2.5. Fear Conditioning and Fear Extinction Test

The fear conditioning and extinction experimental parameters were set according to previous publications with modifications [27,28,29]. In our experiments, the conditioned stimulus (CS) were sound (80 dB intensity, 360 Hz frequency, for 40 s) and light (80 Cd intensity, 505 Hz optical frequency, for 27 s); the followed unconditioned stimulus (US) was scrambled foot-shock treatment (0.5 mA intensity, for 3 s). Freezing was defined as complete immobility of the body and head with no interaction with the environment. Fear memory was evaluated by measuring freezing time [29].

Procedure for fear conditioning training (CS + US): each male mouse was placed into a box with an environmental adaptation period of 180 s before the experiment; then the sound stimulus started at 0 s; the light stimulus started at 10s; the electric foot shock started at 37 s; the experiment cycle was 60 s; 2 continuous cycles was performed once a day. The mice were stimulated with CS + US for 3 consecutive days.

Procedure for re-exposure to fear conditioning training: the mice were stimulated with CS + US on day 1–3, and after 72 hours, they were stimulated with CS + US on day 6–7.

Procedure for fear extinction training: after the mice were stimulated with CS + US on day 1–2, they were stimulated with CS for 3 or 4 consecutive days.

Procedure for combination of fear conditioning, re-exposure and extinction training: The mice were stimulated with CS + US on days 1–3, and after the test was stopped for 6 days, they were stimulated with CS + US on days 9–10 and CS stimulation on days 11–14.

The experiment box was cleaned with 75% ethanol to remove all odors before entering each mouse. The high-definition camera in the experiment box was used to track the behavior, and the real-time data was analyzed by Shanghai Xin soft software (Shanghai XinRuan Information Technology Co., Ltd., Shanghai, China).