EEG recording and sleep data analysis

Adult C57BL/6 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, 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 two-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 and EMG were recorded continuously for 7 days. Two cohorts of six and eight mice, respectively, underwent 1 day of BL recording and 3 days of SD recording, with 48 hours of recovery in between. Cohort 1 underwent SD at ZT4 to ZT8, ZT12 to ZT16, and ZT16 to ZT20. Cohort 2 underwent SD at ZT0 to ZT4, ZT8 to ZT12, and ZT20 to ZT24. SD was performed by gentle handling as described previously (67). 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, <–35 dB at 128 Hz), sampled with 512 Hz, digitally filtered (EEG, low-pass FIR filter: 25 Hz; EMG, band-pass FIR filter: 20 to 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 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: nonrapid eye movement (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 MathWorks, Inc., Natick, MA, USA). Relative frontal SWA was calculated relative to the mean SWA at ZT8 to ZT12 during the BL day. Sleep loss was calculated by comparing NREM sleep amount in each 4-h SD slot with the sleep amount in the same time of day of the corresponding BL day [p < 0.05, one-way analysis of variance (ANOVA)]. Sleep latency was analyzed by measuring the time each mouse stayed awake after the end of each 4-h SD until it slept for >1 min (p < 0.05, one-way ANOVA).