous nient.
• L'incarnation et le présent de l'œuvre : Les œuvres, qu'elles soient antiques ou contemporaines, existent au présent pour le lecteur. L'écrivain donne corps aux personnages, les rendant plus réels que certaines personnes.

Citations marquantes :

• "Le gaslighting, c'est l'art de faire taire les femmes."
• "Les films nous regardent."
• "Le livre [Gaslighting] est autant une enquête qu'une méthode de combat contre les pratiques de gaslighting contemporaines."
• "La question c'est pas Célan et ce poète grec, la question c'est le lien en fait." (Anne Carson)
• "Est-ce que je peux pleurer pour toi ?" (Something's Got to Give) – l'épigraphe de Trois femmes disparaissent.
• "La voix d'une femme est une partie intime." (Les talibans, écho à Aristote)
• "Le silence est le cosmos de la femme." (Sophocle)
• "Le plus grand film jamais réalisé sur le mariage, c'est un film d'horreur." (Sur Gaslight de Cukor)
• "L'intentionnalité, ça nourrit des pensées du péché, c'est pas mon sujet. Et par ailleurs, je ne m'intéresse pas à la psychologie, et George Cukor non plus. Parce que la psychologie nous éloigne de la politique."
• "Il faut être deux pour commencer à parler, sinon on est fou." (Wittgenstein)
• "Écrire c'est pas mettre des mots les uns à côté des autres comme on tisse un collier de perles." (Henry James)
• "La question c'est est-ce que ce qu'elle [Monique Wittig] va dire est intéressant ou pas." (Deleuze)

En résumé, Hélène Frappat propose une exploration riche et transversale du gaslighting, en le dépeignant comme une violence fondamentale qui s'exerce sur le langage, la perception du réel et la subjectivité, particulièrement celle des femmes.

Son approche, qui embrasse philosophie, cinéma, littérature et histoire, vise à déconstruire ces mécanismes pour mieux les combattre, en plaçant la forme, le lien, l'empathie et la résistance de la voix au cœur de sa démarche intellectuelle et créative.

Document de Synthèse : Sociologie de la Défiance et de l'Adhésion aux Croyances dans le Domaine Scientifique

Ce document explore la sociologie de la défiance et de l'adhésion aux croyances scientifiques, en se concentrant sur l'affaire Wakefield et ses répercussions sur la confiance vaccinale.

L'analyse met en lumière le rôle des médias, les normes scientifiques, le contexte social et la diffusion des informations sur internet.

1. Cadre Sociologique et Point de Vue de l'Auteure

• Romy Sauvayre, l'auteure, se positionne dans une approche sociologique qui étudie "les raisons pour lesquelles les acteurs sociaux vont faire ce qu'ils font, croire ce qu'ils croient".

Elle ne cherche pas à déterminer ce qui est "vrai" ou "faux" en soi, mais plutôt ce que les scientifiques considèrent comme tel à un moment donné, reconnaissant que cette perception peut évoluer.

Son analyse se concentre sur "la relation entre empirique et perception qu'on a de recherche scientifique". * 2. L'Affaire Wakefield : Une Chronologie et ses Effets

L'affaire Wakefield est un cas d'étude central pour comprendre la défiance vaccinale.

• Origine (1998) : Le Dr. Andrew Wakefield, gastro-entérologue, publie un article dans la revue The Lancet suggérant un lien entre le vaccin ROR (rougeole, oreillons, rubéole) et l'inflammation intestinale, et potentiellement l'autisme. Il est important de noter que l'article était publié sous l'étiquette "article de prévention", indiquant qu'il s'agissait d'une recherche préliminaire et non d'une preuve définitive. Les co-auteurs eux-mêmes n'ont pas établi de lien direct entre l'autisme et les vaccins, seulement entre le vaccin et la maladie intestinale.
• Contexte Préexistant à la Publication :
• Crise de la Vache Folle (ESB) : De 1985 à 2000, la crise de la vache folle a fortement érodé la confiance du public britannique envers les scientifiques et le gouvernement. "La science elle dit soyez sûrs c'est un cas de déviation britannique qui est appelé chance dans la science". Cela a créé un terrain fertile pour la méfiance envers les déclarations scientifiques officielles.
• Retrait de Vaccins ROR (1992) : Deux vaccins ROR avaient déjà été retirés du marché six ans plus tôt en raison d'effets secondaires, renforçant les craintes préexistantes.
• Rôle des Médias (1998-2004) :
• Soutien Initiale : Les médias britanniques, en particulier le Sunday Times et le Panorama de la BBC (émission "MMR: What The Doctors Won't Tell You" en 2002), ont "promptement soutenu la thèse de Wakefield". Ils ont accordé une grande visibilité à Wakefield, en partie parce qu'il était un expert reconnu et avait un statut académique important (76 publications, senior researcher).
• Biais de Diffusion : Les journalistes ont "davantage diffusé la thème de Wakefield que toute autre thèse", en privilégiant les études qui abondaient dans son sens et en "ne médiatisant pas" les études remettant en cause son hypothèse. Le pic de médiatisation a eu lieu en 2002, suite au documentaire de la BBC.
• Conséquence Directe : Cette médiatisation a eu un "impact énorme sur la vaccination des génies". On observe une "chute conséquente du taux de vaccination" après la publication de l'article de Wakefield en février 1998, et pendant toute la période de médiatisation. Le taux de vaccination ROR est passé de 92% à 83% dans certaines régions du Royaume-Uni.

3. Point de Vue des Scientifiques et Contestations

Bien que les médias aient initialement soutenu Wakefield, la communauté scientifique n'était "pas unanime" et des contestations ont rapidement émergé.

• Critiques Méthodologiques (Dès 1998) :
• Taille de l'Échantillon : L'étude de Wakefield portait sur seulement 12 enfants, un nombre jugé insuffisant pour tirer des conclusions générales. "12 c'est quand même pas bon".
• Manque de Groupe Témoin et de Double Aveugle : La méthodologie ne comprenait pas de groupe de contrôle ni de protocole en double aveugle, des normes aujourd'hui "très valorisée" en recherche médicale.
• Biais de Sélection et Conflits d'Intérêts : Le recrutement des enfants s'est fait via un "réseau d'association de personnes plaignantes qui pensaient que le vaccin contre la rougeole posait l'autisme", suggérant un biais de sélection. Il a été révélé plus tard que Wakefield avait des "conflits d'intérêt" et des "fins pour pouvoir apporter des preuves en faveur de son hypothèse".
• Données Partielles et Antécédents : Les données rapportées étaient partielles, et les travaux précédents de Wakefield, bien que reçus, contenaient déjà des "problèmes méthodologiques" qui rendaient difficile la détection d'un virus dans l'intestin.
• Réponse des Institutions Scientifiques :
• Académie des Sciences : L'Académie des Sciences a initialement déclaré ne pas avoir "les moyens de falsifier l'hypothèse de Wakefield", ce qui, selon l'auteure, n'aurait pas dû être interprété comme une validation. En sciences médicales, on ne se prononce qu'avec des preuves suffisantes.
• Institute of Medicine (2004) : En 2004, l'Institute of Medicine a publié un rapport concluant qu'il n'y avait "pas de danger à inoculer le vaccin ROR", marquant un tournant.

4. Le Rôle de l'Investigation Journalistique et la Rétractation

Le changement de perception médiatique est dû à une enquête journalistique approfondie.

• Enquête du Sunday Times (2004) : Brian Deer, un journaliste d'investigation du Sunday Times, a mené une enquête minutieuse, rencontrant tous les acteurs et consultant les dossiers d'éthique. Il a "constaté qu'il y a des examens invasifs sur les enfants" (colonoscopies, fonctions d'anglais) qui n'étaient "pas possibles" ou "inacceptables" d'un point de vue éthique. Cette révélation a alerté le conseil de l'ordre.
• Arrêt du Soutien Médiatique : Suite à ces révélations, "les médias arrêtent de soutenir" Wakefield.
• Radiation de Wakefield (2010) : Le Conseil Général Médical (General Medical Council) a mené une longue enquête (2004-2010), culminant par le "retrait le droit d'exercer" à Wakefield en 2010. L'article original a été "retiré en 2010" par The Lancet. Les motifs incluaient des "conflits d'intérêt" et la réalisation d'"examens organisés pour les fonds et sur le peuple sans collectivité".

5. L'Ère d'Internet et la Persistance de la Défiance

Malgré la radiation de Wakefield et le retrait de son article, la défiance vaccinale a persisté, amplifiée par les plateformes numériques.

• Diffusion de Théories Conspirationnistes : Wakefield a "publié un qui s'appelle conspirationnisme" après sa radiation. Il a affirmé que ses découvertes étaient si importantes que l'industrie pharmaceutique tentait de le "faire taire". "Bref il était donc sur le groupe d'une machination pour le feu terre".
• Documentaire "Vaxxed" (2016) : En 2016, Wakefield a publié un documentaire "Vaxxed" sur les réseaux sociaux, qui "tient pour consiste le conspirationnisme de santé CDC", affirmant que le CDC (Centers for Disease Control and Prevention) aurait "caché des dans son et il continue à diffuser ça".
• Rôle des Réseaux Sociaux : Les réseaux sociaux ont permis une "division très massive de ces antivaccinisme". La défiance vaccinale a été "d'abord puis sur les différentes réseaux sociaux". L'auteure souligne que le manque de validation scientifique ne freine pas la diffusion : "On est tous des cobages, c'est un maxin expérimental voilà ce qui se sur les réseaux sociaux".
• Influence sur la Perception : La persistance de l'antivaccinisme est également liée à des mécanismes psychologiques : "si quelqu'un est gentil avec vous curieusement vous lui faites" confiance. La diffusion de ces idées est alimentée par des "personnes" qui "disent des choses comme ça affirmative", tandis que les scientifiques, dans leur rôle, "doivent toujours parler avec précaution" car les preuves sont "toujours sous une" incertitude jusqu'à confirmation par de multiples études.
• Conséquences Durables : Malgré les efforts pour rétablir la confiance, "la défiance qu'il n'y avait à monde se sur les réseaux sociaux se retrouve avec une plus grande population de défis qui n'ont pas". En France, une partie de la population (environ 35%) "continue à marquer vacc".

6. Leçons Apprises et Précautions

Nécessité de la Précaution Scientifique : "Il est nécessaire d'être près que pressionner avec des études". La science exige une multiplicité de preuves et des méta-analyses pour valider les conclusions. Critique des Sources : L'auteure conseille de se méfier des informations sur les réseaux sociaux et de privilégier les sources fiables comme Google Scholar pour les questions médicales, qui "font des analyses sur plein de sujets libres" et permettent d'"assurer d'avoir une information juste".

Responsabilité des Journalistes : Les journalistes ont une "part de responsabilité" dans la diffusion d'études minoritaires et contestées. Ils devraient "diffuser davantage l'information que la précaution" quand ils se basent sur un consensus scientifique majoritaire.

En conclusion, l'affaire Wakefield illustre la complexité de la diffusion des croyances scientifiques, l'impact majeur du contexte socio-historique, le rôle ambivalent des médias (initialement facilitateurs de la désinformation, puis correcteurs par l'investigation), et la capacité d'internet à perpétuer et amplifier la défiance, même face à un consensus scientifique établi.

Compte-rendu détaillé : Labels et marques - leviers de différenciation

• Contexte du Webinaire : Ce webinaire, animé par Débora Récher de Logitourisme, s'inscrit dans le parcours "Expérience client" pour Moselle Académie.

L'objectif est d'éclairer les professionnels du tourisme, de la culture et de la gastronomie sur la distinction et les bénéfices des labels et marques comme outils de différenciation dans un marché concurrentiel.

1. Distinction entre Labels et Marques

Il est crucial de bien différencier un label d'une marque, car ils remplissent des fonctions distinctes :

• Label : Un label est un "signe officiel ou privé... reconnaissant une qualité ou un engagement selon un cahier des charges bien précis." (Débora Récher). Il certifie le respect de critères spécifiques.
• Exemples cités : Gîtes de France, Clé Vacances (hébergement), Qualité Tourisme (qui deviendra Destination d'Excellence), Accueil Vélo, Tourisme et Handicap.
• Caractéristique clé : Basé sur une grille de critères, il permet ou non d'être labellisé.
• Marque : Une marque est "une identité d'une organisation, c'est d'un territoire, c'est porteuse de valeur et d'image. Très souvent la marque c'est du marketing, c'est ce qui permet d'être d'être clairement identifié." (Débora Récher). Elle représente une identité forte, souvent déposée auprès de l'INPI pour se protéger de la concurrence.
• Exemples cités : Nike (marque de consommation courante), Esprit Parc National, Moselle (marque territoriale), Normandie Impressionniste.
• Caractéristique clé : Axée sur le marketing et la communication de valeurs et d'une image.

2. Pourquoi choisir un Label ou une Marque ?

Le choix d'un label ou d'une marque est volontaire et vise à :

• Se différencier dans un marché concurrentiel : Le tourisme en France est un secteur "extrêmement concurrentiel" (Débora Récher), avec une offre pléthorique. Labels et marques permettent de se distinguer.
• Garantir le sérieux et la qualité : Ils offrent "une gage de sérieux et de qualité" pour le consommateur, qui doit "avoir compris ce qu'il y avait derrière ce label et que ce label a du sens." (Débora Récher).
• Bénéficier d'une reconnaissance :Officielle/Institutionnelle : Comme l'aide à la création d'hébergements conditionnée à l'affiliation à un label.
• Territoriale : Une marque territoriale partagée et connue, pouvant être "prescripteur, ambassadeur et fasse vivre une marque" (Débora Récher).
• Valoriser des engagements spécifiques : Qualité, durabilité, accueil spécifique (ex: Accueil Vélo pour la clientèle cycliste, Tourisme et Handicap pour l'inclusivité). Cela "rassure" le client.
• Profiter d'un réseau et d'une promotion collective : Travailler en réseau est "beaucoup plus simple que travailler tout seul dans son dans son coin" (Débora Récher), offrant une dynamique collective, promotion et parfois formations.
• Renforcer la confiance des clientèles : C'est un objectif primordial.

3. Pièges à éviter et Précautions

• Ne pas accumuler les labels : "Plus c'est pas parce que plus vous en avez que plus vous serez visible, parfois ça brouille complètement la lisibilité on ne sait plus qui vous êtes et ce que vous êtes." (Débora Récher).
• Cohérence avec les valeurs et la clientèle cible : Un label doit être "cohérent avec vos valeurs et votre clientèle cible" (Débora Récher). Une communication "qui va pour tout le monde à tout va... vous communiquez dans le vide." (Débora Récher).
• Engagement sincère : Au-delà de l'opportunisme, il doit y avoir un "engagement sincère". Si les clients ne retrouvent pas les engagements du label (ex: écolabels), cela sera "contreproductif" et entraînera insatisfaction, notamment via les réseaux sociaux.

4. Comment choisir son Label ou sa Marque ?

• Le choix doit être stratégique et réfléchi :
• Définir les objectifs : Qualité, visibilité, accompagnement.
• Identifier les valeurs à mettre en avant : "Les valeurs que vous voulez vous mettre en avant on en a parlé ça c'est très important" (Débora Récher).
• Cibler la clientèle : À qui s'adresse le produit ou le service ?
• Évaluer la notoriété et le dynamisme du label/marque : Est-il reconnu par la clientèle ? Quelles sont les actions menées par les porteurs du label ?
• Estimer l'effort et l'engagement requis : Êtes-vous prêt à modifier des pratiques ou des équipements ?
• Le coût de la cotisation : Ne doit pas être le facteur de choix principal, car "le prix est une notion relative pour peu que derrière vous ayez le service attendu." (Débora Récher). L'important est la stratégie et le bénéfice escompté.

