Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides new insights into how cancer cells adapt their metabolism under nutrient-deprived conditions. They find cells respond differentially to serine and lipid deprivation via oxidising the cell redox state, which enables biomass synthesis and cell proliferation. They identified mitochondrial respiration as the major mechanism that dictates the endogenous NAD+/NADH ratio. By incorporating a dual stress paradigm, serine and lipid deprivation, the study further suggests that the NAD+/NADH ratio can serve as a link to orchestrate the complex interplay between multiple nutrient changes in the tumour microenvironment.

Strengths:

A novel aspect of this study is the idea that cancer cells are not uniformly passive victims of nutrient limitation; some can actively invoke endogenous NAD+ regeneration to combat nutrient stress. The conclusion is well-supported by comparing multiple cell lines from different tissues and genetic backgrounds, which improves generalizability. While most of the smaller conclusions align with common reasoning and expectations, the step-by-step deduction that leads to a novel 'big picture' is commendable. Another notable strength is the integration of dual stress (lipid and serine deprivation), which better mimics the complex tumor microenvironment with multiple nutrient fluctuations, raising the translational potential of these findings. The observation that lipid-deprived cells can stimulate serine synthesis and support proliferation in a subset of cancer cell lines offers a novel perspective on metabolic plasticity under starvation conditions.

Weaknesses:

Although the authors derive a novel and valuable overarching concept, the presentation of this "big picture" is not clearly articulated, making it less accessible to readers outside the immediate field. It would greatly enhance the manuscript to include a clearer summary of the overarching model and its implications. Additionally, discussing the potential clinical significance and applications of the findings would increase the relevance and broader impact of the work. Finally, the manuscript's clarity and credibility are undermined by inconsistent figure labeling and the lack of statistical analysis, particularly for the Western blot data.

While this study identifies changes in serine synthesis, mitochondrial respiration, PHGDH protein levels, and NAD+/NADH ratio in different cell lines, some of these relationships appear correlative rather than causally established (Figure 2; Figure 5; Figure 6). Some claims are thus overinterpreted. For example, the co-occurrence of increased NAD+/NADH ratio and citrate levels under lipid deprivation in A549 cells does not establish causality (Figure 5). Direct perturbation experiments that manipulate NAD+/NADH and assess downstream effects on citrate synthesis would substantially strengthen the conclusions.

The study focuses predominantly on mitochondrial respiration as a source of NAD+ regeneration. However, it will also be interesting to check other significant pathways, such as NAD+ salvage, which have been implicated in supporting serine biosynthesis. In addition, the subcellular distribution of NAD+ may distinguish whether some cells are truly redox-unresponsive. Mitochondrial NAD+ regeneration might counteract the cytosolic NAD+ consumption, rendering a relatively stable intracellular NAD+/NADH ratio. The malate-aspartate shuttle can be an interesting aspect.

The authors should acknowledge the limitations of short-term isotope tracing in their experimental design. Differences in metabolic rates across cell lines can affect the kinetics of metabolite labeling, limiting the direct comparability of metabolic fluxes between them. As a result, observed changes may reflect transient adaptations rather than stable metabolic reprogramming. It is important to clarify that the study primarily captures short-term responses, and the conclusions may not extrapolate to longer-term adaptations or protein-level changes under sustained nutrient stress.

Reviewer #1 (Public review):

Summary:

Weiss et. al. seek to delineate the mechanisms by which antigen-specific CD8+ T cells outcompete bystanders in the epidermis when active TGF-b is limiting, resulting in selective retention of these cells and more complete differentiation into the TRM phenotype.

Strengths:

They begin by demonstrating that at tissue sites where cognate antigen was expressed, CD8+ T cells adopt a more mature TRM transcriptome than cells at tissue sites where cognate antigen was never expressed. By integrating their scRNA-Seq data on TRM with the much more comprehensive ImmGenT atlas, the authors provide a very useful resource for future studies in the field. Furthermore, they conclusively show that these "local antigen-experienced" TRM have increased proliferative capacity and that TCR avidity during TRM formation positively correlates with their future fitness. Finally, using an elegant experimental strategy, they establish that TCR signaling in CD8+ T cells in epidermis induces TGFBRIII expression, which likely contributes to endowing them with a competitive advantage over antigen-inexperienced TRM.

Weaknesses:

The main weakness in this paper lies in the authors' reliance on a single model to derive conclusions on the role of local antigen during the acute phase of the response by comparing T cells in model antigen-vaccinia virus (VV-OVA) exposed skin to T cells in contralateral skin exposed to DNFB 5 days after the VV-OVA exposure. In this setting, antigen-independent factors may contribute to the difference in CD8+ T cell number and phenotype at the two sites. For example, it was recently shown that very early memory precursors (formed 2 days after exposure) are more efficient at seeding the epithelial TRM compartment than those recruited to skin at later times (Silva et al, Sci Immunol, 2023). DNFB-treated skin may therefore recruit precursors with reduced TRM potential. In addition, TRM-skewed circulating memory precursors have been identified (Kok et al, JEM, 2020), and perhaps VV-OVA exposed skin more readily recruits this subset compared to DNFB-exposed skin. Therefore, when the DNFB challenge is performed 5 days after vaccinia virus, the DNFB site may already be at a disadvantage in the recruitment of CD8+ T cells that can efficiently form TRM. In addition, CD8+ T cell-extrinsic mechanisms may be at play, such as differences in myeloid cell recruitment and differentiation or local cytokine and chemokine levels in VV-infected and DNFB-treated skin that could account for differences seen in TRM phenotype and function between these two sites. Although the authors do show that providing exogenous peptide antigen at the DNFB-site rescues their phenotype in relation to the VV-OVA site, the potential antigen-independent factors distinguishing these two sites remain unaddressed. In addition, there is a possibility that peptide treatment of DNFB-treated initiates a second phase of priming of new circulatory effectors in the local-draining lymph nodes that are then recruited to form TRM at the DFNB-site, and that the effect does not solely rely on TRM precursors at the DNFB-treated skin site at the time of peptide treatment.

Secondly, although the authors conclusively demonstrate that TGFBRIII is induced by TCR signals and required for conferring increased fitness to local-antigen-experienced CD8+ TRM compared to local antigen-inexperienced cells, this is done in only one experiment, albeit repeated 3 times. The data suggest that antigen encounter during TRM formation induces sustained TGFBRIII expression that persists during the antigen-independent memory phase. It remains unclear why only the antigen encounter in skin, but not already in the draining lymph nodes, induces sustained TGFBRIII expression. Further characterizing the dynamics of TGFBRIII expression on CD8+ T cells during priming in draining lymph nodes and over the course of TRM formation and persistence may shed more light on this question. Probing the role of this mechanism at other sites of TRM formation would also further strengthen their conclusions and enhance the significance of this finding.

Reviewer #2 (Public review):

Summary:

The authors set out to dissect the mechanistic basis of their previously published finding that encountering cutaneous antigen augments the persistence of CD8+ memory T cells that enter skin (TRM) (Hirai et al., 2021, Immunity). Here they use the same murine model to study the fate of CD8+ T cells after antigen-priming in the lymph nodes, (1) those that re-encounter antigen in the skin via vaccinia virus (VV) versus (2) those that do not encounter antigen in skin but rather are recruited via topical dinitrofluorobenzene (DNFB) (so-called "bystander TRM"). The authors' previous publication establishes that this first group of CD8+ TRM has a persistence advantage over bystander TRM under TGFb-limiting conditions. The current paper advances this finding by elucidating the role of TGFBR3 in regulating CD8+ TRM skin persistence upon topical antigen exposure. Key novelty of the work lies in the generation and use of the CD8+ T cell-specific TGFBR3 knockout model, which allows them to demonstrate the role of TGFBR3 in fine-tuning the degree of CD8+ T cell skin persistence and that TGFBR3 expression is promoted by CD8+ TRM encountering their cognate antigen upon initial skin entry. Future work directly measuring active TGFb in the skin under different conditions would help identify physiologic scenarios that yield active TGFb-limiting conditions, thus establishing physiologic relevance.

Strengths:

Technical strengths of the paper include (1) complementary imaging and flow cytometry analyses, (2) integration of their scRNA-seq data with the existing CD8+ TRM literature via pathway analysis, and (3) use of orthogonal models where possible. Using a vaccina virus (VV) model, with and without ovalbumin (OVA), the authors investigate how topical antigen exposure and TCR strength regulate CD8+ TRM skin recruitment and retention. The authors use both FTY720 and a Thy1.1 depleting antibody to demonstrate that skin CD8+ TRM expand locally following both a primary and secondary recall response to topical OVA application.

A conceptual strength of the paper is the authors' observation that TCR signal strength upon initial TRM tissue entry helps regulate the extent of their local re-expansion on subsequent antigen re-exposure. They achieved this by applying peptides of varying affinity for the OT-I TCR on the DNFB-exposed flank in tandem with initial VV-OVA + DNFB treatment. They then measured TRM expansion after OVA peptide rechallenge, revealing that encountering a higher-affinity peptide upon skin entry leads to greater subsequent re-expansion. Additionally, by generating an OT-I Thy1.1+ E8i-creERT2 huNGFR Tgfbr3fl/fl (Tgfbr3∆CD8) mouse, the authors were able to elucidate a unique role for TGFBR3 in CD8+TRM persistence when active TGFb in skin is limited.

Weaknesses:

Overall, the authors' conclusions are well supported, although there are some instances where additional controls, experiments, or clarifications would add rigor. The conclusions regarding skin-localized TCR signaling leading to increased skin CD8+ TRM proliferation in-situ and increased TGFBR3 expression would be strengthened by assessing skin CD8+ TRM proliferation and TGFBR3 expression in models of high versus low avidity topical OVA-peptide exposure. The authors could further increase the novelty of the paper by exploring whether TGFBR3 is regulated at the RNA or protein level. To this end, they could perform analysis of their single-cell RNA sequencing data (Figure 1), comparing Tgfbr3 mRNA in DNFB versus VV-treated skin.

For clarity, when discussing antigen exposure throughout the paper, it would be helpful for the authors to be more precise that they are referring to the antigen in the skin rather than in the draining lymph node. A more explicit summary of some of the lab's previous work focused on CD8+ TRM and the role of TGFb would also help readers better contextualize this work within the existing literature on which it builds.

For rigor, it would be helpful where possible to pair flow cytometry quantification with the existing imaging data. Additional controls, namely enumerating TRM in the opposite, untreated flank skin of VV-only-treated mice and the treated flank skin of DNFB-only treated mice, would help contextualize the results seen in dually-treated mice in Figure 1. In figure legends, we suggest clearly reporting unpaired T tests comparing relevant metrics within VV or DNFB-treated groups (for example, VV-OVA PBS vs VV-OVA FTY720 in Figure 3F). Finally, quantifying right and left skin draining lymph node CD8+ T cell numbers would clarify the skin specificity and cell trafficking dynamics of the authors' model.

Reviewer #1 (Public review):

Summary

The manuscript presents EIDT, a framework that extracts an "individuality index" from a source task to predict a participant's behaviour in a related target task under different conditions. However, the evidence that it truly enables cross-task individuality transfer is not convincing.

Strengths

The EIDT framework is clearly explained, and the experimental design and results are generally well-described. The performance of the proposed method is tested on two distinct paradigms: a Markov Decision Process (MDP) task (comparing 2-step and 3-step versions) and a handwritten digit recognition (MNIST) task under various conditions of difficulty and speed pressure. The results indicate that the EIDT framework generally achieved lower prediction error compared to baseline models and that it was better at predicting a specific individual's behaviour when using their own individuality index compared to using indices from others.

Furthermore, the individuality index appeared to form distinct clusters for different individuals, and the framework was better at predicting a specific individual's behaviour when using their own derived index compared to using indices from other individuals.

Weaknesses

(1) Because the "source" and "target" tasks are merely parameter variations of the same paradigm, it is unclear whether EIDT achieves true cross-task transfer. The manuscript provides no measure of how consistent each participant's behaviour is across these variants (e.g., two- vs three-step MDP; easy vs difficult MNIST). Without this measure, the transfer results are hard to interpret. In fact, Figure 5 shows a notable drop in accuracy when transferring between the easy and difficult MNIST conditions, compared to transfers between accuracy-focused and speed-focused conditions. Does this discrepancy simply reflect larger within-participant behavioural differences between the easy and difficult settings? A direct analysis of intra-individual similarity for each task pair - and how that similarity is related to EIDT's transfer performance - is needed.

(2) Related to the previous comment, the individuality index is central to the framework, yet remains hard to interpret. It shows much greater within-participant variability in the MNIST experiment (Figure S1) than in the MDP experiment (Figure 3). Is such a difference meaningful? It is hard to know whether it reflects noisier data, greater behavioural flexibility, or limitations of the model.

(3) The authors suggests that the model's ability to generalize to new participants "likely relies on the fact that individuality indices form clusters and individuals similar to new participants exist in the training participant pool". It would be helpful to directly test this hypothesis by quantifying the similarity (or distance) of each test participant's individuality index to the individuals or identified clusters within the training set, and assessing whether greater similarity (or closer proximity) to the clusters in the training set is associated with higher prediction accuracy for those individuals in the test set.

Reviewer #2 (Public review):

This paper introduces a framework for modeling individual differences in decision-making by learning a low-dimensional representation (the "individuality index") from one task and using it to predict behaviour in a different task. The approach is evaluated on two types of tasks: a sequential value-based decision-making task and a perceptual decision task (MNIST). The model shows improved prediction accuracy when incorporating this learned representation compared to baseline models.

The motivation is solid, and the modelling approach is interesting, especially the use of individual embeddings to enable cross-task generalization. That said, several aspects of the evaluation and analysis could be strengthened.

(1) The MNIST SX baseline appears weak. RTNet isn't directly comparable in structure or training. A stronger baseline would involve training the GRU directly on the task without using the individuality index-e.g., by fixing the decoder head. This would provide a clearer picture of what the index contributes.

(2) Although the focus is on prediction, the framework could offer more insight into how behaviour in one task generalizes to another. For example, simulating predicted behaviours while varying the individuality index might help reveal what behavioural traits it encodes.

(3) It's not clear whether the model can reproduce human behaviour when acting on-policy. Simulating behaviour using the trained task solver and comparing it with actual participant data would help assess how well the model captures individual decision tendencies.

(4) Figures 3 and S1 aim to show that individuality indices from the same participant are closer together than those from different participants. However, this isn't fully convincing from the visualizations alone. Including a quantitative presentation would help support the claim.

(5) The transfer scenarios are often between very similar task conditions (e.g., different versions of MNIST or two-step vs three-step MDP). This limits the strength of the generalization claims. In particular, the effects in the MNIST experiment appear relatively modest, and the transfer is between experimental conditions within the same perceptual task. To better support the idea of generalizing behavioural traits across tasks, it would be valuable to include transfers across more structurally distinct tasks.

(6) For both experiments, it would help to show basic summaries of participants' behavioural performance. For example, in the MDP task, first-stage choice proportions based on transition types are commonly reported. These kinds of benchmarks provide useful context.

(7) For the MDP task, consider reporting the number or proportion of correct choices in addition to negative log-likelihood. This would make the results more interpretable.

(8) In Figure 5, what is the difference between the "% correct" and "% match to behaviour"? If so, it would help to clarify the distinction in the text or figure captions.

(9) For the cognitive model, it would be useful to report the fitted parameters (e.g., learning rate, inverse temperature) per individual. This can offer insight into what kinds of behavioural variability the individuality index might be capturing.

(10) A few of the terms and labels in the paper could be made more intuitive. For example, the name "individuality index" might give the impression of a scalar value rather than a latent vector, and the labels "SX" and "SY" are somewhat arbitrary. You might consider whether clearer or more descriptive alternatives would help readers follow the paper more easily.

(11) Please consider including training and validation curves for your models. These would help readers assess convergence, overfitting, and general training stability, especially given the complexity of the encoder-decoder architecture.

Reviewer #3 (Public review):

Summary:

This work presents a novel neural network-based framework for parameterizing individual differences in human behavior. Using two distinct decision-making experiments, the authors demonstrate the approach's potential and claims it can predict individual behavior (1) within the same task, (2) across different tasks, and (3) across individuals. While the goal of capturing individual variability is compelling and the potential applications are promising, the claims are weakly supported, and I find that the underlying problem is conceptually ill-defined.

Strengths:

The idea of using neural networks for parameterizing individual differences in human behavior is novel, and the potential applications can be impactful.

Weaknesses:

(1) To demonstrate the effectiveness of the approach, the authors compare a Q-learning cognitive model (for the MDP task) and RTNet (for the MNIST task) against the proposed framework. However, as I understand it, neither the cognitive model nor RTNet is designed to fit or account for individual variability. If that is the case, it is unclear why these models serve as appropriate baselines. Isn't it expected that a model explicitly fitted to individual data would outperform models that do not? If so, does the observed superiority of the proposed framework simply reflect the unsurprising benefit of fitting individual variability? I think the authors should either clarify why these models constitute fair control or validate the proposed approach against stronger and more appropriate baselines.

(2) It's not very clear in the results section what it means by having a shorter within-individual distance than between-individual distances. Related to the comment above, is there any control analysis performed for this? Also, this analysis appears to have nothing to do with predicting individual behavior. Is this evidence toward successfully parameterizing individual differences? Could this be task-dependent, especially since the transfer is evaluated on exceedingly similar tasks in both experiments? I think a bit more discussion of the motivation and implications of these results will help the reader in making sense of this analysis.

(3) The authors have to better define what exactly he meant by transferring across different "tasks" and testing the framework in "more distinctive tasks". All presented evidence, taken at face value, demonstrated transferring across different "conditions" of the same task within the same experiment. It is unclear to me how generalizable the framework will be when applied to different tasks.

(4) Conceptually, it is also unclear to me how plausible it is that the framework could generalize across tasks spanning multiple cognitive domains (if that's what is meant by more distinctive). For instance, how can an individual's task performance on a Posner task predict task performance on the Cambridge face memory test? Which part of the framework could have enabled such a cross-domain prediction of task performance? I think these have to be at least discussed to some extent, since without it the future direction is meaningless.

(5) How is the negative log-likelihood, which seems to be the main metric for comparison, computed? Is this based on trial-by-trial response prediction or probability of responses, as what usually performed in cognitive modelling?

(6) None of the presented evidence is cross-validated. The authors should consider performing K-fold cross-validation on the train, test, and evaluation split of subjects to ensure robustness of the findings.

(7) The authors excluded 25 subjects (20% of the data) for different reasons. This is a substantial proportion, especially by the standards of what is typically observed in behavioral experiments. The authors should provide a clear justification for these exclusion criteria and, if possible, cite relevant studies that support the use of such stringent thresholds.

(8) The authors should do a better job of creating the figures and writing the figure captions. It is unclear which specific claim the authors are addressing with the figure. For example, what is the key message of Figure 2C regarding transfer within and across participants? Why are the stats presentation different between the Cognitive model and the EIDT framework plots? In Figure 3, it's unclear what these dots and clusters represent and how they support the authors' claim that the same individual forms clusters. And isn't this experiment have 98 subjects after exclusion, this plot has way less than 98 dots as far as I can tell. Furthermore, I find Figure 5 particularly confusing, as the underlying claim it is meant to illustrate is unclear. Clearer figures and more informative captions are needed to guide the reader effectively.

(9) I also find the writing somewhat difficult to follow. The subheadings are confusing, and it's often unclear which specific claim the authors are addressing. The presentation of results feels disorganized, making it hard to trace the evidence supporting each claim. Also, the excessive use of acronyms (e.g., SX, SY, CG, EA, ES, DA, DS) makes the text harder to parse. I recommend restructuring the results section to be clearer and significantly reducing the use of unnecessary acronyms.

Reviewer #1 (Public review):

Summary:

This work presents a formalism for the relationship between neural signals and pooled signals (e.g., voxel estimates in fMRI) and explores why correlation-based and mean-removed Euclidean RDMs perform well in practice. The key assumption is that the pooled estimates are weighted averages, with i.i.d. non-negative weights. Two sets of simulations are used to support the theoretical findings: one based on fully simulated neural data and another that reverse-engineers neural data from an RDM estimated from real macaque data. The authors also discuss limitations of their simulations, particularly concerning the i.i.d. assumption of the weights.

Strengths:

The strengths of this work include its mathematical rigor and the clear connection that is drawn between the derivations and empirical observations. The simulations were well-designed and easy to follow. One small suggestion: a brief explanation of what is meant by "sparse" in Figure 3 would help orient the reader without requiring them to jump ahead to the methods. Overall, I found the work engaging and insightful.

Weaknesses:

Although I appreciate the effort to explore *why* certain dissimilarity measures perform well, it wasn't clear how these findings would inform the practical choices of researchers conducting RDM-based analyses. Many researchers likely already use correlation-based or mean-removed Euclidean distance measures, given their popularity. In that case, how do these results provide additional value or guidance beyond current practice?

Another aspect that could benefit from further clarification is the core assumption underlying the work - that channel-based activity reflects a non-negative weighted average of neural activity. Is this widely accepted as the most plausible model, or are there alternative relationships that researchers should consider? While this may seem intuitive, it's not something I would expect all readers to be familiar with, and only a single reference was provided to support it (which I unfortunately didn't have time to read). That said, I did appreciate the discussion of the i.i.d. assumption in the discussion section. Can more be said to educate researchers as to when the i.i.d. assumption might be violated?

I didn't find the "Simulations based on neural data" section added much, and it risks being misinterpreted. The main difference here is that neural data were reverse-engineered from a macaque RDM and then used in simulations similar to those in the previous section. What is the added value of using a real RDM to generate simulated data? Were the earlier simulations lacking in some way? There's also a risk of readers mistakenly inferring that human dissimilarities have been reconstructed from macaque data, an assumption that goes beyond the paper's core message, which focuses on linking neural and channel-based signals from the *same* source. If this section is retained, the motivation should be clarified, and the implied parallel in Figure 6, between the human data and simulated data, should be reconsidered.

Reviewer #2 (Public review):

Summary:

The paper is a methodological contribution to multivariate pattern analysis and, in particular, the analysis of representational geometry via pairwise representational distances, sometimes called representational dissimilarity analysis (RDA). The authors investigate through theoretical analysis and simulations how true representational distances (defined on the neural level) give rise to representational distances estimated from neurophysiological data, including fMRI and cell recordings. They demonstrate that, due to the way measurements sample neural activity, the activity common to all sampled neurons can be amplified in the representational geometry derived from these measurements, and therefore, an empirical representational geometry may deviate substantially from the true representational geometry. The authors propose to modify the obtained representational structure by removing the dimension corresponding to that common activity, and argue that such a removal of a single dimension does not relevantly affect the representational structure, again underpinned by mathematical analysis and simulation.

Importance:

The paper may at first sight be tackling a specific problem within a specific subfield of cognitive neuroscience methods. However, understanding the structure of representations is a fundamental goal of cognitive psychology and cognitive neuroscience, and the fact that methods of representational geometry are not yet routinely used by the wider community may at least partially be due to uncertainty regarding the reliability of these methods. This paper is an important step towards clarifying and improving reliability, and therefore towards more widespread adoption of representational geometry methods.

Strengths:

The paper makes its argument generally well, relying on previous work by the authors as well as others to support assumptions about neural sampling by neurophysiological measurements. Their main points are underpinned by both detailed mathematical analysis and simulations, and the latter also produces intuitively accessible illustrations of the authors' argument. The authors discuss in detail under which exact circumstances common neural activity distorts the representational geometry, and therefore, when exactly the removal of the common dimension is necessary to minimize that distortion.

Weaknesses:

(1) The argument around the Johnson-Lindenstrauss lemma on pages 5 & 6 is somewhat confused, and also not really convincing.

First, the correct reference for the lemma seems to be not [20] = Johnson et al. (1986), but Johnson & Lindenstrauss (1984). Moreover, as far as I can tell, Johnson et al. (1986) do not discuss random projections, and while they play a role in Johnson & Lindenstrauss (1984), that is only as a proof device. The paper text suggests that the lemma itself is probabilistic, while actually it is a statement of existence.

Second, the authors correctly state that the lemma implies that "the number of measurement channels required for a good approximation does not depend on the number of neurons and grows only logarithmically with the number of stimuli", but it is not clear what the relevance of this statement for this paper is, considering that distances between N points can be exactly preserved within an N − 1 dimensional subspace, irrespective of the number of dimensions of the original space, and since in cognitive neuroscience the number of measurement channels is usually (much) larger than the number of experimental stimuli.

The actually centrally important statement is not the Johnson-Lindenstrauss lemma, but one about the metric-preserving properties of random projections with zero-mean weights. It is this statement that needs to be backed up by the correct references, which, as far as I can tell, are neither the cited Johnson et al. (1986) nor even Johnson & Lindenstrauss (1984) for the lemma.

(2) The detailed mathematical analyses and simulations focus on the effect of non-zero-mean sampling weights, and that is justified by the result that such sampling leads to a distorted representational geometry. However, there is another assumption which seems to be used almost everywhere in both mathematical analyses and simulations, and which I suspect may have a relevant effect on the observed representational geometry: statistical independence between weights. In particular, in fMRI, the existence of a naturally limited spatial resolution (due to MRI technology or vasculature) makes it unlikely that the weights with which a given neuron affects different voxels are independent.

Reviewer #3 (Public review):

Summary:

This manuscript investigates the conditions under which representational distances estimated from brain-activity measurements accurately mirror the true geometry of the underlying neural representations. Using a theoretical framework and simulations, the authors show that (i) random weighted sampling of individual neurons preserves representational distances; (ii) the non-negative pooling characteristic of fMRI stretches the geometry along the population-mean dimension; and (iii) subtracting the across-channel mean from each activity pattern removes this distortion, explaining the well-known success of correlation-based RSA. They further argue that a mean-centred, squared Euclidean (or Mahalanobis) distance retains this corrective benefit while avoiding some pitfalls of variance normalisation.

Strengths:

(1) Theoretical clarity and novelty:<br /> The paper offers an elegant and convincing proof of how linear measurement models affect representational geometry and pinpoints the specific condition (non-zero-mean sampling weights) under which voxel pooling introduces a systematic bias. This quantitative explanation of why mean removal is effective in RSA is new and valuable.

(2) Simulations:<br /> Experiments on both synthetic high-dimensional fMRI data and macaque-IT-inspired embeddings corroborate the mathematics, providing practical insights into the theoretical reasoning outlined by the authors.

(3) Actionable recommendations:<br /> The work summarises the results into clear guidelines: random single-unit sampling is "safe" (the estimated geometry is undistorted); fMRI voxel data with unstructured or single-scale codes should be mean-centred; and multi-scale cortical maps require explicit forward modelling. These guidelines are clear, and useful for future research.

Weaknesses:

(1) Simplistic assumptions:<br /> The assumption that measurement-channel weights are drawn independently and identically distributed (i.i.d.) from a univariate distribution is a significant idealisation for fMRI data. Voxels have spatially structured responses (and noise), meaning they do not sample neurons with i.i.d. weights. The extent to which the conclusions (especially the "exact recovery" with mean centring) hold when this assumption is violated needs more discussion. While the paper states that the non-negative IWLCS model is a best-case scenario, the implications of deviations from this best case could be elaborated.

(2) Random-subpopulation model for electrophysiology:<br /> Similarly, the "random subpopulation model" is presented as an idealisation of single-cell recordings. In reality, electrophysiological sampling is often biased (e.g., towards larger, more active neurons or neurons in accessible locations). The paper acknowledges biased sampling as a challenge that requires separate modelling, but the gap between this idealised model and actual practice should be highlighted more strongly when interpreting the optimistic results.

(3) Noise as an "orthogonal issue":<br /> The theoretical derivations largely ignore measurement noise, treating it as an orthogonal problem solvable by cross-validation. Although bias from noise is a well-known problem, interactions between noise and sampling-induced distortions (especially the down-scaling of orthogonal dimensions) could complicate the picture. For instance, if a dimension is already heavily down-scaled by averaging, it might become more susceptible to being obscured by noise. Addressing or highlighting these points more explicitly would make the limitations of this theoretical framework more transparent.

(4) Simulation parameters and generalizability:<br /> The random ground-truth geometries were generated from a Gaussian mixture in 5-D and then embedded into 1,024-D, with ≈25 % of the variance coming from the mean dimension. The sensitivity of the findings to these specific parameters (initial dimensionality, geometry complexity, proportion of mean variance, and sample size) could be discussed. How would the results change if the true neural geometry had a much higher or lower intrinsic dimensionality, or if the population-mean component were substantially smaller or larger? If the authors' claims are to generalise, more scenarios should be considered.

(5) Mean addition to the neural-data simulation:<br /> In simulations based on neural data from Kiani et al., a random mean was added to each pattern to introduce variation along the mean dimension. This was necessary because the original patterns had identical mean activation. However, the procedure might oversimplify how population means vary naturally and could influence the conclusions, particularly regarding the impact of the population-mean dimension. While precisely modelling how the mean varies across conditions is beyond the manuscript's scope, this point should be stated and discussed more clearly.

(6) Effect of mean removal on representational geometry:<br /> As noted, the benefits of mean removal hold "under ideal conditions". Real data often violates these assumptions. A critical reader might ask: What if conditions differ in overall activation and in more complex ways (e.g., differing correlation structures across neurons)? Is it always desirable to remove population-mean differences? For example, if a stimulus truly causes a global increase in firing across the entire population (perhaps reflecting arousal or salience), subtracting the mean would treat this genuine effect as a nuisance and eliminate it from the geometry. Prior literature has cautioned that one should interpret RSA results after demeaning carefully. For instance, Ramírez (2017) dubbed this problem "representational confusion", showing that subtracting the mean pattern can change the relationships between conditions in non-intuitive ways. These potential issues and previous results should be discussed and properly referenced by the authors.

Appraisal, Impact, and Utility:

The authors set out to identify principled conditions under which measured representational distances faithfully reflect the underlying neural geometry and to provide practical guidance for RSA across modalities. Overall, I believe they achieved their goals. Theoretical derivations identify the bias-inducing factors in linear measurement models, and the simulations verify the analytic claims, demonstrating that mean-pattern subtraction can indeed correct some mean-related geometric distortions. These conclusions strongly rely on idealised assumptions (e.g., i.i.d. sampling weights and negligible noise), but the manuscript is explicit about them, and the reasoning from evidence to claim is sound. A deeper exploration of how robust each conclusion is to violations of these assumptions, particularly correlated voxel weights and realistic noise, would make the argument even stronger.

Beyond their immediate aims, the authors offer contributions likely to shape future work. Its influence is likely to influence both analysis decisions and the design of future studies exploring the geometry of brain representations. By clarifying why correlation-based RSA seems to work so robustly, they help demystify a practice that has so far been adopted heuristically. Their proposal to adopt mean-centred Euclidean or Mahalanobis distances promises a straightforward alternative that better aligns representational geometry with decoding-based interpretations.

In sum, I see this manuscript as a significant and insightful contribution to the field. The theoretical work clarifying the impact of sampling schemes and the role of mean removal is highly valuable. However, the identified concerns, primarily regarding the idealized nature of the models (especially for fMRI), the treatment of noise, and the need for more nuanced claims, suggest that some revisions are necessary. Addressing these points would substantially strengthen the paper's conclusions and enhance its impact on the neuroscience community by ensuring the proposed methods are robustly understood and appropriately applied in real-world research settings.

Reviewer #1 (Public review):

Kaller et al. (2025) explore the impact of environmental enrichment (EE) on the developing mouse brain, specifically during the perinatal period. The authors use high-resolution MRI to examine structural brain changes in neonates (postnatal day 7, P7) and compare these changes to those observed in adulthood. A key aspect of the study is the investigation of maternal care as a potential mediating factor in the effects of perinatal EE on neonatal brain development.

The work exhibits the following notable strengths:

(1) The study addresses a significant gap in the literature by investigating the effects of perinatal EE on whole-brain structure in neonates. Previous research has primarily focused on the effects of EE on the adult brain or specific aspects of early development, such as the visual system.

(2) The authors employ a combination of high-resolution MRI and behavioral analysis of maternal care, providing a comprehensive view of the effects of EE.

(3) The study reveals that EE affects brain structure as early as P7, with distinct regional changes compared to adulthood. The finding that maternal care influences neonatal brain structure and correlates with the effects of EE is particularly noteworthy.

(4) The paper is clearly written, well-organized, and easy to follow. The figures and tables are informative and effectively illustrate the key findings.

However, some weaknesses should be addressed to improve the quality of this study:

(1) While the study includes an assessment of maternal care, the observational period is relatively short. A more extended or continuous assessment of maternal behavior could provide a more comprehensive understanding of its role in mediating the effects of EE.

(2) The study primarily focuses on structural brain changes. Investigating the functional consequences of these changes could provide further insights into the long-term impact of perinatal EE.

(3) The study demonstrates a correlation between maternal care and neonatal brain structure but does not elucidate the underlying mechanisms. Future studies could explore potential molecular or cellular mechanisms involved in these effects.

Reviewer #2 (Public review):

This paper by Kaller and colleagues combines an interesting replication of findings on the importance of maternal behavior on brain development in the offspring with a state-of-the-art MRI analysis and a novel comparison between such perinatal and early postnatal enrichment via the activity of the mother and a classical enriched environment in the adult. In general, the observations are as one would have expected. Early postnatal enrichment and adult enrichment have differential effects, which is plausible because, as the source of these changes is environmental, and environmental means very different things at these different stages. The three data sets presented are really interesting, and while the comparison between them might not always be as straightforward as it seems, the cross-sectional phenotyping with MRI already provides very important material and allows for interesting insight. Most interesting is possibly the massive effect of housing conditions at P7.

In particular, the role of individual behavior differs. The authors highlight this role of the interaction with the environment, rather than the environment alone. Maternal care is a process that involves the pup.

Importantly, the study shows that being born into an enriched environment predates certain changes that are still available after exposure at a later stage, but that there are also important differences. Detailed interpretation of these effects is not easy, however.

Notably, the study does not include a condition of enrichment from birth into adulthood, and no analysis of the perinatal enrichment effects at an adult age. The timeline can be guessed from Figure 1b, but the authors might in places be more explicit about the fact that, indirectly and sometimes directly, animals of different ages (young adult versus adult) are compared. There is obviously no experience of maternal care in adulthood and no active exploration, etc in childhood. In part, this is what this paper is about, but it requires some thought for the reader to separate the more trivial from the more profound conclusions. Some more guidance would probably be welcome here. In general, Figure 4 is a great idea (and visually very appealing), but the content is not quite clear. "Adults born in EE vs. switched to EE in adulthood": this has, as far as I can tell, not been studied. What is compared are EE effects at two different time-points with two supposedly different mechanisms.

From such a more mechanistic side, the authors might, for example, want to relate the observed patterns to what is known about the developmental (and plastic) dynamics in the respective brain regions at the given time. But age is a confounder here.

