CHRONIC MILD STRESS

The chronic mild stress (CMS) animal model of depression has been used extensively to study the pathophysiology of depression in rodents (Frisbee et al., 2015; Willner et al., 1992). This model is based on the fundamental concept that chronic exposure to stressors is an important cause for the development of depression in humans. Animals were exposed to a randomized series of mild stressors on a daily basis. Specifically, two stressors per day for 8 weeks were delivered before behavioral screening. Mice experienced one stressor during the day and a different stressor during the night. Well-validated and approved standard stressors (Frisbee et al., 2015; Willner et al., 1992; Tye et al., 2012) were randomly chosen from the following list so that they are unpredictable for the subjects: cage tilt on a 45° angle for 12 to 16 h; food deprivation for 12 to 16 h; strobe light illumination for 2 to 6 h; crowded housing for 2 to 6 h; cage shaking (100 RPM) for 2 to 6 h; individual housing for 2 to 6 h; continuous illumination for 24 to 36 h; continuous darkness for 24 to 36 h; water deprivation for 12 to 16 h; damp bedding (200 mL water poured into bedding) for 12 to 16 h; bedding removal for 12 to 16 h. When not undergoing food or water deprivation stressors, water and food were available ad libitum. Non-CMS (control, CTRL) animals were housed for 8 weeks under standard housing conditions with access to food and water ad libitum. Most CTRL and CMS mice underwent three behavioral screening tests on three consecutive days in the following order: 1. Elevated plus maze (EPM), 2. Sucrose Preference Test (SPT), 3. Tail Suspension Test (TST). However, some mice shown in Figure 1A–1C did not experience all three behavioral screening tests, and some animals also underwent a social interaction test (SIT; Figure S1). In addition, for the data shown in Figures 6 and S6, all mice also underwent an open field locomotor test (OFT), which was performed on the fourth day.