Individual differences drive social hierarchies in mouse societies

Reviewer #1 (Public review):

Summary:

The goal of the study was to address the question of the degree to which social position in a group is a stable trait that persists across conditions. Reinwald et al. use a custom-built cage system with automated tracking and continuous testing for social dominance that does not require intervention by the experimenter. Remixing of individuals from different groups revealed that social position was rather stable and not really predictable from other measures that were taken. The authors conclude that social position is multifaceted but dependent on characteristics like personality traits.

Strengths:

(1) Reductionistic, highly controlled setting that allows for the control of many confounding variables.

(2) Very interesting and important question.

(3) Confirms the emergence of inter-individual behavior-driven differences in inbred mice in a shared environment.

(4) Innovative paradigm and experimental setup.

(5) Fresh perspective on an old question that makes the best use of modern technology.

(6) Intelligent use of behavioral and cognitive covariables to generate a non-social context.

(7) Bold and almost provocative conclusion, inviting discussion and further elaboration.

Weaknesses:

(1) Reductionistic, highly controlled setting that blends out much of the complexity of social behavior in a community.

(2) The motivation to enter the test tube is not "trait" (or at least not solely a trait) but the basic need to reach food and water; chasing behavior would be less dependent on this stimulus.

(3) Dominance is only one aspect of sociality, social structure is reduced to rank. The information that might lie in the chasing behavior is not optimally used to explain social behavior beyond the rank measure.

(4) Focus on rank bears the risk of overgeneralization for readers not familiar with the context.

(5) Conclusion only valid for the reductionistic setting, in which environment, social and non-social changes only within narrow limits, and in which the mouse population does not face challenges

(6) Animals are not naive at the beginning of the experiment, but are already several weeks old.

In summary, this is a wonderful study, but not one that is easy to interpret. The bold conclusion is valid only within the constraints of the study, but nevertheless points in an important direction. The paradigm is clever and could be used for many interesting follow-ups.

To define social position as a personality trait will elicit strong opposition and much debate; the nuances of the paper might be lost on many readers and call for the (re)-consideration of many concepts that are touched. I find this attitude a strength of the paper, but the approach bears the risk of misunderstanding.

Reviewer #2 (Public review):

Summary:

This manuscript presents the "NoSeMaze", a novel automated platform for studying social behavior and cognitive performance in group-housed male mice. The authors report that mice form robust, transitive dominance hierarchies in this environment and that individual social rank remains largely stable across multiple group compositions. They further demonstrate that social dominance and aggressive behaviors, like chasing, are partially dissociable and that dominance traits are independent of non-social cognitive performance. The study includes a genetic manipulation of oxytocin receptor expression in the anterior olfactory nucleus, which showed only transient effects on social rank.

Strengths:

(1) Innovative Methodology:
The NoSeMaze platform is a technically elegant and conceptually well-integrated system that enables fully automated, long-term monitoring of both social and cognitive behaviors in large groups of group-housed mice. It combines tube-test-like dominance contests, voluntary chase-escape interactions, and an embedded operant olfactory discrimination task within a single, ethologically relevant environment. This modular design allows for high-throughput, minimally invasive behavioral assessment without the need for repeated handling or artificial isolation.

(2) Experimental Scale and Rigor:
The study includes 79 male mice and over 4,000 mouse-days of observation across multiple group reshufflings. The use of RFID-based identification, automated data logging, and longitudinal design enables robust quantification of individual trait stability and group-level social structure.

(3) Multidimensional Behavioral Profiling:
The integration of social (tube dominance, proactive chasing), physical (body weight), and cognitive (olfactory learning task) measures offers a rich, multi-dimensional profile of each individual mouse. The authors' finding that social dominance traits and non-social cognitive performance are largely uncorrelated reinforces emerging models of orthogonal behavioral trait axes or "animal personalities".

(4) Clarity and Data Analysis:
The analytical framework is well-suited to the study's complexity, with appropriate use of dominance metrics, mixed-effects models, and permutation tests. The analyses are clearly explained, statistically rigorous, and supported by transparent supplementary materials.

