Reviewer #2 (Public review):

Summary:

The authors have made microfluidic arrays of pores and obstacles with a complex shape and studied the swimming of multicellular magnetotactic bacteria through this system. They provide a comprehensive discussion of the relevant parameters of this system and identify one dimensionless parameter, which they call the scattering number and which depends on the swimming speed and magnetic moment of the bacteria as well as the magnetic field and the size of the pores, as the most relevant. They measure the effective speed through the array of pores and obstacles as a function of that parameter, both in their microfluidic experiments and in simulations, with good agreement between the two. They find an optimal scattering number, which they estimate to reflect the parameters of the studied multicellular bacteria in their natural environment. They finally use this knowledge to compare different species. Despite the variability of bacteria parameters, they estimate the scattering number to be rather narrowly distributed, suggesting that their results apply to a broad range of species.

Strengths:

This is a beautiful experimental approach and the observation of an optimal scattering number (likely reflecting an optimal magnetic moment) is very convincing. The results here improve on similar previous work in two respects: On the one hand, the tracking of bacteria does not have the limitations of previous work, and on the other hand, the effective motility is quantified. Both features are enabled by choices of the experimental system: the use the multicellular bacteria which are larger than the usual single-celled magnetotactic bacteria and the design of the obstacle array which allows the quantification of transition rates due to the regular organization as well as the controlled release of bacteria into this array through a clever mechanism.

Weaknesses:

Some of the key experimental choices on which the strength of the approach is based also come at a price and impose some limitations, namely the use of a non-culturable organism and the regular, somewhat unrealistic artificial obstacle array, but the advantages of these choices outweigh the drawbacks.

Comments on revisions:

The paper has been improved with respect to presentation and content. In particular, I appreciate the new plots comparing the simulation and experiments directly and the estimate of the scattering number for different species. In my opinion, all issues raised by the reviewers have been addressed in a productive way.

Reviewer #1 (Public review):

Summary:

This manuscript reports the investigation of PriC activity during DNA replication initiation in Escherichia coli. It is reported that PriC is necessary for growth and control of DNA replication initiation under diverse conditions where helicase loading is perturbed at the chromosome origin oriC. A model is proposed where PriC loads helicase onto ssDNA at the open complex formed by DnaA at oriC. Reconstituted helicase loading assays in vitro are consistent with the model.

Strengths:

The complementary combination of genetics in vivo and reconstituted assays in vitro provide solid evidence to support the role of PriC at a replication origin.

The manuscript is well written and has a logical narrative.

The data provide new insight to how bacteria might load helicase at the replication origin when the wild-type DnaA-dependent loading pathway is perturbed.

Weakness:

It has not yet been established whether PriC localises at oriC in vivo under the conditions tested.

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins is suboptimal. Here highlighted by mutations in dnaA or dnaC.

This a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second in loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.<br /> This puts into questions the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support initiation of replication and cell viability only in the presence of PriC. Such mutants are capable to generate single strand opening but may fail to load the helicase in absence of PriC. Similarly, dnaA mutants may generate aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

I have a few specific questions/comments

The partial complementation of the dnaC2 strain by PriC seems quite straightforward since this particular mutation leads to initiation arrest at the open complex stage and this sets the stage for PriC to load the helicase. The situation is somewhat different for dnaA46. Why is this mutation partly complemented by PriC at 37C? DnaA46 binds neither ATP nor ADP, yet it functions in initiation at permissive temperature. At nonpermssive temperature, it binds oriC as well but does not lead to initiation. Does the present data imply that the true initiation defect of DnaA46 lies in helicase loading? The authors need to comment on this in the text.

Relating to the above. In Fig. 3 it is shown that the pFH plasmid partly complement dnaA46 in a PriC dependent manner. Again, it would be nice to know the nature of the DnaA46 protein defect. It would be interesting to see how a pING1-dnaA46 plasmid performs in the experiment presented in Fig. 3.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. The conclusions drawn supported by the evidence provided are compelling.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Reviewer #1 (Public review):

Summary:

This paper focuses on understanding how covalent inhibitors of peroxisome proliferator-activated receptor-gamma (PPARg) show improved inverse agonist activities. This work is important because PPARg plays essential roles in metabolic regulation, insulin sensitization, and adipogenesis. Like other nuclear receptors, PPARg, is a ligand-responsive transcriptional regulator. Its important role, coupled with its ligand-sensitive transcriptional activities, makes it an attractive therapeutic target for diabetes, inflammation, fibrosis, and cancer. Traditional non-covalent ligands like thiazolininediones (TZDs) show clinical benefit in metabolic diseases, but utility is limited by off-target effects and transient receptor engagement. In previous studies, the authors characterized and developed covalent PPARg inhibitors with improved inverse agonist activities. They also showed that these molecules engage unique PPARg ligand binding domain (LBD) conformations whereby the c-terminal helix 12 penetrates into the orthosteric binding pocket to stabilize a repressive state. In the nuclear receptor superclass of proteins, helix 12 is an allosteric switch that governs pharmacologic responses, and this new conformation was highly novel. In this study, the authors did a more thorough analysis of how two covalent inhibitors, SR33065 and SR36708 influence the structural dynamics of PPARg LBD.

Strengths:

(1) The authors employed a compelling integrated biochemical and biophysical approach.

(2) The cobinding studies are unique for the field of nuclear receptor structural biology, and I'm not aware of any similar structural mechanism described for this class of proteins.

(3) Overall, the results support their conclusions.

(4) The results open up exciting possibilities for the development of new ligands that exploit the potential bidirectional relationship between the covalent versus non-covalent ligands studied here.

Weaknesses:

(1) The major weakness in this work is that it is hard to appreciate what these shifting allosteric ensembles actually look like on the protein structure. Additional graphical representations would really help convey the exciting results of this study.

Reviewer #2 (Public review):

Summary:

The authors use ligands (inverse agonists, partial agonists) for PPAR, and coactivators and corepressors, to investigate how ligands and cofactors interact in a complex manner to achieve functional outcomes (repressive vs. activating).

Strengths:<br /> The data (mostly biophysical data) are compelling from well-designed experiments. Figures are clearly illustrated. The conclusions are supported by these compelling data. These results contribute to our fundamental understanding of the complex ligand-cofactor-receptor interactions.

Weaknesses:

This is not the weakness of this particular paper, but the general limitation in using simplified models to study a complex system.

Reviewer #1 (Public review):

Summary:

This manuscript by Harris and Gallistel investigates how the rate of learning and strength of conditioned behavior post learning depend on the various temporal parameters of Pavlovian conditioning. They replicate results from Gibbon and Balsam (1981) in rats to show that the rate of learning is proportional to the ratio between the cycle duration and the cue duration. They further show that the strength of conditioned behavior post learning is proportional to the cue duration, and not the above ratio. The overall findings here are interesting, provide context to many conflicting recent results on this topic, and are supported by reasonably strong evidence. Nevertheless, there are some major weaknesses in the evidence presented for some of the stronger claims in the manuscript.

Strengths:

This manuscript has many strengths including a rigorous experimental design, several different approaches to data analysis, careful consideration of prior literature, and a thorough introduction and discussion. The central claim-that animals track the rates of events in their environment, and that the ratio of two rates determine the rate of learning-is supported with solid evidence.

Weaknesses:

Despite the above major strengths, some key aspects of the paper need major improvement. These are listed below.

(1) A key claim made here is that the same relationship (including the same parameter) describes data from pigeons by Gibbon and Balsam (1981) and the rats in this study. I think the evidence for this claim is weak as presented here. First, the exact measure used for identifying trials to criterion makes a big difference in Fig 3. As best as I understand, the authors do not make any claims about which of these approaches is the "best" way. Second, the measure used for identifying trials to criterion in Fig 1 appears different from any of the criteria used in Fig 3. If so, to make the claim that the quantitative relationship is one and the same in both datasets, the authors need to use the same measure of learning rate on both datasets and show that the resultant plots are statistically indistinguishable. Currently, the authors simply plot the dots from the current dataset on the plot in Fig 1 and ask the readers to notice the visual similarity. This is not at all enough to claim that both relationships are the same. In addition to the dependence of the numbers on the exact measure of learning rate used, the plots are in log-log axis. Slight visual changes can mean a big difference in actual numbers. For instance, between Fig 3 B and C, the highest information group moves up only "slightly" on the y-axis but the difference is a factor of 5. The authors need to perform much more rigorous quantification to make the strong claim that the quantitative relationships obtained here and in Gibbon and Balsam 1981 are identical.

(2) Another interesting claim here is that the rates of responding during ITI and the cue are proportional to the corresponding reward rates with the same proportionality constant. This too requires more quantification and conceptual explanation. For quantification, it would be more convincing to calculate the regression slope for the ITI data and the cue data separately and then show that the corresponding slopes are not statistically distinguishable from each other. Conceptually, I am confused why the data used to the test the ITI proportionality come from the last 5 sessions. Specifically, if the model is that animals produce response rates during the ITI (a period with no possible rewards) based on the overall rate of rewards in the context, wouldn't it be better to test this before the cue learning has occurred? Before cue learning, the animals would presumably only have attributed rewards in the context to the context and thus, produce overall response rates in proportion to the contextual reward rate. After cue learning, the animals could technically know that the rate of rewards during ITI is zero. Why wouldn't it be better to test the plotted relationship for ITI before cue learning has occurred? Further, based on Fig 1, it seems that the overall ITI response rate reduces considerably with cue learning. What is the expected ITI response rate prior to learning based on the authors' conceptual model? Why does this rate differ pre and post cue learning? Finally, if the authors' conceptual framework predicts that ITI response rate after cue learning should be proportional to contextual reward rate, why should the cue response rate be proportional to cue reward rate instead of cue reward rate plus contextual reward rate?

(3) I think there was a major conceptual disconnect between the gradual nature of learning shown in Figs 7 and 8 and the information theoretic model proposed by the authors. To the extent that I understand the model, the animals should simply learn the association once the evidence crosses a threshold (nDKL > threshold) and then produce behavior in proportion to the expected reward rate. If so, why should there be a gradual component of learning as shown in these figures? In terms of the proportional response rule to rate of rewards, why is it changing as animals go from 10% to 90% of peak response? I think the manuscript would be much strengthened if these results are explained within the authors' conceptual framework. If these results are not anticipated by the authors' conceptual framework, please do explicitly state this in the manuscript.

(4) I find the idea stated in the Conclusion section that any model considering probability of reinforcement cannot be correct because it doesn't have temporal units to be weak. I think the authors might mean that existing models based on probability do not work and not that no possible model can work. For any point process, the standard mathematical treatment of continuous time is to compute the expected count of events as p*dt where p is the probability of occurrence of the event in that time bin and dt is an infinitesimal time bin. There is obviously a one-to-one mapping between probability of an event in a point process and its rate. Existing models use an arbitrary time bin/trial and thus, I get the authors' argument in the discussion. However, I think their conclusion is overstated.

(5) The discussion states that the mutual information defined in equation 1 does not change during partial reinforcement. I am confused by this. The mean delay between reinforcements increases in inverse proportion to the probability of reinforcement, but doesn't the mean delay between cue and next reinforcement increase by more than this amount (next reinforcement is greater than or equal to the cue-to-cue interval away from the cue for many trials)? Why is this ratio invariant to partial reinforcement?

Comments on revisions:

Update following revision

(1) This point is discussed in more detail in the attached file, but there are some important details regarding the identification of the learned trial that require more clarification. For instance, isn't the original criterion by Gibbon et al. (1977) the first "sequence of three out of four trials in a row with at least one response"? The authors' provided code for the Wilcoxon signed rank test and nDkl thresholds looks for a permanent exceeding of the threshold. So, I am not yet convinced that the approaches used here and in prior papers are directly comparable. Also, there's still no regression line fitted to their data (Fig 3's black line is from Fig 1, according to the legends). Accordingly, I think the claim in the second paragraph of the Discussion that the old data and their data are explained by a model with "essentially the same parameter value" is not yet convincing without actually reporting the parameters of the regression. Related to this, the regression for their data based on my analysis appears to have a slope closer to -0.6, which does not support strict timescale invariance. I think that this point should be discussed as a caveat in the manuscript.

(2) The authors report in the response that the basis for the apparent gradual/multiple step-like increases after initial learning remains unclear within their framework. This would be important to point out in the actual manuscript. Further, the responses indicating the fact that there are some phenomena that are not captured by the current model would be important to state in the manuscript itself.

(3) There are several mismatches between results shown in figures and those produced by the authors' code, or other supplementary files. As one example, rat 3 results in Fig 11 and Supplementary Materials don't match and neither version is reproduced by the authors' code. There are more concerns like this, which are detailed in the attached review file.

Reviewer #2 (Public review):

A long-standing debate in the field of Pavlovian learning relates to the phenomenon of timescale invariance in learning i.e. that the rate at which an animal learns about a Pavlovian CS is driven by the relative rate of reinforcement of the cue (CS) to the background rate of reinforcement. In practice, if a CS is reinforced on every trial, then the rate of acquisition is determined by the relative duration of the CS (T) and the ITI (C = inter-US-interval = duration of CS + ITI), specifically the ratio of C/T. Therefore, the point of acquisition should be the same with a 10s CS and a 90s ITI (T = 10; C = 90 + 10 = 100, C/T = 100/10 = 10) and with a 100s CS and a 900s ITI (T = 100; C = 900 + 100 = 1000, C/T = 1000/100 = 10). That is to say, the rate of acquisition is invariant to the absolute timescale as long as this ratio is the same. This idea has many other consequences, but is also notably different from more popular prediction-error based associative learning models such as the Rescrola-Wagner model. The initial demonstrations that the ratio C/T predicts the point of acquisition across a wide range of parameters (both within and across multiple studies) was conducted in Pigeons using a Pavlovian autoshaping procedure. What has remained under contention is whether or not this relationship holds across species, particularly in the standard appetitive Pavlovian conditioning paradigms used in rodents. The results from rodent studies aimed at testing this have been mixed, and often the debate around the source of these inconsistent results focuses on the different statistical methods used to identify the point of acquisition for the highly variable trial-by-trial responses at the level of individual animals.<br /> The authors successfully replicate same effect found in pigeon autoshaping paradigms decades ago (with almost identical model parameters) in a standard Pavlovian appetitive paradigm in rats. They achieve this through a clever change the experimental design, using a convincingly wide range of parameters across 14 groups of rats, and by a thorough and meticulous analysis of these data. It is also interesting to note that the two author's have published on opposing sides of this debate for many years, and as a result have developed and refined many of the ideas in this manuscript through this process.

Main findings

(1) The present findings demonstrate that the point of initial acquisition of responding is predicted by the C/T ratio.

(2) The terminal rates of responding to the CS appears to be related to the reinforcement rate of the CS (T; specifically, 1/T) but not its relation to the reinforcement rate of the context (i.e. C or C/T). In the present experiment, all CS trials were reinforced so it is also the case that the terminal rate of responding was related to the duration of the CS.

(3) An unexpected finding was that responding during the ITI was similarly related to the rate of contextual reinforcement (1/C). This novel finding suggests that the terminal rate of responding during the ITI and the CS are related to their corresponding rates of reinforcement. This finding is surprising as it suggests that responding during the ITI is not being driven by the probability of reinforcement during the ITI.

(4) Finally, the authors characterised the nature of increased responding from the point of initial acquisition until responding peaks at a maximum. Their analyses suggest that nature of this increase was best described as linear in the majority of rats, as opposed to the non-linear increase that might be predicted by prediction error learning models (e.g. Rescorla-Wagner). However, more detailed analyses revealed that these changes can be quite variable across rats, and more variable when the CS had lower informativeness (defined as C/T).

Strengths and Weaknesses:

There is an inherent paradox regarding the consistency of the acquisition data from Gibbon & Balsam's (1981) meta-analysis of autoshaping in pigeons, and the present results in magazine response frequency in rats. This consistency is remarkable and impressive, and is suggestive of a relatively conserved or similar underlying learning principle. However, the consistency is also surprising given some significant differences in how these experiments were run. Some of these differences might reasonably be expected to lead to differences in how these different species respond. For example:

- The autoshaping procedure commonly used in the pigeons from these data were pretrained to retrieve rewards from a grain hopper with an instrumental contingency between head entry into the hopper and grain availability. During Pavlovian training, pecking the key light also elicited an auditory click feedback stimulus, and when the grain hopper was made available the hopper was also illuminated.

- In the present experimental procedure, the rats were not given contextual exposure to the pellet reinforcers in the magazine (e.g. a magazine training session is typically found in similar rodent procedures). The Pavlovian CS was a cue light within the magazine itself.

These design features in the present rodent experiment are clearly intentional. Pretraining with the reinforcer in the testing chambers would reasonably alter the background rate of reinforcement (parameter), so it make sense not to include this but differs from the paradigm used in pigeons. Having the CS inside the magazine where pellets are delivered provides an effective way to reduce any potential response competition between CS and US directed responding and combines these all into the same physical response. This makes the magazine approach response more like the pecking of the light stimulus in the pigeon autoshaping paradigm. However, the location of the CS and US is separated in pigeon autoshaping, raising questions about why the findings across species are consistent despite these differences.

Intriguingly, when the insertion of a lever is used as a Pavlovian cue in rodent studies, CS directed responding (sign-tracking) often develops over training such that eventually all animals bias their responding towards the lever than towards the US (goal-tracking at the magazine). However, the nature of this shift highlights the important point that these CS and US directed responses can be quite distinct physically as well as psychologically. Therefore, by conflating the development of these different forms of responding, it is not clear whether the relationship between C/T and the acquisition of responding describes the sum of all Pavlovian responding or predominantly CS or US directed responding.

Another interesting aspect of these findings is that there is a large amount of variability that scales inversely with C/T. A potential account of the source of this variability is related to the absence of preexposure to the reward pellets. This is normally done within the animals' homecage as a form of preexposure to reduce neophobia. If some rats take longer to notice and then approach and finally consume the reward pellets in the magazine, the impact of this would systematically differ depending on the length of the ITI. For animals presented with relatively short CSs and ITIs, they may essentially miss the first couple of trials and/or attribute uneaten pellets accumulating in the magazine to the background/contextual rate of reinforcement. What is not currently clear is whether this was accounted for in some way by confirming when the rats first started retrieving and consuming the rewards from the magazine.

While the generality of these findings across species is impressive, the very specific set of parameters employed to generate these data raise questions about the generality of these findings across other standard Pavlovian conditioning parameters. While this is obviously beyond the scope of the present experiment, it is important to consider that the present study explored a situation with 100% reinforcement on every trial, with a variable duration CS (drawn form a uniform distribution), with a single relatively brief CS (maximum of 122s) CS and a single US. Again, the choice of these parameters in the present experiment is appropriate and very deliberately based on refinements from many previous studies from the authors. This includes a number of criteria used to define magazine response frequency that includes discarding specific responses (discussed and reasonably justified clearly in the methods section). Similarly, the finding that terminal rates of responding are reliably related to 1/T is surprising, and it is not clear whether this might be a property specific to this form of variable duration CS, the use of a uniform sampling distribution, or the use of only a single CS. However, it is important to keeps these limitations in mind when considering some of the claims made in the discussion section of this manuscript that go beyond what these data can support.

The main finding demonstrating the consistent findings across species is presented in Figure 3. In the analysis of these data, it is not clear why the correlations between C, T, and C/T and the measure of acquisition in Figure 3A were presented as r values, whereas the r2 values were presented in the discussion of Figure 3B, and no values were provided in discussing Figure 3C. The measure of acquisition in Figure 3A is based on a previously established metric, whereas the measure in Figure 3B employs the relatively novel nDKL measure that is argued to be a better and theoretically based metric. Surprisingly, when r and r2 values are converted to the same metric across analyses, it appears that this new metric (Figure 3B) does well but not as well as the approach in Figure 3A. This raises questions about why a theoretically derived measure might not be performing as well on this analysis, and whether the more effective measure is either more reliable or tapping into some aspect of the processes that underlie acquisition that is not accounted for by the nDKL metric. Unfortunately, the new metric is discussed and defined at great length but its utility is not considered.<br /> An important analysis issue that is unclear in the present manuscript is exactly how the statistics were run (how the model was defined, were individual subjects or group medians used, what software was used etc...). For example, it is not clear whether the analyses conducted in relation to Figure 3 used the data from individual rats or the group medians. Similarly, it appears that each rat contributes four separate data points, and a single regression line was fit to all these data despite the highly likely violation of the assumption independent observations (or more precisely, the assumption of uncorrelated errors) in this analysis. Furthermore, it is claimed that the same regression line fit the IT and CS period data in this figure, however this<br /> If the data in figure 3 were analyzed with log(ITI) or log(C/ITI) i.e. log(C/(T-C)), would this be a better fit for these data? Is it the case that the ratio of C/T the best predictor of the trial/point of acquisition, or is it the case that another metric related to reinforcement rates provides a better fit?

Based on the variables provided in Supplementary file 3, containing the acquisition data, I was unable to reproduce the values reported in the analysis of Figure 3.<br /> In relation to Figure 3: I am curious about whether the authors would be able to comment on whether the individual variability in trials to acquisition would be expected to scale differently based on C/T, or C, or (if a less restricted range was used) T?<br /> It is not clear why Figure 3C is presented but not analyzed, and why the data presented in Figure 4 to clarify the spread of the distribution of the data observed across the plots in Figure 3 uses the data from Figure 3C. This would seem like the least representative data to illustrate the point of Figure 4. It also appears to my eye that the data actually plotted in Figure 4 correspond to Figure 3A and 3B rather than the odds 10:1 data indicated in text.

What was the decision criteria used to decide on averaging the final 5 conditioning sessions as terminal responding for the analyses in Figure 5? This is an oddly specific number. Was this based on consistency with previous work, or based on the greatest number of sessions where stable data for all animals could be extracted?<br /> In the analysis corresponding to Figures 7-8: If I understand the description of this analysis correctly, for each rat the data are the cumulative response data during the CS, starting from the trial on which responding to the CS > ITI (t = 1), and ending at the trial on which CS responding peaked (maximum over 3 session moving average window; t = end). This analysis does not seem to account for changes (decline) in the ITI response rates over this period of acquisition, and it is likely that responding during the ITI is still declining after t=1. Are the 4 functions that were fit to these data to discriminate between different underlying generative processes still appropriate on total CS responding instead of conditional CS responding after accounting for changes in baseline response rates during ITI?

Page 27, Procedure, final sentence: The magazine responding during the ITI is defined as the 20s period immediately before CS onset. The range of ITI values (Table 1) always starts as low as 15s in all 14 groups. Even in the case of an ITI on a trial that was exactly 20s, this would also mean that the start of this period overlaps with the termination of the CS from the previous trial and delivery (and presumably consumption) of a pellet. Please indicate if the definition of the ITI period was modified on trials where the preceding ITI was <20s, and if any other criteria were used to define the ITI.

Were the rats exposed to the reinforcers/pellets in their home cage prior to acquisition? Please indicate whether rats where pre-exposed to the reward pellets in their home cages e.g. as is often done to reduce neophobia. Given the deliberate absence of a magazine-training phase, this information is important when assessing the experienced contingency between the CS and the US.

For all the analyses, please provide the exact models that were fit and the software used. For example, it is not necessarily clear to the reader (particularly in the absence of degrees of freedom) that the model fits discussed in Figure 3 are fit on the individual subject data points or the group medians. Similarly, in Figure 6 there is no indication of whether a single regression model was fit to all the plotted data or whether tests of different slopes for each of the conditions were compared. With regards to the statistics in Figure 6, depending on how this was run, it is also a potential problem that the analyses does not correct for the potentially highly correlated multiple measurements from the same subjects i.e. each rat provides 4 data points which are very likely not to be independent observations.

A number of sections of the discussion are speculative or not directly supported by the present experimental data (but may well be supported by previous findings that are not the direct focus of the present experiment). For example, Page 19, Paragraph 2: this entire paragraph is not really clearly explained and is presenting an opinion rather than a strong conclusion that follows directly from the present findings. Evidence for an aspect of RET in the present paper (i.e. the prediction of time scale invariance on the initial point of acquisition, but not necessarily the findings regarding the rate of terminal acquisition) - while supportive - does not necessarily provide unconditional evidence for this theory over all the alternatives.

Similarly, the Conclusion section (Page 23) makes the claim that "the equations have at most one free parameter", which may be an oversimplification that is conditionally true in the narrow context of the present experiment where many things were kept constant between groups and run in a particular way to ensure this is the case. While the equations do well in this narrow case, it is unlikely that additional parameters would not need to be added to account for more general learning situations. To clarify, I am not contending that this kind of statement is necessarily untrue, merely that it is being presented in a narrow context and may require a deeper discussion of much more of the literature to qualify/support properly - and the discussion section of the present experiment/manuscript may not be the appropriate place for this.

- Consider taking advantage of an "Ideas and Speculation" subsection within the Discussion that is supported by eLife [ https://elifesciences.org/inside-elife/e3e52a93/elife-latest-including-ideas-and-speculation-in-elife-papers ]. This might be more appropriate to qualify the tone of much of the discussion from page 19 onwards.

It seems like there are entire analyses and new figures being presented in the discussion e.g. Page 20: Information-Theoretic Contingency. These sections might be better placed in the methods section or a supplementary section/discussion.

Reviewer #1 (Public review):

Summary:

Systemic and partial Tcf7l2 repression is effective in protecting cancer mice from cachexia-induced death. Hence, this is a promising treatment strategy for cancer patients suffering from cachexia.

Strengths:

The method is well-designed and clearly explained.

Weaknesses:

(1) Abbreviations should be mentioned in full terms for the first time.

(2) Relatively old or even very old references in the Introduction and Discussion.

(3) The result section contains discussion with references, as well.

(4) The number of mice in individual groups is relatively small (3 mice in some groups).

Reviewer #2 (Public review):

Summary:

This study by Leong and colleagues examines the role of the TCF7L2 transcription factor in the Wnt signaling pathway as a regulator of colon/small intestinal cancers and cachexia. Investigators utilize a Tet off repressor genetic system in mice under Dox regulation to silence TCF7L2. Results show DSS-treated APCMin/+ mice lose body weight that can be rapidly rescued by Dox treatment and suppression of TCF7L2 expression. Reduction of TCF7L2 rescues features of cachexia, including body weight, gastrocnemius muscle and adipose mass, as well as molecular markers of cachexia such as the E3 Ub ligases, MuRF1, and Atrogin-1. The most significant finding in the study is that loss of TCF7L2 reduces but does not eliminate tumor progression, as tumors go from adenomas to adenocarcinomas over time while mice are treated with Dox, yet cachexia persists. This implies that TCF7L2 has a direct effect on cachexia. Overall, the study provides insight into the role of TCFL2 in the development of intestinal cancers and muscle atrophy in cachexia.

Strengths:

The study uses an elegant genetic mouse model to provide significant new insight into the role of TCFL2 in colon and small intestinal cancers. In addition, the authors describe the role of TCF7L2 as a regulator of muscle wasting in cachexia. This, too, can be viewed as a new finding for the cachexia field.

Weaknesses:

However, in its current form, the study lacks sufficient data to support the authors' claim regarding the relevance of TCF7L2 as a regulator of cachexia.

Reviewer #1 (Public review):

This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

Reviewer #2 (Public review):

Summary:

The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

Major strengths and weaknesses of the methods and results:

The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

Reviewer #3 (Public review):

Summary:

Here, Guo et al. (2025) propose that the IFN-induced GTPase GVIN1 forms a coat on cytosolic Burkholderia thailandensis, blocking actin tail formation through a mechanism analogous to GBP1-mediated restriction of Shigella motility.

Their study was prompted by the intriguing observation that IFNγ priming and GBP1 coat formation fail to inhibit B. thailandensis actin-based motility in HeLa cells, yet IFNγ restricts the motility of Burkholderia in T24 cells. Further investigation revealed that IFNγ restricts B. thailandensis motility in T24 cells via both GBP1-dependent and -independent mechanisms, suggesting that HeLa cells lack a critical GBP1 co-factor required to inhibit actin tail formation.

To identify the GBP1-independent mechanism, the authors performed an siRNA screen of interferon-stimulated genes (ISGs) and identified GVIN1, a large IFN-induced GTPase, as essential for restricting B. thailandensis motility. To identify the GBP1-independent mechanism, perform a knock-down screen for ISGs and find that the loss of GVIN, a very large IFN-induced GTPase, results in higher actin tail-positive B. thailandensis in T24 cells. They further demonstrate that GVIN forms coats on the surface of B. thailandensis, which prevent the polar localization of BimA and thus actin tail formation. In summary, the data reveal two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent, each relying on distinct host co-factors.

Global assessment:

This is a well-executed study that convincingly demonstrates how GVIN1 restricts the actin-based motility of B. thailandensis through the assembly of coatomers. The results are clearly described, the manuscript is easy to follow, and the data are overall compelling and well presented. I have only a few suggestions on how the manuscript could be further improved.

Reviewer #1 (Public review):

Summary:

This manuscript describes a novel magnetic steering technique to target human adipose derived mesenchymal stem cells (hAMSC) or induce pluripotent stem cells to the TM (iPSC-TM). The authors show delivery of the stem cells lowered IOP, increased ouflow facility, and increased TM cellularity.

Strengths:

The technique is novel and shows promise as a novel therapeutic to lower in IOP in glaucoma. hAMSC are able to lower IOP below baseline as well as increase outflow facility above baseline with no tumorigenicity. These data will have a positive impact on the field and will guide further research using hAMSC in glaucoma models.

Weaknesses:

The transgenic mouse model of glaucoma the authors used did not show ocular hypertensive phenotypes as previously reported; therefore, the Tg-MYOCY437H model should be used with caution in the future. However, the results presented here clearly show magnetically steered cell therapy as a viable treatment strategy to lower intraocular pressure even from baseline. Future studies are needed to demonstrate the effects in ocular hypertensive eyes.

Reviewer #2 (Public review):

This observational study investigates the efficacy of intracameral injected human stems cells as a means to re-functionalize the trabecular meshwork for the restoration of intraocular pressure homeostasis. Using a murine model of glaucoma, human adipose-derived mesenchymal stem cells are shown to be biologically safer and functionally superior at eliciting a sustained reduction in intraocular pressure (IOP). The authors conclude that the use of magnetically-steered human adipose-derived mesenchymal stem cells has potential for long-term treatment of ocular hypertension in glaucoma.

Comments on revisions: Previously noted concerns have been thoughtfully and sincerely considered by the authors and are now clearly addressed in the revised manuscript. No further concerns/comments.

Reviewer #1 (Public review):

The authors attempted to replicate previous work showing that counterconditioning leads to more persistent reduction of threat responses, relative to extinction. They also aimed to examine the neural mechanisms underlying counterconditioning and extinction. They achieved both of these aims, and were able to provide some additional information, such as how counterconditioning impacts memory consolidation. Having a better understanding of which neural networks are engaged during counterconditioning may provide novel pharmacological targets to aid in therapies for traumatic memories. It will be interesting to follow up by examining the impact of varying amounts of time between acquisition and counterconditioning phases, to enhance replicability to real world therapeutic settings.

Major strengths

• This paper is very well written and attempts to comprehensively assess multiple aspects counterconditioning and extinction processes. For instance, the addition of memory retrieval tests is not core to the primary hypotheses, but provides additional mechanistic information on how episodic memory is impacted by counterconditioning. This methodical approach is commonly seen in animal literature, but less so in human studies.

• The Group x Cs-type x Phase repeated measure statistical tests with 'differentials' as outcome variables are quite complex, however the authors have generally done a good job of teasing out significant F test findings with post hoc tests and presenting the data well visually. It is reassuring that there is convergence between self-report data on arousal and valence and the pupil dilation response. Skin conductance is a notoriously challenging modality, so it is not too concerning that this was placed in the supplementary materials. Neural responses also occurred in logical regions with regards to reward learning.

• Strong methodology with regards to neuroimaging analysis, and physiological measures.

• The authors are very clear on documenting where there were discrepancies from their pre-registration and providing valid rationales for why.

Major Weaknesses

• The statistics showing that counterconditioning prevents differential spontaneous recovery are the weakest p values of the paper (and using one tailed tests, although this is valid due to directions being pre-hypothesised). This may be due to relatively small number of participants and some variability in responses.

Reviewer #2 (Public review):

Summary:

The present study sets out to examine the impact of counterconditioning (CC) and extinction on conditioned threat responses in humans, particularly looking at neural mechanisms involved in threat memory suppression. By combining behavioral, physiological, and neuroimaging (fMRI) data, the authors aim to provide a clear picture of how CC might engage unique neural circuits and coding dynamics, potentially offering a more robust reduction in threat responses compared to traditional extinction.

Strengths:

One major strength of this work lies in its thoughtful and unique design - integrating subjective, physiological, and neuroimaging measures to capture the variouse aspects of counterconditiong (CC) in humans. Additionally, the study is centered on a well-motivated hypothesis and the findings have potentials for improving the current understanding of pathways associated with emotional and cognitive control.

The data presentation is systematic, and the results on behavioral and physiological measures fit well with the hypothesized outcomes. The neuroimaging results also provide strong support for distinct neural mechanisms underlying CC versus extinction.

Weaknesses:

Overall, this study is a well-conducted and thought-provoking investigation into counterconditioning, with strong potential to advance our understanding of threat modulation mechanisms. Two minor weaknesses concern the scope and decisions regarding analysis choices. First, while the findings are solid, the topic of counterconditioning is relatively niche and may have limited appeal to a broader audience. Expanding the discussion to connect counterconditioning more explicitly to widely studied frameworks in emotional regulation or cognitive control would enhance the paper's accessibility and relevance to a wider range of readers. This broader framing could also underscore the generalizability and broader significance of the results. In addition, detailed steps in the statistical procedures and analysis parameters seem to be missing. This makes it challenging for readers to interpret the results in light of potential limitations given the data modality and/or analysis choices.

Comments on revisions: My previous concerns and questions have been sufficiently addressed.

Reviewer #3 (Public review):

In this manuscript, Wirz et al use neuroimaging (fMRI) to show that counterconditioning produces a longer lasting reduction in fear conditioning relative to extinction and appears to rely on the nucleus accumbens rather than the ventromedial prefrontal cortex. These important findings are supported by convincing evidence and will be of interest to researchers across multiple subfields, including neuroscientists, cognitive theory researchers, and clinicians.

In large part, the authors achieved their aims of giving a qualitative assessment of the behavioural mechanisms of counterconditioning versus extinction, as well as investigating the brain mechanisms. The results support their conclusions and give interesting insights into the psychological and neurobiological mechanisms of the processes that underlie the unlearning, or counteracting, of threat conditioning.

Strengths:

* Clearly written with interesting psychological insights<br /> * Excellent behavioural design, well-controlled and tests for a number of different psychological phenomena (e.g. extinction, recovery, reinstatement, etc).<br /> * Very interesting results regarding the neural mechanisms of each process.<br /> * Good acknowledgement of the limitations of the study.

Weaknesses:

* I am not sure that the memories tested were truly episodic<br /> * Twice as many female participants than males

Comments on revisions: I have no remaining concerns

Reviewer #1 (Public review):

Summary:

Audio et al. present an interesting study examining cerebral blood volume (CBV) across cortical areas and layers in non-human primates (NHPs) using high-resolution MRI. While with contrast agents are frequently employed to improve fMRI sensitivity in NHP research, its application for characterizing baseline CBV distribution is less common. This study quantifies large-vessel distribution as well as regional and laminar CBV variations, comparing them with other metrics.

Strengths:

(1) Noninvasive mapping of relative cerebral blood volume is novel for non-human primates.<br /> (2) A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV.<br /> (3) The measured relative CBV values correlated with previously reported neuronal and receptor densities, potentially providing valuable physiological insights.

Weaknesses:

(1) A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels is not fully validated, especially on a laminar scale.<br /> (2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels. However, this approach depends on multiple factors, including spatial resolution, contrast agent dosage, and data processing methods. This raises concerns about the generalizability of these findings to other experimental setups or populations.<br /> (3) Baseline R2* is sensitive to baseline R2, vascular volume, iron content, and susceptibility gradients. Additionally, it is sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Although baseline R2* correlates with several physiological parameters, drawing direct physiological inferences from it remains challenging.<br /> (4) CBV-weighted deltaR2*, which depends on both CBV and contrast agent dose, correlates with various metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While such correlations may be useful for exploratory analyses, all comparisons depend on measurement accuracy. A fundamental question remains whether CBV-weighted ΔR2* can provide reliable and biologically meaningful insights into these metrics, particularly in diseased or abnormal brain states.

Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based, measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with known distribution of different types of neurons, markers of metabolic load and others. While the presented methodology captures and estimated 30% of the vasculature, the authors corroborated previous findings regarding lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non-invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to captures the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work, begins by characterizing the spacing of penetrating arteries and ascending veins using vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 is computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to other brain areas is therefore not possible without further exploration with independent methodologies.

Please note that these are comments on the revised version.

Reviewer #1 (Public review):

Summary:

This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.

Strengths:

This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.

Reviewer #2 (Public review):

Summary:

The authors generated three mouse lines harboring ASD, Schizophrenia, and Bi-polar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bi-polar disorder development.

Strengths:

The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bi-polar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was carefully examined using an impressive host of methods. The authors achieved their goal of identifying behavioral, physiological, and molecular alterations that are disorder/variant specific. The impact of this work is extremely high given the growing appreciation of TRIO dysfunction in a large number of brain-related disorders. This work is very interesting in that it begins to identify the unique and subtle ways brain function is altered in ASD, Schizophrenia, and Bi-polar disorder.

Weaknesses:

(1) Most assays were performed in older animals and perhaps only capture alterations that result from homeostatic changes resulting from prodromal pathology that may look very different.

(2) Identification of upregulated (potentially compensating) genes in response to these disorder specific Trio variants is extremely interesting. However, a functional demonstration of compensation is not provided.

(3) There are instances where data is not shown in the manuscript. See "data not shown". All data collected should be provided even if significant differences are not observed.

I consider weaknesses 1 and 2 minor. While they would very interesting to explore, these experiments might be more appropriate for a follow up study. The missing data in 3 should be provided in the supplemental material.

Revised Manuscript:

All of my above concerns were well addressed by the authors in the revised submission.

Reviewer #2 (Public review):

Summary:

This manuscript is about using different analytical approaches to allow ancestry adjustments to GWAS analyses amongst admixed populations. This work is a follow-on from the recently published ITHGC multi-population GWAS (https://doi.org/10.7554/eLife.84394), with the focus on the admixed South African populations. Ancestry adjustment models detected a peak of SNPs in the class II HLA DPB1, distinct from the class II HLA DQA1 loci signficant in the ITHGC analysis.

Strengths:

Excellent demonstration of GWAS analytical pipelines in highly admixed populations. Particularly the utility of ancestry adjustment to improve study power to detect novel associations. Further confirmation of the importance of the HLA class II locus in genetic susceptibility to TB.

Weaknesses:

Limited novelty compared to the group's previous existing publications and the body of work linking HLA class II alleles with TB susceptibility in South Africa or other African populations. This work includes only ~100 new cases and controls from what has already been published. High resolution HLA typing has detected significant signals in both the DQA1 and DPB1 regions identified by the larger ITHGC and in this GWAS analysis respectively (Chihab L et al. HLA. 2023 Feb; 101(2): 124-137).<br /> Despite the availability of strong methods for imputing HLA from GWAS data (Karnes J et Plos One 2017), the authors did not confirm with HLA typing the importance of their SNP peak in the class II region. This would have supported the importance of this ancestry adjustment versus prior ITHGC analysis.

