Rank- and Threat-Dependent Social Modulation of Innate Defensive Behaviors

Reviewer #1 (Public review):

Summary:

This study presents an interesting behavioral paradigm and reveals interactive effects of social hierarchy and threat type on defensive behaviors. However, addressing the aforementioned points regarding methodological detail, rigor in behavioral classification, depth of result interpretation, and focus of the discussion is essential to strengthen the reliability and impact of the conclusions in a revised manuscript.

Strengths:

The paper is logically sound, featuring detailed classification and analysis of behaviors, with a focus on behavioral categories and transitions, thereby establishing a relatively robust research framework.

Weaknesses:

Several points require clarification or further revision.

(1) Methods and Terminology Regarding Social Hierarchy:

The study uses the tube test to determine subordinate status, but the methodological description is quite brief. Please provide a more detailed account of the experimental procedure and the criteria used for determination.

The dominance hierarchy is established based on pairs of mice. However, the use of terms like "group cohesion" - typically applied to larger groups - to describe dyadic interactions seems overstated. Please revise the terminology to more accurately reflect the pairwise experimental setup.

(2) Criteria and Validity of Behavioral Classification:

The criteria for classifying mouse behaviors (e.g., passive defense, active defense) are not sufficiently clear. Please explicitly state the operational definitions and distinguishing features for each behavioral category.

How was the meaningfulness and distinctness of these behavioral categories ensured to avoid overlap? For instance, based on Figure 3E, is "active defense" synonymous with "investigative defense," involving movement to the near region followed by return to the far region? This requires clearer delineation.

The current analysis focuses on a few core behaviors, while other recorded behaviors appear less relevant. Please clarify the principles for selecting or categorizing all recorded behaviors.

(3) Interpretation of Key Findings and Mechanistic Insights:

Looming exposure increased the proportion of proactive bouts in the dominant zone but decreased it in the subordinate zone (Figure 4G), with a similar trend during rat exposure. Please provide a potential explanation for this consistent pattern. Does this consistency arise from shared neural mechanisms, or do different behavioral strategies converge to produce similar outputs under both threats?

(4) Support for Claims and Study Limitations:

The manuscript states that this work addresses a gap by showing defensive responses are jointly shaped by threat type and social rank, emphasizing survival-critical behaviors over fear or stress alone. However, it is possible that the behavioral differences stem from varying degrees of danger perception rather than purely strategic choices. This warrants a clear description and a deeper discussion to address this possibility.

The Discussion section proposes numerous brain regions potentially involved in fear and social regulation. As this is a behavioral study, the extensive speculation on specific neural circuitry involvement, without supporting neuroscience data, appears insufficiently grounded and somewhat vague. It is recommended to focus the discussion more on the implications of the behavioral findings themselves or to explicitly frame these neural hypotheses as directions for future research.

Reviewer #2 (Public review):

Summary:

The authors investigate how dominance hierarchy shapes defensive strategies in mice under two naturalistic threats: a transient visual looming stimulus and a sustained live rat. By comparing single versus paired testing, they report that social presence attenuates fear and that dominant and subordinate mice exhibit different patterns of defensive and social behaviors depending on threat type. The work provides a rich behavioral dataset and a potentially useful framework for studying hierarchical modulation of innate fear.

Strengths:

(1) The study uses two ecologically meaningful threat paradigms, allowing comparison across transient and sustained threat contexts.

(2) Behavioral quantification is detailed, with manual annotation of multiple behavior types and transition-matrix level analysis.

(3) The comparison of dominant versus subordinate pairs is novel in the context of innate fear.

(4) The manuscript is well-organized and clearly written.

(5) Figures are visually informative and support major claims.

Weaknesses:

Lack of neural mechanism insights.

Reviewer #3 (Public review):

Summary:

This study examines how dominance hierarchy influences innate defensive behaviors in pair-housed male mice exposed to two types of naturalistic threats: a transient looming stimulus and a sustained live rat. The authors show that social presence reduces fear-related behaviors and promotes active defense, with dominant mice benefiting more prominently. They also demonstrate that threat exposure reinforces social roles and increases group cohesion. The work highlights the bidirectional interaction between social structure and defensive behavior.

