2.7. Passive avoidance test

A shuttle‐box apparatus was used to analysis associative learning and memory. The maze consisted of two parts (a light and a dark chamber) with a sliding guillotine door. Each animal was first habituated to the test by placement in the light chamber (door closed) for 10 s. Then the door was opened and the animal was allowed to enter the dark chamber and was explored for 30 s. In the learning trial, each mouse was located in light chamber of the apparatus and then the door was opened the mouse was allowed to enter into the dark cavity. Then, the gate was closed and an electric shock (0.5 mA, 50 Hz) delivered to the animal for 2 s. This last step was repeated at 30 min intervals until the animal learned to avoid the dark chamber. The number of learning trials to get efficient learning was recorded. After 30 min, the same test was conducted again, and if the mice did not enter the dark chamber by 300 s, the successful acquisition of passive avoidance response was recorded (shock number) and the number of allowable shocks was 5. Twenty‐four hours after learning phase, the retention trial was performed to assess memory. In this step, the animals were placed in the light compartment of the apparatus. After 10 s, the gate was opened and the time before the first entry of the animal to the dark chamber was considered as step‐through latency (STL) during a 300 s interval (Nazeri et al., 2014a).