Three-chamber Social Interaction Test

Three chambered boxes were used to assess sociability and social novelty preference (Yang et al., 2011). The apparatus is made of 3 chambers separated by walls, each having a vertical opening allowing the mice to travel between chambers. The left and right chambers have weighted cups in the middle of the chamber. The test was divided into 3 segments, each lasting 10 min: habituation, sociability, and social novelty preference. The mouse started each segment in the middle chamber. During habituation, both cups were empty. During the sociability segment, one of the cups had a conspecific sex- and age-matched stranger and the other one had an object about the same size as a mouse. During the social novelty preference segment, the object was replaced by a novel stranger. Testing was done in dim light, between ZT2 and ZT8.

The night before the experiment, stranger mice were habituated to the apparatus. During the habituation of stranger mice, a mouse was put under each weighted cup for 10 min. The mouse under the left cup was then allowed to discover the apparatus for 10 min. This mouse was put back under the cup and the other mouse was allowed to discover the apparatus for 10 min.

Tracking was done using a stopwatch program coded using Wing101, for this experiment. The time spent interacting with the weighted cups, defined as time at which the mouse is directly interacting with the cups by sniffing or walking on them, was used to assess sociality and social novelty preference:

Mice that jumped out of the apparatus were excluded from the analysis, starting from the trial during which they jumped out (n_males: WT CJL = 6, Sdy CJL = 5; n_females: Sdy LD 12:12 = 1, Sdy CJL = 1). Furthermore, significant outliers according to the ROUT test during sociability were excluded from both the sociability and social novelty analysis (n_males: WT CJL = 1, Sdy LD 12:12 = 1, Sdy CJL = 1; n_females: WT LD 12:12 = 2, Sdy CJL = 1).