Stereotype of mouse social competency and status revealed by a novel competition paradigm in combination with available paradigms

Reviewer #1 (Public review):

Summary:

The authors present a new protocol to assess social dominance in pairs and triads of C57BL/6j mice, based on a competition to access a hidden food pellet. Using this new protocol, the authors have been able to identify stable ranking among male and female pairs, while reporting more fluctuant hierarchies among triads of males. Ranking readouts identified with this new apparatus were compared to the outcomes obtained with the same animals competing in the tube and in the warm spot tests, which have been both commonly used during the last decade to identify social ranks in rodents under laboratory conditions.

Strengths:

FPCT allows for easy and fast identification of a winner and a loser in the context of food competition. The apparatus and the protocol are relatively easy and quick to implement in the lab and free from any complex post-processing/analysis, which qualifies it for wide distribution, particularly within laboratories that do not have the resources to implement more sophisticated protocols. Hierarchical readouts identified through the FPCT correlate with social ranks identified with the tube and the warm spot tests, which have been widely adopted during the last decade and allow for study comparison.

Weaknesses:

While the FPCT is validated by the tube and the warm spot test, this paper would have gained strength by providing a more ethologically based validation. Tube and warm spot tests have been shown to provide conflicting results and might not been a sufficient measurement for social ranking (see Varholik et al, Scientific reports, 2019; Battivelli et al, Biological psychiatry, 2024). Instead, a general consensus pushing toward more ethological approaches for neuroscience studies is emerging.
Other papers already successfully identified social ranks dyadic food competition, using relatively simple scoring protocol (see for example Merlot et al., 2006), within a more naturalistic set-up, allowing the 2 opponents to directly interact while competing for the food. A potential issue with the FPCT, is that the opponents being isolated from each other, the normal inhibition expected to appear in subordinates in the presence of a dominant to access food, could be diminished, and usually avoiding subordinates could be more motivated to push for the access to the food pellet.

There are issues with use of the English language throughout the text. Some sentences are difficult to understand and should be clarified and/or synthesized.

Open question:
Is food restriction mandatory? Palatable food pellet is not sufficient to trigger competition? Food restriction has numerous behavioral and physiological consequences that would be better to prevent to be able to clearly interpret behavioral outcomes in FPCT (see for example Tucci et al., 2006).

Conclusive remarks:
Although this protocol attempts to provide a novel approach to evaluate social ranks in mice, it is not clear how it really brings a significant advance in neuroscience research. The FPCT dynamic is very similar to the one observed in the tube test, where mice compete to navigate forward in a narrow space, constraining the opponent to go backward. The main difference between the FPCT and the tube test is the presence of food between the opponents. In the tube test, a food reward was initially used to increase motivation to cross the tube and push the opponent upon the testing day. This component has been progressively abandoned, precisely because it was not necessary for the mice to compete in the tube.

This paper would really bring a significant contribution to the field by providing a neuronal imaging or manipulation correlate to the behavioral outcome obtained by the application of the FPCT.

Reviewer #2 (Public review):

Summary:

In this study, the authors have devised a novel assay to measure relative social rank in mice that is aimed at incorporating multiple aspects of social competition while minimizing direct contact between animals. Forming a hierarchy often involves complex social dynamics related to competitive drives for different fundamental resources including access to food, water, territory, and sexual mates. This makes the study of social dominance and its neural underpinnings hard, warranting the development of new tools and methods that can help understand both social functions as well as dysfunction.

Strengths:

This study showcases an assay called the Food Pellet Competition Test where cagemate mice compete for food, without direct contact, by pushing a block in a tube from opposite directions. The authors have attempted to quantify motivation to obtain the food independent of other factors such as age, weight, sex, etc. by running the assay under two conditions: one where the food is accessible and one where it isn't. This assay results in an impressive outcome consistency across days for females and males paired housed and for male groups of three. Further, the determined social ranks correlate strongly with two common assays: the tube test and the warm spot test.

Weaknesses:

This new assay has limited ethological validity since mice do not compete for food without touching each other with a block in the middle. In addition, the assay may only be valid for a single trial per day making its utility for recording neural recordings and manipulations limited to a single sample per mouse. Although the authors attempt to measure motivation as a factor driving who wins the social competition, the data is limited. This novel assay requires training across days with some mice reaching criteria before others. From the data reported, it is unclear what effects training can have on the outcome of social competition. Beyond the data shown, the language used throughout the manuscript and the rationale for the design of this novel assay is difficult to understand.

