The authors would like to thank Dr. Christina Ginkel (German Center for Neurodegenerative Diseases, DZNE), Dr. Michaela M&#246;hring (DZNE) and Dr. Robert Stark (DZNE) for assistance in animal breeding and animal health care. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut f&#252;r Arzneimittel und Medizinprodukte, BfArM) Bonn, Germany.

&#160;Ethics Statement: All animal experimentation was performed according to the guidelines of the local and institutional Council on Animal Care (University of Bonn, BfArM, LANUV, Germany). In addition, all animal experimentation was carried out in accordance with superior legislation, e.g., the European Communities Council Directive of 24 November 1986 (86/609/EEC) or individual regional or national legislation. Specific effort is made to minimize the number of animals used and their suffering.
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Implantable EEG radiotelemetry is of central relevance as it is a non-restraining technique allowing experimental animals to perform their full repertoire of behavior1,3. This is of major interest as the telemetric approach enables not only spontaneous EEG recordings but also recordings under cognitive tasks and circadian analytical setups, such as T-maze, radial maze, water maze, sleep deprivation tasks or whenever an EEG recording is necessary or helpful during complex cognitive or motor activity.
<p cla...

Radiotelemetry is a most valuable methodological approach for measuring a variety of behavioral and physiological parameters in conscious, unrestrained animals of various sizes, particularly in the context of EEG, ECG, EMG, blood pressure, body core temperature or activity measurements 1-7. Theoretically, any species can be analyzed using implantable EEG radiotelemetry from laboratory rodents such as mice and rats to cats, dogs, pigs and primates 3,8. Even fish, reptiles and amphibians are subject to radiotelemetric investigation 9. Over the last two decades, implantable EEG radiotelemetry has proven to be valuable in the characterization of various transgenic animal models of human diseases, such as epilepsies, sleep disorders, neurodegenerative and neuropsychiatric disorders 7,10-12. In the past, numerous methodological approaches collecting physiological data including biopotentials from mice and rats have been described. Worn in jacket recorder systems, physical restraint methods, non-implanted radiotransmitters and tethered systems have received the main attention in the past 13,14. Nowadays, various systems for radiotelemetric implantation are commercially available. However, a literature screen also revealed 29 publications that describe the development of self-made radiotelemetric systems 15-40. Whereas home-made systems are likely to be less expensive and more user adapted, commercially available systems are straight forward, relatively easy to install and can be setup quickly.
Implantable EEG radiotelemetry has a number of advantages compared to competing techniques such as physical restraint methods, worn in jacket systems or tethered approaches. The latter are restraining by definition, i.e., the animal is unable to move or its normal behavior is impaired. It might even be necessary to anesthetize the animal for acquisition of reliable data. Modern tethered systems however are likely to be less restraining, but this needs to be scientifically validated. Radiotelemetry on the other hand allows animals to exhibit their full repertoire of behavior without spatiotemporal restrictions and thus, is thought to be superior to restraining approaches and be more predictive of the results that could be acquired in humans 1,3. It is known for quite a while that restraining approaches can dramatically alter fundamental physiological parameters, e.g., food intake, body core temperature, blood pressure and heart rate and physical activity for example 3. Tethered systems represent one still widely used classical restraining approach 13,14. The electrodes which are either epidural or deep electrodes are generally connected to a miniature socket which is anchored to the skull. The socket itself is exposed for attachment of a cable that allows relatively free movement of the animal. Although nowadays tethered systems have become extremely filigree and highly flexible, one of its major disadvantages is, that it is still semi-restraining. Besides, there might be a risk of infection at the electrode implantation site as the animals tend to manipulate any external devices originating from their body (head). Although wireless radiotelemetry technology in various species has already been described in the late 60s and has thus existed for decades, it has only recently become affordable, reliable, and relatively easy-to-use 10,41,42, particularly in small laboratory rodents such as mice and rats. Small, miniature implantable EEG transmitters are now commercially available and can be implanted in mice greater than 20 g (~10 weeks). Thus, the electrophysiological characterization of transgenic mouse models in particular has become a predominant field of application of implantable EEG radiotelemetry these days. Animal size is no longer an absolute experimental restriction whereas the life-span of the transmitters&#39; battery indeed is. Despite its limited life-time, implantable transmitter systems are capable of minimizing most disadvantages related to potential recording-associated stress by restraining systems. Rodents can present their complete armamentarium of physiological behavior including resting, locomotor activity (exploration) and sleep (REM, slow-wave sleep) 43,44. Importantly, implantable radiotelemetry can strongly reduce animal use 3. Currently, there is an intense discussion on how to limit the number of experimental animals in science and reduce their suffering. Clearly, animal experimentation and animal models of human and animal diseases are essential for our understanding of the bottom-line pathophysiology and subsequent progress in therapy. Furthermore, animal experiments are critical in drug research and development. They do substantially contribute to preclinical/toxicological studies in drug licensing thus committing to both human and animal care. It&#39;s noteworthy, that currently no alternatives are yet available to animal research to understand the complex pathophysiological mechanisms which would be otherwise impossible to be elicited. At the same time, the 3R, i.e., replacement, reduction and refinement strategy in the EU and the USA strongly encourages research into complementary and alternative methods. Radiotelemetry is an important example of a successful 3R strategy as it can reduce the number of experimental animals and their suffering compared to other techniques.
Here we provide a detailed and contiguous step-by-step approach to perform a subcutaneous pouch implantation of a radiofrequency transmitter in both mice and rats. This first sequence is followed by a description of stereotaxic epidural and deep intracerebral EEG electrode positioning. Special attention is paid to housing conditions, anesthesia, peri- and postoperative pain management and possible anti-infective treatment. The focus is on the computerized 3D stereotaxic approach to reliably target epidural and deep intracerebral structures. We also comment on frequent experimental pitfalls in EEG electrode implantation and strategies for reduction of trauma and optimization of pain management during postoperative recovery. Finally, examples of surface and deep EEG recordings are presented.

