analyzing sleep behavior in mice through electroencephalogram and electromyogram recordings

Analyzing Sleep Behavior in Mice through Electroencephalogram and Electromyogram Recordings

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&#39;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&#39; 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.

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JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

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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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare a cable for connecting the electrode with the slip ring as described below. This cable can be reused.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Insert each crimp socket into a 4-position crimp housing.</li>
		<li>Completely cover the crimp sockets with epoxy adhesive by using a thin wooden stick or toothpick.</li>
	</ol>
	</li>
</ol>
2. Implantation of Electrodes in the Mouse Head (Duration: Approx. 20 min)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>By using a jeweler&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Recording and Acquiring EEG/EMG Data
<ol>
	<li>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 &#177; 1 &#176;C and automatically control cycles of 12-hr light/12-hr dark (lights on at 08:00, illumination intensity ~ 100 lux).</li>
	<li>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.</li>
	<li>Habituate the mouse for 2 - 3 days in the recording chamber.</li>
	<li>Subsequently, start EEG/EMG recording software.
	<ol>
		<li>Select the &#39;Data file information&#39; tab and click the box next to the file name. Enter a file name and click &#39;Save&#39;.</li>
		<li>Select the &#39;Recording condition&#39; tab and select all EEG/EMG channels which will be recorded.</li>
		<li>Select the sampling frequency (128 Hz) in the &#39;Recording condition&#39; tab.</li>
		<li>Check if the selected channels are displayed properly in the &#39;Channel information&#39; tab.</li>
		<li>Select the &#39;Timer setting&#39; tab and click &#39;Monitor&#39; to display EEG and EMG.</li>
		<li>Check if EEG/EMG signals are displayed correctly.</li>
		<li>Select the &#39;Timer setting&#39; tab and set the clock time for the beginning and end of the recording in the &#39;Main Timer&#39; area.</li>
		<li>Click &#39;Monitor&#39; in the &#39;Timer setting&#39; tab to start the recording.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-pin header</td>
			<td>Hirose</td>
			<td>A3B-4PA-2DSA(71)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Meiji Seika</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog-to-digital converter</td>
			<td>Contec</td>
			<td>AD16-16U(PCIEV)</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbide cutter</td>
			<td>Minitor</td>
			<td>B1055</td>
			<td></td>
		</tr>
		<tr>
			<td>Crimp housing</td>
			<td>Hirose</td>
			<td>DF11-4DS-2C</td>
			<td></td>
		</tr>
		<tr>
			<td>Crimp socket</td>
			<td>Hirose</td>
			<td>DF11-30SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement (Toughron Rebase)</td>
			<td>Miki Chemical Product</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy adhesive</td>
			<td>Konishi</td>
			<td>#16351</td>
			<td></td>
		</tr>
		<tr>
			<td>FFC/FPC connector</td>
			<td>Honda Tsushin Kogyo</td>
			<td>FFC-10BMEP1(B)</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat cable</td>
			<td>Hitachi Cable</td>
			<td>20528-ST LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant glue (Aron Alpha A)</td>
			<td>Toagosei</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer Ingelheim</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Kyoritsu Seiyaku</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal amplifier</td>
			<td>Biotex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sleep recording chamber</td>
			<td>APL</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SleepSign software</td>
			<td>Kissei Comtec</td>
			<td>N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr>
			<td>Slip ring</td>
			<td>Biotex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel screw</td>
			<td>Yamazaki</td>
			<td>N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>Cooner Wire</td>
			<td>AS633</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Oishi, Y., et&#160; al. <a href="https://app.jove.com/v/53678">Polygraphic Recording Procedure for Measuring Sleep in Mice.</a> J. Vis. Exp. (2016).
This video demonstrates a protocol for implanting an electroencephalogram (EEG) and electromyogram (EMG) electrode assembly in an anesthetized mouse. The process involves securing the mouse on a stereotaxic frame, making a midline incision in the skull, drilling holes for the electrodes, and fixing the electrode unit for signal recording. After recovery, the mouse&#39;s sleep-wake behavior is analyzed using the recorded signals.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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.&#160;

91 조회수. 뇌파 및 근전도 기록을 통한 마우스의 수면 행동 분석

비디오: 뇌파 및 근전도 기록을 통한 마우스의 수면 행동 분석 - 실험

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

침술에 의해 쥐와 마우스에서 간질 Electrocorticograms (ECoGs)의 조작 및 절전

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

연구

JoVE Journal

행동분석학

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

새로운 방법으로 자유롭게 움직이는 신생아 쥐의 뇌파 기록

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

신경과학

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

마우스에서 수면/각성 상태를 모니터링하는 동안 신경 회로의 광유전학 적 조작

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Analyzing Sleep Behavior in Mice through Electroencephalogram and Electromyogram Recordings

내레이션 대본

Analyzing Sleep Behavior in Mice through Electroencephalogram and Electromyogram Recordings