Sex–specific Single Transcript Level Atlas of Vasopressin and its Receptor (AVPR1a) in the Mouse Brain
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eLife Assessment
This work presents a brain-wide atlas of vasopressin (Avp) and vasopressin receptor 1A (Avpr1a) mRNA expression in mouse brains using high-resolution RNAscope in situ hybridization. The single-transcript approach provides precise localization and identifies additional brain regions expressing Avpr1a, creating a valuable resource for the field. The revised manuscript is clearer and more impactful, with improved figures, stronger data organization, and enhanced scholarship through added context and citations. Overall, the evidence is compelling, and the atlas should be broadly of use to researchers studying vasopressin signaling and related neural circuits.
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Abstract
Vasopressin (AVP), a nonapeptide synthesized predominantly by magnocellular hypothalamic neurons, is conveyed to the posterior pituitary via the pituitary stalk, where AVP is secreted into the circulation. Known to regulate blood pressure and water homeostasis, it also modulates diverse social behaviors, such as pair–bonding, social recognition and cognition in mammals including humans. Importantly, AVP modulates social behaviors in a sex–specific manner, perhaps, due to sex differences in the distribution in the brain of AVP and its main receptor AVPR1a. There is a corpus of integrative studies for the expression of AVP and AVPR1a in various brain regions, and their functions in modulating central and peripheral actions. In order to purposefully address sexually dimorphic and novel roles of AVP on central and peripheral functions through its AVPR1a, we utilized RNAscope to map Avp and Avpr1a single transcript expression in the mouse brain. As the most comprehensive atlas of AVP and AVPR1a in the mouse brain, this compendium highlights the importance of newly identified AVP/AVPR1a neuronal nodes that may stimulate further functional studies.
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eLife Assessment
This work presents a brain-wide atlas of vasopressin (Avp) and vasopressin receptor 1A (Avpr1a) mRNA expression in mouse brains using high-resolution RNAscope in situ hybridization. The single-transcript approach provides precise localization and identifies additional brain regions expressing Avpr1a, creating a valuable resource for the field. The revised manuscript is clearer and more impactful, with improved figures, stronger data organization, and enhanced scholarship through added context and citations. Overall, the evidence is compelling, and the atlas should be broadly of use to researchers studying vasopressin signaling and related neural circuits.
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Reviewer #1 (Public review):
Summary:
Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains.
Strengths:
This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.
The authors have previously adequately addressed all of my concerns. I have no further questions or concerns.
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Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised …
Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.
Strengths:
A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.
[Editors' note: The authors have substantially addressed all the reviewers' concerns and comments.]
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Author response:
The following is the authors’ response to the previous reviews
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The authors have adequately addressed all of my concerns. I have no further questions or concerns.
We thank the Reviewer #1.
Reviewer #2 (Recommendations for the authors):
We thank the Reviewer #2 for thoughtful recommendations.
(1) Figure 1A, 1B, 2B, 2C, etc.: The Y-axis label is confusing. I assume the intention was to make big numbers small by dividing by 1000. The comma makes the label confusing. Perhaps, make the label more "mathematical" as in "Avp density ((transcript/µm2) * 10-3)" or rearrange the math to be clearer as in "Avp density (transcript/1000 per µm2)".
Great suggestion and done exactly as suggested in Figures 1, 2 and 4.
(2) Figure 1B and 1C: The figure and …
Author response:
The following is the authors’ response to the previous reviews
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The authors have adequately addressed all of my concerns. I have no further questions or concerns.
We thank the Reviewer #1.
Reviewer #2 (Recommendations for the authors):
We thank the Reviewer #2 for thoughtful recommendations.
(1) Figure 1A, 1B, 2B, 2C, etc.: The Y-axis label is confusing. I assume the intention was to make big numbers small by dividing by 1000. The comma makes the label confusing. Perhaps, make the label more "mathematical" as in "Avp density ((transcript/µm2) * 10-3)" or rearrange the math to be clearer as in "Avp density (transcript/1000 per µm2)".
Great suggestion and done exactly as suggested in Figures 1, 2 and 4.
(2) Figure 1B and 1C: The figure and legend do not match up. Either switch the figures or the legends. Currently, legend 1B describes image 1C.
Agreed and done as suggested.
(3) Figure 2A is broken up into separate pages/panels. It could be integrated better or separated to make A and B, then shift B and C to C and D.
Great suggestion and we have done exactly as suggested.
(4) Figure 2 legend: I recommend putting the scale bar info with (A) rather than at the end. The stars used in the figure are not explained in the legend.
Good points. We have made all necessary changes as suggested.
(5) Supplementary Figure 1B: The legend states that the data are the number of transcript-containing cells, but the figure states transcript number.
