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).