Weaknesses:

(1) Conceptual Novelty and Prior Work:
While the study is carefully executed and methodologically innovative, several of its core findings reaffirm concepts already established in the literature. The emergence of stable, transitive social hierarchies, the persistence of individual differences in social behavior, and the presence of non-despotic social structures have all been previously reported in mice, including under semi-naturalistic conditions (e.g., Fan et al., 2019; Forkosh et al., 2019). Although this work extends those findings with greater behavioral resolution and scale, the manuscript would benefit from a clearer articulation of what is genuinely novel at the conceptual level, beyond the technological advance.

(2) Role of OXTR Deletion:
The inclusion of the OXTR manipulation feels somewhat disconnected from the manuscript's central aims. The effects were minimal and transient, and the authors defer full interpretation to a separate study.

(3) Scope Limitations (Sex and Age):
The study is limited to male mice, and although this is acknowledged, the title and overall framing imply broader generalizability. This sex-specific focus represents a common but problematic bias. Additionally, results from the older mouse cohort are under-discussed; if age had no effect, this should be explicitly stated.

(4) Ambiguity of Dominance as a Construct:
While the study robustly quantifies social rank and hierarchy structure, the broader functional meaning of "dominance" remains unclear. As in prior work (e.g., Varholick et al., 2019), dominance rank here shows only weak associations with physical attributes (e.g., body weight), cognitive strategy, or neuromodulatory manipulation (OXTR deletion). This recurring pattern, where rank metrics are reliably established yet poorly predictive of other behavioral or biological traits, raises important questions about what such measures actually capture. In particular, it challenges the assumption that outcomes in paradigms like the tube test or chase frequency necessarily reflect dominance per se, rather than other constructs.

Reviewer #3 (Public review):

Reinwald et al. present the NoSeMaze, a semi-natural behavioral system designed to track social behaviors alongside reinforcement-learning in large groups of mice. Accumulating more than 4,000 days of behavioral monitoring, the authors demonstrate that social rank (determined by tube competitions) is a stable trait across shuffled cohorts and correlated with active chasing behaviors. The system also provides a solid platform for long-term measurements of reinforcement learning, including flexibility, response adaptation, and impulsiveness. Yet, the authors show that social ranking and chasing are mostly independent of these cognitive traits, and both seem mostly independent of oxytocin signaling in the AON.

Strengths:

(1) The neuroethological approach for automated tracking of several mice under semi-natural conditions is still rare in social behavioral research and should be encouraged.

(2) The assessment of dominance by two independent measures, i.e., spontaneous tube competitions and proactive chasing, is innovative and valuable.

(3) The integration of a long-term reinforcement-learning module into the semi-natural system provides novel opportunities to combine cognitive traits into social personality assessments.

(4) The open-source system provides a valuable resource for the scientific community.

Limitations:

(1) Apparent ambiguity and inconsistency in age structure and cohort participation across rounds, raising concerns about uncontrolled confounds.

(2) Chasing behavior appears more stable than tube-test competitions (Figure 4D vs. Figure 3D), which challenges the authors' decision to treat tube competitions as the primary basis for hierarchy determination.

Major concerns:

(1) Unclear and inconsistent handling of age groups and repeated sampling. The manuscript repeatedly refers to "younger" and "older" adults, but it is unclear whether age was ever controlled for or included in models. Some mice completed only one round, others 2-5 rounds, without explanation of the criteria or balancing.

(2) Stability of chasing appears stronger than the stability of tube competitions. Figure 4D shows highly consistent chasing behavior across weeks, while Figure 3D shows weaker and more variable correlations for tube-based David scores. This is also evident from Figure 5A-B,D. Thus, it appears that chasing, which serves to quantify dominance in similar semi-natural setups, may be a more reliable and behaviorally meaningful measure of dominance than the incidental tube competitions.

(3) Unbalanced participation across rounds compromises stability analyses. Stability analyses (e.g., ICCs, round-to-round correlations) assume comparable sampling across individuals. However, some mice contribute 1 round, others 2, 3, 4, and even 5 rounds. This imbalance may inflate stability estimates or confound group reshuffling effects, and the rationale for variable participation is not explained.