Reviewer #2 (Public review):

In this study, the authors aim to investigate habituation, the phenomenon of increasing reduction in activity following repeated stimuli, in the context of its information-theoretic advantage. To this end, they consider a highly simplified three-species reaction network where habituation is encoded by a slow memory variable that suppresses the receptor and therefore the readout activity. Using analytical and numerical methods, they show that in their model the information gain, the difference between the mutual information between the signal and readout after and before habituation, is maximal for intermediate habituation strength. Furthermore, they demonstrate that the Pareto front corresponds to an optimization strategy that maximizes the mutual information between signal and readout in the steady state, minimizes some form of dissipation, and also exhibits similar intermediate habituation strength. Finally, they briefly compare predictions of their model to whole-brain recordings of zebrafish larvae under visual stimulation.

The author's simplified model might serve as a solid starting point for understanding habituation in different biological contexts as the model is simple enough to allow for some analytic understanding but at the same time exhibits all basic properties of habituation in sensory systems. Furthermore, the author's finding of maximal information gain for intermediate habituation strength via an optimization principle is, in general, interesting. However, the following points remain unclear or are weakly explained:

(1) Is it unclear what the meaning of the finding of maximal information gain for intermediate habituation strength is for biological systems? Why is information gain as defined in the paper a relevant quantity for an organism/cell? For instance, why is a system with low mutual information after the first stimulus and intermediate mutual information after habituation better than one with consistently intermediate mutual information? Or, in other words, couldn't the system try to maximize the mutual information acquired over the whole time series, e.g., the time series mutual information between the stimulus and readout?

(2) The model is very similar to (or a simplification of previous models) for adaptation in living systems, e.g., for adaptation in chemotaxis via activity-dependent methylation and demethylation. This should be made clearer.

(3) It remains unclear why this optimization principle is the most relevant one. While it makes sense to maximize the mutual information between stimulus and readout, there are various choices for what kind of dissipation is minimized. Why was \delta Q_R chosen and not, for instance, \dot{\Sigma}_int or the sum of both? How would the results change in that case? And how different are the results if the mutual information is not calculated for the strong stimulation input statistics but for the background one?

(4) The comparison to the experimental data is not too strong of an argument in favor of the model. Is the agreement between the model and the experimental data surprising? What other behavior in the PCA space could one have expected in the data? Shouldn't the 1st PC mostly reflect the "features", by construction, and other variability should be due to progressively reduced activity levels?

Reviewer #3 (Public review):

The authors use a generic model framework to study the emergence of habituation and its functional role from information-theoretic and energetic perspectives. Their model features a receptor, readout molecules, and a storage unit, and as such, can be applied to a wide range of biological systems. Through theoretical studies, the authors find that habituation (reduction in average activity) upon exposure to repeated stimuli should occur at intermediate degrees to achieve maximal information gain. Parameter regimes that enable these properties also result in low dissipation, suggesting that intermediate habituation is advantageous both energetically and for the purpose of retaining information about the environment.

A major strength of the work is the generality of the studied model. The presence of three units (receptor, readout, storage) operating at different time scales and executing negative feedback can be found in many domains of biology, with representative examples well discussed by the authors (e.g. Figure 1b). A key takeaway demonstrated by the authors that has wide relevance is that large information gain and large habituation cannot be attained simultaneously. When energetic considerations are accounted for, large information gain and intermediate habituation appear to be a favorable combination.

While the generic approach of coarse-graining most biological detail is appealing and the results are of broad relevance, some aspects of the conducted studies, the problem setup, and the writing lack clarity and should be addressed:

(1) The abstract can be further sharpened. Specifically, the "functional role" mentioned at the end can be made more explicit, as it was done in the second-to-last paragraph of the Introduction section ("its functional advantages in terms of information gain and energy dissipation"). In addition, the abstract mentions the testing against experimental measurements of neural responses but does not specify the main takeaways. I suggest the authors briefly describe the main conclusions of their experimental study in the abstract.

(2) Several clarifications are needed on the treatment of energy dissipation.<br /> - When substituting the rates in Eq. (1) into the definition of δQ_R above Eq. (10), "σ" does not appear on the right-hand side. Does this mean that one of the rates in the lower pathway must include σ in its definition? Please clarify.<br /> - I understand that the production of storage molecules has an associated cost σ and hence contributes to dissipation. The dependence of receptor dissipation on , however, is not fully clear. If the environment were static and the memory block was absent, the term with would still contribute to dissipation. What would be the nature of this dissipation?<br /> - Similarly, in Eq. (9) the authors use the ratio of the rates Γ_{s → s+1} and Γ_{s+1 → s} in their expression for internal dissipation. The first-rate corresponds to the synthesis reaction of memory molecules, while the second corresponds to a degradation reaction. Since the second reaction is not the microscopic reverse of the first, what would be the physical interpretation of the log of their ratio? Since the authors already use σ as the energy cost per storage unit, why not use σ times the rate of producing S as a metric for the dissipation rate?

(3) Impact of the pre-stimulus state. The plots in Figure 2 suggest that the environment was static before the application of repeated stimuli. Can the authors comment on the impact of the pre-stimulus state on the degree of habituation and its optimality properties? Specifically, would the conclusions stay the same if the prior environment had stochastic but aperiodic dynamics?

(4) Clarification about the memory requirement for habituation. Figure 4 and the associated section argue for the essential role that the storage mechanism plays in habituation. Indeed, Figure 4a shows that the degree of habituation decreases with decreasing memory. The graph also shows that in the limit of vanishingly small Δ⟨S⟩, the system can still exhibit a finite degree of habituation. Can the authors explain this limiting behavior; specifically, why does habituation not vanish in the limit Δ⟨S⟩ -> 0?

Reviewer #1 (Public review):

Summary:

The paper develops a phase method to obtain the excitatory and inhibitory afferents to certain neuron populations in the brainstem. The inferred contributions are then compared to the results of voltage clamp and current clamp experiments measuring the synaptic contributions to post-I, aug-E, and ramp-I neurons.

Strengths:

The electrophysiology part of the paper is sound and reports novel features with respect to earlier work by JC Smith et al 2012, Paton et al 2022 (and others) who have mapped circuits of the respiratory central pattern generator. Measurements on ramp-I neurons, late-I neurons, and two types of post-I neurons in Figure 2 besides measurements of synaptic inputs to these neurons in Figure 5 are to my knowledge new.

Weaknesses:

The phase method for inferring synaptic conductances fails to convince. The method rests on many layers of assumptions and the inferred connections in Figure 4 remain speculative. To be convincing, such a method ought to be tested first on a model CPG with known connectivity to assess how good it is at inferring known connections back from the analysis of spatio-temporal oscillations. For biological data, once the network connectivity has been inferred as claimed, the straightforward validation is to reconstruct the experimental oscillations (Figure 2) noting that Rybak et al (Rybak, Paton Schwaber J. Neurophysiol. 77, 1994 (1997)) have already derived models for the respiratory neurons.

The transformation from time to phase space, unlike in the Kuramoto model, is not justified here (Line 94) and is wrong. The underpinning idea that "the synaptic conductances depend on the cycle phase and not on time explicitly" is flawed because synapses have characteristic decay times and delays to response which remain fixed when the period of network oscillations increases. Synaptic properties depend on time and not on phase in the network. One major consequence relevant to the present identification of excitatory or inhibitory behaviour, is that it cannot account for change in the behaviour of inhibitory synapses - from inhibitory to excitatory action - when the inhibitory decay time becomes commensurable to the period of network oscillations (Wang & Buzsaki Journal of Neuroscience 16, 6402 (1996), van Vreeswijk et al. J. Comp. Neuroscience 1,313 (1994), Borgers and Kopell Neural Comput. 15, 2003). In addition, even small delays in the inhibitory synapse response relative to the pre-synaptic action potential also produce in-phase synchronization (Chauhan et al., Sci. Rep. 8, 11431 (2018); Borgers and Kopell, Neural Comput. 15, 509 (2003)). The present assumptions are way too simplistic because you cannot account for these commensurability effects with a single parameter like the network phase. There is therefore little confidence that this model can reliably distinguish excitatory from inhibitory synapses when their dynamic properties are not properly taken into account.

Line 82, Equation 1 makes extremely crude assumptions that the displacement current (CdV/dt) is negligible and that the ion channel currents are all negligible. Vm(t) is also not defined. The assumption that the activation/inactivation times of all ion channels are small compared to the 10-20ms decay time of synaptic currents is not true in general. Same for the displacement current. The leak conductance is typically g~0.05-0.09ms/cm^2 while C~1uF/cm^2. Therefore the ratio C/g leak is in the 10-20ms range - the same as the typical docking neurotransmitter time in synapses.

Models of brainstem CPG circuits have been known to exist for decades: JC Smith et al 2012, Paton et al 2022, Bellingham Clin. Exp. Pharm. And Physiol. 25, 847 (1998); Rubin et al., J. Neurophysiol. 101, 2146 (2009) among others. The present paper does not discuss existing knowledge on respiratory networks and gives the impression of reinventing the wheel from scratch. How will this paper add to existing knowledge?

Reviewer #2 (Public review):

Summary:

By measuring intracellular changes in membrane voltage from a single neuron of the medulla the authors describe a method for determining the balance of excitatory and inhibitory synaptic drive onto a single neuron within this important brain region.

Strengths:

This approach could be valuable in describing the microcircuits that generate rhythms within this respiratory control centre. This method could more generally be used to enable microcircuits to be studied without the need for time-consuming anatomical tracing or other more involved electrophysiological techniques.

Weaknesses:

This approach involves assuming the reversal potential that is associated with the different permeant ions that underlie the excitation and inhibition as well as the application of Ohms law to estimate the contribution of excitation and inhibitory conductance. My first concern is that this approach relies on a linear I-V relationship between the measured voltage and the estimated reversal potential. However, open rectification is a feature of any I-V relationship generated by asymmetric distributions of ions (see the GHK current equation) and will therefore be a particular issue for the inhibition resulting from asymmetrical Cl- ion gradients across GABA-A receptors. The mixed cation conductance that underlies most synaptic excitation will also generate a non-linear I-V relationship due to the inward rectification associated with the polyamine block of AMPA receptors. Could the authors please speculate what impact these non-linearities could have on results obtained using their approach?

This approach has similarities to earlier studies undertaken in the visual cortex that estimated the excitatory and inhibitory synaptic conductance changes that contributed to membrane voltage changes during receptive field stimulation. However, these approaches also involved the recording of transmembrane current changes during visual stimulation that were undertaken in voltage-clamp at various command voltages to estimate the underlying conductance changes. Molkov et al have attempted to essentially deconvolve the underlying conductance changes without this information and I am concerned that this simply may not be possible. The current balance equation (1) cited in this study is based on the parallel conductance model developed by Hodgkin & Huxley. However, one key element of the HH equations is the inclusion of an estimate of the capacitive current generated due to the change in voltage across the membrane capacitance. I would always consider this to be the most important motivation for the development of the voltage-clamp technique in the 1930's. Indeed, without subtraction of the membrane capacitance, it is not possible to isolate the transmembrane current in the way that previous studies have done. In the current study, I feel it is important that the voltage change due to capacitive currents is taken into consideration in some way before the contribution of the underlying conductance changes are inferred.

Studies using acute slicing preparations to examine circuit effects have often been limited to the study of small microcircuits - especially feedforward and feedback interneuron circuits. It is widely accepted that any information gained from this approach will always be compromised by the absence of patterned afferent input from outside the brain region being studied. In this study, descending control from the Pons and the neocortex will not be contributing much to the synaptic drive and ascending information from respiratory muscles will also be absent completely. This may not have been such a major concern if this study was limited to demonstrating the feasibility of a methodological approach. However, this limitation does need to be considered when using an approach of this type to speculate on the prevalence of specific circuit motifs within the medulla (Figure 4). Therefore, I would argue that some discussion of this limitation should be included in this manuscript.

Reviewer #1 (Public review):

Summary:

The authors aimed to investigate the interaction between tissue-resident immune cells (microglia) and circulating systemic neutrophils in response to acute, focal retinal injury. They induced retinal lesions using 488 nm light to ablate photoreceptor (PR) outer segments, then utilized various imaging techniques (AOSLO, SLO, and OCT) to study the dynamics of fluorescent microglia and neutrophils in mice over time. Their findings revealed that while microglia showed a dynamic response and migrated to the injury site within a day, neutrophils were not recruited to the area despite being nearby. Post-mortem confocal microscopy confirmed these in vivo results. The study concluded that microglial activation does not recruit neutrophils in response to acute, focal photoreceptor loss, a scenario common in many retinal diseases.

Strengths:

The primary strength of this manuscript lies in the techniques employed.

In this study, the authors utilized advanced Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) to document immune cell interactions in the retina accurately. AOSLO's micron-level resolution and enhanced contrast, achieved through near-infrared (NIR) light and phase-contrast techniques, allowed visualization of individual immune cells without extrinsic dyes. This method combined confocal reflectance, phase-contrast, and fluorescence modalities to reveal various cell types simultaneously. Confocal AOSLO tracked cellular changes with less than 6 μm axial resolution, while phase-contrast AOSLO provided detailed views of vascular walls, blood cells, and immune cells. Fluorescence imaging enabled the study of labeled cells and dyes throughout the retina. These techniques, integrated with conventional histology and Optical Coherence Tomography (OCT), offered a comprehensive platform to visualize immune cell dynamics during retinal inflammation and injury.

Weaknesses:

One significant weakness of the manuscript is the use of Cx3cr1GFP mice to specifically track GFP-expressing microglia. While this model is valuable for identifying resident phagocytic cells when the blood-retinal barrier (BRB) is intact, it is important to note that recruited macrophages also express the same marker following BRB breakdown. This overlap complicates the interpretation of results and makes it difficult to distinguish between the contributions of microglia and infiltrating macrophages, a point that is not addressed in the manuscript.

Another major concern is the time point chosen for analyzing the neutrophil response. The authors assess neutrophil activity 24 hours after injury, which may be too late to capture the initial inflammatory response. This delayed assessment could overlook crucial early dynamics that occur shortly after injury, potentially impacting the overall findings and conclusions of the study.

Reviewer #2 (Public review):

Summary:

This study uses in vivo multimodal high-resolution imaging to track how microglia and neutrophils respond to light-induced retinal injury from soon after injury to 2 months post-injury. The in vivo imaging finding was subsequently verified by an ex vivo study. The results suggest that despite the highly active microglia at the injury site, neutrophils were not recruited in response to acute light-induced retinal injury.

Strengths:

An extremely thorough examination of the cellular-level immune activity at the injury site. In vivo imaging observations being verified using ex vivo techniques is a strong plus.

Weaknesses:

This paper is extremely long, and in the perspective of this reviewer, needs to be better organized.

Study weakness: though the finding prompts more questions and future studies, the findings discussed in this paper are potentially important for us to understand how the immune cells respond differently to different severity levels of injury.

Reviewer #3 (Public review):

Summary:

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature, and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation at the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience. However, there are some issues with the conclusions drawn from the data and analysis that can be addressed to further bolster the manuscript.

Strengths:

Adaptive optics imaging of the murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is beneficial for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, (a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, (b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. While not directly shown in the article, this model would potentially allow for controlling the size, depth, and severity of the laser injury opening interesting avenues for future study.

Weaknesses:

(1) It is unclear based on the current data/study to what extent the mild laser damage phenotype is generalizable to disease phenotypes. The outer nuclear cell loss of 28% and a complete recovery in 2 months would seem quite mild, thus the generalizability in terms of immune-mediated response in the face of retinal remodeling is not certain, specifically whether the key finding regarding the lack of neutrophil recruitment will be maintained with a stronger laser ablation.

(2) Mice numbers and associated statistics are insufficient to draw strong conclusions in the paper on the activity of neutrophils, some examples are below :

a) 2 catchup mice and 2 positive control EAU mice are used to draw inferences about immune-mediated activity in response to injury. If the goal was to show 'feasibility' of imaging these mouse models for the purposes of tracking specific cell type behavior, the case is sufficiently made and already published by the authors earlier. It is possible that a larger sample size would alter the conclusion.

b) There are only 2 examples of extravasated neutrophils in the entire article, shown in the positive control EAU model. With the rare extravasation events of these cells and their high-speed motility, the chance of observing their exit from the vasculature is likely low overall, therefore the general conclusions made about their recruitment or lack thereof are not justified by these limited examples shown.

c) In Figure 3, the 3-day time point post laser injury shows an 18% reduction in the density of ONL nuclei (p-value of 0.17 compared to baseline). In the case of neutrophils, it is noted that "Control locations (n = 2 mice, 4 z-stacks) had 15 {plus minus} 8 neutrophils per sq.mm of retina whereas lesioned locations (n = 2 mice, 4 z-stacks) had 23 {plus minus} 5 neutrophils per sq.mm of retina (Figure 10b). The difference between control and lesioned groups was not statistically significant (p = 0.19)." These data both come from histology. While the p-values - 0.17 and 0.19 - are similar, in the first case a reduction in ONL cell density is concluded while in the latter, no difference in neutrophil density is inferred in the lesioned case compared to control. Why is there a difference in the interpretation where the same statistical test and methodology are used in both cases? Besides this statistical nuance, is there an alternate possibility that there is an increased, albeit statistically insignificant, concentration of circulating neutrophils in the lesioned model? The increase is nearly 50% (15 {plus minus} 8 vs. 23 {plus minus} 5 neutrophils per sq.mm) and the reader may wonder if a larger animal number might skew the statistic towards significance.

(2) The conclusions on the relative activity of neutrophils and microglia come from separate animals. The reader may wonder why simultaneous imaging of microglia and neutrophils is not shown in either the EAU mice or the fluorescently labeled catchup mice where the non-labeled cell type could possibly be imaged with phase-contrast as has been shown by the authors previously. One might suspect that the microglia dynamics are not substantially altered in these mice compared to the CX3CR1-GFP mice subjected to laser lesions, but for future applicability of this paradigm of in vivo imaging assessment of the laser damage model, including documenting the repeatability of the laser damage model and the immune cell behavior, acquiring these data in the same animals would be critical.

(3) Along the same lines as above, the phase contrast ONL images at time points from 3-day to 2-month post laser injury are not shown and the absence of this data is not addressed. This missing data pertains only to the in vivo imaging mice model but are conducted in histology that adequately conveys the time-course of cell loss in the ONL. It is suggested that the reason be elaborated for the exclusion of this data and the simultaneous imaging of microglia and neutrophils mentioned above. Also, it would be valuable to further qualify and check the claims in the Discussion that "ex vivo analysis confirms in vivo findings" and "Microglial/neutrophil discrimination using label-free phase contrast"

Reviewer #1 (Public review):

Summary:

Characterizing the molecular and spatial organization of dendritically localized RNAs is an important endeavor as the authors nicely articulate in their abstract and introduction. In particular, identifying patterns of mRNA distribution and colocalization between groups of RNAs could characterize new mechanisms of transport and/or reveal new functional relationships between RNAs. However, it's not clear to me how much the current study addresses those gaps in knowledge. The manuscript by Kim et al uses 8 overlapping combinations of 3-color fluorescence in situ hybridization to characterize the spatial distributions and pairwise colocalizations of six previously uncharacterized dendritically localized RNAs in cultured neurons (15 DIV). The strength of the work is in the graph-based analyses of individual RNA distances from the soma, but the conclusions reached, that spatial distributions vary per dendritic RNA, has been well known since early 2000s (as reviewed in Schuman and Steward, 2001 & 2003), but paradoxically the authors show that dendritic length can account for these differences. It's not clear to me the significance of the spatial distribution relationship with dendritic morphology as distinct spatial distribution patterns (i.e. proximal expression then drop off) have been clearly shown in intact circuits with homogeneity in dendrite length governed by neuropil laminae. The colocalization results are intriguing but as currently presented they lack sufficient control analyses and contextualization to be compelling. In general, the results of the manuscript are potentially interesting but unnecessarily difficult to follow both in text and figure presentation.

Major comments:

The authors state that their data expand upon our understanding of dendritic RNA spatial distributions by adding high-resolution data for six newly characterized dendritic RNAs. While this is true, without including data for a well-known/previously characterized RNA, it makes it difficult for the reader to contextualize how these new data on six dendritic RNAs fit in with our understanding of the dendritic RNAs with well-described spatial distributions and colocalization analyses (Camk2a, Actb, Map1b, etc). For example, how do we interpret the 7-fold higher colocalization values between RNAs in this manuscript compared to the results of Batish et al (as referred to in the paper)-is it because these RNAs are fundamentally different, or is it because of other experimental factors/conditions? The spatial distribution patterns described in this manuscript differ from those of Fonkeu et al, but an alternative explanation is that Fonkeu et al modeled based on Camk2a, not the six genes studied here. Is it possible that these six RNAs have similar distribution patterns (as shown) whereby dendritic morphology impacts distribution more than individual differences but inclusion of dendritic RNAs with demonstrably different distributions (Camk2a/distal localization vs Map2/proximal localization) would alter the results?

Reviewer #2 (Public review):

In the manuscript by Kim et al titled, "Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution," the authors perform multiplex single-molecule FISH in cultured neurons, along with analysis and modeling, to show the spatial features, including differing mRNA densities between soma and dendrites, dendritic length-related distributions and clustering, of multiple mRNAs in dendrites. Although the clustering analyses and modeling are intriguing and offer previously underappreciated spatial association within and across mRNA molecules, the data is difficult to interpret and the conclusions lack novelty in their current form. There is a need for a stronger rationale as to why the methodology employed in the manuscript is better suited to characterize the clustering of mRNA in dendrites compared to previously published works and how such clustering or declustering can affect dendritic/neuronal function.

(1) Validation of mRNA labeling, detection, and quantification is necessary. Single-molecule fluorescence in situ hybridization (smFISH) is the gold standard to detect RNA inside cells. The method utilizes multiple fluorescent probes (~48) designed to hybridize along a single RNA, resulting in a population of diffraction-limited fluorescent puncta with varying intensities. A histogram of cytoplasmic smFISH puncta intensities should reveal a normally distributed population with a single major peak, where the upper and lower tails indicate the maximum probe binding and the lower detection limit, respectively. Once single molecule detection (and limits) have been established, smFISH should be performed for each gene individually to obtain ground truth of detection under identical experimentally-defined conditions using the same fluorophore. Total RNA counts from different probe combinations (Figure S1A) or total mRNA density (Figure 2A) is not sufficient to inform individual gene labeling efficiency or detection. It is difficult to interpret whether observed variabilities across different probe combinations are of significance. For example, the mRNA densities of Adap2 and Dtx3L in soma seem to vary even after normalization with the pixel area (Figure 2A).

Absolute counts and normalized counts for each gene detected should be included in the results or in supplementary data/table to provide the reader with a reference point for evaluation.

As a control, it is recommended to perform smFISH against beta-actin or aCaMKII, which are the two most abundant mRNA in dendrites, and serve as internal validation that the technique, detection, and quantification are consistent with previously published works.

(2) The rationale for single dendrite selection is unclear. To suggest that dendrite length, as a feature of dendritic morphology, may affect mRNA localization in dendrites, the authors manually selected segments of dendrites that have no branching or overlap, 'biased for shorter dendrites,' resulting in a subset of dendritic segments that changes mRNA distribution in raw distances (Figure S3A) into the normalized distance (Figure 4A). As a result, the distribution appears to convert from a monotonic- or exponential-decay to a more even distribution of mRNA (plateau). The rationale for this normalization is unclear, as manual curation of dendritic segments can incorporate experimenter bias. Moreover, the inclusion of short dendritic segments can stretch out their mRNA distributions following distance normalization which can give the appearance of an even distribution of mRNAs when aggregated.

Next, the authors use pairwise Jensen-Shannon distance cluster analysis to identify 4 different patterns of clustering among mRNAs. Although the patterns are quite intriguing, the distributions of mRNA clusters were i) difficult to interpret and ii) compared to Fonkeu et al (2019) protein distribution is not a sufficient explanation for the observed clustering. For example, the clustering patterns (C1-4) are quite striking and even if the authors' analyses were an improvement in identifying mRNA clustering in dendrites, the authors need to provide better justification or modeling on what role such clustering can play on dendritic function or cellular physiology. This is important and necessary as the authors are suggesting that their analysis is different from mRNA distributions previously observed or modeled by Buxbaum et al (2014) and Fonkeu et al (2019), respectively.<br /> Of note, the identity-independent and dendritic length-dependent aspect of spatial distributions of mRNAs is striking (Figure S3E-F, Figure 4), and this length-related feature is one of the major take-home points in the first part of the manuscript. However, it is evident that some mRNAs (e.g. Adap2 and Dtx3L) or probe combinations (e.g. Colec12-Adap2-Nsmf) disproportionally make up the mRNA distribution clusters (Figure 4D and Figure S3F). It seems plausible that the copy numbers of mRNAs can differentially affect clusters' distribution patterns. Appropriate statistical tests among the cluster groups, therefore, will help to strengthen the interpretation of the results provided in the supplementary figures (Figures S3E and S3F).

(3) It is not clear how Figure 5 GradCAM analysis helps the point that the authors put forth in previous sections or forthcoming sections. Unless this section and figure are more effectively linked to the general theme of the paper - the morphological features as a determinant of mRNA distribution or clustering of mRNA molecules, it may be included in the supplementary figure section.

(4) Clustering of mRNA remains an exception rather than the rule. From their high-resolution triple smFISH data, the authors make some interesting findings regarding colocalization in dendrites. Among the six genes tested, the authors found higher incidents of colocalization between pair-wise genes (up to 23%) than previously reported (5-10%). Also, they report higher levels of colocalization within the same gene (17-23%) than previously reported (5-10%). First, to better evaluate this increased colocalization efficiency overall, the histograms of smFISH puncta intensity are necessary (as stated in 1) to determine whether a second peak is present in the population. Second, even though 23% is higher than previously reported, it remains that 77% do not colocalize and does not suggest that colocalization is the rule but remains the exception. Given the results in Table 1, it is likely that the increased colocalization could be a gene-specific effect and not transcriptome-wide as the majority of values between genes are below 10%, consistent with previous findings. Third, labeling of a control gene (i.e. b-actin or aCaMKII) would provide higher confidence that the detection and colocalization comparisons are consistent with previous findings.

It is recommended to refrain from concluding that mRNA is 'co-transported' from smFISH results. Typically co-transport is best identified through observations in live cells where two fluorescent particles of different colors are moving together. Although stationary particles positioned in close proximity to one another could potentially be co-transported, there has been very little evidence to support this.

The use of Ripley's K-function is an interesting way to look at clustering neighborhoods within a single or pairwise sets of genes. Previous studies from the Singer group have looked at mRNA clustering and have observed that mRNA in living cells tends to cluster within a 6-micron range for b-actin and for both b-actin and Arc after local stimulation. What was intriguing in the results in Figure 7 was that there was an exclusion zone 2-4 microns away from the area of colocalization that may suggest that mRNA are able to avoid over-clustering and maintain an even distribution throughout the dendrite--perhaps with a goal of not devoting too many resources (mRNA) to a single dendritic area. Modeling how mRNAs avoid over-clustering to a specific 2-micron segment of dendrites could provide an explanation on how dendrites can respond to multiple or simultaneous synaptic activity at different sites along the same dendrite.

Reviewer #3 (Public review):

Summary:

The paper by Kim et al utilizes smFISH method to probe for six genes to understand the spatial distribution of the mRNAs in dendrites and identify the spatial relationships between the transcripts. While they have delved into a high-resolution characterization of the dendritic transcripts and compared their data with existing datasets, the analysis needs more robustness, and therefore the findings are inconclusive. The rationale of the study and choosing these genes is not clear - it appears more like a validation of some of the datasets without much biological significance.

Overall, several conclusions for spatial distribution of dendritic RNAs were based on correlations and it is difficult to understand whether this represents a true biological phenomenon or if it is an artifact of the imaging and morphological heterogeneity of neurons and difficulties in dendritic segmentation.

Strengths:

The authors have performed an extensive analysis of the smFISH datasets and quantified the precise localization patterns of the dendritic mRNAs in relation to the dendritic morphology. Their images and the analysis pipeline can be a resource for the community.

Weaknesses:

(1) The authors have attempted to identify general patterns of mRNA distribution as a function of distance, proximal vs distal, however, in many of the cases the results are a bit redundant and the size of the neurons or the length of the dendrites or image segmentation artifacts turn out to be the determining factors. A better method to normalize the morphological differences is needed to make meaningful conclusions about RNA distribution patterns.

(2) Another concerning factor is that there are many redundancies throughout the paper. For example, to begin with, all analysis should have been done as RNA density measurements (and not absolute numbers of mRNAs) and with proper normalization and accounting for differences in length. Some of these were done only in the latter half of the paper, for example in Figure 4.

(3) Images for the smFISH are missing. It is important to show the actual images, and the quality of the images is a crucial factor for all subsequent analyses.

(4) The parameters used for co-localization analysis are very relaxed (2 - 6 microns), particularly the distances of interactions far exceed feasible interactions between the biomolecules. Typically, transport granules are significantly smaller than the length scales used.

Reviewer #1 (Public review):

Summary:

The work of Zhou's team is to perform bioinformatics analysis of single-cell transcriptomes (scRNA), spatial transcriptomic (ST) data, and bulk RNA-seq data from Gene Expression Omnibus (GEO) datasets, published or not in different journals from other teams, about spinal cord injury and/or microglia cells derived human iPSC. Based on their analysis, the authors claim that innate microglial cells are inhibited. They postulate that TGF beta signaling pathways play a role in the regulation of migration to enhance SCI recovery and that Trem2 expression contributes to neuroinflammation response by modulating cell death in spinal cord injury. Finally, they suggest a therapeutic strategy to inhibit Trem2 responses and transplant iPSC-derived microglia with long-term TGF beta stimulation.

Although the idea of using already available data and reanalyzing them is remarkable, I have major concerns about the paper. The authors have used data from different models of injury, regions, as well as IPSC. It is not possible to mix and draw conclusions when the models used are different. This raises doubts about the authors' expertise in the field of spinal cord injury. Furthermore, the innovativeness of the results is of little significance, especially as no hypothesis is confirmed by experimental data.

Strengths:

Analysis of already large-scale existing data.

Weaknesses:

Mixing data from different models, unfounded conclusions, and over-interpretations, little expertise in the field of spinal cord injury.

Reviewer #2 (Public review):

Summary:

The authors present an intriguing study utilizing datasets from spinal cord injury (SCI) research to identify potential microglial genes involved in SCI-induced neuronal damage. They identify that inhibiting TREM2 and enhancing the TGF-b signal pathway can inhibit reactive microglia-mediated neuroinflammation. Microglia transplantation using iPSC-derived microglia could also be beneficial for SCI recovery.

Strengths:

This research aims to identify potential genes and signaling pathways involved in microglia-mediated inflammation in spinal cord injury (SCI) models. Meanwhile, analyzing transplanted microglia gene expression provides an extra layer of potential in SCI therapy. The approach represents a good data mining strategy for identifying potential targets to combat neurological diseases.

Weaknesses:

Microglial gene expression patterns may vary significantly between these models. Without proper normalization or justification, combining these datasets to draw conclusions is problematic. Moreover, other factors also need to be considered, like the gender of the microglia source. Are there any gender differences? How were the iPSC-derived microglia generated? Different protocols may affect microglia gene expression.

While the concept is interesting, the data presented in this study appears preliminary. Without further experiments to support their findings, the conclusions are not convincing.

Reviewer #3 (Public review):

Summary:

In this study, the authors perform a meta-analysis of existing transcriptomic data describing the responses of cells in the mouse spinal cord to traumatic injury (SCI). They identify two subclasses of microglia, which they term 'innate' and 'reactive' microglia, in the dataset, with the majority of microglia in the uninjured spinal cord being 'innate' and the majority of microglia in the injured region being 'reactive'. The authors propose that, during injury, the population of innate microglia is depleted and replaced by the population of reactive microglia. Using DEG and gene ontology pipelines, the authors suggest that TGF signaling is a positive force that helps recruit healthy microglia to enhance recovery in the context of SCI. In contrast, the microglial phagocytic receptor Trem2 contributes to neuroinflammation and neuronal death. Finally, the authors suggest replacing reactive microglia with innate microglia as a potential therapeutic approach to treat SCI in humans.

Strengths:

The work utilizes numerous and multi-modal datasets describing transcriptomic changes in the mouse CNS following SCI.

The topic is translationally relevant.

Weaknesses:

There is not enough information about how each of the datasets re-analyzed by the authors was obtained and processed both by the group generating the data and by the group re-analyzing it.

The conclusions drawn by the authors are not sufficiently supported by the evidence.

Whether the study represents a significant conceptual advance in our understanding of microglial contributions to SCI is not clear.

My specific concerns and suggestions to address these weaknesses are provided below.

Major comments:

(1) Questions remain about the nature, quality, and features of the datasets re-analyzed in the study. For example, how were these datasets obtained? Were the same animal models and time points used in each? What modality of RNA sequencing was done? What criteria did the authors consider in deciding which datasets to include in the study? Since the study is entirely reliant on data generated elsewhere, a more thorough description of these datasets within the text is needed.

(2) Relatedly, the authors chose to filter out some cells from the datasets based on quality, but this information is incomplete. For example, the authors omit cells with 10% mitochondrial genes, but this value is higher than most investigators use (typically between 1%-5%). Why is 10% the appropriate limit in this particular study? Further, how did the authors ensure the removal of doublets from the dataset?

(3) A principal finding of the paper is that microglia in the uninjured CNS mostly have an 'innate' transcriptomic phenotype, while microglia in the injured CNS mostly have a 'reactive' phenotype. However, there are some issues here that require further discussion. First, while historically microglia were thought to possess distinct 'homeostatic' versus 'activated' profiles which would be consistent with the authors' interpretations here, these differences are now thought of more as changes in a given microglial cell's transcriptomic status. Thus, while the authors interpret their results as meaning that innate microglia are depleted and replaced by a different set of reactive microglia following SCI (or at least this is how the paper is written), it is equally if not more likely that the microglia within the injured regions themselves become more reactive as a result of the insult. The authors should clarify why their interpretation is more likely to be correct.

(4) Related to the above point, the authors base the manuscript on the idea that microglia are mostly 'innate' in the uninjured CNS and 'reactive' after injury, however, the UMAP plots in Figures 1A and 1C suggest that both classes of microglia cluster together and may not actually represent distinct subclasses. Have the authors tried sub-clustering just the myeloid clusters and seeing how well they separate? Even if they do technically represent distinct clusters, the UMAP could be interpreted to mean that their transcriptomic differences are not particularly robust.

(5) I appreciate the authors' use of loss-of-function data to explore the roles of microglial TGF and Trem2 signaling to glean some mechanistic insights into SCI. However, many of the conclusions reached by the authors in the manuscript are insufficiently supported by the data and would require additional experiments to rigorously confirm. A couple of examples are the following:<br /> 5a. Lines 160-162: "Hence, we conclude that the cascade of injury events in SCI significantly influences microglia, leading to the replacement of innate microglial cells by reactive microglia." That SCI influences microglia is well-supported by the study, but whether reactive microglia replace innate microglia, versus whether innate microglia in the region transition to a reactive state, needs to be tested experimentally.<br /> 5b. Lines 321-323: "Taken together, iPSC-derived microglia have the potential to replace the functions of naïve microglial cells, and they perform even more effectively in the in vivo CNS." Again, the first part of the sentence is supported, but whether iPSCs are more effective than other populations in vivo would need to be tested experimentally.

(6) As microglia have long been appreciated as contributors to the CNS injury response, the conceptual advance here isn't particularly clear to me. For example, Gao et al, 2023 (*cited by the authors) describe the role of Trem2+ microglia in SCI versus demyelinating disease with major conceptual overlap with the current study. It would be helpful for the authors to include a discussion of what we now know about SCI based on this study that we did not know (or strongly suspect) before.

Reviewer #1 (Public review):

Summary:

The manuscript co-authored by Pál Barzó et al is very clear and very well written, demonstrating the electrophysiological and morphological properties of human cortical layer 2/3 pyramidal cells across a wide age range, from age 1 month to 85 years using whole-cell patch clamp. To my knowledge, this is the first study that looks at the cross-age differences in biophysical and morphological properties of human cortical pyramidal cells. The community will also appreciate the significant effort involved in recording data from 485 cells, given the challenges associated with collecting data from human tissue. Understanding the electrophysiological properties of individual cells, which are essential for brain function, is crucial for comprehending human cortical circuits. I think this research enhances our knowledge of how biophysical properties change over time in the human cortex. I also think that by building models of human single cells at different ages using these data, we can develop more accurate representations of brain function. This, in turn, provides valuable insights into human cortical circuits and function and helps in predicting changes in biophysical properties in both health and disease.

Strengths:

The strength of this work lies in demonstrating how the electrophysiological and morphological features of human cortical layer 2/3 pyramidal cells change with age, offering crucial insights into brain function throughout life.

Weaknesses:

One potential weakness of the paper is that the methodology could be clearer, especially in how different cells were used for various electrophysiological measurements and the conditions under which the recordings were made. Clarifying these points would improve the study's rigor and make the results easier to interpret.

Reviewer #2 (Public review):

Summary:

In this study, Barzo and colleagues aim to establish an appraisal for the development of basal electrophysiology of human layer 2/3 pyramidal cells across life and compare their morphological features at the same ages.

Strengths:

The authors have generated recordings from an impressive array of patient samples, allowing them to directly compare the same electrophysiological features as a function of age and other biological features. These data are extremely robust and well organised.

Weaknesses:

The use of spine density and shape characteristics is performed from an extremely limited sample (2 individuals). How reflective these data are of the population is not possible to interpret. Furthermore, these data assume that spines fall into discrete types - which is an increasingly controversial assumption.

Many data are shown according to somewhat arbitrary age ranges. It would have been more informative to plot by absolute age, and then perform more rigourous statistics to test age-dependent effects.

Overall, the authors achieve their aims by assessing the physiological and morphological properties of human L2/3 pyramidal neurons across life. Their findings have extremely important ramifications for our understanding of human life and implications for how different neuronal properties may influence neurological conditions.

Reviewer #3 (Public review):

Summary:

To understand the specificity of age-dependent changes in the human neocortex, this paper investigated the electrophysiological and morphological characteristics of pyramidal cells in a wide age range from infants to the elderly.

The results show that some electrophysiological characteristics change with age, particularly in early childhood. In contrast, the larger morphological structures, such as the spatial extent and branching frequency of dendrites, remained largely stable from infancy to old age. On the other hand, the shape of dendritic spines is considered immature in infancy, i.e., the proportion of mushroom-shaped spines increases with age.

Strengths:

Whole-cell recordings and intracellular staining of pyramidal cells in defined areas of the human neocortex allowed the authors to compare quantitative parameters of electrophysiological and morphological properties between finely divided age groups.

They succeeded in finding symmetrical changes specific to both infants and the elderly, and asymmetrical changes specific to either infants or the elderly. The similarity of pyramidal cell characteristics between areas is unexpected.

Weaknesses:

Human L2/3 pyramidal cells are thought to be heterogeneous, as L2/3 has expanded to a high degree during the evolution from rodents to humans. However, the diversity (subtyping) is not revealed in this paper.

Reviewer #1 (Public review):

Summary:

Oor et al. report the potentially independent effects of the spatial and feature-based selection history on visuomotor choices. They outline compelling evidence, tracking the dynamic history effects based on their clever experimental design (urgent version of the search task). Their finding broadens the framework to identify variables contributing to choice behavior and their neural correlates in future studies.

Strengths:

In their urgent search task, the variable processing time of the visual cue leads to a dichotomy in choice performance - uninformed guesses vs. informed choices. Oor et al. did rigorous analyses to find a stronger influence of the location-based selection history on the uninformed guesses and a stronger influence of the feature-based selection history on the informed choices. It is a fundamental finding that contributes to understanding the drivers of behavioral variance. The results are clear.

