Hypothalamic representation of the imminence of predator threat detected by the vomeronasal organ in mice

  1. Quynh Anh Thi Nguyen
  2. Andrea Rocha
  3. Ricky Chhor
  4. Yuna Yamashita
  5. Christian Stadler
  6. Crystal Pontrello
  7. Hongdian Yang
  8. Sachiko Haga-Yamanaka

  • Curated by eLife

    eLife logo

    eLife Assessment

    This valuable study addresses one way in which animals identify predator-associated cues and respond in a manner that reflects the imminence of the potential threat. The report shows that, in mice, fresh saliva from a natural predator (cat) elicits a greater defensive response compared to old cat saliva and implicates the vomeronasal organ and ventromedial hypothalamus as part of a circuit that underlies this process. The evidence supporting the main conclusions is solid. This study will be of interest to those interested in aversive behavior, its processes, and mechanisms.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Animals have the innate ability to select optimal defensive behaviors with appropriate intensity within specific contexts. The vomeronasal organ (VNO) serves as a primary sensory channel for detecting predator cues by relaying signals to the medial hypothalamic nuclei, particularly the ventromedial hypothalamus (VMH), which directly controls defensive behavioral outputs. Here, we demonstrate that cat saliva contains predator cues that signal the imminence of predator threat and modulate the intensity of freezing behavior through the VNO in mice. Cat saliva activates VNO neurons expressing the V2R-A4 subfamily of sensory receptors, and the number of VNO neurons activated in response to saliva correlates with both the freshness of saliva and the intensity of freezing behavior. Moreover, the number of VMH neurons activated by fresh, but not old, saliva positively correlates with the intensity of freezing behavior. Detailed analyses of the spatial distribution of activated neurons, as well as their overlap within the same individual mice, revealed that fresh and old saliva predominantly activate distinct neuronal populations within the VMH. Collectively, this study suggests that there is an accessory olfactory circuit in mice that is specifically tuned to time-sensitive components of cat saliva, which optimizes their defensive behavior to maximize their chance of survival according to the imminence of threat.

Article activity feed

  1. Version published to 10.7554/elife.92982.4 on eLife
  2. Version published to 10.7554/elife.92982 on eLife
  3. eLife

    eLife Assessment

    This valuable study addresses one way in which animals identify predator-associated cues and respond in a manner that reflects the imminence of the potential threat. The report shows that, in mice, fresh saliva from a natural predator (cat) elicits a greater defensive response compared to old cat saliva and implicates the vomeronasal organ and ventromedial hypothalamus as part of a circuit that underlies this process. The evidence supporting the main conclusions is solid. This study will be of interest to those interested in aversive behavior, its processes, and mechanisms.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

    The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

    In this revised version of the manuscript, the authors have made important modifications in the text, inserted new experiments and performed additional data analyses, as recommended. These modifications have significantly improved the quality of the manuscript and addressed all the major concerns detected during the prior submission.

  5. eLife

    Reviewer #2 (Public review):

    In this study, Nguyen et al. showed that cat saliva can robustly induce freezing behavior in mice. This effect is mediated through accessory olfactory system as it requires physical contact and is abolished in Trp2 KO mice. The authors further showed that V2R-A4 cluster is responsive to cat saliva. Lastly, they demonstrated c-Fos induction in AOB and VMHdm/c by the cat saliva. The c-Fos level in the VMHdm/c is correlated with freezing response.

    Strength:

    The study opens an interesting direction. It reveals the potential neural circuit for detecting cat saliva and driving defense behavior in mice. The behavior results and the critical role of accessory olfactory system in detecting cat saliva are clear and convincing.

    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 the cat saliva.

    Here are some specific comments:

    (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.

    (2) AOB does not project to VMH directly. Other known important nodes for the predator defense circuit includes 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 regarding how the brain process cat saliva to induce the behavior change.

    (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 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 level 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.

    (4) Please indicate n in all figure plots and specify what individual dots means. In Figure 4h, there are 7 dots in old saliva group, presumably indicating 7 animals. In Figure 6b, there appear to be more than 7 dots for 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 independent sample. One animal should not contribute more than one dot.

    (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).

