Reviewer #2 (Public review):

Summary:

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

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

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

Strengths:

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

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

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

Major Concerns:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

- The univariate analysis pipeline is solid and well developed.

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

Weaknesses:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

I have no major remaining concerns.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #2 (Public review):

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

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

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

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

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

Comments on revisions:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

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

Strengths:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

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

Reviewer #2 (Public review):

Strengths:

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

Weaknesses:

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

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

Reviewer #3 (Public review):

Summary

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

Strengths

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

Weaknesses

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The authors have addressed my concerns

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

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

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

Strengths:

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

There are no further recommendations for the authors.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Additionally, detailed comments are as follows:

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

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

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

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

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

Comments on revisions:

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Comments on revisions:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

No major weaknesses noted.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

I have mainly two questions for this work.

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

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

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

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

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

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

Reviewer #1 (Public review):

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

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

Comments on revisions:

The authors have adequately addressed my earlier comments and questions.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Comments on revisions:

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

Reviewer #3 (Public review):

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

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

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

Additional points:

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

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

Comments on revisions:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

Reviewer #1 (Public review):

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

Several specific points raised in previous reviews still require attention:

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

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

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

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

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

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

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

Reviewer #2 (Public review):

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

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

Reviewer #1 (Public review):

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

Strengths:

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

Opportunities:

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

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

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

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

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

Reviewer #2 (Public review):

General comment:

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

Comments on revised version:

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

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

Joint Public Review:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

The conclusions are supported by the results.

Reviewer #1 (Public review):

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Major suggestions

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

Data from cell lines is great and solid.

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #3 (Public review):

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses: Two minor comments

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

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

Reviewer #1 (Public review):

Public Review

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Major suggestions

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Reviewer #2 (Public review):

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

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

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

Reviewer #3 (Public review):

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary.

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

Strengths.

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

Weaknesses.

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

Reviewer #2 (Public review):

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

The major strengths of the work are described above.

Weaknesses:

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

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

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

Reviewer #2 (Public review):

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

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

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

Reviewer #1 (Public review):

Summary

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

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

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

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

Strength:

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

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

Weakness:

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

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

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

Reviewer #3 (Public review):

Summary

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

Strengths

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

Weaknesses

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

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

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #2 (Public review):

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

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

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

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

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

Reviewer #1 (Public review):

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

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

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

Strengths:

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

Reviewer #2 (Public review):

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

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

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

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

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

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

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

Reviewer #1 (Public review):

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

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

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

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

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

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

and

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Evaluations:

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

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

Reviewer #2 (Public review):

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

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

Comments on second revised version by editorial team:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

This analysis approach will prove to be useful for the C. elegans community. The application of the method on various age-related videos on various strains presents a good use-case for the approach. The manuscript is well written and presented.

Specific comments:

(1) One of the main motivations as mentioned in the introduction as well as emphasized in the discussion section is that this approach does not require key-point estimation for skeletonization and is also not dependent on the eigenworm approach for pose estimation. However, the eigenworm data has been estimated using the Tierpsy tracker in videos used in this work and stored as metadata. This is subsequently used for interpretation. It is not clear at this point, how else the spatial embedded map may be interpreted without using this kind of pose estimates obtained from other approaches. Please elaborate and comment.

(2) As per the manuscript, the second part of the pipeline is used to estimate the masked sequences of the spatiotemporal behavioral feature. However, it is not clear what the numbers listed in Fig. 2.3 represent?

(3) It is not clear how motion speed is linked to individual poses as mentioned in Figs. 4 (b) and (c).

Reviewer #2 (Public review):

Summary:

The manuscript by Maurice and Katarzyna describes a self-supervised, annotation-free deep-learning approach capable of quantitatively representing complex poses and behaviors of C. elegans directly from video pixel values. Their method overcomes limitations inherent to traditional methods relying on skeletonization or keypoint tracking, which often fail with highly coiled or self-intersecting worms. By applying self-supervised contrastive learning and a Transformer-based network architecture, the authors successfully capture diverse behavioral patterns and depict the aging trajectory of behavioral repertoire. This provides a useful new tool for behavioral research in C. elegans and other flexible-bodied organisms.

Strengths:

Reliable tracking and segmentation of complex poses remain significant bottlenecks in C. elegans behavioral research, and the authors made valuable attempts to address these challenges. The presented method offers several advantages over existing tools, including freedom from manual labeling, independence from explicit skeletonization or keypoint tracking, and the capability to capture highly coiled or overlapping poses. Thus, the proposed method would be useful to the C. elegans research community.

The research question is clearly defined. Methods and results are engagingly presented, and the manuscript is concise and well-organized.

Weaknesses:

(1) In the abstract, the claim of an 'unbiased' approach is not well-supported. The method is still affected by dataset biases, as mentioned in the aging results (section 4.3).<br /> (2) In section 3.2, the rationale behind rotating worm images to a vertical orientation is unclear.<br /> (3) The methods section is clearly written but uses overly technical language, making it less accessible to the audience of eLife, the majority of whom are biologists. Clearer explanations of key methods and the rationale behind their selection are needed. For example, in section 3.3, the authors should briefly explain in simple language what contrastive learning is, why they chose it, and why this method potentially achieves their goal.<br /> (4) The reason why the gray data points could not be resolved by Tierpsy is not quantitatively described. Are they all due to heavily coiled or overlapping poses?<br /> (5) In section 4.1, generating pose representations grouped by genetic strains would provide insights into strain-specific differences resolved by the proposed method.<br /> (6) Fig. 3a requires clarification. Highly bent poses (red points) intuitively should be close to highly coiled poses (gray points). The authors should explain the observed greenish/blueish points interfacing with the gray points.<br /> (7) In Fig. 3a, some colored points overlap with the gray point cloud. Why can Tierpsy resolve these overlapping points representing highly coiled poses? A more systematic quantitative comparison between Tierpsy and the proposed method is required.<br /> (8) The claim in section 4.2 regarding strain separation in pose embedding spaces is unsupported by Fig. 3a, which lacks strain-based distinctions. As mentioned in point #5, showing pose representations grouped by different strains is required.<br /> (9) In section 4.2, how the authors could verify the statement, "This likely occurs since most strains share common behaviors such as simple forward locomotion"?<br /> (10) An important weakness of the proposed method is its low direct interpretability, as it is not based on handcrafted features. To better interpret the pose/behavior embedding space, it would be helpful to compare it against more basic Tierpsy features in Fig. 3 and 4. This comparison could reveal what understandable features were learned by the neural network, thereby increasing human interpretability.<br /> (11) The main conclusion of section 4.3 is not sufficiently tested. Is Fig. 5a generated only from data of N2 animals? To quantitatively verify the statement, "Young individuals appear to display a wide range of behaviors, while as they age their behavior repertoire reduces," the authors should perform a formal analysis of behavioral variability throughout aging.<br /> (12) In Fig. 5a, better visualization of aging trajectories could include plotting the center of mass along with variance of the point cloud over time.<br /> (13) To better reveal aging trajectories of behavioral changes for different genetic backgrounds, it would be meaningful to generate behavior representations for different strains as they age.<br /> (14) As a methods paper, the ease of use for other researchers should be explicitly addressed, and source code and datasets should be provided.

Reviewer #3 (Public review):

Summary:

In this paper, the authors present an unsupervised learning approach to represent C. elegans poses and temporal sequences of poses in low-dimensional spaces by directly using pixel values from video frames. The method does not rely on the exact identification of the worm's contour/midline, nor on the identification of the head and tail prior to analyzing behavioral parameters. In particular, using contrastive learning, the model represents worm poses in low-dimensional spaces, while a transformer encoder neural network embeds sequences of worm postures over short time scales. The study evaluates this newly developed method using a dataset of different C. elegans genetic strains and aging individuals. The authors compared the representations inferred by the unsupervised learning with features extracted by an established approach, which relies on direct identification of the worm's posture and its head-tail direction.

Strengths:

The newly developed method provides a coarse classification of C. elegans posture types in a low-dimensional space using a relatively simple approach that directly analyzes video frames. The authors demonstrate that representations of postures or movements of different genotypes, based on pixel values, can be distinguishable to some extent.

Weaknesses:

- A significant disadvantage of the presented method is that it does not include the direction of the worm's body (e.g., head/tail identification). This highly limits the detailed and comprehensive identification of the worm's behavioral repertoire (on- and off-food), which requires body directionality in order to infer behaviors (for example, classifying forward vs. reverse movements). In addition, including a mix of opposite postures as input to the new method may create significant classification artifacts in the low-dimensional representation-such that, for example, curvature at opposite parts of the body could cluster together. This concern applies both to the representation of individual postures and to the representation of sequences of postures.<br /> - The authors state that head-tail direction can be inferred during forward movement. This is true when individuals are measured off-food, where they are highly likely to move forward. However, when animals are grown on food, head-tail identification can also be based on quantifying the speed of the two ends of the worm (the head shows side-to-side movements). This does not require identifying morphological features. See, for example, Harel et al. (2024) or Yemini et al. (2013).<br /> - Another confounding parameter that cannot be distinguished using the presented method is the size of individuals. Size can differ between genotypes, as well as with aging. This can potentially lead to clustering of individuals based on their size rather than behavior.<br /> - There is no quantitative comparison between classification based on the presented method and methods that rely on identifying the skeleton.

Reviewer #1 (Public review):

Summary:

Using a combination of EEG and behavioural measurements, the authors investigate the degree to which processing of spatially-overlapping targets (coherent motion) and distractors (affective images) are sampled rhythmically and how this affects behaviour. They found that both target processing (via measurement of amplitude modulations of SSVEP amplitude to target frequency) and distractor processing (via MVPA decoding accuracy of bandpassed EEG relative to distractor SSVEP frequency) displayed a pronounced rhythm at ~1Hz, time-locked to stimulus onset. Furthermore, the relative phase of this target/distractor sampling predicted the accuracy of coherent motion detection across participants.

Strengths:

(1) The authors are addressing a very interesting question with respect to sampling of targets and distractors, using neurophysiological measurements to their advantage in order to parse out target and distractor processing.

(2) The general EEG analysis pipeline is sensible and well-described.

(3) The main result of rhythmic sampling of targets and distractors is striking and very clear even on a participant level.

(4) The authors have gone to quite a lot of effort to ensure the validity of their analyses, especially in the Supplementary Material.

(5) It is incredibly striking how the phases of both target and distractor processing are so aligned across trials for a given participant. I would have thought that any endogenous fluctuation in attention or stimulus processing like that would not be so phase aligned. I know there is literature on phase resetting in this context, the results seem very strong here and it is worth noting. The authors have performed many analyses to rule out signal processing artifacts, e.g., the sideband and beating frequency analyses.

Weaknesses:

(1) In general, the representation of target and distractor processing is a bit of a reach. Target processing is represented by SSVEP amplitude, which is most likely going to be related to the contrast of the dots, as opposed to representing coherent motion energy, which is the actual target. These may well be linked (e.g., greater attention to the coherent motion task might increase SSVEP amplitude), but I would call it a limitation of the interpretation. Decoding accuracy of emotional content makes sense as a measure of distractor processing, and the supplementary analysis comparing target SSVEP amplitude to distractor decoding accuracy is duly noted.

(2) Comparing SSVEP amplitude to emotional category decoding accuracy feels a bit like comparing apples with oranges. They have different units and scales and probably reflect different neural processes. Is the result the authors find not a little surprising in this context? This relationship does predict performance and is thus intriguing, but I think this methodological aspect needs to be discussed further. For example, is the phase relationship with behaviour a result of a complex interaction between different levels of processing (fundamental contrast vs higher order emotional processing)?

Reviewer #2 (Public review):

Summary:

In this study, Xiong et al. investigate whether rhythmic sampling - a process typically observed in the attended processing of visual stimuli - extends to task-irrelevant distractors. By using EEG with frequency tagging and multivariate pattern analysis (MVPA), they aimed to characterize the temporal dynamics of both target and distractor processing and examine whether these processes oscillate in time. The central hypothesis is that target and distractor processing occur rhythmically, and the phase relationship between these rhythms correlates with behavioral performance.

Major Strengths:

(1) The extension of rhythmic attentional sampling to include distractors is a novel and interesting question.

(2) The decoding of emotional distractor content using MVPA from SSVEP signals is an elegant solution to the problem of assessing distractor engagement in the absence of direct behavioral measures.

(3) The finding that relative phase (between 1 Hz target and distractor processes) predicts behavioral performance is compelling.

Major Weaknesses and Limitations:

(1) Incomplete Evidence for Rhythmicity at 1 Hz: The central claim of 1 Hz rhythmic sampling is insufficiently validated. The windowing procedure (0.5s windows with 0.25s step) inherently restricts frequency resolution, potentially biasing toward low-frequency components like 1 Hz. Testing different window durations or providing controls would significantly strengthen this claim.

(2) No-Distractor Control Condition: The study lacks a baseline or control condition without distractors. This makes it difficult to determine whether the distractor-related decoding signals or the 1 Hz effect reflect genuine distractor processing or more general task dynamics.

(3) Decoding Near Chance Levels: The pairwise decoding accuracies for distractor categories hover close to chance (~55%), raising concerns about robustness. While statistically above chance, the small effect sizes need careful interpretation, particularly when linked to behavior.

(4) No Clear Correlation Between SSVEP and Behavior: Neither target nor distractor signal strength (SSVEP amplitude) correlates with behavioral accuracy. The study instead relies heavily on relative phase, which - while interesting - may benefit from additional converging evidence.

(5) Phase-analysis: phase analysis is performed between different types of signals hindering their interpretability (time-resolved SSVEP amplitude and time-resolved decoding accuracy).

Appraisal of Aims and Conclusions:

The authors largely achieved their stated goal of assessing rhythmic sampling of distractors. However, the conclusions drawn - particularly regarding the presence of 1 Hz rhythmicity - rest on analytical choices that should be scrutinized further. While the observed phase-performance relationship is interesting and potentially impactful, the lack of stronger and convergent evidence on the frequency component itself reduces confidence in the broader conclusions.

Impact and Utility to the Field:

If validated, the findings will advance our understanding of attentional dynamics and competition in complex visual environments. Demonstrating that ignored distractors can be rhythmically sampled at similar frequencies to targets has implications for models of attention and cognitive control. However, the methodological limitations currently constrain the paper's impact.

Additional Context and Considerations:

(1) The use of EEG-fMRI is mentioned but not leveraged. If BOLD data were collected, even exploratory fMRI analyses (e.g., distractor modulation in visual cortex) could provide valuable converging evidence.

(2) In turn, removal of fMRI artifacts might introduce biases or alter the data. For instance, the authors might consider investigating potential fMRI artifact harmonics around 1 Hz to address concerns regarding induced spectral components.

Reviewer #1 (Public review):

Summary:

Some years ago, Brookshire proposed a method to identify oscillations in behavioural data that controls for effects of aperiodic trends. Such trends can produce false positive results if not controlled for. Although this method successfully controlled for this issue, it was also relatively insensitive to true effects, and it remained unclear whether it was unable to replicate published evidence for behavioural oscillations because they were false positives or the method could not detect them. In simulated data, Harris & Beale show that their revised version of the method proposed by Brookshire is more sensitive to effects and equally unsusceptible to false positives. When applied to available data, this new version indeed revealed evidence for behavioural oscillations. This paper is therefore an important piece in the puzzle of the ongoing debate on behavioural oscillations.

Strengths:

(1) The paper is well written and compact.

(2) The new method proposed is tested thoroughly, and its application in simulated data shows its properties.

(3) It is very important that the code is made publicly available.

(4) The fact that this new version identifies behavioural oscillations in available datasets can resolve the current debate on the existence of such oscillations.

Weaknesses:

I see the following weaknesses as minor.

(1) I wonder whether the frequency-dependent results (e.g., Figures 7 and 8) need to be seen in light of the sampling rate used in the simulations. For example, a lower sampling rate might be sufficient if only low frequencies are of interest in the data and lead to higher sensitivity as the number of trials (per time point) can be increased. Conversely, a higher sampling rate might lead to a higher sensitivity for the detection of effects at higher frequencies.

(2) The behavioural oscillations from individual participants do not need to have common phases for this analysis to reveal an effect. However, this also means that in a scenario where they do have common phases, this similarity remains "unused" by the analysis (e.g., due to similar phases, the oscillation could be easier to identify on the group level as signals that are not phase locked are averaged out). In such a scenario, it remains unclear whether the analysis proposed is the most sensitive one.

Reviewer #2 (Public review):

Summary

Dozens of published studies have investigated rhythms in behavior. These studies have typically tested for oscillations by shuffling the timestamps of the individual observations and comparing the resulting shuffled spectra with the empirical spectrum. However, that shuffling-in-time method leads to strongly inflated rates of false positives. Brookshire (2022) suggested a method that controls the rate of false positives (the "AR-surrogate method"). In the current study, Harris and Beale propose a modification of the AR-surrogate analysis method with the goal of increasing the sensitivity while maintaining a low rate of false positives.

This study is carefully conducted and it addresses an interesting question. However, the simulations were performed in a way that ignores one important source of temporal structure: non-oscillatory patterns that are consistent across subjects. In order to know whether the updated AR-surrogate method would control the rate of false positives in real behavioral data, we need to know whether it controls the rate of false positives when the data includes aperiodic patterns that are consistent across subjects.

Strengths

This study was constructed carefully and written up very clearly. It's a clever idea to analyze the time series separately for each participant. After examining how the updated AR-surrogate method behaves when the simulated data includes consistency across subjects, this will be a useful contribution to the field.

