Env 414

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 apparatus and stimuli used have been previously described [12 (link),13 (link),19 (link)]. Fear conditioning occurred in four Plexiglas chambers measuring 16.5 cm × 12.1 cm × 21.6 cm which were arranged in a 2 × 2 formation on a Plexiglas stand within a fume hood to provide ambient light and background noise (Context A). Each chamber had a grid floor made of 9 stainless steel bars (11.5 cm from the top of the chamber), 0.5 cm in diameter and spaced 1.25 cm apart. The alternate context (Context B) consisted of the same Plexiglas chambers with a convex wire mesh insert that covered the back wall and floor of the chamber and a white paper sleeve that covered the outside walls of the chamber. Footshock was delivered using a shock scrambler (Med Associates, Georgia, VT ENV-414S) connected to the grid floor of the chamber. The fear chambers were cleaned with 5% ammonium hydroxide solution prior to each load of experimental animals. Videos of each session (preexposure, training, testing) were recorded using Freeze Frame 3.0 software (Actimetrics, Wilmette IL) with freezing defined as a bout of 0.75 s or longer without a change in video pixilation as previously described [13 (link)].

Sixteen operant conditioning chambers (28 × 21 × 21 cm; ENV-008; MED Associates, St. Albans, VT) located inside sound attenuating chambers (ENV-018M; MED Associates) were used. The front and back walls of the chambers were made of aluminum, while the side walls were made of Plexiglas. There was a recessed food tray (5 × 4.2 cm) located 2 cm above the floor in the bottom-center of the front wall and was located between two retractable levers. Each lever (4.8 × 0.55 × 1.9 cm) was located 2.1 cm above the floor and required a force of 0.245 N to depress. An infrared photobeam was used to record head entries into the food tray. A 28-V white stimulus light (2.54 cm diameter) was located 6 cm above each response lever. A 28-V white house light was mounted in the center of the back wall of the chamber. The floor of the operant chamber was composed of steel rods connected to a shock generator (ENV-414S; MED Associates) that delivered foot shocks. All responses and scheduled consequences were recorded and controlled by a computer interface. A computer controlled each experimental session using Med-V software.

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.