This work was supported by EU-FP7-ERC-2013-Starting grant (No.337075), the &#39;Momentum&#39; Program of the Hungarian Academy of Sciences (LP2013-62), and the GINOP-2.3.2-15-2016-00018 grant. We thank M&#225;t&#233; Koz&#225;k for documenting the stimulation and recording electrodes and Mih&#225;ly V&#246;r&#246;slakos for the fruitful discussions during protocol design.

All methods described here are in accordance with the directives of the European Communities Council (86/609 ECC) and have been approved by the Ethical Committee for Animal Research at the Albert Szent-Gy&#246;rgyi Medical and Pharmaceutical Center of the University of Szeged (XIV/218/2016).
1. Fabrication of the Stimulation Electrodes
<ol>
	<li>To make one stimulation electrode, cut six 10 cm long pieces of miniature hook-up wires, remove 2 cm of the cladding on one end, and 1 cm on the oth...

<ol>
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F., 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=Entrainment+of+brain+oscillations+by+transcranial+alternating+current+stimulation.">Entrainment of brain oscillations by transcranial alternating current stimulation.</a> Current biology : CB. 24 (3), 333-339 (2014).</li><li>Bikson, 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=Transcranial+direct+current+stimulation+for+major+depression:+a+general+system+for+quantifying+transcranial+electrotherapy+dosage.">Transcranial direct current stimulation for major depression: a general system for quantifying transcranial electrotherapy dosage.</a> Current treatment options in neurology. 10 (5), 377-385 (2008).</li><li>Lefaucheur, J. P., 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=Evidence-based+guidelines+on+the+therapeutic+use+of+transcranial+direct+current+stimulation+(tDCS).">Evidence-based guidelines on the therapeutic use of transcranial direct current stimulation (tDCS).</a> Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology. 128 (1), 56-92 (2017).</li><li>Kuo, M. F., Nitsche, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+transcranial+electrical+stimulation+on+cognition.">Effects of transcranial electrical stimulation on cognition.</a> Clinical EEG and neuroscience. 43 (3), 192-199 (2012).</li><li>Sandrini, M., Fertonani, A., Cohen, L. G., Miniussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+dissociation+of+working+memory+load+effects+induced+by+bilateral+parietal+modulation.">Double dissociation of working memory load effects induced by bilateral parietal modulation.</a> Neuropsychologia. 50 (3), 396-402 (2012).</li><li>Berenyi, A., Belluscio, M., Mao, D., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed-loop+control+of+epilepsy+by+transcranial+electrical+stimulation.">Closed-loop control of epilepsy by transcranial electrical stimulation.</a> Science. 337 (6095), 735-737 (2012).</li><li>Kozak, G., Berenyi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+efficacy+of+closed+loop+electrical+stimulation+for+long-term+treatment+of+absence+epilepsy+in+rats.">Sustained efficacy of closed loop electrical stimulation for long-term treatment of absence epilepsy in rats.</a> Scientific reports. 7 (1), 6300 (2017).</li><li>Fertonani, A., Ferrari, C., Miniussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+you+feel+if+I+apply+transcranial+electric+stimulation?+Safety,+sensations+and+secondary+induced+effects.">What do you feel if I apply transcranial electric stimulation? Safety, sensations and secondary induced effects.</a> Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology. 126 (11), 2181-2188 (2015).</li><li>Marshall, L., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+transcranial+oscillatory+stimulation+to+research+on+neural+networks:+an+emphasis+on+hippocampo-neocortical+rhythms.">Contribution of transcranial oscillatory stimulation to research on neural networks: an emphasis on hippocampo-neocortical rhythms.</a> Frontiers in human neuroscience. 7, 614 (2013).</li><li>Reato, D., Rahman, A., Bikson, M., Parra, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+weak+transcranial+alternating+current+stimulation+on+brain+activity-a+review+of+known+mechanisms+from+animal+studies.">Effects of weak transcranial alternating current stimulation on brain activity-a review of known mechanisms from animal studies.</a> Frontiers in human neuroscience. 7, 687 (2013).</li><li>Thut, G., Miniussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+rhythmic+brain+activity+from+TMS-EEG+studies.">New insights into rhythmic brain activity from TMS-EEG studies.</a> Trends in cognitive sciences. 13 (4), 182-189 (2009).</li><li>Gage, G. J., 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=Surgical+implantation+of+chronic+neural+electrodes+for+recording+single+unit+activity+and+electrocorticographic+signals.">Surgical implantation of chronic neural electrodes for recording single unit activity and electrocorticographic signals.</a> Journal of visualized experiments : JoVE. (60), (2012).</li><li>Vandecasteele, 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=Large-scale+recording+of+neurons+by+movable+silicon+probes+in+behaving+rodents.">Large-scale recording of neurons by movable silicon probes in behaving rodents.</a> Journal of visualized experiments : JoVE. (61), e3568 (2012).</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> Journal of visualized experiments : JoVE. (101), e52554 (2015).</li><li>Ung, K., Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-optic+implantation+for+chronic+optogenetic+stimulation+of+brain+tissue.">Fiber-optic implantation for chronic optogenetic stimulation of brain tissue.</a> Journal of visualized experiments : JoVE. (68), e50004 (2012).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> Journal of visualized experiments : JoVE. (12), (2008).</li></ol>