Weaknesses

When describing their simulations of behavioral data, the authors write: "Each participant's data was produced by creating an independent idealised time-course of 1-second length, sampled at 60 Hz."

Because these simulations generated a totally independent time-course for every subject, they don't capture an important source of aperiodic structure in real behavior: consistent non-oscillatory patterns that occur across subjects. In other words, these simulations do not account for any pattern that remains after averaging across subjects. The literature is rich with patterns that persist across subjects, including all the studies of behavioral oscillations that analyze their data after averaging across subjects (e.g., Landau & Fries, 2012; Fiebelkorn, Saalmann, & Kastner, 2013, etc). As a consequence, I suspect that the reported increase in power comes at the expense of a corresponding increase in false positives, but that the false positives aren't captured here due to the lack of consistency across simulated subjects.

It's therefore possible that the authors' updated AR-surrogate method would mistakenly conclude that behavior oscillates when it only includes aperiodic consistency across subjects. Since that kind of aperiodic structure is ubiquitous, this analysis could lead to very high rates of false positives. Luckily, it's easy to find out whether this is the case - the authors could simulate data using an idealized time-course that is consistent across subjects.

Reviewer #3 (Public review):

Summary:

This work revises the autoregressive surrogate (AR-surrogate) method proposed in 2022 by Brookshire to estimate the oscillatory content of behavioural time series. The main issue raised by Brookshire was the inadequacy of methods used in a series of papers that rely on shuffling the time axis of the behavioural data. Brookshire argued that while this approach tests for temporal structures, it does not differentiate between 1/f activity and true oscillatory signals. The AR-surrogate, on the other hand, removes aperiodic activity and should therefore provide a more accurate representation of oscillatory behaviour.

In this well-written paper, Harris and Beale clearly describe an improvement to Brookshire's method, which has been called into question for its low sensitivity.

Strengths:

The starting point of this work is that oscillatory patterns should be tested at the individual participant level rather than at the group level. This is critical because anyone working with behavioural data will know that averaging across participants generates distorted time series. Averaging also assumes phase consistency across participants, which may not always be valid.

Once freed from this limitation, the results presented here are exciting and convincingly demonstrate a significant improvement over the original implementation.

The authors have devised a series of tests that systematically assess the effects of participant and trial number, and effect size on the accuracy of AR-surrogate results. This is particularly useful, as it may guide researchers in designing appropriate behavioural experiments.

Weaknesses:

The method proposed here is undoubtedly an improvement on the original. However, its biggest limitation is the restriction on the frequencies that can be investigated. This is acknowledged by the authors, who rightly point out that there is still room for improvement. Another issue is that modulation depths below 10-15% may be difficult to detect.

Reviewer #1 (Public review):

The authors note that very premature infants experience the visual world early and, as a consequence, sustain lasting deficits including compromised motion processing. Here they investigate the effects of early eye opening in ferret, choosing a time point after birth when both retinal waves and light traveling through closed lids drive sensory responses. The laboratory has long experience in quantitative studies of visual response properties across development and this study reflects their expertise.

The investigators find little or no difference in mean orientation and direction selectivity, or in spatial frequency tuning, as a result of early eye opening but marked differences in temporal frequency tuning. These changes are especially interesting as they relate to deficits seen in prematurely delivered children. Temporal frequency bandwidth for responses evoked from early-opened contralateral eyes were broader than for controls; this is the case for animals in which either one or both eyes were opened prematurely. Further, when only one eye was opened early, responses to low temporal frequencies were relatively stronger.

The investigators also found changes in firing rate and signs of response to visual stimuli. Premature eye-opening increased spontaneous rates in all test configurations. When only one eye was opened early, firing rates recorded from the ipsilateral cortex were strongly suppressed, with more modest effects in other test cases.

As the authors' discussion notes, these observations are just a starting point for studies underlying mechanism. The experiments are so difficult to perform and so carefully described that the results will be foundational for future studies of how premature birth influences cortical development.

Reviewer #2 (Public review):

In this paper, Griswold and Van Hooser investigate what happens if animals are exposed to patterned visual experience too early, before its natural onset. To this end, they make use of the benefits of the ferret as a well-established animal model for visual development. Ferrets naturally open their eyes around postnatal day 30; here, Griswold and Van Hooser opened either one or both eyes prematurely. Subsequent recordings in the mature primary visual cortex show that while some tuning properties like orientation and direction selectivity developed normally, the premature visual exposure triggered changes in temporal frequency tuning and overall firing rates. These changes were widespread, in that they occurred even for neurons responding to the eye that was not opened prematurely. These results demonstrate that the nature of the visual input well before eye opening can have profound consequences on the developing visual system.

The conclusions of this paper are well supported by the data, but some aspects of the data could be clarified, and the discussion could be extended.

(1) The assessment of the tuning properties is based on fits to the data. Presumably, neurons for which the fits were poor were excluded? It would be useful to know what the criteria were, how many neurons were excluded, and whether there was a significant difference between the groups in the numbers of neurons excluded (which could further point to differences between the groups).

(2) For the temporal frequency data, low- and high-frequency cut-offs are defined, but then only used for the computation of the bandwidth. Given that the responses to low temporal frequencies change profoundly with premature eye opening, it would be useful to directly compare the low- and high-frequency cut-offs between groups, in addition to the index that is currently used.

(3) In addition to the tuning functions and firing rates that have been analyzed so far, are there any differences in the temporal profiles of neural responses between the groups (sustained versus transient responses, rates of adaptation, latency)? If the temporal dynamics of the responses are altered significantly, that could be part of an explanation for the altered temporal tuning.

(4) It would be beneficial for the general interpretation of the results to extend the discussion. First, it would be useful to provide a more detailed discussion of what type of visual information might make it through the closed eyelids (the natural state), in contrast to the structured information available through open eyes. Second, it would be useful to highlight more clearly that these data were collected in peripheral V1 by discussing what might be expected in binocular, more central V1 regions. Third, it would be interesting to discuss the observed changes in firing rates in the context of the development of inhibitory neurons in V1 (which still undergo significant changes through the time period of premature visual experience chosen here).

Reviewer #1 (Public review):

Summary:

This computational modeling study builds on multiple previous lines of experimental and theoretical research to investigate how a single neuron can solve a nonlinear pattern classification task. The authors construct a detailed biophysical and morphological model of a single striatal medium spiny neuron, and endow excitatory and inhibitory synapses with dynamic synaptic plasticity mechanisms that are sensitive to (1) the presence or absence of a dopamine reward signal, and (2) spatiotemporal coincidence of synaptic activity in single dendritic branches. The latter coincidence is detected by voltage-dependent NMDA-type glutamate receptors, which can generate a type of dendritic spike referred to as a "plateau potential." In the absence of inhibitory plasticity, the proposed mechanisms result in good performance on a nonlinear classification task when specific input features are segregated and clustered onto individual branches, but reduced performance when input features are randomly distributed across branches. Interestingly, adding inhibitory plasticity improves classification performance even when input features are randomly distributed.

Strengths:

The integrative aspect of this study is its major strength. It is challenging to relate low-level details such as electrical spine compartmentalization, extrasynaptic neurotransmitter concentrations, dendritic nonlinearities, spatial clustering of correlated inputs, and plasticity of excitatory and inhibitory synapses to high-level computations such as nonlinear feature classification. Due to high simulation costs, it is rare to see highly biophysical and morphological models used for learning studies that require repeated stimulus presentations over the course of a training procedure. The study aspires to prove the principle that experimentally-supported biological mechanisms can explain complex learning.

Weaknesses:

The high level of complexity of each component of the model makes it difficult to gain an intuition for which aspects of the model are essential for its performance, or responsible for its poor performance under certain conditions. Stripping down some of the biophysical detail and comparing it to a simpler model may help better understand each component in isolation.

Reviewer #2 (Public review):

Summary:

The study explores how single striatal projection neurons (SPNs) utilize dendritic nonlinearities to solve complex integration tasks. It introduces a calcium-based synaptic learning rule that incorporates local calcium dynamics and dopaminergic signals, along with metaplasticity to ensure stability for synaptic weights. Results show SPNs can solve the nonlinear feature binding problem and enhance computational efficiency through inhibitory plasticity in dendrites, emphasizing the significant computational potential of individual neurons. In summary, the study provides a more biologically plausible solution to single-neuron learning and gives further mechanical insights into complex computations at the single-neuron level.

Strengths:

The paper introduces a novel learning rule for training a single multicompartmental neuron model to perform nonlinear feature binding tasks (NFBP), highlighting two main strengths: the learning rule is local, calcium-based, and requires only sparse reward signals, making it highly biologically plausible, and it applies to detailed neuron models that effectively preserve dendritic nonlinearities, contrasting with many previous studies that use simplified models.

Reviewer #1 (Public review):

Summary:

In this study, Jeong and Choi examine neural correlates of behavior during a naturalistic foraging task in which rats must dynamically balance resource acquisition (foraging) with the risk of threat. Rats first learn to forage for sucrose reward from a spout, and when a threat is introduced (an attack-like movement from a "LobsterBot"), they adjust their behavior to continue foraging while balancing exposure to the threat, adopting anticipatory withdraw behaviors to avoid encounter with the LobsterBot. Using electrode recordings targeting the medial prefrontal cortex (PFC), they identify heterogenous encoding of task variables across prelimbic and infralimbic cortex neurons, including correlates of distance to the reward/threat zone and correlates of both anticipatory and reactionary avoidance behavior. Based on analysis of population responses, they show that prefrontal cortex switches between different regimes of population activity to process spatial information or behavioral responses to threat in a context-dependent manner. Characterization of the heterogenous coding scheme by which frontal cortex represents information in different goal states is an important contribution to our understanding of brain mechanisms underlying flexible behavior in ecological settings.

Strengths:

As many behavioral neuroscience studies employ highly controlled task designs, relatively less is generally known about how the brain organizes navigation and behavioral selection in naturalistic settings, where environment states and goals are more fluid. Here, the authors take advantage of a natural challenge faced by many animals - how to forage for resources in an unpredictable environment - to investigate neural correlates of behavior when goal states are dynamic. Related to his, they also investigate prefrontal cortex (PFC) activity is structured to support different functional "modes" (here, between a navigational mode and a threat-sensitive foraging mode) for flexible behavior. Overall, an important strength and real value of this study is the design of the behavioral experiment, which is trial-structured, permitting strong statistical methods for neural data analysis, yet still rich enough to encourage natural behavior structured by the animal's volitional goals. The experiment is also phased to measure behavioral changes as animals first encounter a threat, and then learn to adapt their foraging strategy to its presence. Characterization of this adaptation process is itself quite interesting and sets a foundation for further study of threat learning and risk management in the foraging context. Finally, the characterization of single-neuron and population dynamics in PFC in this naturalistic setting with fluid goal states is an important contribution to the field. Previous studies have identified neural correlates of spatial and behavioral variables in frontal cortex, but how these representations are structured, or how they are dynamically adjusted when animals shift their goals, has been less clear. The authors synthesize their main conclusions into a conceptual model for how PFC activity can support mode switching, which can be tested in future studies with other task designed and functional manipulations.

Weaknesses:

While the task design in this study is intentionally stimulus-rich and places minimal constraint on the animal to preserve naturalistic behavior, this also introduces confounds that limit interpretability of the neural analysis. For example, some variables which are the target of neural correlation analysis, such as spatial/proximity coding and coding of threat and threat-related behaviors, are naturally entwined. To their credit, the authors have included careful analyses and control conditions to disambiguate these variables and significantly improve clarity.

The authors also claim that the heterogenous coding of spatial and behavioral variables in PFC is structured in a particular way that depends on the animal's goals or context. As the authors themselves discuss, the different "zones" contain distinct behaviors and stimuli, and since some neurons are modulated by these events (e.g., licking sucrose water, withdrawing from the LobsterBot, etc.), differences in population activity may to some extent reflect behavior/event coding. The authors have included a control analysis, removing timepoints corresponding to salient events, to substantiate the claim that PFC neurons switch between different coding "modes." While this significantly strengthens evidence for their conclusion, this analysis still depends on relatively coarse labeling of only very salient events. Future experiment designs, which intentionally separate task contexts (e.g. navigation vs. foraging), could serve to further clarify the structure of coding across contexts and/or goal states.

Finally, while the study includes many careful, in-depth neural and behavioral analyses to support the notion that modal coding of task variables in PFC may play a role in organizing flexible, dynamic behavior, the study still lacks functional manipulations to establish any form of causality. This limitation is acknowledged in the text, and the report is careful not to over interpret suggestions of causal contribution, instead setting a foundation for future investigations.

Reviewer #2 (Public review):

Summary:

Jeong & Choi (2023) use a semi-naturalistic paradigm to tackle the question of how the activity of neurons in the mPFC might continuously encode different functions. They offer two possibilities: either there are separate dedicated populations encoding each function, or cells alter their activity dependent on the current goal of the animal. In a threat-avoidance task rats procurred sucrose in an area of a chamber where, after remaining there for some amount of time, a 'Lobsterbot' robot attacked. In order to initiate the next trial rats had to move through the arena to another area before returning to the robot encounter zone. Therefore the task has two key components: threat avoidance and navigating through space. Recordings in the IL and PL of the mPFC revealed encoding that depended on what stage of the task the animal was currently engaged in. When animals were navigating, neuronal ensembles in these regions encoded distance from the threat. However, whilst animals were directly engaged with the threat and simultaneously consuming reward, it was possible to decode from a subset of the population whether animals would evade the threat. Therefore the authors claim that neurons in the mPFC switched between two functional modes: representing allocentric spatial information, and representing egocentric information pertaining to the reward and threat. Finally, the authors propose a conceptual model based on these data whereby this switching of population encoding is driven by either bottom-up sensory information or top-down arbitration.

Strengths:

Whilst these multiple functions of activity in the mPFC have generally been observed in tasks dedicated to the study of a singular function, less work has been done in contexts where animals continuously switch between different modes of behaviour in a more natural way. Being able to assess whether previous findings of mPFC function apply in natural contexts is very valuable to the field, even outside of those interested in the mPFC directly. This also speaks to the novelty of the work; although mixed selectivity encoding of threat assessment and action selection has been demonstrated in some contexts (e.g. Grunfeld & Likhtik, 2018) understanding the way in which encoding changes on-the-fly in a self-paced task is valuable both for verifying whether current understanding holds true and for extending our models of functional coding in the mPFC.

The authors are also generally thoughtful in their analyses and use a variety of approaches to probe the information encoded in the recorded activity. In particular, they use relatively close analysis of behaviour as well as manipulating the task itself by removing the threat to verify their own results. The use of such a rich task also allows them to draw comparisons, e.g. in different zones of the arena or different types of responses to threat, that a more reduced task would not otherwise allow. Additional in-depth analyses in the updated version of the manuscript, particularly the feature importance analysis, as well as complimentary null findings (a lack of cohesive place cell encoding, and no difference in location coding dependent on direction of trajectory) further support the authors' conclusion that populations of cells in the mPFC are switching their functional coding based on task context rather than behaviour per se. Finally, the authors' updated model schematic proposes an intriguing and testable implementation of how this encoding switch may be manifested by looking at differentiable inputs to these populations.

Weaknesses:

The main existing weakness of this study is that its findings are correlational (as the authors highlight in the discussion). Future work might aim to verify and expand the authors' findings - for example, whether the elevated response of Type 2 neurons directly contributes to the decision-making process or just represents fear/anxiety motivation/threat level - through direct physiological manipulation. However, I appreciate the challenges of interpreting data even in the presence of such manipulations and some of the additional analyses of behaviour, for example the stability of animals' inter-lick intervals in the E-zone, go some way towards ruling out alternative behavioural explanations. Yet the most ideal version of this analysis is to use a pose estimation method such as DeepLabCut to more fully measure behavioural changes. This, in combination with direct physiological manipulation, would allow the authors to fully validate that the switching of encoding by this population of neurons in the mPFC has the functional attributes as claimed here.

Reviewer #3 (Public review):

Summary:

This study investigates how various behavioral features are represented in the medial prefrontal cortex (mPFC) of rats engaged in a naturalistic foraging task. The authors recorded electrophysiological responses of individual neurons as animals transitioned between navigation, reward consumption, avoidance, and escape behaviors. Employing a range of computational and statistical methods, including artificial neural networks, dimensionality reduction, hierarchical clustering, and Bayesian classifiers, the authors sought to predict from neural activity distinct task variables (such as distance from the reward zone and the success or failure of avoidance behavior). The findings suggest that mPFC neurons alternate between at least two distinct functional modes, namely spatial encoding and threat evaluation, contingent on the specific location.

Strengths:

This study attempt to address an important question: understanding the role of mPFC across multiple dynamic behaviors. The authors highlight the diverse roles attributed to mPFC in previous literature and seek to explain this apparent heterogeneity. They designed an ethologically relevant foraging task that facilitated the examination of complex dynamic behavior, collecting comprehensive behavioral and neural data. The analyses conducted are both sound and rigorous.

Weaknesses:

Because the study still lacks experimental manipulation, the findings remain correlational. The authors have appropriately tempered their claims regarding the functional role of the mPFC in the task. The nature of the switch between functional modes encoding distinct task variables (i.e., distance to reward, and threat-avoidance behavior type) is not established. Moreover, the evidence presented to dissociate movement from these task variables is not fully convincing, particularly without single-session video analysis of movement. Specifically, while the new analyses in Figure 7 are informative, they may not fully account for all potential confounding variables arising from changes in context or behavior.

