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eLife assessment

This work contributes to the study of H3-K27M mutated pediatric gliomas. It convincingly demonstrates that the concomitant targeting of histone deacetylases (HDACs) and the transcription factor MYC results in a notable reduction in cell viability and tumor growth. This reduction is linked to the suppression of critical oncogenic pathways, particularly mTOR signaling, emphasizing the role of these pathways in the disease's pathogenesis. The current version of the manuscript is important because it unveils a vulnerability from dual targeting HDACs and MYC in the context of pediatric gliomas. This work will be of interest to cancer epigenetics and therapeutics research, with a focus on the neuro-oncology field.

Reviewer #2 (Public Review):

This study by Algranati et al. is a important contribution to our understanding of H3-K27M pediatric gliomas. It convincingly demonstrates that the concomitant targeting of histone deacetylases (HDACs) and MYC, through a combination therapy of Sulfopin and Vorinostat, results in a notable reduction in cell viability and tumor growth. This reduction is linked to the suppression of critical oncogenic pathways, particularly mTOR signaling, emphasizing the role of these pathways in the disease's pathogenesis. The manuscript is a step forward in the field, as it unveils a vulnerability from dual targeting HDACs and MYC in the context of pediatric gliomas.

Comments on revised version

The authors have nicely explained their rationale for dose selection, treatment timing, and the relationship between MYC expression and sensitivity to the combined treatment. They have also clarified the experimental conditions for the in vitro and in vivo studies, ensuring consistency across the various analyses.

Overall, the authors have been responsive to the reviewers' comments and have made appropriate revisions to improve the clarity and robustness of their study.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This is an interesting study that utilizes a novel epigenome profiling technology (single molecule imaging) in order to demonstrate its utility as a readout of therapeutic response in multiple DIPG cell lines. Two different drugs were evaluated, singly and in combination. Sulfopin, an inhibitor of a component upstream of the MYC pathway, and Vorinostat, an HDAC inhibitor. Both drugs sensitized DIPG cells, but high (>10 micromolar) concentrations were needed to achieve half-maximal effects. The combination seemed to have some efficacy in vivo, but also produced debilitating side-effects that precluded the measurement of any survival benefit.

We thank the reviewer for deeply evaluating our work and acknowledging the use of multiple experimental strategies to explore the effect of combination therapy on DMG cells. Of note, all mice in our experiment experienced deterioration (including the control mice and those treated with single agents). Thus, it is not the combination of drugs that led to the debilitating side-effects; the mice deteriorated due to the extremely aggressive tumor cells, forming relatively large tumors prior to the treatment onset, calling for further optimization of the therapeutic regime.

We modified the text in the results section to clarify this point (lines 238-241): “This rapid deterioration is likely a result of the aggressiveness of the transplanted tumors and does not represent side effects of the treatment, as mice from all groups, including the non-treated mice, showed similar signs of deterioration”.  

We also elaborate on this in the discussion (lines 272-276): “Notably, despite a significant reduction in tumor size in-vivo, the combined treatment did not increase mice survival. This is perhaps due to the relatively large tumors already formed at the onset of treatment, leading to rapid deterioration of mice in all experimental groups. Thus, further optimization of the modeling system and therapeutic regime is needed.” We truly hope that further studies will allow better assessment of this drug combination in various models.

Strengths:

Interesting use of a novel epigenome profiling technology (single molecule imaging).

Weaknesses:

The use of this novel imaging technology ultimately makes up only a minor part of the study. The rest of the results, i.e. DIPG sensitivity to HDAC and MYC pathway inhibition, have already been demonstrated by others (Grasso Monje 2015; Pajovic Hawkins 2020, among others). The drugs have some interesting opposing effects at the level of the epigenome, demonstrated through CUT&RUN, but this is not unexpected in any way. The drugs evaluated here also didn't have higher efficacy, or efficacy at especially low concentrations, than inhibitors used in previous reports. The combination therapy attempted here also caused severe side effects in mice (dehydration/deterioration), such that an effect on survival could not be determined. I'm not sure this study advances knowledge of targeted therapy approaches in DIPGs, or if it iterates on previous findings to deliver new, or more efficient, mechanistic or therapeutic/pharmaclogic insights. It is a translational report evaluating two drugs singly and in combination, finding that although they sensitise cells in vitro, efficacy in vivo is limited at best, as this particular combination cannot progress to human translation.

We thank the reviewer for pointing out the strengths and weaknesses of our work. As far as we know, while many studies demonstrated upregulation of the MYC pathway in DIPG, this is the first study that shows inhibition of this pathway (via PIN1) as a therapeutic strategy. While it is clear from the literature that MYC inhibition may pose therapeutic benefit, the development of potent MYC inhibitors is highly challenging due to its structure and cellular localization. Of note, in the 2020 paper, Pajovic and colleagues inhibited MYC by transfecting the cells with a plasmid expressing a specific inhibitory MYC peptide (Omomyc); while this strategy works well for cell cultures, the clinical translation requires different delivery strategies. Sulfopin is a small molecule inhibitor that can be used in-vivo and potentially in clinical studies. Thus, we believe that our study offers a novel strategy, as well as mechanistic insights, regarding the potential use of Sulfopin and Vorinostat to treat DIPG.

As noted above, the combination therapy did not cause side effects, but rather the aggressiveness of the tumors. We did not notice specific toxicity in the mice treated with Sulfopin alone, or the combined treatment. Furthermore, Dubiella et al. extensively examined toxicity issues and did not observe adverse effects or weight loss when administrating Sulfopin in a dose of 40 mg kg–1.

Optimization of the model and treatment regime (# of cells injected, treatment starting point, etc.) may have allowed us to reveal survival benefits. Yet, these are highly complicated and expensive experiments; unfortunately, we did not have the resources to perform them within the scope of this revision. Importantly, within the current manuscript, we show the effect of this drug combination in reducing the growth of DMG cells in-vitro and in-vivo, laying the framework for follow-up exploration in future studies. Furthermore, the epigenetic and transcriptomic profiling shed light on the molecular mechanisms that drive these aggressive tumors.

Reviewer #2 (Public Review):

Summary:

The study by Algranati et al. introduces an exciting and promising therapeutic approach for the treatment of H3-K27M pediatric gliomas, a particularly aggressive brain cancer predominantly affecting children. By exploring the dual targeting of histone deacetylases (HDACs) and MYC activation, the research presents a novel strategy that significantly reduces cell viability and tumor growth in patient-derived glioma cells and xenograft mouse models. This approach, supported by transcriptomic and epigenomic profiling, unveils the potential of combining Sulfopin and Vorinostat to downregulate oncogenic pathways, including the mTOR signaling pathway. While the study offers valuable insights, it would benefit from additional clarification on several points, such as the rationale behind the dosing decisions for the compounds tested, the specific contributions of MYC amplification and H3K27me3 alterations to the observed therapeutic effects, and the details of the treatment protocols employed in both in-vitro and in-vivo experiments.

We thank the reviewer for evaluating our work and recognizing its potential for the DMG research field. We address in detail below the important comments regarding the treatment protocols and dosing decisions.

Clarification is needed on how doses were selected for the compounds in Figure S2A and throughout the study. Understanding the basis for these choices is crucial for interpreting the results and their potential clinical relevance. IC50s are calculated for specific patient derived lines, but it is not clear how these are used for selecting the dose.

We thank the reviewer for these important comments. For the epigenetic drugs shown in Figure S2A, we followed published experimental setups; for EPZ6438, GSKJ4, Vorinostat and MM-102 we chose the treating concentrations according to Mohammad et al. 2017, Grasso et al. 2015 and Furth et al. 2022, accordingly. For Sulfopin, we conducted a dedicated dose curve analysis (shown in Figure 1E), indicating only a mild effect on viability and relatively high IC-50 values as a single agent. Since we aimed to test the ability of a combined treatment to additively reduce viability, we used a sub-IC50 concentration for Sulfopin in these experiments. We added this information in lines 123 and 131-132.

Finally, following the results obtained in the experiment shown in Figure S2A, we conducted a full dose-curve analysis of the combined treatment in multiple DMG patient-derived cells (figure 2B and S2C), to identify a combination of concentrations that provides an additive effect (as indicated by BLISS index in figure 2C and S2E). Of note, for downstream analysis of the molecular mechanisms underlying the treatment response (RNAseq and Cut&Run), we intentionally used concentrations that provide an additive BLISS index, but do not completely abolish the culture, to allow for cellular analysis (i.e. 10uM Sulfopin and 1uM Vorinostat).

The introduction mentions MYC amplification in high-grade gliomas. It would be beneficial if the authors could delineate whether the models used exhibit varying degrees of MYC amplification and how this factor, alongside differences in H3K27me3, contributes to the observed effects of the treatment.

The reviewer highlights an important part of our study relating to the MYC-dependent sensitivity of the proposed treatment combination. Since high expression of MYC can be mediated by different molecular mechanisms and not only genomic amplification, we directly quantified mRNA levels of MYC by qPCR (shown in figure S2G) in order to explore its relationship with cellular response to Sulfopin and Vorinostat. Indeed, cultures that express high levels of MYC mRNA were more sensitive to Sulfopin treatment alone (figure S1P) and to the combined treatment (figure 2D-E). We also relate to these findings in lines 103-106 and 142-147 of the results section. Importantly, in cultures that express high levels of MYC (SU-DIPG13 as an example), we see downregulation of MYC targets upon the combined treatment, supporting the notion that this treatment affects viability by attenuation of MYC signaling.

In Figure 2A, the authors outline an optimal treatment timing for their in vitro models, which appears to be used throughout the figure. It would be helpful to know how this treatment timing was selected and also why Sulfopin is dosed first (and twice) before the vorinostat. Was this optimized?

As PIN1 regulates the G2/M transition, its inhibition by Sulfopin delays cell cycle progression (Yeh et al. 2007). Thus, in order to observe a strong viability difference in culture, a prolonged treatment period of 8-9 days is required (Dubiella et al., 2021). To maintain an active concentration of the drug during this long time period, we added a Sulfopin pulse (2nd dose) to achieve a stronger effect on cell viability. We and others noticed that, unlike Sulfopin, the effect of Vorinostat on viability is rapid and can be clearly seen after 2-3 days of treatment. Thus, we added this drug only after the 2nd dose of Sulfopin. We now relate to the mode of action of Sulfopin in lines 79-81.

It should be clarified whether the dosing timeline for the combination drug experiments in Figure 3 aligns with that of Figure 2. This information is also important for interpreting the epigenetic and transcriptional profiling and the timing should be discussed if they are administered sequentially (also shown in Figure 2A).I have the same question for the mouse experiments in Figure 4.

The reviewer is correct that this information is critical for evaluating the results. In order to link the expression changes to the epigenetic changes, we kept the same experimental conditions in both the Cut&Run and RNA-seq experiments (shown in figures 2-3). We added this information to the text in line 184.

For the in-vivo studies of HDAC inhibition (Figure 4), we followed published protocols (Ehteda et al. 2021). In these experiments both drugs were administrated simultaneously every day. We added this information to the text in line 231-232.  It may be that changing the admission regime may improve the efficacy of the drug combination, which remains to be tested in future studies.

The authors mention that the mice all had severe dehydration and deterioration after 18 days. It would be helpful to know if there were differences in the side effects for different treatment groups? I would expect the combination to be the most severe. This is important in considering the combination treatment.

As noted in our response to Reviewer #1, all mice in our experiment experienced deterioration (including the control mice and those treated with single agents- we could not observe any differences between the groups). This is due to the extremely aggressive tumor cells, forming relatively large tumors prior to the treatment onset, calling for further optimization of the system and therapeutic regime (# of cell injected, treatment starting point, etc.). Unfortunately, this model is very challenging (especially the injection of cells to the pons of the mice brains, which requires unique expertise and is associated with mortality of some of the mice). Thus, these are highly complicated and expensive experiments; unfortunately, we did not have the resources to repeat and optimize the treatment protocol within the scope of this revision. Of note, Dubiella et al. extensively examined toxicity issues and did not observe adverse effects or weight loss when administrating Sulfopin in a dose of 40 mg kg–1. In our model, the side effects were caused by the tumors rather than the drugs.

Minor Points:

(1) For Figure 1F, reorganizing the bars to directly compare the K27M and KO cell lines at each dose would improve readability of this figure.

We have changed figure 1F as the reviewer suggested.

(2) In Figure 4D, it would be helpful to know how many cells were included (or a minimum included) to calculate the percentages.

We added the number of H3-K27M positive cells detected per FOV to the figure legend and method section (n=13-198 cells per FOV). Of note, while we analyzed similar-sized FOVs, the number of tumor cells varied between the groups, with the treated group presenting a lower number of H3-K27M cells (due to the effect of the treatment on tumor growth). To account for this difference, we calculated the portion of mTOR-positive cells out of the tumor cells.   

Reviewer #3 (Public Review):

Summary:

The authors use in vitro grown cells and mouse xenografts to show that a combination of drugs, Sulfopin and Vorinostat, can impact the growth of cells derived from Diffuse midline gliomas, in particular the ones carrying the H3 K27M-mutations (clinically classified as DMG, H3 K27M-mutant). The authors use gene expression studies, and chromatin profiling to attempt to better understand how these drugs exert an effect on genome regulation. Their main findings are that the drugs reduce cell growth in vitro and in mouse xenografts of patient tumours, that DMG, H3 K27M-mutant tumours are particularly sensitive, identify potential markers of gene expression underlying this sensitivity, and broadly characterize the correlations between chromatin modification changes and gene expression upon treatment, identifying putative pathways that may be affected and underlie the sensitive (and thus how the drugs may affect the tumour cell biology).

Strengths:<br /> It is a neat, mostly to-the-point work without exploring too many options and possibilities. The authors do a good job not overinterpreting data and speculating too much about the mechanisms, which is a very good thing since the causes and consequences of perturbing such broad epigenetic landscapes of chromatin may be very hard to disentangle. Instead, the authors go straight after testing the performance of the drugs, identifying potential markers and characterizing consequences.

Weaknesses:

If anything, the experiments done on Figure 3 could benefit from an additional replicate.<br />

We thank the reviewer for evaluating our work, and for the positive and insightful comments.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Perhaps a more substantial drug screen, or CRISPR screen, that utilises single molecule imaging as a readout would identify pharmacologic candidates that are either more effective, or novel.

While out of scope for the current study, this is a highly interesting suggestion, which will be considered in future studies. Here, we focused on the potential use of the novel MYC inhibitor, Sulfopin. While the dependency of DMG cells on MYC signaling has been documented, to the best of our knowledge, pharmacological inhibition of MYC has not been tested for this disease due to the severe lack of potent MYC inhibitors. We show preliminary evidence for the use of this inhibitor, in combination with HDAC inhibition, to attenuate DMG growth in-vitro and in-vivo.  

Reviewer #2 (Recommendations For The Authors):

In Figure 1B, it is hard to tell if there are error bars for HSP90 and E2F2. Is there a potential error here? Seems unlikely to not have an error with a RT-qPCR?

We thank the reviewer for the careful evaluation of the figures. We included error bars for all genes shown in Figure 1B. We have now increased the line width with the hope of making this information more accessible. As stated in the figure legend, these error bars represent the standard deviation of two technical repeats.

I noticed that many experiments only had technical replicates. Incorporating biological (independent) replicates, where feasible, would strengthen the study's findings.

We agree with the reviewer regarding the importance of biological replicates. While some of the panels present error estimates based on technical repeats, the main results were repeated independently with complementary approaches or various biological systems for validation.

The RNAseq analysis presented in figure 1 was conducted in triplicates and then independently validated by qPCR (Figure 1A-B). Similarly, the transcriptomic analysis presented in figures 2G-I was verified by both western blot (figure 2J) and qPCR (figure S2O). Of note, this later validation was conducted for two different DMG-patient derived cultures.

To verify the robust effects on cellular viability, we analyzed the response to each drug and the combination on eight different DMG-patient-derived cultures, each representing a completely independent experiment. We show very similar trends in response to treatment between cultures that share the same H3-K27M variant. Thus, while for each culture technical repeats are shown, we provide multiple, independent repeats by examining the different cultures. Similarly, in figure 1F we examined the dependency of Sulfopin treatment on the expression of the H3-K27M oncohistone in two independent isogenic systems.

Reviewer #3 (Recommendations For The Authors):

A few questions and suggestions:

(1) To avoid confusion is important to state if the cells used in each experiment are or not K27M mutants (e.g. SU-DIPG13 on line 63).

We thank the reviewer for pointing this out and have now added this information when appropriate across the manuscript.

2) Line 72 - confirming epigenetic silencing of these genes upon PIN1 inhibition (Fig. 1C, S1D)

Considering that the mechanism of down regulation of MYC targets is likely H3K27me3-independent if it is also happening in DMG H3 K27M-mutants (high H3K27me3 here may rather be a consequence of less MYC binding?), I would strike this sentence out and just point out the correlation between lower expression and higher H3K27me3.

We agree with the reviewer that the exact molecular mechanism mediating the silencing is yet to be characterized. We have modified the text in line 72 accordingly.

3) (line 78) Are MYC targets also down regulated in Sulfopin treated DMG, H3 K27M-mutant lines? Any qPCR or previously done RNA-seq data to use?

In addition to the extensive analysis done on SU-DIPG13 cells (Figure 1 and S1), in light of the reviewer`s comment we examined specific MYC targets in an additional H3-K27M mutant DMG culture (SU-DIPG6) treated with Sulfopin, followed by qPCR. We observed a mild reduction in two prominent targets, E2F2 and mTOR (new figure S1D). Unfortunately, within this study, we only conducted full RNA-sequencing analysis on SU-DIPG13 cells treated with Sulfopin, and thus, we could not examine the global effect of Sulfopin on the transcriptome of other DMG cultures. This will, of course, be of high interest for future studies.

ok

Nice

nice

eLife assessment

The authors report solid evidence for a valuable set of findings in rats performing a new virtual place-preference task. Temporary pharmacological inhibition targeting the dorsal or intermediate hippocampus disrupted navigation to a goal location in the task, and functional inhibition of the intermediate hippocampus was more detrimental than functional inhibition of the dorsal hippocampus. The work provides novel insights into functional differentiation along the dorsal-ventral axis of the hippocampus.

Reviewer #1 (Public Review):

Summary:

The manuscript examines the contribution of dorsal and intermediate hippocampus to goal-directed navigation in a wide virtual environment where visual cues are provided by the scenery on the periphery of a wide arena. Among a choice of 2 reward zones located near the arena periphery, rats learn to navigate from the center of the arena to the reward zone associated with the highest reward. Navigation performance is largely assessed from the rats' body orientation when they leave the arena center and when they reach the periphery, as well as the angular mismatch between reward zone and the site rats reach the periphery. Muscimol inactivation of dorsal and intermediate hippocampus alters rat navigation to the reward zone, but the effect was more pronounced for the inactivation of intermediate hippocampus, with some rat trajectories ending in the zone associated with the lowest reward. Based on these results, the authors suggest that the intermediate hippocampus is critical especially for navigating to the highest reward zone.

Strengths:

- The authors developed an effective approach to study goal-directed navigation in a virtual environment where visual cues are provided by the peripheral scenery.

- In general, text is clearly written and the figures are well designed and relatively straightforward to interpret, even without reading the legends.

- An intriguing result, which would deserve to be better investigated and/or discussed, was that rats tended to rotate always in the counterclockwise direction. Could this be because of a hardware bias making it easier to turn left, some aspect of the peripheral landscape, or a natural preference of rats to turn left that is observable (or reported) in real environment?

- Another interesting observation, which would also deserved to be addressed in the discussion, is the fact that dHP/iHP inactivations produced to some extent consistent shifts in departing and peripheral crossing directions. This is visible from the distributions in Figures 6 and 7, which still show a peak under muscimol inactivation, but this peak is shifted to earlier angles than the correct ones. Such change is not straightforward to interpret, unlike the shortening of the mean vector length.<br /> Maybe rats under muscimol could navigate simply using association of reward zone with some visual cues in the peripheral scene, in brain areas other than the hippocampus, and therefore stopped their rotation as soon as they saw the cues, a bit before the correct angle. While with their hippocampus intact, rats could estimate precisely the spatial relationship between the reward zone and visual cues.

Weaknesses:

- I am not sure that the differential role of dHP and iHP for navigation to high/low reward locations is supported by the data. The current results could be compatible with iHP inactivation producing a stronger impairment on spatial orientation than dHP inactivation, generating more erratic trajectories that crossed by chance the second reward zone.

To make the point that iHP inactivation affects disambiguation of high and low reward locations, the authors should show that the fraction of trajectories aiming at the low reward zone is higher than expected by chance. Somehow we would expect to see a significant peak pointing toward the low reward zone in the distribution of Figures 6-7.

Review of revised manuscript

The experimental paradigm and analyses are interesting/novel and generate some intriguing phenomena such as the animals' preference for counterclockwise rotation and the stereotypical trajectory shifts induced by muscimol inactivation. Understanding better the underlying mechanisms of these phenomena and the navigational strategies involved in this apparatus will be important in the future for correctly interpreting inactivation experiments.

The idea of a differential effect of dMUS and iMUS was toned down in the abstract and other parts of the manuscript, such that the claims now better match the data.

Reviewer #2 (Public Review):

Summary:

The aim of this paper was to elucidate the role of the dorsal HP and intermediate HP (dHP and iHP) in value-based spatial navigation through behavioral and pharmacological experiments using a newly developed VR apparatus. The authors inactivated dHP and iHP by muscimol injection and analyzed the differences in behavior. The results showed that dHP was important for spatial navigation, while iHP was critical for both value judgments and spatial navigation. The present study developed a new sophisticated behavioral experimental apparatus and proposed a behavioral paradigm that is useful for studying value-dependent spatial navigation. In addition, the present study provides important results that support previous findings of differential function along the dorsoventral axis of the hippocampus.

Reviewer #3 (Public Review):

Summary:

The authors established a new virtual reality place preference task. On the task, rats, which were body-restrained on top of a moveable Styrofoam ball and could move through a circular virtual environment by moving the Styrofoam ball, learnt to navigate reliably to a high-reward location over a low-reward location, using allocentric visual cues arranged around the virtual environment.<br /> The authors also showed that functional inhibition by bilateral microinfusion of the GABA-A receptor agonist muscimol, which targeted the dorsal or intermediate hippocampus, disrupted task performance. The impact of functional inhibition targeting the intermediate hippocampus was more pronounced than that of functional inhibition targeting the dorsal hippocampus.<br /> Moreover, the authors demonstrated that the same manipulations did not significantly disrupt rats' performance on a virtual reality task that required them to navigate to a spherical landmark to obtain reward, although there were numerical impairments in the main performance measure and the absence of statistically significant impairments may partly reflect a small sample size (see under Weaknesses, point 3.).

Overall, the study established a new virtual-reality place preference task for rats and established that performance on this task requires the dorsal to intermediate hippocampus. It also established that task performance is more sensitive to the same muscimol infusion when the infusion was applied to the intermediate hippocampus, compared to the dorsal hippocampus. The authors suggest that these differential effects of muscimol infusions reflect that dorsal hippocampus is responsible for 'precise' spatial navigation and intermediate hippocampus for place-value associations, but this idea remains to be tested by further studies. In their first revision to the paper, the authors toned down this claim, but I still think it would be good to consider more explicitly potential alternative explanations for the differential effects of dorsal and intermediate muscimol infusions (see under Weaknesses, point 2.).

Strengths:

(1) The authors established a new place preference task for body-restrained rats in a virtual environment and, using temporary pharmacological inhibition by intra-cerebral microinfusion of the GABA-A receptor agonist muscimol, showed that task performance requires dorsal to intermediate hippocampus.

(2) These findings extend our knowledge about place learning tasks that require dorsal to intermediate hippocampus and add to previous evidence that the intermediate hippocampus may be more important than other parts of the hippocampus, including the dorsal hippocampus, for goal-directed navigation based on allocentric place memory.

(3) The hippocampus-dependent task may be useful for future recording studies examining how hippocampal neurons may support behavioral performance based on place information.

