Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Major Concerns:
(1) An important point that the authors should clarify in this study is whether mice are detecting qualitative or quantitative differences between fresh and old cat saliva. Do the environmental conditions in which the old saliva was maintained cause degradation of Fel d 4, the main protein known for inducing a defensive response in rodents? (see Papes et al, 2010 again). If that is the case, one would expect that a lower concentration of Fel d 4 in the old saliva after protein degradation would result in reduced antipredator responses. Alternatively, if the authors believe that different proteins that are absent in the old saliva are contributing to the increased defensive responses observed with the fresh saliva, further protein quantification experiments should be performed. An important experiment to differentiate qualitative versus quantitative differences between the two types of saliva would be diluting the fresh saliva to verify if the amount of protein, rather than the type of protein, is the main factor regulating the behavioral differences.
We thank the reviewer for their important suggestions. We agree that both the quality and quantity of molecular components in saliva undergo changes after the saliva is kept at room temperature for 4 hours. Our findings indicate that mice detect these changes through the VNO and adjust their defensive response patterns accordingly. For instance, freezing behavior is reduced in response to 4-hour-old saliva compared to fresh saliva. On the other hand, the duration of interaction with saliva (investigation behavior) remains low, and the stress hormone ACTH level is upregulated in both cases. A future study ought to identify the specific molecules—most likely proteins or peptides—in cat saliva responsible for these distinct defensive responses in mice. While Fel d 4 stands as one of the potential candidates as it has been shown to induce a form of defensive behavior in mice (Papes et al., 2010), there exists a possibility of a different molecule or a combination of multiple molecules playing a role. Once the molecules are identified, it is imperative to investigate how their quantity and quality change over time and how these factors correlate with freezing behavior in mice. Such an exploration will provide answers to this ethologically significant question raised by the reviewer. We added a paragraph in Discussion under the “The VNO as the sensor of predator cues that induce fear-related behavior” section to clarify this.
(2) The authors claim that fresh saliva is recognized as an immediate danger by rodents, whereas old saliva is recognized as a trace of danger. However, the study lacks empirical tests to support this interpretation. With the current experimental tests, the behavioral differences between animals exposed to fresh vs. old saliva could be uniquely due to the reduced amount of the exact same protein (e.g., Fel d 4) in the two samples of saliva.
As mentioned in response to comment 1, we agree with the alterations in both the quality and quantity of molecules within saliva after 4 hours. What we would like to emphasize in our current study is that mice detect these time-dependent changes through the VNO and subsequently adjust their defensive response patterns. Identifying the specific molecules responsible for inducing behavioral changes and investigating their time-dependent alterations is crucial in the next step. We added a paragraph in the Discussion under the 'The VNO as the sensor of predator cues that induce fear-related behavior' section to clarify this.
(3) In Figure 4H, the authors state that there were no significant differences in the number of cFos-positive cells between the two saliva-exposed groups. However, this result disagrees with the next result section showing that fresh and old saliva differentially activate the VMH. It is unclear why cFos quantification and behavioral correlations were not performed in other upstream areas that connect the VNO to the VMH (e.g., BNST, MeA, and PMCo). That would provide a better understanding of how brain activity correlates with the different types of behaviors reported with the fresh vs. old saliva.
We greatly appreciate this valuable advice. We added c-Fos immunoreactivity (IR) data in the BNST, MeApv, and PAG, together with the data for VMH as shown in new Figure 4G-J. Upon exposure to both fresh and old saliva, we observed an upregulation trend of cFos in the MeApv, VMH, and dPAG, but not in the BNST, compared to the control stimulus.