Weaknesses:

(1) In this urgent search task, as the authors stated in line 724, the variability in performance was mainly driven by the amount of time available for processing the visual cue. The authors used processing time (PT) as the proxy for this "time available for processing the visual cue." But PT itself is already a measure of behavioral variance since it is also determined by the subject's reaction time (i.e., PT = Reaction time (RT) - Gap). In that sense, it seems circular to explain the variability in performance using the variability in PT. I understand the Gap time and PT are correlated (hinted by the RT vs. Gap in Figure 1C), but Gap time seems to be more adequate to use as a proxy for the (imposed) time available for processing the visual cue, which drives the behavioral variance. Can the Gap time better explain some of the results? It would be important to describe how the results are different (or the same) if Gap time was used instead of PT and also discuss why the authors would prefer PT over Gap time (if that's the case).

(2) The authors provide a compelling account of how the urgent search task affords<br /> (i) more pronounced selection history effects on choice and<br /> (ii) dissociating the spatial and feature-based history effects by comparing their different effects on the tachometric curves. However, the authors didn't discuss the limits of their task design enough. It is a contrived task (one of the "laboratoray tasks"), but the behavioral variability in this simple task is certainly remarkable. Yet, is there any conclusion we should avoid from this study? For instance, can we generalize the finding in more natural settings and say, the spatial selection history influences the choice under time pressure? I wonder whether the task is simple yet general enough to make such a conclusion.

(3) Although the authors aimed to look at both inter- and intra-trial temporal dynamics, I'm not sure if the results reflect the true within-trial dynamics. I expected to learn more about how the spatial selection history bias develops as the Gap period progresses (as the authors mentioned in line 386, the spatial history bias must develop during the Gap interval). Does Figure 3 provide some hints in this within-trial temporal dynamics?

(4) The monkeys show significant lapse rates (enough error trials for further analyses). Do the choices in the error trials reflect the history bias? For example, if errors are divided in terms of PTs, do the errors with short PT reflect more pronounced spatial history bias (choosing the previously selected location) compared to the errors with long PT?

Reviewer #2 (Public review):

Summary:

This is a clear and systematic study of trial history influences on the performance of monkeys in a target selection paradigm. The primary contribution of the paper is to add a twist in which the target information is revealed after, rather than before, the cue to make a foveating eye movement. This twist results in a kind of countermanding of an earlier "uninformed" saccade plan by a new one occurring right after the visual information is provided. As with countermanding tasks in general, time now plays a key factor in the success of this task, and it is time that allows the authors to quantitatively assess the parametric influences of things like previous target location, previous target identity, and previous correctness rate on choice performance. The results are logical and consistent with the prior literature, but the authors also highlight novelties in the interpretation of prior-trial effects that they argue are enabled by the use of their paradigm.

Strengths:

Careful analysis of a multitude of variables influencing behavior

Weaknesses:

Results appear largely confirmatory.

Reviewer #1 (Public review):

Summary:

These authors have asked how lytic phage predation impacts antibiotic resistance and virulence phenotypes in methicillin-resistant Staphylococcus aureus (MRSA). They report that staphylococcal phages cause MRSA strains to become sensitized to b-lactams and to display reduced virulence. Moreover, they identify mutations in a set of genes required for phage infection that may impact antibiotic resistance and virulence phenotypes.

Strengths:

Phage-mediated re-sensitization to antibiotics has been reported previously but the underlying mutational analyses have not been described. These studies suggest that phages and antibiotics may target similar pathways in bacteria.

Weaknesses:

One limitation is the lack of mechanistic investigations linking particular mutations to the phenotypes reported here. This limits the impact of the work.

Another limitation of this work is the use of lab strains and a single pair of phages. However, while incorporation of clinical isolates would increase the translational relevance of this work it is unlikely to change the conclusions.

Reviewer #2 (Public review):

Summary:

The work presented in the manuscript by Tran et al deals with bacterial evolution in the presence of bacteriophage. Here, the authors have taken three methicillin-resistant S. aureus strains that are also resistant to beta-lactams. Eventually, upon being exposed to phage, these strains develop beta-lactam sensitivity. Besides this, the strains also show other changes in their phenotype such as reduced binding to fibrinogen and hemolysis.

Strengths:

The experiments carried out are convincing to suggest such in vitro development of sensitivity to the antibiotics. Authors were also able to "evolve" phage in a similar fashion thus showing enhanced virulence against the bacterium. In the end, authors carry out DNA sequencing of both evolved bacteria and phage and show mutations occurring in various genes. Overall, the experiments that have been carried out are convincing.

Weaknesses:

Although more experiments are not needed, additional experiments could add more information. For example, the phage gene showing the HTH motif could be reintroduced in the bacterial genome and such a strain can then be assayed with wildtype phage infection to see enhanced virulence as suggested. At least one such experiment proves the discoveries regarding the identification of mutations and their outcome. Secondly, I also feel that authors looked for beta-lactam sensitivity and they found it. I am sure that if they look for rifampicin resistance in these strains, they will find that too. In this case, I cannot say that the evolution was directed to beta-lactam sensitivity; this is perhaps just one trait that was observed. This is the only weakness I find in the work. Nevertheless, I find the experiments convincing enough; more experiments only add value to the work.

Reviewer #1 (Public review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being to explain if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's circadian rhythms. This concept is rooted in understanding dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. The study participants self-reported the primary outcome, body weight loss.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

Weaknesses:

The methodology lacks some measurements that are emerging as very relevant in the field of nutritional science, such as data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity). The primary outcome's reliance on self-reported body weight and subsequent measurement biases undermines the reliability of the findings.

Achievement of Objectives and Support for Conclusions:

The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the aforementioned weaknesses. The evidence does not fully support most of the claims due to methodological limitations caused partially by the COVID-19 pandemic.

Impact and Utility:

Despite its innovative approach, the study's utility for practical application is limited by methodological and analytical shortcomings. Nevertheless, it represents a good basis for further research. If these findings were further investigated, they could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

Reviewer #2 (Public review):

The authors tested a dietary intervention focused on improving meal regularity. Participants first utilized a smartphone application to track participants' meal frequencies, participants were then asked to restrict their meal intake to times when they most often eat to enhance meal regularity for six weeks, resulting in significant weight loss despite supposedly no changes in caloric intake.

While the concept is appealing, and the use of a smartphone app in participants' typical everyday environment to regularize food intake is interesting, significant weaknesses severely limit the value of the study due to a lack of rigor, such as the reliance on self-reported food intake which has been discredited in the field. The study's major conclusions are insufficiently supported, particularly that weight loss occurred even though food intake supposedly is not altered. This intervention may merely represent another restrictive diet among countless others that all seem to work for a few weeks to months resulting in a few pounds of weight loss

(1) Unreliable method of caloric intake

The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake. For example, as cited in the revised manuscript, the NEJM paper (DOI: 10.1056/NEJM199212313272701) reported that some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake should have been utilized. Further, the control group was not asked to restrict their diet in any way, and hence, to do that in the treatment group may be sufficient to reduce caloric intake and weight loss. Merely acknowledging the unreliability of self-reported caloric intake is insufficient, as it still leaves the reader with the impression that there is no change in food intake when, in reality, we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced that a more rigorous study would unveil testing this paradigm, this intervention may merely represent another restrictive diet among countless others that show that one may lose weight by going on a diet. Seemingly, any restrictive diet works for a few months.

(2) Lack of objective data regarding circadian rhythm

The assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, is unreliable, and it is unclear why more objective methods like actigraphy was not used.

In the revised version, the authors emphasize these limitations in the manuscript. The study's major conclusions are insufficiently supported, in particular, that weight loss occurred even though food intake supposedly is not altered and that circadian rhythm was improved.

Reviewer #1 (Public review):

Summary:

The report examines the control of the antiviral RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. K3 binds to PKR, hindering its ability to control protein translation by blocking its phosphorylation of the eukaryotic initiation factor EIF2α. Kinase function is probed by saturation mutation of the K3/EIF2α-binding surface on PKR, guided by models of their interaction. The findings identify specific residues at the predicted interface that asymmetrically influence repression by K3 and the phosphorylation of EIF2α. This recognises the potential of PKR alleles to resist control by the viral virulence factor.

Strengths:

The experimentation is diligent, generating and screening many point mutants to identify residues at the interface between PKR and EIF2α or K3 that distinguishes PKR's phosphor control of its substrate from the antithetical interaction with the viral virulence factor.

Weaknesses:

The protein interaction between PKR and K3 has already been well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays. Accordingly, the findings are not pioneering but reinforce and extend what had previously been established.

The authors responded to this comment by pointing out that their more comprehensive screen better defined the extent of the plasticity of the K3/EIF2α-binding surface on PKR.

Also in their response, the authors added the caveat that the equivalent expression of the different PKR mutants has not been verified, added information clarifying the states of the model proteins compared to their determined molecular structures, and provided clarifications or responses to all other questions.

I question eLife's assessment that the development of the yeast-based assay is a key advancement of this report, as this assay has been used for over 30 years.

Reviewer #2 (Public review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the kinase domain of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings: The findings are important with implications to various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, antiviral immunity.

Strength of the evidence: Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths

Systematic and Unbiased Approach: The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.<br /> Identification of Key Sites: The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.<br /> Evolutionary Implications: The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").<br /> Experimental Design: The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas of improvement

Validation of the screen: In the revised version, the authors now provide the results of two independent experiments in a complete yeast growth assay on a handful of candidates to control the screen's results. This strengthens the direct findings from the screen. It would strengthen the study to complement this validation by another method to assess PKR functions; for example, in human PKR-KO cells, because results between yeast and human cells can differ. These limitations are now acknowledged in the revised version.<br /> Evolutionary Data: Beyond residues under positive selection, the screen allows the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites. The authors now discuss the implications for the conserved residues.<br /> Mechanistic insights and viral diversity: While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms and the diversity of viral antagonists. The authors have now acknowledged these limitations in the Discussion and updated the manuscript to be more specific. These exciting research avenues will be the objectives of a next study.

Overall Assessment

The systematic approach, identification of key sites, and evolutionary implications are all notable strengths. While there is room for a stronger validation of the functions and further investigation into the mechanistic details and broader viral diversity, the findings are robust and already provide important advancements. The manuscript is well-written and clear, and the revised figures are informative and improved.

Reviewer #1 (Public Review):

Summary:

This study retrospectively analyzed clinical data to develop a risk prediction model for pulmonary hypertension in high-altitude populations. This finding holds clinical significance as it can be used for intuitive and individualized prediction of pulmonary hypertension risk in these populations. The strength of evidence is high, utilizing a large cohort of 6,603 patients and employing statistical methods such as LASSO regression. The model demonstrates satisfactory performance metrics, including AUC values and calibration curves, enhancing its clinical applicability.

Strengths:

(1) Large Sample Size: The study utilizes a substantial cohort of 6,603 subjects, enhancing the reliability and generalizability of the findings.

(2) Robust Methodology: The use of advanced statistical techniques, including least absolute shrinkage and selection operator (LASSO) regression and multivariate logistic regression, ensures the selection of optimal predictive features.

(3) Clinical Utility: The developed nomograms are user-friendly and can be easily implemented in clinical settings, particularly in resource-limited high-altitude regions.

(4) Performance Metrics: The models demonstrate satisfactory performance, with strong AUC values and well-calibrated curves, indicating accurate predictions.

Weaknesses:

(1) Lack of External Validation: The models were validated internally, but external validation with cohorts from other high-altitude regions is necessary to confirm their generalizability.

(2) Simplistic Predictors: The reliance on ECG and basic demographic data may overlook other potential predictors that could improve the models' accuracy and predictive power.

(3) Regional Specificity: The study's cohort is limited to Tibet, and the findings may not be directly applicable to other high-altitude populations without further validation.

Comments on revised version:

The authors have made revisions in response to the primary concerns raised in the initial review, leading to significant improvements in the manuscript's technical accuracy, formatting consistency, and overall clarity. They have provided a detailed explanation of the selection criteria for the final model variables, which has enhanced the transparency and robustness of the study's methodology. Additionally, the authors have acknowledged the limitation of lacking external validation in cohorts from other high-altitude regions and outlined their plans for future research to address this issue.

Reviewer #1 (Public review):

This study presents a large cohort of plasma-derived extracellular vesicle samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. The authors identified a panel of protein markers for the early detection of pancreatic cancer and validated in an external cohort.

Reviewer #2 (Public review):

This work investigates the use of extracellular vesicles (EVs) in blood as a noninvasive 'liquid biopsy' to aid in differentiation of patients with pancreatic cancer (PDAC) from those with benign pancreatic disease and healthy controls, an important clinical question where biopsies are frequently non-diagnostic. The use of extracellular vesicles as biomarkers of disease has been gaining interest in recent history, with a variety of published methods and techniques, looking at a variety of different compositions ('the molecular cargo') of EVs particularly in cancer diagnosis (Shah R, et al, N Engl J Med 2018; 379:958-966).

This study adds to the growing body of evidence in using EVs for earlier detection of pancreatic cancer, identifying both new and known proteins of interest. Limitations in studying EVs in general include dealing with low concentrations in circulation and identifying the most relevant molecular cargo. This study provides validation of assaying EVs using the novel EVtrap method (Extracellular Vesicles Total Recovery And Purification), which the authors show to be more efficient than current standard techniques and potentially more scalable for larger clinical studies.

The strength of this study is in its numbers - the authors worked with a cohort of 124 cases, 93 of them which were PDAC samples, which considered large for an EV study (Jia, E et al. BMC Cancer 22, 573 (2022)). The benign disease group (n=20, between chronic pancreatitis and IPMNs) and healthy control groups (n=11) were relatively small, but the authors were not only able to identify candidate biomarkers for diagnosis that clearly stood out in the PDAC cohort, but also validate it in an independent cohort of 36 new subjects. Proteins they've identified as associated with pancreatic cancer over benign disease included PDCD6IP, SERPINA12 and RUVBL2. They were even able to identify a set of EV proteins associated with metastasis and poorer prognosis , which include the proteins PSMB4, RUVBL2 and ANKAR and CRP, RALB and CD55. Their 7-EV protein signature yielded an 89% prediction accuracy for the diagnosis of PDAC against a background of benign pancreatic diseases that is compelling and comparable to other studies in the literature (Jia, E. et al. BMC Cancer 22, 573 (2022)).

The limitations of this study are its containment within a single institution - further studies are warranted to apply the authors' 7-EV protein PRAC panel to multiple other cases at other institutions in a larger cohort.

Reviewer #1 (Public review):

Summary:

The manuscript by Mäkelä et al. presents compelling experimental evidence that the amount of chromosomal DNA can become limiting for the total rate of mRNA transcription and consequently protein production in the model bacterium Escherichia coli. Specifically, the authors demonstrate that upon inhibition of DNA replication the rate of RNA transcription and the single-cell growth rate continuously decrease, the latter in direct proportion to the concentration of active ribosomes, as measured indirectly by single-particle tracking. The decrease of ribosomal activity with filamentation is likely caused by a decrease of the concentration of mRNAs, as suggested by an observed plateau of the total number of active RNA polymerases. These observations are compatible with the hypothesis that DNA limits the total rate of transcription and thus, indirectly, translation.

The authors also demonstrate that the decrease of RNAp activity is independent of two candidate stress response pathways, the SOS stress response and the stringent response, as well as an anti-sigma factor previously implicated in variations of RNAp activity upon variations of nutrient sources.

Remarkably, the reduction of growth rate is observed soon after the inhibition of DNA replication, suggesting that the amount of DNA in wild-type cells is tuned to provide just as much substrate for RNA polymerase as needed to saturate most ribosomes with mRNAs. While previous studies of bacterial growth have most often focused on ribosomes and metabolic proteins, this study provides important evidence that chromosomal DNA has a previously underestimated important and potentially rate-limiting role for growth.

Strengths:

This article links the growth of single cells to the amount of DNA, the number of active ribosomes and to the number of RNA polymerases, combining quantitative experiments with theory. The correlations observed during depletion of DNA, notably in M9gluCAA medium, are compelling and point towards a limiting role of DNA for transcription and subsequently for protein production soon after reduction of the amount of DNA in the cell. The article also contains a theoretical model of transcription-translation that contains a Michaelis-Menten type dependency of transcription on DNA availability and is fit to the data.

At a technical level, single-cell growth experiments and single-particle tracking experiments are well described, suggesting that different diffusive states of molecules represent different states of RNAp/ribosome activities, which reflect the reduction of growth.

Apart from correlations in DNA-deplete cells, the article also investigates the role of candidate stress response pathways for reduced transcription, demonstrating that neither the SOS nor the stringent response are responsible for the reduced rate of growth. Equally, the anti-sigma factor Rsd recently described for its role in controlling RNA polymerase activity in nutrient-poor growth media, seems also not involved according to mass-spec data. While other (unknown) pathways might still be involved in reducing the number of active RNA polymerases, the proposed hypothesis of the DNA substrate itself being limiting for the total rate of transcription is appealing.

Finally, the authors confirm the reduction of growth in the distant Caulobacter crescentus, which lacks overlapping rounds of replication and could thus have shown a different dependency on DNA concentration.

Weaknesses:

The study has no apparent weaknesses after review.

Reviewer #2 (Public review):

In this work, the authors uncovered the effects of DNA dilution on E. coli, including a decrease in growth rate and a significant change in proteome composition. The authors demonstrated that the decline in growth rate is due to the reduction of active ribosomes and active RNA polymerases because of the limited DNA copy numbers. They further showed that the change in the DNA-to-volume ratio leads to concentration changes in almost 60% of proteins, and these changes mainly stem from the change in the mRNA levels.

Comments on revised version:

The authors have satisfyingly answered all of our questions.

Reviewer #3 (Public review):

Mäkelä et al. here investigate genome concentration as a limiting factor on growth. Previous work has identified key roles for transcription (RNA polymerase) and translation (ribosomes) as limiting factors on growth, which enable an exponential increase in cell mass. While a potential limiting role of genome concentration under certain conditions has been explored theoretically, Mäkelä et al. here present direct evidence that when replication is inhibited, genome concentration emerges as a limiting factor.

A major strength of this paper is the diligent and compelling combination of experiment and modeling used to address this core question. The use of origin- and ftsZ-targeted CRISPRi is a very nice approach that enables dissection of the specific effects of limiting genome dosage in the context of a growing cytoplasm. While it might be expected that genome concentration eventually becomes a limiting factor, what is surprising and novel here is that this happens very rapidly, with growth transitioning even for cells within the normal length distribution for E. coli. Fundamentally, it demonstrates the fine balance of bacterial physiology, where the concentration of the genome itself (at least under rapid growth conditions) is no higher than it needs to be. A further surprising finding of this study is that susceptibility to this genome-limiting effect is felt differently by different genes, with unstable transcripts more affected and rRNA and many essential genes being more robust to it.

It should be noted that the authors do not identify a "smoking gun" - a gene or small number of genes that mediate the effects of genome concentration-dependent growth limitation. However, what they do achieve is to develop plausible criteria for identifying such a gene - through investigating essential genes that decrease in their abundance more rapidly than others.

Overall, this study provides a fundamental contribution to bacterial physiology by illuminating the relationship between DNA, mRNA, and protein in determining growth rate. While coarse-grained, the work invites exciting questions about how the composition of major cellular components is fine-tuned to a cell's needs and which specific gene products mediate this connection. The work also suggests the presence of buffering mechanisms that allow essential proteins such as RNA polymerase to be robust to fluctuations in genome concentration, which is an exciting area for future exploration. This work has implications not only for biotechnology, as the authors discuss, but potentially also for our understanding of how DNA-targeted antibiotics limit bacterial growth.

Comments on revised version:

Nothing left to add - the authors did a fantastic job addressing my points. In some ways doing so opened up even more interesting questions, but I happily accept that those are best left to future investigations.

Reviewer #1 (Public review):

(1a) Summary:

The author studied metabolic networks for central metabolism, focusing on how system trajectories returned to their steady state. To quantify the response, systematic perturbation was performed in simulation and the maximal destabilization away from steady state (compared with initial perturbation distance) was characterized. The author analyzed the perturbation response and found that sparse network and networks with more cofactors are more "stable", in the sense that the perturbed trajectories have smaller deviation along the path back to the steady state.

(1b) Strengths and major contributions:

The author compared three metabolic models and performed systematic perturbation analysis in simulation. This is the first work characterized how perturbed trajectories deviate from equilibrium in large biochemical systems and illustrated interesting findings about the difference between sparse biological systems and randomly simulated reaction networks.

(1c) Weaknesses:

There are two main weaknesses in this study:

First, the metabolic network in this study is incomplete. For example, amino acid synthesis and lipid synthesis are important for biomass and growth, but they are not included in the three models used in this study. NADH and NADPH are as important as ATP/ADP/AMP, but they are not included in the models. In the future, a more comprehensive metabolic and biosynthesis model is required.

Second, this work does not provide mathematics explanation on the perturbation response χ. Since the perturbation analysis are performed closed to steady state (or at least belongs to the attractor of single steady state), local linear analysis would provide useful information. By complement with other analysis in dynamical systems (described in below) we can gain more logical insights about perturbation response.

(1d) Discussion and impact for the field:

Metabolic perturbation is an important topic in cell biology and has important clinical implication in pharmacodynamics. The computational analysis in this study provides an initiative for future quantitative analysis on metabolism and homeostasis.

Comments on revised version:

The revised version of this manuscript made some clarifications, while I think the analysis of response coefficients is still numerical and model-specific, being unclear under dynamical systems of views.

Reviewer #2 (Public review):

The authors have conducted a valuable comparative analysis of perturbation responses in three nonlinear kinetic models of E. coli central carbon metabolism found in the literature. They aimed to uncover commonalities and emergent properties in the perturbation responses of bacterial metabolism. They discovered that perturbations in the initial concentrations of specific metabolites, such as adenylate cofactors and pyruvate, significantly affect the maximal deviation of the responses from steady-state values. Furthermore, they explored whether the network connectivity (sparse versus dense connections) influences these perturbation responses. The manuscript is reasonably well written.

Comments on revised version:

The authors have addressed my concerns to a large extent. However, a few minor issues remain, as listed below:

(1) The authors identified key metabolites affecting responses to perturbations in two ways: (i) by fixing a metabolite's value and (ii) by performing a sensitivity analysis. It would be helpful for the modeling community to understand better the differences and similarities in the obtained results. Do both methods identify substrate-level regulators? Is freezing a metabolite's dynamics dramatically changing the metabolic response (and if yes, which ones are so different in the two cases)? Does the scope of the network affect these differences and similarities?

(2) Regarding the issues the authors encountered when performing the sensitivity analysis, they can be approached in two ways. First, the authors can check the methods for computing conserved moieties nicely explained by Sauro's group (doi:10.1093/bioinformatics/bti800) and compute them for large-scale networks (but beware of metabolites that belong to several conserved pools). Otherwise, the conserved pools of metabolites can be considered as variables in the sensitivity analysis-grouping multiple parameters is a common approach in sensitivity analysis.

Reviewer #1 (Public review):

This study delineates an important set of uninjured and injured periosteal snRNAseq data that provides an overview of periosteal cell responses to fracture healing. The authors also took additional steps to validate some of the findings using immunohistochemistry and transplantation assays. This study will provide a valuable publicly accessible dataset to reexamine the expression of the reported periosteal stem and progenitor cell markers.

Strengths:

(1) This is the first single-nuclei atlas of periosteal cells that are obtained without enzymatic cell dissociation or targeted cell purification by FACS. This integrated snRNAseq dataset will provide additional opportunities for the community to revisit the expression of many periosteal cell markers that have been reported to date.<br /> (2) The authors delved further into the dataset using cutting-edge algorithms, including CytoTrace, SCENIC, Monocle, STRING and CellChat, to define potential roles of identified cell populations in the context of fracture healing. These additional computation analyses generate many new hypotheses regarding periosteal cell reactions.<br /> (3) The authors also sought to validate some of the computational findings using immunohistochemistry and transplantation assays to support the conclusion.

Weaknesses:

(1) The current snRNAseq datasets contain only a small number of nuclei (1,189 nuclei at day 0, 6,213 nuclei day 0-7 combined). It is possible that these datasets are underpowered to discern subtle biological changes in skeletal stem/progenitor cell populations during fracture healing.<br /> (2) POSTN is expressed in the cambium layer of the periosteum without fracture. The current data do not exclude the possibility that these pre-existing POSTN+ cells are the main responder of fracture healing.

Reviewer #2 (Public review):

Summary:

The authors described cell type mapping was conducted for both WT and fracture types. Through this, unique cell populations specific to fracture conditions were identified. To determine these, the most undifferentiated cells were initially targeted using stemness-related markers and CytoTrace scoring. This led to the identification of SSPC differentiating into fibroblasts. It was observed that the fibroblast cell type significantly increased under fracture conditions, followed by subsequent increases in chondrocytes and osteoblasts.

Strengths:

This study presented the injury-induced fibrogenic cell (IIFC) as a characteristic cell type appearing in the bone regeneration process and proposed that the IIFC is a progenitor undergoing osteochondrogenic differentiation.

Comments on revised version:

The authors have thoroughly addressed the reviewer's comments and have conducted additional experiments.

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Comments on revised version:

The issues I raised have largely been addressed. A minor concern relates to the legend for Figure 2C, where, according to the author's rebuttal, the vertical axis is "The ratio would be (Gsα + oleic acid stimulation) / (Gsα stimulation)" Otherwise, my general evaluation of the importance of the manuscript stands as stated in my initial review, namely, that the manuscript presents data and results that add a new dimension to existing paradigms for AC regulation, and will prompt future research into the role of physiological lipids in isoform-specific activation or inhibition of AC in tissues.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Comments on revised version:

In general, the authors have addressed my comments satisfactorily apart from the suggestion to use a lower ISO concentration in their assay or at least to discuss this issue, cite relevant literature etc. Pending this small amendment I would to fine to proceed.

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, 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 more in-depth study, the conclusions drawn by the authors are often open to questions. Major points concern the fact that there are not enough biological replicates for most experiments and some critical controls and data are lacking. Also, 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 think 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.<br /> (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.<br /> (3) Fig 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 Figs 1A-C to have single-cell and quantitative data as well.<br /> (4) Fig 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.<br /> (5) Fig 2A-C and Fig 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 to generate data with purified nTreg.<br /> (6) Fig 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 (Fig 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 were sorted using beads and not FACS on 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 expression of Nrn1 in iTregs 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+ iTreg phenotype is inhibited in the absence of Nrn1, and the iTregs that do develop appear functionally defective. On the other hand, the differentiation and expansion of Teff cells appears to be enhanced following deletion of Nrn1. With such defects in anergy induction as well as dysregulated Treg and Teff cell survival and function, auto reactive effector T cell activation becomes 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 author's use of both Nrn1-/- T cells as well as anti-Nrn1 neutralizing Ab to achieve similar results is a strength. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well performed and 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 for alterations in the electrical and metabolic state of iTreg and Teff cells when the Nrn1 gene is deleted. Nrn1-/- Tregs and Teff 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. It remains unclear how Nrn1 reinforces the membrane potential and facilitates aerobic glycolysis during and after iTreg differentiation, and yet suppresses the membrane potential and restrains aerobic glycolysis during Teff cell differentiation. Importantly, naive cells lacking Nrn1 expression show normal electrical and metabolic behaviors.

Reviewer #2 (Public review):

Summary:

In this study, the authors combine the study of clinical samples of antibiotic resistant bacteria with experimental evolution and evolutionary genomics to address important questions about the propensity for reversion in two different schema: de novo resistance arising within a patient, and transmitted resistance. The authors' use of a combination of methods help to answer the question outlined in their hypothesis, that de novo resistance mechanisms appear to revert to sensitive phenotypes more readily in a drug-free environment.

Strengths:

This study is exceptionally well-written and organized. The authors state their hypothesis clearly, and follow it up with an impressive effort that is truly translational-they make direct use of clinical samples of bacteria, and combine that with approaches in experimental evolution and evolutionary genomics. The conclusions follow naturally from the results, and there are no irresponsible leaps made.

Weaknesses:

I will divide my criticism into two areas, conceptual (most of my critique), with a very small methodological question.

(1) In the end, the authors offer findings that appear to be correct, and (again) are reported very clearly. However, this study is very surface-level in its theoretical underpinnings and construction, which is puzzling, because the field of antibiotic resistance and adaptation more broadly, is full of relevant studies and explanatory tools. Below I'll identify several areas where this manifests.

For one, the authors do not engage with a large recent literature on reversion, reversal, and compensation. It provides much more conceptual grounding for what the authors observe, much of it compatible with the findings from this study:

To offer two quick examples:<br /> - Avrani S, Katz S, Hershberg R. Adaptations accumulated under prolonged resource exhaustion are highly transient. MSphere. 2020 Aug 26;5(4):10-128.<br /> - Pennings, P.S., Ogbunugafor, C.B. and Hershberg, R., 2022. Reversion is most likely under high mutation supply when compensatory mutations do not fully restore fitness costs. G3, 12(9), p.jkac190.

Examinations of the studies on adaptation and reversion offer a richer mechanistic take on what was observed. But this literature alone is less of a problem than the general offering of different takes for the results. One can turn to a different literature - from ecology - to find a different explanation that is compatible with the findings.

De novo evolution involves the strong selection and rapid fixation of populations that are evolving largely to a relatively simple ecological milieu: their only primary function is to promote replication and survival of populations experiencing the negative fitness effects of drug pressure. Alternatively, transmitted resistant populations must deal with a multitude of selective pressures, working dynamically across time and space. In such a scenario, one would expect populations to locate places on the fitness landscape that are commensurate with survival in both drug-poor and drug-rich environments, as this is the ecological reality of the transmitted resistant bacteria. I could envision selection for "generalism" in this setting, corresponding to populations that have fixed mutations that promote resistance, but also those that ensure replication in drug-free environments. This regime might even reflect selection for "generalism" or "increased niche breadth." That is, transmitted resistance may have adopted a "jack of all trades, master of none" phenotype. The de novo resistance strains, alternatively, are selected for "generalism."

See the following for examples (there are many):

- Kassen R. The experimental evolution of specialists, generalists, and the maintenance of diversity. Journal of evolutionary biology. 2002 Mar 1;15(2):173-90.<br /> - Bell TH, Bell T. Many roads to bacterial generalism. FEMS microbiology ecology. 2021 Jan;97(1):fiaa240.

Note that this classically ecological explanation is only one of several other literatures that offer models for the findings in this study.

To the authors' credit, their study was about the very real-world problem of antibiotic resistance, using a system that is far less tractable than the model systems research that has generated a lot of data and theory. And sure: the study is valuable because it communicates an interesting finding using a combination of methods (impressively). But in some ways, the study almost reads as a descriptive exercise: it offers a good question (does de novo or transmitted resistance revert more readily), and tells you what they found (de novo does). However the explanatory mechanisms do not advance our understanding much. Reporting the presence of unstable and disruptive mutations in the de novo populations is hardly an explanation. That is, alternatively, data in support of a proper explanation. There is nothing magical about de novo evolution that should be selected for disruptive mutations.

The reasons for the different sorts of mutation could have to do with the population genetic particulars of the de novo regime: large populations, strong selective pressure, relatively static fitness landscape. In such a setting, selection marches a population greedily up a peak. Alternatively, a transmitted population arises from a lineage that has observed a multitude of ecologies, across different fitness landscapes and has fixed mutations that confer survival across all of them.

There's a literature that speaks to this:<br /> - Miller CM, Draghi JA. Range expansion can promote the evolution of plastic generalism in coarse-grained landscapes. Evolution Letters. 2024 Apr 1;8(2):322-30.<br /> - Bono LM, Draghi JA, Turner PE. Evolvability costs of niche expansion. Trends in genetics. 2020 Jan 1;36(1):14-23.

The findings are simple enough (a testament to the strong study design and execution) that supporting population genetic simulations, or analytical descriptions (maybe not relevant) could offer insight as to what really happened here.

(2) I recognize the challenge of working with clinical samples. It is very difficult to understand everything about them. But even having considered that, I might be missing something.

My main question here involves the origin of the putatively transmitted strains. The authors state that " Isolates were also obtained from six patients with a putatively transmitted resistant bacteria (hereafter PT), where a daptomycin-resistant, E. faecium bacteremia was identified on their first culture."

This seems like an awfully dubious way to identify transmitted resistance. I suppose I understand the logic (de novo evolution requires the observer to have seen the evolution happen in real-time). But this definition leaves the study wide open for an "apples to oranges" comparison that might render the other aspects questionable.

The de novo strains are being compared to transmitted strains that may have been part of lineages that had passed between many, many patients. If this were true, then we should expect the genomic architecture of the transmitted strains to be far different. The transmitted strains might have undergone more selection in different regimes and genetic drift. Drift might have fixed mutations in transmission bottlenecks, altering the genetic architecture. In such a scenario, one might expect these populations to have a more difficult time unwinding their resistance phenotype.

In the end, I applaud the authors on a well-done and well-written study.

Reviewer #1 (Public review):

Summary:

Tracy and colleagues study the loss of daptomycin resistance in Enterococcus faecium isolates from bloodstream infections using in vitro evolution experiments in the absence of antibiotics. They test the hypothesis that antibiotic resistance arising de novo during treatment will carry a higher fitness cost and will revert more readily than resistance isolates which have been transmitted and have therefore already survived in the absence of antibiotic selection pressure.

Strengths:

This is an important question as a fitness cost to resistance is typically found in lab evolution experiments and assumed in modelling studies, but often not identified in clinical isolates. Here the authors find examples of clinical isolates which do and don't revert to sensitivity in in vitro evolution in the absence of antibiotics. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo, which is nice evidence that these resistance mutations did confer a fitness cost.

Weaknesses:

Although this is an interesting study on an important topic, currently the results are overinterpreted do not justify the title of the paper 'Reversion to sensitivity explains limited transmission of resistance in a hospital pathogen' for several reasons. Firstly, the patient group, e.g. 'putatively transmitted' isolates vs 'de novo' isolates was not a significant predictor of change in MIC. Instead the change in MIC in the absence of antibiotics was significantly associated with the starting MIC of the isolate in the evolution experiments, but this would be expected since isolates with a higher MIC have more potential to decrease in MIC in the evolution experiments. The abstract and some conclusion do not match the results in some instances, for example the abstract states 'resistance that arose de novo within patients was higher level but exhibited greater declines in resistance in vitro'. In the discussion: they state "these findings support our hypothesis that transmitted resistance strains are less likely to revert". However, on page 14 the initial MICs between DNR and PTR were not significantly different and patient group was not a significant predictor of change in MIC. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo. However, there were also some example of mutations in the same genes within the PTR isolates, so it remains unclear if there is a significant difference in behaviour between the DNR and PTR isolates in terms of reversion mutations. Significance testing, controlling for the starting MIC, would help confirm this.

Secondly, the 'putatively transmitted isolates', i.e. isolates that were resistant in the first positive blood culture, do not necessarily represent resistant isolates that have been transmitted between hosts. E. faecium is primarily a commensal of the intestinal tract, but which can cause opportunistic extra-intestinal infections. These bacteremia cases were most likely caused by within-host translocation of a strain already colonizing the intestine to the bloodstream - indeed, it has been shown that antibiotics can lead to Enterococcus overgrowth in the intestine and subsequent bloodstream invasion (DOI: 10.1172/JCI43918). The 'putatively transmitted isolates' may have initially colonised the intestine via between host transmission in an already resistant state, as assumed by the authors, but they may also have evolved resistance de novo within the host's intestine prior to causing bloodstream infections. Since they do not have data on past daptomycin exposure in these individuals it cannot be assumed that these isolates were transmitted with high resistance between hosts. An alternative explanation for any differences between the 'de novo' and 'putatively transmitted' could be the environment where resistance evolved, e.g. the intestine with strong competition from other strains and species, or within the otherwise sterile bloodstream environment. The authors hypothesise that "newly resistant population must continue to transmit between hosts in antibiotic free conditions to ensure its survival" and that "transmission acts as a filter to select for resistance with a lower cost or lower chance of reversion". Rather than transmission per se, it is equally plausible that survival of the newly resistant population within the primary niche, the intestinal microbiota, is the crucial to filter for resistance with a lower cost.

Reviewer #1 (Public review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developping animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

Unfortunately, the revised manuscript does not show significant improvement. While the identification of the receptors is highly convincing, important issues about the biological relevance remain unaddressed.

First, the main point I raised about the first version of this article is that the redundancy and/or specificity of the two receptors should be clarified, even though I understand that it cannot be deeply investigated here. I believe that this point, shared by all reviewers, is highly relevant for the scope of this work. In this revised version, it is still unclear how to reconcile gain and loss-of-function experiments and the different expression profiles of the receptors.

Second, the newly added explanations and pieces of discussion provided about the mild in vivo phenotypes of early pupation upon Cad96ca or Fgfr1 knock-out do not clarify the issue but instead put emphasis on methodological issues. Indeed, it is not clear whether the mild phenotypes reflect the biological role of Cad96ca and Fgfr1, or the redundancy of these two RTKs (and/or others), or some issue with the knock-out strategy (partial efficiency, mosaicism...).

Finally, parts of the updated discussion and the modifications to the figures are confusing.

Reviewer #2 (Public review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identify and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Reviewer #3 (Public review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The authors have provided evidences that the Cad96Ca and FGF1 RTK receptors contribute to JH signaling through CRISPR/Cas9, inducing precocious metamorphosis, although not to the same extent as absence of JH. Therefore, it still remains unclear whether these RTKs are completely required for pathway activation or only necessary for high activation levels during the last larval stage.

While the authors have included some additional data, the mechanism by which different RTKs function in transducing JH signaling in a tissue specific manner is still unclear. As the authors note in the discussion, it is possible that other RTKs may also play a role in facilitating the transduction of JH signaling.

Lastly, the study does not yet explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. Although receptor promiscuity has been suggested as a possible mechanism, future studies could explore whether activation of RTK pathways by their known ligands induces certain levels of JH transducer phosphorylation, which, in the presence of JH, could contribute to full pathway activation without the need for direct JH-RTK binding.

Reviewer #1 (Public review):

Summary:

The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display non-proneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants. However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is non-apoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Strengths:

Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.

Weaknesses:

The authors have addressed all my concerns and questions.

Reviewer #2 (Public review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to the readers.

The revised manuscript addresses my concerns from the first round of review.

Reviewer #3 (Public review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:

The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:

The authors did an adequate job in dealing with the limitations of the reviewed preprint. Although they could have done more, they chose not to for reasons they adequately defended.

Reviewer #1 (Public review):

This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest.

Reviewer #2 (Public review):

Summary:

This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

The manuscript is well-written, and the methods are clearly described and appropriate. Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes. The results are interesting and novel.

Weaknesses:

Analyses were conducted for task-based data rather than resting-state data. The authors report behavioral differences between groups and include behavioral performance as a nuisance regressor in their analysis. This is a good approach to account for behavioral task differences, given the data. Nevertheless, additional work using resting-state functional connectivity could remove the potential confound fully.

The authors have addressed my concerns well.

Reviewer #1 (Public review):

Summary:

This manuscript from So et al. describes what is suggested to be an improved protocol for single-nuclei RNA sequencing (snRNA-seq) of adipose tissue. The authors provide evidence that modifications to the existing protocols result in better RNA quality and nuclei integrity than previously observed, with ultimately greater coverage of the transcriptome upon sequencing. Using the modified protocol, the authors compare the cellular landscape of murine inguinal and perigonadal white adipose tissue (WAT) depots harvested from animals fed a standard chow diet (lean mice) or those fed a high-fat diet (mice with obesity).

Strengths:

Overall, the manuscript is well written, and the data are clearly presented. The strengths of the manuscript rest in the description of an improved protocol for snRNA-seq analysis. This should be valuable for the growing number of investigators in the field of adipose tissue biology that are utilizing snRNA-seq technology, as well as those other fields attempting similar experiments with tissues possessing high levels of RNAse activity.