Joint Public Review:

Editors’ note: This is the third version of this article, and it addresses the points made during the peer review of the second version by performing additional analyses and clarifying some of the limitations of the study.

Comments made during the peer review of the first version, along with author's responses to these comments, are available with previous versions of the article.

The following summary of the article is taken from comments made by Reviewer #1 about version 2 of the article:

In this manuscript, the authors use a large dataset of neuroscience publications to elucidate the nature of self-citation within the neuroscience literature. The authors initially present descriptive measures of self-citation across time and author characteristics; they then produce an inclusive model to tease apart the potential role of various article and author features in shaping self-citation behavior. This is a valuable area of study, and the authors approach it with a rich dataset and solid methodology.

Reviewer #1 (Public review):

Summary:

This study seeks to investigate the role of the transcription factor Bcl11b/Ctip2 in regulating subcerebral projection neuron (SCPN) axon development through both cell-autonomous and non-cell-autonomous mechanisms. The authors demonstrate that Bcl11b is required within SCPNs for axonal outgrowth and proper entry into the internal capsule, while its expression in medium spiny neurons (MSNs) influences SCPN axon fasciculation and pathfinding in a non-cell-autonomous manner. Notably, through transcriptomic analysis, immunocytochemistry, and in vivo growth cone purification, the study identifies Cdh13 as a downstream mediator of Bcl11b function, localizing along axons and at growth cone surfaces to regulate SCPN axonal outgrowth.

Strengths:

To me the most interesting aspect of this study is how common transcriptional programs across neuronal cell types cooperate to facilitate axon pathfinding, this is a very interesting concept.

Overall, it could be of interest to the brain development field.

Weaknesses:

My main concern is that, as presented in the figures, many phenotypes are too subtle to be convincing and would require quantitative analyses to corroborate the claims of the study.

I also think that the growth cones transcription data needs additional validation to be incorporated into the manuscript. In fact, I am not even sure that it really brings anything to the story.

I also think that the CRISPR in utero electroporation experiments lack appropriate controls.

Reviewer #2 (Public review):

Summary:

Itoh et al. investigate the role of the zinc finger transcription factor Bcl11b/Citp2 on sub cerebral projection neurons (SCPN) development. They dissect Bcl11b cell-autonomous and non-cell-autonomous functions on subcerebral projection neurons. In addition, they identify Cdh13 as a downstream target of Bcl11b in the process of SCPN axon outgrowth.

Strengths:

Itoh et al. take advantage of a mouse CRE/Lox genetic system as a powerful tool to distinguish Bcl11b cell-autonomous function on cortical layer V subcerebral projection neurons and its non-cell-autonomous function mediated by the striatal medium spiny neurons (MSN).

Besides the description of the cellular and anatomical defects of the corticofugal projection neurons' outgrowth and fasciculation, they perform a transcriptomic analysis of SCPN somata to identify Bcl11b target genes. As a result, they find that Cdh13, a membrane-anchored cadherin , is downstream of Bcl11b and mediates its cell-autonomous role on axon outgrowth. To validate the role of Cdh13 as a mediator of Bcl11b on SCPN development, they set up a new technique to identify and quantify superficial antigens on growth cone membranes.

Weaknesses:

While the authors shed light on the role of Bcl11b on SCPN development, they lack to contextualize their findings on the previously described interplay between Bcl11a and b.<br /> In addition, this work is another example of the common practice of picking from a list of differentially expressed genes the most likely ones. This approach, while useful, does not allow the identification of new and unknown players.

Reviewer #1 (Public review):

Summary:

Nysten et al. use in vivo 2-photon calcium imaging in behaving mice learning a visual associative memory task to understand how neural dynamics in the postrhinal cortex and medial entorhinal cortex evolve over task learning and through reversal learning. Using a combination of analyses to measure trial-averaged neural responses, regression models, and population decoding methods, the authors argue that both POR and MEC dynamics evolve over learning, with relatively more neurons in MEC becoming responsive. The impact of this study comes from comparing neural dynamics across multiple medial temporal lobe circuits to show how different aspects of task structure are differentially encoded. Below, I have listed several major concerns that need to be addressed to ensure the findings are robust.

Strengths:

(1) The study employs a well-controlled behavioral paradigm alongside powerful cellular-resolution two-photon imaging, enabling high-throughput recordings of hundreds of neurons simultaneously in deep brain structures.

(2) The simplicity of the task allows for a detailed examination of learning dynamics across multiple stages, including early and late learning in the main task, as well as during reversal learning.

(3) The use of sophisticated analysis methods to compare and contrast learning dynamics in large neuronal populations strengthens the study, though additional steps are needed to ensure their robustness (detailed below).

(4) Two-photon imaging enables the investigation of functional topography, further supporting previous findings of functional clustering in MEC across different task and behavioral domains.

Weaknesses:

(1) GLM Robustness & Behavioral Attribution: The current GLM design may misattribute neural activity by lacking appropriate time lags for velocity and not accounting for distinct neural states (e.g., rest vs. run). Given MEC's known speed-invariant coding, the observed decrease in speed-modulated neurons may be an artifact rather than a true learning effect. Additionally, gradual behavioral stabilization over training could influence neural dynamics in ways not fully accounted for.

(2) Licking vs. Movement Encoding: The increase in lick-modulated neurons raises questions about whether these neurons encode reward anticipation or motor execution. Without a detailed analysis of error trials and the timing of licking vs. movement adjustments, it remains unclear whether MEC activity reflects predictive coding of reward or simply motor feedback.

(3) Clustering Interpretation Issues: The functional clustering approach does not control for correlations between behavioral features, making it difficult to determine whether speed modulation plays a role in cluster assignments. The anatomical analysis in Figure 6 relies heavily on clusters that may be predominantly defined by a single regressor, requiring further clarification.

(4) Data Presentation & Statistical Support: Some key claims, particularly the increase in task-modulated neurons with learning (Figure 3), lack statistical quantification.

Reviewer #2 (Public review):

Summary:

The authors examine medial entorhinal cortex (MEC) and postrhinal cortex (POR) responses using Ca imaging during a non-spatial, Go/No-Go visual association task. The authors specifically consider whether MEC encodes stimulus information, as previously seen and hypothesized in POR, as well as other task elements such as reward, and whether and how these responses evolve with learning in both regions. The authors find that, in general, POR encodes task-related information more strongly compared to MEC. In particular, POR encodes the stimulus even before the animal reaches expert performance, whereas MEC shows considerably weaker stimulus encoding that emerges with learning. Both regions also display licking-related coding, although notably this activity reflects choice or licking-preparation, which emerges with learning. Further, despite its overall reduced coding, MEC exhibits greater anatomical clustering of cells with similar functional properties compared to POR.

Strengths:

These data are generally well-presented, both in the description of the experimental paradigm - which is simple yet highly informative - and in the individual results for each section. A major strength is the dataset, which includes many cells, including a subset that are tracked across learning. I found the core findings - (1) that POR has robust stimulus encoding while MEC develops weaker stimulus information with learning, and (2) that both POR and MEC exhibit an increase in lick-modulated cells, although POR has more, and stronger, lick-modulated cells - to be generally well-supported by the data presented. The general question of whether and how MEC encodes non-spatial task-relevant features and how these responses (if they exist) emerge with learning is of general interest. In addition, how MEC activity contrasts with activity in an upstream region, thereby indicating what information MEC gets and what it does with it, is also of broad interest.

Weaknesses:

I perceived two primary weaknesses.

The first was that it was not entirely clear to me what was expected of MEC and POR responses, and whether the observations the authors made were surprising or entirely in line with what would've been predicted based on prior work. In some ways, the results seem expected - POR had visual signals, MEC had few visual signals but some reward signals.

The second is that it took me a long time to extract what I perceived to be the core results of the paper, and in some places, it was a little hard for me to understand all the analyses and results together as one cohesive step forward in our understanding of MEC and POR coding properties.

I think this was most evident in the results presented in Figure 4. Up until Figure 4, it seemed to me that the core results were:<br /> (1) visual (stimulus information) is present in POR responses from very early learning, whereas weak stimulus information develops in MEC with learning, and in both cases, there is a preference for the plus stimulus.<br /> (2), both POR and MEC show an increase in lick-modulated cells with learning, although more cells encode licking at all stages in POR.

This is nicely summarized in my view by Figure 3e. However, I became confused when Figure 4 entered the picture. Here, it seems that by far the most predominant coefficient in the model is the lick response, with stimulus features playing a smaller role - specifically, at the end of learning, 60% of POR cells were characterized as predominantly lick/non lick, compared to 25% defined by their coding to the stimulus. I can appreciate that there might be nuances to these and previous analyses such that all the results sit cohesively together, but I think that needs to be clarified.

A second example - Figure 2b - shows that many (75%) of MEC neurons seem to be selectively active for the plus stimulus, but when doing the GLM analysis with the plus stimulus (and reward/licking) as features, many fewer neurons (35%) are determined to be encoding task information. It was not clear to me what was contributing to the discrepancy between these two results - is it that MEC activity often increases with learning, but doesn't increase by that much?

I think in general this can be helped by specifically pointing out how the results of these different analyses relate to each other, including specifically mentioning where the results might seem unaligned (at least on the surface).

Reviewer #1 (Public review):

Summary:

The manuscript reports that expression of the E. coli operon topAI/yjhQ/yjhP is controlled by the translation status of a small open reading frame, that authors have discovered and named toiL, located in the leader region upstream of the operon. Authors propose the following model for topAI activation: Under normal conditions, toiL is translated but topAI is not expressed because of Rho-dependent transcription termination within the topAI ORF and because its ribosome binding site and start codon are trapped in an mRNA hairpin. Ribosome stalling at various codons of the toiL ORF, prompted in this work by some ribosome-targeting antibiotics, triggers an mRNA conformational switch which allows translation of topAI and, in addition, activation of the operon's transcription because presence of translating ribosomes at the topAI ORF blocks Rho from terminating transcription. The model is appealing and several of the experimental data mainly support it. However, it remains unanswered what is the true trigger of the translation arrest at toiL and what is the physiological role of the induced expression of the topAI/yjhQ/yjhP operon.

Reviewer #2 (Public review):

Summary:

Baniulyte and Wade describe how translation of an 8-codon uORF denoted toiL upstream of the topAI-yjhQP operon is responsive to different ribosome-targeting antibiotics, consequently controlling translation of the TopAI toxin as well as Rho-dependent termination with the gene.

Strengths:

The authors used multiple different approaches such as a genetic screen to identify factors such as 23S rRNA mutations that affect topA1 expression and ribosome profiling to examine the consequences of various antibiotics on toiL-mediated regulation.

Weaknesses: Future experiments will be needed to better understand the physiological role of the toiL-mediated regulation and elucidate the mechanism of specific antibiotic sensing.

The results are clearly described, and the revisions have helped to improve the presentation of the data.

Reviewer #3 (Public review):

In this revised manuscript, the authors provide convincing data to support an elegant model in which ribosome stalling by ToiL promotes downstream topAI translation and prevents premature Rho-dependent transcription termination. However, the physiological consequences of activating topAI-yjhQP expression upon exposure to various ribosome-targeting antibiotics remain unresolved. The authors have satisfactorily addressed all major concerns raised by the reviewers, particularly regarding the SHAPE-seq data. Overall, this study underscores the diversity of regulatory ribosome-stalling peptides in nature, highlighting ToiL's uniqueness in sensing multiple antibiotics and offering significant insights into bacterial gene regulation coordinated by transcription and translation.

Reviewer #1 (Public review):

The manuscript consists of two separate but interlinked investigations: genomic epidemiology and virulence assessment of Salmonella Dublin. ST10 dominates the epidemiological landscape of S. Dublin, while ST74 was uncommonly isolated. Detailed genomic epidemiology of ST10 unfolded the evolutionary history of this common genotype, highlighting clonal expansions linked to each distinct geography. Notably, North American ST10 was associated with more antimicrobial resistance compared to others. The authors also performed long read sequencing on a subset of isolates (ST10 and ST74), and uncovered a novel recombinant virulence plasmid in ST10 (IncX1/IncFII/IncN). Separately, the authors performed cell invasion and cytotoxicity assays on the two S. Dublin genotypes, showing differential responses between the two STs. ST74 replicates better intracellularly in macrophage compared to ST10, but both STs induced comparable cytotoxicity levels. Comparative genomic analyses between the two genotypes showed certain genetic content unique to each genotype, but no further analyses were conducted to investigate which genetic factors likely associated with the observed differences. The study provides a comprehensive and novel understanding on the evolution and adaptation of two S. Dublin genotypes, which can inform public health measures. The methodology included in both approaches were sound and written in sufficient detail, and data analysis were performed with rigour. Source data were fully presented and accessible to readers.

Comments on revised version:

The authors have addressed all the points raised by the reviewer. The manuscript is now much enhanced in clarity and accuracy. The rewritten Discussion is more relevant and brings in comparison with other invasive Salmonella serotypes.

Reviewer #2 (Public review):

This is a comprehensive analysis of Salmonella Dublin genomes that offers insights into the global spread of this pathogen and region-specific traits that are important to understand its evolution. The phenotyping of isolates of ST10 and ST74 also offer insights into the variability that can be seen in S. Dublin, which is also seen in other Salmonella serovars, and reminds the field that it is important to look beyond lab-adapted strains to truly understand these pathogens. This is a valuable contribution to the field. The only limitation, which the authors also acknowledge, is the bias towards S. Dublin genomes from high-income settings. However, there is no selection bias; this is simply a consequence of publicly available sequences.

Reviewer #1 (Public review):

Summary:

This work uses a novel, ethologically relevant behavioral task to explore decision-making paradigms in C. elegans foraging behavior. By rigorously quantifying multiple features of animal behavior as they navigate in a patch food environment, the authors provide strong evidence that worms exhibit one of three qualitatively distinct behavioral responses upon encountering a patch: (1) "search", in which the encountered patch is below the detection threshold; (2) "sample", in which animals detect a patch encounter and reduce their motor speed, but do not stay to exploit the resource and are therefore considered to have "rejected" it; and (3) "exploit", in which animals "accept" the patch and exploit the resource for tens of minutes. Interestingly, the probability of these outcomes varies with the density of the patch as well as the prior experience of the animal. Together, these experiments provide an interesting new framework for understanding the ability of the C. elegans nervous system to use sensory information and internal state to implement behavioral state decisions.

Strengths:

-The work uses a novel, neuroethologically-inspired approach to studying foraging behavior<br /> -The studies are carried out with an exceptional level of quantitative rigor and attention to detail<br /> -Powerful quantitative modeling approaches including GLMs are used to study the behavioral states that worms enter upon encountering food, and the parameters that govern the decision about which state to enter<br /> -The work provides strong evidence that C. elegans can make 'accept-reject' decisions upon encountering a food resource<br /> -Accept-reject decisions depend on the quality of the food resource encountered as well as on internally represented features that provide measurements of multiple dimensions of internal state, including feeding status and time

Reviewer #2 (Public review):

This study provides an experimental and computational framework to examine and understand how C. elegans make decisions while foraging environments with patches of food. The authors show that C. elegans reject or accept food patches depending on a number of internal and external factors.

The key novelty of this paper is the explicit demonstration of behavior analysis and quantitative modeling to elucidate decision-making processes. In particular, the description of the exploring vs. exploiting phases, and sensing vs. non-sensing categories of foraging behavior based on the clustering of behavioral states defined in a multi-dimensional behavior-metrics space, and the implementation of a generalized linear model (GLM) whose parameters can provide quantitative biological interpretations.

The work builds on the literature of C. elegans foraging by adding the reject/accept framework.

Reviewer #3 (Public review):

Summary:

In this study by Haley et al, the authors investigated explore-exploit foraging using C. elegans as a model system. Through an elegant set of patchy environment assays, the authors built a GLM based on past experience that predicts whether an animal will decide to stay on a patch to feed and exploit that resource, instead of choosing to leave and explore other patches.

Strengths:

I really enjoyed reading this paper. The experiments are simple and elegant, and address fundamental questions of foraging theory in a well-defined system. The experimental design is thoroughly vetted, and the authors provide a considerable volume of data to prove their points. My only criticisms have to do with the data interpretation, which I think are easily addressable.

Weaknesses:

History-dependence of the GLM

The logistic GLM seems like a logical way to model a binary choice, and I think the parameters you chose are certainly important. However, the framing of them seem odd to me. I do not doubt the animals are assessing the current state of the patch with an assessment of past experience; that makes perfect logical sense. However, it seems odd to reduce past experience to the categories of recently exploited patch, recently encountered patch, and time since last exploitation. This implies the animals have some way of discriminating these past patch experiences and committing them to memory. Also, it seems logical that the time on these patches, not just their density, should also matter, just as the time without food matters. Time is inherent to memory. This model also imposes a prior categorization in trying to distinguish between sensed vs. not-sensed patches, which I criticized earlier. Only "sensed" patches are used in the model, but it is questionable whether worms genuinely do not "sense" these patches.

