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.