Moreover, the study makes some notable observations that provide the foundation for future investigation. One observation is the correlation between nuclei size and cell size, allowing for the transcriptomes of relatively hypertrophic adipocytes in perigonadal WAT to be examined. Another notable observation is the identification of an adipocyte subcluster (Ad6) that appears "stressed" or dysfunctional and likely localizes to crown-like inflammatory structures where pro-inflammatory immune cells reside.

Weaknesses:

Analogous studies have been reported in the literature, including a notable study from Savari et al. (Cell Metabolism). This somewhat diminishes the novelty of some of the biological findings presented here. This is deemed a minor criticism as the primary goal is to provide a resource for the field.

Reviewer #2 (Public review):

Summary:

In the present manuscript So et al describe an optimized method for nuclei isolation and single nucleus RNA sequencing (snRNA-Seq), which they use to characterize cell populations in lean and obese murine adipose tissues.

Strengths:

The detailed description of the protocol for single-nuclei isolation incorporating VRC may be useful to researchers studying adipose tissues, which contain high levels of RNAses.

While the majority of the findings largely confirm previous published adipose data sets, the authors present a detailed description of a mature adipocyte sub-cluster that appears to represent stressed or dying adipocytes present in obesity, and which is better characterized using the described protocol.

Weaknesses:

The use of VRC to enhance snRNA-seq has been previously published in other tissues, somewhat diminishing the novelty of this protocol.

The snRNA-seq data sets presented in this manuscript, when compared with numerous previously published single-cell analysis of adipose tissue, represent an incremental contribution. The nuclei-isolation protocol may represent an improvement in transcriptional analysis for mature adipocytes, however other stromal populations may be better sequenced using single intact-cell cytoplasmic RNA-Seq.

Reviewer #3 (Public review):

The authors aimed to improve single-nucleus RNA sequencing (snRNA-seq) to address current limitations and challenges with nuclei and RNA isolation quality. They successfully developed a protocol that enhances RNA preservation and yields high-quality snRNA-seq data from multiple tissues, including a challenging model of adipose tissue. They then applied this method to eWAT and iWAT from mice fed either a normal or high-fat diet, exploring depot-specific cellular dynamics and gene expression changes during obesity. Their analysis included subclustering of SVF cells and revealed that obesity promotes a transition in APCs from an early to a committed state and induces a pro-inflammatory phenotype in immune cells, particularly in eWAT. In addition to SVF cells, they discovered six adipocyte subpopulations characterized by a gradient of unique gene expression signatures. Interestingly, a novel subpopulation, termed Ad6, comprised stressed and dying adipocytes with reduced transcriptional activity, primarily found in eWAT of mice on a high-fat diet. Overall, the methodology is sound, and the data presented supports the conclusions drawn. Further research based on these findings could pave the way for potential novel interventions in obesity and metabolic disorders, or for similar studies in other tissues or conditions.

Strengths:

The authors have presented a compelling set of results. They have compared their data with two previously published datasets and provide novel insight into the biological processes underlying mouse adipose tissue remodeling during obesity. The results are generally consistent and robust. The revised Discussion is comprehensive and puts the work in the context of the field.

Weaknesses:

• The adipose tissues were collected after 10 weeks of high-fat diet treatment, lacking the intermediate time points for identifying early markers or cell populations during the transition from healthy to pathological adipose tissue.<br /> • The expansion of the Ad6 subpopulation in obese iWAT and gWAT is interesting. The author claims that Ad6 exhibited a substantial increase in eWAT and a moderate rise in iWAT (Figure 4C). However, this adipocyte subpopulation remains the most altered in iWAT upon obesity. Could the authors elaborate on why there is a scarcity of adipocytes with ROS reporter and B2M in obese iWAT?<br /> • While the study provides extensive data on mouse models, the potential translation of these findings to human obesity remains uncertain.

Revised version: The authors have properly revised the paper in response to the above questions, and I have no other concerns.

Reviewer #1 (Public review):

The blood-brain barrier separates neural tissue from blood-borne factors and is important for maintaining central nervous system health and function. Endothelial cells are the site of the barrier. These cells exhibit unique features relative to peripheral endothelium and a unique pattern of gene expression. There remains much to be learned about how the transcriptome of brain endothelial cells is established in development and maintained throughout life.

The manuscript by Sadanandan, Thomas et al. investigates this question by examining transcriptional and epigenetic changes in brain endothelial cells in embryonic and adult mice. Changes in transcript levels and histone marks for various BBB-relevant transcripts, including Cldn5, Mfsd2a and Zic3 were observed between E13.5 and adult mice. To perform these experiments, endothelial cells were isolated from E13.5 and adult mice, then cultured in vitro, then sequenced. This approach is problematic. It is well-established that brain endothelial cells rapidly lose their organotypic features in culture (https://elifesciences.org/articles/51276). Indeed, one of the primary genes investigated in this study, Cldn1, exhibits very low expression at the transcript level in vivo, but is strongly upregulated in cultured ECs.

(https://elifesciences.org/articles/36187 ; https://markfsabbagh.shinyapps.io/vectrdb/)

This undermines the conclusions of the study. While this manuscript is framed as investigating how epigenetic processes shape BBB formation and maintenance, they may be looking at how brain endothelial cells lose their identity in culture.

An additional concern is that for many experiments, siRNA knockdowns are performed without validation of the efficacy of knockdown.

Some experiments in the paper are promising, however. For example, the knockout of HDAC2 in endothelial cells resulting in BBB leakage was striking. Investigating the mechanisms underlying this phenotype in vivo could yield important insights.

Reviewer #2 (Public review):

Sadanandan et al describe their studies in mice of HDAC and Polycomb function in the context of vascular endothelial cell (EC) gene expression relevant to the blood-brain barrier, (BBB). This topic is of interest because the BBB gene expression program represents an interesting and important vascular diversification mechanism. From an applied point of view, modifying this program could have therapeutic benefits in situations where BBB function is compromised.

The study involves comparing the transcriptomes of cultured CNS ECs at E13 and adult stages and then perturbing EC gene expression pharmacologically in cell culture (with HDAC and Polycomb inhibitors) and genetically in vivo by EC-specific conditional KO of HDAC2 and Polycomb component EZH2.

This reviewer has several critiques of the study.

First, based on published data, the effect of culturing CNS ECs is likely to have profound effects on their differentiation, especially as related to their CNS-specific phenotypes. Related to this, the authors do not state how long the cells were cultured.

Second, the use of qPCR assays for quantifying ChIP and transcript levels is inferior to ChIPseq and RNAseq. Whole genome methods, such as ChIPseq, permit a level of quality assessment that is not possible with qPCR methods. The authors should use whole genome NextGen sequencing approaches, show the alignment of reads to the genome from replicate experiments, and quantitatively analyze the technical quality of the data.

Third, the observation that pharmacologic inhibitor experiments and conditional KO experiments targeting HDAC2 and the Polycomb complex perturb EC gene expression or BBB integrity, respectively, is not particularly surprising as these proteins have broad roles in epigenetic regulation is a wide variety of cell types.

Reviewer #1 (Public review):

Summary:

This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

Strengths:

Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

Weaknesses:<br /> I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims. Additionally, the data about apoptosis is not sufficiently explained.

Reviewer #2 (Public review):

Summary:<br /> In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

Strengths:

(1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.<br /> (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

Weaknesses:

(1) The authors provide a superficial description of the related phenotypes, lacking a mechanistic understanding of how Fmi regulates cell competition. While induction of apoptosis and JNK activation are commonly observed outcomes in various cell competition conditions, it is crucial to determine the specific mechanisms through which they are induced in fmi-depleted clones. Furthermore, it is recommended that the authors utilize the power of fly genetics to conduct a series of genetic epistasis analyses.

Reviewer #3 (Public review):

Summary:

In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

Strengths:

- A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

- One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term)

Weaknesses:

- The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

Reviewer #2 (Public review):

Summary:

This work by Grogan and colleagues aimed to translate animal studies showing that acetylcholine plays a role in motivation by modulating the effects of dopamine on motivation. They tested this hypothesis with a placebo-controlled pharmacological study administering a muscarinic antagonist (trihexyphenidyl; THP) to a sample of 20 adult men performing an incentivized saccade task while undergoing electroencephalography (EEG). They found that reward increased vigor and reduced reaction times (RTs) and, importantly, these reward effects were attenuated by trihexyphenidyl. High incentives increased preparatory EEG activity (contingent negative variation), and though THP also increased preparatory activity, it also reduced this reward effect on RTs.

Strengths:

The researchers address a timely and potentially clinically relevant question with a within-subject pharmacological intervention and a strong task design. The results highlight the importance of the interplay between dopamine and other neurotransmitter systems in reward sensitivity and even though no Parkinson's patients were included in this study, the results could have consequences for patients with motivational deficits and apathy if validated in the future.

Weaknesses:

The main weakness of the study is the small sample size (N=20) that unfortunately is limited to men only. Generalizability and replicability of the conclusions remain to be assessed in future research with a larger and more diverse sample size and potentially a clinically relevant population. The EEG results do not shape a concrete mechanism of action of the drug on reward sensitivity.

Reviewer #3 (Public review):

Summary:

Grogan et al examine a role for muscarinic receptor activation in action vigor in a saccadic system. This work is motivated by a strong literature linking dopamine to vigor, and some animal studies suggesting that ACH might modulate these effects, and is important because patient populations with symptoms related to reduced vigor are prescribed muscarinic antagonists. The authors use a motivated saccade task with distractors to measure the speed and vigor of actions in humans under placebo or muscarinic antagonism. They show that muscarinic antagonism blunts the motivational effects of reward on both saccade velocity and RT, and also modulates the distractibility of participants, in particular by increasing the repulsion of saccades away from distractors. They show that preparatory EEG signals reflect both motivation and drug condition, and make a case that these EEG signals mediate the effects of the drug on behavior.

Strengths:

This manuscript addresses an interesting and timely question and does so using an impressive within subject pharmacological design and a task well designed to measure constructs of interest. The authors show clear causal evidence that ACH affects different metrics of saccade generation related to effort expenditure and their modulation by incentive manipulations. The authors link these behavioral effects to motor preparatory signatures, indexed with EEG, that relate to behavioral measures of interest and in at least one case statistically mediate the behavioral effects of ACH antagonism.

Weaknesses:

A primary weakness of this paper is the sample size - since only 20 participants completed the study. The authors address the sample size in several places and I completely understand the reason for the reduced sample size (study halt due to covid). Nonetheless, it is worth stating explicitly that this sample size is relatively small for the effect sizes typically observed in such studies highlighting the need for future confirmatory studies.

Reviewer #1 (Public review):

Summary:

In this study, the authors address whether the dorsal nucleus of the inferior colliculus (DCIC) in mice encodes sound source location within the front horizontal plane (i.e., azimuth). They do this using volumetric two-photon Ca2+ imaging and high-density silicon probes (Neuropixels) to collect single-unit data. Such recordings are beneficial because they allow large populations of simultaneous neural data to be collected. Their main results and the claims about those results are the following:<br /> (1) DCIC single-unit responses have high trial-to-trial variability (i.e., neural noise);<br /> (2) approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth;<br /> (3) single-trial population responses (i.e., the joint response across all sampled single units in an animal) encode sound source azimuth "effectively" (as stated in the title) in that localization decoding error matches average mouse discrimination thresholds;<br /> (4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus (as stated in the Abstract);<br /> (5) evidence of noise correlation between pairs of neurons exists;<br /> and 6) noise correlations between responses of neurons help reduce population decoding error.<br /> While simultaneous recordings are not necessary to demonstrate results #1, #2, and #4, they are necessary to demonstrate results #3, #5, and #6.

Strengths:<br /> - Important research question to all researchers interested in sensory coding in the nervous system.<br /> - State-of-the-art data collection: volumetric two-photon Ca2+ imaging and extracellular recording using high-density probes. Large neuronal data sets.<br /> - Confirmation of imaging results (lower temporal resolution) with more traditional microelectrode results (higher temporal resolution).<br /> - Clear and appropriate explanation of surgical and electrophysiological methods. I cannot comment on the appropriateness of the imaging methods.

Strength of evidence for the claims of the study:

(1) DCIC single-unit responses have high trial-to-trial variability -<br /> The authors' data clearly shows this.

(2) Approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth -<br /> The sensitivity of each neuron's response to sound source azimuth was tested with a Kruskal-Wallis test, which is appropriate since response distributions were not normal. Using this statistical test, only 8% of neurons (median for imaging data) were found to be sensitive to azimuth, and the authors noted this was not significantly different than the false positive rate. The Kruskal-Wallis test was not reported for electrophysiological data. The authors suggested that low numbers of azimuth-sensitive units resulting from the statistical analysis may be due to the combination of high neural noise and relatively low number of trials, which would reduce statistical power of the test. This is likely true, and highlights a weakness in the experimental design (i.e., relatively small number of trials). The authors went on to perform a second test of azimuth sensitivity-a chi-squared test-and found 32% (imaging) and 40% (e-phys) of single units to have statistically significant sensitivity. However, the use of a chi-squared test is questionable because it is meant to be used between two categorical variables, and neural response had to be binned before applying the test.

(3) Single-trial population responses encode sound source azimuth "effectively" in that localization decoding error matches average mouse discrimination thresholds -<br /> If only one neuron in a population had responses that were sensitive to azimuth, we would expect that decoding azimuth from observation of that one neuron's response would perform better than chance. By observing the responses of more than one neuron (if more than one were sensitive to azimuth), we would expect performance to increase. The authors found that decoding from the whole population response was no better than chance. They argue (reasonably) that this is because of overfitting of the decoder model-too few trials were used to fit too many parameters-and provide evidence from decoding combined with principal components analysis which suggests that overfitting is occurring. What is troubling is the performance of the decoder when using only a handful of "top-ranked" neurons (in terms of azimuth sensitivity) (Fig. 4F and G). Decoder performance seems to increase when going from one to two neurons, then decreases when going from two to three neurons, and doesn't get much better for more neurons than for one neuron alone. It seems likely there is more information about azimuth in the population response, but decoder performance is not able to capture it because spike count distributions in the decoder model are not being accurately estimated due to too few stimulus trials (14, on average). In other words, it seems likely that decoder performance is underestimating the ability of the DCIC population to encode sound source azimuth.

To get a sense of how effective a neural population is at coding a particular stimulus parameter, it is useful to compare population decoder performance to psychophysical performance. Unfortunately, mouse behavioral localization data do not exist. Instead, the authors compare decoder error to mouse left-right discrimination thresholds published previously by a different lab. However, this comparison is inappropriate because the decoder and the mice were performing different perceptual tasks. The decoder is classifying sound sources to 1 of 13 locations from left to right, whereas the mice were discriminating between left or right sources centered around zero degrees. The errors in these two tasks represent different things. The two data sets may potentially be more accurately compared by extracting information from the confusion matrices of population decoder performance. For example, when the stimulus was at -30 deg, how often did the decoder classify the stimulus to a lefthand azimuth? Likewise, when the stimulus was +30 deg, how often did the decoder classify the stimulus to a righthand azimuth?

(4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus -<br /> It is unclear what exactly the authors mean by this statement in the Abstract. There are major differences in the encoding of azimuth between the two neighboring brain areas: a large majority of neurons in the CNIC are sensitive to azimuth (and strongly so), whereas the present study shows a minority of azimuth-sensitive neurons in the DCIC. Furthermore, CNIC neurons fire reliably to sound stimuli (low neural noise), whereas the present study shows that DCIC neurons fire more erratically (high neural noise).

(5) Evidence of noise correlation between pairs of neurons exists -<br /> The authors' data and analyses seem appropriate and sufficient to justify this claim.

(6) Noise correlations between responses of neurons help reduce population decoding error -<br /> The authors show convincing analysis that performance of their decoder increased when simultaneously measured responses were tested (which include noise correlation) than when scrambled-trial responses were tested (eliminating noise correlation). This makes it seem likely that noise correlation in the responses improved decoder performance. The authors mention that the naïve Bayesian classifier was used as their decoder for computational efficiency, presumably because it assumes no noise correlation and, therefore, assumes responses of individual neurons are independent of each other across trials to the same stimulus. The use of a decoder that assumes independence seems key here in testing the hypothesis that noise correlation contains information about sound source azimuth. The logic of using this decoder could be more clearly spelled out to the reader. For example, if the null hypothesis is that noise correlations do not carry azimuth information, then a decoder that assumes independence should perform the same whether population responses are simultaneous or scrambled. The authors' analysis showing a difference in performance between these two cases provides evidence against this null hypothesis.

Minor weakness:<br /> - Most studies of neural encoding of sound source azimuth are done in a noise-free environment, but the experimental setup in the present study had substantial background noise. This complicates comparison of the azimuth tuning results in this study to those of other studies. One is left wondering if azimuth sensitivity would have been greater in the absence of background noise, particularly for the imaging data where the signal was only about 12 dB above the noise.

Reviewer #2 (Public review):

In the present study, Boffi et al. investigate the manner in which the dorsal cortex of the of the inferior colliculus (DCIC), an auditory midbrain area, encodes sound location azimuth in awake, passively listening mice. By employing volumetric calcium imaging (scanned temporal focusing or s-TeFo), complemented with high-density electrode electrophysiological recordings (neuropixels probes), they show that sound-evoked responses are exquisitely noisy, with only a small portion of neurons (units) exhibiting spatial sensitivity. Nevertheless, a naïve Bayesian classifier was able to predict the presented azimuth based on the responses from small populations of these spatially sensitive units. A portion of the spatial information was provided by correlated trial-to-trial response variability between individual units (noise correlations). The study presents a novel characterization of spatial auditory coding in a non-canonical structure, representing a noteworthy contribution specifically to the auditory field and generally to systems neuroscience, due to its implementation of state-of-the-art techniques in an experimentally challenging brain region. However, nuances in the calcium imaging dataset and the naïve Bayesian classifier warrant caution when interpreting some of the results.

Strengths:

The primary strength of the study lies in its methodological achievements, which allowed the authors to collect a comprehensive and novel dataset. While the DCIC is a dorsal structure, it extends up to a millimetre in depth, making it optically challenging to access in its entirety. It is also more highly myelinated and vascularised compared to e.g., the cerebral cortex, compounding the problem. The authors successfully overcame these challenges and present an impressive volumetric calcium imaging dataset. Furthermore, they corroborated this dataset with electrophysiological recordings, which produced overlapping results. This methodological combination ameliorates the natural concerns that arise from inferring neuronal activity from calcium signals alone, which are in essence an indirect measurement thereof.

Another strength of the study is its interdisciplinary relevance. For the auditory field, it represents a significant contribution to the question of how auditory space is represented in the mammalian brain. "Space" per se is not mapped onto the basilar membrane of the cochlea and must be computed entirely within the brain. For azimuth, this requires the comparison between miniscule differences between the timing and intensity of sounds arriving at each ear. It is now generally thought that azimuth is initially encoded in two, opposing hemispheric channels, but the extent to which this initial arrangement is maintained throughout the auditory system remains an open question. The authors observe only a slight contralateral bias in their data, suggesting that sound source azimuth in the DCIC is encoded in a more nuanced manner compared to earlier processing stages of the auditory hindbrain. This is interesting because it is also known to be an auditory structure to receive more descending inputs from the cortex.

Systems neuroscience continues to strive for the perfection of imaging novel, less accessible brain regions. Volumetric calcium imaging is a promising emerging technique, allowing the simultaneous measurement of large populations of neurons in three dimensions. But this necessitates corroboration with other methods, such as electrophysiological recordings, which the authors achieve. The dataset moreover highlights the distinctive characteristics of neuronal auditory representations in the brain. Its signals can be exceptionally sparse and noisy, which provide an additional layer of complexity in the processing and analysis of such datasets. This will undoubtedly be useful for future studies of other less accessible structures with sparse responsiveness.

Weaknesses:

Although the primary finding that small populations of neurons carry enough spatial information for a naïve Bayesian classifier to reasonably decode the presented stimulus is not called into question, certain idiosyncrasies, in particular the calcium imaging dataset and model, complicate specific interpretations of the model output, and the readership is urged to interpret these aspects of the study's conclusions with caution.

I remain in favour of volumetric calcium imaging as a suitable technique for the study, but the presently constrained spatial resolution is insufficient to unequivocally identify regions of interest as cell bodies (and are instead referred to as "units" akin to those of electrophysiological recordings). It remains possible that the imaging set is inadvertently influenced by non-somatic structures (including neuropil), which could report neuronal activity differently than cell bodies. Due to the lack of a comprehensive ground-truth comparison in this regard (which to my knowledge is impossible to achieve with current technology), it is difficult to imagine how many informative such units might have been missed because their signals were influenced by spurious, non-somatic signals, which could have subsequently misled the models. The authors reference the original Nature Methods article (Prevedel et al., 2016) throughout the manuscript, presumably in order to avoid having to repeat previously published experimental metrics. But the DCIC is neither the cortex nor hippocampus (for which the method was originally developed) and may not have the same light scattering properties (not to mention neuronal noise levels). Although the corroborative electrophysiology data largely eleviates these concerns for this particular study, the readership should be cognisant of such caveats, in particular those who are interested in implementing the technique for their own research.

A related technical limitation of the calcium imaging dataset is the relatively low number of trials (14) given the inherently high level of noise (both neuronal and imaging). Volumetric calcium imaging, while offering a uniquely expansive field of view, requires relatively high average excitation laser power (in this case nearly 200 mW), a level of exposure the authors may have wanted to minimise by maintaining a low number of repetitions, but I yield to them to explain. Calcium imaging is also inherently slow, requiring relatively long inter-stimulus intervals (in this case 5 s). This unfortunately renders any model designed to predict a stimulus (in this case sound azimuth) from particularly noisy population neuronal data like these as highly prone to overfitting, to which the authors correctly admit after a model trained on the entire raw dataset failed to perform significantly above chance level. This prompted them to feed the model only with data from neurons with the highest spatial sensitivity. This ultimately produced reasonable performance (and was implemented throughout the rest of the study), but it remains possible that if the model was fed with more repetitions of imaging data, its performance would have been more stable across the number of units used to train it. (All models trained with imaging data eventually failed to converge.) However, I also see these limitations as an opportunity to improve the technology further, which I reiterate will be generally important for volume imaging of other sparse or noisy calcium signals in the brain.

Indeed, in separate comments to these remarks, the authors confirmed that the low number of trials was technically limited, to which I emphasise is to no fault of their own. However, they also do not report this as a typical imaging constraint, such as photobleaching, but rather because the animals exhibited signs of stress and discomfort at longer imaging periods. From an animal welfare perspective, I would encourage the authors to state this in the methods for transparency. It would demonstrate their adherence to animal welfare policies, which I find to be an incredibly strong argument for limiting the number of trials in their study.

Transitioning to the naïve Bayesian classifier itself, I first openly ask the authors to justify their choice of this specific model. There are countless types of classifiers for these data, each with their own pros and cons. Did they actually try other models (such as support vector machines), which ultimately failed? If so, these negative results (even if mentioned en passant) would be extremely valuable to the community, in my view. I ask this specifically because different methods assume correspondingly different statistical properties of the input data, and to my knowledge naïve Bayesian classifiers assume that predictors (neuronal responses) are assumed to be independent within a class (azimuth). As the authors show that noise correlations are informative in predicting azimuth, I wonder why they chose a model that doesn't take advantage of these statistical regularities. It could be because of technical considerations (they mention computing efficiency), but I am left generally uncertain about the specific logic that was used to guide the authors through their analytical journey.

In a revised version of the manuscript, the authors indeed justify their choice of the naïve Bayesian classifier as a conservative approach (not taking into account noise correlations), which could only improve with other models (that do). They even tested various other commonly used models, such as support vector machines and k-nearest neighbours, to name a few, but do not report these efforts in the main manuscript. Interestingly, these models, which I supposed would perform better in fact did not overall - a finding that I have no way of interpreting but nevertheless find interesting. I would thus encourage the authors to include these results in a figure supplement and mention it en passant while justifying their selection of model (but please include detailed model parameters in the methods section).

That aside, there remain other peculiarities in model performance that warrant further investigation. For example, what spurious features (or lack of informative features) in these additional units prevented the models of imaging data from converging? In an orthogonal question, did the most spatially sensitive units share any detectable tuning features? A different model trained with electrophysiology data in contrast did not collapse in the range of top-ranked units plotted. Did this model collapse at some point after adding enough units, and how well did that correlate with the model for the imaging data? How well did the form (and diversity) of the spatial tuning functions as recorded with electrophysiology resemble their calcium imaging counterparts? These fundamental questions could be addressed with more basic, but transparent analyses of the data (e.g., the diversity of spatial tuning functions of their recorded units across the population). Even if the model extracts features that are not obvious to the human eye in traditional visualisations, I would still find this interesting.

Although these questions were not specifically addressed in the revised version of the manuscript, I also admit that I did not indent do assert that these should necessarily fall within the scope of the present study. I rather posed them as hypothetical directions one could pursue in future studies. Finally, further concerns I had with statements regarding the physiological meaning of the findings have been ameliorated by nicely modified statements, thus bringing transparency to the readership, which I appreciate.

In summary, the present study represents a significant body of work that contributes substantially to the field of spatial auditory coding and systems neuroscience. However, limitations of the imaging dataset and model as applied in the study muddles concrete conclusions about how the DCIC precisely encodes sound source azimuth and even more so to sound localisation in a behaving animal. Nevertheless, it presents a novel and unique dataset, which, regardless of secondary interpretation, corroborates the general notion that auditory space is encoded in an extraordinarily complex manner in the mammalian brain.

Reviewer #3 (Public review):

Summary:

Boffi and colleagues sought to quantify the single-trial, azimuthal information in the dorsal cortex of the inferior colliculus (DCIC), a relatively understudied subnucleus of the auditory midbrain. They accomplished this by using two complementary recording methods while mice passively listened to sounds at different locations: calcium imaging that recorded large neuronal populations but with poor temporal precision and multi-contact electrode arrays that recorded smaller neuronal populations with exact temporal precision. DCIC neurons respond variably, with inconsistent activity to sound onset and complex azimuthal tuning. Some of this variably was explained by ongoing head movements. The authors used a naïve Bayes decoder to probe the azimuthal information contained in the response of DCIC neurons on single trials. The decoder failed to classify sound location better than chance when using the raw population responses but performed significantly better than chance when using the top principal components of the population. Units with the most azimuthal tuning were distributed throughout the DCIC, possessed contralateral bias, and positively correlated responses. Interestingly, inter-trial shuffling decreased decoding performance, indicating that noise correlations contributed to decoder performance. Overall, Boffi and colleagues, quantified the azimuthal information available in the DCIC while mice passively listened to sounds, a first step in evaluating if and how the DCIC could contribute to sound localization.

Strengths:

The authors should be commended for collection of this dataset. When done in isolation (which is typical), calcium imaging and linear array recordings have intrinsic weaknesses. However, those weaknesses are alleviated when done in conjunction - especially when the data is consistent. This data set is extremely rich and will be of use for those interested in auditory midbrain responses to variable sound locations, correlations with head movements, and neural coding.

The DCIC neural responses are complex with variable responses to sound onset, complex azimuthal tuning and large inter-sound interval responses. Nonetheless, the authors do a decent job in wrangling these complex responses: finding non-canonical ways of determining dependence on azimuth and using interpretable decoders to extract information from the population.

Weaknesses:

The decoding results are a bit strange, likely because the population response is quite noisy on any given trial. Raw population responses failed to provide sufficient information concerning azimuth for significant decoding. Importantly, the decoder performed better than chance when certain principal components or top ranked units contributed but did not saturate with the addition of components or top ranked units. So, although there is azimuthal information in the recorded DCIC populations - azimuthal information appears somewhat difficult to extract.

Although necessary given the challenges associated with sampling many conditions with technically difficult recording methods, the limited number of stimulus repeats precludes interpretable characterization of the heterogeneity across the population. Nevertheless, the dataset is public so those interested can explore the diversity of the responses.

The observations from Boffi and colleagues raises the question: what drives neurons in the DCIC to respond? Sound azimuth appears to be a small aspect of the DCIC response. For example, the first 20 principal components which explain roughly 80% of the response variance are insufficient input for the decoder to predict sound azimuth above chance. Furthermore, snout and ear movements correlate with the population response in the DCIC (the ear movements are particularly peculiar given they seem to predict sound presentation). Other movements may be of particular interest to control for (e.g. eye movements are known to interact with IC responses in the primate). These observations, along with reported variance to sound onsets and inter-sound intervals, question the impact of azimuthal information emerging from DCIC responses. This is certainly out of scope for any one singular study to answer, but, hopefully, future work will elucidate the dominant signals in the DCIC population. It may be intuitive that engagement in a sound localization task may push azimuthal signals to the forefront of DCIC response, but azimuthal information could also easily be overtaken by other signals (e.g. movement, learning).

Boffi and colleagues set out to parse the azimuthal information available in the DCIC on a single trial. They largely accomplish this goal and are able to extract this information when allowing the units that contain more information about sound location to contribute to their decoding (e.g., through PCA or decoding on their activity specifically). Interestingly, they also found that positive noise correlations between units with similar azimuthal preferences facilitate this decoding - which is unusual given that this is typically thought to limit information. The dataset will be of value to those interested in the DCIC and to anyone interested in the role of noise correlations in population coding. Although this work is first step into parsing the information available in the DCIC, it remains difficult to interpret if/how this azimuthal information is used in localization behaviors of engaged mice.

Reviewer #1 (Public review):

From the Reviewing Editor:

Four reviewers have assessed your manuscript on valence and salience signaling in the central amygdala. There was universal agreement that the question being asked by the experiment is important. There was consensus that the neural population being examined (GABA neurons) was important and the circular shift method for identifying task-responsive neurons was rigorous. Indeed, observing valenced outcome signaling in GABA neurons would considerably increase the role the central amygdala in valence. However, each reviewer brought up significant concerns about the design, analysis and interpretation of the results. Overall, these concerns limit the conclusions that can be drawn from the results. Addressing the concerns (described below) would work towards better answering the question at the outset of the experiment: how does the central amygdala represent salience vs valence.

A weakness noted by all reviewers was the use of the terms 'valence' and 'salience' as well as the experimental design used to reveal these signals. The two outcomes used emphasized non-overlapping sensory modalities and produced unrelated behavioral responses. Within each modality there are no manipulations that would scale either the value of the valenced outcomes or the intensity of the salient outcomes. While the food outcomes were presented many times (20 times per session over 10 sessions of appetitive conditioning) the shock outcomes were presented many fewer times (10 times in a single session). The large difference in presentations is likely to further distinguish the two outcomes. Collectively, these experimental design decisions meant that any observed differences in central amygdala GABA neuron responding are unlikely to reflect valence, but likely to reflect one or more of the above features.

A second weakness noted by a majority of reviewers was a lack of cue-responsive unit and a lack of exploration of the diversity of response types, and the relationship cue and outcome firing. The lack of large numbers of neurons increasing firing to one or both cues is particularly surprising given the critical contribution of central amygdala GABA neurons to the acquisition of conditioned fear (which the authors measured) as well as to conditioned orienting (which the authors did not measure). Regression-like analyses would be a straightforward means of identifying neurons varying their firing in accordance with these or other behaviors. It was also noted that appetitive behavior was not measured in a rigorous way. Instead of measuring time near hopper, measures of licking would have been better. Further, measures of orienting behaviors such as startle were missing.<br /> The authors also missed an opportunity for clustering-like analyses which could have been used to reveal neurons uniquely signaling cues, outcomes or combinations of cues and outcomes. If the authors calcium imaging approach is not able to detect expected central amygdala cue responding, might it be missing other critical aspects of responding?

All reviewers point out that the evidence for salience encoding is even more limited than the evidence for valence. Although the specific concern for each reviewer varied, they all centered on an oversimplistic definition of salience. Salience ought to scale with the absolute value and intensity of the stimulus. Salience cannot simply be responding in the same direction. Further, even though the authors observed subsets of central amygdala neurons increasing or decreasing activity to both outcomes - the outcomes can readily be distinguished based on the temporal profile of responding.

Additional concerns are raised by each reviewer. Our consensus is that this study sought to answer an important question - whether central amygdala signal salience or valence in cue-outcome learning. However, the experimental design, analyses, and interpretations do not permit a rigorous and definitive answer to that question. Such an answer would require additional experiments whose designs would address the significant concerns described here. Fully addressing the concerns of each reviewer would result in a re-evaluation of the findings. For example, experimental design better revealing valence and salience, and analyses describing diversity of neuronal responding and relationship to behavior would likely make the results Important or even Fundamental.

Reviewer #2 (Public review):

In this article, Kong and authors sought to determine the encoding properties of central amygdala (CeA) neurons in response to oppositely valenced stimuli and cues predicting those stimuli. The amygdala and its subregional components have historically been understood to be regions that encode associative information, including valence stimuli. The authors performed calcium imaging of GABA-ergic CeA neurons in freely-moving mice conditioned in Pavlovian appetitive and fear paradigms, and showed that CeA neurons are responsive to both appetitive and aversive unconditioned and conditioned stimuli. They used a variant of a previously published 'circular shifting' technique (Harris, 2021), which allowed them to delineate between excited/non-responsive/inhibited neurons. While there is considerable overlap of CeA neurons responding to both unconditioned stimuli (in this case, food and shock, deemed "salience-encoding" neurons), there are considerably fewer CeA neurons that respond to both conditioned stimuli that predict the food and shock. The authors finally demonstrated that there are no differences in the order of Pavlovian paradigms (fear - shock vs. shock - fear), which is an interesting result, and convincingly presented given their counterbalanced experimental design.

In total, I find the presented study useful in understanding the dynamics of CeA neurons during a Pavlovian learning paradigm. There are many strengths of this study, including the important question and clear presentation, the circular shifting analysis was convincing to me, and the manuscript was well written. We hope the authors will find our comments constructive if they choose to revise their manuscript.

While the experiments and data are of value, I do not agree with the authors interpretation of their data, and take issue with the way they used the terms "salience" and "valence" (and would encourage them to check out Namburi et al., NPP, 2016) regarding the operational definitions of salience and valence which differ from my reading of the literature. To be fair, a recent study from another group that reports experiments/findings which are very similar to the ones in the present study (Yang et al., 2023, describing valence coding in the CeA using a similar approach) also uses the terms valence and salience in a rather liberal way that I would also have issues with (see below). Either new experiments or revised claims would be needed here, and more balanced discussion on this topic would be nice to see, and I felt that there were some aspects of novelty in this study that could be better highlighted (see below).

One noteworthy point of alarm is that it seems as if two data panels including heatmaps are duplicated (perhaps that panel G of Figure 5-figure supplement 2 is a cut and paste error? It is duplicated from panel E and does not match the associated histogram).

Major concerns:

(1) The authors wish to make claims about salience and valence. This is my biggest gripe, so I will start here.<br /> (1a) Valence scales for positive and negative stimuli and as stated in Namburi et al., NPP, 2016 where we operationalize "valence" as having different responses for positive and negative values and no response for stimuli that are not motivational significant (neutral cues that do not predict an outcome). The threshold for claiming salience, which we define as scaling with the absolute value of the stimulus, and not responding to a neutral stimulus (Namburi et al., NPP, 2016; Tye, Neuron, 2018; Li et al., Nature, 2022) would require the lack of response to a neutral cue.<br /> (1b) The other major issue is that the authors choose to make claims about the neural responses to the USs rather than the CSs. However, being shocked and receiving sucrose also would have very different sensorimotor representations, and any differences in responses could be attributed to those confounds rather than valence or salience. They could make claims regarding salience or valence with respect to the differences in the CSs but they should restrict analysis to the period prior to the US delivery.<br /> (1c) The third obstacle to using the terms "salience" or "valence" is the lack of scaling, which is perhaps a bigger ask. At minimum either the scaling or the neutral cue would be needed to make claims about valence or salience encoding. Perhaps the authors disagree - that is fine. But they should at least acknowledge that there is literature that would say otherwise.<br /> (1d) In order to make claims about valence, the authors must take into account the sensory confound of the modality of the US (also mentioned in Namburi et al., 2016). The claim that these CeA neurons are indeed valence-encoding (based on their responses to the unconditioned stimuli) is confounded by the fact that the appetitive US (food) is a gustatory stimulus while the aversive US (shock) is a tactile stimulus.

(2) Much of the central findings in this manuscript have been previously described in the literature. Yang et al., 2023 for instance shows that the CeA encodes salience (as demonstrated by the scaled responses to the increased value of unconditioned stimuli, Figure 1 j-m), and that learning amplifies responsiveness to unconditioned stimuli (Figure 2). It is nice to see a reproduction of the finding that learning amplifies CeA responses, though one study is in SST::Cre and this one in VGAT::cre - perhaps highlighting this difference could maximize the collective utility for the scientific community?

(3) There is at least one instance of copy-paste error in the figures that raised alarm. In the supplementary information (Figure 5- figure supplement 2 E;G), the heat maps for food-responsive neurons and shock-responsive neurons are identical. While this almost certainly is a clerical error, the authors would benefit from carefully reviewing each figure to ensure that no data is incorrectly duplicated.

(4) The authors describe experiments to compare shock and reward learning; however, there are temporal differences in what they compare in Figure 5. The authors compare the 10th day of reward learning with the 1st day of fear conditioning, which effectively represent different points of learning and retrieval. At the end of reward conditioning, animals are utilizing a learned association to the cue, which demonstrates retrieval. On the day of fear conditioning, animals are still learning the cue at the beginning of the session, but they are not necessarily retrieving an association to a learned cue. The authors would benefit from recording at a later timepoint (to be consistent with reward learning- 10 days after fear conditioning), to more accurately compare these two timepoints. Or perhaps, it might be easier to just make the comparison between Day 1 of reward learning and Day 1 of fear learning, since they must already have these data.

(5) The authors make a claim of valence encoding in their title and throughout the paper, which is not possible to make given their experimental design. However, they would greatly benefit from actually using a decoder to demonstrate their encoding claim (decoding performance for shock-food versus shuffled labels) and simply make claims about decoding food-predictive cues and shock-predictive cues. Interestingly, it seems like relatively few CeA neurons actually show differential responses to the food and shock CSs, and that is interesting in itself.

Reviewer #3 (Public review):

Summary:

In their manuscript entitled Kong and colleagues investigate the role of distinct populations of neurons in the central amygdala (CeA) in encoding valence and salience during both appetitive and aversive conditioning. The study expands on the work of Yang et al. (2023), which specifically focused on somatostatin (SST) neurons of the CeA. Thus, this study broadens the scope to other neuronal subtypes, demonstrating that CeA neurons in general are predominantly tuned to valence representations rather than salience.

Strengths:

One of the key strengths of the study is its rigorous quantitative approach based on the "circular-shift method", which carefully assesses correlations between neural activity and behavior-related variables. The authors' findings that neuronal responses to the unconditioned stimulus (US) change with learning are consistent with previous studies (Yang et al., 2023). They also show that the encoding of positive and negative valence is not influenced by prior training order, indicating that prior experience does not affect how these neurons process valence.

Weaknesses:

However, there are limitations to the analysis, including the lack of population-based analyses, such as clustering approaches. The authors do not employ hierarchical clustering or other methods to extract meaning from the diversity of neuronal responses they recorded. Clustering-based approaches could provide deeper insights into how different subpopulations of neurons contribute to emotional processing. Without these methods, the study may miss patterns of functional specialization within the neuronal populations that could be crucial for understanding how valence and salience are encoded at the population level.

Furthermore, while salience encoding is inferred based on responses to stimuli of opposite valence, the study does not test whether these neuronal responses scale with stimulus intensity-a hallmark of classical salience encoding. This limits the conclusions that can be drawn about salience encoding specifically.

In sum, while the study makes valuable contributions to our understanding of CeA function, the lack of clustering-based population analyses and the absence of intensity scaling in the assessment of salience encoding are notable limitations.

Reviewer #4 (Public review):

Summary:

The authors have performed endoscopic calcium recordings of individual CeA neuron responses to food and shock, as well as to cues predicting food and shock. They claim that a majority of neurons encode valence, with a substantial minority encoding salience.