5. Exemples Inspirants par Secteur

Débora Récher a présenté de nombreux exemples de labels et marques, illustrant leur diversité et leur pertinence :

• Tourisme :Qualité d'accueil : Qualité Tourisme, Gîtes de France, Clé Vacances.
• Thématiques : Accueil Vélo, Tourisme et Handicap (inclusif, pour différents types de handicaps, personnes âgées, familles avec poussettes).
• Marques de territoire/destination : OnlyLyon, Esprit Parc, Valeurs Parc, Alsace (avec son logo en forme de bretzel).
• Spécifiques : Famille Plus (destinations adaptées aux familles), Pavillon Bleu (plages et ports propres), Jardin Remarquable (jardins d'exception), Entreprise du Patrimoine Vivant (savoir-faire artisanaux et industriels d'excellence).
• Culture et Patrimoine :Généralistes : Ville et Pays d'Art et d'Histoire (valorise politique culturelle ambitieuse), UNESCO (sites culturels/naturels d'exception).
• Spécifiques : Patrimoine du 20e Siècle (architectures modernes/post-industrielles), Maison des Illustres (lieux liés à personnages célèbres).
• Équipements culturels : Scène Nationale, Musées de France (garantissent conservation, valorisation, expertise scientifique), France Muséum (exportation savoir-faire muséal français).
• Événements : Festivals de musique labellisés, événements éco-engagés (réduction déchets, etc.).
• Itinéraires : Itinéraires culturels européens (ex: Route des Abbayes Cisterciennes).
• Artisanat (compléments) : Qualités Artisan Métier d'Art, MOF, Maître Artisan, France Terre Textile (pour les Vosges).
• Gastronomie et Terroir :Produits : AOP, AOC, IGP (ex: Mirabelle de Lorraine, Vin de Moselle, Quiche Lorraine), Label Rouge (produits de qualité supérieure), Agriculture Biologique.
• Tourisme œnologique et circuits gourmands : Vignobles et Découvertes, Route des Vins de Moselle, Vallée de la Gastronomie.
• Accueil à la ferme : Bienvenue à la Ferme (favorise vente directe et accueil touristique, répond à la demande de "circuit court" et consommation locale).
• Restauration : Collège Culinaire de France, Tables Distinguées, Bistrot de Pays, Écotable (restauration durable et responsable).

6. Le Cas Spécifique de la Moselle

La Moselle offre un exemple intéressant d'imbrication entre marque et label :

• La marque "Moselle" (Moselle Sans Limite) : Lancée par le département en 2017, c'est une "marque territoriale" qui vise à "valoriser l'attractivité globale du territoire" (Débora Récher), incluant le tourisme, l'économie, la gastronomie, l'artisanat et la culture. C'est une bannière pour tous.
• Le label "Qualité Moselle" : Attribué aux produits, savoir-faire, hébergements, artisans et restaurateurs qui respectent un cahier des charges de qualité et ont un "ancrage local fort." (Débora Récher).
• L'un soutient l'autre : on peut utiliser le logo de la marque Moselle sur son site pour communiquer l'identité globale, et aller plus loin en obtenant le label Qualité Moselle pour valoriser un savoir-faire précis et gagner en visibilité commerciale. Cette synergie locale est essentielle : "Plus elle est partagée par tous, c'est-à-dire les prestataires, les professionnels, plus elle va être visible." (Débora Récher). Cela garantit "qualité et identité locale", rassure le client sur l'authenticité et la provenance locale des produits et services.

7. Bilan et Recommandations Clés

• Choisir 1 à 3 labels/marques maximum : Pour maintenir une stratégie "lisible".
• Intégrer la stratégie de labellisation : Le choix doit être intégré à l'offre, la communication et l'équipe. "Travaillez votre marque et travaillez votre label pour en tirer le meilleur profit." (Débora Récher).
• Utiliser les labels/marques dans la communication : Les intégrer dans les supports de communication et la stratégie marketing.
• Suivre les résultats : Ne pas se limiter au retour sur investissement direct. Surveiller l'image améliorée, la notoriété accrue, la visibilité, l'acquisition de nouvelles clientèles et la satisfaction client. Les avis clients en ligne sont des indicateurs "très forts" (Débora Récher), et un label reconnu peut "impacter directement l'image de votre établissement d'un point de vue positif".
• Un label est un outil, pas une fin en soi : "Le label doit servir votre stratégie marketing, votre stratégie de communication." (Débora Récher). Ne pas être déçu si un label n'est pas obtenu, d'autres options peuvent être plus adaptées.
• Nécessité d'une présence en ligne : Un site internet est "rigoureusement nécessaire pour démarrer" (Débora Récher). 80% des touristes préparent leur séjour en ligne et près de la moitié réservent directement. Les clients croisent les informations (ex: Airbnb et site internet propre) pour se rassurer.

Conseils supplémentaires :

Demander les cahiers des charges des labels pertinents. Contacter les institutions (Moselle Attractivité, labels nationaux) pour des renseignements et un accompagnement.

Évaluer les services d'accompagnement individuels souvent proposés par les labels.

Faire du benchmarking : consulter les expériences d'autres professionnels labellisés.

Reviewer #2 (Public review):

Summary:

The manuscript by Jia and Chen addresses the structural basis of voltage-activation of BK channels using computational approaches. Although a number of experimental studies using gating current and patch-clamp recording have analyzed voltage-activation in terms of observed charge movements and the apparent energetic coupling between voltage-sensor movement and channel opening, the structural changes that underlie this phenomenon have been unclear. The present studies use a reduced molecular system comprising the transmembrane portion of the BK channel (i.e. the cytosolic domain was deleted), embedded in a POPC membrane, with either 0 or 750 mV applied across the membrane. This system enabled acquisition of long simulations of 10 microseconds, to permit tracking of conformational changes of the channel. The authors principal findings were that the side chains of R210 and R213 rapidly moved toward the extracellular side of the membrane (by 8 - 10 Å), with greater displacements than any of the other charged transmembrane residues. These movements appeared tightly coupled to movement of the pore-lining helix, pore hydration, and ion permeation. The authors estimate that R210 and R213 contribute 0.25 and 0.19 elementary charges per residue to the gating current, which is roughly consistent with estimates based on electrophysiological measurements that used the full-length channel.

Strengths:

The methodologies used in this work are sound, and these studies certainly contribute to our understanding of voltage-gating of BK channels. An intriguing observation is the strongly coupled movement of the S4, S5, and S6 helices that appear to underlie voltage-dependent opening. Based on Fig 2a-d, the substantial movements of the R210 and R213 side chains occur nearly simultaneously to the S6 movement (between 4 - 5 usec of simulation time). This seems to provide support for a "helix-packing" mechanism of voltage gating in the so-called "non-domain-swapped" voltage-gated K channels.

Weaknesses:

The main limitation is that these studies used a truncated version of the BK channel, and there are likely to be differences in VSD-pore coupling in the context of the full-length channels that will not be resolved in the present work. Nonetheless, the authors provide a strong rationale for their use of the truncated channel, and the results presented will provide a good starting point for future computational studies of this channel.

Author response:

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

Recommendations for the authors:

Reviewing Editor Comments:

The resubmitted version of the manuscript adequately addressed several initial comments made by reviewing editors, including a more detailed analysis of the results (such as those of bilayer thickness). This version was seen by 2 reviewers. Both reviewers recognize this work as being an important contribution to the field of BK and voltage-dependent ion channels in general. The long trajectories and the rigorous/novel analyses have revealed important insights into the mechanisms of voltage-sensing and electromechanical coupling in the context of a truncated variant of the BK channel. Many of these observations are consistent with structural and functional measurements of the channel, available thus far. The authors also identify a novel partially expanded state of the channel pore that is accessed after gating-charge displacement, which informs the sequence of structural events accompanying voltage-dependent opening of BK.

However, there are key concerns regarding the use of the truncated channel in the simulations. While many gating features of BK are preserved in the truncated variant, studies have suggested that opening of the channel pore to voltage-sensing domain rearrangement is impaired upon gating-ring deletion. So the inferences made here might only represent a partial view of the mechanism of electromechanical coupling.

It is also not entirely clear whether the partially expanded pore represents a functionally open, sub-conductance, or another closed state. Although the authors provide evidence that the inner pore is hydrated in this partially open state, in the absence of additional structural/functional restraints, a confident assignment of a functional state to this structure state is difficult. Functional measurements of the truncated channel seem to suggest that not only is their single channel conductance lower than full-length channels, but they also appear to have a voltage-independent step that causes the gates to open. It is unclear whether it is this voltage-independent step that remains to be captured in these MD trajectories. A clean cut resolution of this conundrum might not be feasible at this time, but it could help present the various possibilities to the readers.

We appreciate the positive comments and agree that there will likely be important differences between the mechanistic details of voltage activation between the Core-MT and full-length constructs of BK channels. We also agree that the dilated pore observed in the simulation may not be the fully open state of Core-MT.

Nonetheless, the notion that the simulation may not have captured the full pore opening transition or the contribution of the CTD should not render the current work “incomplete”, because a complete understanding of BK activation would be an unrealistic goal beyond the scope of this work. We respectfully emphasize that the main insights of the current simulations are the mechanisms of voltage sensing (e.g., the nature of VSD movements, contributions of various charged residues, how small charge movements allow voltage sensing, etc.) as well as the role of the S4-S5-S6 interface in VSD-pore coupling. As noted by the Editor and reviewers, these insights represent important steps towards establishing a more complete understanding of BK activation.

Below are the specific comments of the two experts who have assessed the work and made specific suggestions to improve the manuscript.

Reviewer #1 (Recommendations for the authors):

(1) Although the successful simulation of V-dependent K+ conduction through the BK channel pore and analysis of associated state dependent VSD/pore interactions and coupling analysis is significant, there are two related questions that are relevant to the conclusions and of interest to the BK channel community which I think should be addressed or discussed.

One key feature of BK channels is their extraordinarily large conductance compared to other K+ selective channels. Do the simulations of K+ conductance provide any insight into this difference? Is the predicted conductance of BK larger than that of other K+ channels studied by similar methods? Is there any difference in the conductance mechanism (e.g., the hard and soft knock-on effects mentioned for BK)?

The molecular basis of the large conductance of BK channels is indeed an interesting and fundamental question. Unfortunately, this is beyond the scope of this work and the current simulation does not appear to provide any insight into the basis of large conductance. It is interesting to note, though, the conductance is apparently related to the level of pore dilation and the pore hydration level, as increasing hydration level from ~30 to ~40 waters in the pore increases the simulated conductance from ~1.5 to 6 pS (page 8). This is consistent with previous atomistic simulations (Gu and de Groot, Nature Communications 2023; ref. 33) showing that the pore hydration level is strongly correlated with observed conductance. As noted in the manuscript, the conductance mechanism through the filter appears highly similar to previous simulations of other K+ channels (Page 8). Given the limit conductance events observed in the current simulations, we will refrain from discussing possible basis of the large conductance in BK channels except commenting on the role of pore hydration (page 8; also see below in response to #5).

The pore in the MD simulations does not open as wide as the Ca-bound open structure, which (as the authors note) may mean that full opening requires longer than 10 us. I think that is highly likely given that the two 750 mV simulations yielded different degrees of opening and that in BK channels opening is generally much slower than charge movement. Therefore, a question is - do any of the conclusions illustrated in Figures 6, S5, S6 differ if the Ca-bound structure is used as the open state? For example, I expect the interactions between S5 and S6 might at least change to some extent as S6 moves to its final position. In this case, would conclusions about which residues interact, and get stronger or weaker, be the same as in Figures S6 b,c? Providing a comparison may help indicate to what extent the conclusions are dependent on achieving a fully open conformation.

We appreciate the reviewer’s suggestion and have further analyzed the information flow and coupling pathways using the simulation trajectory initiated from the Ca2+-bound cryo-EM structure (sim 7, Table S1). The new results are shown in two new SI Figures S7 and S8, and new discussion has been added to pages 14-15. Comparing Figures 5 and S7, we find that dynamic community, coupling pathways, and information flow are highly similar between simulation of the open and closed states, even though there are significant differences in S5 contacts in the simulated open state vs Ca2+-bound open state (Figure S8). Interestingly, there are significant differences in S4-S5 packing in the simulated and Ca2+-bound open states (Figure S8 top panel), which likely reflect important difference in VSD/pore interactions during voltage vs Ca2+ activation.

(2) P4 Significance -"first, successful direct simulation of voltage-activation"

This statement may need rewording. As noted above Carrasquel-Ursulaez et al.,2022 (reference 39) simulated voltage sensor activation under comparable conditions to the current manuscript (3.9 us simulation at +400 mV), and made some similar conclusions regarding R210, R213 movement, and electric field focusing within the VSD. However, they did not report what happens to the pore or simulate K+ movement. So do the authors here mean something like "first, successful direct simulation of voltage-dependent channel opening"?

We agree with the reviewer and have revised the statement to “ … the first successful direct simulation of voltage-dependent activation of the big potassium (BK) channel, ..”

(3) P5 "We compare the membrane thickness at 300 and 750 mV and the results reveal no significant difference in the membrane thickness (Figure S2)" The figure also shows membrane thickness at 0 mV and indicates it is 1.4 Angstroms less than that at 300 or 750 mV. Whether or not this difference is significant should be stated, as the question being addressed is whether the structure is perturbed owing to the use of non-physiological voltages (which would include both 300 and 750 mV).

We have revised the Figure S2 caption to clarify that one-way ANOVA suggest the difference is not significant.

(4) P7 "It should be noted that the full-length BK channel in the Ca2+ bound state has an even larger intracellular opening (Figure 2f, green trace), suggesting that additional dilation of the pore may occur at longer timescales."

As noted above, I agree it is likely that additional pore dilation may occur at longer timescales. However, for completeness, I suppose an alternative hypothesis should be noted, e.g. "...suggesting that additional dilation of the pore may occur at longer timescales, or in response to Ca-binding to the full length channel."

This is a great suggestion. Revised as suggested.

(5) Since the authors raise the possibility that they are simulating a subconductance state, some more discussion on this point would be helpful, especially in relation to the hydrophobic gate concept. Although the Magleby group concluded that the cytoplasmic mouth of the (fully open) pore has little impact on single channel conductance, that doesn't rule out that it becomes limiting in a partially open conformation. The simulation in Figure 3A shows an initial hydration of the pore with ~15 waters with little conductance events, suggesting that hydration per se may not suffice to define a fully open state. Indeed, the authors indicate that the simulated open state (w/ ~30-40 waters) has 1/4th the simulated conductance of the open structure (w/ ~60 waters). So is it the degree of hydration that limits conductance? Or is there a threshold of hydration that permits conductance and then other factors that limit conductance until the pore widens further? Addressing these issues might also be relevant to understanding the extraordinarily large conductance of fully open BK compared to other K channels.

We agree with the reviewer’s proposal that pore hydration seems to be a major factor that can affect conductance. This is also well in-line with the previous computational study by Gu and de Groot (2023). We have now added a brief discussion on page 8, stating “Besides the limitation of the current fixed charge force fields in quantitively predicting channel conductance, we note that the molecular basis for the large conductance of BK channels is actually poorly understood (78). It is noteworthy that the pore hydration level appears to be an important factor in determining the apparent conductance in the simulation, which has also been proposed in a previous atomistic simulation study of the Aplysia BK channel (33).”

Minor points

(1) P5 "the fully relaxed pore profile (red trace in Figure S1d, top row) shows substantial differences compared to that of the Ca2+-free Cryo-EM structure of the full-length channel." For clarity, I suggest indicating which is the Ca-free profile - "... Ca2+-free Cryo-EM structure of the full-length channel (black trace)."

We greatly appreciate the thoughtful suggestion. Revised as suggested.

(2) P8 "Consistent with previous simulations (78-80), the conductance follows a multi-ion mechanism, where there are at least two K+ ions inside the filter" For clarity, I suggest indicating these are not previous simulations of BK channels (e.g., "previous simulations of other K+ channels ...").

Revised as suggested. Thank you.

(3) Figure 2, S1 - grey traces representing individual subunits are very difficult to see (especially if printed). I wonder if they should be made slightly darker. Similar traces in Figure 3 are easier to see.

The traces in Figure S1 are actually the same thickness in Figure 3 and they appear lighter due to the size of the figure. Figure 2 panels a-c have been updated to improve the resolution.

