The social choice test was carried out in a three-chambered apparatus, as previously described [33 (link),71 (link),72 (link)], that consisted of a center chamber and two end chambers. Before the start of the test and in a counter-balanced sequence, one end chamber was designated the social chamber, into which a stimulus mouse would be introduced, and the other end chamber was designed the nonsocial chamber. Two identical, clear Plexiglas cylinders with multiple holes to allow for air exchange were placed in each end chamber. In the habituation phase of the test (Phase 1), the experimental mouse freely explores the arena with empty cue cylinders in place for 10 min. In the social choice phase of the test (Phase 2), an age-matched stimulus mouse (M1) (adult, gonadectomized A/J mice) was placed in the cylinder in the social chamber while an inanimate object was simultaneously placed into the other cylinder in the nonsocial chamber. The social novelty phase immediately followed. In the social novelty phase (phase 3), the object used in phase 2 was replaced by a novel mouse (M2). The experimental mouse was tracked during a 10 min trial as it explores M2 and the familiarized mouse (M1) used in the choice phase. Image analysis software (ANY-maze) was used to determine the time of cue exploration and visits to each cue (snout of experimental mouse within 1 cm of cue cylinder) in all phases. The data was verified with manual video review and scoring.
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