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116The EEG/EMG signals were amplified using a Grass Instrument model 12 (West Warwick, RI, USA), and digitally filtered (30 Hz Low Pass Filter for EEG; 10-100 Hz Band Pass Filter for EMG). Subsequently, the EEG signals were captured at 256 Hz using data acquisition software (Vital Recorder; Kissei Comtec Co. Ltd., Matsumoto, Japan). EEG signals collected with ipsilateral bipolar EEG electrodes placed over motor and visual cortices, together with the bipolar EMG signals, were recorded for sleep scoring.Sleep recordingWe visually confirmed the sleep/wake stages based on EEG and EMG signals in 10-s epochs, according to our standard criteria using the Sleep-sign software (Kissei Comtec Co. Ltd.) (Figure 1). We required 50% or more of a specific state in each epoch to score the epoch. Firstly, wakefulness was characterized by desynchronized, low-amplitude, and mixed-frequency (>4 Hz) EEG with high EMG activity, which appears as a rhythmic theta/alpha (7-9 Hz) wave. Secondly, non-rapid eye movement (NREM) sleep was characterized by synchronized, high-amplitude, and low-frequency (0.25-4 Hz) EEG with low EMG activity. Thirdly, similar to wakefulness, rapid eye movement (REM) sleep was characterized by desynchronized, low- amplitude, and mixed-frequency EEG, whereas the EMG activity during REM sleep was low compared with that observed during NREM sleep. During REM sleep, some muscle fasciculation may be observed in the EMG trace. Typically, rhythmic theta/alpha (7-9 Hz) waves with reduced EMG activity are dominant. Changes in the sleep state were considered when at least one 10-s epoch was scored as appearing a different sleep stage, and the duration of the state episode was determined as the duration of a continuous single state episode. All sleep scoring was performed by a single observer who was blinded to animal information.Core body temperature and locomotor data analysesThe plastic cages were individually placed on the telemetry receiver (Series 4000, Mini Mitter) that transmitted the signals for core body temperature and locomotor activity every 1 min (Figure 1). Subsequently, the data were acquired using the Vital View software (Mini Mitter, OR). The 0.5- Experimental Procedure for Measuring Sleep in Surgical procedures for telemetry implant and headstageA telemetry implanting device (G2 E-Mitter; Mini Mitter OR, Oakmont, PA, USA) was intraper-itoneally implanted under 3% isoflurane anesthesia to evaluate the core body temperature and loco-motor activity. The mice were surgically prepared for EEG and EMG recordings with a headstage attached to the cable recorder. Two of the four electrodes, which consisted of stainless-steel screws, were implanted into the skull 1.5 mm lateral and 1.5 mm anterior to the bregma (over the motor cortex). The other two electrodes were implanted 3 mm lateral and 1 mm anterior to the lambda (over the visual cortex) for the EEG. Two Teflon-coated stainless-steel wires were inserted into the neck extensor muscles on both sides for the EMG. The six electrodes were attached to one 2 × 3 pin header that was secured to the skull with dental acrylic. Postoperatively, the mice subcutaneously received an analgesic (carprofen, 3 mg/kg) and antibiotic (enrofloxacin, 25 mg/kg), and were allowed to recover for 2 weeks before the experiments.The Procedures of data collectionAfter 2 weeks of the postoperative recovery period, the mice were individually moved into experimental cages specifically modified to include plastic micro- isolation cages fitted with a low-torque slip-ring commutator (Biella Engineering, Irvine, CA, USA). Each cage was placed in the custom-designed recording chamber with individual ventilated compartments. The following day, the headstages of the mice were connected to the EEG/EMG recording cables, which consisted of the slip ring commutator through a 15-20 cm of lightweight six-strand shielded signal cable (NMUF6/30-4046SJ; Cooner Wire, Chatsworth, CA, USA). The commutator output was amplified. The mice had ad libitum access to food and water in the experi-mental cages. The room temperature was main-tained at 23°C ± 1°C. A 12-h light/dark cycle was implemented throughout the experiment. After 1 week of habituation to the experimental conditions, consecutive EEG/EMG, core body temperature, and locomotor activity recordings were performed.Mice