Strengths:

This study makes a valuable contribution to behavioral neuroscience through its well-designed examination of socially modulated fear. A key strength is the use of two ethologically relevant threat paradigms - a transient looming stimulus and a sustained live predator, enabling a nuanced comparison of defensive behaviors. The experimental design is robust, systematically comparing animals tested alone versus with their cage mate to cleanly isolate social effects. The behavioral analysis is sophisticated, employing detailed transition maps that reveal how social context reshapes behavioral sequences, going beyond simple duration measurements. The finding that social modulation is rank-dependent adds significant depth, linking social hierarchy to adaptive defense strategies. Furthermore, the demonstration that threat exposure reciprocally enhances social cohesion provides a compelling systems-level perspective. Together, these elements establish a strong behavioral framework for future investigations into the neural circuits underlying socially modulated innate fear.

Weaknesses:

The study exhibits several limitations. The neural mechanism proposed is speculative, as the study provides no causal evidence.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study presents an interesting behavioral paradigm and reveals interactive effects of social hierarchy and threat type on defensive behaviors. However, addressing the aforementioned points regarding methodological detail, rigor in behavioral classification, depth of result interpretation, and focus of the discussion is essential to strengthen the reliability and impact of the conclusions in a revised manuscript.

Strengths:

The paper is logically sound, featuring detailed classification and analysis of behaviors, with a focus on behavioral categories and transitions, thereby establishing a relatively robust research framework.

Weaknesses:

Several points require clarification or further revision.

(1) Methods and Terminology Regarding Social Hierarchy:

The study uses the tube test to determine subordinate status, but the methodological description is quite brief. Please provide a more detailed account of the experimental procedure and the criteria used for determination.

We will add more details about how the tube test was performed in the revised manuscript.

The dominance hierarchy is established based on pairs of mice. However, the use of terms like "group cohesion" - typically applied to larger groups - to describe dyadic interactions seems overstated. Please revise the terminology to more accurately reflect the pairwise experimental setup.

Thanks for the comment. We agree that the term “group cohesion” can be misleading and will replace it with “social engagement”.

(2) Criteria and Validity of Behavioral Classification:

The criteria for classifying mouse behaviors (e.g., passive defense, active defense) are not sufficiently clear. Please explicitly state the operational definitions and distinguishing features for each behavioral category.

Passive defense was defined as an immobility-based defensive strategy characterized by suppression of locomotor activity. This category included freezing and tail rattling, which in our study involved minimal body displacement aside from rapid tail vibration. Active defense was defined as movement- or posture-dependent defensive strategy, encompassing behaviors that involved locomotor engagement or spatial repositioning relative to the threat, including approach, investigation, withdrawal, and stretch-attend. We will clarify this in the revised manuscript.

How was the meaningfulness and distinctness of these behavioral categories ensured to avoid overlap? For instance, based on Figure 3E, is "active defense" synonymous with "investigative defense," involving movement to the near region followed by return to the far region? This requires clearer delineation.

Defensive behaviors in the rat exposure paradigm were grouped into two categories: passive and active defense, each comprising distinct behaviors. All the manually annotated behaviors were mutually exclusive; that is, each video frame was assigned a single behavioral label to avoid overlap across behaviors. Active defense includes four behaviors: approach, investigation, withdrawal, and stretch-attend. We will clarify these points in the revised manuscript.

The current analysis focuses on a few core behaviors, while other recorded behaviors appear less relevant. Please clarify the principles for selecting or categorizing all recorded behaviors.

Thank you for pointing this out. In the current study, we focused primarily on defensive and social behaviors. We also included several neutral solitary behaviors related to anxiety and defensive state, such as sniffing, grooming, and rearing, which were consistently expressed across animals and closely linked to our main findings. We will clarify this rationale in the revised manuscript.

(3) Interpretation of Key Findings and Mechanistic Insights:

Looming exposure increased the proportion of proactive bouts in the dominant zone but decreased it in the subordinate zone (Figure 4G), with a similar trend during rat exposure. Please provide a potential explanation for this consistent pattern. Does this consistency arise from shared neural mechanisms, or do different behavioral strategies converge to produce similar outputs under both threats?

