Reviewer #1 (Public Review):

The manuscript describes a GAN-based approach that generates parameters for HH-like channels for multiple C. Elengans neurons. The network is trained on generated data to produce parameter sets that, on the one hand, reproduce voltage responses and IV curves, and on the other hand, are indistinguishable from the ground truth parameters, as tested by the discriminator. It is then shown that these generated parameter sets lead to reasonable reproductions of the recorded responses (but see the section "weaknesses" below for some reservations).

Strengths:

In itself, I find the methodology of high interest, particularly in that it can generate parameter sets to construct models of new recordings at a very low computational cost.

Weaknesses:

Nevertheless, I believe there are some weaknesses in the evaluation of the models that should be addressed before the quality of the methodology can be fully assessed. Firstly, at the methodological level, the authors should provide more clarity on the inverse gradient operation they use, as opposed to just simulating the models, as such an inversion depends not only on the parameters but also on the state of the model. How the state is obtained remains unclear here. Secondly, in the evaluation of their models, the authors could provided more information such as IV curves, as whether these would be accurate is difficult to visually infer from their figures. Thirdly, the authors do not address the question of whether all obtained parameter sets are stable when simulated over longer times, while their figures do include hints that this might not be the case for at least some of their models (e.g. voltage traces that do not converge back to the equilibrium after the stimulus, but rather seem to diverge).

Reviewer #2 (Public Review):

Summary:

Generating biophysically detailed computational models that capture the characteristic physiological properties of biological neurons for diverse cell types is an important and difficult problem in computational neuroscience. One major challenge lies in determining the large number of parameters of such models, which are notoriously difficult to fit into experimental data. Thereby, the computational and energy costs can be significant. The study 'ElectroPhysiomeGAN: Generation of Biophysical Neuron Model Parameters from Recorded Electrophysiological Responses' by Kim et al. describes a computationally efficient approach for predicting model parameters of Hodgkin-Huxley neuron models using Generative Adversarial Networks (GANs) trained on simulation data. The method is applied to generate models for 9 non-spiking neurons in C. elegans based on electrophysiological recordings. While the generated models capture the responses of these neurons to some degree, they generally show significant deviations from the empirically observed responses in important features. While interesting, in its current form, the method has not been demonstrated to generate models that faithfully capture empirically observed responses.

Strengths:

The authors work on an important and difficult problem. A noteworthy strength of their approach is that once trained, the GANs can generate models from new empirical data with very little computational effort. The generated models reproduce the average voltage during current injections reasonably well.

Weaknesses:

Major 1: While the models generated with EP-GAN reproduce the average voltage during current injections reasonably well, the dynamics of the response are not well captured. For example, for the neuron labeled RIM (Figure 2), the most depolarized voltage traces show an initial 'overshoot' of depolarization, i.e. they depolarize strongly within the first few hundred milliseconds but then fall back to a less depolarized membrane potential. In contrast, the empirical recording shows no such overshoot. Similarly, for the neuron labeled AFD, all empirically recorded traces slowly ramp up over time. In contrast, the simulated traces are mostly flat. Furthermore, all empirical traces return to the pre-stimulus membrane potential, but many of the simulated voltage traces remain significantly depolarized, far outside of the ranges of empirically observed membrane potentials. While these deviations may appear small in the Root mean Square Error (RMSE), the only metric used in the study to assess the quality of the models, they likely indicate a large mismatch between the model and the electrophysiological properties of the biological neuron.

Major 2: Other metrics than the RMSE should be incorporated to validate simulated responses against electrophysiological data. A common approach is to extract multiple biologically meaningful features from the voltage traces before, during and after the stimulus, and compare the simulated responses to the experimentally observed distribution of these features. Typically, a model is only accepted if all features fall within the empirically observed ranges (see e.g. https://doi.org/10.1371/journal.pcbi.1002107). However, based on the deviations in resting membrane potential and the return to the resting membrane potential alone, most if not all the models shown in this study would not be accepted.

Major 3: Abstract and introduction imply that the 'ElectroPhysiome' refers to models that incorporate both the connectome and individual neuron physiology. However, the work presented in this study does not make use of any connectomics data. To make the claim that ElectroPhysiomeGAN can jointly capture both 'network interaction and cellular dynamics', the generated models would need to be evaluated for network inputs, for example by exposing them to naturalistic stimuli of synaptic inputs. It seems likely that dynamics that are currently poorly captured, like slow ramps, or the ability of the neuron to return to its resting membrane potential, will critically affect network computations.

Reviewer #1 (Public Review):

Summary:

This valuable study by Wu and Zhou combined neurophysiological recordings and computational modelling to investigate the neural mechanisms that underpin the interaction between sensory evaluation and action selection. The neurophysiological results suggest non-linear modulation of decision-related LIP activity by action selection, but some further analysis would be helpful in order to understand whether these results can be generalised to LIP circuitry or might be dependent on specific spatial task configurations. The authors present solid computational evidence that this might be due to projections from choice target representations. These results are of interest for neuroscientists investigating decision-making.

Strengths:

Wu and Zhou combine awake behaving neurophysiology for a sophisticated, flexible visual-motion discrimination task and a recurrent network model to disentangle the contribution of sensory evaluation and action selection to LIP firing patterns. The correct saccade response direction for preferred motion direction choices is randomly interleaved between contralateral and ipsilateral response targets, which allows the dissociation of perceptual choice from saccade direction.<br /> The neurophysiological recordings from area LIP indicate non-linear interaction between motion categorisation decisions and saccade choice direction.

The careful investigation of a recurrent network model suggests that feedback from choice target representations to an earlier sensory evaluation stage might be the source for this non-linear modulation and that it is an important circuit component for behavioural performance.

The paper presents a possible solution to a central controversy about the role of LIP in perceptual decision-making, but see below.

Weaknesses:

The paper presents a possible solution to a central controversy about the role of LIP in perceptual decision-making. However, the authors could be more clear and upfront about their interpretational framework and potential alternative interpretations.<br /> Centrally, the authors' model and experimental data appears to test only that LIP carries out sensory evaluation in its RFs. The model explicitly parks the representation of choice targets outside the "LIP" module receiving sensory input. The feedback from this separate target representation provides then the non-linear modulation that matches the neurophysiology. However, they ignore the neurophysiological results that LIP neurons can also represent motor planning to a saccade target.<br /> The neurophysiological results with a modulation of the direction tuning by choice direction (contralateral vs ipsilateral) are intriguing. However, the evaluation of the neurophysiological results are difficult, because some of the necessary information is missing to exclude alternative explanations. It would be good to see the actual distributions and sizes of the RF, which were determined based on visual responses not with a delayed saccade task. There might be for example a simple spatial configuration, for example, RF and preferred choice target in the same (contralateral) hemifield, for which there is an increase in firing. It is a shame that we do not see what these neurons would do if only a choice target would be put in the RF, as has been done in so many previous LIP experiments. The authors exclude also some spatial task configurations (vertical direction decisions), which makes it difficult to judge whether these data and models can be generalised. The whole section is difficult to follow, partly also because it appears to mix reporting results with interpretation (e.g. "feedback").

The model and its investigation is very interesting and thorough, but given the neurophysiological literature on LIP, it is not clear that the target module would need to be in a separate brain area, but could be local circuitry within LIP between different neuron types.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors recorded activity in the posterior parietal cortex (PPC) of monkeys performing a perceptual decision-making task. The monkeys were first shown two choice dots of two different colors. Then, they saw a random dot motion stimulus. They had to learn to categorize the direction of motion as referring to either the right or left dot. However, the rule was based on the color of the dot and not its location. So, the red dot could either be to the right or left, but the rule itself remained the same. It is known from past work that PPC neurons would code the learned categorization. Here, the authors showed that the categorization signal depended on whether the executed saccade was in the same hemifield as the recorded PPC neuron or in the opposite one. That is, if a neuron categorized the two motion directions such that it responded stronger for one than the other, then this differential motion direction coding effect was amplified if the subsequent choice saccade was in the same hemifield. The authors then built a computational RNN to replicate the results and make further tests by simulated "lesions".

Strengths:

Linking the results to RNN simulations and simulated lesions.

Weaknesses:

Potential interpretational issues due to a lack of evidence on what happens at the time of the saccades.

Reviewer #1 (Public Review):

Summary:

Two important factors in visual performance are the resolving power of the lens and the signal-to-noise ratio of the photoreceptors. These both compete for space: a larger lens has improved resolving power over a smaller one, and longer photoreceptors capture more photons and hence generate responses with lower noise. The current paper explores the tradeoff of these two factors, asking how space should be allocated to maximize eye performance (measured as encoded information).

Strengths:

The topic of the paper is interesting and not well studied. The approach is clearly described and seems appropriate (with a few exceptions - see weaknesses below). In most cases, the parameter space of the models are well explored and tradeoffs are clear.

Weaknesses:

- Light level<br /> The calculations in the paper assume high light levels (which reduces the number of parameters that need to be considered). The impact of this assumption is not clear. A concern is that the optimization may be quite different at lower light levels. Such a dependence on light level could explain why the model predictions and experiment are not in particularly good agreement. The paper would benefit from exploring this issue.

- Discontinuities<br /> The discontinuities and non-monotonicity of the optimal parameters plotted in Figure 4 are concerning. Are these a numerical artifact? Some discussion of their origin would be quite helpful.

- Discrepancies between predictions and experiment<br /> As the authors clearly describe, experimental measurements of eye parameters differ systematically from those predicted. This makes it difficult to know what to take away from the paper. The qualitative arguments about how resources should be allocated are pretty general, and the full model seems a complex way to arrive at those arguments. Could this reflect a failure of one of the assumptions that the model rests on - e.g. high light levels, or that the cost of space for photoreceptors and optics is similar? Given these discrepancies between model and experiment, it is also hard to evaluate conclusions about the competition between optics and photoreceptors (e.g. at the end of the abstract) and about the importance for evolution (end of introduction).

Reviewer #2 (Public Review):

Summary:

In short, the paper presents a theoretical framework that predicts how resources should be optimally distributed between receptors and optics in eyes.

Strengths:

The authors build on the principle of resource allocation within an organism and develop a formal theory for optimal distribution of resources within an eye between the receptor array and the optics. Because the two parts of eyes, receptor arrays and optics, share the same role of providing visual information to the animal it is possible to isolate these from resource allocation in the rest of the animal. This allows for a novel and powerful way of exploring the principles that govern eye design. By clever and thoughtful assumptions/constraints, the authors have built a formal theory of resource allocation between the receptor array and the optics for two major types of compound eye as well as for camera-type eyes. The theory is formalized with variables that are well characterized in a number of different animal eyes, resulting in testable predictions.

The authors use the theory to explain a number of design features that depend on different optimal distribution of resources between the receptor array and the optics in different types of eyes. As an example, they successfully explain why eye regions with different spatial resolution should be built in different ways. They also explain differences between different types of eyes, such as long photoreceptors in apposition compound eyes and much shorter receptors in camera type eyes. The predictive power in the theory is impressive.

To keep the number of parameters at a minimum, the theory was developed for two types of compound eye (neural superposition, and apposition) and for camera-type eyes. It is possible to extend the theory to other types of eyes, although it would likely require more variables and assumptions/constraints to the theory. It is thus good to introduce the conceptual ideas without overdoing the applications of the theory.

The paper extends a previous theory, developed by the senior author, that develops performance surfaces for optimal cost/benefit design of eyes. By combining this with resource allocation between receptors and optics, the theoretical understanding of eye design takes a major leap and provides entirely new sets of predictions and explanations for why eyes are built the way they are.

The paper is well written and even though the theory development in the Results may be difficult to take in for many biologists, the Discussion very nicely lists all the major predictions under separate headings, and here the text is more tuned for readers that are not entirely comfortable with the formalism of the Results section. I must point out though that the Results section is kept exemplary concise. The figures are excellent and help explain concepts that otherwise may go above the head of many biologists.

Reviewer #3 (Public Review):

Summary:

This is a proposal for a new theory for the geometry of insect eyes. The novel cost-benefit function combines the cost of the optical portion with the photoreceptor portion of the eye. These quantities are put on the same footing using a specific (normalized) volume measure, plus an energy factor for the photoreceptor compartment. An optimal information transmission rate then specifies each parameter and resource allocation ratio for a variable total cost. The elegant treatment allows for comparison across a wide range of species and eye types. Simple eyes are found to be several times more efficient across a range of eye parameters than neural superposition eyes. Some trends in eye parameters can be explained by optimal allocation of resources between the optics and photoreceptors compartments of the eye.

Strengths:

Data from a variety of species roughly align with rough trends in the cost analysis, e.g. as a function of expanding the length of the photoreceptor compartment.

New data could be added to the framework once collected, and many species can be compared.

Eyes of different shapes are compared.

Weaknesses:

Detailed quantitative conclusions are not possible given the approximations and simplifying assumptions in the models and poor accounting for trends in the data across eye types.

Reviewer #1 (Public Review):

Summary:

This study uses an online cognitive task to assess how reward and effort are integrated in a motivated decision-making task. In particular the authors were looking to explore how neuropsychiatric symptoms, in particular apathy and anhedonia, and circadian rhythms affect behavior in this task. Amongst many results, they found that choice bias (the degree to which integrated reward and effort affects decisions) is reduced in individuals with greater neuropsychiatric symptoms, and late chronotypes (being an 'evening person').

Strengths:

The authors recruited participants to perform the cognitive task both in and out of sync with their chronotypes, allowing for the important insight that individuals with late chronotypes show a more reduced choice bias when tested in the morning.<br /> Overall, this is a well-designed and controlled online experimental study. The modelling approach is robust, with care being taken to both perform and explain to the readers the various tests used to ensure the models allow the authors to sufficiently test their hypotheses.

Weaknesses:

This study was not designed to test the interactions of neuropsychiatric symptoms and chronotypes on decision making, and thus can only make preliminary suggestions regarding how symptoms, chronotypes and time-of-assessment interact.

Reviewer #2 (Public Review):

Summary:

The study combines computational modeling of choice behavior with an economic, effort-based decision-making task to assess how willingness to exert physical effort for a reward varies as a function of individual differences in apathy and anhedonia, or depression, as well as chronotype. They find an overall reduction in effort selection that scales with apathy and anhedonia and depression. They also find that later chronotypes are less likely to choose effort than earlier chronotypes and, interestingly, an interaction whereby later chronotypes are especially unwilling to exert effort in the morning versus the evening.

Strengths:

This study uses state-of-the-art tools for model fitting and validation and regression methods which rule out multicollinearity among symptom measures and Bayesian methods which estimate effects and uncertainty about those estimates. The replication of results across two different kinds of samples is another strength. Finally, the study provides new information about the effects not only of chronotype but also chronotype by timepoint interactions which are previously unknown in the subfield of effort-based decision-making.

Weaknesses:

The study has few weaknesses. One potential concern is that the range of models which were tested was narrow, and other models might have been considered. For example, the Authors might have also tried to fit models with an overall inverse temperature parameter to capture decision noise. One reason for doing so is that some variance in the bias parameter might be attributed to noise, which was not modeled here. Another concern is that the manuscripts discuss effort-based choice as a transdiagnostic feature - and there is evidence in other studies that effort deficits are a transdiagnostic feature of multiple disorders. However, because the present study does not investigate multiple diagnostic categories, it doesn't provide evidence for transdiagnosticity, per se.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Mehrhof and Nord study a large dataset of participants collected online (n=958 after exclusions) who performed a simple effort-based choice task. They report that the level of effort and reward influence choices in a way that is expected from prior work. They then relate choice preferences to neuropsychiatric syndromes and, in a smaller sample (n<200), to people's circadian preferences, i.e., whether they are a morning-preferring or evening-preferring chronotype. They find relationships between the choice bias (a model parameter capturing the likelihood to accept effort-reward challenges, like an intercept) and anhedonia and apathy, as well as chronotype. People with higher anhedonia and apathy and an evening chronotype are less likely to accept challenges (more negative choice bias). People with an evening chronotype are also more reward sensitive and more likely to accept challenges in the evening, compared to the morning.

Strengths:

This is an interesting and well-written manuscript which replicates some known results and introduces a new consideration related to potential chronotype relationships which have not been explored before. It uses a large sample size and includes analyses related to transdiagnostic as well as diagnostic criteria. I have some suggestions for improvements.

Weaknesses:

(1) The novel findings in this manuscript are those pertaining to transdiagnostic and circadian phenotypes. The authors report two separate but "overlapping" effects: individuals high on anhedonia/apathy are less willing to accept offers in the task, and similarly, individuals tested off their chronotype are less willing to accept offers in the task. The authors claim that the latter has implications for studying the former. In other words, because individuals high on anhedonia/apathy predominantly have a late chronotype (but might be tested early in the day), they might accept less offers, which could spuriously look like a link between anhedonia/apathy and choices but might in fact be an effect of the interaction between chronotype and time-of-testing. The authors therefore argue that chronotype needs to be accounted for when studying links between depression and effort tasks.<br /> The authors argue that, if X is associated with Y and Z is associated with Y, X and Z might confound each other. That is possible, but not necessarily true. It would need to be tested explicitly by having X (anhedonia/apathy) and Z (chronotype) in the same regression model. Does the effect of anhedonia/apathy on choices disappear when accounting for chronotype (and time-of-testing)? Similarly, when adding the interaction between anhedonia/apathy, chronotype, and time-of-testing, within the subsample of people tested off their chronotype, is there a residual effect of anhedonia/apathy on choices or not?<br /> If the effect of anhedonia/apathy disappeared (or got weaker) while accounting for chronotype, this result would suggest that chronotype mediates the effect of anhedonia/apathy on effort choices. However, I am not sure it renders the direct effect of anhedonia/apathy on choices entirely spurious. Late chronotype might be a feature (induced by other symptoms) of depression (such as fatigue and insomnia), and the association between anhedonia/apathy and effort choices might be a true and meaningful one. For example, if the effect of anhedonia/apathy on effort choices was mediated by altered connectivity of the dorsal ACC, we would not say that ACC connectivity renders the link between depression and effort choices "spurious", but we would speak of a mechanism that explains this effect. The authors should discuss in a more nuanced way what a significant mediation by the chronotype/time-of-testing congruency means for interpreting effects of depression in computational psychiatry.

(2) It seems that all key results relate to the choice bias in the model (as opposed to reward or effort sensitivity). It would therefore be helpful to understand what fundamental process the choice bias is really capturing in this task. This is not discussed, and the direction of effects is not discussed either, but potentially quite important. It seems that the choice bias captures how many effortful reward challenges are accepted overall which maybe captures general motivation or task engagement. Maybe it is then quite expected that this could be linked with questionnaires measuring general motivation/pleasure/task engagement. Formally, the choice bias is the constant term or intercept in the model for p(accept), but the authors never comment on what its sign means. If I'm not mistaken, people with higher anhedonia but also higher apathy are less likely to accept challenges and thus engage in the task (more negative choice bias). I could not find any discussion or even mention of what these results mean. This similarly pertains to the results on chronotype. In general, "choice bias" may not be the most intuitive term and the authors may want to consider renaming it. Also, given the sign of what the choice bias means could be flipped with a simple sign flip in the model equation (i.e., equating to accepting more vs accepting less offers), it would be helpful to show some basic plots to illustrate the identified differences (e.g., plotting the % accepted for people in the upper and lower tertile for the SHAPS score etc).

(3) None of the key effects relate to effort or reward sensitivity which is somewhat surprising given the previous literature and also means that it is hard to know if choice bias results would be equally found in tasks without any effort component. (The only analysis related to effort sensitivity is exploratory and in a subsample of N=56 per group looking at people meeting criteria for MDD vs matched controls.) Were stimuli constructed such that effort and reward sensitivity could be separated (i.e., are uncorrelated/orthogonal)? Maybe it would be worth looking at the % accepted in the largest or two largest effort value bins in an exploratory analysis. It seems the lowest and 2nd lowest effort level generally lead to accepting the challenge pretty much all the time, so including those effort levels might not be sensitive to individual difference analyses?

(4) The abstract and discussion seem overstated (implications for the school system and statements on circadian rhythms which were not measured here). They should be toned down to reflect conclusions supported by the data.

Reviewer #1 (Public Review):

Summary:

This manuscript by Shea and Villeda furnishes the field with a valuable scRNAseq data set detailing microglial aging in the mouse hippocampus. They provide clear evidence that changes in microglial attributes begin in mid-life, well before time points when mice are traditionally considered to be "aging." It also adds to a growing body of data in the field demonstrating that there is substantial heterogeneity in microglial responses to aging. Using in vitro experiments and transgenic manipulations in mice, the authors show that transforming growth factor beta (TGFb1)-based signaling can potently impact microglial state, consistent with previous findings in the field. They also demonstrate that manipulation of microglial TGFb1-based signaling can impact hippocampus-dependent behaviors.

Limitations of the study lie primarily in reaching too far with interpretations of the data. The authors argue that changes in microglial transcriptome during midlife represent a type of "checkpoint," after which microglial aging can progress along distinct trajectories depending on the status of TGFb1 signaling. They also posit that a specific intermediate "stress response" state in midlife is mechanistically linked to a translational burst that drives the subsequent progression of microglia to an "inflammatory state." Unequivocal data to support these causal links is lacking, however. similarly, key additional experiments would be needed to demonstrate that TGFb1 signaling and microglial progression through these identified intermediate states are causally linked to cognitive decline.

Guidance for readers along with study strengths and caveats:

The present manuscript provides valuable strengthening and expansion to a growing body of data showing prominent changes in the microglial state during aging. Microarray(1), bulkRNAseq(2-5), scRNAseq(6,7), snRNAseq(8,9), and spatial transcriptomic(10) approaches have been leveraged to map changes in microglial transcriptome during aging in rodents, non-human primates, and humans. A number of these studies include the hippocampus (1,8,9,11) and have highlighted variation across brain regions in microglial transcriptomic changes during aging (1,11). They have also revealed differences across sex (7) as well as increased cell-to-cell heterogeneity (6-10), consistent with the idea that individual microglia can follow distinct aging trajectories. Several of these studies revealed that changes in microglial attributes begin in middle age (1,7,11), supporting similar observations from studies that did not use omics (12-14). The present manuscript utilizes scRNAseq of hippocampal microglia at adulthood (6mo), middle age (12mo), late middle age (18mo) and aging (24mo) to show that aging-induced changes in microglia begin in middle age and that microglia exhibit ample phenotypic heterogeneity during the progression of aging.

To gain further insight into the dynamics of microglial aging in the hippocampus, the authors used a bioinformatics method known as "pseudotime" or "trajectory inference" to understand how cells may progress through different functional states, as defined by cellular transcriptome (15,16). These bioinformatics approaches can reveal key patterns in scRNAseq / snRNAseq datasets and, in the present study, the authors conclude that a "stress response" module characterized by expression of TGFb1 represents a key "checkpoint" in microglial aging in midlife, after which the cells can move along distinct transcriptional trajectories as aging progresses. This is an intriguing possibility. However, pseudotime analyses need to be validated via additional bioinformatics as well as follow-up experiments. Indeed, Heumos et al, in their Nature Genetics "Expert Guidelines" Review, emphasize that "inferred trajectories might not necessarily have biological meaning." They recommend that "when the expected topology is unknown, trajectories and downstream hypotheses should be confirmed by multiple trajectory inference methods using different underlying assumptions."(15) Numerous algorithms are available for trajectory inference (e.g. Monocle, PAGA, Sligshot, RaceID/StemID, among many others) and their performance and suitability depends on the individual dataset and nature of the trajectories that are to be inferred. It is recommended to use dynGuidelines(16) for the selection of optimal pseudotime analysis methods. In the present manuscript, the authors do not provide any justification for their use of Monocle 3 over other trajectory inference approaches, nor do they employ a secondary trajectory inference method to confirm observations made with Monocle 3. Finally, follow-up validation experiments that the authors carry out have their own limitations and caveats (see below). Hence, while the microglial aging trajectories identified by this study are intriguing, they remain hypothetical trajectories that need to be proven with additional follow-up experiments.

To follow up on the idea that TGFb1 signaling in microglia plays a key role in determining microglial aging trajectories, the authors use RNAscope to show that TGFb1 levels in microglia peak in middle age. They also treat primary LPS-activated microglia with TGFb1 and show that this restores expression of microglial homeostatic gene expression and dampens expression of stress response and, potentially, inflammatory genes. Finally, they utilize transgenic approaches to delete TGFb1 from microglia around 8-10mo of age and scRNAseq to show that homeostatic signatures are lost and inflammatory signatures are gained. Hence, findings in this study support the idea that TGFb1 can strongly regulate microglial phenotype. Loss of TGFb1 signaling to microglia in adulthood has already been shown to cause decreased microglial morphological complexity and upregulation of genes typically associated with microglial responses to CNS insults(17-19). TGFb1 signaling to microglia has also been implicated in microglial responses to disease and manipulations to increase this signaling can improve disease progression in some cases(19). In this light, the findings in the present study are largely confirmatory of previous findings in the literature. They also fall short of unequivocally demonstrating that TGFb1 signaling acts as a "checkpoint" for determining subsequent microglial aging trajectory. To show this clearly, one would need to perturb TGFb1 signaling around 12mo of age and carry out sequencing (bulkRNAseq or scRNAseq) of microglia at 18mo and 24mo. Such experiments could directly demonstrate whether the whole microglial population has been diverted to the TGFb1-low aging trajectory (that progresses through a translational burst state to an inflammation state as proposed). Future development of tools to tag TGFb1 high or low microglia could also enable fate tracing type experiments to directly show whether the TGFb1 state in middle age predicts cell state at later phases of aging.

The present study would also like to draw links between features of microglial aging in the hippocampus and a decline in hippocampal-dependent cognition during aging. To this end, they carry out behavioral testing in 8-10mo old mice that have undergone microglial-specific TGFb1 deletion and find deficits in novel object recognition and contextual fear conditioning. While this provides compelling evidence that TGFb1 signaling in microglia can impact hippocampus-dependent cognition in midlife, it does not demonstrate that this signaling accelerates or modulates cognitive decline (see below). Age-associated cognitive decline refers to cognitive deficits that emerge as a result of the normative brain aging process(20-21). For a cognitive deficit to be considered age-associated cognitive decline, it must be shown that the cognitive operation under study was intact at some point earlier in the adult lifespan. This requires longitudinal study designs that determine whether a manipulation impacts the relationship between brain status and cognition as animals age (22-24). Alternatively, cross-sectional studies with adequate sample sizes can be used to sample the variability in cognitive outcomes at different points of the adult lifespan(22-24) and show that this is altered by a particular manipulation. For this specific study, one would ideally demonstrate that hippocampal-based learning/memory was intact at some point in the lifespan of mice with microglial TGFb1 KO but that this manipulation accelerated or exacerbated the emergence of deficits in hippocampal-dependent learning/memory during aging. In the absence of these types of data, the authors should tone down their claims that they have identified a cellular and molecular mechanism that contributes to cognitive decline.

A final point of clarification for the reader pertains to the mining of previously generated data sets within this study. The language in the results section, methods, and figure legends causes confusion about which experiments were actually carried out in this study versus previous studies. Some of the language makes it sound as though parabiosis experiments and experiments using mouse models of Alzheimer's Disease were carried out in this study. However, parabiosis and AD mouse model experiments were executed in previous studies (25,26), and in the present study, RNAseq datasets were accessed for targeted data mining. It is fantastic to see further mining of datasets that already exist in the field. However, descriptions in the results and methods sections need to make it crystal clear that this is what was done.

References:

(1) Grabert, K. et al. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat. Neurosci. (2016). doi:10.1038/nn.4222<br /> (2) Hickman, S. E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. (2013). doi:10.1038/nn.3554<br /> (3) Deczkowska, A. et al. Mef2C restrains microglial inflammatory response and is lost in brain ageing in an IFN-I-dependent manner. Nat. Commun. (2017). doi:10.1038/s41467-017-00769-0<br /> (4) O'Neil, S. M., Witcher, K. G., McKim, D. B. & Godbout, J. P. Forced turnover of aged microglia induces an intermediate phenotype but does not rebalance CNS environmental cues driving priming to immune challenge. Acta Neuropathol. Commun. (2018). doi:10.1186/s40478-018-0636-8<br /> (5) Olah, M. et al. A transcriptomic atlas of aged human microglia. Nat. Commun. (2018). doi:10.1038/s41467-018-02926-5<br /> (6) Hammond, T. R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50, 253-271 (2019).<br /> (7) Li, X. et al. Transcriptional and epigenetic decoding of the microglial aging process. Nat. aging 3, 1288-1311 (2023).<br /> (8) Zhang, H. et al. Single-nucleus transcriptomic landscape of primate hippocampal aging. Protein Cell 12, 695-716 (2021).<br /> (9) Su, Y. et al. A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan. Cell Stem Cell 29, 1594-1610.e8 (2022).<br /> (10) Allen, W. E., Blosser, T. R., Sullivan, Z. A., Dulac, C. & Zhuang, X. Molecular and spatial signatures of mouse brain aging at single-cell resolution. Cell 186, 194-208.e18 (2023).<br /> (11) Soreq, L. et al. Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging. Cell Rep. 18, 557-570 (2017).<br /> (12) Hefendehl, J. K. et al. Homeostatic and injury-induced microglia behavior in the aging brain. Aging Cell (2014). doi:10.1111/acel.12149<br /> (13) Nikodemova, M. et al. Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week. J. Neuroimmunol. 0, 280-288 (2015).<br /> (14) Moca, E. N. et al. Microglia Drive Pockets of Neuroinflammation in Middle Age. J. Neurosci. 42, 3896-3918 (2022).<br /> (15) Heumos, L. et al. Best practices for single-cell analysis across modalities. Nat. Rev. Genet. 24, 550-572 (2023).<br /> (16) Saelens, W., Cannoodt, R., Todorov, H. & Saeys, Y. A comparison of single-cell trajectory inference methods: towards more accurate and robust tools. (2018). doi:10.1101/276907<br /> (17) Zöller, T. et al. Silencing of TGFβ signalling in microglia results in impaired homeostasis. Nat. Commun. 9, (2018).<br /> (18) Bedolla, A. et al. Microglia-derived TGF-β1 ligand maintains microglia homeostasis via autocrine mechanism and is critical for normal cognitive function in adult mouse brain. bioRxiv Prepr. Serv. Biol. (2023). doi:10.1101/2023.07.05.547814<br /> (19) Spittau, B., Dokalis, N. & Prinz, M. The Role of TGFβ Signaling in Microglia Maturation and Activation. Trends Immunol. 41, 836-848 (2020).<br /> (20) L. Nyberg, M. Lövdén, K. Riklund, U. Lindenberger, L. Bäckman, Memory aging and brain maintenance. Trends Cogn. Sci. 16, 292-305 (2012).<br /> (21) L. Luo, F. I. M. Craik, Aging and memory: A cognitive approach. Can. J. Psychiatry 53, 346-353 (2008).<br /> (22) Y. Stern, M. Albert, C. Barnes, R. Cabeza, A. Pascual-Leone, P. Rapp.<br /> A framework for concepts of reserve and resilience in aging. Neurobiol. Aging, 124 (2022), pp. 100-103, 10.1016/j.neurobiolaging.2022.10.015<br /> (23) Y. Stern, C.A. Barnes, C. Grady, R.N. Jones, N. Raz. Brain reserve, cognitive reserve, compensation, and maintenance: operationalization, validity, and mechanisms of cognitive resilience. Neurobiol. Aging, 83 (2019), pp. 124-129, 10.1016/j.neurobiolaging.2019.03.022<br /> (24) R. Cabeza, M. Albert, S. Belleville, F.I.M. Craik, A. Duarte, C.L. Grady, U. Lindenberger, L. Nyberg, D.C. Park, P.A. Reuter-Lorenz, M.D. Rugg, J. Steffener, M.N. Rajah. Maintenance, reserve and compensation: the cognitive neuroscience of healthy ageing. Nat. Rev. Neurosci., 19 (11) (2018), Article 11, 10.1038/s41583-018-0068-2<br /> (25) Palovics, R. et al molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature 603, 309-314 (2022)<br /> (26) Sala Frigerio, C. et al. The major risk factors for Alzheimer's Disease: age, sex, and genes modulate the microglial response to Abeta plaques. Cell Rep, 27, 1293-1306 (2019)

Reviewer #2 (Public Review):

Summary:

The goal of the paper was to trace the transitions hippocampal microglia undergo along aging. ScRNA-seq analysis allowed the authors to predict a trajectory and hypothesize about possible molecular checkpoints, which keep the pace of microglial aging. E.g. TGF1b was predicted as a molecule slowing down the microglial aging path and indeed, loss of TGF1 in microglia led to premature microglia aging, which was associated with premature loss of cognitive ability. The authors also used the parabiosis model to show how peripheral, blood-derived signals from the old organism can "push" microglia forward on the aging path.

Strengths:

A major strength and uniqueness of this work is the in-depth single-cell dataset, which may be a useful resource for the community, as well as the data showing what happens to young microglia in heterochronic parabiosis setting and upon loss of TGFb in their environment.

Weaknesses:

That said, given what we recently learned about microglia isolation for RNA-seq analysis, there is a danger that some of the observations are a result of not age, but cell stress from sample preparation (enzymatic digestion 10min at 37C; e.g. PMID: 35260865). Changes in cell state distribution along aging were made based on scRNA-seq and were not corroborated by any other method, such as imaging of cluster-specific marker expression in microglia at different ages. This analysis would allow confirming the scRNA-seq data and would also give us an idea of where the subsets are present within the hippocampus, and whether there is any interesting distribution of cell states (e.g. some are present closer to stem cells?). Since TGFb is thought to be crucial to microglia biology, it would be valuable to include more analysis of the mice with microglia-specific Tgfb deletion e.g. what was the efficiency of recombination in microglia? Did their numbers change after induction of Tgfb deletion in Cx3cr1-creERT2::Tgfb-flox mice.

Overall:

In general, I think the authors did a good job following the initial observations and devised clever ways to test the emerging hypotheses. The resulting data are an important addition to what we know about microglial aging and can be fruitfully used by other researchers, e.g. those working on microglia in a disease context.

Reviewer #1 (Public Review):

Summary:

Yang, Hu et al. examined the molecular mechanisms underlying astrocyte activation and its implications for multiple sclerosis. This study shows that the glycolytic enzyme PKM2 relocates to astrocyte nuclei upon activation in EAE mice. Inhibiting PKM2's nuclear import reduces astrocyte activation, as evidenced by decreased proliferation, glycolysis, and inflammatory cytokine release. Crucially, the study identifies TRIM21 as pivotal in regulating PKM2 nuclear import via ubiquitination. TRIM21 interacts with PKM2, promoting its nuclear translocation and enhancing its activity, affecting multiple signaling pathways. Confirmatory analyses using single-cell RNA sequencing and immunofluorescence demonstrate TRIM21 upregulation in EAE astrocytes. Modulating TRIM21 expression in primary astrocytes impacts PKM2-dependent glycolysis and proliferation. In vivo experiments targeting this mechanism effectively mitigate disease severity, CNS inflammation, and demyelination in EAE.

The authors supported their claims with various experimental approaches, however, some results should be supported with higher-quality images clearly depicting the conclusions and additional quantitative analyses of Western blots.

Strength:

This study presents a comprehensive investigation into the function and molecular mechanism of metabolic reprogramming in the activation of astrocytes, a critical aspect of various neurological diseases, especially multiple sclerosis. The study uses the EAE mouse model, which closely resembles MS. This makes the results relevant and potentially translational. The research clarifies how TRIM21 regulates the nuclear import of PKM2 through ubiquitination by integrating advanced techniques. Targeting this axis may have therapeutic benefits since lentiviral vector-mediated knockdown of TRIM21 in vivo significantly reduces disease severity, CNS inflammation, and demyelination in EAE animals.

Weaknesses:

The authors reported that PKM2 levels are elevated in the nucleus of astrocytes at different EAE phases compared to cytoplasmic localization. However, Figure 1 also shows elevated cytoplasmic expression of PKM2. The authors should clarify the nuclear localization of PKM2 by providing zoomed-in images. An explanation for the increased cytoplasmic PKM2 expression should provided. Similarly, while PKM2 translocation is inhibited by DASA-58, in addition to its nuclear localization, a decrease in the cytoplasmic localization of PKM2 is also observed. This situation brings to mind the possibility of a degradation mechanism being involved when its nuclear translocation of PKM2 is inhibited.

In Figure 3D, the authors claim that PKM2 expression causes nuclear retention of STAT3, p65, and p50, and inhibiting PKM2 localization with DASA-58 suppresses this retention. The western blot results for the MOG-stimulated group show high levels of STAT3, p50, and p65 in nuclear localization. However, in the MOG and DASA-58 treated group, one would expect high levels of p50, p65, and STAT3 proteins in the cytoplasm, while their levels decrease in the nucleus. These western blot results could be expanded. Additionally, intensity quantification for these results would be beneficial to see the statistical difference in their expressions, especially to observe the nuclear localization of PKM2.

The discrepancy between Figure 7A and its explaining text is confusing. The expectation from the knocking down of TRIM21 is the amelioration of activated astrocytes, leading to a decrease in inflammation and the disease state. The presented results support these expectations, while the images showing demyelination in EAE animals are not highly supportive. Clearly labeling demyelinated areas would enhance readers' understanding of the important impact of TRIM21 knockdown on reducing the disease severity.

Reviewer #2 (Public Review):

This study significantly advances our understanding of the metabolic reprogramming underlying astrocyte activation in neurological diseases such as multiple sclerosis. By employing an experimental autoimmune encephalomyelitis (EAE) mouse model, the authors discovered a notable nuclear translocation of PKM2, a key enzyme in glycolysis, within astrocytes.

Preventing this nuclear import via DASA 58 substantially attenuated primary astrocyte activation, characterized by reduced proliferation, glycolysis, and inflammatory cytokine secretion.<br /> Moreover, the authors uncovered a novel regulatory mechanism involving the ubiquitin ligase TRIM21, which mediates PKM2 nuclear import. TRIM21 interaction with PKM2 facilitated its nuclear translocation, enhancing its activity in phosphorylating STAT3, NFκB, and c-myc. Single-cell RNA sequencing and immunofluorescence staining further supported the upregulation of TRIM21 expression in astrocytes during EAE.

Manipulating this pathway, either through TRIM21 overexpression in primary astrocytes or knockdown of TRIM21 in vivo, had profound effects on disease severity, CNS inflammation, and demyelination in EAE mice. This comprehensive study provides invaluable insights into the pathological role of nuclear PKM2 and the ubiquitination-mediated regulatory mechanism driving astrocyte activation.

The author's use of diverse techniques, including single-cell RNA sequencing, immunofluorescence staining, and lentiviral vector knockdown, underscores the robustness of their findings and interpretations. Ultimately, targeting this PKM2-TRIM21 axis emerges as a promising therapeutic strategy for neurological diseases involving astrocyte dysfunction.

While the strengths of this piece of work are undeniable, some concerns could be addressed to refine its impact and clarity further; as outlined in the recommendations for the authors.