The populations consider active TB and healthy controls (from high-burden presumed exposed communities) and do not provide QFT or other data to identify latent TB infection.

Important methodological points for clarification and for readers to be aware of when reading this paper:

(1) One of the reasons cited for the lack of African ancestry-specific associations or suggestive peaks in the ITHGC study was the small African sample size. The current association test includes a larger African cohort and yields a near-genome-wide significant threshold in the HLA-DPB1 gene originating from the KhoeSan ancestry. Investigation is needed as to whether the increase in power is due to increased African samples and not necessarily the use of the LAAA model as stated on lines 295 and 296?

Authors response - The Manhattan plot in Figure 3 includes the results for all four models: the traditional GWAS model (GAO), the admixture mapping model (LAO), the ancestry plus allelic (APA) model and the LAAA model. In this figure, it is evident that only the LAAA model identified the association peak on chromosome 6, which lends support the argument that the increase in power is due to the use of the LAAA model and not solely due to the increase in sample size.<br /> Reviewer comment - This data supports the authors conclusions that increase power is related to the LAAA model application rather than simply increase sample size.

(2) In line 256, the number of SNPs included in the LAAA analysis was 784,557 autosomal markers; the number of SNPs after quality control of the imputed dataset was 7,510,051 SNPs (line 142). It is not clear how or why ~90% of the SNPs were removed. This needs clarification.

Authors response:<br /> In our manuscript (line 194), we mention that "...variants with minor allele frequency (MAF) < 1% were removed to improve the stability of the association tests." A large proportion of imputed variants fell below this MAF threshold and were subsequently excluded from this analysis.

Reviewers additional comment: The authors should specify the number of SNPs in the dataset before imputation and indicate what proportion of the 784,557 remaining SNPs were imputed. Providing this information might help the reader better understand the rationale behind the imputation process.

(3) The authors have used the significance threshold estimated by the STEAM p-value < 2.5x10-6 in the LAAA analysis. Grinde et al. (2019 implemented their significance threshold estimation approach tailored to admixture mapping (local ancestry (LA) model), where there is a reduction in testing burden. The authors should justify why this threshold would apply to the LAAA model (a joint genotype and ancestry approach).

Authors response: We describe in the methods (line 189 onwards) that the LAAA model is an extension of the APA model. Since the APA model itself simultaneously performs the null global ancestry only model and the local ancestry model (utilised in admixture mapping), we thus considered the use of a threshold tailored to admixture mapping appropriate for the LAAA model.

Reviewers additional comment: While the LAAA model is an extension of the APA model, the authors describe the LAAA test as 'models the combination of the minor allele and the ancestry of the minor allele at a specific locus, along with the effect of this interaction,' thus a joint allele and ancestry effects model. Grinde et al. (2019) proposed the significance threshold estimation approach, STEAM, specifically for the LA approach, which tests for ancestry effects alone and benefits from the reduced testing burden. However, it remains unclear why the authors found it appropriate to apply STEAM to the LAAA model, a joint test for both allele and ancestry effects, which does not benefit from the same reduction in testing burden.

(4) Batch effect screening and correction (line 174) is a quality control check. This section is discussed after global and local ancestry inferences in the methods. Was this QC step conducted after the inferencing? If so, the authors should justify how the removed SNPs due to the batch effect did not affect the global and local ancestry inferences or should order the methods section correctly to avoid confusion.

Authors response: The batch effect correction method utilised a pseudo-case-control comparison which included global ancestry proportions. Thus, batch effect correction was conducted after ancestry inference. We excluded 36 627 SNPs that were believed to have been affected by the batch effect. We have amended line 186 to include the exact number of SNPs excluded due to batch effect.<br /> The ancestry inference by RFMix utilised the entire merged dataset of 7 510 051 SNPs. Thus, the SNPs removed due to the batch effect make up a very small proportion of the SNPs used to conduct global and local ancestry inferences (less than 0.5%). As a result, we do not believe that the removed SNPs would have significantly affected the global and local ancestry inferences. However, we did conduct global ancestry inference with RFMix on each separate dataset as a sanity check. In the tables below, we show the average global ancestry proportions inferred for each separate dataset, the average global ancestry proportions across all datasets and the average global ancestry proportions inferred using the merged dataset. The SAC and Xhosa cohorts are shown in two separate tables due to the different number of contributing ancestral populations to each cohort. The differences between the combined average global ancestry proportions across the separate cohorts does not differ significantly to the global ancestry proportions inferred using the merged dataset.

This is an excellent response and should remain accessible to readers for clarifying this issue.

Comments on revisions:

Thank you for addressing my other recommendations to authors. These have all been satisfactorily addressed.

Reviewer #1 (Public review):

Summary:

This study reveals that TRPV1 signaling plays a key role in tympanic membrane (TM) healing by promoting macrophage recruitment and angiogenesis. Using a mouse TM perforation model, researchers found that blood-derived macrophages accumulated near the wound, driving angiogenesis and repair. TRPV1-expressing nerve fibers triggered neuroinflammatory responses, facilitating macrophage recruitment. Genetic Trpv1 mutation reduced macrophage infiltration, angiogenesis, and delayed healing. These findings suggest that targeting TRPV1 or stimulating sensory nerve fibers could enhance TM repair, improve blood flow, and prevent infections. This offers new therapeutic strategies for TM perforations and otitis media in clinical settings. This is an excellent and high-quality study that provides valuable insights into the mechanisms underlying TM wound healing.

Strengths:

The work is particularly important for elucidating the cellular and molecular processes involved in TM repair. However, there are several concerns about the current version.

Weaknesses:

Major concerns

(1) The method of administration will be a critical factor when considering potential therapeutic strategies to promote TM healing. It would be beneficial if the authors could discuss possible delivery methods, such as topical application, transtympanic injection, or systemic administration, and their respective advantages and limitations for targeting TRPV1 signaling. For example, Dr. Kanemaru and his colleagues have proposed the use of Trafermin and Spongel to regenerate the eardrum.

(2) The authors appear to have used surface imaging techniques to observe the TM. However, the TM consists of three distinct layers: the epithelial layer, the fibrous middle layer, and the inner mucosal layer. The authors should clarify whether the proposed mechanism involving TRPV1-mediated macrophage recruitment and angiogenesis is limited to the epithelial layer or if it extends to the deeper layers of the TM.

Minor concerns

In Figure 8, the schematic illustration presents a coronal section of the TM. However, based on the data provided in the manuscript, it is unclear whether the authors directly obtained coronal images in their study. To enhance the clarity and impact of the schematic, it would be helpful to include representative images of coronal sections showing macrophage infiltration, angiogenesis, and nerve fiber distribution in the TM.

Reviewer #2 (Public review):

Summary:

This study examines the role of TRPV1 signaling in the recruitment of monocyte-derived macrophages and the promotion of angiogenesis during tympanic membrane (TM) wound healing. The authors use a combination of genetic mouse models, macrophage depletion, and transcriptomic approaches to suggest that neuronal TRPV1 activity contributes to macrophage-driven vascular responses necessary for tissue repair.

Strengths:

(1) The topic of neuroimmune interactions in tissue regeneration is of interest and underexplored in the context of the TM, which presents a unique model due to its anatomical features.

(2) The use of reporter mice and bone marrow chimeras allows for some dissection of immune cell origin.

(3) The authors incorporate transcriptomic data to contextualize inflammatory and angiogenic processes during wound healing.

Weaknesses:

(1) The primary claims of the manuscript are not convincingly supported by the evidence presented. Most of the data are correlative in nature, and no direct mechanistic experiments are included to establish causality between TRPV1 signaling and macrophage recruitment or function.

(2) Functional validation of key molecular players (such as Tac1 or Spp1) is lacking, and their roles are inferred primarily from gene expression data rather than experimentally tested.

(3) The reuse of publicly available scRNA-seq data is not sufficiently integrated or extended to yield new biological insights, and it remains largely descriptive.

(4) The macrophage depletion model (CX3CR1CreER; iDTR) lacks specificity, and possible off-target or systemic effects are not addressed.

(5) Several interpretations of the data appear overstated, particularly regarding the necessity of TRPV1 for monocyte recruitment and wound healing.

(6) Overall, the study appears to apply known concepts - namely, TRPV1-mediated neurogenic inflammation and macrophage-driven angiogenesis - to a new anatomical site without providing new mechanistic insight or advancing the field substantially.

Overall:

While the study addresses an interesting topic, the current version does not provide sufficiently strong or novel evidence to support its major conclusions. Additional mechanistic experiments and more rigorous validation would be necessary to substantiate the proposed model and clarify the relevance of the findings beyond this specific tissue context.

Reviewer #1 (Public review):

Summary:

This paper presents results from four independent experiments, each of which tests for rhythmicity in auditory perception. The authors report rhythmic fluctuations in discrimination performance at frequencies between 2 and 6 Hz. The exact frequency depends on the ear and experimental paradigm, although some frequencies seem to be more common than others.

Strengths:

The first sentence in the abstract describes the state of the art perfectly: "Numerous studies advocate for a rhythmic mode of perception; however, the evidence in the context of auditory perception remains inconsistent". This is precisely why the data from the present study is so valuable. This is probably the study with the highest sample size (total of > 100 in 4 experiments) in the field. The analysis is very thorough and transparent, due to the comparison of several statistical approaches and simulations of their sensitivity. Each of the experiments differs from the others in a clearly defined experimental parameter, and the authors test how this impacts auditory rhythmicity, measured in pitch discrimination performance (accuracy, sensitivity, bias) of a target presented at various delays after noise onset.

Weaknesses:

(1) The authors find that the frequency of auditory perception changes between experiments. I think they could exploit differences between experiments better to interpret and understand the obtained results. These differences are very well described in the Introduction, but don't seem to be used for the interpretation of results. For instance, what does it mean if perceptual frequency changes from between- to within-trial pitch discrimination? Why did the authors choose this experimental manipulation? Based on differences between experiments, is there any systematic pattern in the results that allows conclusions about the roles of different frequencies? I think the Discussion would benefit from an extension to cover this aspect.

(2) The Results give the impression of clear-cut differences in relevant frequencies between experiments (e.g., 2 Hz in Experiment 1, 6 Hz in Exp 2, etc), but they might not be so different. For instance, a 6 Hz effect is also visible in Experiment 1, but it just does not reach conventional significance. The average across the three experiments is therefore very useful, and also seems to suggest that differences between experiments are not very pronounced (otherwise the average would not produce clear peaks in the spectrum). I suggest making this point clearer in the text.

(3) I struggle to understand the hypothesis that rhythmic sampling differs between ears. In most everyday scenarios, the same sounds arrive at both ears, and the time difference between the two is too small to play a role for the frequencies tested. If both ears operate at different frequencies, the effects of the rhythm on overall perception would then often cancel out. But if this is the case, why would the two ears have different rhythms to begin with? This could be described in more detail.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures. The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions.

Weaknesses:

(1) Conceptional concerns:

The authors place their research in the context of a rhythmic mode of perception. They also discuss continuous vs rhythmic mode processing. Their study further follows a design that seems to be based on paradigms that assume a recent phase in neural oscillations that subsequently influence perception (e.g., Fiebelkorn et al.; Landau & Fries). In my view, these are different facets in the neural oscillation research space that require a bit more nuanced separation. Continuous mode processing is associated with vigilance tasks (work by Schroeder and Lakatos; reduction of low frequency oscillations and sustained gamma activity), whereas the authors of this study seem to link it to hearing tasks specifically (e.g., line 694). Rhythmic mode processing is associated with rhythmic stimulation by which neural oscillations entrain and influence perception (also, Schroeder and Lakatos; greater low-frequency fluctuations and more rhythmic gamma activity). The current study mirrors the continuous rather than the rhythmic mode (i.e., there was no rhythmic stimulation), but even the former seems not fully fitting, because trials are 1.8 s short and do not really reflect a vigilance task. Finally, previous paradigms on phase-resetting reflect more closely the design of the current study (i.e., different times of a target stimulus relative to the reset of an oscillation). This is the work by Fiebelkorn et al., Landau & Fries, and others, which do not seem to be cited here, which I find surprising. Moreover, the authors would want to discuss the role of the background noise in resetting the phase of an oscillation, and the role of the fixation cross also possibly resetting the phase of an oscillation. Regardless, the conceptional mixture of all these facets makes interpretations really challenging. The phase-reset nature of the paradigm is not (or not well) explained, and the discussion mixes the different concepts and approaches. I recommend that the authors frame their work more clearly in the context of these different concepts (affecting large portions of the manuscript).

(2) Methodological concerns:

The authors use a relatively unorthodox approach to statistical testing. I understand that they try to capture and characterize the sensitivity of the different analysis approaches to rhythmic behavioral effects. However, it is a bit unclear what meaningful effects are in the study. For example, the bootstrapping approach that identifies the percentage of significant variations of sample selections is rather descriptive (Figures 5-7). The authors seem to suggest that 50% of the samples are meaningful (given the dashed line in the figure), even though this is rarely reached in any of the analyses. Perhaps >80% of samples should show a significant effect to be meaningful (at least to my subjective mind). To me, the low percentage rather suggests that there is not too much meaningful rhythmicity present. I suggest that the authors also present more traditional, perhaps multi-level, analyses: Calculation of spectra, binning, or single-trial analysis for each participant and condition, and the respective calculation of the surrogate data analysis, and then comparison of the surrogate data to the original data on the second (participant) level using t-tests. I also thought the statistical approach undertaken here could have been a bit more clearly/didactically described as well.

The authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. In practice, this can be a disadvantage relative to keeping the intensity constant throughout, because, on average, correct trials will be associated with a higher intensity than incorrect trials, potentially making observations of perceptual rhythmicity more challenging. The authors would want to discuss this potential issue. Intensity adjustments could perhaps contribute to the observed rhythmicity effects. Perhaps the rhythmicity of the stimulus intensity could be analyzed as well. In any case, the adaptive procedure may add variance to the data.

Additional methodological concerns relate to Figure 8. Figures 8A and C seem to indicate that a baseline correction for a very short time window was calculated (I could not find anything about this in the methods section). The data seem very variable and artificially constrained in the baseline time window. It was unclear what the reader might take from Figure 8.

Motivation and discussion of eye-movement/pupillometry and motor activity: The dual task paradigm of Experiment 4 and the reasons for assessing eye metrics in the current study could have been better motivated. The experiment somehow does not fit in very well. There is recent evidence that eye movements decrease during effortful tasks (e.g., Contadini-Wright et al. 2023 J Neurosci; Herrmann & Ryan 2024 J Cog Neurosci), which appears to contradict the results presented in the current study. Moreover, by appealing to active sensing frameworks, the authors suggest that active movements can facilitate listening outcomes (line 677; they should provide a reference for this claim), but it is unclear how this would relate to eye movements. Certainly, a person may move their head closer to a sound source in the presence of competing sound to increase the signal-to-noise ratio, but this is not really the active movements that are measured here. A more detailed discussion may be important. The authors further frame the difference between Experiments 1 and 2 as being related to participants' motor activity. However, there are other factors that could explain differences between experiments. Self-paced trials give participants the opportunity to rest more (inter-trial durations were likely longer in Experiment 2), perhaps affecting attentional engagement. I think a more nuanced discussion may be warranted.

Discussion:

The main data in Figure 3 showed little rhythmicity. The authors seem to glance over this fact by simply stating that the same phase is not necessary for their statistical analysis. Previous work, however, showed rhythmicity in the across-participant average (e.g., Fiebelkorn's and similar work). Moreover, one would expect that some of the effects in the low-frequency band (e.g., 2-4 Hz) are somewhat similar across participants. Conduction delays in the auditory system are much smaller than the 0.25-0.5 s associated with 2-4 Hz. The authors would want to discuss why different participants would express so vastly different phases that the across-participant average does not show any rhythmicity, and what this would mean neurophysiologically.

An additional point that may require more nuanced discussion is related to the rhythmicity of response bias versus sensitivity. The authors could discuss what the rhythmicity of these different measures in different frequency bands means, with respect to underlying neural oscillations.

Figures:

Much of the text in the figures seems really small. Perhaps the authors would want to ensure it is readable even for those with low vision abilities. Moreover, Figure 1A is not as intuitive as it could be and may perhaps be made clearer. I also suggest the authors discuss a bit more the potential monoaural vs binaural issues, because the perceptual rhythmicity is much slower than any conduction delays in the auditory system that could lead to interference.

Reviewer #3 (Public review):

Summary:

The finding of rhythmic activity in the brain has, for a long time, engendered the theory of rhythmic modes of perception, that humans might oscillate between improved and worse perception depending on states of our internal systems. However, experiments looking for such modes have resulted in conflicting findings, particularly in those where the stimulus itself is not rhythmic. This paper seeks to take a comprehensive look at the effect and various experimental parameters which might generate these competing findings: in particular, the presentation of the stimulus to one ear or the other, the relevance of motor involvement, attentional demands, and memory: each of which are revealed to effect the consistency of this rhythmicity.

The need the paper attempts to resolve is a critical one for the field. However, as presented, I remain unconvinced that the data would not be better interpreted as showing no consistent rhythmic mode effect. It lacks a conceptual framework to understand why effects might be consistent in each ear but at different frequencies and only for some tasks with slight variants, some affecting sensitivity and some affecting bias.

Strengths:

The paper is strong in its experimental protocol and its comprehensive analysis, which seeks to compare effects across several analysis types and slight experiment changes to investigate which parameters could affect the presence or absence of an effect of rhythmicity. The prescribed nature of its hypotheses and its manner of setting out to test them is very clear, which allows for a straightforward assessment of its results

Weaknesses:

There is a weakness throughout the paper in terms of establishing a conceptual framework both for the source of "rhythmic modes" and for the interpretation of the results. Before understanding the data on this matter, it would be useful to discuss why one would posit such a theory to begin with. From a perceptual side, rhythmic modes of processing in the absence of rhythmic stimuli would not appear to provide any benefit to processing. From a biological or homeostatic argument, it's unclear why we would expect such fluctuations to occur in such a narrow-band way when neither the stimulus nor the neurobiological circuits require it.

Secondly, for the analysis to detect a "rhythmic mode", it must assume that the phase of fluctuations across an experiment (i.e., whether fluctuations are in an up-state or down-state at onset) is constant at stimulus onset, whereas most oscillations do not have such a total phase-reset as a result of input. Therefore, some theoretical positing of what kind of mechanism could generate this fluctuation is critical toward understanding whether the analysis is well-suited to the studied mechanism.

Thirdly, an interpretation of why we should expect left and right ears to have distinct frequency ranges of fluctuations is required. There are a large number of statistical tests in this paper, and it's not clear how multiple comparisons are controlled for, apart from experiment 4 (which specifies B&H false discovery rate). As such, one critical method to identify whether the results are not the result of noise or sample-specific biases is the plausibility of the finding. On its face, maintaining distinct frequencies of perception in each ear does not fit an obvious conceptual framework.

Reviewer #1 (Public review):

Summary:

The study by Cao et al. provides a compelling investigation into the role of mutational input in the rapid evolution of pesticide resistance, focusing on the two-spotted spider mite's response to the recent introduction of the acaricide cyetpyrafen. This well-documented introduction of the pesticide - and thus a clearly defined history of selection - offers a powerful framework for studying the temporal dynamics of rapid adaptation. The authors combine resistance phenotyping across multiple populations, extensive resequencing to track the frequency of resistance alleles, and genomic analyses of selection in both contemporary and historical samples. These approaches are further complemented by laboratory-based experimental evolution, which serves as a baseline for understanding the genetic architecture of resistance across mite populations in China. Their analyses identify two key resistance-associated genes, sdhB and sdhD, within which they detect 15 mutations in wild-collected samples. Protein modeling reveals that these mutations cluster around the pesticide's binding site, suggesting a direct functional role in resistance. The authors further examine signatures of selective sweeps and their distribution across populations to infer the mechanisms - such as de novo mutation or gene flow-driving the spread of resistance, a crucial consideration for predicting evolutionary responses to extreme selection pressure. Overall, this is a well-rounded, thoughtfully designed, and well-written manuscript. It shows significant novelty, as it is relatively rare to integrate broad-scale evolutionary inference from natural populations with experimentally informed bioassays, however, some aspects of the methods and discussion have an opportunity to be clarified and strengthened.

Strengths:

One of the most compelling aspects of this study is its integration of genomic time-series data in natural populations with controlled experimental evolution. By coupling genome sequencing of resistant field populations with laboratory selection experiments, the authors tease apart the individual effects of resistance alleles along with regions of the genome where selection is expected to occur, and compare that to the observed frequency in the wild populations over space and time. Their temporal data clearly demonstrates the pace at which evolution can occur in response to extreme selection. This type of approach is a powerful roadmap for the rest of the field of rapid adaptation.

The study effectively links specific genetic changes to resistance phenotypes. The identification of sdhB and sdhD mutations as major drivers of cyetpyrafen resistance is well-supported by allele frequency shifts in both field and experimental populations. The scope of their sampling clearly facilitated the remarkable number of observed mutations within these target genes, and the authors provide a careful discussion of the likelihood of these mutations from de novo or standing variation. Furthermore, the discovered cross-resistance that these mutations confer to other mitochondrial complex II inhibitors highlights the potential for broader resistance management and evolution.

Weaknesses:

(1) Experimental Evolution:

- Additional information about the lab experimental evolution would be useful in the main text. Specifically, the dose of cyetpyrafen used should be clarified, especially with respect to the LD50 values. How does it compare to recommended field doses? This is expected to influence the architecture of resistance evolution. What was the sample size? This will help readers contextualize how the experimental design could influence the role of standing variation.

- The finding that lab-evolved strains show cross-resistance is interesting, but potentially complicates the story. It would help to know more about the other mitochondrial complex II inhibitors used across China and their impact on adaptive dynamics at these loci, particularly regarding pre-existing resistance alleles. For example, a comparison of usage data from 2013, 2017, and 2019 could help explain whether cyetpyrafen was the main driver of resistance or if previous pesticides played a role. What happened in 2020 that caused such rapid evolution 3 years after launch?

(2) Evolutionary history of resistance alleles:

- It would be beneficial to examine the population structure of the sampled populations, especially regarding the role of migration. Though resistance evolution appears to have had minimal impact on genome-wide diversity (as shown in Supplementary Figure 2), could admixture be influencing the results? An explicit multivariate regression framework could help to understand factors influencing diversity across populations, as right now much is left to the readers' visual acuity.

- It is unclear why lab populations were included in the migration/treemix analysis. We might suggest redoing the analysis without including the laboratory populations to reveal biologically plausible patterns of resistance evolution.

- Can the authors explore isolation by distance (IBD) in the frequency of resistance alleles?

- Given the claim regarding the novelty of the number of pesticide resistance mutations, it is important to acknowledge the evolution of resistance to all pesticides (antibiotics, herbicides, etc.). ALS-inhibiting herbicides have driven remarkable repeatability across species based on numerous SNPs within the target gene.

- Figure 5 A-B. Why not run a multivariate regression with status at each resistance mutation encoded as a separate predictor? It is interesting that focusing on the predominant mutation gives the strongest r2, but it is somewhat unintuitive and masks some interesting variation among populations.

(3) Haplotype Reconstruction (Line 271-):

- We are a bit sceptical of the methods taken to reconstruct these haplotypes. It seems as though the authors did so with Sanger sequencing (this should be mentioned in the text), focusing only on homozygous SNPs. How many such SNPs were used to reconstruct haplotypes, along what length of sequence? For how many individuals were haplotypes reconstructed? Nonetheless, I appreciated that the authors looked into the extent to which the reconstructed haplotypes could be driven by recombination. Can the authors elaborate on the calculations in line 296? Is that the census population size estimate or effective?

(4) Single Mutations and Their Effect (line 312-):

- It's not entirely clear how the breeding scheme resulted in near-isogenic lines. Could the authors provide a clearer explanation of the process and its biological implications?

- If they are indeed isogenic, it's interesting that individual resistance mutations have effects on resistance that vary considerably among lines. Could the authors run a multivariate analysis including all potential resistance SNPs to account for interactions between them? Given the variable effects of the D116G substitution (ranging from 4-25%), could polygenic or epistatic factors be influencing the evolution of resistance?

- Why are there some populations that segregate for resistance mutations but have no survival to pesticides (i.e., the green points in Figure 5)? Some discussion of this heterogeneity seems required in the absence of validation of the effects of these particular mutations. Could it be dominance playing a role, or do the authors have some other explanation?

- The authors mention that all resistance mutations co-localized to the Q-site. Is this where the pesticide binds? This seems like an important point to follow their argument for these being resistance-related.

(5) Statistical Considerations for Allele Frequency Changes (Figure 3):

- It might be helpful to use a logistic regression model to assess the rate of allele frequency changes and determine the strength of selection acting on these alleles (e.g., Kreiner et al. 2022; Patel et al. 2024). This approach could refine the interpretation of selection dynamics over time.

Reviewer #2 (Public review):

Summary:

This paper investigates the evolution of pesticide resistance in the two-spotted spider mite following the introduction of an SDHI acaricide, cyatpyrafen, in China. The authors make use of cyatpyrafen-naive populations collected before that pesticide was first used, as well as more recent populations (both sensitive and resistant) to conduct comparative population genomics. They report 15 different mutations in the insecticide target site from resistant populations, many reported here for the first time, and look at the mutation and selection processes underlying the evolution of resistance, through GWAS, haplotype mapping, and testing for loss of diversity indicating selective sweeps. None of the target site mutations found in resistant populations was found in pre-exposure populations, suggesting that the mutations may have arisen de novo rather than being present as standing variation, unless initially present at very low frequencies; a de novo origin is also supported by evidence of selective sweeps in some resistant populations. Furthermore, there is no significant evidence of migration of resistant genotypes between the sampled field populations, indicating multiple origins of common mutations. Overall, this indicates a very high mutation rate and a wide range of mutational pathways to resistance for this target site in this pest species. The series of population genomic analyses carried out here, in addition to the evolutionary processes that appear to underlie resistance development in this case, could have implications for the study of resistance evolution more widely.

Strengths:

This paper combines phenotypic characterisation with extensive comparative population genomics, made possible by the availability of multiple population samples (each with hundreds of individuals) collected before as well as after the introduction of the pesticide cyatpyrafen, as well as lab-evolved lines. This results in findings of mutation and selection processes that can be related back to the pesticide resistance trait of concern. Large numbers of mites were tested phenotypically to show the levels of resistance present, and the authors also made near-isogenic lines to confirm the phenotypic effects of key mutations. The population genomic analyses consider a range of alternative hypotheses, including mutations arising by de novo mutation or selection from standing genetic variation, and mutations in different populations arising independently or arriving by migration. The claim that mutations most likley arose by multiple repeated de novo mutations is therefore supported by multiple lines of evidence: the direct evidence of none of the mutations being found in over 2000 individuals from naive populations, and the indirect evidence from population genomics showing evidence of selective sweeps but not of significant migration between the sampled populations.

Weaknesses:

As acknowledged within the discussion, whilst evidence supports a de novo origin of the resistance-associated mutations, this cannot be proven definitively as mutations may have been present at a very low frequency and therefore not found within the tested pesticide-naive population samples.

Near-isofemale lines were made to confirm the resistance levels associated with five of the 15 mutations, but otherwise, the genotype-phenotype associations are correlative, as confirmation by functional genetics was beyond the scope of this study.

Reviewer #1 (Public review):

Summary:

Felipe and colleagues try to answer an important question in Sarbecovirus Orf9b-mediated interferon signaling suppression, given that this small viral protein adopts two distinct conformations, a dimeric β-sheet-rich fold and a helix-rich monomeric fold when bound by Tom70 protein. Two Orf9b structures determined by X-ray crystallography and Cryo-EM suggest an equilibrium between the two Orf9b conformations, and it is important to understand how this equilibrium relates to its functions. To answer these questions, the authors developed a series of ordinary differential equations (ODE) describing the Orf9b conformation equilibrium between homodimers and monomers binding to Tom70. They used SPR and a fluorescent polarization (FP) peptide displacement assay to identify parameters for the equilibrium and create a theoretical model. They then used the model to characterize the effect of lipid-binding and the effects of Orf9b mutations in homodimer stability, lipid binding, and dimer-monomer equilibrium. They used their model to further analyze dimerization, lipid binding, and Orf9b-Tom70 interactions for truncated Orf9b, Orf9b fusion mutant S53E (blocking Tom70 binding), and Orf9b from a set of Sars-CoV-2 VOCs. They evaluated the ability of different Orf9b variants for binding Tom70 using Co-IP experiments and assessed their activity in suppressing IFN signaling in cells.

Overall, this work is well designed, the results are of high quality and well-presented; the results support their conclusions.

Strengths:

(1) They developed a working biophysical model for analyzing Orf9b monomer-dimer equilibrium and Tom70 binding based on SPR and FP experiments; this is an important tool for future investigation.

(2) They prepared lipid-free Orf9b homodimer and determined its crystal structure.

(3) They designed and purified obligate Orf9b monomer, fused-dimer, etc., a very important Orf9b variant for further investigations.

(4) They identified the lipid bound by Orf9b homodimer using mass spectra data.

(5) They proposed a working model of Orf9b-Tom70 equilibrium.

Weaknesses:

(1) It is difficult to understand why the obligate Orf9b dimer has similar IFN inhibition activity as the WT protein and obligate Orf9b monomer truncations.

(2) The role of Orf9b homodimer and the role of Orf9b-bound lipid in virus infection, remains unknown.

Reviewer #2 (Public review):

Summary:

This study focuses on Orf9b, a SARS-COV1/2 protein that regulates innate signaling through interaction with Tom70. San Felipe et al use a combination of biophysical methods to characterize the coupling between lipid-binding, dimerization, conformational change, and protein-protein-interaction equilibria for the Orf9b-Tom70 system. Their analysis provides a detailed explanation for previous observations of Orf9b function. In a cellular context, they find other factors may also be important for the biological functioning of Orf9b.

Strengths:

San Felipe et al elegantly combine structural biology, biophysics, kinetic modelling, and cellular assays, allowing detailed analysis of the Orf9b-Tom70 system. Such complex systems involving coupled equilibria are prevalent in various aspects of biology, and a quantitative description of them, while challenging, provides a detailed understanding and prediction of biological outcomes. Using SPR to guide initial estimates of the rate constants for solution measurements is an interesting approach.

Weaknesses:

This study would benefit from a more quantitative description of uncertainties in the numerous rate constants of the models, either through a detailed presentation of the sensitivity analysis or another approach such as MCMC. Quantitative uncertainty analysis, such as MCMC is not trivial for ODEs, particularly when they involve many parameters and are to be fitted to numerous data points, as is the case for this study. The authors use sensitivity analysis as an alternative, however, the results of the sensitivity analysis are not presented in detail, and I believe the authors should consider whether there is a way to present this analysis more quantitatively. For example, could the residuals for each +/-10% parameter change for the peptide model be presented as a supplementary figure, and similarly for the more complex models? Further details of the range of rate constants tested would be useful, particularly for the ka and kB parameters.

The authors build a model that incorporates an α-helix-β-sheet conformational change, but the rate constant for the conversion to the α-helix conformation is required to be second order. Although the authors provide some rationale, I do not find this satisfactorily convincing given the large number of adjustable parameters in the model and the use of manual model fitting. The authors should discuss whether there is any precedence for second-order rate constants for conformational changes in the literature. On page 14, the authors state this rate constant "had to be non-linear in the monomer β-sheet concentration" - how many other models did the authors explore? For example, would αT↔α↔αα↔ββ (i.e., conformational change before dimer dissociation) or α↔βαT↔ββ (i.e., Tom70 binding driving dimer dissociation) be other plausible models for the conformational change that do not require assumptions of second-order rate constants for the conformational change?

Overall, this study progresses the analysis of coupled equilibria and provides insights into Orf9b function.

Reviewer #1 (Public review):

Summary:

In this study, Meunier et al. investigated the functional role of IL-10 in avian mucosal immunity. While the anti-inflammatory role of IL-10 is well established in mammals, and several confirmatory knockout models are available in mice, IL-10's role in avian mucosal immunity is so far correlative. In this study, the authors generated two different models of IL-10 ablation in Chickens. A whole body knock-out model and an enhancer KO model leading to reduced IL10 expression. The authors first performed in vitro LPS stimulation-based experiments, and then in vivo two different infection models employing C. jejuni and E. tenella, to demonstrate that complete ablation of IL10 leads to enhanced inflammation-related pathology and gene expression, and enhanced pathogen clearance. At a steady-state level, however, IL-10 ablation did not lead to spontaneous colitis.

Strengths:

Overall, the study is well executed and establishes an anti-inflammatory role of IL-10 in birds. While the results are expected and not surprising, this appears to be the first report to conclusively demonstrate IL-10's anti-inflammatory role upon its genetic ablation in the avian model. Provided this information is applicable in combating pathogen infection in livestock species in sustainable industries like poultry, the study will be of interest to the field.

Weaknesses:

The study is primarily a confirmation of the already established anti-inflammatory role of IL-10.

Reviewer #2 (Public review):

Summary:

The authors were to investigate the functional role of IL10 on mucosal immunity in chickens. CRISPR technology was employed to generate IL10 knock-out chickens in both exon and putative enhancer regions. IL10 expressions were either abolished (knockout in exon) or reduced (enhancer knock-out). IL-10 plays an important role in the composition of the caecal microbiome. Through various enteric pathogen challenges, deficient IL10 expression was associated with enhanced pathogen clearance, but with more severe lesion scores and body weight loss.

Strengths:

Both in vitro and in vivo knock-out abolished and reduced IL10 expression, and broad enteric pathogens were challenged in vivo, and various parameters were examined to evaluate the functional role of IL10 on mucosal immunity.

Weaknesses:

Overexpression of IL-10 either in vitro or in vivo may further support the findings from this study.

Reviewer #1 (Public review):

Summary:

By applying a laser scanning photostimulation (LSPS) approach to a novel slice preparation, the authors aimed to study the degree of convergence and divergence of cortical inputs to individual striatal projection neurons (SPNs).

Strengths:

The experiments were well-designed and conducted, and data analysis was thorough. The manuscript was well written, and related work in the literature was properly discussed. This work has the potential to advance our understanding of how sensory inputs are integrated into the striatal circuits.

Weaknesses:

This work focuses on the connection strength of the corticostriatal projections, without considering the involvement of synaptic plasticity in sensory integration.

Reviewer #2 (Public review):

Summary:

How corticostriatal synaptic connectivity gives rise to SPN encoding of sensory information is an important and currently unanswered question. The authors utilize a clever slice preparation in combination with electrophysiology and glutamate uncaging to dissect the synaptic connectivity between barrel cortex and individual striatal SPNs. In addition to mapping connectivity across major anatomical axes and cortical layers, the authors provide data showing that SPNs uniquely integrate sparse input from variable stretches across barrel cortex.

Strengths:

The methodology shows impressive rigor, and the data robustly support the authors' conclusions. Overall, the manuscript addresses its core question, provides valuable insights into corticostriatal architecture, and is a welcome addition to the field.

Weaknesses:

A few minor changes to the figures and text could be made to improve clarity.

Reviewer #3 (Public review):

Summary:

The authors explored how individual dorsolateral striatum (DLS) spiny projection neurons (SPNs) receive functional input from whisker-related cortical columns. The authors developed and validated a novel slice preparation and method to which they applied rigorous functional mapping and thorough analysis. They found that individual SPNs were driven by sparse, scattered cortical clusters. Interestingly, while the cortical input fields of nearby SPNs had some degree of overlap, connectivity per SPN was largely distinct. Despite sparse, heterogeneous connectivity, topographical organization was identified. The authors lastly compared direct (D1) vs. indirect (D2) pathway cells, concluding that overall connectivity patterns were the same, but D1 cells received stronger input from L6 and D2 cells from L2/3. The paper thoughtfully addresses the question of whether barrel cortex broadly or selectively innervates SPNs. Their results indicate selective input that is loosely topographic. Their work deepens the understanding of how whisker-related somatosensory signals can drive striatal neurons.

Strengths:

Overall, this is a carefully conducted study, and the major claims are well-supported. The use of a novel ex vivo slice prep that keeps relevant corticostriatal projections intact allows for careful mapping of the barrel cortex to dorsolateral striatum SPNs. Careful reporting of both columnar and layer position, as well as postsynaptic SPN type (D1 or D2), allows the authors to uncover novel details about how the dorsolateral striatum represents whisker-related sensory information.

Weaknesses:

(1) Several factors may contribute to an underestimation of barrel cortex inputs to SPNs (and thus an overestimate of the input heterogeneity among SPNs). First, by virtue of the experiments being performed in an acute slice prep, it is probable that portions of recorded SPN dendritic trees have been dissected (in an operationally consistent anatomical orientation). If afferents happen to systematically target the rostral/caudal projections of SPN dendritic fields, these inputs could be missed. Similarly, the dendritic locations of presynaptic cortical inputs remain unknown (e.g., do some inputs preferentially target distal vs proximal dendritic positions?). As synaptic connectivity was inferred from somatic recordings, it's likely that inputs targeting the proximal dendritic arbor are the ones most efficiently detected. Mapping the dendritic organization of synapses is beyond the scope of this work, but these points could be broached in the text.

(2) In general, how specific (or generalizable) is the observed SPN-specific convergence of cortical barrel cortex projections in the dorsolateral striatum? In other words, does a similar cortical stimulation protocol targeted to a non-barrel sensory (or motor) cortex region produce similar SPN-specific innervation patterns in the dorsolateral striatum?

(3) In general, some of the figure legends are extremely brief, making many details difficult to infer. Similarly, some statistical analyses were either not carried out or not consistently reported.

Joint Public Review:

Following up on their previous work, the authors investigated whether HIV-1 cell-to-cell transmission activates the CARD8 inflammasome in macrophages, a key question given that inflammasome activation in myeloid cells triggers proinflammatory cytokine release. Co-cultures of HIV-infected T cells with macrophages led to viral spreading, resulting in IL1β release and cell death, with CARD8 playing a crucial role in this inflammasome response, triggered by HIV protease. The authors also found that HIV isolates resistant to protease inhibitors showed differences in CARD8 activation and IL1β production, highlighting the clinical relevance of their findings. Overall, this well-written study provides strong evidence for the role of CARD8 in protease-dependent sensing of viral spread, with implications for understanding chronic inflammation in HIV infections and its potential contribution to systemic immune activation, especially under ART. The authors have addressed initial weaknesses and verified effects in cocultures of primary T cells and macrophages. They now also provide evidence that CARD8 is activated by protease from incoming viral particles. Further studies are needed to clarify how much this mechanism contributes to systemic immune activation in untreated infections and whether this mechanism drives chronic inflammation under ART.

Reviewer #1 (Public review):

Summary:

Gruskin and colleagues use twin data from a movie-watching fMRI paradigm to show how genetic control of cortical function intersects with the processing of naturalistic audiovisual stimuli. They use hyperalignment to dissect heritability into the components that can be explained by local differences in cortical-functional topography and those that cannot. They show that heritability is strongest at slower-evolving neural time scales and is more evident in functional connectivity estimates than in response time series.

Strengths:

This is a very thorough paper that tackles this question from several different angles. I very much appreciate the use of hyperalignment to factor out topographic differences, and I found the relationship between heritability and neural time scales very interesting. The writing is clear, and the results are compelling.

Weaknesses:

The only "weaknesses" I identified were some points where I think the methods, interpretation, or visualization could be clarified.