Weaknesses:

(1) The new findings do not strongly support the authors' suggestion that dorsal hippocampus is responsible for precise spatial navigation and intermediate hippocampus for place-value associations (e.g., final sentence of the first paragraph of the Discussion). The authors base this claim on differential effects of the dorsal and intermediate hippocampal muscimol infusions on different performance measures on the virtual reality place preference task. More specifically, dorsal hippocampal muscimol infusion significantly increased perimeter crossings and perimeter crossing deviations, whereas other measures of task performance are not significantly changed, including departure direction and visits to the high-value location. However, these statistical outcomes offer only limited evidence that dorsal hippocampal infusion specifically affected the perimeter crossing, without affecting the other measures. Numerically the pattern of infusion effects is quite similar across these various measures: intermediate hippocampal infusions markedly impaired these performance measures compared to vehicle infusions, and the values of these measures after dorsal hippocampal muscimol infusion were between the values in the intermediate hippocampal muscimol and the vehicle condition (Figs 5-7). In my opinion, these findings could reflect that dorsal and intermediate hippocampus play distinct roles, as suggested by the authors, but the findings are also consistent with the suggestion that intermediate hippocampal muscimol had a quantitatively stronger, but qualitatively similar effect to dorsal hippocampal muscimol. However, I am largely content with the authors acknowledging within the paper that their suggestion would need to be confirmed by additional studies.

Moreover, I do not find it clearly described in the paper which distinct aspects of navigation the departure direction and perimeter crossing deviation measures capture. The authors suggest that departure direction and perimeter crossing deviation are indices of the navigational efficiency and precision of navigation, respectively (e.g., from p. 7, line 195). However, both departure direction and perimeter crossing deviation measure how accurate/precise, in other words 'close to the target', the rat's navigation is. Efficiency of navigation may rather be reflected by the path length taken (a measure that was not reported). It appears to me that a key difference between the two measures is that departure direction measures the rat's direction towards the goal at the beginning of the rat's navigational path, whereas perimeter crossing deviation measures this further toward the end of the navigational path. This would suggest that departure direction may depend more on directional orienting mechanisms early on in the rat's journey, whereas perimeter crossing deviation may also depend on fine-grained place recognition as the rat approaches the goal. Given the fine-grained place representations in the dorsal hippocampus, the latter may, therefore, depend more on the dorsal hippocampus than the former. I think this would fit with the authors' suggestion 'that the dHP represents a fine-scaled spatial map of an environment' (p. 18, first line). If the authors agree with my interpretation of the different measures, they may consider clarifying this in the Results and Discussion sections.

(2) The claim that the different effects of intermediate and dorsal hippocampal muscimol infusions reflect different functions of intermediate and dorsal hippocampus rests on the assumption that both manipulations inhibit similar volumes of hippocampal tissue to a similar extent, but at different levels along the dorso-ventral axis of the hippocampus. However, this is not a foregone conclusion (e.g., drug spread may differ depending on the infusion site or drug effects may differ due to differential distribution or efficiency of GABA-A receptors), and the authors do not provide direct evidence for this assumption. Therefore, an alternative account of the weaker effects of dorsal compared to intermediate hippocampal muscimol infusions on place-preference performance is that the dorsal infusions affect less hippocampal volume or less markedly inhibit neurons within the affected volume than the intermediate infusions (e.g., due to different drug spread following dorsal and intermediate infusions or due to different distribution or effectiveness of GABA-A receptors in dorsal and intermediate hippocampus). I would recommend that the authors explicitly state this limitation in the limitations section of the Discussion. In their response to my original comments, the authors argue that it is unlikely that muscimol exerts stronger effects in intermediate compared to dorsal hippocampus, based on the finding that in vitro paired pulse inhibition is reduced in ventral compared to dorsal hippocampal slices (Papatheodoropoulos et al., 2002). However, this claim is not strongly supported by the in vitro paired-pulse inhibition findings. First, these findings relate to differences between dorsal and ventral hippocampus, whereas differences between dorsal and intermediate hippocampus were not investigated. Second, reduced paired pulse inhibition may not necessarily reflect reduced GABA-A receptor expression/efficiency (which would be likely to reduce muscimol effects), but may also reflect reduced GABAergic input, which would not be expected to reduce muscimol effects.

(3) It is good that the authors included a comparison/control experiment using a spherical beacon-guided navigation task, to examine the specific psychological mechanisms disrupted by the hippocampal manipulations. However, the sample size for the comparison experiment (n=5 rats) was lower than for the main study (n=8 rats, and the data shown in Fig. 8 suggest that the comparison task may be affected by the hippocampal manipulations similarly to the place-preference task, albeit less markedly. This effect may well have been significant if the same sample size had been used as in the main experiment. Therefore, I would recommend that the authors acknowledge this limitation in the Discussion (perhaps, in the Limitation section).

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript examines the contribution of the dorsal and intermediate hippocampus to goal-directed navigation in a wide virtual environment where visual cues are provided by the scenery on the periphery of a wide arena. Among a choice of 2 reward zones located near the arena periphery, rats learn to navigate from the center of the arena to the reward zone associated with the highest reward. Navigation performance is largely assessed from the rats' body orientation when they leave the arena center and when they reach the periphery, as well as the angular mismatch between the reward zone and the site rats reach the periphery. Muscimol inactivation of the dorsal and intermediate hippocampus alters rat navigation to the reward zone, but the effect was more pronounced for the inactivation of the intermediate hippocampus, with some rat trajectories ending in the zone associated with the lowest reward. Based on these results, the authors suggest that the intermediate hippocampus is critical, especially for navigating to the highest reward zone.

Strengths:

-The authors developed an effective approach to study goal-directed navigation in a virtual environment where visual cues are provided by the peripheral scenery.

- In general, the text is clearly written and the figures are well-designed and relatively straightforward to interpret, even without reading the legends.

- An intriguing result, which would deserve to be better investigated and/or discussed, was that rats tended to rotate always in the counterclockwise direction. Could this be because of a hardware bias making it easier to turn left, some aspect of the peripheral landscape, or a natural preference of rats to turn left that is observable (or reported) in a real environment?

Thank you for the insightful question. As the reviewer mentioned, the counterclockwise rotation behavior was intriguing and unexpected. To answer the reviewer’s question properly, we examined whether such stereotypical turning behavior appeared before the rats acquired the task rule and reward zones in the pre-surgical training phase of the task. Data from the last day of shaping and the first day of the pre-surgical main task day showed no significant difference in the number of trials in which the first body-turn was either clockwise or counterclockwise, suggesting that the rats did not have a bias toward a specific side (p=0.46 for Shaping; p=0.76 for the Main task, Wilcoxon signed-rank test). These results excluded the possibility that there was something in the apparatus's hardware that made the rats turn only to the left. Also, since we used the same peripheral landscape for the shaping and main task, we could assume that the peripheral landscape did not cause movement bias.

Author response image 1.

Although it remains inconclusive, we have noticed that some prior studies alluded to a phenomenon similar to this issue, framed as the topic of lateralization or spatial preference by comparing left and right biases. For example, Wishaw et al. (1992) suggested that there was natural lateralization in rats (“Most of the rats displayed either a strong right limb bias or a strong left limb bias.”) but no dominance to a specific side. Andrade et al. (2001) also claimed that “83% of Wistar rats spontaneously showed a clear preference for left or right arms in the T-maze.” However, to the best of our knowledge, there has been no direct evidence that rats have a dominant natural preference only to one side.

Therefore, while the left-turning behavior remains an intriguing topic for further investigation, we find it difficult to pinpoint the reason behind the behavior in the current study. However, we would like to emphasize that this behavior did not interrupt testing our hypothesis. Nonetheless, we agree with the reviewer’s point that the counterclockwise rotation needs to be discussed more, so we revised the manuscript as follows:

“To rule out the potential effect of hardware bias or any particular aspect of peripheral landscape to make rats turn only to one side, we measured the direction of the first body-turn in each trial on the last day of shaping and the first day of the main task (i.e., before rats learned the reward zones). There was no significant difference between the clockwise and counterclockwise turns (p=0.46 for shaping, p=0.76 for main task; Wilcoxon signed-rank test), indicating that the stereotypical pattern of counterclockwise body-turn appeared only after the rats learned the reward locations.” (p.6)

- Another interesting observation, which would also deserve to be addressed in the discussion, is the fact that dHP/iHP inactivations produced to some extent consistent shifts in departing and peripheral crossing directions. This is visible from the distributions in Figures 6 and 7, which still show a peak under muscimol inactivation, but this peak is shifted to earlier angles than the correct ones. Such change is not straightforward to interpret, unlike the shortening of the mean vector length.

Maybe rats under muscimol could navigate simply by using the association of reward zone with some visual cues in the peripheral scene, in brain areas other than the hippocampus, and therefore stopped their rotation as soon as they saw the cues, a bit before the correct angle. While with their hippocampus is intact, rats could estimate precisely the spatial relationship between the reward zone and visual cues.

We agree with the possibility suggested by the reviewer. However, although not described in the original manuscript, we performed several different control experiments in a few rats using various visual stimulus manipulations to test how their behaviors change as a result. One of the experiments was the landmark omission test, where one of the landmarks was omitted. The landmark to be made disappear was pseudorandomly manipulated on a trial-by-trial basis. We observed that the omission of one landmark, regardless of its identity, did not cause a specific behavioral change in finding the reward zones, suggesting that the rats were not relying on a single visual landmark when finding the reward zone.

Author response image 2.

Therefore, it is unlikely that rats used the spatial relationship between the reward zone and a specific visual cue to solve the task in our study. However, the result was based on an insufficient sample size (n=3), not permitting any meaningful statistical testing. Thus, we have now updated this information in the manuscript as an anecdotal result as follows:

“Additionally, to investigate whether the rats used a certain landmark as a beacon to find the reward zones, we conducted the landmark omission test as a part of control experiments. Here, one of the landmarks was omitted, and the landmark to be made disappear was pseudorandomly manipulated on a trial-by-trial basis. The omission of one landmark, regardless of its identity, did not cause a specific behavioral change in finding the reward zones, suggesting that the rats were not relying on a single visual landmark when finding the reward zones. The result can be reported anecdotally only because of an insufficient sample size (n=3), not permitting any meaningful statistical testing.” (p.9)

Weaknesses:

-I am not sure that the differential role of dHP and iHP for navigation to high/low reward locations is supported by the data. The current results could be compatible with iHP inactivation producing a stronger impairment on spatial orientation than dHP inactivation, generating more erratic trajectories that crossed by chance the second reward zone.

To make the point that iHP inactivation affects the disambiguation of high and low reward locations, the authors should show that the fraction of trajectories aiming at the low reward zone is higher than expected by chance. Somehow we would expect to see a significant peak pointing toward the low reward zone in the distribution of Figures 6-7.

We thank the reviewer for the valuable comments. We agree that it is difficult to rigorously distinguish the loss of value representation from spatial disorientation in our experiment. Since the trial ended once the rat touched either reward zone, it was difficult to specify whether they intended to arrive at the location or just moved randomly and arrived there by chance. Moreover, it is possible that the drug infusion did not completely inactivate the iHP but only partially did so.

To investigate this issue further, we checked whether the distribution of the departure direction (DD) differed between the trials in which rats initially headed north (NW, N, NE) and south (SE, S, SW) at the start. In the manuscript, we demonstrated that DD aligned with the high-value zone, indicating that the rat remembered the scenes associated with the high-value zone (p.8). Based on the rats’ characteristic counterclockwise rotation, the reward zone rats would face first upon starting while heading north would be the high-value zone. On the other hand, the rat would face the low-value reward zone when starting while heading south. In this case, normal rats would inhibit leaving the start zone and rotate further until they face the high-value zone before finally departing the start location. If the iHP inactivation caused a more severe impairment in spatial orientation but not in value representation, it is likely that the iHP-inactivated rats in both north- and south-starting trials would behave similarly with the dHP-inactivated rats, but producing a larger deviation from the high-value zone. However, if the iHP inactivation affected the disambiguation of high and low reward locations, north and south-starting trials would show different DD distributions.

The circular plots shown below are the DD distributions of dMUS and iMUS. We could see that when they started facing north, iHP-inactivated rats still aligned themselves towards the high-value zone and thus remained spatially oriented, similar to the dHP inactivation session. However, in the south-starting trials, the DD distribution was completely different from the north-starting trials; the rats failed in body alignment towards the high-value zone. Instead, they departed the start point while heading south in most trials. This pattern was not seen in dMUS sessions, even in their south-starting trials, illustrating the distinct deficit caused by iHP inactivation. Additionally, most of the rats with iHP inactivation visited the low-value zone more in south-headed starting trials than in the north-headed trials, except for one rat.

Author response image 3.

Furthermore, we would like to clarify that we do not limit the effect of iHP inactivation to the impairment in distinguishing the high and low reward zones. It is possible that iHP inactivation resulted in the loss of a global value-representing map, leading to the impairment in distinguishing both reward zones from other non-rewarded areas in the environment. Figures 6 and 7 implicated this possibility by showing that the peaks are not restricted only to the reward zones. Unfortunately, we cannot rigorously address this in the current study because of the limitations of our experimental design mentioned above.

Nonetheless, we agree with the reviewer that this limitation needs to be addressed, so we now added how the current study needs further investigation to clarify what causes the behavioral change after the iHP inactivation in the Limitations section (p.21).

Reviewer #2 (Public Review):

Summary:

The aim of this paper was to elucidate the role of the dorsal HP and intermediate HP (dHP and iHP) in value-based spatial navigation through behavioral and pharmacological experiments using a newly developed VR apparatus. The authors inactivated dHP and iHP by muscimol injection and analyzed the differences in behavior. The results showed that dHP was important for spatial navigation, while iHP was critical for both value judgments and spatial navigation. The present study developed a new sophisticated behavioral experimental apparatus and proposed a behavioral paradigm that is useful for studying value-dependent spatial navigation. In addition, the present study provides important results that support previous findings of differential function along the dorsoventral axis of the hippocampus.

Strengths:

The authors developed a VR-based value-based spatial navigation task that allowed separate evaluation of "high-value target selection" and "spatial navigation to the target." They were also able to quantify behavioral parameters, allowing detailed analysis of the rats' behavioral patterns before and after learning or pharmacological inactivation.

Weaknesses:

Although differences in function along the dorsoventral axis of the hippocampus is an important topic that has received considerable attention, differences in value coding have been shown in previous studies, including the work of the authors; the present paper is an important study that supports previous studies, but the novelty of the findings is not that high, as the results are from pharmacological and behavioral experiments only.

We appreciate the reviewer's insightful comments. In response, we would like to emphasize that a very limited number of studies investigated the function of the intermediate hippocampus, especially in spatial memory tasks. We tested the differential functions of the dorsal and intermediate hippocampus using a within-animal design and used reversible inactivation manipulation (i.e., muscimol injection) to prevent potential compensation by other brain regions when using irreversible manipulation techniques (i.e., lesion). Also, very few studies have analyzed the navigation trajectories of animals as closely as in the current study. We emphasize the novelty of our study by comparing it with prior studies, as shown below in Table 1.

Author response table 1.

Comparison of our study with those from prior studies

Moreover, to the best of our knowledge, the current manuscript is the first to investigate the hippocampal subregions along the long axis in a VR environment using a hippocampal-dependent spatial memory task. Nonetheless, we agree that the current study has a limitation as a behavior-only experiment. We now have added a comment on how other techniques, such as electrophysiology, would develop our findings in the Limitation section (p.21).

Reviewer #3 (Public Review):

Summary:

The authors established a new virtual reality place preference task. On the task, rats, which were body-restrained on top of a moveable Styrofoam ball and could move through a circular virtual environment by moving the Styrofoam ball, learned to navigate reliably to a high-reward location over a low-reward location, using allocentric visual cues arranged around the virtual environment.

The authors also showed that functional inhibition by bilateral microinfusion of the GABA-A receptor agonist muscimol, which targeted the dorsal or intermediate hippocampus, disrupted task performance. The impact of functional inhibition targeting the intermediate hippocampus was more pronounced than that of functional inhibition targeting the dorsal hippocampus.

Moreover, the authors demonstrated that the same manipulations did not significantly disrupt rats' performance on a virtual reality task that required them to navigate to a spherical landmark to obtain reward, although there were numerical impairments in the main performance measure and the absence of statistically significant impairments may partly reflect a small sample size (see comments below).

Overall, the study established a new virtual-reality place preference task for rats and established that performance on this task requires the dorsal to intermediate hippocampus. They also established that task performance is more sensitive to the same muscimol infusion (presumably - doses and volumes used were not clearly defined in the manuscript, see comments below) when the infusion was applied to the intermediate hippocampus, compared to the dorsal hippocampus, although this does not offer strong support for the authors claim that dorsal hippocampus is responsible for accurate spatial navigation and intermediate hippocampus for place-value associations (see comments below).

Strengths:

(1) The authors established a new place preference task for body-restrained rats in a virtual environment and, using temporary pharmacological inhibition by intra-cerebral microinfusion of the GABA-A receptor agonist muscimol, showed that task performance requires dorsal to intermediate hippocampus.

(2) These findings extend our knowledge about place learning tasks that require dorsal to intermediate hippocampus and add to previous evidence that, for some place memory tasks, the intermediate hippocampus may be more important than other parts of the hippocampus, including the dorsal hippocampus, for goal-directed navigation based on allocentric place memory.

(3) The hippocampus-dependent task may be useful for future recording studies examining how hippocampal neurons support behavioral performance based on place information.

Weaknesses:

(1) The new findings do not strongly support the authors' suggestion that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

The authors base this claim on the differential effects of the dorsal and intermediate hippocampal muscimol infusions on different performance measures. More specifically, dorsal hippocampal muscimol infusion significantly increased perimeter crossings and perimeter crossing deviations, whereas dorsal infusion did not significantly change other measures of task performance, including departure direction and visits to the high-value location. However, these statistical outcomes offer only limited evidence that dorsal hippocampal infusion specifically affected the perimeter crossing, without affecting the other measures. Numerically the pattern of infusion effects is quite similar across these various measures: intermediate hippocampal infusions markedly impaired these performance measures compared to vehicle infusions, and the values of these measures after dorsal hippocampal muscimol infusion were between the values in the intermediate hippocampal muscimol and the vehicle condition (Figures 5-7). Moreover, I am not so sure that the perimeter crossing measures really reflect distinct aspects of navigational performance compared to departure direction and hit rate, and, even if they did, which aspects this would be. For example, in line 316, the authors suggest that 'departure direction and PCD [perimeter crossing deviation] [are] indices of the effectiveness and accuracy of navigation, respectively'. However, what do the authors mean by 'effectiveness' and 'accuracy'? Accuracy typically refers to whether or not the navigation is 'correct', i.e. how much it deviates from the goal location, which would be indexed by all performance measures.

So, overall, I would recommend toning down the claim that the findings suggest that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

The reviewer mentioned that the statistical outcomes offer limited evidence as the dHP inactivation results were always positioned between the results of the iHP inactivation and controls. However, we would like to emphasize that, projecting to each other, the two subregions are not completely segregated anatomically. It is highly likely this is also true functionally and there should be some overlap in their roles. Considering such relationships between the dHP and iHP, it could be natural to see an intermediate effect after inactivating the dHP, and that is why we focused on the “magnitude” of behavioral changes after inactivation instead of complete dissociation between the two subregions in our manuscript. Unfortunately, because of the nature of the drug infusion study, further dissociation would be difficult, requiring further investigation with different experimental techniques, such as physiological examinations of the neural firing patterns between the two regions. We mentioned this caveat of the current study in the Limitations as follows:

“However, our study includes only behavioral results and further mechanistic explanations as to the processes underlying the behavioral deficits require physiological investigations at the cellular level. Neurophysiological recordings during VR task performance could answer, for example, the questions such as whether the value-associated map in the iHP is built upon the map inherited from the dHP or it is independently developed in the iHP.” (p.21)

Regarding the reviewer’s comment on the meaning of measuring the perimeter crossing directions, we would like to draw the reviewer’s attention to the individual trajectories during the iMUS sessions described in Figure 5. Particularly when they were not confident with the location of the higher reward, rats changed their heading directions during the navigation, which resulted in a less efficient route to the goal location. Rats showing this type of behavior tended to hit the perimeter of the arena first before correcting their routes toward the goal zone. In contrast, rats showing effective navigation hardly bumped into the wall or perimeter before hitting the goal zone. Thus, their PCDs matched DDs almost always. When considered together with DD, our PCD measure could tell whether rats not hitting the goal zone directly after departure were impaired in either maintaining the correct heading direction to the goal zone at the start location or orienting themselves to the target zone accurately from the start. Our results suggest that the latter is the case. We included the relevant explanation in the Discussion section as follows:

“Particularly, rats changed their heading directions during the navigation when they were not confident with the location of the higher reward, resulting in a less efficient route to the goal location. Rats showing this type of behavior tended to hit the perimeter of the arena first before correcting their routes. Therefore, when considered together with DD, our PCD measure could tell that the rats not hitting the goal zone directly after departure were impaired in orienting themselves to the target zone accurately from the start, not in maintaining the correct heading direction to the goal zone at the start location.” (p.19)

Nonetheless, we agree with the reviewer that the term ‘accuracy’ might be confusing with performance accuracy, so we replaced the term with ‘precision’ throughout the manuscript, referring to the precise targeting of the reward zones.

(2) The claim that the different effects of intermediate and dorsal hippocampal muscimol infusions reflect different functions of intermediate and dorsal hippocampus rests on the assumption that both manipulations inhibit similar volumes of hippocampal tissue to a similar extent, but at different levels along the dorso-ventral axis of the hippocampus. However, this is not a foregone conclusion (e.g., drug spread may differ depending on the infusion site or drug effects may differ due to differential expression of GABA-A receptors in the dorsal and intermediate hippocampus), and the authors do not provide direct evidence for this assumption. Therefore, a possible alternative account of the weaker effects of dorsal compared to intermediate hippocampal muscimol infusions on place-preference performance is that the dorsal infusions affect less hippocampal volume or less markedly inhibit neurons within the affected volume than the intermediate infusions. I would recommend that the authors briefly consider this issue in the discussion. Moreover, from the Methods, it is not clear which infusion volume and muscimol concentration were used for the different infusions (see below, 4.a.), and this must be clarified.

We appreciate these insightful comments from the reviewer and agree that we do not provide direct evidence for the point raised by the reviewer. To the best of our knowledge, most of the behavioral studies on the long axis of the hippocampus did not particularly address the differential expression of GABA-A receptors along the axis. We could not find any literature that specifically introduced and compared the levels of expression of GABA-A receptors or the diffusion range of muscimol in the intermediate hippocampus to the other subregions. However, we found that Sotiriou et al. (2005) made such comparisons with respect to the expression of different GABA-A receptors. They concluded that the dorsal and ventral hippocampi have different levels of the GABA-A receptor subtypes. The a1/b2/g2 subtype was dominant in the dorsal hippocampus, while the a2/b1/g2 subtype was prevalent in the ventral hippocampus. Sotiriou and colleagues also mentioned the lower affinity of GABA-A receptor binding in the ventral hippocampus, and this result is consistent with the Papatheodoropoulos et al. (2002) study that showed a weaker synaptic inhibition in the ventral hippocampus compared to the dorsal hippocampus. Papatheodoropoulos et al. speculated differences in GABA receptors as one of the potential causes underlying the differential synaptic inhibition between the dorsal and ventral hippocampal regions. Based on these findings, the same volume of muscimol is more likely to cause a more severe effect on the ventral hippocampus than the dorsal hippocampus. Therefore, we do not believe that the less significant changes after the dorsal hippocampal inactivation were induced by the expression level of GABA-A receptors. Additionally, we have demonstrated in our previous study that muscimol injections in the dorsal hippocampus impair performance to the chance level in scene-based behavioral tasks (Lee et al., 2014; Kim et al., 2012).

Nonetheless, we mentioned the possibility of differential muscimol expressions between the two target regions. Following the suggestion of the reviewer, we now included this information in the Discussion as follows:

“Although there is still a possibility that the levels of expression of GABA-A receptors might be different along the longitudinal axis of the hippocampus, …” (p.20)

Regarding the drug infusion volume and concentration, we included these details in the Methods. Please see our detailed response to 4.a. below.