We thank the Reviewer for pointing out this typo. We corrected all graph legends in the Supplementary Figure 1.
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eLife Assessment
This work presents a brain-wide atlas of vasopressin (Avp) and vasopressin receptor 1A (Avpr1a) mRNA expression in mouse brains using high-resolution RNAscope in situ hybridization. The single-transcript approach provides precise localization and identifies additional brain regions expressing Avpr1a, creating a valuable resource for the field. The revised manuscript is clearer and more impactful, with improved figures, stronger data organization, and enhanced scholarship through added context and citations. Overall, the evidence is compelling, and the atlas should be broadly of use to researchers studying vasopressin signaling and related neural circuits.
-
Reviewer #1 (Public review):
Summary:
Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains.
Strengths:
This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.
The authors have adequately addressed all of my concerns. I have no further questions or concerns.
-
Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised …
Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.
Strengths:
A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.
Future considerations:
The work contained in the manuscript is substantial and informative. Some questions remain and would be addressed in the current manuscript. How many cells are impacted? Are transcripts spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield?
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1:
We thank the reviewer for great suggestions.
(1) The X-axis labels in some panels in Figure 2C and Supplementary Figure 2B overlap, making them difficult to read. Adjusting the label spacing or formatting would improve clarity.
We thank the reviewer for the comment. All panels including Figure 2C and Supplementary Figure 2B, have now been organized the way in which X-axis labels are easily read.
(2) In the scatter dot plot bar diagrams, it appears that n=3 for most of the data. Does this represent the number of mice used or the number of tissue sections per sample? This should be clarified in the figure legends for better transparency.
Great suggestion. In Results (page 7, lines 135-136), we now clarified that quantification was performed …
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1:
We thank the reviewer for great suggestions.
(1) The X-axis labels in some panels in Figure 2C and Supplementary Figure 2B overlap, making them difficult to read. Adjusting the label spacing or formatting would improve clarity.
We thank the reviewer for the comment. All panels including Figure 2C and Supplementary Figure 2B, have now been organized the way in which X-axis labels are easily read.
(2) In the scatter dot plot bar diagrams, it appears that n=3 for most of the data. Does this represent the number of mice used or the number of tissue sections per sample? This should be clarified in the figure legends for better transparency.
Great suggestion. In Results (page 7, lines 135-136), we now clarified that quantification was performed on every tenth section of the brain from 3 female and 3 male mice. Additionally, in the legends for scatter dot plot bar diagrams we also mentioned that n=3 represents the number of mice used.
(3) In Supplemental Figure 2B, the positive signals are not clearly visible. Providing higher-magnification images is recommended.
Great suggestion. The revised Supplemental Figure 2B, but also Figure 2A, now provide higher magnification inset images with distinctive positive signals.
Reviewer #2:
We thank the reviewer for great and critical suggestions.
(1) Introduction:
Line 58: References should be provided for this statement as it is based on a robust field of research, not on a new concept.
We thank the reviewer for the comment. We have now included relevant references as suggested (page 4, line 58).
(2) Line 100-102: This sentence seems to make new, an idea that has been well-documented since the late 1970s. Posterior pituitary hormones oxytocin and vasopressin have long been known to have multiple peripheral targets, and at least a subset of vasopressin and oxytocin neurons have robust central projections. The central targets have been the focus of study for numerous labs. Reference 34 does not relate to posterior pituitary hormones and seems mis-cited.
We have changed this sentence, excluded the reference that does not relate to posterior pituitary hormones and added 4 further references reporting other non-traditional roles of vasopressin and oxytocin (page 6, lines 100-102).
(3) Lines 102-108: While the regulation of bone is an interesting example of an under-appreciated impact of vasopressin, the example does not build to the rationale for examining central Avp and Avpr1a expression.
We mean no disrespect here, but we have recently reported neural brain-bone connections using the SNS-specific PRV152 virus (Ryu et al., 2024; PMID: 38963696) and submitted Single Transcript Level Atlas of Oxytocin and the Oxytocin Receptor in the Mouse Brain (doi: https://doi.org/10.1101/2024.02.15.580498). Surprisingly, we detected Avpr1a and Oxtr expression in certain brain areas (for example, PVH and MPOM) that connect to both bone and adipose tissue through the SNS—raising an important question regarding a central role of Avpr1a and Oxtr in bodily mass and fat regulation.
(4) Line 111: Avp expression and Avpr1a expression have both been studied using in situ hybridization. Thus, the overall concept is less novel than hinted at in the text. Avp expression has been studied quite extensively. Avpr1a expression has not been studied in an exhaustive fashion.
We thank the reviewer for this comment and absolutely agree that brain AVP expression has been studied extensively. As with the Avpr, we believe that RNAscope probe design and signal amplification system employed in our study allow for more specific and sensitive detection of individual RNA targets at the single transcript level with much cleaner background noise comparing to in situ hybridization method.