(1) On page 16, the authors compare heritability in functional connectivity (FC) and response time series, and find that the heritability effect is larger in FC. In general, I agree with your diagnosis that this is in large part due to the fact that FC captures the covariance structure across parcels, whereas response time series only diverge in terms of univariate time-point-by-time-point differences. Another important factor here is that (within-subject) FC can be driven by intrinsic fluctuations that occur with idiosyncratic timing across subjects and are unrelated to the stimulus (whereas time-locked metrics like ISC and time-series differences cannot, by definition). This makes me wonder how this connectivity result would change if the authors used intersubject functional connectivity (ISFC) analysis to specifically isolate the stimulus-driven components of functional connectivity (Simony et al., 2016). This, to me, would provide a closer comparison to the ISC and response time series results, and could allow the authors to quantify how much of the heritability in FC is intrinsic versus stimulus-driven. I'm not asking that the authors actually perform this analysis, as I don't think it's critical for the message of the manuscript, but it could be an interesting future direction. As the authors discuss on page 17, I also suspect there's something fundamentally shared between response time series and connectivity as they relate to functional topography (Busch et al., 2021) that drives part of the heritability effect.

(2) The observation that regions with intermediate ISC have the largest differences between MZ, DZ, and UR is very interesting, but it's kind of hard to see in Figure 1B. Is there any other way to plot this that might make the effect more obvious? For example, I could imagine three scatter plots where the x- and y-axes are, e.g., MZ ISC and UR ISC, and each data point is a parcel. In this kind of plot, I would expect to see the middle values lifted visibly off the diagonal/unity line toward MZ. The authors could even color the data points according to networks, like in Figure 3C. (They also might not need to scale the ISC axis all the way to r = 1, which would make the differences more visible.)

(3) On page 9, if I understand correctly, the authors regress the vector of ISC values across parcels out of the vector of heritability values across parcels, and then plot the residual heritability values. Do they center the heritability values (or include some kind of intercept) in the process? I'm trying to understand why the heritability values go from all positive (Figure 2A) to roughly balanced between positive and negative (Figure 2B). Important question for me: How should we interpret negative values in this plot? Can the authors explain this explicitly in the text? (I also wonder if there's a more intuitive way to control for ISC. For example, instead of regressing out ISC at the parcel/map level, could they go into a single parcel and then regress the subject-level pairwise ISC values out when computing the heritability score?).

(4) On page 4 (line 155), the authors say "we shuffled dyad labels"- is this equivalent to shuffling rows and columns of the pairwise subject-by-subject matrix combined across groups? I'm trying to make sure their approach here is consistent with recommendations by Chen et al., 2016. Is this the same kind of shuffling used for the kinship matrix mentioned in line 189?

(5) I found panel A in Figure 4 to be a little bit misleading because their parcel-wise approach to hyperalignment won't actually resolve topographic idiosyncrasies across a large cortical distance like what's depicted in the illustration (at the scale of the parcels they are performing hyperalignment within). Maybe just move the green and purple brain areas a bit closer to each other so they could feasibly be "aligned" within a large parcel. Worth keeping in mind when writing that hyperalignment is also not actually going to yield a one-to-one mapping of functionally homologous voxels across individuals: it's effectively going to model any given voxel time series as a linear combination of time series across other voxels in the parcel.

(6) I believe the subjects watched all different movies across the two days, however, for a moment I was wondering "are Day 1 and Day 2 repetitions of the same movies?" Given that Day 1 and Day 2 are an organizational feature of several figures, it might be worth making this very explicit in the Methods and reminding the reader in the Results section.

References:

Busch, E. L., Slipski, L., Feilong, M., Guntupalli, J. S., di Oleggio Castello, M. V., Huckins, J. F., Nastase, S. A., Gobbini, M. I., Wager, T. D., & Haxby, J. V. (2021). Hybrid hyperalignment: a single high-dimensional model of shared information embedded in cortical patterns of response and functional connectivity. NeuroImage, 233, 117975. https://doi.org/10.1016/j.neuroimage.2021.117975

Chen, G., Shin, Y. W., Taylor, P. A., Glen, D. R., Reynolds, R. C., Israel, R. B., & Cox, R. W. (2016). Untangling the relatedness among correlations, part I: nonparametric approaches to inter-subject correlation analysis at the group level. NeuroImage, 142, 248-259. https://doi.org/10.1016/j.neuroimage.2016.05.023

Simony, E., Honey, C. J., Chen, J., Lositsky, O., Yeshurun, Y., Wiesel, A., & Hasson, U. (2016). Dynamic reconfiguration of the default mode network during narrative comprehension. Nature Communications, 7, 12141. https://doi.org/10.1038/ncomms12141

Reviewer #2 (Public review):

Summary:

The authors attempt to estimate the heritability of brain activity evoked from a naturalistic fMRI paradigm. No new data were collected; the authors analyzed the publicly available and well-known data from the Human Connectome Project. The paper has 3 main pieces, as described in the Abstract:

(1) Heritability of movie-evoked brain activity and connectivity patterns across the cortex.

(2) Decomposition of this heritability into genetic similarity in "where" vs. "how" sensory information is processed.

(3) Heritability of brain activity patterns, as partially explained by the heritability of neural timescales.

Strengths:

The authors investigate a very relevant topic that concerns how heritable patterns of brain activity among individuals subjected to the same kind of naturalistic stimulation are. Notably, the authors complement their analysis of movie-watching data with resting-state data.

Weaknesses:

The paper has numerous problems, most of which stem from the statistical analyses. I also note the lack of mapping between the subsections within the Methods section and the subsections within the Results section. We can only assess results after understanding and confirming the methods are valid; here, however, Methods and Results, as written, are not aligned, so we can't always be sure which results are coming from which analysis.

(A) Intersubject correlation (ISC) (section that starts from line 143): "We used non-parametric permutation testing to quantify average differences in ISC for each parcel in the Schaefer 400 atlas for each day of data collection across three groups: MZ dyads, DZ dyads, and unrelated (UR) dyads, where all UR dyads were matched for gender and age in years." ... "some participants contributed to ISC values for multiple dyads (thus violating independence assumptions)"

This is an indirect attempt to demonstrate heritability. And it's also incorrect since, as the authors themselves point out, some subjects contribute to more than one dyad.

Permutation tests don't quantify "average differences", they provide a measure of evidence about whether differences observed are sufficient to reject a hypothesis of no difference.

Matching subjects is also incorrect as it artificially alters the sample; covarying for age and sex, as done in standard analyses of heritability, would have been appropriate.

It isn't clear why the authors went through the trouble of implementing their own non-parametric test if HCP recommends using PALM, which already contains the validated and documented methods for permutation tests developed precisely for HCP data.

The results from this analysis, in their current form, are likely incorrect.

(B) Functional connectivity (FC) (section that starts from line 159): Here the authors compute two 400x400 FC matrix for each subject, one for rest, one for movie-watching, then correlate the correlations within each dyad, then compared the average correlation of correlations for MZ, DZ, and UR. In addition to the same problems as the previous analysis, here it is not clear what is meant by "averaging correlations [...] within a network combination". What is a "network combination"? Further, to average correlations, they need to be r-to-z transformed first. As with the above, the results from this analysis in its current form are likely incorrect.

(C) ISC and FC profile heritability analyses (section that starts from line 175): Here, the authors use first a valid method remarkably similar to the old Haseman-Elston approach to compute heritability, complemented by a permutation test. That is fine. But then they proceed with two novel, ill-described, and likely invalid methods to (1) "compare the heritability of movie and rest FC profiles" and (2) to "determine the sample size necessary for stable multidimensional heritability results". For (1), they permute, seemingly under the alternative, rest and movie-watching timeseries, and (2), by dropping subjects and estimating changes in the distribution.

The (1) might be correct, but there are items that are not clearly described, so the reader cannot be sure of what was done. What are the "153 unique network combinations"? Why do the authors separate by day here, whereas the previous analyses concatenated both days? Were the correlations r-to-z transformed before averaging?

The (2) is also not well described, and in any case, power can be computed analytically; it isn't clear why the authors needed to resort to this ad hoc approach, the validity of which is unknown. If the issue is the possibility that the multidimensional phenotypic correlation matrix is rank-deficient, it suffices that there are more independent measurements per subject than the number of subjects.

(D) Frequency-dependent ISC heritability analysis (from line 216): Here, the authors decompose the timeseries into frequency bands, then repeat earlier analyses, thus bringing here the same earlier problems and questions of non-exchangability in the permutations given the dyads pattern, r-z transforms, and sex/age covariates.

(E) FC strength heritability analysis (from line 236): Here, the authors use the univariate FC to compute heritability using valid and well-established methods as implemented in SOLAR. There is no "linkage" being done here (thus, the statement in line 238 is incorrect in this application. SOLAR already produces SEs, so it's unclear why the authors went out of their way to obtain jackknife estimates. If the issue is non-normality, I note that the assumption of normality is present already at the stage in which parameters themselves are estimated, not just the standard errors; for non-normal data, a rank-based inverse-normal transformation could have been used. Moreover, typically, r-to-z transformed values tend to be fairly normally distributed. So, while the heritabilities might be correct, the standard errors may not be (the authors don't demonstrate that their jackknife SE estimator is valid). The comparison of h2 between dyads raises the same questions about permutations, age/sex covariates, and r-z transforms as above.

(F) Hyperalignment (from line 245): It isn't clear at this point in the manuscript in what way hyperalignment would help to decompose heritability in "where vs. how" (from the Abstract). That information and references are only described much later, from around line 459. The description itself provides no references, and one cannot even try to reproduce what is described here in the Methods section. Regardless, it isn't entirely clear why this analysis was done: by matching functional areas, all heritabilities are going to be reduced because there will be less variance between subjects. Perhaps studying the parameters that drive the alignment (akin to what is done in tensor-based and deformation-based morphometry) could have been more informative. Plus, the alignment process itself may introduce errors, which could also reduce heritability. This could be an alternative explanation for the reduced heritability after hyperalignment and should be discussed. An investigation of hyperaligment parameters, their heritability, and their co-heritability with the BOLD-phenotypes can inform on this.

(G) Relationships between parcel area and heritability (from line 270): As under F), how much the results are distorted likely depends on the accuracy of the alignment, and the error variance (vs heritable variance) introduced by this.

(H) Neural timescale analyses (from line 280): Here, a valid phenotype (NT) is assessed with statistical methods with the same limitations as those previously (exchangability of dyads, age/sex covariates, and r-z transforms). NT values are combined across space and used as covariates in "some multivariate analyses". As a reader, I really wanted to see the results related to NT, something as simple as its heritability, but these aren't clearly shown, only differences between types of dyads.

(I) Significance testing for autocorrelated brain maps and FC matrices (from line 310): Here, the authors suddenly bring up something entirely different: reliability of heritability maps, and then never return to the topic of reliability again. As a reader, I find this confusing. In any case, analyses with BrainSMASH with well-behaved, normally distributed data are ok. Whether their data is well behaved or whether they ensured that the data would be well behaved so that BrainSMASH is valid is not described. As to why Spearman correlations are needed here, Mantel tests, or whether the 1000 "surrogate" maps are valid realizations of the data under the null, remains undemonstrated.

(J) Global signal was removed, and the authors do not acknowledge that this could be a limitation in their analyses, nor offer a side analysis in which the global signal is preserved.

(K) FDR is used to control the error rate, but in many cases, as it's applied to multiple sets of p-values, the amount of false discoveries is only controlled across all tests, but not within each set. The number of errors within any set remains unknown.

(L) Generally, when studying the heritability of a trait, the trait must be defined first. Here, multiple traits are investigated, but are never rigorously defined. Worse, the trait being analyzed changes at every turn.

Reviewer #3 (Public review):

Strengths:

It's sort of novel to study the heritability of movie-watching fMRI data. The methodology the authors used in the paper is also supportive of their findings. Figures are nicely organized and plotted. They finally found that sensory processing in the human brain is under genetic control over stable aspects of brain function (here referring to neural timescale and resting state connectivity).

Weaknesses:

What I am worried about most is the sample size and interpretation of heritability.

(1) Figure 1. I assumed that the authors just calculated the ISC within each group (MZ, DZ, and UR). Of course, you can get different variations between each group. Therefore, there is heritability. Why not calculate ISC across the whole sample, then separate MZ, DZ, and UR?

(2) Heritability scores in the paper are sort of small. If the sample size is small, please consider p-values, which will tell more about the trustworthiness of your heritability.

(3) I don't understand the high-frequency signals in fMRI data. It's always regarded as noise, the band 1 here in particular.

(4) The statement "we show that the heritability of brain activity patterns can be partially explained by the heritability of the neural timescale" should come from Figure 5. However, after controlling for NT, the heritability decreased max. 0.025 in temporal areas. I am not sure this change supports the statement. If the visual cortex is outlined, and combining ISC changes in the visual cortex, I think this would somehow be answered. Instead of delta h2, adding a new model h2 would be obvious to the readers.

(5) Figures 7 and 8, when getting the difference of heritability, please also consider the standard errors of the heritability estimates. Then you can compare across networks/regions.

(6) I think movie VS resting state is a really important result in this paper. However, there is almost no discussion. Discussing this part would be more beneficial for understanding the genetic control over the neuron arousal and excitation circuits.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors report that activation of excitatory DREADDs in the mid-cervical spinal cord can increase inspiratory activity in mice and rats. This is an important first step toward an ultimate goal of using this, or similar, technology to drive breathing in disorders associated with decreased respiratory motor output, such as spinal injury or neurodegenerative disease. Strengths to this study include a comparison of non-specific DREADD expression in the mid-cervical spinal cord versus specific targeting to ChAT-positive neurons, and the measurement of multiple respiratory-related outcomes, including phrenic inspiratory output, diaphragm EMG activity and ventilation. The data show convincingly that DREADDs can be used to drive phrenic inspiratory activity, which in turn increases diaphragm EMG activity and ventilation.

Comments on revisions: All of my prior comments have been sufficiently addressed.

Reviewer #2 (Public review):

Summary:

This study shows that when excitatory DREADD receptors are expressed in the ventral area of the cervical spinal cord containing phrenic motoneurons, systemic administration of the DREADD ligand J60 increases diaphragm EMG activity without altering respiratory rate. The authors took a non-selective expression approach in wild-type mice, as well as a more selective Cre-dependent approach in Chat-Cre mice and Chat-Cre rats to stimulate cervical motoneurons in the spinal cord. This is a proof of principle study that supports the use of DREADD technology to stimulate the motor output to the diaphragm.

Strengths:

The strengths of the study lie in the use of both mice and rats to test whether the chomogenetic activation of phrenic motoneurons with multiple experimental approaches increases diaphragm EMG activity (both tonic and phasic) and tidal volume.

Comments on revisions:

Thanks for addressing my comments. One last comment that could be discussed or addressed is :

Line 295- was the time post-infection, which varies considerably between groups and across samples, taken into consideration when comparison of response was made between ChatCre mice (4-9 weeks post-infection) and WT mice (four to five weeks post-infection)?

Reviewer #1 (Public review):

Summary:

This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.

Strengths:

The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.

Weaknesses were addressed during the revision.

The authors effectively addressed my critiques in the rebuttal letter and made corresponding changes in the manuscript. Specifically, they: 1) emphasized the importance of TO orientation in influencing STB nuclear subtype differentiation by adding text to the introduction; 2) clarified the differences in cluster numbers and names between primary tissue and TO data, explaining that each dataset was analyzed independently with separate clustering algorithms and adding clarifying text to the results section; 3) included additional rationale for using SN over SC sequencing, particularly for studying the multinucleated STB; 4) acknowledged that their original evidence was insufficient to definitively determine STBout nuclei differentiation status and removed language suggesting STB-3 as a terminally differentiated subtype, presenting alternative hypotheses in the discussion; and 5) incorporated new figures and clarifications, including RNA-FISH experiments, to validate subtype-specific marker gene expression. Overall, the authors' revisions strengthened the manuscript and aligned well with my critiques.

Reviewer #1 (Public review):

Summary:

This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.

Strengths:

The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.

Weaknesses were addressed during the revision.

The authors effectively addressed my critiques in the rebuttal letter and made corresponding changes in the manuscript. Specifically, they: 1) emphasized the importance of TO orientation in influencing STB nuclear subtype differentiation by adding text to the introduction; 2) clarified the differences in cluster numbers and names between primary tissue and TO data, explaining that each dataset was analyzed independently with separate clustering algorithms and adding clarifying text to the results section; 3) included additional rationale for using SN over SC sequencing, particularly for studying the multinucleated STB; 4) acknowledged that their original evidence was insufficient to definitively determine STBout nuclei differentiation status and removed language suggesting STB-3 as a terminally differentiated subtype, presenting alternative hypotheses in the discussion; and 5) incorporated new figures and clarifications, including RNA-FISH experiments, to validate subtype-specific marker gene expression. Overall, the authors' revisions strengthened the manuscript and aligned well with my critiques.

Reviewer #3 (Public review):

In this report, Keenen et al. present a thoroughly characterized platform for identifying potential molecular mechanisms regulating syncytiotrophoblast cell functions in placental biology. Application of single cell assessments to identify developmental trajectories of this lineage have been challenging due to the complex, multinucleated structure of the syncytium. The authors provide a comprehensive comparative assessment of term placental tissue and three independent trophoblast organoid models. They use single cell and single nucleus RNA sequencing followed by differential gene expression and pseudotime analyses to identify subpopulations and differentiation trajectories. They further compare the datasets generated in this study to publicly available datasets from first trimester placental tissue. The work is timely as optimization of trophoblast organoids is an evolving topic in placental research. And careful characterization of in vitro models has been noted as essential for model selection and result interpretation in the field.

The study elucidates syncytiotrophoblast nucleus subtypes and proportions in three different organoid models and compares subtypes and gene expression signatures to placental tissues. This work advances the field by demonstrating the utility of different trophoblast organoids to model syncytiotrophoblast differentiation. The in-depth characterization of cell types comprising the different organoid models and how they compare to placental tissue will help to inform model selection for future experimentation in the field. Defining cell composition and cell differentiation trajectories will also aid in data interpretation for data generated by these tissue and model sources. Overall, the conclusions presented in the manuscript are well supported by the data. The figures, as presented, are informative and striking.

The authors present outstanding progress toward their overall aim of identifying, "the underlying control of the syncytiotrophoblast". They identify the chromatin remodeler, RYBP, as well as other regulatory networks that they propose are critical to syncytiotrophoblast development.

The initial study was limited in fully addressing the aim, however, as functional evidence for the contributions of the factors/pathways to syncytiotrophoblast cell development was absent. In a revised version of the manuscript, the authors report the first application of CRISPR-mediated gene silencing in a TO model. They use CRISPR-Cas9-mediated gene targeting to generate RYBP and AFF1 knockout models. Deletion of either RYBP or AFF1 increased STB-2 marker gene expression, as determined using bulk RNA-seq. Future experimentation will assess the distribution of STB nuclear subtypes in the RYBP and AFF1 knockout models and explore the essentiality of RYBP, AFF1, and other identified factors to syncyiotrophoblast development and function.

Localization and validation of the identified factors within tissue and at the protein level will also provide further contextual evidence to address the hypotheses generated. In a revised version of the manuscript, the authors localize STB markers PAPPA2 and ADAMTS6 in TOs using RNA-FISH. Future work will aim to further validate the markers and hypotheses generated from this study.

Reviewer #1 (Public review):

Summary:

The study characterises an RNA polymerase (Pol) I mutant (RPA135-F301S) named SuperPol. This mutant was previously shown to increase yeast ribosomal RNA (rRNA) production by Transcription Run-On (TRO). In this work, the authors confirm this mutation increases rRNA transcription using a slight variation of the TRO method, Transcriptional Monitoring Assay (TMA), which also allows the analysis of partially degraded RNA molecules. The authors show a reduction of abortive rRNA transcription in cells expressing the SuperPol mutant and a modest occupancy decrease at the 5' region of the rRNA genes compared to WT Pol I. These results suggest that the SuperPol mutant displays a lower frequency of premature termination. Using in vitro assays, the authors found that the mutation induces an enhanced elongation speed and a lower cleavage activity on mismatched nucleotides at the 3' end of the RNA. Finally, SuperPol mutant was found to be less sensitive to BMH-21, a DNA intercalating agent that blocks Pol I transcription and triggers the degradation of the Pol I subunit, Rpa190. Compared to WT Pol I, short BMH-21 treatment has little effect on SuperPol transcription activity, and consequently, SuperPol mutation decreases cell sensitivity to BMH-21.

I'd suggest the following points to be taken into consideration:

Major comments:

(1) The differences in the transcriptionally engaged WT Pol I and SuperPol profiles (Figure 2) are very modest, without any statistical analyses. What is the correlation between CRAC replicates? Are they separated in PCA analyses? Please, include more quality control information. In my opinion, these results are not very convincing. Similarly, the effect of BMH-21 on WT Pol I activity (Figure 7) is also very subtle and doesn't match the effect observed in a previous study [1]. Could the author comment on the reasons for these differences? These discrepancies raise concerns about the methodology. In addition, according to the laboratory's previous work [2], Pol I ChIP signal at rDNA is not significantly different in cells expressing WT Pol I and SuperPol. How can these two observations be reconciled? I would suggest using an independent methodology to analyse Pol I transcription, for example, GRO-seq or TT-seq.

(2) While the experiments clearly show SuperPol mutant increases nascent transcription and decreases the production of abortive promoter-proximal transcripts compared to WT Pol I. RPA135-F301S mutation has a minor impact on total rRNA levels, at least those shown in Figure 3B. Are steady-state rRNA levels higher in cells expressing SuperPol mutant? It would be interesting to know if SuperPol mutant produces more functional rRNAs.

Significance:

The work further characterises a single amino acid mutation of one of the largest yeast Pol I subunits (RPA135-F301S). While this mutation was previously shown to increase rRNA synthesis, the current work expands the SuperPol mutant characterisation, providing details of how RPA135-F301S modifies the enzymatic properties of yeast Pol I. In addition, their findings suggest that yeast Pol I transcription can be subjected to premature termination in vivo. The molecular basis and potential regulatory functions of this phenomenon could be explored in additional studies.

Our understanding of rRNA transcription is limited, and the findings of this work may be interesting to the transcription community. Moreover, targeting Pol I activity is an open strategy for cancer treatment. Thus, the resistance of SuperPol mutant to BMH-21 might also be of interest to a broader community, although these findings are yet to be confirmed in human Pol I and with more specific Pol I inhibitors in future.

Reviewer #2 (Public review):

Summary:

This article presents a study on a mutant form of RNA polymerase I (RNAPI) in yeast, referred to as SuperPol, which demonstrates increased rRNA production compared to the wild-type enzyme. While rRNA production levels are elevated in the mutant, RNAPI occupancy as detected by CRAC is reduced at the 5' end of rDNA transcription units. The authors interpret these findings by proposing that the wild-type RNAPI pauses in the external transcribed spacer (ETS), leading to premature transcription termination (PTT) and degradation of truncated rRNAs by the RNA exosome (Rrp6). They further show that SuperPol's enhanced activity is linked to a lower frequency of PTT events, likely due to altered elongation dynamics and reduced RNA cleavage activity, as supported by both in vivo and in vitro data.

The study also examines the impact of BMH-21, a drug known to inhibit Pol I elongation, and shows that SuperPol is less sensitive to this drug, as demonstrated through genetic, biochemical, and in vivo approaches. The authors show that BMH-21 treatment induces premature termination in wild-type Pol I, but only to a lesser extent in SuperPol. They suggest that BMH-21 promotes termination by targeting paused Pol I complexes and propose that PTT is an important regulatory mechanism for rRNA production in yeast.

The data presented are of high quality and support the notion that 1) premature transcription termination occurs at the 5' end of rDNA transcription units; 2) SuperPol has an increased elongation rate with reduced premature termination; and 3) BMH-21 promotes both pausing and termination. The authors employ several complementary methods, including in vitro transcription assays. These results are significant and of interest for a broad audience.

Beyond the minor points listed below, my main criticism concerns the interpretation of data in relation to termination. While it is possible that the SuperPol mutation affects the wild-type Pol I's natural propensity for termination, it is also possible that premature termination is simply a consequence of natural or BMH-21-induced Pol I pausing. SuperPol may elongate more efficiently than the wild-type enzyme, pause less frequently, and thus terminate less often. In this light, the notion that termination "regulates" rRNA production might be an overstatement, with pausing as the primary event. Claiming a direct effect on termination by both the mutation and BMH-21 would require showing that with equivalent levels of pausing, termination occurs more or less efficiently, which would be challenging and should not be expected in this study. The authors address this point in the last two paragraphs of the discussion. My suggestion is to temper the claims regarding termination as a regulatory mechanism.

Significance:

These results are significant and of interest for a basic research audience.

Reviewer #3 (Public review):

Summary:

In the manuscript "Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription", Azouzi and co-authors investigate the regulatory mechanisms of ribosomal RNA (rRNA) transcription by RNA Polymerase I (RNAPI) in the budding yeast S. cerevisiae. They follow up on exploring the molecular basis of a mutant allele of the second largest subunit of RNAPI, RPA135-F301S, also dubbed SuperPol, that they had previously reported (Darrière et al, 2019), and which was shown to rescue Rpa49-linked growth defects, possibly by increasing rRNA production.

Through a combination of genomic and in vitro approaches, the authors test the hypothesis that RNAPI activity could be subjected to a Premature Transcription Termination (PPT) mechanism, akin to what is observed for RNA Polymerase II (RNAPII), and which is suggested to be an important step for the quality control of rRNA transcripts. SuperPol is proposed to lack such a regulatory mechanism, due to an increased processivity. In agreement, SuperPol is shown to be resistant to BMH-21, a drug previously shown to impair RNAPI elongation.<br /> Overall, the experiments are performed with rigor and include the appropriate controls and statistical analysis. Both the figures and the text present the data clearly. The Material and Methods section is detailed enough. The reported results are interesting; however, I am not fully convinced of the existence of PPT of RNAPI, and even less of its utmost importance.

The existence of PPT of RNAPI would entail an intended regulatory mechanism. The authors propose that PPT could serve as quality control step for the UTP-A complex loading on the rRNA 5'-end. While this hypothesis is enticing and cautiously phrased by the authors, the lack of evidence showing a specific regulatory function (such as UTP-A loading checkpoint or else) limits these termination events to possibly abortive actions of unclear significance.

The authors may want to consider comparisons to other processive alleles, such as the rpb1-E1103G mutant of the RNAPII subunit (Malagon et al, 2006) or the G1136S allele of E. coli RNAP (Bar-Nahum et al., 2005). While clearly mechanistically distinct, these mutations result in similarly processive enzymes that achieve more robust transcription, possibly at the cost of decreased fidelity. Indeed, an alternative possibility explaining these transcripts could be that they originate from unsuccessful resumption of transcription after misincorporation (see below).

I suggest reconsidering the study's main conclusions by limiting claims about the regulatory function of these termination events (the title of the manuscript should be changed accordingly). Alternatively, the authors should provide additional investigation on their regulatory potential, for example by assessing if indeed this quality control is linked to the correct assembly of the UTP-A complex. The expectation would be that SuperPol should rescue at least to some extent the defects observed in the absence of UTP-A components.

Moreover, the results using the clv3 substrate suggest the possibility that SuperPol might simply be more able to tolerate mismatches, thus be more processive in transcribing, because not subjected to proof-reading mechanisms, similarly to what observed in Schwank et al., 2022. This could explain many of the observations, and I think it is worth exploring by assessing the fidelity of the enzyme, especially in the frame of suggesting a regulatory function for these termination events.

Significance:

Azouzi and co-authors' work builds on their previous study (Darrière et al, 2019) of RPA135-F301S (SuperPol), a mutant allele of the second largest RNAPI subunit, which was shown to compensate for Rpa49 loss, potentially by increasing rRNA production. The work advances the mechanistic understanding of the the SuperPol allele, demonstrating the increased processivity of this enzyme compared to its wild-type counterpart. Such increased processivity "desensitizes" RNAPI from abortive transcription cycles, the existence of which is clearly shown, though the biological significance of this phenomenon remains unclear. The lack of evidence for a regulatory mechanism behind these early termination events is, in my opinion, a limitation of this study, as it does not allow for differentiation between an intended regulatory process and a byproduct of an imperfect system.

This work is of interest for researchers studying transcription regulation, particularly those interested in understanding RNAPI's role and fidelity. Demonstrating PPT as a regulatory quality control for RNAPI could point to common strategies in between RNAPI and RNAPII regulation, where premature termination has been extensively documented. However, without evidence of a specific regulatory function, these findings may currently be limited to descriptive insights.

Joint public review:

Summary:

This study investigates the hypoxia rescue mechanisms of neurons by non-neuronal cells in the brain from the perspective of exosomal communication between brain cells. Through multi-omics combined analysis, the authors revealed this phenomenon and logically validated this intercellular rescue mechanism under hypoxic conditions through experiments. The study proposed a novel finding that hemoglobin maintains mitochondrial function, expanding the conventional understanding of hemoglobin. This research is highly innovative, providing new insights for the treatment of hypoxic encephalopathy.

Overall, the manuscript is well organized and written, however, the authors have only partially answered the reviewers comments.

Reviewer #1 (Public review):

In this study, the authors introduced an essential role of AARS2 in maintaining cardiac function. They also investigated the underlying mechanism that through regulating alanine and PKM2 translation are regulated by AARS2. Accordingly, a therapeutic strategy for cardiomyopathy and MI was provided.

Comments on revised version:

The authors have completely addressed my concerns.

Reviewer #3 (Public review):

In the present study, the author revealed that cardiomyocyte-specific deletion of mouse AARS2 exhibited evident cardiomyopathy with impaired cardiac function, notable cardiac fibrosis, and cardiomyocyte apoptosis. Cardiomyocyte-specific AARS2 overexpression in mice improved cardiac function and reduced cardiac fibrosis after myocardial infarction (MI), without affecting cardiomyocyte proliferation and coronary angiogenesis. Mechanistically, AARS2 overexpression suppressed cardiomyocyte apoptosis and mitochondrial reactive oxide species production, and changed cellular metabolism from oxidative phosphorylation toward glycolysis in cardiomyocytes, thus leading to cardiomyocyte survival from ischemia and hypoxia stress. Ribo-Seq revealed that AARS2 overexpression increased pyruvate kinase M2 (PKM2) protein translation and the ratio of PKM2 dimers to tetramers that promote glycolysis. Additionally, PKM2 activator TEPP-46 reversed cardiomyocyte apoptosis and cardiac fibrosis caused by AARS2 deficiency. Thus, this study demonstrates that AARS2 plays an essential role in protecting cardiomyocytes from ischemic pressure via fine-tuning PKM2-mediated energy metabolism, and presents a novel cardiac protective AARS2-PKM2 signaling during the pathogenesis of MI.

Comments on revised version:

The authors addressed all the issues, no more comments.

Reviewer #1 (Public review):

Summary:

In this paper, Manley and Vaziri investigate whole-brain neural activity underlying behavioural variability in zebrafish larvae. They combine whole brain (single cell level) calcium imaging during the presentation of visual stimuli, triggering either approach or avoidance, and carry out whole brain population analyses to identify whole brain population patterns responsible for behavioural variability. They show that similar visual inputs can trigger large variability in behavioural responses. Though visual neurons are also variable across trials, they demonstrate that this neural variability does not degrade population stimulus decodability. Instead, they find that the neural variability across trials is in orthogonal population dimensions to stimulus encoding and is correlated with motor output (e.g. tail vigor). They then show that behavioural variability across trials is largely captured by a brain-wide population state prior to the trial beginning, which biases choice - especially on ambiguous stimulus trials. This study suggests that parts of stimulus-driven behaviour can be captured by brain-wide population states that bias choice, independently of stimulus encoding.

Comments on revisions:

The authors have revised their manuscript and provided novel analyses and figures, as well as additions to the text based on our reviewer comments.

As stated in my first review, the strength of the paper principally resides in the whole brain cellular level imaging - using a novel fourier light field microscopy (Flfm) method - in a well-known but variable behaviour.

Many of the authors' answers have provided additional support for their interpretations of results, but the new analysis in Figure 3g - further exploring the orthogonality of e1 and wopt - puts into question the interpretation of a key result: that e1 and wopt are orthogonal in a non-arbitrary way. This needs to be addressed. I have made suggestions below to address this:

Reviewer 3 had correctly highlighted the issue that in high-dimensional data, there is an increasingly high chance of two vectors being orthogonal. The authors address this by shuffling the stimulus labels. They then state (and provide a new panel g in Fig. 3) that the shuffled distribution is wider than the actual distribution, and state that a wilcoxon rank-sum test shows this is significant. Given the centrality of this claim, I would like the authors to clarify what exactly is being done here, as it is not clear to me how this conclusion can be drawn from this analysis:

In lines 449:453 the authors state:<br /> 'While it is possible to observe shuffled vectors which are nearly orthogonal to e1, the shuffled distribution spans a significantly greater range of angles than the observed data (p<0.05, Wilcoxon rank- sum test), demonstrating that this orthogonality is not simply a consequence of analyzing multi-dimensional activity patterns. '<br /> I don't understand how the authors arrive at the p-value using a rank-sum test here. (a) What is the n in this test? Is n the number of shuffles? If so, this violates the assumptions of the test (as n must be the number of independent samples and not the arbitrary number of shuffles). (b) If the shuffling was done once for each animal and compared with actual data with a rank-sum test, how likely is that shuffling result to happen in 10000 shuffle comparisons?<br /> I am highlighting this, as it looks from Figure 3g that the shuffled distribution is substantially overlapping with the actual data (i.e., not outside of the 95 percentile of the shuffled distribution), which would suggest that the angle found between e1 and wept could happen by chance.

I would also suggest the authors instead test whether e1 is consistently aligned with itself when calculated on separate held out data-sets (for example by bootstrapping 50-50 splits of the data). If they can show that there is a close alignment between independently calculated e1's across separate data sets (and do the same for wopt), and then show e1 and wopt are orthogonal, then that supports their statement that e1 and wopt are orthogonal in a meaningful way. Given that e1 captures tail vigor variability (and Wopt appears to not) then I would think this could be the case. But the current answer the authors have given is not supporting their statement.

Reviewer #2 (Public review):

This work by Manley and Vaziri identify brain networks that are associated with trial-to-trial variability during prey-capture and predator avoidance behaviors. However, mixing of signals across space and time make it difficult to interpret the data generated and relate the data to findings from prior work.

Comments on revisions:'

In their response to prior reviewer comments, Manley and Vaziri have now provided helpful methodological clarity and additional analyses. The additional work makes clear that the claims of variability and mixing of sensory, motor, and internal variables at the single-cell level are not well supported.

RESOLUTION<br /> - The new information provided regarding resolution may not be very relevant as this was from an experiment in air. It would be much more informative to show how PSF degrades in the brain with depth.

DEPTH<br /> - It is helpful to see the registered light-field and confocal images. Both appear to provide poor or little information in regions >200 below the surface (like the hypothalamus), making the claim that whole brain data is being collected at cellular resolution difficult to justify.

MERGING<br /> - The typical soma at these ages has a radius of 2.5 microns, which corresponds to a volume of 65 microns^3. Given the close packing of most cells, this means that a typical ROI of 750 microns^3 contains more than 10 neurons. Therefore, the authors should not claim they are reporting activity at cellular resolution.<br /> - Furthermore, the fact that these ROIs contains tens of cells brings into question the degree of variability at the single-cell level. For example, if every cluster of 10 cells has one variable cell, then all clusters might be labeled as exhibiting variability even though only 10% of the cells show variability.

SLOW CALCIUM DYNAMICS<br /> - Convolution/Deconvolution with the inappropriate kernel both have problems, some of which the authors have noted. However, by not deconvolving, the authors are significantly obscuring the interpretation of their data by mixing together signals across time.<br /> - Also, the claim that "neurons highly tuned to a particular stimulus exhibited variability in their responses across multiple presentations of the same stimuli" should be clarified or qualified. It is not clear from what has been shown if the responses are indeed variable, or rather if there is additional activity (or apparent activity) occasionally present that shifts the pre-stimulus baseline around (for example, 3J suggests that in many cases the visual signal from the prior trial is still present when a new trial begins).<br /> - Figure 3A should show when the stimulus occurs, should show some of the prestimulus period, and ideally be off-set corrected so all traces in a given panel start at the same y-value at the beginning of the stimulus period.

ORTHOGONALITY<br /> - It is now clearer that the visual signal and noise vectors were determined for the entire time series with all trials. Therefore, the concern that sources of activation in advance of a given trial were being ignored is alleviated. The concern remains, however, that these sources are being properly accounted for given potential kernel variations and nonlinearity. Nonetheless, it is recognized that the GCaMP filtering most likely would lead to a decrease in the disparity between two populations.<br /> - The authors' clarification that the analyzed ROIs consist of cell clusters raises the trivial possibility that the observed orthogonality between the visual signal and leading noise vectors is explained by noise simply reflecting the activation of different motor or motor-planning related neurons in an ROI, neurons that are separate from visually-encoding neurons in the same cluster.

SOURCES of VARIABILITY<br /> - The data presented in Supplemental Figure 3Ei actually is suggestive that eye movements are a significant contributor to the reported variability. Notice how in (1 4) vs (1 5) and (4 7) vs (4 8) there is a notable difference in the distribution of responses. Adding eye kinematic variables to the analysis of Figure S4 could be clarifying.-

Reviewer #1 (Public review):

Summary:

In this manuscript, Jin et. al., describe SMARTTR, an image analysis strategy optimized for analysis of dual-activity ensemble tagging mouse reporter lines. The pipeline performs cell segmentation, then registers the location of these cells into an anatomical atlas, and finally, calculates the degree of co-expression of the reporters in cells across brain regions. The authors demonstrate the utility of the method by labeling two ensemble populations during two related experiences: inescapable shock and subsequent escapable shock as part of learned helplessness.

Strengths:

- We appreciated that the authors provided all documentation necessary to use their method, and that the scripts in their publicly available repository are well commented. Submission of the package to CRAN will, as the other reviewer pointed out, ensure that the package and its dependencies can be easily installed using few lines of code in the future. Additionally, we particularly appreciate the recently added documentation website and vignettes, which provide guidance on package installation and use cases.<br /> - The manuscript was well-written and very clear, and the methods were generally highly detailed.<br /> - The authors have addressed our previous concerns, and we appreciate their revised manuscript.

Reviewer #2 (Public review):

Summary:

This manuscript describes a workflow and software package, SMARTR, for mapping and analyzing neuronal ensembles tagged using activity-dependent methods. They showcase this pipeline by analyzing ensembles tagged during the learned helplessness paradigm. This is an impressive effort, and I commend the authors for developing open-source software to make whole-brain analyses more feasible for the community. After peer-review, the authors addressed reviewer suggestions and concerns regarding the usability and maintainability of the SMARTTR package, ensuring that the package will be published on CRAN, improving documentation, and including unit tests to ensure code stability. Overall, this software package will prove to have a broad impact on the field.

Reviewer #1 (Public review):

Summary:

This study provides new insights into the role of miR-19b, an oncogenic microRNA, in the developing chicken pallium. Dynamic expression pattern of miR-19b is associated with its role in regulating cell cycle progression in neural progenitor cells. Furthermore, miR-19b is involved in determining neuronal subtypes by regulating Fezf2 expression during pallial development. These findings suggest an important role for miR-19b in the coordinated spatio-temporal regulation of neural progenitor cell dynamics and its evolutionary conservation across vertebrate species.

Strengths:

The authors identified conserved roles of miR-19 in the regulation of neural progenitor maintenance between mouse and chick, and the latter is mediated by the repression of E2f8 and NeuroD1. Furthermore, the authors found that miR-19b-dependent cell cycle regulation is tightly associated with specification of Fezf1 or Mef2c-positive neurons, in spatio-temporal manners during chicken pallial development. These findings uncovered molecular mechanisms underlying microRNA-mediated neurogenic controls.