(3) It is good that the authors included a comparison/control study using a spherical beacon-guided navigation task, to examine the specific psychological mechanisms disrupted by the hippocampal manipulations. However, as outlined below (4.b.), the sample size for the comparison study was lower than for the main study, and the data in Figure 8 suggest that the comparison task may be affected by the hippocampal manipulations similarly to the place-preference task, albeit less markedly. This would raise the question as to which mechanisms that are common to the two tasks may be affected by hippocampal functional inhibition, which should be considered in the discussion.

The sample size for the object-guided navigation task was smaller because we initially did not plan the experiment, but later in the study decided to conduct the control test. Therefore, the object-guided navigation task was added to the study design after finishing the first three rats, resulting in a smaller sample size than the place preference task. We included this detail in the manuscript, as follows:

“Note the smaller sample size in the object-guided navigation task. This was because the task was later added to the study design.” (p.24)

Regarding the mechanism behind the two different tasks, we did not perform the same heading direction analysis here as in the place preference task because the two tasks have different characteristics such as task complexity. The object-guided navigation task is somewhat similar to the visually guided (or cued) version of the water maze task, which is widely known as hippocampal-independent (Morris et al., 1986; Packard et al., 1989; also see our descriptions on p.15). Therefore, we would argue that the two tasks (i.e., place preference task and object-guided navigation task) used in the current manuscript do not share neural mechanisms in common. Additionally, we confirmed that several behavioral measurements related to motor capacity, such as travel distance and latency, along with the direct hit proportion provided in Figure 8, did not show any statistically significant changes across drug conditions.

4. Several important methodological details require clarification:

a. Drug infusions (from line 673):

- '0.3 to 0.5 μl of either phosphate-buffered saline (PBS) or muscimol (MUS) was infused into each hemisphere'; the authors need to clarify when which infusion volume was used and why different infusion volumes were used.

We thank the reviewer for carefully reading our manuscript. We were cautious about side effects, such as suppressed locomotion or overly aggressive behavior, since the iHP injection site was close to the ventricle. We were keenly aware that the intermediate to ventral hippocampal regions are sensitive to the drug dosage from our previous experiments. Thus, we observed the rat’s behavior for 20 minutes after drug injection in a clean cage. We started from 0.5 μl, based on our previous study, but if the injected rat showed any sign of side effects in the cage, we stopped the experiment for the day and tried with a lower dosage (i.e., 0.4 μl first, then 0.3 μl, etc.) until we found the right dosage under which the rat did not show any side effect. This procedure is necessary because cannula tip positions are slightly different from rat to rat. When undergoing this procedure, five out of eight rats received 0.4 μl, two received 0.3 μl, and one received 0.5 μl. Still, there was no significant difference in performance, including the high-value visit percentage, departing and perimeter crossing directions, across all dosages. This information is now added in the Methods section as follows:

“If the rat showed any side effect, particularly sluggishness or aggression, we reduced the drug injection amount in the rat by 0.1 ml until we found the dosage with which there was no visible side effect. As a result, five of the rats received 0.4 ml, two received 0.3 ml, and one received 0.5 ml.” (p.25)

- I could not find the concentration of the muscimol solution that was used. The authors must clarify this and also should include a justification of the doses used, e.g. based on previous studies.

Thank you for the suggestion. We used the drug concentration of 1mg/ml, which was adapted from our previous muscimol study (Lee et al., 2014; Kim et al., 2012). The manuscript is now updated, as follows:

“…or muscimol (MUS; 1mg/ml, dissolved in saline) was infused into each hemisphere via a 33-gauge injection cannula at an injection speed of 0.167 ml/min, based on our previous study (Lee et al., 2014; Kim et al., 2012).” (p.25)

-  Please also clarify if the injectors and dummies were flush with the guides or by which distance they protruded from the guides.

The injection and dummy cannula both protruded from the guide cannula by 1 mm, and this information is now added to the Methods section, as follows:

“The injection cannula and dummy cannula extended 1 mm below the tip of the guide cannula.” (p.25)

b. Sample sizes: The authors should include sample size justifications, e.g. based on considerations of statistical power, previous studies, practical considerations, or a combination of these factors. Importantly, the smaller sample size in the control study using the spherical beacon-guided navigation task (n=5 rats) limits comparability with the main study using the place-preference task (n=8). Numerically, the findings on the control task (Figure 8) look quite similar to the findings on the place-preference task, with intermediate hippocampal muscimol infusions causing the most pronounced impairment and dorsal hippocampal muscimol infusions causing a weaker impairment. These effects may have reached statistical significance if the same sample size had been used in the place-preference study.

We set the current sample size for several reasons. First, based on our previous studies, we assumed that eight, or more than six, would be enough to achieve statistical power in a “within-animal design” study. Also, considering the ethical commitments, we tried to keep the number of animals used in the study to the least. Last, our paradigm required very long training periods (3 months on average per animal), so we could not increase the sample size for practical reasons. Regarding the reasons for the smaller sample size for the object-guided navigation task, please see the previous response to 3 above. The manuscript is now revised as follows:

“Based on our prior studies (Park et al., 2017; Yoo and Lee, 2017; Lee et al., 2014), the sample size of our study was set to the least number to achieve the necessary statistical power in the current within-subject study design for ethical commitments and practical considerations (i.e., relatively long training periods).” (p.22)

c. Statistical analyses: Why were the data of the intermediate and dorsal hippocampal PBS infusion conditions averaged for some of the analyses (Figure 5; Figure 6B and C; Figure 7B and C; Figure 8B) but not for others (Figure 6A and Figure 7A)?

The reviewer is correct that we only illustrated the separate dPBS and iPBS data for Figures 6A and 7A. Since the directional analysis is the main focus of the current manuscript, we tried to provide better visualization and more detailed examples of how the drug infusion changed the behavioral patterns between the PBS and MUS conditions in each region. Except for the visualization of DD and PCD, we averaged the PBS sessions to increase statistical power, as described in p.9. We added a detailed description of the reasons for illustrating dPBS and iPBS data separately in the manuscript, as follows:

“Note that dPBS and iPBS sessions were separately illustrated here for better visualization of changes in the behavioral pattern for each subregion.” (p.12)

Reviewing Editor (Recommendations For The Authors):

The strength of evidence rating in the assessment is currently noted as "incomplete." This can be improved following revisions if you amend your conclusions in the paper, including in the title and abstract, such that the paper's major conclusions more closely match what is shown in the Results.

Following the suggestions of the reviewing editor, we have mentioned the caveats of our study in the Limitations section of our revised manuscript (p.21). In addition, the manuscript has been revised so that the conclusions in the paper match more closely to the experimental results as can been seen in some of the relevant sentences in the abstract and main text as follows:

“Inactivation of both dHP and iHP with muscimol altered efficiency and precision of wayfinding behavior, but iHP inactivation induced more severe damage, including impaired place preference. Our findings suggest that the iHP is more critical for value-dependent navigation toward higher-value goal locations.” (Abstract; p.2)

“Whereas inactivation of the dHP mainly affected the precision of wayfinding, iHP inactivation impaired value-dependent navigation more severely by affecting place preference.” (p.5)

“The iHP causes more damage to value-dependent spatial navigation than the dHP, which is important for navigational precision” (p.12)

However, we haven’t changed the title of the manuscript as it carries what we’d like to deliver in this study accurately.

Reviewer #1 (Recommendations For The Authors):

- What were the dimensions of the environment? What distance did rats typically run to reach the reward zone? A scale bar would be helpful in Figure 1.

We used the same circular arena from the shaping session, which was 1.6 meters in diameter (p.23), and the shortest path between the start location and either reward zone was 0.62 meters. We revised the manuscript for clarification as follows:

“For the pre-training session, rats were required to find hidden reward zones…, on the same circular arena from the shaping session.” (p.23)

“Therefore, the shortest path length between the start position and the reward zone was 0.62 meters.” (p.23)

We also added a scale bar in Figure 1C for a better understanding.

- Line 169: "The scene rotation plot covers the period from the start of the trial to when the rat leaves the starting point at the center and the departure circle (Figure 2B)."

The sentence is unclear. Maybe it should be "... from the start of the trial to when the rat leaves the departure circle”.

The sentence has been revised following the reviewer's suggestion. (p.7)

- Line 147: "First, they learned to rotate the spherical treadmill counterclockwise to move around in the virtual environment (presumably to perform energy-efficient navigation)."

It is not clear from this sentence if rats naturally preferred the counterclockwise direction or if the counterclockwise direction was a task requirement.

We now clarified in our revised manuscript that it was not a task requirement to turn counterclockwise, as follows:

“First, although it was not required in the task, they learned to rotate the spherical treadmill counterclockwise…” (p.6)

- Line 149: "Second, once a trial started, but before leaving the starting point at the center, the animal rotated the treadmill to turn the virtual environment immediately to align its starting direction with the visual scene associated with the high-value reward zone."

The sentence is unclear. Maybe "Second, once a trial started, the animal rotated the treadmill immediately to align its starting direction with the visual scene associated with the high-value reward zone.”

We have updated the description following the suggestion. (p.6)

Reviewer #2 (Recommendations For The Authors):

- There are some misleading descriptions of the conclusion of the results in this paper. In this study, the functions of (a) selection of high-value target and (b) spatial navigation to the target were assessed in the behavioral experiments. The results of the pharmacological experiments showed that dHP inactivation impaired (b) and iHP inactivation impaired both (a) and (b) (Figures 5 B & D). However, the last sentence of the abstract states that dHP is important for the functions of (a) and iHP for (b). There are several other similar statements in the main text. Since the separation of (a) and (b) is an important and original aspect of this study, the description should clearly show the conclusion that dHP is important for (a) and iHP is important for both (a) and (b).

Related to the above, the paragraph title in the Discussion "The iHP may contain a value-associated cognitive map with reasonable spatial resolution for goal-directed navigation (536-537)" is also somewhat misleading: "with reasonable resolution for goal-directed behavior" seems to reflect the results of an object-guided navigation task (Figure 8). However, the term "goal-directed behavior" is also used for value-dependent spatial navigation (i.e., the main task), which causes confusion. I would like to suggest clarifying the wording on this point.

First, we need to correct the reviewer’s statement regarding our descriptions of the results. As the reviewer mentioned, our results indicated that the dHP inactivation impaired (b) but not (a), while the iHP inactivation impaired both (a) and (b). Regarding the iHP inactivation result, we focused on the impairment of (a) since our aim was to investigate spatial-value association in the hippocampus. Also, it was more likely that (a) affected (b), but not the other way, because (a) remained intact when (b) was impaired after dHP inactivation. We emphasized this difference between dHP and iHP inactivation, which was (a). Therefore, we mentioned in the last sentence of the abstract that the dHP is important for (b), which is the precision of spatial navigation to the target location, and the iHP is critical for (a).

Moreover, we would like to clarify that we were not referring to the object-guided navigation task in Figure 8 in the phrase ‘with a reasonable spatial resolution for goal-directed navigation.’ Please note that the object-guided navigation task did not require fine spatial resolution to find the reward. The phrase instead referred to the dHP inactivation result (Figure 5 and 6), where the rats could find the high-value zone even with dHP inactivation, although the navigational precision decreased. Nonetheless, we agree with the reviewer for the confusion that the title might cause, so now have updated the title as follows:

“The iHP may contain a value-associated cognitive map with reasonable spatial resolution for value-based navigation” (p.19)

- As an earlier study focusing on the physiology of iHP, Maurer et al, Hippocampus 15:841 (2005) is also a pioneering and important study, and I suggest citing it.

Thank you for the suggestion. We included the Maurer et al. (2005) study in the Introduction section as follows:

“…Specifically, there is physiological evidence that the size of a place field becomes larger as recordings of place cells move from the dHP to the vHP (Jung et al., 1994; Maurer et al., 2005; Kjelstrup et al., 2008; Royer et al., 2010).” (p.4)

- One of the strengths of this paper is that we have developed a new control system for the VR navigation task device, but I cannot get a very detailed description of this system in the Methods section. Also, no information about the system control has been uploaded to GitHub. I would suggest adding a description of the manufacturer, model number, and size of components, such as a rotary encoder and ball, and information about the software of the control system, with enough detail to allow the reader to reconstruct the system.

We have now added detailed descriptions of the VR system in the Methods section (see “2D VR system). (p.22)

Reviewer #3 (Recommendations For The Authors):

(1) Some comments on specific passages of text:

Lines 87 to 89: 'Surprisingly, beyond the recognition of anatomical divisions, little is known about the functional differentiation of subregions along the dorsoventral axis of the hippocampus. Moreover, the available literature on the subject is somewhat inconsistent.'

I would recommend to rephrase these statements. Regarding the first statement, there is substantial evidence for functional differentiation along the dorso-ventral axis of the hippocampus (e.g., see reviews by Moser and Moser, 1998, Hippocampus; Bannerman et al., 2004, Neurosci Biobehav Rev; Bast, 2007, Rev Neurosci; Bast, 2011, Curr Opin Neurobiol; Fanselow and Dong, 2010, Neuron; Strange et al., 2014, Nature Rev Neurosci). Regarding the second statement, the authors may consider being more specific, as the inconsistencies demonstrated seem to relate mainly to the hippocampal representation of value information, instead of functional differentiation along the dorso-ventral hippocampal axis in general.

We agree with the reviewer that the abovementioned statements need further clarification. The manuscript is now revised as follows:

“Surprisingly, beyond the recognition of anatomical divisions, the available literature on the functional differentiation of subregions along the dorsoventral axis of the hippocampus, particularly in the context of value representation, is somewhat inconsistent.” (p.4)

Lines 92 to 93: 'Thus, it has been thought that the dHP is more specialized for precise spatial representation than the iHP and vHP.'

I think 'fine-grained' may be the more appropriate term here. Also, check throughout the manuscript when referring to the differences of spatial representations along the hippocampal dorso-ventral axis.

Thank you for the insightful suggestion. We changed the term to ‘fine-grained’ throughout the manuscript, as follows:

“Thus, it has been thought that the dHP is more specialized for fine-grained spatial representation than the iHP and vHP.” (p.4)

“Consequently, the fine-grained spatial map present in the dHP…” (p.20)

Line 217: well-'trained' rats?

We initially used the term ‘well-learned’ to focus on the effect of learning, not training. Please note that the rats were already adapted to moving freely in the VR environment during the Shaping sessions, but the immediate counterclockwise body alignment only appeared after they acquired the reward locations for the main task. Nonetheless, we agree that the term might cause confusion, so we revised the manuscript as the reviewer suggested, as follows:

“This implies that well-trained rats aligned their bodies more efficiently…” (p.8)

Lines 309 to 311: 'Taken together, these results indicate that iHP inactivation severely damages normal goal-directed navigational patterns in our place preference task.'

Consider to mention that dHP inactivation also causes impairments, albeit weaker ones.

We thank the reviewer for the suggestion. We revised the manuscript by mentioning dHP inactivation as follows:

“Taken together, these results indicate that iHP inactivation more severely damages normal goal-directed navigational patterns than dHP inactivation in our place-preference task.” (p.11-12)

Lines 550 to 552: 'The involvement of the iHP in spatial value association has been reported in several studies. For example, Bast and colleagues reported that rapid place learning is disrupted by removing the iHP and vHP, even when the dHP remains undamaged (Bast et al., 2009).'

Bast et al. (2009) did not directly show the role of iHP in 'spatial value associations'. They suggested that the importance of iHP for behavioral performance based on rapid, one-trial, place learning may reflect neuroanatomical features of the intermediate region, especially the combination of afferents that could convey the required fine-grained visuo-spatial information with relevant afferent and efferent connections that may be important to translate hippocampal place memory into appropriate behavioral performance (this may include afferents conveying value information). More recent theoretical and empirical research suggests that projections to the (ventral) striatum may be relevant (see Tessereau et al., 2021, BNA and Bauer et al., 2021, BNA).

We appreciate the reviewer for this insightful comment. We agree with the reviewer that Bast et al. (2009) did not directly mention spatial value association; however, learning a new platform location needs an update of value information in the spatial environment. Therefore, we thought the study, though indirectly, suggested how the iHP contributes to spatial value associations. Nonetheless, to avoid confusion, we revised the manuscript, as follows:

“The involvement of the iHP in spatial value association has been reported or implicated in several studies” (p.20)

(2) Figures and legends:

Figure 2B: What do the numbers after novice and expert indicate?

The numbers indicate the rat ID, followed by the session number. We added the details to the Figure legend, as follows:

“The numbers after ‘Novice’ and ‘Expert’ indicate the rat and session number of the example.” (p.34)

Figure 2C: Please indicate units of the travel distance and latency measurements.

The units are now described in the Figure legends, as follows:

“Mean travel distance in meters and latency in seconds are shown below the VR arena trajectory.” (p.34)

Figure 3Aii: Here and in other figures - do the vector lengths have a unit (degree?)?

No, the mean vector length is an averaged value of the resultant vectors, thus having no specific unit.

Figure 5A: Please explain what the numbers on top of the individual sample trajectories indicate.

The numbers are IDs for rats, sessions, and trials of specific examples. We added the explanation to the Figure legends, as follows:

“Numbers above each trajectory indicate the identification numbers for rat, session, and trial.” (p.35)

(3) Additional comments on some methodological details:

a. Why was the non-parametric Wilcoxon signed-rank test used for the planned comparison between intermediate and dorsal hippocampal PBS infusions, whereas parametric ANOVA and post-hoc comparisons were used for other analyses? This probably doesn't make a big difference for the interpretation of the present data (as a parametric pairwise comparison would also not have revealed any significant difference between intermediate and dorsal hippocampal PBS infusions), but it would nevertheless be good to clarify the rationale for this.

We used the non-parametric statistics since our sample size was rather small (n=8) to use the parametric statistics, although we used the parametric ANOVA for some of the results because it is the most commonly known and widely used statistical test in such comparisons. However, we also checked the statistics with the alternatives (i.e., non-parametric Wilcoxon signed-rank test to parametric paired t-test and parametric One-way RM ANOVA with Bonferroni post hoc test to non-parametric Friedman’s test with Dunn’s post hoc test), and the statistical significance did not change with any of the tests. We now added the explanation in the manuscript, as follows:

“Although most of our statistics were based on the non-parametric tests for the relatively small sample size (n=8), we used the parametric RM ANOVA for comparing three groups (i.e., PBS, dMUS, and iMUS) because it is the most commonly known and widely used statistical test in such comparison. However, we also performed statistical tests with the alternatives for reference, and the statistical significances were not changed with any of the results.” (p.26)

b. Single housing of rats:

Why was this chosen? Based on my experience, this is not necessary for studies involving cannula implants and food restriction. Group housing is generally considered to improve the welfare of rats.

We chose single housing of rats because our training paradigm required precise restrictions on the food consumption of individual rats, which could be difficult in group housing.

c. Anesthesia:

Why was pentobarbital used, alongside isoflurane, to anesthetize rats for surgery (line 663)? The use of gaseous anesthesia alone offers very good control of anesthesia and reduces the risk of death from anesthesia compared to the use of pentobarbital.

Why was anesthesia used for the drug infusions (line 674)? If rats are well-habituated to handling by the experimenter, manual restraint is sufficient for intra-cerebral infusions. Therefore, anesthesia could be omitted, reducing the risk of adverse effects on the experimental rats.

I do not think that points b. and c. are relevant for the interpretation of the present findings, but the authors may consider these points for future studies to improve further the welfare of the experimental rats.

We appreciate the reviewer’s careful suggestions. For both the use of pentobarbital during surgery and anesthesia for the drug infusion, we chose to do so to avoid any risk of rats being awake and becoming anxious and to ensure safety during the procedures. They might not be necessary, but they were helpful for the experimenters to proceed with sufficient time to maintain precision. Nonetheless, we agree with the reviewer’s concern, which was the reason why we monitored the rats’ behavior for 20 minutes in the cage after drug infusion to minimize any potential influence on the task performance. We updated the relevant details in the Methods section, as follows:

“The rat was kept in a clean cage to recover from anesthesia completely and monitored for side effects for 20 minutes, then was moved to the VR apparatus for behavioral testing.” (p.25)

ここも翻訳したい

ここも意訳してこうとか

そのユーザーをどこに遷移させるかをDjangoに指定する必要があります。

ここは意訳してもいいかなと思いました。 送り出すとかがちょっとわかりにくいかなと

ユーザーのログインが成功したら、そのユーザーをどこに遷移させるかをDjangoに指定する必要があります。

とか

引数

空文字列 がよいかと

原書も普通のフォントだけど、等幅フォントにしておいた方がよさそう

ここはシンプルに 子孫 だけでよいのでは。 （次の文も 子 だけなので）

原文だと31353677はコード表記されています。

nits: <br /> ちょっと一部に読点が多いように感じるので

大きく異なる点は、出力がそれほど長くないので、フォーマットされた結果の文字列をファイルに書き込まずに出力しているところです❶。

はどうでしょうか（好みかも

typo？　が

typo 「カスタマイズ」

「グラフのほとんどの要素を更新する」のか「グラフのほとんどの要素を、更新するメソッド経由で」なのかわかりづらかったです。

「更新するメソッド」とあるのですが、これは update_...メソッド群のことだと思うので、シンプルに「更新メソッド」と表現した方がイメージが伝わりやすいかなと思いました。

代案:

一旦グラフを作成したら、グラフのほとんどの要素は更新メソッド経由でカスタマイスが可能です。

typo？「を」では。 title_font_sizeのように 「aspects of a chart element」（titleとfontとsize）がunderscoresで繋がれているので。

全体を修正するなら、

Plotlyでは、グラフの要素の特徴をアンダースコアで繋げるという慣例があります。

などでしょうか（aspectsをどう訳すか悩むけど...

代案:

タイトルを追加して各軸にラベルを付けたら、グラフへのスタイル設定をはじめます。

元の原稿だと「タイトルの追加」の後に「ラベルをつけることでグラフにスタイル設定をする」という作業があるように読めるのですが、原文だと by adding a title and labels for each axis で「タイトルの追加」と「ラベルの付与」がセットなので、代案のように読点の位置を変えた方がイメージが伝わりやすいと思います。

直訳すればそうなのですが、日本語としては少し違和感ありました。

たった2行のコードを書くだけで、最初の可視化を行えます。

あるいは

最初の可視化は、たった2行のコードを書くだけです。

などでしょうか...

