The recording room was kept under 12-h light-dark cycles and a constant temperature (24-25 °C). To examine sleep-wake behavior under baseline conditions, EEG/EMG signals were recorded for two consecutive days from the onset of the light phase. EEG/EMG data were visualized and analyzed using a MatLab (MathWorks)-based, custom semi-automated staging program followed by visual inspection. EEG signals were subjected to fast Fourier transform analysis from 1 to 30 Hz with 1-Hz bin using MatLab-based custom software. Epochs containing movement artifacts were included in the state totals but excluded from subsequent spectral analysis. Sleep/wakefulness was staged into wakefulness, NREM sleep and REM sleep. Wakefulness was scored based on the presence of low amplitude, fast EEG and high amplitude, variable EMG.
NREMS was characterized by high amplitude, delta (1-4 Hz) frequency EEG and low EMG tonus, whereas REMS was staged based on theta (6-9 Hz) dominant EEG and EMG atonia. Hourly delta density during NREMS indicates hourly averages of delta density which is the ratio of delta power to total EEG power at each 20-second epoch. For the power spectrum of sleep/wakefulness, EEG power of each frequency bins was expressed as a percentage of the total EEG power over all frequency bins (1-30Hz) and sleep/wakefulness states34 (link),35 . For sleep deprivation, mice were sleep deprived for 2, 4 and 6 hours from the onset of the light phase by gently touching the cages when they started to recline and lower their heads. Food and water were available. To evaluate the effect of sleep deprivation, NREM delta power during the first hour after sleep deprivation was expressed relative to the same zeitgeber time of the basal recording or relative to the mean of the basal recording. For caffeine and modafinil injection experiments, mice were fully acclimatized for intraperitoneal injection before sleep recording. After 24-h baseline recording, mice received intraperitoneally caffeine (Sigma), modafinil (Sigma) or vehicle (0.5% methyl cellulose (Wako)) at ZT0, followed by 12-h recording. Injections were delivered once per week, with each injection followed by a 6-8 day washout period, during which mice remained in the recording chamber. To examine the sleep/wakefulness behavior under constant darkness, after 48-h recording under a LD 12: 12 cycle, EEG/EMG recording continued in constant darkness for 3 days.
NREMS was characterized by high amplitude, delta (1-4 Hz) frequency EEG and low EMG tonus, whereas REMS was staged based on theta (6-9 Hz) dominant EEG and EMG atonia. Hourly delta density during NREMS indicates hourly averages of delta density which is the ratio of delta power to total EEG power at each 20-second epoch. For the power spectrum of sleep/wakefulness, EEG power of each frequency bins was expressed as a percentage of the total EEG power over all frequency bins (1-30Hz) and sleep/wakefulness states34 (link),35 . For sleep deprivation, mice were sleep deprived for 2, 4 and 6 hours from the onset of the light phase by gently touching the cages when they started to recline and lower their heads. Food and water were available. To evaluate the effect of sleep deprivation, NREM delta power during the first hour after sleep deprivation was expressed relative to the same zeitgeber time of the basal recording or relative to the mean of the basal recording. For caffeine and modafinil injection experiments, mice were fully acclimatized for intraperitoneal injection before sleep recording. After 24-h baseline recording, mice received intraperitoneally caffeine (Sigma), modafinil (Sigma) or vehicle (0.5% methyl cellulose (Wako)) at ZT0, followed by 12-h recording. Injections were delivered once per week, with each injection followed by a 6-8 day washout period, during which mice remained in the recording chamber. To examine the sleep/wakefulness behavior under constant darkness, after 48-h recording under a LD 12: 12 cycle, EEG/EMG recording continued in constant darkness for 3 days.
