Tracking software

On PND 42 rats were placed in a plexiglas open-field arena (108 × 108 cm). The floor of the testing arena was covered with a thin layer of corncob bedding since the rats were familiar with the surface covering. The bedding was rearranged after each trial to redistribute odors left by the previous animal. Behaviors were scored automatically or manually in Noldus tracking software (Noldus, Leesburg, VA, United States) as appropriate. A random numbers table was used to determine the order each rat was assessed in the arena. Trials lasted 300 s, during which the duration of freezing, crossing the center of the arena, and rearing responses (internal and external directions) were recorded as well as the duration and frequency of thigmotaxis and total distance traveled (in mm.). All measures were automatically collected and scored by Noldus tracking software; additionally, rearing responses were scored manually by observers within Noldus.

On PND 38, rats were subjected to a social investigation test (SIT) in a glass tank (76 × 32 × 31 cm) with a thin layer of corncob bedding (3 cm deep). The SIT was conducted over two phases. The first phase was habituation to the glass tank arena and an empty plastic holding container (19 × 10 × 10 cm). The second phase introduced a novel conspecific of the same sex and age that was placed in the plastic holding container. The holding container had holes that allowed the test animals to detect olfactory cues emitted by the stimulus animal. The same stimulus animal was used for the consecutive testing of two cages (from different groups), after which, the container was cleaned with water. Noldus tracking software (Noldus, Leesburg, VA, United States) recorded distance traveled, as well as frequency and duration of interactions with the container at each phase. Blind observers recorded the frequency of rears (internal and external) and grooming bouts during each phase.