Three-chamber test (3CT)

To evaluate sociability and social cognition, 3CT was performed in a three-chambered box (62 cm length × 42 cm width × 22 cm height) consisted of the opaque polyvinyl plastic. During the habituation phase, mouse was placed in the middle chamber and allowed to explore three chambers freely for 10 min with one empty vertical metal wire cage in the corner of each extreme chamber. The first session aims to test the sociability, and a stranger (S1) mouse was placed into one of the vertical metal wire cages and the other cage was empty object (O). Then the mouse was placed in the middle chamber and allowed to explore freely all chambers for 10 min. The second session is to test its social cognition, and a new stranger mouse (S2) was placed in the chamber and on the other end was S1. In this session, the mouse was allowed to explore freely for 10 min. S1 and S2 at same age and with same sex to the mice to be tested were used. The time spent in each chamber was recorded and analyzed using Any-Maze software. The sociability was evaluated by the time preference index (S1-O) and the social cognition was calculated by the time preference index (S2-S1).