Moreover, we conducted correlation analyses between the numbers of cFos-positive neurons and the duration of freezing behavior in those neural substrates, which have been added to new Figure 5. The numbers of cFos-IR signals in neurons in the BNST and dPAG did not correlate with the duration of freezing behavior in any of the exposure groups (Figure 5C, F). However, in addition to a significant positive correlation in the VMH for the fresh saliva-exposed group (R2 = 0.5708, 95% CI [-0.1449, 0.9714], p = 0.0412) (Figure 5E), we observed a similar positive correlation trend in the MeApv (R2 = 0.3854, 95% CI [0.3845, 0.9525], p = 0.0942), although it was not statistically significant possibly due to low sample numbers (Figure 5D).
Based on these results, our current circuit model is as follows: different numbers of the VNO sensory neurons activated by fresh and old saliva result in differential excitation levels in mitral cells in the AOB. This, in turn, leads to the differential activation of targeting neural substrates, possibly MeApv, resulting in the differential activation of VMH neurons. This model is depicted in Figure 7 and discussed under the section of 'Differential processing of fresh and old saliva signals in the VNO-to-VMH pathway' in the Discussion."
(4) The interpretation that fresh and old saliva activates different subpopulations of neurons in the VMH based on the observation that cFos positively correlates with freezing responses only with the fresh saliva lacks empirical evidence. To address this question, the authors should use two neuronal activity markers to track the response of the same population of VHM cells within the same animals during exposure to fresh vs. old saliva. Alternatively, they could use single-cell electrophysiology or imaging tools to demonstrate that cat saliva of distinct freshness activates different subpopulations of cells in the VMH. Any interpretation without a direct within-subject comparison or the use of cell-type markers would become merely speculative. Furthermore, the authors assume that differential activations of mitral cells between fresh and old saliva result in the differential activation of VMH subpopulations (page 13, line 3). However, there are intermediate structures between the mitral cells and the VMH, which are completely ignored in this study (e.g., BNST, medial amygdala).
We appreciate this important feedback. We agree that performing a same-animal comparison for fresh and old saliva exposure will offer direct evidence of the differential activation of a sub-population of VMH neurons. However, there is technical difficulties. We have stimulated the same animal with the same or different types of swabs (e.g., Freshcontrol, fresh-fresh, fresh-old, or old-fresh) and observed that once mice were exposed to a saliva-containing swab and exhibited freezing behavior, they no longer made contact with the second swab within the timeframe when two different types of neuroactivity markers can be analyzed. As shown in Figure 2A, direct contact with the saliva swab is necessary for triggering saliva-elicited freezing behavior. Therefore, we concur that conducting further investigations into real-time neural activation responses to both fresh and old saliva within the same subjects, using an appropriate stimulus delivery method into the VNO, as demonstrated in (Bansal et al., 2021; Ben-Shaul et al., 2010; Bergan et al., 2014), would be useful to strengthen our argument.
For the second part of the comment regarding the intermediate structures between the mitral cells and the VMH, please refer to our comment above in response to comment 3.
(5) The authors incorrectly cited the Papes et al., 2010 article on several occasions across the manuscript. In the introduction, the authors cited the Papes et al 2010 study to make reference to the response of rodents to chemical cues, but the Papes et al. study did not use any of the chemical cues listed by the authors (e.g., fox feces, snake skin, cat fur, and cat collars). Instead, the Papes et al. 2010 article used the same chemical cue as the present study: cat saliva. The Papes et al. 2010 article was miscited again in the results section where the authors cited the study to make reference to other sources of cat odor that differ from the cat saliva such as cat fur and cat collars. Because the Papes et al. 2010 article has previously shown the involvement of Trpc2 receptors in the VNO for the detection of cat saliva and the subsequent expression of defensive behaviors by using Trpc2-KO mice, the authors should properly cite this study in the introduction and across the manuscript when making reference to their findings.