It seems more likely that the worm simply has some memory of chemosensation and relative satiety, both of which increase on patches and decrease while off of patches. The magnitudes are likely a function of patch density. That being said, I leave it up to the reader to decide how best to interpret the data.

osm-6

The argument is that osm-6 animals can't sense food very well, so when they sense it, they enter the exploitation state by default. That is what they appear to do, but why? Clearly they are sensing the food in some other way, correct? Are ciliated neurons the only way worms can sense food? Don't they also actively pump on food, and can therefore sense the food entering their pharynx? I think you could provide further insight by commenting on this. Perhaps your decision model is dependent on comparing environmental sensing with pharyngeal sensing? Food intake certainly influences their decision, no? Perhaps food intake triggers exploitation behavior, which can be over-run by chemo/mechanosensory information?

Impact:

I think this work will have a solid impact on the field, as it provides tangible variables to test how animals assess their environment and decide to exploit resources. I think the strength of this research could be strengthened by a reassessment of their model that would both simplify it and provide testable timescales of satiety/starvation memory.

Reviewer #1 (Public review):

Summary:

The use of a multi-omics approach to elucidate the regulatory mechanism underlying parturition and myometrial quiescence adds novelty to the study. The identification of myometrial cis-acting elements and their association with gene expression, particularly the regulation of the PLCL2 gene by PGR opens the door to further investigate the impact of PGR and other regulators.

Strengths:

(1) Multi-Omic Approach: The paper employs a comprehensive multi-omic approach, combining ChIP-Seq, RNA-Seq, and CRISPRa-based Perturb-Seq assays, which allow for a thorough investigation of the regulatory mechanisms underlying myometrial gene expression.<br /> (2) Clinical Relevance: Investigating human myometrial specimens provides direct clinical relevance, as understanding the molecular mechanisms governing parturition and myometrial quiescence can have significant implications for the management of pregnancy-related disorders.<br /> (3) Functional work: For functional screening, They have used CRISPRa-based screening of PLCL2 gene regulation using immortalized human cell-line hTERT-HM and T-hESC to add more dimension to the work which strengthens their finding of PGR-dependent regulation of the PLCL2 gene in the human myometrial cells.

Weaknesses:

(1) Variability in epigenomic mapping: The significant variations in the number and location of H3K27ac-positive intervals across different samples and studies suggest potential challenges in accurately mapping the myometrial epigenome. This variability may introduce uncertainty and complicate the interpretation of results.<br /> (2) Sample specificity: The study focuses on term pregnant nonlabor myometrial specimens, limiting the generalizability of the findings to other stages of pregnancy or labor.<br /> (3) Limited Understanding of Regulatory Mechanisms: While the study identifies potential regulatory programs within super-enhancers, the exact mechanisms by which these enhancers regulate gene expression and cellular functions in the myometrium remain unclear. Further mechanistic studies are needed to elucidate these processes.<br /> (4) Discordant analysis: Why regular enhancers are being understood in terms of motif enrichment of transcription factors and super-enhancers in terms of pathways enriched for active genes? This needs a clear reason.

Reviewer #2 (Public review):

Summary:

In "Assessment of the Epigenomic Landscape in Human Myometrium at Term Pregnancy" the authors generate a number of genome-wide data sets to investigate epigenomic and transcriptomic regulation of the myometrium at term pregnancy. These data provide a useful resource for further evaluation of gene regulatory mechanisms in the myometrium and include the first Hi-C data published for this tissue. There is a comparison to previously published histone modification data and integration with RNA-seq to highlight potential enhancer-gene regulatory relationships. The authors further investigate putative enhancers upstream of the PLCL2 gene and identify a candidate region that may be regulated by the PGR (progesterone receptor) signaling.

Strengths:

The strengths of this study are in the multi-omics nature of the design as several genome-wide data sets are generated from the same patient samples. Extending this type of approach in the future to a larger number of samples will allow for additional investigation into gene regulation as correlation between epigenomic features and gene expression across a larger number of samples can reveal regulatory relationships.

Weaknesses:

One of the most interesting aspects of this study is the generation of the first Hi-C data for the human pregnant myometrium, however, there is minimal description in the results section of the Hi-C data analysis and the only data shown are the number of loops identified and one such loop that includes the PLCL2 promoter shown in figure 3A. The manuscript would benefit from a more extensive analysis of the Hi-C data, for example, the analysis of TADs (topological associating domains) would be interesting to add and could be used to evaluate to what extent H3K27ac domains and putative regulated genes fall within the same TAD.

The authors present some convincing evidence on the transcriptional regulation of the PLCL2 gene using Perturb-Seq to identify putative upstream enhancer regions and PGR over-expression showing PGR can act as an activator. These two experiments on their own are interesting, however, they are not as mechanistically integrated as they could be to clarify the molecular mechanisms. Deletion of the putative enhancer upstream of PLCL2 followed by over-expression of PGR would clarify the mechanistic relationship between the proposed enhancer, PGR and PLCL2 expression. Does PGR act through the proposed enhancer? In addition, reporter assays using this proposed enhancer region with and without increased expression of PGR and mutation of any PRE sequences would also provide mechanistic insight. Although CRISPRa and Perturb-Seq can be used to identify potential regulatory regions, the best approach to verify the requirement for a particular enhancer in regulating a specific gene is a deletion approach.

Comments on revisions:

The authors have addressed my comments that were directly sent to them, however, my comments in the public review, specifically the superficial nature of the Hi-C analysis were not addressed.

In addition, many of the comments to reviewer 3 were unaddressed and declared out of the scope of this study, as these were points of accuracy in the data analysis they are very much in scope.

I hope the authors reconsider presenting a more thorough analysis.

Reviewer #3 (Public review):

In this manuscript, Wu et al. investigate active H3K27ac and H3K4me1 marks in term pregnant nonlabor myometrial biopsies, linking putative enhancers and super enhancers to gene expression levels. Through their findings, they reveal the PGR-dependent regulation of the PLCL2 gene in human myometrial cells via a cis-acting element located 35-kilobases upstream of the PLCL2 gene. By targeting this region using a CRISPR activation system, they were able to increase the elevate the endogenous PLCL2 mRNA levels in immortalized human myometrial cells.

This research offers novel insights into the molecular mechanisms governing gene expression in myometrial tissues, advancing our understanding of pregnancy-related processes.

Major comments:

(1) A more comprehensive analysis of the epigenetic and transcriptomic data would have strengthened the paper, moving beyond basic association studies. Currently, it is challenging to assess the quality and significance of the data as much of the information is lacking.

Strengths:

- The combination of ChIP-Seq, RNA-Seq, and CRISPRa Perturb-Seq approaches to investigate gene regulation and expression in myometrial cells.<br /> - The use of CRISPR activation system to specifically target cis-acting elements.

Weaknesses:

- The manuscript would strongly benefit from a deeper analysis of the Omic datasets. Furthermore, expanding figures/graphs to effectively contextualize these datasets would be greatly beneficial and would add more value to this research.<br /> - Limited sample size, coupled with variability in results and overall lack of details, compromises the robustness of result interpretation.<br /> - Additional efforts are needed to dissect the proposed regulatory mechanisms.<br /> - While the discussion provided helpful context for understanding some of the experiments performed, it lacked interpretation of the results in relation to the existing literature.

Comments on revisions:

The authors have improved the manuscript by enhancing its readability and organization. Tables were added to present key information more clearly, and figures were refined for better visualization. Additionally, more details were included, particularly in the methods and bioinformatics analyses sections, ensuring a more comprehensive and precise presentation of the data.

However, in many cases, reviewers' questions and concerns were addressed in the response to reviewers rather than incorporated into the manuscript, or it was noted that these points would be explored in future studies.

Reviewer #2 (Public review):

Summary:

This study investigates the molecular function of the N-glycan-dependent endoplasmic reticulum protein quality control system (ERQC) in Cryptococcus neoformans and correlates this pathway with key features of C. neoformans virulence, especially those mediated by extracellular vesicle transport. The findings provide valuable insights into the connection between this pathway and the biogenesis of C. neoformans extracellular vesicles.

Strengths:

The strength of this study lies primarily in the careful selection of appropriate and current methodologies, which provide a solid foundation for the authors' results and conclusions across all presented data. All experiments are supported by well-designed and established controls in the study of C. neoformans, further strengthening the validity of the results and conclusions drawn from them. The study presents novel data on this important pathway in C. neoformans, establishing its connection with C. neoformans virulence. Interestingly, the findings led the authors to understand the relationship between this pathway and the transport of key fungal virulence factors via extracellular vesicles. This was demonstrated in the study, paving the way for a deeper understanding of extracellular vesicle biogenesis-a field still filled with gaps but one that this study contributes to with solid data, helping to clarify aspects of this process.

Reviewer #3 (Public review):

Summary:

Cryptococcus neoformans is a global critical threat pathogen and the manuscript by Mota et al demonstrates that the pathogen's N-glycan-dependent protein quality control system regulates the capacity of the fungus to cause disease. The system ensures that glycoproteins are folded correctly. The system is involved in fitness and virulence of the fungus by regulating aspects of cellular robustness and the trafficking of virulence-associated compounds outside of the cell via transport in extracellular vesicles.

Strengths:

The investigators use multiple modalities to demonstrate that the system is involved in cryptococcal pathogenesis. The investigators generated mutant C. neoformans to explore the role of genes involved in the protein folding system. Basic microbiology, genetic analyses, proteomics, fluorescence and transmission microscopy, nanotracking analyses, and murine studies were performed. The validity of the findings are thus very high. Hypotheses are robustly demonstrated.

Reviewer #1 (Public review):

Summary:

This manuscript aims to explain the emergence of grid-like spatial firing patterns. Rather than taking the existence of grid cells as a given and asking what does their properties say about their function, the authors reverse the approach: they begin with a proposed computational function that the brain may need to perform-coding of 2D spatial trajectories using sequences of neural activity-and ask what type of neural code would optimally support this function. They show that, under a set of formal assumptions, such a code leads to the emergence of spatial periodicity and a hexagonal grid pattern. The aim is to provide a normative explanation for the existence of grid cells grounded in functional constraints.

Strengths:

The manuscript presents a mathematically well-defined framework that is internally consistent. The derivation is structured and leads to a hexagonal lattice as the most efficient solution for representing directional trajectories. The authors provide comparisons to experimental observations and extend the model to explain several findings in the grid cell literature. In the revised version, the discussion of foundational assumptions is expanded, and the manuscript better situates itself in relation to prior theoretical work. Overall, this work adds a very interesting view to the broader conversation about the role and origin of grid cells by offering a theoretical alternative grounded in trajectory coding.

Weaknesses:

The model depends on assumptions that, while plausible, should be treated as chosen assumptions. These include the premise that (1) grid function is trajectory coding, (2) that trajectory coding is implemented through sequences of neural activity, and (3) that such sequences are largely independent of spatial position. In the revised manuscript, the authors provide more literature to support these assumptions.

Reviewer #2 (Public review):

Summary:

In this work, the authors consider the required functional properties of neurons that trajectories in 2D space using cell sequences, ultimately linking the required properties to those found in grid cells. In their argument, the authors first introduce a set of definitions and axioms, which then lead to their conclusion that a hexagonal pattern is the most efficient or parsimonious pattern one could use to uniquely label different 2D trajectories using sequences of cells. The authors then go through a set of classic experimental results in the grid cell literature - e.g. that the grid modules exhibit a multiplicative scaling, that the grid pattern expands with novelty or is warped by reward, etc. - and describe how these results are either consistent with or predicted by their theory. Overall, this paper asks a very interesting question and provides an intriguing answer.

Major strengths:

The general idea behind the paper is very interesting - why *does* the grid pattern take the form of a hexagonal grid? This is a question that has been raised many times; finding a truly satisfying answer is difficult but of great interest. The authors' main assertion that the answer to this question has to do with the ability of a hexagonal arrangement of neurons to uniquely encode 2D trajectories is an intriguing suggestion. It is also impressive that the authors considered such a wide range of experimental results in relation to their theory.

Major weaknesses:

One weakness I perceive is that the paper overstates what it delivers. In the introduction, the authors claim to provide "mathematical proof that ... the nature of the problem being solved by grid cells is coding of trajectories in 2-D space using cell sequences. By doing so, we offer a specific answer to the question of why grid cell firing patterns are observed in the mammalian brain." By virtue of the fact that the authors make assumptions about biological function in their claims, this paper does not provide proof of what grid cells are doing to support behavior nor provide the true answer as to why grid patterns are found in the brain. Although I find this study both intriguing and important - and I respect the authors' perspective - as an experimentalist guided by the principle that biological theories are never proven but instead continually supported by data, suggestions of a proof of grid cell function are hard for me to get behind. Regardless, the paper presents a compelling line of reasoning that enhance our understanding of grid cells.

Reviewer #3 (Public review):

Concerning the revised manuscript, the authors are to be commended for carefully addressing the reviewers' comments and updating the references cited.

I will differ with the authors' argument that Gardner et al's paper supports the idea of sequences, except in the most trivial sense, namely that topology implies continuity, and hence movement along the manifold will be continuous, and if one discretizes this movement, one would get a sequence.

This has very little to do with the idea that the authors propose in their manuscript. If the authors were to so choose, their idea will produce an embedded graph, and they could study the topology of this construct---but this would mean going off on a tangent.

Let us not hold up the authors to an impossible standard, namely that their theory should explain everything in the grid cell field. Not every finding under the sun needs be addressed in the discussion.

My own take on the manuscript had been that the authors' interesting idea might be fairly straightforwardly provable. The authors have decided to put another student on this particular project. That is perfectly OK. Just one final note: mathematically, the main focus ought to be on sequences of prime length (many other results will likely follow).

Joint Public Review:

Summary:

In this study, Daniel et al. used three cognitive tasks to investigate behavioural signatures of cerebellar degeneration. In the first two tasks, the authors found that if an equation was incorrect, reaction times slowed significantly more for cerebellar patients than for healthy controls. In comparison, the slowing in the reaction times when the task required more operations was comparable to normal controls. In the third task, the authors show increased errors in cerebellar patients when they had to judge whether a letter string corresponded to an artificial grammar.

Strengths:

Overall, the work is methodologically sound and the manuscript well written. The data do show some evidence for specific cognitive deficits in cerebellar degeneration patients.

Weaknesses:

The current version has some weaknesses in the visual presentation of results. Overall, the study lacks a more precise discussion on how the patterns of deficits relate to the hypothesized cerebellar function.

The reviewers and the editor agreed that the data are interesting and point to a specific cognitive deficit in cerebellar patients. However, in the discussion, we were somewhat confused about the interpretation of the result:

If the cerebellum (as proposed in the introduction) is involved in forming expectations in a cognitive task, should they not show problems both in the expected (1+3 =4) and unexpected (1+3=2) conditions? Without having formed the correct expectation, how can you correctly say "yes" in the expected condition? No increase in error rate is observed - just slowing in the unexpected condition. But this increase in error rate was not observed. If the patients make up for the lack of prediction by using some other strategy, why are they only slowing in the unexpected case?

If the cerebellum is NOT involved in making the prediction, but only involved in detecting the mismatch between predicted and real outcome, why would the patients not show specifically more errors in the unexpected condition?

Reviewer #1 (Public review):

Summary:

This study builds upon a major theoretical account of value-based choice, the 'attentional drift diffusion model' (aDDM), and examines whether and how this might be implemented in the human brain using functional magnetic resonance imaging (fMRI). The aDDM states that the process of internal evidence accumulation across time should be weighted by the decision maker's gaze, with more weight being assigned to the currently fixated item. The present study aims to test whether there are (a) regions of the brain where signals related to the currently presented value are affected by the participant's gaze; (b) regions of the brain where previously accumulated information is weighted by gaze.

To examine this, the authors developed a novel paradigm that allowed them to dissociate currently and previously presented evidence, at a timescale amenable to measuring neural responses with fMRI. They asked participants to choose between bundles or 'lotteries' of food times, which they revealed sequentially and slowly to the participant across time. This allowed modelling of the haemodynamic response to each new observation in the lottery, separately for previously accumulated and currently presented evidence.

Using this approach, they find that regions of the brain supporting valuation (vmPFC and ventral striatum) have responses reflecting gaze-weighted valuation of the currently presented item, whereas regions previously associated with evidence accumulation (preSMA and IPS) have responses reflecting gaze-weighted modulation of previously accumulated evidence.

Strengths:

A major strength of the current paper is the design of the task, nicely allowing the researchers to examine evidence accumulation across time despite using a technique with poor temporal resolution. The dissociation between currently presented and previously accumulated evidence in different brain regions in GLM1 (before gaze-weighting), as presented in Figure 5, is already compelling. The result that regions such as preSMA respond positively to |AV| (absolute difference in accumulated value) is particularly interesting, as it would seem that the 'decision conflict' account of this region's activity might predict the exact opposite result. Additionally, the behaviour has been well modelled at the end of the paper when examining temporal weighting functions across the multiple samples.

Weaknesses:

The results relating to gaze-weighting in the fMRI signal could do with some further explication to become more complete. A major concern with GLM2, which looks at the same effects as GLM1 but now with gaze-weighting, is that these gaze-weighted regressors may be (at least partially) correlated with their non-gaze-weighted counterparts (e.g., SVgaze will correlate with SV). But the non-gaze-weighted regressors have been excluded from this model. In other words, the authors are not testing for effects of gaze-weighting of value signals *over and above* the base effects of value in this model. In my mind, this means that the GLM2 results could simply be a replication of the findings from GLM1 at present. GLM3 is potentially a stronger test, as it includes the value signals and the interaction with gaze in the same model. But here, while the link to the currently attended item is quite clear (and a replication of Lim et al, 2011), the link to previously accumulated evidence is a bit contorted, depending upon the interpretation of a behavioural regression to interpret the fMRI evidence. The results from GLM3 are also, by the authors' own admission, marginal in places.

