Head mount

Wild type mice were anesthetized by 1% Pentobarbital with a dose of 0.12 ml per 25g body weight. Subsequently, mice were placed in the stereotaxic apparatus and fixed by ear bars. Then, a 1 cm rostral-caudal incision through the skin on the top of the mouse head was made to expose the top surface of the skull. 3% hydrogen peroxide was used to clean and disinfect the top surface of the skull. A small amount of cyanoacrylate was put on the bottom of the 6 pore head-mount (Purchased from Pinnacle Technology Inc) for gluing the head-mount on the top of the mouse head. Then, place the head-mount on the top surface of the dry skull of the mouse until the head-mount cannot be moved. Finally, the mouse was put in separate cages to wait for PTZ injection after suturing the incision. Before peritoneal injection of PTZ, the configured preamplifier was connected to the head-mount on the head top of the mouse according to standard protocol provided by Pinnacle Technology Inc (USA) for 1 hour. The 3 channel preamplifier was further connected to computer system through wires to collect EEG signals. The data were stored in computer and were analyzed by software provided by Pinnacle Technology Inc.

Naïve, 10-week-old WT and α2δ-1 KO littermates were implanted for EEG recording based on a Tufts Institutional Animal Care and Use Committee approved protocol. Briefly, animals were anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine). An incision was made on the scalp, the surface of the skull was dried, and four burr holes were drilled with care to avoid puncturing the dura. Four 0.1" stainless steel screws (Pinnacle Technologies) were gently screwed into the drilled holes. A common reference electrode was placed anterior to the left side of the intraparietal bone, and two EEG electrodes were placed in the left front bone (for front/motor cortex) and in the right parietal bone (for somatosensory cortex). A separate ground electrode was also placed anterior to the right side of the intraparietal bone. The screws were then attached to a headmount (Pinnacle Technology), which was fixed using dental cement. Following surgery, the animals were given 7 d to recover. Chronic 24-h EEG recordings were acquired using a 100× gain preamplifier high-pass filtered at 1.0 Hz (Pinnacle Technology) with video monitoring for two weeks. LabChart Pro software (ADInstruments, RRID:SCR_001620) was used for data acquisition and analysis.

For EEG and EMG recordings, mice were implanted with a head mount (Pinnacle Technology Inc., Laurence, KS, USA) parallel to the sagittal suture. Four stainless steel screws were placed 1.5 mm lateral to the sagittal suture, 2.0 mm anterior of bregma, and 4.0 mm posterior to bregma to record EEG signals. EMG signals were acquired by a pair of multi-stranded stainless steel wires inserted into the neck extensor muscles. One week after surgery, animals were habituated for 3 days before EEG/EMG signals were acquired with a 3-channel EEG/EMG tethered system (Pinnacle Technology Inc.) and digitalised by SIRENIA SLEEP PRO^® software (Pinnacle Technology). EEG and EMG data were recorded in 10 s epochs and automatically scored by Sleep Sign^® software (Kissei Comtec, Matsumoto, Japan). Automatically scored data were manually inspected and corrected if required.