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also suggest that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. The work also shows that different VMH populations are activated by fresh and old saliva, demonstrating that these stimuli create qualitatively different neural activity profiles. However, the exact components that differ between fresh and old saliva remain unknown and may be identified in future studies.

    Strengths:

    (1) Predator saliva is a stimulus of high ethological relevance
    (2) The authors performed a careful quantification of fos induction across the anterior-posterior axis
    (3) Authors show that different VMH populations are activated by fresh and old saliva

    Weaknesses:

    (1) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections

    (2) Some of the findings are disconnected from the story. For example, the authors show V2R-A4-expressing cells are activated by predator odors, but the causal role of these cells in generating defensive actions is not shown

  7. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    We greatly appreciate all the reviewers’ constructive comments on our previously revised manuscript. In the current revision, we added several experimental data for answering the reviewers’ comments. Below we describe our point-by-point responses to their comments:

    Reviewer #1 (Public Review):

    Unaddressed and additional concerns (re-submission)

    In this revised version of the manuscript, the authors have made important modifications in the text, inserted new references, and incorporated additional quantifications of cFos immunolabeling in three brain regions, as recommended by the reviewers. While these modifications have significantly improved the quality of the manuscript, other critical concerns raised during the initial submission of the

    manuscript (Major concerns 1, 2, and 4; some of them also raised by the other reviewers) were not properly addressed by the authors. On several occasions, the authors recognize the importance of clarifying the points for the correct interpretation of the results but opt for leaving the open questions to be addressed during future studies. Therefore, the authors might consider adding a new section at the end of the manuscript to include all the caveats and future directions.

    In the current revision, in order to answer the reviewer #1’s original concerns 1, 2, and 4, we added several experimental data.

    Original major concerns 1) and 2): Regarding whether mice are detecting qualitative or quantitative differences between fresh and old cat saliva.

    To address these concerns, as shown in new Figure 1I and J, we measured volumes of saliva contained in in individual swabs and total protein concentrations at the time of behavior tests: Fresh (15 minutes after collection) and Old (4 hours after collection). The saliva volumes at the time of behavioral testing were indistinguishable between fresh and old samples (Figure 1I). In addition, the concentrations of total proteins in both fresh and old saliva were also indiscernible (Figure 1J). Furthermore, we also examined the difference of the amount of Fel d 4 protein, one of the most abundant proteins in cat saliva, between fresh and old saliva by conducting western blotting analyses. As shown in new Supplemental Figure 2, the amount of Fel d 4 was nearly equivalent between fresh and old saliva. Indeed, our analyses using recombinant Fel d 4 protein showed that Fel d 4 does not induce freezing behavior (Supplemental Figure 5). Based on these findings, we believe that the difference between fresh and old cat saliva lies in specific components rather than the total or major saliva content. One possible explanation for this difference is the time-dependent reduction of specific freezing-inducing components in old saliva.

    To investigate such a possibility, we also examined mouse behavior directed toward swabs containing diluted fresh cat saliva. Indeed, exposure to diluted fresh saliva resulted in a shorter duration of freezing behavior. Fresh saliva diluted to 70% induced freezing behavior for a duration equivalent to that of undiluted fresh saliva, while freezing behavior in response to 50% and 30% fresh saliva was significantly reduced to the same duration as that observed with old saliva (Figure 1K). The duration of direct interaction with swabs containing 70% and 50–30% fresh saliva also exhibited a similar trend to that observed with fresh and old saliva swabs, respectively (Figure 1L).

    These new results provide compelling evidence that the differential freezing response of mice to fresh versus old cat saliva is not attributed to quantitative differences, such as total volume, total protein concentration, or the amount of major proteins like Fel d 4. However, when fresh saliva was diluted, we observed a corresponding reduction in freezing behavior, suggesting that specific components within the saliva—those responsible for inducing freezing—may decrease over time.