Reviewer #2 (Public review):

Summary:

In this paper, the authors seek to disentangle brain areas that encode the subjective value of individual stimuli/items (input regions) from those that accumulate those values into decision variables (integrators) for value-based choice. The authors used a novel task in which stimulus presentation was slowed down to ensure that such a dissociation was possible using fMRI despite its relatively low temporal resolution. In addition, the authors leveraged the fact that gaze increases item value, providing a means of distinguishing brain regions that encode decision variables from those that encode other quantities such as conflict or time-on-task. The authors adopt a region-of-interest approach based on an extensive previous literature and found that the ventral striatum and vmPFC correlated with the item values and not their accumulation, whereas the pre-SMA, IPS, and dlPFC correlated more strongly with their accumulation. Further analysis revealed that the pre-SMA was the only one of the three integrator regions to also exhibit gaze modulation.

Strengths:

The study uses a highly innovative design and addresses an important and timely topic. The manuscript is well-written and engaging, while the data analysis appears highly rigorous.

Weaknesses:

With 23 subjects, the study has relatively low statistical power for fMRI.

Reviewer #1 (Public review):

Summary:

This study builds on previous work demonstrating that several beta connexins (Cx26, Cx30, and Cx32) have a carbamylation motif which renders them sensitive to CO2. In response to CO2, hemichannels composed of these connexins open, enabling diffusion of small molecules (such as ATP) between the cytosol and extracellular environment. Here, the authors have identified that an alpha connexin, Cx43, also contains a carbamylation motif, and they demonstrate that CO2 opens Cx43 hemichannels. Most of the study involves using transfected cells expressing wild-type and mutant Cx43 to define amino acids required for CO2 sensitivity. Hippocampal tissue slices in culture were used to show that CO2-induced synaptic transmission was affected by Cx43 hemichannels, providing a physiological context. The authors point out that the Cx43 gene significantly diverges from the beta connexins that are CO2 sensitive, suggesting that the conserved carbamylation motif was present before the alpha and beta connexin genes diverged.

Strengths:

(1) The molecular analysis defining the amino acids that contribute to the CO2 sensitivity of Cx43 is a major strength of the study. The rigor of analysis was strengthened by using three independent assays for hemichannel opening: dye uptake, patch clamp channel measurements, and ATP secretion. The resulting analysis identified key lysines in Cx43 that were required for CO2-mediated hemichannel opening. A double K to E Cx43 mutant produced a construct that produced hemichannels that were constitutively open, which further strengthened the analysis.

(2) Using hippocampal tissue sections to demonstrate that CO2 can influence field excitatory postsynaptic potentials (fEPSPs) provides a native context for CO2 regulation of Cx43 hemichannels. Cx43 mutations associated with Oculodentodigital Dysplasia (ODDD) inhibited CO2-induced hemichannel opening, although the mechanism by which this occurs was not elucidated.

Weaknesses:

(1) Cx43 channels are sensitive to cytosolic pH, which will be affected by CO2. Cytosolic pH was not measured, and how this affects CO2-induced Cx43 hemichannel activity was not addressed.

(2) Cultured cells are typically grown in incubators containing 5% CO2, which is ~40 mmHg. It is unclear how cells would be viable if Cx43 hemichannels are open at this PCO2.

(3) Experiments using Gap26 to inhibit Cx43 hemichannels in fEPSP measurements used a scrambled peptide as a control. Analysis should also include Gap peptides specifically targeting Cx26, Cx30, and Cx32 as additional controls.

(4) The mechanism by which ODDD mutations impair CO2-mediated hemichannel opening was not addressed. Also, the potential roles for inhibiting Cx43 hemichannels in the pathology of ODDD are unclear.

(5) CO2 has no effect on Cx43-mediated gap junctional communication as opposed to Cx26 gap junctions, which are inhibited by CO2. The molecular basis for this difference was not determined.

(6) Whether there are other non-beta connexins that have a putative carbamylation motif was not addressed. Additional discussion/analysis of how the evolutionary trajectory for Cx43 maintaining a carbamylation motif is unique for non-beta connexins would strengthen the study.

Reviewer #2 (Public review):

Summary:

This paper examines the CO2 sensitivity of Cx43 hemichannels and gap junctional channels in transiently transfected Hela cells using several different assays, including ethidium dye uptake, ATP release, whole cell patch clamp recordings, and an imaging assay of gap junctional dye transfer. The results show that raising pCO2 from 20 to 70 mmHg (at a constant pH of 7.3) causes an increase in opening of Cx43 hemichannels but does not block Cx43 gap junctions. This study also showed that raising pCO2 from 20 to 35 mm Hg resulted in an increase in synaptic strength in hippocampal rat brain slices, presumably due to downstream ATP release, suggesting that the CO2 sensitivity of Cx43 may be physiologically relevant. As a further test of the physiological relevance of the CO2 sensitivity of Cx43, it was shown that two pathological mutations of Cx43 that are associated with ODDD caused loss of Cx43 CO2-sensitivity. Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26. To understand the structural changes involved in CO2 sensitivity, a number of mutations were made in Cx43 sites thought to be the equivalent of those known to be involved in the CO2 sensitivity of Cx26, and the CO2 sensitivity of these mutants was investigated.

Strengths:

This study shows that the apparent lack of functional Cx43 hemichannels observed in a number of previous in vitro function studies may be due to the use of HEPES to buffer the external pH. When Cx43 hemichannels were studied in external solutions in which CO2/bicarbonate was used to buffer pH instead of HEPES, Cx43 hemichannels showed significantly higher levels of dye uptake, ATP release, and ionic conductance. These findings may have major physiological implications since Cx43 hemichannels are found in many organs throughout the body, including the brain, heart, and immune system.

Weaknesses:

(1) Interpretation of the site-directed mutation studies is complicated. Although Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26, the results of site-directed mutation studies were inconsistent with a simple model in which K144 and K105 interact following carbamylation to cause the opening of Cx43 hemichannels.

(2) Secondly, although it is shown that two Cx43 ODDD-associated mutations show a loss of CO2 sensitivity, there is no evidence that the absence of CO2 sensitivity is involved in the pathology of ODDD.

Reviewer #3 (Public review):

In this paper, the authors aimed to investigate carbamylation effects on the function of Cx43-based hemichannels. Such effects have previously been characterized for other connexins, e.g., for Cx26, which display increased hemichannel (HC) opening and closure of gap junction channels upon exposure to increased CO2 partial pressure (accompanied by increased bicarbonate to keep pH constant).<br /> The authors used HeLa cells transiently transfected with Cx43 to investigate CO2-dependent carbamylation effects on Cx43 HC function. In contrast to Cx43-based gap junction channels that are reported here to be insensitive to PCO2 alterations, they provide evidence that Cx43 HC opening is highly dependent on the PCO2 pressure in the bath solution, over a range of 20 up to 70 mmHg encompassing the physiologically normal resting level of around 40 mmHg. They furthermore identified several Cx43 residues involved in Cx43 HC sensitivity to PCO2: K105, K109, K144 & K234; mutation of 2 or more of these AAs is necessary to abolish CO2 sensitivity. The subject is interesting and the results indicate that a fraction of HCs is open at a physiological 40 mmHg PCO2, which differs from the situation under HEPES buffered solutions where HCs are mostly closed under resting conditions. The mechanism of HC opening with CO2 gassing is linked to carbamylation, and the authors pinpointed several Lys residues involved in this process.

Overall, the work is interesting as it shows that Cx43 HCs have a significant open probability under resting conditions of physiological levels of CO2 gassing, probably applicable to the brain, heart, and other Cx43 expressing organs. The paper gives a detailed account of various experiments performed (dye uptake, electrophysiology, ATP release to assess HC function) and results concluded from those. They further consider many candidate carbamylation sites by mutating them to negatively charged Glu residues. The paper ends with hippocampal slice work showing evidence for connexin-dependent increases of the EPSP amplitude that could be inhibited by HC inhibition with Gap26 (Figure 10). Another line of evidence comes from the Cx43-linked ODDD genetic disease, whereby L90V as well as the A44V mutations of Cx43 prevented the CO2-induced hemichannel opening response (Figure 11). Although the paper is interesting, in its present state, it suffers from (i) a problematic Figure 3, precluding interpretation of the data shown, and (ii) the poor use of hemichannel inhibitors that are necessary to strengthen the evidence in the crucial experiment of Figure 2 and others.

Reviewer #1 (Public review):

Summary:

In this manuscript, Lau et al reported that KDM5 inhibition in luminal breast cancer cells results in R-loop-mediated DNA damage, reduced cell fitness and an increase in ISG and AP signatures as well as cell surface Major Histocompatibility Complex (MHC) class I, mediated by RNA:DNA hybrid activation of the CGAS/STING pathway.

Strengths:

More importantly, they have shown that KDM5 inhibition does not result in DNA damage or activation of the CGAS/STING pathway in normal breast epithelial cells. This suggests that KDM5 inhibitors may enable a wide therapeutic window in this setting, as compared to STING agonists or Type I Interferons. Their findings provide new insights into the interplay between epigenetic regulation of genomic repeats, R-loop formation, innate immunity, and cell fitness in the context of cancer evolution and therapeutic vulnerability.

Weaknesses:

More thorough analyses would be appreciated.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated how the type-I interferon response (ISG) and antigen presentation (AP) pathways are repressed in luminal breast cancer cells and how this repression can be overcome. They found that a STING agonist can reactivate these pathways in breast cancer cells, but it also does so in normal cells, suggesting that this is not a good way to create a therapeutic window. Depletion of ADAR and inhibition of KDM5 also activate ISG and AP genes. The activation of ISG and AP genes is dependent on cGAS/STING and the JAK kinase. Interestingly, although both ADAR depletion and KDM5 inhibition activate ISG and AP genes, their effects on cell fitness are different. Furthermore, KDM5 inhibitor selectively activates ISG and AP genes in tumor cells but not normal cells, arguing that it may create a larger therapeutic window than the STING agonist. These results also suggest that KDM5 inhibition may activate ISG and AP genes in a way different from ADAR loss, and this process may affect tumor cell fitness independently of the activation of ISG and AP genes.

The authors further showed that KDM5 inhibition increases R-loops and DNA damage in tumor cells, and XPF, a nuclease that cuts R-loops, is required for the activation of ISG and AP genes. Using H3K4me3 CUT&RUN, they found that KMD5 inhibition results in increased H3K4me3 not only at genes, but also at repetitive elements including SINE, LINE, LTR, telomeres, and centromeres. Using S9.6 CUT&TAG, they confirmed that R-loops are increased at SINE, LINE, and LTR repeated with increased H3K4me3. Together, the results of this study suggest that KMD5 inhibition leads to H3K4me3 and R-loop accumulation in repetitive elements, which induces DNA damage and cGAS/STING activation and subsequently activates AP genes. This provides an exciting approach to stimulate the anti-tumor immunity against breast tumors.

KDM5 inhibition activates interferon and antigen presentation genes through R-loops.

Strengths:

Overall, this study was carefully designed and executed. This is a new approach to make breast tumors "hot" for anti-tumor immunity.

Weaknesses:

Future in vivo studies are needed to show the effects of KDM5 inhibitors on the immunotherapy responses of breast tumors.

Reviewer #1 (Public review):

Summary:

In this study, the authors set out to define how arginine availability regulates lipid metabolism and to explore the implications of this relationship in pancreatic ductal adenocarcinoma (PDAC), a tumor type known to exist in an arginine-poor microenvironment. Using a combination of rigorous genetic and metabolomic approaches, they uncover a previously underappreciated role for arginine in maintaining lipid homeostasis. Importantly, they demonstrate that arginine deprivation sensitizes PDAC cells to ferroptosis through lipidome perturbations, which can be exploited therapeutically via co-treatment with aESA and ferroptosis inducers (FINs). These findings have meaningful implications for the field. They not only shed light on the metabolic vulnerabilities created by nutrient restriction in PDAC, but also suggest a practical avenue for combination therapies that exploit ferroptosis sensitivity. This is particularly relevant in the context of pancreatic cancer, which is notoriously resistant to conventional treatments. The methods employed are broadly applicable to other nutrient-stress contexts and may inspire similar investigations in other solid tumor types.

Strengths:

One of the major strengths of the study is the use of complementary and well-controlled approaches-including metabolomic profiling, genetic perturbations, and in vivo models-to support the central hypothesis. The experiments are thoughtfully designed and clearly presented, and the conclusions are, for the most part, well supported by the data. The findings provide mechanistic insight into nutrient-lipid crosstalk and identify a potential therapeutic strategy for targeting arginine-deprived tumors.

Weaknesses:

A key weakness of the study lies in the mechanistic connection between arginine levels and SREBP1 activation. While the authors show that arginine restriction leads to reduced SREBP1 expression, the magnitude of this effect appears modest relative to the substantial changes observed in the lipidome. The study would benefit from a deeper analysis of SREBP1 regulation-particularly whether nuclear translocation or activation is affected. This could be addressed by examining the nuclear pool of SREBP1, using either subcellular fractionation or improved immunofluorescence imaging in both cell lines and tissue samples.

Another area where additional context would strengthen the manuscript is in the transcriptomic profiling of PDAC cells cultured in a tumor interstitial fluid mimic (TIFM). While the study emphasizes lipid-related pathways, highlighting the most significantly upregulated and downregulated pathways in Figure 1B would give readers a broader perspective on how arginine restriction reprograms the PDAC transcriptome. For instance, because polyamines are downstream of arginine and are known to influence lipid metabolism, it would be worth discussing whether these metabolites contribute to the phenotypes observed. Similarly, an evaluation of whether Dgat1/2 expression is altered could help delineate the full scope of lipid metabolic rewiring.

Finally, it is worth noting that the KPC mouse model used in this study is based on conditional deletion of p53, which leads to faster-growing tumors and a distinct tumor microenvironment compared to models harboring the p53^R172H point mutation. Including a brief discussion of this distinction would help readers contextualize the translational relevance of the findings.

Reviewer #2 (Public review):

This study by Jonker et al. examines how the metabolic adaptations to the microenvironment by pancreatic ductal adenocarcinomas (PDAC) present vulnerabilities that could be used for therapeutic purposes. The evidence supporting the claims of the authors is mostly solid, and the multiplicity of models used, as well as the combination of in vitro and in vivo work, are appreciated, but some conclusions would benefit from additional substantiation. This work would be of interest to biologists working on the impact of microenvironment and metabolism in cancer, and especially those investigating pancreatic cancer.

In this study, the authors use mostly "doublings per day" as an indicator of cell death, notably for Figures 4 to 6. However, proliferative arrest (or a decrease in the proliferative rate) is not necessarily synonymous with cell death. It might be nice to complement these experiments with a true measure of cell death (e.g., PI uptake).

The composition of Tumor Interstitial Fluid Medium (TIFM) was published previously, but nonetheless a reminder of the composition of this medium in a Supplemental file of this study might be helpful. In particular, at the start of the Results section, the nature of serum/lipids in the different media should be specifically noted, especially given that the subsequent focus of the work is on lipids/SREBP. It is known that differences in the extracellular availability of lipids can profoundly alter de novo lipid biosynthesis pathways.

Reviewer #3 (Public review):

This important study investigates the impact of nutrient stress in the tumor microenvironment (TME), focusing on lipid metabolism in pancreatic ductal adenocarcinoma (PDAC).

Understanding TME composition is crucial, as it highlights cancer vulnerabilities independent of intracellular mutations, particularly because PDAC tumors are often exposed to limited nutrient availability due to reduced perfusion.

By utilizing a medium that mimics the nutrient conditions of PDAC tumors, the authors convincingly show that TME nutrient stress suppresses SREBP1, leading to reduced lipid synthesis, with low arginine levels identified as a key driver of this suppression. Importantly, mice with arginine-starved pancreatic tumors respond to a polyunsaturated fatty acid-rich diet. This discovery uncovers a synthetic lethal interaction in the tumor microenvironment that could be leveraged through dietary interventions.

The conclusions of this paper are mostly well supported by data; however, below are some aspects that could be further clarified.

This study uses PDAC cells from the LSL-Kras G12D/+ ; Trp53 ; Pdx-1-Cre PDAC model. The authors convincingly demonstrate that the cell-extrinsic stimuli of low arginine availability suppress lipid synthesis and thus exert a dominant effect over the cell-intrinsic oncogenic Ras mutation, which is known to enhance fatty acid synthesis. Could the effect of low arginine on lipid synthesis be specific for certain mutations in PDAC? It would be interesting to investigate or discuss whether different mutations show the same SREBP1 reduction caused by low arginine levels, and whether these low SREBP1 levels can be ameliorated by arginine re-supplementation. Here, Jonker et al. show that human PDAC cells cultured in TIFM have reduced SREBP1 levels (Figure 1 - Figure supplement 1C). It would be further supportive of their conclusions if the authors could show that arginine re-supplementation is sufficient to restore SREBP1 levels in human PDAC cells.

The authors demonstrate that mPDAC cells cultured in RPMI and subsequently implanted into an orthotopic mouse model exhibit reduced expression of SREBP target genes when compared to in vitro cultured mPDAC-RPMI cells. This finding is in line with the observation that culturing PDAC cells in TIFM downregulates SREBP target genes compared to PDAC cells cultured in RPMI. However, caution is needed when directly comparing mPDAC-RPMI cultured cells to those in the orthotopic model, as the latter may include non-tumor cells and additional factors that could confound the results. The authors should explicitly acknowledge this limitation in their study.