There is another interesting point that the authors might discuss more prominently. The inter-individual differences in Z-score are dramatic within essentially all groups. So while the mean effects might still be statistically different, a large proportion of animals are within a range of values that could be found in either experimental group. The same is also true for the effects of maternal care, as depicted in Figure 3. While there is, for this ROI, a clear trend that overall relative volume decreases with maternal contact time at each time point, there is a large range of values for each maternal contact time bin. Consequently, neither genetics nor maternal care per se can be the driver of this variation. Part of it will be technical, but the trend in the data indicates that certainly not all of this is noise and technical error.

This study has some open ends but also provides a very important and interesting direction for future study, corroborating the idea that behavior, maternal and own, does matter.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate the effect of environmental enrichment (EE) during the critical perinatal period on the developing brain structure and compare it with other periods. Different datasets of mice with EE or standard housing (SH) were compared with post-mortem MRI: dataset A (MRI at P96; 13 animals in EE during adulthood P53-P96, 14 animals in SH), dataset P (MRI at P43; 24 animals in EE during perinatal period and adulthood E17-P43, 25 animals in SH) and dataset N (MRI at P7; 52 animals in EE during perinatal period E13-P7, 67 animals in SH / resulting from 5 dams with 2 litters: 4 dams in EE and 6 dams in SH). The study replicated the effects observed during adulthood (main neuroanatomical EE/SH difference in datasets A and P: increase in the hippocampus volume) but also showed that volumetric changes for some regions differ between datasets A and P, suggesting different mechanisms of brain responses to enrichment depending on the period when EE was applied. Results on dataset N further showed that EE leads to lower brain size and differences for various regions: volume reduction in striatum, frontal, parietal, and occipital regions, hippocampus; volume increase for a few thalamic nuclei and hindbrain, suggesting different patterns of perinatal EE effects in datasets P and N. Since mice at P7 show little engagement with their environment, the authors further explored the hypothesis that the dams' behavior and interaction with neonates could be a mediator of brain differences observed at P7 between EE and SH animals. Maternal contact time was related to the P7 volumes for some regions (striatum, brainstem), but the variability and low sample size prevented a clear separation between EE and SH in terms of maternal behaviors.

Strengths:

(1) The question raised by this article is important at a fundamental level for our understanding of the complex interactions between the brain, behavior, and the environment.

(2) This study replicates previous observations on the effects of EE in adult mice.

(3) While some studies have been performed on neonates of dams exposed to EE during gestation, it is the first time that the effects of perinatal EE are investigated, in both the developing and mature brains with MRI. From a translational perspective, this is crucial for our understanding of human neurodevelopment in interaction with the environment.

(4) The analyses carried out are numerous and detailed.

Weaknesses:

(1) The analyses carried out do not allow us to fully assess whether differences in maternal care mediate the effects of EE on brain structure during development. The observations support this causal hypothesis, but a complete mediation analysis would be useful if permitted by the sample size and the variability observed between litters.

(2) The article is quite dense to read, given the number of analyses carried out. It is difficult at first reading to get a global view of the results. Figure 4 could be highlighted earlier to present the hypotheses and tests carried out.

(3) The figures could be more explicit in terms of legends (particularly the supplementary figures).

Reviewer #1 (Public review):

Summary:

The authors present an interesting study using RL and Bayesian modelling to examine differences in learning rate adaptation in conditions of high and low volatility and noise respectively. Through "lesioning" an optimal Bayesian model, they reveal that apparently suboptimal adaptation of learning rates results from incorrectly detecting volatility in the environment when it is not in fact present.

Strengths:

The experimental task used is cleverly designed and does a good job of manipulating both volatility and noise. The modelling approach takes an interesting and creative approach to understand the source of apparently suboptimal adaptation of learning rates to noise, through carefully "lesioning" and optimal Bayesian model to determine which components are responsible for this behaviour.

Weaknesses:

The model space could be more extensive, although the authors have covered the most relevant models for the question at hand.

Comments on revisions: I have no further recommendations for the authors, they have addressed my previous comments very well.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to investigate how humans learn and adapt their behavior in dynamic environments characterized by two distinct types of uncertainty: volatility (systematic changes in outcomes) and noise (random variability in outcomes). Specifically, they sought to understand how participants adjust their learning rates in response to changes in these forms of uncertainty.

To achieve this, the authors employed a two-step approach:

Reinforcement Learning (RL) Model:<br /> They first used an RL model to fit participants' behavior, revealing that the learning rate was context-dependent-it varied based on the levels of volatility and noise. However, the RL model showed that participants misattributed noise as volatility, leading to higher learning rates in noisy conditions, where the optimal strategy would be to be less sensitive to random fluctuations.

Bayesian Observer Model (BOM):<br /> To better account for this context dependency, they introduced a Bayesian Observer Model (BOM), which models how an ideal Bayesian learner would update their beliefs about environmental uncertainty. They found that a degraded version of the BOM, where the agent had a coarser representation of noise compared to volatility, best fit the participants' behavior. This suggested that participants were not fully distinguishing between noise and volatility, instead treating noise as volatility and adjusting their learning rates accordingly.

The authors also aimed to use pupillometry data (measuring pupil dilation) as a physiological marker to arbitrate between models and understand how participants' internal representations of uncertainty influenced both their behavior and physiological responses. Their objective was to explore whether the BOM could explain not just behavioral choices but also these physiological responses, thereby providing stronger evidence for the model's validity.

Overall, the study sought to reconcile approximate rationality in human learning by showing that participants still follow a Bayesian-like learning process, but with simplified internal models that lead to suboptimal decisions in noisy environments.

Strengths:

The generative model presented in the study is both innovative and insightful. The authors first employ a Reinforcement Learning (RL) model to fit participants' behavior, revealing that the learning rate is context-dependent-specifically, it varies based on the levels of volatility and noise in the task. They then introduce a Bayesian Observer Model (BOM) to account for this context dependency, ultimately finding that a degraded BOM-in which the agent has a coarser representation of noise compared to volatility-provides the best fit to the participants' behavior. This suggests that participants are not fully distinguishing between noise and volatility, leading to misattribution of noise as volatility. Consequently, participants adopt higher learning rates even in noisy contexts, where an optimal strategy would involve being less sensitive to new information (i.e., using lower learning rates). This finding highlights a rational but approximate learning process, as described in the paper.

Weaknesses:

While the RL and Bayesian models both successfully predict behavior, it remains unclear how to fully reconcile the two approaches. The RL model captures behavior in terms of a fixed or context-dependent learning rate, while the BOM provides a more nuanced account with dynamic updates based on volatility and noise. Both models can predict actions when fit appropriately, but the pupillometry data offers a promising avenue to arbitrate between the models. However, the current study does not provide a direct comparison between the RL framework and the Bayesian model in terms of how well they explain the pupillometry data. It would be valuable to see whether the RL model can also account for physiological markers of learning, such as pupil responses, or if the BOM offers a unique advantage in this regard. A comparison of the two models using pupillometry data could strengthen the argument for the BOM's superiority, as currently, the possibility that RL models could explain the physiological data remains unexplored.

The model comparison between the Bayesian Observer Model and the self-defined degraded internal model could be further enhanced. Since different assumptions about the internal model's structure lead to varying levels of model complexity, using a formal criterion such as Bayesian Information Criterion (BIC) or Akaike Information Criterion (AIC) would allow for a more rigorous comparison of model fit. Including such comparisons would ensure that the degraded BOM is not simply favored due to its flexibility or higher complexity, but rather because it genuinely captures the participants' behavioral and physiological data better than alternative models. This would also help address concerns about overfitting and provide a clearer justification for using the degraded BOM over other potential models.

Comments on revisions:

The authors have addressed all my questions. Congratulations on the impressive work accomplished by the authors!

Reviewer #1 (Public review):

The chromophore molecule of animal and microbial rhodopsins is retinal which forms a Schiff base linkage with a lysine in the 7-th transmembrane helix. In most cases, the chromophore is positively charged by protonation of the Schiff base, which is stabilized by a negatively charged counterion. In animal opsins, three sites have been experimentally identified, Glu94 in helix 2, Glu113 in helix 3, and Glu181 in extracellular loop 2, where a glutamate acts as the counterion by deprotonation. In this paper, Sakai et al. investigated molecular properties of anthozoan-specific opsin II (ASO-II opsins), as they lack these glutamates. They found an alternative candidate, Glu292 in helix 7, from the sequences. Interestingly, the experimental data suggested that Glu292 is not the direct counterion in ASO-II opsins. Instead, they found that ASO-II opsins employ a chloride ion as the counterion. In case of microbial rhodopsin, a chloride ion serves as the counterion of light-driven chloride pumps. This paper reports the first observation of a chloride ion as the counterion in animal rhodopsin. Theoretical calculation using a QM/MM method supports their experimental data. The authors also revealed the role of Glu292, which serves as the counterion in the photoproduct and is involved in G protein activation.

The conclusions of this paper are well supported by data.

Reviewer #2 (Public review):

Summary:

This work reports the discovery of a new rhodopsin from reef-building corals that is characterized experimentally, spectroscopically, and by simulation. This rhodopsin lacks a carboxylate-based counterion, which is typical for this family of proteins. Instead, the authors find that a chloride ion stabilizes the protonated Schiff base and thus serves as a counterion.

Strengths:

This work focuses on the rhodopsin Antho2a, which absorbs in the visible spectrum with a maximum at 503 nm. Spectroscopic studies under different pH conditions, including the mutant E292A and different chloride concentrations, indicate that chloride acts as a counterion in the dark. In the photoproduct, however, the counterion is identified as E292.

These results lead to a computational model of Antho2a in which the chloride is modeled in addition to the Schiff base. This model is improved using the hybrid QM/MM simulations. As a validation, the absorption maximum is calculated using the QM/MM approach for the protonated and deprotonated E292 residue as well as the E292A mutant. The results are in good agreement with the experiment. However, there is a larger deviation for ADC(2) than for sTD-DFT. Nevertheless, the trend is robust since the wt and E292A mutant models have similar excitation energies. The calculations are performed at a high level of theory that includes a large QM region.

Reviewer #3 (Public review):

Summary:

The paper by Saito et al. studies the properties of anthozoan-specific opsins (ASO-II) from organisms found in reef-building coral. Their goal was to test if ASO-II opsins can absorb visible light, and if so, what are they key factors involved.

The most exciting aspect of this work is their discovery that ASO-II opsins do not have a counterion residue (Asp or Glu) located at any of the previously known sites found in other animal opsins.

This is very surprising. Opsins are only able to absorb visible (long wavelength light) if the retinal Schiff base is protonated, and the latter requires (as the name implies) a "counter ion". However, the authors clearly show that some ASO-II opsins do absorb visible light.

To address this conundrum, they tested if the counterion could be provided by exogenous chloride ions (Cl-). Their results find compelling evidence supporting this idea, and their studies of ASO-II mutant E292A suggests E292 also plays a role in G protein activation and is a counterion for a protonated Schiff base in the light-activated form.

Strengths:

Overall, the methods are well described and carefully executed, and the results very compelling.

Their analysis of seven ASO-II opsin sequences undoubtedly shows they all lack a Glu or Asp residue at "normal" (previously established) counter-ion sites in mammalian opsins (typically found at positions 94, 113 or 181). The experimental studies clearly demonstrate the necessity of Cl- for visible light absorbance, as do their studies of the effect of altering the pH.

Importantly, the authors also carried out careful QM/MM computational analysis (and corresponding calculation of the expected absorbance effects), thus providing compelling support for the Cl- acting directly as a counterion to the protonated retinal Schiff base, and thus limiting the possibility that the Cl- is simply altering the absorbance of ASO-II opsins through some indirect effect on the protein.

Altogether, the authors clearly achieved their aims, and the results support their conclusions. The manuscript is carefully written, and refreshingly, the results and conclusions not overstated.

This study is impactful for several reasons. There is increasing interest in optogenetic tools, especially those that leverage G protein coupled receptor systems. Thus, the authors demonstration that ASO-II opsins could be useful for such studies is of interest.

Moreover, the finding that visible light absorbance by an opsin does not absolutely require a negatively charged amino acid be placed at one of the expected sites (94, 113 or 181) typically found in animal opsins is very intriguing and will help future protein engineering efforts. The argument that the Cl- counterion system they discover here might have been a preliminary step in the evolution of amino acid based counterions used in animal opsins is also interesting.

Finally, given the ongoing degradation of coral reefs worldwide, the focus on these curious opsins is very timely, as is the authors proposal that the lower Schiff base pKa they discovered here for ASO-II opsins may cause them to change their spectral sensitivity and G protein activation due to changes in their environmental pH.

Reviewer #1 (Public review):

Summary:

This useful work extends a prior study from the authors to observe distance changes within the CNBD domains of a full length CNG channel based on changes in single photon lifetimes due to tmFRET between a metal at an introduced chelator site and a fluorescent non canonical amino acid at another site. The data are excellent and convincingly support the authors' conclusions. In addition to the methodology being of general use for other proteins, the authors show that coupling of the CNBDs to the rest of the channel stabilizes the CNBDs in their active state relative to an isolated CNBD construct.

Strengths:

The manuscript is very well written and clear.

Reviewer #2 (Public review):

The manuscript by Eggan et al. investigates the energetics of conformational transitions in the cyclic nucleotide-gated (CNG) channel SthK. This lab pioneered transition metal FRET (tmFRET), which has previously provided detailed insights into ion channel conformational changes. Here, the authors analyze tmFRET fluorescence lifetime measurements in the time domain, yielding detailed insights into conformational transitions within the cyclic nucleotide binding domains (CNBDs) of the channel. The integration of tmFRET with time-correlated single-photon counting (TCSPC) represents an advancement of this technique.

Reviewer #3 (Public review):

Summary:

This is a lucidly written manuscript describing the use of transition-metal FRET to assess distance changes during functional conformational changes in a CNG channel. The experiments were performed on an isolated C-terminal nucleotide binding domain (CNBD) and on a purified full-length channel, with FRET partners placed at two positions in the CNBD.

The data and quantitative analysis are exemplary, and they provide a roadmap for the use of this powerful approach in other proteins. In particular, the use of the fluorescence-lifetime decay histograms to learn not just the mean distance reported by the FRET, but also the distribution of states with different distances, allows better refinement of hypotheses for the gating motions.

Reviewer #1 (Public review):

This is a convincing description of approximately ten years of funding from the NIH BRAIN initiative. It is of particular value at this moment in history, given the cataclysmic changes in the US government structure and function occurring in early 2025.

The paper contains a fair bit of documentation so that the curious reader can actually parse what this BRAIN program funded. The authors are able to draw on a wealth of real-life experience reviewing, funding, and administering large team projects, and assessing how well they achieve their goals. In revision, the paper has been improved with respect to clarity and by bringing together two separate papers into one stronger piece.

Reviewer #2 (Public review):

Summary:

The authors provide an important summary of ten years of Brain Initiative funding including a description of the historical development of the initiative, the specific funding mechanisms utilized, and examples of grants funded and work produced. The authors also conduct analyses of the impact on overall funding in Systems and Computational Neuroscience, the raw and field normalized bibliographic impact of the work, the social media impact of the funded work, and the popularity of some tools developed.

The authors have improved the presentation by integrating the weaker of the two manuscripts with the stronger, by clarifying terminology and by performing additional analyses.

Reviewer #1 (Public review):

Summary:

The study investigated how individuals living in urban slums in Salvador, Brazil, interact with environmental risk factors, particularly focusing on domestic rubbish piles, open sewers, and a central stream. The study makes use of the step selection functions using telemetry data, which is a method to estimate how likely individuals move towards these environmental features, differentiating among groups by gender, age, and leptospirosis serostatus. The results indicated that women tended to stay closer to the central stream while avoiding open sewers more than men. Furthermore, individuals who tested positive for leptospirosis tended to avoid open sewers, suggesting that behavioral patterns might influence exposure to risk factors for leptospirosis, hence ensuring more targeted interventions.

Strengths:

(1) The use of step selection functions to analyze human movement represents an innovative adaptation of a method typically used in animal ecology. This provides a robust quantitative framework for evaluating how people interact with environmental risk factors linked to infectious diseases (in this case, leptospirosis).

(2) Detailed differentiation by gender and serological status allows for nuanced insights, which can help tailor targeted interventions and potentially improve public health measures in urban slum settings.

(3) The integration of real-world telemetry data with epidemiological risk factors supports the development of predictive models that can be applied in future infectious disease research, helping to bridge the gap between environmental exposure and health outcomes.

Weaknesses:

(1) The sample size for the study was not calculated, although it was a nested cohort study.

(2) The step‐selection functions, though a novel method, may face challenges in fully capturing the complexity of human decision-making influenced by socio-cultural and economic factors that were not captured in the study.

(3) The study's context is limited to a specific urban slum in Salvador, Brazil, which may reduce the generalizability of its findings to other geographical areas or populations that experience different environmental or socio-economic conditions.

(4) The reliance on self-reported or telemetry-based movement data might include some inaccuracies or biases that could affect the precision of the selection coefficients obtained, potentially limiting the study's predictive power.

(5) Some participants with less than 50 relocations within the study area were excluded without clear justification, see line 149.

(6) Some figures are not clear (see Figure 4 A & B).

(7) No statement on conflict of interest was included, considering sponsorship of the study.

Reviewer #2 (Public review):

Summary:

Pablo Ruiz Cuenca et al. conducted a GPS logger study with 124 adult participants across four different slum areas in Salvador, Brazil, recording GPS locations every 35 seconds for 48 hours. The aim of their study was to investigate step-selection models, a technique widely used in movement ecology to quantify contact with environmental risk factors for exposure to leptospires (open sewers, community streams, and rubbish piles). The authors built two different types of models based on distance and based on buffer areas to model human environmental exposure to risk factors. They show differences in movement/contact with these risk factors based on gender and seropositivity status. This study shows the existence of modest differences in contact with environmental risk factors for leptospirosis at small spatial scales based on socio-demographics and infection status.

Strengths:

The authors assembled a rich dataset by collecting human GPS logger data, combined with field-recorded locations of open sewers, community streams, and rubbish piles, and testing individuals for leptospirosis via serology. This study was able to capture fine-scale exposure dynamics within an urban environment and shows differences by gender and seropositive status, using a method novel to epidemiology (step selection).

Weaknesses:

Due to environmental data being limited to the study area, exposure elsewhere could not be captured, despite previous research by Owers et al. showing that the extent of movement was associated with infection risk. Limitations of step selection for use in studying human participants in an urban environment would need to be explicitly discussed.

Reviewer #1 (Public review):

In this study, Nishi et al. claim that the ratio of long-term hematopoietic stem cell (LT-HSC) versus short-term HSC (ST-HSC) determines the lineage output of HSCs and reduced ratio of ST-HSC in aged mice causes myeloid-biased hematopoiesis. Authors used Hoxb5 reporter mice to isolated LT-HSC and ST-HSC and performed molecular analyses and transplantation assays to support their arguments. How hematopoietic system becomes myeloid-biased upon aging is an important question with many implications in disease context as well. However, this study needs more definitive data.

(1) Authors' experimental designs have some caveats to definitely support their claims. Authors claimed that aged LT-HSCs have no myeloid-biased clone expansion using transplantation assays. In these experiments, authors used 10 HSCs and young mice as recipients. Given the huge expansion of old HSC by number and known heterogeneity in immunophenotypically defined HSC populations, it is questionable how 10 out of so many old HSCs (an average of 300,000 up to 500,000 cells per mouse; Mitchell et al., Nature Cell Biology, 2023) can faithfully represent old HSC population. The Hoxb5+ old HSC primary and secondary recipient mice data (Fig. 2C and D) support this concern. In addition, they only used young recipients. Considering the importance of inflammatory aged niche in the myeloid-biased lineage output, transplanting young vs old LT-HSCs into aged mice will complete the whole picture.

In response to the above comments, the authors calculated the required sample size as approximately 384 cells to represent 500,000 HSCs per old mouse. Based on the total 1260 cells used throughout the whole manuscript (Figures 2, 3, 5, 6, S3, and S6), the authors claimed that the data is reflecting old HSC behavior. However, 384 cells represent HSCs from one old mouse. Following the authors' logic, they did only 3.2 mice (1260/384) experiment for the whole manuscript to make their argument. N of 3 is not enough, especially for old mice experiments considering the heterogeneity of aged mice. Also, they did not address the comment regarding inflammatory aged niche effects.

(2) Authors' molecular data analyses need more rigor with unbiased approaches. They claimed that neither aged LT-HSCs nor aged ST-HSCs exhibited myeloid or lymphoid gene set enrichment but aged bulk HSCs, which are just a sum of LT-HSCs and ST-HSCs by their gating scheme (Fig. 4A), showed the "tendency" of enrichment of myeloid-related genes based on the selected gene set (Fig. 4D). Although the proportion of ST-HSCs is reduced in bulk HSCs upon aging, since ST-HSCs do not exhibit lymphoid gene set enrichment based on their data, it is hard to understand how aged bulk HSCs have more myeloid gene set enrichment compared to young bulk HSCs. This bulk HSC data rather suggest that there could be a trend toward certain lineage bias (although not significant) in aged LT-HSCs or ST-HSCs. Authors need to verify the molecular lineage priming of LT-HSCs and ST-HSCs using another comprehensive dataset.

(3) Although authors could not find any molecular evidence for myeloid-biased hematopoiesis from old HSCs (either LT or ST), they argued that the ratio between LT-HSC and ST-HSC causes myeloid-biased hematopoiesis upon aging based on young HSC experiments (Fig. 6). However, old ST-HSC functional data showed that they barely contribute to blood production unlike young Hoxb5- HSCs (ST-HSC) in the transplantation setting (Fig. 2). Is there any evidence that in unperturbed native old hematopoiesis, old Hoxb5- HSCs (ST-HSC) still contribute to blood production? To answer this question, authors performed additional experiments with increased cell number (Fig. S6). Although Fig. S6.D data has a statistical significance, it is questionable how biologically meaningful it is. More fundamental question is back to the representability. Can this cell number used in this experiment represent old HSC (either LT or ST) behavior?

Reviewer #2 (Public review):

Summary:

Nishi et al, investigate the well-known and previously described phenomenon of age-associated myeloid-biased hematopoiesis. Using a previously established HoxB5mCherry mouse model, they used HoxB5+ and HoxB5- HSCs to discriminate cells with long-term (LT-HSCs) and short-term (ST-HSCs) reconstitution potential and compared these populations to immunophenotypically defined 'bulk HSCs' that consists of a mixture of LT-HSC and ST-HSCs. They then isolated these HSC populations from young and aged mice to test their function and myeloid bias in non-competitive and competitive transplants into young and aged recipients. Based on quantification of hematopoietic cell frequencies in the bone marrow, peripheral blood, and in some experiments the spleen and thymus, the authors argue against the currently held belief that myeloid-biased HSCs expand with age.

While aspects of their work are fascinating and might have merit, several issues weaken the overall strength of the arguments and interpretation. Multiple experiments were done with a very low number of recipient mice, showed very large standard deviations, and had no statistically detectable difference between experimental groups. While the authors conclude that these experimental groups are not different, the displayed results seem too variable to conclude anything with certainty. The sensitivity of the performed experiments (e.g. Fig 3; Fig 6C, D) is too low to detect even reasonably strong differences between experimental groups and is thus inadequate to support the author's claims. This weakness of the study is not acknowledged in the text and is also not discussed. To support their conclusions the authors need to provide higher n-numbers and provide a detailed power analysis of the transplants in the methods section.

As the authors attempt to challenge the current model of the age-associated expansion of myeloid-biased HSCs (which has been observed and reproduced by many different groups), ideally additional strong evidence in the form of single-cell transplants is provided.<br /> It is also unclear why the authors believe that the observed reduction of ST-HSCs relative to LT-HSCs explains the myeloid-biased phenotype observed in the peripheral blood. This point seems counterintuitive and requires further explanation.

Based on my understanding of the presented data, the authors argue that myeloid-biased HSCs do not exist, as:<br /> a) they detect no difference between young/aged HSCs after transplant (mind low n-numbers and large std);<br /> b) myeloid progenitors downstream of HSCs only show minor or no changes in frequency and c) aged LT-HSCs do not outperform young LT-HSC in myeloid output LT-HScs in competitive transplants (mind low n-numbers and large std!!!).<br /> However, given the low n-numbers and high variance of the results, the argument seems weak and the presented data does not support the claims sufficiently. That the number of downstream progenitors does not change could be explained by other mechanisms, for instance, the frequently reported differentiation short-cuts of HSCs and/or changes in the microenvironment.

Strengths:

The authors present an interesting observation and offer an alternative explanation of the origins of aged-associated myeloid-biased hematopoiesis. Their data regarding the role of the microenvironment in the spleen and thymus appears to be convincing.

Weaknesses:

"Then, we found that the myeloid lineage proportions from young and aged LT-HSCs were nearly comparable during the observation period after transplantation (Fig. 3, B and C)."<br /> [Comment to the authors]: Given the large standard deviation and low n-numbers, the power of the analysis to detect differences between experimental groups is very low. Experimental groups with too large standard deviations (as displayed here) are difficult to interpret and might be inconclusive. The absence of clearly detectable differences between young and aged transplanted HSCs could thus simply be a false-negative result. The shown experimental results hence do not provide strong evidence for the author's interpretation of the data. The authors should add additional transplants and include a detailed power analysis to be able to detect differences between experimental groups with reasonable sensitivity.

Line 293: "Based on these findings, we concluded that myeloid-biased hematopoiesis observed following transplantation of aged HSCs was caused by a relative decrease in ST-HSC in the bulk-HSC compartment in aged mice rather than the selective expansion of myeloid-biased HSC clones."

[Comment to the authors]: Couldn't that also be explained by an increase in myeloid-biased HSCs, as repeatedly reported and seen in the expansion of CD150+ HSCs? It is not intuitively clear why a reduction of ST-HSCs clones would lead to a myeloid bias. The author should try to explain more clearly where they believe the increased number of myeloid cells comes from. What is the source of myeloid cells if the authors believe they are not derived from the expanded population of myeloid-biased HSCs?

New comment for the authors:

While the authors provide new evidence, clarify the text, and adjust their interpretation, the presented data remain weak and do not convincingly challenge the current paradigm. As myeloid-biased HSC expansion with age has been observed and published by many different groups, the authors need to provide much stronger evidence to challenge the observations of others. Key experiments that might support their claims had been suggested, but as indicated, the authors plan to provide these much more rigorous experiments in future studies. As it stands, the overall conclusions of this manuscript thus remain weak and preliminary.

In an attempt to quantify the absolute cell number of HSPC subpopulations, the authors use a usual readout and quantify "Number of cells per minute of analysis time". This appears to be a quick and dirty reanalysis of already existing flow cytometry data. Unfortunately, this quantification cannot count the absolute number of cells reliably, as the number of cells per minute recorded is heavily influenced by the abundance of other cell populations. Instead, the author should have counted the absolute number of HSCs, MPPs, GMPs, etc. per femur, which is typically done to address this question.

At this point, as authors are seemingly not willing to provide additional hard evidence to support their claims in this study and are instead in the process of preparing additional data for a future manuscript, I believe this study, as it stands (although weak), suggests an interesting alternative model. Despite being highly controversial, this alternative model warrants future investigations and discussions in the field. As always, it will also be important to reproduce these findings independently in other labs. As my concerns and the concerns of the other reviewers are documented and available to read by others, I believe the manuscript should be published in its current form to stimulate critical discussion and future investigations of the current model.

Reviewer #3 (Public review):

In this manuscript, Nishi et al. propose a new model to explain the previously reported myeloid-biased hematopoiesis associated with aging. Traditionally, this phenotype has been explained by the expansion of myeloid-biased hematopoietic stem cell (HSC) clones during aging. Here, the authors question this idea and show how their Hoxb5 reporter model can discriminate long-term (LT) and short-term (ST) HSC and characterized their lineage output after transplant. From these analyses, the authors conclude that changes during aging in the LT/ST HSC proportion explain the myeloid bias observed.

Comments on revisions:

I appreciate the authors' reply to some of my comments. However, there are some key aspects that remain unresolved. Please see below.

- The authors propose a critical change in the way we consider the mechanisms leading to lineage biased hematopoiesis during aging. As Reviewer 2 mentioned, such a strong claim needs to be supported by solid experimental data. Unfortunately, the level of variability in key in vivo experiments (Figure 2 and 3) diminishes the robustness of these results.

The authors argue that even with the low number of mice used in some of these experiments and the high level of variability, differences still reach (or not) statistical significance according to their analysis. I am not an expert on statistics but the only test that is mentioned is their methodology is a Welch's t test, which is only appropriate for data following a normal distribution. A more rigorous statistical analysis should be performed to sustain the claims included in the current manuscript.

- The chosen irradiation regiment might contribute to the uncertainty of the data and influence their interpretation. As the authors show in their response to my "comment to our #3-4 response", there is a considerable (and variable) amount of "radioresistant" CD45.1+CD45.2- cells in their primary recipients, which become concerningly high in the secondary transplant. This is not found in previous publications focused on this topic and, therefore, it makes it difficult to compare those studies with the present manuscript. The inclusion of this aspect in the text is appreciated but definitely reduces the impact of their claims.

- The correction introduced in the main text as an answer to the original comment #3-6 is still misleading. There is an assumption for GMP, CMP and MEP to increase with age if myeloid-biased HSC clones increase with age ("in contrast to what we anticipated"). Again, the link between these two changes could be more complex than just a direct correlation.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

(3) Good, well-researched background section.

Weaknesses:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

Major:

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

(2) The NMR hydroxylation assay.

A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B.<br /> B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec?<br /> C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this?

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

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

Minor:

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

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

Reviewer #3 (Public review):

Summary:

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

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

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of FMRP in the formation and function of RNA granules in mouse brain/cultured hippocampal neurons. Most of their results indicate that FMRP does not have a role in the formation or function of RNA granules with specific mRNAs, but may have some role in distal RNA granules in neurons and their response to synaptic stimulation. This is an important work (though the results are mostly negative) in understanding the composition and function of neuronal RNA granules. The last part of the work in cultured neurons is disjointed from the rest of the manuscript, and the results are neither convincing nor provide any mechanistic insight.

Strengths:

(1) The study is quite thorough, the methods and analysis used are robust, and the conclusion and interpretation are diligent.

(2) The comparative study of Rat and Mouse RNA granules is very helpful for future studies.

(3) The conclusion that the absence of FMRP does not affect the RNA granule composition and many of its properties in the system the authors have chosen to study is well supported by the results.

(4) The difference in the response to DHPG stimulation concerning RNA granules described here is very interesting and could provide a basis for further studies, though it has some serious technical issues.

Weaknesses:

(1) The system used for the study (P5 mouse brain or DIV 8-10 cultured neuron) is surprising, as the majority of defects in the absence of FMRP are reported in later stages (P30+ brain and DIV 14+ neurons). It is important to test if the conclusions drawn here hold good at different developmental stages.

(2) The term 'distal granules' is very vague. Since there is no structural or biochemical characterization of these granules, it is difficult to understand how they are different from the proximal granules and why FMRP has an effect only on these granules.

(3) Since the manuscript does not find any effect of FMRP on neuronal RNA granules, it does not provide any new molecular insight with respect to the function of FMRP

Reviewer #2 (Public review):

In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.

In order to improve this study, our main suggestions are as follows:

(1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."

How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.

(2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.

(3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".

(4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.

(5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.

Reviewer #3 (Public review):

Summary: Li et al describe a set of experiments to probe the role of FMRP in ribosome stalling and RNA granule composition. The authors are able to recapitulate findings from a previous study performed in rats (this one is in mice).

Strengths:

1) The work addresses an important and challenging issue, investigating mechanisms that regulate stalled ribosomes, focusing on the role of FMRP. This is a complicated problem, given the heterogeneity of the granules and the challenges related to their purification. This work is a solid attempt at addressing this issue, which is widely understudied.

2) The interpretation of the results could be interesting, if supported by solid data. The idea that FMRP could control the formation and release of RNA granules, rather than the elongation by stalled ribosomes is of high importance to the field, offering a fresh perspective into translational regulation by FMRP.

3) The authors focused on recapitulating previous findings, published elsewhere (Anadolu et al., 2023) by the same group, but using rat tissue, rather than mouse tissue. Overall, they succeeded in doing so, demonstrating, among other findings, that stalled ribosomes are enriched in consensus mRNA motifs that are linked to FMRP. These interesting findings reinforce the role of FMRP in formation and stabilization of RNA granules. It would be nice to see extensive characterization of the mouse granules as performed in Figure 1 of Anadolu and colleagues, 2023.

4) Some of the techniques incorporated aid in creating novel hypotheses, such as the ribopuromycilation assay and the cryo-EM of granule ribosomes.

Weaknesses:

1) The RNA granule characterization needs to be more rigorous. Coomassie is not proper for this type of characterization, simply because protein weight says little about its nature. The enrichment of key proteins is not robust and seems to not reach significance in multiple instances, including S6 and UPF1. Furthermore, S6 is the only proxy used for ribosome quantification. Could the authors include at least 3 other ribosomal proteins (2 from small, 2 from large subunit)?

2) Page 12-13 - The Gene Ontology analysis is performed incorrectly. First, one should not rank genes by their RPKM levels. It is well known that housekeeping genes such as those related to actin dynamics, molecular transport and translation are highly enriched in sequencing datasets. It is usually more informative when significantly different genes are ranked by p adjust or log2 Fold Change, then compared against a background to verify enrichment of specific processes. However, the authors found no DEGs. I would suggest the removal of this analysis, incorporation of a gene set enrichment analyses (ranked by p adjust). I further suggest that the authors incorporate a dimensionality reduction analysis to demonstrate that the lack of significance stems from biology and not experimental artifacts, such as poor reproducibility across biological replicates.

Reviewer #1 (Public review):

Summary:

ZMAT3 is a p53 target gene that the Lal group and others have shown is important for p53-mediated tumor suppression, and which plays a role in the control of RNA splicing. In this manuscript, Lal and colleagues perform quantitative proteomics of cells with ZMAT3 knockout and show that the enzyme hexokinase HKDC1 is the most upregulated protein. Mechanistically, the authors show that ZMAT3 does not appear to directly regulate the expression of HKDC1; rather, they show that the transcription factor c-JUN was strongly enriched in ZMAT3 pull-downs in IP-mass spec experiments, and they perform IP-western to demonstrate an interaction between c-JUN and ZMAT3. Importantly, the authors demonstrate, using ChIP-qPCR, that JUN is present at the HKDC1 gene (intron 1) in ZMAT3 WT cells and shows markedly enhanced binding in ZMAT3 KO cells. The data best fit a model whereby p53 transactivates ZMAT3, leading to decreased JUN binding to the HKDC1 promoter, and altered mitochondrial respiration.

Strengths:

The authors use multiple orthogonal approaches to test the majority of their findings.

The authors offer a potentially new activity of ZMAT3 in tumor suppression by p53: the control of mitochondrial respiration.

