2.5. Passive Avoidance Test for Learning and Memory Assessment

Learning and memory in animals were tested using a passive avoidance task following completion of the treatment paradigm. The step-through passive avoidance test apparatus (Gemini Avoidance System, San Diego Instruments, San Diego, CA, USA) consisted of illuminated and nonilluminated chambers (25 cm × 20 cm × 20 cm) separated by a guillotine door (5 cm × 5 cm). The floor of the illuminated and nonilluminated chambers is composed of 2 mm diameter stainless steel rods spaced 6 mm apart. During the acquisition test session, mice were gently placed in the illuminated compartment, and the guillotine door between the two chambers was opened 10 s later. When mice entered the nonilluminated chamber, the door automatically closed, and an electrical foot shock (0.9 mA, 3 s) was delivered through the stainless-steel rods. The retention trial session was carried out after 24 h, and the mice were again placed in the illuminated chamber. The step-through latency or the time taken to enter the nonilluminated chamber was then measured. If the mouse did not enter the nonilluminated chamber within 300 s, the retention trial was ended, and the step-through latency time was scored using 300 s as the upper limit.