Weaknesses:

Although the authors in this study claimed striking similarities of miR-19a/b in neurogenesis between mouse and chick pallium, a previous study by Bian et al. revealed that miR-19a contributes the expansion of radial glial cells by suppressing PTEN expression in the developing mouse neocortex, while miR-19b maintains apical progenitors via inhibiting E2f2 and NeuroD1 in chicken pallium. Thus, it is still unclear whether the orthologous microRNAs regulate common or species-specific target genes.

The spatiotemporal expression patterns of miR-19b and several genes are not convincing. For example, the authors claim that NeuroD1 is initially expressed uniformly in the subventricular zone (SVZ) but disappears in the DVR region by HH29 and becomes detectable by HH35 (Figure 1). However, the in situ hybridization data revealed that NeuroD1 is highly expressed in the SVZ of the DVR at HH29 (Figure 4F). Thus, perhaps due to the problem of immunohistochemistry, the authors have not been able to detect NeuroD1 expression in Figure 1D, and the interpretation of the data may require significant modification.

It seems that miR-19b is also expressed in neurons (Figure 1), suggesting the role of miR19-b must be different in progenitors and differentiated neurons. The data on the gain- and loss-of-function analysis of miR-19b on the expression of Mef2c should be carefully considered, as it is possible that these experiments disturb the neuronal functions of miR19b rather than in the progenitors.

The regions of chicken pallium were not consistent among figures: in Figure 1, they showed caudal parts of the pallium (HH29 and 35), while the data in Figure 4 corresponded to the rostral part of the pallium (Figure 4B).

The neurons expressing Fezf2 and Mef2 in the chicken pallium are not homologous neuronal subtypes to mammalian deep and superficial cortical neurons. The authors must understand that chicken pallial development proceeds in an outside-in manner. Thus, Mef2c-postive neurons in a superficial part are early-born neurons, while FezF2-positive neurons residing in deep areas are later-born neurons. It should be noted that the expression of a single marker gene does not support cell type homology, and the authors' description "the possibility of primitive pallial lamina formation in common ancestors of birds and mammals" is misleading.

Overexpression of CDKN1A or Sponge-19b induced ectopic expression of Fezf2 in the ventricular zone (Figure 3C, E). Do these cells maintain progenitor statement or prematurely differentiate to neurons? In addition, the authors must explain that the induction of Fezf2 is also detected in GFP-negative cells.

Reviewer #2 (Public review):

Summary:

This paper investigates the general concept that avian and mammalian pallium specifications share similar mechanisms. To explore that idea, the authors focus their attention on the role of miR-19b as a key controlling factor in the neuronal proliferation/differentiation balance. To do so, the authors checked the expression and protein level of several genes involved in neuronal differentiation, such as NeuroD1 or E2f8, genes also expressed in mammals after conducting their functional gene manipulation experiments. The work also shows a dysregulation in the number of neurons from lower and upper layers when miR-19b expression is altered.

To test it, the authors conducted a series of functional experiments of gain and loss of function (G&LoF) and enhancer-reporter assays. The enhancer-reporter assays demonstrate a direct relationship between miR-19b and NeuroD1 and E2f8 which is also validated by the G&LoF experiments. It´s also noteworthy to mention that the way miR-19b acts is maintaining the progenitor cells from the ventricular zone in an undifferentiated stage, thus promoting them into a stage of cellular division.

Overall, the paper argues that the expression of miR-19b in the ventricular zone promotes the cells in a proliferative phase and inhibits the expression of differentiation genes such as E2f8 and NeurD1. The authors claim that a decrease in the progenitor cell pool leads to an increase and decrease in neurons in the lower and upper layers, respectively.

Strengths:

(1) Novelty Contribution<br /> The paper offers strong arguments to prove that the neurodevelopmental basis between mammals and birds is quite the same. Moreover, this work contributes to a better understanding of brain evolution along the animal evolutionary tree and will give us a clearer idea about the roots of how our brain has been developed. This stands in contrast to the conventional framing of mammal brain development as an independent subject unlinked to the "less evolved species". The authors also nicely show a concept that was previously restricted to mammals - the role of microRNAs in development.

(2) Right experimental approach<br /> The authors perform a set of functional experiments correctly adjusted to answer the role of miR-19b in the control of neuronal stem cell proliferation and differentiation. Their histological, functional, and genetic approach gives us a clear idea about the relations between several genes involved in the differentiation of the neurons in the avian pallium. In this idea, they maintain the role of miR-19b as a hub controller, keeping the ventricular zone cells in an undifferentiated stage to perpetuate the cellular pool.

(3) Future directions<br /> The findings open a door to future experiments, particularly to a better comprehension of the role of microRNAs and pallidal genetic connections. Furthermore, this work also proves the use of avians as a model to study cortical development due to the similarities with mammals.

Weaknesses:

While there are questions answered, there are still several that remain unsolved. The experiments analyzed here lead us to speculate that the early differentiation of the progenitor cells from the ventricular zone entails a reduction in the cellular pool, affecting thereafter the number of latter-born neurons (upper layers). The authors should explore that option by testing progenitor cell markers in the ventricular zone, such as Pax6. Even so, it remains possible that miR-19b is also changing the expression pattern of neurons that are going to populate the different layers, instead of their numbers, so the authors cannot rule that out or verify it. Since the paper focuses on the role of miR-19b in patterning, I think the authors should check the relationship and expression between progenitors (Pax6) and intermediate (Tbr2) cells when miR-19b is affected. Since neuronal expression markers change so fast within a few days (HH24-HH35), I don't understand why the authors stop the functional experiments at different time points.

Reviewer #3 (Public review):

Summary:

This is a timely article that focuses on the molecular machinery in charge of the proliferation of pallial neural stem cells in chicks, and aims to compare them to what is known in mammals. miR19b is related to controlling the expression of E2f8 and NeuroD1, and this leads to a proper balance of division/differentiation, required for the generation of the right number of neurons and their subtype proportions. In my opinion, many experiments do reflect an interaction between all these genes and transcription factors, which likely supports the role of miR19b in participating in the proliferation/differentiation balance.

Strengths:

Most of the methodologies employed are suitable for the research question, and present data to support their conclusions.

The authors were creative in their experimental design, in order to assess several aspects of pallial development.

Weaknesses:

However, there are several important issues that I think need to be addressed or clarified in order to provide a clearer main message for the article, as well as to clarify the tools employed. I consider it utterly important to review and reinterpret most of the anatomical concepts presented here. The way the are currently used is confusing and may mislead readers towards an understanding of the bird pallium that is no longer accepted by the community.

Major Concerns:

(1) Inaccurate use of neuroanatomy throughout the entire article. There are several aspects to it, that I will try to explain in the following paragraphs:

a) Figure 1 shows a dynamic and variable expression pattern of miR19b and its relation to NeuroD1. Regardless of the terms used in this figure, it shows that miR19b may be acting differently in various parts of the pallium and developmental stages. However, all the rest of the experiments in the article (except a few cases) abolish these anatomical differences. It is not clear, but it is very important, where in the pallium the experiments are performed. I refer here, at least, to Figures 2C, E, F, H, I; 3D, E; 4C, D, G, I. Regarding time, all experiments were done at HH22, and the article does not show the native expression at this stage. The sacrifice timing is variable, and this variability is not always justified. But more importantly, we don't know where those images were taken, or what part of the pallium is represented in the images. Is it always the same? Do results reflect differences between DVR and Wulst gene expression modifications? The authors should include low magnification images of the regions where experiments were performed. And they should consider the variable expression of all genes when interpreting results.

b) SVZ is not a postmitotic zone (as stated in line 123, and wrongly assigned throughout the text and figures). On the contrary, the SVZ is a secondary proliferative zone, organized in a layer, located in a basal position to the VZ. Both (VZ and SVZ) are germinative zones, containing mostly progenitors. The only postmitotic neurons in VZ and SVZ occupy them transiently when moving to the mantle zone, which is closer to the meninges and is the postmitotic territory. Please refer to the original Boulder committee articles to revise the SVZ definition. The authors, however, misinterpret this concept, and label the whole mantle zone as it this would be the SVZ. Indeed, the term "mantle zone" does not appear in the article. Please, revise and change the whole text and figures, as SVZ statements and photographs are nearly always misinterpreted. Indeed, SVZ is only labelled well in Figure 4F.

The two articles mentioning the expression of NeuroD1 in the SVZ (line 118) are research in Xenopus. Is there a proliferative SVZ in Xenopus?

For the actual existence of the SVZ in the chick pallium, please refer to the recent Rueda-Alaña et al., 2025 article that presents PH3 stainings at different timepoints and pallial areas.

c) What is the Wulst, according to the authors of the article? In many figures, the Wulst includes the medial pallium and hippocampus, whereas sometimes it is used as a synonym of the hyperpallium (which excludes the medial pallium and hippocampus). Please make it clear, as the addition or not of the hippocampus definitely changes some interpretations.

d) The authors compare the entirety of the chick pallium - including the hippocampus (see above), hyperpallium, mesopallium, nidopallium - to only the neocortex of mammals. This view - as shown in Suzuki et al., 2012 - forgets the specificity of pallial areas of the pallium and compares it to cortical cells. This is conceptually wrong, and leads to incorrect interpretations (please refer to Luis Puelles' commentaries on Suzuki et al results); there are incorrect conclusions about the existence of upper-layer-like and deep-layer-like neurons in the pallium of birds. The view is not only wrong according to the misinterpreted anatomical comparisons, but also according to novel scRNAseq data (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025). These articles show that many avian glutamatergic neurons of the pallium have highly diversified, and are not comparable to mammalian cortical cells. The authors should therefore avoid this incorrect use of terminology. There are not such upper-layer-like and deep-layer-like neurons in the pallium of birds.

(2) From introduction to discussion, the article uses misleading terms and outdated concepts of cell type homology and similarity between chick and pallial territories and cells. The authors must avoid this confusing terminology, as non-expert readers will come to evolutionary conclusions which are not supported by the data in this article; indeed, the article does not deal with those concepts.

a) Recent articles published in Science (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025) directly contradict some views presented in this article. These articles should be presented in the introduction as they are utterly important for the subject of this article and their results should be discussed in the light of the new findings of this article. Accordingly, the authors should avoid claiming any homology that is not currently supported. The expression of a single gene is not enough anymore to claim the homology of neuronal populations.

b) Auditory cortex is not an appropriate term, as there is no cortex in the pallium of birds. Cortical areas require the existence of neuronal arrangements in laminae that appear parallel to the ventricular surface. It is not the case of either hyperpallium or auditory DVR. The accepted term, according to the Avian Nomenclature forum, is Field L.

c) Forebrain, a term overused in the article, is very unspecific. It includes vast areas of the brain, from the pretectum and thalamus to the olfactory bulb. However the authors are not researching most of the forebrain here. They should be more specific throughout the text and title.

(3) In the last part of the results, the authors claim miR19b has a role in patterning the avian pallium. What they see is that modifying its expression induces changes in gene expression in certain neurons. Accordingly, the altered neurons would differentiate into other subtypes, not similar to the wild type example. In this sense, miR19b may have a role in cell specification or neuronal differentiation. However, patterning is a different developmental event, which refers to the determination of broad genetic areas and territories. I don't think miR19b has a role in patterning.

(4) Please add a scheme of the molecules described in this article and the suggested interaction between them.

(5) The methods section is way too brief to allow for repeatability of the procedures. This may be due to an editorial policy but if possible, please extend the details of the experimental procedures.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to understand the neural basis of implicit causal inference, specifically how people infer causes of illness. They use fMRI to explore whether these inferences rely on content-specific semantic networks or broader, domain-general neurocognitive mechanisms. The study explores two key hypotheses: first, that causal inferences about illness rely on semantic networks specific to living things, such as the 'animacy network,' given that illnesses affect only animate beings; and second, that there might be a common brain network supporting causal inferences across various domains, including illness, mental states, and mechanical failures. By examining these hypotheses, the authors aim to determine whether causal inferences are supported by specialized or generalized neural systems.

The authors observed that inferring illness causes selectively engaged a portion of the precuneus (PC) associated with the semantic representation of animate entities, such as people and animals. They found no cortical areas that responded to causal inferences across different domains, including illness and mechanical failures. Based on these findings, the authors concluded that implicit causal inferences are supported by content-specific semantic networks, rather than a domain-general neural system, indicating that the neural basis of causal inference is closely tied to the semantic representation of the specific content involved.

Strengths:

- The inclusion of the four conditions in the design is well thought out, allowing for the examination of the unique contribution of causal inference of illness compared to either a different type of causal inference (mechanical) or non-causal conditions. This design also has the potential to identify regions involved in a shared representation of inference across general domains.

- The presence of the three localizers for language, logic, and mentalizing, along with the selection of specific regions of interest (ROIs), such as the precuneus and anterior ventral occipitotemporal cortex (antVOTC), is a strong feature that supports a hypothesis-driven approach (although see below for a critical point related to the ROI selection).

- The univariate analysis pipeline is solid and well developed.

- The statistical analyses are a particularly strong aspect of the paper.

Weaknesses:

After carefully considering the authors' response, I believe that my primary concern has not been fully addressed. My main point remains unresolved:

The authors attempt to test for the presence of a shared network by performing only the Causal vs. Non-causal analysis. However, this approach is not sufficiently informative because it includes all conditions mixed together and does not clarify whether both the illness-causal and mechanical-causal conditions contribute to the observed results.

To address this limitation, I originally suggested an additional step: using as ROIs the different regions that emerged in the Causal vs. Non-causal decoding analysis and conducting four separate decoding analyses within these specific clusters:<br /> (1) Illness-Causal vs. Non-causal - Illness First<br /> (2) Illness-Causal vs. Non-causal - Mechanical First<br /> (3) Mechanical-Causal vs. Non-causal - Illness First<br /> (4) Mechanical-Causal vs. Non-causal - Mechanical First

This approach would allow the authors to determine whether any of these ROIs can decode both the illness-causal and mechanical-causal conditions against at least one non-causal condition. However, the authors did not conduct these analyses, citing an independence issue. I disagree with this reasoning because these analyses would serve to clarify their initial general analysis, in which multiple conditions were mixed together. As the results currently stand, it remains unclear which specific condition is driving the effects.

My suggestion was to select the ROIs from their general analysis (Causal vs. Non-causal) and then examine in more detail which conditions were driving these results. This is not a case of double-dipping from my perspective, but rather a necessary step to unpack the general findings. Moreover, using ROIs would actually reduce the number of multiple comparisons that need to be controlled for.

If the authors believe that this approach is methodologically incorrect, then they should instead conduct all possible analyses at the whole-brain level to examine the effects of the specific conditions independently.

Reviewer #2 (Public review):

Summary:

In this study, the authors test whether intuitive biological causal knowledge is embedded in domain-specific semantic networks, primarily focusing on the precuneus as part of the animacy semantic network. They do so tanks to an fMRI task, by comparing brain activity elicited by participants' exposure to written situations suggesting a plausible cause of illness with brain activity in linguistically equivalent situations suggesting a plausible cause of mechanical failure or damage and non-causal situations. These contrasts confirm the PC as the main "culprit" in whole-brain and fROIs univariate analyses. In turn, inferring causes of mechanical failure engages mostly the PPA. The authors further test whether the content-specificity has to do with inferences about animates in general, or if there are some distinctions between reasoning about people's bodies versus mental states. To answer this question, the authors localize the mentalizing network and study the relation between brain activity elicited by Illness-Causal > Mech-Causal and Mentalizing > Physical stories. They conclude that inferring about the causes of illness partially differentiates from reasoning about people's states of mind. The authors finally test the alternative yet non-mutually exclusive hypothesis that both types of implicit causal inferences (illness and mechanical) depend on shared neural machinery. Good candidates are language and logic, which justifies the use of a language/logic localizer. No evidence of commonalities across causal inferences versus non-causal situations are found.

Strengths:

(1) This study introduces a useful paradigm and well-designed set of stimuli to test for implicit causal inferences.<br /> (2) Another important methodological advance is the addition of physical stories to the original mentalizing protocol.<br /> These tools pave the way for further investigation of domain-specific causal inference.<br /> (3) The authors have significantly improved the manuscript, addressing previous concerns and incorporating additional analyses that strengthen their conclusions.

Key improvements:<br /> (1) The revised introduction makes the study's contribution more explicit and resolves initial ambiguities regarding its scope.<br /> (2) The rationale for focusing primarily on the precuneus is now clearer and the additional analysis in the fusiform face area provides a valuable comparison.<br /> (3) The revised manuscript now includes a more detailed examination of the searchlight MVPA results, showing that illness and mechanical inferences elicit spatially distinct neural patterns in key regions, including the left PC, anterior PPA, and lateral occipitotemporal cortex.<br /> (4) The authors' justification for using an implicit inference task, arguing that explicit tasks introduce executive function confounds, is convincing.<br /> (5) The authors now acknowledge that while their results support a content-specific neural basis for implicit causal inference, domain-general mechanisms may still play a role in other contexts.

I have no major remaining concerns.

Reviewer #3 (Public review):

Summary:

This study employed an implicit task, showing vignettes to participants while bold signal was acquired. The aim was to capture automatic causal inferences that emerge during language processing and comprehension. In particular, the authors compared causal inferences about illness with two control conditions, causal inferences about mechanical failures and non-causal phrases related to illnesses. All phrases that where employed described contexts with people, to avoid animacy/inanimate confound in the results. The authors had a specific hypothesis concerning the role of the precuneus (PC) being sensitive to causal inferences about illnesses (that was preregistered).<br /> Findings indicate that implicit causal inferences are facilitated by semantic networks specialized for encoding causal knowledge.

Strengths:

The major strength of the study is the clever design of the stimuli (which are nicely matched for a number of features) which can tease apart the role of the type of causal inference (illness-causal or mechanical-causal) and the use of two localizers (logic/language and mentalizing) to investigate the hypothesis that the language and/or logical reasoning networks preferentially respond to causal inference regardless of the content domain being tested (illnesses or mechanical).

I think that authors' revisions of the original manuscript have strengthened the study. Overall, the paper provides an interesting contribution to the (rather new) field of study concerning the neural basis of implicit causal inference.

I see two weaknesses concerning the visualization of the data (which could be improved)

(1) Measures of dispersion are now provided for the average PSC in the critical window. It would be more appropriate to show the variance of the data also for the percentage signal changes (PSC) figures (e.g., 1A by using shaded lines providing SE around the means or boxplots at each timepoint).

(2) The authors could consider showing in Figure 2 the data of supplementary Figure 3. It is not clear why the authors report in the main manuscript the results of a subsample of participants (and only for this figure).

Reviewer #1 (Public review):

Summary:

The paper addresses the knowledge gap between the representation of goal direction in the central complex and how motor systems stabilize movement toward that goal. The authors focused on two descending neurons, DNa01 and 02, and showed that they play different roles in steering the fly toward a goal. They also explored the connectome data to propose a model to explain how these DNs could mediate response to lateralized sensory inputs. They finally used lateralized optogenetic activation/inactivation experiments to test the roles of these neurons in mediating turnings in freely walking flies.

Strengths:

The experiments are well-designed and controlled. The experiment in Figure 4 is elegant, and the authors put a lot of effort into ensuring that ATP puffs do not accidentally activate the DNs. They also have explained complex experiments well. I only have minor comments for the authors.

Comments on revisions:

I am happy with the revised manuscript and authors' response to our concerns. The addition of Figure S8, makes it more transparent and the revised text is now more accessible to the non-experts.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but has minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs is stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals chose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

Comments on revisions:

I am happy that the revised manuscript addresses all reviewers' concerns.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit-flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and show functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1). In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies which have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Fig. 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In-line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2). Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble (PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02 (Fig. 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring-attractor returns to its previous stable point.

(3). The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus directed turns.

Weaknesses:

While this study makes a compelling case for the importance of DNa02 in steering control, the role of DNa01 on the other hand seems unclear based on physiology, optogenetics perturbations as well as connectome analysis. DNa01 still remains a bit mysterious regarding both its role in controlling steering maneuvers as well as what in behavioral context it would be relevant.

Reviewer #3 (Public review):

Summary:

The hippocampal CA3 region is generally considered to be the primary site of initiation of sharp wave ripples-highly synchronous population events involved in learning and memory-although the precise mechanism remains elusive. A recent study revealed that CA3 comprises two distinct pyramidal cell populations: thorny cells that receive mossy fiber input from the dentate gyrus, and athorny cells that do not. That study also showed that it is athorny cells in particular which play a key role in sharp wave initiation. In the present work, Sammons, Masserini and colleagues expand on this by examining the connectivity probabilities among and between thorny and athorny cells. Using whole-cell patch clamp recordings, they find an asymmetrical connectivity pattern, with athorny cells receiving the most synaptic connections from both athorny and thorny cells, and thorny cells receiving fewer.

The authors then use a spiking network model to show how this assymmetrical connectivity is consistent with a preferential role of athorny cells in sharp wave initiation. Essentially, thorny and athorny cells are put into a winner-takes-all scenario in which athorny cells always win initially. Thorny cells can only become active after athorny cells decrease their firing rate due to adaptation, leading to a delay between the activation of athorny and thorny cells. As far as I understand, the initial victory of athorny cells in the winner-takes-all is doubly determined: it is both due to their intrinsic properties (lower rheobase and steeper f-I curve), and due to the bias in connectivity towards them. It appears to me that either of these two mechanisms (i.e., different intrinsic properties and symmetrical self- and cross-connections, or the same intrinsic properties and asymmetrical connectivity) would suffice to explain the sequential activation of the two cell types. From a theoretician's perspective, this overdetermination is not very elegant, but biology often isn't...

Strengths:

The authors provide independent validation of some of the findings by Hunt et al. (2018) concerning the distinction between thorny and athorny pyramidal cells in CA3 and advance our understanding of their differential integration in CA3 microcircuits. The properties of excitatory connections among and between thorny and athorny cells described by the authors will be key in understanding CA3 functions including, but not limited to, sharp wave initiation.

As stated in the paper, the modeling results lend support to the idea that the increased excitatory connectivity towards athorny cells plays an important role in causing them to fire before thorny cells in sharp waves. More generally, the model adds to an expanding pool of models of sharp wave ripples which should prove useful in guiding and interpreting experimental research.

Reviewer #1 (Public review):

Summary:

This study utilises fNIRS to investigate the effects of undernutrition on functional connectivity patterns in infants from a rural population in Gambia. fNIRS resting-state data recording spanned ages 5 to 24 months, while growth measures were collected from birth to 24 months. Additionally, executive functioning tasks were administered at 3 or 5 years of age. The results show an increase in left and right frontal-middle and right frontal-posterior connections with age and, contrary to previous findings in high-income countries, a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months of age. Additionally, the study describes some connectivity patterns, including stronger frontal interhemispheric connectivity, which is associated with better cognitive flexibility at preschool age.

Strengths:

- The study analyses longitudinal data from a large cohort (n = 204) of infants living in a rural area of Gambia. This already represents a large sample for most infant studies, and it is impressive, considering it was collected outside the lab in a population that is underrepresented in the literature. The research question regarding the effect of early nutritional deficiency on brain development is highly relevant and may highlight the importance of early interventions. The study may also encourage further research on different underrepresented infant populations (i.e., infants not residing in Western high-income countries) or in settings where fMRI is not feasible.

- The preprocessing and analysis steps are carefully described, which is very welcome in the fNIRS field, where well-defined standards for preprocessing and analysis are still lacking.

Weaknesses:

- While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level and investigates how restricted growth influences connectivity patterns at 24 months, it does not explore the links between adverse situations and developmental trajectories for functional connectivity. Considering the longitudinal nature of the dataset, it would have been interesting to apply more sophisticated analytical tools to link undernutrition to specific developmental trajectories in functional connectivity. The authors mention that they lack the statistical power to separate infants into groups according to their growing profiles. However, I wonder if this aspect could not have been better explored using other modelling strategies and dimensional reduction techniques. I can think about methods such as partial least squares correlation, with age included as a numerical variable and measures of undernutrition.

- Connectivity was asses in 6 big ROIs. While the authors justify this choice to reduce variability due to head size and optode placement, this also implies a significant reduction in spatial resolution. Individual digitalisation and co-registration of the optodes to the head model, followed by image reconstruction, could have provided better spatial resolution. This is not a weakness specific to this study but rather a limitation common to most fNIRS studies, which typically analyse data at the channel level since digitalisation and co-registration can be challenging, especially in complex setups like this. However, the BRIGHT project has demonstrated that it is possible and that differences in placement affect activation patterns, which become more localised when data is co-registered at the subject level (Collins-Jones et al., 2021). Could the co-registration of individual data have increased sensitivity, particularly given that longitudinal effects are being investigated?

- I believe that a further discussion in the manuscript on the application of global signal regression and its effects could have been beneficial for future research and for readers to better understand the negative correlations described in the results. Since systemic physiological changes affect HbO/HbR concentrations, resulting in an overestimation of functional connectivity, regressing the global signal before connectivity computation is a common strategy in fNIRS and fMRI studies. However, the recommendation for this step remains controversial, likely depending on the case (Murphy & Fox, 2017). I understand that different reasons justify its application in the current study. In addition to systemic physiological changes originating from brain tissue, fNIRS recordings are contaminated by changes occurring in superficial layers (i.e., the scalp and skull). While having short-distance channels could have helped to quantify extracerebral changes, challenges exist in using them in infant populations, especially in a longitudinal study such as the one presented here. The optimal source-detector distance that minimises sensitivity to changes originating from the brain would increase with head size, and very young participants would require significantly shorter source-detector distances (Brigadoi & Cooper, 2015). Thus, having them would have been challenging. Under these circumstances (i.e., lack of short channels and external physiological measures), and considering that the amount the signal is affected by physiological noise (either coming from the brain or superficial tissue) might change through development, the choice of applying global signal regression is justified. Nevertheless, since the method introduces negative correlations in the data by forcing connectivity to average to zero, I believe a further discussion of these points would have enriched the interpretation of the results.

Reviewer #2 (Public review):

Strengths:

The article addresses a topic of significant importance, focusing on early life growth faltering in low-income countries-a key marker of undernutrition-and its impact on brain functional connectivity (FC) and cognitive development. The study's strengths include the laborious data collection process, as well as the rigorous data preprocessing methods employed to ensure high data quality. The use of cutting-edge preprocessing techniques further enhances the reliability and validity of the findings, making this a valuable contribution to the field of developmental neuroscience and global health.

Weaknesses:

The study fails to fully leverage its longitudinal design to explore neurodevelopmental changes or trajectories, as highlighted by all three reviewers. The revised manuscript still primarily focuses on FC values at a single age stage (i.e., 24 months) rather than utilizing the longitudinal data to investigate how FC evolves over time or predicts cognitive development. Although the authors acknowledge that analyzing changes in FC (ΔFC) would reduce degrees of freedom (to ~30) and risk interpretability, they do not report or discuss these results, even as exploratory findings.

Furthermore, the study lacks specificity in identifying which specific brain networks are affected by growth faltering, as the current exploratory analyses mainly provide an overall conclusion that infant brain network development is impacted without pinpointing the precise neural mechanisms or networks involved.

Reviewer #3 (Public review):

Summary

This study aimed to investigate whether the development of functional connectivity (FC) is modulated by early physical growth, and whether these might impact cognitive development in childhood. This question was investigated by studying a large group of infants (N=204) assessed in Gambia with fNIRS at 5 visits between 5 and 24 months of age. Given the complexity of data acquisition at these ages and following data processing, data could be analyzed for 53 to 97 infants per age group. FC was analyzed considering 6 ensembles of brain regions and thus 21 types of connections. Results suggested that: i) compared to previously studied groups, this group of Gambian infants have different FC trajectory, in particular with a change in frontal inter-hemispheric FC with age from positive to null values; ii) early physical growth, measured through weight-for-length z-scores from birth on, is associated with FC at 24 months. Some relationships were further observed between FC during the first two years and cognitive flexibility, in different ways between 4- and 5-year-old preschoolers, but results did not survive corrections for multiple comparisons.

Strengths

The question investigated in this article is important for understanding the role of early growth and undernutrition on brain and behavioral development in infants and children. The longitudinal approach considered is highly relevant to investigate neurodevelopmental trajectories. Furthermore, this study targets a little studied population from a low-/middle-income country, which was made possible by the use of fNIRS outside the lab environment. The collected dataset is thus impressive and it opens up a wide range of analytical possibilities.

Weaknesses

- Data analyses were constrained by the limited number of children with longitudinal data on NIRS functional connectivity. Nevertheless, considering more advanced statistical modeling approaches would be relevant to further explore neurodevelopmental trajectories as well as relationships with early growth and later cognitive development.<br /> - The abstract and end of the discussion should make it clearer that the associations between FC and cognitive flexibility are results that need to be confirmed, insofar as they did not survive correction for multiple comparisons.

Reviewer #1 (Public review):

Summary:

Building on previous in vitro synaptic circuit work (Yamawaki et al., eLife 10, 2021), Piña Novo et al. utilize an in vivo optogenetic-electrophysiological approach to characterize sensory-evoked spiking activity in the mouse's forelimb primary somatosensory (S1) and motor (M1) areas. Using a combination of a novel "phototactile" somatosensory stimuli to the mouse's hand and simultaneous high-density linear array recordings in both S1 and M1, the authors report evoked activity in S1 was biased to middle layers, whereas it was biased to upper layers in M1. They report that M1 responses are delayed and attenuated relative to S1. Further analysis revealed a 20-fold difference in subcortical versus corticocortical propagation speeds. They also find that PV interneurons in S1 are strongly recruited by hand stimulation, and their selective activation can produce a suppression and rebound response similar to "phototactile" stimuli. Silencing S1 through local PV cells was sufficient to reduce M1 response to hand stimulation, suggesting S1 may directly drive M1 responses.

Strengths:

The study was technically well done, with convincing results. The data presented are appropriately analyzed. The author's findings build on a growing body of both in vitro and in vivo work examining the synaptic circuits underlying the interactions between S1 and M1. The paper is well-written and illustrated. Overall, the study will be valuable to those interested in forelimb S1-M1 interactions.

Weaknesses:

The authors have addressed my concerns

Reviewer #2 (Public review):

Summary:

Communication between sensory and motor corticies is likely to be important for many aspects of behavior, and in this study the authors carefully analyse neuronal spiking activity in S1 and M1 evoked by peripheral paw stimulation finding clear evidence for sensory responses in both cortical regions

Strengths:

The experiments and data analyses appear to have been carefully carried out and clearly represented.

Weaknesses:

The revised manuscript addressed the minor weaknesses I noted relating to the first submission.

Reviewer #3 (Public review):

Summary:

This is a solid study of stimulus-evoked neural activity dynamics in the feedforward pathway from mouse hand/forelimb mechanoreceptor afferents to S1 and M1 cortex. The conclusions are generally well supported and match expectations from previous studies of hand/forelimb circuits by this same group (Yamawaki et al., 2021), from the well-studied whisker tactile pathway to whisker S1 and M1, and from the corresponding pathway in primates. The study uses the novel approach of optogenetic stimulation of PV afferents in the periphery, which provides an impulse-like volley of peripheral spikes, which is useful for studying feedforward circuit dynamics. These are primarily proprioceptors, so results could differ for specific mechanoreceptor populations, but this is a reasonable tool to probe basic circuit activation. Mice are awake but not engaged in a somatosensory task, which is sufficient for the study goals.

The main results are: 1) brief peripheral activation drives brief sensory-evoked responses at ~ 15 ms latency in S1 and ~25 ms latency in M1, which is consistent with classical fast propagation on the subcortical pathway to S1, followed by slow propagation on the polysynaptic, non-myelinated pathway from S1 to M1; 2) each peripheral impulse evokes a triphasic activation-suppression-rebound response in both S1 and M1; 3) PV interneurons carry the major component of spike modulation for each of these phases; 4) activation of PV neurons in each area (M1 or S1) drives suppression and rebound both in the local area and in the other downstream area; 5) peripheral-evoked neural activity in M1 is at least partially dependent on transmission through S1.

All conclusions are well-supported and reasonably interpreted. There are no major new findings that were not expected from standard models of somatosensory pathways or from prior work in the whisker system.

Strengths:

This is a well-conducted and analyzed study in which the findings are clearly presented. The optogenetic sensory afferent stimulation method is novel and is well-suited for studying feedforward circuit dynamics. This study provides important baseline knowledge from which studies of more complex sensorimotor processing can build.

There are no further recommendations for the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors discovered MYL3 of marine medaka (Oryzias melastigma) as a novel NNV entry receptor, elucidating its facilitation of RGNNV entry into host cells through macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 pathway.

Strengths:

In this manuscript, the authors have performed in vitro and in vivo experiments to prove that MnMYL3 may serve as a receptor for NNV via macropinocytosis pathway. These experiments with different methods include Co-IP, RNAi, pulldown, SPR, flow cytometry, immunofluorescence assays and so on. In general, the results are clearly presented in the manuscript.

Weaknesses:

For the writing in the introduction and discussion sections, the author Yao et al mainly focus on the viral pathogens and fish in Aquaculture, the meaning and novelty of results provided in this manuscript are limited, not broad in biology. The authors should improve the likely impact of their work on the viral infection field, maybe also in the evolutionary field with fish model.

Additionally, detailed comments are as follows:

(1) Myosin is a big family, why did authors choose MYL3 as a candidate receptor for NNV?

(2) What's the relationship between MmMYL3 and MmHSP90ab1 and other known NNV receptors? Why dose NNV have so many receptors? Which one is supposed to serve as the key entry receptor?

(3) In vivo knockout of MYL3 using CRISPR-Cas9 should be conducted to verify whether the absence of MYL3 really inhibits NNV infection. Although it might be difficult to do it in marine medaka as stated by authors, the introduce of zebrafish is highly recommended, since it has already been reported that zebrafish could be served as a vertebrate model to study NNV (doi: 10.3389/fimmu.2022.863096).

(4) The results shown in Figure 6 are not enough to support the conclusion that "RGNNV triggers macropinocytosis mediated by MmMYL3". Additional electron microscopy of macropinosomes (sizes, morphological characteristics, etc.) will be a more direct evidence.

(5) MYL3 is "predominantly found in muscle tissues, particularly the heart and skeletal muscles". However, NNV is a virus mainly causes necrosis of nervous tissues (brain and retina). If MYL3 really acts as a receptor for NNV, how does it balance this difference so that nervous tissues, rather than muscle tissues, have the highest viral titers?

Comments on revisions:

The authors have addressed most of my concerns in the revised manuscript, but still one question need to further improve to strengthen the study's rationale and conclusions.

Specificity of MYL3 Selection:<br /> My previous question focused on why MYL3 was prioritized over other myosin family members. While the response broadly implicates myosins in viral entry, it does not justify why MYL3 was specifically chosen. For clarity, the "Introduction sections" should explicitly state the unique features of MYL3 (e.g., domain structure, binding affinity, or prior evidence linking it to NNV) that distinguish it from other myosins.

Reviewer #2 (Public review):

Summary:

The manuscript offers an important contribution to the field of virology, especially concerning NNV entry mechanisms. The major strength of the study lies in the identification of MmMYL3 as a functional receptor for RGNNV and its role in macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 signaling axis. This represents a significant advance in understanding NNV entry mechanisms beyond previously known receptors such as HSP90ab1 and HSC70. The data, supported by comprehensive in vitro and in vivo experiments, strongly justify the authors' claims about MYL3's role in NNV infection in marine medaka.

Strengths:

(1) The identification of MmMYL3 as a functional receptor for RGNNV is a significant contribution to the field. The study fills a crucial gap in understanding the molecular mechanisms governing NNV entry into host cells.

(2) The work highlights the involvement of IGF1R in macropinocytosis-mediated NNV entry and downstream Rac1/Cdc42 activation, thus providing a thorough mechanistic understanding of NNV internalization process. This could pave the way for further exploration of antiviral targets.

Comments on revisions:

The authors have addressed the concerns from reviewers. This manuscript can be published in the current form.

Reviewer #3 (Public review):

Summary:

The manuscript presents a detailed study on the role of MmMYL3 in the viral entry of NNV, focusing on its function as a receptor that mediates viral internalization through the macropinocytosis pathway. The use of both in vitro assays (e.g., Co-IP, SPR, and GST pull-down) and in vivo experiments (such as infection assays in marine medaka) adds robustness to the evidence for MmMYL3 as a novel receptor for RGNNV. The findings have important implications for understanding NNV infection mechanisms, which could pave the way for new antiviral strategies in aquaculture.

Strengths:

The authors show that MmMYL3 directly binds the viral capsid protein, facilitates NNV entry via the IGF1R-Rac1/Cdc42 pathway, and can render otherwise resistant cells susceptible to infection. This multifaceted approach effectively demonstrates the central role of MmMYL3 in NNV entry.

Comments on revisions:

The implemented revisions have remarkably improved the manuscript's conceptual clarity, scientific depth, and methodological rigor. Through comprehensive addressing of issue with meticulous attention to detail, the authors have produced a substantially strengthened manuscript that demonstrates enhanced experimental validity and theoretical coherence. No additional revisions appear necessary at this stage.

Reviewer #1 (Public review):

Summary:

Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely to remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.

Strengths:

The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability the move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.

Weaknesses:

No major weaknesses noted.

Reviewer #2 (Public review):

Summary:

In this work, the investigators isolated one Lacticaseibacillus rhamnosus strain (P118), and determined this strain worked well against Salmonella Typhimurium infection. Then, further studies were performed to identify the mechanism of bacterial resistance, and a list of confirmatory assays were carried out to test the hypothesis.

Strengths:

The authors provided details regarding all assays performed in this work, and this reviewer trusted that the conclusion in this manuscript is solid. I appreciate the efforts of the authors to perform different types of in vivo and in vitro studies to confirm the hypothesis.

Weaknesses:

I have mainly two questions for this work.

Main point-1:<br /> The authors provided the below information about the sources from which Lacticaseibacillus rhamnosus was isolated. More details are needed. What are the criteria to choose these samples? Where were these samples originate from? How many strains of bacteria were obtained from which types of samples?

Lines 486-488: Lactic acid bacteria (LAB) and Enterococcus strains were isolated from the fermented yoghurts collected from families in multiple cities of China and the intestinal contents from healthy piglets without pathogen infection and diarrhoea by our lab.

Lines 129-133: A total of 290 bacterial strains were isolated and identified from 32 samples of the fermented yoghurt and piglet rectal contents collected across diverse regions within China using MRS and BHI medium , which consist s of 63 Streptococcus strains, 158 Lactobacillus/ Lacticaseibacillus Limosilactobacillus strains and 69 Enterococcus strains.

Main-point-2:<br /> As probiotics, Lacticaseibacillus rhamnosus has been widely studied. In fact, there are many commercially available products, and Lacticaseibacillus rhamnosus is the main bacteria in these products. There are also ATCC type strain such as 53103.

I am sure the authors are also interested to know if P118 is better as a probiotics candidate than other commercially available strains. Also, would the mechanism described for P118 apply to other Lacticaseibacillus rhamnosus strains?

It would be ideal if the authors could include one or two Lacticaseibacillus rhamnosus which are currently commercially used, or from the ATCC. Then, the authors can compare the efficacy and antibacterial mechanisms of their P118 with other strains. This would open the windows for future work.