(4) Figure 2 - suggest labeling S6 as "S6 313-324" (similar to S4 notation) to indicate it is not the entire segment.

Figure 2 panel d) has been updated as suggested.

(5) Figure 2 legend - "Voltage activation of Core-MT BK channels. a-d)..."

It would be easier to find details corresponding to individual panels if they were referenced individually. For example:

"a-d) results from a 10-μs simulation under 750 mV (sim2b in Table S1). Each data point represents the average of four subunits for a given snapshot (thin grey lines), and the colored thick lines plot the running average. a) z-displacement of key side chain charged groups from initial positions. The locations of charged groups were taken as those of guanidinium CZ atoms (for Arg) and sidechain carboxyl carbons (for Asp/Glu) b) z-displacement of centers-of-mass of VSD helices from initial positions, c) backbone RMSD of the pore-lining S6 (F307-L325) to the open state, and d) tilt angles of all TM helices. Only residues 313-324 of S6 were included inthe tilt angle calculation, and the values in the open and closed Cryo-EM structures are marked using purple dashed lines. "

We appreciate the thoughtful suggestion and have revised the caption as suggested.

(6) Figure S1 - column labels a,b,c, and d should be referenced in the legend.

The references to column labels have been added to Figure S1 caption.

(7) References need to be double-checked for duplicates and formatting.

a) I noticed several duplicate references, but did not do a complete search: Budelli et al 2013 (#68, 100), Horrigan Aldrich 2002 (#22,97), Sun Horrigan 2022 (#40, 86), Jensen et al 2012 (#56,81).

b) Reference #38 is incorrectly cited with the first name spelled out and the last name abbreviated.

We appreciate the careful proofreading of the reviewer. The duplicated references were introduced by mistake due to the use of multiple reference libraries. We have gone through the manuscript and removed a total of 5 duplicated references.

Reviewer #2 (Recommendations for the authors):

This manuscript has been through a previous level of review. The authors have provided their responses to the previous reviewers, which appear to be satisfactory, and I have no additional comments, beyond the caveats concerning interpretations based on the truncated channel, which are noted above.

We greatly appreciate the constructive comments and insightful advice. Please see above response to the Reviewing Editor’s comments for response and changes regarding the caveats concerning interpretations of the current simulations.

Reviewer #2 (Public review):

This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. Single-cell RNA sequencing (scRNA-seq) analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.

Some of the major issues are described below.

(1) The CD31 immunostaining data (Figure 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

(2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Fig. 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

(3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Fig. 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

(4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

(5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

(6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

(7) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

Comments on Revised Version (from BRE):

The manuscript has greatly improved following the revision, and I have no additional comments to offer.

Reviewer #3 (Public review):

Summary:

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with focus on critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cells network in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated heart. Lastly they reported a compensatory collagen expression in long term ablated neonate heart. Overall, this study provides us with important insight on fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM focused research.

Strengths:

The authors utilized good analyzing tools to investigate on multiple database of single cell sequencing and Multi-seq. They identified significant pathways, cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used very strident technique to study fibroblast functions by ablating one of the major cell population of heart. Also, experimental validation of their analyzed downstream pathways will be required eventually.

Comments on Revised Version (from BRE):

The authors did a good job addressing the questions asked at first review. However, I have some minor concerns.

(1) The paper notes that collagen signaling is observed in FB-VasEC in humans, but not in FB-VenCM, unlike mice. Did the authors analyze predictive ligand receptor interaction as they did with control and ablated mice heart? This could add valuable new insights that how FB regulate ventricular CM in human heart.

(2) The authors provided data on Defect in CD31 expression in several models. Did they observe any other phenotypes associated with defective endothelial or vascular system? Such as, blood accumulation in pericardium, larger/smaller capillaries? Did they also examine percentage of Cdh5+ cells?

(3) Please mention the sample age of Figure 2A-C.

(4) Please follow the same style to describe X axis in graphs in Figure 3D (and all similar graphs in the manuscript) as followed in 3G.

(5) It is important to provide echocardiographic M mode images with a comparable number of cardiac cycles in control and ablated (Fig. 6H).

(6) In the long-term neonatal ablation experiments, collagen expressions return to normal. The manuscript attributes this to possible "compensatory expression," Do they have any thoughts how this is regulated? Are other cell types stepping in, or are surviving FBs proliferating?

(7) While collagen is shown to be a dominant signaling molecule, its centrality is inferred primarily from scRNA-seq and ligand-receptor predictions. Did authors try any functional rescue experiment (e.g., exogenous collagen supplementation or receptor blockade) to directly validate this pathway's role in vivo?

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study by Deng et al reports single cell expression analysis of developing mouse hearts and examines the requirements for cardiac fibroblasts in heart maturation. The work includes extensive gene expression profiling and bioinformatic analysis. The prenatal fibroblast ablation studies show new information on the requirement of these cells on heart maturation before birth.

The strengths of the manuscript are the new single cell datasets and comprehensive approach to ablating cardiac fibroblasts in pre and postnatal development in mice. Extensive data are presented on mouse embryo fibroblast diversity and morphology in response to fibroblast ablation. Histological data support localization of major cardiac cell types and effects of fibroblast ablation on cardiac gene expression at different times of development.

A weakness of the study is that the major conclusions regarding collagen signaling and heart maturation are based on gene expression patterns and are not functionally validated.

Reviewer #2 (Public review):

This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. Single-cell RNA sequencing (scRNA-seq) analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.

We thank the reviewer for carefully reading our revised manuscript. All of the questions listed below were raised in the previous round and have been addressed in the current revision. As noted in the “Recommendations for the Authors” section, the reviewer has no additional comments at this time.

Some of the major issues are described below.

(1) The CD31 immunostaining data (Figure 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

(2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Fig. 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

(3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Fig. 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

(4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

(5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

(6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

(7) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

Reviewer #3 (Public review):

Summary:

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with focus on critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cells network in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated heart. Lastly they reported a compensatory collagen expression in long term ablated neonate heart. Overall, this study provides us with important insight on fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM focused research.

Strengths:

The authors utilized good analyzing tools to investigate on multiple database of single cell sequencing and Multi-seq. They identified significant pathways, cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used very strident technique to study fibroblast functions by ablating one of the major cell population of heart. Also, experimental validation of their analyzed downstream pathways will be required eventually.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Most of my comments have been adequately addressed. Additional comments on new data in the revised manuscript are below.

(1) In the new figure S11, it is not really possible to draw major conclusions on mitral valve morphology and maturation since the planes of sections to not seem comparable. Observations regarding attachment to the papillary muscle might be dependent on the particular section being evaluated. However, it is useful to see that the valves are not severely affected in the ablated animals.

We appreciate the reviewer’s comment and agree with the reviewer’s observation. Accordingly, we have updated the manuscript by removing the original conclusion-related statement and instead highlighting that the valves were not severely affected in the ablated animals (page 6).

(2) In the last supplemental figure S19, it is not possible to determine if results are or are not statistically significant for n=2 as shown for FS and EF for the ablated animals and controls. The text says that there is a trend of improved heart function, but evaluation of additional animals is needed to support this conclusion.

We thank the reviewer for the comment and agree that a sample size of n = 2 is too small to draw meaningful conclusions. As previously suggested by the reviewer, we have removed this result from the manuscript (page 10).

Reviewer #2 (Recommendations for the authors):

The manuscript has greatly improved following the revision, and I have no additional comments to offer.

Thanks!

Reviewer #3 (Recommendations for the authors):

Authors did a good job addressing questions asked at first review. However, I have some minor concerns.

(1) The paper notes that collagen signaling is observed in FB-VasEC in humans, but not in FB-VenCM, unlike mice. Did authors analyze predictive ligand receptor interaction as they did with control and ablated mice heart? This could add valuable new insights that how FB regulate ventricular CM in human heart.

Thank you. We have analyzed the predicted ligand-receptor interactions between Fb and Ven_CM, as well as between Fb and Vas_EC, using human scRNA-seq data. The results are provided as a supplemental figure (Fig. S8C).

(2) The authors provided data on Defect in CD31 expression in several models. Did they observed any other phenotypes associated with defective endothelial or vascular system? Such as, blood accumulation in pericardium, larger/smaller capillaries? Did they also examined percentage of Cdh5+ cells?

We thank the reviewer for the questions. We did not observe clear evidence of blood accumulation in the pericardium of the ablated hearts, as shown in figure 3B, 3E, 6B, and 6F. Additionally, we did not perform Cdh5 staining in either the control or ablated hearts.

(3) Please mention the sample age of Figure 2A-C.

These are single-cell mRNA sequencing data from CD1 mice across 18 developmental stages, ranging from E9.5 to P9. We have added this information to the manuscript (page 4).

(4) Please follow the same style to describe X axis in graphs in Figure 3D (and all similar graphs in manuscript) as followed in 3G.

Thank you. We assume the reviewer was referring to the descriptions in the relevant figure legends. We have updated the legend for Figure 3D to ensure consistency with the description provided for Figure 3G (page 15).

(5) It is important to provide echocardiographic M mode images with a comparable number of cardiac cycles in control and ablated (Fig. 6H).

We thank the reviewer for the comment. As explained in our previous response, the echocardiographic data for both control and mutant mice were collected in conscious animals. The differences in their cardiac cycles reflect variations in heart rate, which represent a disease phenotype and cannot be altered. Therefore, we are unable to provide M-mode images with a similar number of cardiac cycles for control and ablated mice.

(6) In the long-term neonatal ablation experiments, collagen expressions return to normal. The manuscript attributes this to possible "compensatory expression," Do they have any thoughts how this is regulated? Are other cell types stepping in, or are surviving FBs proliferating?

We thank the reviewer for the question. As suggested, the compensatory collagen expression could be driven by surviving fibroblasts or other cell types. Since we currently lack evidence to exclude either possibility, we believe both could be contributing factors.

(7) While collagen is shown to be a dominant signaling molecule, its centrality is inferred primarily from scRNAseq and ligand-receptor predictions. Did authors try any functional rescue experiment (e.g., exogenous collagen supplementation or receptor blockade) to directly validate this pathway's role in vivo?

We thank the reviewer for the comment. As noted in our previous revision in response to similar questions from the other two reviewers, we agree that these rescue experiments are of interest but are beyond the scope of the current study. We plan to pursue these investigations in future work and share our findings when available.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript "Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation" by Cho and colleagues investigates de novo tight junction formation during the differentiation of immortalized human HaCaT keratinocytes to granular-like cells, as well as during epithelial remodeling that occurs upon the apoptotic of individual cells in confluent monolayers of the representative epithelial cell line EpH4. The authors demonstrate the involvement of Rho-ROCK with well-conducted experiments and convincing images. Moreover, they unravel the underlying molecular mechanism, with Rho-ROCK activity activating the transmembrane serine protease Matriptase, which in turn leads to the cleavage of EpCAM and TROP2, respectively, releasing Claudins from EpCAM/TROP2/Claudin complexes at the cell membrane to become available for polymerization and de novo tight junction formation. These functional studies in the two different cell culture systems are complemented by localization studies of the according proteins in the stratified mouse epidermis in vivo.

In total, these are new and very intriguing and interesting findings that add important new insights into the molecular mechanisms of tight junction formation, identifying Matriptase as the "missing link" in the cascade of formerly described regulators. The involvement of TROP2/EpCAM/Claudin has been reported recently (Szabo et al., Biol. Open 2022; Bugge lab), and Matriptase had been formerly described to be required for in tight junction formation as well, again from the Bugge lab. Yet, the functional correlation/epistasis between them, and their relation to Rho signaling, had not been known thus far.

However, experiments addressing the role of Matriptase require a little more work.

Strengths:

Convincing functional studies in two different cell culture systems, complemented by supporting protein localization studies in vivo. The manuscript is clearly written and most data are convincingly demonstrated, with beautiful images and movies.

Weaknesses:

The central finding that Rho signaling leads to increased Matriptase activity needs to be more rigorously demonstrated (e.g. western blot specifically detecting the activated version or distinguishing between the full-length/inactive and processed/active version).

First, we thank the reviewer for their fair evaluation of our manuscript and for providing constructive feedback. Regarding the detection of matriptase activation—which Reviewer 1 identified as a weakness—we fully agree that direct validation is crucial. Therefore, in this revision we have carried out additional experiments using the M69 antibody, which specifically recognizes the activated form of matriptase. Details of these new experiments are provided in our point-by-point responses below.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho-mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also for directly controlling tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescent imaging of fixed and live cells together with genetic and drug-mediated interference to show that Rho activation is required and sufficient to form novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease Matriptase which cleaves EpCAM and TROP2, two claudin-binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

Strengths:

The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue-specific expression of EpCAM and TROP2. The authors present careful state-of-the-art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well-written and easy to follow.

Weaknesses:

Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho-mediated Matriptase activation has been nicely demonstrated it remains unclear how Rho activates Matriptase.

Thank you for your valuable feedback on our manuscript. As Reviewer 2 points out, the precise mechanism by which the Rho/ROCK pathway activates matriptase remains unclear. We have discussed the possible molecular mechanisms in the Discussion section. Elucidating the detailed mechanism of matriptase activation will be the focus of our future work.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Comment 1-1 - Matriptase activation by Rho: The authors show activation of Matriptase in western blots by the simple reduction of (full-length?) protein level in Figures 5 and 7. Most publications however show activated Matriptase either by antibodies detecting specifically the active form (including the publication referenced in this manuscript), or the appearance of the activated form next to the inactive form (based on different molecular weights). Therefore, it is not completely clear whether the treatment with Rho activators (Figure 5) results in an overall decrease of Matriptase, or really in an increase in the activated form. Therefore, the authors should show the actual increase of the active form. As a control, the impact of camostat treatment and overexpression of Hai1 on the active form of Matriptase could be included. It also should be indicated in the figure legend how long cells had been treated with the drugs before being subjected to lysis. Moreover, the western blots need to be quantified.

We performed a more rigorous analysis using the M69 antibody, which specifically recognizes the activated form of matriptase and has been widely used in previous studies（e.g. Benaud et al., 2001; Hung et al., 2004; Wang et al., 2009）. We likewise confirmed a significant increase in M69 signals by both western blotting and immunostaining from samples in which matriptase was activated by acid medium treatment (Figure 5A). Crucially, we also observed matriptase activation with the M69 antibody both in Rho/ROCK activator-treated cells (Figure 5A) and in differentiated granular-layer-like cells (Figures 7A and 7D). These findings strongly support the conclusion that matriptase is activated downstream of the Rho/ROCK pathway.

Comment 1-2 - Based on their results, the authors conclude that Matriptase cleaves TROP2 in the SG2 layer of the epidermis, which is a little contradictory to former studies, which have shown Matriptase to be most prominently expressed and active in the basal layer and only little in the spinous layer (e.g Chen et al., Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes. J Invest Dermatol.2013). In this light, one could also argue that inhibiting Matriptase "simply" reduces epidermal differentiation. Can other differentiation markers be tested to rule that the effects on tight junctions are secondary consequences of interferences with earlier / more global steps of keratinocyte differentiation?

As the reviewer noted, previous studies have demonstrated that matriptase is essential for keratinocyte differentiation, and that it cleaves substrates beyond EpCAM and TROP2—any of which could potentially influence the differentiation process. To test this possibility, we chose to monitor maturation of adherens junction (AJ) as an indicator of keratinocyte differentiation into granular-layer cells. Prior work has shown that during differentiation into granular-layer cells, AJs develop and experience increased intercellular mechanical tension, and that this rise in mechanical tension at AJs is critical for subsequent TJ formation (Rübsam et al., 2017). To assess AJ tension, we stained with the α-18 monoclonal antibody, which specifically recognizes the tension-dependent conformational change of α-catenin, a core AJ component. In control cells, differentiation into granular-layer like cells led to a marked increase in α-18 signal at cell–cell adhesion sites. Importantly, when HaCaT cells were treated with Camostat to inhibit matriptase and then induced to differentiate, we observed an equivalent increase in α-18 signal at AJs (Figure 7F). However, we did not detect claudin enrichment at cell-cell contacts under these conditions (Figures 7F and 7H). These results suggest that matriptase inhibition does not impair AJ maturation during granular-layer differentiation, but does profoundly disrupt TJ formation. While we cannot rule out the possibility that matriptase acts more broadly from these results, we judged that a comprehensive substrate survey lies outside the scope of the present manuscript.

