This work was supported by Ministry of Science and Technology (MOST) grants MOST104-2410-H-002-053 &amp; NSC99-2320-B-002-026-MY3. This manuscript was edited and proofread by Mr. Brian Chang, who has experience revising professional documents.

All experimental protocols are approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University.
1. Stereotaxic Surgery for Implanting ECoG Electrodes, EMG Electrodes, Brain Thermistor, and Injection Guide Cannula
<ol>
	<li>For rats (250 - 350 g, 6- to 8-week-old Sprague-Dawley rats)
	<ol>
		<li>Anesthetize the rats by IP injection with 50 mg/kg zoletil. Confirm the proper depth of anesthesia by observing a lack of response after pinching the...

<ol>
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Choosing a feasible epilepsy animal model is essential for each experimental purpose. One of our goals is to elucidate the effects of EA on epilepsy suppression. EA is an alternative medicine that may exhibit therapeutic effect in epilepsy and has been documented in ancient Chinese literature. However, there is a lack of scientific evidence to prove it. To determine the effects of EA on epilepsy, we primarily focused on the effects of EA on mild focal epilepsy, rather than on severe generalized seizure or SE. Our previou...

Epilepsy is a common neurological disorder in which recurrent seizures occur throughout a patient&#39;s lifespan. Most epileptic recurrences can be well-controlled by anti-epileptic drugs (AEDs). However, about 30% of epileptic patients develop refractory epilepsy1. Epilepsy causes sleep disturbances, which can further exacerbate recurrence. Evidence demonstrates that epilepsy may either disrupt sleep at night or may cause excessive daytime sleepiness2,3. Our previous studies further indicate that epilepsy occurring at zeitgeber time (ZT) 0, i.e., the beginning of the light period in the light:dark cycle, decreases sleep; this is mediated by corticotropin-releasing hormone (CRH), a homeostatic factor. Epilepsy at ZT13 (the beginning of the dark period) enhances the expression of another homeostatic factor, interleukin-1 (IL-1), which increases sleep. Sleep circadian rhythms are altered when epilepsy occurs at ZT6, the middle of the light period4,5. On the other hand, sleep problems further exacerbate the progression and recurrence of epilepsy6. Based on the aforementioned evidence, we try to reveal an optimal therapeutic method to simultaneously control epilepsy and prevent sleep disruptions in epilepsy patients. We previously found that electroacupuncture (EA) with a 10-Hz stimulation frequency, in which a certain amount of current is delivered into the acupoint through a stainless-steel needle, successfully suppresses electrocorticogram (ECoG) epileptic activities and epilepsy-induced sleep disturbances7. EA with a 100-Hz stimulation frequency further deteriorates epileptic activities and sleep disruptions in rats8,9. This successful experiment depends on three factors: firstly, a feasible epileptic animal model; secondly, a method for sleep recording and analysis in rodents; and thirdly, the accurate performance of acupuncture and the accuracy of the acupoint locations.
Epilepsy has been categorized into two major types: focal epilepsy and generalized epilepsy. We are interested in focal temporal lobe epilepsy (TLE), generalized epilepsy, status epilepticus (SE), and the recurrence of spontaneous generalized epilepsy. Therefore, different manipulations are applied to create suitable epileptic models for our experiments. To establish focal TLE, a low dose of pilocarpine is administered into the left central nucleus of the amygdala (CeA). To verify this model, six ECoG electrodes are implanted on the frontal (F1 &#38; F2), parietal (P1 &#38; P2), and occipital (O1 &#38; O2) lobes in both the left and right hemispheres, and another two reference electrodes (R1 &#38; R2) are placed over the cerebellum in both hemispheres. An additional microinjection guide cannula is surgically implanted into the left CeA (AP, 2.8 mm from bregma; ML, 4.2 mm; DV, 7.8 mm relative to bregma). The coordinates are adapted from the Paxinos and Watson rat atlas10. If the focal TLE is successfully induced, only the recording from the electrode on left parietal cortex (P1), which is near the left CeA, should acquire the dominant epileptiform ECoGs, with no significant epileptiform ECoGs recorded from the other ECoG electrodes. Intraperitoneal (IP) injections of pilocarpine into rats induce generalized epilepsy and SE, but this can be fatal. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induce spontaneous generalized epilepsy in mice and also ensure the mice&#39;s survival. Two wire ECoG electrodes are implanted into the frontal and parietal cortices in the mice to receive ECoG signals and to verify spontaneously recurrent epilepsy.
