Author response:
The following is the authors’ response to the previous reviews
Public Reviews:
Reviewer #1 (Public review):
This study by Li and colleagues examines how defensive responses to visual threats during foraging are modulated by both reward level and social hierarchy. Using a naturalistic paradigm, the authors test how the availability of water or sucrose, with sucrose being more rewarding than water, shapes escape behavior in mice exposed to looming stimuli of different intensities, which are used to probe perceived threat level and defensive responses. In parallel, the study compares dominant and subordinate animals to assess how social rank biases the trade off between reward seeking and threat avoidance. By combining detailed behavioral analyses with computational modeling, the work addresses how reward level and social context jointly influence escape decisions in an ethologically relevant setting.
Across the different experimental conditions, perceived threat level is the main determinant of behavior. The authors show that looming stimuli associated with higher threat (contrast) consistently elicit faster and more robust escape responses than lower threat stimuli. This effect is particularly evident during early exposures, when animals are highly vigilant and have not yet habituated to the looming stimulus (learned that it is not dangerous). Later they described that as animals gain experience and habituate, behavior becomes more flexible, and reward level begins to exert a graded modulation of the escape response. Importantly, the authors show that under high threat conditions increasing reward value leads to more frequent and faster escape rather than greater reward pursuit. This finding is particularly relevant, as it suggests that highly valued rewards can heighten vigilance and thereby enhance responsiveness to threat, highlighting that reward does not simply compete with defensive behavior but can also reshape it depending on the perceived level of danger, in contrast to low threat conditions, where threat can be more easily outweighed by reward. Thus, an important conceptual contribution of the study is the introduction of vigilance as a useful framework to interpret these effects. Vigilance is treated as a behavioral state reflecting heightened attention to potential danger. In line with what is known from natural foraging, mice initially maintain high vigilance when confronted with an innate threat. This perspective helps clarify a finding that might otherwise appear counterintuitive. One might expect higher rewards to motivate animals to tolerate risk, explore more, and habituate faster in any scenario. Instead, the data suggest that highly rewarding outcomes can elevate vigilance, making animals more responsive to threat and leading to faster or more frequent escape under high threat conditions. In this sense, reward does not simply compete with threat but can also amplify sensitivity to it, depending on the internal state of the animal.
The social results are particularly interesting in this context as well. Dominant mice consistently prioritize avoidance over reward, showing stronger escape responses and slower habituation than subordinates. This behavior is well captured by the vigilance framework proposed by the authors: dominant animals appear to maintain higher vigilance, which biases decisions toward threat avoidance. The authors further suggest that stable social relationships sustain high vigilance and slow habituation, framing this as an evolutionarily conserved strategy that may enhance survival. This interpretation provides a valuable perspective on how social structure shapes defensive behavior beyond immediate physical interactions. At the same time, there are important limitations to this interpretation. All experiments were conducted in male mice, and it is possible that the relationship between social hierarchy, vigilance, and defensive behavior would differ substantially in females. In addition, the idea that stable social relationships maintain elevated vigilance does not straightforwardly align with broader views of social stability as protective for mental health and as a buffer against anxiety and stress. These points do not undermine the findings but suggest that the social effects described here should be interpreted with caution and within the specific context of the task and sex studied.
We thank the reviewer for raising this important point. In the context of repeated looming exposure, slower habituation reflects more sustained vigilance over time. Compared to individually housed mice, group-housed mice exhibit slower habituation (Lenz et al., 2022), and pair-housed mice showed even slower habituation in our current work. Importantly, this pattern does not indicate that pair-housed mice have higher overall vigilance than individually housed animals. Although individually housed mice habituate more quickly, they display higher initial vigilance, as reflected by their increased probability of escaping in response to looming stimuli (Lenz et al., 2022). Thus, pairhoused mice exhibited reduced defensive responses compared to individually housed animals, consistent with a social buffering effect.