Reviewer #4 (Public Review):

Summary:

The authors report the role of the Pyruvate Kinase M2 (PKM2) enzyme nuclear translocation as fundamental in the activation of astrocytes in a model of autoimmune encephalitis (EAE). They show that astrocytes, activated through culturing in EAE splenocytes medium, increase their nuclear PKM2 with consequent activation of NFkB and STAT3 pathways. Prevention of PKM2 nuclear translocation decreases astrocyte counteracts this activation. The authors found that the E3 ubiquitin ligase TRIM21 interacts with PKM2 and promotes its nuclear translocation. In vivo, either silencing of TRIM21 or inhibition of PKM2 nuclear translocation ameliorates the severity of the disease in the EAE model.

Strengths:

This work contributes to the knowledge of the complex action of the PKM2 enzyme in the context of an autoimmune-neurological disease, highlighting its nuclear role and a novel partner, TRIM21, and thus adding a novel rationale for therapeutic targeting.

Weaknesses:

Despite the relevance of the work and its goals, some of the conclusions drawn would require more thorough proof:

I believe that the major weakness is the fact that TRIM21 is known to have per se many roles in autoimmune and immune pathways and some of the effects observed might be due to a PKM2-independent action. Some of the experiments to link the two proteins, besides their interaction, do not completely clarify the issue. On top of that, the in vivo experiments address the role of TRIM21 and the nuclear localisation of PKM2 independently, thus leaving the matter unsolved.

Some experimental settings are not described to a level that is necessary to fully understand the data, especially for a non-expert audience: e.g. the EAE model and MOG treatment; action and reference of the different nuclear import inhibitors; use of splenocyte culture medium and the possible effect of non-EAE splenocytes.

The statement that PKM2 is a substrate of TRIM21 ubiquitin ligase activity is an overinterpretation. There is no evidence that this interaction results in ubiquitin modification of PKM2; the ubiquitination experiment is minimal and is not performed in conditions that would allow us to see ubiquitination of PKM2 (e.g. denaturing conditions, reciprocal pull-down, catalytically inactive TRIM21, etc.).

Reviewer #1 (Public Review):

Summary:

This manuscript by Vogt et al examines how the synaptic composition of AMPA and NMDA receptors changes over sleep and wake states. The authors perform whole-cell patch clamp recordings to quantify changes in silent synapse numbers across conditions of spontaneous sleep, sleep deprivation, and recovery sleep after deprivation. They also perform single nucleus RNAseq to identify transcriptional changes related to AMPA/NMDA receptor composition following spontaneous sleep and sleep deprivation. The findings of this study are consistent with a decrease in silent synapse number during wakefulness and an increase during sleep. However, these changes cannot be conclusively linked to sleep/wake states. Measurements were performed in the motor cortex, and sleep deprivation was achieved by forced locomotion, raising the possibility that recent levels of neuronal activity/induction of plasticity, rather than sleep/wake states, are responsible for the observed results.

Strengths:

This study examines an important question. Glutamatergic synaptic transmission has been a focus of studies in the sleep field, but AMPA receptor function has been the primary target of these studies. Silent synapses, which contain NMDA receptors but lack AMPA receptors, have important functional consequences for the brain. Exploring the role of sleep in regulating silent synapse numbers is important to understanding the role of sleep in brain function. The electrophysiological approach of measuring the failure rate ratio, supported by AMPA/NMDA ratio measurements, is a rigorous tool to evaluate silent synapse numbers.

The authors also perform snRNAseq to identify genes differentially expressed in the spontaneous sleep and sleep deprivation groups. This analysis reveals an intriguing pattern of upregulated genes controlled by HDAC4 and Mef2c, along with synaptic shaping component genes and genes associated with autism spectrum disorder, across cell types in the sleep deprivation group. This unbiased approach identifies candidate genes for follow-up studies.

Weaknesses:

A major weakness of this study is the experimental design. Measurements are made from the motor cortex, and sleep deprivation was achieved using forced locomotion on a treadmill. Therefore, the effects observed could be due to recent high levels of activity or plasticity induction in the motor cortex from locomotion, rather than lack of sleep per se. In support of this interpretation, other groups have failed to find a difference in AMPA/NMDA ratio in mice with different spontaneous sleep/wake histories, although sleep deprivation was not performed (Bridi et al., Neuron 2020).

The electrophysiological measurements are problematic in several ways. First, the methods lack crucial details such as inclusion/exclusion criteria for each cell based on input and series resistance, stability of input/series resistance, polysynaptic responses, etc. that make it difficult to interpret the data. The holding potential (-90mV) used for AMPA receptor current recordings is much more hyperpolarized than typically used for these measurements. The statistical analysis of these experiments is also problematic. The number of mice used is low (3/group) and more should be added to account for inter-animal variability. Comparing the raw data with the statistical tests in supplementary table 1 (FR ratio), it appears that a data point has been dropped from the analysis, but it is unclear why. In addition, a false discovery rate (FDR) correction for multiple comparisons is used to evaluate group differences following the ANOVAs. Correcting for the FDR is less stringent and is typically used when a large number of hypotheses are tested and false positives are more acceptable. In this analysis, few comparisons are made, and the standard approach of correcting for the family-wise error rate is more appropriate.

The snRNAseq data are intriguing, but a more thorough discussion of the candidate genes and pathways that are upregulated during sleep deprivation is warranted. Several genes relevant to the AMPA/NMDA ratio are mentioned, but upregulation of most of these genes would not be expected to increase the AMPA/NMDA ratio based on the literature cited. The model presented in Figure 4C is not consistent with the data (e.g. many candidate genes could alter NMDAR function without receptor insertion/removal), and it is unclear how the current study fits into the model presented in 4D.

Reviewer #2 (Public Review):

Summary:

Here Vogt et al., provide new insights into the need for sleep and the molecular and physiological response to sleep loss. The authors expand on their previously published work (Bjorness et al., 2020) and draw from recent advances in the field to propose a neuron-centric molecular model for the accumulation and resolution of sleep need and the basis of restorative sleep function. While speculative, the proposed model successfully links important observations in the field and provides a framework to stimulate further research and advances on the molecular basis of sleep function. In my review, I highlight the important advances of this current work, and the clear merits of the proposed model, and indicate areas of the model that can serve to stimulate further investigation.

Strengths:

Reviewer comment on new data in Vogt et al., 2024<br /> Using classic slice electrophysiology, the authors conclude that wakefulness (sleep deprivation (SD)) drives a potentiation of excitatory glutamate synapses, mediated in large part by "un-silencing" of NMDAR-active synapses to AMPAR-active synapses. Using a modern single nuclear RNAseq approach the authors conclude that SD drives changes in gene expression primarily occurring in glutamatergic neurons. The two experiments combined highlight the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons. This view is entirely consistent with a large body of extant and emerging literature and provides important direction for future research.

Consistent with prior work, wakefulness/SD drives an LTP-type potentiation of excitatory synaptic strength on principle cortical neurons. It has been proposed that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity. This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon by introducing the concept of silent synapses. The new data show that in mice well rested, a substantial number of synapses are "silent", containing an NMDAR component but not AMPARs. Silent synapses provide a type of reservoir for learning in that activity can drive the un-silencing, increasing the number of functional synapses. SD depletes this reservoir of silent synapses to essentially zero, explaining how SD can exhaust learning capacity. Recovery sleep led to restoration of silent synapses, explaining how recovery sleep can renew learning capacity. In their prior work (Bjorness et al., 2020) this group showed that SD drives an increase in mEPSC frequency onto these same cortical neurons, but without a clear change in pre-synaptic release probability, implying a change in the number of functional synapses. This prediction is now born out in this new dataset.

The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies. First, this conclusion is corroborated by an independent, contemporary snRNAseq analysis recently available as a pre-print (Ford et al., 2023 BioRxiv https://doi.org/10.1101/2023.11.28.569011). A recently published analysis on the effects of SD in drosophila imaged synapses in every brain region in a cell-type dependent manner (Weiss et al., PNAS 2024), concluding that SD drives brain wide increases in synaptic strength almost exclusively in excitatory neurons. Further, Kim et al., Nature 2022, heavily cited in this work, show that the newly described SIK3-HDAC4/5 pathway promotes sleep depth via excitatory neurons and not inhibitory neurons.

The new experiments provided in Fig1-3 are expertly conducted and presented. This reviewer has no comments of concern regarding the execution and conclusions of these experiments.

Reviewer comment on the model in Vogt et al., 2024

In the view of this reviewer the new model proposed by Vogt et al., is an important contribution. The model is not definitively supported by new data, and in this regard should be viewed as a perspective, providing mechanistic links between recent molecular advances, while still leaving areas that need to be addressed in future work. New snRNAseq analysis indicates that SD drives the expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function. SD-induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes. As pointed out by the authors, sleep problems are commonly reported in ASD, but the emphasis has been on sleep amount. This new analysis highlights the need to understand the impact on sleep's functional output (synapses) to fully understand the role of sleep problems in ASD.

Importantly, SD-induced gene expression in excitatory neurons overlaps with genes regulated by the transcription factor MEF2C and HDAC4/5 (Figure 4). In their prior work, the authors show loss of MEF2C in excitatory neurons abolished the SD transcriptional response and the functional recovery of synapses from SD by recovery sleep. Recent advances identified HDAC4/5 as major regulators of sleep depth and duration (in excitatory neurons) downstream of the recently identified sleep-promoting kinase SIK3. In Zhou et al., and Kim et al., Nature 2022, both groups propose a model whereby "sleep-need" signals from the synapse activate SIK3, which phosphorylates HDAC4/5, driving cytoplasmic targeting, allowing for the de-repression and transcriptional activation of "sleep genes". Prior work shows that HDAC4/5 are repressors of MEF2C. Therefore, the "sleep genes" derepressed by HDAC4/5 may be the same genes activated in response to SD by MEF2C. The new model thereby extends the signaling of sleep need at synapses (through SIK3-HDAC4/5) to the functional output of synaptic recovery by expression of synaptic/sleep genes by MEF2C. The model thereby links aspects of the expression of sleep need with the resolution of sleep need by mediating sleep function: synapse renormalization.

Weaknesses:

Areas for further investigation

In the discussion section Vogt et al., explore the links between excitatory synapse strength, arguably the major target of "sleep function", and NREM slow-wave activity (SWA), the most established marker of sleep need. SIK3-HDAC4/5 have major effects on the "depth" of sleep by regulating NREM-SWA. The effects of MEF2C loss of function on NREM SWA activity are less obvious, but clearly impact the recovery of glutamatergic synapses from SD. The authors point out how adenosine signaling is well established as a mediator of SWA, but the links between adenosine and glutamatergic strength are far from clear. The mechanistic links between SIK3/HDAC4/5, adenosine signaling, and MEF2C, are far from understood. Therefore, the molecular/mechanistic links between a synaptic basis of sleep need and resolution with NREM-SWA activity require further investigation.

Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity. The authors point out that constitutively nuclear (cn) HDAC4/5 (acting as a repressor) will mimic MEF2C loss of function. This is reasonable, however, there are notable differences in the reported phenotypes of each. Notably, cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020). These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes. Likely HDAC4/5 have functionally important interactions with other transcription factors, and likewise for MEF2C, suggesting areas for future analysis.

One emerging theme may be that the SIK3-HDAC4/5 axis is a major regulator of the sleep state, perhaps stabilizing the NREM state once the transition from wakefulness occurs. MEF2C is less involved in regulating sleep per se, and more involved in executing sleep function, by promoting restorative synaptic modifications to resolve sleep need.

Finally, advances in the roles of the respective SIK3-HDAC4/5 and MEF2C pathways point towards transcription of "sleep genes", as clearly indicated in the model of Figure 4. Clearly, more work is needed to understand how the expression of such genes ultimately leads to the resolution of sleep need by functional changes at synapses. What are these sleep genes and how do they mechanistically resolve sleep need? Thus, the current work provides a mechanistic framework to stimulate further advances in understanding the molecular basis for sleep need and the restorative basis of sleep function.

Reviewer #1 (Public Review):

The study reports that STN neurons recorded while monkeys performed a random-dot motion task show diverse activation timecourses relative to task events and dependencies on coherence, reaction time, and saccade-choice direction. Different neuron types could be grouped into functional subpopulations, e.g., coherence sensitivity emerging early only in choice-coding neurons. Clustering techniques identified three functionally defined neuron clusters whose dynamic activity profiles related to computational predictions of different decision models in the literature. Microstimulation at different STN recording sites affected behavioral performance in varying but well-conceptualized ways that were captured by the parameters of drift-diffusion models and related to the presence of STN functional clusters at recording sites. The authors conclude that their results validate key aspects of decision models and identify novel aspects of decision-related STN activity.

This is an interesting and high-quality paper that will be of interest across computational and decision neuroscience fields. The recordings and data analyses seem carefully conducted. The study has an attractive theoretical starting point of three specific computational signals that are then mapped onto identified neuron clusters. The combination of single-cell recordings, microstimulation, and computational modelling is a distinct strength of the paper. I only have a few questions and suggestions for clarification.

(1) It would be helpful to explain the criteria for choosing a given number of clusters and for accepting the final clustering solution more clearly. The quantitative results (silhouette plots, Rand index) in Supplementary Figure 2 should perhaps be included in the main figure to justify the parameter choices and acceptance of specific clustering solutions.

(2) It would be helpful to show how the activity profiles in Figure 3 would look like for 3 or 5 (or 6) clusters, to give the reader an impression of how activity profiles recovered using different numbers of clusters would differ.

(3) The authors attempt to link the microstimulation effects to the presence of functional neuron clusters at the stimulation site. How can you rule out that there were other, session-specific factors (e.g., related to the animal's motivation) that affected both neuronal activity and behavior? For example, could you incorporate aspects of the monkey's baseline performance (mean reaction time, fixation breaks, error trials) into the analysis?

(4) Line 84: What was the rationale for not including both coherence and reaction time in one multiple regression model?

Reviewer #2 (Public Review):

This study uses single-unit recordings in the monkey STN to examine the evidence for three theoretical models that propose distinct roles for the STN in perceptual decision-making. Importantly, the proposed functional roles are predictive of unique patterns of neural activity. Using k-means clustering with seeds informed by each model's predictions, the current study identified three neural clusters with activity dynamics that resembled those predicted by the described theoretical models. The authors are thorough and transparent in reporting the analyses used to validate the clustering procedure and the stability of the clustering results. To further establish a causal role for the STN in decision-making, the researchers applied microstimulation to the STN and found effects on response times, choice preferences, and latent decision parameters estimated with a drift diffusion model. Overall, the study provides strong evidence for a functionally diverse population of STN neurons that could indeed support multiple roles involved in perceptual decision-making. The manuscript would benefit from stronger evidence linking each neural cluster to specific decision roles in order to strengthen the overall conclusions.

The interpretation of the results, and specifically, the degree to which the identified clusters support each model, is largely dependent on whether the artificial vectors used as model-based clustering seeds adequately capture the expected behavior under each theoretical model. The manuscript would benefit from providing further justification for the specific model predictions summarized in Figure 1B. Further, although each cluster's activity can be described in the context of the discussed models, these same neural dynamics could also reflect other processes not specific to the models. That is, while a model attributing the STN's role to assessing evidence accumulation may predict a ramping up of neural activity, activity ramping is not a selective correlate of evidence accumulation and could be indicative of a number of processes, e.g., uncertainty, the passage of time, etc. This lack of specificity makes it challenging to infer the functional relevance of cluster activity and should be acknowledged in the discussion.

Additionally, although the effects of STN microstimulation on behavior provide important causal evidence linking the STN to decision processes, the stimulation results are highly variable and difficult to interpret. The authors provide a reasonable explanation for the variability, showing that neurons from unique clusters are anatomically intermingled such that stimulation likely affects neurons across several clusters. It is worth noting, however, that a substantial body of literature suggests that neural populations in the STN are topographically organized in a manner that is crucial for its role in action selection, providing "channels" that guide action execution. The authors should comment on how the current results, indicative of little anatomical clustering amongst the functional clusters, relate to other reports showing topographical organization.

Overall, the association between the identified clusters and the function ascribed to the STN by each of the models is largely descriptive and should be interpreted accordingly. For example, Figure 3 is referenced when describing which cluster activity is choice/coherence dependent, yet it is unclear what specific criteria and measures are being used to determine whether activity is choice/coherence "dependent." Visually, coherence activity seems to largely overlap in panel B (top row). Is there a statistically significant distinction between low and high coherence in this plot? The interpretation of these plots and the methods used to determine choice/coherence "dependence" needs further explanation.

In general, the association between cluster activity and each model could be more directly tested. At least two of the models assume coordination with other brain regions. Does the current dataset include recordings from any of these regions (e.g., mPFC or GPe) that could be used to bolster claims about the functional relevance of specific subpopulations? For example, one would expect coordinated activity between neural activity in mPFC and Cluster 2 according to the Ratcliff and Frank model. Additionally, the reported drift-diffusion model (DDM) results are difficult to interpret as microstimulation appears to have broad and varied effects across almost all the DDM model parameters. The DDM framework could, however, be used to more specifically test the relationships between each neural cluster and specific decision functions described in each model. Several studies have successfully shown that neural activity tracks specific latent decision parameters estimated by the DDM by including neural activity as a predictor in the model. Using this approach, the current study could examine whether each cluster's activity is predictive of specific decision parameters (e.g., evidence accumulation, decision thresholds, etc.). For example, according to the Ratcliff and Frank model, activity in cluster 2 might track decision thresholds.

Reviewer #3 (Public Review):

Summary:

The authors provide compelling evidence for the causal role of the subthalamic nucleus (STN) in perceptual decision-making. By recording from a large number of STN neurons and using microstimulation, they demonstrate the STN's involvement in setting decision bounds, scaling evidence accumulation, and modulating non-decision time.

Strengths:

The study tested three hypotheses about the STN's function and identified distinct STN subpopulations whose activity patterns support predictions from previous computational models. The experiments are well-designed, the analyses are rigorous, and the results significantly advance our understanding of the STN's multi-faceted role in decision formation.

Weaknesses:

While the study provides valuable insights into the STN's role in decision-making, there are a few areas that could be improved. First, the interpretation of the neural subpopulations' activity patterns in relation to the computational models should be clarified, as the observed patterns may not directly correspond to the specific signals predicted by the models. Second, the authors could consider using a supervised learning method to more explicitly model the pattern correlations between the three profiles. Third, a neural population model could be employed to better understand how the STN population jointly contributes to decision-making dynamics. Finally, the added value of the microstimulation experiments should be more directly addressed in the Results section, as the changes in firing patterns compared to the original patterns are not clearly evident.

Reviewer #1 (Public Review):

What neurophysiological changes support the learning of new sensorimotor transformations is a key question in neuroscience. Many studies have attempted to answer this question at the neuronal population level - with varying degrees of success - but few, if any, have studied the change in activity of the apical dendrites of layer 5 cortical neurons. Neurons in layer 5 of the sensory cortex appear to play a key role in sensorimotor transformations, showing important decision and reward-related signals, and being the main source of cortical and subcortical projections from the cortex. In particular, pyramidal track (PT) neurons project directly to subcortical regions related to motor activity, such as the striatum and brainstem, and could initiate rapid motor action in response to given sensory inputs. Additionally, layer 5 cortical neurons have large apical dendrites that extend to layer 1 where different neuromodulatory and long-range inputs converge, providing motor and contextual information that could be used to modulate layer 5 neurons output and/or to establish the synaptic plasticity required for learning a new association.

In this study, the authors aimed to test whether the learning of a new sensorimotor transformation could be supported by a change in the evoked response of the apical dendrites of layer 5 neurons in the mouse whisker primary somatosensory cortex. To do this, they performed longitudinal functional calcium imaging of the apical dendrites of layer 5 neurons while mice learned to discriminate between two multi-whisker stimuli. The authors used a simple conditioning task in which one whisker stimulus (upward or backward air puff, CS+) is associated with a reward after a short delay, while the other whisker stimulus (CS-) is not. They found that task learning (measured by the probability of anticipatory licking just after the CS+) was not associated with a significant change in the average population response evoked by the CS+ or the CS-, nor a change in the average population selectivity. However, when considering individual dendritic tufts, they found interesting changes in selectivity, with approximately equal numbers of dendrites becoming more selective for CS+ and dendrites becoming more selective for CS-.

One of the major challenges when assessing changes in neural representation during the learning of such Go/NoGo tasks is that the movements and rewards themselves may elicit strong neural responses that may be a confounding factor, that is, inexperienced mice do not lick in response to the CS+, while trained mice do. In this study, the authors addressed this issue in three ways: first, they carefully monitored the orofacial movements of mice and showed that task learning is not associated with changes in evoked whisker movements. Second, they show that whisking or licking evokes very little activity in the dendritic tufts compared to whisker stimuli (CS+ and CS-). Finally, the authors introduced into the design of their task a post-conditioning session after the last conditioning session during which the CS+ and the CS- are presented but no reward is delivered. During this post-session, the mice gradually stopped licking in response to the CS+. A better design might have been to perform the pre-conditioning and post-conditioning sessions in non-water-restricted, unmotivated mice to completely exclude any lick response, but the fact that the change in selectivity persists after the mice stopped licking in the last blocks of the post-conditioning session (in mice relying only on their whiskers to perform the task) is convincing.

The clever task design and careful data analysis provide compelling evidence that learning this whisker discrimination task does not result in a massive change in sensory representation in the apical dendritic tufts of layer 5 neurons in the primary somatosensory cortex on average. Nevertheless, individual dendritic tufts do increase their selectivity for one or the other sensory stimulus, likely enhancing the ability of S1 neurons to accurately discriminate the two stimuli and trigger the appropriate motor response (to lick or not to lick).

One limitation of the present study is the lack of evidence for the necessity of the primary somatosensory cortex in the learning and execution of the task. As the authors have strongly emphasized in their previous publications, the primary somatosensory cortex may not be necessary for the learning and execution of simple whisker detection tasks, especially when the stimulus is very salient. Although this new task requires the discrimination between two whisker stimuli, the simplicity and salience of the whisker stimuli used could make this task cortex-independent. Especially when considering that some mice seem to not rely entirely on their whiskers to execute the task.

Nevertheless, this is an important result that shows for the first time changes in the selectivity to sensory stimuli at the level of individual apical dendritic tufts in correlation with the learning of a discrimination task. This study sheds new light on the cortical cellular substrates of reward-based learning and opens interesting perspectives for future research in this area. In future studies, it will be important to determine whether the change in selectivity of dendritic calcium spikes is causally involved in the learning of the task or whether it simply correlates with learning, as a consequence of changes in synaptic inputs caused by reward. The dendritic calcium spikes may be involved in the establishment of synaptic plasticity required for learning and impact the output of layer 5 pyramidal neurons to trigger the appropriate motor response. It would be important also to study the changes in selectivity in the apical dendrite of the identified projection neurons.

Reviewer #2 (Public Review):

Summary:

The authors did not find an increased representation of CS+ throughout reinforcement learning in the tuft dendrites of Rbp4-positive neurons from layer 5B of the barrel cortex, as previously reported for soma from layer 2/3 of the visual cortex.

Alternatively, the authors observed an increased selectivity to both stimuli (CS+ and CS-) during reinforcement learning. This feature:

(1) was not present in repeated exposures (without reinforcement),<br /> (2) was not explained by the animal's behaviour (choice, licking, and whisking), and<br /> (3) was long-lasting, being present even when the mice disengaged from the task.

Importantly, increased selectivity was correlated with learning (% correct choices), and neural discriminability between stimuli increased with learning.

In conclusion, the authors show that tuft dendrites from layer 5B of the barrel cortex increase the representation of conditioned (CS+) and unconditioned stimuli (CS-) applied to the whiskers, during reinforcement learning.

Strengths:

The results presented are very consistent throughout the entire study, and therefore very convincing:

(1) The results observed are very similar using two different imaging techniques (2-photon -planar imaging- and SCAPE-volumetric imaging). Figure 3 and Figure 4 respectively.

(2) The results are similar using "different groups" of tuft dendrites for the analysis (e.g. initially unresponsive and responsive pre- and post-learning). Figure 5.

(3) The results are similar from a specific set of trials (with the same sensory input, but different choices). Figure 7.

(4) Additionally, the selectivity of tuft dendrites from layer 5B of the barrel cortex was higher in the mice that exclusively used the whisker to respond to the stimuli (CS+ and CS-).<br /> The results presented are controlled against a group of mice that received the same stimuli presentation, except for the reinforcement (reward).

Additionally, the behaviour outputs, such as choice, whisking, and licking could not account for the results observed.

Although there are no causal experiments, the correlation between selectivity and learning (percentage of correct choices), as well as the increased neural discriminability with learning, but not in repeated exposure, are very convincing.

Weaknesses:

The biggest weakness is the absence of causality experiments. Although inhibiting specifically tuft dendritic activity in layer 1 from layer 5 pyramidal neurons is very challenging, tuft dendritic activity in layer 1 could be silenced through optogenetic experiments as in Abs et al. 2018. By manipulating NDNF-positive neurons the authors could specifically modify tuft dendritic activity in the barrel cortex during CS presentations, and test if silencing tuft dendritic activity in layer 1 would lead to the lack of selectivity and an impairment of reinforcement learning. Additionally, this experiment will test if the selectivity observed during reinforcement learning is due to changes in the local network, namely changes in local synaptic connectivity, or solely due to changes in the long-range inputs.

Reviewer #1 (Public Review):

Summary:

This study focuses on metabolic changes in the paraventricular hypothalamic (PVH) region of the brain during acute periods of cold exposure. The authors point out that in comparison to the extensive literature on the effects of cold exposure in peripheral tissues, we know relatively little about its effects on the brain. They specifically focus on the hypothalamus, and identify the PVH as having changes in Atgl and Hsl gene expression changes during cold exposure. They then go on to show accumulation of lipid droplets, increased Fos expression, and increased lipid peroxidation during cold exposure. Further, they show that neuronal activation is required for the formation of lipid droplets and lipid peroxidation.

Strengths:

A strength of the study is trying to better understand how metabolism in the brain is a dynamic process, much like how it has been viewed in other organs. The authors also use a creative approach to measuring in vivo lipid peroxidation via delivery of a BD-C11 sensor through a cannula to the region in conjunction with fiber photometry to measure fluorescence changes deep in the brain.

Weaknesses:

Although the topic and findings are of interest, there are a few key weaknesses in the study that would improve the work if addressed. One weakness was many of the experiments were done in a manner that could not distinguish between the contributions of neurons and glial cells, limiting the extent of conclusions that could be made. While this is not easily doable for all experiments, it can be done for some. For example, the Fos experiments in Figure 3 would be more conclusive if done with the labeling of neuronal nuclei with NeuN, as glial cells can also express Fos. To similarly show more conclusively that neurons are being activated during cold exposure, the calcium imaging experiments in Figure S3 can be done with cold exposure. Additionally, many experiments are only done with the minimal three animals required for statistics and could be more robust with additional animals included. Another weakness is that the authors do not address whether manipulating lipid droplet accumulation or lipid peroxidation has any effect on PVH function (e.g. does it change neuronal activity in the region?).

Reviewer #2 (Public Review):

Summary:

Cold-induced lipid metabolism is well-established in adipose tissues. The authors set out to determine whether cold could alter brain lipid metabolism. By QPCR analysis of brain punches after acute cold, they found that mRNA expressions of several lipolysis-related genes were upregulated compared to RT controls. By combining fluorescent sensors and in vivo fiberphotometry, they observed cold-induced lipid peroxidation/lipolysis, which could be blocked by pharmacological inhibitors of neuronal activity (muscimol and kynurenic acid). The brain is not traditionally considered an organ with high lipid metabolism (vs carbohydrate); therefore, the observation and hypothesis proposed by the authors are unexpected and can be interesting. However, the experiments and data were rather preliminary and superficial and did not support the authors' conclusions. In addition, the main hypothesis, in relationship to the role of cold/temperature, remains incoherent and needs a major update.

Strengths:

A set of relatively novel and interesting observations.

Creative use of several in vivo sensors and techniques.

Weaknesses:

(1) The physiological relevance of lipolysis and thermogenesis genes in the PVH. The authors need to provide quantitative and substantial characterizations of lipid metabolism in the brain beyond a panel of qPCRs, especially considering these genes are likely expressed at very low levels. mRNA and protein level quantification of genes in Fig 1, in direct comparison to BAT/iWAT, should be provided. Besides bulk mRNA/protein, IHC/ISH-based characterization should be added to confirm to cellular expression of these genes.

(2) The fiberphotometry work they cited (Chen 2022, Andersen 2023, Sun 2018) used well-established, genetically encoded neuropeptide sensors (e.g., GRABs). The authors need to first quantitatively demonstrate that adapting BD-C11 and EnzCheck for in vivo brain FP could effectively and accurately report peroxidation and lipolysis. For example, the sensitivity, dynamic range, and off-time should all be calibrated with mass spectrometry measurements before any conclusions can be made based on plots in Figures 4, 5, and 6. This is particularly important because the main hypothesis heavily relies on this unvalidated technique.

(3) Generally, the histology data need significant improvement. It was not convincing, for example, in Figure 3, how the Fos+ neurons can be quantified based on the poor IF images where most red signals were not in the neurons.

(4) The hypothesis regarding the direct role of brain temperature in cold-induced lipid metabolism is puzzling. From the introduction and discussion, the authors seem to suggest that there are direct brain temperature changes in responses to cold, which could be quite striking. However, this was not supported by any data or experiments. The authors should consolidate their ideas and update a coherent hypothesis based on the actual data presented in the manuscript.

Reviewer #2 (Public Review):

Summary:

The study has demonstrated how two neurotransmitters and neuromodulators from the same neurons can be regulated and utilized in thermoregulation.

The study utilized electrophysiological methods to examine the characteristics and thermoregulation of Neurotensin (Nts)-expressing neurons in the medial preoptic area (MPO). It was discovered that GABA and Nts may be co-released by neurons in MPO when communicating with their target neurons.

Strengths:

The study has leveraged optogenetic, chemogenetic, knockout, and pharmacological inhibitors to investigate the release process of Nts and GABA in controlling body temperature.

The findings are relevant to those interested in the various functions of specific neuron populations and their distinct regulatory mechanisms on neurotransmitter/neuromodulator activities

Weaknesses:

Key points for consideration include:

(1) The co-release of GABA and Nts is primarily inferred rather than directly proven. Providing more direct evidence for the release of GABA and the co-release of GABA and Nts would strengthen the argument. Further in vitro analysis could strengthen the conclusion regarding this co-releasing process.

(2) The differences between optogenetic and chemogenetic methods were not thoroughly investigated. A comparison of in vitro results and direct observation of release patterns could clarify the mechanisms of GABA release alone or in conjunction with Nts under different stimulation techniques.

(3) Neuronal transcripts were mainly identified through PCR, and alternative methods like single-cell sequencing could be explored.

(4) In Figure 6, the impact of GABA released from Nts neurons in MPO on CBT regulation appears to vary with ambient temperatures, requiring a more detailed explanation for better comprehension.

(5) The model should emphasize the key findings of the study.

Reviewer #3 (Public Review):

Summary:

Understanding the central neural circuits regulating body temperature is critical for improving health outcomes in many disease conditions and in combating heat stress in an ever-warming environment. The authors present important and detailed new data that characterizes a specific population of POA neurons with a relationship to thermoregulation. The new insights provided in this manuscript are exactly what is needed to assemble a neural network model of the central thermoregulatory circuitry that will contribute significantly to our understanding of regulating the critical homeostatic variable of body temperature. These experiments were conducted with the expertise of an investigator with career-long experience in intracellular recordings from POA neurons. They were interpreted conservatively in the appropriate context of current literature.

The Introduction begins with "Homeotherms, including mammals, maintain core body temperature (CBT) within a narrow range", but this ignores the frequent hypothermic episodes of torpor that mice undergo triggered by cold exposure. Although the author does mention torpor briefly in the Discussion, since these experiments were carried out exclusively in mice, greater consideration (albeit speculative) of the potential for a role of MPO Nts neurons in torpor initiation or recovery is warranted. This is especially the case since some 'torpor neurons' have been characterized as PACAP-expressing and a population of PACAP neurons represent the target of MPO Nts neurons.

Reviewer #1 (Public Review):

Summary:

In this technical paper, the authors introduce a useful variation on the fully automated multi-electrode patch-clamp recording technique for probing synaptic connections that they term "patch-walking". The patch-walking approach involves coordinated pipette route-planning and automated pipette cleaning procedures for pipette reuse to improve recording throughput efficiency, which the authors argue can theoretically yield almost twice the number of connections to be probed by paired recordings on a multi-patch electrophysiology setup for a given number of cells compared to conventional manual patch-clamping approaches used in brain slices in vitro. The authors show solid results from recordings in mouse in vitro cortical slices, demonstrating the efficient recording of dozens of paired neurons with a two-patch pipette configuration for paired recordings and detection of synaptic connections. This approach will be of interest and valuable to neuroscientists conducting automated multi-patch in vitro electrophysiology experiments and seeking to increase the efficiency of neuron connectivity detection while avoiding the more complex recording configurations (e.g., 8-10 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Strengths:

(1) The authors introduce the theory and methods and show experimental results for a fully automated electrophysiology dual patch-clamp recording approach, which uses coordinated patch-clamp pipette route-planning and automated pipette cleaning procedures to "patch-walk" across an in vitro brain slice.

(2) The patch-walking approach improves throughput efficiency over manual patch clamp recording approaches, especially for investigators looking to utilize paired patch electrode recordings in electrophysiology experiments in vitro.

(3) Experimental results are presented from in vitro mouse cortical slices demonstrating the efficiency of recording dozens of paired neurons with a two-patch pipette configuration for paired recordings and detecting synaptic connections, demonstrating the feasibility and efficiency of the patch-walking approach.

(4) The authors suggest extensions of their technique while keeping the number of recording pipettes employed and recording rig complexity low, which are important practical technical considerations for investigators wanting to avoid the more complex recording configurations (e.g., 8-10 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Reviewer #2 (Public Review):

Summary:

In this study, the authors aim to combine automated whole-cell patch clamp recording simultaneously from multiple cells. Using a 2-electrode approach, they are able to sample as many cells (and connections) from one slice, as would be achieved with a more technically demanding and materially expensive 8-electrode patch clamp system. They provide data to show that this approach is able to successfully record from 52% of attempted cells, which was able to detect 3 pairs in 71 screened neurons. The authors state that this is a step forward in our ability to record from randomly connected ensembles of neurons.

Strengths:

The conceptual approach of recording multiple partner cells from another in a stepwise manner indeed increases the number of tested connections. An approach that is widely applicable to both automated and manual approaches. Such a method could be adopted for many connectivity studies using dual recording electrodes.

The implementation of automated robotic whole-cell patch-clamp techniques from multiple cells simultaneously is a useful addition to the multiple techniques available to ex vivo slice electrophysiologists.

The approach using 2 electrodes, which are washed between cells is economically favourable, as this reduces equipment costs for recording multiple cells, and limits the wastage of capillary glass that would otherwise be used once.

Weaknesses:

(1) The premise of this article is based upon the fact that even a "skilled" whole-cell electrophysiologist is only capable of recording ~10 cells per day are flawed. Many studies have shown that capable electrophysiologists can record upwards of 50 cells a day, given adequate slice quality and reliable recording conditions with multiple electrodes (e.g. Pastoll et al., 2020 eLife, Booker et al., 2014, JoVE, Peng et al., 2017); often with over 80% success rates for recording. It is not convincing that this approach is a dramatic improvement on such approaches - except when a less skilled researcher is beginning recordings.

Importantly, could the patch walk protocol not be alternatively implemented using manual recording approaches? Yes, the use of a semi-automated robotic system aids recording from many cells by a less experienced colleague, but the inferences about the number of connections tested are common to the approach, not the technique used. This seems like a crucial conceptual point to include.

(2) A key omission of this study is the absence of brain area, cell type, and layer recorded from. It is mentioned in Figure 2 that this is the somatosensory and visual cortices. Which were these, and how were they confirmed?

(3) A comparison of measurements shown in Figure 2 to other methods - e.g. conventional dual patch, 8-electrode patch, single electrode. How do the values obtained for cell quality measurements compare to those expected for the cell population recorded (which is unclear - see point 2)?

(4) What is the reliability of performing outside-out patch configuration to obtain sealed and biocytin-filled cells under these conditions? A key tenet of performing high-throughput paired recordings is the ability to identify the cell types involved in the local microcircuit, and if their axon has been preserved in the slice configuration (which varies between cell types). Not having confirmation of morphological identity and integrity likely leads to a dramatic underestimation of connection probability, given that main axon collaterals could be severed during acute brain slice preparation.

(5) The quality control criteria used in this manuscript require further clarification. An upper limit of 50 MΩ access resistance is extremely high (i.e. 20-30 MΩ is a more typical and stringent cut-off), which is worsened as no real information is given to the degree of resistance change that could be accepted. This is simply listed as "If the seal quality decreased during recording, the cell is excluded from analysis". Indeed, the range of access resistances plotted in Figure 2 is from 10-100 MΩ, which implies that some neurons included in this data did not meet recording criteria. Also, it is widely accepted in the field that a 10-20% change in access during recording is acceptable - within a more defined range. I would consider re-assessing the recorded cells to only include cells with access resistances <30MΩ and those that did not fluctuate by more than 20%.

Appraisal of aims:

The authors certainly established a system that is useful for interrogating synaptic connectivity in an automated manner. However, it remains unclear how widely used this would be in the field, and whether this truly represents an advancement from manual recordings or >4 electrode recordings.

Discussion of impact:

This approach, particularly the conceptual approach to paired testing, is of use to the field. However, in practice, many researchers using conventional dual-electrode paired recording likely implement similar approaches - especially when targeting specific cell types (see Booker et al., 2014 JoVE, Qi et al., 2020 Front Synaptic Neurosci.). This may pave the way for greater implementation of dual and multi-electrode recordings using robotic patch-clamp techniques.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Yip and colleagues incorporated the pipette cleaning technique into their existing dual-patch robotic system, "the PatcherBot", to allow sequential patching of more cells for synaptic connection detection in living brain slices. During dual-patching, instead of retracting all two electrodes after each recording attempt, the system cleaned just one of the electrodes and reused it to obtain another recording while maintaining the other. With one new patch clamp recording attempt, new connections can be probed. By placing one pipette in front of the other in this way, one can "walk" across the tissue, termed "patch-walking." This application could allow for probing additional neurons to test the connectivity using the same pipette in the same preparation.

Strengths:

Compared to regular dual-patch recordings, this new approach could allow for probing more possible connections in brain slices with dual-patch recordings, thus having the potential to improve the efficiency of identifying synaptic connections

Weaknesses:

While this new approach offers the potential to increase efficiency, it has several limitations that could curtail its widespread use.