Polysomnography (PSG) is a comprehensive method to record physiological changes that occur during sleep, and it can objectively classify sleep into different stages of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. PSG records parameters of body functions, including brain waves (electroencephalogram, EEG), eye movements (electrooculogram, EOG), skeletal muscle tones (electromyogram, EMG), heart rhythms (electrocardiogram, ECG), and blood oxygen levels and respiratory parameters. In rats, we record ECoGs, EMGs, cortical temperature, and locomotor activity to classify vigilance states into wakefulness, NREM sleep, and REM sleep. Sleep analysis in mice is conducted using ECoGs, EMGs, and locomotor activity results. Rats are surgically implanted with three ECoG screw electrodes at the frontal, parietal, and contralateral cerebellar cortices by stereotaxic surgery. Post-acquisition determination of the vigilance states (wakefulness, NREM sleep, and REM sleep) is conducted according to the parameters acquired from the ECoGs, EMG, brain temperature, and locomotor activity. Detailed criteria for categorizing the animal&#39;s behavior in both rats and mice are described in the protocol.
Both rats and mice need to be anesthetized with a low dose of zoletil (25 mg/kg), which is half the dosage of anesthetics normally administered during stereotaxic surgery, before performing manual acupuncture or EA. This dosage allows animals to wake up 30 to 35 min after the injection. Either manual acupuncture or EA is performed at the beginning of the dark period, with a constant time period of 30 min, and each animal is consecutively treated for two to three days. Stimulating EA currents are delivered into a particular acupoint through a stainless-steel needle that is inserted into the acupoint. The stimulus current is a train of biphasic square pulses, in which the pulse duration is 150 ms and the stimulation intensity is 1 mA. If a dry needle is used for manual acupuncture, the needle inserted into the acupoints is twitched 10 times every 5 min. The difficult part of manual acupuncture or EA is to localize the acupoints in rodents. The location of acupoints in rats or mice is similar to their anatomical location in humans. For example, the bilateral Fengchi acupoints are located 3 mm away from the posterior median line on the neck, between the two ears, which is similar to its anatomical location in humans11. Furthermore, the acupoints with low impedance on the skin can be further confirmed. Sham acupuncture or sham EA manipulation is necessary for acupuncture or EA experiments. Sham acupuncture or sham EA should be performed at a non-acupoint located close to the acupoint, such as near the axilla12.
To successfully investigate the effects of acupuncture or EA on epilepsy and epilepsy-induced sleep disruptions, the following factors must be in place: a feasible epileptic animal model, the precise analysis of epileptiform ECoGs and the recurrence of epilepsy, a method to classify vigilance states, and the accurate performance of acupuncture or EA in rodents.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Drugs</td>
			<td style="width:277px;"></td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zoletil</td>
			<td>Virbac</td>
			<td></td>
			<td>50 mg/kg i.p.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pilocarpine</td>
			<td>Sigma-Aldrich</td>
			<td>P6503</td>
			<td>300 mg/kg i.p.; 1.2 mg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTZ</td>
			<td>Sigma-Aldrich</td>
			<td>P6500</td>
			<td>0.035 mg/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysporin</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECoG electrode</td>
			<td>Plastics One</td>
			<td>E363/20</td>
			<td>screw electrode for rats</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pedestal</td>
			<td>Plastics One</td>
			<td>MS363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cannula</td>
			<td>Plastics One</td>
			<td>C315G/spc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermistor</td>
			<td>Omega Engineering</td>
			<td align="right">44008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental acrylic</td>
			<td>Tempron</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td></td>
			<td>Dural arms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recording equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECoG amplifier</td>
			<td>Colbourn Instruments</td>
			<td>V75-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A/D Board</td>
			<td>National Instruments</td>
			<td>NI PCI-6033E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infrared-based motion detectors</td>
			<td>Biobserve GmbH</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICELUS</td>
			<td>G-System</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxoScope 10 Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acupuncture needs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless needles</td>
			<td>Shanghai Yanglong Medical Articles Co.</td>
			<td>32 gauge x 1&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Functions Electrical Stimulator</td>
			<td>I.T.O., Japan</td>
			<td>Trio 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcuPen</td>
			<td>Lhasa OMS</td>
			<td>Pointer Excel II</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

