For EEG recording, the rats were divided into 3 groups (control, Chry 100 mg/kg, Chry 200 mg/kg, n = 6). To record EEG and EMG, a 7-pin EEG/EMG cable (363–363; P1 Technologies) was inserted into the implanted pedestal, then connected to a commutator. To allow the rats to get accustomed to the tethered environment, each animal was placed in the experimental apparatus for 3 days prior to the recording. EEG/EMG signals were continuously collected and saved at 200 Hz sampling rates through a recording amplifier (AURA24; Grass-Telefactor, West Warwick, RI, USA) and data acquisition software (Twin 4.5.3; Grass Technologies LTM). Sleep state recordings were made twice, one day before saline (control) or Chry ext treatment (100, and 200 mg/kg) and one hour after the last treatment for 7 days, as shown in the timeline in Figure 1 b. Considering the circadian pattern of rodents, 6 h (10:00 to 16:00) were exported and used for analysis. The sleep–wake states (wakefulness, REM, and NREM sleep) were scored semi-automatically by 5 s epochs of EEG/EMG signals using the open-source software AccuSleep [29 (link)] written in MATLAB (R2021b, The MathWorks Inc., Natick, MA, USA). All results of the AccuSleep scoring were validated after visual analysis to reduce errors. Representative EEG/EMG waveforms are shown in Figure 1 c.
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