最初のグラフに

とかですかね。「初期状態のグラフ」という言い方がちょっと違和感ありました。

代案

もう調査フェーズではなく

nits <br /> 意訳の範囲としてOKな気もしますが、 the data we want. なのでどちらかというと 欲しいデータ かなと思いました

別コメントでも書いたように、結果がset型っぽく受け取れます。 もし実際にset型でないのであれば、

結果が完全な集合か確認する

あるいはシンプルに

完全な結果が得られたか確認する

などの方が日本語としては自然に感じました

あんまりわかってないのですが、「API呼び出しを作成する」という言い回しは日本語でよく使われる表現なのでしょうか（API呼び出しを行うようなオブジェクトを生成してるように聞こえる）

あまりない表現であれば、単純に「API呼び出しを行います」「API呼び出しをします」でも良いのでは、と思いました。

「比較して可視化」だと「比較した後に可視化する」ように読めて違和感ありました。

原文は「relative popularity」なので「相対的な人気を可視化しましょう」か、あるいはやや意訳して「人気を可視化して比較しましょう」の方がイメージが近いかなと思います。

nits

一文が長いので読点が欲しいです

API呼び出しから返される各リポジトリについて、選択した情報を出力するループを書きましょう。

• 文が長いので、読点を入れた方が良い
• 「public-apis」がプログラマーが と が が続くので、「public-apisには...含まれている」と受動態にした方が読みやすいかなと思います
• mightがあるので、「興味のある」ではなく「興味を持つかもしれない」「興味を持ちそうな」では。

代案:

また説明文から、「public-apis」にはプログラマーが興味を持ちそうなフリーのAPIのリストが含まれていることがわかります。

typo？ 最近更新されて

nits ひらがなが続くので、読点があった方が読みやすいかなと思います。

代案:

repo_dict にあるキーのうち、いくつかの値を取り出してみましょう。

「様子がわかるでしょう」という言い回しがちょっと違和感ありました

参考: get a sense of = 〜を感じ取れる、何となく分かる <br /> https://eow.alc.co.jp/search?q=get+a+sense+of

代案:

これらのキーを見渡せば、プロジェクトに関してどんな種類の情報を抽出できるのか把握できるでしょう

あるいは

これらのキーを見渡せば、プロジェクトに関してどんな種類の情報を抽出できるのか何となくわかるでしょう

などはどうでしょうか

追加のデータページ

でしょうか。「データの追加ページ」だと、データを追加するページっぽく感じました。

[nits] <br /> ちょっと一文が長いので読点があった方が良いかと。

各リポジトリの情報をより詳しく見るために、 repo_dicts の最初の要素を取り出して repo_dict に代入します❸。

[nits] <br /> 日本語としては ここでは、 のようなワンクッションおいた表現があった方が自然な流れで読めると思いました。

（原文には「ここ」という表現はないですが、「we print ...」の we が示す「私たちはこうするよ」感を「ここではこうするよ」と意訳する感じ

代案:

ここでは、この値を直接出力するのではなく、逆の値を出力します。

「セット」とカタカナで書かれるとset型のデータっぽく感じるのですが、実際にそうなっているのでしょうか（未確認）。 もしset型でなければ、 結果の集合 と言った表現の方が自然に感じました。

typo？

API呼び出しを作成して、レスポンスを確認する

あるいは

APIを呼び出して、レスポンスを確認する

でしょうか（他のコードにも同様の記述あり

今回の差分の範囲外ですが、ちょっと文意がわかりにくかったです。

This is a good way to make sure we received the information we expected, and to start examining the information we're interested in:

なので、

これは期待通りに情報を取得できたか確認して、関心のある情報について調べ始める上で良い方法です。

などでしょうか（これもちょっと直訳感があるけど...

「ヘッダー定義で使用する」と読めちゃうので、語順を入れ替えた方が誤解が生じないかと思います。

代案:

使用するAPIのバージョンをAPI呼び出しのヘッダー定義で明確に指定し、結果をJSONフォーマットで返します❷

「よう」

(1) <br /> APIへのレスポンス性能

「ユーザに対するAPIのレスポンス性能」あるいは「ユーザへのAPIレスポンス性能」では。原稿の表現だと、APIに向かってレスポンスを返しているようです。

(2) <br /> [nits] ここの文は、前文の「GitHubのプロセスが途中で止まっている」ことを補足するための文なので、語順を入れ換えた方が「プロセスが途中で止まってるのは、実行時間を制限してるから」という流れがわかりやすいかなと思いました（これは好みかも

代案:

GitHubは全てのユーザーに対してAPIのレスポンス性能を維持するために、各クエリの実行時間を制限しています。

代案:

"incomplete_results" の値が true になっており、GitHubがクエリーを完全には処理しなかったことをがわかります❷

でしょうか。 「全プロセスが完了していない」だと「全然処理できなかった（進捗率0%）」のか「全部は処理できなかった（一部はできた可能性がある）」のか判断しづらく感じました。

that's been requestedを「現在選択されている」と訳すのはちょっと違和感がある。リクエストされた、とかでもよいのでは

同じ文章が語順を変えて2回書いてます。どちらかを削除してください。

MUST: topic.text

トル

原書ではイタリックではなく"で囲んでいるので、「web server gateway interface」と書くのはどうでしょうか

で、

と読点を入れたい

訳註として最新のDjangoは5.0.6で、本書のコードを確認したバージョンはN.Nと書きたい。4.2系?

eLife assessment

This paper provides an important method that uses a computational model to predict photoreceptor currents in mammalian photoreceptors. By inverting the model, visual stimuli can be constructed to produce desired photoreceptor current responses. The authors provide compelling evidence that this approach can disentangle the effects of photoreceptor nonlinearities including light adaptation from downstream nonlinear processing, thus facilitating future studies of the higher visual system.

Reviewer #1 (Public Review):

Summary:

This manuscript aims at a quantitative model of how visual stimuli, given as time-dependent light intensity signals, are transduced into electrical currents in photoreceptors of macaque and mouse retina. Based on prior knowledge on the fundamental biophysical steps of the transduction cascade and a relatively small number of free parameters, the resulting model is found to fairly accurately capture measured photoreceptor currents under a range of diverse visual stimuli and with parameters that are (mostly) identical for photoreceptors of the same type.

Furthermore, as the model is invertible, the authors show that it can be used to derive visual stimuli that result in a desired, predetermined photoreceptor response. As demonstrated with several examples, this can be used to probe how the dynamics of phototransduction affect downstream signals in retinal ganglion cells, for example, by manipulating the visual stimuli in such a way that photoreceptor signals are linear or have reduced or altered adaptation. This innovative approach had already previously been used by the same lab to probe the contribution of photoreceptor adaptation to differences between On and Off parasol cells (Yu et al, eLife 2022), but the present paper extends this by describing and testing the photoreceptor model more generally and in both macaque and mouse as well as for both rods and cones.

Strengths:

The presentation of the model is thorough and convincing, and the ability to capture responses to stimuli as different as white noise with varying mean intensity and flashes with a common set of model parameters across cells is impressive. Also, the suggested approach of applying the model to modify visual stimuli that effectively alter photoreceptor signal processing is thought-provoking and should be a powerful tool for future investigations of retinal circuit function. The examples of how this approach can be applied are convincing and corroborate, for example, previous findings that adaptation to ambient light in the primate retina, as measured by responses to light flashes, mostly originates in photoreceptors. Application of the approach by other labs is facilitated by the clear exposition and the listing of obtained optimal parameter values.

Weaknesses:

The model is impressive, but not perfect, including some small systematic differences between model predictions and measurements from held-out cells. The deviations likely (partly) reflect differences between cells used for parameter optimization and test cells, as stated in the text (though this is not fully proven), which has to be kept in mind when applying the model, in particular with the listed parameters.

Reviewer #2 (Public Review):

Summary:

This manuscript proposes a modeling approach to capture nonlinear processes of photocurrents in mammalian (mouse, primate) rod and cone photoreceptors. The ultimate goal is to separate these nonlinearities at the level of photocurrent from subsequent nonlinear processing that occurs in retinal circuitry. The authors devised a strategy to generate stimuli that cancel the major nonlinearities in photocurrents. For example, modified stimuli would generate genuine sinusoidal modulation of the photocurrent, whereas a sinusoidal stimulus would not (i.e., because of asymmetries in the photocurrent to light vs. dark phases of a sinusoidal stimulus); and modified stimuli that could cancel the effects of light adaptation at the photocurrent level. Using these modified stimuli, one could record downstream neurons, knowing that any nonlinearities that emerge must happen beyond the stage of the photocurrent. This could be a useful method for separating nonlinear mechanisms across different stages of retinal processing and may be useful in vivo.

Strengths:

(1) This is a very quantitative and thoughtful approach and addresses a long-standing problem in the field: determining the location of nonlinearities within a complex circuit, including asymmetric responses to different polarities of contrast, adaptation, etc.<br /> (2) The study presents data for two primary models of mammalian retina, mouse and primate, and shows that the basic strategy works in each case.<br /> (3) Ideally, the present results would generalize to the work in other labs and possibly other sensory systems. The authors do provide evidence that a photocurrent model constructed from data in one set of cells can be used in a second set of cells.

Weaknesses:

(1) The model is limited to describing photoreceptor responses at the level of photocurrents, as opposed to the output of the cell, which takes into account voltage-dependent mechanisms, horizontal cell feedback, etc., as the authors acknowledge. It could be interesting to expand the model in the future to include factors that affect photoreceptor output beyond the stage of the photocurrent.<br /> (2). It will be interesting to eventually test the impact of this work for in vivo experiments.

Reviewer #3 (Public Review):

Summary:

The authors propose to invert a mechanistic model of phototransduction in mouse and rod photoreceptors to derive stimuli that compensate for nonlinearities in these cells. They fit the model to a large set of photoreceptor recordings, and show in additional data that the compensation works. This can allow to exclude photoreceptors as a source of nonlinear computation in the retina, as desired to pinpoint nonlinearties in retinal computation. The recordings made by the authors are impressive and I appreciate the simplicity and elegance of the idea. The data support the authors conclusions.

Strengths:

- The authors collected an impressive set of recordings from mouse and primate photoreceptors, which is very challenging to obtain.<br /> - The other proposes to exploit mechanistic mathematical models of a well understood phototransduction to design light stimuli which compensate for nonlinearities.<br /> - The authors demonstrate through additional experiments that their proposed approach works and is useful for offering insights into retinal computation.<br /> - The biophysical modeling approach is well described.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This manuscript aims at a quantitative model of how visual stimuli, given as time-dependent light intensity signals, are transduced into electrical currents in photoreceptors of macaque and mouse retina. Based on prior knowledge of the fundamental biophysical steps of the transduction cascade and a relatively small number of free parameters, the resulting model is found to fairly accurately capture measured photoreceptor currents under a range of diverse visual stimuli and with parameters that are (mostly) identical for photoreceptors of the same type.

Furthermore, as the model is invertible, the authors show that it can be used to derive visual stimuli that result in a desired, predetermined photoreceptor response. As demonstrated with several examples, this can be used to probe how the dynamics of phototransduction affect downstream signals in retinal ganglion cells, for example, by manipulating the visual stimuli in such a way that photoreceptor signals are linear or have reduced or altered adaptation. This innovative approach had already previously been used by the same lab to probe the contribution of photoreceptor adaptation to differences between On and Off parasol cells (Yu et al, eLife 2022), but the present paper extends this by describing and testing the photoreceptor model more generally and in both macaque and mouse as well as for both rods and cones.

Strengths:

The presentation of the model is thorough and convincing, and the ability to capture responses to stimuli as different as white noise with varying mean intensity and flashes with a common set of model parameters across cells is impressive. Also, the suggested approach of applying the model to modify visual stimuli that effectively alter photoreceptor signal processing is thought-provoking and should be a powerful tool for future investigations of retinal circuit function. The examples of how this approach can be applied are convincing and corroborate, for example, previous findings that adaptation to ambient light in the primate retina, as measured by responses to light flashes, mostly originates in photoreceptors.

Weaknesses:

In the current form of the presentation, it doesn't become fully clear how easily the approach is applicable at different mean light levels and where exactly the limits for the model inversion are at high frequency. Also, accessibility and applicability by others could be strengthened by including more details about how parameters are fixed and what consensus values are selected.

Thank you - indeed a central goal of writing this paper was to provide a tool that could be easily used by other laboratories. We have clarified and expanded four points in this regard: (1) we have stated more clearly that mean light levels are naturally part of inversion process, and hence the approach can be applied across a broad range of light levels (lines 292-297); (2) we have expanded our analysis of the high frequency limits to the inversion and added that expanded figure to the main text (new Fig 5); (3) we have included additional detail about our calibration procedures, including our calibration code, to facilitate transfer to other labs; and, (4) we have detailed the procedure for identification of consensus parameters (line 172-182, 191-199 and Methods section starting on line 831).

Reviewer #2 (Public Review):

Summary:

This manuscript proposes a modeling approach to capture nonlinear processes of photocurrents in mammalian (mouse, primate) rod and cone photoreceptors. The ultimate goal is to separate these nonlinearities at the level of photocurrent from subsequent nonlinear processing that occurs in retinal circuitry. The authors devised a strategy to generate stimuli that cancel the major nonlinearities in photocurrents. For example, modified stimuli would generate genuine sinusoidal modulation of the photocurrent, whereas a sinusoidal stimulus would not (i.e., because of asymmetries in the photocurrent to light vs. dark changes); and modified stimuli that could cancel the effects of light adaptation at the photocurrent level. Using these modified stimuli, one could record downstream neurons, knowing that any nonlinearities that emerge must happen post-photocurrent. This could be a useful method for separating nonlinear mechanisms across different stages of retinal processing, although there are some apparent limitations to the overall strategy.

Strengths:

(1) This is a very quantitative and thoughtful approach and addresses a long-standing problem in the field: determining the location of nonlinearities within a complex circuit, including asymmetric responses to different polarities of contrast, adaptation, etc.

(2) The study presents data for two primary models of mammalian retina, mouse, and primate, and shows that the basic strategy works in each case.

(3) Ideally, the present results would generalize to the work in other labs and possibly other sensory systems. How easy would this be? Would one lab have to be able to record both receptor and post-receptor neurons? Would in vitro recordings be useful for interpreting in vivo studies? It would be useful to comment on how well the current strategy could be generalized.

We agree that generalization to work in other laboratories is important, and indeed that was a motivation for writing this as a methods paper. The key issue in such generalization is calibration. We have expanded our discussion of our calibration procedures and included that code as part of the github repository associated with the paper. Figure 10 (previously Figure 9) was added to illustrate generalization. We believe that the approach we introduce here should generalize to in vivo conditions. We have expanded the text on these issues in the Discussion (sections starting on line 689 and 757).

Weaknesses:

(1) The model is limited to describing photoreceptor responses at the level of photocurrents, as opposed to the output of the cell, which takes into account voltage-dependent mechanisms, horizontal cell feedback, etc., as the authors acknowledge. How would one distinguish nonlinearities that emerge at the level of post-photocurrent processing within the photoreceptor as opposed to downstream mechanisms? It would seem as if one is back to the earlier approach, recording at multiple levels of the circuit (e.g., Dunn et al., 2006, 2007).

Indeed the current model is limited to a description of rod and cone photocurrents. Nonetheless, the transformation of light inputs to photocurrents can be strongly nonlinear, and such nonlinearities can be difficult to untangle from those occurring late in visual processing. Hence, we feel that the ability to capture and manipulate nonlinearities in the photocurrents is an important step. We have expanded Figure 10 to show an additional example of how manipulation of nonlinearities in phototransduction can give insight into downstream responses. We have also noted in text that an important next step would be to include inner segment mechanisms (section starting on line 661); doing so will require not only characterization of the current-to-voltage transformation, but also horizontal cell feedback and properties of the cone output synapse.

(2) It would have been nice to see additional confirmations of the approach beyond what is presented in Figure 9. This is limited by the sample (n = 1 horizontal cell) and the number of conditions (1). It would have been interesting to at least see the same test at a dimmer light level, where the major adaptation mechanisms are supposed to occur beyond the photoreceptors (Dunn et al., 2007).

We have added an additional experiment to this figure (now Figure 10) which we feel nicely exemplifies the approach. The approach that we introduce here really only makes sense at light levels where the photoreceptors are adapting; at lower light levels the photoreceptors respond near-linearly, so our “modified” and “original” stimuli as in Figure 10 (previously Figure 9) would be very similar (and post-phototransduction nonlinearities are naturally isolated at these light levels).

Reviewer #3 (Public Review):

Summary:

The authors propose to invert a mechanistic model of phototransduction in mouse and rod photoreceptors to derive stimuli that compensate for nonlinearities in these cells. They fit the model to a large set of photoreceptor recordings and show in additional data that the compensation works. This can allow the exclusion of photoreceptors as a source of nonlinear computation in the retina, as desired to pinpoint nonlinearities in retinal computation. Overall, the recordings made by the authors are impressive and I appreciate the simplicity and elegance of the idea. The data support the authors' conclusions but the presentation can be improved.

Strengths:

-  The authors collected an impressive set of recordings from mouse and primate photoreceptors, which is very challenging to obtain.

-  The authors propose to exploit mechanistic mathematical models of well-understood phototransduction to design light stimuli that compensate for nonlinearities.

-  The authors demonstrate through additional experiments that their proposed approach works.

Weaknesses:

-  The authors use numerical optimization for fitting the parameters of the photoreceptor model to the data. Recently, the field of simulation-based inference has developed methods to do so, including quantification of the uncertainty of the resulting estimates. Since the authors state that two different procedures were used due to the different amounts of data collected from different cells, it may be worthwhile to rather test these methods, as implemented e.g. in the SBI toolbox (https://joss.theoj.org/papers/10.21105/joss.02505). This would also allow them to directly identify dependencies between parameters, and obtain associated uncertainty estimates. This would also make the discussion of how well constrained the parameters are by the data or how much they vary more principled because the SBI uncertainty estimates could be used.

Thank you - we have improved how we describe and report parameter values in several ways. First, the previous text erroneously stated that we used different fitting procedures for different cell types - but the real difference was in the amount of data and range of stimuli we had available between rods and cones. The fitting procedure itself was the same for all cell types. We have clarified this along with other details of the model fitting both in the main text (lines 121-130) and in the Methods (section starting on line 832). We also collected parameter values and estimates of allowed ranges in two tables. Finally, we used sloppy modeling to identify parameters that could covary with relatively small impact on model performance; we added a description of this analysis to the Methods (section starting on line 903).

-  In several places, the authors refer the reader to look up specific values e.g. of parameters in the associated MATLAB code. I don't think this is appropriate, important values/findings/facts should be in the paper (lines 142, 114, 168). I would even find the precise values that the authors measure interesting, so I think the authors should show them in a figure/table. In general, I would like to see also the average variance explained by different models summarized in a table and precise mean/median values for all important quantities (like the response amplitude ratios in Figures 6/9).

We have added two tables with these parameters values and estimates of allowable ranges. We also added points to show the mean (and SD) across cells to the population figures and added those numerical values to the figure legends throughout.

-  If the proposed model is supposed to model photoreceptor adaptation on a longer time scale, I fail to see why this can be an invertible model. Could the authors explain this better? I suspect that the model is mainly about nonlinearities as the authors also discuss in lines 360ff.

For the stimuli that we use we see little or no contribution of slow adaptation in phototransduction. We have expanded the description of this point in the text and referred to Angueyra et al (2022) which looks at this issue in more detail for primate cones (paragraph starting on line 280).

-  The important Figures 6-8 are very hard to read, as it is not easy to see what the stimulus is, the modified stimulus, the response with and without modification, what the desired output looks like, and what is measured for part B. Reworking these figures would be highly recommended.

We have reworked all of the figures to make the traces clearer.

-  If I understand Figure 6 correctly, part B is about quantifying the relative size of the response to the little first flash to the little second flash. While clearly, the response amplitude of the second flash is only 50% for the second flash compared to the first flash in primate rod and cones in the original condition, the modified stimulus seems to overcompensate and result in 130% response for the second flash. How do the authors explain this? A similar effect occurs in Figure 9, which the authors should also discuss.

Indeed, in those instances the modified stimulus does appear to overcompensate. We suspect this is due to differences in sensitivity of the specific cells probed for these experiments and those used in the model construction. We now describe this limitation in more detail (lines 524-526). A similar point comes up for those experiments in which we speed the photoreceptor responses (new FIgure 9B), and we similarly note that the cells used to test those manipulations differed systematically from those used to fit the model (lines 558-560).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

I only have a few minor questions and suggestions for clarification.

It hasn't become fully clear to me how general the model is when different mean light levels (on long-time scales) are considered. Are there slow adaptation processes not captured in the model that affect model performance? And how should one go about setting the mean light level when, for example, probing ganglion cells with a stimulus obtained through model inversion? Should it work to add an appropriate DC component to the current that is provided as input to the inverted model? (Presumably, deriving a stimulus and then just adding background illumination should not work, or could this be a good approximation, given a steady state that is adapted to the background?)

We have clarified in the main text that slow adaptation does not contribute substantially to responses to the range of stimuli we explored (lines 281-289). We have also clarified that the stimulus in the model inversion is specified in isomerizations per second - so the mean value of the stimulus is automatically included in the model inversion (lines 293-298).

Furthermore, a caveat for the model inversion seems to be the potential amplification of high-frequency noise. The suggested application of a cutoff temporal frequency seems appropriate, but data are shown only for a few example cells. Is this consistent across cells? (Given that performance between, e.g., mouse cones can vary considerably according to Fig. 4B?) I would also like to suggest moving the corresponding Supplemental Figure (4.1) into the main part of the manuscript, as it seems quite important.

We have added population analysis to the new Figure 5 (which was Figure 4 - Figure Supplement 1). We have also clarified that the amplification of high frequency noise is an issue only when we try to apply model inversion to measured stimuli. When we use model inversion to identify stimuli that elicit desired responses, the target responses are computed from a linear model that has no noise, so this is not a concern in applications like those in Figures 6-10.

Also, could the authors explain more clearly what the effect of the normalization of the estimated stimulus by the power of the true stimulus is? Does this simply reduce power at high frequency or also affect frequencies below the suggested cutoff (where the stimulus reconstruction should presumably be accurate even without normalization)?

Indeed this normalization reduces high frequency power and has little impact on low frequencies where the inversion is accurate; this is now noted in the text (line 363). As for amplification of high frequency noise (previous comment), the normalization by the stimulus power is only needed when inverting measured responses (i.e. responses with noise) and is omitted when we are identifying stimuli that elicit desired responses (e.g. in Figures 6-10).

While the overall performance of the model to predict photoreceptor currents is impressive, it seems that particular misses occur for flashes right after a step in background illumination and for the white-noise responses at low background illumination (e.g. Figure 1B). Is that systematic, and if so what might be missing in the model?

Indeed the model (at least with fixed parameters across stimuli) appears to systematically miss a few aspects of the photoreceptor responses. These include the latency of the response to a bright flash and the early flashes in the step + flash protocol in Figure 1B. Model errors for the variable mean noise stimulus (Figure 2) showed little dependence on time even when responses were sorted by mean light level and by previous mean level. Model errors did not show a clear systematic dependence on light level; this likely reflects, at least in part, the use of mean-square-error to identify model parameters. We have expanded our discussion of these systematic errors in the text (lines 164-166).

I was also wondering whether this is related to the fact that in Figure 9B, the gain in the modified condition is actually systematically higher when there is more background light. Do the authors think that this could be a real effect or rather an overcompensation from the model? (By the way, is it specified what "Delta-gain" really is, i.e., ratio or normalized difference?)

We suspect this is an issue with the sensitivity of the specific cells for which we did these experiments (i.e. variability in the gamma parameter between cells). This sensitivity varies between cells, and such variations are likely to place the strongest limitation on our ability to use this approach to manipulate responses in different retinas. We now note those issues in the Results (lines 523-526, 557-559 and 591-593) with reference to Figures 9 (previously Figure 8) and 10 (previously Figure 9), and describe this limitation more generally in the Discussion (section starting on line 649). We have also changed delta-gain to response ratio, which seemed more intuitive.

Maybe I missed this, but it seems that the parameter gamma is fitted in a cell-type-specific fashion (e.g. line 163), but then needs to be fixed for held-out cells. How was this done? Is there much variability of gamma between cells?

There is variability in gamma between cells, and this likely explains some of systematic differences between data and model (see above and Methods, lines 902-903). For the consensus models in Figure 2B, gamma was allowed to vary for each cell while the remaining consensus model parameters were fixed. Gamma was set equal to the mean value across cells for model inversion (i.e. for all of the analyses in Figures 4-10). We have described the fitting procedure in considerably more detail in the revised Methods (starting on line 832).

For completeness, it would be nice to have the applied consensus model parameters in the manuscript rather than just in the Matlab code (especially since the code has not been part of the submission). Also, some notes on how the numerical integration of the differential equations was done would be nice (time step size?).

We have added tables with consensus parameters and estimates of the sensitivity of model predictions to each parameter. We have also added additional details about the numerical approaches (including the time step) to Methods.

Similarly, it would be nice to explicitly see the relationships that are used to fix certain model parameters (lines 705ff). And can the constants k and n (lines 709-710) be assumed identical for different species and receptor types?

We have added more details to the model fitting to the methods, including the use of steady-state conditions to hold certain parameters fixed (lines 862 and 866). We are not aware of any direct comparisons of k and n across species and receptor types. We have noted that model performance was not improved by modest changes in these parameters (due to compensation by other model parameters). More generally, we have explained how some parameters trade for others and hence the logic of fixing some even when exact values were not available.

For the previous measurements of m and beta (lines 712-713), is there a reference or source?

We have added references for these values.

Did the authors check for differences in the model parameters between cone types (e.g., S vs. M)?

