Context and Cued Fear Conditioning Protocol

The context and cue fear conditioning assay was performed as previously described^{57 (link)}. The apparatus consists of a sound attenuated chamber with a grid floor capable of delivering an electric shock, and freezing was recorded and measured with an overhead camera and FreezeFrame software (Actimetrics), respectively. On day 1, each mouse was placed into the chamber and baseline freezing behavior was recorded during the first two min. Then, mice were exposed to 80-dB white noise for 30 s as the conditioned stimulus (CS). During the final 2 s of this noise, mice received a mild foot shock (0.5 mA) as the unconditioned stimulus (US). After 1 min, mice were subjected to another CS-US pair stimulus, and then returned to their home cages and homeroom. On day 2, each mouse was returned to the test chamber and freezing behavior was recorded for 5 min (context test). Mice were returned to their home cage and placed in a different room with reduced lighting for at least 1 h. Moreover, environmental and contextual cues were changed. For the cued test, each mouse was placed in the modified chamber for 3 min, and then exposed to the auditory CS and freezing behavior was recorded for another 3 min (cue test). Baseline freezing behavior obtained during training was subtracted from the context or cue tests to control for variability in each animal.