The study conducted by Papes et al. in 2010 (Papes et al., 2010) explored mouse defensive responses triggered by native odors derived from three natural mouse predator species: cat, snake, and rat. These odors were derived from neck fur swabs, shed skin, and urine, respectively. Notably, all three types of samples induced defensive risk assessment and avoidance behaviors in mice. These responses were significantly diminished in Trpc2 knock-out (KO) mice, which lack the Trpc2 transduction channel in their vomeronasal sensory neurons, resulting in an impairment in transmitting sensory signals to the brain. Moreover, Papes et al. (2010) mentioned that, 'we did find cat saliva, a potential source of fur chemosignals, sufficient to induce c-Fos expression in the AOB and initiate defensive behavior.' While Papes et al. reported c-Fos expression in the AOB as well as behavioral responses induced by cat saliva in C57BL/6 mice, they did not provide information regarding the c-Fos expression or the defensive behavioral responses to cat saliva in Trpc2KO mice. Overall, we highly value these findings and explicitly state in the results section of our study that ‘Cat saliva has been considered as a source of predator cues found on cat fur and collars, which induce defensive behaviors in rodents (Engelke et al., 2021; Papes et al., 2010),’ providing the rationale for our utilization of cat saliva in our experimental design.
(6) In the introduction, the authors hypothesized that the VNO detects predator cues and sends sensory signals to the VMH to trigger defensive behavioral decisions and stated that direct evidence to support this hypothesis is still missing. However, the evidence that cat saliva activates the VMH and that activity in the VMH is necessary for the expression of antipredator defensive response in rodents has been previously demonstrated in a study by Engelke et al., 2021 (PMID: 33947849), which was entirely omitted by the authors.
We appreciate this insightful comment. Our original sentence meant that the direct evidence was missing for the hypothesis that the mouse VNO detects predator cues and sends sensory signals to the VMH, triggering appropriate defensive behavioral decisions. To clarify this, we altered the sentence (the last sentence of the second last paragraph in Introduction) to “However, how the sensory signals detected through the VNO-to-VMH circuitry modulate behavioral decisions in specific contexts remains elusive.
The study in Engelke et al., 2021(Engelke et al., 2021) has shown that cat saliva activates the VMH and that activity in the VMH is necessary for the expression of antipredator defensive response, including freezing behavior, in rats. This important paper is now cited at multiple locations; page 4 line 16, page 9 line 8, and page 14 line 17. Interestingly, the vomeronasal receptor genes expressed in cat saliva-responsive VNO neurons, V2R-A4 subfamily genes, seem to have expanded independently within mice and rats, lacking direct V2R-A4 orthologues between mice and rats (Rocha et al. submitted). Therefore, exploring the sensory mechanism behind the induction of defensive behavioral responses in rats by cat saliva would be highly intriguing. Comparing the mechanism operating in rats with that observed in mice could offer valuable insights into understanding how the divergent sensory signaling pathways lead to the VMH-mediated defensive behavioral responses across different species.
(7) In the discussion, the authors stated that their findings suggest that the induction of robust freezing behavior is mediated by a distinct subpopulation of VMH neurons. The authors should cite the study by Kennedy et al., 2020 (PMID: 32939094) that shows the involvement of VMH in the regulation of persistent internal states of fear, which may provide an alternative explanation for why distinct concentrations of saliva could result in different behavioral outcomes.
We appreciate this valuable advice to cite this important paper. It is now cited at page 14 line 17 in the Discussion under “Differential activation of VMH neurons potentially underlying distinct intensities of freezing behavior.” We agree that it is intriguing to hypothesize that different freshness of cat saliva induces different degree of persistence of neural activity in a subpopulation of VMH neurons, which regulates the freezing behavior intensity.
(8) The anatomical connectivity between the olfactory system and the ventromedial hypothalamus (VMH) in the abstract is unclear. The authors should clarify that the VMH does not receive direct inputs from the vomeronasal organ (VNO) nor the accessory olfactory bulb (AOB) as it seems in the current text.