Weaknesses:

Some indication as to whether other c-JUN target genes are also regulated by ZMAT3 would improve the broad relevance of the authors' findings.

Reviewer #2 (Public review):

Summary:

The study elucidates the role of the recently discovered mediator of p53 tumor suppressive activity, ZMAT3. Specifically, the authors find that ZMAT3 negatively regulates HKDC1, a gene involved in the control of mitochondrial respiration and cell proliferation.

Strengths:

Mechanistically, ZMAT3 suppresses HKDC1 transcription by sequestering JUN and preventing its binding to the HKDC1 promoter, resulting in reduced HKDC1 expression. Conversely, p53 mutation leads to ZMAT3 downregulation and HKDC1 overexpression, thereby promoting increased mitochondrial respiration and proliferation. This mechanism is novel; however, the authors should address several points.

Weaknesses:

The authors conduct mechanistic experiments (e.g., transcript and protein quantification, luciferase assays) to demonstrate regulatory interactions between p53, ZMAT3, JUN, and HKDC1. These findings should be supported with functional assays, such as proliferation, apoptosis, or mitochondrial respiration analyses.

Reviewer #3 (Public review):

Summary:

In their manuscript, Kumar et al. investigate the mechanisms underlying the tumor suppressive function of the RNA binding protein ZMAT3, a previously described tumor suppressor in the p53 pathway. To this end, they use RNA-sequencing and proteomics to characterize changes in ZMAT3-deficient cells, leading them to identify the hexokinase HKDC1 as upregulated with ZMAT3 deficiency first in colorectal cancer cells, then in other cell types of both mouse and human origin. This increase in HKDC1 is associated with increased mitochondrial respiration. As ZMAT3 has been reported as an RNA-binding and DNA-binding protein, the authors investigated this via PAR-CLIP and ChIP-seq but did not observe ZMAT3 binding to HKDC1 pre-mRNA or DNA. Thus, to better understand how ZMAT3 regulates HKDC1, the authors used quantitative proteomics to identify ZMAT3-interacting proteins. They identified the transcription factor JUN as a ZMAT3-interacting protein and showed that JUN promotes the increased HKDC1 RNA expression seen with ZMAT3 inactivation. They propose that ZMAT3 inhibits JUN-mediated transcriptional induction of HKDC1 as a mechanism of tumor suppression. This work uncovers novel aspects of the p53 tumor suppressor pathway.

Strengths:

This novel work sheds light on one of the most well-established yet understudied p53 target genes, ZMAT3, and how it contributes to p53's tumor suppressive functions. Overall, this story establishes a p53-ZMAT3-HKDC1 tumor suppressive axis, which has been strongly substantiated using a variety of orthogonal approaches, in different cell lines and with different data sets.

Weaknesses:

While the role of p53 and ZMAT3 in repressing HKDC1 is well substantiated, there is a gap in understanding how ZMAT3 acts to repress JUN-driven activation of the HKDC1 locus. How does ZMAT3 inhibit JUN binding to HKDC1? Can targeted ChIP experiments or RIP experiments be used to make a more definitive model? Can ZMAT3 mutants help to understand the mechanisms? Future work can further establish the mechanisms underlying how ZMAT3 represses JUN activity.

Reviewer #1 (Public review):

In this work, Neiswender and colleagues test the hypothesis that mutations in BicD2 that are associated with SMALED alter BicD2-cargo interactions. To do this, they first establish the WT BicD2 cargo interactome (using a proximity-dependent biotin ligase screen with Turbo-ID on the BicD2 C-terminus). In addition to known cargo interactors, they also identified many proteins in the HOPs complex. Interestingly, they find that the HOPs complex may interact with BicD2 in a different manner than other known cargos. The authors also show that while BicD2 is required for the HOPs complex localization, on average, depletion of BicD2 from HeLa and Cos7 cells causes HOPs and Lysosome mislocalization that is consistent with Kinesin-1 trafficking defects, rather than dynein. The authors also use proximity biotin ligase approaches to define the cargo interactome of three BicD2 variants associated with SMALED. One variant (R747C) has the most altered cargo interactome. The authors highlight one protein, in particular, GRAMD1A, that is only found in the R747C dataset and mislocalizes specifically when R747C is expressed.

The work in this manuscript is of a very high quality and contributes important findings to the field. I have a few questions that, if answered, could increase the impact of this work.

(1) I was surprised at the effect of BicD2 knockdown on LAMP (and VPS41) localization, which really suggests that in HeLa and Cos7 cells, BicD2 regulation of Kinesin-1 (rather than dynein) is the primary driver of lysosome localization. The KIF5B-knockout rescue of the BicD2-overexpression phenotype was a very powerful result that supports this conclusion. Have the authors looked at other cargos, eg, Golgi or centrosomes in G2? Can the authors include more discussion about what this result means or how they imagine dynein and kinesin-1's interaction with BicD2 is regulated?

(2) Have the authors examined if the SMALED mutants show diminished or increased binding to KIF5B? While the authors are correct that the mutations could hyperactivate dynein because they reduce BicD2 autoinhibition, it is possible that the SMALED mutants hyperactivate dynein because they no longer bind kinesin. This would be particularly interesting, given the complex relationship between BicD2 regulation of dynein and kinesin that the authors show in Figure 3.

(3) What is already known about the protein GRAMD1A? Did the authors choose to focus on GRAMD1A because it was the only novel interaction found in the SMALED mutant interactomes, or was this protein interesting for a different reason? Does the known function of GRAMD1A explain the potential dysfunction of cells expressing BICD2_R747C or patients who have this mutation? More discussion of this protein and why the authors focused on it would really strengthen the manuscript.

Reviewer #2 (Public review):

Neiswender et al. investigated the interactomes between wild-type BICD2 and BICD2 mutants that are associated with Spinal Muscular Atrophy with Lower Extremity Predominance (SMALED2). Although BICD2 has previously been implicated in SMALED2, it is unclear how mutations in BICD2 may contribute to disease symptoms. In this study, the authors characterize the interactome of wild-type BICD2 and identify potential new cargos, including the HOPS complex. The authors then chose three SMALED2-associated BICD2 mutants and compared each mutant interactome to that of wild-type BICD2. Each mutant had a change in the interactome, with the most drastic being BICD2_R747C, a mutation in the cargo binding domain of BICD2. This mutant displayed less interaction with a potential new BICD2 cargo, the HOPS complex. Additionally, it displayed more interaction with an ER protein, GRAMD1A.

The data in the paper is generally strong, but the major conclusions of this paper need more evidence to be better supported.

(1) The authors use cells that have been engineered to express the different BICD2 constructs. As shown in Figure 4B, the authors see wide expression of BICD2_WT throughout the cell. However, WT BICD2 usually localizes to the TGN. This widespread localization introduces some uncertainty about the interactome data. The authors should either try to verify the interaction data (specifically with the HOPS complex and GRAMD1A) by immunoprecipitating endogenous BICD2 or by repeating their interactome experiment in Figure 1 using BICD2 knockout cells that express the BICD2_WT construct. This should also be done to verify the immunoprecipitation and microscopy data shown in Figure 7.

(2) The authors conclude that cargo transport defects resulting from BICD2 mutations may contribute to SMALED2 symptoms. However, the authors are unable to determine if BICD2 directly binds to the potential new cargo, the HOPS complex. To address this, the authors could purify full-length WT BICD2 and perform in vitro experiments. Furthermore, the authors were unable to identify the minimal region of BICD2 needed for HOPS interaction. The authors could expand on the experiment attempted with the extended BICD2 C-terminal using a deltaCC1 construct, which could also be used for in vitro experiments.

(3) Again, the authors conclude that BICD2 mutants cause cargo transport defects that are likely to lead to SMALED2 symptoms. This would be better supported if the authors are able to find a protein relevant to SMALED2 and examine if/how its localization is changed under expression of the BICD2 mutants. The authors currently use the HOPS complex and GRAMD1A as indicators of cargo transport defects, but it is unclear if these are relevant to SMALED2 symptoms.

Reviewer #3 (Public review):

Summary:

BicD2 is a motor adapter protein that facilitates cellular transport pathways, which are impacted by human disease mutations of BicD2, causing spinal muscular atrophy with lower extremity dominance (SMALED2). The authors provide evidence that some of these mutations result in interactome changes, which may be the underlying cause of the disease. This is supported by proximity biotin ligation screens, immunoprecipitation, and cell biology assays. The authors identify several novel BicD2 interactions, such as the HOPS complex that participates in the fusion of late endosomes and autophagosomes with lysosomes, which could have important functions. Three BicD2 disease mutants studied had changes in the interactome, which could be an underlying cause for SMALED2. The study extends our understanding of the BicD2 interactome under physiological conditions, as well as of the changes in cellular transport pathways that result in SMALED2. It will be of great interest for the BicD2 and dynein fields.

Strengths:

Extensive interactomes are presented for both WT BicD2 as well as the disease mutants, which will be valuable for the community. The HOPS complex was identified as a novel interactor of BicD2, which is important for fusion of late endosomes and lysosomes, which is of interest, since some of the BicD2 disease mutations result in Golgi-fragmentation phenotypes. The interaction with the HOPS complex is affected by the R747C mutation, which also results in a gain-of-function interaction with GRAMD1A.

Weaknesses:

The manuscript should be strengthened by further evidence of the BicD2/HOPS complex interaction and the functional implications for spinal muscular atrophy by changes in the interactome through mutations. Which functional implications does the loss of the BicD2/HOPS complex interaction and the gain of function interaction with GRAMD1A have in the context of the R747C mutant?

Major points:

(1) In the biotin proximity ligation assay, a large number of targets were identified, but it is not clear why only the HOPS complex was chosen for further verification. Immunoprecipitation was used for target verification, but due to the very high number of targets identified in the screen, and the fact that the HOPS complex is a membrane protein that could potentially be immunoprecipitated along with lysosomes or dynein, additional experiments to verify the interaction of BicD2 with the HOPS complex (reconstitution of a complex in vitro, GST-pull down of a complex from cell extracts or other approaches) are needed to strengthen the manuscript.

(2) In the biotin proximity ligation assay, a large number of BicD2 interactions were identified that are distinct between the mutant and the WT, but it was not clear why, particularly GRAMD1A was chosen as a gain-of-function interaction, and what the functional role of a BicD2/GRAMD1A interaction may be. A Western blot shows a strengthened interaction with the R747C mutant, but GRAMD1A also interacts with WT BicD2.

(3) Furthermore, the functional implications of changed interactions with HOPS and GRAMD1A in the R747C mutant are unclear. Additional experiments are needed to establish the functional implication of the loss of the BicD2/HOPS interaction in the BicD2/R747C mutant. For the GRAMD1A gain of function interaction, according to the authors, a significant amount of the protein localized with BicD2/R747C at the centrosomal region. This changed localization is not very clear from the presented images (no centrosomal or other markers were used, and the changed localization could also be an effect of dynein hyperactivation in the mutant). Furthermore, the functional implication of a changed localization of GRAMD1A is unclear from the presented data.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to understand how Rhino, a chromatin protein essential for small RNA production in fruit flies, is initially recruited to specific regions of the genome. They propose that asymmetric arginine methylation of histones, particularly mediated by the enzyme DART4, plays a key role in defining the first genomic sites of Rhino localization. Using a combination of inducible expression systems, chromatin immunoprecipitation, and genetic knockdowns, the authors identify a new class of Rhino-bound loci, termed DART4 clusters, that may represent nascent or transitional piRNA clusters.

Strengths:

One of the main strengths of this work lies in its comprehensive use of genomic data to reveal a correlation between ADMA histones and Rhino enrichment at the border of known piRNA clusters. The use of both cultured cells and ovaries adds robustness to this observation. The knockdown of DART4 supports a role for H3R17me2a in shaping Rhino binding at a subset of genomic regions.

Weaknesses:

However, Rhino binding at, and piRNA production from, canonical piRNA clusters appears largely unaffected by DART4 depletion, and spreading of Rhino from ADMA-rich boundaries was not directly demonstrated. Therefore, while the correlation is clearly documented, further investigation would be needed to determine the functional requirement of these histone marks in piRNA cluster specification.

The study identify piRNA cluster-like regions called DART4 clusters. While the model proposes that DART4 clusters represent evolutionary precursors of mature piRNA clusters, the functional output of these clusters remains limited. Additional experiments could help clarify whether low-level piRNA production from these loci is sufficient to guide Piwi-dependent silencing.

In summary, the authors present a well-executed study that raises intriguing hypotheses about the early chromatin context of piRNA cluster formation. The work will be of interest to researchers studying genome regulation, small RNA pathways, and the chromatin mechanisms of transposon control. It provides useful resources and new candidate loci for follow-up studies, while also highlighting the need for further functional validation to fully support the proposed model.

Reviewer #2 (Public review):

This study seeks to understand how the Rhino factor knows how to localize to specific transposon loci and to specific piRNA clusters to direct the correct formation of specialized heterochromatin that promotes piRNA biogenesis in the fly germline. In particular, these dual-strand piRNA clusters with names like 42AB, 38C, 80F, and 102F generate the bulk of ovarian piRNAs in the nurse cells of the fly ovary, but the evolutionary significance of these dual-strand piRNA clusters remains mysterious since triple null mutants of these dual-strand piRNA clusters still allows fly ovaries to develop and remain fertile. Nevertheless, mutants of Rhino and its interactors Deadlock, Cutoff, Kipferl and Moonshiner, etc, causes more piRNA loss beyond these dual-strand clusters and exhibit the phenotype of major female infertility, so the impact of proper assembly of Rhino, the RDC, Kipferl etc onto proper piRNA chromatin is an important and interesting biological question that is not fully understood.

This study tries to first test ectopic expression of Rhino via engineering a Dox-inducible Rhino transgene in the OSC line that only expresses the primary Piwi pathway that reflects the natural single pathway expression the follicle cells and is quite distinct from the nurse cell germline piRNA pathway that is promoted by Rhino, Moonshiner, etc. The authors present some compelling evidence that this ectopic Rhino expression in OSCs may reveal how Rhino can initiate de novo binding via ADMA histone marks, a feat that would be much more challenging to demonstrate in the germline where this epigenetic naïve state cannot be modeled since germ cell collapse would likely ensue. In the OSC, the authors have tested the knockdown of four of the 11 known Drosophila PRMTs (DARTs), and comparing to ectopic Rhino foci that they observe in HP1a knockdown (KD), they conclude DART1 and DART4 are the prime factors to study further in looking for disruption of ADMA histone marks. The authors also test KD of DART8 and CG17726 in OSCs, but in the fly, the authors only test Germ Line KD of DART4 only, they do not explain why these other DARTs are not tested in GLKD, the UAS-RNAi resources in Drosophila strain repositories should be very complete and have reagents for these knockdowns to be accessible.

The authors only characterize some particular ADMA marks of H3R17me2a as showing strong decrease after DART4 GLKD, and then they see some small subset of piRNA clusters go down in piRNA production as shown in Figure 6B and Figure 6F and Supplementary Figure 7. This small subset of DART4-dependent piRNA clusters does lose Rhino and Kipferl recruitment, which is an interesting result.

However, the biggest issue with this study is the mystery that the set of the most prominent dual-strand piRNA clusters. 42AB, 38C, 80F, and 102F, are the prime genomic loci subjected to Rhino regulation, and they do not show any change in piRNA production in the GLKD of DART4. The authors bury this surprising negative result in Supplementary Figure 5E, but this is also evident in no decrease (actually an n.s. increase) in Rhino association in Figure 5D. Since these main piRNA clusters involve the RDC, Kipferl, Moonshiner, etc, and it does not change in ADMA status and piRNA loss after DART4 GLKD, this poses a problem with the model in Figure 7C. In this study, there is only a GLKD of DART4 and no GLKD of the other DARTs in fly ovaries.

One way the authors rationalize this peculiar exception is the argument that DART4 is only acting on evolutionarily "young" piRNA clusters like the bx, CG14629, and CG31612, but the lack of any change on the majority of other piRNA clusters in Figure 6F leaves upon the unsatisfying concern that there is much functional redundancy remaining with other DARTs not being tested by GLKD in the fly that would have a bigger impact on the other main dual-strand piRNA clusters being regulated by Rhino and ADMA-histone marks.

Also, the current data does not provide convincing enough support for the model Figure 7C and the paper title of ADMA-histones being the key determinant in the fly ovary for Rhino recognition of the dual-strand piRNA clusters. Although much of this study's data is well constructed and presented, there remains a large gap that no other DARTs were tested in GLKD that would show a big loss of piRNAs from the main dual-strand piRNA clusters of 42AB, 38C, 80F, and 102F, where Rhino has prominent spreading in these regions.

As the manuscript currently stands, I do not think the authors present enough data to conclude that "ADMA-histones [As a Major new histone mark class] does play a crucial role in the initial recognition of dual-strand piRNA cluster regions by Rhino" because the data here mainly just show a small subset of evolutionarily young piRNA clusters have a strong effect from GLKD of DART4. The authors could extensively revise the study to be much more specific in the title and conclusion that they have uncovered this very unique niche of a small subset of DART4-dependent piRNA clusters, but this niche finding may dampen the impact and significance of this study since other major dual-strand piRNA clusters do not change during DART4 GLKD, and the authors do not show data GLKD of any other DARTs. The niche finding of just a small subset of DART-4-dependent piRNA clusters might make another specialized genetics forum a more appropriate venue.

Reviewer #1 (Public review):

The manuscript by Shan et al seeks to define the role of the CHI3L1 protein in macrophages during the progression of MASH. The authors argue that the Chil1 gene is expressed highly in hepatic macrophages. Subsequently, they use Chil1 flx mice crossed to Clec4F-Cre or LysM-Cre to assess the role of this factor in the progression of MASH using a high-fat, high-fructose diet (HFFC). They found that loss of Chil1 in KCs (Clec4F Cre) leads to enhanced KC death and worsened hepatic steatosis. Using scRNA seq, they also provide evidence that loss of this factor promotes gene programs related to cell death. From a mechanistic perspective, they provide evidence that CHI3L serves as a glucose sink and thus loss of this molecule enhances macrophage glucose uptake and susceptibility to cell death. Using a bone marrow macrophage system and KCs they demonstrate that cell death induced by palmitic acid is attenuated by the addition of rCHI3L1. While the article is well written and potentially highlights a new mechanism of macrophage dysfunction in MASH, there are some concerns about the current data that limit my enthusiasm for the study in its current form. Please see my specific comments below.

Major:

(1) The authors' interpretation of the results from the KC ( Clec4F) and MdM KO (LysM-Cre) experiments is flawed. For example, in Figure 2 the authors present data that knockout of Chil1 in KCs using Clec4f Cre produces worse liver steatosis and insulin resistance. However, in supplemental Figure 4, they perform the same experiment in LysM-Cre mice and find a somewhat different phenotype. The authors appear to be under the impression that LysM-Cre does not cause recombination in KCs and therefore interpret this data to mean that Chil1 is relevant in KCs and not MdMs. However, LysM-Cre DOES lead to efficient recombination in KCs and therefore Chil1 expression will be decreased in both KCs and MdM (along with PMNs) in this line.

Therefore, a phenotype observed with KC-KO should also be present in this model unless the authors argue that loss of Chil1 from the MdMs has the opposite phenotype of KCs and therefore attenuates the phenotype. The Cx3Cr1 CreER tamoxifen inducible system is currently the only macrophage Cre strategy that will avoid KC recombination. The authors need to rethink their results with the understanding that Chil1 is deleted from KCs in the LysM-Cre experiment. In addition, it appears that only one experiment was performed, with only 5 mice in each group for both the Clec4f and LysM-Cre data. This is generally not enough to make a firm conclusion for MASH diet experiments.

(2) The mouse weight gain is missing from Figure 2 and Supplementary Figure 4. This data is critical to interpret the changes in liver pathology, especially since they have worse insulin resistance.

(3) Figure 4 suggests that KC death is increased with KO of Chil1. However, this data cannot be concluded from the plots shown. In Supplementary Figure 6 the authors provide a more appropriate gating scheme to quantify resident KCs that includes TIM4. The TIM4 data needs to be shown and quantified in Figure 4. As shown in Supplementary Figure 6, the F4/80 hi population is predominantly KCs at baseline; however, this is not true with MASH diets. Most of the recruited MoMFs also reside in the F4/80 hi gate where they can be identified by their lower expression of TIM4. The MoMF gate shown in this figure is incorrect. The CD11b hi population is predominantly PMNs, monocytes, and cDC,2 not MoMFs (PMID:33997821). In addition, the authors should stain the tissue for TIM4, which would also be expected to reveal a decrease in the number of resident KCs.

(4) While the Clec4F Cre is specific to KCs, there is also less data about the impact of the Cre system on KC biology. Therefore, when looking at cell death, the authors need to include some mice that express Clec4F cre without the floxed allele to rule out any effects of the Cre itself. In addition, if the cell death phenotype is real, it should also be present in LysM Cre system for the reasons described above. Therefore, the authors should quantify the KC number and dying KCs in this mouse line as well.

(5) I am somewhat concerned about the conclusion that Chil1 is highly expressed in liver macrophages. Looking at our own data and those from the Liver Atlas it appears that this gene is primarily expressed in neutrophils. At a minimum, the authors should address the expression of Chil1 in macrophage populations from other publicly available datasets in mouse MASH to validate their findings (several options include - PMID: 33440159, 32888418, 32362324). If expression of Chil1 is not present in these other data sets, perhaps an environmental/microbiome difference may account for the distinct expression pattern observed. Either way, it is important to address this issue.

Reviewer #2 (Public review):

The manuscript from Shan et al., sets out to investigate the role of Chi3l1 in different hepatic macrophage subsets (KCs and moMFs) in MASLD following their identification that KCs highly express this gene. To this end, they utilise Chi3l1KO, Clec4f-CrexChi3l1fl, and Lyz2-CrexChi3l1fl mice and WT controls fed a HFHC for different periods of time.

Firstly, the authors perform scRNA-seq, which led to the identification of Chi3l1 (encoded by Chil1) in macrophages. However, this is on a limited number of cells (especially in the HFHC context), and hence it would also be important to validate this finding in other publicly available MASLD/Fibrosis scRNA-seq datasets. Similarly, it would be important to examine if cells other than monocytes/macrophages also express this gene, given the use of the full KO in the manuscript. Along these lines, utilisation of publicly available human MASLD scRNA-seq datasets would also be important to understand where the increased expression observed in patients comes from and the overall relevance of macrophages in this finding.

Next, the authors use two different Cre lines (Clec4f-Cre and Lyz2-Cre) to target KCs and moMFs respectively. However, no evidence is provided to demonstrate that Chil1 is only deleted from the respective cells in the two CRE lines. Thus, KCs and moMFs should be sorted from both lines, and a qPCR performed to check the deletion of Chil1. This is especially important for the Lyz2-Cre, which has been routinely used in the literature to target KCs (as well as moMFs) and has (at least partial) penetrance in KCs (depending on the gene to be floxed). Also, while the Clec4f-Cre mice show an exacerbated MASLD phenotype, there is currently no baseline phenotype of these animals (or the Lyz2Cre) in steady state in relation to the same readouts provided in MASLD and the macrophage compartment. This is critical to understand if the phenotype is MASLD-specific or if loss of Chi3l1 already affects the macrophages under homeostatic conditions.

Next, the authors suggest that loss of Chi3l1 promotes KC death. However, to examine this, they use Chi3l1 full KO mice instead of the Clec4f-Cre line. The reason for this is not clear, because in this regard, it is now not clear whether the effects are regulated by loss of Chi3l1 from KCs or from other hepatic cells (see point above). The authors mention that Chi3l1 is a secreted protein, so does this mean other cells are also secreting it, and are these needed for KC death? In that case, this would not explain the phenotype in the CLEC4F-Cre mice. Here, the authors do perform a basic immunophenotyping of the macrophage populations; however, the markers used are outdated, making it difficult to interpret the findings. Instead of F4/80 and CD11b, which do not allow a perfect discrimination of KCs and moMFs, especially in HFHC diet-fed mice, more robust and specific markers of KCs should be used, including CLEC4F, VSIG4, and TIM4.

Additionally, while the authors report a reduction of KCs in terms of absolute numbers, there are no differences in proportions. This, coupled with a decrease also in moMF numbers at 16 weeks (when one would expect an increase if KCs are decreased, based on previous literature) suggests that the differences in KC numbers may be due to differences in total cell counts obtained from the obese livers compared with controls. To rule this out, total cell counts and total live CD45+ cell counts should be provided. Here, the authors also provide tunnel staining in situ to demonstrate increased KC death, but as it is typically notoriously difficult to visualise dying KCs in MASLD models, here it would be important to provide more images. Similarly, there appear to be many more Tunel+ cells in the KO that are not KCs; thus, it would be important to examine this in the CLEC4F-Cre line to ascertain direct versus indirect effects on cell survival.

Finally, the authors suggest that Chi3l1 exerts its effects through binding glucose and preventing its uptake. They use ex vivo/in vitro models to assess this with rChi3l1; however, here I miss the key in vivo experiment using the CLEC4F-Cre mice to prove that this in KCs is sufficient for the phenotype. This is critical to confirm the take-home message of the manuscript.

Reviewer #3 (Public review):

This paper investigates the role of Chi3l1 in regulating the fate of liver macrophages in the context of metabolic dysfunction leading to the development of MASLD. I do see value in this work, but some issues exist that should be addressed as well as possible.

Here are my comments:

(1) Chi3l1 has been linked to macrophage functions in MASLD/MASH, acute liver injury, and fibrosis models before (e.g., PMID: 37166517), which limits the novelty of the current work. It has even been linked to macrophage cell death/survival (PMID: 31250532) in the context of fibrosis, which is a main observation from the current study.

(2) The LysCre-experiments differ from experiments conducted by Ariel Feldstein's team (PMID: 37166517). What is the explanation for this difference? - The LysCre system is neither specific to macrophages (it also depletes in neutrophils, etc), nor is this system necessarily efficient in all myeloid cells (e.g., Kupffer cells vs other macrophages). The authors need to show the efficacy and specificity of the conditional KO regarding Chi3l1 in the different myeloid populations in the liver and the circulation.

(3) The conclusions are exclusively based on one MASLD model. I recommend confirming the key findings in a second, ideally a more fibrotic, MASH model.

(4) Very few human data are being provided (e.g., no work with own human liver samples, work with primary human cells). Thus, the translational relevance of the observations remains unclear.

Joint Public Review:

This study presents a valuable contribution to our understanding of ion channel complex assembly by investigating whether BK and CaV1.3 channels begin to form functional associations early in the biosynthetic pathway, prior to reaching the plasma membrane. Using a combination of proximity ligation assays, single-molecule RNA imaging, and super-resolution microscopy, the authors provide convincing evidence that these channels co-localize intracellularly within the ER and Golgi, in both overexpression systems and a relevant endogenous cell model. The study addresses an important and underexplored aspect of membrane protein trafficking and organization, with broader implications for how ion channel signaling complexes are assembled and regulated. The experimental approaches are generally appropriate and the imaging data are clearly presented, with a commendable number of control experiments included. However, several limitations temper the interpretation of the results. The mechanisms underlying mRNA co-localization, and the role of co-translation in complex formation, remain insufficiently defined. Similarly, while intracellular colocalization is convincingly demonstrated, the study does not establish whether such early assembly is the predominant pathway for generating functional complexes at the plasma membrane. More rigorous quantification of channel co-association across compartments, and clarification of key terminology and image analysis methods, would strengthen the overall conclusions. Some of the language in the manuscript would also benefit from a more measured tone to avoid overstating the novelty of the findings. Despite these limitations, the study offers meaningful insights into intracellular ion channel organization and will be of interest to researchers in cell biology, membrane trafficking, and neurophysiology. With focused revisions addressing the outlined points, the manuscript has the potential to make a solid contribution to the field.

Reviewer #1 (Public review):

Summary:

The article by Zdraljevic et al. reports the discovery of a third toxin-antidote (TA) element in C. elegans, composed of the genes mll-1 (toxin) and smll-1 (antidote). Unlike previously characterized TA systems in C. elegans, this element induces larval arrest rather than embryonic lethality. The study identifies three distinct haplotypes at the TA locus, including a hyper-divergent version in the standard laboratory strain N2, which retains a functional toxin but lacks a functional antidote. The authors propose that small RNA-mediated silencing mechanisms, dependent on MUT-16 and PRG-1, suppress the toxicity of the divergent toxin allele. This work provides insights into the evolutionary dynamics of TA elements and their regulation through RNA interference (RNAi).

Overall, there are many things to like about this paper and only a few small quibbles, which will not require more than a little rewriting or relatively minor analyses.

Strengths:

(1) The discovery of a maternally deposited TA element with delayed toxicity due to delayed mRNA translation of the maternally deposited toxin mRNA is a significant addition to the literature on selfish genetic elements in metazoans.

(2) Identifying three haplotypes at the TA locus provides a snapshot of potential evolutionary trajectories for these elements, which are often inferred but rarely demonstrated in naturally occurring strains. The genomic analysis of 550 wild isolates contextualizes the findings within natural populations, revealing geographic clustering and evolutionary pressures acting on the TA locus.

(3) The study employs various techniques, including CRISPR/Cas9 knockouts, FISH, long-read RNA sequencing, and population genomics. The use of inducible systems to confirm toxicity and antidote functionality is particularly robust. This multifaceted approach strengthens the validity of the findings.

(4) The authors provide compelling evidence that small RNA pathways suppress toxin activity in strains lacking a functional antidote. This highlights an alternative mechanism for neutralizing selfish genetic elements.

Weaknesses:

(1) The introduction focuses strongly (for good reason) on bacterial TA systems and then jumps to TA systems in C. elegans. It's unclear why TA systems in other eukaryotes are not discussed.

(2) Similarly, there is a missed opportunity to discuss an analogy between the suppressor mechanism discovered here and the hairpin RNA suppressors of meiotic drive identified by Eric Lai and colleagues. Discussing these will provide a fuller context of the present study's findings and will not affect their novelty.

(3) While the evidence for RNAi-mediated suppression is strong, the claim that positive selection drove diversification at piRNA binding sites requires further discussion and clarification. The elevated dN and dS are unusual (how unusual relative to other genes in vicinity? What is hyper-divergent statistically speaking?), but there is no a priori reason that there would be selection on piRNA binding sites within the mll-1 transcript to facilitate its recognition by endogenous RNAi machinery; what is the selective pressure for mll-1 to do so? Most TA systems would like to avoid being suppressed by the host. One cannot make the argument that this was motivated by the loss of the antidote because the loss of the antidote would be instantly suicidal, so the cadence of events described requiring hypermutation of the mll-1 transcript does not work.

Reviewer #2 (Public review):

Summary:

In the manuscript by Walter-McNeill, Kruglyak, and team, the authors provide solid evidence of another toxin-antidote (TA) system in C. elegans. Generally, TA systems involve selfish and linked genetic elements, one encoding a toxin that kills progeny inheriting it, unless an antidote (the second element) is also present. Currently, only two TA systems have been characterized in this species, pointing to the importance of identifying new instances of such systems to understand their transmission dynamics, prevalence, and functions in shaping worm populations.

Strengths:

This novel TA system (mll-1/smll-1) was identified on LGV in wild C. elegans isolates from the Hawaiian islands, by crossing divergent strains and observing allele frequency distortions by high-throughput genome sequencing after 10 generations. These allele frequency distortions were subsequently confirmed in another set of crosses with a separate divergent strain, and crosses of heterozygous males or hermaphrodites resulted in a pattern of L1 lethality in progeny (with a rod arrest phenotype) that suggested the maternal transmission of this TA system from the XZ1516 genetic background. By elegantly combining the use of near-isogenic lines, CRISPR editing to generate knock-outs, and a transgene rescue of the antidote gene, the authors identified the genes encoding the toxin and the antidote, which they refer to as mll-1 and smll-1. Moreover, the specific mll-1 isoform responsible for the production of the toxin was identified and mll-1 transcripts were observed by FISH in early and late embryos, as well as in larvae. Inducible expression of the toxin in various strains resulted in larval arrest and rod phenotypes. The authors then characterized the genetic variation of 550 wild isolates at the toxin/antidote region on LGV and distinguished three clades: (1) one with the conserved TA system, (2) one having lost the toxin and retaining a mostly functional antidote, and (3) one having lost the antidote and retaining a divergent yet coding toxin (this includes the reference strain Bristol N2, in which the homologous toxin gene has acquired mutations and is known as B0250.8). Further, the authors show that this region is under positive selection. These data are compelling and provide very strong evidence of a new TA system in this species.

Weaknesses:

The question remained as to how one clade, including N2, could retain the toxin gene but not possess a functional antidote. In the second part of the manuscript, the authors hypothesized that small RNA targeting (RNAi) of the toxin transcript could provide the necessary repression to allow worms to survive without the antidote. Through a meta-analysis of multiple small RNA datasets from the literature, the authors found evidence to support this idea, in which the toxin transcript is targeted by 22G siRNAs whose biogenesis is dependent on the Mutator foci protein, MUT-16. They note that from previous studies, mut-16 null mutants displayed a varied penetrance of larval arrest. In their own hands, mut-16 mutants displayed 15% varied larval arrest and 2% rod phenotypes. In an attempt to link B0250.8 to mut-16/siRNAs, they made a double mutant and examined body length as a proxy for developmental stage. Here, they observed a partial rescue of the mut-16 size defect by B0250.8 mutation. Finally, the authors also highlight data from further meta-analysis, which predicts the recognition of B0250.8 by several piRNAs. Also based on existing data from the literature, the authors link loss of Piwi (PRG-1), which binds piRNAs, to a depletion of 22G-RNAs targeting B0250.8 and an upregulation of B0250.8 expression in gonads, suggesting that piRNAs are the primary small RNAs that target B0250.8 for downregulation. The data in this portion of the manuscript are intriguing, but somewhat preliminary and incomplete, as they are based on little primary experimentation and a collection of different datasets (which have been acquired by slightly different methods in most cases). This portion of the study would require subsequent experimentation to firmly establish this mechanistic link. For example, to be able to claim that "the N2 toxin allele has acquired mutations that enable piRNA binding to initiate MUT-16-dependent 22G small RNA amplification that targets the transcript for degradation" the identified piRNA sites should be mutated and protein and transcript levels analysed in wild-type and in the strain with mutated piRNA sites. At a minimum, the protein levels in wild-type and mut-16, prg-1, and/or wago-1 mutants should be measured by western blot and/or by live imaging (introducing a GFP or some other tag to the endogenous protein via CRISPR editing) to show that the toxin is not accumulated as a protein in wt, but increases in levels in these mutants. mRNA levels in Figure S5A suggest there is still some expression of the B0250.8 transcript in a wild-type situation.