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			<td>Carprofen (Rimadyl VET - InjektionA2:D43sl&#246;sung)</td>
			<td>Pfizer</td>
			<td>PZN 0110208</td>
			<td>20 ml</td>
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		<tr>
			<td>binocular surgical magnification microscope&#160;</td>
			<td>Zeiss Stemi 2000</td>
			<td>0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000</td>
			<td></td>
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			<td>bulldog serrefine</td>
			<td>F.S.T.</td>
			<td>18051-28</td>
			<td>28mm</td>
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		<tr>
			<td>cages (Macrolon)</td>
			<td>Techniplast</td>
			<td>1264C, 1290D</td>
			<td></td>
		</tr>
		<tr>
			<td>cold light source</td>
			<td>Schott KL2500 LCD</td>
			<td>9.705&#160;202</td>
			<td>ordered at Th.Geyer</td>
		</tr>
		<tr>
			<td>cotton tip applicators (sterile)</td>
			<td>Carl Roth&#160;</td>
			<td>EH12.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexpanthenole (Bepanthen Wund- und Heilsalbe)</td>
			<td>Bayer</td>
			<td>PZN: 1578818</td>
			<td></td>
		</tr>
		<tr>
			<td>drapes (sterile)</td>
			<td>Hartmann</td>
			<td>PZN 0366787</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Carl Roth&#160;</td>
			<td>9065.5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.3% / 3% hydrogene peroxide solution</td>
			<td>Sigma</td>
			<td>95321</td>
			<td>30% stock solution&#160;</td>
		</tr>
		<tr>
			<td>gloves (sterile)</td>
			<td>Unigloves</td>
			<td>1570</td>
			<td></td>
		</tr>
		<tr>
			<td>dental glas ionomer cement</td>
			<td>KentDental /NORDENTA</td>
			<td>957 321</td>
			<td></td>
		</tr>
		<tr>
			<td>2% glutaraldehyde solution</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps-curved, serrated</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder-Tungsten Carbide</td>
			<td>F.S.T.</td>
			<td>12500-12</td>
			<td>12.5 cm</td>
		</tr>
		<tr>
			<td>heat-based surgical instrument sterilizer</td>
			<td>F.S.T.</td>
			<td>18000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>AEG HK5510</td>
			<td>520010</td>
			<td>ordered at myToolStore</td>
		</tr>
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			<td>high-speed dental drill</td>
			<td>Adeor</td>
			<td>SI-1708</td>
			<td></td>
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		<tr>
			<td>Iris scissors extra thin&#160;</td>
			<td>F.S.T.</td>
			<td>14058-09</td>
			<td>9 cm</td>
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		<tr>
			<td>Inhalation narcotic system (isoflurane)</td>
			<td>Harvard Apparatus GmbH</td>
			<td>34-1352, 10-1340, 34-0422, 34-1041, 34-0401, 34-1067, 72-3044, 34-0426, 34-0387, 34-0415, 69-0230</td>
			<td></td>
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			<td>Isoflurane</td>
			<td>Baxter 250 ml</td>
			<td>PZN 6497131</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pfizer</td>
