Beyond the barrier: an assessment of social behaviour in mice using a modified 3-chamber test

The 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 approach. However, such behaviour may also reflect exploration or investigatory processes, and factors such as sex and social hierarchy are often not explicitly considered.

We examined whether standard 3CT measures reflect an affiliative social approach by incorporating a direct interaction phase and evaluating the influence of sex and social status.

Adult male and female C57BL/6 mice (n = 32) were tested in a standard 3CT, followed by removal of the cage barrier to allow direct interaction. Behavioural measures were quantified, and social status was assessed using the tube test.

Males showed higher levels of social investigation during the indirect phase. Following barrier removal, both sexes showed a negative direct sociability index, indicating a relative shift toward environmental exploration rather than direct interaction. Sex differences were most evident among submissive mice.

These findings suggest that standard 3CT measures primarily capture social approach and investigatory behaviour rather than sustained affiliative interaction. The inclusion of a short direct interaction phase provides complementary insight into behavioural prioritisation under unrestricted conditions and highlights the importance of considering biological and contextual factors, including sex and social status, when interpreting social behaviour in mice.