Loss of Morphological Information: Unlike traditional multi-patch recording, this approach likely loses all detailed morphology of each recorded neuron. This loss is significant because morphology can be crucial for cell type verification and understanding connectivity patterns by morphological cell type.

Spatial Restrictions: The robotic system appears primarily suited to probing connections between neurons with greater spatial separation (~100µm ISD). This means it may not reliably detect connections between neurons in close proximity, a potential drawback given that the connectivity is much higher between spatially close neurons. This limitation could help explain the low connectivity rate (5%) reported in the study.

Limited Applicability: While the approach might be valuable in specific research contexts, its overall applicability seems limited. It's important to consider scenarios where the trade-off between efficiency and specific questions that are asked.

Scalability Challenges: Scaling this method beyond a two-pipette setup may be difficult. Additional pipettes would introduce significant technical and logistical complexities.

Reviewer #1 (Public Review):

Cheng, Yu-Ting, et al. demonstrate the capabilities of three-photon excited fluorescence (3PEF) microscopy for in vivo imaging of the mouse spinal cord. It enables imaging up to ~550 µm in depth, overcoming the limitations of two-photon excited fluorescence (2PEF) microscopy. The authors used 3PEF to visualize and quantify blood flow across different vessel types within the spinal cord and observed the cellular responses following venule occlusion. They showed depth-dependent structural changes in neurites and the behavior of microglia with a high contrast. The findings show that 3PEF can provide high-resolution, multicolor imaging of dynamic cellular interactions and vascular architecture, helping studies of spinal cord physiology and pathology.

The experiments are well done and supported by data but some points need to be clarified:

(1) For the two vs three-photon comparison, the authors should provide more information about how they performed the 2PEF: power and pulse duration. This comparison is primarily focused on imaging depth and signal-to-background ratio (SBR), but imaging speed should also be discussed.

(2) A comparison with state-of-the-art 2PEF would have been more convincing. For instance, the use of adaptive optics, or red-shifted fluorophores allowing better 2PEF SBR, or deeper imaging.

(3) The study focuses on structural imaging and does not provide extensive data on real-time dynamic processes, which could be crucial for understanding rapid cellular responses in the spinal cord.<br /> By addressing these weaknesses, future studies could enhance the applicability and reliability of 3PEF microscopy for spinal cord research.

Reviewer #2 (Public Review):

Summary:

In this work, the authors attempt to advance our capacity to image the intact spinal cord in living mice, with the ultimate goal of allowing optical access to all spinal layers, from the dorsal (sensory-related) to the ventral (motor-related) laminae. They demonstrate the potency of 3-photon excited fluorescence imaging (3PEF) to collect fluorescent signals in anesthetized adult mice to depths of up to 450 µm from the dorsal surface.

Strengths:

• 3PEF is convincingly demonstrated as a significant improvement over previously used 2-photon imaging.

• The images show very good spatial resolution and stable signal-to-noise ratio up to 450 µm from the dorsal surface, providing unprecedented access to intermediate ventral laminae.

Weaknesses:

• The paper in its current form lacks a detailed description of the experimental apparatus used, including its invasiveness (removal of vertebrae and muscles) and its impact on animal behavior. One can hope that, in the future, a similar implantation chamber may be used for awake, freely-moving animals.

• In general, non-optic specialists may find it difficult to appreciate some of the findings due to technical writing at times, and minimally described metrics.

• The possibility that the 3-photon illumination may cause tissue damage, notably by heat induction, is not evaluated or discussed.

• At this stage, no attempt has been made to image cellular activity. The reader should keep in mind that motor neurons, as well as most of their upstream circuits, are located between 500 and 900 µm from the dorsal surface. Hence, although the method is a significant advancement, it still does not allow for the evaluation of morphological (or possibly, activity) changes in the whole spinal cord, particularly excluding motor-related laminae."

Reviewer #1 (Public Review):

Summary:

This manuscript by Meissner and colleagues described a novel take on a classic social cognition paradigm developed for marmosets. The classic pull task is a powerful paradigm that has been used for many years across numerous species, but its analog approach has several key limitations. As such, it has not been feasible to adopt the task for neuroscience experiments. Here the authors capture the spirit of the classic task but provide several fundamental innovations that modernize the paradigm - technically and conceptually. By developing the paradigm for marmosets, the authors leverage the many advantages of this primate model for studies of social brain functions and their particular amenability to freely-moving naturalistic approaches.

Strengths:

The current manuscript describes one of the most exciting paradigms in primate social cognition to be developed in many years. By allowing for freely-moving marmosets to engage in high numbers of trials, while precisely quantifying their visual behavior (e.g. gaze) and recording neural activity this paradigm has the potential to usher in a new wave of research on the cognitive and neural mechanisms underlying primate social cognition and decision-making. This paradigm is an elegant illustration of how naturalistic questions can be adapted to more rigorous experimental paradigms. Overall, I thought the manuscript was well written and provided sufficient details for others to adopt this paradigm. I did have a handful of questions and requests about topics and information that could help to further accelerate its adoption across the field.

Weaknesses:

LN 107 - Otters have also been successful at the classic pull task (https://link.springer.com/article/10.1007/s10071-017-1126-2)

LN 151 - Can you provide a more precise quantification of timing accuracy than the 'sub-second level'. This helps determine synchronization with other devices.

Using this paradigm, the marmosets achieved more trials than in the conventional task (146 vs 10). While this is impressive, given that only ~50 are successful Mutual Cooperation trials it does present some challenges for potential neurophysiology experiments and particular cognitive questions. The marmosets are only performing the task for 20 minutes, presumably because they become sated and are no longer motivated. This seems a limitation of the task and is something worth discussing in the manuscript. Did the authors try other food rewards, reduce the amount of reward, food/water restrict the animals for more than the stated 1-3 hours? How might this paradigm be incorporated into in-cage approaches that have been successful in marmosets? Any details on this would help guide others seeking to extend the number of trials performed each day.

Can you provide more details on the DLC/Anipose procedure? How were the cameras synchronized? What percentage of trials needed to be annotated before the model could be generalized? Did each monkey require its own model, or was a single one applied to all animals?

Will the schematics and more instructions on building this system be made publicly available? A number of the components listed in Table 1 are custom-designed. Although it is stated that CAD files will be made available upon request, sharing a link to these files in an accessible folder would significantly add to the potential impact of this paradigm by making it easier for others to adopt.

In the Discussion, it would be helpful to have some discussion of how this paradigm might be used more broadly. The classic pulling paradigm typically allows one to ask a specific question about social cognition, but this task has the potential to be more widely applied to other social decision-making questions. For example, how might this task be adopted to ask some of the game-theory-type approaches common in this literature? Given the authors' expertise in this area, this discussion could serve to provide a roadmap for the broader field to adopt.

Although this paradigm was developed specifically for marmosets, it seems to me that it could readily be adopted in other species with some modifications. Could the authors speak to this and their thoughts on what may need to be changed to be used in other species? This is particularly important because one of the advantages of the classic paradigm is that it has been used in so many species, providing the opportunity to compare how different species approach the same challenge. For example, though both chimps and bonobos are successful, their differences are notably illuminating about the nuances of their respective social cognitive faculties.

Reviewer #2 (Public Review):

Summary:

This important work by Meisner et al., developed an automated apparatus (MarmoAPP) to collect a wide array of behavioral data (lever pulling, gaze direction, vocalizations) in marmoset monkeys, with the goal of modernizing collection of behavioral data to coincide with the investigation of neurological mechanisms governing behavioral decision making in an important primate neuroscience model. The authors show a variety of "proof-of-principle" concepts that this apparatus can collect a wide range of behavioral data, with higher behavioral resolution than traditional methods. For example, the authors highlight that typical behavioral experiments on primate cooperation provide around 10 trials per session, while using their approach the authors were able to collect over 100 trials per 20-minute session with the MarmoAAP.

Overall the authors argue that this approach has a few notable advantages:<br /> (1) it enhances behavioral output which is important for measuring small or nuanced effects/changes in behavior;<br /> (2) allows for more advanced analyses given the higher number of trials per session;<br /> (3) significantly reduces the human labor of manually coding behavioral outcomes and experimenter interventions such as reloading apparatuses for food or position;<br /> (4) allows for more flexibility and experimental rigor in measuring behavior and neural activity simultaneously.

Strengths:

The paper is well-written and the MarmoAPP appears to be highly successful at integrating behavioral data across many important contexts (cooperation, gaze, vocalizations), with the ability to measure significantly many more behavioral contexts (many of which the authors make suggestions for).

The authors provide substantive information about the design of the apparatus, how the apparatus can be obtained via a long list of information Apparatus parts and information, and provide data outcomes from a wide number of behavioral and neurological outcomes. The significance of the findings is important for the field of social neuroscience and the strength of evidence is solid in terms of the ability of the apparatus to perform as described, at least in marmoset monkeys. The advantage of collecting neural and freely-behaving behavioral data concurrently is a significant advantage.

Weaknesses:

While this paper has many significant strengths, there are a few notable weaknesses in that many of the advantages are not explicitly demonstrated within the evidence presented in the paper. There are data reported (as shown in Figures 2 and 3), but in many cases, it is unclear if the data is referenced in other published work, as the data analysis is not described and/or self-contained within the manuscript, which it should be for readers to understand the nature of the data shown in Figures 2 and 3.

(1) There is no data in the paper or reference demonstrating training performance in the marmosets. For example, how many sessions are required to reach a pre-determined criterion of acceptable demonstration of task competence? The authors reference reliably performing the self-reward task, but this was not objectively stated in terms of what level of reliability was used. Moreover, in the Mutual Cooperation paradigm, while there is data reported on performance between self-reward vs mutual cooperation tasks, it is unclear how the authors measured individual understanding of mutual cooperation in this paradigm (cooperation performance in the mutual cooperation paradigm in the presence or absence of a partner; and how, if at all, this performance varied across social context). What positive or negative control is used to discern gained advantages between deliberate cooperation vs two individuals succeeding at self-reward simultaneously?

(2) One of the notable strengths of this approach argued by the authors is the improved ability to utilize trials for data analysis, but this is not presented or supported in the manuscript. For example, the paper would be improved by explicitly showing a significant improvement in the analytical outcome associated with a comparison of cooperation performance in the context of ~150 trials using MarmoAAP vs 10-12 trials using conventional behavioral approaches beyond the general principle of sample size. The authors highlight the dissection of intricacies of behavioral dynamics, but more could be demonstrated to specifically show these intricacies compared to conventional approaches. Given the cost and expertise required to build and operate the MarmoAAP, it is critical to provide an important advantage gained on this front. The addition of data analysis and explicit description(s) of other analytical advantages would likely strengthen this paper and the advantages of MarmoAAP over other behavioral techniques.

Reviewer #3 (Public Review):

Summary:

The authors set out to devise a system for the neural and behavioral study of socially cooperative behaviors in nonhuman primates (common marmosets). They describe instrumentation to allow for a "cooperative pulling" paradigm, the training process, and how both behavioral and neural data can be collected and analyzed. This is a valuable approach to an important topic, as the marmoset stands as a great platform to study primate social cognition. Given that the goals of such a methods paper are to (a) describe the approach and instrumentation, (b) show the feasibility of use, and (c) quantitatively compare to related approaches, the work is easily able to meet those criteria. My specific feedback on both strengths and weaknesses is therefore relatively limited in scope and depth.

Strengths:

The device is well-described, and the authors should be commended for their efforts in both designing this system but also in "writing it up" so that others can benefit from their R&D.

The device appears to generate more repetitions of key behavior than other approaches used in prior work (with other species).

The device allows for quantitative control and adjustment to control behavior.

The approach also supports the integration of markerless behavioral analysis as well as neurophysiological data.

Weaknesses:

A few ambiguities in the descriptions are flagged below in the "Recommendations for authors".

The system is well-suited to marmosets, but it is less clear whether it could be generalized for use in other species (in which similar behaviors have been studied with far less elegant approaches). If the system could impact work in other species, the scope of impact would be significantly increased, and would also allow for more direct cross-species comparisons. Regardless, the future work that this system will allow in the marmoset will itself be novel, unique, and likely to support major insights into primate social cognition.

Reviewer #1 (Public Review):

The manuscript by Yu et al seeks to investigate the role of neuritin (Nrn1), identified as a marker of anergic cells, in the biology of regulatory (Tregs) and conventional (Tconv) T cells. Although the role of Nrn1 expressed by Tregs has already been explored (Gonzalez-Figueroa 2021 cited in the manuscript), this manuscript shows original new data suggesting that this molecule would be important in promoting Treg function and inhibiting Tconv effector function by acting at the level of membrane potential and molecule transport across the plasma membrane. However, it is disappointing that reading this manuscript leaves an impression of incomplete work done too quickly. Multiple models have been used, but none has been studied thoroughly enough to provide really conclusive and unambiguous data. For example, 5 different models were used to study T cells in vivo. It would have been preferable to use fewer, but to go further in the study of mechanisms. In the absence of a more in-depth study, the conclusions drawn by the authors are often open to question. Major points concern the fact that there are enough biological replicates for most experiments, some critical controls and data are lacking, and the authors have used iTregs rather than nTregs for many experiments (see below). This is unfortunate because the role of neuritin in T cell biology studied here is new and interesting.

Major points (in the order in which they appear in the text):

(1) A real weakness of this work is the fact that in most of the results shown, there are few biological replicates with differences that are often small between Ctrl and Nrn1 -/-. The systematic use of student's t-test may lead to thinking that the differences are significant, which is often misleading given the small number of samples, which makes it impossible to know whether the distributions are Gaussian and whether a parametric test can be used. RNAseq bulk data are based on biological duplicates, which is open to criticism.

(2) The authors use Nrn1+/+ and Nrn1+/- cells indiscriminately as control cells on the basis of similar biology between Nrn1+/+ and Nrn1+/- cells at homeostasis. However, it is quite possible that the Nrn1+/- cells have a phenotype in situations of in vitro activation or in vivo inflammation (cancer, EAE). It would be important to discriminate Nrn1+/- and Nrn1+/+ cells in the data or to show that both cell types have the same phenotype in these conditions too.

(3) Figure 1A-D. Since the authors are using the Nrp1 KO mice, it would be important to confirm the specificity of the anti-Nrn1 mAb by FACS. Once verified, it would be important to add FACS results with this mAb in Figures 1A-C to have single-cell and quantitative data as well.

(4) Figure 1E-H. The authors assume that this immunization protocol induces anergic cells, but they provide no experimental evidence for this. It would be useful to show that T cells are indeed anergic in this model, especially those that are OVA-specific. The lack of IL-2 production by Cltr cells could be explained by the presence of fewer OVA-specific cells, rather than by an anergic status.

(5) Figure 2A-C and Figure 3. The use of iTregs to try to understand what is happening in vivo is problematic. iTregs are cells that have probably no equivalent in vivo, and so may have no physiological relevance. In any case, they are different from pTreg cells generated in vivo. Working with pTreg may be challenging, that is why I would suggest generating data with purified nTreg. Moreover, it was shown in the article of Gonzalez-Figueroa 2021 that Nrn1-/- nTreg retained a normal suppressive function, which would not be what is concluded by the authors of this manuscript. Moreover, we do not even know what the % of Foxp3 cells is in the iTreg used (after differentiation and 20h of re-stimulation) and whether this % is the same between Ctlr and Nrn1 KO cells.

(6) Figure 2D-L. The model is designed to study the role of Nrn1 in nTreg. However, the % of Foxp3+ among CD45.2 nTreg cells fell to 5-15% of CD4+ cells (Figure 2F). Since we do not know what is the % of Foxp3 among the injected cells, we do not know whether this very low % is due to very high Treg instability or to preferential expansion of contaminating Tconvs. It is possible that the % of Tconv contaminant is high since Treg was sorted using beads and not FACS in some experiments. As it is very likely that there are Tconv contaminants that would be Nrn1-/- in the group transferred with Nrn1-/- "nTreg", the higher tumor rejection could be due to an overactivation of Nrn1-/- Tconvs (rather than a defect in Nrn1-/- Treg function).

Reviewer #2 (Public Review):

Summary:

This manuscript explores the role of Nrn1 in T cell tolerance. A previous study has demonstrated that Nrn1 is up-regulated in the Tfr fraction of Foxp3+ T regulatory cells. These authors now confirm the expression of Nrn1 in Tregs as well as report here that Nrn1 is also greatly over-expressed in anergic CD4 T cells, and this is the stepping-off point for this investigation.

Most remarkably, experiments show that anergy induction is defective when T cells cannot express Nrn1. Furthermore, differentiation to a Foxp3+ Treg phenotype is inhibited in the absence of Nrn1, and the Tregs that do develop appear functionally defective. With such defects in the anergy induction and Treg differentiation and function, auto-reactive effector T cell activation is unrestrained, and Nrn1-/- mice are more susceptible to severe EAE development.

Strengths:

The characterizations of T cell Nrn1 expression both in vitro and in vivo are comprehensive and convincing. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well done to strengthen the notion that Nrn1 is an important regulator of CD4 responsiveness.

Weaknesses:

The major weakness of this study stems from a lack of a clear molecular mechanism involving Nrn1. Previous studies of Nrn1 have suggested its role as a soluble molecule involved in intracellular communication, perhaps influencing cellular ion channel function and/or triggering downstream NFAT and mTOR activation. However, a unique receptor for Nrn1 has not been discovered and it remains unclear whether it acts in a cell-intrinsic or cell-extrinsic fashion for any particular cell type.

Data shown here provide evidence of alterations in the electrical and metabolic state of T cells when the Nrn1 gene is deleted. Nrn1-/- Tregs and Teffector cells each express a unique pattern of genes associated with Neurotransmitter receptor, Metal ion transmembrane transport, Amino acid transport, and mTORC1 signaling activities, different than that seen in wild-type mice. Although the biochemical and informatics studies are well-performed, it is my opinion that these results are inconclusive in part due to the absence of key "naive" control groups. This limits my ability to understand the significance of these data.

Specifically, studies of the electrical and metabolic state of Nrn1-/- inducible Treg cells (iTregs) would benefit from similar data collected from wild-type and Nrn1-/- naive CD4 T cells. Even though naive T cells don't express Nrn1, they may be positively influenced by soluble Nrn1. Does deletion of Nrn1 lead to changes in metabolic and electrical state in naive T cells? Is that why Nrn1 deletion in mice blocks naive T cell activation?

Since the loss of Nrn1 inhibits the activation of T cells, are Nrn1-/- iTregs transcriptionally, electrically, and metabolically similar to naive T cells due to their suboptimal activation? Does this account for their persistent functional defects? Or is up-regulation of Nrn1 (and cell-intrinsic Nrn1 signaling) necessary to complete Treg differentiation and to promote T regulatory function (similar to how cell-intrinsic Nrn1 facilitates anergy induction)? The study of naive cells in parallel with iTregs would address these possibilities.

A comparison of Nrn1-/- naive cells to Teffector cells should also be undertaken to reveal how it is that Nrn1-/- Teffector cells regain the capacity to respond effectively to stimulation (e.g. increased mTOR activation) despite their early activation defects.

Joint Public Review:

In this paper Hui and colleagues investigate how the predictive accuracy of a polygenic score (PGS) for body mass index (BMI) changes when individuals are stratified by 62 different covariates. After showing that the PGS has different predictive power across strata for 18 out of 62 covariates, they turn to understanding why these differences and seeing if predictive performance could be improved. First they investigated which types of covariates result in the largest differences in PGS predictive power, finding that covariates with with larger "main effects" on the trait and covariates with larger interaction effects (interacting with the PGS to affect the trait) tend to better stratify individuals by PGS performance. The authors then see if including interactions between the PGS and covariates improves predictive accuracy, finding that linear models only result in modest increases in performance but nonlinear models result in more substantial performance gains.

Overall, the results are interesting and well-supported. The results will be broadly interesting to people using and developing PGS methods, as well as the broader statistical genetics community.

A few of the important points of the paper are:

A major impediment to the clinical use of PGS is the interaction between the PGS and various other routinely measure covariates, and this work provides a very interesting empirical study along these lines. The problem is interesting, and the work presented here is a convincing empirical study of the problem.

The result that PGS accuracy differs across covariates, but in a way that is not well-captured by linear models with interactions is important for PGS method development.

The quantile regression analysis is an interesting approach to explore how and why PGS may differ in accuracy across different strata of individuals.

Reviewer #2 (Public Review):

Summary

The authors developed new tools for isolating PI3K activity and for labeling newly made membrane proteins for monitoring membrane trafficking. They found that PI3K activity alone was able to explain the increased presence of TRPV1 on the membrane independent of other cascades induced by NGF signaling. They also showed an interesting feedback between PI3K and the insulin receptor trafficking to the membrane.

Strengths:

A major strength of the paper is the innovative combination of techniques. The first technique used the optogenetic PhyB/PIF system. They anchored PhyB to the membrane and fused PIF with the interSH2 domain from PI3K. This allowed them to use 650nm light to induce an interaction between the PhyB and PIF resulting in a recruitment of the endogenous PI3K to the membrane through the iSH2 domain without actual activation of an RTK. This allowed them to dissect out one function, just PI3K recruitment/activation from the vast number of RTK downstream cascades.

The second technique was the development of a new non-canonical amino acid that is cell-impermeant. The authors synthesized the sTSO-sulfa-Cy5 compound that will react with the Tet3 ncAA through click chemistry. They showed that the sulfa-Cy5 did not cross the membrane and would be used to track protein production over time, though the reaction rates were slow as noted by the authors. The comparison of the sulfa-Cy5 data with the standard GFP with TIRF showed a clear difference indicating the useful information that is gained with the ncAA.

Another strength comes from the discovery that an isolated PI3K is responsible for increasing TRPV1 and InR trafficking to the plasma membrane.

Weakness:

The discussion does not go into much detail regarding the importance of their discovery of TRPV1 and InR increases trafficking due to PI3K activation. It also jumps to the limitations of in vivo implementation prematurely. These weaknesses are minor however.

The authors achieved their goal of creating the tools needed to separate out one of the many RTK signals and give a strong proof of concept implementation of their tools. Their results support their conclusions and will help understand how TRPV1 is regulated by signals other than the traditional channel activators. The tools developed in the article will be of use to the broader cell biology and biophysics community, not just the channel community. The opto control of the PhyB/PIF system makes it more convenient than other systems since it does not take the typical wavelengths needed for fluorescence. The cell-impermeant ncAA will also be a great tool for those studying membrane proteins, protein trafficking and protein dynamics.

Reviewer #1 (Public Review):

Summary:

This work seeks to isolate the specific effects of phosphoinositide 3-kinase (PI3K) on the trafficking of the ion channel TRPV1, distinct from other receptor tyrosine kinase-activated effectors. It builds on earlier studies by the same group (Stein et al. 2006; Stratiievska et al. 2018), which described the regulatory relationship between PI3K, nerve growth factor (NGF), and TRPV1 trafficking. A central theme of this study is the development of methods that precisely measure the influence of PI3K on TRPV1 trafficking and vice versa. The authors employ a range of innovative methodologies to explore the dynamics between TRPV1 and PI3K trafficking.

Strengths:

A major strength of this study is the application of innovative methods to understand the interaction between PI3K and TRPV1 trafficking. The key techniques presented include:

(1) The optogenetic trafficking system based on phytochrome B, introduced in this research. Its interaction mechanism, dependent on reversible light activation, is comprehensively explained in Figures 1 and 2, with the system's efficacy demonstrated in Figure 3.

(2) An extracellular labeling method using click chemistry, which although not exclusive to this study, introduces specific reagents engineered for membrane impermeability.

The central biological insight presented here is the sufficiency of PI3K activation to guide TRPV1 trafficking to the plasma membrane. An additional notable discovery is the potential regulation of insulin receptors via this mechanism.

The paper's strengths are anchored in its innovative methodologies and the valuable collaboration between groups specializing in distinct areas of research.

Weaknesses:

The paper might benefit from a more streamlined structure and a clearer emphasis on its findings. A possible way to enhance its impact might be to focus more on its methodological aspects. The methodological facets stand out as both innovative and impactful. These experiments are well-executed and align with biological expectations. It's evident how these techniques could be tailored for many protein trafficking studies, a sentiment echoed in the manuscript (lines 287-288). When seen through a purely biological lens, some findings, like those concerning the PI3K-TRPV1 interaction, are very similar to previous work (Stratiievska et al. 2018). A biological focus demands further characterization of this interaction through mutagenesis. Also, the incorporation of insights on the insulin receptor feels somewhat tangential. A cohesive approach could be to reshape the manuscript with a primary focus on methodology, using TRPV1 and InsR as illustrative examples.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Koh, Stratiievska, and their colleagues investigate the mechanism by which TRPV1 channels are delivered to the plasma membrane following the activation of receptor tyrosine kinases, specifically focusing on the NGF receptor. They demonstrate that the activation of the NGF receptor's PI3K pathway alone is sufficient to increase the levels of TRPV1 at the plasma membrane.

Strengths:

The authors employ cutting-edge optogenetic, imaging, and chemical-biology techniques to achieve their research goals. They ingeniously use optogenetics to selectively activate the PI3K pathway without affecting other NGF pathways. Additionally, they develop a novel, membrane-impermeable fluorescent probe for labeling cell-surface proteins through click-chemistry.

Comment on revised version:

We commend the authors on the significant improvements made to the manuscript. They have adequately addressed our comments. Notably, the new control experiments shown in Figure 4E and Figure 5 Fig. Supp 1 convincingly demonstrate the specificity of the NGF and 650 nm light stimuli, respectively. The addition of quantitative analyses strengthens the findings significantly. Furthermore, the manuscript is now presented in a much linear manner, enhancing its clarity and impact.

Reviewer #2 (Public Review):

Summary:

The study tries to connect energy metabolism with immune tolerance during bacterial infection. The mechanism details the role of pyruvate transporter expression via ERRalpha-PGC1 axis, resulting in pro-inflammatory TNF alpha signalling responsible for acquired infection tolerance.

Strengths:

Overall, the study is an excellent addition to the role of energy metabolism during bacterial infection. The mechanism-based approach in dissecting the roles of metabolic coactivator, transcription factor, mitochondrial transporter, and pro-inflammatory cytokine during acquired tolerance towards infections indicates a detailed and well-written study. The in vivo studies in mice nicely corroborate with the cell line-based data, indicating the requirement for further studies in human infections with another bacterial model system.

Weaknesses:

The authors have involved various mechanisms to justify their findings. However, they have missed out on certain aspects which connect the mechanism throughout the paper. For example, they measured ATP and acetyl COA production linked with bacterial re-exposures and added various targets like MCP1, EER alpha, PGC1 alpha and TNF alpha. However, they skipped PGC1 alpha levels, ATP and acetyl COA in various parts of the paper. Including the details would make the work more comprehensive.

The use of public data sets to support their claim on immune tolerance is missing. Including various data sets of similar studies will strengthen the findings independently.

Reviewer #1 (Public Review):

Summary:

Their findings elucidate the mechanisms underlying 2-AA-mediated reduction of pyruvate transport into mitochondria, which impairs the interaction between ERRα and PGC1α, consequently suppressing MPC1 expression and reducing ATP production in tolerized macrophages. While the data presented is intriguing and the paper is well-written, there are several points that warrant consideration. The authors should enhance the clarity, relevance, and impact of their study.

Strengths:

This paper presents a novel discovery regarding the mechanisms through which PA regulates the bioenergetics of tolerized macrophages.

Weaknesses:

The relevance of the in vivo model to support the conclusions is questionable. Further clarification is needed on this point.

Reviewer #1 (Public Review):

Cheng et al explore the utility of analyte ratios instead of relative abundance alone for biological interpretation of tissue in a MALDI MSI workflow. Utilizing the ratio of metabolites and lipids that have complimentary value in metabolic pathways, they show the ratio as a heat map which enhances the understanding of how multiple analytes relate to each other spatially. Normally, this is done by projecting each analyte as a unique color but using a ratio can help clarify visualization and add to biological interpretability. However, existing tools to perform this task are available in open-source repositories, and fundamental limitations inherent to MALDI MSI need to be made clear to the reader. The study lacks rigor and controls, i.e. without quantitative data from a variety of standards (internal isotopic or tissue mimetic models for example), the potential delta in ionization efficiencies of different species subtracts from the utility of pathway analysis using metabolite ratios.

Reviewer #2 (Public Review):

Summary:

In the article, "Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging" the authors describe their software package in R for visualizing metabolite ratio pairs. I think the novelty of this manuscript is overstated and there are several notable issues with the figures that prevent detailed assessment but the work would be of interest to the mass spectrometry community.

Strengths:

The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding of metabolomics data and enhance the identification of critical metabolites.

Weaknesses:

The authors are missing several references and discussion points, particularly about SIMS MSI, where ratio imaging has been previously performed.

There are several misleading sentences about the novelty of the approach and the limitations of metabolite imaging.

Several sentences lack rigor and are not quantitative enough.

The figures are difficult to interpret/ analyze in their current state and lack some critical components, including labels and scale bars.

Reviewer #1 (Public Review):

This important study uses a wide variety of convincing, state-of-the-art neuroimaging analyses to characterize whole-brain networks and relate them to reward-based motor learning. During early learning, the authors found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks. During late learning, they observed the opposite pattern. It remains to be seen whether these changes reflect generic changes in task engagement during learning or are specific to reward-based motor learning. This study is highly relevant for researchers interested in reward-based motor learning and decision-making.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is compelling and goes beyond the current state of the art. The results are of interest to neuroscientists working on motor control and reward-based learning.

Comments on revised version:

The revision has produced a stronger manuscript. Thank you for your thorough responses to the comments and concerns.

Reviewer #3 (Public Review):

Summary:

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field.

Strengths:

This manuscript is written in a clear and comprehensible way. It introduces a rather novel technique of assessing connectivity across lower-dimensional manifold which has hitherto not been applied in this way to the question of reward-based motor learning. Thus, this presents a unique viewpoint on understanding how the brain changes with motor learning. I particularly enjoyed the combination of connectivity-based, followed by further scrutiny of seed-based connectivity analyses, thus providing a more comprehensive viewpoint. Now it also has added a more comprehensive report on the behavioural changes of learning, and the added statistical quantification, which is useful.

Weaknesses:

The main weakness of the manuscript is the lack of direct connection between the reported neural changes and behavioural learning. Namely, most of the results could also be explained by changes in attention allocation during the session, or changes in movement speed (independent of learning). The authors acknowledge some of these potential confounds and argue that factors like attention are important for learning. While this is true, it is nonetheless very limiting if one cannot ascertain whether the observed effects are due to attention (independent of learning) or attention allocated in the pursuit of learning. The only direct analysis linking behavioural changes to neural changes is based on individual differences in learning performance, where the DAN-A shows the opposite trend than group level effects, which they interpret as differences given the used higher-resolution parcellation. However, it could be that these learning effects are indeed much smaller and subtler compared to more dominant group-level attention effects during the task. The lack of a control condition in the task limits the interpretability of results as learning-related.

Reviewer #1 (Public Review):

The authors investigated how global brain activity varied during reward-based motor learning. During early learning, they found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks; during late learning, they found the opposite pattern. Individual learning performance varied only with changes in the dorsal attention network. The authors certainly used a wide variety of valuable, state-of-the-art techniques to interrogate whole-brain networks and extract the key components of learning behavior. However, the findings are incomplete, tempered by potential confounds in the experimental design. As such, the underlying claim regarding how these networks jointly support reward-based motor learning is unclear.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is highly compelling and goes beyond the current state-of-the-art, but it is incomplete with respect to examining different signatures of learning, clarifying probed learning processes, and investigating changes in all relevant subcortical structures is incomplete and would benefit from more rigorous approaches. With the rationale and data presentation strengthened this paper would be of interest to neuroscientists working on motor control and reward-based learning.

Reviewer #3 (Public Review):

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field. The evidence supporting the presented findings is strong, though at times lacking rigorous statistical quantification. Nevertheless, there are several issues that require attention and clarification.

Reviewer #1 (Public Review):

Fang Huang et al found that RBM7 deficiency promotes metastasis by coordinating MFGE8 splicing switch and NF-kB pathway in breast cancer by utilizing clinical samples as well as cell and tail vein injection models.

This study uncovers a previously uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis, and provides evidence supporting RBM7 function in splicing regulation. These findings facilitate the mechanistic understanding of how splicing dysregulation contributes to metastasis in cancer, a direction that has increasingly drawn attention recently, and provides a potentially new prognostic and therapeutic target for breast cancer.

Reviewer #2 (Public Review):

In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

Reviewer #1 (Public Review):

This paper reports the useful discovery of the roles and signaling components of the TOR pathway in vegetative growth, sexual development, stress response, and aflatoxin production in Aspergillus flavus.

While I acknowledge the authors' effort in conducting Southern blot analysis to address my prior concern regarding the presence of dual copies of torA and tapA, I find their current resolution inadequate. Specifically, the simple deletion of the respective result sections for torA and tapA significantly impacts the overall significance of this study. The repeated unsuccessful attempts to generate correct mutants only offer circumstantial evidence, as technical issues may have been a contributing factor. Therefore, instead of merely removing these sections, it is essential for the authors to present more compelling experimental data demonstrating that torA and tapA are indeed vital for the viability of A. flavus. Such data would enhance the overall significance of this study.

Reviewer #2 (Public Review):

In this study, authors identified TOR, HOG and CWI signaling network genes as modulators of the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation. They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially that the conserved site K19 of FKBP3 plays a key role in regulating aflatoxin biosynthesis. In general, the study involved a heavy workload and the findings are potentially interesting and important for understanding or controlling the aflatoxin biosynthesis. However, the findings have not been deeply explored and the conclusions mostly are based on parallel phenotypic observations.

Reviewer #1 (Public Review):

Summary:

The study investigates parafoveal processing during natural reading, combining eye-tracking and MEG techniques, building upon the RIFT paradigm previously introduced by Pan et al. (2021).

The manuscript is well-written with a clear structure, and the data analysis and experimental results are presented in a lucid manner.

Comments on revised version:

I am satisfied with the revisions made by the authors. I believe the study introduces a new research paradigm to the field.

Reviewer #1 (Public Review):

Summary:

The authors study the variability of patient response of NSCLC patients on immune checkpoint inhibitors using single-cell RNA sequencing in a cohort of 26 patients and 33 samples (primary and metastatic sites), mainly focusing on 11 patients and 14 samples for association analyses, to understand the variability of patient response based on immune cell fractions and tumor cell expression patterns. The authors find immune cell fraction, clonal expansion differences, and tumor expression differences between responders and non-responders. Integrating immune and tumor sources of signal the authors claim to improve prediction of response markedly, albeit in a small cohort.

Strengths:

- The problem of studying the tumor microenvironment, as well as the interplay between tumor and immune features is important and interesting and needed to explain the heterogeneity of patient response and be able to predict it.

- Extensive analysis of the scRNAseq data with respect to immune and tumor features on different axes of hypothesis relating to immune response and tumor immune evasion using state-of-the-art methods.

- The authors provide an interesting scRNAseq data set linked to outcomes data.

- Integration of TCRseq to confirm subtype of T-cell annotation and clonality analysis.

- Interesting analysis of cell programs/states of the (predicted) tumor cells and characterization thereof.

Weaknesses:

- Generally, a very heterogeneous and small cohort where adjustments for confounding are hard. Additionally, there are many tests for association with outcome, where necessary multiple testing adjustments would negate signal and confirmation bias likely, so biological takeaways have to be questioned.

- RNAseq is heavily influenced by the tissue of origin (both cell type and expression), so the association with the outcome can be confounded. The authors try to argue that lymph node T-cell and NK content are similar, but a quantitative test on that would be helpful.

- The authors claim a very high "accuracy" performance, however, given the small cohort and lack of information on the exact evaluation it is not clear if this just amounts to overfitting the data.

- Especially for tumor cell program/state analysis the specificity to the setting of ICIs is not clear and could be prognostic.

- Due to the small cohort with a lot of variability, more external validation is needed to be convincingly reproducible, especially when talking about AUC/accuracy of a predictor.

Reviewer #2 (Public Review):

Summary:

The authors have utilised deep profiling methods to generate deeper insights into the features of the TME that drive responsiveness to PD-1 therapy in NSCLC.

Strengths:

The main strengths of this work lie in the methodology of integrating single-cell sequencing, genetic data, and TCRseq data to generate hypotheses regarding determinants of IO responsiveness.

Some of the findings in this study are not surprising and well precedented eg. association of Treg, STAT3, and NFkB with ICI resistance and CD8+ activation in ICI responders and thus act as an additional dataset to add weight to this prior body of evidence. Whilst the role of Th17 in PD-1 resistance has been previously reported (eg. Cancer Immunol Immunother 2023 Apr;72(4):1047-1058, Cancer Immunol Immunother 2024 Feb 13;73(3):47, Nat Commun. 2021; 12: 2606 ) these studies have used non-clinical models or peripheral blood readouts. Here the authors have supplemented current knowledge by characterization of the TME of the tumor itself.

Weaknesses:

Unfortunately, the study is hampered by the small sample size and heterogeneous population and whilst the authors have attempted to bring in an additional dataset to demonstrate the robustness of their approach, the small sample size has limited their ability to draw statistically supported conclusions. There is also limited validation of signatures/methods in independent cohorts, no functional characterisation of the findings, and the discussion section does not include discussion around the relevance/interpretation of key findings that were highlighted in the abstract (eg. role of Th17, TRM, STAT3, and NFKb). Because of these factors, this work (as it stands) does have value to the field but will likely have a relatively low overall impact.

Related to the absence of discussion around prior TRM findings, the association between TRM involvement in response to IO therapy in this manuscript is counter to what has been previously demonstrated (Cell Rep Med. 2020;1(7):100127, Nat Immunol. 2017;18(8):940-950., J Immunol. 2015;194(7):3475-3486.). However, it should be noted that the authors in this manuscript chose to employ alternative markers of TRM characterisation when defining their clusters and this could indicate a potential rationale for differences in these findings. TRM population is generally characterised through the inclusion of the classical TRM markers CD69 (tissue retention marker) and CD103 (TCR experienced integrin that supports epithelial adhesion), which are both absent from the TRM definition in this study. Additional markers often used are CD44, CXCR6, and CD49a, of which only CXCR6 has been included by the authors. Conversely, the majority of markers used by the authors in the cell type clustering are not specific to TRM (eg. CD6, which is included in the TRM cluster but is expressed at its lowest in cluster 3 which the authors have highlighted as the CD8+ TRM population). Therefore, whilst there is an interesting finding of this particular cell cluster being associated with resistance to ICI, its annotation as a TRM cluster should be interpreted with caution.

Reviewer #2 (Public Review):

Summary:

Singh and colleagues employ a methodic approach to reveal the function of the transcription factors Rela and Stat3 in the regulation of the inflammatory response in the intestine.