Furthermore, in a separate study (Rank- and Threat-Dependent Social Modulation of Innate Defensive Behaviors; Li, Gao, Li, 2026, eLife 15:RP109571), we directly compared responses to looming stimuli when mice were tested alone versus in the presence of a social partner and observed clear evidence of social buffering.
Another important limitation is that the neural mechanisms underlying these effects remain speculative. The manuscript includes an extensive discussion of candidate circuits, particularly involving the superior colliculus and downstream structures, but this section is necessarily based on prior literature rather than on data presented in the study. Given the complexity of the circuits involved in integrating internal state, reward, social context, and vigilance, the current work should be viewed as providing a strong behavioral and conceptual framework rather than direct insight into underlying neural mechanisms.
We fully agree that the proposed neural mechanisms remain speculative and that the circuits involved in integrating internal state, reward, and social context are likely far more complex. We have revised the manuscript to acknowledge this limitation.
Methodologically, the behavioral paradigm is well suited for studying escape decisions in socially housed animals, and the machine learning based classification of defensive responses is a clear strength. The computational model provides a useful formalization of how threat level, reward level, and vigilance interact and may be valuable for other laboratories studying escape, approach avoidance, or conflict situations, particularly as a way to classify behavioral outcomes after pose estimation. More generally, the work will be of interest to the neuroethology community for its detailed characterization of escape behavior under naturalistic conditions.
Given the ethological nature of the study and the high inter individual variability reported by the authors, clarity and precision in the methods are especially important for reproducibility. While the revised manuscript addresses many earlier concerns, some aspects remain slightly difficult to follow. For example, the main text states that animals were not water deprived to avoid differences in internal state, whereas parts of the methods describe conditions in which animals were water deprived, suggesting that internal state manipulation may differ across experiments. Clearer separation and explanation of these conditions would further strengthen confidence in the work.
To improve clarity, we have revised the Methods section to clearly distinguish between experimental conditions that involved water deprivation and those that did not.
Overall, this study provides a rich and thoughtful analysis of how reward level and social hierarchy modulate defensive behavior through changes in vigilance. It offers a useful conceptual advance for thinking about escape behavior in naturalistic settings and lays a solid foundation for future work aimed at linking these behavioral states to underlying neural circuits.
Reviewer #2 (Public review):
Zhe Li and colleagues investigate how mice exposed to visual threats and rewards balance their decisions in favour of consuming rewards or engaging in defensive actions. By varying threat intensity and reward value, they first confirm previous findings showing that defensive responses increase with threat intensity and that there is habituation to the threat stimulus. They then find that water-deprived mice have a reduced probability of escaping from low contrast visual looming stimuli when water or sucrose are offered in the environment, but that when the stimulus contrast is high, the presence of sucrose or water increases the probability of escape. By analysing behaviour metrics such as the latency to flee from the threat stimulus, they suggest that this increase in threat sensitivity is due to increased vigilance. Analysis of this behaviour as a function of social hierarchy shows that dominant mice have higher threat sensitivity, which is also interpreted as being due to increased vigilance. These results are captured by a drift diffusion model variant that incorporates threat intensity and reward value.
The main contribution of this work is quantifying how the presence of water or sucrose in water-deprived mice affects escape behaviour. The differential effects of reward between the low and high contrast conditions are intriguing, but I find the interpretation that vigilance plays a major in this process not supported by the data. The idea that reward value exerts some form of graded modulation of the escape response is also not supported by the data. In addition, there is very limited methodological information, which makes assessing the quality of some of the analyses difficult, and there is no quantification on the quality of the model fits.
