Electroencephalogram (EEG) recording and sleep data analysis

Adult wild-type (WT) mice were used for surgery (8 to 10 weeks old at surgery). Mice were implanted epidurally under isoflurane anesthesia for EEG recording. Right before and 24 hours after surgery mice were treated with an analgesic (Temgesic, 0.1 mg/kg, intraperitoneal). Gold-plated miniature screws (0.9 mm diameter) were used as EEG electrodes and positioned on the left hemisphere above the frontal cortex (1.5 mm anterior to bregma, 1.5 mm lateral to the midline) and the parietal cortex (2 mm posterior to bregma and 2 mm lateral to the midline). The reference electrode was placed above the cerebellum (2 mm posterior to lambda, 0 mm lateral to the midline). Screws were connected to copper wires and fixed to the skull with dental cement (Paladur 2-component system). Electromyography (EMG) was recorded using two gold wires (0.2 mm diameter) inserted bilaterally in the neck muscle. After 1 week of recovery EEG-EMG was recorded continuously for 7 days. 2 cohorts of 6 and 8 mice, respectively, underwent a BL days recording and 3 SD days with 48 hours of recovery in between. Cohort 1 underwent SD at ZT4-8, ZT12-16 and ZT 16-20. Cohort 2 underwent SD at ZT0-4, ZT8-12 and ZT20-24. SD was performed by gentle handling as described by (42). Both EEG and EMG signals were amplified (factor 2000), analog filtered (high-pass filter: –3 dB at 0.016 Hz; low-pass filter: –3 dB at 40 Hz, less than –35 dB at 128 Hz), sampled with 512 Hz, digitally filtered (EEG: low-pass FIR filter 25 Hz; EMG: band-pass FIR 20-50 Hz) and stored with a 128 Hz resolution. EEG power spectra were computed for 4-s epochs by a Fast Fourier Transform routine within the frequency range of 0.5 to 25 Hz. Between 0.5 Hz and 5 Hz, 0.5 Hz bins were used, and between 5 and 25 Hz 1 Hz bins were used. The corresponding slow-wave-activity (SWA) was calculated using the raw parietal and frontal EEG, as well as the raw and integrated EMG to visually score three vigilance states [non–rapid eye-movement sleep (NREM) sleep, rapid eye-movement sleep (REM), and wake] for 4-s epochs. Epochs containing artifacts were identified and excluded from the spectral analysis. Data analysis was carried out using MATLAB version R2015a (The Math Works, Natick, MA, USA). Relative frontal SWA was calculated relative to the mean SWA at ZT8-12 during the BL day. Sleep loss was calculated by comparing NREM sleep amount in each 4h SD slot to the sleep amount in the same time of day of the corresponding BL day [1-way analysis of variance (ANOVA), P < 0.05). Sleep latency was analyzed by measuring the time each mouse stayed awake after the end of each 4 hours SD until it slept for more than 1 min (1-way ANOVA, P < 0.05).