EEG/EMG Recording and Sleep Analysis.

EEG/EMG recordings and sleep/wake analysis were performed as described previously (43). In brief, mice were anesthetized with isoflurane, and EEG/EMG electrodes were implanted and fixed to the skull with dental cement. After >7 d of recovery, mice were moved to recording cages, individually housed in the recording room, and acclimatized to the recording environment for >5 d. The mice were allowed to freely move in the cage while connected to the recording wire. EEG/EMG signals were amplified and filtered (EEG, 0.3 to 300 Hz; EMG, 30 to 300 Hz) using an amplifier AB-611J (Nihon Kohden), then digitized at a sampling rate of 250 Hz and acquired using our SleepyDAC software. States for each 20-s epoch were classified as wakefulness (wake), non-rapid eye movement (REM) sleep, or REM sleep, using our SleepyScore software, according to fast Fourier transformation (FFT) and visual inspection.