We did not include S cones here. They are harder to record from and collecting a fairly large data set across a range of stimuli would be challenging. Our previous work shows that S cones have slower responses than L and M cones, and this would certainly be reflected in differences in model parameters. We have noted this in the text (Methods, line 808-810).

For the stated flash responses time-to-peak (lines 183-184), is this for a particular light intensity with no background illumination?

Those are flashes from darkness - now noted in the text.

Figure 2 - Supplement 1 doesn't have panel labels A and B, unlike the legend.

Fixed - thank you.

Reviewer #2 (Recommendations For The Authors):

(1) Fig. 2B - for some cells, the consensus model seems to fit better than the individual model. How is this possible?

This was mostly an error on our part (we inadvertently included responses to more stimuli in fitting the individual models, which slightly hampered their performance). Even with this correction, however, a few cells remain for which the consensus model outperforms and individual model. We believe this is because there is more data to constrain model parameters for the consensus models (since they are fit to all cells at the same time), and that can compensate for improvements associated with customizing parameters to specific cells.

(2) Fig. 2 Supplement 1, it would be useful to see a blow-up of the data in an inset, as in Fig. 2B.

Thanks - added.

(3) Line 400 - this paragraph could include additional quantification and statistics to back up claims re 'substantially reduced', 'considerably lower'.

We quantify that in the next sentence by computing the mean-square-error between responses and sinusoidal fits (also in Figure 7B, which now includes statistics as well). We have made that connection more direct in the text.

(4) Maybe a supplement to Fig. 8 could show the changes to the stimulus required to alter the kinetics in both directions - to give more insight into part B., especially.

Good suggestion - we have added the stimuli to all of the panels of the figure (now Figure 9).

(5) Fig. 8B - in 'Speed response up' condition - there seems to be error in the model for the decay time of the response - especially for the 'original' condition, which is not quantified in 8C. Was it generally difficult to predict responses to flashes?

That seems largely to reflect that the cells used for those experiments had faster initial kinetics than the average cells (responses to the control traces are also faster than model predictions in these cells - black traces in Figure 9B). We have added this to the text.

(6) Line 678, possibly notes that 405 nm equally activates S and M photopigments in mice, since most of the cones co-express the two photopigments (Rohlich et al., 1994; Applebury et al., 2000; Wang et al., 2011).

Thanks - we have added this (lines 827-829).

(7) The discussion could include a broader description of the various approaches to identifying nonlinearities within retinal circuitry, which include (incomplete list): recording at multiple levels of the circuit (e.g., Kim and Rieke 2001; Rieke, 2001; Baccus and Meister, 2002; Dunn et al., 2006; 2007; Beaudoin et al., 2007; Baccus et al., 2008); recording currents vs. spiking responses in a ganglion cell (e.g., Kim and Rieke, 2001; Zaghloul et al., 2005; Cui et al., 2016); neural network modeling approaches (e.g., Maheswaranathan et al., 2023); optogenetic approaches to studying filtering/nonlinear behavior at synapses (e.g., Pottackal et al., 2020; 2021).

Good suggestion - we have added this to the final paragraph of the Discussion.

Reviewer #3 (Recommendations For The Authors):

-  I am personally not a fan of the style: "... as Figure 4A shows..." or comparable and much prefer a direct "We observe that X is the case (Figure 4A)". If the authors agree, they may want to revise their paper in this way.

We have revised the text to avoid the “... as Figure xx shows” construction. We have retained multiple instances which follow a “Figure xx shows that …” construction (which is both active rather than passive and does not use a personal pronoun).

-  I am not a fan of the title. Light-adaption clamp caters only to a very specialized audience.

We have changed the title to “Predictably manipulating photoreceptor light responses to reveal their role in downstream visual responses.”

-  The parameter fitting procedure should not only be described in Matlab code, but in the paper.

Thanks - we have expanded this in the Methods considerably (section starting on line 832).

-  The authors should elaborate on why different fitting procedures were used.

We did not describe that issue clearly. The fitting procedures used across cells were identical, but we had different data available for different cell types due to experimental limitations. We have substantially revised that part of the main text to clarify this issue (paragraph starting on line 121).

-  The authors state in line 126 that the input stimulus is supposed to mimic eye movements mouse, monkey, or human? Please clarify.

Thanks - we have changed this sentence to “abrupt and frequent changes in intensity that characterize natural vision.”

-  Please improve the figure style. For example, labels should be in consistent capitalization and ideally use complete words (e.g. Figure 2B, 4B, and others).

We have made numerous small changes in the figures to make them more consistent.

-  Is the fraction of variance calculated on held-out-data? Linear models should be added to Figure 2B.

The fraction of variance explained was not calculated on held out data because of limitations in the duration of our recordings. Given the small number of free parameters, and the ability of the model to capture held out cells, we believe that the model generalizes well. We have added a supplemental figure with linear model performance (Figure 2 - Figure Supplement 2).

-  Fig. 9A is lacking bipolar cell and amacrine cell labels. Currently, it looks like HC is next to the BC in the schematic.

Thanks - we have updated that figure (now Figure 10A)

-  Maybe I am misunderstanding something, but it seems like the linear model prediction shown in Figure 2A for the rod could be easily improved by scaling it appropriately. Is this impression correct or why not?

We have clarified how the linear model is constructed (by fitting the linear model to low contrast responses of the full model at the mean stimulus intensity). We also added a supplemental figure, following the suggestion above, showing the linear model performance when a free scaling factor is included for each cell.

-  The verification experiment in Fig. 5 is only anecdotal and is elaborated only in Figure 6. If I am not mistaken, this does not necessitate its own figure/section but could rather be merged.

We have kept this figure separate (now Figure 6) as we felt that it was important to highlight the approach in general in a figure before getting into quantification of how well it works.

-  Figure 5 right is lacking labels. What is red and grey?

Thanks for catching that - labels are added now.

-  The end of the Discussion is slightly unusual. Did some text go missing?

Thanks - we have rearranged the Discussion so as not to end on Limitations.

-  There is a bonus figure at the end which seems also not to belong in the manuscript.

Thanks - the bonus figure is removed now.

-  The methods should also describe briefly what kind of routines were used in the Matlab code, e.g. gradient descent with what optimizer?

We’ve added that information as well.

Reviewer #2 (Public Review):

Here I submit my previous review and a great deal of additional information following on from the initial review and the response by the authors.

* Initial Review *

Assessment:

This manuscript is based upon the unprecedented identification of an apparently highly unusual trigeminal nuclear organization within the elephant brainstem, related to a large trigeminal nerve in these animals. The apparently highly specialized elephant trigeminal nuclear complex identified in the current study has been classified as the inferior olivary nuclear complex in four previous studies of the elephant brainstem. The entire study is predicated upon the correct identification of the trigeminal sensory nuclear complex and the inferior olivary nuclear complex in the elephant, and if this is incorrect, then the remainder of the manuscript is merely unsupported speculation. There are many reasons indicating that the trigeminal nuclear complex is misidentified in the current study, rendering the entire study, and associated speculation, inadequate at best, and damaging in terms of understanding elephant brains and behaviour at worst.

Original Public Review:

The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.<br /> The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, the current study differs.

Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others, appears to be just "lateral nuclei" with no suggestion of what might be there instead.

Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occurs. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400 000 and 500 000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

But let's look more specifically at the justification outlined in the current study to support their identification of the unusual located trigeminal sensory nuclei of the brainstem.

(1) Intense cytochrome oxidase reactivity<br /> (2) Large size of the putative trunk module<br /> (3) Elongation of the putative trunk module<br /> (4) Arrangement of these putative modules correspond to elephant head anatomy<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds<br /> (6) Location apparently matches other mammals<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals

Let's examine these justifications more closely.

(1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. In order to obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. It is likely that the histochemical staining observed is background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.<br /> Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

(4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

(6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

(7) The dual to quadruple repetition of rostro-caudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. But, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown is that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

(8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to details to support the major changes indicated by the authors.

So what are these "bumps" in the elephant brainstem?

Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship to the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

* Review of Revised Manuscript *

Assessment:

There is a clear dichotomy between the authors and this reviewer regarding the identification of specific structures, namely the inferior olivary nuclear complex and the trigeminal nuclear complex, in the brainstem of the elephant. The authors maintain the position that in the elephant alone, irrespective of all the published data on other mammals and previously published data on the elephant brainstem, these two nuclear complexes are switched in location. The authors maintain that their interpretation is correct, this reviewer maintains that this interpretation is erroneous. The authors expressed concern that the remainder of the paper was not addressed by the reviewer, but the reviewer maintains that these sequelae to the misidentification of nuclear complexes in the elephant brainstem renders any of these speculations irrelevant as the critical structures are incorrectly identified. It is this reviewer's opinion that this paper is incorrect. I provide a lot of detail below in order to provide support to the opinion I express.

Public Review of Current Submission:

As indicated in my previous review of this manuscript (see above), it is my opinion that the authors have misidentified, and indeed switched, the inferior olivary nuclear complex (IO) and the trigeminal nuclear complex (Vsens). It is this specific point only that I will address in this second review, as this is the crucial aspect of this paper - if the identification of these nuclear complexes in the elephant brainstem by the authors is incorrect, the remainder of the paper does not have any scientific validity.

The authors, in their response to my initial review, claim that I "bend" the comparative evidence against them. They further claim that as all other mammalian species exhibit a "serrated" appearance of the inferior olive, and as the elephant does not exhibit this appearance, that what was previously identified as the inferior olive is actually the trigeminal nucleus and vice versa.

For convenience, I will refer to IOM and VsensM as the identification of these structures according to Maseko et al (2013) and other authors and will use IOR and VsensR to refer to the identification forwarded in the study under review.<br /> The IOM/VsensR certainly does not have a serrated appearance in elephants. Indeed, from the plates supplied by the authors in response (Referee Fig. 2), the cytochrome oxidase image supplied and the image from Maseko et al (2013) shows a very similar appearance. There is no doubt that the authors are identifying structures that closely correspond to those provided by Maseko et al (2013). It is solely a contrast in what these nuclear complexes are called and the functional sequelae of the identification of these complexes (are they related to the trunk sensation or movement controlled by the cerebellum?) that is under debate.

Elephants are part of the Afrotheria, thus the most relevant comparative data to resolve this issue will be the identification of these nuclei in other Afrotherian species. Below I provide images of these nuclear complexes, labelled in the standard nomenclature, across several Afrotherian species.

(A) Lesser hedgehog tenrec (Echinops telfairi)

Tenrecs brains are the most intensively studied of the Afrotherian brains, these extensive neuroanatomical studies undertaken primarily by Heinz Künzle. Below I append images (coronal sections stained with cresol violet) of the IO and Vsens (labelled in the standard mammalian manner) in the lesser hedgehog tenrec. It should be clear that the inferior olive is located in the ventral midline of the rostral medulla oblongata (just like the rat) and that this nucleus is not distinctly serrated. The Vsens is located in the lateral aspect of the medulla skirted laterally by the spinal trigeminal tract (Sp5). These images and the labels indicating structures correlate precisely with that provide by Künzle (1997, 10.1016/S0168- 0102(97)00034-5), see his Figure 1K,L. Thus, in the first case of a related species, there is no serrated appearance of the inferior olive, the location of the inferior olive is confirmed through connectivity with the superior colliculus (a standard connection in mammals) by Künzle (1997), and the location of Vsens is what is considered to be typical for mammals. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 1.

(B) Giant otter shrew (Potomogale velox)

The otter shrews are close relatives of the Tenrecs. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see hints of the serration of the IO as defined by the authors, but we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 2.

(C) Four-toed sengi (Petrodromus tetradactylus)

The sengis are close relatives of the Tenrecs and otter shrews, these three groups being part of the Afroinsectiphilia, a distinct branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see vague hints of the serration of the IO (as defined by the authors), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 3.

(D) Rock hyrax (Procavia capensis)

The hyraxes, along with the sirens and elephants form the Paenungulata branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per the standard mammalian anatomy. Here we see hints of the serration of the IO (as defined by the authors), but we also see evidence of a more "bulbous" appearance of subnuclei of the IO (particularly the principal nucleus), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 4.

(E) West Indian manatee (Trichechus manatus)

The sirens are the closest extant relatives of the elephants in the Afrotheria. Below I append images of cresyl violet (top) and myelin (bottom) stained coronal sections (taken from the University of Wisconsin-Madison Brain Collection, https://brainmuseum.org, and while quite low in magnification they do reveal the structures under debate) through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see the serration of the IO (as defined by the authors). Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 5.

These comparisons and the structural identification, with which the authors agree as they only distinguish the elephants from the other Afrotheria, demonstrate that the appearance of the IO can be quite variable across mammalian species, including those with a close phylogenetic affinity to the elephants. Not all mammal species possess a "serrated" appearance of the IO. Thus, it is more than just theoretically possible that the IO of the elephant appears as described prior to this study.

So what about elephants? Below I append a series of images from coronal sections through the African elephant brainstem stained for Nissl, myelin, and immunostained for calretinin. These sections are labelled according to standard mammalian nomenclature. In these complete sections of the elephant brainstem, we do not see a serrated appearance of the IOM (as described previously and in the current study by the authors). Rather the principal nucleus of the IOM appears to be bulbous in nature. In the current study, no image of myelin staining in the IOM/VsensR is provided by the authors. However, in the images I provide, we do see the reported myelin stripes in all stains - agreement between the authors and reviewer on this point. The higher magnification image to the bottom left of the plate shows one of the IOM/VsensR myelin stripes immunostained for calretinin, and within the myelin stripes axons immunopositive for calretinin are seen (labelled with an arrow). The climbing fibres of the elephant cerebellar cortex are similarly calretinin immunopositive (10.1159/000345565). In contrast, although not shown at high magnification, the fibres forming the Sp5 in the elephant (in the Maseko description, unnamed in the description of the authors) show no immunoreactivity to calretinin.

Review image 6.

Peripherin Immunostaining

In their revised manuscript the authors present immunostaining of peripherin in the elephant brainstem. This is an important addition (although it does replace the only staining of myelin provided by the authors which is unusual as the word myelin is in the title of the paper) as peripherin is known to specifically label peripheral nerves. In addition, as pointed out by the authors, peripherin also immunostains climbing fibres (Errante et al., 1998). The understanding of this staining is important in determining the identification of the IO and Vsens in the elephant, although it is not ideal for this task as there is some ambiguity. Errante and colleagues (1998; Fig. 1) show that climbing fibres are peripherin-immunopositive in the rat. But what the authors do not evaluate is the extensive peripherin staining in the rat Sp5 in the same paper (Errante et al, 1998, Fig. 2). The image provided by the authors of their peripherin immunostaining (their new Figure 2) shows what I would call the Sp5 of the elephant to be strongly peripherin immunoreactive, just like the rat shown in Errant et al (1998), and more over in the precise position of the rat Sp5! This makes sense as this is where the axons subserving the "extraordinary" tactile sensitivity of the elephant trunk would be found (in the standard model of mammalian brainstem anatomy). Interestingly, the peripherin immunostaining in the elephant is clearly lamellated...this coincides precisely with the description of the trigeminal sensory nuclei in the elephant by Maskeo et al (2013) as pointed out by the authors in their rebuttal. Errante et al (1998) also point out peripherin immunostaining in the inferior olive, but according to the authors this is only "weakly present" in the elephant IOM/VsensR. This latter point is crucial. Surely if the elephant has an extraordinary sensory innervation from the trunk, with 400 000 axons entering the brain, the VsensR/IOM should be highly peripherin-immunopositive, including the myelinated axon bundles?! In this sense, the authors argue against their own interpretation - either the elephant trunk is not a highly sensitive tactile organ, or the VsensR is not the trigeminal nuclei it is supposed to be.

Summary:

(1) Comparative data of species closely related to elephants (Afrotherians) demonstrates that not all mammals exhibit the "serrated" appearance of the principal nucleus of the inferior olive.

(2) The location of the IO and Vsens as reported in the current study (IOR and VsensR) would require a significant, and unprecedented, rearrangement of the brainstem in the elephants independently. I argue that the underlying molecular and genetic changes required to achieve this would be so extreme that it would lead to lethal phenotypes. Arguing that the "switcheroo" of the IO and Vsens does occur in the elephant (and no other mammals) and thus doesn't lead to lethal phenotypes is a circular argument that cannot be substantiated.

(3) Myelin stripes in the subnuclei of the inferior olivary nuclear complex are seen across all related mammals as shown above. Thus, the observation made in the elephant by the authors in what they call the VsensR, is similar to that seen in the IO of related mammals, especially when the IO takes on a more bulbous appearance. These myelin stripes are the origin of the olivocerebellar pathway, and are indeed calretinin immunopositive in the elephant as I show.

(4) What the authors see aligns perfectly with what has been described previously, the only difference being the names that nuclear complexes are being called. But identifying these nuclei is important, as any functional sequelae, as extensively discussed by the authors, is entirely dependent upon accurately identifying these nuclei.

(4) The peripherin immunostaining scores an own goal - if peripherin is marking peripheral nerves (as the authors and I believe it is), then why is the VsensR/IOM only "weakly positive" for this stain? This either means that the "extraordinary" tactile sensitivity of the elephant trunk is non-existent, or that the authors have misinterpreted this staining. That there is extensive staining in the fibre pathway dorsal and lateral to the IOR (which I call the spinal trigeminal tract), supports the idea that the authors have misinterpreted their peripherin immunostaining.

(5) Evolutionary expediency. The authors argue that what they report is an expedient way in which to modify the organisation of the brainstem in the elephant to accommodate the "extraordinary" tactile sensitivity. I disagree. As pointed out in my first review, the elephant cerebellum is very large and comprised of huge numbers of morphologically complex neurons. The inferior olivary nuclei in all mammals studied in detail to date, give rise to the climbing fibres that terminate on the Purkinje cells of the cerebellar cortex. It is more parsimonious to argue that, in alignment with the expansion of the elephant cerebellum (for motor control of the trunk), the inferior olivary nuclei (specifically the principal nucleus) have had additional neurons added to accommodate this cerebellar expansion. Such an addition of neurons to the principal nucleus of the inferior olive could readily lead to the loss of the serrated appearance of the principal nucleus of the inferior olive, and would require far less modifications in the developmental genetic program that forms these nuclei. This type of quantitative change appears to be the primary way in which structures are altered in the mammalian brainstem.

eLife assessment

This provocative manuscript from presents valuable comparisons of the morphologies of Archaean bacterial microfossils to those of microbes transformed under environmental conditions that mimic those present on Earth during the same Eon, although the evidence in support of the conclusions is currently incomplete. The reasons include that taphonomy is not presently considered, and a greater diversity of experimental environmental conditions is not evaluated -- which is important because we ultimately do not know much about Earth's early environments. The authors may want to reframe their conclusions to reflect this work as a first step towards an interpretation of some microfossils as 'proto-cells,' and less so as providing strong support for this hypothesis.

Reviewer #1 (Public Review):

Summary:

Microfossils from the Paleoarchean Eon represent the oldest evidence of life, but their nature has been strongly debated among scientists. To resolve this, the authors reconstructed the lifecycles of Archaean organisms by transforming a Gram-positive bacterium into a primitive lipid vesicle-like state and simulating early Earth conditions. They successfully replicated all morphologies and life cycles of Archaean microfossils and studied cell degradation processes over several years, finding that encrustation with minerals like salt preserved these cells as fossilized organic carbon. Their findings suggest that microfossils from 3.8 to 2.5 billion years ago were likely liposome-like protocells with energy conservation pathways but without regulated morphology.

Strengths:

The authors have crafted a compelling narrative about the morphological similarities between microfossils from various sites and proliferating wall-deficient bacterial cells, providing detailed comparisons that have never been demonstrated in this detail before. The extensive number of supporting figures is impressive, highlighting numerous similarities. While conclusively proving that these microfossils are proliferating protocells morphologically akin to those studied here is challenging, we applaud this effort as the first detailed comparison between microfossils and morphologically primitive cells.

Weaknesses:

Although the species used in this study closely resembles the fossils morphologically, it would be beneficial to provide a clearer explanation for its selection. The literature indicates that many bacteria, if not all, can be rendered cell wall-deficient, making the rationale for choosing this specific species somewhat unclear.

While this manuscript includes clear morphological comparisons, we believe the authors do not adequately address the limitations of using modern bacterial species in their study. All contemporary bacteria have undergone extensive evolutionary changes, developing complex and intertwined genetic pathways unlike those of early life forms. Consequently, comparing existing bacteria with fossilized life forms is largely hypothetical, a point that should be more thoroughly emphasized in the discussion.

Another weak aspect of the study is the absence of any quantitative data. While we understand that obtaining such data for microfossils may be challenging, it would be helpful to present the frequencies of different proliferative events observed in the bacterium used. Additionally, reflecting on the chemical factors in early life that might cause these distinct proliferation modes would provide valuable context.

Reviewer #2 (Public Review):

Summary:

In summary, the manuscript describes life-cycle-related morphologies of primitive vesicle-like states (Em-P) produced in the laboratory from the Gram-positive bacterium Exiguobacterium Strain-Molly) under assumed Archean environmental conditions. Em-P morphologies (life cycles) are controlled by the "native environment". In order to mimic Archean environmental conditions, soy broth supplemented with Dead Sea salt was used to cultivate Em-Ps. The manuscript compares Archean microfossils and biofilms from selected photos with those laboratory morphologies. The photos derive from publications on various stratigraphic sections of Paleo- to Neoarchean ages. Based on the similarity of morphologies of microfossils and Em-Ps, the manuscript concludes that all Archean microfossils are in fact not prokaryotes, but merely "sacks of cytoplasm".

Strengths:

The approach of the authors to recognize the possibility that "real" cells were not around in the Archean time is appealing. The manuscript reflects the very hard work by the authors composing the Em-Ps used for comparison and selecting the appropriate photo material of fossils.

Weaknesses:

While the basic idea is very interesting, the manuscript includes flaws and falls short in presenting supportive data. The manuscript makes too simplistic assumptions on the "Archean paleoenvironment". First, like in our modern world, the environmental conditions during the Archean time were not globally the same. Second, we do not know much about the Archean paleoenvironment due to the immense lack of rock records. More so, the Archean stratigraphic sections from where the fossil material derived record different paleoenvironments: shelf to tidal flat and lacustrine settings, so differences must have been significant. Finally, the Archean spanned 2.500 billion years and it is unlikely that environmental conditions remained the same. Diurnal or seasonal variations are not considered. Sediment types are not considered. Due to these reasons, the laboratory model of an Archean paleoenvironment and the life therein is too simplistic. Another aspect is that eucaryote cells are described from Archean rocks, so it seems unlikely that prokaryotes were not around at the same time. Considering other fossil evidence preserved in Archean rocks except for microfossils, the many early Archean microbialites that show baffling and trapping cannot be explained without the presence of "real cells". With respect to lithology: chert is a rock predominantly composed of silica, not salt. The formation of Em-Ps in the "salty" laboratory set-up seems therefore not a good fit to evaluate chert fossils. Formation of structures in sediment is one step. The second step is their preservation. However, the second aspect of taphonomy is largely excluded in the manuscript, and the role of fossilization (lithification) of Em-Ps is not discussed. This is important because Archean rock successions are known for their tectonic and hydrothermal overprint, as well as recrystallization over time. Some of the comparisons of laboratory morphologies with fossil microfossils and biofilms are incorrect because scales differ by magnitudes. In general, one has to recognize that prokaryote cell morphologies do not offer many variations. It is possible to arrive at the morphologies described in various ways including abiotic ones.

eLife assessment

This important work examines the role of blood flow and Ghrelin in influencing the migration speed of adult-born olfactory neurons. The authors present solid evidence that newborn rostral migratory stream (RMS) neurons are closely situated alongside blood vessels, preferentially along arterioles, and that migratory speed is correlated with blood flow. They also provide evidence (in vitro and some in vivo) that Ghrelin from blood is involved in augmenting RMS neuron migration speed. While the data from the imaging experiments are convincing, the evidence for the causal roles of Ghrelin is limited and requires additional experimental clarifications to reach a strong conclusion.

Reviewer #1 (Public Review):

Summary:

This study provides compelling evidence suggesting that ghrelin, a molecule released in the surroundings of the major adult brain neurogenic niche (V-SVZ) by blood vessels with high blood flow, controls the migration of newborn interneurons towards the olfactory bulbs.