We apologize for the confusion caused by our statement in the abstract. The reviewer is correct that the VMH does not receive direct inputs from the VNO and AOB. The abstract now states: 'The vomeronasal organ (VNO) is one of the major sensory input channels through which predator cues are detected with ascending inputs to the medial hypothalamic nuclei, especially to the ventromedial hypothalamus (VMH), through the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST).’
Reviewer #2 (Public Review):
Weakness:
The findings are relatively preliminary. The identities of the receptor and the ligand in the cat saliva that induces the behavior remain unclear. The identity of VMH cells that are activated by the cat saliva remains unclear. There is a lack of targeted functional manipulation to demonstrate the role of V2R-A4 or VMH cells in the behavioral response to cat saliva.
We concur with the reviewer’s comments and agree with the necessity to explore the behavioral response to cat saliva in mice with V2R-A4 receptor(s) knocked out, alongside those with targeted functional manipulations in the VMH. These future studies will allow us to further elucidate the molecular and neural mechanisms underlying this sensory-tohypothalamic circuit.
Reviewer #3 (Public Review):
Weaknesses:
(1) It is unclear if fresh and old saliva indeed alter the perceived imminence predation, as claimed by the authors. Prior work indicates that lower imminence induces anxiety-related actions, such as re-organization of meal patterns and avoidance of open spaces, while slightly higher imminence produces freezing. Here, the authors show that fresh and old predator saliva only provoke different amounts of freezing, rather than changing the topography of defensive behaviors, as explained above. Another prediction of predatory imminence theory would be that lower imminence induced by old saliva should produce stronger cortical activation, while fresh saliva would activate the amygdala, if these stimuli indeed correspond to significantly different levels of predation imminence.
We thank the reviewer for this valuable insight. In our current study, we exclusively compared defensive behavioral responses to 15-minute-old and 4-hour-old cat saliva in mice within their home cages. In future studies, it would be intriguing to expand this investigation by examining behavioral changes in response to saliva collected at additional time points across diverse behavioral settings. Additionally, exploring neural activity in various brain regions in future studies would complement our understanding of these responses.
(2) It is known that predator odors activate and require AOB, VNO, and VMH, thus replications of these findings are not novel, decreasing the impact of this work.
We acknowledge the previous findings mentioned by the reviewer. Our finding in this paper is that cat saliva samples with different freshness predominantly activate different numbers of VNO sensory neurons expressing the same subfamily of sensory receptors, which results in differential activation of the downstream circuit to modulate behavioral outputs.
(3) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing the neural activity of relevant cell bodies and axonal projections, significantly decreasing the mechanistic insights generated by this work.
We thank the reviewer for the valuable comments. We acknowledge that exploring the behavioral effects through the manipulation of specific cell types within defined neural substrates, along with characterizing circuit connectivity, is crucial to understand this circuit more thoroughly in future studies.
(4) The correlation shown in Figure 5c may be spurious. It appears that the correlation is primarily driven by a single point (the green square point near the bottom left corner). All correlations should be calculated using Spearman correlation, which is non-parametric and less likely to show a large correlation due to a small number of outliers. Regardless of the correlation method used, there are too few points in Figure 5c to establish a reliable correlation. Please add more points to 5c.
We thank the reviewer for this important suggestion. We assessed normality of the data using the Shapiro-Wilk and Kolmogorov-Smirnov tests, confirming that the dataset is parametric. We anticipate employing a larger sample size in future studies to further examine rigorous correlation patterns.
(5) Some of the findings are disconnected from the story. For example, the authors show that V2R-A4-expressing cells are activated by predator odors. Are these cells more likely to be connected to the rest of the predatory defense circuit than other VNO cells?
Yes, our hypothesis posits that V2R-A4-expressing VNO sensory neurons serve as receptor neurons for predator cues present in cat saliva. Additionally, we assume that these specific sensory neurons have stronger anatomical connections with the defensive circuit compared to VNO sensory neurons expressing other receptor subfamilies. In our modified Discussion section, we discussed this point under “V2R-A4 subfamily as the receptor for predator cues in cat saliva.”
