Behavior Testing
To assess anxiety and compulsive behavior, we performed marble burying in a standard marble burying condition. A set of 20 glass marbles were evenly spaced in a rat cage on sawdust bedding. For the duration of 20 minutes, mice explored and buried marbles undisturbed. Marbles that were at least two-thirds covered by corn cob bedding at the end of the testing period were counted as buried. The number of marbles buried was scored by a single skilled observer; inter-group differences were assessed by one-way ANOVA (p-value < 0.05).
To assess spatial working memory and cognitive integrity, we performed a Y-maze behavior test(“Purpose and Cognition: The Determiners of Animal Learning.” n.d.; Hughes 2004). Mice were placed in one arm of a standard Y-maze (MazeEngineers, Boston, MA), consisting of a high walled chamber with three arms connected at 120°. Mice were video recorded exploring the maze undisturbed for 10 minutes. After each test, the maze was cleaned with 70% ethanol and then water to eliminate confounding scents for the subsequent trials. The total number of maze arm entries and spontaneous alterations (SA) were scored by a treatment-blinded skilled observer. Spontaneous alternation percentage was calculated as the number of spontaneous alterations ÷ (number of entries -2) × 100. One spontaneous alternation was counted when three consecutive entries into unique arms (e.g., A, B, C) were recorded on video. Arm entries were recorded when the test mouse was positioned with all four feet inside the maze arm. The total number of arm entries was also recorded as a measure of exploration. Results were determined significant by a one-way ANOVA (p-value < 0.05).