Thanks for bringing up this important question. The consistent increase in proactive bouts in dominant mice across both paradigms suggests a consistent rank-dependent reorganization of dyadic interaction under threats. We propose that this convergence reflects a shared neural mechanism that links defensive state with social-rank information, potentially mediated by overlapping hypothalamic and prefrontal circuits. We will expand the Discussion to incorporate this explanation.

(4) Support for Claims and Study Limitations:

The manuscript states that this work addresses a gap by showing defensive responses are jointly shaped by threat type and social rank, emphasizing survival-critical behaviors over fear or stress alone. However, it is possible that the behavioral differences stem from varying degrees of danger perception rather than purely strategic choices. This warrants a clear description and a deeper discussion to address this possibility.

We thank the reviewer for this insightful comment. We agree that, in principle, behavioral differences could arise from variations in perceived danger rather than strategic choice. In humans, decisions can sometimes reflect value-based strategies that override perceived danger. In contrast, under naturalistic threat conditions, mice likely rely predominantly on danger perception to make behavioral decisions, and such responses are expected to be consistent with value-based strategies shaped by natural selection. In the revised manuscript, we will expand the Discussion to address the role of threat perception and its relationship to decision-making in our behavioral paradigms.

The Discussion section proposes numerous brain regions potentially involved in fear and social regulation. As this is a behavioral study, the extensive speculation on specific neural circuitry involvement, without supporting neuroscience data, appears insufficiently grounded and somewhat vague. It is recommended to focus the discussion more on the implications of the behavioral findings themselves or to explicitly frame these neural hypotheses as directions for future research.

We will revise the Discussion to focus more directly on behavioral findings and add explicit neural hypotheses as potential future directions.

Reviewer #2 (Public review):

Summary:

The authors investigate how dominance hierarchy shapes defensive strategies in mice under two naturalistic threats: a transient visual looming stimulus and a sustained live rat. By comparing single versus paired testing, they report that social presence attenuates fear and that dominant and subordinate mice exhibit different patterns of defensive and social behaviors depending on threat type. The work provides a rich behavioral dataset and a potentially useful framework for studying hierarchical modulation of innate fear.

Strengths:

(1) The study uses two ecologically meaningful threat paradigms, allowing comparison across transient and sustained threat contexts.

(2) Behavioral quantification is detailed, with manual annotation of multiple behavior types and transition-matrix level analysis.

(3) The comparison of dominant versus subordinate pairs is novel in the context of innate fear.

(4) The manuscript is well-organized and clearly written.

(5) Figures are visually informative and support major claims.

Weaknesses:

Lack of neural mechanism insights.

The current study focused on behavior. In the revised manuscript, we will incorporate a discussion of potential neural mechanisms and highlight this as an important direction for future work.

Reviewer #3 (Public review):

Summary:

This study examines how dominance hierarchy influences innate defensive behaviors in pair-housed male mice exposed to two types of naturalistic threats: a transient looming stimulus and a sustained live rat. The authors show that social presence reduces fear-related behaviors and promotes active defense, with dominant mice benefiting more prominently. They also demonstrate that threat exposure reinforces social roles and increases group cohesion. The work highlights the bidirectional interaction between social structure and defensive behavior.

Strengths:

This study makes a valuable contribution to behavioral neuroscience through its well-designed examination of socially modulated fear. A key strength is the use of two ethologically relevant threat paradigms - a transient looming stimulus and a sustained live predator, enabling a nuanced comparison of defensive behaviors. The experimental design is robust, systematically comparing animals tested alone versus with their cage mate to cleanly isolate social effects. The behavioral analysis is sophisticated, employing detailed transition maps that reveal how social context reshapes behavioral sequences, going beyond simple duration measurements. The finding that social modulation is rank-dependent adds significant depth, linking social hierarchy to adaptive defense strategies. Furthermore, the demonstration that threat exposure reciprocally enhances social cohesion provides a compelling systems-level perspective. Together, these elements establish a strong behavioral framework for future investigations into the neural circuits underlying socially modulated innate fear.

Weaknesses:

The study exhibits several limitations. The neural mechanism proposed is speculative, as the study provides no causal evidence.

Establishing causal evidence for neural mechanisms is beyond the scope of the current behavioral study. We highlight this as an important direction for future work.