The in vivo evidence demonstrating that PUFA-rich tung oil reduces tumor size is compelling. However, the specific in vitro findings regarding its impact on doubling rates per day, particularly in the context of arginine-dependent PUFA supplementation, require further explanation. To enhance the robustness of their data and conclusions, the authors could consider conducting additional cell viability and proliferation assays. Moreover, it would be valuable to assess whether the observed effects on doubling rates per day remain significant after normalizing the data to the initial doubling time prior to PUFA supplementation. This is in particular important regarding the statement that "Addition of arginine significantly decreases sensitivity to a-ESA" as these cells already start with a higher doubling rate prior to a-ESA treatment.

Overall, this paper presents a compelling study that significantly enhances our understanding of the PDAC tumor microenvironment and its complex interactions with the tumor lipid metabolism.

Reviewer #1 (Public review):

Summary:

The manuscript uses state-of-the-art analysis technology to document the spatio-temporal dynamics of brain activity during the processing of threats. The authors offer convincing evidence that complex spatio-temporal aspects of brain dynamics are essential to describe brain operations during threat processing.

Strengths:

Rigorous complex analyses well suited to the data.

Weaknesses:

Lack of a simple take-home message about discovery of a new brain operation.

Comments on revisions:

The authors have improved the presentation of the work. The overstatements about existing models of brain functions ignoring exogenous components remains largely unaddressed. The clarifications and improvements provided in revision confirm my assessment of the paper.

Reviewer #2 (Public review):

Summary:

This paper by Misra and Pessoa uses switching linear dynamical systems (SLDS) to investigate the neural network dynamics underlying threat processing at varying levels of proximity. Using an existing dataset from a threat-of-shock paradigm in which threat proximity is manipulated in a continuous fashion, the authors first show that they can identify states that each have their own linear dynamical system and are consistently associated with distinct phases of the threat-of-shock task (e.g., "peri-shock", "not near", etc). They then show how activity maps associated with these states are in agreement with existing literature on neural mechanisms of threat processing, and how activity in underlying brain regions alters around state transitions. The central novelty of the paper lies in its analyses of how intrinsic and extrinsic factors contribute to within-state trajectories and between-state transitions. Additional analyses furthermore show how individual brain regions contribute to state dynamics. Finally, the authors show how their findings generalize to another (related) threat paradigm.

Strengths:

The analyses for this study are conducted at a very high level of mathematical and theoretical sophistication. The paper is very well written and effectively communicates complex concepts from dynamical systems. The paper provides valuable neuroscientific insights into threat processing, and the methodology has potential to deepen our understanding at a neurobiological level in future work.

Weaknesses:

I was somewhat disappointed initially by the level of inferences made by the authors based on their analyses at the level of systems neuroscience. After revision this has improved, for instance with inclusion of analyses on the importance of individual brain regions to state dynamics, but I still believe the findings can be made more biologically meaningful, for instance by focusing on what we learn from these sophisticated analyses beyond what is already known from more conventional methodologies. However, the paper as it stands is solid scientific work and such efforts may also be left to future work.

Reviewer #1 (Public review):

Summary:

A cortico-centric view is dominant in the study for the neural mechanisms of consciousness. This investigation represents the growing interest to understand how subcortical regions are involved in conscious perception. To achieve this, the authors engaged an ambitious and rare procedure in humans of directly recording from neurons in the subthalamic nucleus and thalamus. While participants were in surgery for the placement of deep brain stimulation devices for the treatment of essential tremor and Parkinson's disease, they were awakened and completed a perceptual-threshold tactile detection task. The authors identified individual neurons and analyzed single-unit activity corresponding with the task phases and tactile detection/perception. Among the neurons that were perception-responsive, the authors report changes in firing rate beginning ~150 milliseconds from the onset of the tactile stimulation. Curiously, the majority of the perception-responsive neurons had a higher firing rate for missed/not perceived trials. In summary, this investigation is a valuable addition to the growing literature on the role of subcortical regions in conscious perception.

Strengths:

The authors achieve the challenging task of recording human single-unit activity while participants performed a tactile perception task. The methods and statistics are clearly explained and rigorous, particularly for managing false positives and non-normal distributions. The results offer new detail at the level of individual neurons in the emerging recognition for the role of subcortical regions in conscious perception. Also, this study highlights the timing of neural activity linked to conscious perception (approximately 150 millisecond).

Weaknesses:

Due to constraints of testing with this patient population, a standard report-based detection task was administered. This type of task cannot fully exclude motor preparatory and post-perceptual processing as a factor that contributes to distinguishing between perceived versus not perceived stimuli. The authors show sensitivity to this issue by identifying task-selective neurons and their discussion of the results that refers to the confound of post-perceptual processing. Despite this limitation, the results are valuable for contributing to a growing body of literature on the subcortical neural mechanisms of consciousness.

Reviewer #2 (Public review):

The authors have examined subpopulations of individual neurons recorded in the thalamus and subthalamic nucleus (STN) of awake humans performing a simple cognitive task. They have carefully designed their task structure to minimize motor components that could confound their analyses in these subcortical structures, particularly given that the data was collected from patients with Parkinson's disease (PD) and essential tremor (ET). The recorded data represents a valuable contribution to the field. Pereira et al. conclude that their single-neuron recordings indicate task-related activity that is purportedly distinct from previously identified sensory signals.

Despite the significance of the dataset, important limitations arise due to the small number of recorded neurons relative to the high number of participants. That raises concerns about the generalizability of the conclusions drawn from the study.

(1) While I support the work conducted by the authors and their efforts to improve the manuscript, the number of significant neurons is considerably lower than the number of participants studied-approximately 8 neurons, compared to 32 participants. This low number of neurons involved in encoding raises concerns about the strength of the conclusions drawn.

(2) Additionally, the authors state that participants do not need to perform a motor execution, yet they are required to communicate their response verbally. This presents a contradiction, as speech involves the activation of facial muscles, and previous studies have shown that neuronal activity in the ventral premotor cortex can encode such movements in humans (Willet et al., Nature 2023). Clarifying this point would strengthen the argument and ensure consistency in the interpretation of results.

(3) One way to improve the study is to analyze the local field potentials (LFPs) recorded alongside the spikes. By examining different LFP components, particularly the beta band (Haegens et al., PNAS 2011), it may be possible to identify consistent modulation across the 32 recorded participants. This approach could provide additional support for the study's conclusions and help clarify the role of neural activity in the observed phenomena.

Reviewer #3 (Public review):

Summary:

This important study relies on a rare dataset: intracranial recordings within the thalamus and the subthalamic nucleus in awake humans, while they were performing a tactile detection task. This procedure allowed the authors to identify a small but significant proportion of individual neurons, in both structures, whose activity correlated with the task (e.g. their firing rate changed following the audio cue signalling the start of a trial) and/or with the stimulus presentation (change in firing rate around 200 ms following tactile stimulation) and/or with participant's reported subjective perception of the stimulus (difference between hits and misses around 200 ms following tactile stimulation). Whereas most studies interested in the neural underpinnings of conscious perception focus on cortical areas, these results suggest that subcortical structures might also play a role in conscious perception, notably tactile detection.

Strengths:

There are two strongly valuable aspects in this study that make the evidence convincing and even compelling. First, these type of data are exceptional, the authors could have access to subcortical recordings in awake and behaving humans during surgery. Additionally, the methods are solid. The behavioral study meets the best standards of the domain, with a careful calibration of the stimulation levels (staircase) to maintain them around detection threshold, and additional selection of time intervals where the behavior was stable. The authors also checked that stimulus intensity was the same on average for hits and misses within these selected periods, which warrants that the effects of detection that are observed here are not confounded by stimulus intensity. The neural data analysis is also very sound and well conducted. The statistical approach complies to current best practices, although I found that, on some instances, it was not entirely clear which type of permutations had been performed, and I would advocate for more clarity in these instances. Globally, the figures are nice, clear and well presented. I appreciated the fact that the precise anatomical location of the neurons was directly shown in each figure.

Weaknesses:

The results rely on a small number of neurons; it is only the beginning of this exploration! Figure S5 is important for observing the variety of ways the neurons' activity correlated with either stimulus presence, or perception, or both. Interpretations are still very open on these different profiles.

Reviewer #1 (Public review):

This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.

Reviewer #2 (Public review):

Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness on birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used generalized linear mixed models to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite from that hypothesized. The authors have done a very good job of addressing many of the reviewer's concerns, especially by adding two additional replicates.

Reviewer #1 (Public review):

In this study, Tiang et al. explore the role of ubiquitination of non-structural protein 16 (nsp16) in the SARS-CoV-2 life cycle. nsp16, in conjunction with nsp10, performs the final step of viral mRNA capping through its 2'-O-methylase activity. This modification allows the virus to evade host immune responses and protects its mRNA from degradation. The authors demonstrate that nsp16 undergoes ubiquitination and subsequent degradation by the host E3 ubiquitin ligases UBR5 and MARCHF7 via the ubiquitin-proteasome system (UPS). Specifically, UBR5 and MARCHF7 mediate nsp16 degradation through K48- and K27-linked ubiquitination, respectively. Notably, degradation of nsp16 by either UBR5 or MARCHF7 operates independently, with both mechanisms effectively inhibiting SARS-CoV-2 replication in vitro and in vivo. Furthermore, UBR5 and MARCHF7 exhibit broad-spectrum antiviral activity by targeting nsp16 variants from various SARS-CoV-2 strains. This research advances our understanding of how nsp16 ubiquitination impacts viral replication and highlights potential targets for developing broadly effective antiviral therapies.

Strengths:

The proposed study is of significant interest to the virology community because it aims to elucidate the biological role of ubiquitination in coronavirus proteins and its impact on the viral life cycle. Understanding these mechanisms will address broadly applicable questions about coronavirus biology and enhance our overall knowledge of ubiquitination's diverse functions in cell biology. Employing in vivo studies is a strength.

Weaknesses:

Minor comments:<br /> Figure 5A- The authors should ensure that the figure is properly labeled to clearly distinguish between the IP (Immunoprecipitation) panel and the input panel.

Reviewer #3 (Public review):

Summary:

The manuscript "SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7" is an interesting work by Tian et al. describing the degradation/ stability of NSP16 of SARS CoV2 via K48 and K27-linked Ubiquitination and proteasomal degradation. The authors have demonstrated that UBR5 and MARCHF7, an E3 ubiquitin ligase bring about the ubiquitination of NSP16. The concept, and experimental approach to prove the hypothesis looks ok. The in vivo data looks ok with the controls. Overall, the manuscript is good.

Strengths:

The study identified important E3 ligases (MARCHF7 and UBR5) that can ubiquitinate NSP16, an important viral factor.

Comments on revisions:

I had gone through the revised form of the manuscript thoroughly. The authors have addressed all of my concerns. To me, the experimental approach looks convincing that the host E3 ubiquitin ligases (UBR5 and MARCHF7) ubiquitinate NSP16 and mark it for proteasomal degradation via K48- and K27- linkage. The authors have represented the final figure (Fig.8) in a convincing manner, opening a new window to explore the mechanism of capping the vRNA bu NSP16.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigated the effect of chronic activation of dopamine neurons using chemogenetics. Using Gq-DREADDs, the authors chronically activated midbrain dopamine neurons and observed that these neurons, particularly their axons, exhibit increased vulnerability and degeneration, resembling the pathological symptoms of Parkinson's disease. Baseline calcium levels in midbrain dopamine neurons were also significantly elevated following the chronic activation. Lastly, to identify cellular and circuit-level changes in response to dopaminergic neuronal degeneration caused by chronic activation, the authors employed spatial genomics (Visium) and revealed comprehensive changes in gene expression in the mouse model subjected to chronic activation. In conclusion, this study presents novel data on the consequences of chronic hyperactivation of midbrain dopamine neurons.

Strengths:

This study provides direct evidence that the chronic activation of dopamine neurons is toxic and gives rise to neurodegeneration. In addition, the authors achieved the chronic activation of dopamine neurons using water application of clozapine-N-oxide (CNO), a method not commonly employed by researchers. This approach may offer new insights into pathophysiological alterations of dopamine neurons in Parkinson's disease. The authors also utilized state-of-the-art spatial gene expression analysis, which can provide valuable information for other researchers studying dopamine neurons. They also presented a substantial number of intriguing ideas in their discussion, which are worth further investigation.

Weaknesses:

Although not fully supported by data, the authors provided a well-explained rationale and proposed possible mechanisms for dopamine neuron degeneration due to chronic activation in their results and discussion.

Comments on revised version:

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

Reviewer #2 (Public review):

Rademacher et al. present a paper showing that chronic chemogenetic excitation of dopaminergic neurons in the mouse midbrain results in differential degeneration of axons and somas across distinct regions (SNc vs VTA). These findings are important for two reasons: 1. This approach can be used as a mouse model for Parkinson's Disease without the need for the infusion of toxins (e.g. 6-OHDA or MPTP). This mouse model also has the advantage of showing a axon-first degeneration over an experimentally-useful time course (2-4 weeks). 2. The findings that direct excitation of dopaminergic neurons causes differential degeneration sheds light on the mechanisms of dopaminergic neuron selective vulnerability. The evidence that activation of dopaminergic neurons causes degeneration, alters motor behavior, and alters mRNA expression is convincing. This is an exciting and important paper and will have an impact on the Parkinson's Disease field.

Strengths:

This is an exciting and important paper and will have an impact on the Parkinson's Disease field.

It presents a new highly useful mouse model of PD.

The paper compares mouse transcriptomics with human patient data.

It shows that selective degeneration can occur across the midbrain dopaminergic neurons even in the absence of a genetic, prion, or toxin neurodegeneration mechanism.

Weaknesses:

The authors have addressed all my concerns. This is an interesting, important, and carefully-controlled study.

Reviewer #3 (Public review):

Summary:

In this manuscript, Rademacher and colleagues examined the effect on the integrity of the dopamine system in mice of chronically stimulating dopamine neurons using a chemogenetic approach. They find that one to two weeks of constant exposure to the chemogenetic activator CNO leads to a decrease in the density of tyrosine hydroxylase staining in striatal brain sections and to a small reduction of the global population of tyrosine hydroxylase positive neurons in the ventral midbrain. They also report alterations in gene expression in both regions using a spatial transcriptomics approach. Globally, the work is well done and valuable and some of the conclusions are interesting. However, the conceptual advance is perhaps a bit limited in the sense that there is extensive previous work in the literature showing that excessive depolarization of multiple types of neurons associated with intracellular calcium elevations promotes neuronal degeneration. The present work adds to this by showing evidence of a similar phenomenon in dopamine neurons. In terms of the mechanisms explaining the neuronal loss observed after 2 to 4 weeks of chemogenetic activation, it would be important to consider that dopamine neurons are known from a lot of previous literature to undergo a decrease in firing through a depolarization-block mechanism when chronically depolarized. Is it possible that such a phenomenon explains much of the results observed in the present study? It would be important to consider this in the manuscript. The relevance to Parkinson's disease (PD) is also not totally clear because there is not a lot of previous solid evidence showing that the firing of dopamine neurons is increased in PD, either in human subjects or in mouse models of the disease.

Comments on revisions:

The authors have done a good job at revising the manuscript. The revised manuscript better frames the results in the context of previous literature.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigated the effect of chronic activation of dopamine neurons using chemogenetics. Using Gq-DREADDs, the authors chronically activated midbrain dopamine neurons and observed that these neurons, particularly their axons, exhibit increased vulnerability and degeneration, resembling the pathological symptoms of Parkinson's disease. Baseline calcium levels in midbrain dopamine neurons were also significantly elevated following the chronic activation. Lastly, to identify cellular and circuit-level changes in response to dopaminergic neuronal degeneration caused by chronic activation, the authors employed spatial genomics (Visium) and revealed comprehensive changes in gene expression in the mouse model subjected to chronic activation. In conclusion, this study presents novel data on the consequences of chronic hyperactivation of midbrain dopamine neurons.

Strengths:

This study provides direct evidence that the chronic activation of dopamine neurons is toxic and gives rise to neurodegeneration. In addition, the authors achieved the chronic activation of dopamine neurons using water application of clozapine-N-oxide (CNO), a method not commonly employed by researchers. This approach may offer new insights into pathophysiological alterations of dopamine neurons in Parkinson's disease. The authors also utilized state-of-the-art spatial gene expression analysis, which can provide valuable information for other researchers studying dopamine neurons. Although the authors did not elucidate the mechanisms underlying dopaminergic neuronal and axonal death, they presented a substantial number of intriguing ideas in their discussion, which are worth further investigation.

Weaknesses:

Many claims raised in this paper are only partially supported by the experimental results. So, additional data are necessary to strengthen the claims. The effects of chronic activation of dopamine neurons are intriguing; however, this paper does not go beyond reporting phenomena. It lacks a comprehensive explanation for the degeneration of dopamine neurons and their axons. While the authors proposed possible mechanisms for the degeneration in their discussion, such as differentially expressed genes, these remain experimentally unexplored.