Antal Ber&#233;nyi is the owner and founder of Amplipex Ltd, a company manufacturing large-scale multiplexing biosignal amplifiers. G&#225;bor Koz&#225;k and Tam&#225;s F&#246;ldi have nothing to disclose.

The most critical step of this protocol is the gluing of the electrode package on the bone surface. In case of improper sealing, a gap is formed between the electrodes and the bone, and secondary scar tissue can grow into this gap, which lessens the quality of stimulation. The bone surface must be completely dry during the steps of sticking on the package, and in the case of experiencing instability of the electrodes, it must be removed and replaced with a new package to gain the best results.
<p class="jove_content"...

Transcranial electrical stimulation (TES) is a valuable methodological approach to influence brain activity in a temporally constrained manner. Depending on the size and placement of the stimulation electrodes, TES can affect large brain volumes and entrain neuronal populations diffusely1,2,3. Transcranial direct current stimulation is already medically approved for the treatment of major depressive disorder4,5, and many studies focus on showing the cognitive effects of transcranial stimulation in humans6,7. Furthermore, promising results were reported regarding the potential of TES in controlling epileptic seizures8,9.
Despite the intensive research, there are still many open questions regarding the detailed mechanism of action, potential side-effects, and the long-term outcome of applying this method10,11,12. Therefore, it is critically important to have a robust, reproducible protocol to investigate the effects of TES in animal models. Given that many disorders (e.g., depression, epilepsy, and schizophrenia) can only be extensively investigated in awake animals, and the nature of these medical conditions usually necessitate long-term treatment, we provide a protocol for chronic implantation of transcranial electrodes in rats. The method presented here can be used for behavioral studies or can be combined with implantation of recording electrodes (i.e., wires, silicone probes, juxtacellular electrodes) or with chronic cranial windows for electrophysiological experiments and imaging studies, respectively. Depending on the experimental design, the timing of the stimuli can be either random or event-triggered to specific behavioral cues, or to the electrophysiological hallmarks of the particular brain states (seizures, theta oscillations)8,11,13.
It is important to mention that in contrast to the currently used human approach, which uses an embodiment of electrodes placed on the skin, here we show a method that employs subcutaneous implantation right over the surface of the temporal bone, since rats barely tolerate anything placed on their skin which is easily accessible using their paws.
In line with the principles of Replacement, Reduction, and Refinement, due to the chronic nature of implantation, this method helps to reduce the number of animals, since each animal can be recruited in different experimental conditions for months, allowing the use of fewer animals to test various hypotheses.
In the present study, we provide a detailed, step-by-step protocol of transcranial stimulation electrode manufacturing (Figure 1A-B) and demonstrate the chronic implantation of these electrodes over the temporal bones of a six-month-old male Long-Evans rat.