Strengths:

The use of endoscopic imaging is valuable, as it provides the ability to resolve signals from single cells, while also being able to track these cells across time. The recordings appear well-executed, and employ a sophisticated circular shifting analysis to avoid statistical errors caused by correlations between neighboring image pixels.

Weaknesses:

My main critique is that the authors didn't fully test whether neurons encode valence. While it is true that they found CeA neurons responding to stimuli that have positive or negative value, this by itself doesn't indicate that valence is the primary driver of neural activity. For example, they report that a majority of CeA neurons respond selectively to either the positive or negative US, and that this is evidence for "type I" valence encoding. However, it could also be the case that these neurons simply discriminate between motivationally relevant stimuli in a manner unrelated to valence per se. A simple test of this would be to check if neural responses generalize across more than one type of appetitive or aversive stimulus, but this was not done. The closest the authors came was to note that a small number of neurons respond to CS cues, of which some respond to the corresponding US in the same direction. This is relegated to the supplemental figures (3 and 4), and it is not noted whether the the same-direction CS-US neurons are also valence-encoding with respect to different USs. For example, are the neurons excited by CS-food and US-food also inhibited by shock? If so, that would go a long way toward classifying at least a few neurons as truly encoding valence in a generalizable way.

A second and related critique is that, although the authors correctly point out that definitions of salience and valence are sometimes confused in the existing literature, they then go on themselves to use the terms very loosely. For example, the authors define these terms in such a way that every neuron that responds to at least one stimulus is either salience or valence-encoding. This seems far too broad, as it makes essentially unfalsifiable their assertion that the CeA encodes some mixture of salience and valence. I already noted above that simply having different responses to food and shock does not qualify as valence-encoding. It also seems to me that having same-direction responses to these two stimuli similarly does not quality a neuron as encoding salience. Many authors define salience as being related to the ability of a stimulus to attract attention (which is itself a complex topic). However, the current paper does not acknowledge whether they are using this, or any other definition of salience, nor is this explicitly tested, e.g. by comparing neural response magnitudes to any measure of attention.

The impression I get from the authors' data is that CeA neurons respond to motivationally relevant stimuli, but in a way that is possibly more complex than what the authors currently imply. At the same time, they appear to have collected a large and high-quality dataset that could profitably be made available for additional analyses by themselves and/or others.

Lastly, the use of 10 daily sessions of training with 20 trials each seems rather low to me. In our hands, Pavlovian training in mice requires considerably more trials in order to effectively elicit responses to the CS. I wonder if the relatively sparse training might explain the relative lack of CS responses?

Reviewer #1 (Public review):

Summary:

This manuscript by Garbelli et al. investigates the roles of excitatory amino acid transporters (EAATs) in retinal bipolar cells. The group previously identified that EAAT5b and EAAT7 are expressed at the dendritic tips of bipolar cells, where they connect with photoreceptor terminals. The previous study found that the light responses of bipolar cells, measured by electroretinogram (ERG) in response to white light, were reduced in double mutants, though there was little to no reduction in light responses in single mutants of either EAAT5b or EAAT7.

The current study further explores the roles of EAAT5b and EAAT7 in bipolar cells' chromatic responses. The authors found that bipolar cell responses to red light, but not to green or UV-blue light, were reduced in single mutants of both EAAT5b and EAAT7. In contrast, UV-blue light responses were reduced in double mutants. Additionally, the authors observed that EAAT5b, but not EAAT7, is strongly localized in the UV cone-enriched area of the eye, known as the "Strike Zone (SZ)." This led them to investigate the impact of the EAAT5b mutation on prey detection performance, which is mediated by UV cones in the SZ. Surprisingly, contrary to the predicted role of EAAT5b in prey detection, EAAT5b mutants did not show any changes in prey detection performance compared to wild-type fish. Interestingly, EAAT7 mutants exhibited enhanced prey detection performance, though the underlying mechanisms remain unclear.

The distribution of EAAT7 protein in the outer plexiform layer across the eye correlates with the distribution of red cones. Based on this, the authors tested the behavioral performance driven by red light in EAAT5b and EAAT7 mutants. The results here were again somewhat contrary to predictions based on ERG findings and protein localization: the optomotor response was reduced in EAAT5b mutants, but not in EAAT7 mutants.

Strengths:

Although the paper lacks cohesive conclusions, as many results contradict initial predictions as mentioned above, the authors discuss possible mechanisms for these contradictions and suggest future avenues for study. Nevertheless, this paper demonstrates a novel mechanism underlying chromatic information processing.<br /> The manuscript is well-written, the data are well-presented, and the analysis is thorough.

Weaknesses:

I have only a minor comment. The authors present preliminary data on mGluR6b distribution across the eye. Since this result is based on a single fish, I recommend either adding more samples or removing this data, as it does not significantly impact the paper's main conclusions.

Reviewer #2 (Public review):

Garbelli et. al. set out to elucidate the function of two glutamate transporters, EAAT5b and EAAT7, in the functional and behavioral responses to different wavelengths of light. The question is an interesting one, because these transporters are well positioned to affect responses to light, and their distribution in the retina suggests that they could play differential roles in visual behaviors. However, the low resolution of both the functional and behavioral data presented here means that the conclusions are necessarily a bit vague.

In Figure 1, the authors show that the double KO has a decreased ERG response to UV/blue and red wavelengths. However, the individual mutations only affect the response to red light, suggesting that they might affect behaviors such as OMR which typically rely on this part of the visual spectrum. However, there was no significant change in the response to UV/blue light of any intensity, making it unclear whether the mutations could individually play roles in the detection of UV prey. Based on the later behavioral data, it seems likely that at least the EAAT7 KO should affect retinal responses to UV light, but it may be that the ERG does not have the spatial or temporal resolution to detect the difference, or that the presence of blue light overwhelmed any effect of the individual knockouts on the response to UV light.

In Figures 5 and 6, the authors compare the two knockouts to wild-type fish in terms of their sensitivity to UV prey in a hunting assay. The EAAT5b KO showed no significant impairment in UV sensitivity, while the EAAT7 KO fish actually had an increased hunting response to UV prey. However, there is no comparison of the KO and WT responses to different UV intensities, only in bulk, so we cannot conclude that the EAAT7 KO is allowing the fish to detect weaker prey-like stimuli.

In Figure 7, the EAAT5b KO seems to cause a decrease in OMR behavior to red grating stimuli, but only one stimulus is tested, so it is unclear whether this is due to a change in visual sensitivity or resolution.

The conclusions made in the manuscript are appropriately conservative; the abstract states that these transporters somehow influence prey detection and motion sensing, and this is probably true. However, it is unclear to what extent and how they might be acting on these processes, so the conclusions are a bit unsatisfying.

In terms of impact on the field, this work highlights the potential importance of these two transporters to visual processing, but further studies will be required to say how important they are and what they are doing. The methods presented here are not novel, as UV prey and red OMR stimuli and behaviors have previously been described.

Reviewer #1 (Public review):

Summary:

Previous work demonstrated a strong bias in the percept of an ambiguous Shepard tone as either ascending or descending in pitch, depending on the preceding contextual stimulus. The authors recorded human MEG and ferret A1 single-unit activity during presentation of stimuli identical to those used in the behavioral studies. They used multiple neural decoding methods to test if context-dependent neural responses to ambiguous stimulus replicated the behavioral results. Strikingly, a decoder trained to report stimulus pitch produced biases opposite to the perceptual reports. These biases could be explained robustly by a feed-forward adaptation model. Instead, a decoder that took into account direction selectivity of neurons in the population was able to replicate the change in perceptual bias.

Strengths:

This study explores an interesting and important link between neural activity and sensory percepts, and it demonstrates convincingly that traditional neural decoding models cannot explain percepts. Experimental design and data collection appear to have been executed carefully. Subsequent analysis and modeling appear rigorous. The conclusion that traditional decoding models cannot explain the contextual effects on percepts is quite strong.

Weaknesses:

Beyond the very convincing negative results, it is less clear exactly what the conclusion is or what readers should take away from this study. The presentation of the alternative, "direction aware" models is unclear, making it difficult to determine if they are presented as realistic possibilities or simply novel concepts. Does this study make predictions about how information from auditory cortex must be read out by downstream areas? There are several places where the thinking of the authors should be clarified, in particular, around how this idea of specialized readout of direction-selective neurons should be integrated with a broader understanding of auditory cortex.

Reviewer #2 (Public review):

Summary:

This is an elegant study investigating possible mechanisms underlying the hysteresis effect in the perception of perceptually ambiguous Shepard tones. The authors make a fairly convincing case that the adaptation of pitch direction sensitive cells in auditory cortex is likely responsible for this phenomenon.

Strengths:

The manuscript is overall well written. My only slight criticism is that, in places, particularly for non-expert readers, it might be helpful to work a little bit more methods detail into the results section, so readers don't have to work quite so hard jumping from results to methods and back.

The methods seem sound and the conclusions warranted and carefully stated. Overall I would rate the quality of this study as very high, and I do not have any major issues to raise.

Weaknesses:

I think this study is about as good as it can be with the current state of the art. Generally speaking, one has to bear in mind that this is an observational, rather than an interventional study, and therefore only able to identify plausible candidate mechanisms rather than making definitive identifications. However, the study nevertheless represents a significant advance over the current state of knowledge, and about as good as it can be with the techniques that are currently widely available.

Reviewer #2 (Public review):

Summary:

The revised manuscript presents interesting findings on the role of gut microbiota in gout, focusing on the interplay between age-related changes, inflammation, and microbiota-derived metabolites, particularly butyrate. The study provides valuable insights into the therapeutic potential of microbiota interventions and metabolites for managing hyperuricemia and gout. While the authors have addressed many of the previous concerns, a few areas still require clarification and improvements to strengthen the manuscript's clarity and overall impact.

(1) While the authors mention that outliers in the data do not affect the conclusions, there remains a concern about the reliability of some figures (e.g., Figure 2D-G). It is recommended to provide a more detailed explanation of the statistical analysis used to handle outliers. Additionally, the clarity of the Western blot images, particularly IL-1β in Figure 3C, should be improved to ensure clear and supportive evidence for the conclusions.<br /> (2) The manuscript raises a key question about why butyrate supplementation and FMT have different effects on uric acid metabolism and excretion. While the authors have addressed this by highlighting the involvement of multiple bacterial genera, it is still recommended to expand on the differences between these interventions in the discussion, providing more mechanistic insights based on available literature.<br /> (3) It is noted that IL-6 and TNF-α results in foot tissue were requested and have been added to supplementary material. However, the main text should clearly reference these additions, and the supplementary figures should be thoroughly reviewed for consistency with the main findings. The use of abbreviations (e.g., ns for no significant difference) and labeling should also be carefully checked across all figures.<br /> (4) The manuscript presents butyrate as a key molecule in gout therapy, yet there are lingering concerns about its central role, especially given that other short-chain fatty acids (e.g., acetic and propionic acids) also follow similar trends. The authors should consider further acknowledging these other SCFAs and discussing their potential contribution to gout management. Additionally, the rationale for focusing primarily on butyrate in subsequent research should be made clearer.<br /> (5) The full-length uncropped Western blot images should be provided as requested, to ensure transparency and reproducibility of the data.<br /> (6) Despite the authors' revisions, several references still lack page numbers. Please ensure that all references are properly formatted, including complete page ranges.<br /> The manuscript has improved with the revisions made, particularly regarding clarifications on experimental design and the inclusion of supplementary data. However, some concerns about data quality, mechanistic insights, and clarity in the figures remain. Addressing these points will enhance the overall impact of the work and its potential contribution to the understanding of the gut microbiome in gout and hyperuricemia. A final revision, with careful attention to both major and minor points, is highly recommended before resubmission.

Reviewer #1 (Public review):

Summary:

In their manuscript the authors report that fecal transplantation from young mice into old mice alleviates susceptibility to gout. The gut microbiota in young mice is found to inhibit activation of the NLRP3 inflammasome pathway and reduce uric acid levels in the blood in the gout model.

Strengths:

They focused on the butanoate metabolism pathway based on the results of metabolomics analysis after fecal transplantation and identified butyrate as the key factor in mitigating gout susceptibility. In general, this is a well-performed study.

Weaknesses:

The discussion on the current results and previous studies regarding the effect of butyrate on gout symptoms is insufficient. The authors need to provide a more thorough discussion of other possible mechanisms and relevant literature.

Reviewer #1 (Public review):

Tleiss et al. demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis are blocked in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. This effect induces muscular muscle contraction, which is marked by the formation of TARM structures (thoracic ary-related muscles). This muscle contraction-related blocking happens early after infection (15mins). On the other side, the clearance of bacteria is done by the IMD pathway possibly through antimicrobial peptide production while it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense.

In general, this fundamentally important study reveals unique mechanisms in the gut immunity of Drosophila larvae. It also describes a previously understudied structure, TARM, which may play a crucial role in this process. This significant work substantially advances our understanding of pathogen clearance by identifying a new mode of pathogen eradication from the insect gut. The evidence supporting the authors' claims is compelling, and the study opens new avenues for future research in gut immunity.

Reviewer #2 (Public review):

Summary:

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. Use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

Reviewer #1 (Public review):

Summary:

The planarian flatworm Schmidtea mediterranea is widely used as a model system for regeneration because of its remarkable ability to regenerate its entire body plan from very small fragments of tissue, including the complete and rapid regeneration of the CNS. Prior to this study, analysis of CNS regeneration in planaria has mostly been performed on a gross anatomical level. Despite its simplicity compared to vertebrates, the CNS of many invertebrates, including planaria, is nonetheless complex, intricate, and densely packed. Some invertebrate models allow the visualization of individual cellular components of the CNS using transgenic techniques. Until transgenesis becomes commonplace in planaria, the visualization and analysis of detailed CNS anatomy must rely on alternate approaches in order to capitalize on the immense promise of this system as a model for CNS regeneration. Another challenge for the study of the CNS more broadly is how to perform imaging of a complete CNS on a reasonable timescale such that multiple individuals per experimental condition can be imaged.

Strengths:

In this report, Lu et al. describe a careful and detailed analysis of the planarian neuroanatomy and musculature in both the homeostatic and regenerating contexts. To improve the effective resolution of their imaging, the authors optimized a tissue expansion protocol for planaria. Imaging was performed by light sheet microscopy, and the resulting optical sections were tiled to reconstruct whole worms. Labelled tissues and cells were then segmented to allow quantification of neurons and muscle fibers, as well as all cells in individual worms using a DNA dye. The resulting workflow can produce highly detailed and quantifiable 3D reconstructions at a rate that is fast enough to allow the analysis of large numbers of animals.

Weaknesses:

Lu et al. use their workflow to visualize RNA expression of five enzymes that are each involved in the biosynthetic pathway of different neurotransmitters/modulators, namely chat (cholinergeric), gad (GABAergic), tbh (octopaminergic), th (dopaminergic), and tph (serotonergic). In this way, they generate an anatomical atlas of neurons that produce these molecules. Collectively these markers are referred to as the "neuronpool." They overstate when they write, "The combination of these five types of neurons constitutes a neuron pool that enables the labeling of all neurons throughout the entire body." This statement does not accurately represent the state of our knowledge about the diversity of neurons in S. mediterranea. There are several lines of evidence that support the presence of glutamatergic and glycinergic neurons, including the following. The glutamate receptor agonists NMDA and AMPA both produce seizure-like behaviors in S. mediterranea that are blocked by the application of glutamate receptor antagonists MK-801 and DNQX (which antagonize NMDA and AMPA glutamate receptors, respectively; Rawls et al., 2009). scRNA-Seq data indicates that neurons in S. mediterranea express a vesicular glutamate transporter, a kainite-type glutamate receptor, a glycine receptor, and a glycine transporter (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Two AMPA glutamate receptors, GluR1 and GluR2, are known to be expressed in the CNS of another planarian species, D. japonica (Cebria et al., 2002). Likewise, there is abundant evidence for the presence of peptidergic neurons in S. mediterranea (Collins et al., 2010; Fraguas et al., 2012; Ong et al., 2016; Wyss et al., 2022; among others) and in D. japonica (Shimoyama et al., 2016). For these reasons, the authors should not assume that all neurons can be assayed using the five markers that they selected. The situation is made more complex by the fact that many neurons in S. mediterranea appear to produce more than one neurotransmitter/modulator/peptide (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022), which is common among animals (Vaaga et al., 2014; Brunet Avalos and Sprecher, 2021). However the published literature indicates that there are substantial populations of glutamatergic, glycinergic, and peptidergic neurons in S. mediterranea that do not produce other classes of neurotransmission molecule (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Thus it seems likely that the neuronpool will miss many neurons that only produce glutamate, glycine or a neuropeptide.

The authors use their technique to image the neural network of the CNS using antibodies raised vs. Arrestin, Synaptotagmin, and phospho-Ser/Thr. They document examples of both contralateral and ipsilateral projections from the eyes to the brain in the optic chiasma (Figure 1C-F). These data all seem to be drawn from a single animal in which there appears to be a greater than normal number of nerve fiber defasciculatations. It isn't clear how well their technique works for fibers that remain within a nerve tract or the brain. The markers used to image neural networks are broadly expressed, and it's possible that most nerve fibers are too densely packed (even after expansion) to allow for image segmentation. The authors also show a close association between estrella-positive glial cells and nerve fibers in the optic chiasma.

The authors count all cell types, neuron pool neurons, and neurons of each class assayed. They find that the cell number to body volume ratio remains stable during homeostasis (Figure S3C), and that the brain volume steadily increases with increasing body volume (Figure S3E). They also observe that the proportion of neurons to total body cells is higher in worms 2-6 mm in length than in worms 7-9 mm in length (Figure 2D, S3F). They find that the rate at which four classes of neurons (GABAergic, octopaminergic, dopaminergic, serotonergic) increase relative to the total body cell number is constant (Figure S3G-J). They write: "Since the pattern of cholinergic neurons is the major cell population in the brain, these results suggest that the above observation of the non-linear dynamics between neurons and cell numbers is likely from the cholinergic neurons." This conclusion should not be reached without first directly counting the number of cholinergic neurons and total body cells. Given that glutamatergic, glycinergic, and peptidergic neurons were not counted, it also remains possible that the non-linear dynamics are due (in part or in whole) to one or more of these populations.

The authors next assayed the production of different classes of neurons in regenerating post-pharyngeal tail fragments. At 14 dpa, they find significantly reduced proportions of octopaminergic, GABAergic, and dopaminergic neurons in these regenerated animals (Figure 3K). Given that these three neuron classes are primarily found in the brain region (Figure S2A), this suggests that the brains of these animals may not have finished regenerating by 14 dpa.

The authors next applied their imaging and segmentation technique to the musculature using the 6G10 antibody. They find that the body wall muscle fibers from the dorsal and ventral body walls integrate differently at the anterior end (to form a cobweb-like arrangement) compared to the posterior end (Figure 4I). They knock down β-catenin in regenerating head anterior fragments and find that the resulting double-headed worms produce a cobweb-like arrangement at both ends (Figure 4J).

RNAi knockdown of inr-1 is known to produce mobility defects and have elongated bodies relative to control animals (Lei et al., 2016; Figure S6A). To understand the nature of these defects, the authors image the muscle of inr-1 RNAi animals and find increased circular body wall muscle fibers on both dorsal and ventral sides, while β-catenin RNAi animals have increased longitudinal muscle fibers on the dorsal side (Figure 6C). The inr-1 RNAi animals also have reduced cholinergic neurons (Figure S6B), and ectopic expression of the GABAergic marker gad in the periphery (Figure S6B). Lastly the authors simultaneously image muscle and estrella-positive glia and find that these glia lack their typically elaborate stellate morphology in inr-1 RNAi animals (Figure 6E, S6E-K). The combination of this muscle, neuronal, and glial defects may account for the mobility defects observed in inr-1 RNAi worms.

Reviewer #2 (Public review):

Summary:

This manuscript builds on the authors' 2020 study by combining tissue expansion with light sheet microscopy to quantify the organism-wide spatial distribution of various cell types in the planarian.

Strengths:

(1) The quantification of cell types as a function of animal size and regeneration stages could be a useful resource for the planarian research community.

(2) The high-quality images can help clarify some anatomical structures within the planarian tissues.

Weaknesses:

(1) The proprietary nature of the microscope, protected by a patent, limits the technical details provided, making the method hard to reproduce in other labs.

(2) The resolution of the analyses is mostly limited to the cellular level, which does not fully leverage the advantages of expansion microscopy. Previous applications of expansion microscopy have revealed finer nanostructures in the planarian nervous system (see Fan et al. Methods in Cell Biology 2021; Wang et al. eLife 2021). It is unclear whether the current protocol can achieve a comparable resolution.

(3) The data largely corroborate past observations, while the novel claims are insufficiently substantiated.

A few major issues with the claims:

(4) Line 303-304: While 6G10 is a widely used antibody to label muscle fibers in the planarian, it doesn't uniformly mark all muscle types (Scimone at al. Nature 2017). For a more complete view of muscle fibers, it is important to use a combination of antibodies targeting different fiber types or a generic marker such as phalloidin. This raises fundamental concerns about all the conclusions drawn from Figures 4 and 6 about differences between various muscle types. Additionally, the authors should cite the original paper that developed the 6G10 antibody (Ross et al. BMC Developmental Biology 2015).

(5) Lines 371-379: The claim that DV muscles regenerate into longitudinal fibers lacks evidence. Furthermore, previous studies have shown that TFs specifying different muscle types (DV, circular, longitudinal, and intestinal) both during regeneration and homeostasis are completely different (Scimone et al., Nature 2017 and Scimone et al., Current Biology 2018). Single-cell RNAseq data further establishes the existence of divergent muscle progenitors giving rise to different muscle fibers. These observations directly contradict the authors' claim, which is only based on images of fixed samples at a coarse time resolution.

(6) Line 423: The manuscript lacks evidence to claim glia guide muscle fiber branching.

(7) Lines 432/478: The conclusion about neuronal and muscle guidance on glial projections is similarly speculative, lacking functional evidence. It is possible that the morphological defects of estrella+ cells after bcat1 RNAi are caused by Wnt signaling directly acting on estrella+ cells independent of muscles or neurons.

(8) Finally, several technical issues make the results difficult to interpret. For example, in line 125, cell boundaries appear to be determined using nucleus images; in line 136, the current resolution seems insufficient to reliably trace neural connections, at least based on the images presented.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors apply tissue expansion and tiling light sheet microscopy to study allometric growth and regeneration in planaria. They developed image analysis pipelines to help them quantify different neuronal subtypes and muscles in planaria of different sizes and during regeneration. Among the strengths of this work, the authors provide beautiful images that show the potential of the approaches they are taking and their ability to quantify specific cell types in relatively large numbers of whole animal samples. Many of their findings confirm previous results in the literature, which helps validate the techniques and pipelines they have applied here. Among their new observations, they find that the body wall muscles at the anterior and posterior poles of the worm are organized differently and show that the muscle pattern in the posterior head of beta-catenin RNAi worms resembles the anterior muscle pattern. They also show that glial cell processes appear to be altered in beta-catenin or insulin receptor-1 RNAi worms. Weaknesses include some over-interpretation of the data and lack of consideration or citation of relevant previous literature, as discussed below.

Strengths:

This method of tissue expansion will be useful for researchers interested in studying this experimental animal. The authors provide high-quality images that show the utility of this technique. Their analysis pipeline permits them to quantify cell types in relatively large numbers of whole animal samples.

The authors provide convincing data on changes in total neurons and neuronal sub-types in different-sized planaria. They report differences in body wall muscle pattern between the anterior and posterior poles of the planaria, and that these differences are lost when a posterior head forms in beta-catenin RNAi planaria. They also find that glial cell projections are reduced in insulin receptor-1 RNAi planaria.

Weaknesses:

The work would have been strengthened by a more careful consideration of previous literature. Many papers directly relevant to this work were not cited. Such omissions do the authors a disservice because in some cases, they fail to consider relevant information that impacts the choice of reagents they have used or the conclusions they are drawing.

For example, when describing the antibody they use to label muscles (monoclonal 6G10), they do not cite the paper that generated this reagent (Ross et al PMCID: PMC4307677), and instead, one of the papers they do cite (Cebria 2016) that does not mention this antibody. Ross et al reported that 6G10 does not label all body wall muscles equivalently, but rather "predominantly labels circular and diagonal fibers" (which is apparent in Figure S5A-D of the manuscript being reviewed here). For this reason, the authors of the paper showing different body wall muscle populations play different roles in body patterning (Scimone et al 2017, PMCID: PMC6263039, also not cited in this paper) used this monoclonal in combination with a polyclonal antibody to label all body wall muscle types. Because their "pan-muscle" reagent does not label all muscle types equivalently, it calls into question their quantification of the different body wall muscle populations throughout the manuscript. It does not help matters that their initial description of the body wall muscle types fails to mention the layer of thin (inner) longitudinal muscles between the circular and diagonal muscles (Cebria 2016 and citations therein).

Ipsilateral and contralateral projections of the visual axons were beautifully shown by dye-tracing experiments (Okamoto et al 2005, PMID: 15930826). This paper should be cited when the authors report that they are corroborating the existence of ipsilateral and contralateral projections.

The proportional decrease of neurons with growth in S. mediterranea was shown by counting different cell types in macerated planarians (Baguna and Romero, 1981; https://link.springer.com/article/10.1007/BF00026179) and earlier histological observations cited there. These results have also been validated by single-cell sequencing (Emili et al, bioRxiv 2023, https://www.biorxiv.org/content/10.1101/2023.11.01.565140v). Allometric growth of the planaria tail (the tail is proportionately longer in large vs small planaria) can explain this decrease in animal size. The authors never really discuss allometric growth in a way that would help readers unfamiliar with the system understand this.

In some cases, the authors draw stronger conclusions than their results warrant. The authors claim that they are showing glial-muscle interactions, however, they do not provide any images of triple-stained samples labeling muscle, neurons, and glia, so it is impossible for the reader to judge whether the glial cells are interacting directly with body wall muscles or instead with the well-described submuscular nerve plexus. Their conclusion that neurons are unaffected by beta-cat or inr-1 RNAi based on anti-phospho-Ser/Thr staining (Fig. 6E) is unconvincing. They claim that during regeneration "DV muscles initially regenerate into longitudinal fibers at the anterior tip" (line 373). They provide no evidence for such switching of muscle cell types, so it is unclear why they say this.

The authors show how their automated workflow compares to manual counts using PI-stained specimens (Figure S1T). I may have missed it, but I do not recall seeing a similar ground truth comparison for their muscle fiber counting workflow. I mention this because the segmented image of the posterior muscles in Figure 4I seems to be missing the vast majority of circular fibers visible to the naked eye in the original image.

It is unclear why the abstract says, "We found the rate of neuron cell proliferation tends to lag..." (line 25). The authors did not measure proliferation in this work and neurons do not proliferate in planaria.

It is unclear what readers are to make of the measurements of brain lobe angles. Why is this a useful measurement and what does it tell us?

The authors repeatedly say that this work lets them investigate planarians at the single-cell level, but they don't really make the case that they are seeing things that haven't already been described at the single-cell level using standard confocal microscopy.

Reviewer #1 (Public review):

Summary:

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells.

Strengths:

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC.

Weaknesses:

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses.

Main comments

Novelty:<br /> The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234)

Mouse data:<br /> (a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneth cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis.<br /> (b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was low (17 cells, Fig.3), and assuming that these cells are driving the differences seems somewhat far-fetched.<br /> (c) Fig. 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis.<br /> (d) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.?

Human data:<br /> Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage?

Bioinformatic analysis<br /> GEO repositories remain not open (at the time of the re-review) and SRA links for raw data are still unavailable. Without access to raw data, it is not possible to verify the analyses or fully assess the results. A part of the article was made by re-analyzing public data so the authors should make even the raw available and not just the count tables

Reviewer #2 (Public review):

Summary:

The authors propose that DKK2 is necessary for the metastasis of colon cancer organoids. They then claim that DKK2 mediates this effect by permitting the generation of lysozyme-positive Paneth-like cells within the tumor microenvironmental niche. They argue that these lysozyme-positive cells have Paneth-like properties in both mouse and human contexts. They then implicate HNF4A as the causal factor responsive to DKK2 to generate lysozyme-positive cells through Sox9.

Strengths:

The use of a genetically defined organoid line is state-of-the-art. The data in Figure 1 and the dependence of DKK2 for splenic injection and liver engraftment, as well as the long-term effect on animal survival, are interesting and convincing. The rescue using DKK2 administration for some of their phenotype in vitro is good. The inclusion and analysis of human data sets help explore the role of DKK2 in human cancer and help ground the overall work in a clinical context.

Remaining Weaknesses after revision:

(1) The authors have effectively explained the regulation of HNF4A at both mRNA and protein levels. To further strengthen their findings, I recommend using CRISPR technology to generate DKK2 and HNF4A double knockout organoids. This approach would allow the authors to investigate whether the AKP liver metastasis is restored in the double knockout condition. Such an experiment would provide more direct evidence that HNF4A protein stabilization is the crucial mechanism for liver metastasis suppression following DKK2 knockout.

Reviewer #1 (Public review):

Summary:

In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del.. This article is well written but needs improvement in some points.

Strengths:

This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.

Weaknesses:

This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work

Reviewer #2 (Public review):

Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.

Strengths:

The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.

Weaknesses:

The authors have inadequate experiments to support their hypothesis that the decay of type IIa muscle fibers is likely due to muscle degeneration and reduced capillary formation. Further investigation into capillary density and histopathological changes across different muscle fibers is needed, which could clarify the mechanisms behind these observations.

Reviewer #1 (Public review):

Summary:

The authors have assembled a cohort of 10 SiNET, 1 SiAdeno, and 1 lung MiNEN samples to explore the biology of neuroendocrine neoplasms. They employ single-cell RNA sequencing to profile 5 samples (siAdeno, SiNETs 1-3, MiNEN) and single-nuclei RNA sequencing to profile seven frozen samples (SiNET 4-10).

They identify two subtypes of siNETs, characterized by either epithelial or neuronal NE cells, through a series of DE analyses. They also report findings of higher proliferation in non-malignant cell types across both subtypes. Additionally, they identify a potential progenitor cell population in a single-lung MiNEN sample.

Strengths:

Overall, this study adds interesting insights into this set of rare cancers that could be very informative for the cancer research community. The team probes an understudied cancer type and provides thoughtful investigations and observations that may have translational relevance.

Weaknesses:

The study could be improved by clarifying some of the technical approaches and aspects as currently presented, toward enhancing the support of the conclusions:

(1) Methods: As currently presented, it is possible that the separation of samples by program may be impacted by tissue source (fresh vs. frozen) and/or the associated sequencing modality (single cell vs. single nuclei). For instance, two (SiNET1 and SiNET2) of the three fresh tissues are categorized into the same subtype, while the third (SiNET9) has very few neuroendocrine cells. Additionally, samples from patient 1 (SiNET1 and SiNET6) are separated into different subtypes based on fresh and frozen tissue. The current text alludes to investigations (i.e.: "Technical effects (e.g., fresh vs. frozen samples) could also impact the capture of distinct cell types, although we did not observe a clear pattern of such bias."), but the study would be strengthened with more detail.

(2) Results:<br /> Heterogeneity in the SiNET tumor microenvironment: It is unclear if the current analysis of intratumor heterogeneity distinguishes the subtypes. It may be informative if patterns of tumor microenvironment (TME) heterogeneity were identified between samples of the same subtype. The team could also evaluate this in an extension cohort of published SiNET tumors (i.e. revisiting additional analyses using the SiNET bulk RNAseq from Alvarez et al 2018, a subset of single-cell data from Hoffman et al 2023, or additional bulk RNAseq validation cohorts for this cancer type if they exist [if they do not, then this could be mentioned as a need in Discussion])

(3) Proliferation of NE and immune cells in SiNETs: The observed proliferation of NE and immune cells in SiNETs may also be influenced by technical factors (including those noted above). For instance, prior studies have shown that scRNA-seq tends to capture a higher proportion of immune cells compared to snRNA-seq, which should be considered in the interpretation of these results. Could the team clarify this element?

(4) Putative progenitors in mixed tumors: As written, the identification of putative progenitors in a single lung MiNEN sample feels somewhat disconnected from the rest of the study. These findings are interesting - are similar progenitor cell populations identified in SiNET samples? Recognizing that ideally additional validation is needed to confidently label and characterize these cells beyond gene expression data in this rare tumor, this limitation could be addressed in a revised Discussion.

Reviewer #2 (Public review):

Summary:

The research identifies two main SiNET subtypes (epithelial-like and neuronal-like) and reveals heterogeneity in non-neuroendocrine cells within the tumor microenvironment. The study validates findings using external datasets and explores unexpected proliferation patterns. While it contributes to understanding SiNET oncogenic processes, the limited sample size and depth of analysis present challenges to the robustness of the conclusions.

Strengths:

The studies effectively identified two subtypes of SiNET based on epithelial and neuronal markers. Key findings include the low proliferation rates of neuroendocrine (NE) cells and the role of the tumor microenvironment (TME), such as the impact of Macrophage Migration Inhibitory Factor (MIF).

Weaknesses:

However, the analysis faces challenges such as a small sample size, lack of clear biological interpretation in some analyses, and concerns about batch effects and statistical significance.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to profile small intestine neuroendocrine tumors (siNETs) using single-cell/nucleus RNA sequencing, an established method to characterize the diversity of cell types and states in a tumor. Leveraging this dataset, they identified distinct malignant subtypes (epithelial-like versus neuronal-like) and characterized the proliferative index of malignant neuroendocrine cells versus non-malignant microenvironment cells. They found that malignant neuroendocrine cells were far less proliferative than some of their non-malignant counterparts (e.g., B cells, plasma cells, epithelial cells) and there was a strong subtype association such that epithelial-like siNETs were linked to high B/plasma cell proliferation, potentially mediated by MIF signaling, whereas neuronal-like siNETs were correlated with low B/plasma cell proliferation. The authors also examined a single case of a mixed lung tumor (neuroendocrine and squamous) and found evidence of intermediate/mixed and stem-like progenitor states that suggest the two differentiated tumor types may arise from the same progenitor.

Strengths:

The strengths of the paper include the unique dataset, which is the largest to date for siNETs, and the potentially clinically relevant hypotheses generated by their analysis of the data.

Weaknesses:

The weaknesses of the paper include the relatively small number of independent patients (n = 8 for siNETs), lack of direct comparison to other published single-cell NET datasets, mixing of two distinct methods (single-cell and single-nucleus RNA-seq), lack of direct cell-cell interaction analyses and spatially-resolved data, and lack of in vitro or in vivo functional validation of their findings.

The analytical methods applied in this study appear to be appropriate, but the methods used are fairly standard to the field of single-cell omics without significant methodological innovation. As the authors bring forth in the Discussion, the results of the study do raise several compelling questions related to the possibility of distinct biology underlying the epithelial-like and neuronal-like subtypes, the origin of mixed tumors, drivers of proliferation, and microenvironmental heterogeneity. However, this study was not able to further explore these questions through spatially-resolved data or functional experiments.

Reviewer #1 (Public review):

Summary:

This study evaluates whether species can shift geographically, temporally, or both ways in response to climate change. It also teases out the relative importance of geographic context, temperature variability, and functional traits in predicting the shifts. The study system is large occurrence datasets for dragonflies and damselflies split between two time periods and two continents. Results indicate that more species exhibited both shifts than one or the other or neither, and that geographic context and temp variability were more influential than traits. The results have implications for future analyses (e.g. incorporating habitat availability) and for choosing winner and loser species under climate change. The methodology would be useful for other taxa and study regions with strong community/citizen science and extensive occurrence data.

Strengths:

This is an organized and well-written paper that builds on a popular topic and moves it forward. It has the right idea and approach, and the results are useful answers to the predictions and for conservation planning (i.e. identifying climate winners and losers). There is technical proficiency and analytical rigor driven by an understanding of the data and its limitations.

Weaknesses:

(1) The habitat classifications (Table S3) are often wrong. "Both" is overused. In North America, for example, Anax junius, Cordulia shurtleffii, Epitheca cynosura, Erythemis simplicicollis, Libellula pulchella, Pachydiplax longipennis, Pantala flavescens, Perithemis tenera, Ischnura posita, the Lestes species, and several Enallagma species are not lotic breeding. These species rarely occur let alone successfully reproduce at lotic sites. Other species are arguably "both", like Rhionaeschna multicolor which is mostly lentic. Not saying this would have altered the conclusions, but it may have exacerbated the weak trait effects.

(2) The conservative spatial resolution (100 x 100 km) limits the analysis to wide-ranging and generalist species. There's no rationale given, so not sure if this was by design or necessity, but it limits the number of analyzable species and potentially changes the inference.

(3) The objective includes a prediction about generalists vs specialists (L99-103) yet there is no further mention of this dichotomy in the abstract, methods, results, or discussion.

(4) Key references were overlooked or dismissed, like in the new edition of Dragonflies & Damselflies model organisms book, especially chapters 24 and 27.

Reviewer #2 (Public review):

Summary:

This paper explores a highly interesting question regarding how species migration success relates to phenology shifts, and it finds a positive relationship. The findings are significant, and the strength of the evidence is solid. However, there are substantial issues with the writing, presentation, and analyses that need to be addressed. First, I disagree with the conclusion that species that don't migrate are "losers" - some species might not migrate simply because they have broad climatic niches and are less sensitive to climate change. Second, the results concerning species' southern range limits could provide valuable insights. These could be used to assess whether sampling bias has influenced the results. If species are truly migrating, we should observe northward shifts in their southern range limits. However, if this is an artifact of increased sampling over time, we would expect broader distributions both north and south. Finally, Figure 1 is missed panel B, which needs to be addressed.

Reviewer #3 (Public review):

Summary:

In their article "Range geographies, not functional traits, explain convergent range and phenology shifts under climate change," the authors rigorously investigate the temporal shifts in odonate species and their potential predictors. Specifically, they examine whether species shift their geographic ranges poleward or alter their phenology to avoid extreme conditions. Leveraging opportunistic observations of European and North American odonates, they find that species showing significant range shifts also exhibited earlier phenological shifts. Considering a broad range of potential predictors, their results reveal that geographical factors, but not functional traits, are associated with these shifts.

Strengths:

The article addresses an important topic in ecology and conservation that is particularly timely in the face of reports of substantial insect declines in North America and Europe over the past decades. Through data integration the authors leverage the rich natural history record for odonates, broadening the taxonomic scope of analyses of temporal trends in phenology and distribution to this taxon. The combination of phenological and range shifts in one framework presents an elegant way to reconcile previous findings improving our understanding of the drivers of biodiversity loss.

Weaknesses:

The introduction and discussion of the article would benefit from a stronger contextualization of recent studies on biological responses to climate change and the underpinning mechanism.

The presentation of the results (particularly in figures) should be improved to address the integrative character of the work and help readers extract the main results. While the writing of the article is generally good, particularly the captions and results contain many inconsistencies and lack important detail. With the multitude of the relationships that were tested (the influence of traits) the article needs more coherence.

Reviewer #1 (Public review):

Summary:

Chen and Phillips describe the dynamic appearance of cytoplasmic granules during embryogenesis analogous to SIMR germ granules, and distinct from CSR-1-containing granules, in the C. elegans germline. They show that the nuclear Argonaute NRDE-3, when mutated to abrogate small RNA binding, or in specific genetic mutants, partially colocalizes to these granules along with other RNAi factors, such as SIMR-1, ENRI-2, RDE-3, and RRF-1. Furthermore, NRDE-3 RIP-seq analysis in early vs. late embryos is used to conclude that NRDE-3 binds CSR-1-dependent 22G RNAs in early embryos and ERGO-1-dependent 22G RNAs in late embryos. These data lead to their model that NRDE-3 undergoes small RNA substrate "switching" that occurs in these embryonic SIMR granules and functions to silence two distinct sets of target transcripts - maternal, CSR-1 targeted mRNAs in early embryos and duplicated genes and repeat elements in late embryos.

Strengths:

The identification and function of small RNA-related granules during embryogenesis is a poorly understood area and this study will provide the impetus for future studies on the identification and potential functional compartmentalization of small RNA pathways and machinery during embryogenesis.