Reviewer #1 (Public review):

Summary:

This work aims to improve our understanding of the factors that influence female-on-female aggressive interactions in gorilla social hierarchies, using 25 years of behavioural data from five wild groups of two gorilla species. Researchers analysed aggressive interactions between 31 adult females, using behavioural observations and dominance hierarchies inferred through Elo-rating methods. Aggression intensity (mild, moderate, severe) and direction (measured as the rank difference between aggressor and recipient) were used as key variables. A linear mixed-effects model was applied to evaluate how aggression direction varied with reproductive state (cycling, trimester-specific pregnancy, or lactation) and sex composition of the group. This study highlights the direction of aggressive interactions between females, with most interactions being directed from higher- to lower-ranking adult females close in social rank. However, the results show that 42% of these interactions are directed from lower- to higher-ranking females. Particularly, lactating and pregnant females targeted higher-ranking individuals, which the authors suggest might be due to higher energetic needs, which increase risk-taking in lactating and pregnant females. Sex composition within the group also influenced which individuals were targeted. The authors suggest that male presence buffers female-on-female aggression, allowing females to target higher-ranking females than themselves. In contrast, females targeted lower-ranking females than themselves in groups with a larger ratio of females, which supposes a lower risk for the females since the pool of competitors is larger. The findings provide an important insight into aggression heuristics in primate social systems and the social and individual factors that influence these interactions, providing a deeper understanding of the evolutionary pressures that shape risk-taking, dominance maintenance, and the flexibility of social strategies in group-living species.

The authors achieved their aim by demonstrating that aggression direction in female gorillas is influenced by factors such as reproductive condition and social context, and their results support the broader claim that aggression heuristics are flexible. However, some specific interpretations require further support. Despite this, the study makes a valuable contribution to the field of behavioural ecology by reframing how we think about intra-sexual competition and social rank maintenance in primates.

Strengths:

One of the study's major strengths is the use of an extensive dataset that compiles 25 years of behavioural data and 6871 aggressive interactions between 31 adult females in five social groups, which allows for a robust statistical analysis. This study uses a novel approach to the study of aggression in social groups by including factors such as the direction and intensity of aggressive interactions, which offers a comprehensive understanding of these complex social dynamics. In addition, this study incorporates ecological and physiological factors such as the reproductive state of the females and the sex composition of the group, which allows an integrative perspective on aggression within the broader context of body condition and social environment. The authors successfully integrate their results into broader evolutionary and ecological frameworks, enriching discussions around social hierarchies and risk sensitivity in primates and other animals.

Weaknesses:

Although the paper has a novel approach by studying the effect of reproductive state and social environment on female-female aggression, the use of observational data without experimental manipulation limits the ability to establish causation. The authors suggest that the difference observed in female aggression direction between groups with different sex composition might be indicative of male presence buffering aggression, which seems speculative, as no direct evidence of male intervention or support was reported. Similarly, the use of reproductive state as a proxy for energetic need is an indirect measure and does not account for actual energy expenditure or caloric intake, which weakens the authors' claims that female energetic need induces risk-taking. Overall, this paper would benefit from stronger justification and empirical support to strengthen the conclusions of the study about the mechanisms driving female aggression in gorillas.

Reviewer #2 (Public review):

Summary:

The authors' aim in this study is to assess the factors that can shift competitive incentives against higher- or lower-ranking groupmates in two gorilla species.

Strengths:

This is a relevant topic, where important insights could be gained. The authors brought together a substantial dataset: a long-term behavioral dataset representing two gorilla species from five social groups.

Weaknesses:

The authors have not fully shown the data used in the model and explored the potential of the model. Therefore, I remain cautious about the current results and conclusions.

Some specific suggestions that require attention are

(1) The authors described how group size can affect aggression patterns in some species (line 54), using a whole paragraph, but did not include it as an explanation variable in their model, despite that they stated the overall group size can "conflate opposing effects of females and males" (line 85). I suggest underlining the effects of numbers of males or/and females here and de-emphasizing the effect of group size in the Introduction.

(2) There should be more details given about how the authors calculated individual Elo-ratings (line 98). It seems that authors pooled all avoidance/displacement behaviors throughout the study period. But how often was the Elo-rating they included in the model calculated? By the day or by the month? I guess it was by the day, as they "estimate female reproductive state daily" (line 123). If so, it should be made clear in the text.

In addition, all groups were long-term studied, and the group composition seems fluctuant based on the Table 1 in Reference 11. When an individual enters/leaves the group with a stable hierarchy, it takes time before the hierarchy turns stable again. If the avoidance/displacement behaviors used for the rank relationship were not common, it would take a few days or maybe longer. Also, were the aggressive behaviors more common during rank fluctuations? In other words, if avoidance/displacement behaviors and aggressive behaviors occur simultaneously during rank fluctuations, how did the authors deal with it and take it into consideration in the analysis?

The authors emphasized several times in the text that gorillas "form highly stable hierarchical relationships". Also, in Reference 25, they found very high stabilities of each group's hierarchy. However, the number of females involved in that analysis was different from that used here. They need to provide more basic info on each group's dominance hierarchy and verify their statement. I strongly suggest that the authors display Elo-rating trajectories and necessary relevant statistics for each group throughout the study period as part of the supplementary materials.

(3) The authors stated why they differentiated the different stages based on female reproductive status. They also referred to the differences in energetic needs between stages of pregnancy and lactation (lines 127-128). However, in the mixed model, they only compared the interaction score between the female cycling stage and other stages. The model was not well explained, and the results could be expanded. I suggest conducting more pairwise comparisons in the model and presenting the statistics in the text, if there are significant results. If all three pregnancy stages differed significantly from cycling and lactating stages but not from each other, they may be merged as one pregnancy stage. More in-depth analysis would help provide better answers to the research questions.

Reviewer #3 (Public review):

Smit and Robbins' manuscript investigates the dynamics of aggression among female groupmates across five gorilla groups. The authors utilize longitudinal data to examine how reproductive state, group size, presence of males, and resource availability influence patterns of aggression and overall dominance rankings as measured by Elo scores. The findings underscore the important role of group composition and reproductive status, particularly pregnancy, in shaping dominance relationships in wild gorillas. While the study addresses a compelling and understudied topic, I have several comments and suggestions that may enhance clarity and improve the reader's experience.

(1) Clarification of longitudinal data - The manuscript states that 25 years of behavioral data were used, but this number appears unclear. Based on my calculations, the maximum duration of behavioral observation for any one group appears to be 18 years. Specifically: - ATA: 6 years - BIT: 8 years - KYA: 18 years - MUK: 6 years - ORU: 8 years I recommend that the authors clarify how the 25-year duration was derived.

(2) Consideration of group size - The authors mention that group size was excluded from analyses to avoid conflating the opposing effects of female and male group members. While this is understandable, it may still be beneficial to explore group size effects in supplementary analyses. I suggest reporting statistics related to group size and potentially including a supplementary figure. Additionally, given that the study includes both mountain and wild gorillas, it would be helpful to examine whether any interspecies differences are apparent.

(3) Behavioral measures clarification - Lines 112-116 describe the types of aggressive behaviors observed. It would be helpful to clarify how these behaviors differ from those used to calculate Elo scores, or whether they overlap. A brief explanation would improve transparency regarding the methodology.

(4) Aggression rates versus Elo scores - The manuscript uses aggression rates rather than dominance rank (as measured by Elo scores) as the main outcome variable, but there is no explanation on why. How would the results differ if aggression rates were replaced or supplemented with Elo scores? The current justification for prioritizing aggression rates over dominance rank needs to be more clearly supported.

When I read this, I had a hard time because I do understand the argument, from the POV of there being an airline route in the first place, the current framing focusses so much on an indivdual case that you can miss that on the scale of hundreds of people, doubling the people flying will very likely double the emissions.

This is because the key driver of emissions is burning the fuel, and because airlines scaling up and down the frequency of flights happens on a much faster frequency than laying new cable and network infra.

Reviewer #2 (Public review):

Summary:

The authors observed gene ontologies associated with upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells using scRNA-seq and scATAC-seq datasets from the PBMCs of early HIV-1-infected patients, showing immune responses contributing to HIV pathogenesis and novel targets for viral elimination.

Strengths:

The authors carried out detailed transcriptomics profiling with scRNA-seq and scATAC-seq datasets to conclude upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells.

Comments on revisions:

The authors justified my comments.

Reviewer #3 (Public review):

The revised manuscript demonstrates a marked improvement over the previous version. The authors have successfully incorporated feedback, and have moreover expanded their analyses.

The Methods section is now more detailed and meets the requirements for reproducible research. Authors have reprocessed the data, creating an integrated dataset using a previously published single-cell RNA-Seq atlas, which includes both healthy donors and individuals with chronic HIV-1 infection. An additional batch correction step was included into the processing pipeline after the explicit analysis of inter-donor variability within immune subsets, as was suggested.

Several supplementary figures were added, which both improve the understanding of data and address questions raised by the reviewers. The manuscript also provides additional analysis of cell communication inference, as suggested. The study of interactions between NK cells and infected CD4+ T cells, as well as between monocytes and infected CD4+ T cells, is valuable for understanding the influence of cell signaling on antiviral response and the production of HIV-1 transcripts in infected cells.

The authors have addressed all the reviewers' suggestions, and the current version of the manuscript is both more comprehensive and more informative. Additional analysis has strengthened the narrative and the reproducibility of the research.

The resulting manuscript is both more robust and more informative.

Joint Public Review:

Summary:

Malita and colleagues investigated the mechanism by which infections increase sleep in Drosophila. Their work is important because it further supports the idea that the blood brain barrier is involved in brain-body communication, and because it advances the field of sleep research. Using knock-down and knock-out of cytokines and cytokine receptors specifically in the endocrine cells of the gut (cytokines) as well as in the glia forming the blood-brain barrier (BBB) (cytokines receptors), the authors show that cytokines, upd2 and upd3, secreted by entero-endocrine cells in response to infections increase sleep through the Dome receptor in the BBB. They also show that gut-derived Allatostatin (Alst) A promotes wakefulness by inhibiting the Alst A signaling that is mediated by Alst receptors expressed in BBB glia. Their results suggest there may be additional mechanisms that promote elevated sleep during gut inflammation. The evidence supporting most of their claims is compelling. Nevertheless, the activation of the sleep-promoting pathway by infection should be accomplished through bacterial infection of the gut.

Strengths:

The work is, in general, supported by well-designed and well-performed experiments, especially those that show that the endocrine cells from the gut are the sources of the Upd cytokines, the effects of these cytokines on daytime sleep, and that the glial cells of the BBB are the target cell for the Upds action. In addition, the evidence associating the downregulation of Alst receptors in the BBB by Upd and Jak/Stat pathways is compelling.

Weaknesses:

(1) The model of gut inflammation that is used is based on the increase in reactive oxygen species (ROS) that is caused by adding 1% H2O2 to the food. The use of the model is supported rather weakly by two papers (ref. 26 and 27 ). The paper by Jiang et al. (26) shows that the infection by Pseudomonas entomophila induces cytokine responses Upd2 and 3, which are also induced by the Jnk pathway; there is no mention of ROS. Buchon et al. (27) is a review that refers to results that indicate that as part of the immune response to pathogens in the gut, there is production of ROS by the NADPH oxidase DUOX. Thus, there is no strong support for the use of this model.

(2) There is no support for the use of ROS in the food instead a direct infection by pathogenic bacteria. It is known that ROS causes damage in the gut epithelium, which in turn induces the expression of the cytokines studied, which might be independent of infection and confound the results.

Reviewer #1 (Public review):

Summary:

The authors report a study on how stimulation of receptive-field surround of V1 and LGN neurons affects their firing-rates. Specifically, they examine stimuli in which a grey patch covers the classical RF of the cell and a stimulus appears in the surround. Using a number of different stimulus paradigms they find a long latency response in V1 (but not the LGN) which does not depend strongly on the characteristics of the surround grating (drifting vs static, continuous vs discontinuous, predictable grating vs unpredictable pink noise). They find that population responses to simple achromatic stimuli have a different structure that does not distinguish so clearly between the grey patch and other conditions and the latency of the response was similar regardless of whether the center or surround was stimulated by the achromatic surface. Taken together they propose that the surround-response is related to the representation of the grey surface itself. They relate their findings to previous studies which have put forward the concept of an 'inverse RF' based on strong responses to small grey patches on a full-screen grating. They also discuss their results in the context of studies that suggest that surround responses are related to predictions of the RF content or figure-ground segregation.

Strengths:

I find the study to be an interesting extension of the work on surround stimulation and the addition of the LGN data is useful showing that the surround-induced responses are not present in the feed-forward path. The conclusions appear solid, being based on large numbers of neurons obtained through Neuropixels recordings. The use of many different stimulus combinations provides a rich view of the nature of the surround-induced responses.

Weaknesses:

The LGN data comes from a small number of animals (n=2). Statistics are generally pooled across all recording sessions/animals without taking into account the higher covariance of neurons recorded in the same session. This is not a problem for paired comparisons, but for some statistics in the paper a hierarchical approach would have been more appropriate. The authors do present individual session data and the effects appear to be consistent across sessions.

Reviewer #3 (Public review):

Summary:

This paper explores the phenomenon whereby some V1 neurons can respond to stimuli presented far outside their receptive field. It introduces three possible explanations for this phenomenon and it presents experiments that it argues favor the third explanation, which is based on figure/ground segregation.

Strengths:

I found it useful to see that there are three possible interpretations of this finding (prediction error, interpolation, and figure/ground). I also found it useful to see a comparison with LGN responses and to see that the effect there is not only absent but actually opposite: stimuli presented far outside the receptive field suppress rather than drive the neurons. Other experiments presented here may also be of interest to the field.

Weaknesses:

Though the paper has markedly improved, and now has a clearer statement of the hypotheses, it could be streamlined further, to tighten the relation between hypotheses and analyses, and to draw conclusions from those analyses in terms of the hypotheses.

Reviewer #1 (Public review):

The paper is well written and the figures well laid out. The methods are easy to follow, and the rational and logic for each experiment easy to follow. The introduction sets the scene well, and the discussion is appropriate. The summary sentences throughout the text help the reader.

The authors have done a lot of work addressing my previous concerns and those of the other Reviewers.

Reviewer #2 (Public review):

Summary

Le Roy et al quantify wing morphology and wing kinematics across twenty eight and eight hoverfly species, respectively; the aim is to identify how weight support during hovering is ensured across body sizes. Wing shape and relative wing size vary non-trivially with body mass, but wing kinematics are reported to be size-invariant. On the basis of these results, it is concluded that weight support is achieved solely through size-specific variations in wing morphology, and that these changes enabled hoverflies to decrease in size. Adjusting wing morphology may be preferable compared to the alternative strategy of altering wing kinematics, because kinematics may be subject to stronger evolutionary and ecological constraints, dictated by the highly specialised flight and ecology of the hoverflies.

Strengths

The study deploys a vast array of challenging techniques, including flight experiments, morphometrics, phylogenetic analyses, and numerical simulations; it so illustrates both the power and beauty of an integrative approach to animal biomechanics. The question is well motivated, the methods appropriately designed, and the discussion elegantly places the results in broad biomechanical, ecological, and evolutionary context.

Weaknesses

(1) In assessing evolutionary allometry, it is key to pinpoint the variation expected from changes in size alone. The null hypothesis for wing morphology is well-defined (isometry), but the equivalent predictions for kinematic parameters, although specified, are insufficiently justified, and directly contradict classic scaling theory. A detailed justification of the "kinematic similarity" assumption, or a change in the null hypothesis, would substantially strengthen the paper, and clarify its evolutionary implications.

(2) By relating the aerodynamic output force to wing morphology and kinematics, it is concluded that smaller hoverflies will find it more challenging to support their body mass--a scaling argument that provides the framework for this work. This hypothesis appears to stand in direct contrast to classic scaling theory, where the gravitational force is thought to present a bigger challenge for larger animals, due to their disadvantageous surface-to-volume ratios. The same problem ought to occur in hoverflies, for wing kinematics must ultimately be the result of the energy injected by the flight engine: muscle. Much like in terrestrial animals, equivalent weight support in flying animals thus requires a positive allometry of muscle force output. In other words, if a large hoverfly is able to generate the wing kinematics that suffice to support body weight, an isometrically smaller hoverfly should be, too (but not vice versa). Clarifying the relation between the scaling of muscle mechanical input, wing kinematics, and weight support would help resolve the conflict between these two contrasting hypotheses, and considerably strengthen the biomechanical motivation and evolutionary interpretation.

(3) One main conclusion-- that miniaturization is enabled by changes in wing morphology--is insufficiently supported by the evidence. Is it miniaturization or "gigantism" that is enabled by (or drives) the non-trivial changes in wing morphology? To clarify this question, the isolated treatment of constraints on the musculoskeletal system vs the "flapping-wing based propulsion" system needs to be replaced by an integrated analysis: the propulsion of the wings, is, after all, due to muscle action. Revisiting the scaling predictions by assessing what the engine (muscle) can impart onto the system (wings) will clarify whether non-trivial adaptations in wing shape or kinematics are necessary for smaller or larger hovering insects (if at all!).

In many ways, this work provides a blueprint for work in evolutionary biomechanics; the breadth of both the methods and the discussion reflects outstanding scholarship.

Reviewer #3 (Public review):

This paper addresses an important question about how changes in wing morphology vs. wing kinematics change with body size across an important group of high-performance insects, the hoverflies. The biomechanics and morphology convincingly support the conclusions that there is no significant correlation between wing kinematics and size across the eight specific species analyzed in depth and that instead wing morphology changes allometrically. The morphological analysis is enhanced with phylogenetically appropriate tests across a larger data set incorporating museum specimens.

The authors have made very extensive revisions that have significantly improved the manuscript and brought the strength of conclusions in line with the excellent data. Most significantly, they have expanded their morphological analysis to include museum specimens and removed the conclusions about evolutionary drivers of miniaturization. As a result, the conclusion about morphological changes scaling with body size rather than kinematic properties is strongly supported and very nicely presented with a strong complementary set of data. I only have minor textual edits for them to consider.

Reviewer #1 (Public review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically-tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

Strengths:

• Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones

• The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation

• Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'

• Includes multiple follow-up experiments, which leads to tests of internal replication and an impactful mechanistic proposal

• Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionary ancient

Weaknesses:

• As stated in the summary, the authors are attributing the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either

• The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering

• Lack of attribution of previous published work from other research groups that would provide the proper context of the present study

• There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation"

• The experimental design for studying aggression in males has flaws. A standard test like a resident-intruder test should be used.

• While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside

• The statistics comparing "experimental to experimental" and "control to experimental" isn't appropriate

Reviewer #3 (Public review):

Summary:

Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of brain-derived estrogens in the control of sexual and aggressive behavior in medaka. The constitutive deletion of Cyp19a1b markedly reduced brain estrogen content in males and to a lesser extent in females. These effects are accompanied by reduced sexual and aggressive behavior in males and reduced preference for males in females. These effects are reversed by adult treatment with supporting a role for estrogens. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of estrogens in social behavior in the most abundant vertebrate taxon, however the conclusion of brain-derived estrogens awaits definitive confirmation.

Strengths:

• Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxon are more abundant and yet proportionally less studied that the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.

• Results obtained from multiple mutant lines converge to show that estrogen signaling, likely synthesized in the brain drives aspects of male sexual behavior.

• The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.

• The authors have made important corrections to tone down some of the conclusions which are more in line with the results.

Weaknesses:

• No evaluation of the mRNA and protein products of Cyp19a1b and ESR2a are presented, such that there is no proper demonstration that the mutation indeed leads to aromatase reduction. The conclusion that these effects dependent on brain derived estrogens is therefore only supported by measures of E2 with an EIA kit that is not validated. No discussion of these shortcomings is provided in the discussion thus further weakening the conclusion manuscript.

• Most experiments are weakly powered (low sample size).

• The variability of the mRNA content for a same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).

Conclusions:

Overall, the claims regarding role of estrogens originating in the brain on male sexual behavior is supported by converging evidence from multiple mutant lines. The role of brain-derived estrogens on gene expression in the brain is weaker as are the results in females.

Reviewer #1 (Public review):

Summary:

This work investigated whether cytoplasmic poroelastic properties play an important role in cellular mechanical response over length scales and time scales relevant to cell physiology. Overall, the manuscript concludes that intracellular cytosolic flows and pressure gradients are important for cell physiology and that they act of time- and length-scales relevant to mechanotransduction and cell migration.

Strengths:

Their approach integrates both computational and experimental methods. The AFM deformation experiments combined with measuring z-position of beads is a challenging yet compelling method to determine poroelastic contributions to mechanical realization.

The work is quite interesting and will be of high value to the field of cell mechanics and mechanotransduction.

Weaknesses:

However, there are several issues related to the lack of description of theoretical equations, experimental details, and data transparency that should be addressed, including the following:

(1) Some details are not described for experimental procedures. For example, what were the pharmacological drugs dissolved in, and what vehicle control was used in experiments? How long were pharmacological drugs added to cells?

(2) Details are missing from the Methods section and Figure captions about the number of biological and technical replicates performed for experiments. Figure 1C states the data are from 12 beads on 7 cells. Are those same 12 beads used in Figure 2C? If so, that information is missing from the Figure 2C caption. Similarly, this information should be provided in every figure caption so the reader can assess the rigor of the experiments. Furthermore, how heterogenous would the bead displacements be across different cells? The low number of beads and cells assessed makes this information difficult to determine.

(3) The full equation for displacement vs. time for a poroelastic material is not provided. Scaling laws are shown, but the full equation derived from the stress response of an elastic solid and viscous fluid is not shown or described.

Reviewer #2 (Public review):

Summary:

Malboubi et al. present a novel experimental framework to investigate the rheological properties of the cell cytoplasm. Their findings support a model where the cytoplasm behaves as a poroelastic material governed by Darcy's law - a property overlooked in previous literature. They demonstrate that this poroelastic behavior delays the equilibration of hydrostatic pressure gradients within the cytoplasm over timescales of 1 to 10 seconds following a perturbation, likely due to fluid-solid friction within the cytoplasmic matrix. Furthermore, under sustained perturbations such as depressurization, they reveal that pressure gradients can persist for minutes, which they propose might potentially influence physiological processes like mechanotransduction or cell migration typically happening on these timescales.

Strengths:

This article holds significant value within the ongoing efforts of the cell biology and biophysics communities to quantitatively characterize the mechanical properties of cells. The experiments are innovative and thoughtfully contextualized with quantitative estimates and a finite element model that supports the authors' hypotheses.

Comments & Questions:

While the hypothesis of a poroelastic cytoplasm is insightful and supported by the results, certain parts of the paper (detailed below) rely on qualitative arguments. Given the experimental approaches and accompanying modeling, the study has the potential for more in-depth discussions and stronger quantitative evidence. Placing greater emphasis on quantifications and direct comparisons between the model and experimental data would enhance the work. Additionally, exploring the limitations of the proposed model would add valuable depth to the paper.

The authors state, "Next, we sought to quantitatively understand how the global cellular response to local indentation might arise from cellular poroelasticity." However, the evidence presented in the following paragraph appears more qualitative than strictly quantitative. For instance, the length scale estimate of ~7 μm is only qualitatively consistent with the observed ~10 μm, and the timescale 𝜏𝑧 ≈ 500 ms is similarly described as "qualitatively consistent" with experimental observations. Strengthening this point would benefit from more direct evidence linking the short timescale to cell surface tension. Have you tried perturbing surface tension and examining its impact on this short-timescale relaxation by modulating acto-myosin contractility with Y-27632, depolymerizing actin with Latrunculin, or applying hypo/hyperosmotic shocks?

The authors demonstrate that the second relaxation timescale increases (Figure 1, Panel D) following a hyperosmotic shock, consistent with cytoplasmic matrix shrinkage, increased friction, and consequently a longer relaxation timescale. While this result aligns with expectations, is a seven-fold increase in the relaxation timescale realistic based on quantitative estimates given the extent of volume loss?

If the authors' hypothesis is correct, an essential physiological parameter for the cytoplasm could be the permeability k and how it is modulated by perturbations, such as volume loss or gain. Have you explored whether the data supports the expected square dependency of permeability on hydraulic pore size, as predicted by simple homogeneity assumptions? Additionally, do you think that the observed decrease in k in mitotic cells compared to interphase cells is significant? I would have expected the opposite naively as mitotic cells tend to swell by 10-20 percent due to the mitotic overshoot at mitotic entry (see Son Journal of Cell Biology 2015 or Zlotek Journal of Cell Biology 2015).

Based on your results, can you estimate the pore size of the poroelastic cytoplasmic matrix? Is this estimate realistic? I wonder whether this pore size might define a threshold above which the diffusion of freely diffusing species is significantly reduced. Is your estimate consistent with nanobead diffusion experiments reported in the literature?

Do you have any insights into the polymer structures that define this pore size? For example, have you investigated whether depolymerizing actin or other cytoskeletal components significantly alters the relaxation timescale?

There are no quantifications in Figure 6, nor is there a direct comparison with the model. Based on your model, would you expect the velocity of bleb growth to vary depending on the distance of the bleb from the pipette due to the local depressurization? Specifically, do blebs closer to the pipette grow more slowly?

I find it interesting that during depressurization of the interphase cells, there is no observed volume change, whereas in pressurization of metaphase cells, there is a volume increase. I assume this might be a matter of timescale, as the microinjection experiments occur on short timescales, not allowing sufficient time for water to escape the cell. Do you observe the radius of the metaphase cells decreasing later on? This relaxation could potentially be used to characterize the permeability of the cell surface.

I am curious about the saturation of the time lag at 30 microns from the pipette in Figure 4, Panel E for the model's prediction. A saturation which is not clearly observed in the experimental data. Could you comment on the origin of this saturation and the observed discrepancy with the experiments (Figure E panel 2)? Naively, I would have expected the time lag to scale quadratically with the distance from the pipette, as predicted by a poroelastic model and the diffusion of displacement. It seems weird to me that the beads start to move together at some distance from the pipette or else I would expect that they just stop moving. What model parameters influence this saturation? Does membrane permeability contribute to this saturation?

Reviewer #3 (Public review):

Summary:

In this delightful study, the authors use local indentation of the cell surface combined with out-of-focus microscopy to measure the rates of pressure spread in the cell and to argue that the results can be explained with the poroelastic model. Osmotic shock that decreases cytoskeletal mesh size supports this notion. Experiments with water injection and water suction further support it, and also, together with a mechanical model and elegant measurements of decreasing fluorescence in the cell 'flashed' by external flow, demonstrate that the membrane is permeable, and that steady flow and pressure gradient can exist in a cell with water source/sink in different locations. Use of blebs as indicators of the internal pressure further supports the notion of differential cytoplasmic pressure.

Strengths:

The study is very imaginative, interesting, novel and important.

Weaknesses: I have two broad critical comments:

(1) I sense that the authors are correct that the best explanation of their results is the passive poroelastic model. Yet, to be thorough, they have to try to explain the experiments with other models and show why their explanation is parsimonious. For example, one potential explanation could be some mechanosensitive mechanism that does not involve cytoplasmic flow; another could be viscoelastic cytoskeletal mesh, again not involving poroelasticity. I can imagine more possibilities. Basically, be more thorough in the critical evaluation of your results. Besides, discuss the potential effect of significant heterogeneity of the cell.

(2) The study is rich in biophysics but a bit light on chemical/genetic perturbations. It could be good to use low levels of chemical inhibitors for, for example, Arp2/3, PI3K, myosin etc, and see the effect and try to interpret it. Another interesting question - how adhesive strength affects the results. A different interesting avenue - one can perturb aquaporins. Etc. At least one perturbation experiment would be good.

Reviewer #1 (Public review):

Summary:

Selberg et al. present a small but apparently very relevant modification to the existing BUSTED model. The new model allows for a fraction of codons to be assigned to an error class characterized by a very high dN/dS value. This "omega_e" category is constrained to represent no more than 1% of the alignment. The analyses convincingly show that the method performs well and represents a real improvement for genome-wide scans of positive selection. Alignment and sequencing errors are a major concern in molecular evolution. This new method, which shows strong performance, is therefore a very welcome contribution.

Strengths:

By thoroughly reanalyzing four datasets, the manuscript convincingly demonstrates that omega_e effectively identifies genuine alignment errors. Next, the authors evaluate the reduction in power to detect true selection through simulations. This new model is simple, efficient, and computationally fast. It is already implemented and available in HYPHY software.

As a side note, I found it particularly interesting how the authors tested the statistical support for the new method compared to the simpler version without the error class. In many cases, the simpler model could not be statistically rejected in favor of the more complex model, despite producing biologically incorrect results in terms of parameter inference. This highlights a broader issue in molecular evolution and phylogenomics, where model selection often relies too heavily on statistical tests, potentially at the expense of biological realism. The analyses also reveal a trade-off between statistical power and the false positive rate. As with other methods, BUSTED-E cannot distinguish between alignment/sequencing errors and episodes of very strong positive selection. The authors are transparent about this limitation in the discussion.

Weaknesses:

Regarding the structure of the manuscript, the text could be clearer and more precise. Clear, practical recommendations for users could also be provided in the Results section. Additionally, the simulation analyses could be further developed to include scenarios with both alignment errors and positive selection, in order to better assess the method's performance. Finally, the model is evaluated only in the context of site models, whereas the widely used branch-site model is mentioned as possible but not assessed.

Reviewer #2 (Public review):

Summary:

In this paper, Selberg et al present an extension of their widely used BUSTED family of codon models for the detection of episodic ("site-branch") positive selection from coding gene sequences. The extension adds an "error component" to ω (dN/dS) to capture misaligned codons. This ω component is set to an arbitrarily high value to distinguish it from positive selection, which is characterised by ω > 1 but assumed not to be so high.

The new method is tested on several datasets of comparative genomes, characterised by their size and the fact that the authors scanned for positive selection and/or provided filtering of alignment quality. It is also tested on simple simulations.

Overall, the new method appears to capture relatively little of the ω variability in the alignments, although it is often significant. Given the complexity of codon evolution, adding a new parameter is more or less significant, and the question is whether it captures the signal that is intended, preferably in an unbiased manner.

Strengths:

This is an important issue, and I am enthusiastic to see it explicitly modeled within the codon modeling framework, rather than externalised to ad hoc filtering methods. The promise of quantifying the divergence signal from alignment error vs selection is exciting.

The BUSTED family of models is widely used and very powerful for capturing many aspects of codon evolution, and it is thus an excellent choice for this extension.

Weaknesses:

(1) The definition of alignment error by a very large ω is not justified anywhere in the paper. There are known cases of bona fide positive selection with many non-synonymous and 0 synonymous substitutions over branches. How would they be classified here? E.g., lysosyme evolution, bacterial experimental evolution.

Using the power of the model family that the authors develop, I would suggest characterising a more specific error model. E.g., radical amino-acid "changes" clustered close together in the sequence, proximity to gaps in the alignment, correlation of apparent ω with genome quality.

Also concerning this high ω, how sensitive is its detection to computational convergence issues?

(2) The authors should clarify the relation between the "primary filter for gross or large-scale errors" and the "secondary filter" (this method). Which sources of error are expected to be captured by the two scales of filters? What is their respective contribution to false positives of positive selection?

Sources of error in the alignment of coding genes include:

a) Errors in gene models, which may differ between species but also propagate among close species (i.e., when one species is used as a reference to annotate others).

b) Inconsistent choice of alternative transcripts/isoforms.

Both of these lead to asking an alignment algorithm to align non-homologous sequences, which violates the assumptions of the algorithms, yet both are common issues in phylogenomics.

c) Sequencing errors, but I doubt they affect results much here.

d) Low complexity regions of proteins.

e) Aproximations by alignment heuristics, sometimes non-deterministic or dependent on input order.

f) Failure to capture aspects of protein or gene evolution in the optimality criteria used.

For example, Figure 1 seems to correspond to a wrong or inconsistent definition of the final exon of the gene in one species, which I would expect to be classified as "gross or large-scale error".

(3) The benchmarking of the method could be improved both for real and simulated data.

For real data, the authors only analysed sequences from land vertebrates with relatively low Ne and thus relatively low true positive selection. I suggest comparing results with e.g. Drosophila genomes, where it has been reported that 50% of all substitutions are fixed by positive selection, or with viral evolution.

For simulations, the authors should present simulations with or without alignment errors (e.g., introduce non-homologous sequences, or just disturb the alignments) and with or without positive selection, to measure how much the new method correctly captures alignment errors and incorrect positive selection.

I also recommend simulating under more complex models, such as multinucleotide mutations or strong GC bias, and investigating whether these other features are captured by the alignment error component.

Finally, I suggest taking true alignments and perturbing them (e.g., add non-homologous segments or random gaps which shift the alignment locally), to verify how the method catches this. It would be interesting to apply such perturbations to genes which have been reported as strong examples of positive selection, as well as to genes with no such evidence.

(4) It would be interesting to compare to results from the widely used filtering tool GUIDANCE, as well as to the Selectome database pipeline (https://doi.org/10.1093/nar/gkt1065). Moreover, the inconsistency between BUSTED-E and HMMCleaner, and BMGE is worrying and should be better explained.

(5) For a new method such as this, I would like to see p-value distributions and q-q plots, to verify how unbiased the method is, and how well the chi-2 distribution captures the statistical value.

(6) I disagree with the motivation expressed at the beginning of the Discussion: "The imprimatur of "positive selection" has lost its luster. Researchers must further refine prolific candidate lists of selected genes to confirm that the findings are robust and meaningful." Our goal should not be to find a few impressive results, but to measure accurately natural selection, whether it is frequent or rare.

Reviewer #1 (Public review):

Summary:

The authors provide a simple yet elegant approach to identifying therapeutic targets that synergize to prevent therapeutic resistance using cell lines, data-independent acquisition proteomics, and bioinformatic analysis. The authors identify several combinations of pharmaceuticals that were able to overcome or prevent therapeutic resistance in culture models of ovarian cancer, a disease with an unmet diagnostic and therapeutic need.

Strengths:

The manuscript utilizes state-of-the-art proteomic analysis, entailing data-independent acquisition methods, an approach that maximizes the robustness of identified proteins across cell lines. The authors focus their analysis on several drugs under development for the treatment of ovarian cancer and utilize straightforward thresholds for identifying proteomic adaptations across several drugs on the OVSAHO cell line. The authors utilized three independent and complementary approaches to predicting drug synergy (NetBox, GSEA, and Manual Curation). The drug combination with the most robust synergy across multiple cell lines was the inhibition of MEK and CDK4/6 using PD-0325901+Palbociclib, respectively. Additional combinations, including PARPi (rucaparib) and the fatty acid synthase inhibitor (TVB-2640). Collectively, this study provides important insight and exemplifies a solid approach to identifying drug synergy without large drug library screens.

