Beyond the Barrier: An Assessment of Social Behaviour in Mice using a Modified 3-Chamber Test

BackgroundThe three-chamber test (3CT) is widely used to assess social behaviour in mice, based on the assumption that time spent near a conspecific reflects motivation for social contact. However, the design of the task constrains interpretation, as behaviour may reflect exploration, novelty seeking, or territorial investigation rather than affiliative social motivation. In addition, key biological factors such as sex differences and social hierarchy are often overlooked. AimsWe hypothesised that the 3CT overestimates sociability and used a direct-interaction phase to investigate motivation for affiliative social contact. We also integrated social status to determine if this modulated behavioural patterns and interacted with sex. MethodsAdult male and female C57BL/6 mice (n = 32) were tested in a standard 3CT, followed by removal of the cage barrier to permit direct contact. Behavioural parameters were quantified, and social status was determined using the tube test. ResultsMales exhibited higher social interest index scores than females. Once the barrier was removed, both sexes displayed a negative direct sociability index, indicating greater environmental exploration than social engagement. Correlation analysis revealed no association between indirect and direct measures. Sex differences emerged primarily among submissive mice, with submissive males showing greater social investigation than submissive females. ConclusionThese findings suggest that standard 3CT indices reflect exploratory rather than affiliative social motivation. The modified paradigm incorporating direct interaction provides a more realistic assessment of social behaviour and challenges assumptions about intrinsic sociability in mice.