Comment 1-3 - In addition, as in Figure 5, full-length levels of Matriptase in Figure 7A need to be complemented by the active version to demonstrate more convincingly that TROP2 processing coincides with (and is most likely caused by) increased Matriptase activation. In the quantification in 7B, levels actually go up again after 2 and 4 hours. How is that explained, and what would this mean with respect to tight junction formation seen at 24 h of differentiation? The TROP2 cleavage shown in Figure 7A should be quantified.

This comment is related to Comment 1-1. Using the M69 antibody, which specifically recognizes the activated matriptase, we directly demonstrated that matriptase activation occurs during the differentiation of granular layer-like cells (Figures 7A and 7D). Furthermore, we performed quantitative analysis of TROP2 cleavage and found that, compared with undifferentiated cells, differentiation into granular-layer like cells was accompanied by an increase in the cleaved TROP2 fragments (Figures 7A and 7B).

Minor points:

Comment 1-4 - Figure 1B and C: Including orthogonal views would be a nice add-on to appreciate the findings.

In the revised version, we have added the corresponding orthogonal views to Figure 1B and Figure 1C.

Comment 1-5 - Figure 2D: last row: indication of orthogonal view.

We stated that the bottom panels are orthogonal views in the figure legend of Figure 2D.

Comment 1-6 - Figure 3A: quantification is missing. GST-Rhotekin assay is missing in methods.

In the revised manuscript, we have added quantitative analysis for Figure 3A. We have also supplemented the Materials and Methods section with detailed information on the GST–Rhotekin assay used to quantify levels of active RhoA.

Comment 1-7 - Figure 4H: quantification of the Western blot is missing.

In the revised manuscript, we have added quantitative analysis for Figure 4H as Figure 4I.

Comment 1-8 - Figure 5 and 6: Quantifications of Western blots are missing.

In the revised manuscript, we have added quantitative analyses for Figure 5D as Figure 5F and for Figure 6A as Figure 6B.

Comment 1-9 - Figure 7C: quantification of the Western blot is missing.

Figure 7C does not present western blotting data. For the other western blotting results, we have added quantitative analyses as suggested by Reviewer 1.

Comment 1-10 - Figure 8I: Including Hai1 overexpression would be good for a complete picture.

Following Reviewer 1’s suggestion, we have added staining data for Hai1-overexpressing cells to Figure 8J.

Comment 1-11 - Line 377: The authors say they found Matriptase always present in lateral membranes. I did not find evidence for this in the manuscript.

Previous studies have demonstrated that in polarized epithelial cells, matriptase is localized to the basolateral membrane below TJs (Buzza et al., 2010; Wang et al., 2009). We also found that matriptase consistently localizes to the basolateral membrane but more crucially that it becomes activated there during differentiation into granular layer cells. We added these new data as Figures 7C-7E in the revised manuscript. These findings suggest that matriptase activation occurs without a change in its subcellular localization.

Comment 1-12 - Line 381: should most likely say: and ADAM17 but it is not known whether...

We corrected the sentence in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

The authors have added a significant number of quantifications verifying their observations, which was a major comment in a previous version of the manuscript and thus I have only a few minor comments which should be addressed.

Comment 2-1 - It is not required to have scale bars in every image of a panel if the same scale is used.

Unnecessary scale bars were removed. Specifically, scale bars were removed from Figure 1B, 1C, 1F, 8F, 8G, and 8H.

Comment 2-2 - Throughout all figures: Please state for non-quantified images whether this is a representative example and for how many technical or biological repeats this is representative. Also for "N" number, state what the N stands for and if this is what the dots in the graph represent. Are these the number of junctions or technical, experimental or biological repeats?

In the revised manuscript, we have added the number of independent experiments and corresponding “N” values to the Quantification and Statistical Analysis subsection of the Materials and Methods.

Comment 2-3 - Some Zooms have a scale bar (6d), and some do not (e.g. 5b).

The scale bar was removed from the magnified image in Figure 6D.

Reviewer #3 (Public review):

Summary:

The authors investigated the assembly and polar localization of the chemosensory cluster in P. aeruginosa. They discovered that a certain protein (FlhF) is required for the polar localization of the chemosensory cluster while a fully-assembled motor is necessary for the assembly of the cluster. They found that flagella and chemosensory clusters always co-localize in the cell; either at the cell pole in wild type cells or randomly-located in the cell in FlhF mutant cells. They hypothesize that this co-localization is required to keep the level of another protein (CheY-P), which controls motor switching, at low levels as the presence of high-levels of this protein (if the flagella and chemosensory clusters were not co-localized) is associated with high-levels of c-di-GMP and cell aggregations.

Strengths:

The manuscript is clearly written and straightforward. The authors applied multiple techniques to study the bacterial motility system including fluorescence light microscopy and gene editing. In general, the work enhances our understanding of the subtlety of interaction between the chemosensory cluster and the flagellar motor to regulate cell motility.

Weaknesses:

The major weakness for me in this paper is that the authors never discussed how the flagellar genes expression is controlled in P. aeruginosa. For example, in E. coli there is a transcriptional hierarchy for the flagellar genes (early, middle, and late genes, see Chilcott and Hughes, 2000). Similarly, Campylobacter and Helicobacter have a different regulatory cascade for their flagellar genes (See Lertsethtakarn, Ottemann, and Hendrixson, 2011). How does the expression of flagellar genes in P. aeruginosa compare to other species? how many classes are there for these genes? is there a hierarchy in their expression and how does this affect the results of the FliF and FliG mutants? In other words, if FliF and FliG are in class I (as in E. coli) then their absence might affect the expression of other later flagellar genes in subsequent classes (i.e., chemosensory genes). Also, in both FliF and FliG mutants no assembly intermediates of the flagellar motor are present in the cell as FliG is required for the assembly of FliF (see Hiroyuki Terashima et al. 2020, Kaplan et al. 2019, Kaplan et al. 2022). It could be argued that when the motor is not assembled then this will affect the expression of the other genes (e.g., those of the chemosensory cluster) which might play a role in the decreased level of chemosensory clusters the authors find in these mutants.

Comments on revisions:

I believe the authors have performed additional experiments that improved their manuscript and they have answered many of my comments and those of the other reviewers. I am supportive of publishing this manuscript, but I still find the following points that are not clear to me (probably I am misunderstanding some points; the authors can clarify).

(1) In response to reviewer 1, the authors say that they "analyzed and categorized the distribution of the chemotaxis complex in both wild-type and flhF mutant strains into three patterns: precise-polar, near-polar, and mid-cell localization." I can see what they mean by polar and mid-cell, but near-polar sounds a bit elusive? Can they provide examples of this stage and mention how accurately they can identify it? Also, do the pie charts they show in Figure S4 really show "significant alterations"? There is a difference between 98% and 85% as they mention in their response to reviewer 1, but I am not sure that this is significant? Probably they can explain/change the language in the text? Also, the number of cells they counted for FlhF mutant is more than the double of other strains (WT and FlhF FliF mutant)?

(2) One thing that also confused me is the following: One point that the authors stress is that FlhF localizes both the flagellum and the chemoreceptors to the pole. However, if I look at Figure 2B, the flagellum and the chemoreceptors still co-localize together (although not at the pole). If FlhF was responsible for co-localizing both of them to the pole, then wouldn't one expect them to be randomly localized in this mutant and by that I mean that they do not co-localize but that each of them (the flagellum and the chemoreceptors) are located in a different random location of the cell (not co-localized). The fact that they are still co-localized together in this mutant could also be interpreted by, for example, that FlhF localizes the flagellum to the pole and another mechanism localizes the chemoreceptors to the flagellum, hence, they still co-localize in this mutant because the chemoreceptors follow the flagellum by another mechanism to wherever it goes?

(3) In the response to reviewers, the authors mention "suggesting that the assembly of the receptor complex is likely influenced mainly by the C-ring and MS-ring structures rather than by the P ring" . However, in the article, they still write "The complete assembly of the motor serves as a partial prerequisite for the assembly of the chemotaxis complex, and its assembly site is also regulated by the polar anchor protein FlhF" despite their FlgI results which is not in accordance with this statement? Also, As I mentioned in my previous report, in FliG and FliF mutant the motor does not assemble (see Hiroyuki Terashima et al. 2020., and Kaplan et al., 2022).

(4) The authors have said in their response to my point "and currently, there is no evidence that FliA activity is influenced by proteins like FliG". I just want to clarify what I meant in my previous report: In E. coli, FliA binds to FlgM, and when the hook is assembled FlgM is secreted outside the cell allowing FliA to trigger the transcription of class III genes, which include the chemosensory genes (see Figure 5 in Beeby et al, 2020 in FEMS Microbiology, and Chilcott and Hughes, 2000). This implies that if the hook is not built, then late genes (including the chemoreceptors) should not be present. However, in Kaplan et al., 2019, the authors imaged a FliF mutant in Shewanella oneidensis (Figure S3) and still saw that chemoreceptors are present (I believe the authors must highlight this). This suggests that species such as Shewanella and Pseudomonas have a different assembly process than that E. coli, and although the authors say that in the text, I believe they still can refine this part more in the spirit of what I wrote here.

I do not like to ask for additional experiments in the second round of review, so for me if the authors modify the text to tackle these points and allow for probable alternative explanations/ highlight gaps/ modify language used for some claims, then that is fine with me.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The study by Wu et al presents interesting data on bacterial cell organization, a field that is progressing now, mainly due to the advances in microscopy. Based mainly on fluorescence microscopy images, the authors aim to demonstrate that the two structures that account for bacterial motility, the chemotaxis complex and the flagella, colocalize to the same pole in Pseudomonas aeruginosa cells and to expose the regulation underlying their spatial organization and functioning.

Strengths:

The subject is of importance.

Weaknesses:

The conclusions are too strong for the presented data. The lack of statistical analysis makes this paper incomplete. The novelty of the findings is not clear.

We have strengthened the data analysis by including appropriate statistical tests to support our conclusions more convincingly. Additionally, we have refined the description of the research background to better emphasize the novelty and significance of our findings. Please see the detailed responses below for further information.

Major issues:

(1) The novelty is in question since in the Abstract the authors highlight their main finding, which is that both the chemotaxis complex and the flagella localize to the same pole, as surprising. However, in the Introduction they state that "pathway-related receptors that mediate chemotaxis, as well as the flagellum are localized at the same cell pole17,18". I am not a pseudomonas researcher and from my short glance at these references, I could not tell whether they report colocalization of the two structures to the same pole. However, I trust the authors that they know the literature on the localization of the chemotaxis complex and flagella in their organism. See also major issue number 5 on the novelty regarding the involvement of c-di-GMP.

We thank the reviewer for this valuable comment and appreciate the opportunity to clarify our statements.

Kazunobu et al. (ref. 18) used scanning electron microscopy to preliminarily characterize the flagellation pattern of Pseudomonas aeruginosa during cell division, showing that existing flagella are located at the old pole. Zehra et al. (ref. 17), through fluorescence microscopy, observed that CheA and CheY proteins in dividing cells are typically also present at the old pole. Based on these observations, we inferred in the Introduction that the chemotaxis complex and flagellum may localize to the same cell pole.

However, this inference is indirect and lacks direct live-cell evidence of colocalization, leaving its validity to be confirmed. This uncertainty was indeed the starting point and motivation for our study.

In our work, we simultaneously visualized flagellar filaments and core chemoreceptor proteins at the single-cell level in P. aeruginosa. We characterized the assembly and spatial coordination of the chemotaxis network and flagellar motor throughout the cell cycle, providing direct evidence of their colocalization and coordinated assembly. This represents a significant advance beyond prior indirect observations and supports the novelty of our study.

Accordingly, we have revised the relevant statements in lines 71-75 of the manuscript to better reflect the current state of the literature and emphasize the novelty of our direct observations.

(2) Statistics for the microscopy images, on which most conclusions in this manuscript are based, are completely missing. Given that most micrographs present one or very few cells, together with the fact that almost all conclusions depend on whether certain macromolecules are at one or two poles and whether different complexes are in the same pole, proper statistics, based on hundreds of cells in several fields, are absolutely required. Without this information, the results are anecdotal and do not support the conclusions. Due to the importance of statistics for this manuscript, strict statistical tests should be used and reported. Moreover, representative large fields with many cells should be added as supportive information.

We thank the reviewer for this important comment, which significantly improves the rigor and persuasiveness of our manuscript.

For the colocalization analyses presented in Fig. 1D and Fig. 2B, we quantified 145 and 101 cells with fluorescently labeled flagella, respectively, and observed consistent colocalization of the chemoreceptor complexes and flagella in all examined cells (now added in the figure legends). Regarding the distribution patterns of chemoreceptors shown in Fig. 3A, we have now included comprehensive statistical analyses for both wild-type and mutant strains. For each strain, more than 300 cells were analyzed across at least three independent microscopic fields, providing robust statistical power (detailed data are presented in Fig. 3C).

To further strengthen the evidence, statistical tests were applied to confirm the significance and reproducibility of our findings (Fig. 3C). In addition, representative large-field fluorescence images containing numerous cells have been added to the supplementary materials (Fig. S1 and Fig. S3).

The problem is more pronounced when the authors make strong statements, as in lines 157-158: "The results revealed that the chemoreceptor arrays no longer grow robustly at the cell pole (Figure 2A)". Looking at the seven cells shown in Figure 2A, five of them show polar localization of the chemoreceptors. The question is then: what is the percentage of cells that show precise polar, near-polar, or mid cell localization (the three patterns shown here) in the mutant and in the wild type? Since I know that these three patterns can also be observed in WT cells, what counts is the difference, and whether it is statistically significant.

We thank the reviewer for raising this important point. Following the reviewer's suggestion, we have now analyzed and categorized the distribution of the chemotaxis complex in both wild-type and flhF mutant strains into three patterns: precise-polar, near-polar, and mid-cell localization. For each strain, more than 200 cells across three independent fields of view were quantified.

Our statistical analysis shows that in the wild-type strain, approximately 98% of cells exhibit precise polar localization of the chemotaxis complex. In contrast, the ΔflhF mutant displays a clear shift in distribution, with about 5% of cells showing mid-cell localization and 9.5% showing near-polar localization. These differences demonstrate a significant alteration in the spatial pattern upon flhF deletion.

We have revised the relevant text in lines 166-170 accordingly and included the detailed statistical data in the newly added Fig. S4.

Even for the graphs shown in Figures 3C and 3D, where the proportion of cells with obvious chemoreceptor arrays and absolute fluorescence brightness of the chemosensory array are shown, respectively, the questions that arise are: for how many individual cells these values hold and what is the significance of the difference between each two strains?

The number of cells analyzed for each strain is indicated in the original manuscript: 372 wild-type cells (line 123), 221 ΔflhF cells (line 172), 234 ΔfliG cells (line 197), 323 ΔfliF cells (line 200), 672 ΔflhFΔfliF cells (line 202), and 242 ΔmotAΔmotCD cells (line 207). For each strain, data were collected from three independent fields of view. We have now also provided the number of cells in Fig. 3 legend.

We have now performed statistical comparisons using t-tests between strains. Notably, the measured values in Fig. 3C exhibit a clear, monotonic decrease with successive gene knockouts, supporting the robustness of the observed trend.

Regarding the absolute fluorescence intensity shown in the original Fig. 3D, the mutants did not display consistent directional changes compared to the wild type. Reliable comparison of absolute fluorescence intensity requires consistent fluorescent protein maturation levels across strains. Given the likely variability in maturation levels between strains, we concluded that this data may not accurately reflect true differences in protein concentrations. Therefore, we have removed the fluorescence intensity graph from the revised manuscript to avoid potential misinterpretation.

