Env 005

A threat conditioning chamber (26 × 30 × 33 cm, ENV-007CT, MED Associates) with a metal grid floor (ENV-005, MED Associates) connected to a standalone aversive electric shock stimulator (ENV-414S, MED Associates) was used for foot shock delivery. A USB camera (DFK 33GX236, Imagine Source) was connected to a computer, and video tracking software (Ethovision XT, Noldus) was used for shock delivery and behavioral analysis. The chamber was enclosed in a light- and sound-attenuating cubicle (ENV-018MD, MED Associates). The chamber was cleaned with 70% ethanol and double-distilled water after each trial.
For both Miniscope and loss-of-function experiments, mice were placed inside the chamber without habituation. After a 2 min baseline, an electric shock (2 sec, 0.6 mA) was delivered, and behavior was recorded for 2 more min. For loss-of-function experiments, freezing behavior was monitored one day before (habituation), immediately after (conditioning), and one day after the shock (post-test). For the terminal inhibition experiment, procedures were the same as for the whole-population inhibition experiment, except that the laser was turned on throughout the conditioning and post-test periods.

The fear conditioning chamber (ENV-007CT, MED Associates) is an arena (26 × 30 × 33 cm) with two Plexiglass walls, two metal walls, and a metal grid floor to deliver electrical shocks (ENV-005, MED Associates). The chamber was connected to a standalone aversive stimulator (ENV-414S, MED Associates) and enclosed in a light- and sound- attenuating cubicle (ENV-018MD, MED Associates). EthoVision XT 12 software with a GigE USB camera with 25 FPS tracked the animal. A 70% ethanol solution and deionized water were used for cleaning immediately after each test. On days 1 and 2, mice underwent two 6-min habituation sessions in the chamber. On day 3, mice were introduced to the chamber 45–60 min after CNO injection and subsequently received five shocks (2 s, 0.2 mA) with uneven intervals over a 7 min trial. After 24 h, mice were reintroduced to the chamber for a 2-min context-dependent retrieval test. The percentage of time frozen and the total distance moved during each session were calculated by the EthoVision XT 12 software. Freezing behavior was defined as the period during which the velocity of the mouse was less than 1.75 cm/s for at least 3 s. Automatic scoring was validated with manual scoring.