    Our findings indicate that while the overall content of saliva remains consistent over time, specific freezing-inducing components seem to degrade or reduce at a different rate than other components, which alters the composition of saliva over time. The speed of reduction of these freezing-inducing components appears to be different from more stable proteins such as Fel d 4. As a result, the composition of saliva changes over time, leading to a qualitative difference between fresh and old saliva that mice can detect. This ability to discern such subtle chemical changes likely reflects an adaptive sensory mechanism, allowing mice to respond to predator cues to induce optimal defensive behavior in a certain context. Identifying the specific freezing-inducing components through traditional purification processes, such as high-performance liquid chromatography followed by behavioral examination (Haga-Yamanaka et al., 2014; Kimoto et al., 2005), is crucial for a deeper understanding of the mechanisms underlying the observed behavior. Our research team is actively working to isolate these molecules, and we hope to report our findings in future studies.

    (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.

    To address this issue, as shown in the new Figure 7, we performed a double exposure experiment using Fos2A-iCreERT2; Ai9 (TRAP2) mice (Allen et al., 2017; DeNardo et al., 2019). In this experiment, mice were exposed to the first stimulus under the treatment of 4-hydroxytamoxifen (4-OHT). One week after the initial exposure, the same mice were subjected to a second stimulus exposure for one hour. Through this paradigm, neurons activated by the first stimulus were visualized by tdTomato, while ones activated by the second stimulus were detected as cFos-IR (Figure 7A). Quantification of tdTomato and cFos-IR double-positive cells among tdTomato-labeled cells revealed that 43% (mean per animal: 61 / 143) of cells activated by fresh saliva during the first exposure were also activated by fresh saliva during the second exposure, whereas only 16% (17 / 106) of cells activated by old saliva during the first exposure were activated by fresh saliva during the second exposure (p = 7.5e-6, Chi-squared test). The difference in the fraction of overlapping cells between fresh and old saliva exposures was found significant when we compared the two groups of animals (Figure 7D, p = 0.0035, permutation test). Additionally, quantification of tdTomato and cFos-IR double-positive cells among cFos-IR cells indicated that over 27% (61 / 226) of cells activated by fresh saliva during the second exposure were previously activated by fresh saliva, whereas only 15% (17 / 112) of cells activated by fresh saliva during the second exposure were previously activated by old saliva (p = 0.015, Chi-squared test). The difference in the fraction of overlapping cells between fresh and old saliva exposures was also significant in this analysis (Figure 7E,p = 0.0060, permutation test). Together, these results demonstrate that fresh and old cat saliva activate largely different populations of neurons within the VMH. These new results were described on page 11 line 18 – page 12 line 8.

    In addition to these unaddressed concerns, some new issues have emerged in the new version of the manuscript. For example, the following paragraph introduced in the discussion section is not supported by the experimental findings.

    "We assume that such differential activations of the mitral cells between fresh and old saliva result in the differential activation of targeting neural substrates, possibly MeApv, which results in differential activation of VMH neurons (Figure 7)."

    Although the authors did not observe statistical differences in cFos expression in the pvMeA among groups, they claim that the differences in cFos expression in the VMH between fresh vs. old saliva are mediated by differential activation of upstream neurons in the MeApv. The lack of statistical differences may be caused by the reduced number of subjects in each group, as recognized in the text by the

    authors.

    We appreciate the reviewer's thoughtful comment. We agree that the paragraph in the comment, which presented a working hypothesis regarding differential activations of mitral cells and the MeApv between fresh and old saliva exposures, was speculative and not fully supported by our experimental findings. To address this, we have removed the assumptions related to the differential responses of mitral cells and the MeApv from the discussion and have updated the figure accordingly (now presented as new Figure 8).

    Moreover, the authors propose that in addition to fel d 4, multiple molecules present in the cat saliva can be inducing distinct defensive responses in the animals, but they do not provide any reference to support their claim.

    We thank the reviewer for highlighting this point. Our claim regarding the presence of other molecules in cat saliva inducing freezing defensive responses is based on our observation, as shown in the new Supplemental Figure 5, that recombinant Fel d 4 protein alone does not induce freezing behavior. This suggests the existence of other unidentified components in cat saliva that may contribute to freezing behavior. As we agree that identifying these specific freezing-inducing components is important for a more comprehensive understanding of the underlying mechanisms, our research team is actively working to isolate these molecules, and we hope to report our findings in future studies.

