Spontaneous alternation in the Y-maze

A three-arm plexiglass Y-maze (45 cm length × 8 cm width × 12 cm height for each arm) was used to test spontaneous alternation. Animals were brought into the testing room at least 30 min prior to the start of the session to acclimate to the room. Room lights were dimmed to ~10–15 lux. Animals were placed in the start arm facing away from the center. Recording of the session was started when the animal started to move toward the center and the animal was allowed to freely explore the Y-maze for 8 min. The Y-maze was cleaned with 70% ethanol between animals.

Videos of the sessions were analyzed blindly offline. Arms were identified as A, B, C. Starting from the start arm, entry into each arm was recorded as a sequence of arm letters. Entry was recorded if all four paws of the animal entered the arm. The number of alternating entries (triplet sequences with non-repeating letters, e.g. ABC) and total entries (total number of letters) were counted. Alternation percentage was calculated as: (total alternating entries/(total entries-2)) × 100.