<table>
	<tbody>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Henkel</td>
			<td>Loctite 401</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate gel</td>
			<td>Henkel</td>
			<td>Loctite 454</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire for stimulation electrodes</td>
			<td>Phoenix Wire Inc.</td>
			<td>36744MHW - PTFE Microminiature Hook-Up Wire</td>
			<td></td>
		</tr>
		<tr>
			<td>Board spacer</td>
			<td>E-tec Interconnect</td>
			<td>SP1-020-S378/01-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector</td>
			<td>E-tec Interconnect</td>
			<td>P2510I-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape packaging for stimulation electrodes</td>
			<td>Nexperia</td>
			<td>74HC1G00GW</td>
			<td>Tape packaging of any integrated circuit with SOT-353 case can be used</td>
		</tr>
		<tr>
			<td>Grip Cement Industrial Grade</td>
			<td>Caulk Dentsply</td>
			<td>675571 (powder) 675572 (solvent)</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroconductive gel</td>
			<td>Rextra</td>
			<td>ECG Gel</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording electrode wire</td>
			<td>California Fine Wire Co.</td>
			<td>.002 (50 micron) Tungsten 99.95% (CFW Material #: 100-211), HMl-Natural, cut to 3.0 inch pieces, Round, Cut length piece wire</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafine scissors</td>
			<td>Hammacher Instrumente</td>
			<td>Stainless HSB 544-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel tube</td>
			<td>Vita Needle Company</td>
			<td>29 RW, 304SS Tubing, T.I.G. Welded and Plug</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed rotary saw</td>
			<td>Dremel</td>
			<td>Model # 395</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary saw holder</td>
			<td>Dremel</td>
			<td>Model # 220</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary saw cut-off wheel</td>
			<td>Dremel</td>
			<td>Model # 409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular sticks</td>
			<td>Lohmann-Rauscher</td>
			<td>Pro-ophta Ocular Sticks</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet disinfectant</td>
			<td>Egis</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry disinfectant</td>
			<td>Wagner Pharma</td>
			<td>Reseptyl-urea</td>
			<td></td>
		</tr>
		<tr>
			<td>Drilling machine</td>
			<td>NSK-Nakanishi United Kingdom</td>
			<td>Vmax35RV Pack</td>
			<td></td>
		</tr>
		<tr>
			<td>Anchoring screws</td>
			<td>Antrin Miniature Specialties, Inc.</td>
			<td>000-120x1/16 SL BIND MS SS</td>
			<td></td>
		</tr>
	</tbody>
</table>

chronic transcranial electrical stimulation and intracortical recording in rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

The implantation of chronic stimulation electrodes (Figure 1B) can be combined with additional implantation of recording electrodes (Figure 1C-D). Such settings are suitable to form on-demand recording and stimulation systems to interfere with specific brain activities. Here we present representative results of a closed-loop epilepsy detection and intervention system, applied on Long-Evans rats expressing spontan...

Watch this Scientific Journal Video about Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats at JoVE.com

