Analyzing Sleep Behavior in Mice through Electroencephalogram and Electromyogram Recordings

Secure an anesthetized mouse on a stereotaxic frame.

Disinfect the shaved skin, cover the mouse with a drape, and make a midline incision to expose the skull.

Drill two holes into the skull at specific stereotaxic coordinates.

Prepare a recording electrode unit with a four-pin header connected to stainless steel wires.

Use two wires with screws as electroencephalogram, or EEG electrodes to measure brain electrical activity.

Use the other two wires as electromyogram, or EMG electrodes to measure muscle electrical activity.

Screw the EEG electrodes into the holes, and fix the electrode assembly to the skull.

Insert the EMG electrodes into the neck muscles.

Close the incision with sutures and allow the mouse to recover.

Transfer the mouse to a soundproof recording chamber.

Connect the electrode assembly to a recording cable and link it to an amplifier.

Record the EEG and EMG signals to analyze different sleep stages and wakefulness in mice. 

Before starting, sterilize all the surgical tools in a hot-bead sterilizer. Once the mouse is confirmed as anesthetized by a toe pinch, shave off the hair on the head and neck.

Next, secure the mouse to the stereotaxic frame, and fix the head between the two ear bars. Then, apply ophthalmic ointment to the eyes to prevent dryness. Proceed with cleansing the shaved skin with alcohol and iodine-based scrubs.

Then, cut along the midline with a scalpel to expose the skull, and secure the skin to the side using clips to keep the surgical area open. Using a carbide cutter with a 0.8-millimeter drill, make two holes. Put one hole over the frontal cortical area. Put the second hole over the parietal area of the right hemisphere.

Next, using a jeweler's screwdriver, screw stainless steel EEG recording screws into the holes. Insert the screws with just two to two and a half turns to obtain epidural positioning over the cortex. The screws should not wiggle once inserted. This is very important.

Now, fix the electrode assembly with the pins upward to the skull. The electrodes must be attached straight on. Use a cotton swab to help secure the electrodes' position and glue the electrode down using cyanoacrylate, followed by dental cement.

Next, make small holes in the trapezius and insert the EMG electrode into those holes. Then, suture the skin with 0.1-millimeter diameter silk thread. Now remove the mouse from the stereotaxic frame, and place it on a heat pad.

Give the mouse intraperitoneal injections of ampicillin and meloxicam, and monitor the mouse until it regains sternal recumbency. Then, house the mouse alone.

One week after implanting the electrodes, transfer the mouse to an experimental cage in a soundproof recording chamber. Secure the EEG/EMG electrode assembly on the mouse to a recording cable connected to a slip ring.

Connect the AD converter cable to an EEG/EMG signal filter amplifier connected to the slip ring. Use an AD converter to convert the signal to a 128-hertz digital signal and finally, record the signal to a computer.

Habituate the mouse for two to three days in the recording chamber. Start the EEG/EMG recording software. To begin, click on the data file information tab and click the box next to the file name. Enter a file name and click Save. Then, from the recording condition tab, select all the EEG/EMG channels, which need to be recorded. Also, select the sampling frequency under this tab.

Now in the channel information tab, check if the selected channels are displayed properly. Once confirmed, start the record by first selecting the timer setting tab. Then, click Monitor to display EEG and EMG. If the signals are displayed correctly, set the clock time for the beginning and end of the recording in the main timer area. Then, click the monitor button to start the recording.