Strengths of the manuscript include the focus on the function of these transcription factors in hepatocytes and the discovery of their role in the systemic response to experimental colitis. While the systemic response to induce colitis is appreciated, the cellular and molecular mechanisms that drive such systemic response, especially those involving other organs beyond the intestine are an active area of research. As such, this study contributes to this conceptual advance. Additional strengths are the complementary biochemical and metabolomics approaches to describe the activation of these transcription factors in the liver and their requirement - specifically in hepatocytes - for the production of bile acids in response to colitis.

In this revised version, the authors have addressed previously raised questions.

Reviewer #3 (Public Review):

Summary:

The authors try to elucidate the molecular mechanisms underlying the intra-organ crosstalks that perpetuate intestinal permeability and inflammation.

Strengths:

This study identifies a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases via the gut liver axis using both murine models and human samples.

Weaknesses:

(1) The mechanism by which DSS administration induces the activation of the Rela and Stat3 pathways and subsequent modification of the bile acid pathway remains clear. As the authors state, intestinal bacteria are one candidate, and this needs to be clarified. I recommend the authors investigate whether gut sterilization by administration of antibiotics or germ free condition affects 1. the activation of the Rela and Stat3 pathway in the liver by DSS-treated WT mice and 2. the reduction of colitis in DSS-treated relaΔhepstat3Δhep mice.

(2) It has not been shown whether DSS administration causes an increase in primary bile acids, represented by CDCA, in the colon of WT mice following activation of the Rela and Stat3 pathways, as demonstrated in Figure 6.

(3) The implications of these results for IBD treatment, especially in what ways they may lead to therapeutic intervention, need to be discussed.

The above weakness points have been resolved by the revision and additional experiments.

Reviewer #2 (Public Review):

This manuscript describes new methodology to study low water potential (drought) stress responses in agar plates. They devote considerable effort in comparing transcriptome data among various previously published experimental systems, examining how different approaches of reducing water potential impact the Arabidopsis root and shoot transcriptome. Each method purported to reduce water potential in plate-grown seedlings has a different effect on Arabidopsis root transcriptome responses, which is problematic for the field. In this reviewer's view, differences in transcriptome are not as important, and often not as informative as measurement of physiological parameters, which they do very little of in their study.

The focus on transcriptome data to the almost complete exclusion of other types of data is a symptom of a broader over-emphasis on the transcriptome that is quite prevalent in plant science now. We measure transcriptomes because we can, not because it is inherently the most informative thing to do. The important thing is protein amount, and even more so protein activity/function, which we know has an imperfect, at best, correlation with transcript level. This reviewer acknowledges that using Arabidopsis transcriptomics is a commonly employed method, and as such, the outcomes of this study will hold value for a broad audience, even if largely as a cautionary tale. If transcriptomics is used to identify candidate genes for future investigations, an approach that has had some success, then appropriate cautions should be taken in translating expectations about gene, protein, and phenotypic responses in field conditions.

Reviewer #3 (Public Review):

This work compares transcriptional responses of shoots and roots harvested from four plate-based assays that aim to simulate drought and from plants subjected to water deficit in pots using the model plant Arabidopsis thaliana with the goal to select a plate-based assay that best recapitulates transcriptional changes that are observed during water-deficit in pots. For the plate-based assays polyethylene glycol (PEG), mannitol, and sodium chloride (salt) treatments were used as well as a 'hard agar' assay which was newly developed by the authors. In the 'hard agar' assay, less water was added to the solid components of the media leading to an increase in agar strength and nutrient concentration. Plants in pots were grown on vermiculite with the same nutrient mix as used in the plates and drought was induced by withholding watering for five days.

The authors observed a good directional agreement of differential expressed genes for shoots between the plate assays on the vermiculite drying experiment. However, less directional agreement was observed for differential expressed genes of roots, except for their newly developed 'hard agar' assay which had good directional agreement. Testing whether the increase in agar strength or more concentrated nutrients are attributed to this, they found that both factors contributed to the effect of the 'hard agar'. Arabidopsis ecotypes that showed a stronger reduction in shoot size when grown on the 'hard agar' tended to have a lower fitness according to an external study which may indicate that the 'hard agar' assay simulates physiological relevant conditions.

The work highlights that transcriptional responses for simulated drought on plates and drought caused by water deficit are highly variable and dependent on the tissues that are observed. The authors demonstrate that transcriptomics can be used to select a suitable plate assay that most closely recapitulates drought through water deficit for plants grown in pots. Interestingly their newly developed 'hard agar' assay provides an alternative to traditional plate-based assays with improved directional agreement of differential expressed genes in roots in comparison to plants experiencing water deficit in vermiculite. It is promising that the impact of 'hard agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K). In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity. Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

Reviewer #1 (Public Review):

Summary:

The study presented by Atsumi et al. is about using smartphone-driven, community-sourced data to enhance biodiversity monitoring. The idea is to leverage the widespread use of smartphones to gather data from the community quickly, contributing to a more comprehensive understanding of biodiversity. The authors discuss the importance of ecosystem services linked to biodiversity and the threats posed by human activities. It emphasizes the need for comprehensive biodiversity data to implement the Kunming-Montreal Global Biodiversity Framework. The 'Biome' mobile app, launched in Japan, uses species identification algorithms and gamification to gather over 6 million observations since 2019. While community-sourced data may have biases, incorporating it into Species Distribution Models (SDMs) improves accuracy, especially for endangered species. The app covers urban-natural gradients uniformly, enhancing traditional survey data biased towards natural areas. Combining these sources provides valuable insights into species distributions for conservation, protected area designation, and ecosystem service assessment.

Strengths:

The use of a smartphone app ('Biome') for community-driven species occurrence data collection represents an innovative and inclusive approach to biodiversity monitoring, leveraging the widespread use of smartphones. The app has successfully accumulated a large volume of species occurrence data since its launch in 2019, showcasing its effectiveness in rapidly gathering information from diverse locations. Despite challenges with certain taxa, the study highlights high species identification accuracy, especially for birds, reptiles, mammals, and amphibians, making the 'Biome' app a reliable tool for species observation. The integration of community-sourced data into Species Distribution Models (SDMs) improves the accuracy of predicting species distributions. This has implications for conservation planning, including the designation of protected areas and assessment of ecosystem services. The rapid accumulation of data and advancements in machine learning methods open up opportunities for conducting time-series analyses, contributing to the understanding of ecosystem stability and interaction strength over time. The study emphasizes the collaborative nature of the platform, fostering collaboration among diverse stakeholders, including local communities, private companies, and government agencies. This inclusive approach is essential for effective biodiversity assessment and decision-making. The platform's engagement with various stakeholders, including local communities, supports biodiversity assessment, management planning, and informed decision-making. Additionally, the app's role in fostering nature-positive awareness in society is highlighted as a significant contribution to creating a sustainable society.

Weaknesses:

While the studies make significant contributions to biodiversity monitoring, they also have some weaknesses. Firstly, relying on smartphone-driven, community-sourced data may introduce spatial and taxonomic biases. The 'Biome' app, for example, showed lower accuracy for certain taxa like seed plants, molluscs, and fishes, potentially impacting the reliability of the gathered data. Furthermore, the effectiveness of Species Distribution Models (SDMs) relies on the assumption that biases in community-sourced data can be adequately accounted for. The unique distribution patterns of the 'Biome' data, covering urban-natural gradients uniformly, might not fully represent the diversity of certain ecosystems, potentially leading to inaccuracies in the models. Moreover, the divergence in data distribution patterns along environmental gradients between 'Biome' data and traditional survey data raises concerns. The app data shows a more uniform distribution across natural-urban gradients, while traditional data is biased towards natural areas. This discrepancy may impact the representation of certain ecosystems and influence the accuracy of Species Distribution Models (SDMs). While the integration of 'Biome' data into SDMs improves accuracy, the study notes that controlling the sampling efforts is crucial. Spatially-biased sampling efforts in community-sourced data need careful consideration, and efforts to control biases are essential for reliable predictions.

Reviewer #1 (Public Review):

Summary:

Debeuf et al. introduce a new, fast method for the selection of suitable T cell clones to generate TCR transgenic mice, a method claimed to outperform traditional hybridoma-based approaches. Clone selection is based on the assessment of the expansion and phenotype of cells specific for a known epitope following immune stimulation. The analysis is facilitated by a new software tool for TCR repertoire and function analysis termed DALI. This work also introduces a potentially invaluable TCR transgenic mouse line specific for SARS-CoV-2.

Strengths:

The newly introduced method proved successful in the quick generation of a TCR transgenic mouse line. Clone selection is based on more comprehensive phenotypical information than traditional methods, providing the opportunity for a more rational T cell clone selection.

The study provides a software tool for TCR repertoire analysis and its linkage with function.

The findings entail general practical implications in the preclinical study of a potentially very broad range of infectious diseases or vaccination.

A novel SARS-CoV-2 spike-specific TCR transgenic mouse line was generated.

Weaknesses:

The authors attempt to compare their novel method with a more conventional approach to developing TCR transgenic mice. In this reviewer's opinion, this comparison appears imperfect in several ways:

• Work presenting the "traditional" method was inadequate to justify the selection of a suitable clone. It is therefore not surprising that it yielded negative results. More evidence would have been necessary to select clone 47 for further development of the TCR transgenic line, especially considering the significant time and investment required to create such a line.

• The comparison is somewhat unfair, because the methods start at different points: while the traditional method was attempted using a pool of peptides whose immunogenicity does not appear to have been established, the new method starts by utilising tetramers to select T cells specific for a well-established epitope.

• Given the costs and time involved, only a single clone could be tested for either method, intrinsically making a proper comparison unfeasible. Even for their new method, the authors' ability to demonstrate that the selected clone is ideal is limited unless they made different clones with varying profiles to show that a particular profile was superior to others.

In my view, there was no absolute need to compare this method with existing ones, as the proposed method holds intrinsic value.

While having more data to decide on clone selection is certainly beneficial, given the additional cost, it remains unclear whether knowing the expression profiles of different proteins in Figure 2 aids in selecting a candidate. Is a cell expressing more CD69 preferable to a cell expressing less of this marker? Would either have been effective? Are there any transcriptional differences between clonotype 1 and 2 (red colour in Figure 2G) that justify selecting clone 1, or was the decision to select the latter merely based on their different frequency? If all major clones (i.e. by clonotype count) present similar expression profiles, would it have been necessary to know much more about their expression profiles? Would TCR sequencing and an enumeration of clones have sufficed, and been a more cost-effective approach?

Lastly, it appears that several of the experiments presented were conducted only once. This information should have been explicitly stated in the figure legends.

Reviewer #2 (Public Review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning, and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that are already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers, and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity, and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in a variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.

Reviewer #1 (Public Review):

In their manuscript, Gerlevik et al. performed an integrative analysis of clinical, genetic and transcriptomic data to identify MDS subgroups with distinct outcomes. The study was based on the building of an "immunoscore" and then combined with genotype and clinical data to analyze patient outcomes using multi-omics factor analysis.

Strengths: Integrative analysis of RNA-seq, genotyping and clinical data

Weaknesses: Validation of the bioinformatic pipeline is incomplete

Major comments:

(1) This study considered two RNA-seq data sets publicly available and generated in two distinct laboratories. Are they comparable in terms of RNA-seq technique: polyA versus rRNA depletion, paired-end sequencing, fragment length?

(2) Data quality control (figure 1): the authors must show in a graph whether the features (dimensions) of factor 1 were available for each BMMNC and CD34+ samples.

(3) How to validate the importance of "immunoscore"? If GSEA of RNA-seq data was performed in the entire cohort, in the SF3B1-mutated samples or SRSF2-mutated samples (instead of patients having a high versus low level of factor 1 shown in Sup Fig. 4), what would be the ranking of Hallmarks or Reactome inflammatory terms among the others?

(4) To decipher cell-type composition of BMMNC and CD34+ samples, the authors used van Galen's data (2019; supplementary table 3). Cell composition is expressed as the proportion of each cell population among the others. Surprisingly, the authors found that the promonocyte-like score was increased in SF3B1-mutated samples and not in SRSF2-mutated samples, which are frequently co-mutated with TET2 and associated with a CMML-like phenotype. Is there a risk of bias if bone marrow subpopulations such as megakaryocytic-erythroid progenitors or early erythroid precursors are not considered?

(5) Figures 2a and 2b indicated that the nature of retrotransposons identified in BMMNC and CD34+ was different. ERVs were not detected in CD34+ cells. Are ERVs not reactivated in CD34+ cells? Is there a bias in the sequencing or bioinformatic method?

(6) What is the impact of factor 1 on survival? Is it different between BMMNC and CD34+ cells considering the distinct composition of factor 1 in CD34+ and BMMNC?

(7) In Figure 1e, genotype contributed to the variance of in the CD34+ cell analyses more importantly than in the BMMNC. Because the patients are different in the two cohorts, differences in the variance could be explained either by a greater variability of the type of mutations in CD34 or an increased frequency of poor prognosis mutations in CD34+ compared to BMMNC. The genotyping data must be shown.

(8) Fig. 2a-b: Features with high weight are shown for each factor. For factor 9, features seemed to have a low weight (Fig. 1b and 1c). However, factor 9 was predictive of EFS and OS in the BMMNC cohort. What are the features driving the prognostic value of factor 9?

(9) The authors also provided microarray analyses of CD34+ cell. It could be interesting to test more broadly the correlation between features identified by RNA-seq or microarrays.

(10) The authors should discuss the relevance of immunosenescence features in the context of SRSF2 mutation and extend the discussion to the interest of their pipeline for patient diagnosis and follow up under treatments.

Reviewer #2 (Public Review):

The authors performed a Multi-Omics Factor Analysis (MOFA) on analysis of two published MDS patient cohorts-1 from bone marrow mononuclear cells (BMMNCs) and CD34 cells (ref 17) and another from CD34+ cells (ref 15) --with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, Retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to attempt to identify the latent factors with significant impact on MDS prognosis.

SF3B1 was found to be the only mutation among 13 mutations in the BMMNC cohort that indicated a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34+ cohort. The MOFA factor representing inflammation showed a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases showed a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Also, MOFA identified RTE expression as a risk factor for MDS. They proposed that this work showed the efficacy of their integrative approach to assess MDS prognostic risk that 'goes beyond all the scoring systems described thus far for MDS'.

Several issues need clarification and response:

(1) The authors do not provide adequate known clinical and molecular information which demonstrates prognostic risk of their sample cohorts in order to determine whether their data and approach 'goes 'beyond all the scoring systems described thus far for MDS'. For example, what data have the authors that their features provide prognostic data independent of the prior known factors related to prognosis (eg, marrow blasts, mutational, cytogenetic features, ring sideroblasts, IPSS-R, IPSS-M, MDA-SS)?

(2) A major issue in analyzing this paper relates to the specific patient composition from whom the samples and data were obtained. The cells from the Shiozawa paper (ref 17) is comprised of a substantial number of CMML patients. Thus, what evidence have the authors that much of the data from the BMMNCs from these patients and mutant SRSF2 related predominantly to their monocytic differentiation state?

(3) In addition, as the majority of patients in the Shiozawa paper have ring sideroblasts (n=59), thus potentially skewing the data toward consideration mainly of these patients, for whom better outcomes are well known.

(4) Further, regarding this patient subset, what evidence have the authors that the importance of the SF3B1 mutation was merely related to the preponderance of sideroblastic patients from whom the samples were analyzed?

(5) An Erratum was reported for the Shiozawa paper (Shiozawa Y, Malcovati L, Gallì A, et al. Gene expression and risk of leukemic transformation in myelodysplasia. Blood. 2018 Aug 23;132(8):869-875. doi: 10.1182/blood-2018-07-863134) that resulted from a coding error in the construction of the logistic regression model for subgroup prediction based on the gene expression profiles of BMMNCs. This coding error was identified after the publication of the article. The authors should indicate the effect this error may have had on the data they now report.

(6) What information have the authors as to whether the differing RTE findings were not predominantly related to the differentiation state of the cell population analyzed (ie higher in BM MNCs vs CD34, Fig 1)? What control data have the authors regarding these values from normal (non-malignant) cell populations?

(7) The statement in the Discussion regarding the effects of SRSF2 mutation is speculative and should be avoided. Many other somatic gene mutations have known stronger effects on prognosis for MDS.

Reviewer #1 (Public Review):

Summary:

Mao and colleagues re-analysed published spatial, bulk and single-cell transcriptomic datasets from primary colorectal cancers and colorectal-cancer-derived liver metastases. The analyses of paired cancer and non-cancer tissue samples showed that T cells are enriched in tumour tissue, accompanied by a reduction in the fraction of NK cells in the cancer tissue transcriptional datasets. Furthermore, authors claim that tumour tissue has a higher fraction of GZMK+ (resting) NK cells and suggest a correlation between the presence of these cells and poorer prognosis for cancer patients. In contrast, the increased frequency of KIR2DL4+ (activated) NK cells correlates with improved survival of cancer patients.

Strengths:

The authors performed a comprehensive analysis of published datasets, integrating spatial and single-cell transcriptomic data, which allowed them to discover the enrichment of GZMK+ NK cells in cancer tissues.

Weaknesses:

Despite their thorough analysis, the authors did not provide sufficient experimental evidence to support their claim that GZMK+ NK cells contribute to a worse prognosis for cancer patients or promote cancer progression. The terms resting and activated NK cells are used without properly defining the characteristics of these populations other than the gene expression of a handful of genes. Furthermore, the criteria used to quantify the NK cell population in spatial data is not entirely clear. While one can visually observe an increased fraction of GZMK+ NK cells compared to KIR2DL4+ NK cells in cancer tissues, no quantification is shown. They did not present any preclinical (animal model) or clinical data suggesting a causal relationship between NK cells and tumour growth. Thus, while a correlation may exist between the presence of GZMK+ NK cells and poorer tumour prognosis, causation cannot be claimed based on the available evidence. Furthermore, the in vitro data provided is limited to a single NK cell line derived from a lymphoma patient, which does not fully represent the diversity and functionality of human NK cells. Moreover, the in vitro experiments suffer from a lack of required controls and inadequate methodology.

Reviewer #2 (Public Review):

Summary:

This manuscript investigates the role of the abundant NK cells that are observed in colon cancer liver metastasis using sequencing and spatial approaches in an effort to clarify the pro and anti-tumourigenic properties of NK cells. This descriptive study characterises different categories of NK cells in tumour and tumour-adjacent tissues and some correlations. An attempt has been made using pseudotime trajectory analysis but no models around how these NK cells might be regulated are provided.

Strengths:

This study integrates multiomics data to attempt to resolve correlates of protection that might be useful in understanding NK cell diversity and activation.

Weaknesses:

While this work is interesting, the power of such studies is in taking the discovered information and applying this to other cohorts to determine the strength and predictive power of the genes identified. It is also clear that these 'snapshots' analysed poorly take account of the dynamic temporal changes that occur within a tumour. It would have been good to see a proposed model of NK cell regulation as it might occur in the tumour (accounting for turnover and recruitment) beyond the static data.

Reviewer #1 (Public Review):

The detection sensitivity and accuracy are unclear.

In this manuscript, Zhou et al describe a deaminase and reader protein-assisted RNA m5C sequencing method. The general strategy is similar to DART-seq for m6A sequencing, but the difference is that in DART-seq, m6A sites are always followed by C which can be deaminated by fused APOBEC1 to provide a high resolution of m6A sites, while in the case of m5C, no such obvious conserved motifs for m5C sites exist, therefore, the detection resolution is much lower. In addition, the authors used two known m5C binding proteins ALYREF and YBX1 to guide the fused deaminases, but it is not clear whether these two binding proteins can bind most m5C sites and compete with other m5C binding proteins.

It is well known that two highly modified m5C sites exist in 28S RNA and many m5C sites exist in tRNA, the authors should validate their methods first by detecting these known m5C sites and evaluate the possible false positives in rRNA and tRNA. In mRNA, it is not clear what is the overlap between the technical replicates. In Figures 4A and 4C, they detected more than 10K m5C sites, and most of them did not overlap with sites uncovered by other methods. These numbers are much larger than expected and possibly most of them are false positives. Besides, it is not clear what is the detection sensitivity and accuracy since the method is neither single base resolution nor quantitative. There are no experiments to show that the detected m5C sites are responsive to the writer proteins such as NSUN2 and NSUN6, and the determination of the motifs of these writer proteins.

Reviewer #2 (Public Review):

The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications for the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods - like DRAM-seq, that allow the mapping of m5C independently of ex-situ RNA treatment with chemicals - are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question of whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

Comments:

(1) The use of two m5C reader proteins is likely a reason for the high number of edits introduced by the DRAM-Seq method. Both ALYREF and YBX1 are ubiquitous proteins with multiple roles in RNA metabolism including splicing and mRNA export. It is reasonable to assume that both ALYREF and YBX1 bind to many mRNAs that do not contain m5C.

To substantiate the author's claim that ALYREF or YBX1 binds m5C-modified RNAs to an extent that would allow distinguishing its binding to non-modified RNAs from binding to m5C-modified RNAs, it would be recommended to provide data on the affinity of these, supposedly proven, m5C readers to non-modified versus m5C-modified RNAs. To do so, this reviewer suggests performing experiments as described in Slama et al., 2020 (doi: 10.1016/j.ymeth.2018.10.020). However, using dot blots like in so many published studies to show modification of a specific antibody or protein binding, is insufficient as an argument because no antibody, nor protein, encounters nanograms to micrograms of a specific RNA identity in a cell. This issue remains a major caveat in all studies using so-called RNA modification reader proteins as bait for detecting RNA modifications in epitranscriptomics research. It becomes a pertinent problem if used as a platform for base editing similar to the work presented in this manuscript.

(2) Since the authors use a system that results in transient overexpression of base editor fusion proteins, they might introduce advantageous binding of these proteins to RNAs. It is unclear, which promotor is driving construct expression but it stands to reason that part of the data is based on artifacts caused by overexpression. Could the authors attempt testing whether manipulating expression levels of these fusion proteins results in different editing levels at the same RNA substrate?

(3) Using sodium arsenite treatment of cells as a means to change the m5C status of transcripts through the downregulation of the two major m5C writer proteins NSUN2 and NSUN6 is problematic and the conclusions from these experiments are not warranted. Sodium arsenite is a chemical that poisons every protein containing thiol groups. Not only do NSUN proteins contain cysteines but also the base editor fusion proteins. Arsenite will inactivate these proteins, hence the editing frequency will drop, as observed in the experiments shown in Figure 5, which the authors explain with fewer m5C sites to be detected by the fusion proteins.

(4) The authors should move high-confidence editing site data contained in Supplementary Tables 2 and 3 into one of the main Figures to substantiate what is discussed in Figure 4A. However, the data needs to be visualized in another way than an Excel format. Furthermore, Supplementary Table 2 does not contain a description of the columns, while Supplementary Table 3 contains a single row with letters and numbers.

(5) The authors state that "plotting the distribution of DRAM-seq editing sites in mRNA segments (5'UTR, CDS, and 3'UTR) highlighted a significant enrichment near the initiation codon (Figure 3F).", which is not true when this reviewer looks at Figure 3F.

(6) The authors state that "In contrast, cells expressing the deaminase exhibited a distinct distribution pattern of editing sites, characterized by a prevalence throughout the 5'UTR.", which is not true when this reviewer looks at Figure 3F.

(7) The authors claim in the final conclusion: "In summary, we developed a novel deaminase and reader protein assisted RNA m5C methylation approach...", which is not what the method entails. The authors deaminate As or Us close to 5mC sites based on the binding of a deaminase-containing protein.

(8) The authors claim that "The data supporting the findings of this study are available within the article and its Supplementary Information." However, no single accession number for the deposited sequencing data can be found in the text or the supplementary data. Without the primary data, none of the claims can be verified.

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Reviewer #2 (Public Review):

Summary:

The authors showed the applicability and usefulness of a new AlphaFold2 pipeline called PabFold, which can predict linear antibody epitopes (B-cell epitopes) that can be helpful for the selection of reagents to be applied in competitive ELISA assay.

Strengths:

The authors showed the accuracy of the pipeline to identify correctly the binding epitope for three different antibody-antigen systems (Myc, HA, and Sars-Cov2 nucleocapsid protein). The design of scFvs from Fab of the three antibodies to speed up the analysis time is extremely interesting.

Weaknesses:

The article justifies correctly the findings and no great weaknesses are present. However, it could be useful for a broader audience to show in detail how pLDDT was calculated for both Simple-Max approach (per residue-pLDDT) and Consensus analysis ( average pLDDT for each peptide), with associated equations.

Reviewer #1 (Public Review):

Metabotropic glutamate receptors (mGLuRs) play a key role in regulating neuronal activity and related behaviors. In different brain regions these receptors can be expressed presynaptically and postsynaptically in different classes of neurons. Therefore, it is difficult to predict the effects of systemically applied drugs that act on these receptors. Here, the authors harness the power of photopharmacology, applying modulators that can be activated or inactivated by light with spatial precision, to address this problem. Their stated goal is to determine the role of mGluRs in regulating pain behaviors, and the circuit mechanisms driving this regulation. Their findings suggest that mGluRs acting in medial prefrontal cortex and thalamus drive antinociception in animals with neuropathic pain, whereas these receptors drive pronociception when acting in the amygdala. Their circuit analysis suggests that, in the amygdala, mGluRs act by decreasing feedforward inhibition of the output neurons. These findings have the potential to affect the development of targeted treatment for pain and related disorders. The elegant photopharmacological approaches will likely inform future studies attempting to distinguish the action of neuroactive drugs in different brain regions.

Reducing the impact of these studies are several methodological, analytical, and interpretation issues.

- The authors report that "the effect of optical manipulations of photosensitive mGlu5 NAMs in individual brain regions in pain models has been studied before". It is, therefore, not immediately clear what is novel in the present study.<br /> - The reliance only on reflexive measures of pain, especially in a study that examines the role of "affective and cognitive aspects of pain and pain modulation".<br /> - The inclusion of only males is unfortunate because of known, significant sex differences in neuronal circuits driving pain conditions, in both preclinical models (including form work by the authors) and in clinical populations.<br /> - The elegant slice experiments (especially Fig. 3) were designed to probe circuit mechanisms through which mGluRs act in different brain regions. These experiments also provide a control to assess whether the photopharmacological compounds act as advertised. Surprisingly, the effect size produced by these compounds on neuronal activity are rather small (and, at times, seems driven by outliers). How this small effect affects the interpretation of the behavioral findings is not clear.<br /> - These small effect sizes should also be considered when interpreting the circuit actions studied here.<br /> - Some of the sample sizes are as small as n=3. Without an a priori power analysis, it is difficult to assess the validity of the analyses.<br /> - The authors present intriguing data on changes in InsP levels in some (but not all) animals after injury, but not in sham animals. They also report an increase in the expression of mGLuRs expression in some, but not all brain regions. These findings are not discussed. It is not clear how these selective changes in mGluR expression and activity might affect the interpretation of the photopharmacological results.<br /> - The behavioral data seem to represent discrete, and not continuous variables. The statistical tests applied are likely inappropriate for these analyses.<br /> - The authors assume (and state in the abstract) that they can selectively stimulate BLA afferents to the neocortex. This is technically highly unlikely.<br /> - The results from the experiment on rostroventral medulla (RVM) neurons are less than convincing because only a "trend" towards decreased excitation is reported. As above, without consideration of effect size, it is hard to appreciate the significance of these findings. The absence of a demonstration of a classical ON Cell firing pattern is also unfortunate.

Reviewer #2 (Public Review):

In this study, Notartomaso et al. used optical activation of systemic JF-NP-26, a caged, baseline inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices, thalamus, and BLA to investigate the roles of these receptors in various brain regions in pain processing. They found that alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, but this analgesic effect was reversed by light-induced drug inactivation in the prelimbic and infralimbic cortices, and thalamus. In contrast, these pharmacological effects were reversed in the BLA. They further found that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. They thus concluded that mGlu5 receptors had differential effects in distinct brain regions. mGlu5 receptors are important receptors in pain processing, and their regional specificity has not been studied in detail. Further, this is an interesting study regarding the use of optical activation of pro-drugs, and the findings are timely. The combination of in vivo pharmacology, biochemistry, and slice EP provides complementary results.

Reviewer #3 (Public Review):

In this manuscript, Notartomaso, Antenucci et al. use two different light-sensitive metabotropic glutamate receptor negative allosteric modulators (NAMs) to determine how mGlu5 receptor signaling in distinct brain regions of mice influences mechanical sensitivity in chronic constriction injury (CCI) model of neuropathic pain. This is an extension of their previous work using photocaged mGlu5 negative allosteric modulators and incorporates a systemically active NAM that can be locally photoswitched off and on with violet and green light, respectively. The authors found that NAM signaling in the thalamus and prefrontal cortical regions consistently reduced mechanical hypersensitivity. However, they observed divergent effects on these measures in the basolateral amygdala. The authors attempted to solve the discrepancy in behavioral measurements between mGlu5 signaling in the basolateral amygdala by determining how NAMs modulate synaptic transmission or in vivo firing and found that these effects were projection-dependent.

Strengths:

This study demonstrates the importance of local signaling by mGlu5 receptors across multiple pain-processing circuits in the brain, and the use of optical activation and inactivation strategies is very intriguing.

Weaknesses:

One major limitation is the lack of sham surgery groups and vehicle/light-only controls in behavior and physiology experiments, though the authors did test mechanical sensitivity in the contralateral paw. The reliance on a single behavioral measure in these groups is also problematic. Many of these brain regions are known to influence distinct aspects of somatosensory processing or other behaviors entirely, which may be interpreted as hypersensitivity (e.g. fear or anxiety-like behaviors in the basolateral amygdala). Details on the light intensities used is also absent, and it is important to test whether violet light had any unintended effects on these cells/mice.

While the effort to provide some mechanistic understanding using slice physiology and in vivo recordings is appreciated, they ignore any effects that these NAMs have directly on the excitability of the recorded output neuron. In the models, mGlu5 is proposed to exist on some upstream inhibitory (mPFC) or excitatory (BLA) projection, but no evidence of a direct effect on these synaptic inputs was observed. Given the widespread distribution of mGlu5 in these brain regions, the proposed model seems unlikely. Perhaps CCI induces changes in functional coupling of mGlu5 in different cell types, and this could be revealed with appropriate control experiments.

Overall the broad profiling taken here across multiple brain regions lacks controls and some cohesion, making it challenging to conclude how mGlu5 signaling is acutely impacting these circuits and/or specific cell types.

Reviewer #1 (Public Review):

How does the brain respond to the input of different complexity, and does this ability to respond change with age?

The study by Lalwani et al. tried to address this question by pulling together a number of neuroscientific methodologies (fMRI, MRS, drug challenge, perceptual psychophysics). A major strength of the paper is that it is backed up by robust sample sizes and careful choices in data analysis, translating into a more rigorous understanding of the sensory input as well as the neural metric. The authors apply a novel analysis method developed in human resting-state MRI data on task-based data in the visual cortex, specifically investigating the variability of neural response to stimuli of different levels of visual complexity. A subset of participants took part in a placebo-controlled drug challenge and functional neuroimaging. This experiment showed that increases in GABA have differential effects on participants with different baseline levels of GABA in the visual cortex, possibly modulating the perceptual performance in those with lower baseline GABA. A caveat is that no single cohort has taken part in all study elements, ie visual discrimination with drug challenge and neuroimaging. Hence the causal relationship is limited to the neural variability measure and does not extend to visual performance. Nevertheless, the consistent use of visual stimuli across approaches permits an exceptionally high level of comparability across (computational, behavioural, and fMRI are drawing from the same set of images) modalities. The conclusions that can be made on such a coherent data set are strong.

The community will benefit from the technical advances, esp. the calculation of BOLD variability, in the study when described appropriately, encouraging further linkage between complementary measures of brain activity, neurochemistry, and signal processing.

Reviewer #2 (Public Review):

Lalwani et al. measured BOLD variability during the viewing of houses and faces in groups of young and old healthy adults and measured ventrovisual cortex GABA+ at rest using MR spectroscopy. The influence of the GABA-A agonist lorazepam on BOLD variability during task performance was also assessed, and baseline GABA+ levels were considered as a mediating variable. The relationship of local GABA to changes in variability in BOLD signal, and how both properties change with age, are important and interesting questions. The authors feature the following results: 1) younger adults exhibit greater task-dependent changes in BOLD variability and higher resting visual cortical GABA+ content than older adults, 2) greater BOLD variability scales with GABA+ levels across the combined age groups, 3) administration of a GABA-A agonist increased condition differences in BOLD variability in individuals with lower baseline GABA+ levels but decreased condition differences in BOLD variability in individuals with higher baseline GABA+ levels, and 4) resting GABA+ levels correlated with a measure of visual sensory ability derived from a set of discrimination tasks that incorporated a variety of stimulus categories.

Strengths of the study design include the pharmacological manipulation for gauging a possible causal relationship between GABA activity and task-related adjustments in BOLD variability. The consideration of baseline GABA+ levels for interpreting this relationship is particularly valuable. The assessment of feature-richness across multiple visual stimulus categories provided support for the use of a single visual sensory factor score to examine individual differences in behavioral performance relative to age, GABA, and BOLD measurements. Weaknesses of the study include the absence of an interpretation of the physiological mechanisms that contribute to variability in BOLD signal, particularly for the chosen contrast that compared viewing houses with viewing faces. Whether any of the observed effects can be explained by patterns in mean BOLD signal, independent of variability would be useful to know. The positive correlation between resting GABA+ levels and the task-condition effect on BOLD variability reaches significance at the total group level, when the young and old groups are combined, but not separately within each group. This correlation may be explained by age-related differences since younger adults had higher values than older adults for both types of measurements. This is not to suggest that the relationship is not meaningful or interesting, but that it may be conceptualized differently than presented. Two separate dosages of lorazepam were used across individuals, but the details of why and how this was done are not provided, and the possible effects of the dose are not considered. The observation of greater BOLD variability during the viewing of houses than faces may be specific to these two behavioral conditions, and lingering questions about whether these effects generalize to other types of visual stimuli, or other non-visual behaviors, in old and young adults, limit the generalizability of the immediate findings.

The observed age-related differences in patterns of BOLD activity and ventrovisual cortex GABA+ levels along with the investigation of GABA-agonist effects in the context of baseline GABA+ levels are particularly valuable to the field, and merit follow-up. Assessing background neurochemical levels is generally important for understanding individualized drug effects. Therefore, the data are particularly useful in the fields of aging, neuroimaging, and vision research.

Reviewer #3 (Public Review):

The role of neural variability in various cognitive functions is one of the focal contentions in systems and computational neuroscience. In this study, the authors used a large-scale cohort dataset to investigate the relationship between neural variability measured by fMRI and several factors, including stimulus complexity, GABA levels, aging, and visual performance. Such investigations are valuable because neural variability, as an important topic, is by far mostly studied within animal neurophysiology. There is little evidence in humans. Also, the conclusions are built on a large-scale cohort dataset that includes multi-model data. Such a dataset per se is a big advantage. Pharmacological manipulations and MRS acquisitions are rare in this line of research. Overall, I think this study is well-designed, and the manuscript reads well. I listed my comments below and hope my suggestions can further improve the paper.

Strength:<br /> (1) The study design is astonishingly rich. The authors used task-based fMRI, MRS technique, population contrast (aging vs. control), and psychophysical testing. I appreciate the motivation and efforts for collecting such a rich dataset.<br /> (2) The MRS part is good. I am not an expert in MRS so cannot comment on MRS data acquisition and analyses. But I think linking neural variability to GABA in humans is in general a good idea. There has been a long interest in the cause of neural variability, and inhibition of local neural circuits has been hypothesized as one of the key factors.<br /> (3) The pharmacological manipulation is particularly interesting as it provides at least evidence for the causal effects of GABA and deltaSDBOLD. I think this is quite novel.

Weakness:<br /> (1) I am concerned about the definition of neural variability. In electrophysiological studies, neural variability can be defined as Poisson-like spike count variability. In the fMRI world, however, there is no consensus on what neural variability is. There are at least three definitions. One is the variability (e.g., std) of the voxel response time series as used here and in the resting fMRI world. The second is to regress out the stimulus-evoked activation and only calculate the std of residuals (e.g., background variability). The third is to calculate variability of trial-by-trial variability of beta estimates of general linear modeling. It currently remains unclear the relations between these three types of variability with other factors. It also remains unclear the links between neuronal variability and voxel variability. I don't think the computational principles discovered in neuronal variability also apply to voxel responses. I hope the authors can acknowledge their differences and discuss their differences.<br /> (2) If I understand it correctly, the positive relationship between stimulus complexity and voxel variability has been found in the author's previous work. Thus, the claims in the abstract in lines 14-15, and section 1 in results are exaggerated. The results simply replicate the findings in the previous work. This should be clearly stated.<br /> (3) It is difficult for me to comprehend the U-shaped account of baseline GABA and shift in deltaSDBOLD. If deltaSDBOLD per se is good, as evidenced by the positive relationship between brainscore and visual sensitivity as shown in Fig. 5b and the discussion in lines 432-440, why the brain should decrease deltaSDBOLD ?? or did I miss something? I understand that "average is good, outliers are bad". But a more detailed theory is needed to account for such effects.<br /> (4) Related to the 3rd question, can you should the relationship between the shift of deltaSDBOLD (i.e., the delta of deltaSDBOLD) and visual performance?<br /> (5) Are the dataset openly available ?? I didn't find the data availability statement.

Reviewer #1 (Public Review):

The study is thorough and systematic, and in comparing three well-separated hypotheses about the mechanism leading from grid cells to hexasymmetry it takes a neutral stand above the fray which is to be particularly appreciated. Further, alternative models are considered for the most important additional factor, the type of trajectory taken by the agent whose neural activity is being recorded. Different sets of values, including both "ideal" and "realistic" ones, are considered for the parameters most relevant to each hypothesis. Each of the three hypotheses is found to be viable under some conditions, and less so in others. Having thus given a fair chance to each hypothesis, nevertheless, the study reaches the clear conclusion that the first one, based on conjunctive grid-by-head-direction cells, is much more plausible overall; the hypothesis based on firing rate adaptation has intermediate but rather weak plausibility; and the one based on clustering of cells with similar spatial phases in practice would not really work. I find this conclusion convincing, and the procedure to reach it, a fair comparison, to be the major strength of the study.

What I find less convincing is the implicit a priori discarding of a fourth hypothesis, that is, that the hexasymmetry is unrelated to the presence of grid cells. Full disclosure: we have tried unsuccessfully to detect hexasymmetry in the EEG signal from vowel space and did not find any (Kaya, Soltanipour and Treves, 2020), so I may be ranting off my disappointment, here. I feel, however, that this fourth hypothesis should be at least aired, for a number of reasons. One is that a hexasymmetry signal has been reported also from several other cortical areas, beyond entorhinal cortex (Constantinescu et al, 2016); true, also grid cells in rodents have been reported in other cortical areas as well (Long and Zhang, 2021; Long et al, bioRxiv, 2021), but the exact phenomenology remains to be confirmed. Second, as the authors note, the conjunctive mechanism is based on the tight coupling of a narrow head direction selectivity to one of the grid axes. They compare "ideal" with "Doeller" parameters, but to me the "Doeller" ones appear rather narrower than commonly observed and, crucially, they are applied to all cells in the simulations, whereas in reality only a proportion of cells in mEC are reported to be grid cells, only a proportion of them to be conjunctive, and only some of these to be narrowly conjunctive. Further, Gerlei et al (2020) find that conjunctive grid cells may have each of their fields modulated by different head directions, a truly surprising phenomenon that, if extensive, seems to me to cast doubts on the relation between mass activity hexasymmetry and single grid cells.