Reviewer #3 (Public review):

Summary:

The laboratory mouse is an ideal animal to study the neural and psychological underpinnings of social dominance behavior because of its economic cost and the animals' readiness to display dominant and subordinate behaviors in simple and testable environments. Here, a new and novel method for measuring dominance and the individual social status of mice is presented using a food competition assay. Historically, food competition assays have been avoided because they occur in an open arena or the home cage, and it can be difficult to assess who gets priority access to the resource and to avoid aggressive interactions such as bite wounding. Now, the authors have designed a narrow rectangular arena separated in half by a sliding floor-to-ceiling obstacle, where the mice placed at opposite sides of the obstacle compete by pushing the obstacle to gain priority access to a food pellet resting on the arena floor under the obstacle. One can also place the food pellet within the obstacle to restrict priority access to the food and measure the time or effort spent pushing the obstacle back and forth. As hypothesized, the outcomes in the food competition test were significantly consistent with those of the more common tube test (space competition) and warm spot competition test. This suggests that these animals have a stereotypic dominance organization that exists across multiple resource domains (i.e., food, space, and temperature). Only male and female C57 mice in same-sex pairs or triads were tested.

Strengths:

The design of the apparatus and the inclusion of females are significant strengths within the study.

Weaknesses:

There are at least two major weaknesses of the study: neglecting the value of test inconsistency and not providing the mice time to recognize who they are competing with.

Several studies have demonstrated that although inbred mice in laboratory housing share similar genetics and environment, they can form diverse types of hierarchical organizations (e.g., loose, stable, despotic, linear, etc.) and there are multiple resource domains in the home cage that mice compete over (e.g., space, food, water, temperature, etc.). The advantage of using multiple dominance assays is to understand the nuances of hierarchical organizations better. For example, some groups may have clear dominant and subordinate individuals when competing for food, but the individuals may "change or switch" social status when competing for space. Indeed, social relationships are dynamic, not static. Here, the authors have provided another test to measure another dimension of dominance: food competition. Rather than highlight this advantage, the authors highlight that the test is in agreement with the standard tube test and warm spot test and that C57 mice have stereotypic dominance across multiple domains. While some may find this great, it will leave many to continue using the tube test only (which measures the dimension of space competition) and avoid measuring food competition. If the reader looks at Figures 6E, F, and G they will see examples of inconsistency across the food competition test, tube test, and warm spot test in triads of mice. These groups are quite interesting and demonstrate the diversity of social dynamics in groups of inbred mice in highly standardized environmental conditions. Scientists interested in dominance should study groups that are consistent and inconsistent across multiple dimensions of dominance (e.g., space, food, mates, etc.).

Unlike the tube test and warm spot test, the food competition test presented here provides no opportunity for the animals to identify their opponent. That is, they cannot sniff their opponent's fur or anogenital region, which would allow them an opportunity to identify them individually. Thus, as the authors state, the test only measures psychological motivation to get a food reward. Notably, the outcome in the direct and indirect testing of food competition is in agreement, leaving many to wonder whether they are measuring the social relationship or the effort an individual puts forth in attaining a food reward regardless of the social opponent. Specifically, in the direct test, an individual can retrieve the food reward by pushing the obstacle out of the way first. In the indirect test, the animals cannot retrieve the reward and can only push the obstacle back and forth, which contains the reward inside. In Figure 4E, you can see that winners spent more time pushing the block in the indirect test. Thus, whether the test measures a social relationship or just the likelihood of gaining priority access to food is unclear. To rectify this issue, the authors could provide an opportunity for the animals to interact before lowering the obstacle and raising(?) a food reward. They may also create a very long one-sided apparatus to measure the amount of effort an individual mouse puts forth in the indirect test with only one individual - or any situation with just one mouse where the moving obstacle is not pushed back, and the animal can just keep pushing until they stop. This would require another experiment. It also may not tell us much more since it remains unclear whether inbred mice can individually identify one another (see https://doi.org/10.1098/rspb.2000.1057 for more details).

A minor issue is that the write-up of the history of food competition assays and female dominance research is inaccurate. Food competition assays have a long history since at least the 1950s and many people study female dominance now.

Food competition: https://doi.org/10.1080/00223980.1950.9712776, https://psycnet.apa.org/fulltext/1953-03267-001.pdf, https://doi.org/10.1016/j.bbi.2003.11.007, https://doi.org/10.1038/s41586-022-04507-5

Female dominance: https://doi.org/10.1016/0031-9384(87)90269-1, https://doi.org/10.1016/j.cub.2023.03.020, https://doi.org/10.1016/S0031-9384(01)00494-2, https://doi.org/10.1037/0735-7036.99.4.411