Reviewer #2 (Public Review):<br /> <br /> Summary:

Rademacher et al. present a paper showing that chronic chemogenetic excitation of dopaminergic neurons in the mouse midbrain results in differential degeneration of axons and somas across distinct regions (SNc vs VTA). These findings are important. This mouse model also has the advantage of showing a axon-first degeneration over an experimentally-useful time course (2-4 weeks). 2. The findings that direct excitation of dopaminergic neurons causes differential degeneration sheds light on the mechanisms of dopaminergic neuron selective vulnerability. The evidence that activation of dopaminergic neurons causes degeneration and alters mRNA expression is convincing, as the authors use both vehicle and CNO control groups, but the evidence that chronic dopaminergic activation alters circadian rhythm and motor behavior is incomplete as the authors did not run a CNO-control condition in these experiments.

Strengths:<br /> This is an exciting and important paper.<br /> The paper compares mouse transcriptomics with human patient data.<br /> It shows that selective degeneration can occur across the midbrain dopaminergic neurons even in the absence of a genetic, prion, or toxin neurodegeneration mechanism.

Weaknesses:

Major concerns:

(1) The lack of a CNO-positive, DREADD-negative control group in the behavioral experiments is the main limitation in interpreting the behavioral data. Without knowing whether CNO on its own has an impact on circadian rhythm or motor activity, the certainty that dopaminergic hyperactivity is causing these effects is lacking.

(2) One of the most exciting things about this paper is that the SNc degenerates more strongly than the VTA when both regions are, in theory, excited to the same extent. However, it is not perfectly clear that both regions respond to CNO to the same extent. The electrophysiological data showing CNO responsiveness is only conducted in the SNc. If the VTA response is significantly reduced vs the SNc response, then the selectivity of the SNc degeneration could just be because the SNc was more hyperactive than the VTA. Electrophysiology experiments comparing the VTA and SNc response to CNO could support the idea that the SNc has substantial intrinsic vulnerability factors compared to the VTA.

(3) The mice have access to a running wheel for the circadian rhythm experiments. Running has been shown to alter the dopaminergic system (Bastioli et al., 2022) and so the authors should clarify whether the histology, electrophysiology, fiber photometry, and transcriptomics data are conducted on mice that have been running or sedentary.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Rademacher and colleagues examined the effect on the integrity of the dopamine system in mice of chronically stimulating dopamine neurons using a chemogenetic approach. They find that one to two weeks of constant exposure to the chemogenetic activator CNO leads to a decrease in the density of tyrosine hydroxylase staining in striatal brain sections and to a small reduction of the global population of tyrosine hydroxylase positive neurons in the ventral midbrain. They also report alterations in gene expression in both regions using a spatial transcriptomics approach. Globally, the work is well done and valuable and some of the conclusions are interesting. However, the conceptual advance is perhaps a bit limited in the sense that there is extensive previous work in the literature showing that excessive depolarization of multiple types of neurons associated with intracellular calcium elevations promotes neuronal degeneration. The present work adds to this by showing evidence of a similar phenomenon in dopamine neurons. In terms of the mechanisms explaining the neuronal loss observed after 2 to 4 weeks of chemogenetic activation, it would be important to consider that dopamine neurons are known from a lot of previous literature to undergo a decrease in firing through a depolarization-block mechanism when chronically depolarized. Is it possible that such a phenomenon explains much of the results observed in the present study? It would be important to consider this in the manuscript. The relevance to Parkinson's disease (PD) is also not totally clear because there is not a lot of previous solid evidence showing that the firing of dopamine neurons is increased in PD, either in human subjects or in mouse models of the disease. As such, it is not clear if the present work is really modelling something that could happen in PD in humans.

Comments on the introduction:

The introduction cites a 1990 paper from the lab of Anthony Grace as support of the fact that DA neurons increase their firing rate in PD models. However, in this 1990 paper, the authors stated that: "With respect to DA cell activity, depletions of up to 96% of striatal DA did not result in substantial alterations in the proportion of DA neurons active, their mean firing rate, or their firing pattern. Increases in these parameters only occurred when striatal DA depletions exceeded 96%." Such results argue that an increase in firing rate is most likely to be a consequence of the almost complete loss of dopamine neurons rather than an initial driver of neuronal loss. The present introduction would thus benefit from being revised to clarify the overriding hypothesis and rationale in relation to PD and better represent the findings of the paper by Hollerman and Grace.

It would be good that the introduction refers to some of the literature on the links between excessive neuronal activity, calcium, and neurodegeneration. There is a large literature on this and referring to it would help frame the work and its novelty in a broader context.

Comments on the results section:

The running wheel results of Figure 1 suggest that the CNO treatment caused a brief increase in running on the first day after which there was a strong decrease during the subsequent days in the active phase. This observation is also in line with the appearance of a depolarization block.

The authors examined many basic electrophysiological parameters of recorded dopamine neurons in acute brain slices. However, it is surprising that they did not report the resting membrane potential, or the input resistance. It would be important that this be added because these two parameters provide key information on the basal excitability of the recorded neurons. They would also allow us to obtain insight into the possibility that the neurons are chronically depolarized and thus in depolarization block.

It is great that the authors quantified not only TH levels but also the levels of mCherry, co-expressed with the chemogenetic receptor. This could in principle help to distinguish between TH downregulation and true loss of dopamine neuron cell bodies. However, the approach used here has a major caveat in that the number of mCherry-positive dopamine neurons depends on the proportion of dopamine neurons that were infected and expressed the DREADD and this could very well vary between different mice. It is very unlikely that the virus injection allowed to infect 100% of the neurons in the VTA and SNc. This could for example explain in part the mismatch between the number of VTA dopamine neurons counted in panel 2G when comparing TH and mCherry counts. Also, I see that the mCherry counts were not provided at the 2-week time point. If the mCherry had been expressed genetically by crossing the DAT-Cre mice with a floxed fluorescent reported mice, the interpretation would have been simpler. In this context, I am not convinced of the benefit of the mCherry quantifications. The authors should consider either removing these results from the final manuscript or discussing this important limitation.

Although the authors conclude that there is a global decrease in the number of dopamine neurons after 4 weeks of CNO treatment, the post-hoc tests failed to confirm that the decrease in dopamine number was significant in the SNc, the region most relevant to Parkinson's. This could be due to the fact that only a small number of mice were tested. A "n" of just 4 or 5 mice is very small for a stereological counting experiment. As such, this experiment was clearly underpowered at the statistical level. Also, the choice of the image used to illustrate this in panel 2G should be reconsidered: the image suggests that a very large loss of dopamine neurons occurred in the SNc and this is not what the numbers show. A more representative image should be used.

In Figure 3, the authors attempt to compare intracellular calcium levels in dopamine neurons using GCaMP6 fluorescence. Because this calcium indicator is not quantitative (unlike ratiometric sensors such as Fura2), it is usually used to quantify relative changes in intracellular calcium. The present use of this probe to compare absolute values is unusual and the validity of this approach is unclear. This limitation needs to be discussed. The authors also need to refer in the text to the difference between panels D and E of this figure. It is surprising that the fluctuations in calcium levels were not quantified. I guess the hypothesis was that there should be more or larger fluctuations in the mice treated with CNO if the CNO treatment led to increased firing. This needs to be clarified.

Although the spatial transcriptomic results are intriguing and certainly a great way to start thinking about how the CNO treatment could lead to the loss of dopamine neurons, the presented results, the focussing of some broad classes of differentially expressed genes and on some specific examples, do not really suggest any clear mechanism of neurodegeneration. It would perhaps be useful for the authors to use the obtained data to validate that a state of chronic depolarization was indeed induced by the chronic CNO treatment. Were genes classically linked to increased activity like cfos or bdnf elevated in the SNc or VTA dopamine neurons? In the striatum, the authors report that the levels of DARP32, a gene whose levels are linked to dopamine levels, are unchanged. Does this mean that there were no major changes in dopamine levels in the striatum of these mice?

The usefulness of comparing the transcriptome of human PD SNc or VTA sections to that of the present mouse model should be better explained. In the human tissues, the transcriptome reflects the state of the tissue many years after extensive loss of dopamine neurons. It is expected that there will be few if any SNc neurons left in such sections. In comparison, the mice after 7 days of CNO treatment do not appear to have lost any dopamine neurons. As such, how can the two extremely different conditions be reasonably compared?

Comments on the discussion:

In the discussion, the authors state that their calcium photometry results support a central role of calcium in activity-induced neurodegeneration. This conclusion, although plausible because of the very broad pre-existing literature linking calcium elevation (such as in excitotoxicity) to neuronal loss, should be toned down a bit as no causal relationship was established in the experiments that were carried out in the present study.

In the discussion, the authors discuss some of the parallel changes in gene expression detected in the mouse model and in the human tissues. Because few if any dopamine neurons are expected to remain in the SNc of the human tissues used, this sort of comparison has important conceptual limitations and these need to be clearly addressed.

A major limitation of the present discussion is that it does not discuss the possibility that the observed phenotypes are caused by the induction of a chronic state of depolarization block by the chronic CNO treatment. I encourage the authors to consider and discuss this hypothesis. Also, the authors need to discuss the fact that previous work was only able to detect an increase in the firing rate of dopamine neurons after more than 95% loss of dopamine neurons. As such, the authors need to clearly discuss the relevance of the present model to PD. Are changes in firing rate a driver of neuronal loss in PD, as the authors try to make the case here, or are such changes only a secondary consequence of extensive neuronal loss (for example because a major loss of dopamine would lead to reduced D2 autoreceptor activation in the remaining neurons, and to reduced autoreceptor-mediated negative feedback on firing). This needs to be discussed.

There is a very large, multi-decade literature on calcium elevation and its effects on neuronal loss in many different types of neurons. The authors should discuss their findings in this context and refer to some of this previous work. In a nutshell, the observations of the present manuscript could be summarized by stating that the chronic membrane depolarization induced by the CNO treatment is likely to induce a chronic elevation of intracellular calcium and this is then likely to activate some of the well-known calcium-dependent cell death mechanisms. Whether such cell death is linked in any way to PD is not really demonstrated by the present results.

The authors are encouraged to perform a thorough revision of the discussion to address all of these issues, discuss the major limitations of the present model, and refer to the broad pre-existing literature linking membrane depolarization, calcium, and neuronal loss in many neuronal cell types.

Reviewer #2 (Public review):

The aim of the investigation was to find out more about the mechanism(s) by which the structural protein vimentin can facilitate the epithelial-mesenchymal transition in breast cancer cells.

The authors focused on a key amino acid of vimentin, C238, its role in the interaction between vimentin and actin microfilaments, and the downstream molecular and cellular consequences. They model the binding between vimentin and actin in silico to demonstrate the potential involvement of C238, due to its location in a rod domain known to bind beta-actin. The phenotype of a non-metastatic breast cancer cell line MCF7, which doesn't express vimentin, could be changed to a metastatic phenotype when mutant C238S vimentin, but not wild-type vimentin, was expressed in the cells. Expression of vimentin was confirmed at the level of mRNA, protein and microscopically. Patterns of expression of vimentin and actin reflected the distinct morphology of the two cell lines. Phenotypic changes were assessed through assay of cell adhesion, proliferation, migration and morphology and were consistent with greater metastatic potential in the C238S MCF7 cells. Changes in the transcriptome of MCF7 cells expressing wild-type and C238S vimentins were compared and expression of Xist long ncRNA was found to be the transcript most markedly increased in the metastatic cells expressing C238S vimentin. Moreover changes in expression of many other genes in the C238S cells are consistent with an epithelial mesenchymal transition. Tumourigenic potential of MCF7 cells carrying C238S but not wild-type, vimentin was confirmed by inoculation of cells into nude mice. This assay is a measure of stem-cell quality of the cells and not a measure of metastasis. It does demonstrate phenotypic changes that could be linked to metastasis.

shRNA was used to down-regulate vimentin or Xist in the MCF7 C238S cells. The description of the data is limited in parts and data sets require careful scrutiny to understand the full picture. Down-regulation of vimentin reversed the morphological changes to some degree, but down-regulation of Xist didn't. Conversely down-regulation of Xist inhibited cell growth, a sign of reversing metastatic potential, but down-regulation of vimentin had no effect on growth. Down-regulation of either did inhibit cell migration, another sign of metastatic reversal. Most of these findings are consistent with previous work based on ectopic expression of wild-type vimentin in MCF7 cells, but the mechanism of inhibition of cell migration by downregulation of Xist remains speculative. More complete knockdown of vimentin or Xist by CRISPR technology may be helpful.

Overall the study describes an intriguing model of metastasis that is worthy of further investigation, especially at the molecular level to unravel the connection between vimentin and metastasis. The identification of a potential role for Xist in metastasis, beyond its normal role in female cells to inactivate one of the X chromosomes, corroborates the work of others demonstrating increased levels in a variety of tumours in women and even in some tumours in men. It would be of great interest to see where in metastatic cells Xist is expressed and what it binds to.

Comments on revisions:

The revised manuscript incorporates changes in presentation of the data modelling interaction between the region of vimentin including C238 and F-actin. There is also inclusion of an extra citation supporting the role for Xist in cancer stem cell differentiation.

Reviewer #1 (Public review):

Summary:

Hua et al show how targeting amino acid metabolism can overcome Trastuzumab resistance in HER2+ breast cancer.

Strengths:

The authors used metabolomics, transcriptomics and epigenomics approaches in vitro and in preclinical models to demonstrate how trastuzumab resistant cells utilize cysteine metabolism.

Weaknesses:

However, there are some key aspects that needs to be addressed.

Major:

(1) Patient Samples for Transcriptomic Analysis: It is unclear from the text whether tumor tissues or blood samples were used for the transcriptomic analysis. This distinction is crucial, as these two sample types would yield vastly different inferences. The authors should clarify the source of these samples.

(2) The study only tested one trastuzumab-resistant and one trastuzumab-sensitive cell lines. It is unclear whether these findings are applicable to other HER2-positive tumor cell lines, such as HCC1954. The authors should validate their results in additional cell lines to strengthen their conclusions.

(3) Relevance to Metastatic Disease: Trastuzumab resistance often arises in patients during disease recurrence, which is frequently associated with metastasis. However, the mouse experiments described in this paper were conducted only in the primary tumors. This article will have more impact if the authors could demonstrate that the combination of Erastin or cysteine starvation with trastuzumab can also improve outcomes in metastasis models.

Minor:

(1) The figures lack information about the specific statistical tests used. Including this information is essential to show the robustness of the results.

(2) Figure 3K Interpretation: The significance asterisks in Figure 3K do not specify the comparison being made. Are they relative to the DMSO control? This should be clarified.

Comments on revisions:

While the authors acknowledge the limitation of using only a single trastuzumab resistant/sensitive pair, simply stating that additional cell lines will be tested in future work is simply inadequate. The biological heterogeneity of HER2-positive breast cancer demands validation in at least one independent resistant model (e.g., HCC1954 or BT 474R) alongside its parental counterpart. Without demonstrating that SLC7A11 upregulation, cysteine dependency, and sensitivity to Erastin plus trastuzumab extend beyond the original cell line pair, the generalizability and translational relevance of the findings remain uncertain. The authors need to perform and report key functional results (cell viability, apoptosis, and SLC7A11 expression) in an additional resistant and sensitive HER2-positive cell line before this manuscript can be considered robust.

Reviewer #2 (Public review):

In this manuscript, Hua et al. proposed SLC7A11, a protein facilitating cellular cystine uptake, as a potential target for the treatment of trastuzumab resistant HER2 positive breast cancer. If this claim holds true, the finding would be of significance and might be translated to clinical practice. Nevertheless, this reviewer finds that the conclusion was insufficiently supported by the data.

Notably, most of the data (Figures 2-6) were based on two cell lines - JIMT1 as a representative of trastuzumab resistant cell line, and SKBR3 as a representative of trastuzumab sensitive cell line. As such, these findings could be cell line specific while irrelevant to trastuzumab sensitivity at all. Furthermore, the authors' claim of ferroptosis induction is primarily based on lipid peroxidation assays (Figure 3). The rescuing effects of ferroptosis inhibitors on cell viability were missing. The xenograft experiments were also suspicious (Figure 4). Systemic cysteine starvation is known to cause adverse effects, including liver necrosis, and the compound (i.e., erastin) used by the authors is not suitable for in vivo experiments due to low solubility and low metabolic stability. Finally, the authors focus on epigenetic regulations (Figures 5 & 6) without first investigating well-established transcription factors, such as NRF2 and ATF4, which are known to regulate SLC7A11.

To sum up, this reviewer finds that the most valuable data in this manuscript is perhaps Figure 1, which provides unbiased information concerning the metabolic patterns in trastuzumab sensitive and primary resistant HER2 positive breast cancer patients.

Comments on revisions:

(1) Figure 3: The unit of concentration should be "μM". "μm" means micrometer.

(2) Figure S5: Ferroptosis inhibitors should be used in cell viability assays to exclude the off-target effect of RSL3 and erastin. Note that erastin also targets VDAC, while RSL3 may inhibit other selenoproteins at high concentrations. Cell viability assays are critical for demonstrating ferroptosis and should be included in the main figure rather than relegated to the supplemental materials.

(3) Figure 4B & 4C: the data of "H" group and "Erastin" group are inconsistent. In panel B, the tumor size in the "H" group appears smaller than in the "Erastin" group, while in panel C, the opposite trend is observed.

(4) The catalog numbers for the cystine/cysteine-deficient DMEM (from BIOTREE) and diet (from Xietong Bio) should be provided. This information is essential for readers to identify and verify the specific products used in the study.