Finally, a variant of the fourth hypothesis is that the hexasymmetry might be produced by a clustering of head direction preferences across head direction cells similar to that hypothesized in the first hypothesis, but without such cells having to fire in grid patterns. If head direction selectivity is so clustered, who needs the grids? This would explain why hexasymmetry is ubiquitous, and could easily be explored computationally by, in fact, a simplification of the models considered in this study.

Reviewer #2 (Public Review):

Grid cells - originally discovered in single-cell recordings from the rodent entorhinal cortex, and subsequently identified in single-cell recordings from the human brain - are believed to contribute to a range of cognitive functions including spatial navigation, long-term memory function, and inferential reasoning. Following a landmark study by Doeller et al. (Nature, 2010), a plethora of human neuroimaging studies have hypothesised that grid cell population activity might also be reflected in the six-fold (or 'hexadirectional') modulation of the BOLD signal (following the six-fold rotational symmetry exhibited by individual grid cell firing patterns), or in the amplitude of oscillatory activity recorded using MEG or intracranial EEG. The mechanism by which these network-level dynamics might arise from the firing patterns of individual grid cells remains unclear, however.

In this study, Khalid and colleagues use a combination of computational modelling and mathematical analysis to evaluate three competing hypotheses that describe how the hexadirectional modulation of population firing rates (taken as a simple proxy for the BOLD, MEG, or iEEG signal) might arise from the firing patterns of individual grid cells. They demonstrate that all three mechanisms could account for these network-level dynamics if a specific set of conditions relating to the agent's movement trajectory and the underlying properties of grid cell firing patterns are satisfied.

The computational modelling and mathematic analyses presented here are rigorous, clearly motivated, and intuitively described. In addition, these results are important both for the interpretation of hexadirectional modulation in existing data sets and for the design of future experiments and analyses that aim to probe grid cell population activity. As such, this study is likely to have a significant impact on the field by providing a firmer theoretical basis for the interpretation of neuroimaging data. To my mind, the only weakness is the relatively limited focus on the known properties of grid cells in rodent entorhinal cortex, and the network level activity that these firing patterns might be expected to produce under each hypothesis. Strengthening the link with existing neurobiology would further enhance the importance of these results for those hoping to assay grid cell firing patterns in recordings of ensemble-level neural activity.

Reviewer #3 (Public Review):

This is an interesting and carefully carried out theoretical analysis of potential explanations for hexadirectional modulation of neural population activity that has been reported in the human entorhinal cortex and some other cortical regions. The previously reported hexadirectional modulation is of considerable interest as it has been proposed to be a proxy for the activation of grid cell networks. However, the extent to which this proposal is consistent with the known firing properties of grids hasn't received the attention it perhaps deserves. By comparing the predictions of three different models this study imposes constraints on possible mechanisms and generates predictions that can be tested through future experimentation.

Overall, while the conclusions of the study are convincing, I think the usefulness to the field would be increased if null hypotheses were more carefully considered and if the authors' new metric for hexadirectional modulation (H) could be directly contrasted with previously used metrics. For example, if the effect sizes for hexadirectional modulation in the previous fMRI and EEG data could be more directly compared with those of the models here, then this could help in establishing the extent to which the experimental hexadirectional modulation stands out from path hexasymmetry and how close it comes to the striking modulation observed with the conjunctive models. It could also be helpful to consider scenarios in which hexadirectional modulation is independent of grid firing, for example perhaps with appropriate coordination of head direction cell firing.

Reviewer #1 (Public Review):

In this work, Plaza-Alonso et al. present a collection of volume electron microscopy (EM) reconstructions of human postmortem medial entorhinal cortex (MEC), and they measure properties of MEC cytoarchitecture and synapses as a function of neuroanatomical subdivision. The authors generate a sampling of 9 smaller (≲10 µm/side) EM reconstructions per subdivision to avoid prohibitively large (petabyte) EM volumes, using 3 reconstructions for each of 3 brain donors to control for inter-individual variability. Conducting in-depth analyses for 7 subdivisions (63 reconstructions total), the authors find little significant inter-subdivision variability in structural composition (volume fractions of cell bodies vs. neuropil vs. blood vessels) and multiple synapse properties (spatial distribution, density, area, shape, excitatory/inhibitory type, and postsynaptic cell compartment). They conclude that human MEC connectivity is largely homogeneous, with synapses arranged in a generally random spatial distribution and a large fraction of synapses being asymmetric (putatively excitatory). Their other findings include that asymmetric synapses are larger than symmetric/putatively inhibitory synapses; that asymmetric synapses prefer dendritic spines whereas symmetric synapses prefer dendritic shafts; and that a small fraction of synapses have larger, complex shapes that may suggest increased synaptic efficacy. They note that inhomogeneities may include inter-subdivision variation in asymmetric synapse area and complex-shaped synapse prevalence, and for some reconstructions (12/63), possible substructure in synapse distributions.

Strengths:<br /> The authors have carefully conducted this work, using reasonable methods and comparing their findings with previous volume EM reconstructions where possible. It represents a substantial effort, given the challenges of producing and annotating volume EM data and of collecting human postmortem tissue. They have thus contributed a brain-region-specific characterization of human postmortem tissue with value as both a data resource and an examination of postmortem EM reconstruction quality, given that postmortem tissue is less-studied with volume EM but could be an important source of human brain samples (for example in regions that are surgically inaccessible). Further, some of the authors' measurements may be of added value, as they suggest functional correlates for less-studied synapse structures (such as the differing sizes of complex and simple "macular" synapses formed onto dendritic spines vs. shafts).

Weaknesses:<br /> Despite these strengths, the analysis in this work may be impacted by multiple sources of experimental variability that may have contributed to the observed lack of structural variability, and the potential contributions of these should be addressed in making their claims.

(1) The authors' approach to tissue sampling may have resulted in under-sampling, which may have reduced the detection power of their tests. More specifically, each reconstructed EM volume measured ~10 µm x 7 µm x 6 µm (360 - 502 µm^3) and contained ~300-400 synapses (Lines 211-212, 772-773). Per donor, this amounts to a sampling volume of ~1500 µm^3 for each MEC subdivision or ~1x104 µm^3 total. By contrast, the volume of the adult human MEC is ~1x10^12 µm^3, roughly 1x10^8 times larger [1]. Thus, while these EM reconstructions reflect a substantial effort, it is likely that they represent an under-sampling of MEC structure, especially since multiple excitatory and inhibitory neuron types are likely interspersed throughout (the authors also note this possibility in Lines 640-659).

(2) The authors' measurements are combined across three donors who are biologically diverse (Table S11), including in terms of characteristics that themselves may impact neuronal connectivity. Without controlling for these variables, the possible reduction in stochastic, biological inter-individual variability that could be achieved by combining data across donors may be offset by increases in phenotype-related variability, which could reduce the detectability of true, conserved connectivity variations across MEC subdivisions. Specifically, these donors represent a mix of males and females; a mix of ages (40, 53, and 66 years) that suggest differing degrees of aging-related changes in neuronal connectivity (according to previous work, a majority of people >55 years of age are estimated to have Alzheimer's-associated neurofibrillary tangles, regardless of whether they have dementia symptomatology; see for instance [1]); and one death from metastatic cancer, indicating that for one donor cellular/neuronal abnormalities associated either with cancer itself or related therapies could be present.

These two factors could substantially increase the dispersion of the authors' measurements in each MEC subdivision and lead to a situation with no detectable differences between subdivisions. It would be important to address these impacts when determining whether to interpret a lack of significant differences as true biological homogeneity for human MEC.

One helpful approach would be to explicitly show the variance of each measurement obtained for each EM reconstruction. For example, error bars showing the interquartile range could be added to each data point in Fig. 3C, to show how much synapse areas vary per reconstruction and to allow some comparison across donors and MEC subdivisions.

(3) A third potential source of variability relates to the authors' approach for synapse annotation. They appear to annotate active zones and postsynaptic densities by thresholding synapse images at some user-defined pixel intensity value, taking only pixels darker than that threshold as their annotations (Lines 806 - 812). This technique seems like it could be prone to producing noisy annotations, particularly since in the EM images provided (Figs. S11-16) the pixel intensities of active zones/postsynaptic densities and surrounding neuropil do not appear to be highly distinct.

It would be important for the authors to support their findings by quantifying the variability that may be associated with this technique.

[1] Price, C.C. et al., J. Int. Neuropsychol. Soc., (2010), doi: 10.1017/S135561771000072X.

Reviewer #2 (Public Review):

Plaza-Alanso et al. characterize synaptic properties across human medial entorhinal cortex across layers and, importantly, across multiple individuals. Using an impressive collection of post-mortem autopsy samples, they generate high resolution 3d FIB-SEM volumes across layers and MEC subregions and measure features such as synapse density, spatial distribution, size, shape and target location. The use of volumes permits a richer local context to synaptic reconstructions, and the methods used to count and quantify synapses appear thorough and convincing, although with limited descriptions at times. The core findings suggest few differences in most properties across either layers or individuals, with some modest exceptions in layers 1 and 6. A particular strength of the dataset is the large number of high quality synaptic contact reconstructions.<br /> However, because the volumes have no specific labels and are too small to associate axons or dendrites with individual cells or cell types, it is not clear how to extrapolate these findings to new insights toward the stated goal of a better understanding of the networks and connectivity characteristics of the MEC. Broadly speaking, the paper would benefit from a better explanation of why these specific parameters were chosen and what the authors hoped to gain from them. It might be useful to think of what would need to be the case to see something substantially different. Many of the measures here reflect the properties of dendrites passing through a small volume, which depends on the number of cells of different cell types, the length and thickness of their dendritic arbors, synapse density distributions, local and long range afferents, and more. One interpretation of these results is that these neuropil volumes across layers and individuals are effectively fully packed with dendrites, with a similar ratio of excitatory and inhibitory neurons, dendrites with roughly similar thickness and synaptic input density and local E/I balance. Can the authors disentangle these cellular-scale contributions or constrain their inter-individual variability across individuals? The lack of variability is perhaps the main observation here, and understanding this more clearly could be useful for thinking about larger volumes where fewer replicates are currently possible.

Reviewer #1 (Public Review):

Article strengths:

(1) Detailed data: The authors provided a large amount of clinical data as support, making the analysis results more persuasive and credible.<br /> (2) Scientific method: Appropriate statistical methods were used to analyze the data, which can accurately reflect the internal laws and trends of the data.<br /> (3) Clear conclusions: The conclusions drawn in the article are clear and explicit, easy for readers to understand and accept.<br /> (4) High practicality: The research results have important guiding significance for obstetrics and gynecology clinical practice, helping to improve patient treatment outcomes and quality of life.

Article weaknesses:

Limitations of research methods: Although the authors used statistical methods to analyze the data, they may be limited by factors such as data sources and sample size, leading to some limitations in the research results. It is recommended that the authors further expand the data sources and increase the sample size in subsequent studies to improve the accuracy and reliability of the research.

Reviewer #2 (Public Review):

This prospective study advances our understanding of the predictive value of preoperative serum CA125, CA19-9, CA72-4, CEA, and AFP in endometrial cancer. The evidence supporting the conclusions is convincing with rigorous analysis of the association between prognostic values of several serum markers with the clinical data of endometrial cancer patients. However, there are a few areas in which the article may be improved through further validation of the prognostic value of the risk score in patients with endometrial cancer at different stages. Moreover, the authors should provide a more detailed explanation of the choice of statistical methods in the manuscript. The work will be of broad interest to clinicians, medical researchers and scientists working in endometrial cancer.

(1) The groups of patients with endometrial cancer in the manuscript are classified according to age greater than/less than 60. Please explain why 60 years old is chosen as the boundary value of age.<br /> (2) Among the patients with endometrial cancer selected in the manuscript, AFP outliers accounted for a relatively small proportion. The authors chose the clinical detection outliers of CA-125, CA19-9, AFP and CEA as the dividing line, instead of re-selecting the optimal cut-off value in this population, which should be classified and the prognostic value explored.<br /> (3) In cancer research, stage is an important prognostic factor to guide the treatment of patients in clinical work. Patients with different stages of endometrial cancer have obvious prognostic differences. The authors constructed a new prognostic risk score based on serum level of AFP， CEA and CA125, the prognostic value of the risk score should be validated in patients with endometrial cancer at different stages。

Reviewer #3 (Public Review):

The authors of this study aimed to enhance the prognostic assessment of endometrial cancer (EC) by identifying and validating a set of serum tumor markers (CA125, CEA, and AFP) that could reliably predict progression-free survival (PFS) and overall survival (OS) in patients. By employing a robust methodology that included the use of LASSO Cox regression analysis to construct a predictive model, the study sought to provide a more nuanced tool for clinical decision-making in the management of EC.

Major Strengths:

Methodological Rigor: The study's use of advanced statistical methods to analyze a large dataset of EC patients stands out. The inclusion of a validation cohort enhances the credibility of the prognostic model developed.<br /> Clinical Relevance: The identification of CA125, CEA, and AFP as independent prognostic factors and the creation of a risk score based on these markers offer valuable tools for clinicians. The predictive accuracy of this model could significantly impact patient management and treatment planning.<br /> Weaknesses:

Generalizability: The study is based on a cohort from a single institution, which may limit the applicability of the findings across different populations and healthcare settings.<br /> Loss to Follow-Up: As acknowledged by the authors, the loss to follow-up of some patients introduces a potential source of bias, possibly affecting the study's conclusions.<br /> Achievement of Aims and Support for Conclusions:

The study successfully achieves its aim of developing a prognostic model for EC that integrates serum levels of CA125, CEA, and AFP. The evidence presented supports the authors' conclusions that this model is a robust tool for predicting patient outcomes, evidenced by its performance in both the training and validation cohorts.

Impact and Utility:

This work is poised to make a significant contribution to the field of gynecological oncology, particularly in the management of endometrial cancer. The study's findings provide a practical approach to stratifying patients based on their risk, which could be instrumental in tailoring individualized treatment plans. Moreover, the model's ability to predict PFS and OS with considerable accuracy offers a promising avenue for further research and application in clinical settings.

Additional Context:

Understanding the role of tumor markers in cancer prognosis is a rapidly evolving area of oncology research. This study's focus on combining multiple serum markers into a comprehensive risk score model represents a significant step forward in the quest for more personalized cancer care. Future studies could expand on this work by exploring the integration of such markers with other clinical and molecular data to further refine prognostic models.

Reviewer #1 (Public Review):

The current manuscript investigates the role of microRNA cluster 221/222 (miR221/222) in rheumatoid arthritis synovial fibroblasts (RA SFs) prompted by previous evidence that this cluster is upregulated in these cells. The authors employed multiple genetic mouse models and genomic approaches demonstrating that global overexpression of miR221/222 in huTNFtg polyarthritic mice further expanded SF proliferation and exacerbated RA, whereas global deletion reduced SF proliferation and dampened RA. Mechanistically, the authors provide sufficient evidence that these effects are mediated through the regulation of cell cycle inhibitors (p27 and p57) and the epigenetic regulator Smarca1. In general, these studies offer strong evidence that miR221/222 contributes to the pathogenic mechanisms underlying SF function in RA and provide new critical information to advance the understanding of RA pathology. However, certain important aspects are not addressed. Specifically, limited information related to the immune and inflammatory nature of this mechanism is offered, which is further complicated by limitations of using global overexpression and knockout. For example, it remains unknown to what is the extent of contribution by immune and inflammatory cells as well as what are the SF-derived effectors that propagate tissue damage and erosion

Reviewer #2 (Public Review):

This study focuses on the role of miR221/222 in the pathogenesis of rheumatoid arthritis. Through the use of different murine models and genome-wide techniques, the authors individuate a miR221/222 elicited mechanism leading to synovial fibroblast hyperproliferation. These discoveries may provide a rationale for future targeted therapies for RA treatment.

miR-221 and miR-222 have been linked with arthritis in previous studies from this and other laboratories: miR-221 and miR-222 have been found upregulated in SFs derived from the huTNFtg mouse model and RA patients, where their expression correlates with disease activity. The novelty of the present study resides in the analysis of the role of miR-221/miR-222 in an in vivo system and provides insight into cellular and molecular mechanisms linking miR-221/222 to RA progression.

Reviewer #3 (Public Review):

In this study, Roumelioti et al demonstrate the role of miR-221/222 in synovial fibroblasts (SFs) in inflammatory arthritis, applying a plethora of methods in three transgenic mouse models (huTNFtg, TgColVI-miR-221/222, huTNFtg;TgColVI-miR-221/222). miR-221/222 is upregulated in SFs, upon stimulation with TNF, both in early and established disease, while its gene is activated, as shown by scATAC-seq data. Using RNA sequencing and KEGG pathway analysis, authors showed that overexpression of miR-221 and miR-222 exacerbates arthritis, mainly due to SFs proliferation, driven by cell cycling inhibition and extracellular matrix remodeling. Although the authors suggest the potential utility of miR-221/222 targeting in inflammatory arthritis treatment, this was only examined through miR-221/222 -/- mice generation and not by direct silencing of miR-221/222 by administering a miR-221/222 antagonist.

Reviewer #1 (Public Review):

The authors tested the hypothesis that protein consumption decreases with decreasing mass-specific growth during development. This hypothesis is firmly grounded in the logical premise that as animals progress from periods of reduced activity and rapid growth to phases of increased activity and reduced mass-specific growth during their development, they are likely to adjust their nutrient intake, reducing protein and increasing carbohydrate consumption accordingly. The authors tested their hypothesis using the South American locust Schistocerca cancellata, combining field observations with laboratory experiments. This approach allowed them to discern how variations in activity history and metabolism between field- and laboratory-raised locusts influenced their nutrient requirements.<br /> Their findings, indeed reveal the predicted shift from high protein: carbohydrate consumption to lower protein: carbohydrate intake from the first instar to adult locust - a decline that strongly correlated with a decrease in mass-specific growth rate. Their comparison between field- and laboratory-raised locusts, showed that protein demand was not different, however, carbohydrate consumption rate was >50% higher in the field locusts. These results add depth and significance to the study, shedding light on how environmental factors influence nutrient requirements.<br /> What truly amplifies the strength and novelty of the authors' hypothesis is their anticipation that this observed trend in Schistocerca cancellata could extend to all animals. This anticipation is rooted in the expectation that growth rates scale hypometrically across various body sizes and developmental stages, introducing a universal dimension to their findings that holds great promise for broader ecological and evolutionary understanding.<br /> However, while the study is commendable in its methodology and core findings, there is room for improvement in clarifying the implications of the results. The current lack of clarity is evident in the somewhat shallow questions outlined in lines 358 to 363. For instance, the practice of administering age-specific diets has been commonplace in human and livestock management for ages. Thus, its continued utility may not be the most stimulating question. Instead, a more thought-provoking inquiry might delve into whether variations in global protein availability play a pivotal role in driving niche specialization and the biogeography of animal body sizes and ontogeny, especially considering the potential impacts of climate change. Such inquiries would further elevate the significance of the author's work and its broader implications in the field.

Reviewer #2 (Public Review):

How and why nutritional requirements and intake targets change over development and differ between species are significant questions with wide-ranging implications spanning ecology to health. In this manuscript, Talal et al. set out to address these questions in laboratory and field experiments with grasshoppers and in a comparative analysis of different species.

The authors conclude that the target intake of protein to non-protein energy (in this case carbohydrate) (P:C) falls over developmental stages and that this occurs because of a decline in mass-specific intake of protein whereas mass-specific carbohydrate intake remains more constant. The decrease in mass-specific protein consumption rate is tightly correlated with a decline in specific growth rate. Hence, protein consumption directly reflects requirements for growth, with hypometric scaling of protein intake serving as a useful relationship in nutritional ecology.

The laboratory experiments on the locust, Schistocerca cancellata, provide an elegant dataset in which different instars have been provided with one of two nutritionally complementary food pairings differing in protein to carbohydrate (P: C) content, and their self-selected protein to carbohydrate "intake target" measured.

These lab locust results were then compared with independently collected field data for late instar nymphs of the same locust species, and the conclusion is drawn that field insects ingested similar protein but 50-90% more carbohydrate (with only 23% increased mass-specific resting oxygen consumption rates). Numerous uncontrolled variables between the lab and field studies make meaningful conclusions difficult to draw from this observation.

A graph is then provided showing comparative data across a selection of species, making the case that protein consumption scales similarly both developmentally and across taxa. Questions need to be addressed for this to be convincing, including which criteria were used to select the examples in the graph and how comprehensively do these represent the available literature.

Reviewer #3 (Public Review):

The main goal of this study was to test how and why the intake of two important macronutrients ‒protein and carbon‒ often changes with ontogeny and body size. To do this, authors examined protein and carbon intake in a locusts lab population, across each instar and adult stages. Then, authors examined how the optimal balance of carbon and protein intake in a wild locusts population corresponded to that observed in the laboratory population. Results of these experiments showed that with ontogenic growth, locust decreased protein while increasing carbohydrate intake. Authors concluded that such decrease in the protein: carbohydrate intake may result from reductions in specific growth rates (growth within each instar). The protein: carbohydrate intake in the lab population appeared to be consistent with that observed in a wild locust population. Finally, authors combined their data with that from the literature to examine how protein intake scales with body mass throughout development, within and across different species.

Strengths:<br /> To determine how locusts balance protein: carbohydrate intake, authors applied the Geometric Framework (GF) of nutrition, which is a powerful approach for studying effects of nutrition and understanding the rules of compromise associated with balancing dietary unbalances.

Captivity can change behavior and physiology of most organisms, making it difficult to establish the relevance of laboratory experiments to what happens in the real world. A strength of this paper is that it compares behavior/physiology of lab vs. wild locusts. Finally, this study takes a step further by proposing a new scaling rule based on this study's results and data from the literature on various species.

Weaknesses:<br /> Although the paper has strengths, there seems to be several methodological issues that obscure the interpretation/conclusions presented in the manuscript.<br /> It appears that authors are not actually estimating "Intake Targets", as stated throughout the manuscript. According to the geometric framework, the intake target (IT) is estimated as the point in the nutritional landscape under which performance/fitness is optimized. The geometric framework also predicts that animals can reach their intake targets by feeding selectivity when given a choice of diets that differ in nutrient amounts, which is what authors did here. However, because the relationship between fitness/performance with diet was not established, in the choice experiments authors seem to be assuming (but not testing) that locusts are reaching their intake target.

You estimated a mass-specific protein intake for each instar. It is not clear why mass-specific intake and not just intake of protein was used for analysis. While mass (or size) of an individual may influence food consumption, it seems like authors calculated mass-specific consumption using each instar's final mass, which would make mass a result of protein consumption (and not the opposite). Importantly, the comparison between mass-specific protein consumption and specific growth rate may be problematic, as both variables seem to be estimated using final mass.

Reviewer #1 (Public Review):

Summary:

Marshall and coworkers describe the effects of altering metabotropic glutamate receptor 5 activity on locomotion and related activity of D1 receptor expressing spiny projection neurons in dorsolateral striatum. The authors also examine effects of dSPN-specific constitutive mGlu5 deletion in several motor tests. Effects of inhibiting the degradation of the endocannabinoid 2-arachidonoyl glycerol are also examined. Overall, this study provides intriguing new information with relevance to movement disorders and possibly psychosis. However, there are questions about the interpretation of dSPN activity in relation to movement, as well as the analysis approach. Some aspects of the study are also incomplete.

Strengths:

A nice combination of in vivo cellular calcium imaging, pharmacology, receptor knockout and sophisticated movement analysis are used. The authors conclude that mGlu5 expressed in dSPNs contributes to movement through effects on clustered spatial coactivity of dSPNs. Some data suggesting the story may be different in the other major SPN subpopulation (iSPNs) are also presented. The authors also suggest that mGlu5 stimulation of endocannabinoid signaling may play a role in the receptor effects. Overall, this study provides intriguing new information with relevance to movement disorders and possibly psychosis

Weaknesses:

Major Comments:

(1) The relationship between coactivity and movement in this and the previous study from this group is intriguing. Can the authors offer a hypothesis as to how decreased coactivity promotes increased movement velocity (e.g. as indicated by Figures 2l and 3m, and in the previous study)? Is coactivity during rest part of a "movement preparation" SPN program, or is it simply the case that the actual activity of individual dSPNs starts to contribute to different aspects of movement as velocity increases (given that the majority of neurons appear to show increased event rate during movement).

(2) The authors focus on dSPNs until very late in the study and then provide a little intriguing data suggesting that iSPNs show no difference in coactivity in the mGlu5 cKO mice. However, the basic characterization of the relationship between iSPN coactivity and movement is missing, although Figure 5g does seem to suggest a relationship between coactivity and proximity similar to dSPNs. It would be helpful to include the type of analysis shown in Figure 1 for iMSNs.

(3) The use of the Jaccard similarity index in this study is not intuitive and not fully explained by the methods or the diagram in Figure 1. The more detailed explanations in the previous papers from this group seem to indicate cells are listed as "coactive" if they both show an above-threshold fluorescence increase during a one second time frame after converting signals to a binary "on" or "off" status. However, it seems unlikely that the activity of the neurons would be perfectly or even strongly correlated, as there is bound to be variability in the exact traces from cell to cell. Furthermore, it doesn't seem clear how many frames need to show suprathreshold signals for two neurons to be considered coactive (or does this determine the magnitude of the normalized coactivity y-axis, e.g. in Figure 1i). Thus, while the technique appears to capture some index of coactivity, it does not appear to reveal the true temporal correlations in activity that could be obtained with techniques that use all data points to assess correlations. While this technique may be well suited to determining coactivity based on action potentials, or another all-or-none type biological event, it may not be as optimal for relating calcium transients that have more nuanced features.<br /> Another question is how the one second time frame was chosen. Did the authors run a sensitivity analysis to determine the effect of changing the frame duration on coactivity estimates. This might help determine if the analysis was too conservative in identifying coactive neurons.<br /> These comments may reflect a lack of understanding of the approach on the part of this reviewer. Perhaps a more detailed explanation of the method, maybe including examples of the types of calcium transients that are listed as reflecting coactivity or lack thereof, would clarify the suitability of this technique.

(4) The analysis of a possible 2-AG role in the mGlu5 mediated processes is incomplete and does not add much to the story. As the authors admit, inhibiting MGL globally will have widespread effects on many striatal synapses. Perhaps a dSPN-targeted approach, such as knocking out DAG lipase in dSPNs, would be more informative. For example, one might expect that this knockout would prevent the effects of the JNJ mGlu5 PAM on both movement and dSPN activity. The authors also do not provide any evidence of 2-AG involvement in the synaptic changes they report, although admittedly the role of endocannabinoids in DHPG-induced synaptic depression has been reported in several previous studies.

(5) It would seem to be a simple experiment to examine effects of the mGlu5 NAM in the dSPN mGlu5 cKO mice. If effects of the two manipulations occluded one another this would certainly support the hypothesis that the drug effects are mediated by receptors expressed in dSPNs. A similar argument can be made for examining effects of the JNJ PAM in the cKO mice.

Minor Comments:

(i) The use of CsF-based whole-cell internal solutions has caused concern in some past studies due to possible interference with G-protein, phosphatase and channel function (https://www.sciencedirect.com/science/article/abs/pii/S1044743104000296, https://www.jneurosci.org/content/jneuro/6/10/2915.full.pdf). It is reassuring the DHPG-induced LTD was still observable with this solution. However, it might be worth examining this plasticity with a different internal to ensure that the magnitude of the agonist effect is not altered by this manipulation.

(ii) The Kreitzer and Malenka 2007 paper may not be the best to cite in the context of dSPN-related synaptic plasticity, as these authors claimed that DHPG-induced LTD was restricted to iSPNs (an observation that has not generally been supported by subsequent work in several laboratories).

Reviewer #2 (Public Review):

Strengths are that the topic is of significant interest and understudied and the combination of both genetic and pharmacological approaches. However, while there is great enthusiasm for the need to better understand mGluR5 roles in striatal circuitry, in its present form, there are three overarching concerns that significantly limit the impact of this study. First, while a Jaccard method is used to measure the spatiotemporal dynamics of dSPN activity, collectively the data herein do not support the authors' interpretation of the data that mGluR5 is a modulator of spatiotemporal dSPN dynamics. Specifically, pharmacological and genetic manipulations of mGluR5 do not differentially/preferentially modulate the activity of proximal vs distal dSPNs, therefore, it could also be interpreted that mGluR5 is blanketly boosting/suppressing all dSPN activity as opposed to differential proximal/distal spatial relationships. While this is acknowledged in the manuscript (Figure 2i), it leaves open for question the extent to which mGluR5 is modulating other aspects of dSPN activity independent of the spatiotemporal relationship across dSPNs (i.e. amplitude, firing probability, etc.). Second, while it is a strength that mGluR5 NAM, PAM, and D1 Cre mGluR5-cKO were used to bidirectionally manipulate mGluR5 signaling, the manuscript lacks a clear model of where mGluR5 is acting to affect dSPN activity. This concern can be readily addressed by treating D1 Cre mGluR5-cKO mice with the mGluR5 NAM (as described in Ln. 413-416) to determine the extent to which other sources of mGluR5 are contributing to dSPN activity. The authors' working model predicts that the NAM would have no significant effects on the D1 Grm5 cKO model. Third, there are some concerns about the statistical basis for conclusions that are drawn detailed below that when addressed will strengthen the rigor of the conclusions. Addressing these suggestions should strengthen the mechanistic understanding and further allow the authors to present a more clear working model for their findings.

Reviewer #3 (Public Review):

Summary:

The manuscript by Marshall et al. investigates the role mGluR5 in modulating the coactivity of d1 spiny projection neurons (dSPN) in the dorsolateral striatum through calcium imaging and pharmacological i.p. injections or targeted deletion of mGluR5 in dSPNs. They show a bidirectional modulation by negative and positive allosteric modulators respectively (mainly at rest) on dSPN coactivity, the increase in coactivity by the negative modulator showed qualitative similar effects on coactivity as the deletion of mGluR5 in dSPNs.

Strengths:

Overall the study is well written and easy to read, with the data supporting (most of the time) the conclusion. It brings a new perspective on the role of mGluR5 in the modulation of dSPNs coactivity and its correlation with movement.

Weaknesses:

Some of the experiments would strengthen the solidness of the study providing further information and verifying the claims of the main text with the statistics on the figure legends.

Reviewer #1 (Public Review):

The authors design an automated 24-well Barnes maze with 2 orienting cues inside the maze, then model what strategies the mice use to reach the goal location across multiple days of learning. They consider a set of models and conclude that the animals begin with a large proportion of random choices (choices irrespective of the goal location), which over days of experience becomes a combination of spatial choices (choices targeted around the goal location) and serial choices (successive stepwise choices in a given direction). Moreover, the authors show that after the animal has many days of experience in the maze, they still often began each trial with a random choice, followed by spatial or serial choices.

This study is written concisely and the results are presented concisely. The best fit model provides valuable insight into how the animals solve this task, and therefore offers a quantitative foundation upon which tests of neural mechanisms of the components of the behavioral strategy can be performed. These tests will also benefit from the automated nature of the task.

Reviewer #2 (Public Review):

This paper uses a novel maze design to explore mouse navigation behaviour in an automated analogue of the Barnes maze. A major strength is the novel and clever experimental design which rotates the floor and intramaze cues before the start of each new trial, allowing the previous goal location to become the next starting position. The modelling sampling a Markov chain of navigation strategies is elegant, appropriate and solid, appearing to capture the behavioural data well. This work provides a valuable contribution and I'm excited to see further developments, such as neural correlates of the different strategies and switches between them.

Reviewer #3 (Public Review):

The development of an automated Barnes maze allows for more naturalistic and uninterrupted behavior, facilitating the study of spatial learning and memory, as well as the analysis of the brain's neural networks during behavior when combined with neurophysiological techniques. The system's design has been thoughtfully considered, encompassing numerous intricate details. These details include the incorporation of flexible options for selecting start, goal, and proximal landmark positions, the inclusion of a rotating platform to prevent the accumulation of olfactory cues, and careful attention given to atomization, taking into account specific considerations such as the rotation of the maze without causing wire shortage or breakage. When combined with neurophysiological manipulations or recordings, the system provides a powerful tool for studying spatial navigation system.

The behavioral experiment protocols, along with the analysis of animal behavior, are conducted with care, and the development of behavioral modeling to capture the animal's search strategy is thoughtfully executed. It is intriguing to observe how the integration of these innovative stochastic models can elucidate the evolution of mice's search strategy within a variant of the Barnes maze.

Reviewer #1 (Public Review):

Summary:

The evolution of transporter specificity is currently unclear. Did solute carrier systems evolve independently in response to a cellular need to transport a specific metabolite in combination with a specific ion or counter metabolite, or did they evolve specificity from an ancestral protein that could transport and counter transport most metabolites. The present study addresses this question by applying selective pressure to Saccharomyces cerevisiae and studying the mutational landscape of two well characterised amino acid transporters. The data suggest that AA transporters likely evolved from an ancestral transporter and then specific sub families evolved specificity depending on specific evolutionary pressure.

Strengths:

The work is based on sound logic and the experimental methodology is well thought through. The data appear accurate, and where ambiguity is observed (as in the case of citruline uptake by AGP1), in vitro transport assays are carried out. to verify transport function.

Weaknesses:

The revisions have substantially strengthened the conclusions based on the results of this study. Follow up studies will no doubt try to rationalise/identify if specific mutational hot-spots exist within the APC fold that explain the specialisation observed in mammals (neurotransmitter vs. metabolic) for example.

Reviewer #2 (Public Review):

Summary:

This paper describes evolution experiments performed on yeast amino acid transporters aiming at the enlargement of the substrate range of these proteins. Yeast cells lacking 10 endogenous amino acid transporters and thus being strongly impaired to feed on amino acids were again complemented with amino acid transporters from yeast and grown on media with amino acids as the sole nitrogen source.

In the first set of experiments, complementation was done with seven different yeast amino acid transporters, followed by measuring growth rates. Despite most of them having been described before in other experimental contexts, the authors show that many of them have a broader substrate range than initially thought.

Moving to the evolution experiments, the authors used the OrthoRep system to perform random mutagenesis of the transporter gene while it is actively expressed in yeast. The evolution experiments were conducted such that the medium would allow for poor/slow growth of cells expressing the wt transporters, but much better/faster growth if the amino acid transporter would mutate to efficiently take up a poorly transported (as in case of citrulline and AGP1) or non-transported (as in case of Asp/Glu and PUT4) amino acid.

This way and using Sanger sequencing of plasmids isolated from faster-growing clones, the authors identified a number of mutations that were repeatedly present in biological replicates. When these mutations were re-introduced into the transporter using site-directed mutagenesis, faster growth on the said amino acids was confirmed. Growth phenotype were confirmed by uptake experiments using radioactive amino acids; corresponding correlation plots show that the assays based on growth rates versus radioactive uptake assays indeed can explain the effect of the mutations to a large extent.

When mapped to Alphafold prediction models on the transporters, the mutations mapped to the substrate permeation site, which suggests that the changes allow for more favorable molecular interactions with the newly transported amino acids.<br /> Finally, the authors compared growth rates of the evolved transporter variants with those of the wt transporter and found that some variants exhibit a somewhat diminished capacity to transport its original range of amino acids, while other variants were as fit as the wt transporter in terms of uptake of its original range of amino acids.<br /> Based on these findings, the author conclude that transporters can evolve novel substrates through generalist intermediates, either by increasing a weak activity or by establishing a new one.

Strengths:

The study provides evidence in favour of an evolutionary model, wherein a transporter can "learn" to translocate novel substrates without "forgetting" what it used to transport before. This evolutionary concept has been proposed for enzymes before, and this study shows that it also can apply to transporters. The concept behind the study is easy to understand, i.e. improving growth by uptake of more amino acids as nitrogen source. In addition, the study contains a large and extensive characterization of the transporter variants, including growth assays and radioactive uptake measurements. The authors performed experiments as part of the revision to show that the studied mutations do not greatly change surface expression of the transporters. Further they showed that in the absence of the evolutionary pressure, overexpression of the mutants versus the wildtype transporters does not affect growth rates, which is important to assess. Finally, the authors make careful conclusions saying that in real life, the evolutionary landscape is way more complex than under these "reductive" laboratory conditions with a strain lacking ten natively expressed amino acid transporters and being selected on a single amino acid in a defined medium.

Weaknesses:

The authors took a genetic gain-of-function approach based on random mutagenesis of the transporter. While this experimental approach is suited to find some gain-of-function variants for some of the amino acids, it has also its inherent limitations, the most important being that loss-of-function mutants are not sampled (though they might be interesting) and that mutagenesis is entirely random, thus not targeted. These weaknesses cannot be easily overcome other than by restarting the entire study and conducting for example deep mutational scanning experiments. The authors have done what they could do within the scope of this study to make this manuscript as complete and rigorous as possible.

Reviewer #3 (Public Review):

The goal of the current manuscript is to investigate how changes in transporter substrate specificity emerge in response to a novel selective pressure. The authors investigate the APC family of amino acid transporters, a large family with many related transporters that together cover the spectrum of amino acid uptake in yeast.

The authors use a clever approach for their experimental evolutions. By deleting 10 amino acid uptake transporters in yeast, they develop a strain that relies on amino acid import by introduced APC transporters under nitrogen limiting conditions. They can thus evolve transporters towards transport of new substrates if no other nitrogen source is available. The main takeaway from the paper is that it is relatively easy for the spectrum of substrates in a particular transporter of this family to shift, as a number of single mutants are identified that modulate substrate specificity. In general, transporters evolved towards gain-of-function mutations (better or new activities) also confer transport promiscuity, expanding the range of amino acids transported.

The data in the paper support the conclusions, and the outcomes (evolution towards promiscuity) agree with the literature available for soluble enzymes. The authors do a good job in the discussion of relating the lessons of the current study to natural evolution.

Reviewer #1 (Public Review):

Summary:

Nitric oxide (NO) has been implicated as a neuromodulator in the retina. Specific types of amacrine cells (ACs) produce and release NO in a light-dependent manner. NO diffuses freely through the retina and can modulate intracellular levels of cGMP, or directly modify and modulate proteins via S-nitrosylation, leading to changes in gap-junction coupling, synaptic gain, and adaptation. Although these system-wide effects have been documented, it is not well understood how the physiological function of specific neuronal types is affected by NO. This study aims to address this gap in our knowledge.