Strengths:

This study is a tour de force as it provides a solid set of data obtained by time-lapse recordings in vivo. The data demonstrate that the migration and guidance of newborn neurons rely on factors released by selective types of blood vessels.

Weaknesses:

Some intermediate conclusions are weak and may be reinforced by additional experiments.

Reviewer #2 (Public Review):

Summary:

The authors establish a close spatial relationship between RMS neurons and blood vessels. They demonstrated that high blood flow was correlated with migratory speed. In vitro, they demonstrate that Ghrelin functions as a motogen that increases migratory speed through augmentation of actin cup formation. The authors proceed to demonstrate through the knockdown of the Ghrelin receptor that fewer RMS neurons reach the OB. They show the opposite is true when the animal is fasted.

Strengths:

Compelling evidence of close association of RMS neurons with blood vessels (tissue clearing 3D), preferentially arterioles. Good use of 2-photon imaging to demonstrate migratory speed and its correlation with blood flow. In vitro analysis of Ghrelin administration to cultured RMS neurons, actin visualization, Ghsr1KD, is solid and compelling.

Weaknesses:

(1) Novelty of findings attenuated due to prior work, especially Li et al., Experimental Neurology 2014. Here, the authors demonstrated that Ghrelin enhances migration in adult-born neurons in the SVZ and RMS.

(2) The evidence for blood delivery of Ghrelin is not very convincing. Fluorescently-labeled Ghrelin appears to be found throughout the brain parenchyma, irrespective of the distance from vessels. It is also not clear from the data whether there is a link between increased blood flow and Ghrelin delivery.

(3) The in vivo link between Ghsr1KD and migratory speed is not established. Given the strong work to open the study on blood flow and migratory speed and the in vitro evidence that migratory speed is augmented by Ghrelin, the paper would be much stronger with direct measurement of migration speed upon Ghsr1KD. Indeed, blood flow should also be measured in this experiment since it would address concerns in 2. If blood flow and ghrelin delivery are linked, one would expect that Ghsr1KD neurons would not exhibit increased migratory speed when associated with slow or fast blood flow vessels.

eLife assessment

This important study identifies the anti-inflammatory function of PEGylated PDZ peptides that are derived from the ZO-1 protein. Results from cellular and in vivo experiments tracking key inflammatory markers are compelling. Although the mechanism of action needs further investigation, this study provides a proof of concept for developing novel strategies against acute inflammatory conditions such as sepsis.

Reviewer #2 (Public Review):

Summary:

The authors investigated systemic inflammation induced by LPS in various tissues and also examined immune cells of the mice using tight junction protein-based PDZ peptide. They explored the mechanism of anti-systemic inflammatory action of PDZ peptides, which enhanced M1/M2 polarization and induced the proliferation of M2 macrophages. Additionally, they insisted the physiological mechanism that inhibited the production of ROS in mitochondria, thereby preventing systemic inflammation.

Strength

In the absence of specific treatments for septic shock or sepsis, the study demonstrating that tight junction-based PDZ peptides inhibit systemic inflammation caused by LPS is highly commendable. Whereas previous research focused on antibiotics, this study proves that modifying parts of intracellular proteins can significantly suppress symptoms caused by septic shock. The authors expanded the study of localized inflammation caused by LPS or PM2.5 in the respiratory track to systemic inflammation, presenting promising results. They not only elucidated the physiological mechanism by identifying the transcriptome through RNA sequencing but also demonstrated that PDZ peptides inhibit the production of ROS in mitochondria and prevent mitochondrial fission. This research is highly regarded as an excellent study with potential as a treatment for septic shock or sepsis.

Weakness

(1) They Focused intensively on acute inflammation for a short duration instead of chronic inflammation.<br /> (2) LPS was used to induce septic shock, but administrating actual microbes such as E.coli would yield more accurate results.<br /> (3) The authors used pegylated peptides, but future research should utilize the optimized peptides to derive the optimal peptide, and further, PK/PD studies are also necessary.

Author response:

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

Reviewer 1:

(1) Peptides were synthesized with fluorescein isothiocyanate (FITC) and Tat tag, and then PEGylated with methoxy PEG Succinimidyl Succinate.

I have two concerns about the peptide design. First, FTIC was intended "for monitoring" (line 129), but was never used in the manuscript. Second, PEGylation targets the two lysine sidechains on the Tat, which would alter its penetration property.

We conducted an analysis of the cellular trafficking of FITC-tagged peptides following their permeabilization into cells.

Author response image 1.

However, we did not include it in the main text because it is a basic result.

(2) As can be seen in the figure above, after pegylation and permeabilization, the cells were stained with FITC. It appears that this does not affect the ability to penetrate into the cells.

(2) "Superdex 200 increase 10/300 GL column" (line 437) was used to isolate mono/di PEGylated PDZ and separate them from the residual PEG and PDZ peptide. "m-PEG-succinimidyl succinate with an average molecular weight of 5000 Da" (lines 133 and 134).

To my knowledge, the Superdex 200 increase 10/300 GL column is not suitable and is unlikely to produce traces shown in Figure 1B.

As Superdex 200 increase 10/300 GL featrues a fractionation range of 10,000 to 600,000 Da, we used it to fractionate PEGylated products including DiPEGylated PDZ (approx. 15 kDa) and MonoPEGylated PDZ (approx. 10 kDa) from residuals (PDZ and PEG), demonstrating successful isolation of PEGylated products (Figure 1C). Considering the molecular weights of PDZ and PEG are approximately 4.1 kDa and and 5.0 kDa, respectively, the late eluting peaks from SEC were likely to represent a mixed absorbance of PDZ and PEG at 215 nm.

However, as the reviewer pointed out, it could be unreasonable to annotate peaks representing PDZ and PEG, respectively, from mixed absorbance detected in a region (11-12 min) beyond the fractionation range.

In our revised manuscript, therefore, multiple peaks in the late eluting volume (11-12 min) were labeled as 'Residuals' all together. As a reference, the revised figure 1B includes a chromatogram of pure PDZ-WT under the same analytic condition.

Therefore, we changed Fig.1B to new results as followed:

(3) "the in vivo survival effect of LPS and PDZ co-administration was examined in mice. The pretreatment with WT PDZ peptide significantly increased survival and rescued compared to LPS only; these effects were not observed with the mut PDZ peptide (Figure 2a)." (lines 159-160).

Fig 2a is the weight curve only. The data is missing in the manuscript.

We added the survived curve into Fig. 2A as followed:

(4) Table 1, peptide treatment on ALT and AST appears minor.

In mice treated with LPS, levels of ALT and AGT in the blood are elevated, but these levels decrease upon treatment with WT PDZ. However, the use of mut PDZ does not result in significant changes. Figure 3A shows inflammatory cells within the central vein, yet no substantial hepatotoxicity is observed during the 5-day treatment with LPS. Normally, the ranges of ALT and AGT in C57BL6 mice are 16 ~ 200 U/L and 46 ~ 221 U/L, respectively, according to UCLA Diagnostic Labs. Therefore, the values in all experiments fall within these normal ranges. In summary, a 5-day treatment with LPS induces inflammation in the liver but is too short a duration to induce hepatotoxicity, resulting in lower values.

(5) MitoTraker Green FM shouldn't produce red images in Figure 6.

We changed new results (GREEN one) into Figs 6A and B as followed:

(6) Figure 5. Comparison of mRNA expression in PDZ-treated BEAS-2B cells. Needs a clearer and more detailed description both in the main text and figure legend. The current version is very hard to read.

We changed Fig. 5A to new one to understand much easier and added more detailed results and figure legend as followed:

Results Section in Figure 5:

“…we performed RNA sequencing analysis. The results of RNA-seq analysis showed the expression pattern of 24,424 genes according to each comparison combination, of which the results showed the similarity of 51 genes overlapping in 4 gene categories and the similarity between each comparison combination (Figure 5a). As a result, compared to the control group, it was confirmed that LPS alone, WT PDZ+LPS, and mut PDZ+LPS were all upregulated above the average value in each gene, and when LPS treatment alone was compared with WT PDZ+LPS, it was confirmed that they were averaged or downregulated. When comparing LPS treatment alone and mut PDZ+LPS, it was confirmed that about half of the genes were upregulated. Regarding the similarity between comparison combinations, the comparison combination with LPS…”

Figure 5 Legend Section:

“Figure 5. Comparison of mRNA expression in PDZ-treated BEAS-2B cells.

BEAS-2B cells were treated with wild-type PDZ or mutant PDZ peptide for 24 h and then incubated with LPS for 2 h, after which RNA sequencing analysis was performed. (a) The heat map shows the general regulation pattern of about 51 inflammation-related genes that are differentially expressed when WT PDZ and mut PDZ are treated with LPS, an inflammatory substance. All samples are RED = upregulated and BLUE = downregulated relative to the gene average. Each row represents a gene, and the columns represent the values of the control group treated only with LPS and the WT PDZ and mut PDZ groups with LPS. This was used by converting each log value into a fold change value. All genes were adjusted to have the same mean and standard deviation, the unit of change is the standard deviation from the mean, and the color value range of each row is the same. (b) Significant genes were selected using Gene category chat (Fold change value of 2.00 and normalized data (log2) value of 4.00). The above pie chart shows the distribution of four gene categories when comparing LPS versus control, WT PDZ+LPS/LPS, and mut PDZ+LPS/LPS. The bar graph below shows RED=upregulated, GREEN=downregulated for each gene category, and shows the number of upregulated and downregulated genes in each gene category. (c) The protein-protein interaction network constructed by the STRING database differentially displays commonly occurring genes by comparing WT PDZ+LPS/LPS, mut PDZ+LPS/LPS, and LPS. These nodes represent proteins associated with inflammation, and these connecting lines denote interactions between two proteins. Different line thicknesses indicate types of evidence used in predicting the associations.”

Reviewer 2:

(1) In this paper, the authors demonstrated the anti-inflammatory effect of PDZ peptide by inhibition of NF-kB signaling. Are there any results on the PDZ peptide-binding proteins (directly or indirectly) that can regulate LPS-induced inflammatory signaling pathway? Elucidation of the PDZ peptide-its binding partner protein and regulatory mechanisms will strengthen the author's hypothesis about the anti-inflammatory effects of PDZ peptide

As mentioned in the Discussion section, we believe it is crucial to identify proteins that directly interact with PDZ and regulate it. This direct interaction can modulate intracellular signaling pathways, so we plan to express GST-PDZ and induce binding with cellular lysates, then characterize it using the LC-Mass/Mass method. We intend to further research these findings and submit them for publication.

(2) The authors presented interesting insights into the therapeutic role of the PDZ motif peptide of ZO-1. PDZ domains are protein-protein interaction modules found in a variety of species. It has been thought that many cellular and biological functions, especially those involving signal transduction complexes, are affected by PDZ-mediated interactions. What is the rationale for selecting the core sequence that regulates inflammation among the PDZ motifs of ZO-1 shown in Figure 1A?

The rationale for selecting the core sequence that regulates inflammation among the PDZ motifs of ZO-1, as shown in Figure 1A, is grounded in the specific roles these motifs play in signal transduction pathways that are crucial for inflammatory processes. PDZ domains are recognized for their ability to function as scaffolding proteins that organize signal transduction complexes, crucial for modulating cellular and biological functions. The chosen core sequence is particularly important because it is conserved across ZO-1, ZO-2, and ZO-3, indicating a fundamental role in maintaining cellular integrity and signaling pathways. This conservation suggests that the sequence’s involvement in inflammatory regulation is not only significant in ZO-1 but also reflects a broader biological function across the ZO family.

(3) In Figure 3, the authors showed the representative images of IHC, please add the quantification analysis of Iba1 expression and PAS-positive cells using Image J or other software. To help understand the figure, an indication is needed to distinguish specifically stained cells (for example, a dotted line or an arrow).

We added the semi-quantitative results into Figs. 4d,e,f as followed:

Result section: “The specific physiological mechanism by which WT PDZ peptide decreases LPS-induced systemic inflammation in mice and the signal molecules involved remain unclear. These were confirmed by a semi-quantitative analysis of Iba-1 immunoreactivity and PAS staining in liver, kidney, and lung,respectively (Figures 4d, e, and f). To examine whether WT PDZ peptide can alter LPS-induced tissue damage in the kidney, cell toxicity assay was performed (Figure 3g). LPS induced cell damage in the kidney, however, WT PDZ peptide could significantly alleviate the toxicity, but mut PDZ peptide could not. Because cytotoxicity caused by LPS is frequently due to ROS production in the kidney (Su et al., 2023; Qiongyue et al., 2022), ROS production in the mitochondria was investigated in renal mitochondria cells harvested from kidney tissue (Figure 3h)....”

Figure legend section: “Indicated scale bars were 20 μm. (d,e,f) Semi-quantitative analysis of each are positive for Iba-1 in liver and kidney, and positive cells of PAS in lung, respectively. (g) After the kidneys were harvested, tissue lysates were used for MTT assay. (h) After...”

(4) In Figure 6G, H, the authors confirmed the change in expression of the M2 markers by PDZ peptide using the mouse monocyte cell line Raw264.7. It would be good to add an experiment on changes in M1 and M2 markers caused by PDZ peptides in human monocyte cells (for example, THP-1).

We thank you for your comments. To determine whether PDZ peptide regulates M1/M2 polarization in human monocytes, we examined changes in M1 and M2 gene expression in THP-1 cells. As a result, wild-type PDZ significantly suppressed the expression of M1 marker genes (hlL-1β, hIL-6, hIL-8, hTNF-ɑ), while increasing the expression of M2 marker genes (hlL-4, hIL-10, hMRC-1). However, mutant PDZ did not affect M1/M2 polarization. These results suggest that PDZ peptide can suppress inflammation by regulating M1/M2 polarization of human monocyte cells. These results are for the reviewer's reference only and will not be included in the main content.

Author response image 2.

Author response image 3.

Minor point:

The use of language is appropriate, with good writing skills. Nevertheless, a thorough proofread would eliminate small mistakes such as:

- line 254, " mut PDZ+LPS/LPS (45.75%) " → " mut PDZ+LPS/LPS (47.75%) "

- line 296, " Figure 6f " → " Figure 6h "

We changed these points into the manuscript.

The authour gave three reason why people be;lieve in these abductions: 1.Media ? Images , troupes the romantasization of the "image of alien" 2. Time period 3.

Spirtual healing , extrasensory perception like astro projection and communication with the ded all have in common ? " The great beyond seeking answers beyond the physical plane .

From the pre-face its seems like this may be actually based on aliens /or the study of a population of people who belived they have been abducted .(Page 2)

Questions ; Is this abuduction thing an allusion to love or somthing intangelble ?

eLife assessment

This paper presents useful findings on the dysmyelination phenotype of nervous system-specific Spns1 (a lysosomal lipid transporter) knockout mice. While the analysis of the phenotype is solid, the evidence for the underlying mechanisms, especially the molecular function for SPNS1, is incomplete. With more careful interpretation and/or additional experimental data, this work could have implications for understanding lipid transport and lysosomal storage diseases.

Reviewer #1 (Public Review):

Summary:

In this study, the authors studied the roles of SPNS1 which is a lysolipid transporter from the lysosomes in the nervous system using cell and mouse models. The authors tried to show that reduced sphingosine release from the lysosomes via SPNS1 affects myelination.

Strengths:

The authors used knockout models for cells and animals so the results are solid. They also used electron microscopic analysis of the phenotypes of the cells and mouse tissues.

Weaknesses:

The biochemical methods are not fully described at the moment. There is a lack of solid evidence to support the major claim.

If the authors could provide solid evidence that lipids that are released from the lysosomes via SPNS1 are used for myelination, this would be a major finding for the sources of lipids for the formation of axons.

Reviewer #2 (Public Review):

Summary:

Spns1 is a recently identified lysosomal transporter of lysophospholipids and sphingosine and its mutations in humans lead to neurodegeneration with white matter dysplasia. Since global Spns1 deficiency is embryonic lethal, the role of this particular lipid transporter in the nervous system remained unclear. In this study, Ichimura et al generated and analyzed nervous system-specific Spns1 knockout mice. The mutant mice showed epilepsy, growth retardation, demyelination, and early death, with accumulation of various LPC, LPE, and LPI species as well as sphingosine in specific areas of the brain. Probably due to impaired lysosomal efflux of sphingosine, brain levels of sphingolipids (ceramides, sulfatides, and glycolipids), which are main myelin components, were markedly reduced in the KO brain.

Strengths:

This study has provided convincing evidence for the first time that nervous system-specific deletion of Spns1 in mice leads to neurodegeneration, with disturbed lysophospholipid and sphingolipid metabolism in the brain. The results support the idea that the defective transport of lysosomal sphingosine by loss of Spns1 leads to a marked reduction of sphingolipid species required for myelin formation. This study significantly contributes to the research fields of neurodegeneration, lysosomal biology, and lipid biology.

Weaknesses:

It remains unclear why oligodendrocytes but not neurons are specifically damaged and how astroglia are affected by Spns1 deficiency. Lysosomal efflux of lysophospholipids and sphingosine by Spns1 relied solely on the knowledge from published studies and was not addressed in this study. The expression of key lipid-metabolizing genes and molecular markers should be examined more deeply. Several images lack quantification.

Reviewer #3 (Public Review):

Summary:

The authors attempted to understand the effect of Spns1 deficiency in the brain using a brain-specific knockout mouse model. Basic phenotyping of the brain KO line was performed that included mass spectroscopy for lipids, metabolomics, mass spec imaging of brain tissue, and some histology. Similar methods were used for characterising the liver KO model. The main findings supported by the data are that brain KO results in hypomyelinated brains, brain KO mice presented with symptoms akin to epilepsy, and postnatal lethality at 5 weeks of age. In addition, biochemical studies showed that brain KO mice had significant accumulation in whole brain lysates of the lysolipids LPC and LPE and sphingosine with reduced levels of ceramide, sphingomyelin, and sulfatide. Some of the substantial claims made by the authors in an attempt to provide a mechanistic understanding of the data are not strongly supported by experimental data. Some of the major concerns are that the authors claim hypomyelination is not caused by changes in oligodendrocyte differentiation, but experimental evidence to support this was not provided. The authors also claim that hypomyelination and other neurological phenotypes are caused by reduced sphingosine transport by Spns1 leading to reduced sphingolipid synthesis. However, this conclusion is not supported by experimental data and the authors do not address other equally plausible hypotheses.

eLife assessment

This short manuscript uses mutation counts in phylogenies of millions of SARS-CoV-2 genomes to show that mutation rates systematically differ between regions that are paired or unpaired in the predicted RNA secondary structure of the viral genome. Such an effect of pairing state is not unexpected, but its systematic demonstration using millions of viral genomes is valuable and convincing.

Reviewer #1 (Public Review):

Summary:

This very short paper shows a greater likelihood of C->U substitutions at sites predicted to be unpaired in the SARS-CoV-2 RNA genome, using previously published observational data on mutation frequencies in SARS-CoV-2 (Bloom and Neher, 2023).

General comments:

A preference for unpaired bases as a target for APOBEC-induced mutations has been demonstrated previously in functional studies so the finding is not entirely surprising. This of course assumes that A3A or other APOBEC is actually the cause of the majority of C->U changes observed in SARS-CoV-2 sequences.

I'm not sure why the authors did not use the published mutation frequency data to investigate other potential influences on editing frequencies, such as 5' and 3' base contexts. The analysis did not contribute any insights into the potential mechanisms underlying the greater frequency of C->U (or G->U) substitutions in the SARS-CoV-2 genome.

Reviewer #2 (Public Review):

Hensel investigated the implications of SARS-CoV-2 RNA secondary structure in synonymous and nonsynonymous mutation frequency. The analysis integrated estimates of mutational fitness generated by Bloom and Neher (from publicly available patient sequences) and a population-averaged model of RNA basepairing from Lan et al (from DMS mutational profiling with sequencing, DMS-MaPseq).

The results show that base-pairing limits the frequency of some synonymous substitutions (including the most common CT), but not all: GA and AG substitutions seem unaffected by base-pairing.

The author then addressed nonsynonymous CT substitutions at base-paired positions. While there is still a generally higher estimated mutational fitness at unpaired positions, they propose a coarse adjustment to disentangle base-pairing from inherent mutational fitness at a given position. This adjustment reveals that nonsynonymous substitutions at base-paired positions, which define major variants, have higher mutational fitness.

Overall, this manuscript highlights the importance of considering RNA secondary structure in viral evolution studies.

The conclusions of this work are generally well supported by the data presented. Particularly, the author acknowledges most limitations of the analyses, and addresses them. Even though no new sequencing results were generated, the author used available data generated from the analysis of roughly seven million sequenced patient samples. Finally, the author discusses ways to improve the current available models.

There are a number of limitations of this work that should be highlighted, specifically in regard to the secondary structure data used in this paper. The Lan et al. dataset was generated using a multiplicity of infection (MOI) of 0.05, 24 hours post-infection (h.p.i.). At such a low MOI and late timepoint, viral replication is not synchronous and sequencing artifacts might be generated by cell debris and viral RNA degradation, therefore impacting the population-averaged results. In addition, the nonsynonymous base-paired positions in Figure 2 have relatively high population-averaged DMS reactivity, which suggests those positions are dynamic. Therefore, the proposed adjustment could result in an incorrect estimation of their inherent mutational fitness.

Additionally, like all such RNA probing experiments within cells, it remains difficult to deconvolve DMS/SHAPE low reactivity with RNA accessibility (e.g. from protein binding).

This work presents clear methods and an easy-to-access bioinformatic pipeline, which can be applied to other RNA viruses. Of note, it can be readily implemented in existing datasets. Finally, this study raises novel mechanistic questions on how mutational fitness is not correlated to secondary structure in the same way for every substitution.

Overall, this work highlights the importance of studying mutational fitness beyond an immune evasion perspective. On the other hand, it also adds to the viral intrinsic constraints to immune evasion.

eLife assessment

Floeder and colleagues report that dopamine ramps are determined by the duration of the intertrial interval of the task and the presence of dynamic cues that indicate reward proximity. The manuscript provides valuable new information on a topic of active discussion in the dopamine and reinforcement learning field and the strength of the evidence supporting the claims is solid.

Reviewer #1 (Public Review):

Summary:

In this study, Floedder et al report that dopamine ramps in both Pavlovian and Instrumental conditions are shaped by reward interval statistics. Dopamine ramps are an interesting phenomenon because at first glance they do not represent the classical reward prediction errors associated with dopamine signaling. Instead, they seem somewhat to bridge the gap between tonic and phasic dopamine, with an intense discussion still being held in the field about what is their actual behavioral role. Here, in tests with head-fixed mice, and dopamine being recorded with a genetically encoded fluorescent sensor in the nucleus accumbens, the authors find that dopamine ramps were only present when intertrial intervals were relatively short and the structure of the task (Pavlovian cue or progression in a VR corridor) contained elements that indicated progression towards the reward (e.g., a dynamic cue). The authors show that these findings are well explained by their previously published model of Adjusted Net Contingency of Causal Relation (ANCCR).

Strengths:

This descriptive study delineates some fundamental parameters that define dopamine ramps in the studied conditions. The short, objective, and to-the-point format of the manuscript is great and really does a service to potential readers. The authors are very careful with the scope of their conclusions, which is appreciated by this reviewer.

Weaknesses:

The discussion of the results is very limited to the conceptual framework of the authors' preferred model (which the authors do recognize, but it still is a limitation). The correlation analysis presented in panel I of Figure 3 seems unnecessary at best and could be misleading, as it is really driven by the categorical differences between the two conditions that were grouped for this analysis. There are some key aspects of the data and their relationship with each other, the previous literature, and the methods used to collect them, that could have been better discussed and explored.

Reviewer #2 (Public Review):

In this manuscript by Floeder et al., the authors report a correlation between ITI duration and the strength of a dopamine ramp occurring in the time between a predictive conditioned stimulus and a subsequent reward. They found this relationship occurring within two different tasks with mice, during both a Pavlovian task as well as an instrumental virtual visual navigation task. Additionally, they observed this relationship only in conditions when using a dynamic predictive stimulus. The authors relate this finding to their previously published model ANCCR in which the time constant of the eligibility trace is proportionate to the reward rate within the task.

