Social interaction behavior was investigated using an apparatus consisting of a large open field (Fig. 1a , 89×63×60 cm) containing a small wire cage (14×17×14.5 cm). Each focal mouse was introduced into the open field for 3 min to habituate, and we recorded the amount of time the focal mouse spent interacting with the empty wire cage (within 8 cm, see blue box in Fig. 1A ) using a video tracking system (Stoelting, Wood Dale, IL). Next an unfamiliar, same sex virgin mouse was introduced into the wire cage. For 3 min we recorded the amount of time the focal mouse spent interacting with the wire cage. We also measured time spent in the two corners opposite the wire cage (8×8 cm, Fig 1A ) and total distance traveled as an estimate of total activity. After each test the arena was cleaned with 70% ethanol and dried before the next mouse was tested. Social interaction was assessed at 24 hours and 4 weeks after social defeat exposure. Different stimulus mice were used for the two tests. In between the two social interaction tests the mice were undisturbed except for routine cage changes. Immediately after testing at 4 weeks, each focal mouse was anesthetized with isoflurane and euthanized by decapitation (14:45–17:00 PST). Brains were collected immediately after testing to detect changes in phosphorylated CREB and ERK, which we have previously quantified in California mice after 7 min resident-intruder tests [30] (link), [38] (link). Trunk blood was collected in heparinized tubes and centrifuged to collect plasma (see below for corticosterone assay methods). Brains were quickly removed and immersion fixed in 5% acrolein in phosphate buffered saline (PBS). Each female was lavaged post-mortem. Estrous cycle stage was determined by assessing the presence of leukocytes, nucleated epithelial cells, and/or cornified cells [30] (link), [39] (link).
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