Weaknesses:

(1) While the authors acknowledge the following issue, their finding that loss of SIMR granules has no apparent impact on NRDE-3 small RNA loading puts the functional relevance of these structures into question. As they note in their Discussion, it is entirely possible that these embryonic granules may be "incidental condensates." It would be very welcomed if the authors could include some evidence that these SIMR granules have some function; for example, does the loss of these SIMR granules have an effect on CSR-1 targets in early embryos and ERGO-1-dependent targets in late embryos?

(2) The analysis of small RNA class "switching" requires some clarification. The authors re-define ERGO-1-dependent targets in this study to arrive at a very limited set of genes and their justification for doing this is not convincing. What happens if the published set of ERGO-1 targets is used? Further, the NRDE-3 RIP-seq data is used to conclude that NRDE-3 predominantly binds CSR-1 class 22G RNAs in early embryos, while ERGO-1-dependent 22G RNAs are enriched in late embryos. a) The relative ratios of each class of small RNAs are given in terms of unique targets. What is the total abundance of sequenced reads of each class in the NRDE-3 IPs? b) The "switching" model is problematic given that even in late embryos, the majority of 22G RNAs bound by NRDE-3 is in the CSR-1 class (Figure 5D). c) A major difference between NRDE-3 small RNA binding in eri-1 and simr-1 mutants appears to be that NRDE-3 robustly binds CSR-122G RNAs in eri-1 but not in simr-1 in late embryos. This result should be better discussed.

(3) Ultimately, if the switching is functionally important, then its impact should be observed in the expression of their targets. RNA-seq or RT-qPCR of select CSR-1 and ERGO-1 targets should be assessed in nrde-3 mutants during early vs late embryogenesis.

Reviewer #2 (Public review):

Summary:

NRDE-3 is a nuclear WAGO-clade Argonaute that, in somatic cells, binds small RNAs amplified in response to the ERGO-class 26G RNAs that target repetitive sequences. This manuscript reports that, in the germline and early embryos, NRDE-3 interacts with a different set of small RNAs that target mRNAs. This class of small RNAs was previously shown to bind to a different WAGO-clade Argonaute called CSR-1, which is cytoplasmic, unlike nuclear NRDE-3. The switch in NRDE-3 specificity parallels recent findings in Ascaris where the Ascaris NRDE homolog was shown to switch from sRNAs that target repetitive sequences to CSR-class sRNAs that target mRNAs.

The manuscript also correlates the change in NRDE-3 specificity with the appearance in embryos of cytoplasmic condensates that accumulate SIMR-1, a scaffolding protein that the authors previously implicated in sRNA loading for a different nuclear Argonaute HRDE-1. By analogy, and through a set of corelative evidence, the authors argue that SIMR foci arise in embryogenesis to facilitate the change in NRDE-3 small RNA repertoire. The paper presents lots of data that beautifully documents the appearance and composition of the embryonic SIMR-1 foci, including evidence that a mutated NRDE-3 that cannot bind sRNAs accumulates in SIMR-1 foci in a SIMR-1-dependent fashion.

Weaknesses:

The genetic evidence, however, does not support a requirement for SIMR-1 foci: the authors detected no defect in NRDE-3 sRNA loading in simr-1 mutants. Although the authors acknowledge this negative result in the discussion, they still argue for a model (Figure 7) that is not supported by genetic data. My main suggestion is that the authors give equal consideration to other models - see below for specifics.

Reviewer #3 (Public review):

Summary:

Chen and Phillips present intriguing work that extends our view on the C. elegans small RNA network significantly. While the precise findings are rather C. elegans specific there are also messages for the broader field, most notably the switching of small RNA populations bound to an argonaute, and RNA granules behavior depending on developmental stage. The work also starts to shed more light on the still poorly understood role of the CSR-1 argonaute protein and supports its role in the decay of maternal transcripts. Overall, the work is of excellent quality, and the messages have a significant impact.

Strengths:

Compelling evidence for major shift in activities of an argonaute protein during development, and implications for how small RNAs affect early development. Very balanced and thoughtful discussion.

Weaknesses:

Claims on col-localization of specific 'granules' are not well supported by quantitative data.

Reviewer #1 (Public review):

Summary:

Furman et al. reanalyze data from a previous study and investigate alterations of peak alpha frequency (PAF) and alpha power (AP) in the context of prolonged pain with electroencephalography (EEG). Using two experimental pain models (phasic and capsaicin heat pain), they set out to clarify if previously reported changes in alpha activity in chronic pain can already be observed during prolonged pain in healthy human participants. They conclude that PAF is reliably slowed, and AP reliably decreased in response to prolonged pain. From the patterns of their findings, they furthermore deduce that AP changes indicate the presence of ongoing pain while PAF changes reflect pain-associated states like sensitization which can outlast ongoing pain percepts and indicate a potential for experiencing pain. Lastly, they conclude that the reported changes in alpha activity are likely due to specific power decreases in the faster alpha range between 10 and 12 Hz and discuss potential clinical implications of their findings in terms of risk biomarkers and early pain interventions.

Strengths:

The study focuses on a timely topic with potential implications for chronic pain diagnosis and treatment, an area that urgently needs new approaches. The addressed questions nicely build upon and extend the previous work of the authors. The analyzed data set is comprehensive including two different prolonged pain paradigms, two visits following the same experimental procedures, and a total sample size of n = 61 participants. Thereby, it enabled internal replications of findings across both paradigms and visits, which is important to confirm the consistency of findings.

Weaknesses:

One overarching difficulty is the high number of analyses presented by the authors. They were in part developed "on the go", are not always easy to follow, and sidetrack the reader from the main findings. Only a minor part of the analyses is described in the methods section, while many analyses are outlined within the results, the supplementary material, and/or figure legends. In addition, a range of purely descriptive findings are displayed. Overall, the manuscript would clearly benefit from a more streamlined and consistent presentation of the applied methods and results.

Concerning the main findings, the presented evidence for a slowing of PAF and a reduction of AP in the context of both phasic and capsaicin heat pain and across both visits is convincing. The location of the peak of the effect at left frontocentral areas, however, remains puzzling. The authors convincingly show that the effect cannot be explained by activity related to the pain rating procedure and provide evidence that an effect of the same direction can also observed at corresponding electrodes contralateral to pain stimulation. However, further reasons are not discussed.

The conclusion that PAF slowing might be more related to pain-associated states like sensitization rather than the presence of ongoing pain is deduced from a continued slowing of PAF after capsaicin-induced pain has subsided, while AP goes back to baseline values. Although this speculation is interesting, the readers should be aware that this dissociation was unexpected and resulted in changes in the main a-priori-defined statistical contrasts presented in the methods section. Further replications in future studies are needed to strengthen this finding.

The last conclusion made by the authors is that the observed changes in alpha activity are caused by specific changes in the faster alpha range and are the least convincing. If I understand correctly, the only presented statistical evidence corroborating this conclusion is based on the single selected electrode C3 shown in Figure 5 A, D, and E. With the remaining parts of Figure 5 and Figure 6, differences are discussed but Figures do not include statistical results. Unless the discussed findings are backed up more clearly, the degree of mechanistic conclusions concerning the 10-12 Hz power changes throughout the title, abstract, and main manuscript and in relation to the multiple oscillators model seems not justified.

Lastly, it is important to note that the current manuscript was published as a preprint in 2021. Thus, the cited literature still needs to be updated, and the present findings need to be integrated with the work published since. For example, a recent systematic review on potential M/EEG-based biomarkers of chronic pain (Zebhauser et al., 2023, Pain) revealed that previous evidence concerning changes of alpha activity in chronic pain is much less consistent than currently outlined in the manuscript.

Overall:

All in all, the presented findings extend previous knowledge concerning the role of alpha activity in pain and thus represent a valuable contribution towards a better understanding of the mechanisms of pain and potential new treatment targets.

Reviewer #2 (Public review):

Summary:

This study investigated the modulation of alpha oscillations, specifically peak alpha frequency (PAF) and alpha power, during prolonged pain. The findings suggest that the alpha rhythm consists of multiple, independent oscillators, and suggest that the modulation of a "fast" oscillator may represent a promising therapeutic target for ongoing pain management.

Strengths:

EEG data were collected from a relatively large sample of participants, and the experiment was conducted using two prolonged pain models - phasic heat pain and capsaicin heat pain - at two separate testing visits approximately 8 weeks apart. The study produced reliable results across different pain models and at different testing intervals.

Weaknesses:

There are discrepancies between the results and their interpretation, indicating a need for more appropriate data analyses. Additionally, the experimental design does not adequately control for the potential time effects, which cannot be ruled out as a confounding factor.

Reviewer #3 (Public review):

Summary:

Furman et al. investigated how exposure to prolonged pain impacts human alpha oscillations recorded by electroencephalography (EEG). Two experimental models of prolonged pain were employed in healthy participants, phasic heat pain (PHP) and capsaicin heat pain (CHP). 61 participants completed two identical study visits separated by at least 8 weeks. Peak alpha frequency was reliably slowed by exposure to prolonged pain, whereas overall alpha power was reliably reduced. Both effects appeared to reflect a specific decrease in higher frequency (10-12Hz) alpha activity. The authors suggest that slowing of alpha oscillations is a reliable neural correlate of pain exposure and that manipulation of alpha activity may hold promise for treating chronic pain.

Strengths:

The study uses a within-participants design to show that exposure to pain is associated with acute changes in both the power and frequency of alpha oscillations.

By employing two experimental models of pain exposure and two separate testing visits, the authors were able to show that the effects of pain exposure on alpha activity are replicable across models and time.

Rigorous analysis approaches are used throughout.

Weaknesses:

No a priori power analysis is presented and (due to exclusions) most of the analyses conducted included (sometimes considerably) fewer participants than the overall sample size.

It is not clear whether the power and frequency changes represent two sides of the same coin or whether they reflect distinct mechanisms. The authors suggest in the manuscript that both effects may be explained by decreased power in 'fast' (8-12 Hz) alpha activity, but at other times interpret the effects to potentially represent distinct mechanisms. It would be useful for the authors to further clarify their thoughts on this point.

The statistical significance of some of the effects was dependent on analysis choices such as the exact frequency range chosen to identify alpha peaks.

No control condition was used, and I was left wondering if the effects would be specific to painful stimuli, or would also see the same effects for pleasant or neutral somatosensory stimuli?

Reviewer #1 (Public review):

Summary:

The manuscript uses large-scale existing datasets that span almost the full range of human life (5-100 years) to identify two distinct architectural cortical gradients within the visual cortex. These gradients are distinct: in one, cytoarchitecture and myeloarchitecture converge and in the other, they diverge. The authors tested whether these gradients mapped onto known functional properties of the visual cortex, as well as accounting for visual behaviours that are impacted throughout the lifespan. The manuscript also reports the identification of a hitherto unknown cluster of visual field maps in the anterior temporal lobe.

Strengths:

A major strength of the current manuscript is the use of large-scale measurements of human brain structure throughout the lifespan, courtesy of the Human Connectome Project Initiative. The scope of this cross-sectional analysis would be rare, if not impossible to achieve through an individual project.

The approach employed holds promise for assessing the link between large-scale anatomical gradients in the brain and functional/behavioural properties. The current manuscript focuses on the visual cortex but the approach could easily be implemented across the brain in general.

Weaknesses:

While the evidence in favour of the two gradients largely supports the claims, the evidence for a new visual field map cluster in the anterior temporal lobe falls short of the level used historically when identifying visual field maps in the visual cortex and is, at present, not convincing.

More specifically, the progressions of polar angle within the putative anterior lobe cluster are highly variable across subjects. Few subjects have convincing polar angle reversals at either the horizontal or vertical meridians. In other cases, a putative border is shown that spans different polar angles, which does not align with the accepted definitions for visual field maps in the cortex.

Reviewer #2 (Public review):

Summary:

The authors used large MRI data sets of the Human Connectome Project (HCP) and also conducted additional pRF analyses to describe the structural architecture of the human visual cortex in reference to its functional features. By conducting a PCA, they identify 2 components that explain around 50% of the variance, the driven by a positive co-variance between cortical thickness and T1/T2 ratio, the second by their negative co-variance. The first PC spans most early visual cortex and hence shows a relation to pRF size when taking both early and late visual areas into account. The second is more variable in location and does not relate to pRF size or visual hierarchy. The relationship between these two gradients to cell body density using the BigBrain is explored.

Strengths:

The authors make an attempt to describe the overall architectural features of the cortex and link it to features of functional representations, and the underlying histology, using different sets of datasets and methods, including histology. They highlight that investigating the structural architecture of the cortex provides important information on their intrinsic organization and common features.

Weaknesses:

The neurobiological model does not take into consideration present knowledge about the microstructural organization of the visual system. This limits the way the results are interpreted correctly. Critical information on the layer-specific myeloarchitecture and cytoarchitecture (and their relation to cortical thickness), as explored for example by Sereno et al. 2013 Cereb Cortex, is missing. There is no information given with respect to how different visual areas differ in their microstructural profile. It is also not mentioned that cortical parcellation is indeed characterized by sharp boundaries between areas, rather than structural gradients, so it remains unclear why focusing on a gradient is of interest. The authors cite the parcellation atlas by Glasser et al. 2016, but do not discuss the rationale of this publication, which was not the definition of gradients, but the definition of sharp boundaries for cortex parcellation. Indeed (as explained below), the results of the authors seem to a large extent to be driven by cortex parcellation, but instead of acknowledging this fact, the authors write (line 179) that "we hypothesize that these local deviations from the canonical thickness and density of cortex underlie the finer-scale division of visual cortex into categorically distinct regions. That is, does the realization of the cortex into distinct regions involve these regions becoming more distinct from a prototypical cortical sheet (i.e., gradient 1)?" - While the first sentence is reasonable, the second sentence is pure speculation ignoring present knowledge on cortical parcellation of this area according to which there is no "prototypical cortical sheet", but each area has its distinct microstructural profile.

Instead of building on present, detailed knowledge of brain anatomy and in-vivo cortex parcellation of the visual system and its known relation to visual maps, the authors focus on two metrics of cortex architecture (mean T1/T1 over depth and cortical thickness), and conduct a PCA to explore their shared variance. It needs to be clarified if the PCA was conducted correctly. There is no mention of standardizing the variables, which could bias the results. In addition, in a PCA, all possible features are categorized as vector components, and those are scanned through the samples, hence, one such analysis per vertex. But the authors write "in which participants are features and cortical vertices are samples" and "the thickness and tissue density maps were concatenated". This needs clarification. The architecture of the PCA should be visualized better.

Because the PCA only contains two features, PC1 is driven by the positive relationship between cortical thickness and mean T1/T2, whereas PC2 is driven by their negative relationship. Because in the early visual cortex, cortical thickness and mean T1/T2 correlate positively, it naturally follows that PC1 relates to pRF size (but mediated by the actual cortex parcellation). However, it is unclear why this insight is interesting. I also do not share the view that "these findings demonstrate that gradient 1 acts as a global gradient enveloping the entire visual cortex (...) while gradient 2 acts as a local gradient specific to individual visual streams". I think this relationship between cortical thickness and T1/T2 ratio does not have much to do with local and global gradients. But if so, stronger arguments as to why this should be the case should be presented.

What the authors make of this result (particularly the discussion starting line 366) is not clear to me. I cannot follow the line of argumentation, which in my view is too far away from the data.

Reviewer #1 (Public review):

Summary:

In the presented study, the authors aim to explore the role of nociceptors in the fine particulate matter (FPM) mediated Asthma phenotype, using rodent models of allergic airway inflammation. This manuscript builds on previous studies, and identify transciptomic reprogramming and an increased sensitivity of the jugular nodose complex (JNC) neurons, one of the major sensory ganglion for the airways, on exposure to FPM along with Ova during the challenge phase. The authors then use OX-314 a selectively permeable form of lidocaine, and TRPV1 knockouts to demonstrate that nociceptor blocking can reduce airway inflammation in their experimental setup.

The authors further identify the presence of Gfra3 on the JNC neurons, a receptor for the protein Artemin, and demonstrate their sensitivity to Artmein as a ligand. They further show that alveolar macrophages release Artemin on exposure to FPM.

Strengths:

The study builds on results available from multiple previous work, and presents important results which allow insights into the mixed phenotypes of Asthma seen clinically. In addition, by identifying the role of nociceptors, they identify potential therapeutic targets which bear high translational potential.

Weaknesses:

While the results presented in the study are highly relevant, there is a need for further mechanistic dissection to allow better inferences. Currently certain results seem assocaitive. Also, certain visualisations and experimental protocols presented in the manuscript need careful assessment and interpretation.

While Asthma is a chronic disease, the presented results are particularly important to explore Asthma exacerbations in response to acute expsoure to air pollutants. This is relevant in today's age of increasing air pollution and increasing global travel.

Reviewer #2 (Public review):

Summary:

The authors sought to investigate the role of nociceptor neurons in the pathogenesis of pollution-mediated neutrophilic asthma.

Strengths:

The authors utilize TRPV1 ablated mice to confirm effects of intranasally administered QX-314 utilized to block sodium currents.

The authors demonstrate that via artemin, which is upregulated in alveolar macrophages in response to pollution, sensitizes JNC neurons thereby increasing their responsiveness to pollution. Ablation or inactivity of nociceptor neurons prevented the pollution induced increase in inflammation.

Weaknesses:

While neutrophilic, the model used doesn't appear to truly recapitulate a Th2/Th17 phenotype. No IL-17A is visible/evident in the BALF fluid within the model. (Figure 3F).

Unclear of the relevance of the RNAseq dataset, none of the identified DEGs were evaluated in the context of mechanism.

The authors overall achieved the aim of demonstrating that nociceptor neurons are important to the pathogenesis of pollution-exacerbated asthma. Their results support their conclusions overall, although there are ways the study findings can be strengthened. This work further evaluates how nociceptor neurons contribute to asthma pathogenesis important for consideration while proposing treatment strategies for undertreated asthma endotypes.

Reviewer #3 (Public review):

Asthma is a complex disease that includes endogenous epithelial, immune, and neural components that respond awkwardly to environmental stimuli. Small airborne particles with diameters in the range of 2.5 micrometers or less, so-called PM2.5, are generally thought to contribute to some forms of asthma. These forms of asthma may have increased numbers of neutrophils and/or eosinophils present in bronchoalveolar lavage fluid and are difficult to treat effectively as they tend to be poorly responsive to steroids. Here, Wang and colleagues build on a recent model that incorporated PM2.5 which was found to have a neutrophilic component. Wang altered the model to provide an extra kick via the incorporation of ovalbumin. Building on their prior expertise linking nociceptors and inflammation, they find that silencing TRPV1-expressing neurons either pharmacologically or genetically, abrogated inflammation and the accumulation of neutrophils. By examining bronchoalveolar lavage fluid, they found not only that levels of the number of cytokines were increased, but also that artemin, a protein that supports neuronal development and function, was elevated, which did not occur in nociceptor-ablated mice. They also found that alveolar macrophages exposed to PM2.5 particles had increased artemin transcription, suggesting a further link between pollutants, and immune and neural interactions.

There are substantial caveats that must be attached to the suggestions by the authors that targeting nociceptors might provide an approach to the treatment of neutrophilic airway inflammation in pollution-driven asthma in general and wildfire-associated respiratory problems in particular. These caveats include the uncertainty of the relevance of the conventional source of PM2.5, to pollution and asthma. According to the National Institute of Standards and Technology (NIST), the standard reference material (SRM) 2786 is a mix obtained from an air intake system in the Czech Republic. It is not clear exactly what is in the mix, and a recent bioRxiv preprint, https://www.biorxiv.org/content/10.1101/2023.08.18.553903v3.full.pdf reveals the presence of endotoxin. Care should thus be taken in interpreting data using particulate matter. Regarding wildfires, there is data that indicates that such exposure is toxic to macrophages. What impact might that then have on the production of cytokines, and artemin, in humans?

Reviewer #1 (Public review):

Summary:

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

Strengths:

The manuscript is well-written and a pleasure to read. The figures are beautiful and clear. I actually had a hard time coming up with suggestions.

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strength:

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

Weakness:

As you can see in the "Recommendations to the Authors" section below, there are several things in this paper that are not fully explained about experimental methods. Except for this point, this paper appears to me to have no major weaknesses.

Reviewer #3 (Public review):

Summary:

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

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size.

Weaknesses:

The manuscript would benefit from a more detailed explanation of the research advances this work provides. Stating that this is the first deorphanization of an odorant receptor in a clade is insufficient. The introduction primarily reviews termite chemical communication and deorphanization of olfactory receptors previously performed. Although this is essential background, it lacks a good integration into explaining what problem the current study solves.

Selecting target ORs for deorphanization is an essential step in the approach. Unfortunately, the process of choosing these ORs has not been described. Were the authors just lucky that they found the correct OR out of the 50, or was there a specific selection process that increased the probability of success?

The authors assigned antennal sensilla into five categories. Unfortunately, they did not support their categories well. It is not clear how they were able to differentiate SI and SII in their antennal recordings.

The authors used a large odorant panel to determine receptor tuning. The panel included volatile polar compounds and non-volatile non-polar hydrocarbons. Usually, some heat is applied to such non-volatile odorants to increase volatility for receptor testing. It is unclear how it is possible that these non-volatile compounds can reach the tested sensilla without heat application.

Reviewer #1 (Public review):

Summary:

Sattin, Nardin, and colleagues designed and evaluated corrective microlenses that increase the useable field of view of two long (>6mm) thin (500 um diameter) GRIN lenses used in deep-tissue two-photon imaging. This paper closely follows the thread of earlier work from the same group (e.g. Antonini et al, 2020; eLife), filling out the quiver of available extended-field-of-view 2P endoscopes with these longer lenses. The lenses are made by a molding process that appears practical and easy to adopt with conventional two-photon microscopes.

Simulations are used to motivate the benefits of extended field of view, demonstrating that more cells can be recorded, with less mixing of signals in extracted traces, when recorded with higher optical resolution. In vivo tests were performed in the piriform cortex, which is difficult to access, especially in chronic preparations.

The design, characterization, and simulations are clear and thorough, but not exhaustive (see below), and do not break new ground in optical design or biological application. However, the approach shows much promise, including for applications not mentioned in the present text such as miniaturized GRIN-based microscopes. Readers will largely be interested in this work for practical reasons: to apply the authors' corrected endoscopes.

Strengths:

The text is clearly written, the ex vivo analysis is thorough and well-supported, and the figures are clear. The authors achieved their aims, as evidenced by the images presented, and were able to make measurements from large numbers of cells simultaneously in vivo in a difficult preparation.

Weaknesses:

(1) The novelty of the present work over previous efforts from the same group is not well explained. What needed to be done differently to correct these longer GRIN lenses?

(2) Some strong motivations for the method are not presented. For example, the introduction (page 3) focuses on identifying neurons with different coding properties, but this can be done with electrophysiology (albeit with different strengths and weaknesses). Compared to electrophysiology, optical methods more clearly excel at genetic targeting, subcellular measurements, and molecular specificity; these could be mentioned. Another example, in comparing microfabricated lenses to other approaches, an unmentioned advantage is miniaturization and potential application to mini-2P microscopes, which use GRIN lenses.

(3) Some potentially useful information is lacking, leaving critical questions for potential adopters:

How sensitive is the assembly to decenter between the corrective optic and the GRIN lens? What is the yield of fabrication and of assembly?

Supplementary Figure 1: Is this really a good agreement between the design and measured profile? Does the figure error (~10 um in some cases on average) noticeably degrade the image? How do individual radial profiles compare to the presented means?<br /> What is the practical effect of the strong field curvature? Are the edges of the field, which come very close to the lens surface, a practical limitation?

The lenses appear to be corrected for monochromatic light; high-performance microscopes are generally achromatic. Is the bandwidth of two-photon excitation sufficient to warrant optimization over multiple wavelengths?

GRIN lenses are often used to access a 3D volume by scanning in z (including in this study). How does the corrective lens affect imaging performance over the 3D field of view?

(4) The in vivo images (Figure 7D) have a less impressive resolution and field than the ex vivo images (Figure 4B), and the reason for this is not clear. Given the difference in performance, how does this compare to an uncorrected endoscope in the same preparation? Is the reduced performance related to uncorrected motion, field curvature, working distance, etc? Regarding Figure 7, there is no analysis of the biological significance of the calcium signals or even a description of where olfactory stimuli were presented. The timescale of jGCaMP8f signals in Figure 7E is uncharacteristically slow for this indicator (compared to Zhang et al 2023 (Nature)), though perhaps this is related to the physiology of these cells or the stimuli.

(5) The claim of unprecedented spatial resolution across the FOV (page 18) is hard to evaluate and is not supported by references to quantitative comparisons. The promises of the method for future studies (pages 18-19) could also be better supported by analysis or experiment, but these are minor and to me, do not detract from the appeal of the work.

(6) The text is lengthy and the material is repeated, especially between the introduction and conclusion. Consolidating introductory material to the introduction would avoid diluting interesting points in the discussion.

Reviewer #2 (Public review):

In this manuscript, the authors present an approach to correct GRIN lens aberrations, which primarily cause a decrease in signal-to-noise ratio (SNR), particularly in the lateral regions of the field-of-view (FOV), thereby limiting the usable FOV. The authors propose to mitigate these aberrations by designing and fabricating aspherical corrective lenses using ray trace simulations and two-photon lithography, respectively; the corrective lenses are then mounted on the back aperture of the GRIN lens.

This approach was previously demonstrated by the same lab for GRIN lenses shorter than 4.1 mm (Antonini et al., eLife, 2020). In the current work, the authors extend their method to a new class of GRIN lenses with lengths exceeding 6 mm, enabling access to deeper brain regions as most ventral regions of the mouse brain. Specifically, they designed and characterized corrective lenses for GRIN lenses measuring 6.4 mm and 8.8 mm in length. Finally, they applied these corrected long micro-endoscopes to perform high-precision calcium signal recordings in the olfactory cortex.

Compared with alternative approaches using adaptive optics, the main strength of this method is that it does not require hardware or software modifications, nor does it limit the system's temporal resolution. The manuscript is well-written, the data are clearly presented, and the experiments convincingly demonstrate the advantages of the corrective lenses.

The implementation of these long corrected micro-endoscopes, demonstrated here for deep imaging in the mouse olfactory bulb, will also enable deep imaging in larger mammals such as rats or marmosets.

Reviewer #3 (Public review):

Summary:

This work presents the development, characterization, and use of new thin microendoscopes (500µm diameter) whose accessible field of view has been extended by the addition of a corrective optical element glued to the entrance face. Two micro endoscopes of different lengths (6.4mm and 8.8mm) have been developed, allowing imaging of neuronal activity in brain regions >4mm deep. An alternative solution to increase the field of view could be to add an adaptive optics loop to the microscope to correct the aberrations of the GRIN lens. The solution presented in this paper does not require any modification of the optical microscope and can therefore be easily accessible to any neuroscience laboratory performing optical imaging of neuronal activity.

Strengths:

(1) The paper is generally clear and well-written. The scientific approach is well structured and numerous experiments and simulations are presented to evaluate the performance of corrected microendoscopes. In particular, we can highlight several consistent and convincing pieces of evidence for the improved performance of corrected micro endoscopes:<br /> a) PSFs measured with corrected micro endoscopes 75µm from the centre of the FOV show a significant reduction in optical aberrations compared to PSFs measured with uncorrected micro endoscopes.<br /> b) Morphological imaging of fixed brain slices shows that optical resolution is maintained over a larger field of view with corrected micro endoscopes compared to uncorrected ones, allowing neuronal processes to be revealed even close to the edge of the FOV.<br /> c) Using synthetic calcium data, the authors showed that the signals obtained with the corrected microendoscopes have a significantly stronger correlation with the ground truth signals than those obtained with uncorrected microendoscopes.

(2) There is a strong need for high-quality micro endoscopes to image deep brain regions in vivo. The solution proposed by the authors is simple, efficient, and potentially easy to disseminate within the neuroscience community.

Weaknesses:

(1) Many points need to be clarified/discussed. Here are a few examples:

a) It is written in the methods: « The uncorrected microendoscopes were assembled either using different optical elements compared to the corrected ones or were obtained from the corrected probes after the mechanical removal of the corrective lens. »<br /> This is not very clear: the uncorrected microendoscopes are not simply the unmodified GRIN lenses?

b) In the results of the simulation of neuronal activity (Figure 5A, for example), the neurons in the center of the FOV have a very large diameter (of about 30µm). This should be discussed. Also, why is the optical resolution so low on these images?

c) It seems that we can't see the same neurons on the left and right panels of Figure 5D. This should be discussed.

d) It is not very clear to me why in Figure 6A, F the fraction of adjacent cell pairs that are more correlated than expected increases as a function of the threshold on peak SNR. The authors showed in Supplementary Figure 3B that the mean purity index increases as a function of the threshold on peak SNR for all micro endoscopes. Therefore, I would have expected the correlation between adjacent cells to decrease as a function of the threshold on peak SNR. Similarly, the mean purity index for the corrected short microendoscope is close to 1 for high thresholds on peak SNR: therefore, I would have expected the fraction of adjacent cell pairs that are more correlated than expected to be close to 0 under these conditions. It would be interesting to clarify these points.

e) Figures 6C, H: I think it would be fairer to compare the uncorrected and corrected endomicroscopes using the same effective FOV.

f) Figure 7E: Many calcium transients have a strange shape, with a very fast decay following a plateau or a slower decay. Is this the result of motion artefacts or analysis artefacts? Also, the duration of many calcium transients seems to be long (several seconds) for GCaMP8f. These points should be discussed.

g) The authors do not mention the influence of the neuropil on their data. Did they subtract the neuropil's contribution to the signals from the somata? It is known from the literature that the presence of the neuropil creates artificial correlations between neurons, which decrease with the distance between the neurons (Grødem, S., Nymoen, I., Vatne, G.H. et al. An updated suite of viral vectors for in vivo calcium imaging using intracerebral and retro-orbital injections in male mice. Nat Commun 14, 608 (2023). https://doi.org/10.1038/s41467-023-36324-3; Keemink SW, Lowe SC, Pakan JMP, Dylda E, van Rossum MCW, Rochefort NL. FISSA: A neuropil decontamination toolbox for calcium imaging signals. Sci Rep. 2018 Feb 22;8(1):3493. doi: 10.1038/s41598-018-21640-2. PMID: 29472547; PMCID: PMC5823956)<br /> This point should be addressed.

h) Also, what are the expected correlations between neurons in the pyriform cortex? Are there measurements in the literature with which the authors could compare their data?

(2) The way the data is presented doesn't always make it easy to compare the performance of corrected and uncorrected lenses. Here are two examples:

a) In Figures 4 to 6, it would be easier to compare the FOVs of corrected and uncorrected lenses if the scale bars (at the centre of the FOV) were identical. In this way, the neurons at the centre of the FOV would appear the same size in the two images, and the distances between the neurons at the centre of the FOV would appear similar. Here, the scale bar is significantly larger for the corrected lenses, which may give the illusion of a larger effective FOV.

b) In Figures 3A-D it would be more informative to plot the distances in microns rather than pixels. This would also allow a better comparison of the micro endoscopes (as the pixel sizes seem to be different for the corrected and uncorrected micro endoscopes).

(3) There seems to be a discrepancy between the performance of the long lenses (8.8mm) in the different experiments, which should be discussed in the article. For example, the results in Figure 4 show a considerable enlargement of the FOV, whereas the results in Figure 6 show a very moderate enlargement of the distance at which the person's correlation with the first ground truth emitter starts to drop.

a) There is also a significant discrepancy between measured and simulated optical performance, which is not discussed. Optical simulations (Figure 1) show that the useful FOV (defined as the radius for which the size of the PSF along the optical axis remains below 10µm) should be at least 90µm for the corrected microendoscopes of both lengths. However, for the long microendoscopes, Figure 3J shows that the axial resolution at 90µm is 17µm. It would be interesting to discuss the origin of this discrepancy: does it depend on the microendoscope used? Are there inaccuracies in the construction of the aspheric corrective lens or in the assembly with the GRIN lens? If there is variability between different lenses, how are the lenses selected for imaging experiments?

Reviewer #1 (Public review):

Summary:

Park et al. conducted various analyses attempting to elucidate the biological significance of SARS-CoV-2 mutations. However, the study lacks a clear objective. The specific goals of the analyses in each subsection are unclear, as is how the results from these subsections are interconnected. Compiling results from unrelated analyses into a single paper can be confusing for readers. Clarifying the objective and narrowing down the topics would make the paper's purpose clearer.

The logic of the study is also unclear. For instance, the authors developed an evaluation score, APESS, for analyzing viral sequences. Although they state that the APESS score correlates with viral infectivity, there is no explanation in the results section about why this is the case.

The structure of the paper should be reconsidered.

Reviewer #2 (Public review):

Summary:

The authors have developed a machine learning tool AIVE to predict the infectivity of SARS-CoV-2 variants and also a scoring metric to measure infectivity. A large number of virus sequences were used with a very detailed analysis that incorporates hydrophobic, hydrophilic, acid, and alkaline characteristics. The protein structures were also considered to measure infectivity and search for core mutations. The study especially focused on the S protein of SARS-CoV-2. The contents of this study would be of interest to many researchers related to this area and the web service would be helpful to easily analyze such data without in-depth bioinformatics expertise.

Strengths:

- Analysis of large-scale data.

- Experimental validation on a partial set of searched mutations.

- A user-friendly web-based analysis platform that is made public.

Weaknesses:

- Complexity of the research.

Reviewer #1 (Public review):

Summary:

This is a contribution to the field of developmental bioelectricity. How do changes of resting potential at the cell membrane affect downstream processes? Zhou et al. reported in 2015 that phosphatidylserine and K-Ras cluster upon plasma membrane depolarization and that voltage-dependent ERK activation occurs when constitutive active K-RasG12V mutants are overexpressed. In this paper, the authors advance the knowledge of this phenomenon by showing that membrane depolarization up-regulates mitosis and that this process is dependent on voltage-dependent activation of ERK. ERK activity's voltage-dependence is derived from changes in the dynamics of phosphatidylserine in the plasma membrane and not by extracellular calcium dynamics.

Strengths:

Bioelectricity is an important field for areas of cell, developmental, and evolutionary biology, as well as for biomedicine. Confirmation of ERK as a transduction mechanism, and a characterization of the molecular details involved in control of cell proliferation, is interesting and impactful.

Weaknesses:

The functional cell division data need to be stronger. They show that increasing K+ increases proliferation and argue that since a MEK inhibitor (U0126) reduces proliferation in K+ treated cells, K+ induces cell division via ERK. But I don't see statistics to show that the rescue is significant, and I don't see a key U0126-only control. If the U0126 alone reduces proliferation, the combined effect wouldn't prove much.

Also, unless I'm missing something, it looks like every sample in their control has exactly the same number of mitotic cells. I understand that they are normalizing to this column, but shouldn't they be normalizing to the mean, with the independent values scattering around 1? It doesn't seem like it can be paired replicates since there are 6 replicates in the control and 4 replicates in one of the conditions?

Reviewer #2 (Public review):

Sasaki et al. use a combination of live-cell biosensors and patch-clamp electrophysiology to investigate the effect of membrane potential on the ERK MAPK signaling pathway, and probe associated effects on proliferation. This is an effect that has long been proposed, but convincing demonstration has remained elusive, because it is difficult to perturb membrane potential without disturbing other aspects of cell physiology in complex ways. The time-resolved measurements here are a nice contribution to this question, and the perforated patch clamp experiments with an ERK biosensor are fantastic - they come closer to addressing the above difficulty of perturbing voltage than any prior work. It would have been difficult to obtain these observations with any other combination of tools.

However, there are still some concerns as detailed in specific comments below:

Specific comments:<br /> (1) All the observations of ERK activation, by both high extracellular K+ and voltage clamp, could be explained by cell volume increase (more discussion in subsequent comments). There is a substantial literature on ERK activation by hypotonic cell swelling (e.g. https://doi.org/10.1042/bj3090013, https://doi.org/10.1002/j.1460-2075.1996.tb00938.x, among others). Here are some possible observations that could demonstrate that ERK activation by volume change is distinct from the effects reported here:<br /> (i) Does hypotonic shock activate ERK in U2OS cells?<br /> (ii) Can hypotonic shock activate ERK even after PS depletion, whereas extracellular K+ cannot?<br /> (iii) Does high extracellular K+ change cell volume in U2OS cells, measured via an accurate method such as fluorescence exclusion microscopy?<br /> (iv) It would be helpful to check the osmolality of all the extracellular solutions, even though they were nominally targeted to be iso-osmotic.

(2) Some more details about the experimental design and the results are needed from Figure 1:<br /> (i) For how long are the cells serum-starved? From the Methods section, it seems like the G1 release in different K+ concentration is done without serum, is this correct? Is the prior thymidine treatment also performed in the absence of serum?<br /> (ii) There is a question of whether depolarization constitutes a physiologically relevant mechanism to regulate proliferation, and how depolarization interacts with other extracellular signals that might be present in an in vivo context. Does depolarization only promote proliferation after extended serum starvation (in what is presumably a stressed cell state)? What fraction of total cells are observed to be mitotic (without normalization), and how does this compare to the proliferation of these cells growing in serum-supplemented media? Can K+ concentration tune proliferation rate even in serum-supplemented media?

(3) In Figure 2, there are some possible concerns with the perfusion experiment:<br /> (i) Is the buffer static in the period before perfusion with high K+, or is it perfused? This is not clear from the Methods. If it is static, how does the ERK activity change when perfused with 5 mM K+? In other words, how much of the response is due to flow/media exchange versus change in K+ concentration?<br /> (ii) Why do there appear to be population-average decreases in ERK activity in the period before perfusion with high K+ (especially in contrast to Fig. 3)? The imaging period does not seem frequent enough for photobleaching to be significant.

(4) Figure 3 contains important results on couplings between membrane potential and MAPK signaling. However, there are a few concerns:<br /> (i) Does cell volume change upon voltage clamping? Previous authors have shown that depolarizing voltage clamp can cause cells to swell, at least in the whole-cell configuration: https://www.cell.com/biophysj/fulltext/S0006-3495(18)30441-7 . Could it be possible that the clamping protocol induces changes in ERK signaling due to changes in cell volume, and not by an independent mechanism?<br /> (ii) Does the -80 mV clamp begin at time 0 minutes? If so, one might expect a transient decrease in sensor FRET ratio, depending on the original resting potential of the cells. Typical estimates for resting potential in HEK293 cells range from -40 mV to -15 mV, which would reach the range that induces an ERK response by depolarizing clamp in Fig. 3B. What are the resting potentials of the cells before they are clamped to -80 mV, and why do we not see this downward transient?

(5) The activation of ERK by perforated voltage clamp and by high extracellular K+ are each convincing, but it is unclear whether they need to act purely through the same mechanism - while additional extracellular K+ does depolarize the cell, it could also be affecting function of voltage-independent transporters and cell volume regulatory mechanisms on the timescales studied. To more strongly show this, the following should be done with the HEK cells where there is already voltage clamp data:<br /> (i) Measure resting potential using the perforated patch in zero-current configuration in the high K+ medium. Ideally this should be done in the time window after high K+ addition where ERK activation is observed (10-20 minutes) to minimize the possibility of drift due to changes in transporter and channel activity due to post-translational regulation.<br /> (ii) Measure YFP/CFP ratio of the HEK cells in the high K+ medium (in contrast to the U2OS cells from Fig. 2 where there is no patch data).<br /> (iii) The assertion that high K+ is equivalent to changes in Vmem for ERK signaling would be supported if the YFP/CFP change from K+ addition is comparable to that induced by voltage clamp to the same potential. This would be particularly convincing if the experiment could be done with each of the 15 mM, 30 mM, and 145 mM conditions.

