4.3.1. Passive Avoidance (PA)

The passive avoidance procedure produces similar results in both mice and rats [72]. The PA apparatus consisted of a two-compartment acrylic box with a lighted (8 W light) chamber (10 × 13 × 15 cm) and a darkened chamber (25 × 20 × 15 cm) equipped with an electric grid floor. The compartments were separated by guillotine door. Entrance of animals to the dark box was punished by an electric foot shock (0.15 mA for 2 s).

The PA procedure was based on the association formed between an aversive stimulus (foot shock of 0.15 mA intensity and 2 s duration) and a specific environmental context (dark compartment). The experiment consisted of three steps: habituation (PND60), training (PND60), and retention testing (PND61). The procedure was as follows.

All rats were allowed to habituate in the experimental room for at least 30 min prior to the experiments. Next, in the training session, each animal was gently placed in the light compartment of the apparatus and allowed to freely explore. After 30 s, the guillotine door was opened, and the rat was allowed to enter the dark compartment. When the rat entered the dark chamber, the guillotine door was closed and the electric foot-shock duration was delivered immediately to the animal via the grid floor of the dark room by an insulated stimulator. Next, the animal was removed from the apparatus and placed in its home cage. The latency with which the animal crossed into the dark compartment was recorded (TL1). If the rat failed to enter the dark box within 300 s, it was placed into this dark box, the door was closed, and the electric foot-shock was delivered to the animal. In this case, TL1 value was recorded as 300 s. On PND61, 24 h later, the retention test trial was conducted with exactly the same conditions, but without electric shock. The latency with which the animal crossed into the dark compartment was recorded (TL2) and the time spent in the dark compartment was measured up to 300 s. If the animal did not enter the dark chamber within 300 s, the test was terminated and TL2 was recorded as 300 s.

To assess memory-related behaviors, the changes in the PA task were expressed as the difference between training and test latencies and were taken as the latency index (LI). LI was calculated for each animal as the ratio: LI = TL2-TL1/TL1, where: TL1—the time taken to enter the dark compartment during the training and TL2—the time taken to re-enter the dark compartment during the retention [73]. A high value of IL proves the positive influence of the tested substances on learning processes and memory, while low IL indicates disturbances in short-term memory processes and cognitive processes.