(6) Were there other behavioral differences induced by fresh compared to old saliva? Do they provoke differences in stretch-attend risk evaluation postures, number of approaches, the average distance to odor stimulus, the velocity of movements towards and away from the odor stimulus, etc?
We appreciate the reviewer's valuable comments. We have now incorporated an analysis of
stretch-sniff risk assessment behavior, presented in new Figure 1F (graph) and Supplemental Figure 1B (raster plot). Mice exhibited stretch-sniff risk assessment behavior, which remained consistent across control, fresh saliva, and old saliva swabs. Additionally, we have also included a raster plot for direct investigation, previously noted as ‘interaction’ in the original manuscript (Supplemental Figure 1C). Mice exposed to a swab containing either fresh or old saliva significantly avoided directly investigating the swab. In contrast, mice exposed to a clean control swab spent a significant amount of time directly investigating the swab, engaging in behaviors such as sniffing and chewing (Figure 1G). A comparison of temporal behavioral patterns revealed a slightly higher frequency of direct investigation behavior toward old saliva compared to fresh saliva at the beginning of the exposure period (Supplemental Figure 1C).
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(A) In the discussion (page 13, line 13), the authors proposed approaches to isolate receptors among the V2R-A4 subfamily that could be responsible for the detection of predator cues in cat saliva such as mRNA profiling from cells isolated from VNO GCaMP imaging. However, the authors argue that this method can lead to false positive results. The authors should clarify what they mean by this exactly.
We meant that pairing of kairomones and their cognate vomeronasal receptors is overall challenging, and subsequent confirmations by performing loss-of-function, as well as gainof-function studies, are necessary to avoid false positive receptor-ligand pairings. We modified the sentence in the discussion as follows: “…. as well as receptor mRNA profiling from isolated single cells activated by cat saliva in GcaMP imaging using the VNO slices in vitro (Haga-Yamanaka et al., 2014; Wong et al., 2020). Receptor candidates identified using either of the methods can be further confirmed by examining necessity and sufficiency for detecting cat saliva using genetically modified mouse lines.”
(B) In the discussion, the authors mention that imminent predator cues present in the cat saliva activate a specific population of VMN neurons. However, the authors have not demonstrated that imminent predator cues exist and the differences between fresh and old saliva are not simply a matter of concentration and integrity of the same protein (see a similar concern in item 2 above).
In alignment with our responses to the reviewer’s public comments 1 and 2, we acknowledge the changes in both the quality and quantity of molecules in cat saliva when kept at room temperature for 4 hours. Our findings demonstrate that mice detect this timedependent alteration through the VNO, leading to subsequent adjustments in their defensive response patterns. The identification of specific molecules responsible for inducing behavioral changes and an exploration of their time-dependent alterations are crucial steps in our ongoing research. To provide further clarification, we have added a paragraph in the discussion section under 'The VNO as the sensor of predator cues that induce fear-related behavior.’
(C) In the introduction, the authors cite several studies and reviews that investigated sensory neural circuits that mediate behavioral responses to chemical predator cues in mice. However, the majority of these studies used rats. Therefore, it is recommended to instead indicate that these studies focus on using rodent models.
We appreciate this insightful comment. We have now replaced the term 'mice/mouse' with 'rodents' in corresponding parts of the manuscript.
(D)The description of the extended amygdala is unclear and gives the impression that the posteroventral part of the medial amygdala is also part of the extended amygdala (page 3, line 25).
We appreciate the reviewer’s important feedback. We have removed the phrase 'the extended amygdala consisting of' from the text.
(E) The authors should justify why they have focused on the role of V2R-A4 in cat saliva detection. As shown in the Figure 3A schematic, many other receptors within the V2R family could have been evaluated. Additionally, the authors should indicate how many mice were used for calculating the ratio for each receptor in Figure 3C, and a group comparison should be performed.