(1) The main measure of vigilance in this work is reaction time. While reaction time can indeed be affected by vigilance, reaction times can vary as a function of many variables, and be different for the same level of vigilance. For example, a primate performing the random dot motion task exhibits differences in reaction times that can be explained entirely by the stimulus strength. Reaction time is therefore not a sound measure of vigilance, and if a goal of this work is to investigate this parameter, then it should be measured. There is some attempt at doing this for a subset of the data in Figure 3H, by looking at differences in the action of monitoring the visual field (presumably a rearing motion, though this is not described) between the first and second trials in the presence of sucrose. I find this an extremely contrived measure. What is the rationale for analysing only the difference between the first and second trials? Also, the results are only statistically significant because the first trial in the sucrose condition happens to have zero up action bouts, in contrast to all other conditions. I am afraid that the statistics are not solid here. When analysing the effects of dominance, a vigilance metric is the time spent in the reward zone. Why is this a measure of vigilance? More generally, measuring vigilance of threats in mice requires monitoring the position of the eyes, which previous work has shown is biased to the upper visual field, consistent with the threat ecology of rodents.
(2) In both low and high contrast conditions, there are differences in escape behaviour between no reward and water or sucrose presence, but no statistically significant differences between water and sucrose (eg: Figure 3B). I therefore find that statements about reward value are not supported by the data, which only show differences between the presence or absence of reward. Furthermore, there is a confound in these experiments, because according to the methods, mice in the no-reward condition were not water-deprived. It is thus possible that the differences in behaviour arise from differences in the underlying state.
(3) There is very little methodological information on behavioural quantification. For example, what is hiding latency?
Is this the same are reaction time? Time to reach the safe zone? What exactly is distance fled? I don't understand how this can vary between 20 and 100cm. Presumably, the 20cm flights don't reach the safe place, since the threat is roughly at the same location for each trial? How is the end of a flight determined? How is duration measured in reward zone measures, e.g., from when to when? How is fleeing onset determined?
(4) There is little methodological information on how the model was fit (for example, it is surprising that in the no reward condition, the r parameter is exactly 0. What this constrained in any way), and none of the fit parameters have uncertainty measures so it is not possible to assess whether there are actually any differences in parameters that are statistically significant.
These are the public reviews for the original submission. The corresponding authors responses are provided below.
(1) We agree that reaction time can be influenced by multiple factors, including stimulus strength. Consistent with this, reaction times (i.e. latencies to flee) were substantially shorter under high-contrast conditions (Figure 3E). However, even under the same high-contrast condition, reaction times were significantly shorter in the water condition compared to the no-reward condition, suggesting that other factors such as vigilance may contribute.
Upward-directed attention includes rearing, up-stretching, and upward head orientation, which will be clarified in the Method section. To address concerns about statistical validity, we will quantify these behaviors across the first 10 trials rather than limiting the analysis to the first two.
As for the dominance-related results, we interpret them as reflecting both enhanced vigilance and reduced reward-seeking behavior. Time spent in the reward zone is not a measure of vigilance but an indicator of reward-seeking motivation. We will clarify this in the revised manuscript.
(2) In Figure 3B, the difference between water and sucrose conditions did not reach statistical significance (p = 0.08). We plan to collect additional data to determine whether this is due to limited statistical power. It is also possible that some behavioral readouts are more sensitive to the differences between water and sucrose conditions. For example, Figure 3F shows that escape speed was significantly higher in the sucrose than in the water condition under high-contrast stimulation.
Thank you for pointing this out. To control for the potential confounds related to internal state, mice were not water-deprived under any of the three conditions in Figures 3A-3H. We will clarify this in the main text and Methods. For Figures 3I-3M, which compare decision-making under no-reward and water conditions, we will conduct additional experiments using non-deprived mice in the water condition.
(3) Hiding latency was defined as the time from stimulus onset to the animal’s arrival at the safe zone. Reaction time was quantified as the latency to flee, measured from stimulus onset to the initiation of the first flight state. The flight state was defined as locomotion exceeding 10 cm at a speed greater than 10 cm/s. Distance fled was defined as the distance covered between stimulus onset and offset for all trials. However, in trials classified as no reaction or freezing, this measure does not accurately reflect escape behavior. We will therefore rename it as distance under threat to better capture its meaning. The reward zone was defined as the region within 15 cm of the reward port at the end of the arena. Duration in the reward zone was measured as the time spent within this region during the 20 seconds following stimulus onset. In Figure 4E, the percentage of time spent in the reward zone was calculated relative to the total time the mouse remained in the arena during the 2-hour social session.
