EEG/EMG Recording and Analysis

Mice were attached to a recording cable and acclimatized to a sleep recording chamber for at least 3 days. EEG/EMG recordings were performed four times: baseline, 1st week (1W), 2nd week (2W), and 3rd week (3W) during the stress exposure period. For baseline sleep, the EEG/EMG was recorded for 24 h. For sleep following stress exposure, the EEG/EMG was recorded for 45 h after the stress session [Zeitgeber time (ZT) 3.0-] on Day 6 of each week. For the naïve mice, EEG/EMG recordings were performed two times: 3 and 6 weeks after surgery, corresponding to baseline and 3W of the stressed mouse group in terms of the period after surgery. The EEG/EMG data were filtered (band pass 0.5–64 Hz), and collected and digitized at a sampling rate of 128 Hz, and further filtered post hoc by software (EEG: high pass 0.5 Hz). EEG signals were subjected to fast Fourier transform and further analysis using SleepSign (Kissei Comtec, Nagano, Japan). The vigilance state in each epoch was manually classified as wake, NREMS, or REMS by every 4-s epoch based on EEG patterns of delta power (0.5–4.0 Hz), the theta power (6.0–10.0 Hz) to delta power ratio, and the integral of EMG signals. Epochs with high EMG and low delta power were classified as wakefulness. Epochs with high delta power and low EMG were classified as NREMS. Epochs with even lower EMG (suggestive of muscle atonia) and high theta power to delta power ratio were classified as REMS (Figure 2A). If a single epoch contained multiple states, the state with the longest duration was assigned. For EEG spectrum calculation, to avoid the effect of mixed states, any epochs which contained multiple states were excluded. For each individual, the average EEG power spectrum of each state was calculated and normalized using the average absolute value of the total EEG power across all frequencies and across all 24 h.

Diurnal sleep–wake cycles were affected by WIRS. (A) Experimental timeline and example of EEG (above) and EMG (bottom) patterns in each stage of wake (left), NREMS (middle), and REMS (right). (B–D) Daily variation in wake (B), NREMS (C), and REMS (D) every 3 h. Because the baseline recording was only 24 h, the same data were used as the control for the first and second day recordings at 1W, 2W, and 3W. N = 7. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test. Two-way repeated measures ANOVA was applied over the entire 45 h recording period. Data are presented as means ± SEM.