Reviewer #1 (Public review):

Summary:

This study provides new insight into the non-canonical voltage-gating mechanism of BK channels through prolonged (10 μs) MD simulations of the Slo1 transmembrane domain conformation and K+ conduction in response to high imposed voltages (300, 750 mV). The results support previous conclusions based on functional and structural data and MD simulations that the voltage-sensor domain (VSD) of Slo1 undergoes limited conformational changes compared to Kv channels, and predicts gating charge movement comparable in magnitude to experimental results. The gating charge calculations further indicate that R213 and R210 in S4 are the main contributors owing to their large side chain movements and the presence of a locally focused electric field, consistent with recent experimental and MD simulation results by Carrasquel-Ursulaez et al.,2022. Most interestingly, changes in pore conformation and K+ conduction driven by VSD activation are resolved, providing information regarding changes in VSD/pore interaction through S4/S5/S6 segments proposed to underly electromechanical coupling.

Strengths:

Include that the prolonged timescale and high voltage of the simulation allow apparent equilibration in the voltage-sensor domain (VSD) conformational changes and at least partial opening of the pore. The study extends the results of previous MD simulations of VSD activation by providing quantitative estimates of gating charge movement, showing how the electric field distribution across the VSD is altered in resting and activated states, and testing the hypothesis that R213 and R210 are the primary gating charges by steered MD simulations. The ability to estimate gating charge contributions of individual residues in the WT channel is useful as a comparison to experimental studies based on mutagenesis which have yielded conflicting results that could reflect perturbations in structure. Use of dynamic community analysis to identify coupling pathways and information flow for VSD-pore (electromechanical) coupling, as well as analysis of state-dependent S4/S5/S6 interactions that could mediate coupling, provides useful predictions extending beyond what has been experimentally tested.

Weaknesses:

Include that a truncated channel (lacking the C-terminal gating ring) was used for simulations, which is known to have reduced single channel conductance and reduced electromechanical coupling compared to the full-length channel. In addition, as VSD activation in BK channels is much faster than opening, the timescale of simulations was likely insufficient to achieve a fully open state, as supported by differences in the degree of pore expansion in replicate simulations, which are also smaller than observed in Ca-bound open structures of the full-length channel. Taken together, these limitations suggest that the analysis regarding coupling pathways and interactions is incomplete. In addition, while the simulations convincingly demonstrate voltage-dependent channel opening as evidenced by pore expansion, and conduction of K+ and water through the pore, single channel conductance is underestimated by at least an order of magnitude, as in previous studies of other K+ channels. These quantitative discrepancies suggest that MD simulations may not yet be sufficiently advanced to provide insight into mechanisms underlying the extraordinarily large conductance of BK channels.

Reviewer #2 (Public review):

Summary:

This manuscript addresses the structural basis of voltage-activation of BK channels using computational approaches. Although a number of experimental studies using gating current and patch-clamp recording have analyzed voltage-activation in terms of observed charge movements and the apparent energetic coupling between voltage-sensor movement and channel opening, the structural changes that underlie this phenomenon have been unclear. The present studies use a reduced molecular system comprising the transmembrane portion of the BK channel (i.e., the cytosolic domain was deleted), embedded in a POPC membrane, with either 0 or 750 mV applied across the membrane. This system enabled acquisition of long simulations of 10 microseconds, to permit tracking of conformational changes of the channel. The authors' principal findings were that the side chains of R210 and R213 rapidly moved toward the extracellular side of the membrane (by 8 - 10 Å), with greater displacements than any of the other charged transmembrane residues. These movements appeared tightly coupled to the movement of the pore-lining helix, pore hydration, and ion permeation. The authors estimate that R210 and R213 contribute 0.25 and 0.19 elementary charges per residue to the gating current, which is roughly consistent with estimates based on electrophysiological measurements that used the full-length channel.

Strengths:

The methodologies used in this work are sound, and these studies certainly contribute to our understanding of voltage-gating of BK channels. An intriguing observation is the strongly coupled movement of the S4, S5, and S6 helices that appear to underlie voltage-dependent opening. Based on Figures 2a-d, the substantial movements of the R210 and R213 side chains occur nearly simultaneously to the S6 movement (between 4 - 5 usec of simulation time). This seems to provide support for a "helix-packing" mechanism of voltage gating in the so-called "non-domain-swapped" voltage-gated K channels.

Weaknesses:

The main limitation is that these studies used a truncated version of the BK channel, and there are likely to be differences in VSD-pore coupling in the context of the full-length channels that will not be resolved in the present work. Nonetheless, the authors provide a strong rationale for their use of the truncated channel, and the results presented will provide a good starting point for future computational studies of this channel.

Reviewer #1 (Public review):

In this work, Ligneul and coauthors implemented diffusion-weighted MRS in young rats to follow longitudinally and in vivo the microstructural changes occurring during brain development. Diffusion-weighted MRS is here instrumental in assessing microstructure in a cell-specific manner, as opposed to the claimed gold-standard (manganese-enhanced MRI) that can only probe changes in brain volume. Differential microstructure and complexification of the cerebellum and the thalamus during rat brain development were observed non-invasively. In particular, lower metabolite ADC with increasing age were measured in both brain regions, reflecting increasing cellular restriction with brain maturation. Higher sphere (representing cell bodies) fraction for neuronal metabolites (total NAA, glutamate) and total creatine and taurine in the cerebellum compared to the thalamus were estimated, reflecting the unique structure of the cerebellar granular layer with a high density of cell bodies. Decreasing sphere fraction with age was observed in the cerebellum, reflecting the development of the dendritic tree of Purkinje cells and Bergmann glia. From morphometric analyses, the authors could probe non-monotonic branching evolution in the cerebellum, matching 3D representations of Purkinje cells expansion and complexification with age. Finally, the authors highlighted taurine as a potential new marker of cerebellar development.

From a technical standpoint, this work clearly demonstrates the potential of diffusion-weighted MRS at probing microstructure changes of the developing brain non-invasively, paving the way for its application in pathological cases. Ligneul and coauthors also show that diffusion-weighted MRS acquisitions in neonates are feasible, despite the known technical challenges of such measurements, even in adult rats. They also provide all necessary resources to reproduce and build upon their work, which is highly valuable for the community.

From a biological standpoint, claims are well supported by the microstructure parameters derived from advanced biophysical modelling of the diffusion MRS data.

Specific strengths:

(1) The interpretation of dMRS data in terms of cell-specific microstructure through advanced biophysical modelling (e.g. the sphere fraction, modelling the fraction of cell bodies versus neuronal or astrocytic processes) is a strong asset of the study, going beyond the more commonly used signal representation metrics such as the apparent diffusion coefficient, which lacks specificity to biological phenomena.<br /> (2) The fairly good data quality despite the complexity of the experimental framework should be praised: diffusion-weighted MRS was acquired in two brain regions (although not in the same animals) and longitudinally, in neonates, including data at high b-values and multiple diffusion times, which altogether constitutes a large-scale dataset of high value for the diffusion-weighted MRS community.<br /> (3) The authors have shared publicly data and codes used for processing and fitting, which will allow one to reproduce or extend the scope of this work to disease populations, and which goes in line with the current effort of the MR(S) community for data sharing.

Specific weaknesses:

Ligneul and coauthors have convincingly addressed and included my comments in their revised manuscript.

I believe the following conceptual concerns, which are inherent to the nature of the study and do not require further adjustments of the manuscript, remain:

(1) Metabolite compartmentation in one cell type or the other has often been challenged and is currently impossible to validate in vivo. Here, Ligneul and coauthors did not use this assumption a priori and supported their claims also with non-MR literature (eg. for Taurine), but the interpretation of results in that direction should be made with care.

(2) Longitudinal MR studies of the developing brain make it difficult to extract parameters with an "absolute" meaning. Indirect assumptions used to derive such parameters may change with age and become confounding factors (brain structure, cell distribution, concentrations normalizing metabolites (here macromolecules), relaxation times...). While findings of the manuscript are convincing and supported with literature, the true underlying nature of such changes might be difficult to access.

(3) Diffusion MRI in addition to diffusion MRS would have been complementary and beneficial to validate some of the signal contributions, but was unfeasible in the time constraints of experiments on young animals.

Reviewer #2 (Public review):

This second revision has partially addressed criticisms previously raised; however, substantial inadequacies, particularly concerning rigorous validation and model justification, remain unresolved. While recognizing evident strength, novelty, and technical complexity of this work, the authors have yet to fully resolve key major concerns explicitly pointed out during revision in a satisfactory manner. As currently written, the manuscript does not yet provide sufficiently robust validation, methodological rigour, or clarity required for complete acceptance in a top-tier scientific journal.

Summary of Authors' Aim:

In this revised version, the authors aimed to address prior reviewer critiques harshly pinpointing the need for greater clarity in the manuscript's logical flow, rigorous external validation, clearer explanation of methodological normalization choices, and deeper elaboration of diffusion MRI method relevance and potential translation. The authors present a diffusion-weighted MRS approach paired with complex biophysical modelling to elucidate differential developmental trajectories of cellular structures in cerebellum and thalamus in rat neonates, providing a novel, non-invasive avenue for monitoring cellular microstructure.

Major Comments:

Rigorous Validation (Reviewer #1 - point R1.1, Reviewer #2 - point R2.2):

The major concern previously raised and reiterated here is the insufficient external cross-validation of the dMRS-derived interpretations about cellular changes, including the particularly speculative interpretation that taurine undergoes compartment switching between neuronal and glial compartments in the thalamus. The authors acknowledge this important shortcoming (R1.1, R2.2) but attempt to mitigate these concerns merely through additional contextual comparisons from existing literature (page 23, lines 877-878, Figure S11, Table S2). While better contextualization is welcome, the modified manuscript still falls notably short of the level of rigour necessary to validate such striking switches in compartmentalization. To justify claims of metabolites changing cellular compartments, explicit verification against independent molecular/histological data, ideally with additional immunohistochemical staining for cellular markers (e.g., glial fibrillary acidic protein, NeuN), is necessary. The mere presence of literature correlations (such as the reported visual comparisons to morphometric reconstructions, page 24, lines 883-884) does not constitute rigorous validation at the required standard for high-impact publication. The revised manuscript remains fundamentally weakened without such validation. To properly improve, the authors must consider incorporating independent ex vivo experiments or, if this is no longer feasible, extensively temper their compartment-switching claims, acknowledging explicitly and prominently the speculative nature of current interpretations.

Normalization of Metabolite Concentrations (Reviewer #1 - point R1.3):

The authors clearly responded to a reviewer wish for justification of metabolite normalisation to macromolecular concentrations (page 13, lines 493-503, Figure S2). However, the rationale provided remains only partially convincing. While the authors appropriately acknowledge the unusual nature of their methodological choice and possible confounding factors, they opt to supplement rather than substitute this approach with a more standard method (normalisation by water) in the main body of the manuscript. The additional supplementary Figure S2 is helpful, yet the conclusions derived with macromolecular normalization still remain potentially confounded by age-dependent macromolecular changes (Tkac et al., 2003). The justification given in the revised manuscript remains vague, unsatisfactory, and somewhat contradictory-authors accept macromolecules changes likely with age, yet largely overlook this effect. At least, the comparison between normalization by macromolecules and water should be explicitly discussed in the main text, and conclusions drawn from macromolecular normalization must be cautiously framed.

Choice and Justification of Biophysical Model (Reviewer #1 - point R1.4):

The reviewers questioned model assumptions, particularly ignoring macroscopic anisotropy effects due to white matter presence, myelination, and fibre orientation dispersion in the cerebellar voxel. Authors provided newly included DTI data and acknowledged this limitation explicitly (R1.4, Figure S8, page 25, lines 921-924). However, the addition of these poor-quality DTI data with limited interpretability paradoxically weakens rather than strengthens the manuscript as a whole, since the authors now present unclear supplementary results with little additional interpretative value. Recognizing poor data quality in this scenario, although intellectually honest, does not substantially increase the current robustness of their chosen model nor improve justification. To address this fully, either higher-quality data should be collected to robustly probe anisotropy or fibre dispersion effects, or the authors must much further restrict their interpretations in view of this clear limitation. Currently, the solution proposed is incomplete and insufficient to clarify the consequences of their chosen model.

Logical Flow and Clarity (Reviewer #2 - points R2.1 and R2.3):

The authors attempted to respond to reviewer comments on logical flow and accessibility (page 3, introduction restructuring). While the manuscript readability has improved, the introduction and discussion remain overly intricate, and at times, detail-oriented without clear links into central claims. In particular, the biological rationale for choosing the specific metabolite markers (especially tCho, Ins, Tau, etc.) and their known relevance must be further streamlined and simplified to increase accessibility and directness. Although some helpful restructuring was carried out, further careful paragraph-level revision for logical flow and readability remains necessary.

Translation to Human Studies (Reviewer #2 - point R2.4):

The authors have extended contextual discussion on translational potential regarding taurine as a developmental marker in humans (pages 24-25, lines 906-917). However, mention remains vague and cursory, without presenting sufficiently solid arguments nor drawing from human developmental studies adequately. Translational potential must be assessed within the realistic limitations inherent in clinical translation of MRS studies, particularly given the technical complexities clearly identified even in preclinical studies of this paper. Discussion remains relatively superficial, and if retained, must be expanded to fully discuss realistic human translational hurdles and requirements.

Reviewer #1 (Public review):

Summary:

This study by Torok et al. takes a creative approach to studying circuit perturbations in a sensorimotor region for vocalization control, in a songbird species, the zebra finch. By expressing the light chain of tetanus toxin in neurons in a sensorimotor region HVC, the authors constrain neural firing and study the resulting degradation and then recovery of song, after a protracted (> 70-day) period. Recording data suggest a form of synaptic homeostasis emergent in both HVC and RA as a result of the profound loss of (inhibitory?) tone in HVC. The methods to analyze changes in song are particularly strong here, using dimension reduction and visualization techniques. Single-cell sequencing data showed accompanying changes in microglia abundance, as well as several other markers that were not observed in control viral injections. LFP analyses in birds during the tetanus onset phase showed clear dysregulation of typical voltage deflections and spectral power, each of which showed recovery in parallel with song recovery. Lastly, the authors present data indicating that the anterior forebrain region LMAN is not critical for the song degradation process, pointing instead to the direct relationship between HVC and RA in song plasticity in adults. The methods are generally well established, but my main concerns regard the validation of the viral construct, the lack of direct confirmation of tetanus toxin on inhibitory neurons or E/I balance in HVC, and a missed opportunity to look at song syllable sequence degradation and recovery.

Strengths:

The species under investigation is the premier model for the neural basis of vocal learning, and the telencephalic brain regions investigated are well mapped out for their control of vocal learning behavior. The methods for electrophysiology recording and analysis, song analysis, scRNAseq, and in situ hybridization pose no concern as they are well established for this group of co-authors.

Weaknesses:

The introduction lays out a case for pursuing long-term E/I imbalances, vis-à-vis transient perturbations that have shown effects on the behavior. However, the rationale is not clearly stated. Why should the reader care that "prolonged E/I imbalances" may occur? Do they occur naturally or in some disease states (as alluded to in the first paragraph)? Without this rationale, the reader is left with an impression that the experiments were done because of a technical capability rather than a conceptual thrust.

The cited works for the statement the "AAV viral vector expressing TeNT undre the human dlx promoter, which is selective for HVC inhibitory interneurons" (reference 5 Kosche et al., 2016; and reference 10 Vallentin et al 2016) do not substantiate the targeting of this dlx5 promoter for interneurons in zebra finch HVC. Neither of these cited studies used viral vectors, and so this is a misattribution of the dlx5 promoter as targeting HVC inhibitory interneurons. However, the original development of this enhancer by Gord Fishell and others did have solid expression in HVC (Dimidschstein et al., 2016, Nature Neuroscience), and the enhancer was used to successfully target inhibitory neurons in nearby nidopallium NCM (Spool et al., 2022, Curr Biol). Citing these two studies would improve the standing of this viral approach. Nevertheless, the specific construct used here is not the same as the published studies mentioned above (AAV9-dlx-TeNT). The authors therefore need to show expression of the virus using some histological confirmation to cement the idea that they are indeed targeting inhibitory interneurons with this manipulation. The methods statement "a single injection (~100 nL) in the center of HVC was sufficient to label enough cells" is not convincing in the absence of quantified photomicrographs.

The authors present no physiological confirmation of TeNT on E/I balance directly, and so we don't have a clear picture of how/whether HVC interneurons are physiologically altered by this manipulation. That said, the Npix recordings show that there was a tremendous increase in gamma power following TeNT manipulation, which subsides as the protracted song recovery unfolds. This finding is somewhat counterintuitive, given that gamma oscillations are typically driven by inhibitory neurons in many systems (including songbird pallium) while the TeNT manipulation is purported to cause *reductions* in inhibitory neurotransmitter release within HVC. Some interpretation of these incongruent results would be useful in the Discussion.

The degradation and recovery of song is based mainly on the measures of duration of syllables and inter-syllable intervals, but HVC is also a key locus for song syllable sequence coding. The supplementary figures show some changes in sequences. It would improve the interpretation of both the degradation and recovery of the song to know whether syllable sequences (iiiABCCDDEF) truly recovered or were morphed in some way (e.g., iiiCDDDBEF). The PCA analyses (that the authors conducted) for these two potential outcomes would likely be very similar, but the actual songs would differ greatly under these two scenarios in terms of syllable sequence. From the representative spectrograms, it appears that the song syllable sequence does indeed recover well in these examples (perhaps less so in Supplementary Figure 3). A simple Markov-chain analysis of the syllable sequences across birds in the study would provide important confirmation of these insights.