Polygraphic Recording Procedure for Measuring Sleep in Mice

The overall goal of this polygraphic recording procedure is to determine the vigilant state of mice and measure the time spent in wakefulness, rapid eye movement sleep or non-rapid eye movement sleep. This method can help answer key questions in nearly every neuroscience field that seeks to correlate behavior and physiology with the activity of cortical neurons in behaving animals. The main advantage of this technique is that it can record a lot of nodes, electroencephalogram and electromyogram and can be combined with other brain implants such as optic fibers and infusion cannulas.

Visual demonstration of this method is very important because the implantation of the electrode needs to be performed properly and the quality of the electrode is extremely critical to judge the physical states. Demonstrating the procedure will be Yohko Takata, a postdoc from my laboratory. 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 house 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 electrode's 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. If the EEG/EMG recording includes drug administrations, gently handle the mouse on each day as those there was a drug administration being given. Subsequently, 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.

Now, record the signals under baseline conditions and different treatment conditions over several days. When the experiment is finished, euthanize the mouse with pentobarbital. Start the software for EEG/EMG analysis.

Open the EEG/EMG raw data, which is a KCD file. Click the Sleep tab and set the Epoch time to 10 seconds. Then, select Multi-screening to automatically score all ten second epochs into three stages on the basis of the amplitudes of the EEG and the EMG and based on the power spectral analysis of the EEG.

Next, click on the FFT condition for EEG tab. There, set the parameters for the power spectral analysis. Use 256 datum points corresponding to every two seconds of EEG, use the hamming window function, and use an average of five spectra per epoch.

Then click Ok.Now, click Start Screening to begin the automatic screening. Then, open the stored data, which is saved in a RAF file format and check the results of the automatic screening. As needed, correct the results if they do not meet the standard criteria.

To make a correction, click and hold the left mouse button on an incorrectly scored epoch and drag the cursor across the string of incorrectly scored epochs. Release the left mouse button and select the correct behavioral state in the popup window. Under baseline conditions, the mice exhibited a clear circadian sleep-wake rhythm consistent with their nocturnal nature.

During the 12 hour light period, the mice averaged 6.7 hours of NREM sleep and 0.9 hours of REM sleep. Whereas, during their 12 hour dark period, wakefulness was predominant. Other measurements were also recorded.

These included the sleep epoch distribution, their mean durations and the stage transitions for each vigilant state. The EEG power spectrums for NREM and REM sleep show a strong EEG power density in the frequency range of 0.5 to four hertz for NREM sleep compared to a power density from six to 10 hertz for REM sleep. To assess the impact of caffeine on sleep, C57 black 6 mice were treated with a vehicle on one day and with caffeine on the next day.

Intraperitoneal injections of caffeine were given early in the light cycle. As expected, the recording revealed the caffeine increased the amount of wakefulness in the mice. A three-fold increase in wakefulness occurred for three hours after the injection.

Once mastered, the electrode implantation procedure can be done in less than 20 minutes. Scoring and correcting the EEG data for a 24 hour period can be done in about 30 minutes. After watching this video, you should have a good understanding of how to set up recording of electroencephalogram and electromyogram in mice and how to assess if and how long an animal is awake or asleep.