Name: recording eeg in freely moving neonatal rats using a novel method
Uploaded: 2017-05-29T12:30:00
Description: Watch this Scientific Journal Video about Recording EEG in Freely Moving Neonatal Rats Using a Novel Method at JoVE.com

This work was supported by the Natural Science Foundation of China (31171355) and the Natural Science Foundation of Guangdong (S2011010003403, 2014A030313440).

Animal care, surgical procedure, and recording procedures were in accordance with the guidelines for the South China Normal University Animal Care and Use Committee.
1. Electrode Preparation (Figure 1A-C)
NOTE: Computer pin loci is simply a pronged contact as a part of signal interface in communication devices. It consists of a male connector that plugs into the female connector.
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	<li>Carefully separate the male and female pins from the computer pin loci (<st...

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	<li>Pereda, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+synapses+and+their+functional+interactions+with+chemical+synapses.">Electrical synapses and their functional interactions with chemical synapses.</a> Nat Rev Neurosci. 15 (4), 250-263 (2014).</li><li>Chorev, E., Epsztein, J., Houweling, A. R., Lee, A. K., Brecht, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+behaving+animals--going+beyond+spikes.">Electrophysiological recordings from behaving animals--going beyond spikes.</a> Curr Opin Neurobiol. 19 (5), 513-519 (2009).</li><li>Freeborn, D. L., McDaniel, K. L., Moser, V. C., Herr, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electroencephalography+(EEG)+to+assess+CNS+changes+produced+by+pesticides+with+different+modes+of+action:+effects+of+permethrin,+deltamethrin,+fipronil,+imidacloprid,+carbaryl,+and+triadimefon.">Use of electroencephalography (EEG) to assess CNS changes produced by pesticides with different modes of action: effects of permethrin, deltamethrin, fipronil, imidacloprid, carbaryl, and triadimefon.</a> Toxicol Appl Pharmacol. 282 (2), 184-194 (2015).</li><li>Werhahn, K. J., Hartl, E., Hamann, K., Breimhorst, M., Noachtar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+of+interictal+epileptiform+discharges+in+long-term+EEG+recordings+in+epilepsy+patients.">Latency of interictal epileptiform discharges in long-term EEG recordings in epilepsy patients.</a> Seizure. 29, 20-25 (2015).</li><li>Staba, R. J., Stead, M., Worrell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+biomarkers+of+epilepsy.">Electrophysiological biomarkers of epilepsy.</a> Neurotherapeutics. 11 (2), 334-346 (2014).</li><li>Nimmrich, V., Draguhn, A., Axmacher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Network+Oscillations+in+Neurodegenerative+Diseases.">Neuronal Network Oscillations in Neurodegenerative Diseases.</a> Neuromolecular Med. 17 (3), 270-284 (2015).</li><li>Zayachkivsky, A., Lehmkuhle, M. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Continuous+EEG+Monitoring+in+Small+Rodent+Models+of+Human+Disease+Using+the+Epoch+Wireless+Transmitter+System.">Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System.</a> J Vis Exp. (101), e52554 (2015).</li><li>Zayachkivsky, A., Lehmkuhle, M. J., Fisher, J. H., Ekstrand, J. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+EEG+in+immature+rats+with+a+novel+miniature+telemetry+system.">Recording EEG in immature rats with a novel miniature telemetry system.</a> J Neurophysiol. 109 (3), 900-911 (2013).</li><li>Dzhala, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKCC1+transporter+facilitates+seizures+in+the+developing+brain.">NKCC1 transporter facilitates seizures in the developing brain.</a> Nat Med. 11 (11), 1205-1213 (2005).</li><li>Tucker, A. M., Aquilina, K., Chakkarapani, E., Hobbs, C. E., Thoresen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+amplitude-integrated+electroencephalography+and+interburst+interval+in+the+rat.">Development of amplitude-integrated electroencephalography and interburst interval in the rat.</a> Pediatr Res. 65 (1), 62-66 (2009).</li><li>Savard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+neuronal+IL-1beta+in+acquired+brain+lesions+in+a+rat+model+of+neonatal+encephalopathy.">Involvement of neuronal IL-1beta in acquired brain lesions in a rat model of neonatal encephalopathy.</a> J Neuroinflammation. 10, 110 (2013).</li><li>Cuaycong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+the+study+of+hypoxia-ischemia-induced+clinical+and+subclinical+seizures+in+the+neonatal+rat.">A novel approach to the study of hypoxia-ischemia-induced clinical and subclinical seizures in the neonatal rat.</a> Dev Neurosci. 33 (3-4), 241-250 (2011).</li></ol>

Here, we report surgical and recording procedures to acquire EEG in freely moving neonatal rat pups by wired method (Figure 3). It has been suggested that the P7-P12 rat pup is at the developmental age that corresponds to a full-term human neonate 10,11. It is technically difficult to obtain high-quality EEG recording data when working with rat pups in this age group. In addition, it requires specialized handling and great care ...

