4.3.6. Three-Chamber Social Test

The three-chambered apparatus was composed of three Plexiglas chambers, each measuring 20 × 40 × 22 cm, and dividing walls with small square entrances (10 × 5 cm) that allowed free movement to each chamber. Both side chambers contained a cylindrical plastic cage (17 cm in height and a bottom diameter of 8 cm, with bars spaced 1 cm apart) in the corner that was used to confine the stranger mice. The three-chambered social test was then executed according to a previous study [75]. First, the subject mice were placed in the apparatus to freely explore all three chambers, with an empty plastic cage in each side chamber (5 min habituation period). For sociability testing, an unfamiliar C57BL/6J mouse (Stranger 1, “S1”) was confined in the cylindric plastic cage in one of the side chambers, and an empty cylindrical plastic cage (Empty “E”) was kept on the other side of the chamber. The subject mouse was then placed in the center chamber and allowed to freely explore all three chambers for 10 min. For the social novelty test, the empty plastic cage was replaced with another unfamiliar C57BL/6J mouse (Stranger 2, “S2”) and the subject mouse was again allowed to freely explore all the three chambers for 10 min. All stranger mice were of the same sex and age as the subject mice and were habituated to plastic cages. The time spent in the chamber, distance traveled, and heat-maps were measured using EthoVision^® XT16 software.