All definitions and additional details on behavioral quantification will be included in the revised Methods section.
(4) We appreciate the comment and agree that further clarification is needed. We will provide a more detailed description of the model fitting procedure in the revised Methods section. Specifically, the drift rate parameter (r), which reflects the perceived reward value, was constrained to zero in the no-reward condition. To enable statistical comparison across conditions, we will report uncertainty measures for all fit parameters.
Comments on the revised manuscript:
The manuscript has been revised and improved significantly by the addition of methodological details and new analysis. I remain, however, unconvinced by the argument that increased vigilance in the presence of reward leads to heightened escape behaviour.
In response to my criticism that the work does not measure vigilance directly, the authors have included measures of foraging interval and foraging speed, which they state are "two direct behavioral analyses of vigilance". I disagree - like reaction time, foraging speed and foraging interval can be modulated, for example, by changes in threat sensitivity. Increased threat sensitivity comes with diverse behavioral changes that may well include increased vigilance, but foraging interval and foraging speed can certainly change without the animal expressing increased vigilance behaviors. A bigger issue I still have though, is with the conclusion that the presence of reward increases "direct escape behaviors". Comparing the no reward, water and sucrose groups indeed shows a difference (which is now clear after the split into early and late phases), but the issue is that these are different mice. As the text is written, is sounds like introducing reward will acutely increase escape. But if we look at the raw data show in Figure 2C, what I think is happening is that the presence of reward is decreasing habituation to the stimulus. The data for trials 1 and 10 in the three conditions show this - there is habituation with no reward (reaction times are all shifting to the right), a bit less with water and very little with sucrose. This is interesting in its own right and we can speculate why it might be happening, but I think this is conceptually different from what the authors are proposing.
We agree that vigilance is not directly observable as a single variable. Our intent was not to claim that foraging speed and foraging interval provide a direct measure of vigilance, but rather to suggest that they may serve as indirect behavioral correlates.
We also considered an alternative interpretation: these two measures could reflect perceived reward value under high-threat conditions across distinct reward types. If that were the case, animals would be expected to exhibit shorter intervals and faster speeds across no reward, water, and sucrose conditions. However, our data do not support this interpretation (Figures 3L and 3M), suggesting that these measures are more likely correlated with vigilance.
Furthermore, it is unlikely that changes in foraging interval and speed are driven by altered threat sensitivity, as animals could not see the threat during most of the foraging bout and only encountered it at the end.
Regarding the conclusion that the presence of reward increases direct escape behaviors, our interpretation is that increased reward value reduces habituation, thereby maintaining higher vigilance during the late phase. This was discussed in the second-to-last paragraph of the "Economic and social modulations of innate decision-making under threat" subsection in the Discussion.
Reviewer #3 (Public review):
Male mice were tested in a classic behavioral "flee the looming stimulus" paradigm. This is a purely behavioral study; no neural analyses were done. Mice were housed socially, but faced the looming stimulus individually, using an elegant automated tunnel (see videos for clarity).
The additional changes made to the paper clarify the work done. While there are some limitations (male mice, weird stimulus), the general results are interesting and a valuable addition to the experimental literature. The main claim of the paper is that the different rewards (none, water, sucrose) did not change the escape properties early in learning, but did late, particularly that in the late (already experienced) conditions, reward value (assuming sucrose > water > no reward) interacted with the salience of the looming stimulus (light gray, dark gray). (Panels 3D, 3G, 3K, 3N).
For readers, I want to note that one of the most interesting results is actually in Figure S2, where they find that a looming stimulus behind the mouse still makes a mouse run to the nest. In these conditions, the mouse runs past the looming stimulus to get to safety! (I also do love the video of the mouse running around the barriers like a snake to get home.)