As shown in Supplemental Figure 2 and Figure 3C, our initial investigation involved assessing the co-localization of pS6 signals with signals derived from in situ hybridization probes for all V2R subfamilies. Each probe was designed to recognize all the receptor genes within the subfamily under the tested conditions. This examination led to the identification of V2R-A4, whose probe signals overlap with pS6 signals induced by exposure to cat saliva. In Figure 3C, the percentage of total overlap between the in situ probe and pS6 signals in VNO sections was examined from n=3-6 animals, which is now mentioned in the modified figure legend.
(F) The authors should make it clear to readers at the very beginning of the manuscript that the behavioral differences between fresh and old saliva are not caused by the inefficiency of the old cat saliva to induce defensive responses. Thus, other antipredator behavioral responses should be also quantified (e.g., avoidance time, number and time of investigations to the cat saliva source, risk-assessment, etc.)
We appreciate this valuable comment from the reviewer. In the original version of our manuscript, we used the term 'interaction' to indicate 'direct interaction with the swab for investigation.' We have now replaced the term 'interaction' with 'direct investigation' and added the temporal patterns of these behavioral episodes in Supplemental Figure 1C. Our observations indicate that mice avoid directly investigating both fresh and old saliva compared to the control (Figure 1G). However, there is a slight increase in investigation behavior toward old saliva at the beginning of exposure compared to fresh saliva (Supplemental Figure 1C). Furthermore, we have included the duration (Figure 1F) and temporal patterns (Supplemental Figure 1B) of stretch-sniff risk assessment behavior. Notably, stretch-sniff behavior did not differ towards control, fresh, and old saliva swabs.
(G) The selected representative images for Gαo- and pS6-labeled neurons in Figure 2 should have similar levels of DAPI labeling. Further, the plot depicting the duration of freezing as a function of pS6-IR signals in the VNO (Figure 2H) is difficult to follow. The authors should indicate on the graph which data points represent fresh or old cat saliva exposure, similar to the style used in Figure 5 plots.
We have replaced the representative image in Figure 2E to align the DAPI intensity. Additionally, we updated the data points in Figure 2H and introduced a color code to indicate saliva types.
(H) The schematic in Figure 4 is misleading because the AOB does not directly project to the VMH. The authors should explain which regions are conveying indirect predator information from AOB to VMH (see a similar concern in item 7 above).
We thank the reviewer’s important feedback. We modified the image in Figure 4A to show the entire defensive behavior circuit initiated from the VNO.
Reviewer #2 (Recommendations For The Authors):
(1) This result suggests that V2R-A4 may be the dominant VR for mice to detect cat saliva.
Future studies should determine the identity of the receptor and the ligand in the cat saliva. Additionally, the functional importance of V2R-A4 remains unclear. It is important to knockout the receptor and test changes in cat saliva-induced freezing.
We concur with the reviewer’s comments and recognize the necessity of exploring the behavioral response to cat saliva in mice with V2R-A4 receptor(s) knocked out. Moreover, the identification of the ligand in cat saliva is critical for a deeper understanding of the molecular mechanisms in future studies.
(2) AOB does not project to VMH directly. Other known important nodes for the predator defense circuit include MeApv, BNST, PMd, AHN, and PAG. It will be helpful to provide c-Fos data in those regions (especially MEA and BNST as they are between AOB and VMH) to provide a complete picture of how the brain processes cat saliva to induce the behavior change.
