2.3. Place conditioning procedure

Two weeks after social defeat stress or control handling, conditioned place aversion (CPA) was conducted as described in Robles et al. [14] (Fig. 1). The apparatus consisted of three visually distinct interconnected standard sized mouse cages (28 × 17.5 × 11 cm). The center cage (black and white horizontal stripe background) was connected to a left cage (black and white vertical stripe background) and a right cage (black dots on a white background and textured plastic floor). After each test the apparatus was cleaned with Quatricide (1:64, Quatricide PV in water, Pharmacal Research Labs, Inc). Clear polypropylene lids were used to cover the apparatus during experiments to allow for video recording.

Timeline for conditioned place aversion experiments.

Conditioned place aversion was conducted over 4 days during the light cycle. The protocol was designed so that the mice learn to associate vehicle or drug injection with a given chamber. On day 1 (pre-test) mice were placed in the apparatus and allowed to explore freely for 30 min while being tracked in real time with a visual tracking system (Any-maze Stoelting). For each mouse, initial place biases were corrected for by assigning drug conditioning to the preferred side chamber (Robles et al., 2014; McLaughlin et al., 2003).

Mice were randomly assigned to be conditioned with either vehicle (10% Tween 80 in sterile PBS), 2.5 mg/kg, or 10 mg/kg of (±)U50,488 (Tocris, Ellisville, MO, USA) administered i.p. On days 2 and 3, each mouse received two training sessions. In the morning, each mouse received an i.p. injection of vehicle and was placed in the unconditioned chamber for 30 min. Afterwards mice were returned to their home cages. Three hours later, each mouse received an injection of either vehicle, 2.5 mg/kg, or 10 mg/kg of U50,488 and was placed in the conditioned chamber for 30 min. During training sessions, the entrance to the center chamber was closed so mice were confined to a single chamber. On day 4, each mouse was given free access to the entire apparatus for 30 min (post-test) and was tracked with the video tracking system. The time spent in each cage and the total distance traveled were both recorded. On day 5 mice were injected i.p. with their assigned conditioned drug, and, 1 hour later, anesthetized with isoflurane and euthanized by decapitation. Brains were immediately collected and fixed in 5% acrolein in PBS overnight at 4°C. Brains were then transferred to 20% sucrose in PBS overnight at 4°C and then frozen and stored at −40°C.