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

The overall goal of this procedure is to show a step-by-step protocol to fabricate and implant chronic transcranial stimulation electrodes. This method can help to answer key questions in the neuro-stimulation field such as the long term effects of temporally targeted diffused transcranial electrical stimulation. The main advantage of this technique is that the small size of the implant makes this method suitable to be combined with regular electro-physiological optogenetic and imaging techniques in freely moving animals.The implications of this technique is centered toward the therapy of epilepsy, depression and another neuro-psychological disorders because it is capable to promptly influence large scale brain networks. To make a stimulation electrode, cut six 10 centimeter long pieces of miniature hookup wires. Remove two centimeters of the cladding on one end, and one centimeter on the other end.Next, twist two cables together and solder them with the shorter peeled side. Afterward, acquire a strip of three tape packaging of any integrated circuit with SOT-353 case. Stitch the twisted wires through the three holes of the package.The longer peeled segment of the wires should be in the cavities of the package. Position the cable so that the end of the insulation is in line with the edge of the holes. Then, apply dental cement on the holes to fix the wires.Once the cement is solidified, flip the package to input a thin layer of super glue into the hole to form a water-type seal towards the porous cement. Avoid gluing the peeled wires. After that, twist the cables together on the shorter peeled side, and solder them.Grab the longer peeled segment of the wires with the tip of fine tweezers. Roll them around, and wrap them into the cavities. Cut excess cable if necessary.To make a recording triplet electrode, cut a one centimeter long piece of stainless steel tube using a high-speed rotary saw. Remove the fin from the ends of the tube with a sharp needle and ensure that the tube is free of debris and completely permeable. Bend the gold-plated board spacer pin with the minimum length of three centimeters to form a J-shaped holder.Cut the longer leg of the J-shape and solder it together to get back the original shape to make a detachable joint between the long linear part, which will be held by the stereotaxic device, and the U-shape, which will hold the electrode and serve as an anchoring point. Subsequently, glue the tube and the shorter leg of the J-holder together. Glue some additional one centimeter long bars if necessary for stability.Next, cut three pieces of 2.5 centimeter long electrode wires with stainless steel ultra-fine scissors. Make sure that it is a clear and sharp cut and the insulation is intact at the circular surface of the electrode wire. Also, make sure that the cut tip is not bent due to the force applied.Bend one or two millimeters on one end of each of the wires at different angles to make them distinguishable. Afterward, fill the tube with the wires and choose an appropriate spacing of the electrodes for the experiment. Note which wire corresponds to which depth for maintaining a proper channel order.Once the wires align right next to each other and are parallel with the tube, fix the electrode wires by placing a single drop of liquid super glue at both ends of the tube with a sharp needle. Make sure that the glue is flowing into the tube due to the capillary effect but not towards the recording sites at the protruding ends. Prepare an electrode interface board with a proper micro-connector compatible with a recording system to be used.Then, introduce the bent ends of the wires into the holes of the electrode interface board corresponding to the desired recording channel. When all the wires are in position, push the gold pins into the holes with tweezers. In this procedure, remove most of the hair from the animal scalp with a hair clipper.Next, apply depilatory cream on the scalp. Spread it evenly on the surface, and wait a few minutes. Use a spatula to gently remove the cream and the remaining hair.Rinse the skin with disinfectant. Inject 1 to 2%lidocaine subcutaneously to numb the skin. Then, apply a single drop of vet ointment on each eye.Make a thorough and a long sagittal incision along the midline with a scalpel from the forehead to the neck. Subsequently, dissect the tissues including the periosteum from the skull. Then using a chisel or tooth tweezers, clean the area between the cristae of the two temporal bones.Keep the skull exposed by retracting the dissected skin using four bulldogs. Gently place the fine tweezers between the steep edge of the temporal bone and the muscles and separate them. Make jiggling movements to expose as much of the large surface of the temporal bone as possible, preferably from the edge of the occipital bone to the plane of the coronal sutures without damaging the muscles.Following that, place the retractors bi-temporally to keep the temporal bones exposed. Rinse the surface of the skull with one to two milliliters of 3%hydrogen peroxide. Then wash it with one to two milliliters of water very carefully and leave the surface of the temporal bones to mop up the moisture with ocular sticks.Next, test if the stimulation electrodes fit on to the cleaned vertical skull surface. The upper edge of the stimulation electrode should be in line with the edge of the crista of the temporal bone. Readjust the bulldogs and the retractors, or shape the stimulation electrodes with scissors, if necessary.Following that, fill the cavities of the stimulation electrodes with electro-conductive gel. And put a thin layer of glue on to the rim of the electrodes. Place the stimulation electrode on the dry surface of the temporal bone with one accurate movement and hold it firmly in place for one minute with fine tweezers.Make sure that no moisture is in contact with the glue, and mop up with ocular sticks if necessary. Make sure that the stimulation electrode is evenly glued to the dry temporal bone. Mop out the moisture frequently, and retract the surrounding tissues carefully.Ask for assistance if necessary. After that, apply dental cement over the edges of the stimulation electrode while the leaking moisture of the tissues is continuously dried up with ocular sticks. Cover the whole stimulation electrode with cement.After the cement has completely hardened, repeat these steps on the contra-lateral side. Subsequently, using a 10%smaller diameter drill head compared to the screw diameter, drill some holes all over the skull for anchoring screws. Drive miniatures screws into the holes, and apply dental cement over them.Then, continue with implantation of the recording electrodes. Solder the connectors and anchor them to the construct at the end. Afterward, wash the exposed tissues abundantly with disinfectant.Inject 1 to 2%lidocaine subcutaneously. Then, to breed wound edges, enclose them with simple interrupted sutures around the connector implant. Subsequently, disinfect the wound with povidone iodine.Here, we show the representative results of a closed loop epileptic seizure detection and intervention system, applied on Long-Evans rats, expressing spontaneous seizures. This train is known to show the behavioral symptoms in electrographic patters of absence epilepsy, called spike and wave discharge. Shown here are the LFP traces of seizure intervention, demonstrating the stable performance of the temporally targeted stimuli in interrupting seizures for 16 weeks.This demonstrates the robustness and reliability of the implanted stimulation electrodes. To put these results in context, here is the recording of an aborted experiment where secondary tissue grew between the temporal bone and electrode surface, due to the improper sealing and cementing of the electrodes. After watching this video, you should have a good understanding of how to implant transcranial stimulation electrodes.Following this procedure, other methods like implantation of recording electrodes or cranial windows can be performed in order to record different modalities during stimulation.

chronic-transcranial-electrical-stimulation-intracortical-recording

Research

JoVE Journal

Neuroscience

8.6K Görüntüleme.  University of Szeged. The overall goal of this procedure is to show a step-by-step protocol to fabricate and implant chronic transcranial stimulation electrodes. This method can help to answer key questions in the neuro-stimulation field such as the long term effects of temporally targeted diffused transcranial electrical stimulation. The main advantage of this technique is that the small size of the implant makes this method suitable to be combined with regular electro-physiological optogenetic and imaging techni...