2 | METHODS
2.1. Animals
Specific pathogen-free (SPF) male C57BL/6J mice (WT) were purchased from Japan SLC. Cry1 −/− and Cry2 −/− mice of C57BL/6J background (CryDKO) were generated by Takasu et al. (2015). Animals were maintained in the animal facility of Aichi Medical University. The experimental protocols used in this study were approved by the Committee for Animal Research at Aichi Medical University.
2.2. Behavioral rhythm monitoring
Mice aged 2-12 months were housed individually, and their behaviors were monitored under a 12 h light (fluorescent light, ~100 lux) - 12 h dark cycle (LD) or constant darkness (DD). The light-off time was defined as Zeitgeber Time (ZT) 12. The locomotor activity of the mice was monitored using passive (pyroelectric) infrared sensors (PS-3241; EK-Japan, Japan). Data were collected and analyzed as previously described (Masubuchi et al., 2005 Ikegami et al., 2020) using The Chronobiology kit (Stanford Software Systems, Stanford, CA, USA). Mice were entrained to the LD for more than two weeks and used for experiments. Circasemidian/circadian periods of activity were identified using a chi-square periodogram (Masubuchi et al., 2001). Differences in periods between WT with and without caffeine administration were evaluated using Student’s t-test. Period values of Balb/c mice before and after MAP reported by Masubuchi et al. (2001) were re-evaluated by Student’s t-test. Period changes caused by caffeine administration and withdrawal in WT were evaluated using one-way ANOVA and Tukey HSD. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).
2.3. Caffeine administration
For chronic caffeine treatment, caffeine (FUJIFILM Wako 031-06792) was dissolved in distilled water (0.05%) (Nagasawa et al., 2001) and administered via drinking water.