There are different rat and mouse models to satisfy the needs of different epilepsy types. To induce focal TLE, 0.5 &#181;L of pilocarpine (2.4 mg/&#181;L) is administered into the left CeA. The predominant epileptiform ECoGs are acquired from the ECoG electrode on the parietal lobe of the left hemisphere (Figure 1A: b), and rare epileptic activities are picked up from the rest of the ECoG electrodes (Figure 1A: a, c, <st...

Watch this Scientific Journal Video about Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture at JoVE.com

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.

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 ...

The overall goal of this procedure is to assess epilepsy, sleep and the effects of acupuncture in an animal model. This method can help answer key questions in neuroscience field about how to establish epilepsy, analyze sleep and perform acupuncture in a small animal model. The main advantages of this technique are that the lateral cortical and the lateral myogram electrodes in that semester can be implanted quickly and easily by stereotaxic surgery.Demonstrating this procedure will be Yi-Jou Wu, a graduate student from my laboratories. After confirming the proper depth of sedation by a lack of response to a toe pinch, apply eye ointment to a six-to eight-week-old 250 to 350 gram Spregg Dolly rat from the first group. Use clippers to remove the hair from the top of the skull.Disinfect the skin with sequential applications of povidone iodine solution and 75 percent ethanol, followed by antibiotic injection to prevent infection. Next, mount the animal onto a stereotaxis machine and then place an ear bar into the ear canal. Using a scalpel, make an approximately two centimeter midline caudal incision along the skull between the ears.Then use hemostats to clip the skin flaps, exposing the skull, and use a scalpel to remove the tissue. When the skull is clean, use a rotary tool to drill eight approximately 0.7 millimeter diameter holes into the frontal, parietal, and occipital lobes and the cerebellum in both the left and right hemispheres of the exposed bone. Then screw eight electrocorticogram electrodes into the holes for focal epilepsy detection.In a separate group of rats, drill three holes and place two screw electroencephalogram electrodes over the right frontal and parietal lobes of the cortices with the same coordinates as in the first group. Next, place a third electroencephalogram electrode over the left cerebellum to ground the animal and reduce the signal artifacts. Separate the neck skin and muscle and insert two electromyogram electrodes into the neck muscle.Drill another hole on the skull and place a microinjection guide canula into the left central nucleus of the amygdala. Then, drill a 1.6 millimeter hole in the skull and insert a calibrated 30 kilovolt thermoster on the surface of the parietal cortex to monitor the cortical temperature. Route the insulated electrocortico and electromyogram electrode leads to a pedestal.Using dental acrylic to cement the pedestal, guide the canula to the skull and then connect the thermoster to the tether. Then, treat the incision topically with polysporin to prevent infection and give the animals water supplemented with ibuprofin and penicillin G for one week after surgery. To induce focal temporal lobe epilepsy, use a microinjection pump to administer 0.5 microliters of pilocarpine into the left central nucleus of the amygdala through the injection guide canula at 0.2 microliters per minute.To induce generalized epilepsy with recurrent status epilepticus, inject 300 milligrams per kilogram of pilocarpine IP.For acupuncture, first manually locate the acupoints. For example, the feng chi acupoints are located three millimeters away from the posterior median line between the ears on the neck. After confirming a low skin impedance at the acupoints of interest, insert stainless steel needles into the acupoints at a two millimeter depth.Then, using a functional electrical stimulator, deliver a 10 hertz train of 150 millisecond byphasic pulses with a one milliamp intensity to the acupoints through the needle. Here, a predominant epileptiform electrocorticogram acquired from the electrocorticogram electrode on the parietal lobe of the left hemisphere is shown. After pilocarpine administration, rare epileptic activities are picked up from the rest of the electrocorticogram electrodes, indicating a successful focal temporal lobe epilepsy induction.Within five minutes, severe cholinergic effects on behavior occur, gradually increasing in severity until an electrocorticogram generalized seizure occurs followed by a status epilepticus event. No epileptiform electrocorticograms are observed during the dark period after the first pentilene tetrasol injection, or during the second dark period on an off-day for injection until the fifth pentilene tetrasol injection, along with spontaneously and recurrently generalized epilepsy. Wakefulness exhibits a high amplitude electromyogram and an abundance of locomotor activity, with a gradual increase in the cortical temperature as the vigilant state transits from either non-REM or REM sleep to wakefulness.Non-REM sleep is characterized by synchronized electrocorticograms with a high amplitude and low frequency, with a gradual electromyogram amplitude decline and no locomotor activity, as well as a decrease in the cortical temperature as the vigilant state transits from wakefulness into NREM sleep. During REM sleep, the electrocorticogram wave is desynchronized with a reduced amplitude. The predominant electroencaphalogram power density occurs within the theta frequency.The electromyogram activity is the lowest, phasic body twitch is observed, and the cortical temperature rapidly increases. Administration of 100 hertz of electroacupuncture into the bilateral feng chi acupoints exacerbates the pilocarpine induced epileptiform electrocorticograms, while 10 hertz of electroacupuncture suppresses the epileptiform electrocorticograms. Further, pilocarpine administration into the left central nucleus of the amygdala also suppresses non-REM sleep during the light period of the 12-hour light/dark cycle.100 hertz of electroacupuncture further deteriorates the pilocarpine induced suppression of non-REM sleep. In contrast, a 10 hertz electroacupuncture into the bilateral feng chi acupoints increases non-REM sleep per se during the dark period, and blocks the pilocarpine-induced reduction of non-REM sleep during the light period. Once mastered, this technique can be completed in one and a half hours if it is performed properly.Before attempting this procedure, it is important to practice the skill of stereotaxic surgery until proficiency. After its development, this technique paves the way for the researchers in the field of neuroscience to explore the effects of acupuncture in epilepsy and sleep disturbance. After watching this video, you should have a good understanding of how to surgically implant electroencephalo and electromyogram electrodes, a guide canula, and a semester to assess sleep patterns and other effects of acupuncture on brain activity.

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Research

JoVE Journal

Behavior

9.4K Views.  Aletheia University, Tainan Campus. The overall goal of this procedure is to assess epilepsy, sleep and the effects of acupuncture in an animal model. This method can help answer key questions in neuroscience field about how to establish epilepsy, analyze sleep and perform acupuncture in a small animal model. The main advantages of this technique are that the lateral cortical and the lateral myogram electrodes in that semester can be implanted quickly and easily by stereotaxic surgery.Demonstrating this procedure will be...