(6) Line 170: "ERK activity was reduced with a fast time course (within 1 minute) after repolarization to -80 mV." I don't see this in the data: in Fig. 3C, it looks like ERK remains elevated for > 10 min after the electrical stimulus has returned to -80 mV

Reviewer #3 (Public review):

Summary:

This paper demonstrates that membrane depolarization induces a small increase in cell entry into mitosis. Based on previous work from another lab, the authors propose that ERK activation might be involved. They show convincingly using a combination of assays that ERK is activated by membrane depolarization. They show this is Ca2+ independent and is a result of activation of the whole K-Ras/ERK cascade which results from changed dynamics of phosphatidylserine in the plasma membrane that activates K-Ras. Although the activation of the Ras/ERK pathway by membrane depolarization is not new, linking it to an increase in cell proliferation is novel.

Strengths

A major strength of the study is the use of different techniques - live imaging with ERK reporters, as well as Western blotting to demonstrate ERK activation as well as different methods for inducing membrane depolarization. They also use a number of different cell lines. Via Western blotting the authors are also able to show that the whole MAPK cascade is activated.

Weaknesses

A weakness of the study is the data in Figure 1 showing that membrane depolarization results in an increase of cells entering mitosis. There are very few cells entering mitosis in their sample in any condition. This should be done with many more cells to increase confidence in the results. The study also lacks a mechanistic link between ERK activation by membrane depolarization and increased cell proliferation.

The authors did achieve their aims with the caveat that the cell proliferation results could be strengthened. The results for the most part support the conclusions.

This work suggests that alterations in membrane potential may have more physiological functions than action potential in the neural system as it has an effect on intracellular signalling and potentially cell proliferation.

Reviewer #1 (Public Review):

This manuscript by Capitani et al. extends previous studies of ion channel expression in triple-negative breast cancer cell lines. Probing four phenotypically different breast cancer cell lines, they used co-IP and confocal immunofluorescence (IF) colocalization to reveal that beta1 integrin forms a complex with the neonatal form of the Na+ channel NaV1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1 in addition to previously reported hERG1. They used siRNA to show that silencing beta1 results in a co-depletion of hERG and Nav1.5, further supporting the conclusion that they form a complex; a complementary enhancement of Na current with increased hERG expression was also demonstrated. These data compellingly describe a complex of membrane proteins unregulated in breast cancer and thus present novel potential targets for treatment.

There are several concerns with experimental approaches. How fluorescence measurements were compared and controlled among experiments was not described, and masks drawn to define membrane expression seemed arbitrary, and included in some cases large sections of cytoplasm. There are issues associated with the use of channel blocking agents and a bifunctional small-chain antibody that are not well rationalized. Why are they being used, to test what hypotheses or disrupt what processes? The extremely high concentrations of E-4031 (4000x IC50 for block), e.g., are not expected to have selective actions. The effects of E-4031 at high concentrations altering cytoskeleton properties associated with invasiveness (and thus cancer progression) are questionable. There are numerous problems with co-IPs together carried out together with knock-down, which in one case depleted the protein targeted by the primary IP antibody. Western blots (WB) were quantified by comparing treatment to control, which does not control for loading errors. The control and treated signals should be divided by the respective tubulin signals to control for loading errors. Then the treated value can be compared with the control.

Reviewer #2 (Public Review):

The manuscript by Chiara Capitani and Annarosa Arcangeli reports the identification of a complex comprising NHE1,hERG1, β1 integrin, and NaV1.5 on the plasma membrane of breast cancer cells. The authors further investigated the mutual regulatory interactions among these proteins using Western blotting and co-immunoprecipitation assays. They also examined the downstream signaling pathways associated with this complex and assessed its impact on the malignant behavior of breast cancer cells.

Strengths

The manuscript used different breast cancer cell lines and combined Western blot, immunostaining, and electrophysiology to provide evidence for the proposed complex. The inhibitors are also used to test the requirement of channel activity to function in the development of breast cancer cells with in-vitro studies.

Weaknesses

The data shown in this manuscript include the western blots that are cropped and imaged separately to draw conclusions about protein levels and changes in immunoprecipitation. These cannot be done on separate, cropped blots but must be imaged together to make these comparisons.

Antibodies used for hERG, NaV1.5 and β1 integrin must be validated to work for IP using KO or KD cell lines for the respective proteins to demonstrate specificity. The same goes for all the immunofluorescence imaging shown in the manuscript as these are all key pieces of data to support the conclusions.

Reviewer #1 (Public Review):

The authors examined the hypothesis that plasma ApoM, which carries sphingosine-1-phosphate (S1P) and activates vascular S1P receptors to inhibit vascular leakage, is modulated by SGLT2 inhibitors (SGLTi) during endotoxemia. They also propose that this mechanism is mediated by SGLTi regulation of LRP2/ megalin in the kidney and that this mechanism is critical for endotoxin-induced vascular leak and myocardial dysfunction. The hypothesis is novel and potentially exciting. However, the author's experiments lack critical controls, lack rigor in multiple aspects, and overall does not support the conclusions.

Reviewer #2 (Public Review):

Apolipoprotein M (ApoM) is a plasma carrier for the vascular protective lipid mediator sphingosine 1-phospate (S1P). The plasma levels of S1P and its chaperones ApoM and albumin rapidly decline in patients with severe sepsis, but the mechanisms for such reductions and their consequences for cardiovascular health remain elusive. In this study, Ripoll and colleagues demonstrate that the sodium-glucose co-transporter inhibitor dapagliflizin (Dapa) can preserve serum ApoM levels as well as cardiac function after LPS treatment of mice with diet-induced obesity. They further provide data to suggest that Dapa preserves serum ApoM by increasing megalin-mediated reabsorption of ApoM in renal proximal tubules and that ApoM improves vascular integrity in LPS treated mice. These observations put forward a potential therapeutic approach to sustain vascular protective S1P signaling that could be relevant to other conditions of systemic inflammation where plasma levels of S1P decrease. However, although the authors are careful with their statements, the study falls short of directly implicating megalin in ApoM reabsorption and of ApoM/S1P depletion in LPS-induced cardiac dysfunction and the protective effects of Dapa.

The observations reported in this study are exciting and potentially of broad interest. The paper is well written and concise, and the statements made are mostly supported by the data presented. However, the mechanism proposed and implied is mostly based on circumstantial evidence, and the paper could be substantially improved by directly addressing the role of megalin in ApoM reabsorption and serum ApoM and S1P levels and the importance of ApoM for the preservation for cardiac function during endotoxemia. Some observations that are not necessarily in line with the model proposed should also be discussed.

The authors show that Dapa preserves serum ApoM and cardiac function in LPS-treated obese mice. However, the evidence they provide to suggest that ApoM may be implicated in the protective effect of Dapa on cardiac function is indirect. Direct evidence could be sought by addressing the effect of Dapa on cardiac function in LPS treated ApoM deficient and littermate control mice (with DIO if necessary).

The authors also suggest that higher ApoM levels in mice treated with Dapa and LPS reflect increased megalin-mediated ApoM reabsorption and that this preserves S1PR signaling. This could be addressed more directly by assessing the clearance of labelled ApoM, by addressing the impact of megalin inhibition or deficiency on ApoM clearance in this context, and by measuring S1P as well as ApoM in serum samples.

Methods: More details should be provided in the manuscript for how ApoM deficient and transgenic mice were generated, on sex and strain background, and on whether or not littermate controls were used. For intravital microscopy, more precision is needed on how vessel borders were outland and if this was done with or without regard for FITC-dextran. Please also specify the type of vessel chosen and considerations made with regard to blood flow and patency of the vessels analyzed. For statistical analyses, data from each mouse should be pooled before performing statistical comparisons. The criteria used for choice of test should be outlined as different statistical tests are used for similar datasets. For all data, please be consistent in the use of post-tests and in the presentation of comparisons. In other words, if the authors choose to only display test results for groups that are significantly different, this should be done in all cases. And if comparisons are made between all groups, this should be done in all cases for similar sets of data.

Reviewer #3 (Public Review):

The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for this agonist in most but not all the benefits of Dapa. A strength of the paper is the identification of the pathway responsible for the cardioprotective effects of SGLT2is that may yield additional therapeutic targets. There are some weaknesses in the paper, such as, studying only male mice, as well as providing a power analysis to justify the number of animals used throughout their experimentation. Overall, the paper should have a significant impact on the scientific community because the SGLT2i drugs are likely to find many uses in inflammatory diseases and metabolic diseases. This paper provides support for an important mechanism by which they work in conditions of severe sepsis and hemodynamic compromise.

Reviewer #1 (Public Review):

This paper proposes a novel framework for explaining patterns of generalization of force field learning to novel limb configurations. The paper considers three potential coordinate systems: cartesian, joint-based, and object-based. The authors propose a model in which the forces predicted under these different coordinate frames are combined according to the expected variability of produced forces. The authors show, across a range of changes in arm configurations, that the generalization of a specific force field is quite well accounted for by the model.

The paper is well-written and the experimental data are very clear. The patterns of generalization exhibited by participants - the key aspect of the behavior that the model seeks to explain - are clear and consistent across participants. The paper clearly illustrates the importance of considering multiple coordinate frames for generalization, building on previous work by Berniker and colleagues (JNeurophys, 2014). The specific model proposed in this paper is parsimonious, but there remain a number of questions about its conceptual premises and the extent to which its predictions improve upon alternative models.

A major concern is with the model's premise. It is loosely inspired by cue integration theory but is really proposed in a fairly ad hoc manner, and not really concretely founded on firm underlying principles. It's by no means clear that the logic from cue integration can be extrapolated to the case of combining different possible patterns of generalization. I think there may in fact be a fundamental problem in treating this control problem as a cue-integration problem. In classic cue integration theory, the various cues are assumed to be independent observations of a single underlying variable. In this generalization setting, however, the different generalization patterns are NOT independent; if one is true, then the others must inevitably not be. For this reason, I don't believe that the proposed model can really be thought of as a normative or rational model (hence why I describe it as 'ad hoc'). That's not to say it may not ultimately be correct, but I think the conceptual justification for the model needs to be laid out much more clearly, rather than simply by alluding to cue-integration theory and using terms like 'reliability' throughout.

A more rational model might be based on Bayesian decision theory. Under such a model, the motor system would select motor commands that minimize some expected loss, averaging over the various possible underlying 'true' coordinate systems in which to generalize. It's not entirely clear without developing the theory a bit exactly how the proposed noise-based theory might deviate from such a Bayesian model. But the paper should more clearly explain the principles/assumptions of the proposed noise-based model and should emphasize how the model parallels (or deviates from) Bayesian-decision-theory-type models.

Another significant weakness is that it's not clear how closely the weighting of the different coordinate frames needs to match the model predictions in order to recover the observed generalization patterns. Given that the weighting for a given movement direction is over-parametrized (i.e. there are 3 variable weights (allowing for decay) predicting a single observed force level, it seems that a broad range of models could generate a reasonable prediction. It would be helpful to compare the predictions using the weighting suggested by the model with the predictions using alternative weightings, e.g. a uniform weighting, or the weighting for a different posture. In fact, Fig. 7 shows that uniform weighting accounts for the data just as well as the noise-based model in which the weighting varies substantially across directions. A more comprehensive analysis comparing the proposed noise-based weightings to alternative weightings would be helpful to more convincingly argue for the specificity of the noise-based predictions being necessary. The analysis in the appendix was not that clearly described, but seemed to compare various potential fitted mixtures of coordinate frames, but did not compare these to the noise-based model predictions.

Reviewer #2 (Public Review):

Leib & Franklin assessed how the adaptation of intersegmental dynamics of the arm generalizes to changes in different factors: areas of extrinsic space, limb configurations, and 'object-based' coordinates. Participants reached in many different directions around 360{degree sign}, adapting to velocity-dependent curl fields that varied depending on the reach angle. This learning was measured via the pattern of forces expressed in upon the channel wall of "error clamps" that were randomly sampled from each of these different directions. The authors employed a clever method to predict how this pattern of forces should change if the set of targets was moved around the workspace. Some sets of locations resulted in a large change in joint angles or object-based coordinates, but Cartesian coordinates were always the same. Across three separate experiments, the observed shifts in the generalized force pattern never corresponded to a change that was made relative to any one reference frame. Instead, the authors found that the observed pattern of forces could be explained by a weighted combination of the change in Cartesian, joint, and object-based coordinates across test and training contexts.

In general, I believe the authors make a good argument for this specific mixed weighting of different contexts. I have a few questions that I hope are easily addressed.

Movements show different biases relative to the reach direction. Although very similar across people, this function of biases shifts when the arm is moved around the workspace (Ghilardi, Gordon, and Ghez, 1995). The origin of these biases is thought to arise from several factors that would change across the different test and training workspaces employed here (Vindras & Viviani, 2005). My concern is that the baseline biases in these different contexts are different and that rather the observed change in the force pattern across contexts isn't a function of generalization, but a change in underlying biases. Baseline force channel measurements were taken in the different workspace locations and conditions, so these could be used to show whether such biases are meaningfully affecting the results.

Experiment 3, Test 1 has data that seems the worst fit with the overall story. I thought this might be an issue, but this is also the test set for a potentially awkwardly long arm. My understanding of the object-based coordinate system is that it's primarily a function of the wrist angle, or perceived angle, so I am a little confused why the length of this stick is also different across the conditions instead of just a different angle. Could the length be why this data looks a little odd?

The manuscript is written and organized in a way that focuses heavily on the noise element of the model. Other than it being reasonable to add noise to a model, it's not clear to me that the noise is adding anything specific. It seems like the model makes predictions based on how many specific components have been rotated in the different test conditions. I fear I'm just being dense, but it would be helpful to clarify whether the noise itself (and inverse variance estimation) are critical to why the model weights each reference frame how it does or whether this is just a method for scaling the weight by how much the joints or whatever have changed. It seems clear that this noise model is better than weighting by energy and smoothness.

Are there any force profiles for individual directions that are predicted to change shape substantially across some of these assorted changes in training and test locations (rather than merely being scaled)? If so, this might provide another test of the hypotheses.

I don't believe the decay factor that was used to scale the test functions was specified in the text, although I may have just missed this. It would be a good idea to state what this factor is where relevant in the text.

Reviewer #3 (Public Review):

The author proposed the minimum variance principle in the memory representation in addition to two alternative theories of the minimum energy and the maximum smoothness. The strength of this paper is the matching between the prediction data computed from the explicit equation and the behavioral data taken in different conditions. The idea of the weighting of multiple coordinate systems is novel and is also able to reconcile a debate in previous literature.

The weakness is that although each model is based on an optimization principle, but the derivation process is not written in the method section. The authors did not write about how they can derive these weighting factors from these computational principles. Thus, it is not clear whether these weighting factors are relevant to these theories or just hacking methods. Suppose the author argues that this is the result of the minimum variance principle. In that case, the authors should show a process of how to derive these weighting factors as a result of the optimization process to minimize these cost functions.

In addition, I am concerned that the proposed model can cancel the property of the coordinate system by the predicted variance, and it can work for any coordinate system, even one that is not used in the human brain. When the applied force is given in Cartesian coordinates, the directionality in the generalization ability of the memory of the force field is characterized by the kinematic relationship (Jacobian) between the Cartesian coordinate and the coordinate of interest (Cartesian, joint, and object) as shown in Equation 3. At the same time, when a displacement (epsilon) is considered in a space and a corresponding displacement is linked with kinematic equations (e.g., joint displacement and hand displacement in 2 joint arms in this paper), the generated variances in different coordinate systems are linked with the kinematic equation each other (Jacobian). Thus, how a small noise in a certain coordinate system generates the hand force noise (sigma_x, sigma_j, sigma_o) is also characterized by the kinematics (Jacobian). Thus, when the predicted forcefield (F_c, F_j, F_o) was divided by the variance (F_c/sigma_c^2, F_j/sigma_j^2, F_o/sigma_o^2, ), the directionality of the generalization force which is characterized by the Jacobian is canceled by the directionality of the sigmas which is characterized by the Jacobian. Thus, as it has been read out from Fig*D and E top, the weight in E-top of each coordinate system is always the inverse of the shift of force from the test force by which the directionality of the generalization is always canceled. Once this directionality is canceled, no matter how to compute the weighted sum, it can replicate the memorized force. Thus, this model always works to replicate the test force no matter which coordinate system is assumed. Thus, I am suspicious of the falsifiability of this computational model. This model is always true no matter which coordinate system is assumed. Even though they use, for instance, the robot coordinate system, which is directly linked to the participant's hand with the kinematic equation (Jacobian), they can replicate this result. But in this case, the model would be nonsense. The falsifiability of this model was not explicitly written.

Reviewer #1 (Public Review):

Padilha et al. aimed to find prospective metabolite biomarkers in serum of children aged 6-59 months that were indicative of neurodevelopmental outcomes. The authors leveraged data and samples from the cross-sectional Brazilian National Survey on Child Nutrition (ENANI-2019), and an untargeted multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) approach was used to measure metabolites in serum samples (n=5004) which were identified via a large library of standards. After correlating the metabolite levels against the developmental quotient (DQ), or the degree of which age-appropriate developmental milestones were achieved as evaluated by the Survey of Well-being of Young Children, serum concentrations of phenylacetylglutamine (PAG), cresol sulfate (CS), hippuric acid (HA) and trimethylamine-N-oxide (TMAO) were significantly negatively associated with DQ. Examination of the covariates revealed that the negative associations of PAG, HA, TMAO and valine (Val) with DQ were specific to younger children (-1 SD or 19 months old), whereas creatinine (Crtn) and methylhistidine (MeHis) had significant associations with DQ that changed direction with age (negative at -1 SD or 19 months old, and positive at +1 SD or 49 months old). Further, mediation analysis demonstrated that PAG was a significant mediator for the relationship of delivery mode, child's diet quality and child fiber intake with DQ. HA and TMAO were additional significant mediators of the relationship of child fiber intake with DQ.

Strengths of this study include the large cohort size and study design allowing for sampling at multiple time points along with neurodevelopmental assessment and a relatively detailed collection of potential confounding factors including diet. The untargeted metabolomics approach was also robust and comprehensive allowing for level 1 identification of a wide breadth of potential biomarkers. Given their methodology, the authors should be able to achieve their aim of identifying candidate serum biomarkers of neurodevelopment for early childhood. The results of this work would be of broad interest to researchers who are interested in understanding the biological underpinnings of development and also for tracking development in pediatric populations, as it provides insight for putative mechanisms and targets from a relevant human cohort that can be probed in future studies. Such putative mechanisms and targets are currently lacking in the field due to challenges in conducting these kind of studies, so this work is important.

However, in the manuscript's current state, the presentation and analysis of data impede the reader from fully understanding and interpreting the study's findings. Particularly, the handling of confounding variables is incomplete. There is a different set of confounders listed in Table 1 versus Supplementary Table 1 versus Methods section Covariates versus Figure 4. For example, Region is listed in Supplementary Table 1 but not in Table 1, and Mode of Delivery is listed in Table 1 but not in Supplementary Table 1. Many factors are listed in Figure 4 that aren't mentioned anywhere else in the paper, such as gestational age at birth or maternal pre-pregnancy obesity.

The authors utilize the directed acrylic graph (DAG) in Figure 4 to justify the further investigation of certain covariates over others. However, the lack of inclusion of the microbiome in the DAG, especially considering that most of the study findings were microbial-derived metabolite biomarkers, appears to be a fundamental flaw. Sanitation and micronutrients are proposed by the authors to have no effect on the host metabolome, yet sanitation and micronutrients have both been demonstrated in the literature to affect microbiome composition which can in turn affect the host metabolome.

Additionally, the authors emphasized as part of the study selection criteria the following,<br /> "Due to the costs involved in the metabolome analysis, it was necessary to further reduce the sample size. Then, samples were stratified by age groups (6 to 11, 12 to 23, and 24 to 59 months) and health conditions related to iron metabolism, such as anemia and nutrient deficiencies. The selection process aimed to represent diverse health statuses, including those with no conditions, with specific deficiencies, or with combinations of conditions. Ultimately, through a randomized process that ensured a balanced representation across these groups, a total of 5,004 children were selected for the final sample (Figure 1)."

Therefore, anemia and nutrient deficiencies are assumed by the reader to be important covariates, yet, the data on the final distribution of these covariates in the study cohort is not presented, nor are these covariates examined further.

The inclusion of specific covariates in Table 1, Supplementary Table 1, the statistical models, and the mediation analysis is thus currently biased as it is not well justified.

Finally, it is unclear what the partial-least squares regression adds to the paper, other than to discard potentially interesting metabolites found by the initial correlation analysis.

Reviewer #2 (Public Review):

A strength of the work lies in the number of children Padilha et al. were able to assess (5,004 children aged 6-59 months) and in the extensive screening that the Authors performed for each participant. This type of large-scale study is uncommon in low-to-middle-income countries such as Brazil.<br /> The Authors employ several approaches to narrow down the number of potentially causally associated metabolites.<br /> Could the Authors justify on what basis the minimum dietary diversity score was dichotomized? Were sensitivity analyses undertaken to assess the effect of this dichotomization on associations reported by the article? Consumption of each food group may have a differential effect that is obscured by this dichotomization.<br /> Could the Authors specify the statistical power associated with each analysis?<br /> Could the Authors describe in detail which metric they used to measure how predictive PLSR models are, and how they determined what the "optimal" number of components were?<br /> The Authors use directed acyclic graphs (DAG) to identify confounding variables of the association between metabolites and DQ. Could the dataset generated by the Authors have been used instead? Not all confounding variables identified in the literature may be relevant to the dataset generated by the Authors.<br /> Were the systematic reviews or meta-analyses used in the DAG performed by the Authors, or were they based on previous studies? If so, more information about the methodology employed and the studies included should be provided by the Authors.<br /> Approximately 72% of children included in the analyses lived in households with a monthly income superior to the Brazilian minimum wage. The cohort is also biased towards households with a higher level of education. Both of these measures correlate with developmental quotient. Could the Authors discuss how this may have affected their results and how generalizable they are?<br /> Further to this, could the Authors describe how inequalities in access to care in the Brazilian population may have affected their results? Could they have included a measure of this possible discrepancy in their analyses?<br /> The Authors state that the results of their study may be used to track children at risk for developmental delays. Could they discuss the potential for influencing policies and guidelines to address delayed development due to malnutrition and/or limited access to certain essential foods?

Reviewer #3 (Public Review):

The ENANI-2019 study provides valuable insights into child nutrition, development, and metabolomics in Brazil, highlighting both challenges and opportunities for improving child health outcomes through targeted interventions and further research.

Strengths of the methods and results:<br /> (1) The study utilizes data from the ENANI-2019 cohort, which was already existing. This cohort choice allows for longitudinal assessments and exploration of associations between metabolites and developmental outcomes. In addition, there was conservation of resources which are scanty in all settings in the current scenario.<br /> (2) The study aims to investigate the relationship between circulating metabolites (exposure) and early childhood development (outcome), specifically developmental quotient (DQ). The objectives are clearly stated, which facilitates focused research questions and hypotheses. The population that is studied is clearly mentioned.<br /> (3) The study accessed a large number of children under five years, with blood collected from a final sample size of 5,004 children. The exclusion of infants under six months due to venipuncture challenges and lack of reference values highlights practical considerations in research design.<br /> The study sample reflects a diverse range of children in terms of age, sex distribution, weight status, maternal education, and monthly family income. This diversity enhances the generalizability of findings across different sociodemographic groups within Brazil.<br /> (4) The study uses standardized measures (e.g., DQ assessments) and chronological age. Confounding variables, such as child's age, diet quality, and nutritional status, are carefully considered and incorporated into analyses through a Directed Acyclic Graph (DAG). The mean DQ of 0.98 indicates overall developmental norms among the studied children, with variations noted across different demographic factors such as age, region, and maternal education. The prevalence of Minimum Dietary Diversity (MDD) being met by 59.3% of children underscores dietary patterns and their potential impact on health outcomes. The association between nutritional status (weight-for-height z-scores) and developmental outcomes (DQ) provides insights into the interplay between nutrition and child development.<br /> The study identified key metabolites associated with developmental quotient (DQ):<br /> Component 1: Branched-chain amino acids (Leucine, Isoleucine, Valine).<br /> Component 2: Uremic toxins (Cresol sulfate, Phenylacetylglutamine).<br /> Component 3: Betaine and amino acids (Glutamine, Asparagine).<br /> The study focused on several serum metabolites like PAG (phenylacetylglutamine), CS (p-cresyl sulfate), HA (hippuric acid), TMAO (trimethylamine-N-oxide), MeHis (methylhistidine), and Crtn (creatinine). These metabolites are implicated in various metabolic pathways linked to gut microbiota activity, amino acid metabolism, and dietary factors.<br /> These metabolites explained a significant portion of both metabolite variance (39.8%) and DQ variance (4.3%). The study suggests that these metabolites can be used as proxy measures of the gut microbiome in children.<br /> (5) The use of partial least square regression (PLSR) with cross-validation (80% training, 20% testing) which is a robust approach to identify metabolites predictive of DQ, which minimizes overfitting. This model allows for outliers to remain outliers for transparency.<br /> The Directed Acyclic Graph (DAG) identifies and adjusts for confounding variables (e.g., child's diet quality, nutritional status) and strengthens the validity of findings by controlling for potential biases. Developmental and gender differences were studied by testing interactions with the age of the child and the sex.<br /> Mediation analysis exploring metabolites as potential mediators provides insights into underlying pathways linking exposures (e.g., diet, microbiome) with DQ.<br /> The use of Benjamini-Hochberg correction for multiple comparisons and bootstrap tests (5,000 iterations) enhances the reliability of results by controlling false discovery rates and assessing significance robustly.

Significant correlations between serum metabolites and DQ, particularly negative associations with certain metabolites like PAG and CS, suggest potential biomarkers or pathways influencing developmental outcomes. Notably, these associations varied with age, suggesting different metabolic impacts during early childhood development.

Weaknesses:<br /> (1) The data collected was incomplete especially those related to breastfeeding history and birth weight. These have been mentioned in the limitations of the study but yet might have been potential confounders or even factors leading to the particular identified metabolite state of the population.<br /> (2) Other tests than mediation analysis might have been used to ensure reliability and robustness of the data. How data was processed, data cleaning methods, how outliers were handled and sensitivity analyses would ensure robustness of the findings.<br /> (3) The generalizability of the data is not sound especially considering the children mostly belonged to a higher socioeconomic group in Brazil with mother or caregiver education being above a certain level. Comparative studies with children from other socio-economic groups and other cohorts might have been useful. Consideration of sample size adequacy and power analysis might have helped in generalizing the findings.<br /> (4) Caution is needed in interpreting causality from this data because of the nature of the study design Discussing alternative explanations and potential confounding factors in more depth could strengthen the conclusions.

Appraisal<br /> (1) The aims of the study were to identify associations between children's serum metabolome and Early Childhood development. This aim was met. The results do confirm their conclusions.<br /> Impact of the work on the field

(1) Unless actual gut microbiome of children in this age group from gut bacteria examination or gastrointestinal examination of the gut of children, the causality of gut metabolome on early childhood development cannot be established with certainty. Because this may not be possible in every situation, proxy methods such as the one elucidated here might be useful, considering the risk-benefit ratio.<br /> (2) More research is needed on this theme through longitudinal studies to validate these findings and explore underlying pathways involving gut-brain interactions and metabolic dysregulation.<br /> Other readings: Readers are advised to read other research from other countries and other languages to understand the connection between gut microbiome, metabolite spectra, and child development. In addition to study the effect of these factors on child mental development too.

Readers might consider the following questions:<br /> (1) Should investigators study the families through direct observation of diet and other factors to look for a connection between food taken in and gut microbiome and child development?<br /> (2) Can an examination of the mother's gut microbiome influence the child's microbiome? Can the mother or caregiver's microbiome influence early childhood development?<br /> (3) Is developmental quotient enough to study early childhood development? Is it comprehensive enough?

Reviewer #1 (Public Review):

In this manuscript, El Amri et al. are exploring the role of Marcks and Marcksl1 proteins during spinal cord development and regeneration in Xenopus. Using two different techniques to knockdown their expressions, they argue that these proteins are important for neural progenitors proliferation and neurites outgrowth in both contexts. Finally, using a pharmalogical approach, they suggest that Marcks and Marcksl1 work by modulating the activity of PLD and the levels of PIP2 whilst PKC could modulate Marcks activity.<br /> The strength of this manuscript resides in the ability of the authors to knockdown the expression of 4 different genes using 2 different methods to assess the role of this protein family during early development and regeneration at the late tadpole stage. This has always been a limiting factor in the field as the tools to perform conditional knockouts in Xenopus are very limited. However, this will not really be applicable to essential genes as it relies on the general knockdown of protein expression. The generation of antibodies able to detect endogenous Marcks/Marcksl1 is also a powerful tool to assess the extent to which the expression of these proteins is down-regulated.<br /> Whilst there is a great amount of data provided in this manuscript and there is strong evidence to show that Marcks are important for spinal cord development and regeneration, their roles in both contexts is not explored fully. The description of the effect of knocking down Marcks/Marcksl1 on neurons and progenitors is rather superficial and the evidence for the underlying mechanism underpinning their roles is not very convincing.

Reviewer #2 (Public Review):

M. El Amri et al., investigated the functions of Marcks and Marcks like 1 during spinal cord (SC) development and regeneration in Xenopus laevis. The authors rigorously performed loss of function with morpholino knock-down and CRISPR knock-out combining rescue experiments in developing spinal cord in embryo and regeneration in tadpole stage.

For the assays in the developing spinal cord, a unilateral approach (knock-down/out only one side of the embryo) allowed the authors to assess the gene functions by direct comparing one-side (e.g. mutated SC) to the other (e.g. wild type SC on the other side). For the assays in regenerating SC, the authors microinject CRISPR reagents into 1-cell stage embryo. When the embryo (F0 crispants) grew up to tadpole (stage 50), the SC was transected. They then assessed neurite outgrowth and progenitor cell proliferation. The validation of the phenotypes was mostly based on the quantification of immunostaining images (neurite outgrowth: acetylated tubulin, neural progenitor: sox2, sox3, proliferation: EdU, PH3), that are simple but robust enough to support their conclusions. In both SC development and regeneration, the authors found that Marcks and Marcksl1 were necessary for neurite outgrowth and neural progenitor cell proliferation.<br /> The authors performed rescue experiments on morpholino knock-down and CRISPR knock-out conditions by Marcks and Marcksl1 mRNA injection for SC development and pharmacological treatments for SC development and regeneration. The unilateral mRNA injection rescued the loss-of-function phenotype in the developing SC. To explore the signalling role of these molecules, they rescued the loss-of-function animals by pharmacological reagents They used S1P: PLD activator, FIPI: PLD inhibitor, NMI: PIP2 synthesis activator and ISA-2011B: PIP2 synthesis inhibitor. The authors found the activator treatment rescued neurite outgrowth and progenitor cell proliferation in loss of function conditions. From these results, the authors proposed PIP2 and PLD are the mediators of Marcks and Marcksl1 for neurite outgrowth and progenitor cell proliferation during SC development and regeneration. The results of the rescue experiments are particularly important to assess gene functions in loss of function assays, therefore, the conclusions are solid. In addition, they performed gain-of-function assays by unilateral Marcks or Marcksl1 mRNA injection showing that the injected side of the SC had more neurite outgrowth and proliferative progenitors. The conclusions are consistent with the loss-of-function phenotypes and the rescue results. Importantly, the authors showed the linkage of the phenotype and functional recovery by behavioral testing, that clearly showed the crispants with SC injury swam less distance than wild types with SC injury at 10-day post surgery.<br /> Prior to the functional assays, the authors analyzed the expression pattern of the genes by in situ hybridization and immunostaining in developing embryo and regenerating SC. They confirmed that the amount of protein expression was significantly reduced in the loss of function samples by immunostaining with the specific antibodies that they made for Marcks and Marcksl1. Although the expression patterns are mostly known in previous works during embryo genesis, the data provided appropriate information to readers about the expression and showed efficiency of the knock-out as well.

MARCKS family genes have been known to be expressed in the nervous system. However, few studies focus on the function in nerves. This research introduced these genes as new players during SC development and regeneration. These findings could attract broader interests from the people in nervous disease model and medical field. Although it is a typical requirement for loss of function assays in Xenopus laevis, I believe that the efficient knock-out for four genes by CRISPR/Cas9 was derived from their dedication of designing, testing and validation of the gRNAs and is exemplary.

Weaknesses,<br /> 1) Why did the authors choose Marcks and Marcksl1?<br /> The authors mentioned that these genes were identified with a recent proteomic analysis of comparing SC regenerative tadpole and non-regenerative froglet (Line (L) 54-57). However, although it seems the proteomic analysis was their own dataset, the authors did not mention any details to select promising genes for the functional assays (this article). In the proteomic analysis, there must be other candidate genes that might be more likely factors related to SC development and regeneration based on previous studies, but it was unclear what the criteria to select Marcks and Marcksl1 was.

2) Gene knock-out experiments with F0 crispants,<br /> The authors described that they designed and tested 18 sgRNAs to find the most efficient and consistent gRNA (L191-195). However, it cannot guarantee the same phenotypes practically, due to, for example, different injection timing, different strains of Xenopus laevis, etc. Although the authors mentioned the concerns of mosaicism by themselves (L180-181, L289-292) and immunostaining results nicely showed uniformly reduced Marcks and Marcksl1 expression in the crispants, they did not refer to this issue explicitly.

3) Limitations of pharmacological compound rescue<br /> In the methods part, the authors describe that they performed titration experiments for the drugs (L702-704), that is a minimal requirement for this type of assay. However, it is known that a well characterized drug is applied, if it is used in different concentrations, the drug could target different molecules (Gujral TS et al., 2014 PNAS). Therefore, it is difficult to eliminate possibilities of side effects and off targets by testing only a few compounds.

Reviewer #3 (Public Review):

El Amri et al conducted an analysis on the function of marcks and marcksl in Xenopus spinal cord development and regeneration. Their study revealed these proteins are crucial for neurite outgrowth and cell proliferation, including Sox2+ progenitors. Furthermore, they suggested these genes may act through the PLD pathway. The study is well-executed with appropriate controls and validation experiments, distinguishing it from typical regeneration research by including behavioral assays. The manuscript is commendable for its quantifications, literature referencing, careful conclusions, and detailed methods. Conclusions are well-supported by the experiments performed in this study. Overall, this manuscript contributes to the field of spinal cord regeneration and sets a good example for future research in this area.

Reviewer #1 (Public review):

Summary:

This is a short self-contained study with a straightforward and interesting message. The paper focuses on settling whether PKA activation requires dissociation of the catalytic and regulatory subunits. This debate has been ongoing for ~ 30 years, with renewed interest in the question following a publication in Science, 2017 (Smith et al.). Here, Xiong et al demonstrate that fusing the R and C subunits together (in the same way as Smith et al) prevents the proper function of PKA in neurons. This provides further support for the dissociative activation model - it is imperative that researchers have clarity on this topic since it is so fundamental to building accurate models of localised cAMP signalling in all cell types. Furthermore, their experiments highlight that C subunit dissociation into spines is essential for structural LTP, which is an interesting finding in itself. They also show that preventing C subunit dissociation reduces basal AMPA receptor currents to the same extent as knocking down the C subunit. Overall, the paper will interest both cAMP researchers and scientists interested in fundamental mechanisms of synaptic regulation.

Strengths:

The experiments are technically challenging and well executed. Good use of control conditions e.g untransfected controls in Figure 4.

Weaknesses:

The novelty is lessened given the same team has shown dissociation of the C subunit into dendritic spines from RIIbeta subunits localised to dendritic shafts before (Tillo et al., 2017). Nevertheless, the experiments with RII-C fusion proteins are novel and an important addition.

Reviewer #2 (Public review):

Summary:

PKA is a major signaling protein which has been long studied and is vital for synaptic plasticity. Here, the authors examine the mechanism of PKA activity and specifically focus on addressing the question of PKA dissociation as a major mode of its activation in dendritic spines. This would potentially allow to determine the precise mechanisms of PKA activation and address how it maintains spatial and temporal signaling specificity.

Strengths:

The results convincingly show that PKA activity is governed by the subcellular localization in dendrites and spines and is mediated via subunit dissociation. The authors make use of organotypic hippocampal slice cultures, where they use pharmacology, glutamate uncaging, and electrophysiological recordings.

Overall, the experiments and data presented are well executed. The experiments all show that at least in the case of synaptic activity, distribution of PKA-C to dendritic spines is necessary and sufficient for PKA mediated functional and structural plasticity.<br /> The authors were able to persuasively support their claim that PKA subunit dissociation is necessary for its function and localization in dendritic spines. This conclusion is important to better understand the mechanisms of PKA activity and its role in synaptic plasticity.

Weaknesses:

While the experiments are indeed convincing and well executed, the data presented is similar to previously published work from the Zhong lab (Tillo et al., 2017, Zhong et al 2009). This reduces the novelty of the findings in terms of re-distribution of PKA subunits, which was already established, at least to some degree.

Reviewer #3 (Public review):

Summary:

Xiong et al. investigated the debated mechanism of PKA activation using hippocampal CA1 neurons under pharmacological and synaptic stimulations. Examining all major PKA-R isoforms in these neurons, they found that a portion of PKA-C dissociates from PKA-R and translocate into dendritic spines following norepinephrine bath application. Additionally, their use of a non-dissociable form of PKA demonstrates its essential role in structural long-term potentiation (LTP) induced by two-photon glutamate uncaging, as well as in maintaining normal synaptic transmission, as verified by electrophysiology. This study presents a valuable finding on the activation-dependent re-distribution of PKA catalytic subunits in CA1 neurons, a process vital for synaptic functionality. The robust evidence provided by the authors makes this work particularly relevant for biologists seeking to understand PKA activation mechanisms, its downstream effects, and synaptic plasticity.

Strengths:

The study is methodologically robust, particularly in the application of two-photon imaging and electrophysiology. The experiments are well-designed with effective controls and a comprehensive analysis. The credibility of the data is further enhanced by the research team's previous works in related experiments. The study provides sufficient evidence to support the classical model of PKA activation via dissociation in neurons.

Weaknesses:

No specific weaknesses are noted in the current study; future research could provide additional insights by exploring PKA dissociation under varied physiological conditions, particularly in vivo, to further validate and expand upon these findings.

Reviewer #1 (Public review):

Summary:

The authors examined the salt-dependent phase separation of the low-complexity domain of hnRN-PA1 (A1-LCD). Using all-atom molecular dynamics simulations, they identified four distinct classes of salt dependence in the phase separation of intrinsically disordered proteins (IDPs), which can be predicted based on their amino acid composition. However, the simulations and analysis, in their current form, are inadequate and incomplete.

Strengths:

The authors attempt to unravel the mechanistic insights into the interplay between salt and protein phase separation, which is important given the complex behavior of salt effects on this process. Their effort to correlate the influence of salt on the low-complexity domain of hnRNPA1 (A1-LCD) with a range of other proteins known to undergo salt-dependent phase separation is an interesting and valuable topic.

Weaknesses:

Based on the reviewer's assessment of the manuscript, the following points were raised:

(1) The simulation duration is too short to draw comprehensive conclusions about phase separation.<br /> (2) There are concerns regarding the convergence of the simulations, particularly as highlighted in Figure 2A.<br /> (3) The simulation begins with a protein concentration of 3.5 mM ("we built an 8-copy model for the dense phase (with an initial concentration of 3.5 mM)"), which is high for phase separation studies. The reviewer questions the use of the term "dense phase" and suggests that the authors conduct a clearer analysis depicting the coexistence of both the dilute and dense phases to represent a steady state. Without this, the realism of the described phenomena is doubtful. Commenting on phase separation under conditions that don't align with typical phase separation parameters is not acceptable.<br /> (4) The inference that "Each Arg sidechain often coordinates two Cl- ions simultaneously, but each Lys sidechain coordinates only one Cl- ion" is questioned. According to Supplementary Figure 2A, Lys seems to coordinate with Cl- ions more frequently than Arg.<br /> (5) The authors are requested to update the figure captions for Supplementary Figures 2 and 3, specifying which system the analyses were performed on.<br /> (6) It is difficult to observe a clear trend due to irregularities in the data. Although the authors have included a red dotted line in the figures, the trend is not monotonic. The reviewer expresses concerns about significant conclusions drawn from these figures (e.g., Figure 2C, Figure 5A, Supplementary Figure 1).<br /> (7) Given the error in the radius of gyration (Rg) calculations, the reviewer questions the validity of drawing conclusions from this data.<br /> (8) The pair correlation function values in Figure 5E and supplementary figure 4 show only minor differences, and the reviewer questions whether these differences are significant.<br /> (9) Previous reports suggest that, upon self-assembly, protein chains extend within the condensate, leading to a decrease in intramolecular contacts. However, the authors show an increase in intramolecular contacts with increasing salt concentration (Figure 2C), which contradicts prior studies. The reviewer advises the authors to carefully review this and provide justification.<br /> (10) A systematic comparison of estimated parameters with varying salt concentrations is required. Additionally, the authors should provide potential differences in salt concentrations between the dilute and condensed phases.<br /> (11) The reviewer finds that the majority of the data presented shows no significant alteration with changes in salt concentration, yet the authors have made strong conclusions regarding salt activity.

