Three-chamber social test

The three-chamber test was performed to assess the sociability and social novelty of the animals as previously described ⁴² . In brief, the test consisted of three 10-minute stages, including habituation, sociability, and novelty stages. In the first 10 minutes, adult mice were allowed to freely explore a 60 × 40 × 23 cm Plexiglass arena with three equally sized, interconnected chambers (left, center, and right). The left and right chamber each contained an empty wire-cup. During the next 10 minutes, the mice were placed back in the center, and another age- and gender-matched stranger animal was introduced to a randomly chosen wire-cup. In the last 10 minutes, a second stranger mouse was introduced into the remaining empty cup. Time spent interacting with each cup or mouse was recorded using a camera and the automated AnyMaze software and manually scored to include only the active interactions (sniffing, crawling upon, etc.). Sociability was evaluated by preference to animal versus empty cup, and social novelty was examined by preference to stranger versus familiar mouse.