It is well established that continuous communication between neurons is required to obtain normal brain function. The interneuronal communication primarily takes place at synapses, where information from one neuron is conveyed to a second neuron. This synaptic transmission is mediated by two types of dedicated structural arrangements: electrical or chemical synapses 1. Electrophysiology is the field that captures the electrical potential produced during interneuronal communication that control overall body functions and behavior 2. EEG is the most commonly used method among many electrophysiological techniques.
EEG is a technique used to detect changes in electrical signals produced by internal or external stimuli. Moreover, it is an essential test for clinical diagnosis and outcome prediction of various neurological conditions such as epilepsy, Parkinson&#39;s and Alzheimer&#39;s disease, as well as effects of pharmacological and toxicological agents 3. Generally, an epileptic patient shows hyperexcitability and impaired functional connectivity within the brain; these are summarized as interictal epileptiform discharges (IEDs) and can be recorded by EEG in the form of sharp, transient spikes; sharp waves; spike-wave complexes; or polyspikes 4. The main feature of the epileptic brain is the spontaneous occurrence of epileptic seizures, which can be recorded either from the scalp or from the brain parenchyma in order to locate the brain area responsible for the seizures 5. Furthermore, EEG also has very important implications in neurodegenerative disorders like Alzheimer&#39;s disease (AD). Research suggests that altered EEG recordings and impaired oscillatory networks in AD patients are common. However, our knowledge about the pathophysiology of network oscillations in neurodegenerative diseases is surprisingly incomplete and needs to be further explored 6.
In this protocol, we have designed a simple electrode with which one can record EEG to understand the electrical communication in both the normal and the pathological brain. The surgical implantation in this method is cheaper than other available procedures 7. Moreover, this method can be used to record high-quality and stable EEG signals for longer timeframes (i.e., 2-4 h every day for 1 week). In addition, we used lighter electrodes (weighing approximately 26 mg) that enable the animals to behave more naturally 8. This method is widely applicable to the study of EEG in neonatal rat pups that requires the amplifier and digitizer, commonly used in electrophysiology lab and does not require any additional devices.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Computer pin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pincer</td>
			<td>DELI Group Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>502 super glue</td>
			<td>DELI Group Co., Ltd.</td>
			<td>7144</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Boxun</td>
			<td>GZX-9140MBE</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>RWD Life Science</td>
			<td>902-0000-522</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>900-0068-507</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia apparatus</td>
			<td>RWD Life Science</td>
			<td>902-0000-510</td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic Heating Device</td>
			<td>Harvard Apparatus</td>
			<td>K 024509</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier Model 3000&#160;</td>
			<td>A-M Systems</td>
			<td>61558</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro1401 Analog Digital converter</td>
			<td>Cambridge Electronic Design Ltd.</td>
			<td>4383</td>
			<td>Data acquisition unit</td>
		</tr>
		<tr>
			<td>Spike2</td>
			<td>Cambridge Electronic Design Ltd.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

If the above surgical procedures are conducted properly, one channel rat neonatal pup EEG recording will be successfully performed. 10 min after the KA injection, a regular pattern of behavioral signs emerged in the form of irregular movements and scratching, tremors, and loss of balance. Figure 2 shows the representative raw EEG traces and interictal, ictal-tonic, and ictal-clonic expanded traces. Recurrent interictal and ictal EEG discharge patterns started 15-60 min (30 &#177;5.2 min; ...

Watch this Scientific Journal Video about Recording EEG in Freely Moving Neonatal Rats Using a Novel Method at JoVE.com

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

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.

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 ...

