Three chamber test for sociability

At 25 days of life, rats were tested in an apparatus composed of three chambers (two side chamber and a centre chamber with doorways to both side chambers, all of equal dimensions (42.5 cm, length; 17.5 cm, width; 23 cm height). As a modification of the original apparatus (61) to accommodate the juvenile rats, inverted cups were replaced by creating two small chambers within the side chambers as previously described (62). The two small chambers (10 cm, length; 17.5 cm width; 23 cm height) were created with perforated plexiglass partitions that allowed rats to see, hear and smell across the walls. The evaluation of sociability consisted of two 10 min test sessions, a habitation phase and a test phase performed in dim lighting conditions (15 lux). During the habituation phase, each test subject was placed in the centre chamber and allowed to freely explore the apparatus for 10 min. Test subjects were then allowed to return to the centre chamber and the entrances to the side chambers were blocked with plexiglass walls. Either a conspecific novel wild-type partner rat of the same genetic background, age and sex (purchased from SAGE Labs/Horizon, USA), or a novel object (a light grey LEGO block, 6 cm, length; 6 cm, width; 4 cm, height) was immediately placed behind the plexiglass partitions of the side chambers. The walls obstructing the entrances to the chambers were lifted and the test phase was initiated in which test subjects were allowed to freely investigate all three chambers. The entire test was video recorded, and time spent in each chamber (partner side, centre and object side), as well as time spent actively investigating the wild-type partner or novel object (sniffing at the partition) were scored by an experimenter blinded to genotype using a hand-held computer as previously described Psion Observer 3, Noldus, USA).(62).