    Reviewer #2 (Public Review):

    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 the cat saliva.

    We thank the reviewer’s important insight on the need for further investigation into the molecular and neural mechanisms underlying the behavioral response to cat saliva. We recognize the importance of conducting studies involving V2R-A4 receptor knockouts and targeted functional manipulations within the VMH using neural circuit perturbation approaches.

    However, the V2R-A4 subfamily consists of 25 Vmn2r genes, most of which are closely grouped together, forming a V2R-A4 gene cluster within a 2.5-megabase chromosomal region. As we described in our recent review article (Rocha et al., 2024), the Vmn2r genes within the V2R-A4 subfamily display a high degree of homology, with nucleotide and amino acid identities among the several Vmn2rs surpassing 97-99%, suggesting possible redundancy among these receptor genes. This is in stark contrast to the diversity typically observed within other V2R subfamilies. Consequently, knockout strategies targeting a single receptor gene, which have been successful for other vomeronasal receptors, may not be effective for V2R-A4 receptor genes. The most appropriate strategy for examining the necessity of V2R-A4 receptors would be knocking out the entire V2R-A4 gene cluster, spanning a 2.5-megabase chromosomal region. Due to the technical challenges involved, addressing this issue is not feasible in the foreseeable future. Moreover, in our current study, we aimed to establish the foundational relationship between predator cues in cat saliva and defensive behaviors. We view our findings as an important first step that sets the stage for these more targeted and mechanistic studies involving the neural circuit perturbation experiments, such as optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), in the next step.

    Reviewer #3 (Public Review):

    Weaknesses:

    (1) It is unclear if fresh and old saliva indeed alter the perceived imminence of 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 amygdala, if these stimuli indeed correspond to significantly different levels of predation imminence.

    We appreciate the reviewer’s insightful comments regarding the perceived imminence of predation and the behavioral responses to fresh and old saliva. Our study specifically focused on comparing the defensive behaviors of mice in response to 15-minute-old and 4-hour-old cat saliva, particularly within the context of freezing behavior in their home cages. We chose these specific time points to capture the potential variation in behavioral intensity rather than the full spectrum of defensive behaviors. While a more comprehensive analysis—including varying time points, different types of defensive behaviors, and broader neural activation patterns (e.g., cortical versus amygdala activation)—might provide further insights into predation imminence theory, these aspects were beyond the scope of our current study. Future research could certainly address these points by examining behavioral and neural responses across additional saliva aging intervals and in varied behavioral contexts. Such studies would complement and extend the findings presented here, further elucidating the relationship between predator cue characteristics and defensive behaviors.

    (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.

    As the reviewer mentioned, the activation of the AOB, VNO, and VMH by predator odors has been established in prior studies. However, our study provides new insights by demonstrating that defensive freezing behavior in response to predator odors is mediated through the vomeronasal organ (VNO) sensory circuit, which has not been previously shown. The novelty of our work lies in two key findings: 1) the introduction of a new behavioral paradigm that assesses freezing responses to predator cues based on the freshness of chemosensory signals in cat saliva, and 2) the demonstration that the vomeronasal sensory circuit mediates defensive freezing behavior in response to cat saliva.

    Additionally, our results show that cat saliva of different freshness levels differentially activates VNO sensory neurons that express the same subfamily of sensory receptors. This differential activation subsequently modulates the downstream neural circuits, leading to varied freezing behavioral outcomes. We believe these findings provide a novel conceptual advance over previous studies by elucidating a more detailed mechanism of how predator-derived cues influence defensive behaviors through the accessory olfactory system.

    (3) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections, significantly decreasing the mechanistic insights generated by this work

    We thank the reviewer for this valuable comment. Investigating the behavioral effects of manipulating specific cell types and axonal projections, as well as characterizing circuit connectivity, is essential for a more comprehensive understanding of the underlying neural circuits. These approaches, such as modulating neural activity in defined cell populations and dissecting circuit pathways, using optogenetics, DREADD, etc., would provide deeper mechanistic insights. In our current study, however, we aimed to establish the foundational relationship between predator cues in cat saliva and defensive behaviors. We view our findings as an important first step that sets the stage for these more targeted and mechanistic studies in the future.

