Analyzing Sleep Behavior in Mice through Electroencephalogram and Electromyogram Recordings

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Electrodes and Cables for EEG/EMG Recordings

1. Prepare EEG/EMG recording electrode according to the following procedure.
   Note: The electrode is disposable and can be used only for 1 animal. Plan carefully the wiring configuration for all connectors. Place marks on the connectors for the correct orientation.
   1. Solder each pin of a 4-pin header to a 2-cm stainless steel wire. In brief, hold one end of the wire to the pin, place a hot soldering iron onto the wire-pin joint, and melt some solder to ensure that just enough solder runs smoothly into the joint. Be careful not to apply too much heat to the pin; otherwise, the plastic around the pins will melt.
   2. Solder the free end of each of 2 wires attached to the pin header to the head of a 1.0-mm-diameter stainless steel screw. In brief, hold the free end of the wire to the thread below the screw head, place a hot soldering iron onto the wire-screw joint, and melt some solder to ensure that just enough solder runs smoothly into the joint. The 2 wires with screws serve as EEG recording electrodes, whereas the 2 wires without screws serve as EMG recording electrodes.
   3. Use scissors to strip off 1 mm of the insulation at the end of the EMG electrodes to increase the quality of the EMG signal.
   4. Completely cover all soldered pins with epoxy adhesive by using a thin wooden stick or tooth pick to reduce the electrical noise during EEG/EMG recordings.
2. Prepare a cable for connecting the electrode with the slip ring as described below. This cable can be reused.
   1. Solder each pin of a 4-pin FFC/FPC connector with a wire of a 30-cm flat cable. In brief, hold the stripped end of the wire to the pin, place a hot soldering iron onto the wire-pin joint, and melt some solder to ensure that just enough solder runs smoothly into the joint.
      Note: Choose the length of the flat cable which is appropriate for the height of the experimental animal cage used for EEG/EMG recordings.
   2. Solder crimp sockets to a tip of the wires on the other end of the flat cable. In brief, hold a crimp socket to the stripped free end of the wire, place a hot soldering iron onto the wire-socket joint, and melt some solder to ensure that just enough solder runs smoothly into the joint.
   3. Insert each crimp socket into a 4-position crimp housing.
   4. Completely cover the crimp sockets with epoxy adhesive by using a thin wooden stick or toothpick.

2. Implantation of Electrodes in the Mouse Head (Duration: Approx. 20 min)

1. Sterilize all surgical tools in a hot bead sterilizer before the surgery. Anesthetize a male mouse (10 – 20 weeks old, 20 – 30 g) with an intraperitoneal injection of pentobarbital (50 mg/kg). After checking that the mouse is deeply anesthetized by pinching a toe, shave the hair on the head and neck with clippers.
2. Move the mouse to the stereotaxic frame and fix the head between the 2 ear bars. Apply petroleum jelly on the eyes to prevent dryness. Cleanse the shaved skin with alcohol and cut it along the midline with a scalpel to expose the skull. Clip the skin to keep the surgical area open.
3. By using a carbide cutter (drill size: 0.8-mm diameter), drill 2 holes into the skull, one over the frontal cortical area (1 mm anterior to bregma, 1.5 mm lateral to the midline) and the other over the parietal area (1 mm anterior to lambda, 1.5 mm lateral to midline) of the right hemisphere, according to stereotaxic coordinates of Paxinos and Franklin.
4. By using a jeweler's screwdriver, place stainless steel EEG recording screws in the holes and make 2 – 2.5 turns for each screw for epidural positioning over the cortex.
   Note: Do not insert screws too deep to prevent damage to the brain tissue. Check that the screws are tightly fixed on the skull. This is important to have stable EEG signals during a long period of multiple recordings (typically, more than 1 month). Wiggly screws produce EEG artifacts and may come off before the end of the experimental schedule.
5. Fix the electrode assembly (cf. Section 1.1, pins turned upward) with instant glue to the skull and cover it with dental cement. Make bilaterally small holes with forceps in the trapezius (neck) muscles and insert the stainless-steel wires that serve as EMG electrodes into the holes. Suture the skin with a silk thread (0.1 mm diameter) to avoid exposure of the muscle.
6. Remove the mouse from the stereotaxic frame. Administer ampicillin (100 mg/kg) and meloxicam (1 mg/kg) intraperitoneally to the mouse to avoid bacterial infection and to reduce post-surgical pain, respectively. Keep the mouse on a heat pad and monitor it until it has regained sufficient consciousness to maintain sternal recumbency. House the mouse individually during recovery to avoid removal of electrodes by other animals and administer meloxicam (1 mg/kg) intraperitoneally on the first day after the surgery.

3. Recording and Acquiring EEG/EMG Data

1. After a 1-week recovery period, house each mouse individually in an experimental cage placed in a soundproof recording chamber. Maintain an ambient temperature of 23 ± 1 °C and automatically control cycles of 12-hr light/12-hr dark (lights on at 08:00, illumination intensity ~ 100 lux).
2. Connect the EEG/EMG electrode assembly on the mouse head to a recording cable. Ensure that the recording cable is connected to a slip ring (which is designed so that movements of the mouse are not restricted by twisting of the cable) and an EEG/EMG signal amplifier. Filter EEG/EMG signals (EEG, 0.5-64 Hz; EMG, 16-64 Hz), digitize at a sampling rate of 128 Hz by an analog-to-digital converter (A/D) and finally record on a computer running EEG/EMG recording software.
3. Habituate the mouse for 2 – 3 days in the recording chamber.
4. Subsequently, start EEG/EMG recording software.
   1. Select the 'Data file information' tab and click the box next to the file name. Enter a file name and click 'Save'.
   2. Select the 'Recording condition' tab and select all EEG/EMG channels which will be recorded.
   3. Select the sampling frequency (128 Hz) in the 'Recording condition' tab.
   4. Check if the selected channels are displayed properly in the 'Channel information' tab.
   5. Select the 'Timer setting' tab and click 'Monitor' to display EEG and EMG.
   6. Check if EEG/EMG signals are displayed correctly.
   7. Select the 'Timer setting' tab and set the clock time for the beginning and end of the recording in the 'Main Timer' area.
   8. Click 'Monitor' in the 'Timer setting' tab to start the recording.
5. Record EEG/EMG signals under baseline (i.e., sleep/wake behavior of a freely behaving mouse). Euthanize the mouse with an intraperitoneal injection of pentobarbital (200 mg/kg) after the last experimental day.