We appreciate this important feedback by the reviewer. We have now added c-Fos expression analysis data in the BNST, MeApv, and PAG, in addition to the VMH. Upon exposure to fresh and old saliva, we observed the upregulation of cFos in the MeApv, VMH, and dPAG, but not in the BNST, compared to the control stimulus. The data are now shown in Figure 4G-J. Moreover, we also added correlation analyses between the numbers of cFospositive neurons and the duration of freezing behavior in those neural substrates to Figure 5. The numbers of cFos-IR signals in neurons in the BNST and dPAG, did not correlate with the duration of freezing behavior in any of the exposure groups (Figure 5C, F). However, in addition to a significant positive correlation in the fresh saliva-exposed group in the VMH (R2 = 0.5708, 95% CI [-0.1449, 0.9714], p = 0.0412) (Figure 5E), we observed a similar positive correlation trend in the MeApv (R2 = 0.3854, 95% CI [-0.3845, 0.9525], p = 0.0942), although it was not statistically significant possibly due to low sample numbers (Figure 5D). Based on these results, our current circuit model is as follows: different numbers of the VNO sensory neurons activated by fresh and old saliva result in differential excitation levels in mitral cells in the AOB. Differential excitation of mitral cells leads to the differential activation of targeting neural substrates, possibly MeApv, which results in differential activation of VMH neurons. This model is depicted in Figure 7 and discussed under the section of “Differential processing of fresh and old saliva signals in the VNO-toVMH pathway” in Discussion.
(3) It is interesting that activation level difference in the VNO by old and fresh cat saliva does not transfer to AOB. It could be informative to examine the correlation between VNO and AOB p6/c-Fos cell number and AOB and VMH c-Fos cell number across animals to understand whether the activation levels across those regions are related. If they are not correlated, it could be helpful to add a discussion regarding potential reasons, e.g. neuromodulatory inputs to the AOB.
We agree that analyzing the number of pS6/cFos-positive cells from all the regions in the same animals are ideal; however, due to technical difficulties, we were unable to collect the entire set of neural substrates from the same animals.
(4) Please indicate n in all figure plots and specify what individual dots mean. In Figure 4h, there are 7 dots in the old saliva group, presumably indicating 7 animals. In Figure 6b, there appear to be more than 7 dots for the old cat saliva group. Are there more than 7 animals? If so, why are they not included in Figure 4h? If not, what does each dot mean? Note that each dot should represent an independent sample. One animal should not contribute more than one dot.
We apologize for the confusion about Figure 6b. Each of these dots indicates the number of cFos signals in a single VMH hemisphere sample. The data used for this analysis were the same as the ones for the VMH used in Figure 4. This is now clarified in the figure legends.
(5) The identification of a cluster of VMHdm cells uniquely activated by fresh cat saliva urine is interesting. It will be important to identify the molecular handle of the cells to facilitate further investigation. This could be achieved using either activity-dependent RNAseq or double in situ of saliva-induced c-Fos and candidate genes (candidate gene may be identified based on the known gene expression pattern).
We agree that these experiments are very valuable. We would like to perform those experiments in future studies.
Reviewer #3 (Recommendations For The Authors):
(1) Please cite recent relevant papers showing VMH activity induced by predators, such as https://pubmed.ncbi.nlm.nih.gov/33115925/ and https://pubmed.ncbi.nlm.nih.gov/36788059/
We thank the reviewer’s suggestion to cite these important papers. https://pubmed.ncbi.nlm.nih.gov/33115925/ (Esteban Masferrer et al., 2020) and https://pubmed.ncbi.nlm.nih.gov/36788059/ (Tobias et al., 2023) are now cited at page 14 line 17 in the Discussion under “Differential activation of VMH neurons potentially underlying distinct intensities of freezing behavior.”
(2) Add complete statistical information in the figure legends of all figures, which should include n, name of test used, and exact p values.
We included statistical analysis results in figure legends; for Figure 6B, we provided statistical analysis results in Supplemental Table 1.
(3) Please paste all figure legends directly below their corresponding figure to make the manuscript easier to read.
We have added figure legends directly below their corresponding figures.
Editor's note:
Should you choose to revise your manuscript, please include full statistical reporting including exact p-values wherever possible alongside the summary statistics (test statistic and df) and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Statistics analysis results have been included in figure legends and supplemental table 1.
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