Forced alternation tests were conducted using a symmetrical Y-maze made of a grey steel bottom plate with grey Perspex® walls (Stoelting Co, Wood Dale, IL). Each arm of the Y-maze was 35 cm long, 5 cm wide, and 10 cm high, and the wall at the end of each arm was marked with a different black and white pattern. To reduce anxiety in the animals, light in the testing area was dimmed to 30 ± 5 lux.
The protocol for the forced alternation test was modified from Melnikova et al. [21 (link)]. Mice were handled for three days before testing. The test consisted of a 5 min sample trial (T1) followed by a 5 min retrieval trial (T2). For the scopolamine experiment, mice were dosed with scopolamine or vehicle 30 min before T1. In T1, the mouse was placed into the end of the start arm, facing the wall and away from the center. The mouse was then allowed to explore two arms of the Y-maze, while entry into the third arm was blocked. After the sample trial, the mouse was returned to its home cage for a 30 min inter-trial interval. In T2, the block in arm 3 was removed, the mouse was again placed into the start arm, and then allowed to access all three arms of the maze. If a mouse climbed on the maze wall, it was immediately returned into the abandoned maze arm. After each animal and between T1 and T2, the maze was wiped with a Quatricide® dilution to prevent odor cues. An arm entry was recorded when 85% of a mouse’s body entered the arm. Time in Novel Arm [%] was defined as the time spent in the novel arm divided by the time spent in all arms during the first minute of the retrieval trial T2. Forced Alternation [%] was defined as the percent of mice entering first the novel arm during T2 [22 (link)]. Mice with less than three arm entries in the first minute of T2 were excluded from the analysis.
The protocol for the forced alternation test was modified from Melnikova et al. [21 (link)]. Mice were handled for three days before testing. The test consisted of a 5 min sample trial (T1) followed by a 5 min retrieval trial (T2). For the scopolamine experiment, mice were dosed with scopolamine or vehicle 30 min before T1. In T1, the mouse was placed into the end of the start arm, facing the wall and away from the center. The mouse was then allowed to explore two arms of the Y-maze, while entry into the third arm was blocked. After the sample trial, the mouse was returned to its home cage for a 30 min inter-trial interval. In T2, the block in arm 3 was removed, the mouse was again placed into the start arm, and then allowed to access all three arms of the maze. If a mouse climbed on the maze wall, it was immediately returned into the abandoned maze arm. After each animal and between T1 and T2, the maze was wiped with a Quatricide® dilution to prevent odor cues. An arm entry was recorded when 85% of a mouse’s body entered the arm. Time in Novel Arm [%] was defined as the time spent in the novel arm divided by the time spent in all arms during the first minute of the retrieval trial T2. Forced Alternation [%] was defined as the percent of mice entering first the novel arm during T2 [22 (link)]. Mice with less than three arm entries in the first minute of T2 were excluded from the analysis.
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