    (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 appreciate the reviewer’s suggestion regarding the correlation analysis in Figure 5E. We assessed the normality of our data using both the Shapiro-Wilk and Kolmogorov-Smirnov tests, which confirmed that the dataset is parametric, justifying the use of a parametric correlation method in this context. However, we acknowledge the concern about the limited number of data points and the influence of potential outliers on the observed correlation. Increasing the sample size might provide a more robust assessment of correlation patterns and reduce the potential impact of any single data point. While this would be an important direction for future research, such as with larger sample sizes, it is beyond the scope of the current study.

    (5) 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 16 line 10 in Discussion under “Differential activation of VMH neurons potentially underlying distinct intensities of freezing behavior.”

    (6) 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.

    (7) Some of the findings are disconnected from the story. For example, the authors show 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.”

    (8) 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.

    (9) 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, average distance to odor stimulus, velocity of movements towards and away 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).

    Reviewer #3 (Recommendations For The Authors):

    The authors have partially addressed several important points raised in the prior review, increasing the strength of the manuscript. However, 2 key questions already raised previously, were not addressed:

    (1) Is old saliva qualitatively different from new saliva, or is it the same as a smaller amount of new saliva? As Reviewer 1 wrote: "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."

    Since one of the author's main points is that fresh and old saliva elicit different perceived threat imminences, it is crucial to show that these two stimuli are somehow qualitatively different.

    One way to investigate this could be to show that animals perform different behaviors when exposed to smaller among of new saliva vs old saliva, or that the cfos activation patterns are different in these two conditions.

    The answers to these concerns are provided in the Public review Comment from Reviewer #1.

    (2) The other key question is if different VMH populations are activated by new vs old saliva.

    The answer to this concern is provided in the Public Review comment from Reviewer #1.

    Lastly, although the new analysis and text changes improved the manuscript, many issues raised were addressed with some variation of 'future studies will be done', or 'we concur with the Reviewer'. However, the extra experiments required to answer these questions were not done. For this reason, even though the authors have numerous exciting pieces of data, overall the work is still incomplete. I highlight below some examples in which the authors agree with the Reviewer, but do not answer the question with the new work that would be required, or propose to do the work in future studies.

    In this revised manuscript, we have conducted several additional experiments to address key concerns raised by the reviewers that are directly relevant to our claims. Specifically, we have examined: 1) whether qualitative or quantitative differences between fresh and old cat saliva are detected by mice to modulate behavior (NEW Figure 1I, J, K, and L, and NEW Supplemental Figure 2); 2) the involvement of Fel d 4 in freezing behavior (NEW Supplemental Figure 5); and 3) whether different VMH populations are activated by fresh versus old saliva (NEW Figure 7). However, some concerns raised by the reviewers fall outside the scope of the current manuscript. These include: 1) identifying the specific components that induce freezing, 2) examining the necessity of V2R-A4 receptors, 3) conducting neural circuit perturbations, and 4) performing a comprehensive analysis—including varying time points, different types of defensive behaviors, and broader neural activation patterns (e.g., cortical versus amygdala activation)—of the mouse’s defensive response to different levels of predator threat imminence. As these aspects are beyond the focus of our current manuscript, we have noted in the Public Review comments.

    References:

    Allen WE, DeNardo LA, Chen MZ, Liu CD, Loh KM, Fenno LE, Ramakrishnan C, Deisseroth K, Luo L. 2017. Thirst-associated preoptic neurons encode an aversive motivational drive. Science 357:1149– 1155.

    DeNardo LA, Liu CD, Allen WE, Adams EL, Friedmann D, Fu L, Guenthner CJ, Tessier-Lavigne M, Luo L. 2019. Temporal evolution of cortical ensembles promoting remote memory retrieval. Nat Neurosci 22:460–469.

    Haga-Yamanaka S, Ma L, He J, Qiu Q, Lavis LD, Looger LL, Yu CR. 2014. Integrated action of pheromone signals in promoting courtship behavior in male mice. Elife 3:e03025.

    Kimoto H, Haga S, Sato K, Touhara K. 2005. Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons. Nature 437:898–901.