Reviewer #1 (Public review):

Shin et al. conduct extensive electrophysiological and behavioral experiments to study the mechanisms of short-term synaptic plasticity at excitatory synapses in layer 2/3 of the rat medial prefrontal cortex. The authors interestingly find that short-term facilitation is driven by progressive overfilling of the readily releasable pool, and that this process is mediated by phospholipase C/diacylglycerol signaling and synaptotagmin-7 (Syt7). Specifically, knockdown of Syt7 not only abolishes the refilling rate of vesicles with high fusion probability, but it also impairs the acquisition of trace fear memory.

Overall, the authors offer novel insight to the field of synaptic plasticity through well-designed experiments that incorporate a range of techniques.

Comments on revisions:

The authors have adequately addressed my earlier comments and questions.

Reviewer #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model and is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks. The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths:

This is a very thorough study, addressing the research question from many different angles and the experimental execution is superb. The impact of the work is high, as it applies recent models of short term plasticity behavior to in vivo circuits further providing insights how synapses provide dynamic control to enable working memory related behavior through non-permanent changes in synaptic output.

Weaknesses:

While this work is carefully examined and the results are presented and discussed in a detailed manner, the reviewer is still not fully convinced that regulation of release probability is not a putative contributor to the observed behavior. No additional work is needed, but in the moment, I am not convinced that changes in release probability are not in play. One solution may be to extend the discussion of changes in rules probability as an alternative.

Fig 3. I am confused about the interpretation of the Mean Variance analysis outcome. Since the data points follow the curve during induction of short term plasticity, doesn't these suggests that release probability and not the pool size increases? Related, to measure the absolute release probability and failure rate using the optogenetic stimulation technique is not trivial as the experimental paradigm bias the experiment to a given output strength, and therefore a change in release probability cannot be excluded.

Fig. 4B interprets the phorbol ester stimulation to be the result of pool overfilling, however, phorbol ester stimulation has also been shown to increase release probability without changing the size of the readily releasable pool. The high frequency of stimulation may occlude a increased paired pulse depression in presence of OAG, that others have interpreted in mammalian synapses as an increase in release probability.

The literature on Syt7 function is still quite controversial. An observation in the literature that loss of Syt7 function in the fly synapse leads to an increase of release probability. Thus the observed changes in short term plasticity characteristics in the Syt7 KD experiments may contain a release probability component. Can the authors really exclude this possibility? Figure 5 shows for the Syt7 KD group a very prominent depression of the EPSC/IPSC with the second stimulus, particularly for the short interpulse intervals, usually a strong sign of increased release probability, as lack of pool refilling can unlikely explain the strong drop in synaptic output.

Comments on revisions:

I am satisfied with the reply of the authors and I do not have any further points of concern.

Reviewer #3 (Public review):

The results are consistent with the main claim that facilitation is caused by overfilling a readily releasable pool, but alternative interpretations continue to seem more likely, especially when the current results are taken together with previous studies. Key doubts could be resolved with a single straightforward experiment (see below).

The central issue is the interpretation of paired pulse depression that occurs when the interval between action potentials is 25 ms, but not when 50. To summarize: a similar phenomenon was observed at Schaffer collateral synapses (Dobrunz and Stevens, 1997), but was interpreted as evidence for a decrease in pv. Ca2+-channel inactivation was proposed as the mechanism, but this was not proven. The key point for evaluating the current study is that Dobrunz and Stevens specifically ruled out the kind of decrease in pocc that is the keystone premise of the current study because the depression occurred independently of whether or not the first action potential elicited exocytosis. Of course, the mechanism might be different at layer 2/3 cortical synapses. But, it seems reasonable to hope that the older hypothesis would be ruled out for the cortical synapses before concluding that the new hypothesis must be correct.

The old and new hypotheses could be distinguished from each other cleanly with a straightforward experiment. Most/maybe all central synapses strengthen a great amount when extracellular Ca2+ is increased from 1.3 to 2 mM, even when intracellular Ca2+ is buffered with EGTA. According to the authors' model, this is only possible when pv is low, and so could not occur at synapses between layer 2/3 neurons. Because of this, confirmation that increasing extracellular Ca2+ does not change synaptic strength would support the hypothesis that baseline pv is high, as the authors claim, and the support would be impressive because large changes have been seen at every other type of synapse where this has been studied (to my knowledge at least). In contrast, the Ca2+ imaging experiment that has been added to the new version of the manuscript does not address the central issue because a wide range of mechanisms could, in principle, decrease release without involving prior exocytosis or altering bulk Ca2+ signals, including: a small decrease in nano-domain Ca2+, which wouldn't be detected because nano-domains contribute a minuscule amount to the bulk signal during Ca2+-imaging; or even very fast activity-dependent undocking of synaptic vesicles, which was reported in the same Kusick et al, 2020 study that is central to the LS/TS terminology adopted by the authors.

Additional points:

(1) A new section in the Discussion (lines 458-475) suggests that previous techniques employed to show that augmentation and facilitation are caused by increases in pv did not have the resolution to distinguish between pv and pocc, but this is misleading. The confusion might be because the terminology has changed, but this is all the more reason to clarify this section. The previous evidence for increases in pv - and against increases in pocc - is as follows: The residual Ca2+ that drives augmentation decreases the latency between the onset of hypertonic solution and onset of the postsynaptic response by about 150 ms, which is large compared to the rise time of the response. The decrease indicates that the residual Ca2+ drives a decrease in the energy barrier that must be overcome before readily releasable vesicles can undergo exocytosis, which is precisely the type of mechanism that would enhance pv. In contrast, an increase in pocc could change the rise time, but not the latency. There is a small change in the rise time, but this could be caused by changes in either pv or pocc, and one of the studies (Garcia-Perez and Wesseling, 2008) showed that augmentation occluded facilitation, even at times when pocc was reduced by a factor of 3, which would seem to argue against parallel increases in both pv and pocc.

(2) Similar evidence from hypertonic stimulation indicates that Phorbol esters increase pv, but I am not aware of evidence ruling out a parallel increase in pocc.

Comments on revisions:

There are at least two straightforward ways to address the main concern.

The first would be experiments analogous to those in Dobrunz and Stevens that show that - unlike at Schaffer collateral synapses - paired pulse depression at L2/3 synapses requires neurotransmitter release. I proposed this in the first round, but realized since that a simpler and more powerful strategy would be to test directly that pv is/is-not near 1.0 in 1.2 mM Ca2+ simply by increasing to 2 mM Ca2+ (and showing that synaptic strength does-not/does change). This would be powerful because the increase in Ca2+ greatly increases synaptic strength at Schaffer collaterals by about 2.5-fold. Concerns about a confounding elevation in the basal intracellular Ca2+ concentration could be easily neutralized by pre-treating with EGTA-AM, which the authors have already done for other experiments.

Reviewer #1 (Public review):

Summary:

This study provides comprehensive instructions for using the chromatophore tracking software, Chromas, to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. This software addresses a long-standing challenge faced by many researchers who study these soft-bodied creatures, known for their remarkable ability to change colour rapidly. The updated software features a user-friendly interface that can be applied to a wide range of applications, making it an essential tool for biologists focused on animal dynamic signalling. It will also be of interest to professionals in the fields of computer vision and image analysis.

Strengths:

This work provides detailed instructions for this toolkit along with examples for potential users to try. The Gitlab inventory hosts the software package, installation documentation, and tutorials, further helping potential users with a less steep learning curve.

Weaknesses:

The evidence supporting the authors' claims is solid, particularly demonstrated through the use of cuttlefish and squid. However, it may not be applicable to all coleoid cephalopods yet, such as octopuses, which have an incredibly versatile ability to change their body forms.

Reviewer #2 (Public review):

Summary:

The authors developed a computational pipeline named CHROMAS to track and analyze chromatophore dynamics, which provides a wide range of biological analysis tools without requiring the user to write code.

Strengths:

(1) CHROMAS is an integrated toolbox that provides tools for different biological tasks such as: segment, classify, track and measure individual chromatophores, cluster small groups of chromatophores, analyze full-body patterns, etc.

(2) It could be used to investigate different species. The authors have already applied it to analyze the skin of the bobtail squid Euprymna berryi and the European cuttlefish Sepia officinalis.

(3) The tool is open-source and easy to install. The paper describes in detail the command format to complete each task and provides relevant sample figures.

Weaknesses:

(1) The generality and robustness of the proposed pipeline need to be verified through more experimental evaluations. For example, the implementation algorithm depends on relatively specific or obvious image features, clean backgrounds, and objects that do not move too fast.

(2) The pipeline lacks some kind of self-correction mechanism. If at one moment there is a conflicting match with the previous frames, how does the system automatically handle it to ensure that the tracking results are accurate over a long period of time?

Reviewer #1 (Public review):

This paper presents a set of tools that will pave the way for a comprehensive understanding of the circuits that control wing motion in flies during flight or courtship. These tools are mainly focused on wing motor neurons and interneurons, as well as a few motor neurons of the haltere. This paper and the library of driver lines described within it will serve as a crucial resource in the pursuit of understanding how neural circuits give rise to behavior. Overall, I found the paper well-written, the figures are quite nice, and the data from the functional experiments convincing. I do not have many major concerns, but a few suggestions that I think will make the paper easier to understand.

I think the introduction could use some reorganization, as right now I found it quite difficult to follow. For example, lines 85-88 seem to fit more naturally at the end of the next paragraph, compared to the current location of those sentences, which feels rather disjointed. I would suggest introducing the organization of the wing motor system (paragraphs 3 and 4) and then discussing the VNC (paragraph 2) before moving on to describe the neurons within the VNC that may control wing motion. Additionally, lines 141-144, which describe the broad subdivisions of the VNC, can be moved up to where the VNC is first introduced.

One of my major takeaways from the paper is the call to examine the premotor circuits that govern wing motion. For that reason, I was surprised that there was little mention of the role of sensory input to these circuits. As the authors point out in the discussion, the haltere, for example, provides important input to the wing steering system. I recognize that creating driver lines for the sensory neurons that innervate the VNC is well beyond the scope of this project. I would just like some clarification in the text of the role these inputs play in structuring wing motion, especially as some act at rapid timescales that possibly forgo processing by the very circuits detailed here. This brings up a related issue: if the roles of the interneurons that are presynaptic to the wing motor neurons are "largely unexplored," with how much confidence can we say that they are the key for controlling behavior? To be sure, this has been demonstrated quite nicely in the case of courtship, but in flight, I think the evidence supporting this argument is less clear. I suggest the authors rephrase their language here.

Reviewer #2 (Public review):

Summary:

In this study, the researchers generated an impressive collection of sparse split GAL4 driver lines that target wing-relevant cell types. They then characterized the cell types according to function, development, and morphology. This resource is a necessary companion to the fly ventral nerve cord connectomes. The fly connectomes enable biologically-constrained hypothesis generation, but we need genetic reagents like the ones generated here in order to test those hypotheses and understand the biological limits of what we can learn from connectomes. This project identifies wing-relevant cell types and generates a library of driver lines to provide genetic access to small populations of these cell types. The study also characterizes these cell types according to developmental lineage and morphology, and performs functional analyses on some of the cell types, including single pairs of motor neurons.

Strengths:

The genetic toolkit that the authors produce is rigorous and well-documented, and will be broadly useful. Further, they bolster the utility of the resource by characterizing cell types according to developmental lineage, morphology, and connectome nomenclature. The authors successfully produce a foundation for future studies of the functional organization of neural circuits. In particular, the driver lines created in this study match the specificity of the connectome, providing a necessary resource to functionally test predictions from the connectomes.

Weaknesses:

The manuscript includes several broad statements about certain questions being "unexplored" (e.g., lines 71, 129). However, the authors cite papers (e.g., Harris 2015 and Lillvis 2024) that directly address these topics. To better support the narrative, it would be helpful to more accurately summarize the key findings from these prior studies. For example, the Harris paper found behavioral correlates of hemilineage activation. By using the sparse toolkit you have created, it may be possible to dissect behavior into finer-grained modules or specific movements, providing deeper insight into how complex behaviors are produced by the nervous system.

There are a few places where the current manuscript does not acknowledge the post-connectome universe it now exists in. For example, line 600: the morphology of DVMns in Drosophila had never been described, and line 762: revealed diversity within hemilineages which had not previously been reported. Although this manuscript was in progress before the VNC connectomes were released, they are now published, and the current manuscript should reflect this development.

The authors focus on some well-characterized "Named Neurons" e.g., ChINs and the PSI. This focused approach makes sense, but the authors miss the opportunity to point out a major strength of the toolkit they have produced: we are now less constrained by studying only these Named Neurons. With this new resource, we have genetic access to sparse sets of neurons that are likely just as important but were previously inaccessible.

Reviewer #3 (Public review):

Summary:

This paper provides a catalogue of 195 well-documented Drosophila strains with sparse and cell-type-specific GAL-4 expression in the adult ventral nerve cord (VNC). The focus is on motor neurons, interneurons, and modulatory unpaired neurons in the dorsal VNC neuropils that drive motor control of the wings. Intersegmental sensory and interneurons are not included. The expression patterns of all 195 fly strains are exceptionally well-characterized and catalogized. Compelling links to hemi-lineages and connectomics data are well documented, and some solid functional data demonstrate the applicability of the GAL4 strains in genetic silencing and optogenetic activation experiments. In sum, this catalogue provides a fantastic toolkit for future functional analyses of motor control centers in the dorsal VNC.

Strengths:

Particularly noteworthy is that the authors did a tremendous job in identifying and catalogizing the correspondences between the neurons in their catalogue and the MANC connectomics dataset, as well as with the respective hemi-lineages. The catalogue has been generated with exquisite care and is impressive.

Weaknesses:

There are no significant weaknesses. I have only minor recommendations on definitions and naming of neuron types, the text on the optogenetic experiments, and the comparison to other insects.

Reviewer #1 (Public review):

Summary:

The authors validate the contribution of RAP2A to GB progression. RAp2A participates in asymmetric cell division, and the localization of several cell polarity markers, including cno and Numb.

Strengths:

The use of human data, Drosophila models, and cell culture or neurospheres is a good scenario to validate the hypothesis using complementary systems.

Moreover, the mechanisms that determine GB progression, and in particular glioma stem cells biology, are relevant for the knowledge on glioblastoma and opens new possibilities to future clinical strategies.

Weaknesses:

While the manuscript presents a well-supported investigation into RAP2A's role in GBM, several methodological aspects require further validation. The major concern is the reliance on a single GB cell line (GB5), which limits the generalizability of the findings. Including multiple GBM lines, particularly primary patient-derived 3D cultures with known stem-like properties, would significantly enhance the study's relevance.

Additionally, key mechanistic aspects remain underexplored. Further investigation into the conservation of the Rap2l-Cno/aPKC pathway in human cells through rescue experiments or protein interaction assays would be beneficial. Similarly, live imaging or lineage tracing would provide more direct evidence of ACD frequency, complementing the current indirect metrics (odd/even cell clusters, Numb asymmetry).

Several specific points raised in previous reviews still require attention:

(1) The specificity of Rap2l RNAi needs further confirmation. Is Rap2l expressed in neuroblasts or intermediate neural progenitors? Can alternative validation methods be employed?

(2) Quantification of phenotypic penetrance and survival rates in Rap2l mutants would help determine the consistency of ACD defects.

(3) The observations on neurosphere size and Ki-67 expression require normalization (e.g., Ki-67+ cells per total cell number or per neurosphere size). Additionally, apoptosis should be assessed using Annexin V or TUNEL assays.

(4) The discrepancy in Figures 6A and 6B requires further discussion.

(5) Live imaging of ACD events would provide more direct evidence.

(6) Clarification of terminology and statistical markers (e.g., p-values) in Figure 1A would improve clarity.

(7) Given the group's expertise, an alternative to mouse xenografts could be a Drosophila genetic model of glioblastoma, which would provide an in vivo validation system aligned with their research approach.

Reviewer #2 (Public review):

This study investigates the role of RAP2A in regulating asymmetric cell division (ACD) in glioblastoma stem cells (GSCs), bridging insights from Drosophila ACD mechanisms to human tumor biology. They focus on RAP2A, a human homolog of Drosophila Rap2l, as a novel ACD regulator in GBM is innovative, given its underexplored role in cancer stem cells (CSCs). The hypothesis that ACD imbalance (favoring symmetric divisions) drives GSC expansion and tumor progression introduces a fresh perspective on differentiation therapy. However, the dual role of ACD in tumor heterogeneity (potentially aiding therapy resistance) requires deeper discussion to clarify the study's unique contributions against existing controversies. Some limitations and questions need to be addressed.

(1) Validation of RAP2A's prognostic relevance using TCGA and Gravendeel cohorts strengthens clinical relevance. However, differential expression analysis across GBM subtypes (e.g., MES, DNA-methylation subtypes ) should be included to confirm specificity.

(2) Rap2l knockdown-induced ACD defects (e.g., mislocalization of Cno/Numb) are well-designed. However, phenotypic penetrance and survival rates of Rap2l mutants should be quantified to confirm consistency.

(3) While GB5 cells were effectively used, justification for selecting this line (e.g., representativeness of GBM heterogeneity) is needed. Experiments in additional GBM lines (especially the addition of 3D primary patient-derived cell lines with known stem cell phenotype) would enhance generalizability.

(4) Indirect metrics (odd/even cell clusters, NUMB asymmetry) are suggestive but insufficient. Live imaging or lineage tracing would directly validate ACD frequency.

(5) The initial microarray (n=7 GBM patients) is underpowered. While TCGA data mitigate this, the limitations of small cohorts should be explicitly addressed and need to be discussed.

(6) Conclusions rely heavily on neurosphere models. Xenograft experiments or patient-derived orthotopic models are critical to support translational relevance, and such basic research work needs to be included in journals.

(7) How does RAP2A regulate NUMB asymmetry? Is the Drosophila Rap2l-Cno/aPKC pathway conserved? Rescue experiments (e.g., Cno/aPKC knockdown with RAP2A overexpression) or interaction assays (e.g., Co-IP) are needed to establish molecular mechanisms.

(8) Reduced stemness markers (CD133/SOX2/NESTIN) and proliferation (Ki-67) align with increased ACD. However, alternative explanations (e.g., differentiation or apoptosis) must be ruled out via GFAP/Tuj1 staining or Annexin V assays.

(9) The link between low RAP2A and poor prognosis should be validated in multivariate analyses to exclude confounding factors (e.g., age, treatment history).

(10) The broader ACD regulatory network in GBM (e.g., roles of other homologs like NUMB) and potential synergies/independence from known suppressors (e.g., TRIM3) warrant exploration.

(11) The figures should be improved. Statistical significance markers (e.g., p-values) should be added to Figure 1A; timepoints/culture conditions should be clarified for Figure 6A.

(12) Redundant Drosophila background in the Discussion should be condensed; terminology should be unified (e.g., "neurosphere" vs. "cell cluster").

Reviewer #1 (Public review):

Summary:

Cording et al. investigated how deletion of CNTNAP2, a gene associated with autism spectrum disorder, alters corticostriatal engagement and behavior. Specifically, the authors present slice electrophysiology data showing that striatal projection neurons (SPNs) are more readily driven to fire action potentials in response to stimulation of corticostriatal afferents, and this is due to increases in SPN intrinsic excitability rather than changes in excitatory or inhibitory synaptic inputs. Specifically, these changes seem to be due to preferential reduction of Kv1.2 in dSPNs. The authors separately show that CNTNAP2 mice display repetitive behaviors, enhanced motor learning and cognitive inflexibility. Overall, the authors' conclusions are supported by their data, but a few claims could use some more evidence to be convincing.

Strengths:

The use of multiple behavioral techniques, both traditional and cutting-edge machine learning-based analyses, provides a powerful means of assessing repetitive behaviors and behavioral transitions/rigidity. Characterization of both excitatory and inhibitory synaptic responses in slice electrophysiology experiments offers a broad survey of the synaptic alterations that may lead to increased corticostriatal engagement of SPNs.

Weaknesses:

As it stands, the reported changes in dorsolateral striatum SPN excitability are only correlative with reported changes in repetitive behaviors, motor learning and cognitive flexibility. The authors do broach this in the text (particularly in "Limitations and future directions").

Reviewer #2 (Public review):

Summary:

This is an important study characterizing striatal dysfunction and behavioral deficits in Cntnap2-/- mice. There is growing evidence suggesting that striatal dysfunction underlies core symptoms of ASD but the specific cellular and circuit level abnormalities disrupted by different risk genes remain unclear. This study addresses how deletion of Cntnap2 affects the intrinsic properties and synaptic connectivity of striatal spiny projection neurons (SPN) of the direct (dSPN) and indirect (iSPN) pathways. Using Thy1-ChR2 mice and optogenetics the authors found increased firing of both types of SPNs in response to cortical afferent stimulation. However, there was no significant difference in the amplitude of optically-evoked excitatory postsynaptic currents (EPSCs) or spine density between Cntnap2-/- and WT SPNs, suggesting that the increased corticostriatal coupling might be due to changes in intrinsic excitability. Indeed, the authors found Cntnap2-/- SPNs, particularly dSPNs, exhibited higher intrinsic excitability, reduced rheobase current and increased membrane resistance compared to WT SPNs. The enhanced spiking probability in Cntnap2-/- SPNs is not due to reduced inhibition. Despite previous reports of decreased parvalbumin-expressing (PV) interneurons in various brain regions of Cntnap2-/- mice, the number and function (IPSC amplitude and intrinsic excitability) of these interneurons in the striatum were comparable to WT controls.

This study also includes a comprehensive behavioral analysis of striatal related behaviors. Cntnap2-/- mice demonstrated increased repetitive behaviors (RRBs), including more grooming bouts, increased marble burying, and increased nose poking in the holeboard assay. MoSeq analysis of behavior further showed signs of altered grooming behaviors and sequencing of behavioral syllables. Cntnap2-/- mice also displayed cognitive inflexibility in a four-choice odor-based reversal learning assay. While they performed similarly to WT controls during acquisition and recall phases, they required significantly more trials to learn a new odor-reward association during reversal, consistent with potential deficits in corticostriatal function.

Strengths:

This study provides significant contributions to the field. The finding of altered SPN excitability, the detailed characterization of striatal inhibition, and the comprehensive behavioral analysis are novel and valuable to understand the pathophysiology of Cntnap2-/- mice.

Weaknesses:

All my concerns were addressed in the revised version of the manuscript

Reviewer #3 (Public review):

Summary:

The authors analyzed Cntnap2 KO mice to determine whether loss of the ASD risk gene CNTNAP2 alters the dorsal striatum's function.

Strengths:

The results demonstrate that loss of Cntnap2 results in increased excitability of striatal projection neurons (SPNs) and altered striatal-dependent behaviors, such as repetitive, inflexible behaviors. Unlike other brain areas and cell types, synaptic inputs onto SPNs were normal in Cntnap2 KO mice. The experiments are well-designed, and the results support the authors' conclusions.

Weaknesses:

The mechanism underlying SPN hyperexcitability was not explored, and it is unclear whether this cellular phenotype alone can account for the behavioral alterations in Cntnap2 KO mice. No clear explanation emerges for the variable phenotype in different brain areas and cell types.

Comments on revisions:

The authors have appropriately addressed all my comments. In my opinion, no further changes are required.

Reviewer #1 (Public review):

I was glad to see that the other reviewer and I had similar takeaways on the subjects of historical literature and paedomorphism. While the authors have adequately considered a more historical body of literature, they have not addressed the concerns we had with statements about paedomorphism. I'm inclined to agree with the other reviewer that the discussion on paedomorphism should be cut entirely. My comments below are to seek clarity and make sure you are saying what you intend to say.

Strengths:

Table 1 is the beginning of a useful glossary and possible character definitions with character states that can be coded for phylogenetic analyses. This is particularly important because the goal of the paper is to define terms for chondrichthyan skeletal features in order to unify research questions in the field, and add novel data on how these features might be distributed among chondrichthyan clades, starting with ratfish and little skate.

Opportunities:

Table 1 should be translated into a format reflecting 0s and 1s etc that can be coded and referred back to the matrix that is in Figure 7 (or a standalone matrix as an appendix). Right now, they do not correspond and thus it is challenging to follow and interpret the mapped characters on the tree. You are presuming reversals when you could just list the state and let the data show you the possibility of character transitions.

Figure 1 essentially shows two datapoints Holocephali and Elasmobranchi where holocephali have low TMD and Elasmobranchi have high TMD, therefore nothing directional from an ancestral to derived state. Also, because you drop the catshark from later figures/analysis, you are treating the ratfish and the little skate as sister taxa so you cannot determine which is paedomorphic and which is peramorphic. Unfortunately, the position of where the characters were mapped on Figure 7 is not able to help you determine ancestral states and therefore actually test for paedomorphism. Two sister taxa with two different conditions and no outgroup doesn't explain the TMD in Ratfishes is statistically different from that of little skates. But there is no direction. So you need to be able to reconstruct that state.

Paedomorphosis implies juvenile ancestral organization in actual existing adult stages of modern descendants. You haven't shown that yet. Only that there might be different rates of mineralization in little skates. I suggested that you datamine the literature for other stages if you think you can fill in gaps.

In the response to reviewers, the authors stated that: "... we had reported that the TMD of centra from little skate did significantly increase between stage 32 and 33. Supporting our argument that ratfish had features of little skate embryos, TMD of adult ratfish centra was significantly lower than TMD of adult skate centra (Fig 1). Also, it was significantly higher than stage 33 skate centra, but it was statistically indistinguishable from that of stage 33 and juvenile stages of skate centra. While we do agree that more samples from these and additional groups would bolster these data, we feel they are sufficiently powered to support our conclusions for this current paper."

I will respond to that. In Figure 6L, yes, A little skate stage 33 is significantly different than stage 32, though the SD bars for ratfish appear to overlap with the range for little skate 32. Also, ratfish values are not significantly different than state 33 or juvenile ratfish. You can add the adult little skate data from figures 1 to 6L and then state, "centra of adult ratfish have a TDM within the range of juvenile LS33 little skates." As per conversation earlier, it still doesn't account for paedomorphism, however, it does indicate different amounts of mineralization, and could indicate you hypothesize about rates of mineralization. I think you have a different discussion waiting to replace this one.

Reviewer #2 (Public review):

General comment:

This is a very valuable and unique comparative study. An excellent combination of scanning and histological data from three different species is presented. Obtaining the material for such a comparative study is never trivial. The study presents new data and thus provides the basis for an in-depth discussion about chondrichthyan mineralised skeletal tissues.

Comments on revised version:

The manuscript has been revised and improved and can be published. A very nice manuscript, indeed. My only recommendation (point of discussion, not a requirement) would still be to think about the claim of paedomorphosis in a holocephalan.

Within the chondrichthyes, how distant holocephali are in relation to elasmobranchii remains uncertain, holocephali are quite a specialised group. Holocephali are also older than Batoidea and Selachii. As paedomorphosis is a derived character, I imagine it is difficult to establish that development in an extant holocephalan is derived compared to development in elasmobranchii. If this type of development would have been typical for the "older" holocephali it would not be paedomorphic. Also, the uncertainty how distant holocephali are from elasmobranchii makes it difficult to identify paedomorphosis with reference to chondrichthyes.

Joint Public Review:

Carabalona and colleagues investigated the role of the membrane-deforming cytoskeletal regulator protein Abba (MTSS1L/MTSS2) in cortical development to better understand the mechanisms of abnormal neural stem cell mitosis. The authors used short hairpin RNA targeting Abba20 with a fluorescent reporter coupled with in utero electroporation of E14 mice to show changes to neural progenitors. They performed flow cytometry for in-depth cell cycle analysis of Abba-shRNA impact to neural progenitors and determined an accumulation in S phase. Using culture rat glioma cells and live imaging from cortical organotypic slides from mice in utero electroporated with Abba-shRNA, the authors found Abba played a prominent role in cytokinesis. They then used a yeast-two-hybrid screen to identify three high confidence interactors: Beta-Trcp2, Nedd9, and Otx2. They used immunoprecipitation experiments from E18 cortical tissue coupled with C6 cells to show Abba requirement for Nedd9 localization to the cleavage furrow/cytokinetic bridge. The authors performed an shRNA knockdown of Nedd9 by in utero electroporation of E14 mice and observed similar results as with the Abba-shRNA. They tested a human variant of Abba using in utero electroporation of cDNA and found disorganized radial glial fibers and misplaced, multipolar neurons, but lacked the impact of cell division seen in the shRNA-Abba model.

[Editors' note: the authors have responded to two sets of reviews, which can be found here, https://doi.org/10.7554/eLife.92748.2, and here, https://doi.org/10.7554/eLife.92748.1]

Reviewer #1 (Public review):

Summary:

Qi and colleagues investigated the role of Kallistatin pathway in increasing hippocampal amyloid-β plaques accumulation and tau hyperpholphorylation in Alzheimer's disease, linking the increased Kallistatin level in diabetic patients with a higher risk of Alzheimer's disease development. A Kallistatin overexpressing animal model was utilized, and memory impairment was assessed using Morris water maze and Y-maze. Kallistatin-related pathway protein levels were measured in the hippocampus, and phenotypes were rescued using fenofibrate and rosiglitazone. The current study provides evidence of a novel molecular mechanism linking diabetes and Alzheimer's disease, and suggests the potential use of fenofibrate to alleviate memory impairment. However, several issues need to be addressed before further consideration.

Strengths:

The finding of this study is novel. The finding will have great impacts on diabetes and AD research. The studies were well conducted, and results convincing.

Weaknesses:

(1) The mechanism by which fenofibrate rescues memory loss in Kallistatin-transgenic mice is unclear. As a PPARα agonist, does fenofibrate target the Kallistatin pathway directly or indirectly? Please provide discussion based on literature supporting either possibility.<br /> (2) The current study exclusively investigated hippocampus. What about other cognitive memory-related regions, such as prefrontal cortex? Including data from these regions or discussing the possibility of their involvement could provide a more comprehensive understanding of the role of Kallistatin in memory impairment.<br /> (3) Fenofibrate rescued phenotypes in Kallistatin-transgenic mice while rosiglitazone, a PPARα agonist, did not. This result contradicts the manuscript's emphasis on a PPARα-associated mechanism. Please address this inconsistency.<br /> (4) Most of the immunohistochemistry images are unclear. Inserts have similar magnification to the original representative images, making judgments difficult. Please provide larger inserts with higher resolution.<br /> (5) The immunohistochemistry images in different figures were taken from different hippocampal subregions with different magnifications. Please maintain consistency, or explain why CA1, CA3 or DG was analyzed in each experiment.<br /> (6) Figure 5B is missing a title. Please add a title to maintain consistency with other graphs.<br /> (7) Please list statistical methods used in the figure legends, such as t-test or One way ANOVA with post-hoc tests.

Comments on revisions:

The authors have addressed the issues raised from the review. The manuscript has been revised accordingly.

Reviewer #2 (Public review):

Summary:

The study links Alzheimer's disease (AD) with metabolic disorders through elevated Kallistatin levels in AD patients. Kallistatin-overexpressing mice show cognitive decline, increased Aβ and tau pathology, and impaired hippocampal function. Mechanistically, Kallistatin enhances Aβ production via Notch1 and promotes tau phosphorylation through GSK-3β activation. Fenofibrate improves cognitive deficits by reducing Aβ and tau phosphorylation in these mice, suggesting therapeutic potential in AD linked to metabolic syndromes.

Strengths:

This study presents a novel insights into Alzheimer's disease (AD) pathogenesis and provides strong evidences about the mechanistic roles of Kallistatin and the therapeutic potential of fenofibrate in AD.

It was suggested that Kallistatin is primarily produced by the liver. The study demonstrates increased Kallistatin levels in the hippocampus tissue of AD mice. They also found that Kallistatin is also increased in the liver of AD mice.

They also showed that Kallistatin directly binds to Notch1 and contributes to the activation of the Noch1-HES1 signaling pathway

Reviewer #3 (Public review):

Summary:

The authors investigated the role of kallistatin in metabolic abnormalities associated with AD. They found that Kallistatin promotes Aβ production by binding to the Notch1 receptor and upregulating BACE1 expression. They identified that Kallistatin is a key player that mediates Aβ accumulation and tau hyperphosphorylation in AD.

Strengths:

This manuscript not only provides novel insights into the pathogenesis of AD, but also indicates that the hypolipidemic drug fenofibrate attenuates AD-like pathology in Kallistatin transgenic mice.

Weaknesses:

The authors did not illustrate whether the protective effect of fenofibrate against AD depends on kallistatin.

The conclusions are supported by the results.

Reviewer #1 (Public review):

The authors investigated the role of the C. elegans Flower protein, FLWR-1, in synaptic transmission, vesicle recycling, and neuronal excitability. They confirmed that FLWR-1 localizes to synaptic vesicles and the plasma membrane and facilitates synaptic vesicle recycling at neuromuscular junctions. They observed that hyperstimulation results in endosome accumulation in flwr-1 mutant synapses, suggesting that FLWR-1 facilitates the breakdown of endocytic endosomes. Using tissue-specific rescue experiments, the authors showed that expressing FLWR-1 in GABAergic neurons restored the aldicarb-resistant phenotype of flwr-1 mutants to wild-type levels. By contrast, cholinergic neuron expression did not rescue aldicarb sensitivity at all. They also showed that FLWR-1 removal leads to increased Ca2+ signaling in motor neurons upon photo-stimulation. From these findings, the authors conclude that FLWR-1 helps maintain the balance between excitation and inhibition (E/I) by preferentially regulating GABAergic neuronal excitability in a cell-autonomous manner.

Overall, the work presents solid data and interesting findings, however the proposed cell-autonomous model of GABAergic FLWR-1 function may be overly simplified in my opinion.

Most of my previous comments have been addressed; however, two issues remain.

(1) I appreciate the authors' efforts conducting additional aldicarb sensitivity assays that combine muscle-specific rescue with either cholinergic or GABergic neuron-specific expression of FLWR-1. In the revised manuscript, they conclude, "This did not show any additive effects to the pure neuronal rescues, thus FLWR-1 effects on muscle cell responses to cholinergic agonists must be cell-autonomous." However, I find this interpretation confusing for the reasons outlined below.

Figure 1 - Figure Supplement 3B shows that muscle-specific FLWR-1 expression in flwr-1 mutants significantly restores aldicarb sensitivity. However, when FLWR-1 is co-expressed in both cholinergic neurons and muscle, the worms behave like flwr-1 mutants and no rescue is observed. Similarly, cholinergic FLWR-1 alone fails to restore aldicarb sensitivity (shown in the previous manuscript). These observations indicate a non-cell-autonomous interaction between cholinergic neurons and muscle, rather than a strictly muscle cell-autonomous mechanism. In other words, FLWR-1 expressed in cholinergic neurons appears to negate or block the rescue effect of muscle-expressed FLWR-1. Therefore, FLWR-1 could play a more complex role in coordinating physiology across different tissues. This complexity may affect interpretations of Ca2+ dynamics and/or functional data, particularly in relation to E/I balance, and thus warrants careful discussion or further investigation.

[Editor's note: The authors edited the text of the manuscript to acknowledge potential complexities in the interpretations of these results.]

(2) The revised manuscript includes new GCaMP analyses restricted to synaptic puncta. The authors mention that "we compared Ca2+ signals in synaptic puncta versus axon shafts, and did not find any differences," concluding that "FLWR-1's impact is local, in synaptic boutons." This is puzzling: the similarity of Ca2+ signals in synaptic regions and axon shafts seems to indicate a more global effect on Ca2+ dynamics or may simply reflect limited temporal resolution in distinguishing local from global signals due to rapid Ca2+ diffusion. The authors should clarify how they reached the conclusion that FLWR-1 has a localized impact at synaptic boutons, given that synaptic and axonal signals appear similar. Based on the presented data, the evidence supporting a local effect of FLWR-1 on Ca2+ dynamics appears limited.

[Editor's note: The authors acknowledged that some wording in the previous version was misleading and inaccurate. In the revised version, the authors have withdrawn the conclusion that FLWR-1 function is local in synaptic boutons.]

Reviewer #2 (Public review):

Summary:

The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.

Strengths:

A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.

The authors have adequately addressed most of my previous concerns, however, I recommend minor revisions to further strengthen the study's rigor and interpretation:

Major suggestions

(1) This study relies heavily on aldicarb assays to support its conclusions. While these assays are valuable, their results may not fully align with direct assessment of neurotransmitter release from motor neurons. For instance, prior work has shown that two presynaptic modulators identified through aldicarb sensitivity assays exhibited no corresponding electrophysiological defects at the neuromuscular junction (Liu et al., J Neurosci 27: 10404-10413, 2007). Similarly, at least one study from the Kaplan lab has noted discrepancies between aldicarb assays and electrophysiological analyses. The authors should consider adding a few sentences in the Discussion to acknowledge this limitation and the potential caveats of using aldicarb assays, especially since some of the aldicarb assay results in this study are not easily interpretable.

[Editor's note: The authors added a sentence in the first paragraph of the Discussion to acknowledge these complexities.]

(2) The manuscript states, "Elevated Ca2+ levels were not further enhanced in a flwr-1;mca-3 double mutant." (lines 549-550). However, Figure 7C does not include statistical comparisons between the single and double mutants of flwr-1 and mca-3. Please add the necessary statistical analysis to support this statement.

[Editor's note: In response, the authors noted that these comparisons were indeed carried out. As mentioned in the figure legend, the graph shows only those comparisons that indicated statistical significance.]

(3) The term "Ca2+ influx" should be avoided, as this study does not provide direct evidence (e.g. voltage-clamp recordings of Ca2+ inward currents in motor neurons) for an effect of the flwr-1 mutation of Ca2+ influx. The observed increase in neuronal GCaMP signals in response to optogenetic activation of ChR2 may result from, or be influenced by, Ca2+ mobilization from of intracellular stores. For example, optogenetic stimulation could trigger ryanodine receptor-mediated Ca2+ release from the ER via calcium-induced calcium release (CICR) or depolarization-induced calcium release (DICR). It would be more appropriate to describe the observed increase in Ca2+ signal as "Ca2+ elevation" rather than increased "Ca2+ influx".

[Editor's note: The authors revised their terminology to avoid ambiguities associated with the word "influx".]

Reviewer #1 (Public review):

Summary:

In this study, Xiao et al. conducted a comprehensive analysis of retroperitoneal liposarcoma (RPLS) by classifying patients into two distinct molecular subgroups based on whole transcriptome sequencing data from 88 cases. The G1 subgroup demonstrated a metabolic activation signature, whereas the G2 subgroup was characterized by enhanced cell cycle regulation and DNA damage repair pathways. Notably, the G2 subgroup exhibited more aggressive molecular profiles and poorer clinical prognosis compared to the G1 subgroup. Through the application of machine learning algorithms, the authors established a streamlined classification system, identifying LEP and PTTG1 as pivotal molecular biomarkers for differentiating between these two RPLS subgroups. The manuscript presents a well-structured and methodologically sound study, with particular significance attributed to its substantial sample size and the development of a clinically applicable classification framework. This innovative model holds considerable promise for advancing personalized treatment strategies and improving clinical outcomes for RPLS patients.

Comments on revisions:

The authors have adequately addressed all my concerns, and I have no further comments.

Reviewer #1 (Public review):

Summary:

In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The migration of these cells which are found in two nearby groups of cells normally happens unidirectionally along the dorsal trunk towards the posterior. Here, the authors study how this directionality is regulated. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells plays a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest that trafficking and vesicular transport of Upd2 from the fat body towards the tracheal cells might be important.

