2.8. Passive Avoidance Test (PAT)

The PAT test was performed to evaluate cognitive changes and passive avoidance learning in mice. Given that mice had a tendency to avoid light and prefer darkness, the device contained a light and a dark chamber (280 × 155 × 160 mm, XR-XB110, XinRuan Information Technology Co., Ltd., Shanghai, China), both of which were divided by a door in the middle.

The first day of the experiment was set as adaptation stage. Each mouse was placed in the light chamber with the door between two chambers open, allowing the mice to explore the whole space freely for 3 min. The training stage started 24 h after the adaptation stage. The mice were still placed in the light chamber with the door open. However, whenever the mice entered into the dark chamber, they immediately received electrical stimulation (0.3 mA, 2 s), and the latency and frequency for each mouse to enter the dark chamber were recorded. The training stage lasted for 3 min, and 24 h after the end of training, the retention test was performed, which lasted for 5 min. The procedure was the same as mentioned above, except that the mice would not receive electrical stimulation. The time when the mouse reentered the dark box was recorded as latency, and the number of times a mouse entered into the dark box was recorded as error times.