The manuscript lacks sufficient scientific details of the calculations.

Reviewer #2 (Public review):

This is an interesting computational study addressing how salt affects the assembly of biomolecular condensates. The simulation data are valuable as they provide a degree of atomistic details regarding how small salt ions modulate interactions among intrinsically disordered proteins with charged residues, namely via Debye-like screening that weakens the effective electrostatic interactions among the polymers, or through bridging interactions that allow interactions between like charges from different polymer chains to become effectively attractive (as illustrated, e.g., by the radial distribution functions in Supplementary Information). However, this manuscript has several shortcomings: (i) Connotations of the manuscript notwithstanding, many of the authors' concepts about salt effects on biomolecular condensates have been put forth by theoretical models, at least back in 2020 and even earlier. Those earlier works afford extensive information such as considerations of salt concentrations inside and outside the condensate (tie-lines). But the authors do not appear to be aware of this body of prior works and therefore missed the opportunity to build on these previous advances and put the present work with its complementary advantages in structural details in the proper context. (ii) There are significant experimental findings regarding salt effects on condensate formation [which have been modeled more recently] that predate the A1-LCD system (ref.19) addressed by the present manuscript. This information should be included, e.g., in Table 1, for sound scholarship and completeness. (iii) The strengths and limitations of the authors' approach vis-à-vis other theoretical approaches should be discussed with some degree of thoroughness (e.g., how the smallness of the authors' simulation system may affect the nature of the "phase transition" and the information that can be gathered regarding salt concentration inside vs. outside the "condensate" etc.).

Comments on revised version:

The authors have adequately addressed my previous concerns and suggestions. The manuscript is now significantly improved. The new results and analyses provided by the authors represent a substantial advance in our understanding of the role of electrostatics in the assembly of biomolecular condensates.

Reviewer #3 (Public review):

Summary:

This study investigates the salt-dependent phase separation of A1-LCD, an intrinsically disordered region of hnRNPA1 implicated in neurodegenerative diseases. The authors employ all-atom molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which salt influences A1-LCD phase separation. Contrary to typical intrinsically disordered protein (IDP) behavior, A1-LCD phase separation is enhanced by NaCl concentrations above 100 mM. The authors identify two direct effects of salt: neutralization of the protein's net charge and bridging between protein chains, both promoting condensation. They also uncover an indirect effect, where high salt concentrations strengthen pi-type interactions by reducing water availability. These findings provide a detailed molecular picture of the complex interplay between electrostatic interactions, ion binding, and hydration in IDP phase separation.

Strengths:

• Novel Insight: The study challenges the prevailing view that salt generally suppresses IDP phase separation, highlighting A1-LCD's unique behavior.<br /> • Rigorous Methodology: The authors utilize all-atom MD simulations, a powerful computational tool, to investigate the molecular details of salt-protein interactions.<br /> • Comprehensive Analysis: The study systematically explores a wide range of salt concentrations, revealing a nuanced picture of salt effects on phase separation.<br /> • Clear Presentation: The manuscript is well-written and logically structured, making the findings accessible to a broad audience.

Weaknesses:

• Limited Scope: The study focuses solely on the truncated A1-LCD, omitting simulations of the full-length protein. This limitation reduces the study's comparative value, as the authors note that the full-length protein exhibits typical salt-dependent behavior. However, given the much larger size of the full-length protein, it is acceptable to omit it given the current computing resources available.

Overall, this manuscript represents a significant contribution to the field of IDP phase separation. The authors' findings provide valuable insights into the molecular mechanisms by which salt modulates this process, with potential implications for understanding and treating neurodegenerative diseases.

Reviewer #1 (Public review):

Summary:

Crosslinking mass spectrometry has become an important tool in structural biology, providing information about protein complex architecture, binding sites and interfaces, and conformational changes. One key challenge of this approach represents the quantitation of crosslinking data to interrogate differential binding states and distributions of conformational states.

Here, Luo and Ranish present a novel class of isobaric crosslinkers ("Qlinkers"), conduct proof-of-concept benchmarking experiments on known protein complexes, and show example applications on selected target proteins. The data are solid and this could well be an exciting, convincing new approach in the field if the quantitation strategy is made more comprehensive and the quantitative power of isobaric labeling is fully leveraged as outlined below. It's a promising proof-of-concept, and potentially of broad interest for structural biologists.

Strengths:

The authors demonstrate the synthesis, application, and quantitation of their "Q2linkers", enabling relative quantitation of two conditions against each other. In benchmarking experiments, the Q2linkers provide accurate quantitation in mixing experiments. Then the authors show applications of Q2linkers on MBP, Calmodulin, selected transcription factors, and polymerase II, investigating protein binding, complex assembly, and conformational dynamics of the respective target proteins. For known interactions, their findings are in line with previous studies, and they show some interesting data for TFIIA/TBP/TFIIB complex formation and conformational changes in pol II upon Rbp4/7 binding.

Weaknesses:

This is an elegant approach but the power of isobaric mass tags is not fully leveraged in the current manuscript.

First, "only" Q2linkers are used. This means only two conditions can be compared. Theoretically, higher-plexed Qlinkers should be accessible and would also be needed to make this a competitive method against other crosslinking quantitation strategies. As it is, two conditions can still be compared relatively easily using LFQ - or stable-isotope-labeling based approaches. A "Q5linker" would be a really useful crosslinker, which would open up comprehensive quantitative XLMS studies.

Second, the true power of isobaric labeling, accurate quantitation across multiple samples in a single run, is not fully exploited here. The authors only show differential trends for their interaction partners or different conformational states and do not make full quantitative use of their data or conduct statistical analyses. This should be investigated in more detail, e.g. examine Qlinker quantitation of MBP incubated with different concentrations of maltose or Calmodulin incubated with different concentrations of CBPs. Does Qlinker quantitation match ratios predicted using known binding constants or conformational state populations? Is it possible to extract ratios of protein populations in different conformations, assembly, or ligand-bound states?

With these two points addressed this approach could be an important and convincing tool for structural biologists.

Comments on latest version:

I raised only two points which they have not addressed: Higher multiplexing of Qlinkers (1) and experiments to assess the statistical power of their quantitation strategy (2).

I can see that point (1) requires substantial experimental efforts and synthesis of novel Qlinkers would be months of work. This is an editorial decision if the limited quantitative power of the "2-plex" approach they have right now is sufficient to support publication in eLife. While I like the approach, I feel it falls short of its potential in its current form.

For point (2), the authors did not do any supporting experiments. They claim "higher plex Qlinkers" would need to be available, but I suggested experiments that can be done even with Q2linkers: Using one of the two channels as a reference channel (similar the Super-SILAC strategy published in 2010 by Geiger et al; using an isotope-labeled channel as a stable reference channel between different experiments and LC-MS runs), they could do time-courses or ligand-concentration-series with the other channel and then show that Qlinkers allow quantitative monitoring of the different populations (e.g. conformations or ligand-bound proteins).

As an additional point, I was a bit surprised to read that the quantitation evaluation in Figure 1 is based on a single experiment (reviewer response document page 6, line 2 in the authors' reply). I strongly suggest this to be repeated a few times so a proper statistical test on experimental reproducibiltiy of Qlinkers can be conducted.

In summary, the authors declined to do any experimental work to address my concerns.

Reviewer #2 (Public review):

The regulation of protein function heavily relies on the dynamic changes in the shape and structure of proteins and their complexes. These changes are widespread and crucial. However, examining such alterations presents significant challenges, particularly when dealing with large protein complexes in conditions that mimic the natural cellular environment. Therefore, much emphasis has been put on developing novel methods to study protein structure, interactions, and dynamics. Crosslinking mass spectrometry (CSMS) has established itself as such a prominent tool in recent years. However, doing this in a quantitative manner to compare structural changes between conditions has proven to be challenging due to several technical difficulties during sample preparation. Luo and Ranish introduce a novel set of isobaric labeling reagents, called Qlinkers, to allow for a more straightforward and reliable way to detect structural changes between conditions by quantitative CSMS (qCSMS).

The authors do an excellent job describing the design choices of the isobaric crosslinkers and how they have been optimized to allow for efficient intra- and inter-protein crosslinking to provide relevant structural information. Next, they do a series of experiments to provide compelling evidence that the Qlinker strategy is well suited to detect structural changes between conditions by qCSMS. First, they confirm the quantitative power of the novel-developed isobaric crosslinkers by a controlled mixing experiment. Then they show that they can indeed recover known structural changes in a set of purified proteins (complexes) - starting with single subunit proteins up to a very large 0.5 MDa multi-subunit protein complex - the polII complex.

The authors give a very measured and fair assessment of this novel isobaric crosslinker and its potential power to contribute to the study of protein structure changes. They show that indeed their novel strategy picks up expected structural changes, changes in surface exposure of certain protein domains, changes within a single protein subunit but also changes in protein-protein interactions. However, they also point out that not all expected dynamic changes are captured and that there is still considerable room for improvement (many not limited to this crosslinker specifically but many crosslinkers used for CSMS).

Taken together the study presents a novel set of isobaric crosslinkers that indeed open up the opportunity to provide better qCSMS data, which will enable researchers to study dynamic changes in the shape and structure of proteins and their complexes.

Comments on latest version:

The authors have not really addressed most of the concerns. They have added minimal discussion points to the text. This is okay from my perspective as eLife's policy is to leave it up to the authors of how strongly to consider the reviewers' comments. I should add that I do fully agree with the other reviewer that the quantitative assessment from Figure 1 should have been done in triplicates at least and that this would actually be essential.

Reviewer #1 (Public review):

Summary:

In this study, Nandy and colleagues examine neural, physiological and behavioral correlates of perceptual variability in monkeys performing a visual change detection task. They used a laminar probe to record from area V4 while two macaque monkeys detected a small change in stimulus orientation that occurred at a random time in one of two locations, focusing their analysis on stimulus conditions where the animal was equally likely to detect (hit) or not-detect (miss) a briefly presented orientation change (target). They discovered two behavioral and physiological measures that are significantly different between hit and miss trials - pupil size tends to be slightly larger on hits vs. misses, and monkeys are more likely to miss the target on trials in which they made a microsaccade shortly before target onset. They also examined multiple measures of neural activity across the cortical layers and found some measures that are significantly different between hits and misses.

Strengths:

Overall the study is well executed and the analyses are appropriate (with some possible caveats discussed below).

Weaknesses:

I have two remaining concerns. First, with the exception of the pre-target microsaccades, the correlates of perceptual variability (differences between hits and misses) appear to be weak and disconnected. The GLM analysis of the predictive power of trial outcome based on the behavioral and neural measures is only discussed at the end of the paper. This analysis shows that some of the measures have no significant predictive power, while others cannot be examined using the GLM analysis because these measures cannot be estimated in single trials. Given these weak and disconnected effects, my overall sense is that the current results provide a limited advance to our understanding of the neural basis of perceptual variability.

In addition, because the authors combine data across stimulus contrasts, I am somewhat uneasy about the possible confounding effect of contrast. As expected, stimulus contrast affected the probability of hits vs. misses. Independently, contrast may have affected some of the physiological measurements. Therefore, showing that contrast is not the source of the covariations between the physiological/behavioral measurements and perception can be challenging, and I am not convinced that the authors have ruled this out as a possible confound. It is unclear why the authors had to vary contrast in the first place, and why the analyses had to be done by combining the data across contrasts or by ignoring contrast as a variable (e.g., in the GLM analysis).

Reviewer #1 (Public review):

In this manuscript, Saeb et al reported the mechanistic roles of the flexible stalk domain in sTREM2 function using molecular dynamics simulations. They have reported some interesting molecular bases explaining why sTREM2 shows protective effects during AD, such as partial extracellular stalk domain promoting binding preference and stabilities of sTREM2 with its ligand even in the presence of known AD-risk mutation, R47H. Furthermore, they found that the stalk domain itself acts as the site for ligand binding by providing an "expanded surface", known as 'Expanded Surface 2' together with the Ig-like domain. Also, they observed no difference in the binding free energy of phosphatidyl-serine with wild TREM2-Ig and mutant TREM2-Ig, which is a bit inconsistent with the previous report with experiment studies by Journal of Biological Chemistry 293, (2018), Alzheimer's and Dementia 17, 475-488 (2021), Cell 160, 1061-1071 (2015).

Perhaps the authors made significant efforts to run a number of simulations for multiple models, which is nearly 17 microseconds in total; none of the simulations has been repeated independently at least a couple of times, which makes me uncomfortable to consider this finding technically true. Most of the important conclusions that authors claimed, including the opposite results from previous research, have been made on the single run, which raises the question of whether this observation can be reproduced if the simulation has been repeated independently. Although the authors stated the sampling number and length of MD simulations in the current manuscript as a limitation of this study, it must be carefully considered before concluding rather than based on a single run.

sTREM2 shows a neuroprotective effect in AD, even with the mutations with R47H, as evidenced by authors based on their simulation. sTREM2 is known to bind Aβ within the AD and reduce Aβ aggregation, whereas R47H mutant increases Aβ aggregation. I wonder why the authors did not consider Aβ as a ligand for their simulation studies. As a reader in this field, I would prefer to know the protective mechanism of sTREM2 in Aβ aggregation influenced by the stalk domain.

In a similar manner, why only one mutation is considered "R47H" for the study? There are more server mutations reported to disrupt tethering between these CDRs, such as T66M. Although this "T66M" is not associated with AD, I guess the stalk domain protective mechanism would not be biased among different diseases. Therefore, it would be interesting to see whether the findings are true for this T66M.

In most previous studies, the mechanism for CDR destabilization by mutant was explored, like the change of secondary structures and residue-wise interloop interaction pattern. While this is not considered in this manuscript, neither detailed residue-wise interaction that changed by mutant or important for 'ligand binding" or "stalk domain".

The comparison between the wild and mutant and other different complex structures must be determined by particular statistical calculations to state the observed difference between different structures is significant. Since autocorrelation is one of the major concerns for MD simulation data for predicting statistical differences, authors can consider bootstrap calculations for predicting statistical significance.

Reviewer #2 (Public review):

Significance:

TREM2 is an immunomodulatory receptor expressed on myeloid cells and microglia in the brain. TREM2 consists of a single immunoglobular (Ig) domain that leads into a flexible stalk, transmembrane helix, and short cytoplasmic tail. Extracellular proteases can cleave TREM2 in its stalk and produce a soluble TREM2 (sTREM2). TREM2 is genetically linked to Alzheimer's disease (AD), with the strongest association coming from an R47H variant in the Ig domain. Despite intense interest, the full TREM2 ligand repertoire remains elusive, and it is unclear what function sTREM2 may play in the brain. The central goal of this paper is to assess the ligand-binding role of the flexible stalk that is generated during the shedding of TREM2. To do this, the authors simulate the behavior of constructs with and without stalk. However, it is not clear why the authors chose to use the isolated Ig domain as a surrogate for full-length TREM2. Additionally, experimental binding evidence that is misrepresented by the authors contradicts the proposed role of the stalk.

Summary and strengths:

The authors carry out MD simulations of WT and R47H TREM2 with and without the flexible stalk. Simulations are carried out for apo TREM2 and for TREM2 in complex with various lipids. They compare results using just the Ig domain to results including the flexible stalk that is retained following cleavage to generate sTREM2. The computational methods are well-described and should be reproducible. The long simulations are a strength, as exemplified in Figure 2A where a CDR2 transition happens at ~400-600 ns. The stalk has not been resolved in structural studies, but the simulations suggest the intriguing and readily testable hypothesis that the stalk interacts with the Ig domain and thereby contributes to the stability of the Ig domain and to ligand binding. I suspect biochemists interested in TREM2 will make testing this hypothesis a high priority.

Weaknesses:

Unfortunately, the work suffers from two fundamental flaws.

(1) The authors state that reported differences in ligand binding between the TREM2 and sTREM2 remain unexplained, and the authors cite two lines of evidence. The first line of evidence, which is true, is that there are differences between lipid binding assays and lipid signaling assays. However, signaling assays do not directly measure binding. Secondly, the authors cite Kober et al 2021 as evidence that sTREM2 and TREM2 showed different affinities for Abeta1-42 in a direct binding assay. Unfortunately, when Kober et al measured the binding of sTREM2 and Ig-TREM2 to Abeta they reported statistically identical affinities (Kd = 3.8 {plus minus} 2.9 µM vs 5.1 {plus minus} 3.7 µM) and concluded that the stalk did not contribute measurably to Abeta binding.

(2) The authors appear to take simulations of the Ig domain (without any stalk) as a surrogate for the full-length, membrane-bound TREM2. They compare the Ig domain to a sTREM2 model that includes the stalk. While it is fully plausible that the stalk could interact with and stabilize the Ig domain, the authors need to demonstrate why the full-length TREM2 could not interact with its own stalk and why the isolated Ig domain is a suitable surrogate for this state.

Reviewer #1 (Public review):

Summary:

PPARgamma is a nuclear receptor that binds to orthosteric ligands to coordinate transcriptional programs that are critical for adipocyte biogenesis and insulin sensitivity. Consequently, it is a critical therapeutic target for many diseases but especially diabetes. The malleable nature and promiscuity of the PPARgamma orthosteric ligand binding pocket has confounded the development of improved therapeutic modulators. Covalent inhibitors have been developed but they show unanticipated mechanisms of action depending on which orthosteric ligands are present. In this work, Shang and Kojetin present a compelling and comprehensive structural, biochemical, and biophysical analysis that shows how covalent and noncovalent ligands can co-occupy the PPARgamma ligand binding pocket to elicit distinctive preferences of coactivator and corepressor proteins. Importantly, this work shows how the covalent inhibitors GW9662 and T0070907 may be unreliable tools as pan-PPARgamma inhibitors despite their wide-spread use.

Strengths:

- Highly detailed structure and functional analyses provide a comprehensive structure-based hypothesis for the relationship between PPARgamma ligand binding domain co-occupancy and allosteric mechanisms of action.<br /> - Multiple orthogonal approaches are used to provide high resolution information on ligand binding poses and protein dynamics.<br /> - The large number of x-ray crystal structures solved for this manuscript should be applauded along with their rigorous validation and interpretation.

Reviewer #2 (Public review):

Summary:

The flexibility of the ligand binding domain (LBD) of NRs allows various modes of ligand binding leading to various cellular outcomes. In the case of PPARγ, it's known that two ligands can cobind to the receptor. However, whether a covalent inhibitor functions by blocking the binding of a non-covalent ligand, or cobind in a manner that weakens the binding of a non-covalent ligand remains unclear. In this study, the authors first used TR-FRET and NMR to demonstrate that covalent inhibitors (such as GW9662 and T0070907) weaken but do not prevent non-covalent synthetic ligands from binding, likely via an allosteric mechanism. The AF-2 helix can exchange between active and repressive conformations, and covalent inhibitors shift the conformation toward a transcriptionally repressive one to reduce orthosteric binding of the non-covalent ligands. By co-crystal studies, the authors further reveal the structural details of various non-covalent ligand binding mechanisms in a ligand specific manner (e.g., an alternate binding site, or a new orthosteric binding mode by alerting covalent ligand binding pose).

Strengths:

The biochemical and biophysical evidence as presented is strong and convincing.

Additional comments:

The co-crystal studies were performed by soaking a non-covalent ligand to LBD pre-crystalized with a covalent inhibitor. Since the covalent inhibitors would shift the LBD toward transcriptionally repressive conformation which reduces orthosteric binding of non-covalent ligands, one might ask if the sequence was reversed (i.e., soaking a covalent inhibitor to LBD pre-crystalized with a non-covalent ligand), would similar conclusion be drawn? The authors have reasonably speculated that it might be difficult to soak a covalent inhibitor into preformed crystals where the PPARγ LBD is already bound to a non-covalent ligand, because the larger non-covalent ligand could block the covalent inhibitor to gain access to the region of the orthosteric pocket required for covalent modification.

Reviewer #1 (Public review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Reviewer #2 (Public review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that localization of USP8 to early endosomes are disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although that Rabex5 are accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidences to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members in the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell that whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion. Note that the authors have provided convincing evidence about the effects on Rab proteins in the revised manuscript.<br /> - The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient of both factors displayed similar defects in late endosomes/lysosomes. But the authors didn't confirm whether and/or to which extent that USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model. Note that the authors have provided convincing evidence about the role of USP8-Rabx5 axis in the revised manuscript.

Reviewer #3 (Public review):

Summary:

The authors elucidated the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of abnormal enlarged vesicles (aberrant early endosomes) when USP8 function was lost. They showed that USP8 interacts with Rabx5 to dissociate it from early endosomes promoting the recruitment of the Rab7 GEF SAND-1/Mon1 and the maturation of the endosomes. The authors provided evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells.

Strengths:

The use of two models, C. elegans and a mammalian cell line to describe a similar mechanism.

Reviewer #1 (Public review):

Summary:<br /> In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e and h) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

Strengths:<br /> Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:<br /> The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

The manuscript has improved with the new corrections. I appreciate the authors' attention to the minor comments, which have been fully solved. The authors have not, however, provided additional experimental evidence that uORF-mediated translation of Raf-1 mRNA depends on an intact eIF3 complex, nor have they addressed the consequences of such regulation for cell physiology. While I understand that this is a subject of follow-up research, the authors could have at least included their explanations/ speculations regarding major comments 2-4, which in my opinion could have been useful for the reader.

Reviewer #2 (Public review):

Summary:<br /> mRNA translation regulation permits cells to rapidly adapt to diverse stimuli by fine tuning gene expression. Specifically, the 13-subunit eukaryotic initiation factor 3 (eIF3) complex is critical for translation initiation as it aids in 48S PIC assembly to allow for ribosome scanning. In addition, eIF3 has been shown to drive transcript-specific translation by binding mRNA 5' cap structures through the eIF3d subunit. Dysregulation of eIF3 has been implicated in oncogenesis, however the precise eIF3 subunit contributions are unclear. Here, Herrmannová et al. aim to investigate how eIF3 subcomplexes, generated by knock down (KD) of either eIF3e, eIF3d or eIF3h, affect the global translatome. Using Ribo-seq and RNA-seq, the authors identified a large number of genes that exhibit altered translation efficiency upon eIF3d/e KD, while translation defects upon eIF3h KD were mild. eIF3d/eKD share multiple dysregulated transcripts, perhaps due to both subcomplexes lacking eIF3d. Both eIF3d/e KD increase translation efficiency (TE) of transcripts encoding lysosomal, ER and ribosomal proteins, suggesting a role of eIF3 in ribosome biogenesis and protein quality control. Many transcripts encoding ribosomal proteins harbor a TOP motif, and eIF3d KD and eIF3e KD cells exhibit a striking induction of these TOP-modified transcripts. On the other hand, eIF3d KD and eIF3e KD leads to a reduction of MAPK/ERK pathway proteins. Despite this downregulation, eIF3d KD and eIF3e KD activates MAPK/ERK signaling as ERK1/2 and c-Jun phosphorylation was induced. Finally, in all three knockdowns, MDM2 and ATF4 protein levels are reduced. This is notable because MDM2 and ATF4 both contain short uORFs upstream of the start codon, and further supports a role of eIF3 in reinitiation. Altogether, Herrmannová et al. have gained key insights to precise eIF3-mediated translational control as it relates to key signaling pathways implicated in cancer.

Strengths:<br /> The authors have provided a comprehensive set of data to analyze RNA and ribosome footprinting upon perturbation of eIF3d, eIF3e, and eIF3h. As described above in the summary, these data present many interesting starting points to understand additional roles of the eIF3 complex and specific subunits in translational control.

Weaknesses:<br /> - The differences between eIF3e and eIF3d knockdown are difficult to reconcile, especially since eIF3e knockdown leads to reduction in eIF3d levels.<br /> - The paper would be strengthened by experiments directly testing what RNA determinants allow for transcript-specific translation regulation by the eIF3 complex. This would allow the paper to be less descriptive.<br /> - The paper would have more biological relevance if eIF3 subunits were perturbed to mimic naturally occurring situations where eIF3 is dysregulated. For example, eIF3e is aberrantly upregulated in certain cancers, and therefore an overexpression and profiling experiment would have been more relevant than a knockdown experiment.

The first review is unchanged as no additional experiments were provided to address the first review.

Reviewer #3 (Public review):

Summary:<br /> In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the multisubunit eukaryotic translation initiation factor 3 is disrupted by knocking down individual subunits. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding modification enzymes in the ER and components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down, but retain elevated levels of activity.

Strengths:<br /> The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what transcripts undergo translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes. Changes observed by both ribosome profiling and western blotting supports their claims that eIF3 functions in mRNA-specific control of translation.

Weaknesses:<br /> (1) The paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is noted that the authors plan to address such outcomes of eIF3 dysregulation in future work, which will be of interest.

(2) The paper could also be strengthened if some of the experiments were performed in at least one other cell type to determine whether changes observed are general or cell-type specific. The authors discuss this issue and provide a literature citation to support a more general mechanism.

Reviewer #1 (Public review):

Summary:

How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting viral spread. The authors then go on to show that CPK3 diffusion in the membrane is reduced after viral infection and study the interaction between CPK3 and the remorins, which are a group of scaffold proteins important in nano domain organisation. The authors conclude that there is an interdependence between CPK3 and remorins to control their dynamics during viral infection in plants.

Strengths:

The dissection of which CPK was involved in the viral propagation was masterful and very conclusive. Identifying CPK3 through knockout time course monitoring of viral movement was very convincing. The inclusion of overexpression, constitutively active and point mutation non-functioning lines further added to that.

Weaknesses:

I would like to thank the researchers for including some additional work suggested in the previous round of peer review. However, I still have concerns over this work which are two fold.

(1) Firstly, the imaging described and shown is not sufficient to support the claims made. The PM localisation and its non-PM localised form look similar and with no PM stain or marker construct used to support this. In addition, the quality of lots of the confocal based imaging (including new figure on colocalisation) is simply not sufficient. The images are too noisy and no clear conclusions can be made. The point made previously, the system this data was collected on has an Airyscan detector capable of 120nm resolution and as such NDs can be resolved. The sptPALM data conclusions are nice and fit the narrative. The inclusion of sptPALM movies is useful for the reader and tracks numbers is highly beneficial. But they do not show a high signal to noise ratio compared to other work in the field (see work from Alex Martineire) and the mEOS prticles are only just observable over the detector noise in some videos. As such, I worry about the data quality on which the analysis is based on. In addition, in some of the videos the conversion laser seems too high as it is difficult to separate some of the single particles as they emerge which would again, hinder the analysis.

(2) Secondly, remorins are involved in a lot of nano domain controlled processes at the PM. The authors have not conclusively demonstrated that during viral infection the remorin effects seen are solely due to its interaction with CPK3. The sptPALM imaging of REM1.2 in a cpk3 knockout line goes part way to solve this and the inclusion of CPK3-CA also strengthens the authors claims. But to propose a kiss and go model bearing in mind the differences in diffusion between CPK3 and REM3 and differential changes to diffusion between the two proteins after PIAMV infection without two colour imaging of both proteins at the same time, the claims are much stronger than the evidence. Negative control experiments are required here utilising other PM localised proteins which have no role during viral infection (such as Lti6B).

Overall, I think this work has the potential to be a very strong manuscript but additional evidence supporting interaction claims would significantly strengthen the work and make it exceptional.

Reviewer #3 (Public review):

Summary:

This study examined the role that the activation and plasma membrane localisation of a calcium dependent protein kinase (CPK3) plays in plant defence against viruses.<br /> The authors clearly demonstrate that the ability to hamper the cell-to-cell spread of the virus P1AMV is not common to other CPKs which have roles in defence against different types of pathogens, but appears to be specific to CPK3 in Arabidopsis. Further, they show that lateral diffusion of CPK3 in the plasma membrane is reduced upon P1AMV infection, with CPK3 likely present in nano-domains. This stabilisation however, depends on one of its phosphorylation substrates a Remorin scaffold protein REM1-2. However, when REM1-2 lateral diffusion was tracked, it showed an increase in movement in response to P1AMV infection. These contrary responses to P1AMV infection were further demonstrated to be interdependent, which led the authors to propose a model in which activated CPK3 is stabilised in nano-domains in part by its interaction with REM1.2, which it binds and phosphorylates, allowing REM1-2 to diffuse more dynamically within the membrane.

The likely impact of this work is that it will lead to closer examination of the formation of nano-domains in the plasma membrane and dissection of their role in immunity to viruses, as well as further investigation into the specific mechanisms by which CPK3 and REM1-2 inhibit the cell-to-cell spread of viruses, including examination of their roles in cytoskeletal dynamics.

Strengths:

The paper provided compelling evidence about the roles of CPK3 and REM1-2 through a combination of logical reverse genetics experiments and advanced microscopy techniques, particularly in single particle tracking.

Weaknesses:

There is limited discussion or exploration of the role that CPK3 has in cytoskeletal organisation and whether this may play a role in the plant's defence against viral propagation. Further. although the authors show that there is no accumulation of CPK3/Rem1.2 at plasmodesmata, it would be interesting to investigate whether the demonstrated reduction of viral propagation is due to changes in PD permeability.

Reviewer #1 (Public review):

Summary:

This paper provides a computational model of a synthetic task in which an agent needs to find a trajectory to a rewarding goal in a 2D-grid world, in which certain grid blocks incur a punishment. In a completely unrelated setup without explicit rewards, they then provide a model that explains data from an approach-avoidance experiment in which an agent needs to decide whether to approach or withdraw from, a jellyfish, in order to avoid a pain stimulus, with no explicit rewards. Both models include components that are labelled as Pavlovian; hence the authors argue that their data show that the brain uses a Pavlovian fear system in complex navigational and approach-avoid decisions.

In the first setup, they simulate a model in which a component they label as Pavlovian learns about punishment in each grid block, whereas a Q-learner learns about the optimal path to the goal, using a scalar loss function for rewards and punishments. Pavlovian and Q-learning components are then weighed at each step to produce an action. Unsurprisingly, the authors find that including the Pavlovian component in the model reduces the cumulative punishment incurred, and this increases as the weight of the Pavlovian system increases. The paper does not explore to what extent increasing the punishment loss (while keeping reward loss constant) would lead to the same outcomes with a simpler model architecture, so any claim that the Pavlovian component is required for such a result is not justified by the modelling.

In the second setup, an agent learns about punishments alone. "Pavlovian biases" have previously been demonstrated in this task (i.e. an overavoidance when the correct decision is to approach). The authors explore several models (all of which are dissimilar to the ones used in the first setup) to account for the Pavlovian biases.

Strengths:

Overall, the modelling exercises are interesting and relevant and incrementally expand the space of existing models.

Weaknesses:

I find the conclusions misleading, as they are not supported by the data.

First, the similarity between the models used in the two setups appears to be more semantic than computational or biological. So it is unclear to me how the results can be integrated.

Secondly, the authors do not show "a computational advantage to maintaining a specific fear memory during exploratory decision-making" (as they claim in the abstract). Making such a claim would require showing an advantage in the first place. For the first setup, the simulation results will likely be replicated by a simple Q-learning model when scaling up the loss incurred for punishments, in which case the more complex model architecture would not confer an advantage. The second setup, in contrast, is so excessively artificial that even if a particular model conferred an advantage here, this is highly unlikely to translate into any real-world advantage for a biological agent. The experimental setup was developed to demonstrate the existence of Pavlovian biases, but it is not designed to conclusively investigate how they come about. In a nutshell, who in their right mind would touch a stinging jellyfish 88 times in a short period of time, as the subjects do on average in this task? Furthermore, in which real-life environment does withdrawal from a jellyfish lead to a sting, as in this task?

Crucially, simplistic models such as the present ones can easily solve specifically designed lab tasks with low dimensionality but they will fail in higher-dimensional settings. Biological behaviour in the face of threat is utterly complex and goes far beyond simplistic fight-flight-freeze distinctions (Evans et al., 2019). It would take a leap of faith to assume that human decision-making can be broken down into oversimplified sub-tasks of this sort (and if that were the case, this would require a meta-controller arbitrating the systems for all the sub-tasks, and this meta-controller would then struggle with the dimensionality j).

On the face of it, the VR task provides higher "ecological validity" than previous screen-based tasks. However, in fact, it is only the visual stimulation that differs from a standard screen-based task, whereas the action space is exactly the same. As such, the benefit of VR does not become apparent, and its full potential is foregone.

If the authors are convinced that their model can - then data from naturalistic approach-avoidance VR tasks is publicly available, e.g. (Sporrer et al., 2023), so this should be rather easy to prove or disprove. In summary, I am doubtful that the models have any relevance for real-life human decision-making.

Finally, the authors seem to make much broader claims that their models can solve safety-efficiency dilemmas. However, a combination of a Pavlovian bias and an instrumental learner (study 1) via a fixed linear weighting does not seem to be "safe" in any strict sense. This will lead to the agent making decisions leading to death when the promised reward is large enough (outside perhaps a very specific region of the parameter space). Would it not be more helpful to prune the decision tree according to a fixed threshold (Huys et al., 2012)? So, in a way, the model is useful for avoiding cumulatively excessive pain but not instantaneous destruction. As such, it is not clear what real-life situation is modelled here.

A final caveat regarding Study 1 is the use of a PH associability term as a surrogate for uncertainty. The authors argue that this term provides a good fit to fear-conditioned SCR but that is only true in comparison to simpler RW-type models. Literature using a broader model space suggests that a formal account of uncertainty could fit this conditioned response even better (Tzovara et al., 2018).

Reviewer #2 (Public review):

Summary:

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.).

Strengths:

(1) Simplicity of the model which can at the same time model rather complex environments.

(2) Introduction of a flexible omega parameter.

(3) Direct application to a rather advanced VR task.

(4) The paper is extremely well written. It was a joy to read.

Weaknesses:

Almost none! In very few cases, the explanations could be a bit better.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study.

Weaknesses:

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse.

Reviewer #1 (Public review):

Summary:

The study significantly advances our understanding of how exosomes regulate filopodia formation. Filopodia play crucial roles in cell movement, polarization, directional sensing, and neuronal synapse formation. McAtee et al. demonstrated that exosomes, particularly those enriched with the protein THSD7A, play a pivotal role in promoting filopodia formation through Cdc42 in cancer cells and neurons. This discovery unveils a new extracellular mechanism through which cells can control their cytoskeletal dynamics and interaction with their surroundings. The study employs a combination of rescue experiments, live-cell imaging, cell culture, and proteomic analyses to thoroughly investigate the role of exosomes and THSD7A in filopodia formation in cancer cells and neurons. These findings offer valuable insights into fundamental biological processes of cell movement and communication and have potential implications for understanding cancer metastasis and neuronal development.

Weaknesses:

The conclusions of this study are in most cases supported by data, but some aspects of data analysis need to be better clarified and elaborated. Some conclusions need to be better stated and according to the data observed.

Reviewer #2 (Public review):

Summary:

The authors show that small EVs trigger the formation of filopodia in both cancer cells and neurons. They go on to show that two cargo proteins, endoglin, and THSD7A, are important for this process. This possibly occurs by activating the Rho-family GTPase CDC42.

Strengths:

The EV work is quite strong and convincing. The proteomics work is well executed and carefully analyzed. I was particularly impressed with the chick metastasis assay that added strong evidence of in vivo relevance.

Weaknesses:

The weakest part of the paper is the Cdc42 work at the end of the paper. It is incomplete and not terribly convincing. This part of the paper needs to be improved significantly

Reviewer #3 (Public review):

Summary:

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism.

Strengths:

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel.

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function.

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes.

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia.

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings.

Weaknesses:

(1) A better characterization of the nature of the small EV population is missing:

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations.

(2) Data analysis and quantification should be performed with increased rigor:

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A:

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells?

Reviewer #1 (Public review):

Summary:

The authors perform an analysis of the relationship between the size of an LMM and the predictive performance of an ECoG encoding model made using the representations from that LMM. They find a logarithmic relationship between model size and prediction performance, consistent with previous findings in fMRI. They additionally observe that as the model size increases, the location of the "peak" encoding performance typically moves further back into the model in terms of percent layer depth, an interesting result worthy of further analysis into these representations.

Strengths:

The evidence is quite convincing, consistent across model families, and complementary to other work in this field. This sort of analysis for ECoG is needed and supports the decade-long enduring trend of the "virtuous cycle" between neuroscience and AI research, where more powerful AI models have consistently yielded more effective predictions of responses in the brain. The lag analysis showing that optimal lags do not change with model size is a nice result using the higher temporal resolution of ECoG compared to other methods like fMRI.

Weaknesses:

I would have liked to have seen the data scaling trends explored a bit too, as this is somewhat analogous to the main scaling results. While better performance with more data might be unsurprising, showing good data scaling would be a strong and useful justification for additional data collection in the field, especially given the extremely limited amount of existing language ECoG data. I realize that the data here is somewhat limited (only 30 minutes per subject), but authors could still in principle train models on subsets of this data.

Separately, it would be nice to have better justification of some of these trends, in particular the peak layerwise encoding performance trend and the overall upside-down U-trend of encoding performance across layers more generally. There is clearly something very fundamental going on here, about the nature of abstraction patterns in LLMs and in the brain, and this result points to that. I don't see the lack of justification here as a critical issue, but the paper would certainly be better with some theoretical explanation for why this might be the case.

Lastly, I would have wanted to see a similar analysis here done for audio encoding models using Whisper or WavLM as this is the modality where you might see real differences between ECoG and other slower scanning approaches. Again, I do not see this omission as a fundamental issue, but it does seem like the sort of analysis for which the higher temporal resolution of ECoG might grant some deeper insight.

Reviewer #2 (Public review):

Summary:

This paper investigates whether large language models (LLMs) of increasing size more accurately align with brain activity during naturalistic language comprehension. The authors extracted word embeddings from LLMs for each word in a 30-minute story and regressed them against electrocorticography (ECoG) activity time-locked to each word as participants listened to the story. The findings reveal that larger LLMs more effectively predict ECoG activity, reflecting the scaling laws observed in other natural language processing tasks.

Strengths:

(1) The study compared model activity with ECoG recordings, which offer much better temporal resolution than other neuroimaging methods, allowing for the examination of model encoding performance across various lags relative to word onset.

(2) The range of LLMs tested is comprehensive, spanning from 82 million to 70 billion parameters. This serves as a valuable reference for researchers selecting LLMs for brain encoding and decoding studies.

(3) The regression methods used are well-established in prior research, and the results demonstrate a convincing scaling law for the brain encoding ability of LLMs. The consistency of these results after PCA dimensionality reduction further supports the claim.

Weaknesses:

(1) Some claims of the paper are less convincing. The authors suggested that "scaling could be a property that the human brain, similar to LLMs, can utilize to enhance performance", however, many other animals have brains with more neurons than the human brain, making it unlikely that simple scaling alone leads to better language performance. Additionally, the authors claim that their results show 'larger models better predict the structure of natural language.' However, it remains unclear to what extent the embeddings of LLMs capture the "structure" of language better than the lexical semantics of language.

