Social interaction test

Social interaction testing was conducted as previously described¹¹. Male and female experimental rats were placed into a two-compartment testing chamber for a 30-minute habituation period, followed by a ten-minute interaction period with an unfamiliar social partner of the same age and sex from a separate litter that did not undergo any prenatal exposure and which had no previous interaction with the experimental animal. One set of litters was used for initial social interaction testing across age, while a separate set of litters was used for social behavior testing with chemogenetic manipulation (Fig. 1). Animals were tested only once at a specific age point to avoid testing interactions. All behaviors were video recorded and saved for later scoring by a trained researcher blind to experimental group. Behaviors scored included frequencies of social investigation (sniffing of the partner), contact behavior (grooming of and climbing over/under the partner), and play behavior (pouncing, following/chasing, pinning). Social motivation via social preference/avoidance was measured as crossovers from one compartment of the testing apparatus toward (preference) or away from (avoidance) the partner, and a social preference/avoidance coefficient was calculated [coefficient (%)=(crossovers toward partner–crossovers away from the partner)/(total crossovers toward/away)x100]. Scoring of social investigation, contact behavior, and play behavior were scored by the same researcher for each experiment, while social motivation and locomotor behavior for each experiment were scored by a separate researcher.