Social Interaction Test

The social interaction test is a two-trial procedure (24), conducted under red light conditions. In the first 2.5 min session, the experimental c57BL/6 mouse is allowed to freely explore an open field arena (40 cm length × 40 cm width × 40 cm height; Fig. S1C). Along one side of the arena is a circular (7 cm diameter) wire cage (Stoelting Co., Wood Dale, IL) that remains empty during the first trial (target-absent condition). The experimental c57BL/6 mouse is then removed from the testing arena for 30 sec (into a separate holding cage), and a novel CD1 male mouse is placed into the wire cage. In the second 2.5 min trial (target-present condition), the experimental c57BL/6 mouse is reintroduced into this arena now containing a social target (unfamiliar CD1 male mouse) within the circular wire cage. In order to determine whether social interaction levels would be influenced by the strain/sex of the social target itself, a different group of ES-females was exposed to an unfamiliar female of the same strain during the social interaction test. Also, to examine the salience of the visual stimuli in mediating ES-induced avoidance, we conducted an experiment where white-opaque-dividers without holes (Fig. S1B) were used to prevent females from witnessing the resident-intruder interaction – removing visual sensory cues related to the defeat of the male conspecific. In all cases (social target being an unfamiliar male CD1, or an unfamiliar female c57BL/6 mouse) a social interaction ratio was used to evaluate the effects of stress on social behavior – where the time (sec) spent in the interaction zone (8 cm wide corridor surrounding the wire cage) in the presence of the social target is divided by the time spent in the interaction zone in the absence of the target (25, 27). Lastly, we recorded the distance traveled (cm) during the first 2.5 min of the social interaction test (target-absent condition) to examine whether basal locomotor activity or exploratory behavior was influenced by stress exposure.