(2) The study lacks control LLMs with randomly initialized weights and control regressors, such as word frequency and phonetic features of speech, making it unclear what the baseline is for the model-brain correlation.

(3) The finding that peak encoding performance tends to occur in relatively earlier layers in larger models is somewhat surprising and requires further explanation. Since more layers mean more parameters, if the later layers diverge from language processing in the brain, it raises the question of what aspects of the larger models make them more brain-like.

Reviewer #3 (Public review):

This manuscript studies the connection between neural activity collected through electrocorticography and hidden vector representations from autoregressive language models, with the specific aim of studying the influence of language model size on this connection. Neural activity was measured from subjects who listened to a segment from a podcast, and the representations from language models were calculated using the written transcription as the input text. The ability of vector representations to predict neural activity was evaluated using 10-fold cross-validation with ridge regression models.

The main results are that (as well summarized in section headings):

(1) Larger models predict neural activity better.

(2) The ability of language model representations to predict neural activity differs across electrodes and brain regions.

(3) The layer that best predicts neural activity differs according to model size, with the "SMALL" model showing a correspondence between layer number and the language processing hierarchy.

(4) There seems to be a similar relationship between the time lag and the ability of language model representations to predict neural activity across models.

Strengths:

(1) The experimental and modeling protocols generally seem solid, which yielded results that answer the authors' primary research question.

(2) Electrocorticography data is especially hard to collect, so these results make a nice addition to recent functional magnetic resonance imaging studies.

Weaknesses:

(1) The interpretation of some results seems unjustified, although this may just be a presentational issue.

a) Figure 2B: The authors interpret the results as "a plateau in the maximal encoding performance," when some readers might interpret this rather as a decline after 13 billion parameters. Can this be further supported by a significance test like that shown in Figure 4B?

b) Figure S1A: It looks like the drop in PCA max correlation is larger for larger models, which may suggest to some readers that the same trend observed for ridge max correlation may not hold, contra the authors' claim that all results replicate. Why not include a similar figure as Figure 2B as part of Figure S1?

(2) Discussion of what might be driving the main result about the influence of model size appears to be missing (cf. the authors aim to provide an explanation of what seems to drive the influence of the layer location in Paragraph 3 of the Discussion section). What explanations have been proposed in the previous functional magnetic resonance imaging studies? Do those explanations also hold in the context of this study?

(3) The GloVe-based selection of language-sensitive electrodes (at least to me) isn't explained/motivated clearly enough (I think a more detailed explanation should be included in the Materials and Methods section). If the electrodes are selected based on GloVe embeddings, then isn't the main experiment just showing that representations from larger language models track more closely with GloVe embeddings? What justifies this methodology?

(4) (Minor weakness) The main experiments are largely replications of previous functional magnetic resonance imaging studies, with the exception of the one lag-based analysis. Is there anything else that the electrocorticography data can reveal that functional magnetic resonance imaging data can't?

Reviewer #1 (Public review):

Summary:

The Notch signaling pathway plays important roles in many developmental and disease processes. Although well-studied there remain many puzzling aspects. One is the fact that as well as activating the receptor through a trans-activation, the transmembrane ligands can interact with receptors present in the same cell. These cis-interactions are usually inhibitory, but in some cases, as in the assays used here, they may also be activating. With a total of 6 ligands and 4 receptor there are potentially a wide array of possible outcomes when different combinations are co-expressed in vivo. Here the authors set out to make a systematic analysis of the qualitative and quantitative differences in the signaling output from different receptor ligand combinations, generating sets of "signaling" (ligand expressing) and "receiving" (receptor +/- ligand expressing cells).

The readout of pathway activity is transcriptional, relying on the fusion of GAL4 in the intracellular part of the receptor. Positive ligand interactions result in proteolytic release of Gal4 that turns on expression of H2B-citrine. As an indicator of ligand and receptor expression levels, they are linked via TA to H2B mCherry and H2B mTurq expression respectively. The authors also manipulate expression of the glycosyltransferase Lunatic-Fringe (LFng) that modifies the EGF repeats in the extracellular domains impacting on their interactions. The testing of multiple ligand receptor combinations at varying expression levels is a tour de force, with over 50 stable cell lines generated, and yields valuable insights although as a whole, the results are quite complex.

Strengths:

Taking a reductionist approach to test systematically differences in the signaling strength, binding strength and cis-interactions from the different ligands in the context of the Notch1 and Notch 2 receptors (they justify well they choice of players to test via this approach) produces a baseline understanding of the different properties and leads to some unexpected and interesting findings. Notably:<br /> - Jag1 ligand expressing cells failed to activate Notch1 receptor although were capable of activating Notch2. Conversely, Jag2 cells elicited the strongest activation of both receptors. The results with Jag1 are surprising also because it exhibits some of the strongest binding to plate bound ligands. The failure to activate Notch1 has major functional significance and it will be important in future to understanding the mechanistic basis.<br /> - Jagged ligands have the strongest ciis-inhibitory effects and the receptors differ in their sensitivity to cis-inhibition by Dll ligands. These observations are in keeping with earlier in vivo and cell culture studies. More referencing of those would better place the work in context but it nicely supports and extends previous studies that were conducted in different ways.<br /> - Responses to most trans-activating ligands showed a degree of ultrasensitivity but this was not the case for cis-interactions where effects were more linear. This has implications for the way the two mechanisms operate and for how the signaling levels will be impacted by ligand expression levels.<br /> - Qualitatively similar results are obtained in a second cell line, suggesting they reflect fundamental properties of the ligands/receptors.

Weaknesses:

One weakness is that the methods used to quantify the expression of ligands and receptors rely on co-translation of tagged nuclear H2B proteins. These may not accurately capture surface levels/correctly modified transmembrane proteins. In general, the multiple conditions tested partly compensate for the concerns - for example as Jag1 cells do activate Notch2 even if they do not activate Notch1 some Jag1 must be getting to the surface. But even with Notch2, Jag1 activities are on the lower side, making it important to clarify, especially given the different outcomes with the plated ligands. Similarly, is the fact that all ligands "signalled strongest to Notch2" an inherent property or due to differences in surface levels Notch 2 compared to Notch1?.. The results would be considerably strengthened by calibration of the ligand/receptor levels (and ideally their sub-cellular localizations). Assessing the membrane protein levels would be relatively straightforward to perform on som eof the basic conditions because their ligand constructs contain Flag tags, making it plausible to relate surface protein to H2B, and there are antibodies available for Notch1 and Notch2

In the revised version this has been addressed to some extent. A figure showing the relationship between co-translated mTurquiose and surface receptor expression for some clones (Figure 1-figure supplement 1B) goes some way to address the concerns that differences in Notch1 and Notch 2 could be due to the receptor levels. The data analyzing surface ligand levels is more equivocal, (a Western blot for biotinylated surface proteins), as the levels detected vary substantially between Dll1 and Dll4 (the latter barely detectable). But as a signal for surface expression of Jag1 was obtained this rules-out one concern that this ligand was failing to reach the surface. A discussion of the caveats of the approach is warranted, to make clear the limitations.

Cis-activation as a mode of signaling has only emerged from these synthetic cell culture assays raising questions about its physiological relevance. Cis-activation is only seen at the higher ligand (Dll1, Dll4) levels, how physiological are the expression levels of the ligands/receptors in these assays? Is it likely that this would make a major contribution in vivo? Is it possible that the cells convert themselves into "signaling" and "receiving" sub-populations within the culture by post-translational mechanism. Again some analysis of the ligand/receptors in the cultures would be a valuable addition to show whether or not there are major heterogeneities.

It is hard to appreciate how much cell to cell variability in the "output" there is. For example, low "outputs" could arise from fewer cells becoming activated or from all cells being activated less. As presented, only the latter is considered. That maybe already evident in their data, but not easy for the reader to distinguish from the way they are presented. For example, in many of the graphs, data have been processed through multiple steps of normalization. Some discussion/consideration this point is needed.

Impact:<br /> Overall, cataloguing of the outcomes from the different ligand-receptor combinations, both in cis and trans, yields a valuable baseline for those investigating their functional roles in different contexts. There is still a long way to go before it will be possible to make a predictive model for outcomes based on expression levels, but this work gives an idea about the landscape and the complexities. This is especially important now that signaling relationships are frequently hypothesised based on single cell transcriptomic data. The results presented here demonstrate that the relationships are not straightforward when multiple players are involved.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors extend their previous studies on trans-activation, cis-inhibition (PMID: 25255098) and cis-activation (PMID: 30628888) of the Notch pathway. Here they create a large number of cell lines using CHO-K1 and C2C12 cells expressing either Notch1-Gal4 or Notch2-Gal4 receptors which express a fluorescent protein upon receptor activation (receiver cells). For cis-inhibition and cis-activation assays, these cells were engineered to express one of the four canonical Notch ligands (Dll1, Dll4, Jag1, Jag2) under tetracycline control. Some of the receiver cells were also transfected with a Lunatic fringe (Lfng) plasmid to produce cells with a range of Lfng expression levels. Sender cells expressing all of the canonical ligands were also produced. Cells were mixed in a variety of co-culture assays to highlight trans-activation, cis-activation, and cis-inhibition. All four ligands were able to trans-activate Notch1 and Notch 2, although Jag1 transactivated Notch1 weakly. Lfng enhanced trans-activation of both Notch receptors by Dll1 and Dll4, and inhibited both receptors by Jag 1 and Jag2. Cis-expression of all four ligands were predominantly inhibitory, but Dll1 and Dll4 showed strong cis-activation of Notch2. Interestingly, cis-ligands preferentially inhibited trans-activation by the same ligand, with varying effects on other trans-ligands.

Strengths:

This represents the most comprehensive and rigorous analysis of the effects of canonical ligands on cis- and trans-activation, and cis-inhibition, of Notch1 and Notch2 in the presence or absence of Lfng so far. Studying cis-inhibition and cis-activation is difficult in vivo due to the presence of multiple Notch ligands and receptors (and Fringes) that often occur in single cells. The methods described here are a step towards generating cells expressing more complex arrays of ligands, receptors and Fringes to better mimic in vivo effects on Notch function.

In addition, the fact that their transactivation results with most ligands on Notch1 and 2 in the presence or absence of Lfng were largely consistent with previous publications provides confidence that the author's assays are working properly.

Weaknesses:

In the original version, there was a major concern about quantifying the amount of Notch receptors and ligands on the cell surface (especially Jag1) based on total fluorescence. The authors have added data to demonstrate that most of the receptors and ligands are on the cell surface, allaying most of these concerns.

Reviewer #1 (Public Review):

Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive action of these two parasite molecules, whereas the third family member does not bind and consequently has a shorter half-life and may function via diffusion.

Reviewer #2 (Public Review):

Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo.

Reviewer #1 (Public review):

Summary:

In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some of them are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further show that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2 or the mitotic exit network (MEN) component Bub2.

Strengths:

The manuscript dissects the specific role of different components of the DDC and the SAC during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution for the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are generally well supported by the experimental data. Their observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:

The main weakness of the study is that it is fundamentally based on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This widely used method allows an efficient depletion of proteins in the cell. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic, and hence their capacity to induce a strong checkpoint response might be compromised. A corroboration of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript analyzes and attempts to discriminate genetic requirements for DNA damage-induced cell cycle checkpoint induction, maintenance, and adaptation in budding yeast bearing one or two unrepairable DNA double strand breaks using auxin-induced degradation (AID) of key DNA damage response (DDR) factors. The study paid particular attention to solving a puzzle regarding how yeasts bearing two unrepaired DNA breaks fail to engage in "adaptation" whereas those with a single unrepairable break eventually resume cell cycling after a prolonged (up to 12 h) G2 arrest.

The key findings are: 1. Genetic requirements for the entry and the maintenance of DDC are separable. For instance, Dun1 is partially required for the entry but not the DDC maintenance whereas Chk1 is only required for maintenance. 2. Cells with two unrepairable breaks respond to DDR only up to a certain time (~12-15 h post damage) and beyond this point, depend on spindle assembly checkpoint (SAC) and mitotic exit network (MEN) to halt cell cycling. 3. The authors also propose an interesting concept that the location of DNA breaks and their distance to centromeres are important factors dictating the effect of SAC/MEN on the duration of cell cycle arrest after prolonged arrest (and cells become "deaf" to persistent arrest signals) and yeast's adaptability following DNA damage. The results provide most compelling evidence to date on the role of SAC/MEN in DNA damage response and cell cycle arrest albeit its impact might be limited to the handful of model systems due to the vastly different centromeric elements and far larger chromosome sizes in metazoan cells. The study albeit briefly discussed the basis of transitions from entry, maintenance, and adaptation ( ex. changes in centromeric architectures), it does not offer detailed explanations or a testable hypothesis to this topic.

Overall, the conclusion of the study is well supported by the elegant set of genetic experimental data and employed multiple readouts on DDC factor depletion on checkpoint integrity and cell cycle status. Although the study simply measures Rad53 phosphorylation as the primary metric to assess checkpoint status, it successfully demonstrated how the signaling is modified through the different stages and that eventually cells become recalcitrant to DDC signaling after a prolonged arrest. The results are clear, and rigorously tested and carefully interpreted with good discussion on the possible limitations. The revision provided detailed responses to the reviewers' comments and addressed a few key concerns, one of which is universally raised by the reviewers on the full functionality of AID tagged DDC factors, by simply expressing excess Rad9-AID to restore more normal looking checkpoint response. It will be interesting if the excess expression of other DDC factors could overcome suboptimal checkpoints in cells after 24 h post damage.

Reviewer #3 (Public review):

Summary:

The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:

The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1 and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs. In their revision, the authors have now improved the Rad9-AID methodology to place Rad9 in the context of DDC adaptation, as well as widening the association between adaptation and proximity to centromeres.

Weaknesses:

Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. Conclusions about the maintenance of G2/M in several mutant combinations could have been strengthened by adding simple microscopy experiments with DAPI staining. No clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block is given.

Reviewer #1 (Public review):

Summary:

The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths:

The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

Weaknesses:

Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation. Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Reviewer #1 (Public review):

The authors previously showed in cell culture that Su(H), the transcription factor mediating Notch pathway activity in Drosophila, was phosphorylated on S269 and they found that a phospho-deficient Su(H) allele behaves as a moderate gain of Notch activity in flies, notably during blood cell development. Since downregulation of Notch signaling is important for the production of specialized blood cell types (lamellocytes) in response to wasp parasitism, the authors hypothesized that Su(H) phosphorylation might be involved in this cellular immune response.<br /> Consistent with their hypothesis, the authors now show that Su(H)S269A knock-in flies display a reduced response to wasp parasitism and that Su(H) is phosphorylated upon infestation. Using in vitro kinase assays and a genetic screen, they identify the PKCa family member Pkc53E as the putative kinase involved in Su(H) phosphorylation and they show that Pkc53E can bind Su(H). They further show that Pkc53E deficit or its knock-down in larval blood cells results in similar blood cell phenotypes as Su(H)S269A and their epistatic analyses indicate that Pkc53E acts upstream of Su(H). Finally, they show that Pkc53E mutants aslo display a compromised immune response to wasp parasitism.

Strengths

The manuscript is well presented and the experiments are sound, with a good combination of genetic and biochemical approaches and several clear phenotypes backing the main conclusions. Notably Su(H)S269A mutation strongly reduces lamellocyte production. Moreover, the epistatic data are convincing, notably concerning the relationship between Notch/Su(H) and Pkc53E for crystal cell production.<br /> Even though it is not fully established, the overall model is credible and interesting. In addition, it opens further avenues of research to study the activation of Pkc in response to an immune challenge.

Weaknesses

Apparently, the hypothesis that Pkc53E is required for Su(H) phosphorylation in vivo could not be directly tested due to the lack of an appropriate tool (the specificity and sensitivity of the current anti-pS269 antibody was insufficient).<br /> Also, the poor immune response of Pkc53E mutant might rather be linked to their constitutively reduced circulating blood cell number than to a deficit in Notch/Su(H) down-regulation following wasp infestation.

Reviewer #2 (Public review):

The current draft by Deischel et.al., describes the role of Pkc53E in the phosphorylation of Su(H) to down regulate its transcriptional activity to mount a successful immune response upon parasitic wasp-infection. Overall, I find the study interesting and relevant especially the identification of Pkc53E in phosphorylation of Su(H) is very nice. The authors have proved the central idea linking phosphorylation of Su(H) via Pkc53E to implying its modulation of Notch activity to mount a robust immune response is now well addressed in its entirety and I find the paper indeed very interesting.

Comments on revised version:

The authors have addressed all pending concerns and I have no further comments. I indeed complement the authors for their wonderful piece of work.

Reviewer #3 (Public review):

Diechsel et al. provide important and valuable insights into how Notch signaling is shut down in response to parasitic wasp infestation in order to suppress crystal cell fate and favor lamellocyte production. The study shows that CSL transcription factor Su(H) is phosphorylated at S269A in response to parasitic wasp infestation and this inhibitory phosphorylation is critical for shutting down Notch. The authors go on to perform a screen for kinases responsible for this phosphorylation and have identified Pkc53E as the specific kinase acting on Su(H) at S269A. Using analysis of mutants, RNAi and biochemistry-based approaches the authors convincingly show how Pkc53E-Su(H) interaction is critical for remodeling hematopoiesis upon wasp challenge. I find the study interesting, and the data presented supports the overall conclusions made by the authors. The authors have addressed all my comments satisfactorily in the revised submission.

Strengths:

The manuscript is well presented, and the conclusions made are backed by genetic, biochemical and molecular biology-based approaches. Overall, the authors convincingly demonstrate how Pkc53E mediated phosphorylated of Su(H) shuts down Notch signaling during wasp infestation in Drosophila.

Weaknesses:

The exact molecular trigger for activation of Pkc53E is still uncharacterized and it would be interesting to know how Pkc53E gets activated during wasp infestation and whether Pkc53E gets activated turning down Notch in other stress induced scenarios.

The authors have addressed comments satisfactorily. Overall, I think the findings are interesting and would be useful to the field of developmental biology and immunology and address an important gap in the field. The most significant conclusion from the work is how Notch acts as a molecular switch during parasitic wasp infestation.

Reviewer #1 (Public review):

In this work, the authors study the dynamics of fast-adapting pathogens under immune pressure in a host population with prior immunity. In an immunologically diverse population, an antigenically escaping variant can perform a partial sweep, as opposed to a sweep in a homogeneous population. In a certain parameter regime, the frequency dynamics can be mapped onto a random walk with zero mean, which is reminiscent of neutral dynamics, albeit with differences in higher order moments. Next, they develop a simplified effective model of time dependent selection with expiring fitness advantage, and posit that the resulting partial sweep dynamics could explain the behaviour of influenza trajectories empirically found in earlier work (Barrat-Charlaix et al. Molecular Biology and Evolution, 2021). Finally, the authors put forward an interesting hypothesis: the mode of evolution is connected to the age of a lineage since ingression into the human population. A mode of meandering frequency trajectories and delayed fixation has indeed been observed in one of the long-established subtypes of human influenza, albeit so far only over a limited period from 2013 to 2020. The paper is overall interesting and well-written.

In the revised version, the authors have addressed questions on the role of clonal interference by new simulations in the SI, clarified the connection between the SIR model and vanishing-fitness models, and placed their analysis into the broader context of consumer resource dynamics.

However, the general conclusion, as stated in the abstract, that variant trajectories become unpredictable as a consequence of the SIR dynamics remains somewhat misleading. Two aspects contribute to this problem. (1) The empirical observation of ``quasi-neutrality', i.e. the absence of a net frequency increase inferred as an average of many trajectories at intermediate frequencies, does not imply that individual trajectories are neutral (i.e., fully stochastic and unpredictable) over the time span of observation. Rather, it just says that some have a positive and some have a negative selection coefficient over that time span. (2) As stated by the authors, the observation of average quasi-neutrality is indeed incompatible with the travelling wave model, where initially successful new variants are assumed to retain a fixed, positive selection coefficient from origination to fixation. This observation also limits predictions by extrapolation, where a positive selection coefficient inferred at small frequency is assumed to remain the same at later times and higher frequencies. However, predictions derived from Gog and Grenfell's multi-strain SIR model, as used by several authors, do not make the assumption of fixed selection coefficients and incorporate trajectory-specific, time-dependent expiration effects into their model predictions. This distinction remains blurred throughout the text of the paper.

Reviewer #3 (Public review):

In this work the authors present a multi-strain SIR model in which viruses circulate in a heterogeneous population with different groups characterized by different cross-immunity structures. They reformulate the qualitative features of these SIR dynamics as a random walk characterized by new variants saturating at intermediate frequencies. Then they recast their microscopic description to an effective formalism in which viral strains lose fitness independently from one another. They study several features of this process numerically and analytically, such as the average variants frequency, the probability of fixation, and the coalescent time. They compare qualitatively the dynamics of this model to variants dynamics in RNA viruses such as flu and SARS-CoV-2

The idea that vanishing fitness mechanisms that produce partial sweeps may explain important features of flu evolution is very interesting. Its simplicity and potential generality make it a powerful framework. As noted by the authors, this may have important implications for predictability of virus evolution and such a framework may be beneficial when trying to build predictive models for vaccine design. The vanishing fitness model is well analyzed and produces interesting structures in the strains coalescent. Even though the comparison with data is largely qualitative, this formalism would be helpful when developing more accurate microscopic ingredients that could reproduce viral dynamics quantitatively.<br /> This general framework has the potential to be more universal than human RNA viruses, in situations where invading mutants would saturate at intermediate frequencies.

The qualitative connection between the coarse-grained features of these vanishing fitness dynamics and structured SIR processes offers additional intuition relevant to host-pathogens interactions, although as noted by the authors other ecological processes could drive similar evolutionary patterns. The additions in the revised manuscript, substantiating more thoroughly the connection between the SIR and the vanishing fitness description, are important to better appreciate the scope of the work.

Reviewer #1 (Public review):

Summary:

The authors used a novel multi-dimensional experience sampling (mDES) approach to identify data-driven patterns of experience samples that they use to interrogate fMRI data collected during naturalistic movie-watching data. They identify a set of multi-sensory features of a set of movies that delineate low-dimensional gradients of BOLD fMRI signal patterns that have previously been linked to fundamental axes of cortical organization.

Strengths:

* The novel solution to challenges associated with experience sampling offer potential access to aspects of experience that have been challenging to assess.

Weaknesses:

* The lack of direct interrogation of individual differences/reliability of the mDES scores warrants some pause.

Reviewer #2 (Public review):

Summary:

The present study explores how thoughts map onto brain activity, a notoriously challenging question because of the dynamic, subjective, and abstract nature of thoughts. To tackle this question, the authors collected continuous thought ratings from participants watching a movie, and additionally made use of an open-source fMRI dataset recorded during movie watching as well as five established gradients of brain variation as identified in resting state data. Using a voxel-space approach, the results show that episodic knowledge, verbal detail, and sensory engagement of thoughts commonly modulate visual and auditory cortex, while intrusive distraction modulates the frontoparietal network. Additionally, sensory engagement mapped onto a gradient from primary to association cortex, while episodic knowledge mapped onto a gradient from the dorsal attention network to visual cortex. Building on the association between behavioral performance and neural activation, the authors conclude that sensory coupling to external input and frontoparietal executive control are key to comprehension in naturalistic settings.

The manuscript stands out for its methodological advancements in quantifying thoughts over time and its aim to study the implementation of thoughts in the brain during naturalistic movie watching. However, the conceptualization of thoughts remains vague, limiting the study's insights into brain function.

Strengths:

(1) The study raises a question that has been difficult to study in naturalistic settings so far but is key to understanding human cognition, namely how thoughts map onto brain activation.<br /> (2) The thought ratings introduce a novel method for continuously tracking thoughts, promising utility beyond this study.<br /> (3) The authors used diverse data types, metrics, and analyses to substantiate the effects of thinking from multiple perspectives.

Weaknesses:

(1) The distinction between thinking and stimulus processing (in the sense of detecting and assigning meaning to features, modulated by factors such as attention) remains unclear. Is "thinking" a form of conscious access or a reportable read-out from sensory and higher-level stimulus processing? Or does it simply refer to the method used here to identify different processing states?<br /> (2) The dimensions of thought appear to be directly linked to brain areas traditionally associated with core faculties of perception and cognition. For example, superior temporal cortex codes for speech information, which is also where thought reports on verbal detail localize in this study. This raises the question of whether the present study truly captures mechanisms specific to thinking and distinct from processing, especially given that individual variations in reports were not considered and movie-specific features were not controlled for.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

This paper is framed as presenting a new paradigm but it does little to discuss what this paradigm serves, what are its limitations and how it should have been tested. The novelty appears to be in using experience sampling from 1 sample to model the responses of a second sample.

What are the considerations for treating high-order thought patterns that occur during film viewing as stable enough to use across participants? What would be the limitations of this method? (Do all people reading this paper think comparable thoughts reading through the sections?) This is briefly discussed in the revised manuscript and generally treated as an opportunity rather than as a limitation.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly. It fails to discuss and establish the utility and appropriateness of its proposed method.

Reviewer #1 (Public Review):

In this manuscript, the authors study the effects of synaptic activity on the process of eye-specific segregation, focusing on the role of caspase 3, classically associated with apoptosis. The method for synaptic silencing is elegant and requires intrauterine injection of a tetanus toxin light chain into the eye. The authors report that this silencing leads to increased caspase 3 in the contralateral eye (Figure 1) and demonstrate evidence of punctate caspase 3 that does not overlap neuronal markers like map2. However, the quantifications showing increased caspase 3 in the silenced eye (done at P5) are complicated by overlap with the signal from entire dying cells in the thalamus. The authors also show that global caspase 3 deficiency impairs the process of eye-specific segregation and circuit refinement (Figures 3-4).

The authors also report that "synapse weakening-induced caspase-3 activation determines the specificity of synapse elimination mediated by microglia but not astrocytes" (abstract). They report that microglia engulf fewer RGC axon terminals in caspase 3 deficient animals (Figure 5), and that this preferentially occurs in silenced terminals, but this preferential effect is lost in caspase 3 knockouts. Based on this, the authors conclude that caspase 3 directs microglia to eliminate weaker synapses. However, a much simpler and critical experiment that the authors did not perform is to eliminate microglia and show that the caspase 3 dependent effects go away. Without this experiment, there is no reason to assume that microglia are directing synaptic elimination.

Finally, the authors also report that caspase 3 deficiency alters synapse loss in 6-month-old female APP/PS1 mice, but this is not really related to the rest of the paper.

Reviewer #2 (Public Review):

Summary:

This manuscript by Yu et al. demonstrates that activation of caspase-3 is essential for synapse elimination by microglia, but not by astrocytes. This study also reveals that caspase 3 activation-mediated synapse elimination is required for retinogeniculate circuit refinement and eye-specific territories segregation in dLGN in an activity-dependent manner. Inhibition of synaptic activity increases caspase-3 activation and microglial phagocytosis, while caspase-3 deficiency blocks microglia-mediated synapse elimination and circuit refinement in the dLGN. The authors further demonstrate that caspase-3 activation mediates synapse loss in AD, loss of caspase-3 prevented synapse loss in AD mice. Overall, this study reveals that caspase-3 activation is an important mechanism underlying the selectivity of microglia-mediated synapse elimination during brain development and in neurodegenerative diseases.

Strengths:

A previous study (Gyorffy B. et al., PNSA 2018) has shown that caspase-3 signal correlates with C1q tagging of synapses (mostly using in vitro approaches), which suggests that caspase-3 would be an underlying mechanism of microglial selection of synapses for removal. The current study provides direct in vivo evidence demonstrating that caspase-3 activation is essential for microglial elimination of synapses in both brain development and neurodegeneration.

The paper is well-organized and easy to read. The schematic drawings are helpful for understanding the experimental designs and purposes.

Weaknesses:

It seems that astrocytes contain large amounts of engulfed materials from ipsilateral and contralateral axon terminals (Figure S11B) and that caspase-3 deficiency also decreased the volume of engulfed materials by astrocytes (Figures S11C, D). So the possibility that astrocyte-mediated synapse elimination contributes to circuit refinement in dLGN cannot be excluded.

Does blocking single or dual inactivation of synapse activity (using TeTxLC) increase microglial or astrocytic engulfment of synaptic materials (of one or both sides) in dLGN?

Reviewer #1 (Public review):

Summary:

This paper is an incremental follow-up to the authors' recent paper which showed that Purkinje cells make inhibitory synapses onto brainstem neurons in the parabrachial nucleus which project directly to the forebrain. In that precedent paper, the authors used a mouse line that expresses the presynaptic marker synaptophysin in Purkinje cells to identify Purkinje cell terminals in the brainstem and they observed labeled puncta not only in the vestibular and parabrachial nuclei, as expected, but also in neighboring dorsal brainstem nuclei, prominently the central pontine grey. The present study, motivated by the lack of thorough characterization of PC projections to the brainstem, uses the same mouse line to anatomically map the density and a PC-specific channelrhodopsin mouse line to electrophysiologically assess the strength of Purkinje cell synapses in dorsal brainstem nuclei. The main findings are (1) the density of Purkinje cell synapses is highest in vestibular and parabrachial nuclei and correlates with the magnitude of evoked inhibitory synaptic currents, and (2) Purkinje cells also synapse in the central pontine grey nucleus but not in the locus coeruleus or mesencephalic nucleus.

Strengths:

The complementary use of anatomical and electrophysiological methods to survey the distribution and efficacy of Purkinje cell synapses on brainstem neurons in mouse lines that express markers and light-sensitive opsins specifically in Purkinje cells is the major strength of this study. By systematically mapping presynaptic terminals and light-evoked inhibitory postsynaptic currents in the dorsal brainstem, the authors provide convincing evidence that Purkinje cells do synapse directly onto pontine central grey and nearby neurons but do not synapse onto trigeminal motor or locus coeruleus neurons. Their results also confirm previously documented heterogeneity of Purkinje cell inputs to the vestibular nucleus and parabrachial neurons.

Weaknesses:

Although the study provides strong evidence that Purkinje cells do not make extensive synapses onto LC neurons, which is a helpful caveat given previous reports to the contrary, it falls short of providing the comprehensive characterization of Purkinje cell brainstem synapses which seemed to be the primary motivation of the study. The main information provided is a regional assessment of PC density and efficacy, which seems of limited utility given that we are not informed about the different sources of PC inputs, variations in the sizes of PC terminals, the subcellular location of synaptic terminals, or the anatomical and physiological heterogeneity of postsynaptic cell types. The title of this paper would be more accurate if "characterization" were replaced by "survey".

Several of the study's conclusions are quite general and have already been made for vestibular nuclei, including the suggestions in the Abstract, Results, and Discussion that PCs selectively influence brainstem subregions and that PCs target cell types with specific behavioral roles.

Reviewer #2 (Public review):

Summary:

While it is often assumed that the cerebellar cortex connects, via its sole output neuron, the Purkinje cell, exclusively to the cerebellar nuclei, axonal projections of the Purkinje cells to dorsal brainstem regions have been well documented. This paper provides comprehensive mapping and quantification of such extracerebellar projections of the Purkinje cells, most of which are confirmed with electrophysiology in slice preparation. A notable methodological strength of this work is the use of highly Purkinje cell-specific transgenic strategies, enabling selective and unbiased visualization of Purkinje terminals in the brainstem. By utilizing these selective mouse lines, the study offers compelling evidence challenging the general assumption that Purkinje cell targets are limited to the cerebellar nuclei. While the individual connections presented are not entirely novel, this paper provides a thorough and unambiguous demonstration of their collective significance. Regarding another major claim of this paper, "characterization of direct Purkinje cell outputs (Title)", however, the depth of electrophysiological analysis is limited to the presence/absence of physiological Purkinje input to postsynaptic brainstem neurons whose known cell types are mostly blinded. Overall, conceptual advance is largely limited to confirmatory or incremental, although it would be useful for the field to have the comprehensive landscape presented.

Strengths:

(1) Unsupervised comprehensive mapping and quantification of the Purkinje terminals in the dorsal brainstem are enabled, for the first time, by using the current state-of-the-art mouse lines, BAC-Pcp2-Cre and synaptophysin-tdTomato reporter (Ai34).

(2) Combinatorial quantification with vGAT puncta and synaptophysin-tdTomato labeled Purkinje terminals clarifies the anatomical significance of the Purkinje terminals as an inhibitory source in each dorsal brainstem region.

(3) Electrophysiological confirmation of the presence of physiological Purkinje synaptic input to 7 out of 9 dorsal brainstem regions identified.

(4) Pan-Purkinje ChR2 reporter provides solid electrophysiological evidence to help understand the possible influence of the Purkinje cells onto LC.

Weaknesses:

(1) The present paper is largely confirmatory of what is presented in a previous paper published by the author's group (Chen et al., 2023, Nat Neurosci). In this preceding paper, the author's group used AAV1-mediated anterograde transsynaptic strategy to identify postsynaptic neurons of the Purkinje cells. The experiments performed in the present paper are, by nature, complementary to the AAV1 tracing which can also infect retrogradely and thus is not able to demonstrate the direction of synaptic connections between reciprocally connected regions. Anatomical findings are all consistent with the preceding paper. The likely absence of robust physiological connections from the Purkinje to LC has also been evidenced in the preceding paper by examining c-Fos response to Purkinje terminal photoinhibition at the PBN/LC region.

(2) Although the authors appear to assume uniform cell type and postsynaptic response in each of the dorsal brainstem nuclei (as noted in the Discussion, "PCs likely function similarly to their inputs to the cerebellar nuclei, where a very brief pause in firing can lead to large and rapid elevations in target cell firing"), we know that the responses to the Purkinje cell input are cell type dependent, which vary in neurotransmitter, output targets, somata size, and distribution, in the cerebellar and vestibular nuclei (Shin et al., 2011, J Neurosci; Najac and Raman, 2015, J Neurosci; Özcan et al., 2020, J Neurosci). This consideration impacts the interpretation of two key findings: (a) "Large ... PC-IPSCs are preferentially observed in subregions with the highest densities of PC synapses (Abstract)". For example, we know that the terminal sparse regions reported in the present paper do contain Floccular Targeted Neurons that are sparse yet have dense somatic terminals with profound postinhibitory rebound (Shin et al.). Despite their sparsity, these postsynaptic neurons play a distinct and critical role in proper vestibuloocular reflex. Therefore, associating broad synaptic density with "PC preferential" targets, as written in the Abstract, may not fully capture the behavioral significance of Purkinje extracerebellar projections. (b) "We conclude ... only a small fraction of cell. This suggests that PCs target cell types with specific behavioral roles (Abstract, the last sentence)". Prior research has already established that "PCs target cell types with specific behavioral roles in brainstem regions". Also, whether 23 % (for PCG), for example, is "a small fraction" would be subjective: it might represent a numerically small but functionally important cell type population. The physiological characterization provided in the present cell type-blind analysis could, from a functional perspective, even be decremental when compared to existing cell type-specific analyses of the Purkinje cell inputs in the literature.

(3) The quantification analyses used to draw conclusions about<br /> (a) the significance of PC terminals among all GABAergic terminals and<br /> (b) the fractions of electrophysiologically responsive postsynaptic brainstem neurons may have potential sampling considerations:.<br /> (b.i) this study appears to have selected subregions from each brainstem nucleus for quantification (Figure 2). However, the criteria for selecting these subregions are not explicitly detailed, which could affect the interpretation of the results.<br /> (b.ii) the mapping of recorded cells (Figure 3) seems to show a higher concentration in terminal-rich regions of the vestibular nuclei.

Reviewer #3 (Public review):

Summary:

The manuscript by Chen and colleagues explores the connections from cerebellar Purkinje cells to various brainstem nuclei. They combine two methods - presynaptic puncta labeling as putative presynaptic markers, and optogenetics, to test the anatomical projections and functional connectivity from Purkinje cells onto a variety of brainstem nuclei. Overall, their study provides an atlas of sorts of Purkinje cell connectivity to the brainstem, which includes a critical analysis of some of their own data from another publication. Overall, the value of this work is to both provide neural substrates by which Purkinje cells may influence the brainstem and subsequent brain regions independent of the deep cerebellar nuclei and also, to provide a critical analysis of viral-based methods to explore neuronal connectivity.

Strengths:

The strengths lie in the simplicity of the study, the number of cells patched, and the relationship between the presence of putative presynaptic puncta and electrophysiological results. This type of study is important and should provide a foundation for future work exploring cerebellar inputs and outputs. Overall, I think that the critique of viral-based methods to define connectivity, and a more holistic assessment of what connectivity is and how it should be defined is timely and warranted, as I think this is under-appreciated by many groups and overall, there is a good deal of research being published that do not properly consider the issues that this manuscript raises about what viral-based connectivity maps do and do not tell us.

Weaknesses:

While I overall liked the manuscript, I do have a few concerns that relate to interpretation of results, and discussion of technological limitations. The main concerns I have relate to the techniques that the authors use, and an insufficient discussion of their limitations. The authors use a Cre-dependent mouse line that expresses a synaptophysin-tomato marker, which the authors confidently state is a marker of synapses. This is misleading. Synaptophysin is a vesicle marker, and as such, labels axons, where vesicles are present in transit, and likely cell bodies where the protein is being produced. As such, the presence of tdtomato should not be interpreted definitively as the presence of a synapse. The use of vGAT as a marker, while this helps to constrain the selection of putative pre-synaptic sites, is also a vesicle marker and will likely suffer the same limitations (though in this case, the expression is endogenous and not driven by the ROSA locus). A more conservative interpretation of the data would be that the authors are assessing putative pre-synaptic sites with their analysis. This interpretation is wholly consistent with their findings showing the presence of tdtomato in some regions but only sparse connectivity - this would be expected in the event that axons are passing through. If the authors wish to strongly assert that they are specifically assessing synapses, a marker better restricted to synapses and not vesicles may be more appropriate.

Similarly, while optogenetics/slice electrophysiology remains the state of the art for assessing connectivity between cell populations, it is not without limitations. For example, connections that are not contained within the thickness of the slice (here, 200 um, which is not particularly thick for slice ephys preps) will not be detected. As such, the absence of connections is harder to interpret than the presence of connections. Slices were only made in the coronal plane, which means that if there is a particular topology to certain connections that is orthogonal to that plane, those connections may be under-represented. As such, all connectivity analyses likely are under-representations of the actual connectivity that exists in the intact brain. Therefore, perhaps the authors should consider revising their assessments of connections, or lack thereof, of Purkinje cells to e.g., LC cells. While their data do make a compelling case that the connections between Purkinje cells and LC cells are not particularly strong or numerous, especially compared to other nearby brainstem nuclei, their analyses do indicate that at least some such connections do exist. Thus, rather than saying that the viral methods such as rabies virus are not accurate reflections of connectivity - perhaps a more circumspect argument would be that the quantitative connectivity maps reported by other groups using rabies virus do not always reflect connectivity defined by other means e.g., functional connections with optogenetics. In some cases, the authors do suggest this (e.g."Together, these findings indicate that reliance on anatomical tracing experiments alone is insufficient to establish the presence and importance of a synaptic connection"), but in other cases, they are more dismissive of viral tracing results (e.g. "it further suggests that these neurons project to the cerebellum and were not retrogradely labeled"). Furthermore, some statements are a bit misleading e.g., mentioning that rabies methods are critically dependent on starter cell identity immediately following the citation of studies mapping inputs onto LC cells. While in general, this claim has merit, the studies cited (19-21) use Dbh-Cre to define LC-NE cells which does have good fidelity to the cells of interest in the LC. Therefore, rewording this section in order to raise these issues generally without proximity to the citations in the previous sentence may maintain the authors' intention without suggesting that perhaps the rabies studies from LC-NE cells that identified inputs from Purkinje cells were inaccurate due to poor fidelity of the Cre line. Overall, this manuscript would certainly not be the first report indicating that the rabies virus does not provide a quantitative map of input connections. In my opinion, this is still under-appreciated by the broad community and should be explicitly discussed. Thus, an acknowledgment of previous literature on this topic and how their work contributes to that argument is warranted.