Weaknesses:

The manuscript supports their findings by describing the biological function(s) of targets using referenced literature. While this is valuable, the number of downstream targets for each initial target is extensive, thus, the current work does not attempt to elucidate the mechanism of their drug synergy. Responses to drugs are quantified 72 hours after treatment and exclusively focused on cell viability and protein expression levels. The discovery phase of experimentation was solely performed on the OVSAHO cell line. An additional cell line(s) would increase the impact of how the authors went about identifying synergistic targets using bioinformatics. Ovarian cancer is elusive to treatment as primary cancer will form spheroids within ascites/peritoneal fluids in a state of pseudo-senescence to overcome environmental stress. The current manuscript is executed in 2D culture, which has been demonstrated to deviate from 3D, PDX, and primary tumours in terms of therapeutic resistance (DOI: 10.3390/cancers13164208). Collectively, the manuscript is insufficient in providing additional mechanistic insight beyond the literature, and its interpretation of data is limited to 2D culture until further validated.

Reviewer #2 (Public review):

Summary:

Franz and colleagues combined proteomics analysis of OVSAHO cell lines treated with 6 individual drugs. The quantitative proteomics data were then used for computational analysis to identify candidates/modules that could be used to predict combination treatments for specific drugs.

Strengths:

The authors present solid proteomics data and computational analysis to effectively repeat at the proteomics level analysis that have previously been done predominantly with transcriptional profiling. Since most drugs either target proteins and/or proteins are the functional units of cells, this makes intuitive sense.

Weaknesses:

Considering the available resources of the involved teams, performing the initial analysis in a single HGSC cell is certainly a weakness/limitation.

The data also shows how challenging it is to correctly predict drug combinations. In Table 2 (if I read it correctly), the majority of the drug combinations predicted for the initial cell line OVSAHO did not result in the predicted effect. It also shows how variable the response was in the different HGSC cell lines used for the combination treatment. The success rate will most likely continue to drop as more sophisticated models are being used (i.e., PDX). Human patients are even more challenging.

It would most likely be useful to more directly mention/discuss these caveats in the manuscript.

Reviewer #1 (Public review):

Van Arsdale and colleagues evaluated whether human-HPV DNA junctions could be detected in serum, cell-free DNA from 16 patients with cervical cancer by hybrid capture and Illumina sequencing. Junctions were identified in seven patients, and these junctions were concordant with junctions identified in tumor DNA except for one patient, suggesting that, in most cases, the cfDNA is originating from a clone of the primary tumor. Junction detection at 6 months was found to be statistically significant prognostic for recurrence. The study further validates that type-specific E7 DNA, which is essential for tumorigenesis, was detectable by PCR for most patient sera, but had no association with recurrence. Furthermore, the study provides additional evidence that tumors harboring non-alpha-9 clade HPVs had shorter recurrence-free survival and overall worse outcome from the study's patients, as well as reanalysis of TCGA data. However, these findings need to be more extensively discussed in the context of previous publications. One identified limitation of this approach is the detection of non-tumor HPVs, but this was only seen in one patient. The major shortcoming of this study is the limited number of patients that were evaluated, but for a retrospective study, this is a reasonable number of patients evaluated, and the findings are appropriately not overstated. The design, execution, and detailed analysis of the sequencing data are a major strength. This study provides important foundational evidence for further evaluating the clinical utility of HPV DNA detection from cfDNA and specifically assessing for integration junctions.

Reviewer #2 (Public review):

Summary:

The authors set out to identify cell-free HPV breakpoint junctions and assess their utility in identifying cervical cancer recurrence as a surrogate, tumor-specific assay. They added unrelated findings about a potential relationship between various viral types and cancer recurrence frequencies, concluding that clade alpha 9 types recurred at a lower rate than did non-alpha 9 viral types.

Strengths:

The authors analyzed 16 cervical cancer samples and matched serum samples collected initially or upon clinical treatments. An association between virus types and cancer recurrence frequencies is a novel finding that will likely induce further insights into HPV pathogenic mechanisms.

Weaknesses:

The main claims of this manuscript are only partially supported by the data as presented, because the sequencing data are not quantified and were not analyzed in a statistically adequate way. First, only one or at most two breakpoints are presented per tumor (Table 1). This finding is discrepant from many extensive, published genomics studies of HPV-positive cancers, in which many unique breakpoints are found frequently in individual cancers, ranging from 1 or 2 up to more than 100. Second, no information is provided about likely correlations between genomic DNA copy number at rearranged loci and breakpoint-identifying sequencing read counts. Third, no direct comparison is presented between supporting read counts from cancer samples and read counts from circulating cell-free DNA samples. Fourth, many of the initial cancer samples harbored no insertional breakpoints, so no correlation with breakpoints in the serum samples would be possible. Fifth, no mention was made about tumor heterogeneity, where a given breakpoint may not be present in every cell of the tumor. Previous literature about the general topic of using cell-free DNA breakpoints as a surrogate for cancer cells is not cited adequately. Findings about potential correlations between various viral types and variable recurrence rates are not well-supported by the authors' own data, because of the limited sample numbers studied. This section of the paper is relatively unrelated to the main thrust, which is about breakpoint detection.

Reviewer #1 (Public review):

Summary:

Y-family polymerases, such as polymerases eta, iota, and kappa, have low fidelity relative to other polymerases involved in DNA replication and repair. This is believed to be due to their active sites being less constrained than those of other polymerases. Paradoxically, work by this lab and others shows that in vivo, these Y-family polymerases are more error-free (less error-prone) during DNA damage bypass than would be expected given their low fidelity. For this reason, the authors have been focusing on other cellular factors that may increase the fidelity of Y-family polymerases. The current paper focuses on two such factors: WRN, which possesses exonuclease and helicase activities, and WRNIP1, which possesses a DNA-dependent ATPase.

Previously, this group showed that defects in the exonuclease function of WRN lead to a loss in the fidelity of polymerases eta and iota during DNA damage bypass, presumably by removing nucleotide misinsertions. The current paper extends this work by considering the ATPase activities of WRN and WRNIP1. The authors looked at the impact of various amino acid substitutions in these proteins on the fidelity of DNA damage bypass by Y-family polymerases. They did this by both measuring the mutation frequencies of these cell lines as well as the mutation spectra observed in them. They showed that the ATPase activities of both WRN and WRNIP1, as well as the exonuclease activities of WRN, are necessary high fidelity of Y-family polymerases in cells. They specifically examined the bypass of cyclobutene pyrimidine dimers by polymerase eta, the bypass of 6-4 photoproducts by polymerases eta and iota, and the bypass of ethenoadenine by polymerase iota. Moreover, they showed that WRNIP1 ATPase defects impair the WRN exonuclease from removing misinsertions by polymerase iota at thymine glycol lesions. These defects generally do not affect the efficiency of the bypass, only its fidelity.

Strengths:

The manuscript by Yoon et al is the latest in a series of important and impactful papers by this research group examining the cellular factors that enhance the fidelity of translesion synthesis by Y-family polymerases in human cell lines. Overall, the study is well designed, the data are clearly presented, and the conclusions are well supported and convincing. The authors also discuss a reasonable possibility that complex formation between the WRN and WRNIP1 proteins and Y-family polymerases could tighten the active sites of these polymerases to improve fidelity. Further studies are required to demonstrate this model, but it is a very exciting model that is well supported by the current data.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

The authors of the present study are responsible for a previous study, which also showed that in response to DNA damage, Werner syndrome protein WRN, WRN interacting protein WRNIP1, and Rev1 assemble together with Y-family Pols (Polη, Polι, or Polκ), and that they are indispensable for Trans-Lesion-Synthesis (TLS) (Genes Dev 2024). They also identified a role of WRN's 3'→5' exonuclease activity in the high in vivo fidelity of TLS by Y-family, through UV-induced CPDs by Polη, through N6 ethenodeoxyadenosine (εdA) by Polι, through thymine glycol by Polκ, and through UV-induced (6-4) photoproducts by Polη and Polι. Thus, by removing nucleotides misinserted opposite DNA lesions by the Y-family Pols, WRN's 3'→5' exonuclease activity improves the fidelity of TLS by these Pols. The present work, which follows up on this previous work, reports the crucial role also of the ATPase activities of WRN and WRNIP1 in raising the fidelity of TLS by Y family Pols, in addition to the exonuclease activity, with an entirely different mechanism, which normally consists in unwinding of DNA containing secondary structures.

By using adequate cell line models and methodologies, notably DNA fiber, TLS, and mutation analyses assays, as well as specific ATPase point mutations, they found that progression of the replication forks through UV lesions was not affected in cells lacking the WRN exonuclease activity as well as the WRN and WRNIP1 ATPase activities, but occurs with a vast increase in error-prone TLS, notably through CPDs by Polη, with differential impacts on the nature of mutations between WRN ATPase and WRNIP1 ATPase. The relative contributions of these activities (exonuclease and ATPase) to the fidelity of TLS Pols, however, vary, depending upon the DNA lesion and the TLS Pol involved. Additionally, defects in these ATPase activities cause mutational hot spot formation in different sequence contexts. The authors provide evidence that the combined action of WRN and WRNIP1 ATPases, along with WRN 3' to 5' exonuclease, confers an enormous rise in the fidelity of TLS by Y-family Pols. They identify the means by which these otherwise highly error-prone TLS Pols have been adapted to function in an error-free manner. They suggest that WRNIP1 ATPases prevent misincorporations while WRN exonuclease removes misinserted nucleotides. This combination confers a vast increase in the fidelity of Y-family Pols, essential for genome stability.

Overall, this is a comprehensive and thoughtful manuscript, and all the findings reported are convincing and well supported. The data cannot be considered as entirely novel, as they follow-up on the recent 2024 publication by the same authors who unveiled that the exonuclease activity of WRN and WRNIP1 confers accuracy of TLS. The experimental methods are multiple and rigorous.

Reviewer #3 (Public review):

Summary:

Replication through DNA lesions such as UV-induced pyrimidine dimers is mainly performed by Y-family pols. These translesion synthesis (TLS) pols are intrinsically error-prone. However, in living cells, TLS must be conducted in an error-free manner. This manuscript demonstrated that WRN and WRNIP1 ATPases play an important role in addition to WRN 3'>5' exonuclease in human cells.

Strengths:

The authors made use of WT human fibroblasts and WRN-deficient cell line for TLS assays in human cells and siRNA knock-down experiments to analyze TLS efficiency. For the cII mutation assay, the big blue mouse embryonic fibroblasts were used. These materials, as well as other Materials and Methods, had already been well established by this group or other groups. The authors used Pol eta, iota, kappa, and theta as TLS pols, and used UV-induced CPD, (6-4)PP, epsilon dA, and thymine glycol as DNA lesions. Thus, the authors examined the generality of their results in terms of TLS pols and DNA lesions.

Weaknesses:

Although the main part of this manuscript is the impact of the deficiencies of WRN and WRNIP1 ATPases on TLS by Y-family DNA polymerases, especially on TLS efficiency and mutation spectrum, many readers would be interested in how these ATPases could change molecular structure of Pol eta, because the structure of it have been studied for some time.

Reviewer #3 (Public review):

Summary: In this paper the authors examined the effects of strip cropping, a relatively new agricultural technique of alternating crops in small strips of several meters wide, on ground beetle diversity. The results show an increase in species diversity (i.e. abundance and species richness) of the ground beetle communities compared to monocultures.

Strengths: The article is well written; it has an easily readable tone of voice without too much jargon or overly complicated sentence structure. Moreover, as far as reviewing the models in depth without raw data and R scripts allows, the statistical work done by the authors looks good. They have well thought out how to handle heterogenous, unbalanced and taxonomically unspecific yet spatially and temporarily correlated field data. The models applied and the model checks performed are appropriate for the data at hand. Combining RDA and PCA axes together is a nice touch. Moreover, after the first round of reviews, the authors have done a great job at rewriting the paper to make it less overstated, more relevant to the data at hand and more solid in the findings. Many of the weaknesses noted in the first review have been dealt with. The overall structure of the paper is good, with a clear introduction, hypotheses, results section and discussion.

Weaknesses: The weaknesses that remain are mainly due to a difficult dataset and choices that could have stressed certain aspects more, like the relationship between strip cropping and intercropping. The mechanistic understanding of strip cropping is what is at stake here. Does strip cropping behave similar to intercropping, a technique which has been proven to be beneficial to biodiversity because of added effects due to increased resource efficiency and greater plant species richness.

Unfortunately, the authors do not go into this in the introduction or otherwise and simply state that they consider strip cropping a form of intercropping.

I also do not like the exclusive focus on percentages, as these are dimensionless. I think more could have been done to show underlying structure in the data, even after rarefaction.

A further weakness is a limited embedding into the larger scientific discourses other than providing references. But this may be a matter of style and/or taste

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field, elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a prioritization for generating behavior that supports hawkmoth safety rather than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Reviewer #2 (Public review):

Summary

Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight. The authors linked their behavioral results to visual scene statistics in the hawkmoths' natural environment. The partition of ventral and dorsal visuomotor pathways is well in line with differences in visual cue frequencies. The response hierarchy, however, seems to be dominated by dorsal features, that are less frequent, but presumably highly relevant for the animals' flight safety.

Strengths

The data are very interesting and unique. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

Weaknesses

While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

I find the majority of the data, which are also the data supporting the main claims of the paper, compelling. However, the measurements of flight height are less solid than the rest and I think these data should be interpreted more carefully.

Reviewer #3 (Public review):

The authors have significantly improved the paper in revising to make its contributions distinct from their prior paper. They have also responded to my concerns about quantification and parameter dependency of the integration conclusion. While I think there is still more that could be done in this capacity, especially in terms of the temporal statistics and quantification of the conflict responses, they have a made a case for the conclusions as stated. The paper still stands as an important paper with solid evidence a bit limited by these concerns.

[Editors' note: Due to very minor revisions, the paper was not sent to reviewers for an additional round of review.]

Reviewer #1 (Public review):

Summary:

The aim of the experiment reported in this paper is to examine the nature of the representation of a template of an upcoming target. To this end, participants were presented with compound gratings (consisting of tilted to the right and tilted to the left lines) and were cued to a particular orientation - red left tilt or blue right tilt (counterbalanced across participants). There are two directly compared conditions: (i) no ping: where there was a cue, that was followed by a 5.5-7.5s delay, then followed by a target grating in which the cued orientation deviated from the standard 45 degrees; and (ii) ping condition in which all aspects were the same with the only difference that a ping (visual impulse presented for 100ms) was presented after the 2.5 seconds following the cue. There was also a perception task in which only the 45 degrees to the right or to the left lines were presented. It was observed that during the delay, only in the ping condition, were the authors able to decode the orientation of the to-be-reported target using the cross-task generalization. Attention decoding, on the other hand, was decoded in both ping and non-ping conditions. It is concluded that the visual system has two different functional states associated with a template during preparation: a predominantly non-sensory representation for guidance and a latent sensory-like for prospective stimulus processing.

Strengths:

There is so much to be impressed with in this report. The writing of the manuscript is incredibly clear. The experimental design is clever and innovative. The analysis is sophisticated and also innovative - the cross-task decoding, the use of Mahalanobis distance as a function of representational similarity, the fact that the question is theoretically interesting, and the excellent figures.

Weaknesses:

While I think that this is an interesting study that addresses an important theoretical question, I have several concerns about the experimental paradigm and the subsequent conclusions that can be drawn.

(1) Why was V1 separated from the rest of the visual cortex, and why the rest of the areas were simply lumped into an EVC ROI? It would be helpful to understand the separation into ROIs.

(2) It would have been helpful to have a behavioral measure of the "attended" orientation to show that participants in fact attended to a particular orientation and were faster in the cued condition. The cue here was 100% valid, so no such behavioral measure of attention is available here.

(3) As I was reading the manuscript I kept thinking that the word attention in this manuscript can be easily replaced with visual working memory. Have the authors considered what it is about their task or cognitive demand that makes this investigation about attention or working memory?

(4) If I understand correctly, the only ROI that showed a significant difference for the cross-task generalization is V1. Was it predicted that only V1 would have two functional states? It should also be made clear that the only difference where the two states differ is V1.

(5) My primary concern about the interpretation of the finding is that the result, differences in cross-task decoding within V1 between the ping and no-ping condition might simply be explained by the fact that the ping condition refocuses attention during the long delay thus "resharpening" the template. In the no-ping condition during the 5.5 to 7.5 seconds long delay, attention for orientation might start getting less "crisp." In the ping condition, however, the ping itself might simply serve to refocus attention. So, the result is not showing the difference between the latent and non-latent stages, rather it is the difference between a decaying template representation and a representation during the refocused attentional state. It is important to address this point. Would a simple tone during the delay do the same? If so, the interpretation of the results will be different.

(6) The neural pattern distances measured using Mahalanobis values are really great! Have the authors tried to use all of the data, rather than the high AMI and low AMI to possibly show a linear relationship between response times and AMI?

(7) After reading the whole manuscript I still don't understand what the authors think the ping is actually doing, mechanistically. I would have liked a more thorough discussion, rather than referencing previous papers (all by the co-author).

Comments on revisions:

I am impressed with the thoroughness with which the authors addressed my concerns. I don't have any further concerns and think that this paper makes an interesting and significant contribution to our understanding of VWM. I would only suggest adding citations to the newly added paragraph where the authors state "It could be argued that preparatory attention relies on the same mechanisms as working memory maintenance." They could cite work by Bettencourt and Xu, 2016; and Sheremata, Somers, and Shomstein (2018).

Reviewer #2 (Public review):

Summary:

In the present study, the authors investigated the nature of attentional templates during preparatory period of goal-directed attention. By combing the use of 'pinging' the neural activity with a visual impulse and fMRI-based multivariate decoding, the authors found that the nature of the neural representations of the prospective feature target during preparatory period was contingent on the presence of the 'pinging' impulse. While the preparatory representations contained highly similar information content as the perceptual representations when the pinging impulse was introduced, they fundamentally differed from perceptual representations in the absence of the pinging impulse. Based on these findings, the authors proposed a dual-format mechanism in which both a "non-sensory" template and a latent "sensory" template coexisted during attentional preparation. The former actively guides activity in the preparatory state, and the latter is utilized for future stimulus processing.

Strengths:

Overall, I think that the authors' revision has addressed most, if not all, of my major concerns noted in my previous comments.

Weaknesses:

The results appear convincing and I do not have additional comments.

Reviewer #3 (Public review):

This paper discusses how non-sensory and latent, sensory-like attentional templates are represented during attentional preparation. Using multivariate pattern analysis, they found that visual impulses can enhance the decoding generalization from perception to attention tasks in the preparatory stage in the visual cortex. Furthermore, the emergence of the sensory-like template coincided with enhanced information connectivity between V1 and frontoparietal areas and was associated with improved behavioral performance. It is an interesting paper with supporting evidence for the latent, sensory-like attentional template.

(1) The authors addressed most of my previous concerns and provided additional data analysis. They conducted further analyses to demonstrate that the observed changes in network communication are associated with behavioral RTs, supporting the idea that the impulse-driven sensory-like template enhances informational connectivity between sensory and frontoparietal areas, and relates to behavior.

(2) I would like to further clarify my previous points regarding the definition of the two types of templates and the evidence for their coexistence. The authors stated that the sensory-like template likely existed in a latent state and was reactivated by visual pings, proposing that sensory and non-sensory templates coexist. However, it remains unclear whether this reflects a dynamic switch between formats or true coexistence. If the templates are non-sensory in nature, what exactly do they represent? Are they meant to be abstract or conceptual representations, or, put simply, just "top-down attentional information"? If so, why did the generalization analyses-training classifiers on activity during the stimulus selection period and testing on preparatory activity-fail to yield significant results? While the stimulus selection period necessarily encodes both target and distractor information, it should still contain attentional information. I would appreciate more discussion from this perspective.

Reviewer #1 (Public review):

This study is part of an ongoing effort to clarify the effects of cochlear neural degeneration (CND) on auditory processing in listeners with normal audiograms. This effort is important because ~10% of people who seek help for hearing difficulties have normal audiograms and current hearing healthcare has nothing to offer them.

The authors identify two shortcomings in previous work that they intend to fix. The first is a lack of cross-species studies that make direct comparisons between animal models in which CND can be confirmed and humans for which CND must be inferred indirectly. The second is the low sensitivity of purely perceptual measures to subtle changes in auditory processing. To fix these shortcomings, the authors measure envelope following responses (EFRs) in gerbils and humans using the same sounds, while also performing histological analysis of the gerbil cochleae, and testing speech perception while measuring pupil size in the humans.

The study begins with a comprehensive assessment of the hearing status of the human listeners. The only differences found between the young adult (YA) and middle aged (MA) groups are in thresholds at frequencies > 10 kHz and DPOAE amplitudes at frequencies > 5 kHz. The authors then present the EFR results, first for the humans and then for the gerbils, showing that amplitudes decrease more rapidly with increasing envelope frequency for MA than for YA in both species. The histological analysis of the gerbil cochleae shows that there were, on average, 20% fewer IHC-AN synapses at the 3 kHz place in MA relative to YA, and the number of synapses per IHC was correlated with the EFR amplitude at 1024 Hz.

The study then returns to the humans to report the results of the speech perception tests and pupillometry. The correct understanding of keywords decreased more rapidly with decreasing SNR in MA than in YA, with a noticeable difference at 0 dB, while pupillary slope (a proxy for listening effort) increased more rapidly with decreasing SNR for MA than for YA, with the largest differences at SNRs between 5 and 15 dB. Finally, the authors report that a linear combination of audiometric threshold, EFR amplitude at 1024 Hz, and a few measures of pupillary slope is predictive of speech perception at 0 dB SNR.

I only have two questions/concerns about the specific methodologies used:

(1) Synapse counts were made only at the 3 kHz place on the cochlea. But the EFR sounds were presented at 85 dB SPL, which means that a rather large section of the cochlea will actually be excited. Do we know how much of the EFR actually reflects AN fibers coming from the 3 kHz place? And are we sure that this is the same for gerbils and humans given the differences in cochlear geometry, head size, etc.?

[Note added after revision: the authors have added new data, references, and discussion that have answered my initial questions].

(2) Unless I misunderstood, the predictive power of the final model was not tested on held out data. The standard way to fit and test such model would be to split the data into two segments, one for training and hyperparameter optimization, and one for testing. But it seems that the only spilt was for training and hyperparameter optimization.

[Note added after revision: the authors now make it clear in their response that the modeling tells us how much of the current data can be explained but not necessary about generalization to other datasets.]

While I find the study to be generally well executed, I am left wondering what to make of it all. The purpose of the study with respect to fixing previous methodological shortcomings was clear, but exactly how fixings these shortcomings has allowed us to advance is not. I think we can be more confident than before that EFR amplitude is sensitive to CND, and we now know that measures of listening effort may also be sensitive to CND. But where is this leading us?

I think what this line of work is eventually aiming for is to develop a clinical tool that can be used to infer someone's CND profile. That seems like a worthwhile goal but getting there will require going beyond exploratory association studies. I think we're ready to start being explicit about what properties a CND inference tool would need to be practically useful. I have no idea whether the associations reported in this study are encouraging or not because I have no idea what level of inferential power is ultimately required.

[Note added after revision: the authors have added to the Discussion to put their work into a broader perspective.]

That brings me to my final comment: there is an inappropriate emphasis on statistical significance. The sample size was chosen arbitrarily. What if the sample had been half the size? Then few, if any, of the observed effects would have been significant. What if the sample had been twice the size? Then many more of the observed effects would have been significant (particularly for the pupillometry). I hope that future studies will follow a more principled approach in which relevant effect sizes are pre-specified (ideally as the strength of association that would be practically useful) and sample sizes are determined accordingly.

[Note added after revision: my intention with this comment was not to make a philosophical or nitty-gritty point about statistics. It was more of a follow on to the previous point. Because I don't know what sort of effect size is big enough to matter (for whatever purpose), I don't find the statistical significance (or lack thereof) of the effect size observed to be informative. But I don't think there is anything more that the authors can or should do in this regard.]

So, in summary, I think this study is a valuable but limited advance. The results increase my confidence that non-invasive measures can be used to infer underlying CND, but I am unsure how much closer we are to anything that is practically useful.

Reviewer #2 (Public review):

Summary:

This paper addresses the bottom-up and top-down causes of hearing difficulties in middle-aged adults with clinically-normal audiograms using a cross-species approach (humans vs. gerbils, each with two age groups) mixing behavioral tests and electrophysiology.. The study is not only a follow-up of Parthasarathy et al (eLife 2020), since there are several important differences. Parthasarathy et al. (2020) only considered a group of young normal-hearing individuals with normal audiograms yet with high complaints for hearing in noisy situations. Here, this issue is considered specifically regarding aging, using a between-subject design comparing young NH and older NH individuals recruited from the general population, without additional criterion (i.e. no specifically high problems of hearing in noise). In addition, this is a cross-species approach, with the same physiological EFR measurements with the same stimuli deployed on gerbils.

This article is of very high quality. It is extremely clear, and the results show clearly a decrease of neural phase-locking to high modulation frequencies in both middle-aged humans and gerbils, compared to younger groups/cohorts. In addition, pupillometry measurements conducted during the QuickSIN task suggest increased listening efforts in middle-aged participants, and a statistical model including both EFRs and pupillometry features suggest that both factors contribute to reduced speech-in-noise intelligibility evidenced in middle-aged individuals, beyond their slight differences in audiometric thresholds (although they were clinically normal in both groups).

These provide strong support to the view that normal aging in humans leads to auditory nerve synaptic loss (cochlear neural degeneration - CND- or, put differently, cochlear synaptopathy) as well as increased listening effort, before any clearly visible audiometric deficits as defined in current clinical standards. This result is very important for the community, since we are still missing direct evidence that cochlear synaptopathy might likely underly a significant part of hearing difficulties in complex environments for listeners with normal thresholds, such as middle-aged and senior listeners. This paper shows that these difficulties can be reasonably well accounted for by this sensory disorder (CND), but also that listening effort, i.e. a top-down factor, further contributes to this problem. The methods are sound, well described and I would like to emphasize that they are presented concisely yet in a very precise manner, so that they can be understood very easily - even for a reader that is not familiar with the employed techniques. I believe this study will be of interest to a broad readership. I have some comments and questions which I think would make the paper even stronger once addressed.

Main comments:

(1) Presentation of EFR analyses / Interpretation of EFR differences found in both gerbils and humans

a) Could you comment further on why you think you found a significant difference only at the highest mod. frequency of 1024 Hz in your study? Indeed, previous studies employing SAM or RAM tones very similar to the ones employed here were able to show age effects already at lower modulation freqs. of ~100H; e.g. there are clear age effects reported in human studies of Vasilikov et al. (2021) or Mepani et al. (2021), and also in animals ( see Garrett et al. bioRxiv : https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf)

Furthermore, some previous EEG experiments in humans that SAM tones with modulation freqs. of ~100Hz showed that EFRs do not exhibit a single peak, i.e. there are peaks not only at fm but also for the first harmonics (e.g. 2fm or 3fm) see e.g. Garrett et al. bioXiv https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf

Did you try to extract EFR strength by looking at the summed amplitude of multiple peaks (Vasilikov Hear Res. 2021), in particular for the lower modulation frequencies? (Indeed, there will be no harmonics for the higher mod. freqs).

b) How the present EFR results relate to FFR results, where effects of age are already at low carrier freqs? (e.g. Märcher-Rørsted et al., Hear. Res., 2022 for pure tones with freq < 500 Hz) Do you think it could be explained by the fact that this is not the same cochlear region, and that synapses die earlier in higher compared to lower CFs. This should be discussed. Beyond the main group effect of age, there were no negative correlations of EFRs with age in your data?

(2) Size of the effects / comparing age effects between two species: Although the size of the age effect on EFRs cannot be directly compared between humans and gerbils - the comparison remains qualitative - could you a least provide references regarding the rate of synaptic loss with aging in both humans and gerbils, so that we understand that the yNH/MA difference can be compared between the two age groups used for gerbils; it would have been critical in case of a non-significant age effect in one species.

Equalization / control of stimuli differences across the two species: For measuring EFRs, SAM stimuli were presented at 85 dB SPL for humans vs. 30 dB above detection threshold (inferred from ABRs) for gerbils - I do not think the results strongly depend on this choice, but it would be good to comment on why you did not choose also to present stimuli 30 dB above thresholds in humans.

Simulations of EFRs using functional models could have been used to understand (at least in humans) how the differences in EFRs obtained between the two groups are quantitatively compatible with the differences in % of remaining synaptic connections known from histopathological studies for their age range (see the approach in Märcher-Rørsted et al., Hear. Res., 2022)

(3) Synergetic effects of CND and listening effort Could you test whether there is an interaction between CNR and listening effort? (e.g. one could hypothesize that MA subjects with largest CND have also the higher listening effort)

Comments on revised version:

The authors did well to address all the points raised in my review. This paper will make an important contribution to our assessment of the sources of age-related auditory processing deficits beyond the cochlea that impair speech intelligibility.

Reviewer #1 (Public review):

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

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

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. report that DopR1 and Dop2R, but not DopEcR, are involved in the mating failure-induced suppression of sugar sensitivity in these neurons. Further investigation is needed to clarify how dopamine selectively engages different receptor types depending on internal state.

The data in this revised version are largely convincing and support the authors' conclusions. However, I remain concerned about the results shown in Figure 6E. The authors show that knocking down DopR1 or Dop2R in Gr5a+ neurons restores sucrose-evoked activity in Failed flies to levels seen in Naive and Satisfied animals. This appears to contradict the proposed model, in which these receptors positively modulate Gr5a+ activity through dopaminergic input. If dopamine signaling is reduced in Failed flies, further receptor knockdown should have no effect or further reduce activity-not restore it. I encourage the authors to clarify this apparent inconsistency and, if possible, provide a mechanistic explanation.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

The authors updated missing literature in the manuscript and performed additional experiments regarding behavior, but also to further prove the functional connectivity between TH neurons and GR5a neurons.

I have no further recommendations.

Reviewer #3 (Public review):

Summary

This study by Wang et al. explores a compelling link between two fundamental innate behaviors in Drosophila melanogaster, mating and feeding, demonstrating that repeated sexual failure in male flies leads to a transient yet reversible decrease in sweet taste perception. The authors show that this modulation is mediated by dopamine signaling from a specific subset of dopaminergic neurons in the subesophageal zone (SEZ) that directly influence Gr5a⁺ sweet-sensing neurons.

Aims of the Study

The authors aimed to understand whether unsuccessful mating attempts could affect sensory processing of sweet stimuli and thus feeding behavior in male fruit flies. They further sought to dissect the neural circuitry and molecular pathways underlying this behavioral plasticity, with a particular focus on dopaminergic modulation.

Major Strengths and Weaknesses

Strengths:

• Novelty: The idea that reproductive experience modulates gustatory perception adds a new dimension to our understanding of cross-modal behavioral integration.

• Experimental approach: The study uses a broad array of genetic, pharmacological, imaging, and behavioral assays to demonstrate a causal relationship between sexual failure and reduced sweet perception, mediated by specific dopaminergic pathways.

• Methodological design: The authors link behavioral outcomes (reduced proboscis extension reflex) with neural activity (calcium imaging of Gr5a⁺ neurons) and molecular specificity (dopamine receptor subtype roles), providing a robust multi-level framework.

Weaknesses:

• Ecological relevance: While the laboratory conditions are well controlled, the adaptive value or natural context of this taste modulation following mating failure remains speculative.

Achievement of Aims and Support for Conclusions

The authors have convincingly achieved their central aim. The results support the conclusion that sexual failure reduces sweet taste sensitivity through dopamine signaling. The reduced activity in Gr5a⁺ neuron after courtship rejection, its rescue by dopamine or successful copulation, and the requirement of specific dopamine receptors support the proposed model.

Impact and Utility

This work advances the field's understanding of how motivational states shaped by social experiences can directly influence sensory perception and behavior. It underscores the role of the dopaminergic system not only in reward but in integrating internal states across distinct behavioral responses. The experimental approach, including courtship conditioning paradigms and in vivo imaging methods, provides a valuable foundation for related studies in sensory modulation and behavioral plasticity.

Additional Context

This study supports a growing body of literature suggesting that insects possess emotion-like internal states that influence their behavior across contexts. The findings resonate with prior work on how stressors like social isolation or courtship failure lead to compensatory changes in other reward-seeking behaviors (e.g., ethanol consumption). Moreover, the concept that neural systems underlying basic drives like hunger and mating are dynamically interconnected may be conserved across phyla, suggesting broader relevance to understanding internal state-dependent modulation of behavior.

The authors addressed all the comments of previous reviews. The changes increased the clarity of the manuscript, the interpretation of the results and reinforce the conclusion.

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed on Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

Reviewer #1 (Public review):

Summary:

Fallah and colleagues characterize the connectivity between two basal ganglia output nuclei, the SNr and GPe, and a the pedunculopontine nucleus, a brainstem nucleus that is part of the mesencephalic locomotor region. Through a series of systematic electrophysiological studies, they find that these regions target and inhibit different populations of neurons, with anatomical organization. Overall, SNr projects to PPN and inhibits all major cell types, while the GPe inhibits glutamatergic and GABAergic PPN neurons, and preferentially in the caudal part of the nucleus. Optogenetic manipulation of these inputs in the had opposing effects on behavior - SNr terminals in the PPN drove place aversion, while GPe terminals drove place preference.

Strengths:

This work is thorough and systematic characterization of a set of relatively understudied circuits. They build on the classic notions of basal ganglia connectivity and suggest a number of interesting future directions to dissect motor control and valence processing in brainstem systems.

Limitations:

All the cell type recording studies showing subtle differences in the degree of inhibition and anatomical organization of that inhibition suggest a complex effect of general optogenetic manipulation of SNr or GPe terminals in the PPN. It will be important to determine if SNr or GPe inputs onto a particular cell type in PPN are more or less critical for the how the locomotion and valence effects demonstrated here.

Reviewer #2 (Public review):

Strengths:

Fallah et al carefully dissect projections from SNr and GPe - two key basal ganglia nuclei - to the PPN, an important brainstem nucleus for motor control. They consider inputs from these two areas onto 3 types of downstream PPN neurons: GABAergic, glutamatergic, and cholinergic neurons. They also carefully map connectivity along the rostrocaudal axis of the PPN. They provide important and convincing data on PPN connectivity with two important input structures, which will provide a foundation for many future studies. They also consider the behavioral relevance of these different PPN inputs for controlling movement and reinforcement, showing convincing evidence that SNr and GPe inputs have opposing effects on behavior.

Weaknesses:

The optogenetics and behavioral studies are intriguing, although more work will be required to fit these data together into a specific model of circuit function and to distinguish the locomotor and reinforcement effects. Interestingly, stimulation of SNr axons in the rostral vs caudal PPN likely differs (as predicted by slice experiments), indicating an area for future investigation and dissection of pathways.

Reviewer #3 (Public review):

The study by Fallah et al. provides a thorough characterization of the effects of two basal ganglia output pathways, the SNr and the GPe, on cholinergic, glutamatergic, and GABAergic neurons of the PPN. Using a combination of optogenetics-assisted electrophysiology and behavioral assays in genetically defined mouse lines, the authors show that SNr projections broadly inhibit all PPN subtypes along the rostrocaudal axis, whereas GPe projections are mostly restricted to the caudal PPN and predominantly target glutamatergic neurons, with a lesser effect on GABAergic neurons. Activation of these inputs in vivo revealed opposing behavioral effects: SNr stimulation increased locomotion and caused avoidance in the real-time place preference (RTPP) task, while GPe stimulation reduced locomotion and increased time spent in the stimulation zone.