(3) The authors conclude that "Motor structural integrity is a prerequisite for chemoreceptor self-assembly" based on the reduction in cells with chemoreceptor clusters in mutants deleted for flagellar genes, despite the proper polar localization of the chemotaxis protein CheY. They show that the level of CheY in the WT and the mutant strains is similar, based on Western blot, which in my opinion is over-exposed. "To ascertain whether it is motor integrity rather than functionality that influences the efficiency of chemosensory array assembly", they constructed a mutant deleted for the flagella stator and found that the motor is stalled while CheY behaves like in WT cells. The authors further "quantified the proportion of cells with receptor clusters and the absolute fluorescence intensity of individual clusters (Figures 3C-D)". While Figure 3DC suggests that, indeed, the flagella mutants show fewer cells with a chemotaxis complex, Figure 3D suggests that the differences in fluorescence intensity are not statistically significant. Since it is obvious that the regulation of both structures' production and localization is codependent, I think that it takes more than a Western blot to make such a decision.

We thank the reviewer for the suggestions. To further clarify that the assembly of flagellar motors and chemoreceptor clusters occurs in an orderly manner rather than being merely codependent, we performed additional experiments. Specifically, we constructed a ΔcheA mutant strain, in which chemoreceptor clusters fail to assemble. Using in vivo fluorescent labeling of flagellar filaments, we observed that the proportion of cells with flagellar filaments in the ΔcheA strain was comparable to that of the wild type (Fig. S5).

In contrast, mutants lacking complete motor structures, such as ΔfliF and ΔfliG, showed a significant reduction in the proportion of cells with obvious receptor clusters (Fig. 3C). Based on these results, we conclude that the structural integrity of the flagellar motor is, to a certain extent, a prerequisite for the self-assembly of chemoreceptor clusters.

Accordingly, we have revised the relevant statement in lines 213-217 of the manuscript to reflect this clarification.

(4) I wonder why the authors chose to label CheY, which is the only component of the chemotaxis complex that shuttles back and forth to the base of the flagella. In any case, I think that they should strengthen their results by repeating some key experiments with labeled CheW or CheA.

We thank the reviewer for this valuable suggestion. In our study, we initially focused on the positional relationship between chemoreceptor clusters and flagella, then investigated factors influencing cluster distribution and assembly efficiency. The physiological significance of motor and cluster co-localization was ultimately proposed with CheY as the starting point.

Previous work by Harwood's group demonstrated that both CheY-YFP and CheA-GFP localize to the old poles of dividing Pseudomonas aeruginosa cells. Since our physiological hypothesis centers on CheY, we chose to label CheY-EYFP in our experiments.

To further strengthen our conclusions, we constructed a plasmid expressing CheA-CFP and introduced it into the cheY-eyfp strain via electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP (Fig. S2), confirming that CheY-EYFP accurately marks the location of the chemoreceptor complex.

We have revised the manuscript accordingly (lines 119-123) and added these data as Fig. S2.

(5) The last section of the results is very problematic, regarding the rationale, the conclusions, and the novelty. As far as the rationale is concerned, I do not understand why the authors assume that "a spatial separation between the chemoreceptors and flagellar motors should not significantly impact the temporal comparison in bacterial chemotaxis". Is there any proof for that?

We apologize for the lack of clarity in our original explanation. The rationale behind the statement was initially supported by comparing the timescales of CheY-P diffusion and temporal comparison in chemotaxis. Specifically, the diffusion time for CheY-P to traverse the entire length of a bacterial cell is approximately 100 ms (refs 39&40), whereas the timescale for bacterial chemotaxis temporal comparison is on the order of seconds (ref 41).

To clarify and strengthen this argument, we have expanded the discussion as follows:

The diffusion coefficient of CheY in bacterial cells is about 10 µm2/s, which corresponds to an estimated end-to-end diffusion time on the order of 100 ms (refs 40&41). If the chemotaxis complexes were randomly distributed rather than localized, diffusion times would be even shorter. In contrast, the timescale for the chemotaxis temporal comparison is on the order of seconds (ref. 42). Additionally, a study by Fukuoka and colleagues reported that intracellular chemotaxis signal transduction requires approximately 240 ms beyond CheY or CheY-P diffusion time (ref. 41). Moreover, the intervals of counterclockwise (CCW) and clockwise (CW) rotation of the P. aeruginosa flagellar motor under normal conditions are 1-2 seconds, as determined by tethered cell or bead assays (refs. 30&43).

Taken together, these indicate that for P. aeruginosa, which moves via a run-reverse mode, the potential 100 ms reduction in response time due to co-localization of the chemotaxis complex and motor has a limited effect on overall chemotaxis timing.

We have revised the corresponding text accordingly (lines 238-245) to better explain this rationale.

More surprising for me was to read that "The signal transduction pathways in E. coli are relatively simple, and the chemotaxis response regulator CheY-P affects only the regulation of motor switching". There are degrees of complexity among signal transduction pathways in E. coli, but the chemotaxis seems to be ranked at the top. CheY is part of the adaptation. Perfect adaptation, as many other issues related to the chemotaxis pathway, which include the wide dynamic range, the robustness, the sensitivity, and the signal amplification (gain), are still largely unexplained. Hence, such assumptions are not justified.

We apologize for the confusion and imprecision in our original statements. Our intention was to convey that the chemotaxis pathway in E. coli is relatively simple compared to the more complex chemosensory systems in P. aeruginosa. We did not mean to generalize this simplicity to all signal transduction pathways in E. coli.

We acknowledge that E. coli chemotaxis is a highly sophisticated system, involving processes such as perfect adaptation, wide dynamic range, robustness, sensitivity, and signal amplification, many aspects of which remain incompletely understood. CheY indeed plays a crucial role in adaptation and motor switching regulation.

Accordingly, we have revised the original text (lines 249-255) to avoid any misunderstanding.

More perplexing is the novelty of the authors' documentation of the effect of the chemotaxis proteins on the c-di-GMP level. In 2013, Kulasekara et al. published a paper in eLife entitled "c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility". In the same year, Kulasekara published a paper entitled "Insight into a Mechanism Generating Cyclic di-GMP Heterogeneity in Pseudomonas aeruginosa". The authors did not cite these works and I wonder why.

We apologize for having been unaware of these important references and thank the reviewer for bringing them to our attention. We have now cited the eLife paper and the PhD thesis titled "Insight into a Mechanism Generating Cyclic di-GMP Heterogeneity in Pseudomonas aeruginosa" by Kulasekara et al.

Regarding novelty, there are key differences between our findings and those reported by Kulasekara et al. While they proposed that CheA influences c-di-GMP heterogeneity through interaction with a specific phosphodiesterase (PDE), our results demonstrate that overexpression of CheY leads to an increase in intracellular c-di-GMP levels.

We have revised the original text accordingly (lines 358-362) to clarify these distinctions.

(6) Throughout the manuscript, the authors refer to foci of fluorescent CheY as "chemoreceptor arrays". If anything, these foci signify the chemotaxis complex, not the membrane-traversing chemoreceptors.

We thank the reviewer for this clarification. We have revised the manuscript accordingly to refer to the fluorescent CheY foci as representing the chemotaxis complex rather than the chemoreceptor arrays.

Conclusions:

The manuscript addresses an interesting subject and contains interesting, but incomplete, data.

Reviewer #2 (Public Review):

Summary:

Here, the authors studied the molecular mechanisms by which the chemoreceptor cluster and flagella motor of Pseudomonas aeruginosa (PA) are spatially organized in the cell. They argue that FlhF is involved in localizing the receptors-motor to the cell pole, and even without FlhF, the two are colocalized. FlhF is known to cause the motor to localize to the pole in a different bacterial species, Vibrio cholera, but it is not involved in receptor localization in that bacterium. Finally, the authors argue that the functional reason for this colocalization is to insulate chemotactic signaling from other signaling pathways, such as cyclic-di-GMP signaling.

Strengths:

The experiments and data look to be high-quality.

Weaknesses:

However, the interpretations and conclusions drawn from the experimental observations are not fully justified in my opinion.

I see two main issues with the evidence provided for the authors' claims.

(1) Assumptions about receptor localization:

The authors rely on YFP-tagged CheY to identify the location of the receptor cluster, but CheY is a diffusible cytoplasmic protein. In E. coli, CheY has been shown to localize at the receptor cluster, but the evidence for this in PA is less strong. The authors refer to a paper by Guvener et al 2006, which showed that CheY localizes to a cell pole, and CheA (a receptor cluster protein) also localizes to a pole, but my understanding is that colocalization of CheY and CheA was not shown. My concern is that CheY could instead localize to the motor in PA, say by binding FliM. This "null model" would explain the authors' observations, without colocalization of the receptors and motor. Verifying that CheY and CheA are colocalized in PA would be a very helpful experiment to address this weakness.

We thank the reviewer for this valuable suggestion. We agree that verifying the colocalization of CheY and CheA would strengthen our conclusions. To address this, we constructed a plasmid expressing CheA-CFP and introduced it into the CheY-EYFP strain by electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP signals, indicating that CheY-EYFP indeed marks the location of the chemoreceptor complex rather than the flagellar motor.

We have revised the manuscript accordingly (lines 118-123) and included these results in the new Fig. S2.

(2) Argument for the functional importance of receptor-motor colocalization at the pole:

The authors argue that colocalization of the receptors and motors at the pole is important because it could keep phosphorylated CheY, CheY-p, restricted to a small region of the cell, preventing crosstalk with other signaling pathways. Their evidence for this is that overexpressing CheY leads to higher intracellular cdG levels and cell aggregation. Say that the receptors and motors are colocalized at the pole. In E. coli, CheY-p rapidly diffuses through the cell. What would prevent this from occurring in PA, even with colocalization?

We appreciate the reviewer's insightful question. The colocalization of both the signaling source (the kinase) and sink (the phosphatase) at the chemoreceptor complex at the cell pole results in a rapid decay of CheY-P concentration within approximately 0.2 µm from the cell pole, leading to a nearly uniform distribution elsewhere in the cell, as demonstrated by Vaknin and Berg (ref. 46). This spatial arrangement effectively confines high CheY-P levels to the pole region. When the motor is also localized at the cell pole, this reduces the need for elevated CheY-P concentrations throughout the cytoplasm, thereby minimizing potential crosstalk with other signaling pathways.

We have revised the manuscript accordingly (lines 280-286) to clarify this point.

Elevating CheY concentration may increase the concentration of CheY-p in the cell, but might also stress the cells in other unexpected ways. It is not so clear from this experiment that elevated CheY-p throughout the cell is the reason that they aggregate, or that this outcome is avoided by colocalizing the receptors and motor at the same pole. If localization of the receptor array and motor at one pole were important for keeping CheY-p levels low at the opposite pole, then we should expect cells in which the receptors and motor are not at the pole to have higher CheY-p at the opposite pole. According to the authors' argument, it seems like this should cause elevated cdG levels and aggregation in the delta flhF mutants with wild-type levels of CheY. But it does not look like this happened. Instead of varying CheY expression, the authors could test their hypothesis that receptor-motor colocalization at the pole is important for preventing crosstalk by measuring cdG levels in the flhF mutant, in which the motor (and maybe the receptor cluster) are no longer localized in the cell pole.

We thank the reviewer for raising the important point regarding potential cellular stress caused by elevated CheY concentrations, as well as for the suggestion to test the hypothesis using ΔflhF mutants.

First, as noted above, CheY-P concentration rapidly decreases away from the receptor complex. While deletion of flhF alters the position of the receptor complex, thereby shifting the region of high CheY-P concentration, it does not increase CheY-P levels elsewhere in the cell. Importantly, in the ΔflhF strain, the receptor complex and the motor still colocalize, so this mutant may not effectively test the role of receptor-motor colocalization in preventing crosstalk as suggested.

Regarding the possibility that elevated CheY levels stress the cells independently of CheY-P signaling, prior work in <i.E. coli by Cluzel et al. (ref. 11) showed that overexpressing CheY several-fold did not cause phenotypic changes, indicating that simple CheY overexpression alone may not be generally stressful. Furthermore, our data indicate that the increase in c-di-GMP levels and subsequent cell aggregation upon CheY overexpression is not an all-or-none switch but occurs progressively as CheY concentration rises.

To further confirm that CheY overexpression promotes aggregation through increased c-di-GMP levels, we performed additional experiments co-overexpressing CheY and a phosphodiesterase (PDE) from E. coli to reduce intracellular c-di-GMP. These experiments showed that PDE expression mitigates cell aggregation caused by CheY overexpression (Fig. S8).

We have revised the manuscript accordingly (lines 290-294) and added these new results in Fig. S8.

Reviewer #3 (Public Review):

Summary:

The authors investigated the assembly and polar localization of the chemosensory cluster in P. aeruginosa. They discovered that a certain protein (FlhF) is required for the polar localization of the chemosensory cluster while a fully-assembled motor is necessary for the assembly of the cluster. They found that flagella and chemosensory clusters always co-localize in the cell; either at the cell pole in wild-type cells or randomly-located in the cell in FlhF mutant cells. They hypothesize that this co-localization is required to keep the level of another protein (CheY-P), which controls motor switching, at low levels as the presence of high levels of this protein (if the flagella and chemosensory clusters were not co-localized) is associated with high-levels of c-di-GMP and cell aggregations.

Strengths:

The manuscript is clearly written and straightforward. The authors applied multiple techniques to study the bacterial motility system including fluorescence light microscopy and gene editing. In general, the work enhances our understanding of the subtlety of interaction between the chemosensory cluster and the flagellar motor to regulate cell motility.

Weaknesses:

The major weakness in this paper is that the authors never discussed how the flagellar gene expression is controlled in P. aeruginosa. For example, in E. coli there is a transcriptional hierarchy for the flagellar genes (early, middle, and late genes, see Chilcott and Hughes, 2000). Similarly, Campylobacter and Helicobacter have a different regulatory cascade for their flagellar genes (See Lertsethtakarn, Ottemann, and Hendrixson, 2011). How does the expression of flagellar genes in P. aeruginosa compare to other species? How many classes are there for these genes? Is there a hierarchy in their expression and how does this affect the results of the FliF and FliG mutants? In other words, if FliF and FliG are in class I (as in E. coli) then their absence might affect the expression of other later flagellar genes in subsequent classes (i.e., chemosensory genes). Also, in both FliF and FliG mutants no assembly intermediates of the flagellar motor are present in the cell as FliG is required for the assembly of FliF (see Hiroyuki Terashima et al. 2020, Kaplan et al. 2019, Kaplan et al. 2022). It could be argued that when the motor is not assembled then this will affect the expression of the other genes (e.g., those of the chemosensory cluster) which might play a role in the decreased level of chemosensory clusters the authors find in these mutants.

We thank the reviewer for the insightful comments. P. aeruginosa possesses a four-tiered transcriptional regulatory hierarchy controlling flagellar biogenesis. Within this system, fliF and fliG belong to class II genes and are regulated by the master regulator FleQ. In contrast, chemotaxis-related genes such as cheA and cheW are regulated by intracellular free FliA, and currently, there is no evidence that FliA activity is influenced by proteins like FliG.

To verify that the expression of core chemotaxis proteins was not affected by deletion of fliG, we performed Western blot analyses to compare CheY levels in wild-type, ΔfliF, and ΔfliG strains. We observed no significant differences, indicating that the reduced presence of receptor clusters in these mutants is not due to altered expression of chemotaxis proteins.

Accordingly, we have revised the manuscript (lines 341-348) and updated Fig. 3B to reflect these findings.

Recommendations for the authors:

Reviewing Editor (Recommendations For The Authors):

The reviewers comment on several important aspects that should be addressed, namely: the lack of statistical analysis; the need for clarifications regarding assumptions made regarding receptor localization; the functional importance of receptor-motor colocalization; and the need for an elaborate discussion of flagellar gene expression. Also, two reviewers pointed out the need to prove the co-localization of CheY and CheA; This is important since CheY is dynamic, shuttling back and forth from the chemotaxis complex to the base of the flagella, whereas CheA (or cheW or, even better, the receptors) is considered less dynamic and an integral part of the chemotaxis complex.