(5) Results:
Line 143: RNAscope is indeed a powerful method of detecting mRNA at the single transcript level. However, using that single transcript resolution only to provide transcript per brain region analysis, losing all of the nuance of the individual transcript expression, seems like a poor use of the method potential.
This is a good point and we did notice that Avpr1a transcript expression in several brain nuclei displayed individual pattern of expression versus more ubiquitous expression in most of the other brain areas. We noted this finding in Results (page 9, lines 164-168); however, because of the word limits in Discussion, we are not sure what would be dropped to make more room and whether this is truly necessary.
(6 &7) Line 135: Sections were coded from 3 males and 3 females. I would argue that there is not enough statistical power to make inferences regarding sex differences or regional differences. In fact, the authors did not provide any statistical analysis in the manuscript at all, even though they stated they had completed statistical tests on the methods.
150-157: All statements regarding sex differences in expression are made without statistical analyses, which, if conducted, would be underpowered. Given the limitations of performing and analyzing RNAscope data en masse a low n is understandable, but it requires a much more precise description of the data and a more careful look at how the results can be interpreted.
We thank the reviewer for these comments. We mean no disrespect here, but while statistical analysis for main brain regions is relevant, it is not meaningful as far as nuclei, sub-nuclei and regions are concerned. It is noteworthy to mention that RNAscope data analysis in the whole mouse brain is an extremely drawn-out process requiring almost 2 months to conduct exhaustive manual counting of single Avpr1a transcripts in a single mouse brain—authors analyzed 6 brains. That said, statistical tests have been performed and exact P values are now shown in graphs.
(8) Line 146: I am flagging this instance, but it should be corrected everywhere it occurs. Since we cannot know the gender of a given mouse, I would recommend referring to the mouse's "sex" rather than its "gender."
Good suggestion. We made adequate changes throughout the manuscript.
(9) Line 153: The authors switch to discussing cell numbers. Why is this data relegated to the supplemental material?
Main figures in the manuscript report Avp and Avpr1a transcript density which has more important biological significance in terms of signal efficiency and cellular response dynamics. Due to the graph abundancy in the main text, we included all graphs with Avp and Avpr1a transcript counts in the supplemental material.
(10) Methods:
Line 369: "For simplicity and clarity, exact test results and exact P values are not presented." Simplicity or clarity is not a scientific rationale not to provide accurate statistics.
We now provide exact P values in the graphs and the sentence in line 369 has been corrected accordingly (page 18, lines 379-380).
(11) Line 362: The description of how data were analyzed is inadequate. More detail is needed.
Agreed. We now included a detailed description on how data was analyzed (page 18, lines 365-374).
(12) Discussion:
Line 321: "This contrasts the rudimentary attribution of a single function per brain area." While brain function is often taught in such rudimentary terms to make the information palatable to students, I do not think the scientific literature on vasopressin function published over the past 50 years would suggest that we are so naïve in interpreting the functional role of vasopressin in the brain. Clearly, vasopressin is involved in numerous brain functions that likely cross behavioral modalities.
Agreed and we removed this sentence.
(13) Line 322: "The approach of direct mapping of receptor expression in the brain and periphery provides the groundwork." On its face, this statement is true, but the present data build on the groundwork laid by others (multiple papers from Ostrowski et al. in the early 1990s).
Agreed.
(14) Figures:
Figure 1: 1B, I do not know the purpose of creating graphs with single bars (3V, ic, pir-male, and pir-female); there are no comparisons made in the graph. In the graphs with many brain regions, very little data can be effectively represented with the scale as it is. I recommend using tables to provide the count/density data and making graphs of only the most robust areas. In addition, although there is no statistical comparison, combining males and females in the same graphs might be beneficial to make a visual comparison easier. Why were cell counts only included in the supplemental material? Is that data not relevant?
We thank the reviewer for this comment. Now all figures are presented in a more effective and aesthetically pleasing way.
(15) There is a real missed opportunity to highlight some of the findings. For example, cell counts and density measures are provided for regions in the hippocampus, thalamus, and cortex that are not typically reported to contain vasopressin-expressing cells. Photomicrographs of these locations showing the RNAscope staining would be far more impactful in reporting these data. Are there cells expressing Avp, or is the Avp mRNA in these areas contained in fibers projecting to these areas from hypothalamic and forebrain sources?
Great suggestion. We now see in Figure 1D showing novel Avp transcript expression in the hippocampus, thalamus and cortex. Based on counterstained hematoxylin staining, Avp mRNA transcripts were found in somata.