The relationship between ITI duration and the extent of a dopamine ramp which the authors have reported is very intriguing and certainly provides an important constraint for models for dopamine function. As such, these findings are potentially highly impactful to the field. I do have a few questions for the authors which are written below.

(1) I was surprised to see a lack of counterbalance within the Pavlovian design for the order of the long vs short ITI. Ramping of the lick rate does increase from the long-duration ITIs to the short-duration ITI sessions. Although of course, this increase in ramping of the licking across the two conditions is not necessarily a function of learning, it doesn't lend support to the opposite possibility that the timing of the dynamic CS hasn't reached asymptotic learning by the end of the long-duration ITI. The authors do reference papers in which overtraining tends to result in a reduction of ramping, which would argue against this possibility, yet differential learning of the dynamic CS would presumably be required to observe this effect. Do the authors have any evidence that the effect is not due to heightened learning of the timing of the dynamic CS across the experiment?

(2) The dopamine response, as measured by dLight, seems to drop after the reward is delivered. This reduction in responding also tends to be observed with electrophysiological recordings of dopamine neurons. It seems possible that during the short ITI sessions, particularly on the shorter ITI duration trials, that dopamine levels may still be reduced from the previous trial at the onset of the CS on the subsequent trial. Perhaps the authors can observe the dynamics of the recovery of the dopamine response following a reward delivery on longer-duration ITIs in order to determine how quickly dopamine is recovering following a reward delivery. Are the trials with very short ITIs occurring within this period that dopamine is recovering from the previous trial? If so, how much of the effect may be due to this effect? It should be noted that the lack of observance of a ramp on the condition of short-duration ITIs with fixed CSs provides a potential control for this effect, yet the extent to which a natural ramp might occur following sucrose deliveries should be investigated.

(3) The authors primarily relate the finding of the correlation between the ITI and the slope of the ramp to their ANCCR model by suggesting that shorter time constants of the eligibility trace will result in more precisely timed predictors of reward across discrete periods of the dynamic cue. Based on this prediction, would the change in slope be more gradual, and perhaps be more correlated with a broader cumulative estimate of reward rate than just a single trial?

Reviewer #3 (Public Review):

Summary:

Floeder and colleagues measure dopamine signaling in the nucleus accumbens core using fiber photometry of the dLight sensor, in Pavlovian and instrumental tasks in mice. They test some predictions from a recently proposed model (ANCCR) regarding the existence of "ramps" in dopamine that have been seen in some previous research, the characteristics of which remain poorly understood.

They find that cues signaling a progression toward rewards (akin to a countdown) specifically promote ramping dopamine signaling in the nucleus accumbens core, but only when the intertrial interval just experienced was short. This work is discussed in the context of ongoing theoretical conceptions of dopamine's role in learning.

Strengths:

This work is the clearest demonstration to date of concrete training factors that seem to directly impact whether or not dopamine ramps occur. The existence of ramping signals has long been a feature of debates in the dopamine literature and this work adds important context to that. Further, as a practical assessment of the impact of a relatively simple trial structure manipulation on dopamine patterns, this work will be important for guiding future studies. These studies are well done and thoughtfully presented.

Weaknesses:

It remains somewhat unclear what limits are in place on the extent to which an eligibility trace is reflected in dopamine signals. In the current study, a specific set of ITIs was used, and one wonders if the relative comparison of ITI/history variables ("shorter" or "longer") is a factor in how the dopamine signal emerges, in addition to the explicit length ("short" or "long") of the ITI. Another experimental condition, where variable ITIs were intermingled, could perhaps help clarify some remaining questions.

In both tasks, cue onset responses are larger, and longer on long ITI trials. One concern is that this larger signal makes seeing a ramp during the cue-reward interval harder, especially with a fluorescence method like photometry. Examining the traces in Figure 1i - in the long, dynamic cue condition the dopamine trace has not returned to baseline at the time of the "ramp" window onset, but the short dynamic trace has. So one wonders if it's possible the overall return to baseline trend in the long dynamic conditions might wash out a ramp.

Not a weakness of this study, but the current results certainly make one ponder the potential function of cue-reward interval ramps in dopamine (assuming there is a determinable function). In the current data, licking behavior was similar on different trial types, and that is described as specifically not explaining ramp activity.

eLife assessment

This report details the creation and wide-scale utilization of "Repix", a new technique for chronic neurophysiological recordings using and re-using Neuropixels probes in freely behaving mice and rats. The authors include data and feedback from a variety of labs and researchers who have used this technique, setting an example for open science and reproducibility, and providing convincing evidence that this approach can be employed for chronic Neuropixels recordings. However, evidence is currently incomplete for claims about the advantages of this design over previous approaches and for cell yield and stability claims. This important work will have an impact on a broad range of neuroscientists seeking a straightforward methodology for chronic Neuropixels recordings and will facilitate ethologically relevant experimental designs.

Reviewer #1 (Public Review):

Summary:

Horan et al. present a system for the chronic implantation of Neuropixels probes in mice and rats that allows the repeated cycles of implantation, explant, and reuse. A detailed protocol of the procedure, along with technical drawings for the parts of the system are provided, for potential users to undertake the technique in their own laboratory. The authors documented the adoption of this system in ten laboratories, demonstrating that the technique can be widely deployed. Yields in the number of neurons recorded over time are reported to indicate that the technique can achieve stable yields over time.

Strengths:

The authors provide compelling evidence that their technique can be widely deployed and acquired by different laboratories by documenting in detail the success rates at each step of the procedure and the common failure modes across ten laboratories. This is important because an impediment for a laboratory to try out a new technique is a lack of assurance about whether that technique would be successful outside the environment where the technique was originally developed. It is helpful that the authors show that even users who were not directly trained by the original developer of the technique can acquire the technique by receiving only the protocol and the technical drawings.

Weaknesses:

I would have liked to see more evidence demonstrating the purported advantages of the Repix design ("We found that the key advantage of Repix is robustness and simplicity.") relative to other techniques already available for chronic implantation allowing for reuse (Juavinett 2019, Luo 2020, van Daal 2021, Bimbard 2023, Melin 2023). While it is commendable that the authors demonstrate the durability of their design during social interactions, I would have liked to see evidence demonstrating that aluminum construction (compared to plastic) is necessary for "rough-and-tumble fights of male mice."

Aluminum parts are typically more expensive than plastic parts, and because machining aluminum parts is typically slower than 3D printing in plastic, the commitment to aluminum can greatly slow down the adaptation of the Repix design for specific experimental needs or for newer versions of Neuropixels probes to be released in the future. Also, as the authors stated, aluminum parts are a bit heavier than plastic parts. In addition, I remain not fully convinced that the Repix design is significantly simpler than the existing designs, and I would be more convinced if the authors could quantify the number of modular components of the Repix system relative to existing designs, or perhaps provide a time estimate of assembling a Repix system compared to assembling an existing design.

The possibility of achieving greater yield using dexamethasone is intriguing, but the authors only show this for rats and one brain region. Were the surgeries done using dexamethasone performed after the surgeries not using dexamethasone? If so, could the improved yield simply be due to improvement in surgical technique? As such, it remains unclear whether dexamethasone actually helps to achieve greater yields.

Reviewer #2 (Public Review):

Summary:

This report describes a new "Repix" device for collecting stable, long-term recordings from chronically implanted Neuropixels probes in freely behaving rodents. The device follows the "docking module with payload" design of other similar devices that allows probe explantation and reuse but requires minimal components and is robust to a wide range of rodent behaviors. The docking module is a set of metal posts that are screwed into the payload module (cassette carrying the probe) at one end and cemented to the skull of the animal during surgery at the other end to reversibly anchor the probe to the skull. Loosening of the screws allows the cassette to travel off the posts for explantation. An additional headstage holder and cover are also available for further protection of the implant from mechanical damage during freely moving behaviors. Usage data from almost 200 procedures across multiple labs and users showcase high success rates at all stages of implementation (implantation, data collection, and explantation), even from users without direct training from the original developer of Repix. Device proficiency, defined by the authors as three successive full procedures without failure, was typically achieved within five attempts. Hundreds of neurons were consistently recorded from multiple brain regions, irrespective of animal behavior, Neuropixels probe type, and probe reuse. Impressively, neurophysiological data using Repix has already been published in two studies (one in mice and the other in rats). These findings demonstrate the intended functioning of the device as well as its ease of adoption. The effort to make the Repix system as straightforward as possible (e.g., minimal components and detailed protocols) is evident and will likely be appreciated by new adopters. Furthermore, the cell yield and procedures-to-proficiency data collected from a variety of experiments provide useful data for new adopters to plan their own studies with realistic expectations.

Strengths:

The main claims that the Repix device is "reliable, reusable, [and] versatile" are well-supported.

Weaknesses:

(1) The methodology used to quantify cell yields is concerning, potentially leading to an overestimation of "good" units and a misleading amount of "total" units. The authors define "good" unit yield as the amount of simultaneously recorded neurons labeled "good" by the automated spike sorter Kilosort without post-hoc manual curation. This definition was used to standardize cell yield between users who would otherwise manually curate cells and introduce individual variability as to what is considered a "good" unit. However, manual curation of spike sorted output is typically necessary to eliminate false positive units and "merge" spikes belonging to the same neuron that Kilosort identified as belonging to two separate neurons (i.e., spikes that share a refractory period, waveform shape, and localized to the same channels). As such, one may reasonably expect the yield for actual "good" units to be lower than what is reported. Furthermore, including units labeled by Kilosort as multi-unit activity in the "total" yield does not lend itself, by definition, to accurate quantification of individual neurons.

(2) For transparency's sake, restatement of whether the cell yield data came from mice or rats, and from one lab or multiple labs, in the figure or figure captions would be helpful. Based on the introduction of the paper, one gets the impression that the Repix system was designed for mice and rats and, therefore, that data from mice and rats were to be roughly equally represented. This is not the case, as only 1/3 of the reported Repix users were implanted in rats, and cell yield data was shown for only two brain regions in rats (compared with four in mice). The authors state that Repix was designed "... to record neural activities during social interaction of mice" in the Discussion section. It would be helpful for this statement to appear in the Introduction so that it is clear to the reader that Repix was designed for mice but also works well for rats.

(3) Regarding Figure 2, it would be informative to separate this data by species. Does Repix fail more in a procedural stage depending on whether the user is working with mice or rats?

Reviewer #3 (Public Review):

Summary:

Recent work in systems neuroscience has highlighted the importance of studying the populations of neurons during naturalistic behaviors, which necessitates the use of cutting-edge devices in freely moving animals. However, it has been costly and experimentally difficult to conduct such experiments. In response to this need, Horan et al. developed and thoroughly tested a system called Repix which allows neuroscientists to record from multiple brain areas in freely moving rodents over many days, even weeks. The authors show that this device enables reasonably stable long-term recordings and that the probe can be reused for different experiments.

Strengths:

I deeply appreciated how thoroughly the authors have tested this across labs and different versions of Neuropixels probes (and even other probes). This is unlike many other papers that describe similar devices, which have almost always only been developed and tested in one lab. As such, I think that the Repix device and procedure are very likely to be adopted by even more labs given the robustness of the evidence provided here. The willingness of the authors to allow others to test their device, iterate on the design, and obtain feedback from users is a shining example of how open science and publication should be conducted: with patience and diligence. I'm grateful to the authors for providing this example to the research community.

On a related note, in the discussion, the authors nicely summarize their focus on ease-of-adoption and highlight other examples from the community that have been successful. I would encourage the authors to think about what else - culturally, economically, etc. -- has been helpful in the open science adoption of software and hardware for electrophysiology, and to think critically about what these movements are still lacking or missing. Given the authors' collective experience in this effort, I believe the broader community would benefit from their perspective.

The final strength of this manuscript is the highly detailed protocol that has itself been peer-reviewed by many users and can be adapted for multiple use cases. The authors also provide specific protocols from individual labs in the main manuscript.

Weaknesses:

(1) Claims about longevity. Given the clear drop-off in units in the amygdala and V1, I felt that the claims about long-term stability (particularly at the one-year mark) were oversold. Readers should note the differences between the length of the curves in Figure 3B, and take these differences into consideration when setting expectations on the durability of these probes for recordings in V1 or the amygdala (and possibly nearby areas).

(2) Clarity around curve fitting, statistics, and impact of surgical procedures. I believe the manuscript could benefit from more detail around the curve fitting that was implemented, as well as some of the statistical tests, particularly related to the dexamethasone experiments. It seems the authors fit exponential decay to the unit curves over time, but it is not clear that this kind of fit makes sense given the data, which is a bit hard to see. Relatedly, there is a claim on page 10 about the similarity between mouse and rat decay constants in the amygdala which is hard to evaluate without quantitative evidence.

It is very useful to know that dexamethasone (an anti-inflammatory used by many labs) could improve stability, however, a more thorough explanation of these experiments is warranted. For example, it should be noted that the dexamethasone animals start with a much higher unit yield. Also, the decay in Figure 5e looks similar between dex and non-dex animals despite the claims in the text that the "decay of unit numbers was slower." Additional details about the curve fitting and statistical tests are needed for readers to evaluate this claim.

Nas leituras que realizei sobre ensino híbrido pude concluir o ensino híbrido, apesar de ter recebido um grande impulso com a pandemia, se tornou uma necessidade do processo educativo do século XXI por vivermos num período em que as crianças e jovens são efetivamente nativos digitais (Link, Washington & Lopes,2022) Em virtude desta característica dos nosso alunos, mas também à resposta aos desafios da sociedade contemporânea e à urgência de desenvolvermos o aluno enquanto cidadão global ativo, o ensino híbrido tornou-se um forma de promover um processo de ensino-aprendizagem em que o aluno é o protagonista. Parte-se do interesse dos alunos enquanto nativos digitais para promover um processo educativo integrado e significativo.

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I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

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Referee #3

Evidence, reproducibility and clarity

In this manuscript Ochner et al. report the 3.2 Å cryo-EM structure of the type IV pilus (minus PilY1 adhesin) from P. aeruginosa PAO1. The authors demonstrate that the conserved N-terminal helix of pilin subunits (PilA) form a tubular arrangement within the hydrophobic core of the pilus whereas the divergent C-terminal pilin globular domain decorates the periphery of the pilus. Comparisons are then made against T4P structures from other organisms, highlighting interesting differences including a shorter rod diameter and lack of solvent-accessible loops for which the authors propose reduces proteolysis of the T4P compared to other organisms.

Major comments:

The results of this manuscript are convincing. The models and cryo-EM volumes, which are already accessible from the PDB and EMDB, are of good quality with no obvious issues. The conclusions drawn from the model are not speculative. While extensive mutagenesis experiments could help delineate critical residues involved in T4P assembly and clarify involvement in adhesion/biofilm formation, these would have to be done in the native organism, would require a significant amount of time and effort, and would be beyond the scope of the current manuscript.

Minor comments:

The figures are excellent and clear, and the text is well-written, with results easy to interpret.

One of the strengths of this paper is the comparative analyses across current bacterial T4P structures. In this respect, I would have liked a more thorough analysis here: - While differences in helical parameters, rod diameter, and rod length are presented, a figure showing comparison of surface electrostatics and/or hydrophobicity could help delineate differences (if any) across these species, which may reflect the different environments these bacteria inhabit.<br /> - A consurf representation of PilA is shown in Fig. 3h. It would be helpful to include either the sequence alignment used for this analysis or a sequence alignment for all the species presented in the manuscript, to show precisely which residues are absolutely conserved across these species.<br /> - A panel showing their full T4P as a surface with Consurf coloring would be informative to show conservation across the entire pilus and not just a PilA subunit. - The authors state that the models in Fig. 3 were aligned based on the matchmaker function in Chimera. Wouldn't the poor sequence conservation of the C-terminal globular domain of PilA drive the alignment towards the N-terminal helix? In that case, wouldn't using a comparative alignment strategy that focuses on the model itself (LSQ) or secondary structure elements (SSM) which would drive the alignment more towards the globular domain be more reflective of the full pilin subunit? - Related to the point above, it would be useful to include a table highlighting pairwise RMSDs across all models presented in this manuscript.

Significance

The authors rightfully highlight the importance of P. aeruginosa T4P in the development of biofilms; structural analyses of these pili are of clinical importance and of interest to researchers involved in bacterial motility.

To date, various structures of T4P and T4P subunits across a variety of bacterial have been solved by X-ray crystallography and cryo-EM (PDB: 9EWX (this study), 6GV9, 5VXX, 6XXD, 6VK9, 8TJ2). It appears that another group has recently published a slightly lower resolution (3.6 Å vs 3.2 Å) cryo-EM structure (PDB: 8TUM) of the T4P of P. aeruginosa PAO1 (Thongchol et al., Science, 2024). The model from this latter publication appears to be identical to the model presented in this manuscript. Since this work has now been published (with models being released mid-March 2024), and since Ochner et al.'s manuscript only appeared on Biorxiv on April 9th 2024, I feel it would have been appropriate and necessary to cite this paper. And while the Thongchol publication reduces the novelty of Ochner et al.'s manuscript, there is some merit in the comparative analyses performed, which if expanded upon, could further strengthen this manuscript enough to stand on its own.

Field of expertise: cryo-EM, bacterial secretion, membrane proteins

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Referee #2

Evidence, reproducibility and clarity

The manuscript by Ochner et al. reports the cryo-EM structure of the Type 4 pili of Pseudomonas aeruginosa at decent 3.2 resolution with fully resolved pilin fold. It is a straightforward report, using state-of-the-art microscopy and data processing approaches as usual for the group and the figures and data representation are clear. The main findings of the work is that it visualizes the assembled pilus of an important pathogen (Pseudomonas aeruginosa is one of the ESKAPE pathogens with particularly impressive adaptability and Type IV pili are important for substrate colonization and biofilm formation). The PilA pilin fold is not far from that of a previously crystallized isolated homolog from the PAK strain (core hydrophobic N-helix, globular b-sheet-containing exposed CTD) and presents a central melting of the core helix also observed among multiple other PilA homologs from solved G- pilin structures. The main difference for the PAO1 pilus is the tight packing in a significantly thinner filament, which lacks protruding loops or other secondary structure insertions in the core pilin fold. The authors propose that this could lead to increased stability such as to proteases.

Again, the study is quite straightforward and besides the standard and well-executed EM workflow, it uses the classical approaches for pilus overexpression and purification (a PilT mutant that cannot retract and presents more T4P; well established mechanical shearing protocol for surface release, etc.). The structure is at decent resolution allowing full backbone tracing and side-chain resolution for confident model building, etc. The figures are clear, even if I would encourage some more vivid or at least contrasting colors for the cartoon model in Fig. 2 and some more detailed surface and conservation analyses, especially in terms of packing and surface exposure.

Minor comment: The electron density maps, atomic models and validation reports should be available for the review process. The refinement statistics in the table are very good and the figures and supplementary movie present clear densities but this should be standard protocol and could help with constructive suggestions from the reviewers. Large map files, etc can be provided via a link if too big for upload directly through the manuscript tracking system.

Significance

My main concern with this work is that it is quite minimalistic in terms of biology/physiology, especially in light of the many G- pili structures available, some of which the authors nicely review in terms of specific structural parameters. The hypothesis of increased protease or perhaps mechanical resistance is tantalizing but how the compact pilus fold actually affects Pseudomonas aeruginosa in its physiology is unclear. Are there any other differences in the surface properties relative to other pili (charge, surface motif conservation, etc.) and could they have relevance in terms of interactions with the substrate, another matrix component or a peculiar niche within the host? As a PilA mutant should be easy to get from a number of laboratories, how would a Pseudomonas delta-PilA mutant behave in terms of twitching motility, surface attachment and biofilm formation if complemented with PaO1 PilA vs. other pilins from Table S2 or a pilin with an engineered hybrid architecture (e.g. a loop or b-hairpin insertion)? If such heterologous/engineered pili are incubated with a mild protease mix, would they indeed exhibit increased fragmentation relative to the wt PAO1 pili? To me, most of these assays are relatively easy to be attempted in terms of molecular biology, phenotypic assays and in vitro biochemistry (e.g. plasmid-based complementation if full genetics are judged beyond the scope/time available, twitching, pellicle formation, SDS-PAGE of protease-treated sheared pili) and could really shed more specific insights light into the peculiarities of Pseudomonas T4P function, rather than just present the next resolved filament among the multiple other T4P already out there.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Referee #1

Evidence, reproducibility and clarity

Summary

The study titled "Structure of the Pseudomonas aeruginosa PAO1 Type IV pilus" by Ochner and colleagues utilised cryo-electron microscopy (cryo-EM) to determine and describe the atomic structure of a complete type IV pilus (T4P) filament from Pseudomonas aeruginosa in its native state at an impressive resolution of 3.2 Å. The authors use state-of-the-art cryo-EM methodology, and the detailed description of their procedures allows for an adequate replication of the results. The T4P are essential for the virulence of P. aeruginosa, which is a clinically important human pathogen, as they play a crucial role in biofilm formation, a major factor in its resistance to antibiotics and ability to cause infections. Therefore, understanding the molecular mechanisms behind the ability of these bacteria to establish infections is vital, and this high-resolution structure of T4P provides valuable insight into this process.

Overall, this reviewer acknowledges the importance of the structure presented here and its significance to our understanding of P. aeruginosa infection and persistence, and hence is positive about the publication of the results, however, at its current state the manuscript raises the major questions outlined below, which must be addressed and corrected.

Major comments

The authors propose a model where T4P interact with the type IV secretion systems (T4SS) in the P. aeruginosa membrane. However, there is no current evidence in the literature to support a direct interaction between T4P and T4SS as these are functional and structural distinct secretion systems. T4P biogenesis is mediated by a specialised secretion complex (homologous to type II secretion systems), spanning both bacterial membranes and consisting of the outer membrane secretin subcomplex, the alignment subcomplex, and the inner membrane motor complex. This reviewer recommends that authors refer to the comprehensive review by Hospenthal and colleagues [PMID: 28496159] that details the T4P biogenesis and Craig and colleagues [PMID: 30988511] that provides an in-depth analysis of T4P secretin architecture. This reviewer recommends the authors to remove any misleading claims regarding a T4P-T4SS interaction. Furthermore, the introduction would benefit from a brief overview of the T4P biogenesis and secretin architecture to prevent any further confusion. While this study offers a higher resolution structure of P. aeruginosa T4P (3.2 Å) compared to the previously described work on the P. aeruginosa T4P (8 Å) described by Wang and colleagues [PMID: 28877506], the manuscript fails to convey the significance of this improvement. The authors should directly compare the new structure with the previously obtained cryo-EM structure, similarly to how they tackled the comparison to the X-ray crystallography structure (Figure S6). A dedicated figure visualising the key differences and benefits associated with the higher resolution is necessary to highlight the manuscript's significance. Furthermore, the authors should specify that the reported T4P belongs to the type IVa category and the "globular domain" of PilA should be further differentiated into the αβ loop and D region - widely accepted motifs present in the structures of type IV pilins [PMID: 31784891]. Highlighting them is crucial due to their roles in receptor binding, microcolony formation, and antigenic variation, warranting their inclusion in the manuscript. A more detailed display of intersubunit interactions, including the types and numbers of interactions is also recommended, however optional. Previous studies [PMID: 27698424, PMID: 28609682] hypothesize that disordered loops might be involved in significant T4P stretching, the authors should address how the lack of these structures in their model might affect the filament dynamics. Lastly, the study lacks experimental validation of the structure, either within the study or referenced from the existing literature and very weakly connects the structure to T4P's biological functions, such as twitching motility or DNA acquisition. For instance, a comparison could be drawn between the surface charge of the pili and its DNA binding capacity. Additionally, the T4P secretin complex of P. aeruginosa documented in [PMID: 27705815] should be modelled alongside the obtained T4P structure to compare the structure diameter with the PilQ secretin lumen. These revisions will strengthen the manuscript by addressing crucial points and highlighting the significance of the high-resolution T4P structure.