    Rocha A, Nguyen QAT, Haga-Yamanaka S. 2024. Type 2 vomeronasal receptor-A4 subfamily: Potential predator sensors in mice. Genesis 62:e23597.

  8. Version published to 10.7554/elife.92982.3 on eLife
  9. Version published to 10.1101/2023.09.27.559655v3 on bioRxiv
  10. Version published to 10.7554/elife.92982.2 on eLife
  11. eLife

    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.

    References

    Bansal R, Nagel M, Stopkova R, Sofer Y, Kimchi T, Stopka P, Spehr M, Ben-Shaul Y. 2021. Do all mice smell the same? Chemosensory cues from inbred and wild mouse strains elicit stereotypic sensory representations in the accessory olfactory bulb. BMC Biol 19:133.

    Ben-Shaul Y, Katz LC, Mooney R, Dulac C. 2010. In vivo vomeronasal stimulation reveals sensory encoding of conspeciic and allospeciic cues by the mouse accessory olfactory bulb. Proc Natl Acad Sci U S A 107:5172‒5177.

    Bergan JF, Ben-Shaul Y, Dulac C. 2014. Sex-speciic processing of social cues in the medial amygdala. Elife 3:e02743.

    Engelke DS, Zhang XO, OʼMalley JJ, Fernandez-Leon JA, Li S, Kirouac GJ, Beierlein M, Do-Monte FH. 2021. A hypothalamic-thalamostriatal circuit that controls approachavoidance conlict in rats. Nat Commun 12:2517.

    Esteban Masferrer M, Silva BA, Nomoto K, Lima SQ, Gross CT. 2020. Differential Encoding of Predator Fear in the Ventromedial Hypothalamus and Periaqueductal Grey. J Neurosci 40:9283‒9292.

    Papes F, Logan DW, Stowers L. 2010. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell 141:692‒703.

    Tobias BC, Schuette PJ, Maesta-Pereira S, Torossian A, Wang W, Sethi E, Adhikari A. 2023. Characterization of ventromedial hypothalamus activity during exposure to innate and conditioned threats. Eur J Neurosci 57:1053‒1067.

  12. eLife

    eLife assessment

    This valuable study addresses one way in which animals identify predator-associated cues and respond in a manner that reflects the imminence of the potential threat. The report shows that, in mice, fresh saliva from a natural predator (cat) elicits a greater defensive response compared to old cat saliva and implicates the vomeronasal organ and ventromedial hypothalamus as part of a circuit that underlies this process. While the study has potential, the results are somewhat preliminary, and as such support for the primary conclusions is incomplete.

  13. eLife

    Reviewer #1 (Public Review):

    Summary:

    Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

    The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

    Major Concerns:

    (1) An important point that the authors should clarify in this study is whether mice are detecting qualitative or quantitative differences between the fresh and old cat saliva. Do the environmental conditions in which the old saliva was maintained cause a 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.

    (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.

    (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.

    (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).

    (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.

    (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.

    (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.

    (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.

    UNADDRESSED AND ADDITIONAL CONCERNS (RE-SUBMISSION)

    In this revised version of the manuscript, the authors have made important modifications in the text, inserted new references, and incorporated additional quantifications of cFos immunolabeling in three brain regions, as recommended by the reviewers. While these modifications have significantly improved the quality of the manuscript, other critical concerns raised during the initial submission of the manuscript (Major concerns 1, 2, and 4; some of them also raised by the other reviewers) were not properly addressed by the authors. On several occasions, the authors recognize the importance of clarifying the points for the correct interpretation of the results but opt for leaving the open questions to be addressed during future studies. Therefore, the authors might consider adding a new section at the end of the manuscript to include all the caveats and future directions.

    In addition to these unaddressed concerns, some new issues have emerged in the new version of the manuscript. For example, the following paragraph introduced in the discussion section is not supported by the experimental findings.

    "We assume that such differential activations of the mitral cells between fresh and old saliva result in the differential activation of targeting neural substrates, possibly MeApv, which results in differential activation of VMH neurons (Figure 7)."

