Behavioral Testing: Marble Burying Test

This test was performed as previously described with slight modifications.66, 67, 68 Mice were removed from gnotobiotic isolators and allowed to acclimate to the testing room without being disturbed for 1 hour before testing. Lighting conditions and noise levels were kept constant across all tests. Clean transparent polycarbonate boxes (30 × 30 cm) were filled with a 5-cm layer of fresh, unscented, wood chip bedding. Twenty-five black glass marbles were placed lightly on top of the bedding, spaced equidistantly (5-cm apart) in a 5 × 5 arrangement. Animals (n = 12–13 mice per group) were placed gently in the corner of the testing cage and left undisturbed for 13 minutes, with the researcher out of the room. At the end of the test the mouse was removed promptly, with care taken to not disturb the marbles. The number of marbles buried during the test was recorded by 2 independent blinded observers. Marbles were considered buried when >60% was covered by bedding material. Scores from each observer for each mouse were averaged, and are presented as the percentage buried (number buried/total number of marbles in the cage). Marbles were rinsed in 70% ethanol, then in water, and dried before the next test. Bedding in the cage was flattened using a sterile cage bottom between tests. Cage bedding was changed between sexes and between groups of different colonization status.