Minor comments

Figure 2 For consistency, the colours of PilA subunits between panels (a) and (d) should match.

Figure 3 For clarity, pilins should be coloured by domain.

L41 The word "surfaces" or "target receptors" rather than just "substrates" would be more accurate.

L87 Rather than "other bacteria" consider using "wild-type strains".

L145-147 For clarity, residues C134 and C147 that form a disulfide bond in the C-terminal loop should be displayed in the figure.

L371 For consistency, "h" should be in brackets, following the authors' style.

Significance

General assessment:

The significance of the study stems from a resolution improvement from the previously reported type IV pilus of P. aeruginosa by Wang and colleagues [PMID: 28877506] and complements well the X-ray crystallography data obtained previously by Craig and colleagues [PMID: 12769840]. Due to the role of T4P in the virulence of P. aeruginosa, the structure provides important biological information about the molecular mechanism of its niche establishment. Moreover, the structure can be used in subsequent drug design against P. aeruginosa infections.

Nature of advance:

The nature of the advance provided by this study is in the added structural detail of the T4P due to the obtained higher resolution of the map. Usually, the highest resolution structure is used to derive the conclusions about the biological functions of the filament, hence the structure provided here will be referenced as the final P. aeruginosa T4P structure in further studies.

Audience:

The higher resolution structure compared to the previously described will be interesting to the translational/clinical drug discovery audiences, which require a high-resolution structure for accurate drug design.

Field of expertise:

Type IV secretion systems, bacterial conjugation, conjugative pili.

test

Direct line secrect 和 Drect line toke 是不一样的。

Justement. Le coupable, on le connaît. C'est Lancelot, personne ne met en doute qu'il a tué Gaheriet. Mais "il n'a pas fait exprès". C'est tout le drame dans cette Mort Artu, les choses avancent sans qu'on le veuille.

On travaille ici avec des catégories modernes dans une perspective qui n'est peut-être pas très authentique dans la mesure où personne vers 1230 n'était "inquiet" (au sens où l'on l'entend ici)

Ce début relève un peu du postulat. La "fin" de l'histoire était quand même connue de tout le monde puisque c'est en gros celle qui figure dans la tradition des chroniques, Wace et, surtout, l'Historia Regum Britanniae, de Geoffroy de Monmouth, cette dernière conservée dans plus de 250 manuscrits...

Editors Assessment:

Oxford nanopore direct RNA sequencing (DRS) is a relatively new sequencing technology enabling measurements of RNA modifications. In vitro transcription (IVT)-based negative controls (i.e. modification-free transcripts) are a practical and targeted control for this direct sequencing, providing a baseline measurement for canonical nucleotides within a matched and biologically-derived sequence context. This work presents exactly this type of a long-read, multicellular, poly-A RNA-based, IVT-derived, unmodified transcriptome dataset. Review flagging more statistical analyses needed be performed for the data quality, and this was provided. The resulting data providing a resource to the direct RNA analysis community, helping reduce the need for expensive IVT library preparation and sequencing for human samples. And also serving as a framework for RNA modification analysis in other organisms.

This evaluation refers to version 1 and 2 of the preprint

This work has been published in GigaByte Journal under a CC-BY 4.0 license (https://doi.org/10.46471/gigabyte.129), and has published the reviews under the same license. These reviews are as follows:

Reviewer 1. Joshua Burdick

Is the language of sufficient quality?

Yes. In line 284, "bioinformatic" may be more often used than "BioInformatic", but the meaning is clear.

Are the data and metadata consistent with relevant minimum information or reporting standards?

Yes. Presumably the files (e.g. eventalign data) which are not in SRA will need to be uploaded to the GigaByte site.

Is there sufficient detail in the methods and data-processing steps to allow reproduction?

Yes. Line 177 should presumably be "nanopolish evenetalign".

Is there sufficient data validation and statistical analyses of data quality?

Yes. In my opinion, Figure 3(A) nicely illustrates the uncertainty in current nanopore data, which is useful.

Additional Comments:

The RNA samples, and nanopore sequencing data, should be useful as a negative control. Sequencing these IVT RNA samples using the newer ONT RNA004 pore and kit might also be useful.

Reviewer 2. Jiaxu Wang

Is there sufficient data validation and statistical analyses of data quality?

No. The authors ran DSR for the in vitro transcribed transcriptional RNAs from 6 cell lines to remove the possible natural modifications. The data can be used as a control RNA pool for natural or artificial modification studies. however, more statistical analyses should be performed for the data quality. see comments below: (1) For more possible usage of this data, some QC analysis is better to be provided to confirm the quality of these sequencing data. For example: 1) What is the correlation between in vitro transcribed transcriptional RNAs and original DSR for each cell line? 2) how many genes have been captured in each cell line? (2) In Figure 2B, the author provides 3 conditions for ‘exclude’ and ‘include’, some statistical analysis should be performed to confirm how many cases in condition 1, condition 2, and condition 3. How many mismatches are showing in only 1 cell line, some cell lines or all the cell lines? The shared correct genes may be more confident references for the modification analysis. (3) Different reads of the same gene could have different mismatches in the IVT RNAs due to RT-PCR bias or other reasons (especially for the lower expressed RNAs), for example, there are 100 reads in total, 90 reads are the correct nucleotide at a given position, 10 reads have a mismatch in the IVT sample, then how to define the signal as the control reference? Given that the nature modification is low in RNA, some threshold should be applied for the confident result, for example, what is the lowest expression threshold that could be used as a confident control reference?

Is there sufficient information for others to reuse this dataset or integrate it with other data?

No. For more possible usage of this data, more QC data should be performed, please refer to my above comments.

Re-review: I am happy to see the changes. Thanks!

Acabei de ler o capítulo sobre e-atividades em ambientes digitais do documento fornecido. Achei muito interessante como as e-atividades são destacadas como essenciais para promover uma aprendizagem online ativa e participativa.

O texto enfatiza a importância de diversificar os formatos para atender a diferentes estilos de aprendizagem e a necessidade de oferecer feedback imediato aos alunos.

Além disso, a flexibilidade e acessibilidade dessas atividades são pontos fortes, especialmente para estudantes com agendas mais apertadas.

Gostaria de perguntar como podemos implementar estas práticas de forma eficaz nos nossos próprios cursos. Alguém já tem experiência com isso e poderia partilhar insights?

Cumprimentos, André Calvinho

Acabei de ler o capítulo sobre e-atividades em ambientes digitais do documento fornecido. Achei muito interessante como as e-atividades são destacadas como essenciais para promover uma aprendizagem online ativa e participativa.

O texto enfatiza a importância de diversificar os formatos para atender a diferentes estilos de aprendizagem e a necessidade de oferecer feedback imediato aos alunos.

Além disso, a flexibilidade e acessibilidade dessas atividades são pontos fortes, especialmente para estudantes com agendas mais apertadas.

Gostaria de perguntar como podemos implementar estas práticas de forma eficaz nos nossos próprios cursos. Alguém já tem experiência com isso e poderia partilhar insights?

Cumprimentos, André Calvinho

Nishant says: 2x Output for 1x input...

His formula for mastery: 1. Learn (input -- focus, singletasking) 2. Reflect (output, pause... what is the main takeaway, how to use?) 3. Implement (output, apply) 4. Share (output, teach the material)

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These principles are great... Obviously they are not comprehensive as they do not necessarily reflect higher order learning. See Bloom's and Solo's, nor take foundation of Cognitive Load Theory for example... It's understandable though since you can't mention everything in a 20 minute talk XD.

The argument I'd make is that the 3 subsequent steps are a part of learning. So the first step should not be called learn but rather encode, since that is literally the process of forming the initial cognitive schemas and putting them into long-term memory...

To be fair, for the picture argument... When you have seen a person only once it's more likely you remember their name, not their face. Additionally, if you have not seen someone for a very very long time the same is true.

I get the sentiment though and I agree.

According to Nishant, what I agree with, the truly successful people are MASTERS in their craft. They have committed to lifelong learning.

"Your learning capability decides your earning capacity."

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See also: Ultralearning, Scott H. Young, and Deep Work, Cal Newport... The argument is the same: your ability to adapt in a complex rapidly changing information economy, and to master material determines how much you can earn.

Nishant Kasibhatla memorizes a 30 digit "random" number at the beginning of the video and recalls it correctly, in reverse, at the end of the video.

He uses number visualization of combinations to do this. (every 2 digit number has an image in his mind that he has practiced substantially in relation)... Similar to Mind Palace.

He did make a few mistakes in the normal recall at the beginning... But it is safe to assume that he did it on purpose (for what reason I do not know), because he has a lot of expertise in it.

Introducing humor is very good for giving a speech or presenting.

با استفاده از این label می تونیم برای این checkbox بیای و label تعیین کنی

Nice snippets but need to be converted to LuaSnip. Also some of them (such as code block) are better derived from markdown plugins aka mkdx. Some are even better implemented in a non-snippet plugins (images, tables)

https://www.theguardian.com/environment/article/2024/jul/02/biden-extreme-heat-proposaal

https://www.theguardian.com/business/article/2024/jun/27/bp-imposes-hiring-freeze-and-halts-new-offshore-wind-projects

https://www.theguardian.com/business/article/2024/jul/02/shell-to-pause-construction-of-huge-biodiesel-plant-in-rotterdam

https://www.theguardian.com/world/article/2024/jul/01/hurricane-beryl-caribbean-islands-climate-change

https://www.theguardian.com/environment/article/2024/jul/01/labour-will-take-global-lead-on-climate-action-ed-miliband-vows

https://www.theguardian.com/technology/article/2024/jul/02/google-ai-emissions

Redistribution (cultural anthropology)

https://www.youtube.com/watch?v=ic09paZ4BlE

for - podcast - great simplification - Nate Hagen - guest - Daniel Schmachtenberger - topic progress

July 30, 2023 - Serotonin, or 5-hydroxytryptamine (5-HT), is a neurotransmitter with an integral physiological role in the human body; it regulates various activities, including behavior, mood, memory, and gastrointestinal homeostasis.[1][2] Serotonin is synthesized in the raphe nuclei of the brainstem and ...

https://www.wired.com/story/hurricane-beryl-category-5-storm-extreme-weather-summer-2024/?utm_source=facebook&utm_social-type=owned&utm_brand=wired&utm_medium=social&mbid=social_facebook

Главная идея в том, чтоб в какой-то момент жизни, достать элементы, которые важнее остальных. по сути, можно сказать, когда стоишь в больнице на приёме к травматологу в очереди. и вдруг приходит человек у которого топор в спине, или рука висит, как У гарри поттера после игры в квидич, когда пантовый колдун решил помочь ему вылечить руку. он же сразу проходит без очереди и пофиг, что ты уже час ждёшь

New firms could be better than incumbents as emissions are ring fenced for incumbents

Good point

Résumé de la vidéo [00:00:10][^1^][1] - [00:24:31][^2^][2] : Cette vidéo présente une session des Jeudis de l'expérience usagers, où Stéphanie Metou de l'ASP (Agence de Service et de Paiement) explique comment sensibiliser à l'expérience utilisateur par le jeu. Elle introduit le concept d'andragogie et la gamification, détaille la création d'un jeu de cartes éducatif et partage des témoignages sur son efficacité.

Points forts : + [00:00:10][^3^][3] Introduction à l'expérience utilisateur * Présentation des Jeudis de l'expérience usagers * Objectif de sensibiliser à l'expérience utilisateur par le jeu + [00:01:04][^4^][4] Stéphanie Metou et l'ASP * Introduction de Stéphanie Metou et de son rôle à l'ASP * Explication des missions de l'ASP et de son importance + [00:07:05][^5^][5] Création du jeu de cartes * Processus de création d'un jeu de cartes pour apprendre le lexique de l'expérience utilisateur * Utilisation de l'andragogie et de la gamification pour l'éducation des adultes + [00:20:35][^6^][6] Témoignages et impact du jeu * Partage de témoignages sur l'utilisation du jeu * Impact positif sur l'apprentissage et la communication interne

Résumé de la vidéo [00:24:33][^1^][1] - [00:39:27][^2^][2]:

Cette vidéo présente une session des Jeudis de l'expérience usagers, axée sur la sensibilisation à l'expérience utilisateur par le jeu. Elle explique comment un jeu de cartes peut être utilisé pour faciliter l'apprentissage et encourager la communication et la collaboration au sein des équipes.

Points forts: + [00:24:33][^3^][3] L'apprentissage par le jeu * Favorise les échanges et la coopération * Sert d'outil d'accompagnement au changement * Aide à l'acquisition de compétences + [00:25:01][^4^][4] Le jeu comme reflet du parcours utilisateur * Représente les cinq étapes du parcours utilisateur * Intègre le lexique de l'expérience utilisateur * Encourage la communication et la collaboration + [00:27:10][^5^][5] L'andragogie par le jeu * A fait ses preuves dans la formation * Constitue un support précieux pour l'accompagnement au changement * Permet de réfléchir à d'autres modalités de formation ludiques + [00:31:03][^6^][6] La conception du jeu * Basée sur le besoin d'un langage commun * Implique différentes équipes dans sa création * Vise à rendre les agents acteurs de leur apprentissage

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:10][^2^][2]:

Cette vidéo présente une formation pour lutter contre les incivilités et les agressions envers les agents publics. Elle aborde l'importance de la formation, les rôles des agents et des encadrants, et les moyens de prévention et de réaction face aux incivilités.

Points forts: + [00:00:00][^3^][3] Introduction à la formation * Présentation du thème de la formation * Importance de protéger les agents publics + [00:01:26][^4^][4] Plan de protection des agents publics * Lancement du plan par le ministre * Objectif de mieux accompagner les agents + [00:09:00][^5^][5] Raisons de la formation * Augmentation des incivilités et agressions * Besoin d'appui pour les agents + [00:10:00][^6^][6] Contenu de la formation * Deux piliers : distantiel et présentiel * Neuf modules en ligne pour sensibiliser et former + [00:16:00][^7^][7] Rôle des encadrants * Soutien des encadrants aux agents * Responsabilité collective dans la prévention + [00:20:00][^8^][8] Après les incivilités * Importance de signaler et de parler * Écoute et soutien aux agents affectés

Résumé de la vidéo [00:24:11][^1^][1] - [00:46:02][^2^][2]:

Cette partie de la vidéo aborde la formation et l'accompagnement des agents publics pour lutter contre les incivilités et les agressions. Elle souligne l'importance de la qualité de service comme premier levier de prévention et la nécessité d'une réaction appropriée face aux comportements incivils.

Points forts: + [00:24:11][^3^][3] Formation sur la gestion des conflits * Mise à disposition d'un état de l'art sur la gestion des situations conflictuelles * Formation téléchargeable intégrant cet état de l'art + [00:25:35][^4^][4] Table ronde avec des représentants de divers secteurs * Échange sur des situations pratiques et opérationnelles * Participation de représentants de Pôle Emploi, CHU de Poitiers, La Poste, etc. + [00:27:01][^5^][5] Lancement de la formation par la DITP * Présentation par le ministre Stanislas Guini et Marcel Guoun * Formation disponible sur la plateforme Mentor + [00:29:40][^6^][6] Enjeux des incivilités et agressions * Impact sur la qualité de service et la sécurité des agents * Importance de la formation et de l'accompagnement pour améliorer la relation service-public

Résumé de la vidéo [00:46:05][^1^][1] - [01:07:22][^2^][2]:

Cette vidéo aborde la lutte contre les incivilités et les agressions, en mettant l'accent sur la formation et l'accompagnement des agents publics. Elle souligne l'importance de définir collectivement les seuils de tolérance et les définitions des incivilités, ainsi que la nécessité de former les agents pour qu'ils soient bien armés pour accompagner les démarches du public.

Points forts: + [00:46:05][^3^][3] Définition collective des incivilités * Importance de confronter les ressentis et les points de vue * Accord sur les seuils de tolérance et les définitions + [00:50:01][^4^][4] Recensement et analyse des agressions * Les agents doivent raconter ce qui leur arrive * Identification des bureaux de poste les plus exposés aux agressions + [00:54:00][^5^][5] Mesure de la satisfaction et de l'impact des actions * Mise en place d'indicateurs de succès * Évaluation de l'efficacité des actions et adaptation continue + [01:00:02][^6^][6] Formation et prévention des situations difficiles * Formation obligatoire pour les nouveaux agents * Analyse des cas concrets et partage des expériences vécues

Résumé de la vidéo [01:07:24][^1^][1] - [01:21:42][^2^][2]:

Cette vidéo aborde la formation et l'accompagnement des agents publics pour lutter contre les incivilités et les agressions. Elle souligne l'importance de ne pas banaliser ces comportements et de fournir aux agents les outils nécessaires pour y faire face. La formation vise à rompre l'isolement des agents, à favoriser l'échange d'expériences et à renforcer le soutien hiérarchique.

Points forts: + [01:07:24][^3^][3] L'importance de la formation * Prévenir la banalisation des incivilités * Utiliser la pédagogie pour sensibiliser * Insister sur l'évaluation et l'ajustement des dispositifs + [01:10:01][^4^][4] Le rôle de la hiérarchie et de la collectivité * Soutenir les agents et partager les expériences * Reconnaître l'importance de l'endossement hiérarchique * Encourager la communication et le débriefing au sein des équipes + [01:14:32][^5^][5] Les clés de la gestion des incivilités * Il n'y a pas de solution unique; l'approche doit être adaptée * L'importance de la responsabilité d'équipe et hiérarchique * Verbaliser et prendre du recul est essentiel pour les agents + [01:20:01][^6^][6] La solidarité et la mobilisation collective * La formation est une œuvre collective au service des agents * L'usager est une partie de la solution et du respect mutuel * La nécessité d'une mobilisation continue pour soutenir les agents

水锤问题

how to get a random real number in the range [x,y)?

信息是一种无序、未经加工或理解的数据或事实，而知识则是建立在信息之上的有意义和系统化的理解和认知。

How smFISH works?

• multiple probes are designed to target adjacent segments of the mRNA transcript. Each probe is around 50 nucleotides long.

• these probes are labelled with fluorophores, which emit fluorescence when excited by a specific wavelength of light.

Visualising individual transcripts - When probes hybridise their target mRNA, the combined fluorescence makes the individual mRNA molecules visible as distinct puntca under a fluorescence microscope - The intensity of each punctum's fluorescence corresponds to the number of fluorophores, providing a quantitative measure of the transcript

50 nucleotides long

Early methods with electron microscopy - before the development of smFISH, transmission electron microscopy was used to visualise individual mRNA molecules in fibroblasts. - This involved labelling of the poly-A tail of the mRNA with a single large colloidal gold particle and labelling in-situ reverse transcribed cDNA with smaller gold particles

• Reintegração: Esbulho
• Manutenção: Turbação
• Interdito Proibitório: justo receio de molestar posse.

Vide art. 1.210/CC

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SÚMULA 637/STJ - O ente público detém legitimidade e interesse para intervir, incidentalmente, na ação possessória entre particulares, podendo deduzir qualquer matéria defensiva, inclusive, se for o caso, o domínio.

不想在本地安装坚果云,打算把本地同步到nas再通过nas同步到坚果云

Signalr API Support Server - to - client 的 Broadcast 类型的。

编程

DOI: 10.1016/j.celrep.2021.108876

Resource: (Cell Signaling Technology Cat# 3285, RRID:AB_2269034)

Curator: @Naa003

SciCrunch record: RRID:AB_2269034

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What is this?

DOI: 10.1016/j.celrep.2021.108876

Resource: (Cell Signaling Technology Cat# 3285, RRID:AB_2269034)

Curator: @Naa003

SciCrunch record: RRID:AB_2269034

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 49084,RRID:BDSC_49084)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_49084

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 6596,RRID:BDSC_6596)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_6596

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 9750,RRID:BDSC_9750)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_9750

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 25709,RRID:BDSC_25709)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_25709

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 55134,RRID:BDSC_55134)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_55134

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 44275,RRID:BDSC_44275)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_44275

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 42748,RRID:BDSC_42748)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_42748

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: BDSC_80067

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_80067

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 5140,RRID:BDSC_5140)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_5140

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 23647,RRID:BDSC_23647)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_23647

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 6926,RRID:BDSC_6926)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_6926

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 52547,RRID:BDSC_52547)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_52547

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: BDSC_52814

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_52814

---

What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: (BDSC Cat# 41253,RRID:BDSC_41253)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_41253

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What is this?

DOI: 10.1007/s12264-022-00966-y

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

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What is this?

DOI: 10.1093/nar/gkae311

Resource: NIH NeuroBioBank (RRID:SCR_003131)

Curator: @evieth

SciCrunch record: RRID:SCR_003131

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What is this?

DOI: 10.1126/sciadv.adi8287

Resource: NIH NeuroBioBank (RRID:SCR_003131)

Curator: @evieth

SciCrunch record: RRID:SCR_003131

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What is this?

DOI: 10.1101/2024.06.24.600382

Resource: (RCB Cat# RCB1979, RRID:CVCL_3526)

Curator: @mzhang007

SciCrunch record: RRID:CVCL_3526

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What is this?

DOI: 10.1038/s41380-024-02441-8

Resource: NIH NeuroBioBank (RRID:SCR_003131)

Curator: @evieth

SciCrunch record: RRID:SCR_003131

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What is this?

DOI: 10.3390/pharmaceutics16070870

Resource: (ECACC Cat# 88062428, RRID:CVCL_0131)

Curator: @mzhang007

SciCrunch record: RRID:CVCL_0131

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What is this?

DOI: 10.3390/pharmaceutics16070870

Resource: (NIH-ARP Cat# 2188-324, RRID:CVCL_0022)

Curator: @mzhang007

SciCrunch record: RRID:CVCL_0022

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What is this?

DOI: 10.20944/preprints202401.2131.v1

Resource: BRAIN Initiative Cell Atlas Network (RRID:SCR_022794)

Curator: @bandrow

SciCrunch record: RRID:SCR_022794

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What is this?

DOI: 10.3390/brainsci14040372

Resource: BRAIN Initiative Cell Atlas Network (RRID:SCR_022794)

Curator: @bandrow

SciCrunch record: RRID:SCR_022794

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What is this?

DOI: 10.1101/2024.04.15.589472

Resource: BRAIN Initiative Cell Atlas Network (RRID:SCR_022794)

Curator: @bandrow

SciCrunch record: RRID:SCR_022794

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What is this?

DOI: 10.1101/2024.06.20.599687

Resource: BRAIN Initiative Cell Atlas Network (RRID:SCR_022794)

Curator: @bandrow

SciCrunch record: RRID:SCR_022794

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What is this?

DOI: 10.1038/s41380-024-02620-7

Resource: BRAIN Initiative Cell Atlas Network (RRID:SCR_022794)

Curator: @bandrow

SciCrunch record: RRID:SCR_022794

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What is this?

DOI: 10.26508/lsa.202201540

Resource: (BDSC Cat# 30909,RRID:BDSC_30909)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_30909

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What is this?

DOI: 10.26508/lsa.202201540

Resource: (BDSC Cat# 7376,RRID:BDSC_7376)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_7376

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What is this?

DOI: 10.26508/lsa.202201540

Resource: BDSC_57327

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_57327

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What is this?

DOI: 10.26508/lsa.202201540

Resource: (BDSC Cat# 23651,RRID:BDSC_23651)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_23651

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What is this?

DOI: 10.26508/lsa.202201540

Resource: (BDSC Cat# 31423,RRID:BDSC_31423)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_31423

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What is this?

DOI: 10.26508/lsa.202201540

Resource: BDSC_7195

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_7195

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What is this?

DOI: 10.1016/j.isci.2022.105598

Resource: BDSC_67300

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_67300

---

What is this?

DOI: 10.1016/j.isci.2022.105598

Resource: BDSC_7155

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_7155

---

What is this?

DOI: 10.1016/j.isci.2022.105598

Resource: BDSC_18814

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_18814

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What is this?

DOI: 10.1016/j.isci.2022.105598

Resource: (BDSC Cat# 6326,RRID:BDSC_6326)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_6326

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What is this?

DOI: 10.1016/j.isci.2022.105598

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

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What is this?

DOI: 10.7554/eLife.82459

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

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What is this?