Strengths:

The manuscript is well written. This novel work demonstrates a likely link between Upd2-JAK/STAT signalling in the fat body and tracheal stem cells and the control of unidirectional cell migration of tracheal stem cells. The authors show that hid+rpr or Upd2RNAi expression in a fat body or Dome RNAi, Hop RNAi, or STAT92E RNAi expression in tracheal stem cells results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. Moreover, the authors hypothesise and provide some supporting evidence that extracellular vesicle transport of Upd2 might be involved in this Upd2-JAK/STAT signalling in the fat body and tracheal stem cells, which is quite interesting. Overall, the work presented here provides some novel insights into the mechanism that ensures unidirectional migration of tracheal stem cells that prevents bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration.

Weaknesses:

It remains somewhat unclear how Upd2 transported in extracellular vesicles would bind to the Dome receptor found on the surface of the tracheal cells? How Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway?

Reviewer #1 (Public review):

Summary:

In their manuscript, Li and colleagues introduce a pioneering investigation into the molecular and epigenetic foundations of neuroendocrine transdifferentiation in prostate cancer. By employing a genetically engineered cellular reprogramming approach, they elucidate the pivotal roles of ASCL1 and NeuroD1 as pioneer transcription factors that suppress AR signaling and orchestrate lineage plasticity toward NEPC. Their integrative multi-omics methodology delineates dynamic transcriptional and chromatin reorganization processes, offering profound insights into mechanisms of therapeutic resistance.

Strengths:

(1) The development of a reproducible in vitro reprogramming platform to transition ARPC cells into NEPC represents a significant technical achievement. This model enables high-resolution temporal analysis of NEtD, addressing constraints inherent in traditional PDX systems.

(2) The authors reveal that ASCL1 and NeuroD1 suppress AR signaling through chromatin structural modifications at somatically amplified AR enhancers, a significant discovery that clarifies the longstanding ambiguity surrounding AR pathway inactivation during lineage plasticity.

(3) The integration of RNA sequencing, CUT&RUN, and single-cell multiomic profiling delivers a holistic perspective on dynamic epigenetic and transcriptional reprogramming during NEtD. Their observation that AR suppression precedes NE marker activation provides chronological insights into this process.

(4) By delineating the distinct roles of ASCL1/NeuroD1-driven NE lineage programs versus REST inactivation, the study critiques the excessive dependence on limited immunohistochemical indicators for NEPC classification, directly informing improvements in molecular diagnostics.

(5) The association of ASCL1/NeuroD1 with MHC class I suppression mediated by PRC2 unveils opportunities for combining agents targeting epigenetic modifiers with immune-based therapies to counteract immune evasion in NEPC.

Weaknesses:

While the study is methodologically robust, a modest limitation lies in its primary reliance on established cell lines for mechanistic exploration. Although key observations are corroborated with clinical samples, additional validation in PDX models or organoid systems could enhance translational applicability. Furthermore, while the role of ASCL1/NeuroD1 in AR enhancer silencing is convincingly demonstrated, the upstream regulatory mechanisms governing ASCL1/NeuroD1 induction under therapeutic stress remain unaddressed, a compelling avenue for future research.

Reviewer #2 (Public review):

Summary:

This manuscript reported that the pioneer factors ASCL1 and NeuroD1 in neuroendocrine transdifferentiation of Neuroendocrine prostate cancer (NEPC) and uncovered their abilities to silence AR expression by remodeling chromatin at the somatically acquired AR enhancer and global AR binding sites with enhancer activity. It also elucidated the dynamic temporal changes in the transcriptomic and epigenomic landscapes of cells undergoing acute lineage conversion from AR-active prostate cancer to NEPC which should inform future therapeutic development.

Strengths:

Data from cell lines is great and solid.

Weaknesses:

The paper would be better if some clinical data could be added.

Reviewer #3 (Public review):

Summary:

This study investigates the molecular mechanisms underlying the transdifferentiation of androgen receptor-active prostate cancer (ARPC) to neuroendocrine prostate cancer (NEPC) in prostate cancer (PC). Using a cellular reprogramming strategy, the research team successfully converted ARPC cell lines into NEPC cell lines and explored key molecular mechanisms driving this transformation. The work demonstrates the pivotal role of neurogenic pioneer transcription factors ASCL1 and NeuroD1 in NEPC transdifferentiation, which silence AR expression and signaling by remodeling chromatin architecture while inducing NEPC-associated gene programs. Additionally, the study reveals dynamic transcriptomic and epigenomic changes during NEPC transformation, as well as downregulation of the MHC class I antigen processing and presentation pathway in NEPC cell lines.

Strengths:

(1) The study introduces a novel genetically defined cellular reprogramming strategy to directly convert ARPC to NEPC. This approach circumvents previous limitations by starting from AR-active cells, thereby addressing a critical gap in the field.<br /> (2) The study provides a comprehensive characterization of the dynamic changes in the transcriptomic and epigenomic landscapes during the NEPC transdifferentiation process.

Weaknesses:

(1) What was the rationale for selecting these specific candidate factors (e.g., ASCL1, NeuroD1) to drive neuroendocrine transdifferentiation (NEtD)? Was a comprehensive screening process conducted to identify additional potential drivers of this phenotypic shift?

(‌2) The AR bypass assay employed an AR response element-driven FKBP-Casp8 fusion protein for negative selection. How was the specificity and efficiency of this system validated? Are there additional validation experiments (e.g., orthogonal AR activity assays) to confirm the complete bypass of AR signaling?

(3‌) While extensive omics data (RNA-seq, ATAC-seq, CUT&RUN) are presented, have these datasets been deposited in public repositories (e.g., GEO, SRA) to enable validation and reuse by the scientific community?

(‌4) What criteria guided the selection of time points for analyzing dynamic changes during NEtD? Would denser time-point sampling (e.g., intermediate time courses) enhance resolution of critical transitional events?

(5‌) Were multiple hypothesis testing corrections (e.g., Benjamini-Hochberg) applied during differential expression and pathway enrichment analyses? How was the statistical significance of chromatin accessibility changes and super-enhancer reconfiguration rigorously validated?

Reviewer #1 (Public review):

Summary:

This is an interesting manuscript by Kirk and colleagues describing a highly valuable knock-down system that leverages CRISPRi in order to further elucidate the role of the Kruppel-Like Factor (KLF) transcription factor family in regulating the maturation of postnatal cortical projection neurons. The authors firstly use RNA-Seq and ATAC-Seq data in order to identify the KLF TF family as a potential regulator of cortical neuron maturation in the postnatal brain and subsequently knock down four KLF family members; KLF9, KL13, KLF6 and KLF7, in order to ascertain the functions of specific KLF genes in the developing cortex. The described CRISPRi knock down strategy is highly robust and penetrant as evidenced by a KD efficiency > 95% (assessed by both qPCR and single molecule FISH) and demonstrates that KLF6 and KLF7 play an activating role in driving the expression of target genes relating to axonal growth whereas KLF9 and 13 play a repressive role that inhibits the expression of overlapping gene targets. Together, the authors propose a model where the KLF TF family acts as a regulatory "switch" from activation to repression in the postnatal cortex as a mechanism to control a shift in projection neuron function from axonal growth to circuit refinement. The findings and conclusions of the manuscript offer a valuable contribution to the field of postnatal cortical development and further our understanding of the regulatory mechanisms that govern neuron maturation.

The conclusions of this manuscript are generally supported by the data, but some aspects of the data collection and analysis require some further clarification. Specifically:

(1) The authors comprehensively assess the molecular effects of KLF TF knock-down, however, the authors do not deeply address the cellular effects of these knock-downs. The authors conclude that knockdown of KLF6/7 and KLF9/13 cause downregulation and upregulation, respectively, of a common set of genes involved in cytoskeletal or axon regulation such as Tubb2 and Dpysl3. How is the morphology of the cells affected by these knockdowns? For example, does KLF9/13 knockdown cause neurite/axonal outgrowth? The authors should perform some basic experiments to assess changes in cell morphology following KLF TF KD. This is the one key point that needs addressing, in my opinion.

(2) The authors identify 374 DEGs in P10 Klf6/7 KD neurons and 115 DEGs at P20 (figure 6B). Have the authors looked to see what proportion of these DEGs are upregulated in the KLF9/13 KDs in order to get a more global understanding of the degree of overlap in the genes regulated by the KLF family members? Along similar lines, the authors later indicate that there are 144 shared targets between the KLF activator and repressor pairs (Figure 7C). What percentage does this represent of the total number of DEGs between the KLF pairs. This could further illustrate the degree to which the KLF pairs regulate the same set of genes. If it is already indicated in the manuscript, it should be made a bit more clear to the reader.

(3) Figures 5B and 6D2 are very interesting as they relate the changes in gene expression over time in neurons from P2 to P30 to the functions of KLF9/13 and KLF6/7, respectively. I would be curious to see how these two forms of analyses overlap with one another. For example, in Figure 6D2, where would the KLF9/13 upregulated genes fall on the plot shown in Figure 6D2? And would those overlapping genes fit a similar correlation?

(4) Figure 7E shows expression levels of shared KLF TF targets in control or KD conditions. Interestingly, the expression of Tubb2b, shows higher expression in ScrGFP P10 when compared to KLF9/13 P20, suggesting that derepression of KLF9/13 does not fully restore the expression level of Tubb2b seen at P10. This may suggest that other repressive regulators may be involved in the downregulation of Tubb2b from P10 to P20. Can the authors further comment on this, perhaps in the discussion, and speculate if there are other regulatory factors at play that may be controlling some of the shared targets by KLF6/7 and KLF9/13?

Reviewer #2 (Public review):

Summary:

Kirk et al. use RNA-Seq and CRISPRi to provide evidence that KLF family transcription factors regulate postnatal neuronal maturation of pyramidal neurons. The genetic programs regulating postnatal neuronal maturation are not well understood. The authors first analyzed chromatin accessibility and gene expression data from layer 4 and 6 pyramidal neurons and found that KLF TFs are predicted regulators of postnatal neuronal maturation. They then use CRISPRi knockdown and find that KLF activators first activate genes and then this is followed by KLF repressors repressing genes. Interestingly, some genes, such as those with cytoskeletal functions, are shared targets of KLF activators and repressors.

Strengths:

The study is well-executed and the paper is well-written. A major strength of this study is the application of state-of-the-art transgenic approaches. The CRISPRi approach used to knock down multiple KLFs is compelling. The genomic data generated appears to be high quality and is carefully analyzed. The presented findings provide important insights into the genetic programs that regulate postnatal maturation in cortical pyramidal neurons. The discovery that KLF family activators/repressors regulate gene expression changes during this critical step of neuronal development fills an important gap in the field.

Weaknesses:

A limitation of the current study is that the functional importance of KLF for postnatal neuronal maturation is unclear. Although the authors find that KLFs regulate some of the gene expression changes during postnatal neuronal maturation, it is still unclear whether such gene expression changes mediate the postnatal changes in morphology and physiology. While beyond the scope of the current study, future studies should investigate the contributions of KLFs on postnatal morphological and physiological changes.

Reviewer #3 (Public review):

Summary:

In their manuscript "Multiplexed CRISPRi Reveals a Transcriptional Switch Between KLF Activators and Repressors in the Maturing Neocortex", Kirk and colleagues seek to dissect the developmentally regulated pan-neuronal gene programs that control the postnatal maturation of cortical neurons. For this, the authors analyzed newly generated and existing RNA-seq and ATAC-seq of Layer 4 and Layer 6 cortical pyramidal neurons at postnatal day 2 (P2) and day 30 (P30), and identified thousands of shared developmentally regulated genes and genomic (promoter) regions, including genes involved in axon growth (tend to be downregulated) and synaptic function (tend to be upregulated). Motif enrichment analysis of promoters of differentially regulated genes revealed a strong presence of KLF/Sp family binding motifs, pointing to Krüppel-Like Factors (KLFs) as key transcriptional regulators of cortical maturation. Expression profiling showed a developmental switch from activating KLFs (Klf6, Klf7) expressed neonatally to repressive KLFs (Klf9, Klf13) upregulated during maturation. Using an elegant in vivo multiplexed CRISPR interference (CRISPRi) system, the authors achieved efficient, cell-type-specific knockdown of these TFs and showed that Klf9 and Klf13 repress a set of genes that includes cytoskeletal regulators such as Tubb2b, Dpysl3, and Rac3. Conversely, Klf6 and Klf7 promoted the expression of these same genes in the early postnatal period, and their knockdown led to reduced expression of these genes, particularly at P10 when their activating influence is strongest. Since promoters of shared KLF targets were enriched for KLF/Sp motifs but showed little change in chromatin accessibility, the authors propose a model in which distinct KLF family members function either as transcriptional repressors and activators that compete at constitutively accessible promoters and thereby act as a developmental transcriptional switch that coordinates the downregulation of axon growth programs and upregulation of synaptic maturation genes during cortical development.

Strengths:

The study addresses an interesting question and advances our understanding of the transcriptional regulation underlying postnatal cortical development. A major strength of the study lies in the innovative use of in vivo multiplexed CRISPR interference (CRISPRi), which allows for cell-type-specific, combinatorial knockdown of redundant TFs - this an elegant solution to a long-standing challenge in transcription factor research, and should be useful also for other neuroscience studies that require local and cell-type-specific gene loss-of-function. Also, the integration of RNA-seq and ATAC-seq across developmental time points provides a robust foundation for identifying direct targets of the KLF family, and the findings are reinforced by cross-species conservation and the identification of targets with clear neurodevelopmental relevance.

Weaknesses:

The major weakness of the study lies in its relatively narrow scope: the study focuses primarily on transcriptional mechanisms and largely lacks functional validation of the neuronal phenotypes that are predicted by the gene expression data (e.g. axonal morphology). For example, the authors analyzed the effects of KLF9/13 KD on the neurons' excitability and excitatory inputs, but did not assess the effects on inhibitory inputs and E/I-ratio or morphological parameters such as axonal length and axonal target fields - the manuscript would be strengthened considerably by such analyses (axonal projections could be analyzed e.g. via local injections of the gRNA AAVs and subsequent immunolabeling of brain sections). Similarly, the chromatin-based mechanisms underlying KLF activity remain relatively speculative, and the transcriptional mechanisms upstream of the KLFs remain unexplored (this could be addressed by analyzing existing datasets; see "Additional Point 1" below). Finally, the manuscript is too long (e.g., nearly five pages in the Discussion section are devoted to discussing various misregulated genes) and would benefit from presenting the Results and Discussion sections more concisely. However, despite these limitations, the paper offers an interesting model for a transcriptional switch during neuronal maturation in the cortex and establishes a powerful methodological framework for dissecting redundant gene networks in vivo.

Reviewer #1 (Public review):

It is well established that many potivirids (viruses in the Potiviridae family), particularly potyviruses (viruses in the Potyvirus genus), recruit (selectively) either eIF4E or eIF(iso)4E, while some others can use both of them to ensure a successful infection. CBSD caused by two potyvirids, i.e., ipomoviruses CBSV and UCBSV, severely impedes cassava production in West Africa. In a previous study (PBI, 2019), Gomez and Lin (co-first authors), et al. reported that cassava encodes five eIF4E proteins, including eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, nCBP-1 and nCBP-2, and CBSV VPg interacts with all of them (Co-IP data). Simultaneous CRISPR/Cas9-mediated editing of nCBp-1 and -2 in cassava significantly mitigates CBSD symptoms and incidence. In this study, Lin et al further generated all five eIF4E family single mutants as well as both eIF(iso)4E-1/-2 and nCBP-1/-2 double mutants in a farmer-preferred casava cultivar. They found that both eIF(iso)4E and nCBP double mutants show reduced symptom severity, and the latter is of better performance. Analysis of mutant sequences revealed one important point mutation, L51F of nCBP-,2 that may be essential for the interaction with VPg. The authors suggest that the introduction of the L51F mutation into all five eIF4E family proteins may lead to strong resistance. Overall I believe this is an important study enriching knowledge about eIF4E as a host factor/susceptibility factor of potyvirids and proposing new information for the development of high CBSD resistance in cassava. I suggest the following two major comments for authors to consider for improvement:

(1) As eIF(iso)4e-1/-2 or nCBP-1/-2 double mutants show resistance, why not try to generate a quadruple mutant? I believe it is technically possible through conventional breeding.

(2) I agree that L51F mutation may be important. But more evidence is needed to support this idea. For example, the authors may conduct a quantitative Y2H assay on the binding of VPg to each of the eIF4E (L51F) mutants. Such data may add as additional evidence to support your claim.

Reviewer #2 (Public review):

Summary:

The authors generated single and double knockout mutants for the eIF4E family members eIF4E, iso4E1, iso4E2, nCBP1, and nCBP2 in cassava. While a single knockout of these eIF4E genes did not abolish viral infection, the nCBP1/nCBP2 double knockout mutant displayed the weakest symptoms and viral infection. Through yeast two-hybrid screening, the nCBP-2 L51F mutant was identified, and the mutant was unable to interact with VPg, yet the nCBP-2 L51F mutant could complement the eIF4E yeast mutant. This L51F is a potentially important editing site for eIF4E.

Strengths:

This study systematically generated single and double knockout mutants for the eIF4E family members and investigated their antiviral activity. It also identified a L51F site as a potentially important antiviral editing site in eIF4E, however, its antiviral genetic evidence remains to be validated.

Weaknesses:

(1) The symptoms of the iso4E1 & iso4E2 double-knockout mutant are slightly alleviated, and those of the nCBP1 & nCBP2 double-knockout mutant are alleviated the most. If the iso4E1 & iso4E2 and nCBP1 & nCBP2 mutants are crossed to obtain quadruple-knockout mutant plants, whether the resistance of the quadruple mutant will be more excellent should be further investigated.

(2) Although the yeast two-hybrid identified the nCBP-2 L51F mutant, there is no direct biological evidence demonstrating its antiviral function. While the 6-amino acid deletion mutant (including L51F) showed attenuated symptoms, this deletion might be sufficient to cause loss-of-function of nCBP-2. These indirect observations cannot definitively establish that the L51F mutation specifically confers antiviral activity.

(3) Given that nCBP-2 can rescue yeast eIF4E mutants, introducing wild type and L51F nCBP2 into the Arabidopsis iso4e mutant viral infectious clones into yeast systems could clarify whether the L51F mutation (and the same mutations in eIF4E, iso4E1, iso4E2) abrogates their roles as viral susceptibility factors - critical genetic evidence currently missing.

Reviewer #3 (Public review):

In the manuscript, the authors generated several mutant plants defective in the eIF4E family proteins and detected cassava brown streak viruses (CBSVs) infection in these mutant plants. They found that CBSVs induced significantly lower disease scores and virus accumulation in the double mutant plants. Furthermore, they identified important conserved amino acid for the interaction between eIF4E protein and the VPg of CBSVs by yeast two hybrid screening. The experiments are well designed, however, some points need to be clarified:

(1) The authors reported that the ncbp1 ncbp2 double mutant plants were less sensitive to CBSVs infection in their previous study, and all the eIF4E family proteins interact with VPg. In order to identify the redundancy function of eIF4E family proteins, they generated mutants for all eIF4E family genes, however, these mutants are defective in different eIF4E genes, they did not generate multiple mutants (such as triple, quadruple mutants or else) except several double mutant plants, it is hard to identify the redundant function eIF4E family genes.

(2) The authors identified some key amino acids for the interaction between eIF4E and VPg such as the L51, it is interesting to complement ncbp1 ncbp2 double mutant plants with L51F form of eIF4E and double check the infection by CBSVs.

Reviewer #1 (Public review):

Summary:

In this manuscript the authors test the hypothesis that gonadal steroid signaling influences the transcriptional development of specific neurons in the mPOA during adolescence, and that such adolescent development of the mPOA is necessary for mating behaviors.

Strengths:

The authors establish a role GABAergic-Esr1 neurons in mating behaviors of both male and female mice. Differentially expressed genes are compared across adolescent development and between sexes. Single-cell sequencing is used to resolve clusters of cells based on transcript levels, and in situ hybridization is used to visualize anatomical expression patterns. The research presented is thorough and rigorous and contributes new insight into hormone-sensitive transcriptional profiles within genetically defined neuron clusters in the mPOA during adolescence.

Weaknesses: Two minor comments

(1) Fig 4 (hormone treatment): In this experiment, testosterone is given to males, yet in Sup Fig 6 it is argued that Esr1 is more influential in driving transcriptional changes compared to AR. Does DHT treatment have the same outcome as testosterone? Or, does estrogen treatment in males have the same outcome as testosterone?

(2) Fig 3i: There appears to be an age-dependent transcriptional change in male Vgat HR-low cells. Can the authors comment on age-dependent (hormone-independent) transcriptional changes in males versus females.

Reviewer #2 (Public review):

Summary:

An abundant literature documents molecular changes in the rodent hypothalamus that occur during the transition from prepubertal to mature reproductive physiology. Equally well documented is the role of sex steroids and their receptors during this important period of reproductive development, as well as the importance of GABAergic and glutamatergic neurons. The medial preoptic area (MPOA) is known to play a central role in expression of sexually dimorphic reproductive function and previously reported sexually dimorphic patterns of gene expression are consistent with this role. The present manuscript extends this knowledge base and reports the results of a detailed evaluation of transcriptional dynamics in the MPOA during the adolescent transition to maturity with a particular focus on the role of the estrogen receptor gene (Esr1). Both single cell RNA sequencing (scRNseq) and multiplex in situ hybridization methods were employed and the results subjected to detailed computational analyses to demonstrate that the transcriptomic structure of MPOA neurons displays both sex and cell type specific expression profiles. In addition, both hormonal and genetic manipulations of Esr1 signaling during puberty altered the transcriptional profiles of MPOA neurons, and these changes aligned with maturation of hormone-dependent reproductive function. The authors provide this evidence to illustrate Esr1 dependent control of gene regulatory networks required for normal expression of reproductive behaviors expressed during the transition from adolescence to adulthood. The results presented in this manuscript are extensive and represent the most comprehensive evaluation of transcriptomic changes during reproductive maturation to date. The methods appear strong and the results provide a rich data set that will support a good deal of future analysis. Despite these strengths, the authors are encouraged to revise their manuscript to address significant gaps in their presentation, as well as clarify or improve their conclusions.

Strengths:

(1) The major strength of this manuscript is the extensive set of images and graphs that illustrate molecular changes that occur in MPOA neurons during adolescence, although additional spatial detail as to locations of the source neurons would be welcome in order to place the changes in the proper circuitry context.

(2) Targeting Esr1 deletion to MPOA GABA neurons is a good choice, given how these cells have been implicated in sexual differentiation of reproductive behavior previously, and the lack of comparable responses in glutamatergic neurons is convincing. The AAV-frtFlex-Cre virus created by the investigators is a most useful tool for such studies. Profiling distinct transcriptomic trajectories in GABA and glutamatergic neurons during reproductive maturation is impressive and leads to some of the best supported conclusions in this paper.

(3) Cellular and molecular resolution of the transcriptomics data appears excellent, however, because the source tissue for the scRNAseq analysis was obtained by bulk dissection of the MPOA anatomical resolution is limited. This problem is addressed to some extent by careful comparison of scRNAseq results with previously published spatial transcriptomics data. The HM-HCR-FISH analysis clearly documents spatially restricted changes in gene expression, but it is hard to discern where these changes occur based on the images presented or the descriptions included in the Results. The anatomical schematic included in Figure 4 suggests that investigators are not familiar with components of the MPOA (see Allen Mouse Brain Atlas).

Weaknesses:

(1) A major conceptual flaw is that the authors do not distinguish between genetically determined sex differences in patterns of gene expression and differences caused by the fact that MPOA neurons are exposed to different endocrine environments in adolescent males and females, which can cause different transcriptional trajectories independent of genetic sex. This issue does not render their results invalid, but their terminology should address the issue in the discussion and "limitations" section. At the very least the endocrine status of "intact females" should be included.

(2) A major technical flaw is that the MPOA is treated as a functionally distinct brain region (block dissections) with uniform distribution of cell types (FISH data are not illustrated or reported with sufficient spatial detail). Thus, an enormous amount of molecular data is provided that cannot be mapped to distinct neural circuits, thereby limiting the neurobiological impact. This is also a weakness of the FISH data, which is presented with only small regions illustrated without anatomical detail. In fact, some images are compared that appear to illustrate different MPOA structures, although it is impossible to be certain of this due to the lack of morphological landmarks. The analysis of how Esr1 orchestrates regulatory gene networks is impressive and interesting, but the fact that many of the observed transcriptional events occur in neural circuits that do not overlap confounds interpretation.

(3) The locations of the AAV injections should be characterized because deleting Esr1 in multiple distinct parts of the MPOA will likely confound interpretation. This is especially problematic given the limited number of mice used for parts of the RNAscope analysis.

(4) Although the focus of these experiments on adolescence is welcome, neither the Introduction nor the Discussion do a good job of placing these studies in the context of what is already known about brain maturation during puberty. It is true that this is very much a results-focused manuscript, but the scholarship can be improved. Simply stating that your results are consistent with previous reports places an undue burden on the reader to go figure out what is new.

(5) Throughout the manuscript the authors utilize obscure abbreviations, which often makes reading their text overly cumbersome. This is certainly justified in certain instances where complex names of analytical methods are used repeatedly, but the authors are encouraged to try and simplify their use of non-standard abbreviations.

Reviewer #3 (Public review):

Summary:

Hashikawa and colleagues analyze estrogen signaling in the medial preoptic area using scRNA-sequencing, RNA in situ hybridization, and specific disruption of Esr1 in glutamatergic or GABAergic neurons. They conclude that Esr1 "plays a pivotal role in the transcriptional maturation of GABAergic neurons within the MPOA during adolescence". Overall, the findings are mostly consistent with previous literature but bring additional molecular evidence and timing effects that focus on adolescence rather than the perinatal period. The most surprising results are the lack of effects of Esr1 or adolescence-associated hormone changes in glutamatergic neurons, but this seems like it could be due to limited behavioral and physiological phenotyping as well as limited transcriptomic sampling.

Strengths:

Strengths of this paper are the multiple complementary approaches and the spatially specific disruption of Esr1 in two different neuronal populations of the MPOA. These data add more molecular insights to our understanding of how this region is shaped by hormone changes during adolescence.

The idea that Esr1 regulates "transcriptional maturation" is interesting. This term should be explicitly defined (as well as "arrested adolescent transcriptional progression") and distinguished from general effects of steroid signaling. To what degree does Esr1 disruption narrowly affect genes indicative of transcriptional maturation? The paper highlights specific neuropeptide genes (e.g., Nts, Pdyn, Tac1) that might be estrogen-dependent rather than broad indicators of transcriptional maturation.

Weaknesses:

We already know that Esr1 is important within GABAergic but not glutamatergic neurons for mating behavior. However, there is not enough data to support the claim that disrupting Esr1 in glutamatergic MPOA neurons "had no observable effect." The MPOA is involved in many behaviors and physiologies that were not investigated. More assays would be required to report "no observable effect."

The small number of cells included in the transcriptional studies is a general concern, as noted by the authors. This is a particular concern for conclusions related to the role of adolescence in glutamatergic MPOA neurons. The paper reports 24,627 neurons across all treatment groups, which include 3 timepoints, 2 sexes, and GDX conditions. It seems likely that not much was detected in the glutamatergic neurons because of insufficient power.

Esr1 knockout is initiated in adolescence, not restricted to adolescence. Do we know that the effects on mating behavior are due to what is happening in adolescence vs. the function of Esr1 in adults? Are the effects different if Esr1 is knocked out in mature adults? This comparison would be important to demonstrate that adolescence is a critical time window for Esr1 function.

Reviewer #1 (Public review):

Summary:

This study presents a valuable contribution of NO signaling in zebrafish retinal regeneration in larval animals. The data on NO signaling are solid; however, the link to cxcl118b is inadequate. There are significant concerns that the RNA-seq studies largely repeat the work of a previous study done in adult animals, which is a more relevant biological variable for translational insights.

Strengths:

New data on NO signaling are valuable to the field, but may be limited to larval "regeneration".

Weaknesses:

(1) The authors state that more is known about glial reactivation than cell-cycle re-entry. They are confusing many points here. More gene networks that require cell-cycle re-entry are known. Some of the genes listed for "reactivation" are, in fact, required for cell cycle re-entry/proliferation. And the authors confuse gliosis vs glial reactivation.

(2) A major weakness of the approach is testing cone ablation and regeneration in early larval animals. For example, cones are ablated starting the day that they are born. MG that are responding are also very young, less than 48 hrs old. It is also unclear whether the immune response of microglia is a mature response. All of these assays would be of higher significance if they were performed in the context of a mature, fully differentiated, adult retina. All analysis in the paper is negatively affected by this biological variable.

(3) Related to the above point, the clonal analysis of cxcl18b+ MG is complicated by the fact that new MG are still being born in the CMZ (as are new cones for that matter).

(4) A near identical study was already done by Hoang et al., 2020, in adult zebrafish, a more relevant biological timepoint. Did the authors check this published RNA-seq database for their gene(s) of interest?

(5) KD of cxcl18b did not affect MG proliferation or any other defined outcome. And yet the authors continually state such phrases as "microglia-mediated inflammation is critical for activating the cxcl18b-defined transitional states that drive MG proliferation." This is false. Cxcl18b does not drive MG proliferation at all.

(6) A technical concern is that intravitreal injections are not routinely performed in larval fish.

Reviewer #2 (Public review):

Summary:

In this manuscript, Ye et al. examine the sequence of events that occur in the damaged zebrafish Muller glia (MG) in states between quiescence and the onset of proliferation. Using an inducible metronidazole (MTZ) and nitroreductase system to ablate red/green cones in larval zebrafish, they identify a novel transitional MG state that is characterized by the expression of cxcl18b. Using trajectory analysis from single-cell RNA-seq datasets, they find that cxcl18b is expressed before MG expression PCNA and becomes proliferative. They find that cxcl18b expression peaks in MG at approximately 24 hours post injury (hpi) and rapidly declines as MG proliferate following injury. In a most interesting finding, the authors find a link between nos2b-dependent nitric oxide signaling and cxcl18b-mediated proliferation. Mutagenesis of nos2b decreases MG proliferation. The mechanism linking NO signaling to proliferation was suggested to function via notch signaling as pharmacological inhibition of nitric oxidate signaling resulted in elevated Notch activity, thus preventing MG proliferation. The authors suggest a model whereby cxcl18b induces autocrine NO signaling in MG to reduce the activity of Notch3, thereby promoting MG proliferation. While this model is appealing, there are several limitations and inconsistencies within the data that raise concerns. Several conclusions regarding the role of nos2b rely on low-quality in situ hybridization data and RT-PCR results that are inconsistent with some single-cell RNA-seq data also provided. The temporal sequence of events lacks adequate rigor, as many conclusions are based on transgene expression in the Tg(cxcl18b:GFP) lines, where persistence of the GFP fluorescence may not reflect endogenous cxcl18b. Are cognate cxcl18b receptors found on MG to support an autocrine signaling pathway?

Strengths:

The authors utilize a number of sophisticated transgenic approaches and generate novel lines that will have value to the field. The identification of a novel cxcl18b transition state is exciting, and the putative link between NO signaling and Notch activity would provide new insight into the drivers of Muller glia proliferation.

Weaknesses:

While this model is appealing, there are several limitations and inconsistencies within the data that raise concerns. Several conclusions regarding the role of nos2b rely on low-quality in situ hybridization data and RT-PCR results that are inconsistent with some single-cell RNA-seq data also provided. The temporal sequence of events lacks adequate rigor, as many conclusions are based on transgene expression in the Tg(cxcl18b:GFP) lines, where persistence of the GFP fluorescence may not reflect endogenous cxcl18b. Are cognate cxcl18b receptors found on MG to support an autocrine signaling pathway? The images are generally well organized, although a number of typographical errors exist, and some sentences and phrases appear confusing. Additional proof-reading is strongly recommended.

Reviewer #1 (Public review):

Summary:

In this paper, the authors developed a chemical labeling reagent for P2X7 receptors, called X7-uP. This labeling reagent selectively labels endogenous P2X7 receptors with biotin based on ligand-directed NASA chemistry (Ref. 41). After labeling the endogenous P2X7 receptor with biotin, the receptor can be fluorescently labeled with streptavidin-AlexaFluor647. The authors carefully examined the binding properties and labeling selectivity of X7-uP to P2X7, characterized the labeling site of P2X7 receptors, and demonstrated fluorescence imaging of P2X7 receptors. The data obtained by SDS-PAGE, Western blot, and fluorescence microscopy clearly show that X7-uP labels the P2X7 receptor. Finally, the authors fluorescently labeled the endogenous P2X7 in BV2 cells, which are a murine microglia model, and used dSTORM to reveal a nanoscale P2X7 redistribution mechanism under inflammatory conditions at high resolution.

Strengths:

X7-uP selectively labels endogenous P2X7 receptors with biotin. Streptavidin-AlexaFluor647 binds to the biotin labeled to the P2X7 receptor, allowing visualization of endogenous P2X7 receptors.

Weaknesses:

Weaknesses & Comments<br /> (1) The P2X7 receptor exists in a trimeric form. If it is not a monomer under the conditions of the pull-down assay in Figure 2C, the quantitative values may not be accurate.<br /> (2) In Figure 3, GFP fluorescence was observed in the cell. Are all types of P2X receptors really expressed on the cell surface ?<br /> (3) The reviewer was not convinced of the advantages of the approach taken in this paper, because the endogenous receptor labeling in this study could also be done using conventional antibody-based labeling methods.<br /> (4) Although P2X7 was successfully labeled in this paper, it is not new as a chemistry. There is a need for more attractive functional evaluation such as live trafficking analysis of endogenous P2X7.<br /> (5) The reviewer has concerns that the use of the large-size streptavidin to label the P2X7 receptor may perturbate the dynamics of the receptor.<br /> (6) It is better to directly label Alexa647 to the P2X7 receptor to avoid functional perturbation of P2X7.<br /> (7) In all imaging experiments, the addition of streptavidin, which acts as a cross-linking agent, may induce P2X7 receptor clustering. This concern would be dispelled if the receptors were labeled with a fluorescent dye instead of biotin and observed.<br /> (8) There are several mentions of microglia in this paper, even though they are not used. This can lead to misunderstanding for the reader. The author conducted functional analysis of the P2X7 receptor in BV-2 cells, which are a model cell line but not microglia themselves. The text should be reviewed again and corrected to remove the misleading parts that could lead to misunderstanding.<br /> e.g. P8. lines 361-364. First, it combines N-cyanomethyl NASA chemistry with the high-affinity AZ10606120 ligand, enabling rapid labeling in microglia (within 10 min)<br /> P8. lines 372-373. Our results not only confirm P2X7 expression in microglia, as previously reported (6, 26-33), but also reveal its nanoscale localization at the cell surface using dSTORM.

Reviewer #2 (Public review):

Summary:

In this manuscript, Arnould et. al. develop an unbiased, affinity-guided reagent to label P2X7 receptor and use super-resolution imaging to monitor P2X7 redistribution in response to inflammatory signaling.

Strengths:

I think the X7-uP probe that they developed is very useful for visualizing localization of P2X7 receptor. They convincingly show that under inflammatory conditions, there is a reorganization of P2X7 localization into receptor clusters. Moreover, I think they have shown a very clever way to specifically label any receptor of interest. This has broad appeal

Weaknesses:

Overall, the manuscript is novel and interesting. However, I do have some suggestions for improvement.

(1) While the authors state that chemical modification of AZ10606120 to produce the X7-UP reagent has "minimal impact" on the inhibition of P2X7, we can see from Figure 2A and 2B that it does not antagonize P2X7 as effectively as the original antagonist. For the sake of completeness and quantitation, I think it would be great if the authors could determine the IC50 for X7-uP and compare it to the IC50 of AZ10606120.

(2) Do the authors know whether modification of the lysines with biotin affects the receptor's affinity for ATP (or ability to be activated by ATP)? What about P2X7 that has been modified with biotin and then labeled with Alexa 647? For the sake of completeness and quantitation, I think it would be great if the authors could determine the EC50 of biotinylated P2X7 for ATP as well as biotinylated and then Alexa 647 labeled P2X7 for ATP and compare these values to the affinity of unmodified WT P2X7 for ATP.

(3) It is a little misleading to color the fluorescence signal from mScarlet green (for example, in Figure 3 and Figure 4). The fluorescence is not at the same wavelength as GFP. In fact, the wavelength (570 nm - 610 nm) for emission is closer to orange/red than to green. I think this color should be changed to differentiate the signal of mScarlet from the GFP signal used for each of the other P2X receptor subtypes.

(4) It is my understanding that P2X6 does not form homotrimers. Thus, I was a little surprised to see that the density and distribution of P2X6-GFP in Figure 3 looks very similar to the density and distribution of the other P2X subtypes. Do the authors have an explanation for this? Are they looking at P2X6 protomers inserted into the plasma membrane? Does the cell line have endogenous P2X receptor subtypes? Is Figure 3 showing heterotrimers with P2X6 receptor? A little explanation might be helpful.

(5) It is easy to overlook the fact that the antagonist leaves the binding pocket once the biotin has been attached to the lysines. It might be helpful if the authors made this a little more apparent in Figure 1 or in the text describing the NASA chemistry reaction.

Reviewer #3 (Public review):

Summary:

This manuscript describes the development of a covalent labeling probe (X7-uP) that selectively targets and tags native P2X7 receptors at the plasma membrane of BV2 microglial cells. Using super-resolution imaging (dSTORM), the authors demonstrate that P2X7 receptors form nanoscale clusters upon microglial activation by lipopolysaccharide (LPS) and ATP, correlating with synergistic IL-1β release. These findings advance understanding of P2X7 reorganization during inflammation and provide a generalizable labeling strategy for monitoring endogenous P2X7 in immune cells.

Strengths:

(1) The authors designed X7-uP by coupling a high-affinity, P2X7-specific antagonist (AZ10606120) with N-cyanomethyl NASA chemistry to achieve site-directed biotinylation. This approach offers high specificity, minimal off-target reactivity, and a straightforward pull-down/imaging readout.<br /> (2) The results connect P2X7's nanoscale clustering directly with IL-1β secretion in microglia, reinforcing the role of P2X7 in inflammation. By localizing endogenous P2X7 at single-molecule resolution, the authors reveal how LPS priming and ATP stimulation synergistically reorganize the receptor.<br /> (3) The authors systematically validate their method in recombinant systems (HEK293 cells) and in BV2 cells, showing selective inhibition, mutational confirmation of the binding site, and Western blot pulldown experiments.

Weaknesses:

(1) While the data strongly indicate that P2X7 clustering contributes to IL-1β release, the manuscript would benefit from additional experiments (if feasible) or discussion on how receptor clustering interfaces with downstream inflammasome assembly. Clarification of whether the P2X7 clusters physically colocalize with known inflammasome proteins would solidify the mechanism.<br /> (2) The authors might expand on the scope of X7-uP in other native cells that endogenously express P2X7 (e.g., macrophages, dendritic cells). Although they mention the possibility, demonstrating the probe's applicability in at least one other primary immune cell type would strengthen its general utility.<br /> (3) The authors do include appropriate negative controls, yet providing additional details (e.g., average single-molecule on-time or blinking characteristics) in supplementary materials could help readers assess cluster calculations.

Reviewer #1 (Public review):

Although the use of antimony has been discontinued in India, the observation that Leishmania parasites resistant to antimony are in circulation has been cited as evidence that these resistant parasites are now a distinct strain with properties that ensure their transmission and persistence. It is of interest to determine the properties that favor the retention of their drug resistance phenotype even in the absence of the selective pressure that the drug would otherwise exert. The hypothesis that these authors set out to test is that these parasites have developed a new capacity to acquire and utilize lipids, especially cholesterol, which enables them to grow robustly in infected hosts. The authors present compelling evidence that supports their hypothesis. However, the genetic basis for the parasite's gluttony for lipids remains unresolved.