Reviewer #1 (Recommendations For The Authors):

Minor points:

Line 43: "ubiquitous" - I would choose another word.

We changed "ubiquitous" to "widespread".

Line 49: "order" - change to organize.

We changed "order" to "organize".

Line 52: "To grow and colonize within the host, bacteria have evolved a mechanism for migrating...". Motility "towards more favorable environments" is an important survival strategy of bacteria in various ecological niches, not only within the host.

We revised it to "grow and colonize in various ecological niches".

Line 72: Define F6 in "F6 pathway-related receptors".

The proteins encoded by chemotaxis-related genes collectively constitute the F6 pathway, which we have now explained in the manuscript text.

Line 72-73: Do references 17 &18 really report colocalization of the chemotaxis receptor and flagella to the same pole? If these or other reports document such colocalization, then the sentence in the Abstract "Surprisingly, we found that both are located at the same cell pole..." is not correct.

Kazunobu et al. (ref. 18) used scanning electron microscopy to preliminarily characterize the flagellation pattern of Pseudomonas aeruginosa during cell division, showing that existing flagella are located at the old pole. Zehra et al. (ref. 17), through fluorescence microscopy, observed that CheA and CheY proteins in dividing cells are typically also present at the old pole. Based on these observations, we inferred in the Introduction that the chemotaxis complex and flagellum may localize to the same cell pole.

However, this inference is indirect and lacks direct live-cell evidence of colocalization, leaving its validity to be confirmed. This uncertainty was indeed the starting point and motivation for our study.

In our work, we simultaneously visualized flagellar filaments and core chemoreceptor proteins at the single-cell level in P. aeruginosa. We characterized the assembly and spatial coordination of the chemotaxis network and flagellar motor throughout the cell cycle, providing direct evidence of their colocalization and coordinated assembly. This represents a significant advance beyond prior indirect observations and supports the novelty of our study.

Accordingly, we have revised the relevant statements in lines 71-75 of the manuscript to better reflect the current state of the literature and emphasize the novelty of our direct observations.

Line 108: "CheY has been shown to colocalize with chemoreceptors". The authors rely here (reference 29) and in other places on findings in E. coli. However, in the Introduction, they describe the many differences between the motility systems of P. aeruginosa and E. coli, e.g., the number of chemosensory systems and their spatial distribution (E. coli is a peritrichous bacterium, as opposed to the monotrichous bacterium P. aeruginosa). There seem to be proofs for colocalization of the Che and MCP proteins in P. aeruginosa, which should be cited here.

Thank you for pointing this out. Harwood's group reported that a cheY-YFP fusion strain exhibited bright fluorescent spots at the cell pole, which disappeared upon knockout of cheA or cheW-genes encoding structural proteins of the chemotaxis complex. This strongly suggests colocalization of CheY with MCP proteins in P. aeruginosa. We have now cited this study as reference 17 in the manuscript.

Figure 1B: Please replace the order of the schematic presentations, so that the cheY-egfp fusion, which is described first in the text, is at the top.

We have modified the order of related images in Fig. 1B.

Line 127: "by introducing cysteine mutations". Replace either by "by introducing cysteines" or by "by substituting several residues with cysteines".

We changed the relevant statement to "by introducing cysteines".

Line 144-145: "Given that the physiological and physical environments of both cell poles are nearly identical.". I think that also the physical, but certainly the physiological environment of the two poles is not identical. First, one is an old pole, and the other a new pole. Second, many proteins and RNAs were detected mainly or only in one of the poles of rod-shaped Gram-negative bacteria that are regarded as symmetrically dividing. Although my intuition is that the authors are correct in assuming that "it is unlikely that the unipolar distribution of the chemoreceptor array can be attributed to passive regulatory factors", relating it to the (false) identity between the poles is incorrect.

We thank the reviewer for this important correction. We agree that the physiological environments of the two poles are not identical, given that one is the old pole and the other the new pole, and that many proteins and RNAs show polar localization in rod-shaped Gram-negative bacteria. Accordingly, we have revised the original text (lines 150-152) to read:

“Despite potential differences in the physical and especially physiological environments at the two cell poles, it is unlikely that the unipolar distribution of the chemotaxis complex can be attributed to passive regulatory factors.”

Lines 151-154: "Considering the consistent colocalization pattern between chemosensory arrays and flagellar motors in P. aeruginosa". Does the word consistent relate to different reports on such colocalization or to the results in Figure 1D? In case it is the latter, then what is the word consistent based on? All together only 7 cells are presented in the 5 micrographs that compose Figure 1D (back to statistics...).

We thank the reviewer for raising this point. To clarify, the word "consistent" refers to the observation of colocalization shown in Figure 1D & Figure S3. As noted in the revised figure legend for Figure 1D, a total of 145 cells with labeled flagella were analyzed, all exhibiting consistent colocalization between flagella and chemosensory arrays. Additionally, we have included a new image showing a large field of co-localization in the wild-type strain as Figure S3 to better illustrate this consistency.

Figure 2A: Omit "Subcellular localization of" from the beginning of the caption.

We removed the relevant expression from the caption.

Reviewer #2 (Recommendations For The Authors):

I strongly recommend checking that CheY localizes to the receptor cluster in PA. This could be done by tagging cheA with a different fluorophore and demonstrating their colocalization. It would also be helpful to check that they are colocalized in the delta flhF mutant.

We thank the reviewer for this valuable suggestion. We constructed a plasmid expressing CheA-CFP and introduced it into the CheY-EYFP strain by electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP signals, indicating that CheY-EYFP indeed marks the location of the chemoreceptor complex.

We have revised the manuscript accordingly (lines 118-123) and included these results in the new Fig. S2.

The experiments under- and over-expressing CheY part seemed too unrelated to receptor-motor colocalization. I think the authors should think about a more direct way of testing whether colocalization of the motor and receptors is important for preventing signaling crosstalk. One way would be to measure cdG levels in WT and in delta flhF mutants and see if there is a significant difference.

We thank the reviewer for raising the important point regarding potential cellular stress caused by elevated CheY concentrations, as well as for the suggestion to test the hypothesis using flhF mutants.

First, as noted in the response to your 2nd comment in Public Review, CheY-P concentration rapidly decreases away from the receptor complex. While deletion of flhF alters the position of the receptor complex, thereby shifting the region of high CheY-P concentration, it does not increase CheY-P levels elsewhere in the cell. Importantly, in the ΔflhF strain, the receptor complex and the motor still colocalize, so this mutant may not effectively test the role of receptor-motor colocalization in preventing crosstalk as suggested.

Regarding the possibility that elevated CheY levels stress the cells independently of CheY-P signaling, prior work in E. coli by Cluzel et al. (ref. 11) showed that overexpressing CheY several-fold did not cause phenotypic changes, indicating that simple CheY overexpression alone may not be generally stressful. Furthermore, our data indicate that the increase in c-di-GMP levels and subsequent cell aggregation upon CheY overexpression is not an all-or-none switch but occurs progressively as CheY concentration rises.

To further confirm that CheY overexpression promotes aggregation through increased c-di-GMP levels, we performed additional experiments co-overexpressing CheY and a phosphodiesterase (PDE) from E. coli to reduce intracellular c-di-GMP. These experiments showed that PDE expression mitigates cell aggregation caused by CheY overexpression (Fig. S8).

We have revised the manuscript accordingly (lines 290-294) and added these new results in Fig. S8.

Reviewer #3 (Recommendations For The Authors):

(1) Can the authors elaborate more on the hierarchy of flagellar gene expression in P. aeruginosa and how this relates to their work?

We thank the reviewer for the suggestion. We have now described the hierarchy of flagellar gene expression in P. aeruginosa in lines 341-348.

(2) I would suggest that the authors check other flagellar mutants (than FliF and FliG) where the motor is partially assembled (e.g., any of the rod proteins or the P-ring protein), together with FlhF mutant, to see how a partially assembled motor affects the assembly of the chemosensory cluster.

We thank the reviewer for this valuable suggestion. The P ring, primarily composed of FlgI, acts as a bushing for the peptidoglycan layer, and its absence leads to partial motor assembly. We constructed a ΔflgI mutant and observed that the proportion of cells exhibiting distinct chemotactic complexes was similar to that of the wild-type strain, suggesting that the assembly of the receptor complex is likely influenced mainly by the C-ring and MS-ring structures rather than by the P ring. We have revised the original text accordingly (lines 217-220) and added the corresponding data as Figure S6.

(3) I would suggest that the authors check the levels of CheY in cells induced with different concentrations of arabinose (i.e., using western blotting just like they did in Figure 3B).

We have assessed the levels of CheY in cells induced with different concentrations of arabinose using western blotting, as suggested. The results have been incorporated into the manuscript (lines 274-275) and are presented in Figure S7.

(4) To my eyes, most of the foci in FliF-FlhF mutant in Figure 3A are located at the pole (which is unlike the FlhF mutant in Figure 2). Is this correct? I would suggest that the authors also investigate this to see where the chemosensory cluster is located.

We thank the reviewer for pointing this out. The distribution of the chemotaxis complex in the ΔflhFΔfliF strain was investigated and showed in Fig. S4. Indeed, most of the chemoreceptor foci in this mutant are located at the pole. This probably suggests that, in the absence of both FlhF and an assembled motor, the position of the receptor complex may be largely influenced by passive factors such as membrane curvature. This interesting possibility warrants further investigation in future studies.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary

In this work, the authors recorded the dynamics of the 5-HT with fiber photometry from CA1 in one hemisphere and LFP from CA1 in the other hemisphere. They observed an ultra-slow oscillation in the 5-HT signal during both wake fulness and NREM sleep. The authors have studied different phases of the ultra-slow oscillation to examine the potential difference in the occurrence of some behavioral state-related physiological phenomena hippocampal ripples, EMG, and inter-area coherence).

Strengths

The relation between the falling/rising phase of the ultra-slow oscillation and the ripples is sufficiently shown. There are some minor concerns about the observed relations that should be addressed with some further analysis.

Systematic observations have started to establish a strong relation between the dynamics of neural activity across the brain and measures of behavioral arousal. Such relations span a wide range of temporal scales that are heavily inter-related. Ultra-slow time-scales are specifically under-studied due to technical limitations and neuromodulatory systems are the strongest mechanistic candidates for controlling/modulating the neural dynamics at these time-scales. The hypothesis of the relation between a specific time-scale and one certain neuromodulator (5-HT in this manuscript) could have a significant impact on the understanding of the hierarchy in the temporal scales of neural activity.

Weaknesses:

One major caveat of the study is that different neuromodulators are strongly correlated across all time scales and related to this, the authors need to discuss this point further and provide more evidence from the literature (if any) that suggests similar ultra-slow oscillations are weaker or lack from similar signals recorded for other neuromodulators such as Ach and NA.

The reviewer is correct to point out that the levels of different neuromodulators are often correlated. For example, most monoaminergic neurons, including serotonergic neurons of the raphe nuclei, show similar firing rates across behavioral states, firing most during wake behavior, less during NREM, and ceasing firing during ‘paradoxical sleep’ or REM (Eban-Rothschild et al 2018). Notably, other neuromodulators, such as acetylcholine (ACh), show the opposite pattern across states, with highest levels observed during REM, an intermediate level during wake behavior, and the lowest level during NREM (Vazquez et al. 2001). Despite these differences, ultraslow oscillations of both monoaminergic and non-monoaminergic neuromodulators, have been described, albeit only during NREM sleep (Zhang et al. 2021, Zhang et al. 2024, Osorio-Ferero et al. 2021, Kjaerby et al. 2022). How ultraslow oscillations of different neuromodulators are related has been only recently explored (Zhang et al. 2024). In this study, dual recording of oxytocin (Oxt) and ACh with GRAB sensors showed that the levels of the two neuromodulators were indeed correlated at ultraslow frequencies with a 2 s temporal shift. Furthermore, this shift could be explained by a hippocampal-to-lateral septum intermediate pathway, in which the level of ACh causally impacts hippocampal activity, which then in turn controls Oxt levels. Given the known temporal relationship between ripples, ACh and Oxt, and now with our work, between ripples and 5-HT, one could infer the relative timing of ultraslow oscillations of ACh, Oxt and 5-HT. While dual recordings of norepinephrine (NE) and 5-HT have not been performed, a similar correlation with temporal shift could be hypothesized given the parallel relationships between NE and spindles (OsorioFerero et al. 2021), and 5-HT and ripples, with the known temporal delay between ripples and spindles (Staresina et al. 2023). The fact that the locus coerulus receives particularly dense projections from the dorsal raphe nucleus (Kim et al. 2004) further suggests that 5-HT ultraslow oscillations could drive NE oscillations. How exactly ultraslow oscillations of serotonin are related to ultraslow oscillations of different neuromodulators in different brain regions remains to be studied.

We have further addressed this question and how it relates to the issue of causality in the Discussion section of the manuscript (p. 13):

“In addition to the difficulties involved with typical causal interventions already mentioned, the fact that the levels of different neuromodulators are interrelated and affected by ongoing brain activity makes it very hard to pinpoint ultraslow oscillations of one specific neuromodulator as controlling specific activity patterns, such as ripple timing. While a recent paper purported to show a causative effect of norepinephrine levels on ultraslow oscillations of sigma band power, the fact that optogenetic inhibition of locus coerulus (LC) cells, but also excitation, only caused a minor reduction of the ultraslow sigma power oscillation suggests that other factors also contribute (Osorio-Forero et al., 2021). Generally, it is thought that many neuromodulators together determine brain states in a combinatorial manner, and it is probable that the 5-HT oscillations we measure, like the similar oscillations in NE, are one factor among many.

Nevertheless, given the known effects of 5-HT on neurons, it is not unlikely that the 5-HT fluctuations we describe have some impact on the timing of ripples, MAs, hippocampal-cortical coherence, or EMG signals that correlate with either the rising or descending phase. In fact, causal effects of 5-HT on ripple incidence (Wang et al. 2015, ul Haq et al. 2016 and Shiozaki et al. 2023), MA frequency (Thomas et al. 2022), sensory gating (Lee et al. 2020), which is subserved by inter-areal coherence (Fisher et al. 2020), and movement (Takahashi et al. 2000, Alvarez et al. 2022, Jacobs et al. 1991 and Luchetti et al. 2020) have all been shown. Our added findings that serotonin affects ripple incidence in hippocampal slices in a dose-dependent manner (Figure S1) further suggests that the relationship between ultraslow 5-HT oscillations and ripples we report may indeed result, at least in part, from a direct effect of serotonin on the hippocampal network.

Whether these ‘causal’ relationships between 5-HT and the different activity measures we describe can be used to support a causal link between ultraslow 5-HT oscillations and the correlated activity we report remains an open question. To that point, some studies have described changes in ultraslow oscillations due to manipulation of serotonin signaling. Specifically, reduction of 5-HT1a receptors in the dentate gyrus was recently shown to reduce the power of ultraslow oscillations of calcium activity in the same region (Turi et al. 2024). Furthermore, psilocin, which largely acts on the 5-HT2a receptor, decreased NREM episode length from around 100 s to around 60 s, and increased the frequency of brief awakenings (Thomas et al. 2022). While ultraslow oscillations were not explicitly measured in this study, the change in the rhythmic pattern of NREM sleep episodes and brief awakenings, or microarousals, suggests an effect of psilocin on ultraslow oscillations during NREM. Although these studies do not necessarily point to an exclusive role for 5-HT in controlling ultraslow oscillations of different brain activity patterns, they show that changes in 5-HT can contribute to changes in brain activity at ultraslow frequencies.”

A major question that has been left out from the study and discussion is how the same level of serotonin before and after the peak could be differentially related to the opposite observed phenomenon. What are the possible parallel mechanisms for distinguishing between the rising and falling phases? Any neurophysiological evidence for sensing the direction of change in serotonin concentration (or any other neuromodulator), and is there any physiological functionality for such mechanisms?