Strengths:

The evidence for functional connectivity between SNr and GPe inputs and specific PPN cell types is solid and highlights a prominent influence of SNr across the PPN. The identification of a GPe projection that selectively targets caudal glutamatergic PPN neurons is unexpected and highly relevant to understanding basal ganglia-brainstem interactions. The study stands out for its systematic cell-type-specific approach and the combination of electrophysiological and behavioral data. Importantly, the authors addressed key concerns from the initial review by performing new analyses and adding important controls:

Motor activity was re-analyzed at higher temporal resolution, revealing more nuanced effects of stimulation (Fig. S2).

The concern that motor effects might confound RTPP performance was mitigated by analyzing unstimulated test sessions, which showed that place preference or aversion persisted in the absence of stimulation (Fig. 7G).

The potential recruitment of SNc dopaminergic projections was directly tested using DAT-Cre mice, confirming that dopaminergic axon stimulation drives locomotion and reward but does not explain the aversive effect seen with broader SNr activation (Fig. S3).

Weaknesses:

While the revised analyses and added data strengthen the conclusions, the interpretation of the behavioral effects remains somewhat limited by the use of RTPP, which can be influenced by motor changes, even with unilateral stimulation. Nonetheless, the additional controls and thorough discussion now acknowledge and address these caveats appropriately.

Some minor clarifying edits would enhance the manuscript's precision and readability, including improvements to terminology, data presentation, figure referencing, and the organization of behavioral and statistical reporting.

Conclusion:

This is a strong and compelling study that provides a detailed and novel characterization of basal ganglia inputs to the PPN and their behavioral relevance. The authors were responsive to reviewer feedback, and the revised manuscript is significantly improved. The findings advance our understanding of how basal ganglia output pathways engage brainstem circuits to modulate locomotion and valence.

Reviewer #1 (Public review):

Summary:

This work studies representations in a network with one recurrent layer and one output layer that needs to path-integrate so that its position can be accurately decoded from its output. To formalise this problem, the authors define a cost function consisting of the decoding error and a regularisation term. They specify a decoding procedure that, at a given time, averages the output unit center locations, weighted by the activity of the unit at that time. The network is initialised without position information, and only receives a velocity signal (and a context signal to index the environment) at each timestep, so to achieve low decoding error it needs to infer its position and keep it updated with respect to its velocity by path integration.

The authors take the trained network and let it explore a series of environments with different geometries while collecting unit activities to probe learned representations. They find localised responses in the output units (resembling place fields) and border responses in the recurrent units. Across environments, the output units show global remapping and the recurrent units show rate remapping. Stretching the environment generally produces stretched responses in output and recurrent units. Ratemaps remain stable within environments and stabilise after noise injection. Low-dimensional projections of the recurrent population activity forms environment-specific clusters that reflect the environment's geometry, which suggests independent rather than generalised representations. Finally, the authors discover that the centers of the output unit ratemaps cluster together on a triangular lattice (like the receptive fields of a single grid cell), and find significant clustering of place cell centers in empirical data as well.

The model setup and simulations are clearly described, and are an interesting exploration of the consequences of a particular set of training requirements - here: path integration and decodability. But it is not obvious to what extent the modelling choices are a realistic reflection of how the brain solves navigation. Therefore, it is not clear whether the results generalize beyond the specifics of the setup here.

Strengths:

The authors introduce a very minimal set of model requirements, assumptions, and constraints. In that sense, the model can function as a useful 'baseline', that shows how spatial representations and remapping properties can emerge from the requirement of path integration and decodability alone. Moreover, the authors use the same formalism to relate their setup to existing spatial navigation models, which is informative.

The global remapping that the authors show is convincing and well-supported by their analyses. The geometric manipulations and the resulting stretching of place responses, without additional training, are interesting. They seem to suggest that the recurrent network may scale the velocity input by the environment dimensions so that the exact same path integrator-output mappings remain valid (but maybe there are other mechanisms too that achieve the same).

The simulations and analyses in the appendices serve as insightful controls for the main results.

The clustering of place cell peaks on a triangular lattice is intriguing, given there is no grid cell input. It could have something to do with the fact that a triangular lattice provides optimal coverage of 2d space? The included comparison with empirical data is valuable as a first exploration, showing a promising example, but doesn't robustly support the modelling results.

Reviewer #2 (Public review):

Summary:

The authors proposed a neural network model to explore the spatial representations of the hippocampal CA1 and entorhinal cortex (EC) and the remapping of these representations when multiple environments are learned. The model consists of a recurrent network and output units (a decoder) mimicking the EC and CA1, respectively. The major results of this study are: the EC network generates cells with their receptive fields tuned to a border of the arena; the decoder develops neuron clusters arranged in a hexagonal lattice. Thus, the model accounts for entrohinal border cells and CA1 place cells. It suggests that the remapping of place cells occurs between different environments through state transitions corresponding to unstable dynamical modes in the recurrent network.

Strengths:

The authors found a spatial arrangement of receptive fields similar to their model's prediction in experimental data recorded from CA1. Thus, the model proposes plausible mechanisms to generate hippocampal spatial representations without relying on grid cells. The model also suggests an interesting possibility that path integration is not the speciality of grid cells.

Weaknesses:

The role of grid cells in the proposed view, i.e., the boundary-to-place-to-grid model, remains elusive. The model can generate place cells without generating entorhinal grid cells. Moreover, the model can generate hexagonal grid patterns of place cells in a large arena. Whether and how the proposed model is integrated into the entire picture of the hippocampal-entorhinal meｍory processing remains elusive.

Reviewer #3 (Public review):

Summary:

The authors used recurrent neural network modelling of spatial navigation tasks to investigate border and place cell behaviour during remapping phenomena.

Strengths:

The neural network training seemed for the most part (see comments later) well-performed, and the analyses used to make the points were thorough.

The paper and ideas were well-explained.

Figure 4 contained some interesting and strong evidence for map-like generalisation as environmental geometry was warped.

Figure 7 was striking and potentially very interesting.

It was impressive that the RNN path-integration error stayed low for so long (Fig A1), given that normally networks that only work with dead-reckoning have errors that compound. I would have loved to know how the network was doing this, given that borders did not provide sensory input to the network. I could not think of many other plausible explanations... It would be even more impressive if it was preserved when the network was slightly noisy.

Update:

The analysis of how the RNN remapped, using a context signal to switch between largely independent maps, and the examination of the border like tuning in the recurrent units of the RNN, were both thorough and interesting. Further, in the updated response I appreciated the additional appendix E which helped substantiate the claim that the RNN neurons were border cells.

Reviewer #1 (Public review):

Summary:

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

Strengths:

The manuscript is well written and a pleasure to read.

Weaknesses:

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

Comments on revisions:

I appreciate how the authors have replied to my comments and I have the feeling that also the other reviewers' comments have been dealt with carefully. I therefore support the acceptance of this very nice and interesting manuscript.

Reviewer #2 (Public review):

Summary:

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

Strength:

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

Weakness:

This revised manuscript appears to me to have no major weaknesses.

Reviewer #3 (Public review):

Summary:

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

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size. (I cannot comment on protein modeling and docking due to a lack of expertise in this area)

Weaknesses:

None.

Reviewer #1 (Public review):

The authors examine how probabilistic reversal learning is affected by dopamine by studying the effects of methamphetamine (MA) administration. Based on prior evidence that the effects of pharmacological manipulation depend on baseline neurotransmitter levels, they hypothesized that MA would improve learning in people with low baseline performance. They found this effect, and specifically found that MA administration improved learning in noisy blocks, by reducing learning from misleading performance, in participants with lower baseline performance. The authors then fit participants' behavior to a computational learning model and found that an eta parameter, responsible for scaling learning rate based on previously surprising outcomes, differed in participants with low baseline performance on and off MA.

Questions:

(1) It would be helpful to confirm that the observed effect of MA on the eta parameter is responsible for better performance in low baseline performers. If performance on the task is simulated for parameters estimated for high and low baseline performers on and off MA, does the simulated behavior capture the main behavioral differences shown in Figure 3?

(2) In Figure 4C, it appears that the main parameter difference between low and high baseline performance is inverse temperature, not eta. If MA is effective in people with lower baseline DA, why is the effect of MA on eta and not IT?

Also, this parameter is noted as temperature but appears to be inverse temperature as higher values are related to better performance. The exact model for the choice function is not described in the methods.

Comments on revisions:

Thanks to the authors for their thorough responses and revisions. One typo to note: in the Methods, the "drug effects" paragraph is repeated.

Reviewer #2 (Public review):

Summary:

Kirschner and colleagues test whether methamphetamine (MA) alters learning rate dynamics in a validated reversal learning task. They find evidence that MA can enhance performance for low-performers, and that the enhancement reflects a reduction in the degree to which these low-performers dynamically up-regulate their learning rates when they encounter unexpected outcomes. The net effect is that poor performers show more volatile learning rates (e.g. jumping up when they receive misleading feedback), when the environment is actually stable, undermining their performance over trials.

Strengths:

The study has multiple strengths, including a large sample size, placebo control, double-blind randomized design, and rigorous computational modeling of a validated task. Additionally, the analytic methods are rigorous and offer new types of analyses for people interested in exploring learning as a function of dynamically changing volatility.

Weaknesses:

The limitations, which are acknowledged, include that the drug they use, methamphetamine, can influence multiple neuromodulatory systems including catecholamines and acetylcholine, all of which have been implicated in learning rate dynamics. They also do not have any independent measures of any of these systems, so it is impossible to know which is having an effect.

Another limitation which they should acknowledge is that the fact that participants were aware of having different experiences in the drug sessions means that their blinding was effectively single-blind (to the experimenters) and not double-blind. That said, the authors do provide some evidence that subjective effects of drugs (e.g. arousal, mood, etc.) did not drive differences in performance.

Comments on revisions:

The authors have done an outstanding job responding to, and allaying my prior concerns about their analyses.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

Bonnifet et al. sought to characterize the expression pattern of L1 ORF1p expression across the entire mouse brain, in young and aged animals and to corroborate their characterization with Western blotting for L1 ORF1p and L1 RNA expression data from human samples. They also queried L1 ORF1p interacting partners in the mouse brain by IP-MS.

Strengths:

A major strength of the study is the use of two approaches: a deep-learning detection method to distinguish neuronal vs. non-neuronal cells and ORF1p+ cells vs. ORF1p- cells across large-scale images encompassing multiple brain regions mapped by comparison to the Allen Brain Atlas, and confocal imaging to give higher resolution on specific brain regions. These results are also corroborated by Western blotting on six mouse brain regions. Extension of their analysis to post-mortem human samples, to the extent possible, is another strength of the paper. The identification of novel ORF1p interactors in brain is also a strength in that it provides a novel dataset for future studies.

Weaknesses:

The main weakness of the study is that cell type specificity of ORF1p expression was not examined beyond neuron (NeuN+) vs non-neuron (NeuN-). Indeed, a recent study (Bodea et al. 2024, Nature Neuroscience) found that ORF1p expression is characteristic of parvalbumin-positive interneurons, and it would be very interesting to query whether other neuronal subtypes in different brain regions are distinguished by ORF1p expression. The data suggesting that ORF1p expression is increased in aged mouse brains is intriguing, although it seems to be based upon modestly (up to 27%, dependent on brain region) higher intensity of ORF1p staining rather than a higher proportion of ORF1+ neurons. Indeed, the proportion of NeuN+/Orf1p+ cells actually decreased in aged animals. It is difficult to interpret the significance and validity of the increase in intensity, as Hoechst staining of DNA, rather than immunostaining for a protein known to be stably expressed in young and aged neurons, was used as a control for staining intensity. The main weakness of the IP-MS portion of the study is that none of the interactors were individually validated or subjected to follow-up analyses. The list of interactors was compared to previously published datasets, but not to ORF1p interactors in any other mouse tissue.

The authors achieved the goals of broadly characterizing ORF1p expression across different regions of the mouse brain, and identifying putative ORF1p interactors in the mouse brain. However, findings from both parts of the study are somewhat superficial in depth.

This provides a useful dataset to the field, which likely will be used to justify and support numerous future studies into L1 activity in the aging mammalian brain and in neurodegenerative disease. Similarly, the list of ORF1p interacting proteins in the brain will likely be taken up and studied in greater depth.

Comments on revisions:

The co-staining of Orf1p with Parvalbumin (PV) presented in Supplemental Figure S5 is a welcome addition exploring the cell type-specificity of Orf1p staining, and broadly corroborates the work of Bodea et al. while revealing that Orf1p also is expressed in non-PV+ cells, consistent with L1 activity across a range of neuronal subtypes. The authors also have strengthened their findings regarding the increased intensity of ORF1p staining in aged compared to young animals, and the newly presented results are indeed more convincing. The prospect of increased neuronal L1 activity with age is exciting, and the results in this paper have provided the groundwork for ongoing discoveries in this area. While it is disappointing that no Orf1p interactors were followed up, this is understandable and the data are nonetheless valuable and will likely prove useful to future studies.

Reviewer #1 (Public review):

Summary:

The thalamus is a central subcortical structure consisting of that receives anatomical connections from various cortical areas, each displaying a unique pattern. Previous studies have suggested that certain cortical areas may establish more extensive connections within the thalamus, influencing distributed information flow. Despite these suggestions, a quantitative understanding of cortical areas' anatomical connectivity patterns within the thalamus is lacking. In this study, the researchers addressed this gap by employing diffusion magnetic resonance imaging (dMRI) on a large cohort of healthy adults from the Human Connectome Project. Using brain-wide probabilistic tractography, a framework was developed to measure the spatial extent of anatomical connections within the thalamus for each cortical area. Additionally, the researchers integrated resting-state functional MRI, cortical myelin, and human neural gene expression data to investigate potential variations in anatomical connections along the cortical hierarchy. The results unveiled two distinct cortico-thalamic tractography motifs: 1) a sensorimotor cortical motif featuring focused thalamic connections to the posterolateral thalamus, facilitating fast, feed-forward information flow; and 2) an associative cortical motif characterized by diffuse thalamic connections targeting the anteromedial thalamus, associated with slower, feed-back information flow. These motifs exhibited consistency across human subjects and were corroborated in macaques, underscoring cross-species generalizability. In summary, the study illuminates differences in the spatial extent of anatomical connections within the thalamus for sensorimotor and association cortical areas, potentially contributing to functionally distinct cortico-thalamic information flow.

Strengths:

Quantitative Approach: The study employs diffusion magnetic resonance imaging (dMRI) and probabilistic tractography on a substantial sample size of 828 healthy adults, providing a robust quantitative analysis of anatomical connectivity patterns within the thalamus.

Multi-Modal Integration: By incorporating resting-state functional MRI, cortical myelin, and human neural gene expression data, the study offers a comprehensive approach to understanding anatomical connections, enriching the interpretation of findings and enhancing the study's overall validity.

Cross-Species Generalizability: The identification of consistent cortico-thalamic tractography motifs in both human subjects and macaques demonstrates the robustness and cross-species generalizability of the findings, strengthening the significance and broader applicability of the study.

Supplementary Analyses: There are numerous, excellent examples of clear surrogates used to test the major claims of the paper. This is exemplary work.

Weaknesses:

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

Comments on revised version:

The authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

This paper by Howell and colleagues focuses on describing macro patterns of anatomical connections between cortical areas and the thalamus in the human brain. This research topic poses significant challenges due to the inability to apply the gold standard of mapping anatomical connections, viral tracing, to humans. Moreover, when applied to animal models, viral tracing often has limited scope and resolution. As a result, the field has thus far lacked a comprehensive and validated description of thalamocortical anatomical connectivity in humans.

The paper focuses on an intriguing question: whether anatomical connections from the cortex to the thalamus exhibit a diffuse pattern, targeting multiple thalamic sub-regions, or a more focal pattern, selectively targeting specific thalamic subregions. This novel and significant question holds substantial implications for our understanding of thalamocortical information processing. The authors have developed a sophisticated and innovative quantitative metric to address this question. The study revealed two main patterns: a focal pattern originating from sensorimotor cortical regions to the posterior thalamus and a more diffuse pattern from associative cortical regions to the anterior-medial thalamus. These findings are then framed within the context of thalamocortical motifs involved in feedforward versus feedback processing.

While this paper has several strengths, including its significance and methodological sophistication, its extension to non-human primates and other forms of data for testing hierarchy, there are important limitations. These limitations, discussed in more detail below, primarily concern tracking accuracy and the known limitations of using diffusion data to track thalamocortical connections in humans. These limitations may potentially introduce systematic biases into the results, weakening their support. Addressing these limitations through better validation is crucial, though some may remain unresolved due to the fundamental constraints of diffusion imaging.

Strengths:

This research holds significant basic, clinical, and translational importance as it contributes to our understanding of how thalamocortical anatomical connectivity is organized. Its relevance spans across cognitive, systems, and clinical neuroscientists in various subfields.

The central question addressed in this study, concerning whether cortico-thalamic projections are focal or diffuse, is both novel and previously unexplored to the best of my knowledge. It offers valuable insights into the potential capabilities of the thalamocortical system in terms of parallel or integrative processing.

The development of quantitative metrics to analyze anatomical connectivity is highly innovative and suitable for addressing the research questions at hand.

The findings are not only interesting but also robust, aligning with data from other sources that suggest a hierarchical organization in the brain.

Using PCA to integrate results across a range of thresholds is innovative.

The study's sophisticated integration of a diverse range of data and methods provides strong, converging support for its main findings, enhancing the overall credibility of the research.

Weaknesses:

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

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

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

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

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

Comments on revised version:

I appreciate the additional supplementary figures and responses from the authors. I think this is an important study, and the review I wrote should provide important context for readers to digest their responses.

Reviewer #3 (Public review):

Summary:

In the current work, Howell et al studied the connectivity between cortex and thalamus using DTI tractography per parcel to all voxels in the thalamus. Following they performed various dimensional reduction techniques to uncover how differences in connectivity to the thalamus vary across cortical parcels. Following they explore the spatial correlation of these variations with cortical myelin and functional organization, thalamic nuclei, gene expression derived core-matrix cell differentiation, and extend the model towards macaques. Overall, the authors find a differentiation between sensory and association areas in terms of the association with the thalamus, which reflects differences in cortical microstructure and function, and links to core-matrix differences and can be replicated in macaques.

Strengths:

A clear strength of the current work is the combination of different models and approaches to study the link between the cortex and the thalamus. This approach nicely bridges different approaches to describe the role of the thalamus in cortical organisation using a diffusion-based approach. Especially the extension of the model to the macaque is quite nice.

Appraisal:

The aim of the study: 'to investigate the spatial extent of anatomical connectivity patterns within the thalamus in both humans and non-human primates and determine if such patterns differ between sensorimotor and association cortical areas' has been met. Further work may continue to investigate other implications of this finding.

Discussion:

Overall, I think the study is an intriguing addition to a growing literature studying the anatomical connectivity between thalamus and cortex and its functional implications.

Comments on revised version:

Thank you for the responses.

Reviewer #1 (Public review):

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

Strengths:

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

Weaknesses:

None after thoughtful revision.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

No major weaknesses.

Reviewer #1 (Public review):

Summary

This manuscript reports preliminary evidence of successful optogenetic activation of single retinal ganglion cells (RGCs) through the eye of a living monkey using adaptive optics (AO).

Strengths

The eventual goals of this line of research have an enormous potential impact in that they will probe the perceptual impact of activating single RGCs. While I think more data should be included, the four examples shown look quite convincing.

Weaknesses

While this is undoubtedly a technical achievement and an important step along this group's stated goal to measure the perceptual consequences of single-RGC activations, the presentation lacks the rigor that I would expect from what is really a methods paper. In my view, it is perfectly reasonable to publish the details of a method before it has yielded any new biological insights, but in those publications, there is a higher burden to report the methodological details, full data sets, calibrations, and limitations of the method. There is considerable room for improvement in reporting those aspects. Specifically, more raw data should be shown for activations of neighboring RGCs to pinpoint the actual resolution of the technique, and more than two cells (one from each field of view) should be tested. Some information about the density of labeled RGCs in these animals would also be helpful to provide context for how many well-isolated target cells exist per animal.

Reviewer #2 (Public review):

Murphy et al. expressed ChrimsonR and GCaMP6s in retinal ganglion cells of a living macaque. They recorded calcium responses and stimulated individual cells, optically. Neurons targeted for stimulation were activated strongly whereas neighboring neurons were not.

The ability to record from neuronal populations while simultaneously stimulating a subset in controlled way is a high priority for systems neuroscience, and this has been particularly challenging in primates. This study marks an important milestone in the journey towards this goal.

Reviewer #3 (Public review):

This paper reports a considerable technical achievement: the optogenetic activation of single retinal ganglion cells in vivo in monkeys. As clearly specified in the paper, this is an important step towards causal tests of the role of specific ganglion cell types in visual perception. The paper is brief, and it will be important to follow this work with a more detailed methodological description to guide related work, to explore limitations, and to build confidence in the specificity of the approach.

Reviewer #1 (Public review):

The authors set out to understand the role played by a key global metabolic regulator called Crp/cAMP in the formation of persister Escherichia coli that survive antibiotic treatment without acquiring genetic mutations.

In order to achieve this aim, the authors employ an interdisciplinary approach integrating standard microbiology assays with cutting-edge genomic, metabolomic and proteomics screening.

The data presented by the authors convincingly demonstrate that the deletion of two key genes that are part of the Crp/cAMP complex (i.e. crp and cyaA) leads to a significant decrease in the number of E. coli.

The authors have carried out additional experiments to further validate this point by using the well characterised hipA7 E. coli mutant.

The data presented also demonstrate that deletion of the crp gene leads to an overall decrease in energy metabolism and an overall increase in anabolic metabolism at the population level. The deletion of cyaA has an opposite effect on cAMP concentration compared to crp deletion, the authors presented a possible hypotheses but did not test it.

The authors have now explicitly acknowledged in their discussion that the data presented in this study are obtained at the whole population level rather than at the level of the persister subpopulation and therefore should be considered with caution.

Finally, the authors convincingly show that the persisters they investigated are non-growing and have a higher redox activity and that the deletion of key genes involved in energy metabolism leads to a decrease in the number of persisters.

These data will be important for future investigations on the biochemical mechanisms that allow bacteria to adapt to stressors such as nutrient depletion or exposure to antibiotics. As such this work will likely have an impact in a variety of fields such as bacterial biochemistry, antimicrobial resistance research and environmental microbiology.

Strengths:

Interdisciplinary approach.

Excellent use of replication and ensuring reproducibility.

Excellent understanding and presentation of the biochemical mechanisms underpinning bacterial physiology via an integrated genomic, metabolomic and proteomic screening.

Weaknesses:

There is no tested mechanisms explaining why the deletion of cyaA has an opposite effect on cAMP concentration compared to crp deletion.

Metabolomics, proteomics and metabolic activity data are obtained at the whole population level rather than at the level of the persister sub-population.

Reviewer #1 (Public Review):

In this manuscript, Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

Reviewer #2 (Public Review):

Raymond Laboy et.al explored how transcriptional Mondo/Max-like complex (MML-1/MXL-2) is regulated by glucose metabolic signals using germ-line removal longevity model. They believed that MML-1/MXL-2 integrated multiple longevity pathways through nutrient sensing and therefore screened the glucose metabolic enzymes that regulated MML-1 nuclear localization. Hexokinase 1 and 2 were identified as the most vigorous regulators, which function through mitochondrial beta-oxidation and the pentose phosphate pathway (PPP), respectively. MML-1 localized to mitochondria associated with lipid droplets (LD), and MML-1 nuclear localization was correlated with LD size and metabolism. Their findings are interesting and may help us to further explore the mechanisms in multiple longevity models. The data support their proposed working model.

Comments on Revised Version (from the Reviewing Editor):

The authors have addressed the remaining concerns from both reviewers, adding textual information for reviewer 1 and testing the roles of hxk-1 and lipid oxidation in regulating lipid droplets for reviewer 2. Specifically, they find that knockdown of acs-2 and hxk-1 acs-2 double knockdown each result in a mild but significant increase in LD size. This result supports that the two hexokinases regulate MML-1 via distinct mechanisms, and is reflected in the updated model.

Reviewer #1 (Public review):

SMC5/6 is a highly conserved complex able to dynamically alter chromatin structure, playing in this way critical roles in genome stability and integrity that include homologous recombination and telomere maintenance. In the last years, a number of studies have revealed the importance of SMC5/6 in restricting viral expression, which is in part related to its ability to repress transcription from circular DNA. In this context, Oravcova and colleagues recently reported how SMC5/6 is recruited by two mutually exclusive complexes (orthologs of yeast Nse5/6) to SV40 LT-induced PML nuclear bodies (SIMC/SLF2) and DNA lesions (SLF1/2). In this current work, the authors extend this study, providing some new results. However, as a whole, the story lacks unity and does not delve into the molecular mechanisms responsible for the silencing process. One has the feeling that the story is somewhat incomplete, putting together not directly connected results.

(1) In the first part of the work, the authors confirm previous conclusions about the relevance of a conserved domain defined by the interaction of SIMC and SLF2 for their binding to SMC6, and extend the structural analysis to the modelling of the SIMC/SLF2/SMC complex by AlphaFold. Their data support a model where this conserved surface of SIMC/SLF2 interacts with SMC at the backside of SMC6's head domain, confirming the relevance of this interaction site with specific mutations. These results are interesting but confirmatory of a previous and more complete structural analysis in yeast (Li et al. NSMB 2024). In any case, they reveal the conservation of the interaction. My major concern is the lack of connection with the rest of the article. This structure does not help to understand the process of transcriptional silencing reported later beyond its relevance to recruit SMC5/6 to its targets, which was already demonstrated in the previous study.

(2) In the second part of the work, the authors focus on the functionality of the different complexes. The authors demonstrate that SMC5/6's role in transcription silencing is specific to its interaction with SIMC/SLF2, whereas SMC5/6's role in DNA repair depends on SLF1/2. These results are quite expected according to previous results. The authors already demonstrated that SLF1/2, but not SIMC/SLF2, are recruited to DNA lesions. Accordingly, they observe here that SMC5/6 recruitment to DNA lesions requires SLF1/2 but not SIMC/SLF2. Likewise, the authors already demonstrated that SIMC/SLF2, but not SLF1/2, targets SMC5/6 to PML NBs. Taking into account the evidence that connects SMC5/6's viral resistance at PML NBs with transcription repression, the observed requirement of SIMC/SLF2 but not SLF1/2 in plasmid silencing is somehow expected. This does not mean the expectation has not to be experimentally confirmed. However, the study falls short in advancing the mechanistic process, despite some interesting results as the dispensability of the PML NBs or the antagonistic role of the SV40 large T antigen. It had been interesting to explore how LT overcomes SMC5/6-mediated repression: Does LT prevent SIMC/SLF2 from interacting with SMC5/6? Or does it prevent SMC5/6 from binding the plasmid? Is the transcription-dependent plasmid topology altered in cells lacking SIMC/SLF2? And in cells expressing LT? In its current form, the study is confirmatory and preliminary. In agreement with this, the cartoons modelling results here and in the previous work look basically the same.

(3) There are some points about the presented data that need to be clarified.

Reviewer #2 (Public review):

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

Other comments

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #3 (Public review):

This paper, with a slightly modified title from the initial version, presents the cognitive implications of claims made in two accompanying papers (Berger et al. 2023, 2024) about the creation of rock engravings, the intentional disposal of the dead, and fire use by Homo naledi. The importance of the paper, therefore, still relies on the validity of the claims for the presence of socio-culturally complex and cognitively demanding behaviors that are presented in the associated papers. Given the archaeological, hominin, and taphonomic analyses in the associated papers are not adequate to enable the exceptional claims for naledi-associated complex behaviors, the inferences made in this paper are currently incomplete.

The claimed behaviors are widely recognized as complex and even quintessential to Homo sapiens. The implications of their unequivocal association with such a small-brained Middle Pleistocene hominin are thus far reaching. Accordingly, the main thrust of the paper is to highlight that greater cognition and complex socio-cultural behaviors were not necessarily associated with a positively encephalized brain. This argument begs the obvious question of whether absolute brain size and/or encephalization quotient (i.e., the actual brain volume of a given species relative the expected brain size for a species of the same average body size) can measure cognitive capacity and the complexity of socio-cultural behaviors among late Middle Pleistocene hominins.

Claims for a positive correlation between absolute and/or relative brain size and cognitive ability are not common in discussions surrounding the evolution of Middle- and Late Pleistocene hominin behavior. Currently, the bulk of the evidence for early complex technological and social behaviors derives from multiple sites across South Africa and postdates the emergence of H. sapiens by more than 100,000 years. Such lag in the expression of complex technologies and behaviors within our species renders the brain size-implies-cognitive capacity argument moot. Instead, a rich body of research over the past several decades has focused on aspects related to socio-cultural, environmental, and even the wiring of the brain in order to understand factors underlying the expression of the capacity for greater behavioral variability. In this regard, even if the claimed evidence for complex behaviors among the small-brained naledi populations proves valid, the exploration of the specific/potential socio-cultural, neuro-structural, ecological and other factors will be more informative than the emphasis on absolute/relative brain size.

The paper presents as supporting evidence previous claims for the appearance of similar complex behaviors predating the emergence of our species, H. sapiens, although it does acknowledge their controversial nature. It then uses the current claims for the association of such behaviors with H. naledi as decisive. Given the inadequate analyses in the accompanying papers, and the lack of evidence for stone tools in the naledi sites, the present claims for the expression of culturally and symbolically mediated behaviors by this small-brained hominin must be adequately established. The importance of the paper thus rests on the validity of the claimed evidence-including contextual aspects-for rock engraving, mortuary practices, and the use of fire presented in the associated two papers.

Reviewer #1 (Public review):

In this study, Setogawa et al. employ an auditory discrimination task in freely moving rats, coupled with small animal imaging, electrophysiological recordings, and pharmacological inhibition/lesioning experiments to better understand the role of two striatal subregions: the anterior Dorsal Lateral Striatum (aDLS) and the posterior Ventrolateral Striatum (pVLS), during auditory discrimination learning. Attempting to better understand the contribution of different striatal subregions to sensory discrimination learning strikes me as a highly relevant and timely question, and the data presented in this study are certainly of major interest to the field. The authors have set up a robust behavioral task, systematically tackled the question about a striatal role in learning with multiple observational and manipulative techniques. Additionally, the structured approach the authors take by using neuroimaging to inform their pharmacological manipulation experiments and electrophysiological recordings is a strength.

Reviewer #2 (Public review):

The study by Setogawa et al. aims to understand the role that different striatal subregions belonging to parallel brain circuits have in associative learning and discrimination learning (S-O-R and S-R tasks). Strengths of the study are the use of multiple methodologies to measure and manipulate brain activity in rats, from microPET imaging to excitotoxic lesions and multielectrode recordings across anterior dorsolateral (aDLS), posterior ventral lateral (pVLS)and dorsomedial (DMS) striatum.

Reviewer #1 (Public review):

Summary:

In this work, van Paassen et al. have studied how CD8 T cell functionality and levels predict HIV DNA decline. The article touches on interesting facets of HIV DNA decay, but ultimately comes across as somewhat hastily done and not convincing due to the major issues.

(1) The use of only 2 time points to make many claims about longitudinal dynamics is not convincing. For instance, the fact that raw data do not show decay in intact, but do for defective/total, suggests that the present data is underpowered. The authors speculate that rising intact levels could be due to patients who have reservoirs with many proviruses with survival advantages, but this is not the parsimonious explanation vs the data simply being noisy without sufficient longitudinal follow-up. n=12 is fine, or even reasonably good for HIV reservoir studies, but to mitigate these issues would likely require more time points measured per person.

1b) Relatedly, the timing of the first time point (6 months) could be causing a number of issues because this is in the ballpark for when the HIV DNA decay decelerates, as shown by many papers. This unfortunate study design means some of these participants may already have stabilized HIV DNA levels, so earlier measurements would help to observe early kinetics, but also later measurements would be critical to be confident about stability.

(2) Statistical analysis is frequently not sufficient for the claims being made, such that overinterpretation of the data is problematic in many places.

2a) First, though plausible that cd8s influence reservoir decay, much more rigorous statistical analysis would be needed to assert this directionality; this is an association, which could just as well be inverted (reservoir disappearance drives CD8 T cell disappearance).

2b) Words like "strong" for correlations must be justified by correlation coefficients, and these heat maps indicate many comparisons were made, such that p-values must be corrected appropriately.

(3) There is not enough introduction and references to put this work in the context of a large/mature field. The impacts of CD8s in HIV acute infection and HIV reservoirs are both deep fields with a lot of complexity.

Reviewer #2 (Public review):

Summary:

This study investigated the impact of early HIV specific CD8 T cell responses on the viral reservoir size after 24 weeks and 3 years of follow-up in individuals who started ART during acute infection. Viral reservoir quantification showed that total and defective HIV DNA, but not intact, declined significantly between 24 weeks and 3 years post-ART. The authors also showed that functional HIV-specific CD8⁺ T-cell responses persisted over three years and that early CD8⁺ T-cell proliferative capacity was linked to reservoir decline, supporting early immune intervention in the design of curative strategies.

Strengths:

The paper is well written, easy to read, and the findings are clearly presented. The study is novel as it demonstrates the effect of HIV specific CD8 T cell responses on different states of the HIV reservoir, that is HIV-DNA (intact and defective), the transcriptionally active and inducible reservoir. Although small, the study cohort was relevant and well-characterized as it included individuals who initiated ART during acute infection, 12 of whom were followed longitudinally for 3 years, providing unique insights into the beneficial effects of early treatment on both immune responses and the viral reservoir. The study uses advanced methodology. I enjoyed reading the paper.

Weaknesses:

All participants were male (acknowledged by the authors), potentially reducing the generalizability of the findings to broader populations. A control group receiving ART during chronic infection would have been an interesting comparison.

Reviewer #1 (Public review):

Summary:

The authors extended a previous study of selective response to herbivory in Arabidopsis, in order to look specifically for selection on induced epigenetic variation ("Lamarckian evolution"). They found no evidence. In addition, the re-examined result from a previously published study arguing that environmentally induced epigenetic variation was common, and found that these findings were almost certainly artifactual.

Strengths:

The paper is very clearly written, there is no hype, and the methods used are state-of-the-art.

Weaknesses:

The result is negative, so the best you can do is put an upper bound on any effects.

Significance:

Claims about epigenetic inheritance and Lamarckian evolution continue to be made based on very shaky evidence. Convincing negative results are therefore important. In addition, the study presents results that, to this reviewer, suggest that the 2024 paper by Lin et al. [26] should probably be retracted.