An issue raised in the initial review was the insufficient detail in the discussion of experiments in which parasitophorous vacuoles were isolated from infected cells and their molecular content was investigated. In this new version, the authors have provided more details of those experiments, including the relative enrichment of the preparations.

A puzzling observation for which they provide compelling evidence is the capacity of LD-R parasites to undergo logarithmic growth between 4 and 24 hours in infected cells. Interestingly, after logarithmic growth within the macrophages at those early times, parasite growth slows down inexplicably after 24 hours. One is left to imagine what the consequences of such growth within an infected host will be.

They made the novel observation that Lamp1 expression increases as early as 4 hours after infection with LD-R and by 12 hours after infection with both LD-S and LD-R. Interestingly, transcription analysis did not provide evidence for increased Lamp1 transcripts, leaving open the possibility that parasite infection may exert control over host cell biology at the translational level. How this might be achieved and what genes would be targets for such controls are open questions.

The dynamic changes in lipid biosynthesis pathways, in comparison to lipid uptake and the formation of lipid droplets in infected cells, are tracked. The basis for the beneficial effect of aspirin to stifle parasite resistance to antimony is explored.

Reviewer #2 (Public review):

Summary:

Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identified EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts central pattern generator (CPG) function necessary for motor coordination.

Strengths:

The main strength of this manuscript is the human genetic data, which provide convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.

Weaknesses:

The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not fully supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. Nevertheless, the data showing correlations between spine curvature and abnormal neural patterning, neuronal firing, and swimming in eph4a loss-of-function mutant larvae are sound.

Comments on revisions:

I think the authors misunderstood my point about genetic nomenclature for the zebrafish alleles. The nomenclature guidelines are described at ZFIN, and ZFIN will ask for appropriate allele designations.

Reviewer #1 (Public review):

Public Review

The authors investigated the role of the C. elegans Flower protein, FLWR-1, in synaptic transmission, vesicle recycling, and neuronal excitability. They confirmed that FLWR-1 localizes to synaptic vesicles and the plasma membrane and facilitates synaptic vesicle recycling at neuromuscular junctions. They observed that hyperstimulation results in endosome accumulation in flwr-1 mutant synapses, suggesting that FLWR-1 facilitates the breakdown of endocytic endosomes. Using tissue-specific rescue experiments, the authors showed that expressing FLWR-1 in GABAergic neurons restored the aldicarb-resistant phenotype of flwr-1 mutants to wild-type levels. By contrast, cholinergic neuron expression did not rescue aldicarb sensitivity at all. They also showed that FLWR-1 removal leads to increased Ca2+ signaling in motor neurons upon photo-stimulation. From these findings, the authors conclude that FLWR-1 helps maintain the balance between excitation and inhibition (E/I) by preferentially regulating GABAergic neuronal excitability in a cell-autonomous manner.

Overall, the work presents solid data and interesting findings, however the proposed cell-autonomous model of GABAergic FLWR-1 function may be overly simplified in my opinion.

Most of my previous comments have been addressed; however, two issues remain.

(1) I appreciate the authors' efforts conducting additional aldicarb sensitivity assays that combine muscle-specific rescue with either cholinergic or GABergic neuron-specific expression of FLWR-1. In the revised manuscript, they conclude, "This did not show any additive effects to the pure neuronal rescues, thus FLWR-1 effects on muscle cell responses to cholinergic agonists must be cell-autonomous." However, I find this interpretation confusing for the reasons outlined below.

Figure 1 - Figure Supplement 3B shows that muscle-specific FLWR-1 expression in flwr-1 mutants significantly restores aldicarb sensitivity. However, when FLWR-1 is co-expressed in both cholinergic neurons and muscle, the worms behave like flwr-1 mutants and no rescue is observed. Similarly, cholinergic FLWR-1 alone fails to restore aldicarb sensitivity (shown in the previous manuscript). These observations indicate a non-cell-autonomous interaction between cholinergic neurons and muscle, rather than a strictly muscle cell-autonomous mechanism. In other words, FLWR-1 expressed in cholinergic neurons appears to negate or block the rescue effect of muscle-expressed FLWR-1. Therefore, FLWR-1 could play a more complex role in coordinating physiology across different tissues. This complexity may affect interpretations of Ca2+ dynamics and/or functional data, particularly in relation to E/I balance, and thus warrants careful discussion or further investigation.

(2) The revised manuscript includes new GCaMP analyses restricted to synaptic puncta. The authors mention that "we compared Ca2+ signals in synaptic puncta versus axon shafts, and did not find any differences," concluding that "FLWR-1's impact is local, in synaptic boutons." This is puzzling: the similarity of Ca2+ signals in synaptic regions and axon shafts seems to indicate a more global effect on Ca2+ dynamics or may simply reflect limited temporal resolution in distinguishing local from global signals due to rapid Ca2+ diffusion. The authors should clarify how they reached the conclusion that FLWR-1 has a localized impact at synaptic boutons, given that synaptic and axonal signals appear similar. Based on the presented data, the evidence supporting a local effect of FLWR-1 on Ca2+ dynamics appears limited.

Reviewer #2 (Public review):

Summary:

The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.

Strengths:

A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.

The authors have adequately addressed most of my previous concerns, however, I recommend minor revisions to further strengthen the study's rigor and interpretation:

Major suggestions

(1) This study relies heavily on aldicarb assays to support its conclusions. While these assays are valuable, their results may not fully align with direct assessment of neurotransmitter release from motor neurons. For instance, prior work has shown that two presynaptic modulators identified through aldicarb sensitivity assays exhibited no corresponding electrophysiological defects at the neuromuscular junction (Liu et al., J Neurosci 27: 10404-10413, 2007). Similarly, at least one study from the Kaplan lab has noted discrepancies between aldicarb assays and electrophysiological analyses. The authors should consider adding a few sentences in the Discussion to acknowledge this limitation and the potential caveats of using aldicarb assays, especially since some of the aldicarb assay results in this study are not easily interpretable.

(2) The manuscript states, "Elevated Ca2+ levels were not further enhanced in a flwr-1;mca-3 double mutant." (lines 549-550). However, Figure 7C does not include statistical comparisons between the single and double mutants of flwr-1 and mca-3. Please add the necessary statistical analysis to support this statement.

(3) The term "Ca2+ influx" should be avoided, as this study does not provide direct evidence (e.g. voltage-clamp recordings of Ca2+ inward currents in motor neurons) for an effect of the flwr-1 mutation of Ca2+ influx. The observed increase in neuronal GCaMP signals in response to optogenetic activation of ChR2 may result from, or be influenced by, Ca2+ mobilization from of intracellular stores. For example, optogenetic stimulation could trigger ryanodine receptor-mediated Ca2+ release from the ER via calcium-induced calcium release (CICR) or depolarization-induced calcium release (DICR). It would be more appropriate to describe the observed increase in Ca2+ signal as "Ca2+ elevation" rather than increased "Ca2+ influx".

Reviewer #1 (Public review):

Summary:

In this study from Belato, Knight and co-workers, the authors investigated the Rec domain of a thermophilic Cas9 from Geobacillus stearothermophilus (GeoCas9). The authors investigated three constructs, two individual subdomains of Rec (Rec1 and Rec2) and the full Rec domain. This domain is involved in binding to the guide RNA of Cas9, as well as the RNA-DNA duplex that is formed upon target binding. The authors performed RNA binding and relaxation experiments using NMR for the wild-type domain as well as two-point mutants. They observed differences in RNA binding activities as well as the flexibility of the domain. The authors also performed molecular dynamics and functional experiments on full-length GeoCas9 to determine whether these biophysical differences affect the RNA binding or cleavage activity. Although the authors observed some changes in the thermal stability of the mutant GeoCas9-gRNA complex, they did not observe substantial differences in the guide RNA binding or cleavage activities of the mutant GeoCas9 variants.

Overall, this manuscript provides a detailed biophysical analysis of the GeoCas9 Rec domain. The NMR assignments for this construct should prove very useful, and can serve as the basis for future similar studies of GeoCas9 Rec domain mutants. While the two mutants tested in the study did not produce significant differences from wild-type GeoCas9, the study rules out the possibility that analogous mutations can be translated between type II-A and II-C Cas9 orthologs. Together, these findings may provide the grounds for future engineering of higher fidelity variants of GeoCas9

Reviewer #2 (Public review):

The manuscript from Belato et al., used advanced NMR approaches and a mutagenesis campaign probe the conformational dynamics of the recognition lobe (Rec) of the CRISPR Cas9 enzyme from G. stearothermophilus (GeoCas9). Using truncated and full-length constructs they assess the impacts of two different point mutations have on the redistribution and timescale of these motions and assess gRNA recognition and specificity. Single point mutations in the Rec domain in a Cas9 from a related species had profound impacts on- and off-target DNA editing, therefore the authors reasoned analogous mutations in GeoCas9 would have similar effects. However, despite a redistribution of local motions and changes in global stability, their chosen mutations had little impact on DNA editing in the context of the full-length enzyme.

In their revised manuscript, the authors were highly responsive to the reviewer's comments incorporating new experimental results including molecular dynamics simulations and RNA binding data using full-length GeoCas9, as well as reframing their discussion and conclusions in consideration of the new data. They were receptive to suggestions for clarification in both the text and methods section. With these changes, the manuscript has been significantly improved.

Their studies highlight the species-specific complexity of interdomain communication and allosteric mechanisms used by these multi-domain endonucleases. The noted strengths of the article remain, and despite the negative results, their approach will garner interest from investigators interested in understanding how the activity and specificity of these enzymes can be engineered to tune activity and limit off-target cleavage by these enzymes. Generally, the manuscript highlights the challenges of studying the effect of allosteric networks on protein function, particularly in multidomain proteins, and thus will be of broad interest to the community.

Reviewer #3 (Public review):

The authors explore the role of Rec domains in a thermophilic Cas9 enzyme. They report on the crystal structure of part of the recognition lobe, its dynamics from NMR spin relaxation and relaxation-dispersion data, its interaction mode with guide RNA, and the effect of two single-point mutations hypothesised to enhance specificity. They find that mutations have small effects on Rec domain structure and stability but lead to significant rearrangement of micro- to milli-second dynamics which does not translate into major changes in guide RNA affinity or DNA cleavage specificity, illustrating the inherent tolerance of GeoCas9. The work can be considered as a first step towards understanding motions in GeoCas9 recognition lobe, although no clear hotspots were discovered with potential for future rational design of enhanced Cas9 variants.

Strengths:

- Detailed biophysical and structural investigation, despite a few technical limitations inherent with working with complex targets, provides converging evidence that molecular dynamics embedded in the recognition lobes allow GeoCas9 to operate on a broad range of substrates.<br /> - Since the authors and others have shown that substrate specificity is dictated by equivalent hotspot mutations in other Cas9 variants, we are one step closer to understanding this phenomenon.

Weaknesses:

- Since the mutations investigated here do not significantly affect substrate binding or enzymatic activity, it is difficult to rationalize anything for enzyme engineering at this point.<br /> - Further investigation of the determinants of the observed dynamic modes, and follow-up with rationally designed mutations would hopefully allow to create a real model of the mechanism, but I do understand that this goes beyond the scope of this study.

Reviewer #1 (Public review):

Summary.

The authors goal was to map the neural circuitry underlying cold sensitive contraction in Drosophila. The circuitry underlying most sensory modalities has been characterized but noxious cold sensory circuitry has not been well studied. Importantly, they analyze all downstream partner neurons connected to the Class III sensory neurons. The authors achieve their goal and map out sensory and post-sensory neurons involved in this behavior.

Strengths.

The manuscript provides compelling evidence for sensory and post sensory neurons involved in noxious cold sensitive behavior. They use both connectivity data and functional data to identify these neurons. This work is a clear advance in our understanding of noxious cold behavior. The experiments are done with a high degree of experimental rigor.

Weaknesses.

I find no major weaknesses in this work. It is a massive amount of data that clearly shows the role of the Class III neurons in cold-induced larval body contraction.

Reviewer #2 (Public review):

Patel et al perform the analysis of neurons in a somatosensory network involved in responses to noxious cold in Drosophila larva. Using a combination of behavioral experiments, Calcium imaging, optogenetics and synaptic connectivity analysis in the Drosophila larval they assess the function of circuit elements in the somatosensory network downstream of multimodal somatosensory neurons involved in innocuous and noxious stimuli sensing and probe their function in noxious cold processing, Consistent with their previous findings they find the multidendritic class III neurons , to be the key cold sensing neurons that are both required and sufficient for the CT behaviors response (shown to evoked by noxious cold). They further investigate the downstream neurons identified based on literature and connectivity from EM at different stages of sensory processing characterize the different phenotypes upon activating/silencing those neurons and monitor their responses to noxious cold. The work reveals diverse phenotypes for the different neurons studied and provides the groundwork for understanding how information is processed in the nervous system from sensory input to motor output and how information from different modalities is processed by neuronal networks. However, at times the writing could be clearer and some results interpretations more rigorous

Reviewer #3 (Public review):

Summary:

The authors follow up on prior studies where they have argued for the existence of cold nociception in Drosophila larvae. In the proposed pathway, mechanosensitive Class III multidendritic neurons are the noxious cold responding sensory cells. The current study attempts to explore the potential roles of second and third order neurons, based on information of the Class III neuron synaptic outputs that has been obtained from the larval connectome.

Strengths:

The major strength of the manuscript is the detailed discussion of the second and third order neurons that are downstream of the mechanosensory Class III multidendritic neurons. These will be useful in further studies of gentle touch mechanosensation and mechanonociception both of which rely on sensory input from these cells. Calcium imaging experiments on Class III activation with optogenetics support the wiring diagram.

Weaknesses:

The scientific premise is that a full body contraction in larvae that are exposed to noxious cold is a sensorimotor behavioral pathway. This premise is, to start with, questionable. A common definition of behavior is a set of "orderly movements with recognizable and repeatable patterns of activity produced by members of a species (Baker et al., 2001)." In the case of nociception behaviors, the patterns of movement are typically thought to play a protective role and to protect from potential tissue damage.

Does noxious cold elicit a set of orderly movements with a recognizable and repeatable pattern in larvae? Can the patterns of movement that are stimulated by noxious cold allow the larvae to escape harm? Based on the available evidence, the answer to both questions is seemingly no. In response to noxious cold stimulation many, if not all, of the muscles in the larva, simultaneously contract (Turner et al., 2016) and as a result the larva becomes stationary. In response to cold, the larva is literally "frozen" in place and it is incapable of moving away. This incapacitation by cold is the antithesis of what one might expect from a behavior that protects the animals from harm.

An extensive literature has investigated the physiological responses of insects to cold (reviewed in Overgaard and MacMillan, 2017). In numerous studies of insects across many genera (excluding cold adapted insects such as snow flies), exposure to very cold temperatures quickly incapacitates the animal and induces a state that is known as a chill coma. During a chill coma the insect becomes immobilized by the cold exposure, but if the exposure to cold is very brief the insect can often be revived without apparent damage. Indeed, it is common practice for many laboratories that use adult Drosophila for studies of behavior to use a brief chilling on ice as a form of anesthesia because chilling is less disruptive to subsequent behaviors than the more commonly used carbon dioxide anesthesia. If flies were to perceive cold as a noxious nociceptive stimulus, then this "chill coma" procedure would likely be disruptive to behavioral studies, but is not. Furthermore, there is no evidence to suggest that larval sensation of "noxious cold" is aversive.

The insect chill coma literature has investigated the effects of extreme cold on the physiology of nerves and muscle and the consensus view of the field is that the paralysis that results from cold is due to complex and combined action of direct effects of cold on muscle and on nerves (Overgaard and MacMillan, 2017). Electrophysiological measurements of muscles and neurons find that they are initially depolarized by cold, and after prolonged cold exposure they are unable to maintain potassium homeostasis and this eventually inhibits the firing of action potentials (Overgaard and MacMillan, 2017). The very small thermal capacitance of a Drosophila larva means that its entire neuromuscular system will be quickly exposed to the effect of cold in the behavioral assays under consideration here. It would seem impossible to disentangle the emergent properties of a complex combination of effects on physiology (including neuronal, glial, and muscle homeostasis) on any proposed sensorimotor transformation pathway.

Nevertheless, the manuscript before us makes a courageous attempt at attempting this. A number of GAL4 drivers tested in the paper are found to affect parameters of contraction behavior (CT) in cold exposed larvae in silencing experiments. However, notably absent from all of the silencing experiments are measurements of larval mobility following cold exposure. Thus, it is not known from the study if these manipulations are truly protecting the larvae from paralysis following cold exposure, or if they are simply reducing the magnitude of the initial muscle contraction that occurs immediately following cold (ie reducing CT). The strongest effect of silencing occurs with the 19-12-GAL4 driver which targets Class III neurons (but is not completely specific to these cells).

Optogenetic experiments for Class III neurons relying on the 19-12-GAL4 driver combined with a very strong optogenetic acuator (ChETA) show the CT behavior that was reported in prior studies. It should be noted that this actuator drives very strong activation, and other studies with milder optogenetic stimulation of Class III neurons have shown that these cells produce behavioral responses that resemble gentle touch responses (Tsubouchi et al 2012 and Yan et al 2013). As well, these neurons express mechanoreceptor ion channels such as NompC and Rpk that are required for gentle touch responses.

A major weakness of the study is that none of the second or third order neurons (that are downstream of CIII neurons) are found to trigger the CT behavioral responses even when strongly activated with the ChETA actuator (Figure 2 Supplement 2). These findings raise major concerns for this and prior studies and it does not support the hypothesis that the CIII neurons drive the CT behaviors.

Later experiments in the paper that investigate strong CIII activation (with ChETA) in combination with other second and third order neurons does support the idea activating those neurons can facilitate the body-wide muscle contractions. But many of the co-activated cells in question are either repeated in each abdominal neuromere or they project to cells that are found all along the ventral nerve cord, so it is therefore unsurprising that their activation would contribute to what appears to be a non-specific body-wide activation of muscles along the AP axis. As well, if these neurons are already downstream of the CIII neurons the logic of this co-activation approach is not particular clear. A more convincing experiment would be to silence the different classes of cells in the context of the optogenetic activation of CIII neurons to test for a block of the effects, a set of experiments that is notably absent from the study.

The authors argument that the co-activation studies support "a population code" for cold nociception is a very optimistic interpretation of a brute force optogenetics approach that ultimately results in an enhancement of a relatively non-specific body-wide muscle convulsion.

Comments on revisions:

The resubmitted version of this manuscript suffers from the same weaknesses that were raised in the prior round of review. The authors claim that muscles have been removed from the electrophysiological preparations of prior studies is overstated. A small subset of muscles are removed during their recording procedures and this does not rule out the possibility that mechanical forces that are generated by the remaining muscles are being sensed by the mechanosensory neurons.

Reviewer #1 (Public review):

Summary:

In this study, Nakagawa and colleagues report the observation that YAP is differentially localized, and thus differentially transcriptionally active, in spheroid cultures versus monolayer cultures. YAP is known to play a critical role in the survival of drug-tolerant cancer cells, and as such, the higher levels of basally activated YAP in monolayer cultures lead to higher fractions of surviving drug-tolerant cells relative to spheroid culture (or in vivo culture). The findings of this study, revealed through convincing experiments, are elegantly simple and straightforward, yet they add significantly to the literature in this field by revealing that monolayer cultures may actually be a preferential system for studying residual cell biology simply because the abundance of residual cells in this format is much greater than in spheroid or xenograft models. The potential linkage between matrix density and stiffness and YAP activation, while only speculated upon in this manuscript, is intriguing and a rich starting point for future studies.

Although this work, like any important study, inspires many interesting follow-on questions, I am limiting my questions to only a few minor ones, which may potentially be explored either in the context of the current study or in separate, follow-on studies.

Strengths:

The major strengths of the work are described above.

Weaknesses:

Rather than considering the following points as weaknesses, I instead prefer to think of them as areas for future study:

(1) Given the field's intense interest in the biology and therapeutic vulnerabilities of residual disease cells, I suspect that one major practical implication of this work could be that it inspires scientists interested in working in the residual disease space to model it in monolayer culture. However, this relies upon the assumption that drug-tolerant cells isolated in monolayer culture are at least reasonably similar in nature to drug-tolerant cells isolated from spheroid or xenograft systems. Is this true? An intriguing experiment that could help answer this question would be to perform gene expression profiling on a cell line model in the following conditions: monolayer growth, drug tolerant cells isolated from monolayer growth conditions, spheroid growth, drug tolerant cells isolated from spheroid growth conditions, xenograft tumors, and drug tolerant cells isolated from xenograft tumors. What are the genes and programs shared between drug-tolerant cells cultured in the three conditions above? Which genes and programs differ between these conditions? Data from this exercise could help provide additional, useful context with which to understand the benefits and pitfalls of modeling residual tumor cell growth in monolayer culture.

(2) In relation to the point above, there is an interesting and established connection between mesenchymal gene expression and YAP/TAZ signaling. For example, analyses of gene expression data from human tumors and cell lines demonstrate an extremely strong correlation between these two gene expression programs. Further, residual persister cancer cells have often been characterized as having undergone an EMT-like transition. From the analysis above, is there evidence that residual tumor cells with increased YAP signaling also exhibit increased mesenchymal gene expression?

Reviewer #2 (Public review):

The manuscript by Nakagawa R, et al describes a mechanism of how NSCLC cells become resistant to EGFR and KRAS G12C inhibition. Here, the authors focus on the initial cellular changes that occur to confer resistance and identify YAP activation as a non-genetic mechanism of acute resistance.

The authors performed an initial xenograft study to identify YAP nuclear localization as a potential mechanism of resistance to EGFRi. The increase in the stromal component of the tumors upon Afatinib treatment leads the authors to explore the response to these inhibitors in both 2D and 3D culture. The authors extend their findings to both KRAS G12C and BRAF inhibitors, suggesting that the mechanism of resistance may be shared along this pathway.

The paper would benefit from additional cell lines to determine the generalizability of the findings they presented. While the change in the localization of YAP upon Afatinib treatment was identified in a xenograft model, the authors do not return to animal models to test their potential mechanism, and the effects of the hyperactivated S127A YAP protein on Afatinib sensitivity in culture are modest. Also, combination studies of YAP inhibitors and EGFR/RAS/RAF inhibitors would have strengthened the studies.

Reviewer #1 (Public review):

Summary

Olfactory sensory neurons (OSNs) in the olfactory epithelium detect myriads of environmental odors that signal essential cues for survival. OSNs are born throughout life and thus represent one of the few neurons that undergo life-long neurogenesis. Until recently, it was assumed that OSN neurogenesis is strictly stochastic with respect to subtype (i.e. the receptor the OSN chooses to express). However, a recent study showed that olfactory deprivation via naris occlusion selectively reduced birthrates of only a fraction of OSN subtypes and indicated that these subtypes appear to have a special capacity to undergo changes in birthrates in accordance with the level of olfactory stimulation. These previous findings raised the interesting question of what type of stimulation influences neurogenesis, since naris occlusion does not only reduce the exposure to potentially thousands of odors, but also to more generalized mechanical stimuli via preventing airflow.

In this study, the authors set out to identify the stimuli that are required to promote the neurogenesis of specific OSN subtypes. Specifically, they aim to test the hypothesis if discrete odorants selectively stimulate the same OSN subtypes whose birthrates are affected. This would imply a highly specific mechanism in which exposure to certain odors can "amplify" OSN subtypes responsive to those odors suggesting that OE neurogenesis serves, in part, an adaptive function.

To address this question, the authors focused on a family of OSN subtypes that had previously been identified to respond to musk-related odors and that exhibit higher transcript levels in the olfactory epithelium of mice exposed to males compared to mice isolated from males. First, the authors confirm via a previously established cell birth dating assay in unilateral naris occluded mice that this increase in transcript levels actually reflects a stimulus-dependent birthrate acceleration of this OSN subtype family. In a series of experiments (in unilateral occluded and non-occluded mice) using the same birth dating assay, they show that several subtypes of this OSN family, but not other "control" subtypes exhibit increased birthrates in response to adolescent male exposure, but not to female exposure.

In the core experiment of the study, they expose unilaterally naris occluded and non-occluded mice to two musk-related odors and two "control" odors (that do not activate musk-responsive OSN subtypes) to test if these odors specifically accelerate the birth rates of OSN types that are responsive to these odors. This experiment reveals that (for the tested odors and OSN subtypes) indeed birthrates are only affected by discrete odorants that stimulate these OSN subtypes (with a complex relationship between birth rate acceleration and odor concentrations) suggesting that OE neurogenesis may serve, in part, as an adaptive function

Strength:

The scientific question is valid and opens an interesting direction. The previously established cell birth dating assay in naris occluded and non-occluded mice is well performed and accompanied by several control experiments addressing potential other interpretations of the data.

In this revised version, the authors added several new experiments addressing the previous concern that only the effect of one specific odor (muscone) on musk-responsive OSN subtypes had been tested to make the general claim that discrete odors specifically accelerate the birth rate of OSN subtypes they stimulate. Now the authors demonstrate that another musk-related odor (ambretone) also induces this effect and that other non-musk odors do not. In addition, they show that two other OSN subtypes that do not respond to musk-related odors are not affected. These experiments further substantiate the above claim.

Weakness:

(1) The main research question of this study was to test if discrete odors specifically accelerate the birth rate of OSN subtypes they stimulate, i.e. does muscone only accelerate the birth rate of OSNs that express muscone-responsive ORs, or vice versa is the birthrate of muscone-responsive OSNs only accelerated by odors they respond to?<br /> As mentioned under "strength" the authors added several experiments to further substantiate their claim. While these controls are very important to show that the observed effect is indeed specific for musk-related odors on musk-responsive OSN subtypes, these experiments still only focus on one closely related family of musk-responsive OSN subtypes. To understand if this phenomenon is a more generalized mechanism and plays a role for other OSN subtypes beyond this small family of related receptors, further experiments showing this effect for other OSN subtypes are critical.

(2) Previous concerns (#2, #4, #5 and #6) about a lack of increase in UNO effect size for olfr1440 under any muscone concentrations, strong fluctuations of newborn neurons on the closed side as well as a seemingly contradicting statement that overstimulation possibly reflects reduced survival have been addressed by adding potential explanations to the text.

In addition, the previous remark (#3) that certain phrases gave the misleading impression that musk-related odors are indeed excreted into male mouse urine at certain concentrations was addressed not only by re-phrasing, but by performing additional experiments. Although these did not deliver clear results (because of technical difficulties), interesting possibilities are discussed.

Reviewer #3 (Public review):

Summary

Neurogenesis in the mammalian olfactory epithelium continues throughout the animal's lifespan, replacing damaged or dying olfactory sensory neurons. It has been tacitly assumed that the replacement of olfactory receptor (OR) subtypes occurs randomly. However, anecdotal evidence has suggested otherwise. In this study, Santoro and colleagues challenge this assumption by systematically exploring three key questions: Is there enrichment of specific OR subtypes during neurogenesis? Is this enrichment influenced by sensory stimuli? Does enrichment result from differential generation of OR types or differential cell death regulated by neural activity? The authors present convincing evidence that muscone stimulus selectively enhances the neurogenesis of OSNs expressing muscone receptors, suggesting that the selection of ORs in regenerating neurons is not random.

Strengths

This study is the first in formulating and systematically testing the selective promotion hypothesis. It is a comprehensive and systematic examination of multiple musk receptors under conditions of unilateral naris occlusion and various stimuli. The controls are properly done. The quality of in situ hybridization and immunofluorescent staining is high, allowing the authors to effectively estimates the number of OR types. The data convincingly demonstrate that increased expression of musk receptors in response to male odor or muscone stimulation.

Weaknesses

The revised version has addressed most initial weaknesses. However, some inconsistencies remain, raising questions about the proposed model. For instance, in the unilateral naris occlusion experiment, although average expression of non-musk receptors shows no significant change, receptors seem to fall into two groups with one subset increasing and another decreasing (Fig. 1E). This suggests naris occlusion may regulate OR expression independently of odor-induced activity, a possibility that remains unaddressed.

There is curiosity regarding receptors for the male odor SBT, olfr912, and olfr1295, which exhibit increased expression on the occluded side. The explanation that SBT exposure shortens these neurons' lifespan lacks substantiation and is inconsistent with later data. For example, Figure 3-figure supplement 3 I does not show olfr912 changes on the UNO side, and Figure 4-figure supplement 4 shows no significant decrease in olfr912 expression in SBT-exposed mice. Additionally, single-cell RNASeq experiments did not examine olfr912 and olfr1295 expression.

While the study convincingly demonstrates muscone's selective stimulation of olfr235-expressing OSNs, it lacks exploration of the mechanisms underlying this specificity. The discussion of signaling pathways remains too generic.

In summary, while the study offers significant insights into neurogenesis and OR subtype enrichment, further investigation into underlying mechanisms and addressing existing inconsistencies would strengthen its conclusions.

Reviewer #1 (Public review):

Summary:

In this study, Le et al.. aimed to explore whether AAV-mediated overexpression of Oct4 could induce neurogenic competence in adult murine Müller glia, a cell type that, unlike its counterparts in cold-blooded vertebrates, lacks regenerative potential in mammals. The primary goal was to determine whether Oct4 alone, or in combination with Notch signaling inhibition, could drive Müller glia to transdifferentiate into bipolar neurons, offering a potential strategy for retinal regeneration.

The authors demonstrated that Oct4 overexpression alone resulted in the conversion of 5.1% of Müller glia into Otx2+ bipolar-like neurons by five weeks post-injury, compared to 1.1% at two weeks. To further enhance the efficiency of this conversion, they investigated the synergistic effect of Notch signaling inhibition by genetically disrupting Rbpj, a key Notch effector. Under these conditions, the percentage of Müller glia-derived bipolar cells increased significantly to 24.3%, compared to 4.5% in Rbpj-deficient controls without Oct4 overexpression. Similarly, in Notch1/2 double-knockout Müller glia, Oct4 overexpression increased the proportion of GFP+ bipolar cells from 6.6% to 15.8%.

To elucidate the molecular mechanisms driving this reprogramming, the authors performed single-cell RNA sequencing (scRNA-seq) and ATAC-seq, revealing that Oct4 overexpression significantly altered gene regulatory networks. They identified Rfx4, Sox2, and Klf4 as potential mediators of Oct4-induced neurogenic competence, suggesting that Oct4 cooperates with endogenously expressed neurogenic factors to reshape Müller glia identity.

Overall, this study aimed to establish Oct4 overexpression as a novel and efficient strategy to reprogram mammalian Müller glia into retinal neurons, demonstrating both its independent and synergistic effects with Notch pathway inhibition. The findings have important implications for regenerative therapies as they suggest that manipulating pluripotency factors in vivo could unlock the neurogenic potential of Müller glia for treating retinal degenerative diseases.

Strengths:

(1) Novelty: The study provides compelling evidence that Oct4 overexpression alone can induce Müller glia-to-bipolar neuron conversion, challenging the conventional view that mammalian Müller glia lacks neurogenic potential.<br /> (2) Technological Advances: The combination of Muller glia-specific labeling and modifying mouse line, AAV-GFAP promoter-mediated gene expression, single-cell RNA-seq, and ATAC-seq provides a comprehensive mechanistic dissection of glial reprogramming.<br /> (3) Synergistic Effects: The finding that Oct4 overexpression enhances neurogenesis in the absence of Notch signaling introduces a new avenue for retinal repair strategies.

Weaknesses:

(1) In this study, the authors did not perform a comprehensive functional assessment of the bipolar cells derived from Müller glia to confirm their neuronal identity and functionality.<br /> (2) Demonstrating visual recovery in a bipolar cell-deficiency disease model would significantly enhance the translational impact of this work and further validate its therapeutic potential.

Comments on revisions:

The author answered all my questions and corrected the minor comments, so I have no more comments on the manuscript.

Reviewer #2 (Public review):

Summary:

The authors harness single cell RNAseq data from zebrafish and mice to identify Oct4 as a candidate driver of neurogenesis. They then use adeno-associated virus vectors to show that while Oct4 overexpression alone converts rare adult Müller glia (MG) to bipolar cells, it synergizes with Notch pathway inhibition to cause this neurogenesis (achieved by Cre-mediated knockout of Rbpj floxed allele). Importantly, they genetically lineage-mark adult MG using a GLAST-CreER transgene and a Sun-GFP reporter, so that any non-MG cells that convert can be identified unambiguously. This is crucial because several high-profile papers made erroneous claims using short promoters in the viral delivery vector itself to mark MG, but those promoters are leaky and mark other non-MG cell types, making it impossible to definitively state whether manipulations studied were actually causing neurogenesis, or were merely the result of expression in pre-existing neurons. Once the authors establish Oct4 + RbpjKO synergy they use snRNAseq/ATACseq to identify known and novel transcription factors that could play a role in driving neurogenesis.

Strengths:

The system to mark MG is stringent, so the authors are studying transdifferentiation, not artifactual effects due to leaky viral promoters. The synergy between Oct4 and Notch pathway blockade is notable. The single cell results add the potential involvement of new players such as Rfx4 in adult-MG-neurogenesis.

Weaknesses:

The revised version is clear and there are no major weaknesses.

Overall, the authors achieved what they set out to do, and have made new insights into how neurogenesis can be stimulated in MG. Ultimately, a major long-term goal in the field is to replace lost photoreceptors as this is most relevant to many human visual disorders, and while this paper (like all others before it) does not generate rods or cones, it opens new strategies to coax MG to form a related neuronal cell type. Their approach underscores the benefits of using a gold standard approach for lineage tracing.

Reviewer #2 (Public review):

This study by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, using a powerful screen of secreted mammalian proteins, they find that SLPI interacts directly B. burgdorferi.

The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result but it has not previously been demonstrated and could spur further study. A limitation that is unaddressed experimentally is potential contribution of the greater bacterial burden to the enhanced inflammation, leaving open the question of whether greater immunologic stimulus or a defect in the regulation of inflammation is responsible for the observed enhanced disease. Answering this question would better justify the statement in the abstract that "These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease."

Although the finding of SLPI binding to bacteria is potentially quite interesting the biological relevance of this interaction is not addressed. Readers of only the abstract, which describes the direct interaction of SLPI with bacteria, may mistakenly conclude that the authors demonstrate that recruitment of this immunoregulatory factor to the bacterial surface enhances inflammation of infected tissues. This attractive possibility has not been demonstrated in this study; such assertion would require comparison of bacteria that either bind or do not bind SLPI in a mouse infection model.

Finally, the investigators take advantage of clinical samples to ask if serum SLPI levels a diminished in Lyme disease patients relative to healthy controls. The assessment of human samples is interesting and generally to be lauded, but here the comparison is limited by: (a) a small sample number, with only 5 healthy control samples (which should not be difficult to obtain); and (b) the inclusion of samples from 4 patients with erythema migrans rather than Lyme arthritis, which was the manifestation tracked in the mouse studies. Moreover, of the 3 Lyme arthritis patients, serum samples from multiple blood draws were included, resulting in 5 data points; similarly, of the 4 erythema migrans patients, 13 separate samples were included. The multiple samplings from some but not all subjects could result in differential "weighting" of samples. Therefore, although the investigators provide a statistical analysis of these data, it is difficult to evaluate the validity of this apparent difference.

In summary, this is an interesting study that provides new information regarding infection in a host deficient in SLPI and, using a state-of-the-art screen of the mammalian secretome to show that B. burgdorferi binds SLPI, raising the attractive possibility that this pathogen utilizes a host immune regulator to enhance inflammation. The conclusions that SLPI enhances inflammation directly due to its immunoregulatory activity and that SLPI levels are diminished in human Lyme disease patients, as well as the implication that SLPI binding by the bacterium has pathogenic significance, each require further study.

Reviewer #1 (Public review):

The paper proposes an interesting perspective on the spatio-temporal relationship between FC in fMRI and electrophysiology. The study found that while similar networks configurations are found in both modalities, there is a tendency for the networks to spatially converge more commonly at synchronous than asynchronous timepoints.

My confidence in the findings and their interpretation has been improved by the addition of some basic simulations. It helps give confidence in the measure being used to distinguish between scenarios.

Of course, there may be other scenarios that are problematic that are not covered by the current simulations - this highlights the difficulty of making a claim based on a heuristic measure.

That said, with the simulations included and if the caveat above is acknowledged, then I think the paper is in good shape.

Reviewer #2 (Public review):

Summary:

The study investigates the brain's functional connectivity (FC) dynamics across different timescales using simultaneous recordings of intracranial EEG/source-localized EEG and fMRI. The primary research goal was to determine which of three convergence/divergence scenarios is the most likely to occur.

The results indicate that despite similar FC patterns found in different data modalities, the timepoints were not aligned, indicating spatial convergence but temporal divergence.

The researchers also found that FC patterns in different frequencies do not overlap significantly, emphasizing the multi-frequency nature of brain connectivity. Such asynchronous activity across frequency bands supports the idea of multiple connectivity states that operate independently and are organized into a multiplex system.

Strengths:

The data supporting the authors' claims are convincing and come from simultaneous recordings of fMRI and iEEG/EEG, which has been recently developed and adapted.

The analysis methods are solid and involved a novel approach to analyzing the co-occurrence of FC patterns across modalities (cross-modal recurrence plot, CRP) and robust statistics, including replication of the main results using multiple operationalizations of the functional connectome (e.g., amplitude, orthogonalized, and phase-based coupling).

In addition, the authors provided a detailed interpretation of the results, placing them in the context of recent advances and understanding of the relationships between functional connectivity and cognitive states.

The authors also did a control analysis and verified the effect of temporal window size or different functional connecvitity operationalizations. I also applaud their effort to make the analysis code open-sourced.

Comments on revisions:

The authors addressed all my concerns in the previous round of review.

Reviewer #1 (Public review):

Summary:

Huntington's disease (HD) is characterized by the expansion of polyglutamine repeats in huntingtin protein (HTT), leading to the formation of aggresomes composed of mutant huntingtin (mHTT). This study investigates the potential therapeutic strategy of enhancing autophagy to clear mHTT. The authors' evaluation of the autophagic-lysosomal pathway (ALP) in human HD brains shows that, in early stages, there is upregulated lysosomal biogenesis and relatively normal autophagy flux, while late-stage brains exhibit impaired autolysosome clearance, suggesting that early intervention may be beneficial. The authors cross the Q175 HD knock-in model with the TRGL autophagy reporter mouse to investigate ALP dynamics in vivo. In these models, mHTT is detected in autophagic vacuoles and colocalizes with autophagy receptors p62/SQSTM1 and ubiquitin. Although ALP alterations in the Q175 model are milder and later onset compared to human HD, they do show lysosome depletion and impaired autophagic flux. Treatment with an mTOR inhibitor in 6-month-old TRGL/Q175 mice normalized lysosome numbers, alleviated aggresome pathology, and reduced mHTT, p62, and ubiquitin levels. These findings suggest that autophagy modulation during the early stages of disease progression may offer potential therapeutic interventions for HD pathology.

Strengths:

Provide supportive animal evidence for mTOR inhibition in enhancing autophagy and reducing toxicity in HD animal models.