We have added a paragraph in the discussion to address how this differentiation of the 5-HT signal may be carried out (Discussion, paragraph #3, p. 10):

“In order for the ultraslow oscillation phase to segregate brain activity, as we have observed, the hippocampal network must somehow be able to sense the direction of change of serotonin levels. While single-cell mechanisms related to membrane potential dynamics are typically too fast to explain this calculation, a theoretical work has suggested that feedback circuits can enable such temporal differentiation, also on the slower timescales we observe (Tripp and Eliasmith, 2010). Beyond the direction of change in serotonin levels, temporal differentiation could also enable the hippocampal network to discern the steeper rising slope versus the flatter descending slope that we observe in the ultraslow 5-HT oscillations (Figure S2), which may also be functionally relevant (Cole and Voytek, 2017). The distinction between the rising and falling phase of ultraslow oscillations is furthermore clearly discernible at the level of unit responses, with many units showing preferences for either half of the ultraslow period (Figure S6). Another factor that could help distinguish the rising from the falling phase is the level of other neuromodulators, as it is likely the combination of many neuromodulators at any given time that defines a behavioral substate. Given the finding that ACh and Oxt exhibit ultraslow oscillations with a temporal shift (Zhang et al. 2024), one could posit that distinct combinations of different levels of neuromodulators could segregate the rising from the falling phase via differential effects of the combination of neuromodulators on the hippocampal network.”

Functionally, the ability to distinguish between the rising and falling phases of an oscillatory cycle is a form of phase coding. A well-known example of this can be seen in hippocampal place cells, which fire relative to the ongoing theta oscillations. The key advantage of phase coding is that it introduces an additional dimension, i.e. phase of firing, beyond the simple rate of neural firing. This allows for the multiplexing of information (Panzeri et al., 2010), enabling the brain to encode more complex patterns of activity. Moreover, phase coding is metabolically more efficient than traditional spike-rate coding (Fries et al., 2007).

Reviewer #2 (Public review):

Summary:

In their study, Cooper et al. investigated the spontaneous fluctuations in extracellular 5-HT release in the CA1 region of the hippocampus using GRAB5-HT3.0. Their findings revealed the presence of ultralow frequency (less than 0.05 Hz) oscillations in 5-HT levels during both NREM sleep and wakefulness. The phase of these 5-HT oscillations was found to be related to the timing of hippocampal ripples, microarousals, electromyogram (EMG) activity, and hippocampal-cortical coherence. In particular, ripples were observed to occur with greater frequency during the descending phase of 5-HT oscillations, and stronger ripples were noted to occur in proximity to the 5-HT peak during NREM. Microarousal and EMG peaks occurred with greater frequency during the ascending phase of 5-HT oscillations. Additionally, the strongest coherence between the hippocampus and cortex was observed during the ascending phase of 5-HT oscillations. These patterns were observed in both NREM sleep and the awake state, with a greater prevalence in NREM. The authors posit that 5-HT oscillations may temporally segregate internal processing (e.g., memory consolidation) and responsiveness to external stimuli in the brain.

Strengths:

The findings of this research are novel and intriguing. Slow brain oscillations lasting tens of seconds have been suggested to exist, but to my knowledge they have never been analyzed in such a clear way. Furthermore, although it is likely that ultra-slow neuromodulator oscillations exist, this is the first report of such oscillations, and the greatest strength of this study is that it has clarified this phenomenon both statistically and phenomenologically.

Weaknesses:

As with any paper, this one has some limitations. While there is no particular need to pursue them, I will describe ten of them below, including future directions:

(1) Contralateral recordings: 5-HT levels and electrophysiological recordings were obtained from opposite hemispheres due to technical limitations. Ipsilateral simultaneous recordings may show more direct relationships.

Although we argue that bilateral symmetry defines both the serotonin system and many hippocampal activity patterns (Methods: Dual fiber photometry and silicon probe recordings), we agree that ipsilateral recordings would be superior to describe the link between serotonin and electrophysiology in the hippocampus. In addition to noting that a recent study has adopted the same contralateral design (Zhang et al. 2024), we add a reference further supporting bilateral hippocampal synchrony, specifically of dentate spikes (Farrell et al. 2024). However, as functional lateralization has been recently proposed to underlie certain hippocampal functions in the rodent (Jordan 2020), future studies should ideally include both imaging and electrophysiology in a single hemisphere to guarantee local correlations rather than assuming inter-hemispheric synchrony. This could be accomplished using an integrated probe with attached optical fibers, as described in Markowitz et al. 2018, which is however technically more challenging and has, to our knowledge, not yet been implemented with fiber photometry recordings with GRAB sensors. Given the required separation of a few hundred micrometers between the probe shanks and the optical fiber cannula, it is important to consider whether the recordings are capturing the same neuronal populations. For example, there is a risk of recording electrical activity from dorsal hippocampal neurons while simultaneously measuring light signals from neurons in the intermediate hippocampus, which are functionally distinct populations (Fanselow and Dong 2009).

(2) Sample size: The number of mice used in the experiments is relatively small (n=6). Validation with a larger sample size would be desirable.

While larger sample sizes generally reduce the influence of random variability and minimize the impact of outliers on conclusions, our use of mixed-effects models mitigates these concerns by accounting for both inter-session and inter-mouse variability. With this approach, we explicitly model random effects, such as the variability between individual mice and sessions, alongside fixed effects (such as treatment), which ensures that our results are not driven by random fluctuations in a few individual mice or sessions. Furthermore, the inclusion of random intercepts and slopes in the models allows for the possibility that different animals and/or sessions have different baseline characteristics and respond to different degrees of magnitude to the treatment. In summary, while validating these findings with a larger sample size would certainly help detect more subtle effects, we are confident in the robustness of the conclusions presented.

(3) Lack of causality: The observed associations show correlations, not direct causal relationships, between 5-HT oscillations and neural activity patterns.

We agree that the data we present in this study is largely correlational and generally avoid claims of causality in the manuscript. In the Discussion section, we discuss barriers to interpreting typical causal interventions in vivo, such as optogenetic activation of raphe nuclei: “The two previously mentioned in vivo studies showing reduced ripple incidence…”(paragraph #10, pg. 12), as well as an added section on further causality considerations in the Discussion section of the manuscript (paragraph #12, pg. 13): “In addition to the difficulties involved with…”

Due to these barriers, as a first step, we wanted to describe how physiological changes in serotonin levels are correlated to changes in the hippocampal activity. Equipped with a deeper understanding of physiological serotonin dynamics, future studies could explore interventions that modulate serotonin in keeping with the natural range of serotonin fluctuations for a given state. On that point, another challenge which we have not mentioned in the manuscript is that modulating serotonin, or any neuromodulator’s levels, has the potential, depending on the degree of modulation, to transition the brain to an entirely different behavioral state. This then complicates interpretation, as one is not sure whether effects observed are due to the changes in the neuromodulator itself, or secondary to changes in state. At the same time, 5-HT activity drives networks which in return can change the release of other neurotransmitters, leading to indirect effects.

The results of our in vitro experiments suggest that a causal relationship between serotonin and ripples is possible (Figure S1). Though the hippocampal slice preparation is clearly an artificial model, it provides a controlled environment to isolate the effects of serotonin manipulation on the hippocampal formation, without the confounding influence of systemic 5-HT fluctuations in other brain regions. Notably, the dose-dependent effects of serotonin (5-HT) wash-in on ripple incidence observed in vitro closely mirror the inverted-U dose-response curve seen in our in vivo experiments across states, where small increases in serotonin lead to the highest ripple incidence, and both lower and higher levels correspond to reduced ripple activity. This parallel suggests that the gradual washing of serotonin in our in vitro system may mimic the tonic firing changes in serotonergic neurons that occur during state transitions in vivo. These findings underscore the importance of studying how different dynamics of serotonin modulation can differentially affect hippocampal network activity.

(4) Limited behavioral states: The study focuses primarily on sleep and quiet wakefulness. Investigation of 5-HT oscillations during a wider range of behavioral states (e.g., exploratory behavior, learning tasks) may provide a more complete understanding.

We agree that future studies should investigate a broader range of behavioral states. For this study, as we were focused on general sleep and wake patterns, our recordings were done in the home cage, and we limited ourselves to the basic behavioral states described in the paper. Future studies should be designed to investigate ultraslow 5-HT oscillations during different behaviors, such as continuous treadmill running. Specifically, a finer segregation of extended wake behaviors by level of arousal could greatly add to our understanding of the role of ultraslow serotonin oscillations.

(5) Generalizability to other brain regions: The study focuses on the CA1 region of the hippocampus. It's unclear whether similar 5-HT oscillation patterns exist in other brain regions.

Given the reported ultraslow oscillations of population activity in serotonergic neurons of the dorsal raphe nucleus (Kato et al. 2022) as well as the widespread projections of the serotonergic nuclei, we would expect a broad expression of ultraslow 5-HT oscillations throughout the brain. So far, ultraslow 5-HT oscillations have been described in the basal forebrain, as well as in the dentate gyrus, in addition to what we have shown in CA1 (Deng et al. 2024 and Turi et al. 2024). Furthermore, our results showing that hippocampal-cortical coherence changes according to the phase of hippocampal ultraslow 5-HT oscillations suggests that 5-HT can affect oscillatory activity either indirectly by modulating hippocampal cells projecting to the cortical network or directly by modulating the cortical postsynaptic targets. Given the heterogeneity in projection strength, as well as in pre- and postsynaptic serotonin receptor densities across brain regions (de Filippo & Schmitz, 2024), it would be interesting to see whether local ultraslow 5-HT oscillations are differentially modulated, e.g. in terms of oscillation power. Future studies investigating different brain regions via implantation of multiple optic fibers in different brain areas or using the mesoscopic imaging approach adopted in Deng et al. 2024, will be needed to examine the extent of spatial heterogeneity in this ultraslow oscillation.

(6) Long-term effects not assessed: Long-term effects of ultra-low 5-HT oscillations (e.g., on memory consolidation or learning) were not assessed.

While beyond the scope of our current study, we agree that an important next step would involve modulating the ultraslow serotonin oscillation after learning, and then examining potential effects on memory consolidation, presumably via changes in ripple dynamics, though many possibilities could explain potential effects. There, our results suggest it would be important to isolate effects due to the change in ultraslow oscillation features, rather than simply overall levels of 5-HT. To that end, it would be important to test different modulation dynamics, specifically modulating the oscillation strength, around a constant mean 5-HT level by carefully timed optogenetic stimulation/inhibition. Afterwards, showing a clear correlation between the strength of the 5-HT modulation and memory performance would be important to establishing the relationship, as done in Lecci et al 2017, where more prominent ultraslow oscillations of sigma power in the cortex during sleep, alongside a higher density of spindles, were correlated with better memory consolidation. Given the tight coupling of spindles and ripples during sleep, it is possible that a similar effect on memory consolidation would be observed following changes in ultraslow 5-HT oscillation power.

(7) Possible species differences: It's uncertain whether the findings in mice apply to other mammals, including humans.

We agree that the experiments should ultimately be replicated in humans. In the 2017 study by Lecci et al., the authors highlighted the shared functional requirements for sleep across species, despite apparent differences, such as variations in sleep volume. To explore these commonalities, the researchers conducted parallel experiments in both mice and humans, aiming to identify a universal organizing structure. They discovered that the ultraslow oscillation of sigma power serves this role, enabling both species to balance the competing demands of arousability and sleep imperviousness. Based on this finding, it is plausible that ultraslow oscillations of serotonin, which similarly modulate activity according to arousal levels, would serve a comparable function in humans.

(8) Technical limitations: The temporal resolution and sensitivity of the GRAB5-HT3.0 sensor may not capture faster 5-HT dynamics.

The kinetics of the GRAB5-HT3.0 sensor used in this study limit the range of serotonin dynamics we can observe. However, the ultraslow oscillations we measure reflect temporal changes on the scale of 20 s and greater, whereas the GRAB sensor we use has sub-second on kinetics and below 2 s off kinetics (Deng et al. 2024). Therefore, the sensor is capable of reporting much faster activity than the ultraslow oscillations we observe, indicating that the ultraslow 5-HT signal accurately reflects the dynamics on this time scale. Furthermore, the presence of ultraslow oscillations in spiking activity—observed in the hippocampal formation (Gonzalo Cogno et al., 2024; Aghajan et al., 2023; Penttonen et al., 1999) and in the dorsal raphe (Mlinar et al., 2016), which are not affected by the same temporal smoothing, suggests that the oscillations we record are not likely due to signal aliasing, but instead reflect genuine oscillatory activity. Of course, this does not preclude that other, faster serotonin dynamics are also present in our signal, some of which may be too fast to be observed. For instance, rapid serotonin signaling via the ionotropic 5-HT3a receptors could be missed in our recordings. Additionally, with the fiber photometry approach we adopted, we are limited to capturing spatially broad trends in serotonin levels, potentially overlooking more localized dynamics.

(9) Interactions with other neuromodulators: The study does not explore interactions with other neuromodulators (e.g., norepinephrine, acetylcholine) or their potential ultraslow oscillations.

We agree that the interaction between neuromodulators in the context of ultraslow oscillations is an important issue, which we have addressed in our response to reviewer #1 under ‘Weaknesses.’

(10) Limited exploration of functional significance: While the study suggests a potential role for 5-HT oscillations in memory consolidation and arousal, direct tests of these functional implications are not included.

We agree and reference our answer to (6) regarding memory consolidation. Regarding arousal, direct tests of arousability to different sensory stimuli during different phases of the ultraslow 5-HT oscillation during sleep would be beneficial, in addition to the indirect measures of arousal we examine in the current study, e.g. degree of movement (icEMG) and long range coherence. In line with what we have shown, Cazettes et al. (2021) has demonstrated a direct relationship between 5-HT levels and pupil size, an indicator of arousal level, which like our findings, is consistent across behavioral states.

Reviewer #3 (Public review):

Summary:

The activity of serotonin (5-HT) releasing neurons as well as 5-HT levels in brain structures targeted by serotonergic axons are known to fluctuate substantially across the animal's sleep/wake cycle, with high 5-HT levels during wakefulness (WAKE), intermediate levels during non-REM sleep (NREM) and very low levels during REM sleep. Recent studies have shown that during NREM, the activity of 5HT neurons in raphe nuclei oscillates at very low frequencies (0.01 - 0.05 Hz) and this ultraslow oscillation is negatively coupled to broadband EEG power. However, how exactly this 5-HT oscillation affects neural activity in downstream structures is unclear.

The present study addresses this gap by replicating the observation of the ultraslow oscillation in the 5-HT system, and further observing that hippocampal sharp wave-ripples (SWRs), biomarkers of offline memory processing, occur preferentially in barrages on the falling phase of the 5-HT oscillation during both wakefulness and NREM sleep. In contrast, the raising phase of the 5-HT oscillation is associated with microarousals during NREM and increased muscular activity during WAKE. Finally, the raising 5-HT phase was also found to be associated with increased synchrony between the hippocampus and neocortex. Overall, the study constitutes a valuable contribution to the field by reporting a close association between raising 5-HT and arousal, as well as between falling 5-HT and offline memory processes.

Strengths:

The study makes compelling use of the state-of-the-art methodology to address its aims: the genetically encoded 5-HT sensor used in the study is ideal for capturing the ultraslow 5-HT dynamics and the novel detection method for SWRs outperforms current state-of-the-art algorithms and will be useful to many scientists in the field. Explicit validation of both of these methods is a particular strength of this study.

The analytical methods used in the article are appropriate and are convincingly applied, the use of a general linear mixed model for statistical analysis is a particularly welcome choice as it guards against pseudoreplication while preserving statistical power.

Overall, the manuscript makes a strong case for distinct sub-states across WAKE and NREM, associated with different phases of the 5-HT oscillation.

Weaknesses:

All of the evidence presented in the study is correlational. While the study mostly avoids claims of causality, it would still benefit from establishing whether the 5-HT oscillation has a direct role in the modulation of SWR rate via e.g. optogenetic activation/inactivation of 5-HT axons. As it stands, the possibility that 5-HT levels and SWRs are modulated by the same upstream mechanism cannot be excluded.