The overall goal of this method is to record EEGs from freely moving neonatal rat pups, using a novel electrode set up that is stable for a week or longer. This method will have answered key questions in the field of neuroscience and will be beneficial in the study of a neonatal epilepsy. By using this technique, one can record high quality and stable EEG signals for longer timeframes.Which will help in the diagnosis of various neuro-pathological disorders. Generally, individuals new to this method will struggle because neonatal rat at p8 to p15 are very difficult to handle and require great care. The electrode requires a pronged contact to function as the signal interface with the communication devices.A computer pin lo sai fills this role. Carefully separate the male and female pins from a computer pin lo sai using pliers. Then, connect the male and female pins together to form an electrode, and secure the connection with cyanoacrylate.Make several such electrodes. Once the bonds have hardened, put the electrodes into a beaker filled with distilled water for ten minutes. Then, place them into an ultrasonic cleaner to clean the electrodes.Then, transfer the beaker to an oven set to 45 degrees Celsius, and heat them for half an hour. Lastly, sterilize the electrodes with a 30-minute UV exposure. In a bio-safety hood, first set up the sterilized surgical tools, and a sterilized stereotaxic apparatus for the surgery.Then, anesthetize the neonatal rat pup using two point five percent isoflurane. When the pup is deeply anesthetized, reduce the isoflurane to one percent. Prior to proceeding, use a toe pinch to ensure that the mouse is sufficiently anesthetized.Next, fix the head of the pup in the stereotaxic apparatus. Position the ear bars in the ear canals and gently tighten the bars. Be careful:the neonatal skull is very soft.Now, make a fifteen-millimeter midline incision into the skull. Then, with forceps, gently pull apart the scalp to widen the incision. To keep the incision open, use a piece of saline-soaked cotton.Now, locate the bregma and lambda, and mark them with a pencil. The following electrode implantation steps are critical and must be executed precisely. First, using a 26-gauge needle, make a burr hole into the prefrontal cortex, two millimeters anterior to the bregma and within zero point five millimeters of the midline.Next, make a burr hole into the hippocampus at one point eight millimeters posterior to the bregma and zero point five millimeters lateral of the midline. The needle should not penetrate more than two millimeters below the cortical surface to minimize brain damage. Now, using forceps, insert the reference electrode in the cortex hole, and insert the recording electrodes into the hippocampus hole.Then, apply erythromycin ointment around the electrodes, and fix the electrodes in place with cyanoacrylate. While the glue bonds, prepare dental cement at a gluey and viscous consistency. The viscosity of the dental cement is critical.If the dental cement is too thin, it may result in building up an undesired insulating layer under the electrode. If the cement is too dense, the electrodes will be more easily detached because of the excess mass. Before applying the cement, thoroughly dry the skull and the electrodes.Then, secure the electrodes to the skull with dental cement. Following this, apply some picric acid onto the electrodes to add another layer of protection to them. Now, remove the animal from the stereotaxic frame, and inject 300 microliters of ten percent glucose solution subcutaneously.Then, transfer the animal to a heated blanket to keep it at 37 degrees Celsius until it is ambulatory. Once ambulatory, administer buprenorphine to mediate post-surgical pain, and transfer the pup to its home cage with its dam. Before using the implant, wait at least two days for the pup to recover.After the recovery period, house the pup alone to make recordings for about ten minutes so that the pup may become familiar with the environment. Connect the electrodes to an amplifier, and connect the amplifier to an AD converter attached to a computer. Be careful not to tangle these connections.Next, on the data acquisition unit, select a sampling rate of at least 10, 000 hertz for recording, and check for proper data transmission. Collect a baseline recording. After taking a baseline recording, inject the pup intraperitoneally with kainic acid to induce epileptic seizures.Fifteen minutes after the injection, observe and record the epileptic discharges. Save the digitized data, and analyze it using signal-processing software. Reveal the power level of different frequency components in the neonatal EEGs using power spectrum analysis.Calculate the power from one-minute timeframes by finding root mean square amplitude from one to one hundred hertz, which is the EEG band. Ten minutes after the kainic acid injection, recurrent interictal and ictal EEG discharge patterns were recorded. The most common seizures recorded were the tonic clonic type.The ictal tonic duration lasted about fifteen seconds. It was also observed that tonic discharges were followed by clonic bursts. After watching this video, you should have a good understanding of how to implant electrodes and obtain high-quality EEG recordings from p8 to p15 neonatal rat pups.Once mustered, the cervical implantation can be done in less than ten minutes depending on the complexity of the surgery.

Research

JoVE Journal

Neuroscience

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