			<td>PZN 07506004</td>
			<td></td>
		</tr>
		<tr>
			<td>lactated Ringer&#8217;s solution (sterile)</td>
			<td>Braun</td>
			<td>L7502</td>
			<td></td>
		</tr>
		<tr>
			<td>Lexar-Baby Scissors-straight, 10 cm</td>
			<td>F.S.T.</td>
			<td>14078-10</td>
			<td>10 cm</td>
		</tr>
		<tr>
			<td>Nissl staining solution</td>
			<td>Armin Baack</td>
			<td>BAA31712159</td>
			<td></td>
		</tr>
		<tr>
			<td>non-absorbable suture material 5-0/6-0 (sterile)</td>
			<td>SABANA (Sabafil)</td>
			<td>N-63123-45</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Covidien (Sofsilk)</td>
			<td>S1172, S1173</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Needle Holder</td>
			<td>F.S.T.</td>
			<td>12001-13</td>
			<td>13 cm</td>
		</tr>
		<tr>
			<td>pads (sterile)</td>
			<td>ReWa Krankenhausbedarf</td>
			<td>2003/01</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% saline (NaCl, sterile)</td>
			<td>Braun</td>
			<td>PZN:8609255</td>
			<td></td>
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		<tr>
			<td>scalpel blades with handle (sterile)</td>
			<td>propraxis</td>
			<td>2029/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>F.S.T.</td>
			<td>11000-12, 11000-14</td>
			<td>12 cm and 14.5 cm length</td>
		</tr>
		<tr>
			<td>Steel and tungsten electrodes parylene coated&#160;</td>
			<td>FHC Inc., USA)</td>
			<td>UEWLGESEANND</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic frame</td>
			<td>Neurostar</td>
			<td>51730M</td>
			<td>ordered at Stoelting</td>
		</tr>
		<tr>
			<td>(Stereo Drive-New Motorized Stereotaxic)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tapes (sterile)</td>
			<td>BSN medical GmbH &#38; Co. KG</td>
			<td>626225</td>
			<td></td>
		</tr>
		<tr>
			<td>TA10ETA-F20&#160;</td>
			<td>DSI</td>
			<td>270-0042-001X</td>
			<td>Radiofrequency transmitter 3.9 g,&#160; 
			3.9 g, 1.9 cc, input voltage range &#177; 2.5 mV, 
			channel bandwidth (B) 1-200 Hz,&#160; 
			nominal sampling rate (f) 1000 Hz (f = 5B) 
			temperature operating range 34-41 &#176;C 
			warranted battery life 4 months</td>
		</tr>
		<tr>
			<td>TL11M2-F20EET&#160;</td>
			<td>DSI</td>
			<td>270-0124-001X</td>
			<td>Radiofrequency transmitter&#160; 
			3.9 g, 1.9 cc, input voltage range &#177; 1.25 mV, 
			channel bandwidth (B) 1-50 Hz,&#160; 
			nominal sampling rate (f) 250 Hz (f = 5B) 
			temperature operating range 34-41 &#176;C 
			warranted battery life 1.5 months</td>
		</tr>
		<tr>
			<td>Tissue Forceps- 1x2 Teeth 12 cm</td>
			<td>F.S.T.</td>
			<td>11021-12</td>
			<td>12 cm length</td>
		</tr>
		<tr>
			<td>Tungsten carbide iris scissors</td>
			<td>F.S.T.</td>
			<td>14558-11</td>
			<td>11.5 cm</td>
		</tr>
		<tr>
			<td>Vibroslicer 5000 MZ</td>
			<td>Electron Microscopy Sciences</td>
			<td>5000-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>PZN: 1320422</td>
			<td></td>
		</tr>
	</tbody>
</table>