Strengths:

NO was expected to produce small effects, and considerable effort was expended in validating the system to ensure that any effects of NO would not be confounded by changes in the state of the preparation. The authors used a paired stimulus protocol to control for changes in the sensitivity of the retina during the extended recording periods. The approach potentially increases the sensitivity of the measurements and allows more subtle effects to be observed.

Neural activity was initially measured by Ca-imaging. Responsive ganglion cells were grouped into 32 types using a clustering analysis. Initial control experiments demonstrated that the cell-types revealed here largely recapitulate those from their earlier landmark study using the same approach (Fig. 2).

Application of NO to the retina strongly modulated responses of a single cluster of cells, labeled G32, while having little effect on the remaining 31 clusters. This result is evident in Fig. 3e.

Separate experiments measured ganglion cell spiking activity on a multi-electrode array (MEA). Clustering analysis of the peri-stimulus spike-time histograms (PSTHs) obtained from the MEA data also revealed 32 clusters. The PSTHs for each cluster were aligned to the Ca-imaging data using a convolution approach. The higher temporal resolution of the MEA recordings indicated that NO increased the speed of sub-cluster 2 responses but had no effect on receptive field size. The physiological significance of the small change in kinetics remains unclear.

Weaknesses:

The G32 cluster was further divided into three sub-types using Bayesian Information Criterion (BIC) based on the temporal properties of the Ca-responses. This sub-clustering result seems questionable due to the small difference in the BIC parameter between 2 and 3 clusters. Three sub-clusters of the G32 cluster were also revealed for the PSTH data, however, the BIC analysis was not applied to further validate this result.

The alignment of sub-clusters 1, 2, and 3 identified in the Ca-imaging and the MEA recordings seemed questionable, because the temporal properties of clusters did not align well, nor did the effects of NO.

The title of the paper indicates that nitric oxide modulates contrast suppression in a subset of mouse retinal ganglion cells, however, this result appears to be inferred from previous results showing that G32 is identified as a "suppressed-by-contrast" cell. The present study does not explicitly evaluate the amount of contrast-suppression in G32 cells.

In its current form, the work is likely to have limited impact, since the morphological and functional properties of the affected sub-cluster remain unknown. The finding that there can be cell-specific adaptation effects during experiments on in vitro retina is important new information for the field.

Reviewer #2 (Public Review):

Neuromodulators are important for circuit function, but their roles in the retinal circuitry are poorly understood. This study by Gonschorek and colleagues aims to determine the modulatory effect of nitric oxide on the response properties of retinal ganglion cells. The authors used two photon calcium imaging and multi-electrode arrays to classify and compare cell responses before and after applying a NO donor DETA-NO. The authors found that DETA-NO selectively increases activity in a subset of contrast-suppressed RGC types. In addition, the authors found cell-type specific changes in light response in the absence of pharmacological manipulation in their calcium imaging paradigm. While this study focuses on an important question and the results are interesting, the following issues need further clarification for better interpretation of the data.

(1) Design of the calcium imaging experiments: the control-control pair has a different time course from the control-drug pair (Fig 1e). First, the control-control pair has a 10 minute interval while the control-drug pair has a 25 minute interval. Second, Control 1 Field 2 was imaged 10 min later than Control 1 Field 1 since the start of the calcium imaging paradigm.

Given that the control dataset is used to control for time-dependent adaptational changes throughout the experiment, I wonder why the authors did not use the same absolute starting time of imaging and the same interval between the first and second round of imaging for both the control-control and the control-drug pairs. This can be readily done in one of the two ways: 1. In a set of experiment, add DETA/NO between "Control 1 Field 1 and "Control 2 Field 1" in Fig. 1e as the drug group; or 2. Omit DETA/NO in the Fig. 1e protocol as the control group to monitor the time course of adaptational changes.

Related to the concern above, to determine NO-specific effect, the authors used the criterion that "the response changes observed for control (ΔR(Ctrl2−Ctrl1)) and NO (ΔR(NO−Ctrl1)) were significantly different". This criterion assumes that without DETA-NO, imaging data obtained at the time points of "Control 1 Field 2" and "DETA/NO Field 2" would give the same value of ΔR as ΔR(Ctrl2−Ctrl1) for all RGC types. It is not obvious to me why this should be the case, because of the unknown time-dependent trajectory of the adaptational change for each RGC type. For example, a RGC type could show stable response in the first 30 min and then change significantly in the following 30 min. DETA/NO may counteract this adaptational change, leading to the same ΔR as the control condition (false negative). Alternatively, DETA/NO may have no effect, but the nonlinear time-dependent response drift can give false positive results.

I also wonder why washing-out, a standard protocol for pharmacological experiments, was not done for the calcium protocol since it was done in the MEA experiments. A reversible effect by washing in and out DETA/NO in the calcium protocol would provide a much stronger support that the observed NO modulation is due to NO and not to other adaptive changes.

(2) Effects of Strychnine: In lines 215-219, " In the light-adapted retina, On-cone BCs boost light-Off responses in Off-cone BCs through cross-over inhibition (83, 84) and hence, strychnine affects Off-response components in RGCs - in line with our observations (Fig. S2)" However, Fig. S2 doesn't seem to show a difference in the Off-response components. Rather, the On response is enhanced with strychnine. In addition, suppressed-by-contrast cells are known to receive glycinergic inhibition from VGluT3 amacrine cells (Tien et al., 2016). However, the G32 cluster in Fig. S2 doesn't seem to show a change with strychnine. More explanation on these discrepancies will be helpful.

(3) This study uses DETA-NO as an NO donor for enhancing NO release. However, a previous study by Thompson et al., Br J Pharmacol. 2009 reported that DETA-NO can rapidly and reversible induce a cation current independent of NO release at the 100 uM used in the current study, which could potentially cause the observed effect in G32 cluster such as reduced contrast suppression and increased activity. This potential caveat should at least be discussed, and ideally excluded by showing the absence of DETA-NO effects in nNOS knockout mice, and/or by using another pharmacological reagent such as the NO donor SNAP or the nNOS inhibitor l-NAME.

(4) Clarification of methods: In the Methods, lines 1119-1127, the authors describe the detrending, baseline subtraction, and averaging. Then, line 1129, " the mean activity r(t) was computed and then traces were normalized such that: max t(|r(t)|) = 1. How is the normalization done? Is it over the entire recording (control and wash in) for each ROI? Or is it normalized based on the mean trace under each imaging session (i.e. twice for each imaging field)?

As for the clustering of RGC types, I assume that each ROI's cluster identity remains unchanged through the comparison. If so, it may be helpful to emphasize this in the text.

Reviewer #1 (Public Review):

Previous studies have used a randomly induced label to estimate the number of hematopoietic precursors that contribute to hematopoiesis. In particular, the McKinney-Freeman lab established a measurable range of precursors of 50-2500 cells using random induction of one of the 4 fluorescent proteins (FPs) of a Confetti reporter in the fetal liver to show that hundreds of precursors establish lifelong hematopoiesis. In the presented work, Liu and colleagues aim to extend the measurable range of precursor numbers previously established and enable measurement in a variety of contexts beyond embryonic development. To this end, the authors investigated whether the random induction of a given Confetti FP follows the principles of binomial distribution such that the variance inversely correlates with the precursor number. They tested their hypothesis using a simplified 2-color in vitro system, paying particular attention to minimizing sources of experimental error (elimination of outliers, sample size, events recorded, etc.) that may obscure the measurement of variance. As a result, the data generated are robust and show that the measurable range of precursors can be extended up to 105 cells. They use tamoxifen-inducible Scl-CreER, which is active in hematopoietic stem and progenitor cells (HSPCs) to induce Confetti labeling, and investigated whether they could extend their model to cell numbers below 50 with in vivo transplantation of high versus low numbers of Confetti total bone marrow (BM) cells. The premise of binomial distribution requires that the number of precursors remains constant within a group of mice. The rare frequency of HSPCs in the BM means that the experimentally generated "low" number recipient animals showed some small variability of seeding number, which does not follow the requirement for binomial distribution. While variance due to differences in precursor numbers still dominates, it is unclear how accurate estimated numbers are when precursor numbers are low (<10).

The authors then apply their model to estimate the number of hematopoietic precursors that contribute to hematopoiesis in a variety of contexts including adult steady state, fetal liver, following myeloablation, and a genetic model of Fanconi anemia. Their modeling shows:

-thousands of precursors (~2400-2600) contribute to adult myelopoiesis, which is in line with results from a previous study (Sun et al, 2014).<br /> -myeloablation (single dose 5-FU), while reducing precursor numbers of myeloid progenitors and HSPCs, was not associated with a reduction in precursor numbers of LT-HSCs.<br /> -no major expansion of precursor number in the fetal liver derived from labeling at E11.5 versus E14.5, consistent with recent findings from Ganuza et al, 2022.<br /> -normal precursor numbers in Fancc-/- mice at steady state and from competitive transplantation of young Fancc-/- BM cells, suggesting that reduced Fancc-/- cell proliferation may underlie the reduced chimerism upon transplantation.<br /> -reduced number of lymphoid precursors following transplantation of BM cells from 9-month-old Fancc-/- animals (beyond this age animals have decreased survival).

Although this system does not permit the tracing of individual clones, the modeling presented allows measurements of clonal activity covering nearly the entire HSPC population (as recently estimated by Cosgrove et al, 2021) and can be applied to a wide range of in vivo contexts with relative ease. The conclusions are generally sound and based on high-quality data. Nevertheless, some results could benefit from further explanation or discussion:

-The estimated number of LT-HSCs that contribute to myelopoiesis is not specifically provided, but from the text, it would be calculated to be 1958/5 = ~391. Data from Busch et al, 2015 suggest that the number of differentiation-active HSCs is 5.2x103, which is considered the maximum limit. There is nevertheless a more than 10-fold difference between these two estimates, and it is unclear how this discrepancy arises.<br /> -Similarly, in Figure 3E, the estimated number of precursors is highest in MPP4, a population typically associated with lymphoid potential and transient myeloid potential, whereas the numbers of MPP3, traditionally associated with myeloid potential, tend to be higher but are not significantly different than those found in HSCs.<br /> -The requirement for estimating precursor numbers at stable levels of Confetti labeling is not well explained. As a result, it is unclear how accurate the estimates of B cell precursors upon transplantation of Fancc-/- cells are. In previous experiments on normal Confetti mice (Figure 3B), the authors do not estimate precursors of lymphopoiesis because Confetti labeling of B cells is not saturated, and this appears to be the case in Fanc-/- animals as well (Fig. 5B).<br /> -Do 9-month-old Fanc-/- animals have reduced lymphoid precursors as well?

Reviewer #2 (Public Review):

Summary:

This manuscript by Liu et al. uses Confetti labeling of hematopoietic stem and progenitor cells in situ to infer the clonal dynamics of adult hematopoiesis. The authors apply a new mathematical framework to analyze the data, allowing them to increase the range of applicability of this tool up to tens of thousands of precursors. With this tool, they (1) provide evidence for the large polyclonality of adult hematopoiesis, (2) offer insights on the expansion dynamics in the fetal liver stage, (3) assess the clonal dynamics in a Fanconi anemia model (Fancc), which has engraftment defects during transplantation.

Strengths:

The manuscript is well written, with beautiful and clear figures, and both methods and mathematical models are clear and easy to understand.

Since 2017, Mikel Ganuza and Shannon McKinney-Freeman have been using these Confetti approaches that rely on calculating the variance across independent biological replicates as a way to infer clonal dynamics. This is a powerful tool and it is a pleasure to see it being implemented in more labs around the world. One of the cool novelties of the current manuscript is using a mathematical model (based on a binomial distribution) to avoid directly regressing the Confetti labeling variance with the number of clones (which only has linearity for a small range of clone numbers). As a result, this current manuscript of Liu et al. methodologically extends the usability of the Confetti approach, allowing them more precise and robust quantification.

They then use this model to revisit some questions from various Ganuza et al. papers, validating most of their conclusions. The application to the clonal dynamics of hematopoiesis in a model of Fanconi anemia (Fancc mice) is very much another novel aspect, and shows the surprising result that clonal dynamics are remarkably similar to the wild-type (in spite of the defect that these Fancc HSCs have during engraftment).<br /> Overall, the manuscript succeeds at what it proposes to do, stretching out the possibilities of this Confetti model, which I believe will be useful for the entire community of stem cell biologists, and possibly make these assays available to other stem cell regenerating systems.

Weaknesses:

My main concern with this work is the choice of CreER driver line, which then relates to some of the conclusions made. Scl-CreER succeeds at being as homogenous as possible in labeling HSC/MPPs... however it is clear that it also labels a subcompartment of HSC clones that become dominant with time... This is seen as the percentage of Confetti-recombined cells never ceases to increase during the 9-month chase of labeled cells, suggesting that non-labeled cells are being replaced by labeled cells. The reason why this is important is that then one cannot really make conclusions about the clonal dynamics of the unlabeled cells (e.g. for estimating the total number of clones, etc.).

I am not sure about the claims that the data shows little precursor expansion from E11 to E14. First, these experiments are done with fewer than 5 replicates, and thus they have much higher error, which is particularly concerning for distinguishing differences of such a small number of clones. Second, the authors do see a ~0.5-1 log difference between E11 and E14 (when looking at months 2-3). When looking at months 5+, there is already a clear decline in the total number of clones in both adult-labeled and embryonic-labeled, so these time points are not as good for estimating the embryonic expansion. In any case, the number of precursors at E11 (which in the end defines the degree of expansion) is always overestimated (and thus, the expansion underestimated) due to the effects of lingering tamoxifen after injection (which continues to cause Confetti allele recombination as stem cell divide). Thus, I think these results are still compatible with expansion in the fetal liver (the degree of which still remains uncertain to me).

Reviewer #3 (Public Review):

Summary:

Liu et al. focus on a mathematical method to quantify active hematopoietic precursors in mice using Confetti reporter mice combined with Cre-lox technology. The paper explores the hematopoietic dynamics in various scenarios, including homeostasis, myeloablation with 5-fluorouracil, Fanconi anemia (FA), and post-transplant environments. The key findings and strengths of the paper include (1) precursor quantification: The study develops a method based on the binomial distribution of fluorescent protein expression to estimate precursor numbers. This method is validated across a wide dynamic range, proving more reliable than previous approaches that suffered from limited range and high variance outside this range; (2) dynamic response analysis: The paper examines how hematopoietic precursors respond to myeloablation and transplantation; (3) application in disease models: The method is applied to the FA mouse model, revealing that these mice maintain normal precursor numbers under steady-state conditions and post-transplantation, which challenges some assumptions about FA pathology. Despite the normal precursor count, a diminished repopulation capability suggests other factors at play, possibly related to cell proliferation or other cellular dysfunctions. In addition, the FA mouse model showed a reduction in active lymphoid precursors post-transplantation, contributing to decreased repopulation capacity as the mice aged. The authors are aware of the limitation of the assumption of uniform expansion. The paper assumes a uniform expansion from active precursor to progenies for quantifying precursor numbers. This assumption may not hold in all biological scenarios, especially in disease states where hematopoietic dynamics can be significantly altered. If non-uniformity is high, this could affect the accuracy of the quantification. Overall, the study underscores the importance of precise quantification of hematopoietic precursors in understanding both normal and pathological states in hematopoiesis, presenting a robust tool that could significantly enhance research in hematopoietic disorders and therapy development. The following concerns should be addressed.

Major Points:

• The authors have shown a wide range of seeded cells (1 to 1e5) (Figure 1D) that follow the linear binomial rule. As the standard deviation converges eventually with more seeded cells, the authors need to address this limitation by seeding the number of cells at which the assumption fails.<br /> • Line 276: This suggests myelopoiesis is preferred when very few precursors are available after irradiation-mediated injury. Did the authors see more myeloid progenitors at 1 month post-transplantation with low precursor number? The authors need to show this data in a supplement.

Minor Points:

• Please cite a reference for line 40: a rare case where a single HSPC clone supports hematopoiesis.<br /> • Line 262-263: "This discrepancy may reflect uneven seeding of precursors to the BM throughout the body after transplantation and the fact that we only sampled a part of the BM (femur, tibia, and pelvis)." Consider citing this paper (https://doi.org/10.1016/j.cell.2023.09.019) that explores the HSPCs migration across different bones.<br /> • Lines 299 and 304. Misspellings of RFP.<br /> • The title is misleading as the paper's main focus is the precursor number estimator using the binomial nature of fluorescent tagging. Using a single-copy cassette of Confetti mice cannot be used to measure clonality.

Reviewer #1 (Public Review):

Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animals to guide a genuine navigational task. The sun and moon have long been celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on freely navigating ants 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern.

The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show that nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.

Reviewer #2 (Public Review):

Summary:

The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

Strengths:

The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

Weaknesses:

The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be reworked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.

Impact:

The authors have discovered that nocturnal bull ants while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this is the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

Reviewer #3 (Public Review):

Summary:

This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.

Strengths:

The study was conducted carefully and is clearly explained here.

Weaknesses:

I have only a few comments and suggestions, that I hope will make the manuscript clearer and easier to understand.

Time compensation or periodic snapshots

In the introduction, the authors compare their discovery with that in dung beetles, which have only been observed to use lunar skylight to hold their course, not to travel to a specific location as the ants must. It is not entirely clear from the discussion whether the authors are suggesting that the ants navigate home by using a time-compensated lunar compass, or that they update their polarization compass with reference to other cues as the pattern of lunar skylight gradually shifts over the course of the night - though in the discussion they appear to lean towards the latter without addressing the former. Any clues in this direction might help us understand how ants adapted to navigate using solar skylight polarization might adapt use to lunar skylight polarization and account for its different schedule. I would guess that the waxing and waning moon data can be interpreted to this effect.

Effects of moon fullness and phase on precision

As well as the noted effect on shift magnitudes, the distributions of exit headings and reorientations also appear to differ in their precision (i.e., mean vector length) across moon phases, with somewhat shorter vectors for smaller fractions of the moon illuminated. Although these distributions are a composite of the two distributions of angles subtracted from one another to obtain these turn angles, the precision of the resulting distribution should be proportional to the original distributions. It would be interesting to know whether these differences result from poorer overall orientation precision, or more variability in reorientation, on quarter moon and crescent moon nights, and to what extent this might be attributed to sky brightness or degree of polarization.

N.B. The Watson-Williams tests for difference in mean angle are also sensitive to differences in sample variance. This can be ruled out with another variety of the test, also proposed by Watson and Williams, to check for unequal variances, for which the F statistic is = (n2-1)*(n1-R1) / (n1-1)*(n2-R2) or its inverse, whichever is >1.

Reviewer #1 (Public Review):

Summary:

Hartman and Satija's manuscript constitutes a significant contribution to the field of imaging-based spatial transcriptomics (ST) through their comprehensive comparative analysis of six multiplexed in situ gene expression profiling technologies. Their findings provide invaluable insights into the practical considerations and performance of these methods, offering robust evidence for researchers seeking optimal ST technologies. However, given the simultaneous availability of similar preprints, readers should exercise caution when comparing findings to ensure reliable information. Therefore, the authors should revise their manuscript to ensure consistency among all ST technologies compared, considering findings from other preprints as well if possible.

Strengths:

(1) The manuscript offers a comprehensive and systematic comparison of six in situ gene expression profiling technologies, including both commercially available and academically developed methods, which is the most extensive study in this field.

(2) Novel metrics have been proposed by the authors to mitigate molecular artifacts and off-target signals, enhancing the accuracy of sensitivity and specificity comparisons across datasets. By emphasizing the significance of evaluating both sensitivity and specificity, the study addresses the challenge of comparing standard metrics like the number of unique molecules detected per cell, given variations in panel composition and off-target molecular artifacts. This feature is directly connected to their development of novel cell segmentation methods to improve the specificity.

(3) As a result of the analysis performed earlier, the authors illustrate how molecular false positives can distort spatially-aware differential expression analysis, underscoring the necessity for caution in interpreting downstream results.

(4) Offering guidance for the selection, processing, and interpretation of in situ spatial technologies, the study equips researchers in the field with valuable insights.

Weaknesses:

(1) Although focusing on mouse brain datasets broadens the comparison of technologies, it confines the study to a single biological context. Discussing the potential limitations of this approach and advocating for future studies in diverse tissue types would enrich the manuscript, especially for clinical FFPE applications.

(2) Providing more explicit details on the criteria used to select datasets for each technology would ensure a fair and unbiased comparison. Otherwise, it may look like the Hall of Fame for champion data sets to advertise a certain commercial product.

(3) Improving the discussion part by discussing the origins of non-specific signals and molecular artifacts, alongside the challenges related to cell segmentation across different tissue types and cell morphologies, would enrich its content. Note that all of these experimental sets have been obtained from thin mouse brain slices, which are actually 3D although they are thin like 10-20 um. As a result, there might be a chance to have partial cell overlap in the z-axis, potentially leading to transcript mixing. Additionally, many cells are probably cut so their actual transcriptomes are inherently partial information, which makes direct comparison to scRNA-seq unfair. These aspects should be included for fair comparison issues.

(4) Expanding on the potential implications of the findings for developing new computational methods to address non-specific biases in downstream analyses would augment the manuscript's impact and relevance.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Hartman et al. present a detailed comparison of 6 distinct multiplexed in situ gene expression profiling technologies, including both academic and commercial systems.

The main concept of the study is to evaluate publicly accessible mouse brain datasets provided by the platforms' developers, where optimal performance in showcasing their technologies is expected. The authors stress the difficulty of making a comparison with standard metrics, e.g., the count of total molecules per cell, considering the differences in gene panel sizes across platforms. To make a fair comparison, the authors conceived a metric of specificity performance, which is called "MECR", an average of mutually exclusive gene co-expression rates in the sample. The authors found that the rate mainly depends on the choice of cell segmentation method, thus reanalyzed 5 of these datasets (excluding STARmap PLUS, due to the lack of molecule location information) with an independent cell segmentation algorithm (i.e., Baysor). Based on the reanalysis, the authors clearly suggest the best-performing platform at the end of the manuscript.

Strengths:

I consider that the paper is a valuable contribution to the community, for the following two reasons:

(1) As the authors mentioned, I fully agree that the spatial transcriptomics community indeed needs better metrics in terms of comparison across technologies, rather than traditional metrics, e.g., molecule counts per cell. In that regard, I believe introducing a new metric, MECR, is quite valuable.

(2) This work highlights the differences in results based on the choice of cell segmentation used for each platform, which suggests a need for trying out different segmentation algorithms to derive the right results. I believe this is an urgent warning that should be widespread in the community as soon as possible.

Weaknesses:

I disagree with the conclusion of the manuscript where the authors compare the technologies and suggest the best-performing ones, because of the following major points:

(1) As the authors mentioned, MECR is a measure of "specificity" not "sensitivity". Still, the comparison of sensitivity was done with the mean counts per cell (Figure 3e). However, I strongly disagree with using the mean counts per cell as a measure of sensitivity because the comparison was done with different gene panels. The counts per cell can be highly dependent on the choice of genes, especially due to optical crowding.

(2) The authors compared sensitivity based on the Baysor cell segmentation, but in fact, Baysor uses spatial gene expression for cell segmentation, which depends on the sensitivity of the platform. Thus, a comparison of sensitivity based on an algorithm that is based on sensitivity seems to be nonsensical.

Reviewer #3 (Public Review):

This paper concerns whether synaptic scaling (or homeostatic synaptic plasticity; HSP) occurs similarly at GABA and Glu synapses and comes to the surprising conclusion that these can be regulated independently. In fact, under the conditions used in this study, only the GABAergic synapses show HSP and the glutamatergic synapses don't change. This is surprising because these were thought to be co-regulated during HSP and in fact, the major mechanisms thought to underlie downscaling (TTX or CNQX driven), retinoic acid and TNF, have been shown to regulate both GABARs and AMPARs directly. Thus, the main result, that GABA HSP is dissociable from Glu HSP, is novel and exciting. This suggests either different mechanisms underlie the two processes, or that under certain conditions, another mechanism is engaged that scales one type of synapse and not the other. Given that glutamatergic synapses are unchanged in their conditions, that later seems more likely - a novel form of HSP exists that only scale GABAergic synapses. Whether glutamatergic and GABAergic synapses scale independently during HSP affecting both types of synapses remains to be addressed. It would be necessary to demonstrate the dissociation in the same system, under conditions where both types of synapses are changing. But because the form of HSP studied here appears different than that studied in Fong et al., the authors should be careful when comparing the two results. There seems to be an implicit underlying assumption that there is a simple form of HSP, when the overall literature (and the two studies from this lab) supports the idea of many forms of HSP.

The homeostatic changes at GABAergic synapses do seem to be more consistent in amplitude across the bulk of the synapses, which does suggest that true scaling (a proportional change to all synapses on a cell) is occurring. This may represent a major difference in how homeostatic changes occur at the two types of synapses.

The second finding is that this form of HSP seems more regulated by action potential firing than conventional HSP - previous work from this lab had shown that restoring AP firing during AMPA receptor blockade did not prevent scaling of glutamatergic synapses (it should be noted these experiments were done in rat cultures, not mouse, used a higher concentration of CNQX, and used a different optogenetic stimulation paradigm). Restoring AP firing rates under the conditions used here (and thus the form of HSP only affecting GABA synapses), on the other hand, did prevent the homeostatic response. This suggests that this GABA-only form of HSP is more attuned to spiking rates than other forms.

However, details in the data may suggest that spiking is not the (or the only) homeostat, as TTX and CNQX causes identical changes in mIPSC amplitude but have different effects on spiking (although TTX may be driving a different form of HSP). Further, in Fig 5, CTZ had a minimal effect on spiking but a large effect on mIPSCs. Similar issues appear in Fig 6, where the induction of increased spiking is highly variable, with many cells showing control levels or lower spiking rates. Yet the synaptic changes are robust, across all cells. Overall, more will need to be done to conclude that spiking is the homeostat for GABA synapses.

The paper also suggests that the GABA changes are leading to the recovery of the spiking rates, but while they have the time course of the spiking changes and recovery, they only have the 24h time point for synaptic changes. It is not yet possible to conclude how the time courses align without more data, nor can we assume that cells that did not recover to control firing rates would do so eventually.

Reviewer #1 (Public Review):

The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.

Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:

(1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.

(2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.

Here, the authors' goal is to directly test if the auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.

First the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.

The authors have revised their manuscript and adequately addressed the major concerns. Although more in depth analyses of these rich datasets are definitely possible, the current results nevertheless stand on their own. Indeed, the work serves as a beacon to move away from the idea that cortico-collicular projections function primarily to impart behavioral relevance upon auditory midbrain neurons. This knowledge inspires a search for alternative explanations as to the role of auditory cortico-collicular synapses in behavior.

Reviewer #2 (Public Review):

Summary:

This study takes a new approach to studying the role of corticofugal projections from auditory cortex to inferior colliculus. The authors performed two-photon imaging of cortico-recipient IC neurons during a click detection task in mice with and without lesions of auditory cortex. In both groups of animals, they observed similar task performance and relatively small differences in the encoding of task-response variables in the IC population. They conclude that non-cortical inputs to the IC can provide substantial task-related modulation, at least when AC is absent.

Strengths:

This study provides valuable new insight into big and challenging questions around top-down modulation of activity in the IC. The approach here is novel and appears to have been executed thoughtfully. Thus, it should be of interest to the community.

Weaknesses:

Analysis of single unit activity is limited in its scope.

Reviewer #3 (Public Review):

Summary:

This study aims to demonstrate that cortical feedback is not necessary to signal behavioral outcome to shell neurons of the inferior colliculus during a sound detection task. The demonstration is achieved in a very clear manner by the observation of the activity of cortico-recepient neurons in animals which have received lesions of the auditory cortex. The experiment shows that neither behavior performance nor neuronal responses are significantly impacted by cortical lesions except for the case of partial lesions which seem to have a disruptive effect on behavioral outcome signaling.

Strengths:

The demonstration of the main conclusions is based on state-of-the-art, carefully controlled methods and is highly convincing. There is an in depth discussion of the different effects of auditory cortical lesions on sound detection behavior.

Weaknesses:

The description of feedback signals could be more detailed although it is difficult to achieve good temporal resolution with the calcium imaging technique necessary for targeting cortico-recipient neurons.

Reviewer #1 (Public Review):

Summary:

Through an unbiased genomewide KO screen, the authors identified loss of DBT to suppress MG132-mediated death of cultured RPE cells. Further analyses suggested that DBT reduces ubiquitinated proteins by promoting autophagy. Mechanistic studies indicated that DBT loss promotes autophagy via AMPK and its downstream ULK and mTOR signaling. Furthermore, loss of DBT suppresses polyglutamine- or TDP-43-mediated cytotoxicity and/or neurodegeneration in fly models. Finally, the authors showed that DBT proteins are increased in ALS patient tissues, compared to non-neurological controls.

Strengths:

The idea is novel, the evidence is convincing, and the data are clean. The findings have implications for human diseases.

Weaknesses:

None.

Reviewer #2 (Public Review):

Summary:

Hwang, Ran-Der et al utilized a CRISPR-Cas9 knockout in human retinal pigment epithelium (RPE1) cells to evaluate for suppressors of toxicity by the proteasome inhibitor MG132 and identified that knockout of dihydrolipoamide branched chain transacylase E2 (DBT) suppressed cell death. They show that DBT knockout in RPE1 cells does not alter proteasome or autophagy function at baseline. However, with MG132 treatment, they show a reduction in ubiquitinated proteins but with no change in proteasome function. Instead, they show that DBT knockout cells treated with MG132 have improved autophagy flux compared to wildtype cells treated with MG132. They show that MG132 treatment decreases ATP/ADP ratios to a greater extent in DBT knockout cells, and in accordance causes activation of AMPK. They then show downstream altered autophagy signaling in DBT knockout cells treated with MG132 compared to wild-type cells treated with MG132. Then they express the ALS mutant TDP43 M337 or expanded polyglutamine repeats to model Huntington's disease and show that knockdown of DBT improves cell survival in RPE1 cells with improved autophagic flux. They also utilize a Drosophila models and show that utilizing either a RNAi or CRISPR-Cas9 knockout of DBT improves eye pigment in TDP43M337V and polyglutamine repeat-expressing transgenic flies. Finally, they show evidence for increased DBT in postmortem spinal cord tissue from patients with ALS via both immunoblotting and immunofluorescence.

Strengths:

This is a mechanistic and well-designed paper that identifies DBT as a novel regulator of proteotoxicity via activating autophagy in the setting of proteasome inhibition. Major strengths include careful delineation of a mechanistic pathway to define how DBT is protective. These conclusions are well-justified.

Weaknesses:

None

Reviewer #1 (Public Review):

Summary:

The study introduces and validates the Cyclic Homogeneous Oscillation (CHO) detection method to precisely determine the duration, location, and fundamental frequency of non-sinusoidal neural oscillations. Traditional spectral analysis methods face challenges in distinguishing the fundamental frequency of non-sinusoidal oscillations from their harmonics, leading to potential inaccuracies. The authors implement an underexplored approach, using the auto-correlation structure to identify the characteristic frequency of an oscillation. By combining this strategy with existing time-frequency tools to identify when oscillations occur, the authors strive to solve outstanding challenges involving spurious harmonic peaks detected in time-frequency representations. Empirical tests using electrocorticographic (ECoG) and electroencephalographic (EEG) signals further support the efficacy of CHO in detecting neural oscillations.

Strengths:

The paper puts important emphasis on the 'identity' question of oscillatory identification. The field primarily identifies oscillations through frequency, space (brain region), and time (length, and relative to task or rest). However, more tools that claim to further characterize oscillations by their defining/identifying traits are needed, in addition to data-driven studies about what the identifiable traits of neural oscillations are beyond frequency, location, and time. Such tools are useful for potentially distinguishing between circuit mechanistic generators underlying signals that may not otherwise be distinguished. This paper states this problem well and puts forth a new type of objective for neural signal processing methods.

The paper uses synthetic data and multimodal recordings at multiple scales to validate the tool, suggesting CHO's robustness and applicability in various real-data scenarios. The figures illustratively demonstrate how CHO works on such synthetic and real examples, depicting in both time and frequency domains. The synthetic data are well-designed, and capable of producing transient oscillatory bursts with non-sinusoidal characteristics within 1/f noise. Using both non-invasive and invasive signals exposes CHO to conditions which may differ in the extent and quality of harmonic signal structure. An interesting follow-up question is whether the utility demonstrated here holds for MEG signals, as well as source-reconstructed signals from non-invasive recordings.

This study is accompanied by open-source code and data for use by the community.

Weaknesses:

The criteria that the authors use for neural oscillations embody some operating assumptions underlying their characteristics, perhaps informed by immediate use cases intended by the authors (e.g., hippocampal bursts). The extent to which these assumptions hold in all circumstances should be investigated. For instance, the notion of consistent auto-correlation breaks down in scenarios where instantaneous frequency fluctuates significantly at the scale of a few cycles. Imagine an alpha-beta complex without harmonics (Jones 2009). If oscillations change phase position within a timeframe of a few cycles, it would be difficult for a single peak in the auto-correlation structure to elucidate the complex time-varying peak frequency in a dynamic fashion. Likewise, it is unclear whether bounding boxes with a pre-specified overlap can capture complexes that manoeuvre across peak frequencies.

This method appears to lack the implementation of statistical inferential techniques for estimating and interpreting auto-correlation and spectral structure. In standard practice, auto-correlation functions and spectral measures can be subjected to statistical inference to establish confidence intervals, often helping to determine the significance of the estimates. Doing so would be useful for expressing the likelihood that an oscillation and its harmonic has the same auto-correlation structure and fundamental frequency, or more robustly identifying harmonic peaks in the presence of spectral noise. Here, the authors appear to use auto-correlation and time-frequency decomposition more as a deterministic tool rather than an inferential one. Overall, an inferential approach would help differentiate between true effects and those that might spuriously occur due to the nature of the data. Ultimately, a more statistically principled approach might estimate harmonic structure in the presence of noise in a unified manner transmitted throughout the methodological steps.

Reviewer #2 (Public Review):

Summary:

A new toolbox is presented that builds on previous toolboxes to distinguish between real and spurious oscillatory activity, which can be induced by non-sinusoidal waveshapes. Whilst there are many toolboxes that help to distinguish between 1/f noise and oscillations, not many tools are available that help to distinguish true oscillatory activity from spurious oscillatory activity induced in harmonics of the fundamental frequency by non-sinusoidal waveshapes. The authors present a new algorithm which is based on autocorrelation to separate real from spurious oscillatory activity. The algorithm is extensively validated using synthetic (simulated) data, and various empirical datasets from EEG, and intracranial EEG in various locations and domains (i.e. auditory cortex, hippocampus, etc.).

Strengths:

Distinguishing real from spurious oscillatory activity due to non-sinusoidal waveshapes is an issue that has plagued the field for quite a long time. The presented toolbox addresses this fundamental problem which will be of great use for the community. The paper is written in a very accessible and clear way so that readers less familiar with the intricacies of Fourier transform and signal processing will also be able to follow it. A particular strength is the broad validation of the toolbox, using synthetic, scalp EEG, EcoG, and stereotactic EEG in various locations and paradigms.

Weaknesses:

A weakness is that the algorithm seems to be quite conservative in identifying oscillatory activity which may render it only useful for analyzing very strong oscillatory signals (i.e. alpha), but less suitable for weaker oscillatory signals (i.e. gamma).

Reviewer #2 (Public Review):

Summary:

The authors set out to non-invasively track neuronal development in rat neonates, which they achieved with notable success. However, the direct relationship between the results and broader conclusions regarding developmental biology and potential human implications is somewhat overstretched without further validation.

Strengths:

If adequately revised and validated, this work could have a significant impact on the field, providing a non-invasive tool for longitudinal studies of brain development and neurodevelopmental disorders in preclinical settings.

Weaknesses:

(1) Consistency and Logical Flow:

- The manuscript suffers from a lack of strategic flow in some sections. Specifically, transitions between major findings and methodological discussions need refinement to ensure a logical progression of ideas. For example, the jump from the introduction of developmental trajectories and the technicalities of MRS (Magnetic Resonance Spectroscopy) processing on page 3 could benefit from a bridging paragraph that explicitly states the study's hypotheses based on existing literature gaps.

(2) Scientific Rigour:

- While the novel application of diffusion-weighted MRS is commendable, there's a notable gap in the rigorous validation of this approach against gold-standard histological or molecular techniques. Particularly, the assertions regarding the sphere fraction and morphological changes inferred from biophysical modelling mandates direct validation to solidify the claims made. A study comparing these in vivo findings with ex vivo confirmation in at least a subset of samples would significantly enhance the reliability of these conclusions.

(3) Clarity and Novelty:

- The manuscript often delves deeply into technical specifics at the expense of accessibility to readers not deeply familiar with MRS technology. The introduction and discussions would benefit from a clearer elucidation of why these specific metabolite markers were chosen and their known relevance to neuronal and glial cells, placing this in the context of what is novel compared to existing literature.<br /> - The novelty aspect could be reinforced by a more structured discussion on how this method could change the current understanding or practices within neurodevelopmental research, compared to the current state of the art.

(4) Completeness:

- The Discussion section requires expansion to offer a more comprehensive interpretation of how these findings impact the broader field of neurodevelopment and psychiatric disorders. Specifically, the implications for human studies or clinical translation are touched upon but not fully explored.<br /> - Further, while supplementary material provides necessary detail on methodology, key findings from these analyses should be summarized and discussed in the main text to ensure the manuscript stands complete on its own.

(5) Grammar, Style, Orthography:

- There are sporadic grammatical and typographical errors throughout the text which, while minor, detract from the overall readability. For example, inconsistencies in metabolite abbreviations (e.g., tCr vs Cr+PCr) should be standardized.

(6) References and Additional Context:

- The current reference list is extensive but lacks integration into the narrative. Direct comparisons with existing studies, especially those with conflicting or supportive findings, are scant. More dedicated effort to contextualize this work within the existing body of knowledge would be beneficial.

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. The assumption of metabolite compartmentation, forming the basis of cell-specific microstructure interpretation of dMRS data, remains debated and should be considered with care (Rae, Neurochem Res, 2014, https://doi.org/10.1007/s11064-013-1199-5). External cross-validation of some of the authors' claims, in particular taurine in the thalamus switching from neurons to astrocytes during brain development, would be a highly valuable addition to this study.