I have a few minor clarification questions and a few notes that I think would be useful additions for authors and readers to think about.
Dominance: What does the mouse social science literature say about the "test tube" test? What can we conclude from this test? This would be useful when trying to understand what is causing the dominance/submissive difference in responses. Figure 4 shows that the dominant mice are more risk-averse than the submissive mice. Is "dominance" in the test-tube actually a measure of risk-seeking? Is the issue that the submissive mice don't think they can get back to the food-site easily, so they are less willing to sacrifice the current (if dangerous) foraging opportunity? Is the issue that the submissive mice can't get back to the nest? As I understand it, the nest was always available to all the mice, so I suspect inability to get to the nest is an unlikely hypotheses. Is the issue that the submissive mice also don't feel safe in the nest?
The tube test is a widely used assay in the rodent social behavior literature to assess dominance hierarchies, operationally defined by the ability of one animal to force its opponent to retreat from a narrow tube. Importantly, this assay does not directly measure risk-seeking or anxiety-related traits, but rather competitive outcomes during social conflict. Furthermore, our data indicate that the behavioral responses of subordinate mice to looming stimuli are primarily driven by the visual threat itself rather than by social avoidance. This point was elaborated in the second paragraph of the “Social modulation of innate decision-making” subsection in the Results section.
Limitations of the study: There is an acknowledged limitation to male mice, and the limitations of the small data sets that are typical of such experiments. In addition, however, it is also worth noting the strangeness of the looming stimulus, which is revealed clearly in the videos. The stimulus is a repeating growing circle, growing in a single location within the environment. The stimulus repeats 10 times, once per second. This is not what an attacking hawk or owl would look like. (I now have this image of an owl diving down, and then teleporting up and diving down again.) Note - I am fine with this stimulus. It produces an interesting experiment and interesting results. I do not think the authors need to change anything in their paper, but readers need to recognize that this is not a "looming predator".
These "limitations" are better seen as "caveats" when folding these results in with the rest of the literature that has gone before and the literature to come. (Generally, I do not believe that science works by studies making discoveries that change how we think about problems - instead, science works by studies adding to the literature that we integrate in with the rest of the literature.) Thus, these caveats should not be taken as problems with the study or as fixes that need to be done. Instead, they are notes for future researchers to notice if differences are found in any future studies.
Thus, my only suggestion is that I think authors could write a more careful paper by using the past and subjunctive tense appropriately. Experimental observations should be in past tense, as in "the influence of reward was contextdependent and emerged in the late phase" instead of "the influence of reward is context-dependent and emerges in the late phase" - it emerged in the late phase this once - it might not in future experiments, not due to any fault in this experiment nor due to replicability problems, but rather due to unexpected differences between this and those future experiments. At which point, it will be up to those future experiments to determine the difference. Similarly, large conclusions should be in the subjunctive tense, as in "these data suggest that threat intensity is likely to be the primary determinant of decision making" rather than "threat intensity is the primary determinant of decision making", because those are hypotheses not facts.
We thank the reviewer for the helpful suggestions and have revised the Abstract accordingly.
Recommendations for the authors:
Reviewer #3 (Recommendations for the authors):
Figure 5: The points in panel 5G and 5H are unreadable. What are these stars and symbols supposed to mean? They are also too small to see without zooming way in.
We have increased the symbol size.
Figure 5: What is the final panel of 5J? I did not understand this panel at all. The first three panels of 5J (threat-based detection, reward-based detection, vigilance-based detection) are, I believe, three patterns we should look for in the data. But then what is the "experimental results" section? It contains all three, but they don't overlap? Shouldn't we have an experimental results section for each condition?
Panel 5J was to compare three hypothesized decision patterns with the experimentally observed data. To make this distinction explicit, we have revised the panel titles to: “H1: Threat-based decisions,” “H2: Reward-based decisions,” “H3: Vigilance-based decisions,” and “Experimental results.”
Thank you for including the videos. They made the task construction and the stimulus much clearer.