    Although the authors did not observe statistical differences in cFos expression in the pvMeA among groups, they claim that the differences in cFos expression in the VMH between fresh vs. old saliva are mediated by differential activation of upstream neurons in the MeApv. The lack of statistical differences may be caused by the reduced number of subjects in each group, as recognized in the text by the authors. Moreover, the authors propose that in addition to fel d 4, multiple molecules present in the cat saliva can be inducing distinct defensive responses in the animals, but they do not provide any reference to support their claim.

  14. eLife

    Reviewer #2 (Public Review):

    In this study, Nguyen et al. showed that cat saliva can robustly induce freezing behavior in mice. This effect is mediated through the accessory olfactory system as it requires physical contact and is abolished in Trp2 KO mice. The authors further showed that V2R-A4 cluster is responsive to cat saliva. Lastly, they demonstrated c-Fos induction in AOB and VMHdm/c by the cat saliva. The c-Fos level in the VMHdm/c is correlated with the freezing response.

    Strength:

    The study opens an interesting direction. It reveals the potential neural circuit for detecting cat saliva and driving defense behavior in mice. The behavior results and the critical role of the accessory olfactory system in detecting cat saliva are clear and convincing.

    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 the cat saliva.

  15. eLife

    Reviewer #3 (Public Review):

    Summary:

    Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also show that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. Though interesting, the findings are not all integrated into a single narrative, and some of the results are only replications of earlier findings using modern methods. Overall, these findings provide incremental advance.

    Strengths:

    (1) Predator saliva is a stimulus of high ethological relevance

    (2) The authors performed a careful quantification of fos induction across the anterior-posterior axis in figure 6

    Weaknesses:

    (1) It is unclear if fresh and old saliva indeed alter the perceived imminence of 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 amygdala, if these stimuli indeed correspond to significantly different levels of predation imminence.

    (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.

    (3) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections, significantly decreasing the mechanistic insights generated by this work

    (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.

    (5) 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/

    (6) Add complete statistical information in the figure legends of all figures, which should include n, name of test used and exact p values.

    (7) Some of the findings are disconnected from the story. For example, the authors show 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?

    (8) Please paste all figure legends directly below their corresponding figure to make the manuscript easier to read

    (9) 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, average distance to odor stimulus, velocity of movements towards and away the odor stimulus, etc?

  16. Version published to 10.1101/2023.09.27.559655v2 on bioRxiv
  17. Version published to 10.7554/elife.92982.1 on eLife
  18. eLife

    eLife assessment

    This valuable study shows that, in mice, fresh cat saliva elicits a greater defensive response compared to old cat saliva. Additionally, the authors implicate the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) as part of a circuit that underlies this process. While the study has potential, the results are somewhat preliminary, and as such the evidence presented is incomplete.

  19. eLife

    Reviewer #1 (Public Review):

    Summary:
    Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

    The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

    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.

    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.

    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.

    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).

    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.

    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.

    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.

    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.

  20. eLife

    Reviewer #2 (Public Review):

    In this study, Nguyen et al. showed that cat saliva can robustly induce freezing behavior in mice. This effect is mediated through the accessory olfactory system as it requires physical contact and is abolished in Trp2 KO mice. The authors further showed that V2R-A4 cluster is responsive to cat saliva. Lastly, they demonstrated c-Fos induction in AOB and VMHdm/c by the cat saliva. The c-Fos level in the VMHdm/c is correlated with the freezing response.

    Strength:
    The study opens an interesting direction. It reveals the potential neural circuit for detecting cat saliva and driving defense behavior in mice. The behavior results and the critical role of the accessory olfactory system in detecting cat saliva are clear and convincing.

    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.

  21. eLife

    Reviewer #3 (Public Review):

    Summary:
    Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also show that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. Though interesting, the findings are not all integrated into a single narrative, and some of the results are only replications of earlier findings using modern methods. Overall, these findings provide incremental advance.

    Strengths:
    1 Predator saliva is a stimulus of high ethological relevance
    2 The authors performed a careful quantification of fos induction across the anterior-posterior axis in Figure 6.

    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.

    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.

    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.

    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.

    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?

    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?

  22. Version published to 10.1101/2023.09.27.559655v1 on bioRxiv