Reviewer #2 (Public review):

In this paper, the authors examine the extent to which epigenetic variation acquired during a selection treatment (as opposed to standing epigenetic variation) can contribute to adaptation in Arabidopsis. They find weak evidence for such adaptation and few differences in DNA methylation between experimental groups, which contrasts with another recent study (reference 26) that reported extensive heritable variation in response to the environment. The authors convincingly demonstrate that the conclusions of the previous study were caused by experimental error, so that standing genetic variation was mistaken for acquired (epigenetic) variation. Given the controversy surrounding the possible role of epigenetic variation in mediating phenotypic variation and adaptation, this is an important, clarifying contribution.

I have a few specific comments about the analysis of DNA methylation:

(1) The authors group their methylation analysis by sequence context (CG, CHG, CHH). I feel this is insufficient, because CG methylation can appear in two distinct forms: gene body methylation (gbM), which is CG-only methylation within genes, and transposable element (TE) and TE-like methylation (teM), which typically involves all sequence contexts and generally affects TEs, but can also be found within genes. GbM and teM have distinct epigenetic dynamics, and it is hard to know how methylation patterns are changing during the experiment if gbM and teM are mixed. This can also have downstream consequences (see point below).

(2) For GO analysis, the authors use all annotated genes as a control. However, most of the methylation differences they observe are likely gbM, and gbM genes are not representative of all genes. The authors' results might therefore be explained purely as a consequence of analyzing gbM genes, and not an enrichment of methylation changes in any particular GO group.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

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

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

Weaknesses:

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

Major points

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

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

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

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

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

Joint Public Review:

Summary:

The authors present a metabolic imaging study of pyruvate metabolism in a mouse model of repetitive traumatic brain injury in the chronic recovery stage. They measure pyruvate metabolism with hyperpolarised 13C magnetic resonance spectroscopic imaging. This is acquired alongside semi-quantitative MR imaging metrics, a behavioural measure, and postmortem measures of relevant enzyme activity and expression of metabolic transporter proteins. They find that the MRSI-measured cortical lactate/pyruvate ratio (and signal from pyruvate and lactate independently) can differentiate the rTBI group from the sham group. They additionally find that postmortem, cortical pyruvate dehydrogenase activity is a statistically significant discriminator. All other metrics (MRI and enzyme/transporter measures) are not significantly different between groups. Finally, using a machine learning approach, the authors investigate the predictive power of combinations of all measures.

Strengths:

The primary strength of this work is the likely robustness of the primary finding - that hyperpolarised 13C lactate/pyruvate metabolite ratios are perturbed in this chronic rTBI model compared to the sham control.

Weaknesses:

Focal alterations in blood-brain-barrier permeability may affect the primary lactate/pyruvate measures. Whilst 13C urea measures perfusion, urea remains purely extracellular; whilst in the metabolism of the healthy brain, pyruvate must be transported through two levels of monocarboxylate transporters (MCTs) - in the endothelium surrounding the capillary bed and then into the parenchyma. By mechanically disrupting the brain, tight junctions in the BBB may be disrupted, therefore increasing the flux of pyruvate across the BBB and increasing pyruvate availability. In this case, lac/pyr would be a poor measure of metabolism as "delivery" has changed. While the authors assess perfusion using HP urea, it is unclear whether or how this metric would change in the presence of BBB disruption in relatively large and well-vascularised voxels.

The finding that "HP [1-13C]pyruvate levels were 1.05 fold higher" indicates that delivery of pyruvate might be increased. It is unclear if normalisation to the combined amplitude of lactate and pyruvate is fair in the case that the volume fraction in the voxel might have increased. Ideally, the authors would estimate polarisation separately as a normalisation.

No estimate of uncertainty is provided for the primary metabolic measures. Note that the lactate-pyruvate ratio is not normally distributed (see doi: 10.1002/mrm.26615), and this should be accounted for when carrying out statistical tests.

All metabolic maps are shown masked to the brain and interpolated to the structural MRI resolution (around 20 times). Nor is there any characterisation of the spectroscopic imaging's voxel volume, including the effect of the point spread function. It is, therefore, hard to have confidence in any spatial effects or potential partial volume effects from the tissue surrounding the brain.

The t2-weighted and SWI MRI measures used in this work are not quantitative. Normalisation in each case is carried out without regard to any spatially variable transmit and receive coil sensitivities (B1{plus minus}), which vary per subject. This adds intersubject variance, which could mask any effect between groups. No quality metrics (SNR or uncertainty estimates) are given for the MRI metrics.

Spectroscopic imaging was conducted 16 s after injection. Given the high heart rate of a mouse, measures of perfusion (using urea) could , therefore, be considered in a steady state, lowering sensitivity to any changes in perfusion or metabolite delivery. Furthermore, it is unclear how any changes in BBB permeability would manifest with the relatively low spatial resolution of MRSI. Would signal always be dominated by vascular compartments?

There is no apparent attempt to understand if an immune response occurs at this chronic time point. Macrophages are glycolytic and could affect the pyruvate measurement. Furthermore, is there any evidence for cellular changes in this model, namely density, cell type fraction, or microstructure? Are there any expected changes in glucose uptake?

There is no information or references provided for the accuracy or precision of the postmortem assays or their correlation with in vivo processes. What is the effect of cell density changes after injury on the assay kits?

The proposed interpretation of T1 as a measure of oxidative stress would seem to ignore the many confounding interpretations of T1.

Aims and impact:

In summary, the authors broadly achieve one aim, which is to find that HP 13C measured lac/pyruvate is a biomarker for the chronic effects of rTBI in a mouse model. As the authors themselves highlight in the discussion, the interpretation of this finding is tricky alongside their post-mortem assay results. The MR imaging in this work seems inconclusive, given the potential for inter-subject variance in the normalisation method.

The work, therefore, continues to highlight that HP 13C MRSI is a highly promising avenue of investigation to identify, characterise, and understand traumatic brain injury. It suggests that HP 13C MRSI is more promising in this sense than some standard MRI contrasts. The work currently fails to convincingly interpret the HP 13C MR results in conjunction with the other metrics.

Reviewer #1 (Public review):

Summary:

The manuscript by Ray et al. provides a theoretical framework to study tissue mechanics and the solid-to-fluid transition phenomenon observed in many tissues. The authors advanced previous models by directly incorporating cell-cell adhesion in force calculation with flexible cell geometries. They performed an in-depth analysis of the model and found that reducing cell-cell adhesion in near-confluent tissues can result in spontaneous cell rearrangements and transition to tissue fluidity. This is in contrast with previous predictions of Vertex models, which require higher adhesion for solid-to-fluid transition.

Strengths:

The authors provided a more general formulation of a 2D active foam model by directly incorporating cell-cell adhesion and performed a careful analysis of cell dynamics and cell shape in their simulations. They measured various quantities such as the mean-squared displacement of the cell center and shape index, which was introduced in previous studies to analyze jamming transition in tissues. By careful analysis of their simulations, they found a universal length scale in their simulations, explaining the observed heterogeneity. They provided a qualitative connection to previous experimental observations, where a reduction in cell adhesion caused tissue fluidity.

Weaknesses:

The phenomenon of tissue fluidity is an important and open question in biology. While theoretical models provide guidance to study such complex phenomena, the details in these models should go hand-in-hand with quantitative comparison with experiments. The study by Ray et al. indeed provided a more detailed description of deformable and adhesive cell collectives, but without a quantitative comparison with experiment, it is not clear if one needs all these details, or maybe more is needed. For example, do we need a more detailed mechanical model of the vertices, how the friction with substrate should be incorporated in such models, and is there a feedback between cell dynamics and its internal cytoskeleton organization?

While the manuscript by Ray et al. is an interesting theoretical study, without a quantitative comparison with experiments, it is not clear if it truly advances our understanding of tissue mechanics.

Reviewer #2 (Public review):

Summary:

Ray and coworkers introduce a discrete model of cellular layers aimed at investigating the role of inter-cellular adhesion in collective cell migration. The model combines aspects of particle-based models, in which cells are treated as simple point-particles with pair-interactions, and "morphological models", where interactions primarily depend on the cellular shape. In this case, cells are modeled as rings of beads connected by springs, thus allowing for exploration of the role of cell morphology while treating intercellular interactions as particle-like. Upon exploring the parameter space of this model, the authors recover physical behaviors reminiscent of reconstituted cell layers, including the onset of collective cell migration, when the forces leading to cell propulsion overweight inter-cellular adhesion, and various signatures of glassy dynamics.

Strengths:

The model presented in the article is simple, easy to implement, and scalable. The analysis appears solid and delivers a number of clear physical properties that could be tested in more depth in experiments and future numerical studies (e.g., distribution of displacements, etc.). The authors make an appreciable effort to make contact with other models and share their ideas for further investigations.

Weaknesses:

I found two main weaknesses in the original version of this manuscript, which I strongly encourage the authors to address.

(1) The manuscript explicitly aims at resolving an apparent contradiction of tessellation-based models, such as the Vertex and the Voronoi model. Both models used the so-called shape index p0 - i.e. the ratio between the preferential perimeter and the preferential area of the cells - to drive a solid/liquid phase transition in the presence of Brownian and/or rotational noise. Specifically, for sufficiently large p0 values, these in silico cell layers undergo a transition to a state of collective migration, where a rigid junction network becomes unstable to T1 events. Because p0 is often interpreted as "adhesion strength", this leads to the paradoxical conclusion that cell intercalation is favored by intercellular adhesion. The paradox, however, only lies in this interpretation, which assigns to the shape index p0 a biophysical role that is too specific. To illustrate this concept, let us consider the energy of an individual cell of area A and perimeter P: i.e. e = (a-1)^2+c*(p-p0)^2, where a=A/A_0, with A_0 the preferred area, p=P/sqrt(A_0) and p_0 = P_0/sqrt(A_0), with P_0 the preferred perimeter. Expanding the square in the second term gives e ~ p^2 - 2p_0 p. Thus, increasing p_0, favors longer cell junctions, from which it appears reasonable to interpret p0 as a dimensionless measure of intercellular adhesion. Such an increase in the length of the junctions is, however, only a byproduct of the effect of p0 on the overall shape of the cell, which becomes progressively less rounded as p0 is increased (e.g., for a circle, p0≈3.55, for an equilateral triangle, p0≈4.56). The roundness of an individual cell, on the other hand, cannot single-handedly be ascribed to intercellular adhesion, despite intercellular adhesion being undoubtedly one of the biophysical properties affecting this geometrical feature. Moreover, the shape index p0 ​enters the energy functional at the single-cell level, implying that even in isolation, without intercellular adhesion, an increase in p0 leads to a less rounded cell morphology. These peculiarities of the Vertex/Voronoi model do raise questions about its accuracy and validity, thus justify seeking for alternative cell-resolved models such as that introduced here by Ray et al., but, on the other hand, make the interpretation of p0 as an exclusive measure of adhesion evidently dubious.

(2) The spring-bead model by Ray and coworkers has at least two predecessors in the recent literature, none of which have been cited in the present manuscript. These are Boromand et al., Phys. Rev. Lett. 121, 248003 (2018) and Pasupalak et al. arXiv:2409.16128 (2024). The former paper investigates the packing of flexible polygons and is not specific to epithelial layers, while the latter is specifically designed to address various outstanding problems in tissue mechanics, including collective migration and wound healing. While none of these models is identical to that by Ray et al., it would be fair to present the latter as a member of the family rather than the first one of its kind and possibly comment about the differences and similarities with these previous models.

Reviewer #3 (Public review):

Summary:

This is a very focused and well-performed study that uses a somewhat less common approach in the field of tissue mechanics, a deformable particle model, to propose a solution to some important phenomenological inconsistencies between the standard vertex- and SPV-model approaches and experiments. The authors' focus in their study is on the role of adhesion in glassy dynamics and solid-fluid transition of epithelia.

Strengths:

It is a carefully performed study with an important technical edge compared to "mainstream" vertex and SPV models: the ability to describe cell-cell boundaries with two distinct membranes. This may have an important implication for the phenomenology, like the role of adhesion in solid-fluid transition.

Weaknesses:

Apart from some specific suggestions for improvement and clarification, I believe the authors could do a better job in comparing their results and their approach to other similar models, such as the one by Kim et al (Reference 7).

Reviewer #1 (Public review):

Summary:

In this manuscript, Emperador-Melero et al. seek to determine whether recruitment of endocytic machinery to the periactive zone is activity-dependent or tethered to delivery of active zone machinery. They use genetic knockouts and pharmacological block in two model synapses - cultured mouse hippocampal neurons and Drosophila neuromuscular junctions - to determine how well endocytic machinery localizes after chronic inhibition or acute depolarization by super-resolution imaging. They find that acute depolarization in both models has minimal to no effect on the localization of endocytic machinery at the periactive zone, suggesting that these proteins are constitutively maintained rather than upregulated in response to transient activity. Interestingly, chronic inhibition slightly increases endocytic machinery levels, implying a potential homeostatic upregulation in preparation for rebound depolarization. Using genetic knockouts, the authors show that localization of endocytic machinery to periactive zones occurs independently of proper active zone assembly, even in the absence of upstream organizers like Liprin-α.

Overall, they propose that the constitutive deployment of endocytic machinery reflects its critical role in facilitating rapid and reliable membrane internalization during synaptic functions beyond classical endocytosis, such as regulation of the exocytic fusion pore and dense-core vesicle fusion. Although many experiments reveal limited changes in the localization or abundance of endocytic machinery, the findings are thorough, and data substantially support a model in which endocytic components are organized through a pathway distinct from that of the active zone. This work advances our understanding of synaptic dynamics by supporting a model in which endocytic machinery is constitutively recruited and regulated by distinct upstream organizers compared to active zone proteins. It also highlights the utility of super-resolution imaging across diverse synapse types to uncover functionally conserved elements of synaptic biology.

Strengths:

The study's technical strengths, particularly the use of super-resolution microscopy and rigorous image analyses developed by the group, bolster their findings.

Weaknesses:

One notable limitation, however, is the absence of interrogation of endocytic proteins previously suggested to be recruited in an activity-dependent manner, in particular, endophilin.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

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

Weaknesses:

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

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

This study examines how synaptic endocytic zones are positioned using a combination of cultured neurons and the Drosophila neuromuscular junction. The authors test whether neuronal activity, active zone assembly, or liprin-α function is required to localize endocytic zone markers, including Dynamin, Amphiphysin, Nervous Wreck, PIPK1γ, and AP-180. None of the manipulations tested caused a coordinated disruption in the localization or abundance of these markers, leading to the conclusion that endocytic zones form independently of synaptic activity and active zone scaffolds.

Strengths:

The work is systematic and carefully executed, using multiple manipulations and two complementary model systems. The authors consistently examine multiple molecular markers, strengthening the interpretation that endocytic zone positioning is robust to changes in activity and structural assembly.

Weaknesses:

The main limitation is that the study does not test whether the methods used are sensitive enough to detect subtle functional disruption, and no condition tested produces clear disorganization of the endocytic zone. As a result, the conclusion that these zones assemble independently is supported by negative data, without a strong positive control for disassembly or mislocalization.

This paper addresses a longstanding question in synaptic biology and provides a well-supported boundary on the types of mechanisms that are likely to govern endocytic zone localization. The conclusions are well justified by the data, though additional evidence would be needed to define the assembly mechanism itself.

Reviewer #1 (Public review):

Summary:

In this study, the authors sought to define a molecular pathway that mediates the transformation of an aggregate of cells into a sheet known as the nucleus laminaris, a key site for auditory processing. The data offer a comprehensive view of the sequence of developmental events and suggest possible roles for FGF signaling, the transcription factor Mafb, and the cell surface adhesive molecule Cadherin-23 in this process.

Strengths:

The description of nL development is thorough and well-done, with extensive quantification of the overall structure of the nucleus and also of neuron number. Additionally, the study implicates several molecules in nL development, starting with a clear description of when and where FGF8, Mafb, and several cadherins are expressed, including antibody stains suggesting that one cadherin, cdh2, is localized to the neuronal dendrites. A series of perturbation experiments supports the idea that these three molecules play a role in nL formation. The computational model is an interesting addition that helps to conceptualize how cadherin-mediated adhesion might influence nL morphogenesis.

Weaknesses:

A number of weaknesses limit the impact of this work.

One problem is how the data is interpreted. The logic is often circular in that the same molecules are used both as markers of nL and also as players in its development. An independent measure of nL formation is needed. Along the same lines, while the experiments implicate each molecule, the data do not actually demonstrate that FGF directly modulates Mafb, which in turn modulates cadherin expression, especially as overexpression of cdh2 has no effect on FGF8 expression or lamina organization, and no manipulations of cdh22 are presented.

The other type of problem relates to how the experiments were performed and analyzed. Important details about the experiments, as well as key controls, are missing throughout. Sample sizes are rarely presented, and there is no evidence that either dominant negative construct actually acts as proposed. Some results are not well quantified, which further undermines the strength of the conclusions. For instance, the changes in mafb and cdh22 expression (Figure 7) are subtle and were not quantified for any of the conditions. Likewise, the claim that FGF8 has a dose-dependent effect on lamina size and neuron number needs to be supported by statistics.

There are also some questions about the quality of the data. Much of the histology is of poor quality and does not always show the same piece of brain in the same orientation from experiment to experiment, which makes it challenging to interpret the results. In particular, the quality of the in situ hybridization varies, with much more background in some cases than others, which makes it hard to know what signal is real.

Finally, there are some misstatements and problems with citations that weaken the scholarly nature of the paper. FGF signaling has been studied extensively in the hindbrain and even in auditory nucleus development (Abraira et al., 2007), but this literature is not discussed at all.

Due to these weaknesses, the authors have achieved their aims only in part. The data are suggestive, but the results do not yet fully support their conclusions.

Few labs study how populations of neurons assemble into spatially organized structures. This work has the potential to be very interesting to other developmental neuroscientists studying brain morphogenesis.

Reviewer #2 (Public review):

Summary:

The overall goal of this study by Smith et al. was to understand the mechanisms through which groups of cells form specific nuclei during development. These cell groupings may have importance for the development of nervous system connections. Smith et al. have taken advantage of the ordered structure of the nucleus laminaris of the chick, which plays an important role in sound source localization. They used a candidate gene approach to both mark cells in nL and to test for signaling pathways that regulate nucleogenesis. They found that MafB, FGF8, and cadherins were expressed in the auditory hindbrain at the critical ages. They used in ovo electroporation to test gene function effects on nL lamina formation. They found that both increasing and decreasing FGF signaling (through introduction of mouse FGF8 and expression of a dominant negative FGF receptor, respectively) reduced lamina formation in the nL. An optimal concentration of FGF needed for this process was obtained using cultured hindbrain slices. Misexpression of cadherins also perturbed the normal lamina formation. The authors showed that FGF regulates MafB expression, which in turn regulates cadherin expression, suggesting a pathway that shapes lamina development. They constructed computational models of adhesion on the development of nL cells and found that laminar formation is favored by nL cells modeled as bipolar adhesive units. Overall, the study has demonstrated the importance of these adhesion pathways for the formation of the nucleus laminaris, and the findings likely have significance for the development of other nuclei as well.

Strengths:

The experiments have used in situ hybridization, immunofluorescence, electroporation, and brainstem slice cultures to test their hypotheses, which were based on well-selected candidate molecules. The modeling adds to the rigor of the studies, particularly in light of the observation that cadherin expression is localized to nL dendrites.

Weaknesses:

(1) Some references should be considered more carefully for accuracy, and additional references may be needed (introduction and results).

(2) Information on animal numbers and statistical tests should be added.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

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

Concerns and suggestions:

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

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

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

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

This manuscript examined the neural correlates of the temporal-spatial dynamics of emotional processing while participants were watching short movie clips (each 12.5 s long) from the movie "Forrest Gump". Participants not only watched each film clip, but also gave emotional responses, followed by a brief resting period. Employing fMRI to track the BOLD responses during these stages of emotional processing, the authors found four large-scale brain networks (labeled as IC0,1,2,4) were differentially involved in emotional processing. Overall, this work provides valuable information on the neurodynamics of emotional processing.

Strengths:

This work employs a naturalistic movie watching paradigm to elicit emotional experiences. The authors used a slice-based fMRI method to examine the temporal dynamics of BOLD responses. Compared to previous emotional research that uses static images, this work provides some new data and insights into how the brain supports emotional processing from a temporal dynamics view.

Weaknesses:

Some major conclusions are unwarranted and do not have relevant evidence. For example, the authors seemed to interpret some neuroimaging results to be related to emotion regulation. However, there were no explicit instructions about emotional regulation, and there was no evidence suggesting participants regulated their emotions. How to best interpret the corresponding results thus requires caution.

Relatedly, the authors argued that "In turn, our findings underscore the utility of examining temporal metrics to capture subtle nuances of emotional processing that may remain undetectable using standard static analyses." While this sentence makes sense and is reasonable, it remains unclear how the results here support this argument. In particular, there were only three emotional categories: sad, happy, and fear. These three emotional categories are highly different from each other. Thus, how exactly the temporal metrics captured the "subtle nuances of emotional processing" shall be further elaborated.

The writing also contained many claims about the study's clinical utility. However, the authors did not develop their reasoning nor elaborate on the clinical relevance. While examining emotional processing certainly could have clinical relevance, please unpack the argument and provide more information on how the results obtained here can be used in clinical settings.

Importantly, how are the temporal dynamics of BOLD responses and subjective feelings related? The authors showed that "the time-to-peak differences in IC2 ("response") align closely with response latency results, with sad trials showing faster response latencies and earlier peak times". Does this mean that people typically experience sad feelings faster than happy or fear? Yet this is inconsistent with ideas such that fear detection is often rapid, while sadness can be more sustained. Understandably, the study uses movie clips, which can be very different from previous work, mostly using static images (e.g., a fearful or a sad face). But the authors shall explicitly discuss what these temporal dynamics mean for subjective feelings.

Reviewer #1 (Public review):

Summary:

This paper describes how CoA can overcome suppression of OXPHOS in TLR3 signaling, acting as what the authors term a 'metabolic adjuvant'. Supplementing with CoA enhances TLR signaling, reverses tolerance, and promotes OXPHOS. It promotes histone acetylation, leading to epigenetic modulation of target genes. CoA is further shown to have adjuvant effects in vivo, in anti-tumor immunity, and also in host defense.

Strengths:

Something of a tour-de-force - impressive methodologies and the conclusions are well supported by the data.

Weaknesses:

I was unable to follow the basis for some experiments and have a question around the data on itaconate, since this metabolite should limit IL-1beta production. Also, this is a very wordy manuscript - editing should help the reader.

Reviewer #2 (Public review):

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

This paper by Karimian et al proposes an oscillator model tuned to implement binding by synchrony (BBS*) principles in a visual task. The authors set out to show how well these BBS principles explain human behavior in figure-ground segregation tasks. The model is inspired by electrophysiological findings in non-human primates, suggesting that gamma oscillations in early visual cortex implement feature-binding through a synchronization of feature-selective neurons. The psychophysics experiment involves the identification of a figure consisting of gabor annuli, presented on a background of gabor annuli. The participants' task is to identify the orientation of the figure. The task difficulty is varied based on the contrast and density of the gabor annuli that make up the figure. The same figures (without the background) are used as inputs to the oscillator model. The authors report that both the discrimination accuracy in the psychophysics experiment and the synchrony of the oscillators in the proposed model follow a similar "Arnold Tongue" relationship when depicted as a function of the texture-defining features of the figure. This finding is interpreted as evidence for BBS/gamma synchrony being the underlying mechanism of the figure-ground segregation.

• Note that I chose to use "BBS" over gamma synchrony (used by the authors) in this review, as I am not convinced that the authors show evidence for synchronization in the gamma-band.

Strengths:

The design of the proposed model is well-informed by electrophysiological findings, and the idea of using computational modeling to bridge between intracranial recordings in non-human primates and behavioral results in human participants is interesting. Previous work has criticized the BBS synchrony theory based on the observation that synchronization in the gamma-band is highly localized and the frequency of the oscillation depends on the visual features of the stimulus. I appreciate how the authors demonstrate that frequency-dependence and local synchronization can be features of BBS, and not contradictory to the theory. As such, I feel that this work has the potential to contribute meaningfully to the debate on whether BBS is a biophysically realistic model of feature-binding in visual cortex.

Weaknesses:

I have several concerns regarding the presented claims, assessment of meaning and size of the presented effects, particularly with regard to the absence of a priori defined effect sizes.

Firstly, the paper makes strong claims about the frequency-specificity (i.e., gamma synchrony) and anatomical correlates (early visual cortex) of the observed effects. These claims are informed by previous electrophysiological work in non-human primates but are not directly supported by the paper itself. For instance, the title contains the word "gamma synchrony", but the authors do not demonstrate any EEG/MEG or intracranial data in from their human subjects supporting such claims, nor do they demonstrate that the frequencies in the oscillator model are within the gamma band. I think that the paper should more clearly distinguish between statements that are directly supported by the paper (such as: "an oscillator model based on BBS principles accounts for variance in human behavior") and abstract inferences based on the literature (such as "these effects could be attributed to gamma oscillations in early visual cortex, as the model was designed based on those principles").

Secondly, unlike the human participants, the model strictly does not perform figure-ground segregation, as it only receives the figure as an input. Finally, it is unclear what effect sizes the authors would have expected a priori, making it difficult to assess whether their oscillator model represents the data well or poorly. I consider this a major concern, as the relationship between the synchrony of the oscillatory model and the performance of the human participants is confounded by the visual features of the figure. Specifically, the authors use the BBS literature to motivate the hypothesis that perception of the texture-defined figure is related to the density and contrast heterogeneity of the texture elements (gabor annuli) of the figure. This hypothesis has to be true regardless of synchrony, as the figure will be easier to spot if it consists of a higher number of high-contrast gabors than the background. As the frequency and phase of the oscillators and coupling strength between oscillators in the grid change as a function of these visual features, I wonder how much of the correlation between model synchrony and human performance is mediated by the features of the figure. To interpret to what extent the similarity between model and human behavior relies on the oscillatory nature of the model, the authors should find a way to estimate an empirical threshold that accounts for these confounding effects. Alternatively, it would be interesting to understand whether a model based on competing theories (e.g., Binding by Enhanced Firing, Roelfsema, 2023) would perform better or worse at explaining the data.

Reviewer #2 (Public review):

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

Strengths:

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

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

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

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

Weaknesses:

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

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

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

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

Achievement of Aims and Support for Conclusions:

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

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

Likely Impact and Utility:

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

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

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

Additional Context:

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

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

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

Reviewer #3 (Public review):

Summary:

The manuscript Kroon et al. described two algorithms, which when combined achieve high throughput automation of "martinizing" protein structures with selected protonation states and post-translational modifications.

The authors have addressed all of my concerns as provided previously. Specifically, Figure S2 will be a very useful guideline for future improvement (e.g., parallelization) of the code.

Reviewer #2 (Public review):

This work introduces a Vermouth library framework to enhance software development within the Martini community. Specifically, it presents a Vermouth-powered program, Martinize2, for generating coarse-grained structures and topologies from atomistic structures. In addition to introducing the Vermouth library and the Martinize2 program, this paper illustrates how Martinize2 identifies atoms, maps them to the Martini model, generates topology files, and identifies protonation states or post-translational modifications. Compared with the prior version, the authors provide a new figure to show that Martinize2 can be applied to various molecules, such as proteins, cofactors, and lipids. To demonstrate the general application, Martinize2 was used for converting 73% of 87,084 protein structures from the template library, with failed cases primarily blamed on missing coordinates.

I appreciate the changes that the authors made to clarify the novelty. I have no doubt this paper will receive attention and citations.

Reviewer #1 (Public review):

Summary:

In this study, the authors provide a new computational platform called Vermouth to automate topology generation, a crucial step that any biomolecular simulation starts with. Given a wide arrange of chemical structures that need to be simulated, varying qualities of structural models as inputs obtained from various sources, and diverse force fields and molecular dynamics engines employed for simulations, automation of this fundamental step is challenging, especially for complex systems and in case that there is a need to conduct high-throughput simulations in the application of computer-aided drug design (CADD). To overcome this challenge, the authors develop a programing library composed of components that carry out various types of fundamental functionalities that are commonly encountered in topological generation. These components are intended to be general for any type of molecules and not to depend on any specific force field and MD engines. To demonstrate the applicability of this library, the authors employ those components to reassemble a pipeline called Martinize2 used in topology generation for simulations with a widely used coarse-grained model (CG) MARTINI. This pipeline can fully recapitulate the functionality of its original version Martinize but exhibit greatly enhanced generality, as confirmed by the ability of the pipeline to faithfully generate topologies for two high-complexity benchmarking sets of proteins.

Strengths:

The main strength of this work is the use of concepts and algorithms associated with induced subgraph in graph theory to automate several key but non-trivial steps of topology generation such as the identification of monomer residue units (MRU), the repair of input structures with missing atoms, the mapping of topologies between different resolutions, and the generation of parameters needed for describing interactions between MRUs. In addition, the documentation website provided by the authors is very informative, allowing users to get quickly started with Vermouth.

Weaknesses:

Although the Vermouth library can work for different force fields, exhibiting certain generality, its application has been demonstrated only with GROMACS. The extension of the library to other major MD engines could be future directions for improvement but may not be needed for this study.

Reviewer #1 (Public review):

Summary:

Work by Brosseau et. al. combines NMR, biochemical assays, and MD simulations to characterize the influence of the C-terminal tail of EmrE, a model multi-drug efflux pump, on proton leak. The authors compare the WT pump to a C-terminal tail deletion, delta_107, finding that the mutant has increased proton leak in proteoliposome assays, shifted pH dependence with a new titratable residue, faster alternating access at high pH values, and reduced growth, consistent with proton leak of the PMF.

Strengths:

The work combines thorough experimental analysis of structural, dynamic, and electrochemical properties of the mutant relative to WT proteins. The computational work is well aligned in vision and analysis. Although all questions are not answered, the authors lay out a logical exploration of the possible explanations.

Weaknesses:

A few analyses that were missing in the first submission were included/corrected in the revision.

Reviewer #2 (Public review):

Summary:

This manuscript explores the role of the C-terminal tail of EmrE in controlling uncoupled proton flux. Leakage occurs in the wild-type transporter under certain conditions but is amplified in the C-terminal truncation mutant D107. The authors use an impressive combination of growth assays, transport assays, NMR on WT and mutants with and without key substrates, classical MD, and reactive MD to address this problem. Overall, I think that the claims are well supported by the data, but I am most concerned about the reproducibility of the MD data, initial structures used for simulations, and the stochasticity of the water wire formation. These can all be addressed in a revision with more simulations as I point out below. I want to point out that the discussion was very nicely written, and I enjoyed reading the summary of the data and the connection to other studies very much.

Strengths:

The Henzler-Wildman lab is at the forefront of using quantitative experiments to probe the peculiarities in transporter biophysics, and the MD work from the Voth lab complements the experiments quite well. The sheer number of different types of experimental and computational approaches performed here is impressive.

Weaknesses:

The primary weaknesses are related to the reproducibility of the MD results with regard to the formation of water wires in the WT and truncation mutant. This could be resolved with simulations starting from structures built using very different loops and C-terminal tails.

The water wire gates identified in the MD should be tested experimentally with site-directed mutagenesis to determine if those residues do impact leak.

Comments on revisions:

Having reviewed the latest version of the manuscript, I continue to believe that this is a solid paper with important results. I find the new data regarding the computational pKa estimate of E14 compelling.

Reviewer #1 (Public review):

Summary:

This study addresses the roles of polyunsaturated fatty acids (PUFAs) in animal physiology and membrane function. A C. elegans strain carrying the fat-2(wa17) mutation possesses a very limited ability to synthesize PUFAs and there is no dietary input because the E. coli diet consumed by lab grown C. elegans does not contain any PUFAs. The fat-2 mutant strain was characterized to confirm that the worms grow slowly, have rigid membranes, and have a constitutive mitochondrial stress response. The authors showed that chemical treatments or mutations known to increase membrane fluidity did not rescue growth defects. A thorough genetic screen was performed to identify genetic changes to compensate for the lack of PUFAs. The newly isolated suppressor mutations that compensated for FAT-2 growth defects included intergenic suppressors in the fat-2 gene, as well as constitutive mutations in the hypoxia sensing pathway components EGL-9 and HIF-1, and loss of function mutations in ftn-2, a gene encoding the iron storage protein ferritin. Taken together, these mutations lead to the model that increased intracellular iron, an essential cofactor for fatty acid desaturases, allows the minimally functional FAT-2(wa17) enzyme to be more active, resulting in increased desaturation and increased PUFA synthesis.

Strengths:

(1) This study provides new information further characterizing fat-2 mutants. The authors measured increased rigidity of membranes compared to wild type worms, however this rigidity is not able to be rescued with other fluidity treatments such as detergent or mutants. Rescue was only achieved with polyunsaturated fatty acid supplementation.

(2) A very thorough genetic suppressor screen was performed. In addition to some internal fat-2 compensatory mutations, the only changes in pathways identified that are capable of compensating for deficient PUFA synthesis was the hypoxia pathway and the iron storage protein ferritin. Suppressor mutations included an egl-9 mutation that constitutively activates HIF-1, and Gain of function mutations in hif-1 that are dominant. This increased activity of HIF conferred by specific egl-9 and hif-1 mutations lead to decreased expression of ftn-2. Indeed, loss of ftn-2 leads to higher intracellular iron. The increased iron apparently makes the FAT-2 fatty acid desaturase enzyme more active, allowing for the production of more PUFAs.

(3) The mutations isolated in the suppressor screen show that the only mutations able to compensate for lack of PUFAs were ones that increased PUFA synthesis by the defective FAT-2 desaturase, thus demonstrating the essential need for PUFAs that cannot be overcome by changes in other pathways. This is a very novel study, taking advantage of genetic analysis of C. elegans, and it confirms the observations in humans that certain essential PUFAs are required for growth and development.

(4) Overall, the paper is well written, and the experiments were carried out carefully and thoroughly. The conclusions are well supported by the results.

Weaknesses:

Overall, there are not many weaknesses. The main one I noticed is that the lipidomic analysis shown in Figs 3C, 7C, S1 and S3. While these data are an essential part of the analysis and provide strong evidence for the conclusions of the study, it is unfortunate that the methods used did not enable the distinction between two 18:1 isomers. These two isomers of 18:1 are important in C. elegans biology, because one is a substrate for FAT-2 (18:1n-9, oleic acid) and the other is not (18:1n-7, cis vaccenic acid). Although rarer in mammals, cis-vaccenic acid is the most abundant fatty acid in C. elegans and is likely the most important structural MUFA. The measurement of these two isomers is not essential for the conclusions of the study.