non-restraining eeg radiotelemetry: epidural and deep intracerebral stereotaxic eeg electrode placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

This section illustrates examples obtained from surface and deep, intracerebral EEG recordings. Initially it should be stated that baseline recordings under physiological conditions are mandatory prior to subsequent recordings following e.g., pharmacological treatment. Such baseline recordings may provide valuable information about functional interdependence of brain rhythmicity with different behavioral states or sleep / circadian rhythmicity. Here, we show examples of recorded ...

Watch this Scientific Journal Video about Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement at JoVE.com

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

The overall goal of this experimental procedure is to record long-term electroencephalograms with stereotaxic epidural, and intracerebral electrode placement in different brain regions of freely moving rodents using non-restraining EEG radiotelemetry. This method can have answered key questions in the systemic integrative and translation in neuro-science field, such as dysrhythmia and neurological and neuropsychiatric diseases, as well as epilepsy. The main advantage of this technique is that it allows for epidural and deep intracerebral EEG recordings from freely moving rodents that serve as animal models of human diseases.Julien Soos, a graduate student from our laboratory will be demonstrating the technique. To prepare the epidural electrode, pull out of the silicone isolation, the tip of the transmitter lead with the forceps so it will be bent to a 90 degree angle. Shorten the tip of the lead.To prepare the deep electrode expose the stainless steel helix of the transmitter lead by pulling it out of the silicone isolation. Mechanically insert the deep electrode into the lead of the transmitter. Rewire the transmitter lead to the deep brain electrode to ensure a stable connection.Then bend the electrode to a 90 degree angle and shorten it to the required length. To begin this procedure, shave the head of a fully anesthetized mouse. Then disinfect the shaved area using 70%ethanol and an iodin based scrub.Place the animal in a prone position on a heating blanket to maintain it's body temperature during anesthesia. Using a scalpel, make a mid-line incision on the scalp from the forehead to the neck. Then open a subcutaneous pouch starting from the nuchal incision site along the lateral flank of the animal by blunt dissection with surgical scissors.Subsequently, inject one milliliter of 0.9%sodium chloride in the subcutaneous pouch. Afterward, place a transmitter with the sensing leads oriented crainially into the subcutaneous pocket at the flank close to the ventral abdominal region. In this procedure, place the anesthetized animal on the stereotaxic frame.Carefully position it's head with ear bars and a nose clamp so that the brahma and lambda cranium metric landmarks of the skull are at the same level. Next, clean the periosteum with cotton tips without damaging the temporal and occipital muscles. After that, apply hydrogen peroxide to the skull to exemplify the cranial suture, brahma and lambda.Prepare the 3D stereotaxic system for target coordinates. Drill the target holes at maximum velocity without applying pressure. Place the recording electrode on the motor cortex surface for recording EEG.Then fix the electrodes with dental cement to provide strong adhesion to the underlining neurocranium and leave the cement to dry for five minutes. Place the epidural reference electrode on the cerebellar cortex as it is an electroencephalographically silent region and fix it with cement as well. After having drilled the holes at the coordinates of interest, using the stereotaxic system attach the deep electrode to the vertical arm of the stereotaxic device.Place the deep electrode in the CA1 region. Subsequently, fix the electrode with dental cement to provide strong adhesion to the underlying neurocranium. Place the epidural reference electrode on the cerebellar cortex and fix it with dental cement as well.Leave the cement to dry for five minutes before closing the incision with sutures. This schematic shows that one epidural differential electrode is placed on the motor cortex. An additional intrahippocampal differential electrode is placed in the CA1 region of the hippocampus.Both pseudo reference electrodes are localized on the cerebellum. This coronal section illustrates the localization of the deep intracranial electrode for recording the electrohipppocampagram. And shown here, is the close up of the deep EEG electrode, the sensing lead of the radio frequency transmitter and their arrangement on top of the mirroring skull.These intrahippocampal CA1 recordings are taken from mice following acute treatment with kainate acid at day one, three, and five post-injection. This figure illustrates the examples of simultaneous epidural and deep EEGs in the pilocarpine model of mesial temporal lobe epilepsy in rats. Once mastered, this technique can be done within 30 to 45 minutes, depending on the number of electrodes to be implanted and the target regions if it is performed properly.While attempting this procedure, special attention has to be paid to the vital signs of the anesthetized animals. As transgenic and pharmacological mouse and rat models of human diseases can exhibit differences in anesthesia susceptibilities. Following this procedure, other behavioral, cognitive and electrophysiological masses, such as, T maze, regular maze or evoke potential recordings can be performed in order to address additional questions like, how rhythmicity depends on different functional stages or changes upon repetitive stimulus exposure.After watching this video, you should have a good understanding of how to perform accurate surface and deep EEG electrode placement in mice and rats using a three dimensional stereotaxic device and implantable EEG radiotelemetry.

non-restraining-eeg-radiotelemetry-epidural-deep-intracerebral

Research

JoVE Journal

Neuroscience

18.8K visualizações.  Federal Institute for Drugs and Medical Devices (Bundesinstitut für Arzneimittel und Medizinprodukte, BfArM). The overall goal of this experimental procedure is to record long-term electroencephalograms with stereotaxic epidural, and intracerebral electrode placement in different brain regions of freely moving rodents using non-restraining EEG radiotelemetry. This method can have answered key questions in the systemic integrative and translation in neuro-science field, such as dysrhythmia and neurological and neuropsychiatric diseases, as well as epilepsy. The main advantage of this technique is that...