Standard conditioning chamber

All animals were habituated to the context for 10 min which consisted of a standard conditioning chamber with a metal grid floor (Coulbourn Instruments). Preexposure to the context on Day 1 and a 5 min placement in the context before the first shock on Day 2 were used to reduce any potential behavioral differences between sexes (Wiltgen et al., 2001 (link)). The next day (Day 2), animals were placed in the conditioning chamber for 10 min and received 3 unsignaled footshocks (0.5mA, 1 sec). The first footshock was delivered after 5 min in the conditioning chamber. The timing of second and third footshocks was random with at least 45 seconds between each shock. Animals in the “no-shock” group were also habituated to the chamber on Day 1. On Day 2, they were placed in conditioning chambers for 10 minutes but did not receive shocks. 24 hours later (Day 3), animals were returned to the conditioning chamber for 10 min. Total time spent freezing to the context was manually scored offline for each animal by an observer blind to group assignment. 45 minutes after the completion of behavioral testing, animals were given an overdose of sodium pentobarbital (100mg/kg) and perfused transcardially with 0.1M PBS and 4% paraformaldehyde in 0.1M PB. Animals designated as “home-cage controls” were never exposed to the conditioning chambers.

On day 1, rats were habituated to the context for 10 min which consisted of a standard conditioning chamber with a metal grid floor (Coulbourn Instruments). On day 2, animals in the “shock” group were placed in the conditioning chamber for 8 min and received 3 footshocks (0.5mA, 1 sec). Animals in the “no-shock” group were placed in conditioning chambers for the same 8 minutes on day 2 but did not receive shocks. On day 3, shock and no-shock animals were returned to the conditioning chamber for 10 min. Total time spent freezing before the shock (baseline, 30 seconds), after the shock (30 seconds, post shock) during conditioning and to the context during testing (freezing time per minute over 10 minutes) was manually scored offline for each animal by an observer blind to group assignment. Preexposure to the context on Day 1 and a 5 min placement in the context before the first shock on Day 2 were used to reduce any potential behavioral differences between sexes (Wiltgen et al., 2001 (link)). A third group of animals received no behavioral manipulation and were naïve “home cage” controls (n=5 males, n=5 females).