Chronic Stress-Induced Wheel-Running Behavior

Five-week-old male C3H/HeN mice (Japan SLC Inc., Hamamatsu, Japan) were individually housed in plastic cages containing paper-chip bedding and running wheels (SW-15; Melquest Y.K, Toyama, Japan). The mice had free access to a standard diet (AIN-93G: Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water under a 12 h light-12 h dark cycle (lights on at 08:00) for four weeks until daily wheel-running activity reached a plateau. The CSD mice were then exposed to psychophysiological stress for one week to induce CSD^{23 (link)}. Briefly, paper-chip bedding was replaced with water to a depth of 1.5 cm, which caused the mice to remain on the wheels throughout every day of the study. Wheel-running activity was continuously recorded at 1-min intervals using Chronobiology Kits (Stanford Software Systems, Stanford, CA, USA) and activity data are displayed as actograms. All protocols complied with the guidelines for animal experiments published by the National Institute of Advanced Industrial Science and Technology (AIST) and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The AIST Animal Care and Use Committee approved all experimental protocols (Permission No: #2021-338).