(16) Figure 1C legend suggests images of the hippocampus and cortex, but all images are from the hypothalamus. Abbreviations are not defined.
Thank you for the comment. We corrected Figure 1C legend and separately included Figure 1D showing novel Avp mRNA expression in the hippocampus and cortex.
(17) Figure 2: The analysis of Avpr1a suffers from some of the same issues as the Avp analysis. In Figure 2A, the photomicrographs do not do a very good job of illustrating representative staining. The central canal image does not appear to have any obvious puncta, but the density of Avpr1a puncta suggests something different. The sex difference in the arcuate is also not clearly apparent in representative images. There is minimal visualization of the data for a project that depends so heavily on the appearance of puncta in tissue, coupled with the lack of clarity in the images provided, greatly diminished the overall enthusiasm for the data presentation. The figures in 2C would be more useful as tables with graphs used to highlight specific regions; as is, most of the data points are lost against the graph axis. Photomicrographs would also provide a better understanding of the data than graphs.
Great suggestion. The revised Figure 2A but also Supplemental Figure 2B now provide higher magnification inset images with distinctive positive signals. As with Figures 2C, we arranged all graphs in a more effective and aesthetically pleasing manner.
(18) Figure 3: Given the low number of animals and, therefore, low statistical power, I do not think that illustrating the ratios of male to female is a statistically valid comparison.
Please see response to Point 6 & Point 7.
(19) Figure 4: Pituitary is an interesting choice to analyze. However, why was only the posterior pituitary analyzed? Were Avp transcripts contained in terminals of vasopressin neuron axons or other cells? Was Avpr1a transcript present in blood vessel cells where Avp is released? A different cell type? Why not examine the anterior pituitary, which also expresses Avp receptors (although the literature suggests largely Avpr1b)?
Thank you for the great comment. We included only posterior pituitary because there were no positive Avp/Avpr1a transcripts found in the anterior pituitary. Unfortunately, we have not performed cell type-specific staining, which would have enabled greater variation in AVP and its receptor expression across various cell types.
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eLife Assessment
This work presents an atlas of vasopressin (AVP) and its receptor AVPR1a in mouse brains using RNAscope to map single transcript expressions of Avp and Avpr1a across various brain regions in males and females. The findings are valuable in that they identify brain regions expressing Avpr1a mRNA transcript. The impact of findings is decreased by incomplete analysis of the data due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference.
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Reviewer #1 (Public review):
Summary:
Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains. The findings are important, given that: (1) a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking, and (2) the study offers valuable new insights into Avpr1a expression across the mouse brain. The findings are solid, and with improved data presentation and analysis, this work could serve as an important resource for the neuroscience community.
Strengths:
This well-executed study provides valuable new insights into gender …
Reviewer #1 (Public review):
Summary:
Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains. The findings are important, given that: (1) a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking, and (2) the study offers valuable new insights into Avpr1a expression across the mouse brain. The findings are solid, and with improved data presentation and analysis, this work could serve as an important resource for the neuroscience community.
Strengths:
This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.
Weaknesses:
A few concerns remain to be addressed. The primary weakness of this manuscript lies in the robustness of its data presentation and analysis.
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Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, …
Reviewer #2 (Public review):
Summary:
The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, limited use of photomicrographs, and low number of subjects all combine to limit the impact of the dataset. The finding of Avp-expressing cells outside of the hypothalamus and extended amygdala is poorly documented but would be novel. The Avpr1a data suggest expression in numerous brain regions. However, the data presented are difficult to interpret, with every value being an extremely small density value for a large swath of the brain. How many cells are impacted? Are puncta spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield? For a descriptive study, there is minimal statistical inference and relatively little description. The authors make a case for the novel nature of the work but do not seem, at times, to recognize a robust literature developed over the last 50 years. In conclusion, the experimental data are important and informative; however, the low number of subjects, lack of statistical power, limited description of individual brain regions, and poor quality and design of data figures reduce the overall impact.
Strengths:
A survey of Avpr1a expression in the mouse brain is an important tool for exploring the function of vasopressin in the mammalian brain and developing hypotheses about cell - and circuit-level function.
Weaknesses:
(1) The style and type of data presentation, focusing on the density of individual mRNA transcript across a whole brain region, seemed incomplete in so far as the data presentation did not provide a clear visualization of the distribution of Avpr1a-expressing cells or transcript itself. However, knowing which brain regions do express transcript is itself informative.
(2) The manuscript strongly emphases on the possibility of sex differences in Avp and Avpr1a expression. However, the low number of animals used does not provide adequate statistical power to make strong inferences regarding sex differences in the data.
(3) The manuscript's methods are minimal but adequate to understand data acquisition. The description of how quantitative analyses were conducted is inadequate and would be impossible to replicate beyond identifying the program used.
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