The CUS procedure was modified referring to the published procedures.29–31 After 7 days of habituation, rats in model group and three VOZB treatment groups were housed individually and exposed to the following stressors for 14 days: wet bedding for 12 hours; 30-min restraint in a restraining device which was made of plexiglass and flexible nylon immobilization belts, thus restricting movement but allowing free respiration and air circulation; 12-h food and water deprivation (19:00–7:00); 10-min forced swim at 18°C in a cylindrical tank (60 cm height × 30 cm diameter) filled with fresh water to a 30 cm depth; reversing the light and dark cycle; 10-min tail pinch with a clothes-pin 1 cm from the base of the tail after being restrained in previous device; 15-min electric footshock (1.5mA, 30 s on and 150 s off). In order to maximize the unpredictability of stressors, all stressors were applied in a random order and at varying times, and the same stressors were not applied on two consecutive days. The CUS experimental schedule is outlined in Table 1. After daily CUS procedure, rats were placed in their home cages with fresh bedding and returned to the housing room until the next stress session. The normal group rats were handled daily for 2 min in the housing room, but not stressed.

Chronic Unpredictable Stress Experimental Schedule

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