Fear Conditioning Protocol for Mice

Fear-conditioning experiments were conducted as previously described (Temme et al., 2014 (link)). Fear-conditioning chambers were composed of clear acrylic backs and doors, aluminum sides, stainless steel grid floors with one-eighth inch spaces, and stainless steel drop pans (Med Associates). Throughout experimentation, chambers and floor pans were cleaned with 70% ethanol. Chambers were illuminated using room lights set at 150 W. Footshocks were administered through the grid via solid-state shock scramblers and electronic constant-current shock sources. Shocks were controlled by a desktop PC running Actimetrics FreezeFrame software. The behavior of each mouse was recorded and digitized using individual cameras mounted above each chamber using the Actimetrics FreezeFrame software. Mice were fear conditioned to a context using one training session per day for 2 d. Each fear-conditioning session consisted of 3 min of context exposure to the training chamber followed by three unsignaled footshocks (0.5 mA, 2 s) with 30 s between footshocks. Mice were removed from the training chamber 30 s after the last footshock. Twenty-four hours after the last training session, mice were tested for fear of the trained context using 5 min of context exposure.