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:

(1) This work lacks an introduction and a discussion about diffusion MRI, which is already a validated technique to assess brain development non-invasively. Although water lacks cell-specificity compared to metabolites, several studies have reported a decrease in water ADC and increased fractional anisotropy with brain maturation, associated with the myelination process and decreased water content (overview in Hüppi, Chapt. 30 of "Diffusion MRI: Theory, Methods, and Applications", Oxford University Press, 2010). Interestingly, the same observations are found in this work (decreased ADC with age for most metabolites in both brain regions), which should have been commented on. Moreover, the authors could have reported water diffusion properties in addition to metabolites', as I believe the water signal, used for coil combination and/or Eddy currents corrections, is usually naturally acquired during diffusion-weighted MRS scans.<br /> (2) It is unclear why the authors have normalized metabolite concentrations (measured from low b-values diffusion-weighted MRS spectra) to the macromolecule concentrations. First, it is not specified whether in vivo macromolecules were acquired at each age or just at one time point. Second, such ratios are not standard practice in the MRS community so this choice should have been explained. Third, the macromolecule content was reported to change with age (Tkac et al., Magn Reson Med, 2003), therefore a change in metabolite to macromolecule ratio with age cannot be interpreted unequivocally.<br /> (3) Some discussion is missing about the choice of the analytical biophysical model (although a few are compared in Supplementary Materials), in particular: is a model of macroscopic anisotropy relevant in cerebellum, made of a large fraction of oriented white matter tracks, and does the model remain valid at different ages given white matter maturation and the ongoing myelination process?

Joint Public Review:

Summary:

The authors of the study investigated the generalization capabilities of a deep learning brain age model across different age groups within the Singaporean population, encompassing both elderly individuals aged 55 to 88 years and children aged 4 to 11 years. The model, originally trained on a dataset primarily consisting of Caucasian adults, demonstrated a varying degree of adaptability across these age groups. For the elderly, the authors observed that the model could be applied with minimal modifications, whereas for children, significant fine-tuning was necessary to achieve accurate predictions. Through their analysis, the authors established a correlation between changes in the brain age gap and future executive function performance across both demographics. Additionally, they identified distinct neuroanatomical predictors for brain age in each group: lateral ventricles and frontal areas were key in elderly participants, while white matter and posterior brain regions played a crucial role in children. These findings underscore the authors' conclusion that brain age models hold the potential for generalization across diverse populations, further emphasizing the significance of brain age progression as an indicator of cognitive development and aging processes.

Strengths:

(1) The study tackles a crucial research gap by exploring the adaptability of a brain age model across Asian demographics (Chinese, Malay, and Indian Singaporeans), enriching our knowledge of brain aging beyond Western populations.<br /> (2) It uncovers distinct anatomical predictors of brain aging between elderly and younger individuals, highlighting a significant finding in the understanding of age-related changes and ethnic differences.

Weaknesses:

(1) Clarity in describing the fine-tuning process is essential for improved comprehension.<br /> (2) The analysis often limits its findings to p-values, omitting the effect sizes crucial for understanding the relationship with cognition.<br /> (3) Employing a predictive framework for cognition using brain age could offer more insight than mere statistical correlations.<br /> (4) Expanding the study's scope to evaluate the model's generalisability to unseen Caucasian samples is vital for establishing a comparative baseline.

In summary, this paper underscores the critical need to include diverse ethnicities in model testing and estimation.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors attempt to reconstitute some active zone properties by introducing synaptic ribbon proteins into HEK cells. This "ground-up" approach can be valuable for assessing the necessity of specific proteins in synaptic function. Here, the authors co-transfect a membrane-targeted bassoon, RBP2, calcium channel subunits and Ribeye to generate what they call "synthetic ribbons". The resultant structures show an ability to cluster calcium channels (Figure 4B) and a modest ability to concentrate calcium entry locations (figure 7J). At the light level, the ribeye aggregates look spherical and localize to the membrane through its interaction with the membrane-targeted bassoon. It is a nice proof-of-principle in establishing a useful experimental system for studying calcium channel localization. However, the impact of the study is modest. No new biology is discovered and to call these structures "synthetic ribbons" is an overstatement in the absence of an ultrastructural analysis.

Strengths:

(1) The authors establish a new experimental system for the study of calcium channel localization to active zones.<br /> (2) The clustering of calcium channels to bassoon via RBP2 is a nice confirmation of a previously described interaction between bassoon and calcium channels in a cell-based system<br /> (3) The "ground-up" approach is an attractive one and theoretically allows one to learn a lot about the essential interactions for building a ribbon structure.

Weaknesses:

(1) Are these truly "synthetic ribbons". The ribbon synapse is traditionally defined by its morphology at the EM level. To what extent these structures recapitulate ribbons is not shown. It has been previously shown that Ribeye forms aggregates on its own. Do these structures look any more ribbon-like than ribeye aggregates in the absence of its binding partners?<br /> (2) No new biology is discovered here. The clustering of channels is accomplished by taking advantage of previously described interactions between RBP2, Ca channels and bassoon. The localization of Ribeye to bassoon takes advantage of a previously described interaction between the two. Even the membrane localization of the complexes required the introduction of a membrane-anchoring motif.<br /> (3) The only thing ribbon-specific about these "syn-ribbons" is the expression of ribeye and ribeye does not seem to participate in the localization of other proteins in these complexes. Bsn, Cav1.3 and RBP2 can be found in other neurons.<br /> (4) As the authors point out, RBP2 is not necessary for some Ca channel clustering in hair cells, yet seems to be essential for clustering to bassoon here.<br /> (5) The difference in Ca imaging between SyRibbons and other locations is extremely subtle.<br /> (6) The effect of the expression of palm-Bsn, RBP2 and the combination of the two on Ca-current is ambiguous. It appears that while the combination is larger than the control, it probably isn't significantly different from either of the other two alone (Fig 5). Moreover, expression of Ribeye + the other two showed no effect on Ca current (Figure 7). Also, why is the IV curve right shifted in Figure 7 vs Figure 5?<br /> (7) While some of the IHC is quantified, some of it is simply shown as single images. EV2, EV3 and Figure 4a in particular (4b looks convincing enough on its own, but could also benefit from a larger sample size and quantification)

Reviewer #2 (Public Review):

Summary:

The authors show that co-expression of bassoon, RIBEYE, Cav1.3-alpha1, Cav-beta3, Cav-alpha2delta1, and RBP2 in a heterologus system (HEK293 cells) is sufficient to generate a protein complex resembling a presyanptic ribbon-type active zone both in morphology and in function (in clustering voltage-gated Ca channels and creating sites for localized Ca2+ entry). If the 3 separate Cav gene products are taken as a single protein (i.e. a Ca channel), the conclusion is that the core of a ribbon synapse comprises 4 proteins: bassoon holds the RIBEYE-containing ribbon to the plasma membrane, and RPB2 binds to bassoon and Ca channels, tethering the Ca channels to the presynaptic active zone.

Strengths:

Good use of a heterologous system with generally appropriate controls provides convincing evidence that a presynaptic ribbon-type active zone (without the ability to support exocytosis), with the ability to support localized Ca2+ entry (a key feature of ribbon-type pre-synapses) can be assembled from a few proteins.

Weaknesses:

(1) Relies on over-expression, which almost certainly diminishes the experimentally-measured parameters (e.g. pre-synapse clustering, localization of Ca2+ entry).<br /> (2) Are HEK cells the best model? HEK cells secrete substances and have a studied-endocytitic pathway, but they do not create neurosecretory vesicles. Why didn't the authors try to reconstitute a ribbon synapse in a cell that makes neurosecretory vesicles like a PC12 cell?<br /> (3) Related to 1 and 2: the Ca channel localization observed is significant but not so striking given the presence of Cav protein and measurements of Ca2+ influx distributed across the membrane. Presumably, this is the result of overexpression and an absence of pathways for pre-synaptic targeting of Ca channels. But, still, it was surprising that Ca channel localization was so diffuse. I suppose that the authors tried to reduce the effect of over-expression by using an inducible Cav1.3? Even so, the accessory subunits were constitutively over-expressed.

Reviewer #3 (Public Review):

Summary:

Ribbon synapses are complex molecular assemblies responsible for synaptic vesicle trafficking in sensory cells of the eye and the inner ear. The Ca2+-dependent exocytosis occurs at the active zone (AZ), however, the molecular mechanisms orchestrating the structure and function of the AZs of ribbon synapses are not well understood. To advance in the understanding of those mechanisms, the authors present a novel and interesting experimental strategy pursuing the reconstitution of a minimal active zone of a ribbon synapse within a synapse-naïve cell line: HEK293 cells. The authors have used stably transfected HEK293 cells that express voltage-gated Ca2+ channels subunits (constitutive -CaV beta3 and CaV alpha2 beta1- and inducible CaV1.3 alpha1). They have expressed in those cells several proteins of the ribbon synapse active zone: (1) RIBEYE, (2) a modified version of Bassoon that binds to the plasma membrane through artificial palmitoylation (Palm-Bassoon) and (3) RIM-binding protein 2 (RBP2) to induce the formation of a minimal active zone that they called SyRibbons. The formation of such structures is convincing, however, the evidence of such structures having an impact enhancing Ca2+-currents, as the authors claim, is rather weak in the present version of the study.

Strengths of the study:

(1) The study is carefully carried out using a remarkable combination of (1) superresolution microscopy, to analyze the formation and subcellular distribution of molecular assemblies and (2) functional assessment of voltage-gated Ca2+ channels using patch-clamp recording of Ca2+-currents and fluorometry to correlate Ca2+ influx with the molecular assemblies formed by AZ proteins. The results are of high quality and are in general accompanied of required control experiments.<br /> (2) The method opens new opportunities to further investigate the minimal and basic properties of AZ proteins that are difficult to study using in vivo systems. The cells that operate through ribbon synapses (e.g. photoreceptors and hair cells) are particularly difficult to manipulate, so setting up and validating the use of a heterologous system more suitable for molecular manipulations is highly valuable.<br /> (3) The structures formed by RIBEYE and Palm-Bassoon in HEK293 cells identified by STED nanoscopy are strikingly similar to the AZs of ribbon synapses found in rat inner hair cells (Figure 2).

Weaknesses of the study:

(1) The results obtained in a heterologous system (HEK293 cells) need to be interpreted with caution. They will importantly speed the generation of models and hypothesis that will, however, require in vivo validation.<br /> (2) The authors analyzed the distribution of RIBEYE clusters in different membrane compartments and correctly conclude that RIBEYE clusters are not trapped in any of those compartments, but it is soluble instead. The authors, however, did not carry out a similar analysis for Palm-Bassoon. It is therefore unknown if Palm-Bassoon binds to other membrane compartments besides the plasma membrane. That could occur because in non-neuronal cells GAP43 has been described to be in internal membrane compartments. This should be investigated to document the existence of ectopic internal Synribbons beyond the plasma membrane because it might have implications for interpreting functional data in case Ca2+-channels become part of those internal Synribbons.<br /> (3) The co-expression of RBP2 and Palm-Bassoon induces a rather minor but significant increase in Ca2+-currents (Figure 5). Such an increase does not occur upon expression of (1) Palm-Bassoon alone, (2) RBP2 alone or (3) RIBEYE alone (Figure 5). Intriguingly, the concomitant expression of Palm-Bassoon, RBP2 and RIBEYE does not translate into an increase of Ca2+-currents either (Figure 7).<br /> (4) The authors claim that Ca2+-imaging reveals increased CA2+-signal intensity at synthetic ribbon-type AZs. That claim is a subject of concern because the increase is rather small and it does not correlate with an increase in Ca2+-currents.

Reviewer #1 (Public Review):

Summary:

Protein conformational changes are often critical to protein function, but obtaining structural information about conformational ensembles is a challenge. Over a number of years, the authors of the current manuscript have developed and improved an algorithm, qFit protein, that models multiple conformations into high resolution electron density maps in an automated way. The current manuscript describes the latest improvements to the program, and analyzes the performance of qFit protein in a number of test cases, including classical statistical metrics of data fit like Rfree and the gap between Rwork and Rfree, model geometry, and global and case-by-case assessment of qFit performance at different data resolution cutoffs. The authors have also updated qFit to handle cryo-EM datasets, although the analysis of its performance is more limited due to a limited number of high-resolution test cases and less standardization of deposited/processed data.

Strengths:

The strengths of the manuscript are the careful and extensive analysis of qFit's performance over a variety of metrics and a diversity of test cases, as well as careful discussion of the limitations of qFit. This manuscript also serves as a very useful guide for users in evaluating if and when qFit should be applied during structural refinement.

Reviewer #2 (Public Review):

Summary

The manuscript "Uncovering Protein Ensembles: Automated Multiconformer Model building for X-ray Crystallography and Cryo-EM" by Wankowicz et al. describes updates to qFit, an algorithm for the characterization of conformational heterogeneity of protein molecules based on X-ray diffraction of Cryo-EM data. The work provides a clear description of the algorithm used by qFit. The authors then proceed to validate the performance of qFit by comparing to deposited X-ray entries in the PDB in the 1.2-1.5 Å resolution range as quantified by Rfree, Rwork-Rfree, detailed examination of the conformations introduced by qFit, and performance on stereochemical measures (MolProbity scores). To examine the effect of experimental resolution of X-ray diffraction data, they start from an ultra high-resolution structure (SARS-CoV2 Nsp3 macrodomain) to determine how the loss of resolution (introduced artificially) degrades the ability of qFit to correctly infer the nature and presence of alternate conformations. The authors observe a gradual loss of ability to correctly infer alternate conformations as resolution degrades past 2 Å. The authors repeat this analysis for a larger set of entries in a more automated fashion and again observe that qFit works well for structures with resolutions better than 2 Å, with a rapid loss of accuracy at lower resolution. Finally, the authors examine the performance of qFit on cryo-EM data. Despite a few prominent examples, the authors find only a handful (8) of datasets for which they can confirm a resolution better than 2.0 Å. The performance of qFit on these maps is encouraging and will be of much interest because cryo-EM maps will, presumably, continue to improve and because of the rapid increase in the availability of such data for many supramolecular biological assemblies. As the authors note, practices in cryo-EM analysis are far from uniform, hampering the development and assessment of tools like qFit.

Strengths

qFit improves the quality of refined structures at resolutions better than 2.0 A, in terms of reflecting true conformational heterogeneity and geometry. The algorithm is well-designed and does not introduce spurious or unnecessary conformational heterogeneity. I was able to install and run the program without a problem within a computing cluster environment. The paper is well-written and the validation thorough.<br /> I found the section on cryo-EM particularly enlightening, both because it demonstrates the potential for discovery of conformational heterogeneity from such data by qFit, and because it clearly explains the hurdles towards this becoming common practice, including lack of uniformity in reporting resolution, and differences in map and solvent treatment.

Weaknesses

Due to limitations of past software engineering, the paper lacks a careful comparison to past versions of qFit. In light of the extensive assessment of the current version of qFit, this is a minor concern.

Although qFit can handle supramolecular assemblies and bound organic molecules, analysis in the manuscript is limited to single-chain X-ray structures. I look forward to demonstration of its utility in such cases in future work.

Appraisal & Discussion

Overall, the authors convincingly demonstrate that qFit provides a reliable means to detect and model conformational heterogeneity within high-resolution X-ray diffraction datasets and (based on a smaller sample) in cryo-EM density maps. This represents the state of the art in the field and will be of interest to any structural biologist or biochemist seeking to attain an understanding of the structural basis of the function of their system of interest, including potential allosteric mechanisms-an area where there are still few good solutions. That is, I expect qFit to find widespread use.

Reviewer #3 (Public Review):

Summary:

The authors address a very important issue of going beyond a single-copy model obtained by the two principal experimental methods of structural biology, macromolecular crystallography and cryo electron microscopy (cryo-EM). Such multiconformer model is based on the fact that experimental data from both these methods represent a space- and time-average of a huge number of the molecules in a sample, or even in several samples, and that the respective distributions can be multimodal. Differently from structure prediction methods, this approach is strongly based on accurate high-resolution experimental information and requires validated single-copy high-quality models as input. In overall, the results support the authors' conclusions.

In fact, the method addresses two problems which could be considered separately:

- an automation of construction of multiple conformations when they can be identified visually;<br /> - a determination of multiple conformations when their visual identification is difficult or impossible.

The former is a known problem, when missing alternative conformations may cost a few percent in R-factors. While these conformations are relatively easy to detect and build manually, the current procedure may save significant time being quite efficient, as the test results show. It is an indisputably useful tool for such a goal. The second problem is important from the physical point of view and has been considered first thirty years ago by Burling & Brünger. The manuscript does not specify clearly how much the current tool addresses the second case. To model such maps, the authors introduced errors in structure factors, however, being independent, as in this work, such errors, even quite high, may leave the maps reasonably well interpretable. Obviously, it is impossible to model all kinds of errors and this modeling of noise is appreciated but it would helpful for understanding if the manuscript shows, for example, the worst map when the procedure was successful.

The new procedure deals with a second-order variation in the R-factors, of about 1% or less, like placing riding hydrogen atoms, modeling density deformation or variation of the bulk solvent. In such situations, it is hard to justify model improvement. Keeping Rfree values or their marginal decreasing can be considered as a sign that the model does not overfit data but hardly as a strong argument in favor of the model.

In general, global targets are less appropriate for this kind of problems and local characteristics may be better indicators. Improvement of the model geometry is a good choice. Indeed, yet Cruickshank (1956) showed that averaged density images may lead to a shortening of covalent bonds when interpreting such maps by a single model. However, a total absence of geometric outliers is not necessarily required for the structures solved at a high resolution where diffraction data should have a more freedom to place the atoms where the experiments "see" them.

The key local characteristic for multicomformer models is a closeness of the model map to the experimental one. Actually, the procedure uses a kind of such measure, the Bayesian information criteria (BIC). Unfortunately, the manuscript does not describe how sharply it identifies the best model and how much it changes between the initial and final models; in general, there is no feeling about its values. The Q-score (page 17) can be an appropriate tool for the first problem where the multiple conformations and individual atomic images are clearly separated and not for the second problem where the contributions from neighboring conformations and atoms are merged. In addition to BIC or to even more conventional global target functions such as LS or map correlation, the extreme values of the local difference maps may help to validate, or not, the model.

This described method with the results presented is a strong argument for a need in experimental data and information they contain, differently from a pure structure prediction. This tool is important to produce user-unbiased multiconformer models rapidly and automatically. At the same time, absence of strong density-based validation components may limit its impact.

Strengths:<br /> Addressing an important problem and automatisation of model construction for alternative conformations using high-resolution experimental data.

Weaknesses:<br /> An insufficient validation of the models when no discrete alternative conformations visible and insufficiency of local real-space validation indicators.

Reviewer #1 (Public Review):

Summary:

The authors try to use a gene therapy approach to cure urofacial symptoms in an HSPE2 mutant mouse model.

Strengths:

The authors have convincingly shown the expression of AAV9/HSPE2 in pelvic ganglion and liver tissues. They have also shown the defects in urethra relaxation and bladder muscle contraction in response to EFS in mutant mice, which were reversed in treated mice.

Weaknesses:

It is easy to understand that high expression levels of HPSE2 in the bladder tissue lead to bladder dysfunction in human patients, however, the undetectable level of HPSE2 in AAV9 transfected mice bladders is a big question for the functional correction in those HPSE2 mutated mice.

Reviewer #2 (Public Review):

In this study, Lopes and colleagues provide evidence to support the potential for gene therapy to restore expression of heparanase-2 (Hpse2) in mice mutant for this gene, as occurs in urofacial syndrome. Building on prior studies describing the nature of urinary tract dysfunction in Hpse2 mutant mice, the authors applied a gene therapy approach to determine whether gene replacement could be achieved, and if so, whether restoration of HPSE2 expression could mitigate the urinary tract dysfunction. Using a viral vector-based strategy, shown to be successful for gene replacement in humans, the authors demonstrated dose-dependent viral transduction of pelvic ganglia and liver in wild type mice. No impact on body weight or liver health was noted suggesting the approach was safe. Administration of AAV9/HPSE2 to Hpse2 mutant mice was associated with similar transduction of pelvic ganglia and a corresponding increase in heparanase-2 protein expression in this site. Analysis of bladder outflow tract and bladder body physiology using organ bath studies showed that re-expression of heparanase-2 in Hpse2 mutant mice was associated with restored neurogenic relaxation of the outflow tract and nerve-evoked contraction of the bladder body, albeit with notable variability in the response at lower frequencies across replicates. Differences were noted in the evoked response to carbachol with bladders from Hpse2 mutant male mice showing increased sensitivity upon HPSE2 replacement compared to wild type, but bladders from female mice showing no difference. Based on these findings the authors concluded that AAV9-based HPSE2 replacement is feasible and safe, mitigates some physiological deficits in outflow tract and bladder tissue from Hpse2 mutant mice and provides proof-of-principle for gene replacement approaches for other genes implicated in lower urinary tract disorders. Strengths include a solid experimental design and data in support of some of the conclusions, and discussion of limitations of the approach. Weaknesses include the variability, albeit acknowledged, in some of the functional assessments, and the limited investigation of bladder tissue morphology in Hpse2 mutant mice.

Reviewer #1 (Public Review):

Summary:

This paper suggests to apply intrinsically-motivated exploration for the discovery of robust goal states in gene regulatory networks.

Strengths:

The paper is well written. The biological motivation and the need for such methods are formulated extraordinarily well. The battery of experimental models is impressive.

Weaknesses:

(1) The proposed method is compared to the random search. That says little about the performance with regard to the true steady-state goal sets. The latter could be calculated at least for a few simple ODE (e.g., BIOMD0000000454, `Metabolic Control Analysis: Rereading Reder'). The experiment with 'oscillator circuits' may not be directly interpolated to the other models.

The lack of comparison to the ground truth goal set (attractors of ODE) from arbitrary initial conditions makes it hard to evaluate the true performance/contribution of the method. A part of the used models can be analyzed numerically using JAX, while there are models that can be analyzed analytically.

"...The true versatility of the GRN is unknown and can only be inferred through empirical exploration and proxy metrics....": one could perform a sensitivity analysis of the ODEs, identifying stable equilibria. That could provide a proxy for the ground truth 'versatility'.

(2) The proposed method is based on `Intrinsically Motivated Goal Exploration Processes with Automatic Curriculum Learning', which assumes state action trajectories [s_{t_0:t}, a_{t_0:t}], (2.1 Notations and Assumptions' in the IMGEP paper). However, the models used in the current work do not include external control actions, but rather only the initial conditions can be set. It is not clear from the methods whether IMGEP was adapted to this setting, and how the exploration policy was designed w/o actual time-dependent actions. What does "...generates candidate intervention parameters to achieve the current goal...."<br /> mean considering that interventions 'Sets the initial state...' as explained in Table 2?

(3) Fig 2 shows the phase space for (ERK, RKIPP_RP) without mentioning the typical full scale of ERK, RKIPP_RP. It is unclear whether the path from (0, 0) to (~0.575, ~3.75) at t=1000 is significant on the typical scale of this phase space. is it significant on the typical scale of this phase space?

(4) Table 2:<br /> (a) Where is 'effective intervention' used in the method?<br /> (b) In my opinion 'controllability', 'trainability', and 'versatility' are different terms. If there correspondence is important I would suggest to extend/enhance the column "Proposed Isomorphism". otherwise, it may be confusing. I don't see how this table generalizes generalizes "concepts from dynamical complex systems and behavioral sciences under a common navigation task perspective".

Reviewer #2 (Public Review):

Summary:

Etcheverry et al. present two computational frameworks for exploring the functional capabilities of gene regulatory networks (GRNs). The first is a framework based on intrinsically motivated exploration, here used to reveal the set of steady states achievable by a given gene regulatory network as a function of initial conditions. The second is a behaviorist framework, here used to assess the robustness of steady states to dynamical perturbations experienced along typical trajectories to those steady states. In Figs. 1-5, the authors convincingly show how these frameworks can explore and quantify the diversity of behaviors that can be displayed by GRNs. In Figs. 6-9, the authors present applications of their framework to the analysis and control of GRNs, but the support presented for their case studies is often incomplete.

Following revision, my overall perspective of the paper remains unchanged. The first half of the paper provides solid evidence to support an important conceptual framework. The evidence presented for the use cases in the latter half is incomplete; as the authors note, they are preliminary and meant to be built on in future work. I have included my first round comments below.

Strengths:

Overall, the paper presents an important development for exploring and understanding GRNs/dynamical systems broadly, with solid evidence supporting the first half of their paper in a narratively clear way.

The behaviorist point of view for robustness is potentially of interest to a broad community, and to my knowledge introduces novel considerations for defining robustness in the GRN context.

Some specific weaknesses, mostly concerning incomplete analyses in the second half of the paper:

(1) The analysis presented in Fig. 6 is exciting but preliminary. Are there other appropriate methods for constructing energy landscapes from dynamical trajectories in gene regulatory networks? How do the results in this particular case study compare to other GRNs studied in the paper?

Additionally, it is unclear whether the analysis presented in Fig. 6C is appropriate. In particular, if the pseudopotential landscapes are constructed from statistics of visited states along trajectories to the steady state, then the trajectories derived from dynamical perturbations do not only reflect the underlying pseudo-landscape of the GRN. Instead, they also include contributions from the perturbations themselves.

(2) In Fig. 7, I'm not sure how much is possible to take away from the results as given here, as they depend sensitively on the cohort of 432 (GRN, Z) pairs used. The comparison against random networks is well-motivated. However, as the authors note, comparison between organismal categories is more difficult due to low sample size; for instance, the "plant" and "slime mold" categories each only has 1 associated GRN. Additionally, the "n/a" category is difficult to interpret.

(3) In Fig. 8, it is unclear whether the behavioral catalog generated is important to the intervention design problem of moving a system in one attractor basin to another. The authors note that evolutionary searches or SGD could also be used to solve the problem. Is the analysis somehow enabled by the behavioral catalog in a way that is complementary to those methods? If not, comparison against those methods (or others e.g. optimal control) would strengthen the paper.

(4) The analysis presented in Fig. 9 also is preliminary. The authors note that there exist many algorithms for choosing/identifying the parameter values of a dynamical system that give rise to a desired time series. It would be a stronger result to compare their approach to more sophisticated methods, as opposed to random search and SGD. Other options from the recent literature include Bayesian techniques, sparse nonlinear regression techniques (e.g. SINDy), and evolutionary searches. The authors note that some methods require fine-tuning in order to be successful, but even so, it would be good to know the degree of fine-tuning which is necessary compared to their method. [second round: the authors have included a comparison against CMA-ES, an evolutionary algorithm]

Reviewer #1 (Public Review):

The mechanisms underlying the generation and maintenance of LLPCs have been one of the unresolved issues. In the last few years, several groups have independently generated new genetic tools or models and addressed how LLPCs are generated or maintained in homeostatic conditions or upon immunization or infection. Here, Jing et al. have also established a new PC time stamping system and tried to address the issues above. The authors have found that LLPCs accumulated in the BM PC pool, along with aging, and that LLPCs had unique sufacetome, transcriptome, and BCR clonality. These observations have already been made by other groups (Xu et al. 2020, Robinson et al. 2022, Liu et al. 2022, Koike et al. 2023, Robinson et al. 2023, plus Tellier et al., 2024), therefore it is hard to find significant conceptual advances there. In my opinion, however, genetic analysis of the role of CXCR4 on PC localization or survival in BM (Figure 4 and 5) provided new aspects which have not been addressed in previous studies. Importantly, CXCR4 was required for the maintenance of plasma cells in bone marrow survival niches, conditional loss of which led to rapid mobilization from the bone marrow, reduced plasma cell survival, and reduced antibody titer. Thus, these data suggest that CXCR4-CXCL12 axis is not only important for plasma cell recruitment to the bone marrow but also essential for their lodging on the niches. I think the study is of high quality and the findings should be widely shared in the field.

Reviewer #2 (Public Review):

In this study by Jing, Fooksman, and colleagues, a Blimp1-CreERT2-based genetic tracing study is employed to label plasma cells. Over the course of several months post-tamoxifen treatment, the only remaining labeled cells are long-lived plasma cells. This system provides a way to sort live long-lived plasma cells and compare them to unlabeled plasma cells, which contain a range of short-to-long-lived cells. From this analysis, several observations are made: 1) the turnover rate of plasma cells is greater in the spleen than in the bone marrow; 2) the turnover rate is highest early in life; 3) subtle transcriptional and cell surface marker differences distinguish long- from shorter-lived plasma cells; 4) long-lived plasma cells in the bone marrow are sessile and localize in clusters with each other; 5) CXCR4 is required for plasma cell retention in these clusters and in the bone marrow; 6) Repertoire analysis hints that the selection of long-lived plasma cells is not random for any cell that lands in the bone marrow.

Strengths:

(1) The genetic timestamping approach is a clever and functional way to separate plasma cells of differing longevities.

(2) This approach led to the identification of several markers that could help prospective separation of long-lived plasma cells from others.

(3) Functional labeling of long-lived plasma cells allowed for a higher resolution analysis of transcriptomes and motility than was previously possible.

(4) The genetic system allowed for a revisitation of the importance of CXCR4 in plasma cell retention and survival.

Weaknesses:

(1) Most of the labeling studies, likely for practical reasons, were done on polyclonal rather than antigen-specific plasma cells. The triggers of these responses could vary based on age at the time of exposure, anatomical sites, etc. How these differences might influence markers and transcriptomes, independently of longevity, is not completely known.

(2) The fraction of long-lived plasma cells in the unlabeled fraction varies with age, potentially diluting differences between long- and short-lived plasma cells.

(3) The authors suggest their data favors a model by which plasma cells compete for niche space. Yet there is no evidence presented here that these niches are limiting. While a finite number of plasma cells may occupy a single niche (Figure 2), it may be that these niches overall are abundant in the bone marrow and do not restrict LLPC numbers. Robinson...Tarlinton and colleagues (Immunity, 2023) in fact provide experimental evidence against an extrinsic limit.

(4) The functional importance of the observed transcriptome differences between long- and shorter-lived plasma cells is unknown. An assessment as to whether these differences are conserved in human long- and short-lived bone marrow plasma cells might provide circumstantial supporting evidence that these changes are important for longevity.

Reviewer #3 (Public Review):

Summary:

Long-lived PCs are maintained in a CXCR4-dependent manner.

Strengths:

The reporter mice for fate-mapping can clearly distinguish long-lived PCs from total PCs and greatly contribute to the identification of long-lived PCs.

Reviewer #1 (Public Review):

Summary:

In this study, a chromosome-level genome of the rose-grain aphid M. dirhodum was assembled with high quality, and A-to-I RNA-editing sites were systematically identified. The authors then demonstrated that: 1) Wing dimorphism induced by crowding in M. dirhodum is regulated by 20E (ecdysone signaling pathway); 2) an A-to-I RNA editing prevents the binding of miR-3036-5p to CYP18A1 (the enzyme required for 20E degradation), thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring.

Strengths:

The authors present both genome and A-to-I RNA editing data. An interesting finding is that a A-to-I RNA editing site in CYP18A1 ruin the miRNA binding site of miR-3036-5p. And loss of miR-3036-5p regulation lead to less 20E and winged offspring.

Weaknesses:

How crowding represses the miR-3036-5p is still unclear.

Reviewer #2 (Public Review):

Summary:

Environmental influences on development are ubiquitous, affecting many phenotypes in organisms. However molecular genetic and cellular mechanisms transducing environmental signals are still only barely understood. This study examines part of one such intracellular mechanism in a polyphenic (or dimorphic) aphid.

Strengths:

While other published reports have linked phenotypic plasticity to RNA editing before, this study reports such an interaction in insects. The study uses a wide array of molecular tools to identify connections upstream and downstream of the RNA editing to elucidate the regulatory mechanism, which is illuminating.

Weaknesses:

While this system is intriguing, this report does not foster confidence in its conclusions. Many of the analyses seem based on very small sample sizes. It is itself problematic that sample sizes are not obvious in most figures, although based on Methods section covering RNAseq, they seem to be either 3, 6 or 9, depending on whether stages were pooled, but that point is not made clear. With such small sample sizes, statistical tests of any kind are unreliable. Besides the ambiguity on sample sizes, it's unclear what error bars or whiskers show in plots throughout this study. When sample sizes are small estimates of variance are not reliable. Student's t-test is not appropriate for comparisons with such small sample sizes. Presently, it is not possible to replicate the tests shown in Figures 3, 4 and 6. (Besides the HT-seq reads, other data should also be made publicly available, following the journal's recommendations.) Regardless, effect sizes in some comparisons (Fig 3J, 4A-C, 6E,H) are clearly not large, making confidence in conclusions low. The authors should be cautious about over-interpreting these data.

Reviewer #2 (Public Review):

Summary:

The dominant paradigm in the past decade for modeling the ventral visual stream's response to images has been to train deep neural networks on object classification tasks and regress neural responses from units of these networks. While object classification performance is correlated to variance explained in the neural data, this approach has recently hit a plateau of variance explained, beyond which increases in classification performance do not yield improvements in neural predictivity. This suggests that classification performance may not be a sufficient objective for building better models of the ventral stream. Lindsey & Issa study the role of factorization in predicting neural responses to images, where factorization is the degree to which variables such as object pose and lighting are represented independently in orthogonal subspaces. They propose factorization as a candidate objective for breaking through the plateau suffered by models trained only on object classification. They show the degree of factorization in a model captures aspects of neural variance that classification accuracy alone does not capture, hence factorization may be an objective that could lead to better models of ventral stream. I think the most important figure for a reader to see is Fig. 6.

Strengths:

This paper challenges the dominant approach to modeling neural responses in the ventral stream, which itself is valuable for diversifying the space of ideas.

This paper uses a wide variety of datasets, spanning multiple brain areas and species. The results are consistent across the datasets, which is a great sign of robustness.

The paper uses a large set of models from many prior works. This is impressively thorough and rigorous.

The authors are very transparent, particularly in the supplementary material, showing results on all datasets. This is excellent practice.

Weaknesses:

The authors have addressed many of the weaknesses in the original review. The weaknesses that remain are limitations of the work that cannot be easily addressed. In addition to the limitations stated at the end of the discussion, I'll add two:

(1) This work shows that factorization is correlated with neural similarity, and notably explains some variance in neural similarity that classification accuracy does not explain. This suggests that factorization could be used as an objective (along with classification accuracy) to build better models of the brain. However, this paper does not do that - using factorization to build better models of the brain is left to future work.

Reviewer #3 (Public Review):

Summary:

Object classification serves as a vital normative principle in both the study of the primate ventral visual stream and deep learning. Different models exhibit varying classification performances and organize information differently. Consequently, a thriving research area in computational neuroscience involves identifying meaningful properties of neural representations that act as bridges connecting performance and neural implementation. In the work of Lindsey and Issa, the concept of factorization is explored, which has strong connections with emerging concepts like disentanglement [1,2,3] and abstraction [4,5]. Their primary contributions encompass two facets: (1) The proposition of a straightforward method for quantifying the degree of factorization in visual representations. (2) A comprehensive examination of this quantification through correlation analysis across deep learning models.

To elaborate, their methodology, inspired by prior studies [6], employs visual inputs featuring a foreground object superimposed onto natural backgrounds. Four types of scene variables, such as object pose, are manipulated to induce variations. To assess the level of factorization within a model, they systematically alter one of the scene variables of interest and estimate the proportion of encoding variances attributable to the parameter under consideration.

The central assertion of this research is that factorization represents a normative principle governing biological visual representation. The authors substantiate this claim by demonstrating an increase in factorization from macaque V4 to IT, supported by evidence from correlated analyses revealing a positive correlation between factorization and decoding performance. Furthermore, they advocate for the inclusion of factorization as part of the objective function for training artificial neural networks. To validate this proposal, the authors systematically conduct correlation analyses across a wide spectrum of deep neural networks and datasets sourced from human and monkey subjects. Specifically, their findings indicate that the degree of factorization in a deep model positively correlates with its predictability concerning neural data (i.e., goodness of fit).

Strengths:

The primary strength of this paper is the authors' efforts in systematically conducting analysis across different organisms and recording methods. Also, the definition of factorization is simple and intuitive to understand.

Weaknesses:

Comments on revised version:

I thank the authors for addressing the weaknesses I brought up regarding the manuscript.

Reviewer #1 (Public Review):

Summary:

The authors of the study are trying to show that RNAseq can be used for neoantigen prediction and that the machine learning approach to the prediction can reveal very useful information for the selection of neoantigens for personalized antitumor vaccination.

Strengths:

The authors demonstrated that RNA expression of a neoantigen is a very important factor in the selection of peptides for the creation of personalized vaccines. They proved in vivo that in silico-predicted neoantigens can trigger an antitumor response in mice.

Weaknesses:

The selection of the peptides for vaccination is not clear. Some peptides were selected before and some after processing. What processing is also not clear. The authors didn't provide the full list of peptides before and after processing, please add those. And it wasn't clear that these peptides were previously published. Looking at the previously published table with peptide from B16 F10 (https://www.nature.com/articles/s41598-021-89927-5/tables/3), there are other genes with high expression, e.g. Tab2, Tm9sf3 that have higher expression than Herc6, please clarify the choice.

It's not clear how many mice were used for each group in each experiment, please add this information to the text and figures. It would be good to add this, to aid the understanding of a broader audience.

Please provide information about what software was used for statistical analysis.

Reviewer #2 (Public Review):

Summary:

The authors develop a new neoantigen prediction tool (NAP-CNB) which primarily predicts neoantigens based on expression (RNAseq) and ranks mutations using binding affinity. The validated predicted neoantigens in mice demonstrate that neoantigens with higher expression (but not necessarily the highest immunogenicity) lead to the greatest tumor control.

Strengths:

There is in vivo validation of the neoantigens.<br /> Demonstrates comparability to other prediction algorithms that are commonly used.<br /> Demonstrates that expression holds a higher value than T-cell responses in actual tumor control.

Weaknesses:

Binding affinity does not always predict immune responses or tumor control in vivo which is used as part of the selection criteria.

Reviewer #1 (Public Review):

In this paper, Wu et al. investigated the physiological roles of CCDC113 in sperm flagellum and HTCA stabilization by using CRISPR/Cas knockouts mouse models, co-IP, and single sperm imaging. They find that CCDC113 localizes in the linker region among radial spokes, the nexin-dynein regulatory complex (N-DRC), and doublet microtubules (DMTs) RS, N-DRC, and DMTs and interacts with axoneme-associated proteins CFAP57 and CFAP91, acting as an adaptor protein that facilitates the linkage between RS, N-DRC, and DMTs within the sperm axoneme. They show the disruption of CCDC113 produced spermatozoa with disorganized sperm flagella and CFAP91, DRC2 could not colocalize with DMTs in Ccdc113-/- spermatozoa. Interestingly, the data also indicate that CCDC113 could localize on the HTCA region, and interact with HTCA-associated proteins. The knockout of Ccdc113 could also produce acephalic spermatozoa. By using Sun5 and Centlein knockout mouse models, the authors further find SUN5 and CENTLEIN are indispensable for the docking of CCDC113 to the implantation site on the sperm head. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. Furthermore, the study's findings offer valuable insights into the physiological and developmental roles of CCDC113 in the male germ line, which can provide insight into impaired sperm development and male infertility. The conclusions of this paper are mostly well supported by data, but some points need to be clarified and discussed.<br /> (1) In Figure 1, a sperm flagellum protein, which is far away from CCDC113, should be selected as a negative control to exclude artificial effects in co-IP experiments.<br /> (2) Whether the detachment of sperm head and tail in Ccdc113-/- mice is a secondary effect of the sperm flagellum defects? The author should discuss this point.<br /> (3) Given that some cytoplasm materials could be observed in Ccdc113-/- spermatozoa (Fig. 5A), whether CCDC113 is also essential for cytoplasmic removal?<br /> (4) Although CCDC113 could not bind to PMFBP1, the localization of CCDC113 in Pmfbp1-/- spermatozoa should be also detected to clarify the relationship between CCDC113 and SUN5-CENTLEIN-PMFBP1.