We agree that causality claims cannot be made with our data, and acknowledge the interest in exploring causal interactions between ultraslow serotonin oscillations and the correlated activity we measure. We address this point in depth in our answer to Reviewer #2, Weaknesses #3.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

One major question in the presented data is the nature of the asymmetrical shape of the targeted slow events. How much does it reflect the 5-HT concentration and how much is this shape affected by the dynamics of the designed 5-HT sensor? This needs to be addressed in more detail referencing the original paper for the used sensor.

We have added a paragraph in the Results section of the manuscript to address the asymmetric waveform of the ultraslow 5-HT oscillations and whether it could be affected by the asymmetric kinetics of the GRAB sensor we use: “The waveform of these ultraslow 5-HT oscillations…” (Results, paragraph #4, pg. 5). We include an extended answer to the question here:

Indeed, the GRAB5-HT3.0 sensor we use in the study shows activation response kinetics which are faster than their deactivation time, with time constants at 0.25 s and 1.39 s, respectively (Deng et al. 2024). Likewise, the slope of the rising phase of the ultraslow serotonin oscillation we measure is faster than the slope of the falling phase, and the ratio of time spent in the rising phase versus the falling phase is less than 1, indicating longer falling phases (Figure S2). Although we cannot completely rule out that the asymmetric shape of the ultraslow serotonin oscillations we record is affected by this asymmetry in the 5-HT sensor kinetics, we believe this is unlikely, as the 5-HT signal clearly contains reductions in 5-HT levels that are much faster than the descending phase of the ultraslow oscillation. Although it is difficult to directly compare the different-sized signals, the reported timescales of off kinetics, on the order of a few seconds (Deng et al. 2024), are far below the tens of seconds timescale of the ultraslow oscillation. Furthermore, the finding that some dorsal raphe neurons modulate their firing rate at ultraslow frequencies, and moreover that all examples of such ultraslow oscillations shown display clear asymmetry in rising time versus decay, suggests that the asymmetry we observe in our data could be due to neural activity rather than temporal smoothing by the sensor (Mlinar et al. 2016). In this same direction, another study found similar asymmetry in extracellular 5-HT levels measured with fast scan cyclic voltammetry (FSCV), a technique with greater temporal resolution (sampling rate of 10 Hz) than GRAB sensors, after single pulse stimulation (Bunin and Wightman 1998). In this study, 5-HT was shown to be released extrasynaptically, making the longer clearing time compared to the release time intuitive. Finally, the observation that the onsets and offsets of ripple clusters, recorded with a sampling rate of 20 kHz, are precisely aligned with the peaks and troughs of ultraslow serotonin oscillations (Figure 1, H1-2, columns 2-3) suggests that the duration of the falling phase is not artificially distorted by the temporal smoothing of the sensor dynamics.

Regardless of the dynamics of the serotonin concentration, it should be noted that the elicited neuronal effect might have different dynamics compared to the 5-HT concentration that need to be more studied: to address this one can either examine the average of the broadband LFP (not high passfiltered by the amplifier) or the distribution of simultaneously recorded spiking activity around the peak of ultra-slow oscillations.

We have added Figure S6, showing unit activity relative to the phase of ultraslow serotonin oscillations.

From this analysis, we uncover three groups of units which are largely preserved across states (Figure S6, E vs. F), albeit with a slight temporal shift rightward from NREM to WAKE (Figure S6, C vs. D). Namely, some units spike preferentially during the rising phase, some during the falling phase, and a third group have no clear phase preference. Unit activity during the falling phase is unsurprising, as it is where ripples largely occur, which themselves are associated with spike bursts. During the rising phase, the unit activity we observe could correspond to firing of the hippocampal subpopulation known to be active during NREM interruption states (Jarosiewicz et al. 2002, Miyawaki et al. 2017). While the units’ phase preference was tested based on the category of rising vs. falling phase, as this division described most variation in the data, a few units in the ‘No preference’ group showed heightened activity near the oscillation peak. However, given the very small number of units with this preference, more unit data is needed to describe this group, ideally with high-density recordings. Overall, most units showed a falling vs. rising phase preference, indicating a phase coding of hippocampal activity by 5-HT ultraslow oscillations.

Related to the previous point, it would be helpful to show the average cycle shape of these oscillations (relative to the phase 0 extracted in Figure 3) and do the shape comparison across sessions and also wake/NREM

We agree, and to this end we have added Figure S2. From this waveform analysis, we show that the ultraslow serotonin oscillation is asymmetric, with the rising phase having a greater slope, but shorter length, than the falling phase. While this asymmetry is observed both in NREM and WAKE, the slope difference and length ratio difference in rising vs. falling phase is greater in NREM (Figure S2. B).

In Figure 3D, there seem to be oscillatory rhythms with faster cycles on top of the targeted oscillations. That would make the phase estimation less accurate, e.g. in the left panel, in the second cycle, it is not clear if there are two faster cycles or it is one slow cycle as targeted, and if noted in the rising phase of the second fast cycle there are no ripples. This might suggest that regardless of specific oscillation frequency whenever 5-HT is started to get released, the ripples are suppressed and once the 5-HT is not synaptically effective anymore the ripples start to get generated while the photometry signal starts to wane with the serotonin being cleared. Still, if there is any rhythmicity between bouts of no ripple, it would suggest an ultra-slow regularity in the 5-HT release.

The reviewer is correct to point out that some faster increases in serotonin, which occur on top of the ultraslow oscillations we measure, seem to be associated with decreased ripple incidence, as in the example referenced. The dominance of ultraslow frequencies in the power spectrum of the 5-HT signal suggests, however, that oscillations faster than the ultraslow oscillations we describe are far less prevalent in the data. While there may be some coupling of ripples and other measures to serotonin oscillations of different frequencies, this may be hard or impossible to detect with phase analysis based on their infrequent occurrence and nonstationary nature. In fact, we show in Figure S3 that the strongest phase modulation of ripples by ultraslow serotonin oscillations is observed in the frequencies we use (0.01-0.06 Hz). Methodologically, phase analysis indeed assumes stationary signals, which are rare if not absent in physiological data (Lo et al. 2009), however generally the narrower the frequency band, the better the phase estimation. The narrow frequency band we use provides phase estimates that are largely robust and unaffected by the presence of faster oscillations, as can be seen in the example phase traces shown in Figure 4.

The hypothesis that the rising phase burst of synaptic serotonin is what silences ripples, and that with the clearing of serotonin from the synapses, ripples recover, is a possible explanation of our findings. However, if this were the case, one could expect the ripple rate to increase over the course of the falling phase of ultraslow 5-HT oscillations, as 5-HT decreases, and peak at the trough. This is at odds with what we observe, namely a fairly uniform distribution of ripples along the falling phase (Figure 3F2,F4). Furthermore, the Mlinar et al. 2016 study describes a subpopulation of raphe neurons whose firing rates themselves oscillate at ultraslow frequencies, rather than on-off bursting at ultraslow frequencies, which would argue against this hypothesis. However, as this study looks at a small number of neurons in slices, further in vivo experiments examining firing rates of median raphe neurons are required to understand how the ultraslow oscillation of extracellular serotonin that we measure is generated as well as how it is related to ripple rates.

In Figure 3B, it is not clear why IRI is z-scored. It would be informative to have the actual value of IRI. What is the z relative to? Is it the mean value of IRI in each recording session? Is this to reduce the variability across sessions?

We have now included in Figure 3D a box plot displaying the IRI distributions across different states and sessions. To minimize inter-session variability, data were z-scored within each session for visualization purposes. However, all general linear models were based on raw data, and as a result, the raw differences in IRI are shown in Figure 3C.

Figure 3E, panel labels don't match with the caption

We are grateful to the reviewer for pointing out this mistake, which we have corrected in the updated version of the manuscript.

In the text related to Figure 3E, the related analysis can be more clearly described. "phase preference of individual ripples" does not immediately suggest that the occurring phase of each ripple relative to the targeted oscillation is extracted. I suggest performing this analysis individually for each session and summarizing the results across the sessions.

We have reworded the sentence in Results: 5-HT and ripples to better reflect the analysis performed: “Next, we calculated the ultraslow 5-HT phases at which individual ripples occurred during both NREM and WAKE (3E-F) ...”. Regarding session-level data, we have added Figure S3, which shows session level mean phase vectors, as well as the grand mean across sessions for both NREM and WAKE. Included in this figure are session level means for frequency bands outside of the ultraslow band we used in our study, intended to show that ripples are most strongly timed by the ultraslow band (0.01-0.06 Hz), reflected by the greater amplitude of the mean phase vector for this band.

Figure 3E2, based on the result of ripple-triggered 5-HT in left panels of 2H1-2, one would expect to see a preferred phase closer to 180 (toward the end of the falling phase), it would be helpful to compare and discuss the results of these two analyses.

The reviewer is correct to point out the apparent discrepancy in where the mean ripple falls with respect to the ongoing serotonin oscillation between the two figures mentioned. We have addressed this point in Results: 5-HT and ripples, paragraph #4: “This result appear to be at odds with…”.

Regarding the analysis in 3F, please also compare the power distribution of ripples between NREM and wake. This will help to better understand the potential difference behind the observed difference: how much the strong ripples are comparable between wake and NREM. It is also necessary to report the ripple detection failure rate across ripples with different strengths.

We have added a figure showing analysis done on a subset of the data in which ripples were manually curated in order to evaluate the performance of the ripple detection model (Figure S7) and explanatory text in Methods: Model performance: ‘To ensure that our model …’. In summary, while missed ripples did tend to have lower power than correctly detected ripples, including them did not change the distribution of ripples by the phase of the ultraslow serotonin oscillation (Figure S7C). We would also note that while the phase preference is noisier than what is presented in Figure 3F because this analysis was done with a small subset of all recorded ripples, the fact that ripples occur more clearly on the falling phase is visible for both detected ripples and detected + false negative ripples.

The mixed-effects model examining the influence of 5-HT ultraslow oscillation phase on ripple power revealed no significant effect of state (p = 0.088). This indicates that whether the data were collected during NREM or wake periods did not significantly impact ripple power and that the lack of a significant effect (in Figure 3G,H) in WAKE is probably not due to a difference in the distribution of ripple power between states.

4D, y label is z?

We are grateful for the reviewer to point that out, yes, the y label should be ‘z-score’, as the two traces represent z-scored 5-HT (blue) and z-scored shuffled data (orange). Figure 4D2 and Figure 2H1-2, which show similar data, have been corrected to address this oversight.

Relating to Figure 4, EMG comparison across phases of the oscillations is insightful. Two related and complementary analyses are to compare the theta and gamma power between the falling and rising phases.

We have addressed this suggestion in Figure S5 A-C. While low gamma, high gamma and theta power are modulated identically in NREM, with higher power observed during the falling phase than the rising phase, during WAKE, different patterns can be seen. Specifically, low gamma power shows no phase preference, while high gamma shows a peak near the center of the ultraslow 5-HT oscillation. Theta power, as in NREM, is higher during the falling phase of ultraslow 5-HT oscillations. Increased power across many frequency bands was shown to coincide with decreases in DRN population activity during NREM, which matches with what we report here (Kato et al. 2022). In summary, while NREM patterns are consistent in all frequency bands tested, aligning with the pattern of ripple incidence, in WAKE low and high gamma power show different relationships to ultraslow 5-HT phase.

In the manuscript, we have used the data in both Figure S5 and S6 (unit activity relative to ultraslow 5-HT oscillations), to argue against the idea that our coherence findings result from a lack of activity in the rising phase (see next question), which would have the effect of ‘artificially’ reducing coherence in the falling phase relative the rising phase. The text can be found in Results: 5-HT and hippocampal cortical coherence, paragraph #2.

The results presented in Figure 5 could be puzzling and need to be further discussed: if the ripple band activity is weak during the rising phase, in what circumstances the coherence between cortex and CA1 is specifically very strong in this band?

As mentioned in the previous answer, we have addressed this concern in Results: 5-HT and hippocampal-cortical coherence, paragraph #2. In summary, it is true that the higher coherence in rising phase than in the falling phase for the highest frequency band (termed ‘high frequency oscillation’ (HFO), 100-150 Hz) could be unexpected, given that ripples occur largely during the falling phase. A few points could help explain this finding. Firstly, it should be noted that power in the 100-150 Hz band can arise from physiological activity outside of ripples, such as filtered non-rhythmic spike bursts (Liu et al. 2022), whose coherent occurrence in the rising phase could explain the coherence findings. Secondly, coherence is a compound measure which is affected by both phase consistency and amplitude covariation (Srinath and Ray 2014), thus from only amplitude one cannot predict coherence. Furthermore, HFO power in the cortex is highest near the peak of ultraslow 5-HT oscillations (Figure S5D), as opposed to the falling phase peak in the hippocampus. This shows a lack of covariation in amplitude by phase between the hippocampus and cortex at this frequency band. An alternative explanation of our findings regarding coherence could be that in the rising phase, there is simply little to no activity, which is easier to ‘synchronize’ than bouts of high activity. Hippocampal unit activity in the rising phase (Figure S6) suggests however, that it is not likely to be the absence of activity supporting higher coherence in the rising phase across frequencies. Additional experiments using high density recordings should be conducted to examine 5-HT ultraslow oscillations and their role in gating activity across brain regions, though these results strongly suggest some role exists.

Reviewer #2 (Recommendations for the authors):

I would like to offer two comments. I believe that these are not unusual requests, and thus I would like the authors to respond.

(1) It would be prudent to investigate the possibility that the observed correlation between ultraslow and hippocampal ripples/microarousals is merely superficial and that there are unidentified confounding factors at play. For example, it would be beneficial to provide evidence that administering a serotonin receptor inhibitor result in the disappearance of the slow oscillation of ripples and microarousals, or that the correlation with ultraslow is no longer present. Please note that the former experiments do not require GRAB5-HT3.0 imaging.

We agree that causality claims cannot be made with our data and acknowledge the interest in exploring causal interactions between ultraslow serotonin oscillations and the correlated activity we measure. We address this point in depth in our answer to Reviewer #2, Weaknesses #3. We would further like to note that given the large number of serotonin receptors and the lack of selectivity of many serotonin receptor antagonists, a pharmacological approach would be difficult, though the results certainly useful. Finally, we highlight the psilocin study, which reported changes in the rhythmic occurrence of microarousals, and therefore likely ultraslow oscillations, after administering a 5-HT2a receptor agonist, suggesting a potential causal effect of 5-HT (via 5-HT2a receptor) on MA occurrence (Thomas et al. 2022).

(2) The slow frequency appears to be associated with the default mode network as observed in fMRI signals. The neural basis of the default mode network remains unclear; therefore, a more detailed examination of this possibility would be beneficial.

We agree that it would be interesting to investigate the role of 5-HT in the neural basis of the DMN.

The DMN as described in humans (Raichle et al. 2001) and rodents (Lu et al. 2012) may indeed include some parts of the hippocampus and perhaps some of our neocortical recordings could also be considered part of the DMN. The fact that the activity across the inter-connected brain structures of the DMN is correlated at ultraslow time scales (Gutierrez-Barragan et al. 2019, Mantini et al. 2007), as well as serotonin’s ability to modulate the DMN is intriguing (Helmbold et al. 2016). Further studies simultaneously recording DMN activity via fMRI and electrical activity via silicon probes, as done in Logothetis et al. 2001, could elucidate further a potential link between ultraslow oscillations and the DMN, with serotonergic modulation as a means to understand any potential contribution of serotonin.

Reviewer #3 (Recommendations for the authors):

(1) The impact of the study would benefit from an experiment causally testing the effect of hippocampal 5-HT levels on hippocampal physiology, e.g. using optogenetic manipulations.

We agree that causality claims cannot be made with our data and acknowledge the interest in exploring causal interactions between ultraslow serotonin oscillations and the correlated activity we measure. We address this point in depth in our answer to Reviewer #2, Weaknesses #3.

(2) Data presentation: the figures are of poor resolution, making some diagram details and, more importantly, some example traces (e.g. Figure 1A, right) impossible to see. This should be corrected by either increasing figure resolution or making important figure elements large enough to be readable.