Reviewer #2 (Public review):

Summary:

The authors use a genetic screen in C. elegans to investigate the physiological roles of polyunsaturated fatty acids (PUFAs). They screen for mutations that rescue fat-2 mutants, which have strong reductions in PUFAs. As a result, either mutations in fat-2 itself or mutations in genes involved in the HIF-1 pathway were found to rescue fat-2 mutants. Mutants in the HIF-1 pathway rescue fat-2 mutants by boosting their catalytic activity (via upregulated Fe2+). Thus, the authors show that in the context of fat-2 mutation, the sole genetic means to rescue PUFA insufficiency is to restore PUFA levels.

Strengths:

As C. elegans can produce PUFAs de novo as essential lipids, the genetic model is well-suited to study the fundamental roles of PUFAs. The genetic screen finds mutations in convergent pathways, suggesting that it has reached near-saturation. The authors extensively validate the results of the screening and provide sufficient mechanistic insights to show how PUFA levels are restored in HIF-1 pathway mutants. As many of the mutations found to rescue fat-2 mutants are of gain-of-function, it is unlikely that similar discoveries could have been made with other approaches like genome-wide CRISPR screenings, making the current study distinctive. Consequently, the study provides important messages. First, it shows that PUFAs are essential for life. The inability to genetically rescue PUFA deficiency, except for mutations that restore PUFA levels, suggests that they have pleiotropic essential functions. In addition, the results suggest that the most essential functions of PUFAs are not in fluidity regulation, which is consistent with recent reviews proposing that the importance of unsaturation goes beyond fluidity (doi: 10.1016/j.tibs.2023.08.004 and doi: 10.1101/cshperspect.a041409). Thus, the study provides fundamental insights about how membrane lipid composition can be linked to biological functions.

Weaknesses:

The authors put in a lot of effort to answer the questions that arose through peer review, and now all the claims seem to be supported by experimental data. Thus, I do not see obvious weaknesses. Of course, it remains unclear what PUFAs do beyond fluidity regulation, but this is something that cannot be answered from a single study.

Reviewer #1 (Public review):

Summary:

In this manuscript the authors present a novel CRISPR/Cas9-based genetic tool for the dopamine receptor dop1R2. Based on the known function of the receptor in learning and memory, they tested the efficacy of the genetic tool by knocking out the receptor specifically in mushroom body neurons. The data suggest that dop1R2 is necessary for longer lasting memories through its action on ⍺/ß and ⍺'/ß' neurons but is dispensable for short-term memory and thus in ɣ neurons. The experiments impressively demonstrate the value of such a genetic tool and illustrate the specific function of the receptor in subpopulations of KCs for longer-term memories.

Reviewer #2 (Public review):

Summary:

This manuscript examines the role of the dopamine receptor, Dop1R2, in memory formation. This receptor has complex roles in supporting different stages of memory, and the neural mechanisms for these functions is poorly understood. The authors are able to localize Dop1R2 function to the vertical lobes of the mushroom body, revealing a role in later (presumably middle-term) aversive and appetitive memory. In general the experimental design is rigorous, and statistics are appropriately applied. The manuscript provides a thorough assessment of how Dop1R2 functions within the mushroom bodies to regulate protein-synthesis dependent and independent memory, and provides a valuable new tool for the community.

Strengths:

(1) The FRT lines generated provide a novel tool for temporal and spatially precise manipulation of Dop1R2 function. This tool will be valuable to study the role of Dop1R2 in memory and other behaviors potentially regulated by this gene.

(2) Given the highly conserved role of Dop1R2 in memory and other processes, these findings have high potential to translate to vertebrate species.

Reviewer #3 (Public review):

Summary:

Kaldun et al. investigated the role of Dopamine Receptor Dop1R2 in different types and stages of olfactory associative memory in Drosophila melanogaster. Dop1R2 is a type 1 Dopamine receptor that can act both through Gs-cAMP and Gq-ERCa2+ pathways. The authors first developed a sophisticated tool where tissue-specific knock-out mutants can be generated using Crispr/Cas9 technology in combination with the Gal4/UAS gene-expression toolkit. They direct the K.O. mutation to intrinsic neurons of the main associative memory centre fly brain: the mushroom body (MB). There are three main types of MB-neurons, or Kenyon cells, according to their axonal projections: a/b; a'/b' and g neurons.

Kaldun et al. found that, while not required for short-term memory, dop1R2 is necessary in a/b and a'/b' but not in gamma neurons to display normal appetitive and aversive middle-term (2h) and long-term (24h) memory. These results showcase a compartmentalized role of Dop1R2 in specific neuronal subtypes of the main memory centre of the fly brain for the expression of middle and long-term memories.

The conclusions of this paper are very well supported by the data, and the authors systematically addressed the requirement of a very interesting type of dopamine receptor in both appetitive and aversive memories. These findings are important for the fields of learning and memory and dopaminergic neuromodulation, among others.

Importantly, the authors of this paper produced a tool to generate tissue-specific knock out mutants of dop1R2. Although reports on the requirement of this gene in different memory phases exist, the genetic tools used here represent the most sophisticated approach to induce a loss of function phenotypes in neurons of interest.

Overall, the authors generated a very useful tool to study dopamine neuromodulation in any given circuit when used in combination with the powerful genetic toolkit available in Drosophila. The reports on this paper confirmed a previously described role of Dop1R2 in the expression of aversive and appetitive LTM providing spatio-temporal resolution and additionally, they mapped these effects to two types of memory neurons in the fly brain, shedding light into the intricate modulation of dopamine in memory circuits.

Reviewer #1 (Public review):

Summary:

The study by Deng et al reports single cell expression analysis of developing mouse hearts and examines the requirements for cardiac fibroblasts in heart maturation. The work includes extensive gene expression profiling and bioinformatic analysis. The prenatal fibroblast ablation studies show new information on the requirement of these cells on heart maturation before birth.

The strengths of the manuscript are the new single cell datasets and comprehensive approach to ablating cardiac fibroblasts in pre and postnatal development in mice. Extensive data are presented on mouse embryo fibroblast diversity and morphology in response to fibroblast ablation. Histological data support localization of major cardiac cell types and effects of fibroblast ablation on cardiac gene expression at different times of development.

A weakness of the study is that the major conclusions regarding collagen signaling and heart maturation are based on gene expression patterns and are not functionally validated.

Comments on Revised Version (from BRE):

Most of my comments have been adequately addressed. Additional comments on new data in the revised manuscript are below.

(1) In the new figure S11, it is not really possible to draw major conclusions on mitral valve morphology and maturation since the planes of sections to not seem comparable. Observations regarding attachment to the papillary muscle might be dependent on the particular section being evaluated. However, it is useful to see that the valves are not severely affected in the ablated animals.

(2) In the last supplemental figure S19, it is not possible to determine if results are or are not statistically significant for n=2 as shown for FS and EF for the ablated animals and controls. The text says that there is a trend of improved heart function, but evaluation of additional animals is needed to support this conclusion.

Reviewer #2 (Public review):

This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. Single-cell RNA sequencing (scRNA-seq) analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.

Some of the major issues are described below.

(1) The CD31 immunostaining data (Figure 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

(2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Fig. 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

(3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Fig. 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

(4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

(5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

(6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

(7) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

Comments on Revised Version (from BRE):

The manuscript has greatly improved following the revision, and I have no additional comments to offer.

Reviewer #3 (Public review):

Summary:

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with focus on critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cells network in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated heart. Lastly they reported a compensatory collagen expression in long term ablated neonate heart. Overall, this study provides us with important insight on fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM focused research.

Strengths:

The authors utilized good analyzing tools to investigate on multiple database of single cell sequencing and Multi-seq. They identified significant pathways, cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used very strident technique to study fibroblast functions by ablating one of the major cell population of heart. Also, experimental validation of their analyzed downstream pathways will be required eventually.

Comments on Revised Version (from BRE):

The authors did a good job addressing the questions asked at first review. However, I have some minor concerns.

(1) The paper notes that collagen signaling is observed in FB-VasEC in humans, but not in FB-VenCM, unlike mice. Did the authors analyze predictive ligand receptor interaction as they did with control and ablated mice heart? This could add valuable new insights that how FB regulate ventricular CM in human heart.

(2) The authors provided data on Defect in CD31 expression in several models. Did they observe any other phenotypes associated with defective endothelial or vascular system? Such as, blood accumulation in pericardium, larger/smaller capillaries? Did they also examine percentage of Cdh5+ cells?

(3) Please mention the sample age of Figure 2A-C.

(4) Please follow the same style to describe X axis in graphs in Figure 3D (and all similar graphs in the manuscript) as followed in 3G.

(5) It is important to provide echocardiographic M mode images with a comparable number of cardiac cycles in control and ablated (Fig. 6H).

(6) In the long-term neonatal ablation experiments, collagen expressions return to normal. The manuscript attributes this to possible "compensatory expression," Do they have any thoughts how this is regulated? Are other cell types stepping in, or are surviving FBs proliferating?

(7) While collagen is shown to be a dominant signaling molecule, its centrality is inferred primarily from scRNA-seq and ligand-receptor predictions. Did authors try any functional rescue experiment (e.g., exogenous collagen supplementation or receptor blockade) to directly validate this pathway's role in vivo?

Reviewer #2 (Public review):

Summary:

In this study, authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. Authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been then tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.

Strengths:

The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their application for immunostaining and immunoblotting experiments. Data are convincing and the manuscript is well-written, clear and easy to read.

Weaknesses:

When using membrane-tethered forms of the antigens to test the affinity of nanobodies identified by ELISA, many nanobodies fail to recognize the antigens. While authors suggested a low affinity of these nanobodies for their antigens, this hypothesis has not been tested in the manuscript.

Improving the protocol at each step for nanobody selection would greatly increase a successful rate for nanobodies discovery with high affinity.

Reviewer #1 (Public review):

This study uses MEG to test for a neural signature of the trial history effect known as 'serial dependence.' This is a behavioral phenomenon whereby stimuli are judged to be more similar than they really are, in feature space, to stimuli that were relevant in the recent past (i.e., the preceding trials). This attractive bias is prevalent across stimulus classes and modalities, but a neural source has been elusive. This topic has generated great interest in recent years, and I believe this study makes a unique contribution to the field.

Specifically, while previous neuroimaging studies have found apparent reactivations of previous information, or repulsive biases that may indirectly relate to serial dependence, here Fischer at al. find an attractive bias in neural activity patterns that aligns with the direction of the behavioral effect. Moreover, the data show that the bias emerges later in a trial, after perceptual encoding, which speaks to an ongoing debate about whether such biases are perceptual or decisional.

The revised preprint thoroughly addresses many of the initial concerns, but the results are still open to interpretation. For instance, the model training/testing regime allows that some training data timepoints may be inherently noisier than others (e.g., delay period more so than encoding), and potentially more (or differently) susceptible to bias. The S1 and S2 epochs show no attractive bias, but they may also be based on more high fidelity training sets (i.e., encoding), and therefore less susceptible to the bias that is evident in the retrocue epoch. So, the results could reflect that serial dependence is indeed a post-perceptual process, or it may instead be that the WM representations, as detected with these MEG analyses, become noisier and more subject to reveal the attractive bias over time.

The results are intriguing, but the study was not powered to examine whether there is any feature-specificity to the neural bias (e.g., whether it matches the behavioral pattern that biases are amplified within a particular range of feature distances between stimuli). Nor do analyses get at temporally precise information about when attractive and repulsive biases appear, which would help to better reconcile the work with previous findings. As in, the reconstructions average across coarse trial epochs. The S1 and S2 reconstructions show no attractive bias, and appear to show subtle repulsion, but if the timing were examined more precisely, we might see repulsion magnified at earlier timepoints that shift toward attraction at later time points, thereby counteracting the effect. That is to say that the averaging approach, across feature values and timepoints, still leaves these important theoretical questions unresolved.

Nonetheless, the work marks an important step in identifying the neurophysiological bases of serial dependence. Ideally, all of the data, including the eye-tracking, would be made available so that others might try to address some of these follow-up questions.

Reviewer #2 (Public review):

Summary:

The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

Strengths:

The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (prev. studies reported repulsive biases), to my knowledge, it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

Weaknesses:

The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which, together with previous studies on eye movement-related confounds in neural decoding, justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the attractive shift in reconstructed motion direction. However, the authors' approach of quantifying stimulus-dependent eye movements only considers gaze angle and not gaze amplitude, and thus potentially misses important features of eye movements that could manifest in the MEG data. Moreover, it is unclear whether the gaze consistency metric should correlate with attractive history biases in neural decoding, if there were a confound. These two concerns could be potentially addressed by (1) directly decoding stimulus motion direction from x-y gaze coordinates and relating this decoding performance to neural reconstruction fidelity, and (2) investigating whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (2) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC and dlPFC have been implicated in serial biases. This begs the question which brain areas contribute to the serial dependencies observed in the current study? For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

Reviewer #2 (Public review):

Summary:

The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

Strengths:

The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (prev. studies reported repulsive biases), to my knowledge, it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

Weaknesses:

The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which, together with previous studies on eye movement-related confounds in neural decoding, justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the attractive shift in reconstructed motion direction. However, the authors' approach of quantifying stimulus-dependent eye movements only considers gaze angle and not gaze amplitude, and thus potentially misses important features of eye movements that could manifest in the MEG data. Moreover, it is unclear whether the gaze consistency metric should correlate with attractive history biases in neural decoding, if there were a confound. These two concerns could be potentially addressed by (1) directly decoding stimulus motion direction from x-y gaze coordinates and relating this decoding performance to neural reconstruction fidelity, and (2) investigating whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (2) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC and dlPFC have been implicated in serial biases. This begs the question which brain areas contribute to the serial dependencies observed in the current study? For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

Reviewer #1 (Public review):

Summary:

Participants in this study completed three visits. In the first, participants received experimental thermal stimulations which were calibrated to elicit three specific pain responses (30, 50, 70) on a 0-100 visual analogue scale (VAS). Experimental pressure stimulations were also calibrated at an intensity to the same three pain intensity responses. In the subsequent two visits, participants completed another pre-calibration check (Visit 2 of 3 only). Then, prior to the exercise NALOXONE or a SALINE placebo-control was administered intravenously. Participants then completed 1 of 4 blocks of HIGH (100%) or LOW (55%) intensity cycling which was tailored according to a functional threshold power (FTP) test completed in Visit 1. After each block of cycling lasting 10 minutes, participants entered an MRI scanner and were stimulated with the same thermal and pressure stimulations that corresponded to 30, 50, and 70 pain intensity ratings from the calibration stage. Therefore, this study ultimately sought to investigate whether aerobic exercise does indeed incur a hypoalgesia effect. More specifically, researchers tested the validity of the proposed endogenous pain modulation mechanism. Further investigation into whether the intensity of exercise had an effect on pain and the neurological activation of pain-related brain centres were also explored. Results show that in the experimental visits (Visit 2 and 3), when participants exercised at two distinct intensities as intended. Power output, heart rate, and perceived effort ratings were higher during the HIGH versus LOW intensity cycling. In particular. HIGH intensity exercise was perceived as "hard" / ~15 on the Borg (1974, 1998) scale, whereas LOW intensity exercise was perceived as "very light" / ~9 on the same scale.

The fMRI data from Figure 1 indicates that the anterior insula, dorsal posterior insula and middle cingulate cortex show pronounced activation as stimulation intensity and subsequent pain responses increased, thus linking these brain regions with pain intensity and corroborating what many studies have shown before.

Results also showed that participants rated a higher pain intensity in the NALOXONE condition at all three stimulation intensities compared to the SALINE condition. Therefore, the expected effect of NALOXONE in this study seemed to occur whereby opioid receptors were "blocked" and thus resulted in higher pain ratings compared to a SALINE condition where opioid receptors were "not blocked". When accounting for participant sex, NALOXONE had negligible effects at lower experimental nociceptive stimulations for females compared to males who showed a hyperalgesia effect to NALOXONE at all stimulation intensities (peak effect at 50 VAS). Females did show a hyperalgesia effect at stimulation intensities corresponding to 50 and 70 VAS pain ratings. The fMRI data showed that the periaqueductal gray (PAG) showed increased activation in the NALOXONE versus SALINE condition at higher thermal stimulation intensities. The PAG is well-linked to endogenous pain modulation.

When assessing the effects of NALOXONE and SALINE after exercise, results showed no significant differences in subsequent pain intensity ratings.

When assessing the effect of aerobic exercise intensity on subsequent pain intensity ratings, authors suggested that aerobic exercise in the form of a continuous cycling exercise tailored to an individual's FTP is not effective at eliciting an exercise-induced hypoalgesia response -irrespective of exercise intensity. This is because results showed that pain responses did not differ significantly between HIGH and LOW intensity exercise with (NALOXONE) and without (SALINE) an opioid antagonist. Therefore, authors have also questioned the mechanisms (endogenous opioids) behind this effect.

Strengths:

Altogether, the paper is great piece of work that has provided some truly useful insight into the neurological and perceptual mechanisms associated with pain and exercise-induced modulation of pain. The authors have gone to great lengths to delve into their research question(s) and their methodological approach is relatively sound. The study has incorporated effective pseudo-randomisation and conducted a rigorous set of statistical analysis to account for as many confounds as possible. I will particularly credit the authors on their analysis which explores the impact of sex and female participants' stage of menses on the study outcomes. It would be particularly interesting for future work to pursue some of these lines of research which investigate the differences in the endogenous opioid mechanism between sexes and the added interaction of stage of menses or training status - all of which the authors point out in their discussion.

There are certainly many other areas that this article contributes to the literature due to the depth of methods the research team have used. For example, the authors provide much insight into: the impact of exercise intensity on the exercise-induced hypoalgesia effect; the impact of sex on the endogenous opioid modulation mechanism; and the impact of exercise intensity on the neurological indices associated with endogenous pain modulation and pain processing. All of which, the researchers should be credited for due to the time and effort they have spent completing this study. Indeed, their in-depth analysis of many of these areas provides ample support for the claims they make in relation to these specific questions. As such, I consider their evidence concerning the fMRI data to be very convincing (and interesting).

Weaknesses:

Although the authors have their own view of their results, I, however, do still maintain a slightly different take on what the post-exercise pain ratings seem to show and its implications for judging whether an exercise-induced hypoalgesia effect is present or not and whether this is related to the opioid system.

For example, my basic assumptions relate to data which appears to show that there is an exercise-induced hypoalgesia effect as average pain ratings are ~30% lower than pre-calibrated/resting pain ratings within the SALINE condition at the same temperature of stimulation. Then, it appears there is evidence for the endogenous opioid mechanism as the NALOXONE condition demonstrates a minimal hypoalgesia effect after exercise. I.e., NALOXONE indeed blocked the opioid receptors, and such inhibition prevented the endogenous opioid system from taking effect.

However, through a comprehensive revision of their work, the authors have addressed many areas that myself and my fellow reviewer have questioned and provided a comprehensive set of responses and edits about this. So while I may have some opposing views on the mechanisms at play, I believe that each reader can decide and interpret the data for themselves which has been presented well by the authors.

Reviewer #2 (Public review):

Summary:

This interesting study compared two different intensities of aerobic exercise (low-intensity, high-intensity) and their efficacy in inducing a hypoalgesic reaction (i.e. exercise-induced hypoalgesia; EIH). fMRI was used to identify signal changes in the brain, with infusion of naloxone used to identify hypoalgesia mechanisms. No differences were found in post exercise pain perception between the high-intensity and low-intensity conditions, with naloxone infusion causing increased pain perception across both conditions which was mirrored by activation in the medial frontal cortex (identified by fRMI).

Strengths:

• The use of fMRI and naloxone provides a strong approach by which to identify possible mechanisms of EIH.

• The infusion of naloxone to maintain a stable concentration helps to ensure a consistent effect and that the time-course of the protocol won't affect consistency of changes in pain perception

• The manipulation checks (differences in intensity of exercise, appropriate pain induction) are approached in a systematic way.

• The interactions for fitness level and sex provide some interesting findings which should be explored further.

Weaknesses:

• Given the absence of a baseline/control condition (for exercise), the efficacy of high/low intensity exercise on EIH cannot be assessed. Providing this would have extended and strengthened the findings/conclusions.

• Whilst the exercise test (functional threshold power) used to set the intensity of the low/high exercise bouts set participants to exercise at different intensities, this method does not ensure that they exercised above/below particular thresholds (i.e. within either heavy or severe domains). This could have created very different relative challenges between participants.

Reviewer #2 (Public review):

The work has significant implications for understanding immune evasion and nutrient uptake mechanisms in trypanosomes.

While the experimental rigor is commendable, revisions are needed to clarify methodological limitations and to broaden the discussion of functional consequences.

The authors argue that prior studies missed surface-localized TfR due to harsh washing/fixation (e.g., methanol). While this is plausible, additional evidence would strengthen the claim.

It remains unclear how centrifugation steps of various lengths (as in previous publications) can equally and quantitatively redistribute TfR into the flagellar pocket. If this were the case, it should be straightforward for the authors to test this experimentally.

If TfR is distributed over the cell surface, live-cell imaging with fluorescent transferrin should be performed as a control. Modern detection limits now reach the single-molecule level, and transient immobilization of live trypanosomes has been established, which would exclude hydrodynamic surface clearance as a confounding factor.

In most images, TfR is not evenly distributed on the surface but rather appears punctate. Could this reflect localization to membrane domains? Immuno-EM with high-pressure frozen parasites could resolve this question and is relatively straightforward.

The authors might consider discussing whether differences in parasite life cycle stages (procyclic versus bloodstream forms) or culture conditions (e.g., cell density) affect localization. The developmentally regulated retention of GPI-anchored procyclin in the flagellar pocket might be worth mentioning.

Reviewer #1 (Public review):

Summary:

An interesting manuscript from the Carrington lab is presented investigating the behavior of single vs double GPI-anchored nutrient receptors in bloodstream form (BSF) T. brucei. These include the transferrin receptor (TfR), the HpHb receptor (HpHbR), and the factor H receptor (FHR). The central question is why these critical proteins are not targeted by host-acquired immunity. It has generally been thought that they are sequestered in the flagellar pocket (FP), where they are subject to rapid endocytosis - any Ab:receptor complexes would be rapidly removed from the cell surface. This manuscript challenges that assumption by showing that these receptors can be found all over the outer cell body and flagella surfaces, if one looks in an appropriate manner (rapid direct fixation in culture media).

The main part of the manuscript focuses on TfR, typically a GPI1 heterodimer of very similar E6 (GPI anchored) and E7 (truncated, no GPI) subunits. These are expressed coordinately from 15 telomeric expression sites (BES), of which only one can be transcribed at a time. The authors identify a native E6:E7 pair in BES7 in which E7 is not truncated and therefore forms a GPI2 heterodimer. By in situ genetic manipulation, they generate two different sets of GPI1:GPI2 TfR combinations expressed from two different BESs (BES1 and BES7). Comparative analyses of these receptors form the bulk of the data.

The main findings are:

(1) Both GPI1 and GPI2 TfR can be found on the cell body/flagellar surface. (2) Both are functional for Tf binding and uptake. (3) GPI2 TfR is expressed at ~1.5x relative to GPI1 TfR. (4) Ultimate TfR expression level (protein) is dependent on the BES from which it is expressed.

Most of these results are quite reasonably explained in light of the hydrodynamic flow model of the Engstler lab and the GPI valence model of the Bangs lab. Additional experiments, again by rapid fixation, with HpHbR and FHR, show that these GPI1 receptors can also be seen on the cell surface, in contrast to published localizations.

It is quite interesting that the authors have identified a native GPI2 TfR. However, essentially all of the data with GPI2 TfR are confirmatory for the prior, more detailed studies of Tiengwe et al. (2017). That said, the suggestion that GPI2 was the ancestral state makes good evolutionary sense, and begs the question of why trypanosomes prefer GPI1 TfR in 14 of 15 ESs (i.e., what is the selection pressure?).

Strengths and weaknesses:

(1) BES7 TfR subunit genes (BES7_Tb427v10): There are actually three (in order 5'-3'): E7gpi, E6.1 and E6.2. E6.1 and E6.2 have a single nucleotide difference. This raises the issue of coordinate expression. If overall levels of E6 (2 genes) are not down-regulated to match E7 (1 gene), this will result in a 2x excess of E6 subunits. The most likely fate of these is the formation of non-functional GPI2 homodimers on the cell surface, as shown in Tiengwe et al. (2017), which will contribute to the elevated TfR expression seen in BES7.

(2) Surface binding studies: This is the most puzzling aspect of the entire manuscript. That surface GPI2 TfR should be functional for Tf binding and uptake is not surprising, as this has already been shown by Tiengwe et al. (2017), but the methodology for this assay raises important questions. First, labeled Tf is added at 500 nM to live cells in complete media containing 2.5 uM unlabeled Tf - a 5x excess. It is difficult to see how significant binding of labeled TfR could occur in as little as 15 seconds under these conditions. Second, Tiengwe et al. (2017) found that trypanosomes taken directly from culture could not bind labeled Tf in direct surface labeling experiments. To achieve binding, it was necessary to first culture cells in serum-free media for a sufficient time to allow new unligated TfR to be synthesized and transported to the surface. This result suggests that essentially all surface TfR is normally ligated and unavailable to the added probe. Third, the authors have themselves argued previously, based on binding affinities, that all surface-exposed TfR is likely ligated in a natural setting (DOI: 10.1002/bies.202400053). Could the observed binding actually be non-specific due to the high levels of fixative used?

(3) Variable TfR expression in different BESs: It appears that native TfR is expressed at higher levels from BES7 compared to BES1, and even more so when compared to BES3. This raises the possibility that the anti-TfR used in these experiments has differential reactivity with the three sets of TfRs. The authors discount this possibility due to the overall high sequence similarities of E6s and E7s from the various ESs. However, their own analyses show that the BES1, BES3, and BES7 TfRs are relatively distal to each other in the phylogenetic trees, and this Reviewer strongly suspects that the apparent difference in expression is due to differential reactivity with the anti-TfR used in this work. In the grand scheme, this is a minor issue that does not impact the other major conclusions concerning TfR localization and function, nor the behavior of HpHbR and FHR. However, the authors make very strong conclusions about the role of BESs in TfR expression levels, even claiming that it is the 'dominant determinant' (line 189).

(4) Surface immuno-localization of receptors: These experiments are compelling and useful to the field. To explain the difference with essentially all prior studies, the authors suggest that typical fixation procedures allow for clearance of receptor:ligand complexes by hydrodynamic flow due to extended manipulation prior to fixation (washing steps). Despite the fact that these protocols typically involve ice-cold physiological buffers that minimize membrane mobility, this is a reasonable possibility. Have the authors challenged their hypothesis by testing more typical protocols themselves? Other contributing factors that could play a role are the use of deconvolution, which tends to minimize weak signals, and also the fact that investigators tend to discount weak surface signals as background relative to stronger internal signals.

(5) Shedding: A central aspect of the GPI valence model (Schwartz et al., 2005, Tiengwe et al., 2017) is that GPI1 reporters that reach the cell body surface are shed into the media because a single dimyristoylglycerol-containing GPI anchor does not stably associate with biological membranes. As the authors point out, this is a major factor contributing to higher steady-state levels of cell-associated GPI2 TfR relative to GPI1 TfR. Those studies also found that the size/complexity of the attached protein correlated inversely with shedding, suggesting exit from the flagellar pocket as a restricting factor in cell body surface localization. The amount of newly synthesized TfR shed into the media was ~5%, indicating that very little actually exits the FP to the outer surface. In this regard, is it possible to know the overall ratio of cell surface:FP:endosomal localized receptors? Could these data not be 'harvested' from the 3D structural illumination imaging?

Reviewer #1 (Public review):

Summary:

In the manuscript by Winke et al, the authors present evidence that fear-induced analgesia is mediated by somatostatin projection cells from the vlPAG to the RVM. This study uses a mouse model of fear-induced analgesia, and incorporates optogenetic circuit manipulation with behaviour and electrophysiology to gain a meaningful insight into a novel circuit involved in fear-induced analgesia.

Strengths:

(1) This is a well-constructed study with appropriate controls and analyses.

(2) Alternative interpretations of the data are systematically considered and eliminated via rational experiments. The authors are commended for a nice piece of experimental work.

(3) The vlPAG is a known region of pain modulation, and this study adds valuable insight to the circuit involved in fear-associated analgesia.

Weaknesses:

(1) Only male mice are included in this study.

(2) Animals are excluded from analyses based on clearly defined criteria, but it is not clear how many mice were excluded from each group.

(3) The authors implement a pain sensitivity assay that involves a hot plate with progressively increasing temperature. The time to nociceptive responses is reported. Without reporting the actual temperature at which the mice respond, it makes it difficult to compare nociceptive responses to previously published work (which typically use a defined and static hotplate temperature).

(4) The authors present evidence that inhibition of SST vlPAG cells enhances spinal nociceptive electrophysiological responses, but the corresponding pain sensitivity is not altered (Figure 2, CS- condition). The reason for the discrepancy between electrophysiological and behavioural responses is not clear.

Reviewer #2 (Public review):

Summary:

Wenke et al. investigated the role of vlPAG somatostatin-expressing neurons in the mediation of analgesia during defensive states. A newly developed paradigm of cued fear-conditioned analgesia, which consists of a combination of an auditory fear retrieval session and a pain test, was used to evaluate this cell population's contribution to fear-mediated analgesia. Optogenetic manipulation of vlPAG SST+ neurons modulated the responses to a nociceptive cue (Hot Plate) presented concomitantly with an aversively conditioned tone. At the same time, alterations in the freezing levels could be observed during optogenetic activation of vlPAG SST+ neurons. In order to disentangle the impact of these cells on analgesia from their impact on the expression of defensive behaviors, the authors performed electrophysiological recordings from the dorsal horn in the spinal cord of anesthetized mice. A vlPAG-RVM-DH pathway was identified to trigger nociceptive C-fibers upon optic activation of the RVM. Finally, pathway-specific activation of SST+ vlPAG-RVM neurons could abolish CS-induced analgesia.

Strengths:

The study addresses a relevant topic, that is, brainstem circuits for pain-modulatory mechanisms as part of defensive states evoked by threat. This is important because the circuit mechanisms underlying pain are still not fully understood, and defining molecular markers of cellular circuit substrates may support the identification of potential pharmaceutical targets in treating pain. The authors confirm a previous study in that a somatostatin-positive cellular population presents a crucial vlPAG circuit element mediating anti-nociceptive effects. Key novelty aspects of the present study are the demonstration that these neurons seem to play a role specifically in threat-induced analgesia. This was possible by the elegant design and application of a novel fear analgesia paradigm, combined with cell- and pathway-specific optogenetics.

Weaknesses:

Despite the convincing and rigorous experimental approach, the study leaves some interpretational room when it comes to the proposed circuit mechanism. This could either be addressed by additional experiments or by more discussion of alternative circuit layouts.

Major Comments:

(1) The paper by Zhang et al. (https://pubmed.ncbi.nlm.nih.gov/36641028/), which identified a role for vlPAG SOM+ neurons in mediating anti-nociception in neuropathic pain, needs to be referenced and its results discussed, if not reconciled. While functionally, both studies find an analgetic role of vlPAG SOM+ neurons projecting to the RVM, Zhang et al., using slice physiology, characterize those neurons as glutamatergic. In Figure 4E of Zhang et al. they find general (fear-independent) analgetic effects with PAG-RVM specificity by performing chemogenetic experiments.

It can be argued that in addition to the two functionally distinct inhibitory SOM subtypes hypothesized by Winke et al., there is another, excitatory subpopulation. Also, the different experimental conditions (chronic vs. acute pain, non-threat vs. fearful cues/contexts may recruit different vlPAG SOM+ populations. All of this is conceivable, yet I wonder whether the contrasting findings could more parsimoniously be reconciled. The author's own results presented here in Supplementary Figure 3 suggests that SOM+ vlPAG cells are co-localizing with glutamate and thus could also be excitatory. In addition to this rather complementary piece of evidence, a more extensive characterization of vlPAG neurons using IHC and slice physiology would be needed to justify the unambiguous identification of their inhibitory nature.

In the absence of a direct identification of these cells exclusively releasing GABA, an alternative explanation should be considered. What about looking at vlPAG SOM+ neurons as a putatively mixed bag of local, inhibitory interneurons and long-range, RVM-projecting excitatory cells? This model would then open up interesting questions as to the actual function of somatostatin as a modulator of vlPAG circuit activity and associated function, and from my perspective, would nicely fit into the view of PAG circuits as integrators of complex survival responses.

(2) "Our data indicate that the optogenetic inhibition of SST+ vlPAG cells promotes analgesia irrespective of the animal's defensive state. In contrast, the optogenetic activation of long-range SST+ vlPAG cells that project to the rostral ventromedial medulla (RVM) abolishes the analgesia mediated by fear behavior." (lines 32-35). Consider toning down these conclusions, as contrasting activation with inhibition of two different (though overlapping) populations cannot be fully conclusive. Alternatively, a pathway-specific (vlPAG-RVM) inhibitory experiment could help to fully understand the circuit mechanism and verify the necessity of these neurons.

(3) Despite an overall very thorough reporting style, some information is missing from the manuscript:

a) In Figures 2d and f, what are the freezing levels during optogenetic manipulation? From Figure 3d, one can expect that freezing is inhibited during the hot plate test, which could bias the NC response towards shorter latencies. b) In Figure 5, the histological experiment showing the vlPAG-to-RVM pathway is presented by a qualitative image only. Here, some quantification would strengthen the finding. c) In Figures 6 c and d "Consistently, activation of the SST+ vlPAG-RVM pathway during CFCA had no impact on CS-presentation, whereas the same manipulation performed during CS+ blocked the increase in NC response latency compared to GFP controls." (line 194-196). Is it possible that the NC response cannot be any lower than the one during CS-, thus constituting a floor effect? d) Connected to major point 1- this experiment is important for defining the circuit mode and therefore should be as convincing as possible. However, for the colocalization experiment in Supplementary Figure 3, the methodological description is missing and thus makes it hard to comprehend how this data set was generated (how many data points, etc.). The visual depiction of the results is non-standard and not easily graspable. Consider e.g., a Venn diagram.

Reviewer #3 (Public review):

Summary:

Conditioned analgesia refers to the ability of a learned fear cue to suppress pain-related behavior and neural activity. Understudied, the authors developed a novel conditioned analgesia procedure in which a cue that had been paired or unpaired with shock was played while a hot plate increased temperature. Compared to several control conditions, the authors found increased latency to a nociceptive response (paw licking). The authors identified somatostatin neurons in the periaqueductal gray as a likely mediator of the behavior. They then showed that: (1) stimulating vlPAG-SST neurons blocked nociceptive response latency increases to the CS+, (2) stimulating vlPAG-SST neurons suppressed fear retrieval freezing, (3) stimulating vs. inhibiting vlPAG-SST neurons drove opposing modulation of c-fibers and Aδ-fibers, (4) direct-projecting vlPAG SST neurons modulate freezing while RVM-projecting vlPAG SST neurons modulate conditioned analgesia.