Reviewer #2 (Public Review):

Summary:

In the present study, the authors select the coiled-coil protein CCDC113 and revealed its expression in the stages of spermatogenesis in the testis as well as in the different steps of spermiogenesis with expression also mapped in the different parts of the epididymis. Gene deletion led to male infertility in CRISPR-Cas9 KO mice and PAS staining showed defects mapped in the different stages of the seminiferous cycle and through the different steps of spermiogenesis. EM and IF with several markers of testis germ cells and spermatozoa in the epididymis indicated defects in flagella and head-to-tail coupling for flagella as well as acephaly. The authors' co-IP experiments of expressed CCDC113 in HEK293T cells indicated an association with CFAP91 and DRC2 as well as SUN5 and CENTLEIN.

The authors propose that CCDC113 connects CFAP91 and DRC2 to doublet microtubules of the axoneme and CCDC113's association with SUN5 and CENTLEIN to stabilize the sperm flagellum head-to-tail coupling apparatus. Extensive experiments mapping CCDC13 during postnatal development are reported as well as negative co-IP experiments and studies with SUN5 KO mice as well as CENTLEIN KO mice.

Strengths:

The authors provide compelling observations to indicate the relevance of CCDC113 to flagellum formation with potential protein partners. The data are relevant to sperm flagella formation and its coupling to the sperm head.

Weaknesses:

The authors' observations are consistent with the model proposed but the authors' conclusions for the mechanism may require direct demonstration in sperm flagella. The Walton et al paper shows human CCDC96/113 in cilia of human respiratory epithelia. An application of such methodology to the proteins indicated by Wu et al for the sperm axoneme and head-tail coupling apparatus is eagerly awaited as a follow-up study.

Reviewer #1 (Public Review):

Summary:

The authors provide a genome annotation resource of 33 insects using a motif-blind prediction method for tissue-specific cis-regulatory modules. This is a welcome addition that may facilitate further research in new laboratory systems, and the approach seems to be relatively accurate, although it should be combined with other sources of evidence to be practical.

Strengths:

The paper clearly presents the resource, including the testing of candidate enhancers identified from various insects in Drosophila. This cross-species analysis, and the inherent suggestion that training datasets generated in flies can predict a cis-regulatory activity in distant insects, is interesting. While I can not be sure this approach will prevail in the future, for example with approaches that leverage the prediction of TF binding motifs, the SCRMShaw tool is certainly useful and worth consideration for the large community of genome scientists working on insects.

Weaknesses:

While the authors made the effort to provide access to the SCRMShaw annotations via the RedFly database, the usefulness of this resource is somewhat limited at the moment. First, it is possible to generate tables of annotated elements with coordinates, but it would be more useful to allow downloads of the 33 genome annotations in GFF (or equivalent) format, with SCRMshaw predictions appearing as a new feature. Also, I should note that unlike most species some annotations seem to have issues in the current RedFly implementation. For example, Vcar and Jcoen turn empty.

Reviewer #2 (Public Review):

Summary:

The ability of researchers to identify and compare enhancers across different species is an important facet of understanding gene regulation across development and evolution. Many traditional methods of enhancer identification involve sequence alignments and manual annotations, limiting the ability to expand the scope of regulatory investigations into many species. In order to overcome this obstacle, the authors apply a previously published machine learning method called SCRMshaw to predict enhancers across 33 insect species, using D. melanogaster as a reference. SCRMshaw operates through the selection of a few dozen training loci in a reference genome, marking genomic loci in other species that are significantly enriched with similar k-mer distributions relative to randomly selected genomic backgrounds. Upon identification of predicted enhancer regions, the authors perform post-processing step filtering and identify the most likely predicted enhancer candidates based on the proximity of an orthologous target gene. They then perform reporter gene analysis to validate selected predicted enhancers from other species in D. melanogaster. The analysis of the expression patterns returned variable results across the selected predicted regions.

Strengths:

The authors provide annotations of predicted regions across dozens of insect species, with the intention of expanding and refining the annotations for use by the scientific field. This is useful, as researchers will be able to use the identified annotations for their own work or as a benchmark for future methods. This work also showcases the flexible and versatile nature of SCRMshaw, which can readily obtain predictions using training sets of genomic loci requiring only a few dozen annotations as input. SCRMshaw does not require sequence alignments of the enhancers and can operate without prior knowledge of the cis-regulatory sequence rules such as transcription factor binding motifs, making it a useful tool to explore the evolution of enhancers in further distant and less well-studied species.

Weaknesses:

This work provides predicted enhancer annotations across many insect species, with reporter gene analysis being conducted on selected regions to test the predictions. However, the code for the SCRMshaw analysis pipeline used in this work is not made available, making reproducibility of this work difficult. Additionally, while the authors claim the predicted enhancers are available within the REDfly database, the predicted enhancer coordinates are currently not downloadable as Supplementary Material or from a linked resource.

The authors do not validate or benchmark the application of SCRMshaw against other published methods, nor do they seek to apply SCRMshaw under a variety of conditions to confirm the robustness of the returned predicted enhancers across species. Since SCRMshaw relies on an established k-mer enrichment of the training loci, its performance is presumably highly sensitive to the selection of training regions as well as the statistical power of the given k-mer counts. The authors do not justify their selection of training regions by which they perform predictions.

While there is an attempt made to report and validate the annotated predicted enhancers using previously published data and tools, the validation lacks the depth to conclude with confidence that the predicted set of regions across each species is of high quality. In vivo, reporter assays were conducted to anecdotally confirm the validity of a few selected regions experimentally, but even these results are difficult to interpret. There is no large-scale attempt to assess the conservation of enhancer function across all annotated species.

Lastly, it is suggested that predicted regions are derived from the shared presence of sequence features such as transcription factor binding motifs, detected through k-mer enrichment via SCRMshaw. This assumption has not been examined, although there are public motif discovery tools that would be appropriate to discover whether SCRMshaw is assigning predicted regions based on previously understood motif grammar, or due to other sequence patterns captured by k-mer count distributions. Understanding the sequence-derived nature of what drives predictions is within the scope of this work and would boost confidence in the predicted enhancers, even if it is limited to a few training examples for the sake of clarity of interpretation.

Reviewer #3 (Public Review):

Summary:

In this ambitious paper, the authors develop an unparalleled community resource of insect genome regulatory annotations spanning five insect orders. They employ their previously-developed SCRMshaw method for computational cross-species enhancer prediction, drawing on available training datasets of validated enhancer sequence and expression from Drosophila melanogaster, which had been previously shown to perform well across select holometabolous insects (representing 160-345MY divergence). In this work, they expand regulatory sequence annotation to 33 insect genomes spanning Holometabola and Hemiptera, which is even more distantly related to the fly model. They perform multiple downstream analyses of sets of predicted enhancers to assess the true-positive rate of predictions; the independent comparisons of real predictions with simulated predictions and with chromatin accessibility data, as well as the functional validation through reporter gene analysis, strengthen their conclusions that their annotation pipeline achieves a high true-positive rate and can be used across long divergence times to computationally annotate regulatory genome regions, an ability that has been previously inaccessible for non-model insects and now is possible across the many newly-sequenced insect scaffold-level genomes.

Strengths:

This work fills a large gap in current methods and resources for predicting regulatory regions of the genome, a task that has long lagged behind that of coding region prediction and analysis.

Despite technical constraints in working outside of well-developed model insect systems, the authors creatively draw on existing resources to scaffold a pipeline and independently assess the likelihood of prediction validity.

The established database will be a welcome community resource in its current state, and even more so as the authors continue to expand their annotations to more insect genomes as they indicate. Their available analysis pipeline itself will be useful to the community as well for research groups that may want to undertake their own regulatory genome annotation.

Weaknesses:

The rates of predicted true positive enhancer identification vary widely across the genomes included here based on the simulations and comparison to datasets of accessible chromatin in a manner that doesn't map neatly onto phylogenetic distance. At this point, it is unclear why these patterns may arise, although this may become more clear as regulatory annotation is undertaken for more genomes.

Functional assessment of predicted enhancers was performed through reporter gene assays primarily in Drosophila melanogaster imaginal discs, a system amenable to transgenics. Unfortunately, this mode of canonical imaginal disc development is only representative of a subset of all holometabolous insects; therefore, it is difficult to interpret reporter gene expression in a fly imaginal disc as evidence of a true positive enhancer that would be active in its native species whose adult appendages develop differently through the larval stage (for example, Coleopteran and Lepidopteran legs). However, the reporter gene assays from other tissues do offer strong evidence of true positive enhancer detection, and constraints on transgenic experiments in other systems mean that this approach is the best available.

Reviewer #3 (Public Review):

Overall:

ExoIII has been described and commercialized as a dsDNA specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g., addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).

Comments:

(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting with the FAM and/or BHQ1 ends as well as the DNA. Could this cause quenching? Would this represent real ssDNA activity? Is this figure/data necessary for the manuscript?<br /> (2) Based on the descriptions in the text, it seems there is activity with some of the other nucleases in 1C, 1F, and 1I other than ExoIII and Cas12a. Can this be plotted on a scale that allows the reader to see these relative to one other?<br /> (3) The sequence alignment in Figure 2N and corresponding text indicate a region of ExoIII lacking in APE1 that may be responsible for their differences in substrate specificity in regards to ssDNA. Does the mutational analysis support this hypothesis?

Reviewer #2 (Public Review):

Summary:

This paper describes some experiments addressing 3' exonuclease and 3' trimming activity of bacterial exonuclease III. The quantitative activity is in fact very low, despite claims to the contrary. The work is of low interest with regard to biology, but possibly of use for methods development. Thus the paper seems better suited to a methods forum.

Strengths:

Technical approaches.

Comments on revised version:

All concerns have been addressed.

Reviewer #1 (Public Review):

Summary:

Ciliary rootlet is a structure associated with the ciliary basal body (centriole) with beautiful striation observed by electron microscopy. It has been known for more than a century, but its function and protein arrangement is still unknown. This work reconstructed near-atomic resolution 3D structure of the rootlet using cryo-electron tomography, discovered a number of interesting filamentous structures inside and built molecular model of the rootlet.

Strengths:

The authors exploited the current possible ability of cryo-ET and used it appropriately to describe 3D structure of the rootlet. They carefully conducted subtomogram averaging and classification, which enabled an unprecedented detailed view of this structure. The dual use of (nearly) intact rootlet from cilia and extracted (demembraned) rootlet enabled them to describe with confidence how D1/D2/A bands form periodic structures and cross with longitudinal filaments, which are likely coiled-coil.

Weaknesses:

Some more clarifications in the method and indications in figures were needed in the original version. The authors addressed them in the revision.

Reviewer #3 (Public Review):

Summary:

The study offers a compelling molecular model for the organization of rootlets, a critical organelle that links cilia to the basal body. Striations have been observed in rootlets, but their assembly, composition, and function remain unknown. While previous research has explored rootlet structure and organization, this study delivers an unprecedented level of resolution, valuable to the centrosome and cilia field. The authors isolated rootlets from mice's eyes. They apply EM to partially purified rootlets (first negative stain, then cryoET). From these micrographs, they observed striations along the membranes along the rootlet but no regular spacing was observed.

The thickness of the sample and membranes prevented good contrast in the tomograms. Thus they further purified the rootlets using detergent, which allowed them to obtain cryoET micrographs of the rootlets with greater details. The tomograms were segmented and further processed to improve the features of the rootlet structures. From their analysis, they described 3 regular cross-striations and amorphous densities, which are connected perpendicularly to filaments along the length of the rootlets. They propose that various proteins provide the striations and rootletin (mouse homolog of human c-nap1) forms parallel coiled coils that run along the rootlet. Overall their data provide a detailed model for the molecular organization of the rootlet.

The major strength is that this high-quality study uses state-of-the-art cryo-electron tomography, sub-tomogram averaging, and image analysis to provide a model of the molecular organization of rootlets. The micrographs are exceptional, with excellent contrast and details, which also implies the sample preparation was well optimized to provide excellent samples for cryo-ET. The manuscript is also clear and accessible.

This research marks a significant step forward in our understanding of rootlets' molecular organization.

Reviewer #1 (Public Review):

Summary:

This finding shows a connection between cancer associated beta-catenin mutations extracellular vesicle secretion. A link between the beta-catenin mutation and expression of trafficking and exocytosis machinery. They used a multidisciplinary approach to explore expression levels of relevant proteins and single particle imaging to directly explore the release of extracellular vesicles. These results suggest a role of extracellular vesicles in immune evasion in liver cancer with the role needing to be further explored in other forms of cancer. I find this work to be compelling and of strong significance.

Strengths:

This paper uses multidisciplinary methods to demonstrate a compelling role of beta-catenin mutations in suppressing EV secretion in tumors. The results and imaging are extremely convincing and compelling.

Reviewer #2 (Public Review):

Summary:

Dantzer and colleagues are investigating the pivotal role of ß-catenin, a gene that undergoes mutation in various cancer cells, and its influence on promoting the evasion of immune cells. In their initial experiments, the authors developed a HepG2 mutated ß-catenin KD model, conducting transcriptional and proteomic analyses. The results revealed that the silencing of mutated ß-catenin in HepG2 cells led to an up-regulation in the expression of exosome biogenesis genes.

Furthermore, the researchers verified that these KD cells exhibited an increased production of exosomes, with the mutant form of ß-catenin concurrently decreasing the expression of SDC4 and Rab27a. Intriguingly, applying a GSK inhibitor to the cells resulted in reduced expression of SDC4 and Rab27a. Subsequent findings indicated that mutated ß-catenin actively facilitates immune escape through exosomes, and silencing exosome biogenesis correlates with a decrease in immune cell infiltration.<br /> In a crucial clinical correlation, the study demonstrated that patients with ß-catenin mutations exhibited low levels of exosome biogenesis.

Strengths:

Overall, the data robustly supports the outlined conclusions, and the study is commendably designed and executed. However, there are a few suggestions for manuscript improvement.

Weaknesses: No weakness

Reviewer #3 (Public Review):

Summary:

In this very important study by Dantzer et al., 'Emerging role of oncogenic b-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma' the authors define a role for oncogenic b-catenin on exosome biology and explore the link between reduce exosome secretion and tumor immune cell evasion. Using transcriptional and proteomic analysis of hepatocellular carcinoma cells with either oncogenic or wildtype b-catenin the authors find that oncogenic b-catenin negatively regulates exosome biogenesis.

The authors can provide compelling evidence that oncogenic b-catenin in different hepatocellular carcinoma cells negatively regulates exosome biogenesis and secretion, by downregulation of, amongst others, SDC4 and RAB27A, two proteins involved in exosome biogenesis. The authors corroborate these results by inducing b-catenin activation using CHIR99021 in a hepatocarcinoma cell line with non-oncogenic bCatenin (Huh7 cells). The authors can further demonstrate convincingly that reduction in exosome release by hepatocarcinoma spheroids leads to a reduction in immune cell infiltration into the tumor spheroid.

Strengths:

This is a very important and well-conceived study, that appeals to a readership beyond the field of hepatocarcinoma. The authors demonstrate a compelling link between oncogenic bCatenin and exosome biogenesis. Their results are convincing and with well-designed control experiments. The authors included various complementary lines of investigation to verify their findings.

Weaknesses:

One limitation of this study is that the mechanistic relationship of exosome release and how they affect immune cells remains to be elucidated. In this context, the authors conclusions rest on the assumption that hepatocarcinoma immune evasion is based exclusively on the reduced number of exosomes. However, the authors do not analyze exosome composition between exosomes of wildtype and oncogenic background, which could be different.

Reviewer #1 (Public Review):

Summary:

The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

Strengths:

The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

Comments on revised version:

The authors have satisfactorily addressed my comments and this has significantly improved the clarity of the manuscript.

The only point that I would like to inquire about is the role of EL4 in modulating Na+ entry. In the simulations do the authors see no role of EL4 in controlling Na+ entry. It is particularly intriguing as some studies in the recent past displayed charged mutations in EL4 of dDAT, SERT and GAT1 as being detrimental for substrate entry/uptake. It would therefore be nice to add a small discussion if there is any role for EL4 in Na+ entry.

Reviewer #2 (Public Review):

Summary

Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

Strengths

- MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

- A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

- The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

Weaknesses

- Assessing the effects of Na+ binding on the large scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to a AF2 model that's not well packed or really a feature of the open outward conformation.

- Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

Reviewer #1 (Public Review):

Summary:

This study investigated the phosphoryl transfer mechanism of the enzyme adenylate kinase, using SCC-DFTB quantum mechanical/molecular mechanical (QM/MM) simulations, along with kinetic studies exploring the temperature and pH dependence of the enzyme's activity, as well as the effects of various active site mutants. Based on a broad free energy landscape near the transition state, the authors proposed the existence of wide transition states (TS), characterized by the transferring phosphoryl group adopting a meta-phosphate-like geometry with asymmetric bond distances to the nucleophilic and leaving oxygens. In support of this finding, kinetic experiments were conducted with Ca2+ ions at different temperatures and pH, which revealed a reduced entropy of activation and unique pH-dependence of the catalyzed reaction.

Strengths:

A combined application of simulation and experiments is a strength.

Weaknesses:

The conclusion that the enzyme-catalyzed reaction involves a wide transition state is not sufficiently clarified with some concerns about the determined free energy profiles compared to the experimental estimate. (See Recommendations for the authors.)

Reviewer #2 (Public Review):

Summary:

The authors report results of QM/MM simulations and kinetic measurements for the phosphoryl-transfer step in adenylate kinase. The main assertion of the paper is that a wide transition state ensemble is a key concept in enzyme catalysis as a strategy to circumvent entropic barriers. This assertion is based on observation of a "structurally wide" set of energetically equivalent configurations that lie along the reaction coordinate in QM/MM simulations, together with kinetic measurements that suggest a decrease of the entropy of activation.

Strengths:

The study combines theoretical calculations and supporting experiments.

Weaknesses:

The current paper hypothesizes a "wide" transition state ensemble as a catalytic strategy and key concept in enzyme catalysis. Overall, it is not clear the degree to which this hypothesis is fully supported by the data. The reasons are as follows:

(1) Enzyme catalysis reflects a rate enhancement with respect to a baseline reaction in solution. In order to assert that something is part of a catalytic strategy of an enzyme, it would be necessary to demonstrate from simulations that the activation entropy for the baseline reaction is indeed greater and the transition state ensemble less "wide". Alternatively stated, when indicating there is a "wide transition state ensemble" for the enzyme system - one needs to indicate that is with respect to the non-enzymatic reaction. However, these simulations were not performed and the comparisons not demonstrated. The authors state "This chemical step would take about 7000 years without the enzyme" making it impossible to measure; nonetheless, the simulations of the nonenzymatic reaction would be fairly straight forward to perform in order to demonstrate this key concept that is central to the paper. Rather, the authors examine the reaction in the absence of a catalytically important Mg ion.

(2) The observation of a "wide conformational ensemble" is not a quantitative measure of entropy. In order to make a meaningful computational prediction of the entropic contribution to the activation free energy, one would need to perform free energy simulations over a range of temperatures (for the enzymatic and non-enzymatic systems). Such simulations were not performed, and the entropy of activation was thus not quantified by the computational predictions. The authors instead use a wider TS ensemble as a proxy for larger entropy, and miss an opportunity to compare directly to the experimental measurements.

Reviewer #3 (Public Review):

Summary:

By conducting QM/MM free energy simulations, the authors aimed to characterize the mechanism and transition state for the phosphoryl transfer in adenylate kinase. The qualitative reliability of the QM/MM results has been supported by several interesting experimental kinetic studies. However, the interpretation of the QM/MM results is not well supported by the current calculations.

Strengths:

The QM/MM free energy simulations have been carefully conducted. The accuracy of the semi-empirical QM/MM results was further supported by DFT/MM calculations, as well as qualitatively by several experimental studies.

Weaknesses:

(1) One key issue is the definition of the transition state ensemble. The authors appear to define this by simply considering structures that lie within a given free energy range from the barrier. However, this is not the rigorous definition of transition state ensemble, which should be defined in terms of committor distribution. This is not simply an issue of semantics, since only a rigorous definition allows a fair comparison between different cases - such as the transition state in an enzyme vs in solution, or with and without the metal ion. For a chemical reaction in a complex environment, it is also possible that many other variables (in addition to the breaking and forming P-O bonds) should be considered when one measures the diversity in the conformational ensemble.

In the revised ms, the authors included committor analysis. However, the discussion of the result is very brief. In particular, if we use the common definition of the transition state ensemble (TSE) as those featuring the committor around 0.5, the reaction coordinate of the TSE would span a much narrower range than those listed in Table 1. This point should be carefully addressed.

(2) While the experimental observation that the activation entropy differs significantly with and without the Ca2+ ion is interesting, it is difficult to connect this result with the "wide" transition state ensemble observed in the QM/MM simulations so far. Even without considering the definition of the transition state ensemble mentioned above, it is unlikely that a broader range of P-O distances would explain the substantial difference in the activation entropy measured in the experiment. Since the difference is sufficiently large, it should be possible to compute the value by repeating the free energy simulations at different temperatures, which would lead to a much more direct evaluation of the QM/MM model/result and the interpretation.

Reviewer #1 (Public Review):

Continuous attractor networks endowed with some sort of adaptation in the dynamics, whether that be through synaptic depression or firing rate adaptation, are fast becoming the leading candidate models to explain many aspects of hippocampal place cell dynamics, from hippocampal replay during immobility to theta sequences during run. Here, the authors show that a continuous attractor network endowed with spike frequency adaptation and subject to feedforward external inputs is able to account for several previously unaccounted aspects of theta sequences, including (1) sequences that move both forwards and backwards, (2) sequences that alternate between two arms of a T-maze, (3) speed modulation of place cell firing frequency, and (4) the persistence of phase information across hippocampal inactivations.

I think the main result of the paper (findings (1) and (2)) are likely to be of interest to the hippocampal community, as well as to the wider community interested in mechanisms of neural sequences. In addition, the manuscript is generally well written and the analytics are impressive. However, several issues should be addressed, which I outline below.

Major comments:

In real data, population firing rate is strongly modulated by theta (i.e., cells collectively prefer a certain phase of theta - see review paper Buzsaki, 2002) and largely oscillates at theta frequency during run. With respect to this cyclical firing rate, theta sweeps resemble "Nike" check marks, with the sweep backwards preceding the sweep forwards within each cycle before the activity is quenched at the end of the cycle. I am concerned that (1) the summed population firing rate of the model does not oscillate at theta frequency, and (2) as the authors state, the oscillatory tracking state must begin with a forward sweep. With regards to (1), can the authors show theta phase spike preference plots for the population to see if they match data? With regards to (2), can the authors show what happens if the bump is made to sweep backwards first, as it appears to do within each cycle?

I could not find the width of the external input mentioned anywhere in the text or in the table of parameters. The implication is that it is unclear to me whether, during the oscillatory tracking state, the external input is large compared to the size of the bump, so that the bump lives within a window circumscribed by the external input and so bounces off the interior walls of the input during the oscillatory tracking phase, or whether the bump is continuously pulled back and forth by the external input, in which case it could be comparable to the size of the bump. My guess based on Fig 2c is that it is the latter. Please clarify and comment.

I would argue that the "constant cycling" of theta sweeps down the arms of a T-maze was roughly predicted by Romani & Tsodyks, 2015, Figure 7. While their cycling spans several theta cycles, it nonetheless alternates by a similar mechanism, in that adaptation (in this case synaptic depression) prevents the subsequent sweep of activity from taking the same arm as the previous sweep. I believe the authors should cite this model in this context and consider the fact that both synaptic depression and spike frequency adaptation are both possible mechanisms for this phenomenon. But I certainly give the authors credit for showing how this constant cycling can occur across individual theta cycles.

The authors make an unsubstantiated claim in the paragraph beginning with line 413 that the Tsodyks and Romani (2015) model could not account for forwards and backwards sweeps. Both the firing rate adaptation and synaptic depression are symmetry breaking models that should in theory be able to push sweeps of activity in both directions, so it is far from obvious to me that both forward and backward sweeps are not possible in the Tsodyks and Romani model. The authors should either prove that this is the case (with theory or simulation) or excise this statement from the manuscript.

The section on the speed dependence of theta (starting with line 327) was very hard to understand. Can the authors show a more graphical explanation of the phenomenon? Perhaps a version of Fig 2f for slow and fast speeds, and point out that cells in the latter case fire with higher frequency than in the former?

I had a hard time understanding how the Zugaro et al., (2005) hippocampal inactivation experiment was accounted for by the model. My intuition is that while the bump position is determined partially by the location of the external input, it is also determined by the immediate history of the bump dynamics as computed via the local dynamics within the hippocampus (recurrent dynamics and spike rate adaptation). So that if the hippocampus is inactivated for an arbitrary length of time, there is nothing to keep track of where the bump should be when the activity comes back on line. Can the authors please explain more how the model accounts for this?

Can the authors comment on why the sweep lengths oscillate in the bottom panel of Fig 5b during starting at time 0.5 seconds before crossing the choice point of the T-maze? Is this oscillation in sweep length another prediction of the model? If so, it should definitely be remarked upon and included in the discussion section.

Perhaps I missed this, but I'm curious whether the authors have considered what factors might modulate the adaptation strength. In particular, might rat speed modulate adaptation strength? If so, would have interesting predictions for theta sequences at low vs high speeds.

I think the paper has a number of predictions that would be especially interesting to experimentalists but are sort of scattered throughout the manuscript. It would be beneficial to have them listed more prominently in a separate section in the discussion. This should include (1) a prediction that the bump height in the forward direction should be higher than in the backward direction, (2) predictions about bimodal and unimodal cells starting with line 366, (3) prediction of another possible kind of theta cycling, this time in the form of sweep length (see comment above), etc.

Reviewer #2 (Public Review):

In this work, the authors elaborate on an analytically tractable, continuous-attractor model to study an idealized neural network with realistic spiking phase precession/procession. The key ingredient of this analysis is the inclusion of a mechanism for slow firing-rate adaptation in addition to the otherwise fast continuous-attractor dynamics. The latter continuous-attractor dynamics classically arises from a combination of translation invariance and nonlinear rate normalization.

For strong adaptation/weak external input, the network naturally exhibits an internally generated, travelling-wave dynamics along the attractor with some characteristic speed. For small adaptation/strong external stimulus, the network recovers the classical externally driven continuous-attractor dynamics. Crucially, when both adaptation and external input are moderate, there is a competition with the internally generated and externally generated mechanisms leading to an oscillatory tracking regime. In this tracking regime, the population firing profile oscillates around the neural field tracking the position of the stimulus. The authors demonstrate by a combination of analytical and computational arguments that oscillatory tracking corresponds to realistic phase precession/procession. In particular the authors can account for the emergence of unimodal and bimodal cells, as well as some other experimental observations with respect the dependence of phase precession/procession on the animal's locomotion.

The strengths of this work are at least three-fold: 1) Given its simplicity, the proposed model has a surprisingly large explanatory power of the various experimental observations. 2) The mechanism responsible for the emergence of precession/procession can be understood as a simple yet rather illuminating competition between internally driven and externally driven dynamical trends. 3) Amazingly, and under some adequate simplifying assumptions, a great deal of analysis can be treated exactly, which allows for a detailed understanding of all parametric dependencies. This exact treatment culminates with a full characterization of the phase space of the network dynamics, as well as the computation of various quantities of interest, including characteristic speeds and oscillating frequencies.

As mentioned by the authors themselves, the main limitation of this work is that it deals with a very idealized model and it remains to see how the proposed dynamical behaviors would persists in more realistic models. For example, the model is based on a continuous attractor model that assumes perfect translation-invariance of the network connectivity pattern. Would the oscillating tracking behavior persist in the presence of connection heterogeneities? Another limitation is that the system needs to be tuned to exhibit oscillation within the theta range and that this tuning involves a priori variable parameters such as the external input strength. Is the oscillating-tracking behavior overtly sensitive to input strength variations? The author mentioned that an external pacemaker can serve to drive oscillation within the desired theta band but there is no evidence presented supporting this. A final and perhaps secondary limitation has to do with the choice of parameter, namely the time constant of neural firing which is chosen around 3ms. This seems rather short given that the fast time scale of rate models (excluding synaptic processes) is usually given by the membrane time constant, which is typically about 15ms. I suspect this latter point can easily be addressed.

Reviewer #1 (Public Review):

This study exploits novel agent (IMT) that inhibits mitochondrial activity in combination with venetoclax. While the concept is not novel, the agent is novel (inhibitor of the mitochondrial RNA polymerase, described in Nature in other tumor models), and quest for safe mitochondrial inhibitors is highly warranted. The strength is in vivo activity data shown in CLDX and in one of the two AML PDX models tested, and the apparent safety of the combination. However, the impact on survival is impressive in CLDX but not in PDX, and unclear why Ven-sensitive PDX is resistant to combination (opposite what cell line data show). The paper is lacking mechanistic data beyond Seahorse and standard apoptosis assays, and even transcriptome analysis from PDX cells is poorly analyzed. There is no real evidence that this agent overcome Ven resistance, which could be done for example in primary AML cells. Finally, no on-target pharmacodynamic endpoints are measured in vivo to support the activity of the compound on mitochondrial activity at the doses used (which are safe). These multiple weaknesses significantly reduce my enthusiasm for this manuscript.

The cell line data show additive/synergistic effects of IMT and Ven on cell viability in p53-WT cells. However, no mechanisms of synergy beyond OCR are shown, which is a missed opportunity.

No data are shown in primary AML cells in vitro. This could address venetoclax-resistant AML cells with distinct genomic profiles.

The in vivo CLDX model (MV4;11) data is quite impressive, showing reduction of tumor burden and meaningful extension of survival in combination cohort. It is unclear why venetoclax used at highest dose normally sued in vivo (100mg/kg) did not show any impact on survival in this Ven-sensitive model. It is disappointing that no biomarkers of mitochondrial activity (for example, simple pAMPK, or levels of mitochondrial subunits) are shown to support on-target pharmacodynamic activity. However, efficacy in human PDX is less impressive, for example in Fig 6C the combination has extended survival from 96 to 112 days, possibly due to early stopping of treatment (around day 30); and no extension of survival is seen in another PDX in Fig 7. Still, this is indicative of combinatorial activity in TP53-mutant PDX. There is however discrepancy with in vitro studies that show no impact of combination in TP53 mutant cells and synergy in TP53-wt cells, and the opposite findings in vivo, which is not explained. Overall, the activity of the combination is modest. The safety is encouraging, but again, no pharmacodynamic measurements are shown to support that IMT at least partially inhibited mitochondrial activity in AML cells.

In Discussion the statement that inhibition of POLRMT can overcome venetoclax resistance is not supported by the data, as no additive effects are seen in vitro in TP53 mutant cells, and no other resistant models (such as primary AML cells) are tested. In vivo as stated above there is some activity in TP53 mutant PDX but this alone cannot be sued to justify this strong statement. Also, the sentence that "...we were able to reduce the tumor burden in all (cell- and patient-derived) xenografted mice treated with a combination of IMT and venetoclax" is not supported by data in Fig 7.

Reviewer #2 (Public Review):

Summary:

The manuscript by Arabanian and colleagues presents studies showing how inhibition of mitochondrial transcription and replication with a novel inhibitor of the mitochondrial polymerase, IMT, can promote AML cell death in combination with the Bcl2 inhibitor venetoclax. They further show that this combinatorial efficacy is evident in vivo in both the AML cell line MV411 and in a PDX model. Given the multiple studies showing the importance of Oxphos in maintaining AML cell survival, the current studies provide an additional strategy to inhibit Oxphos and thus improve the therapeutic management of AML.

Strengths:

A novel aspect of this work is that IMT is a new class of mitochondrial inhibitor that acts by inhibiting the mitochondrial polymerase. In addition, the demonstration of therapeutic efficacy both in vitro and in vivo (including with PDX), together with some data showing minimal toxicity, adds to the impact of this work. Their overall conclusion that IMT increases the potency of Vex in treating AMLs is supported.

Weaknesses:

There are several deficiencies that should be addressed to substantiate the rigor and impact of this study. Of most importance, they need to show that IMT actually inhibits the mitochondrial polymerase in AML cells, and there are additional concerns with their models that if addressed would improve the ability of IMT to be developed clinically.

Reviewer #1 (Public Review):

Rebecca R.G. et al. set to determine the function of grid cells. They present an interesting case claiming that the spatial periodicity seen in the grid pattern provides a parsimonious solution to the task of coding 2D trajectories using sequential cell activation. Thus, this work defines a probable function grid cells may serve (here, the function is coding 2D trajectories), and proves that the grid pattern is a solution to that function. This approach is somewhat reminiscent in concept to previous works that defined a probable function of grid cells (e.g., path integration) and constructed normative models for that function that yield a grid pattern. However, the model presented here gives clear geometric reasoning to its case.

Stemming from 4 axioms, the authors present a concise demonstration of the mathematical reasoning underlying their case. The argument is interesting and the reasoning is valid, and this work is a valuable addition to the ongoing body of work discussing the function of grid cells.

However, the case uses several assumptions that need to be clearly stated as assumptions, clarified, and elaborated on: Most importantly, the choice of grid function is grounded in two assumptions:<br /> (1) that the grid function relies on the activation of cell sequences, and<br /> (2) that the grid function is related to the coding of trajectories. While these are interesting and valid suggestions, since they are used as the basis of the argument, the current justification could be strengthened (references 28-30 deal with the hippocampus, reference 31 is interesting but cannot hold the whole case).

The work further leans on the assumption that sequences in the same direction should be similar regardless of their position in space, it is not clear why that should necessarily be the case, and how the position is extracted for similar sequences in different positions. The authors also strengthen their model with the requirement that grid cells should code for infinite space. However, the grid pattern anchors to borders and might be used to code navigated areas locally. Finally, referencing ref. 14, the authors claim that no existing theory for the emergence of grid cell firing that unifies the experimental observations on periodic firing patterns and their distortions under a single framework. However, that same reference presents exactly that - a mathematical model of pairwise interactions that unifies experimental observations. The authors should clarify this point.

Reviewer #2 (Public Review):

Summary:

In this work, the authors consider why grid cells might exhibit hexagonal symmetry - i.e., for what behavioral function might this hexagonal pattern be uniquely suited? The authors propose that this function is the encoding of spatial trajectories in 2D space. To support 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. However, the theory appears to be extremely flexible and very similar to ideas that have been previously proposed regarding grid cell function.

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 to many in the field. 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 major weakness I perceive is that the paper overstates what it delivers, to an extent that I think it can be a bit confusing to determine what the contributions of the paper are. 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." This paper does not provide proof of what grid cells are doing to support behavior or provide the true answer as to why grid patterns are found in the brain. The authors offer some intriguing suggestions or proposals as to why this might be based on what hexagonal patterns could be good for, but I believe that the language should be clarified to be more in line with what the authors present and what the strength of their evidence is.

Relatedly, the authors claim that they find a teleological reason for the existence of grid cells - that is, discover the function that they are used for. However, in the paper, they seem to instead assume a function based on what is known and generally predicted for grid cells (encode position), and then show that for this specific function, grid cells have several attractive properties.

There is also some other work that seems very relevant, as it discusses specific computational advantages of a grid cell code but was not cited here: https://www.nature.com/articles/nn.2901.

A second major weakness was that some of the claims in the section in which they compared their theory to data seemed either confusing or a bit weak. I am not a mathematician, so I was not able to follow all of the logic of the various axioms, remarks, or definitions to understand how the authors got to their final conclusion, so perhaps that is part of the problem. But below I list some specific examples where I could not follow why their theory predicted the experimental result, or how their theory ultimately operated any differently from the conventional understanding of grid cell coding. In some cases, it also seemed that the general idea was so flexible that it perhaps didn't hold much predictive power, as extra details seemed to be added as necessary to make the theory fit with the data.

I don't quite follow how, for at least some of their model predictions, the 'sequence code of trajectories' theory differs from the general attractor network theory. It seems from the introduction that these theories are meant to serve different purposes, but the section of the paper in which the authors claim that various experimental results are predicted by their theory makes this comparison difficult for me to understand. For example, in the section describing the effect of environmental manipulations in a familiar environment, the authors state that the experimental results make sense if one assumes that sequences are anchored to landmarks. But this sounds just like the classic attractor-network interpretation of grid cell activity - that it's a spatial metric that becomes anchored to landmarks.

It was not clear to me why their theory predicted the field size/spacing ratio or the orientation of the grid pattern to the wall.

I don't understand how repeated advancement of one unit to the next, as shown in Figure 4E, would cause the change in grid spacing near a reward.

I don't follow how this theory predicts the finding that the grid pattern expands with novelty. The authors propose that this occurs because the animals are not paying attention to fine spatial details, and thus only need a low-resolution spatial map that eventually turns into a higher-resolution one. But it's not clear to me why one needs to invoke the sequence coding hypothesis to make this point.

The last section, which describes that the grid spacing of different modules is scaled by the square root of 2, says that this is predicted if the resolution is doubled or halved. I am not sure if this is specifically a prediction of the sequence coding theory the authors put forth though since it's unclear why the resolution should be doubled or halved across modules (as opposed to changed by another factor).

Reviewer #3 (Public Review):

The manuscript presents an intriguing explanation for why grid cell firing fields do {\em not} lie on a lattice whose axes aligned to the walls of a square arena. This observation, by itself, merits the manuscript's dissemination to the journals audience.

The presentation is quirky (but keep the quirkiness!).

But let me recast the problem presented by the authors as one of combinatorics. Given repeating, spatially separated firing fields across cells, one obtains temporal sequences of grid cells firing. Label these cells by integers from $[n]$. Any two cells firing in succession should uniquely identify one of six directions (from the hexagonal lattice) in which the agent is currently moving.

Now, take the symmetric group $\Sigma$ of cyclic permutations on $n$ elements.<br /> We ask whether there are cyclic permutations of $[n]$ such that

So, for instance, $(4,2,3,1)$ would not be counted as a valid permutation of $(1,2,3,4)$, as $(2,3)$ and $(1,4)$ are adjacent.

Furthermore, given $[n]$, are there two distinct cyclic permutations such that {\em no} adjacencies are preserved when considering any pair of permutations (among the triple of the original ordered sequence and the two permutations)? In other words, if we consider the permutation required to take the first permutation into the second, that permutation should not preserve any adjacencies.

{\bf Key question}: is there any difference between the solution to the combinatorics problem sketched above and the result in the manuscript? Specifically, the text argues that for $n=7$ there is only {\em one} solution.

Ideally, one would strive to obtain a closed-form solution for the number of such permutations as a function of $n$.