Weaknesses:

Lacks animal behavior and survival rate data, particularly regarding whether the extent of motor dysfunction in TRGL/Q175 mice is comparable to that in Q175 mice and whether the administration of mTORi INK improves these symptoms.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors have explored the beneficial effect of autophagy upregulation in the context of HD pathology in a disease stage-specific manner. The authors have observed functional autophagy lysosomal pathway (ALP) and its machineries at the early stage in HD mouse model, whereas impairment of ALP has been documented at the later stages of the disease progression. Eventually, the authors have taken advantage of operational ALP pathway at the early stage of HD pathology, in order to upregulate ALP and autophagy flux by inhibiting mTORC1 in vivo, which ultimately reverted back multiple ALP-related abnormalities and phenotypes. Therefore, this manuscript is a promising effort to shed light on the therapeutic interventions with which HD pathology can be treated at the patient level in future.

Strengths:

The study has shown alteration of ALP in HD mouse model in a very detailed manner. Such stage dependent in vivo study will be informative and has not been done before. Also, this research provides possible therapeutic intervention in patients in future.

Weaknesses:

In this revised version of the manuscript, the authors have satisfactorily addressed all the concerns raised by the reviewers. They have also provided futuristic viewpoints towards tackling neurodegenerative disorder, especially Huntington Disease (HD).

Reviewer #1 (Public review):

This report addresses a compelling topic. However, I have significant concerns, which necessitate a reassessment of the report's overall value.

Anatomical Specificity and Stimulation Site:<br /> While the authors clarify that the ventral MGB (MGv) was the intended stimulation target, the electrode track (Fig. 1A) and viral spread (Fig. 2E) suggest possible involvement of the dorsal MGB (MGd) and broader area. Given that MGv-AI and MGd-AC pathways have distinct-and sometimes opposing-effects on plasticity, the reported LTP values (with unusually small standard deviations) raise concerns about the specificity of the findings. Additional anatomical verification would help resolve this issue.

Statistical Rigor and Data Variability:<br /> The remarkably low standard deviations in LTP measurements are unexpected based on established variability in thalamocortical plasticity. The authors' response confirms these values are accurate, but further justification, such as methodological controls or replication-would bolster confidence in these results. Additionally, the comparison of in vivo vs. in vitro LTP variability requires more substantive support.

Viral Targeting and Specificity:<br /> The manuscript does not clearly address whether cortical neurons were inadvertently infected by AAV9. Given the potential for off-target effects, explicit confirmation (e.g., microphotograph of stimulation site) would strengthen the study's conclusions.

Integration of Prior Literature:<br /> The discussion of existing work is adequate but could be more comprehensive. A deeper engagement with contrasting findings would provide better context for the study's contributions.

Therapeutic Implications:<br /> The authors' discussion of therapeutic potential is now appropriately cautious and well-reasoned.

Conclusion:<br /> While the study presents intriguing findings, the concerns outlined above must be addressed to fully establish the validity and impact of the results. I appreciate the authors' efforts thus far and hope they can provide additional data or clarification to resolve these issues. With these revisions, the manuscript could make a valuable contribution to the field.

Reviewer #2 (Public review):

Summary:

This work used multiple approaches to show that CCK is critical for long-term potentiation (LTP) in the auditory thalamocortical pathway. They also showed that the CCK mediation of LTP is age-dependent and supports frequency discrimination. This work is important because is opens up a new avenue of investigation of the roles of neuropeptides in sensory plasticity.

Strengths:

The main strength is the multiple approaches used to comprehensively examine the role of CCK in auditory thalamocortical LTP. Thus, the authors do provide a compelling set of data that CCK mediates thalamocortical LTP in an age-dependent manner.

Weaknesses:

There are some details that should be addressed, primarily regarding potential baseline differences in comparison groups. The behavioral assessment is relatively limited, but may be fleshed out in future work.

Reviewer #3 (Public review):

Summary:

Cholecystokinin (CCK) is highly expressed in auditory thalamocortical (MGB) neurons and CCK has been found to shape cortical plasticity dynamics. In order to understand how CCK shapes synaptic plasticity in the auditory thalamocortical pathway, they assessed the role of CCK signaling across multiple mechanisms of LTP induction with the auditory thalamocortical (MGB - layer IV Auditory Cortex) circuit in mice. In these physiology experiments that leverage multiple mechanisms of LTP induction and a rigorous manipulation of CCK and CCK-dependent signaling, they establish an essential role of auditory thalamocortical LTP on the co-release of CCK from auditory thalamic neurons. By carefully assessing the development of this plasticity over time and CCK expression, they go on to identify a window of time that CCK is produced throughout early and middle adulthood in auditory thalamocortical neurons to establish a window for plasticity from 3 weeks to 1.5 years in mice, with limited LTP occurring outside of this window. The authors go on to show that CCK signaling and its effect on LTP in the auditory cortex is also capable of modifying frequency discrimination accuracy in an auditory PPI task. In evaluating the impact of CCK on modulating PPI task performance, it also seems that in mice <1.5 years old CCK-dependent effects on cortical plasticity is almost saturated. While exogenous CCK can modestly improve discrimination of only very similar tones, exogenous focal delivery of CCK in older mice can significantly improve learning in a PPI task to bring their discrimination ability in line with those from young adult mice.

Strengths:

(1) The clarity of the results, along with the rigor multi-angled approach, provide significant support for the claim that CCK is essential for auditory thalamocortical synaptic LTP. This approach uses a combination of electrical, acoustic, and optogenetic pathway stimulation alongside conditional expression approaches, germline knockout, viral RNA downregulation and pharmacological blockade. Through the combination of these experimental configures the authors demonstrate that high-frequency stimulation-induced LTP is reliant on co-release of CCK from glutamatergic MGB terminals projecting to the auditory cortex.

(2) The careful analysis of the CCK, CCKB receptor, and LTP expression is also a strength that puts the finding into the context of mechanistic causes and potential therapies for age-dependent sensory/auditory processing changes. Similarly, not only do these data identify a fundamental biological mechanism, but they also provide support for the idea that exogenous asynchronous stimulation of the CCKBR is capable of restoring an age-dependent loss in plasticity.

(3) Although experiments to simultaneously relate LTP and behavioral change or identify a causal relationship between LTP and frequency discrimination are not made, there is still convincing evidence that CCK signaling in the auditory cortex (known to determine synaptic LTP) is important for auditory processing/frequency discrimination. These experiments are key for establishing the relevance of this mechanism.

Weaknesses:

(1) Given the magnitude of the evoked responses, one expects that pyramidal neurons in layer IV are primarily those that undergo CCK-dependent plasticity, but the degree to which PV-interneurons and pyramidal neurons participate in this process differently is unclear.

(2) While these data support an important role for CCK in synaptic LTP in the auditory thalamocortical pathway, perhaps temporal processing of acoustic stimuli is as or more important than frequency discrimination. Given the enhanced responsivity of the system, it is unclear whether this mechanism would improve or reduce the fidelity of temporal processing in this circuit. Understanding this dynamic may also require consideration of cell type as raised in weakness #1.

(3) In Figure 1, an example of increased spontaneous and evoked firing activity of single neurons after HFS is provided. Yet it is surprising that the group data are analyzed only for the fEPSP. It seems that single neuron data would also be useful at this point to provide insight into how CCK and HFS affect temporal processing and spontaneous activity/excitability, especially given the example in 1F.

(4) The circuitry that determines PPI requires multiple brain areas, including the auditory cortex. Given the complicated dynamics of this process, it may be helpful to consider what, if anything, is known specifically about how layer IV synaptic plasticity in the auditory cortex may shape this behavior.

Comments on revisions:

The manuscript is much improved and many of the issues or questions have been addressed. Ideally, evidence for the degree of transsynaptic spread for AAV9-Syn-ChrimsonR-tdTomato would also be provided in some form since in the authors' response in sounds like some was observed, as expected.

Reviewer #1 (Public review):

Summary:

The manuscript provides a novel method for the automated detection of scent marks from urine and feces in rodents. Given the importance of scent communication in these animals and their role as model organisms, this is a welcome tool.

Strengths:

The method uses a single video stream to allow for the distinction between urine and feces. It is automated.

Weaknesses:

The accuracy is decent but not perfect and may be too low to detect some effects that are biologically real but subtle (e.g. less than 10% differences). For many assays, however, this tools will be useful.

Reviewer #1 (Public review):

About R squared in the plots:<br /> The authors have used a z-scored R squared in the main ridge regression plots. While this may be interpretable, it seems non-standard and overly complicated. The authors could use a simple Pearson r to be most direct and informative (and in line with similar work, including Goldstein et al. 2022 which they mentioned). This way the sign of the relationships is preserved.

About the new TRF analysis:<br /> The new TRF analysis is a necessary addition and much appreciated. However, it is missing the results for the acoustic regressors, which should be there analogous to the HM-LSTM ridge analysis. The authors should also specify which software they have utilized to conduct the new TRF analysis. It also seems that the linguistic predictors/regressors have been newly constructed in a way more consistent with previous literature (instead of using the HM-LSTM features); these specifics should also be included in the manuscript (did it come from Montreal Forced Aligner, etc.?). Now that the original HM-LSTM can be compared to a more standard TRF analysis, it is apparent that the results are similar.

The authors' wording about this suggests that these new regressors have a nonzero sample at each linguistic event's offset, not onset. This should also be clarified. As the authors know, the onset would be more standard, and using the offset has implications for understanding the timing of the TRFs, as a phoneme has a different duration than a word, which has a different duration from a sentence, etc.

About offsets:<br /> TRFs can still be interpretable using the offset timings though; however, the main original analysis seems to be utilizing the offset times in a different, more confusing way. The authors still seem to be saying that only the peri-offset time of the EEG was analyzed at all, meaning the vast majority of the EEG trial durations do not factor into the main HM-LSTM response results whatsoever. The way the authors describe this does not seem to be present in any other literature, including the papers that they cite. Therefore, much more clarification on this issue is needed. If the authors mean that the regressors are simply time-locked to the EEG by aligning their offsets (rather than their onsets, because they have varying onsets or some such experimental design complexity), then this would be fine. But it does not seem to be what the authors want to say. This may be a miscommunication about the methods, or the authors may have actually only analyzed a small portion of the data. Either way, this should be clarified to be able to be interpretable.

Reviewer #2 (Public review):

This study presents a valuable finding on the neural encoding of speech in listeners with normal hearing and hearing impairment, uncovering marked differences in how attention to different levels of speech information is allocated, especially when having to selectively attend to one speaker while ignoring an irrelevant speaker. The results overall support the claims of the authors, although a more explicit behavioural task to demonstrate successful attention allocation would have strengthened the study. Importantly, the use of more "temporally continuous" analysis frameworks could have provided a better methodology to assess the entire time course of neural activity during speech listening. Despite these limitations, this interesting work will be useful to the hearing impairment and speech processing research community.

The study compares speech-in-quiet vs. multi-talker scenarios, allowing to assess within-participant the impact that the addition of a competing talker has on the neural tracking of speech. Moreover, the inclusion of a population with hearing loss is useful to disentangle the effects of attention orienting and hearing ability. The diagnosis of high-frequency hearing loss was done as part of the experimental procedure by professional audiologists, leading to a high control of the main contrast of interest for the experiment. Sample size was big, allowing to draw meaningful comparisons between the two populations.

An HM-LSTM model was employed to jointly extract speech features spanning from the stimulus acoustics to word-level and phrase-level information, represented by embeddings extracted at successive layers of the model. The model was specifically expanded to include lower level acoustic and phonetic information, reaching a good representation of all intermediate levels of speech.

Despite conveniently extracting all features jointly, the HM-LSTM model processes linguistic input sentence-by-sentence, and therefore only allows to assess the corresponding EEG data at sentence offset. If I understood correctly, while the sentence information extracted with the HM-LSTM reflects the entire sentence - in terms of its acoustic, phonetic and more abstract linguistic features - it only gives a condensed final representation of the sentence. As such, feature extraction with the HM-LSTM is not compatible with a continuous temporal mapping on the EEG signal, and this is the main reason behind the authors' decision to fit a regression at nine separate time points surrounding sentence offsets.

While valid and previously used in the literature, this methodology, in the particular context of this experiment, might be obscuring important attentional effects impacted by hearing-loss. By fitting a regression only around sentence-final speech representations, the method might be overlooking the more "online" speech processing dynamics, and only assessing the permanence of information at different speech levels at sentence offset. In other words, the acoustic attentional bias between Attended and Unattended speech might exist even in hearing-impaired participants but, due to a lower encoding or permanence of acoustic information in this population, it might only emerge when using methodologies with a higher temporal resolution, such as Temporal Response Functions (TRFs). If a univariate TRF fit simply on the continuous speech envelope did not show any attentional bias (different trial lengths should not be a problem for fitting TRFs), I would be entirely convinced of the result. For now, I am unsure on how to interpret this finding.

Despite my doubts on the appropriateness of condensed speech representations and single-point regression for acoustic features in particular, the current methodology allows the authors to explore their research questions, and the results support their conclusions.

This work presents an interesting finding on the limits of attentional bias in a cocktail-party scenario, suggesting that fundamentally different neural attentional filters are employed by listeners with high-frequency hearing loss, even in terms of the tracking of speech acoustics. Moreover, the rich dataset collected by the authors is a great contribution to open science and will offer opportunities for re-analysis.

Reviewer #1 (Public review):

I want to reiterate my comment from the first round of reviews: that I am insufficiently familiar with the intricacies of Maxwell's equations to assess the validity of the assumptions and the equations being used by WETCOW. The work ideally needs assessing by someone more versed in that area, especially given the potential impact of this method if valid.

Effort has been made in these revisions to improve explanations of the proposed approach (a lot of new text has been added) and to add new simulations.

However, the authors have still not compared their method on real data with existing standard approaches for reconstructing data from sensor to physical space. Refusing to do so because existing approaches are deemed inappropriate (i.e. they "are solving a different problem") is illogical.

Similarly, refusing to compare their method with existing standard approaches for spatio-temporally describing brain activity, just because existing approaches are deemed inappropriate, is illogical.

For example, the authors say that "it's not even clear what one would compare [between the new method and standard approaches]". How about:

(1) Qualitatively: compare EEG activation maps. I.e. compare what you would report to a researcher about the brain activity found in a standard experimental task dataset (e.g. their gambling task). People simply want to be able to judge, at least qualitatively on the same data, what the most equivalent output would be from the two approaches. Note, both approaches do not need to be done at the same spatial resolution if there are constraints on this for the comparison to be useful.

and

(2) Quantitatively: compare the correlation scores between EEG activation maps and fMRI activation maps

The abstract claims that there is a "direct comparison with standard state-of-the-art EEG analysis in a well-established attention paradigm", but no actual comparison appears to have been completed in the paper.

Reviewer #2 (Public review):

Summary:

The manuscript claims to present a novel method for direct imaging of electric field networks from EEG data with higher spatiotemporal resolution than even fMRI. Validation of the EEG reconstructions with EEG/FMRI, EEG, and iEEG datasets are presented. Subsequently, reconstructions from a large EEG datasets of subjects performing a gambling task are presented.

Strengths:

If true and convincing, the proposed theoretical framework and reconstruction algorithm can revolutionise the use of EEG source reconstructions.

Weaknesses:

There is very little actual information in the paper about either the forward model or the novel method of reconstruction. Only citations to prior work by the authors are given with absolutely no benchmark comparisons, making the manuscript difficult to read and interpret in isolation to their prior body of work.

Comments on revisions:

This is a major rewrite of the paper. The authors have improved the discourse vastly. There is now a lot of didactics included but they are not always relevant to the paper. The section on Maxwell's equation does a disservice to the literature in prior work in bioelectromagnetism and does not even address the issues raised in classic text books by Plonsey et al. There is no logical "backwardness" in the literature. They are based on the relative values of constants in biological tissues. Several sections of the appendix discuss in terms of weather predictions and could just be written specifically for the problem here. There are reinventions of many standard ideas in terms of physics discourses, like Bayesian theory or PCA etc. I think that the paper remains quite opaque and many of the original criticisms remain, especially as they relate to multimodal datasets. The overall algorithm still remains poorly described. The comparisons to benchmark remain unaddressed and the authors state that they couldn't get Loreta to work and so aborted that. The figures are largely unaltered, although they have added a few more, and do not clearly depict the ideas. Again, no benchmark comparisons are provided to evaluate the results and the performance in comparison to other benchmarks.

Reviewer #2 (Public review):

Summary:

In this present Mendelian randomization-phenome-wide association study, the authors found BMI to be positively associated with many health-related conditions, such as heart disease, heart failure, and hypertensive heart disease. They also found sex differences in some traits, such as cancer, psychological disorders, and ApoB.

Strengths:

The use of the UK-biobank study with detailed phenotype and genotype information.

Comments on revisions:

I believe the authors have presented convincing arguments for the novelty and interpretation of their study. I have no additional comments.

Reviewer #1 (Public review):

Summary:

The study identifies two types of activation: one that is cue-triggered and non-specific to motion directions, and another that is specific to the exposed motion directions but occurs in a reversed manner. The finding that activity in the medial temporal lobe (MTL) preceded that in the visual cortex suggests that the visual cortex may serve as a platform for the manifestation of replay events, which potentially enhance visual sequence learning.

Evaluations:

Identifying the two types of activation after exposure to a sequence of motion directions is very interesting. The experimental design, procedures and analyses are solid. The findings are interesting and novel.

In the original submission, it was not immediately clear to me why the second type of activation was suggested to occur spontaneously. The procedural differences in the analyses that distinguished between the two types of activation need to be a little better clarified. However, this concern has been satisfactorily addressed in the revision.

Reviewer #2 (Public review):

This paper shows and analyzes an interesting phenomenon. It shows that when people are exposed to sequences of moving dots (That is moving dots in one direction, followed by another direction etc.), that showing either the starting movement direction, or ending movement direction causes a coarse-grained brain response that is similar to that elicited by the complete sequence of 4 directions. However, they show by decoding the sensor responses that this brain activity actually does not carry information about the actual sequence and the motion directions, at least not on the time scale of the initial sequence. They also show a reverse reply on a highly-compressed time scale, which is elicited during the period of elevated activity, and activated by the first and last elements of the sequence, but not others. Additionally, these replays seem to occur during periods of cortical ripples, similar to what is found in animal studies.

These results are intriguing. They are based on MEG recordings in humans, and finding such replays in humans is novel. Also, this is based on what seems to be sophisticated statistical analysis. The statistical methodology seems valid, but due to its complexity it is not easy to understand. The methods especially those described in figures 3 and 4 should be explained better.

Comments on second revised version by editorial team:

In response to the reviewer, the authors have substantially expanded and clarified their description of the methodology in this version of the manuscript.

Reviewer #1 (Public review):

Summary:

The submitted article reports the development of an unsupervised learning method that enables quantification of behaviour and poses of C. elegans from 15 minute long videos and presents a spatial map of both. The entire pipeline is a two part process, with the first part based on contrastive learning that represents spatial poses onto an embedded space, while the second part uses a transformer encoder to enable estimation of masked parts in a spatiotemporal sequence.

Strengths:

This analysis approach will prove to be useful for the C. elegans community. The application of the method on various age-related videos on various strains presents a good use-case for the approach. The manuscript is well written and presented.

Specific comments:

(1) One of the main motivations as mentioned in the introduction as well as emphasized in the discussion section is that this approach does not require key-point estimation for skeletonization and is also not dependent on the eigenworm approach for pose estimation. However, the eigenworm data has been estimated using the Tierpsy tracker in videos used in this work and stored as metadata. This is subsequently used for interpretation. It is not clear at this point, how else the spatial embedded map may be interpreted without using this kind of pose estimates obtained from other approaches. Please elaborate and comment.

(2) As per the manuscript, the second part of the pipeline is used to estimate the masked sequences of the spatiotemporal behavioral feature. However, it is not clear what the numbers listed in Fig. 2.3 represent?

(3) It is not clear how motion speed is linked to individual poses as mentioned in Figs. 4 (b) and (c).

Reviewer #2 (Public review):

Summary:

The manuscript by Maurice and Katarzyna describes a self-supervised, annotation-free deep-learning approach capable of quantitatively representing complex poses and behaviors of C. elegans directly from video pixel values. Their method overcomes limitations inherent to traditional methods relying on skeletonization or keypoint tracking, which often fail with highly coiled or self-intersecting worms. By applying self-supervised contrastive learning and a Transformer-based network architecture, the authors successfully capture diverse behavioral patterns and depict the aging trajectory of behavioral repertoire. This provides a useful new tool for behavioral research in C. elegans and other flexible-bodied organisms.

Strengths:

Reliable tracking and segmentation of complex poses remain significant bottlenecks in C. elegans behavioral research, and the authors made valuable attempts to address these challenges. The presented method offers several advantages over existing tools, including freedom from manual labeling, independence from explicit skeletonization or keypoint tracking, and the capability to capture highly coiled or overlapping poses. Thus, the proposed method would be useful to the C. elegans research community.

The research question is clearly defined. Methods and results are engagingly presented, and the manuscript is concise and well-organized.

Weaknesses:

(1) In the abstract, the claim of an 'unbiased' approach is not well-supported. The method is still affected by dataset biases, as mentioned in the aging results (section 4.3).<br /> (2) In section 3.2, the rationale behind rotating worm images to a vertical orientation is unclear.<br /> (3) The methods section is clearly written but uses overly technical language, making it less accessible to the audience of eLife, the majority of whom are biologists. Clearer explanations of key methods and the rationale behind their selection are needed. For example, in section 3.3, the authors should briefly explain in simple language what contrastive learning is, why they chose it, and why this method potentially achieves their goal.<br /> (4) The reason why the gray data points could not be resolved by Tierpsy is not quantitatively described. Are they all due to heavily coiled or overlapping poses?<br /> (5) In section 4.1, generating pose representations grouped by genetic strains would provide insights into strain-specific differences resolved by the proposed method.<br /> (6) Fig. 3a requires clarification. Highly bent poses (red points) intuitively should be close to highly coiled poses (gray points). The authors should explain the observed greenish/blueish points interfacing with the gray points.<br /> (7) In Fig. 3a, some colored points overlap with the gray point cloud. Why can Tierpsy resolve these overlapping points representing highly coiled poses? A more systematic quantitative comparison between Tierpsy and the proposed method is required.<br /> (8) The claim in section 4.2 regarding strain separation in pose embedding spaces is unsupported by Fig. 3a, which lacks strain-based distinctions. As mentioned in point #5, showing pose representations grouped by different strains is required.<br /> (9) In section 4.2, how the authors could verify the statement, "This likely occurs since most strains share common behaviors such as simple forward locomotion"?<br /> (10) An important weakness of the proposed method is its low direct interpretability, as it is not based on handcrafted features. To better interpret the pose/behavior embedding space, it would be helpful to compare it against more basic Tierpsy features in Fig. 3 and 4. This comparison could reveal what understandable features were learned by the neural network, thereby increasing human interpretability.<br /> (11) The main conclusion of section 4.3 is not sufficiently tested. Is Fig. 5a generated only from data of N2 animals? To quantitatively verify the statement, "Young individuals appear to display a wide range of behaviors, while as they age their behavior repertoire reduces," the authors should perform a formal analysis of behavioral variability throughout aging.<br /> (12) In Fig. 5a, better visualization of aging trajectories could include plotting the center of mass along with variance of the point cloud over time.<br /> (13) To better reveal aging trajectories of behavioral changes for different genetic backgrounds, it would be meaningful to generate behavior representations for different strains as they age.<br /> (14) As a methods paper, the ease of use for other researchers should be explicitly addressed, and source code and datasets should be provided.

Reviewer #3 (Public review):

Summary:

In this paper, the authors present an unsupervised learning approach to represent C. elegans poses and temporal sequences of poses in low-dimensional spaces by directly using pixel values from video frames. The method does not rely on the exact identification of the worm's contour/midline, nor on the identification of the head and tail prior to analyzing behavioral parameters. In particular, using contrastive learning, the model represents worm poses in low-dimensional spaces, while a transformer encoder neural network embeds sequences of worm postures over short time scales. The study evaluates this newly developed method using a dataset of different C. elegans genetic strains and aging individuals. The authors compared the representations inferred by the unsupervised learning with features extracted by an established approach, which relies on direct identification of the worm's posture and its head-tail direction.

Strengths:

The newly developed method provides a coarse classification of C. elegans posture types in a low-dimensional space using a relatively simple approach that directly analyzes video frames. The authors demonstrate that representations of postures or movements of different genotypes, based on pixel values, can be distinguishable to some extent.

Weaknesses:

- A significant disadvantage of the presented method is that it does not include the direction of the worm's body (e.g., head/tail identification). This highly limits the detailed and comprehensive identification of the worm's behavioral repertoire (on- and off-food), which requires body directionality in order to infer behaviors (for example, classifying forward vs. reverse movements). In addition, including a mix of opposite postures as input to the new method may create significant classification artifacts in the low-dimensional representation-such that, for example, curvature at opposite parts of the body could cluster together. This concern applies both to the representation of individual postures and to the representation of sequences of postures.<br /> - The authors state that head-tail direction can be inferred during forward movement. This is true when individuals are measured off-food, where they are highly likely to move forward. However, when animals are grown on food, head-tail identification can also be based on quantifying the speed of the two ends of the worm (the head shows side-to-side movements). This does not require identifying morphological features. See, for example, Harel et al. (2024) or Yemini et al. (2013).<br /> - Another confounding parameter that cannot be distinguished using the presented method is the size of individuals. Size can differ between genotypes, as well as with aging. This can potentially lead to clustering of individuals based on their size rather than behavior.<br /> - There is no quantitative comparison between classification based on the presented method and methods that rely on identifying the skeleton.

Reviewer #1 (Public review):

Summary:

Using a combination of EEG and behavioural measurements, the authors investigate the degree to which processing of spatially-overlapping targets (coherent motion) and distractors (affective images) are sampled rhythmically and how this affects behaviour. They found that both target processing (via measurement of amplitude modulations of SSVEP amplitude to target frequency) and distractor processing (via MVPA decoding accuracy of bandpassed EEG relative to distractor SSVEP frequency) displayed a pronounced rhythm at ~1Hz, time-locked to stimulus onset. Furthermore, the relative phase of this target/distractor sampling predicted the accuracy of coherent motion detection across participants.

Strengths:

(1) The authors are addressing a very interesting question with respect to sampling of targets and distractors, using neurophysiological measurements to their advantage in order to parse out target and distractor processing.

(2) The general EEG analysis pipeline is sensible and well-described.

(3) The main result of rhythmic sampling of targets and distractors is striking and very clear even on a participant level.

(4) The authors have gone to quite a lot of effort to ensure the validity of their analyses, especially in the Supplementary Material.

(5) It is incredibly striking how the phases of both target and distractor processing are so aligned across trials for a given participant. I would have thought that any endogenous fluctuation in attention or stimulus processing like that would not be so phase aligned. I know there is literature on phase resetting in this context, the results seem very strong here and it is worth noting. The authors have performed many analyses to rule out signal processing artifacts, e.g., the sideband and beating frequency analyses.

Weaknesses:

(1) In general, the representation of target and distractor processing is a bit of a reach. Target processing is represented by SSVEP amplitude, which is most likely going to be related to the contrast of the dots, as opposed to representing coherent motion energy, which is the actual target. These may well be linked (e.g., greater attention to the coherent motion task might increase SSVEP amplitude), but I would call it a limitation of the interpretation. Decoding accuracy of emotional content makes sense as a measure of distractor processing, and the supplementary analysis comparing target SSVEP amplitude to distractor decoding accuracy is duly noted.

(2) Comparing SSVEP amplitude to emotional category decoding accuracy feels a bit like comparing apples with oranges. They have different units and scales and probably reflect different neural processes. Is the result the authors find not a little surprising in this context? This relationship does predict performance and is thus intriguing, but I think this methodological aspect needs to be discussed further. For example, is the phase relationship with behaviour a result of a complex interaction between different levels of processing (fundamental contrast vs higher order emotional processing)?

Reviewer #2 (Public review):

Summary:

In this study, Xiong et al. investigate whether rhythmic sampling - a process typically observed in the attended processing of visual stimuli - extends to task-irrelevant distractors. By using EEG with frequency tagging and multivariate pattern analysis (MVPA), they aimed to characterize the temporal dynamics of both target and distractor processing and examine whether these processes oscillate in time. The central hypothesis is that target and distractor processing occur rhythmically, and the phase relationship between these rhythms correlates with behavioral performance.

Major Strengths:

(1) The extension of rhythmic attentional sampling to include distractors is a novel and interesting question.

(2) The decoding of emotional distractor content using MVPA from SSVEP signals is an elegant solution to the problem of assessing distractor engagement in the absence of direct behavioral measures.

(3) The finding that relative phase (between 1 Hz target and distractor processes) predicts behavioral performance is compelling.

Major Weaknesses and Limitations:

(1) Incomplete Evidence for Rhythmicity at 1 Hz: The central claim of 1 Hz rhythmic sampling is insufficiently validated. The windowing procedure (0.5s windows with 0.25s step) inherently restricts frequency resolution, potentially biasing toward low-frequency components like 1 Hz. Testing different window durations or providing controls would significantly strengthen this claim.

(2) No-Distractor Control Condition: The study lacks a baseline or control condition without distractors. This makes it difficult to determine whether the distractor-related decoding signals or the 1 Hz effect reflect genuine distractor processing or more general task dynamics.

(3) Decoding Near Chance Levels: The pairwise decoding accuracies for distractor categories hover close to chance (~55%), raising concerns about robustness. While statistically above chance, the small effect sizes need careful interpretation, particularly when linked to behavior.

(4) No Clear Correlation Between SSVEP and Behavior: Neither target nor distractor signal strength (SSVEP amplitude) correlates with behavioral accuracy. The study instead relies heavily on relative phase, which - while interesting - may benefit from additional converging evidence.

(5) Phase-analysis: phase analysis is performed between different types of signals hindering their interpretability (time-resolved SSVEP amplitude and time-resolved decoding accuracy).

Appraisal of Aims and Conclusions:

The authors largely achieved their stated goal of assessing rhythmic sampling of distractors. However, the conclusions drawn - particularly regarding the presence of 1 Hz rhythmicity - rest on analytical choices that should be scrutinized further. While the observed phase-performance relationship is interesting and potentially impactful, the lack of stronger and convergent evidence on the frequency component itself reduces confidence in the broader conclusions.

Impact and Utility to the Field:

If validated, the findings will advance our understanding of attentional dynamics and competition in complex visual environments. Demonstrating that ignored distractors can be rhythmically sampled at similar frequencies to targets has implications for models of attention and cognitive control. However, the methodological limitations currently constrain the paper's impact.

Additional Context and Considerations:

(1) The use of EEG-fMRI is mentioned but not leveraged. If BOLD data were collected, even exploratory fMRI analyses (e.g., distractor modulation in visual cortex) could provide valuable converging evidence.

(2) In turn, removal of fMRI artifacts might introduce biases or alter the data. For instance, the authors might consider investigating potential fMRI artifact harmonics around 1 Hz to address concerns regarding induced spectral components.

Reviewer #1 (Public review):

Summary:

Some years ago, Brookshire proposed a method to identify oscillations in behavioural data that controls for effects of aperiodic trends. Such trends can produce false positive results if not controlled for. Although this method successfully controlled for this issue, it was also relatively insensitive to true effects, and it remained unclear whether it was unable to replicate published evidence for behavioural oscillations because they were false positives or the method could not detect them. In simulated data, Harris & Beale show that their revised version of the method proposed by Brookshire is more sensitive to effects and equally unsusceptible to false positives. When applied to available data, this new version indeed revealed evidence for behavioural oscillations. This paper is therefore an important piece in the puzzle of the ongoing debate on behavioural oscillations.

Strengths:

(1) The paper is well written and compact.

(2) The new method proposed is tested thoroughly, and its application in simulated data shows its properties.

(3) It is very important that the code is made publicly available.

(4) The fact that this new version identifies behavioural oscillations in available datasets can resolve the current debate on the existence of such oscillations.

Weaknesses:

I see the following weaknesses as minor.

(1) I wonder whether the frequency-dependent results (e.g., Figures 7 and 8) need to be seen in light of the sampling rate used in the simulations. For example, a lower sampling rate might be sufficient if only low frequencies are of interest in the data and lead to higher sensitivity as the number of trials (per time point) can be increased. Conversely, a higher sampling rate might lead to a higher sensitivity for the detection of effects at higher frequencies.

(2) The behavioural oscillations from individual participants do not need to have common phases for this analysis to reveal an effect. However, this also means that in a scenario where they do have common phases, this similarity remains "unused" by the analysis (e.g., due to similar phases, the oscillation could be easier to identify on the group level as signals that are not phase locked are averaged out). In such a scenario, it remains unclear whether the analysis proposed is the most sensitive one.

Reviewer #2 (Public review):

Summary

Dozens of published studies have investigated rhythms in behavior. These studies have typically tested for oscillations by shuffling the timestamps of the individual observations and comparing the resulting shuffled spectra with the empirical spectrum. However, that shuffling-in-time method leads to strongly inflated rates of false positives. Brookshire (2022) suggested a method that controls the rate of false positives (the "AR-surrogate method"). In the current study, Harris and Beale propose a modification of the AR-surrogate analysis method with the goal of increasing the sensitivity while maintaining a low rate of false positives.

This study is carefully conducted and it addresses an interesting question. However, the simulations were performed in a way that ignores one important source of temporal structure: non-oscillatory patterns that are consistent across subjects. In order to know whether the updated AR-surrogate method would control the rate of false positives in real behavioral data, we need to know whether it controls the rate of false positives when the data includes aperiodic patterns that are consistent across subjects.

Strengths

This study was constructed carefully and written up very clearly. It's a clever idea to analyze the time series separately for each participant. After examining how the updated AR-surrogate method behaves when the simulated data includes consistency across subjects, this will be a useful contribution to the field.

Weaknesses

When describing their simulations of behavioral data, the authors write: "Each participant's data was produced by creating an independent idealised time-course of 1-second length, sampled at 60 Hz."

Because these simulations generated a totally independent time-course for every subject, they don't capture an important source of aperiodic structure in real behavior: consistent non-oscillatory patterns that occur across subjects. In other words, these simulations do not account for any pattern that remains after averaging across subjects. The literature is rich with patterns that persist across subjects, including all the studies of behavioral oscillations that analyze their data after averaging across subjects (e.g., Landau & Fries, 2012; Fiebelkorn, Saalmann, & Kastner, 2013, etc). As a consequence, I suspect that the reported increase in power comes at the expense of a corresponding increase in false positives, but that the false positives aren't captured here due to the lack of consistency across simulated subjects.

It's therefore possible that the authors' updated AR-surrogate method would mistakenly conclude that behavior oscillates when it only includes aperiodic consistency across subjects. Since that kind of aperiodic structure is ubiquitous, this analysis could lead to very high rates of false positives. Luckily, it's easy to find out whether this is the case - the authors could simulate data using an idealized time-course that is consistent across subjects.

Reviewer #3 (Public review):

Summary:

This work revises the autoregressive surrogate (AR-surrogate) method proposed in 2022 by Brookshire to estimate the oscillatory content of behavioural time series. The main issue raised by Brookshire was the inadequacy of methods used in a series of papers that rely on shuffling the time axis of the behavioural data. Brookshire argued that while this approach tests for temporal structures, it does not differentiate between 1/f activity and true oscillatory signals. The AR-surrogate, on the other hand, removes aperiodic activity and should therefore provide a more accurate representation of oscillatory behaviour.

In this well-written paper, Harris and Beale clearly describe an improvement to Brookshire's method, which has been called into question for its low sensitivity.

Strengths:

The starting point of this work is that oscillatory patterns should be tested at the individual participant level rather than at the group level. This is critical because anyone working with behavioural data will know that averaging across participants generates distorted time series. Averaging also assumes phase consistency across participants, which may not always be valid.

Once freed from this limitation, the results presented here are exciting and convincingly demonstrate a significant improvement over the original implementation.

The authors have devised a series of tests that systematically assess the effects of participant and trial number, and effect size on the accuracy of AR-surrogate results. This is particularly useful, as it may guide researchers in designing appropriate behavioural experiments.

Weaknesses:

The method proposed here is undoubtedly an improvement on the original. However, its biggest limitation is the restriction on the frequencies that can be investigated. This is acknowledged by the authors, who rightly point out that there is still room for improvement. Another issue is that modulation depths below 10-15% may be difficult to detect.

Reviewer #1 (Public review):

The authors note that very premature infants experience the visual world early and, as a consequence, sustain lasting deficits including compromised motion processing. Here they investigate the effects of early eye opening in ferret, choosing a time point after birth when both retinal waves and light traveling through closed lids drive sensory responses. The laboratory has long experience in quantitative studies of visual response properties across development and this study reflects their expertise.

The investigators find little or no difference in mean orientation and direction selectivity, or in spatial frequency tuning, as a result of early eye opening but marked differences in temporal frequency tuning. These changes are especially interesting as they relate to deficits seen in prematurely delivered children. Temporal frequency bandwidth for responses evoked from early-opened contralateral eyes were broader than for controls; this is the case for animals in which either one or both eyes were opened prematurely. Further, when only one eye was opened early, responses to low temporal frequencies were relatively stronger.

The investigators also found changes in firing rate and signs of response to visual stimuli. Premature eye-opening increased spontaneous rates in all test configurations. When only one eye was opened early, firing rates recorded from the ipsilateral cortex were strongly suppressed, with more modest effects in other test cases.

As the authors' discussion notes, these observations are just a starting point for studies underlying mechanism. The experiments are so difficult to perform and so carefully described that the results will be foundational for future studies of how premature birth influences cortical development.

Reviewer #2 (Public review):

In this paper, Griswold and Van Hooser investigate what happens if animals are exposed to patterned visual experience too early, before its natural onset. To this end, they make use of the benefits of the ferret as a well-established animal model for visual development. Ferrets naturally open their eyes around postnatal day 30; here, Griswold and Van Hooser opened either one or both eyes prematurely. Subsequent recordings in the mature primary visual cortex show that while some tuning properties like orientation and direction selectivity developed normally, the premature visual exposure triggered changes in temporal frequency tuning and overall firing rates. These changes were widespread, in that they occurred even for neurons responding to the eye that was not opened prematurely. These results demonstrate that the nature of the visual input well before eye opening can have profound consequences on the developing visual system.

The conclusions of this paper are well supported by the data, but some aspects of the data could be clarified, and the discussion could be extended.

(1) The assessment of the tuning properties is based on fits to the data. Presumably, neurons for which the fits were poor were excluded? It would be useful to know what the criteria were, how many neurons were excluded, and whether there was a significant difference between the groups in the numbers of neurons excluded (which could further point to differences between the groups).

(2) For the temporal frequency data, low- and high-frequency cut-offs are defined, but then only used for the computation of the bandwidth. Given that the responses to low temporal frequencies change profoundly with premature eye opening, it would be useful to directly compare the low- and high-frequency cut-offs between groups, in addition to the index that is currently used.

(3) In addition to the tuning functions and firing rates that have been analyzed so far, are there any differences in the temporal profiles of neural responses between the groups (sustained versus transient responses, rates of adaptation, latency)? If the temporal dynamics of the responses are altered significantly, that could be part of an explanation for the altered temporal tuning.

(4) It would be beneficial for the general interpretation of the results to extend the discussion. First, it would be useful to provide a more detailed discussion of what type of visual information might make it through the closed eyelids (the natural state), in contrast to the structured information available through open eyes. Second, it would be useful to highlight more clearly that these data were collected in peripheral V1 by discussing what might be expected in binocular, more central V1 regions. Third, it would be interesting to discuss the observed changes in firing rates in the context of the development of inhibitory neurons in V1 (which still undergo significant changes through the time period of premature visual experience chosen here).