Passive Avoidance Test

The passive avoidance testing was done in an automatically operated commercial Passive Avoidance Apparatus (step-through cage 7550; Ugo Basile, Comerio, Italy). The passive avoidance step-through cage was divided into two equal size compartments (insight dimension 22 cm long × 21 cm wide × 22 cm high, each): START (white and illuminated by a 24 V–10 W bulb) and ESCAPE (black and dark). The two compartments are divided by a partition which embodies an automatically operated sliding door at the floor level. On day 1, each rat was exposed to the exploration trial, by placing it in the START chamber (door closed and shock disconnected) and allowed to explore it for 100 s. After that the door was opened, the rat was allowed to enter into the ESCAPE chamber, and when all four paws were in, the automated slide door was closed. The maximum latency to pass from the START to the ESCAPE compartment was set to 60 s. After 10 s, the rat was removed from the ESCAPE compartment and returned to its home cage. On day 2 (acquisition trial), when the rat entered into the ESCAPE compartment, the door was closed and a 1.0 mA shock was delivered for 5 s. Ten seconds later on, the rat was removed from the ESCAPE compartment, drug administered (saline, verapamil, scopolamine, or verapamil followed by scopolamine treatment), and returned to its home cage. According to the latency period to enter into the ESCAPE compartment on day 1 and sensitivity to the shock (vocalization and jumping response) on day 2, the animals were assigned into 11 groups; thus, there were no significant differences between groups. Eight animals were assigned in each tested group. Forty-eight hours after the acquisition trial, the retention trial was carried out. The test was performed in a similar way to the acquisition trial, but no shock was given. The cage catch pan, grid floor, and side walls were cleaned with 70% ethanol before each animal was tested. The most often cut-off times used in the passive avoidance test are 180, 300, and 600 s. In the present study, the cut-off time for the entrance of the rat into the dark compartment was 9 min (maximum time allowed by the used apparatus). The longer cut-off time is when the individual differences become more apparent (Sahgal, 1993). Therefore, for better drug effect discrimination, three cut-off times (180, 300, and 540 s) were analyzed for each animal and group.