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 other end.</li>
	<li>Twist two cables together and solder them at the shorter peeled side.</li>
	<li>Acquire a strip of three tape packaging of any integrated circuit with SOT-353 case.</li>
	<li>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 cables so that the end of the insulation is in line with the edge of the holes.</li>
	<li>Put the dental cement on the holes to fix the wires. 
	Caution: The dental cement must be dense enough to avoid leaking into the cavity of the package.</li>
	<li>Once the cement is solidified (minimum 10 min), flip the package and put a thin layer of superglue into the hole to form a watertight seal towards the porous cement. Avoid gluing the peeled wires.</li>
	<li>Twist the cables together on the shorter peeled side and solder them (Figure 1A).</li>
	<li>Grab the longer peeled segment of the wires with the tip of fine tweezers, roll them around, and wrap them into the cavities (Figure 1B). Cut excess cable, if necessary.</li>
</ol>
2. Fabrication of the Recording Electrodes
<ol>
	<li>To make one recording triplet electrode (Figure 1C-D), cut a one cm long piece of stainless steel tube using high-speed rotary saw. Remove the fin from the ends of the tube with a sharp needle and ensure that the tube is clean of debris and completely permeable. 
	NOTE: Do not use pliers, as they will distort the tube.</li>
	<li>Bend a gold-plated board spacer pin with a minimum length of 3 cm to form a J-shape holder. Cut the longer leg of the &#39;J&#39; 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 &#39;U&#39; shape, which will hold the electrode, and serve as an anchoring point. Glue the tube and the shorter leg of the &#39;J&#39; holder together. Glue some additional 1 cm long bars, if necessary, for stability.</li>
	<li>Cut 3 pieces of 2.5 cm long electrode wires with stainless steel, ultrafine 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 1 or 2 mm on one end of each of the wires at different angles (e.g., one left straight, one 45&#176;, and one right angle) to make them distinguishable.</li>
	<li>Fill the tube with the wires, choose an appropriate spacing of the electrodes for the experiment (e.g., for cortical local field potential (LFP) and current source density (CSD) registration, the recommended protrusion of the tips of the wires from the opening of the tube: 4.5 mm, 4.1 mm, and 3.7 mm, respectively). Note which wire corresponds to which depth for maintaining a proper channel order.</li>
	<li>Once the wires align right next to each other and are parallel with the tube, to fix the electrode wires, place a single drop of liquid superglue 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.</li>
	<li>Prepare an electrode interface board with a proper microconnector compatible with the recording system to be used.</li>
	<li>Introduce the non-recording, 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. 
	NOTE: Gold pins may be fixed in place using dental cement, but in most cases, it is stable enough even without this safety step.</li>
</ol>
3. Anesthesia
<ol>
	<li>Place the animal in a sealed anesthesia induction chamber and fill it with 4 - 5% of isoflurane in 2 L/min medical air.</li>
	<li>When the rat is recumbent, remove the animal from the chamber and place it in a stereotaxic frame with an appropriate ventilation nose piece.</li>
	<li>Set the isoflurane level to 2%, though the rate of airflow should remain the same. Check the level of anesthesia. If there is an absence of the paw withdrawal reflex in response to pinching, continue, otherwise increase the depth of anesthesia.</li>
	<li>Monitor and maintain the rat&#39;s body temperature at 37 &#177; 0.5 &#176;C with a homeothermic monitoring system. Apply a droplet of paraffin ointment on the eyes to prevent desiccation of the cornea. Repeat this procedure during the surgery several times, if necessary.</li>
	<li>Subcutaneously inject 0.3 mg/kg atropine to avoid mucus formation in the airways. Repeat this every four hours in case of longer surgeries. Monitor breathing and adjust vaporizer, if needed.</li>
</ol>
4. Implantation of the Stimulation and Recording Electrodes
NOTE: Autoclave all the necessary surgical instruments and carefully follow the general rules of asepsis and antisepsis during the whole procedure. Avoid touching nonsterile areas outside of the surgical area.&#160;Immerse the electrodes in ethyl alcohol (70%) for 30 min before implantation.
<ol>
	<li>Remove most of the hair from the scalp with a hair clipper. 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 water, then with disinfectant.</li>
	<li>Inject 1 - 2% lidocaine (do not exceed a total dose of 7 mg/kg) subcutaneously to numb the skin. Apply a single drop of vet ointment for the eyes (e.g., paraffin).</li>
	<li>Make a thorough and a long (~2 cm) sagittal incision in the midline with a scalpel, from the forehead to the neck. 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.</li>
	<li>Gently place fine tweezers between the steep edge of the temporal bone and the muscles and separate them. Make jigging 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.</li>
	<li>Place retractors bitemporally to keep the temporal bones exposed.</li>
	<li>Rinse the surface of the skull with 1 - 2 mL 3% H2O2, then wash it with 1 - 2 mL water.</li>
	<li>Air-dry the surface of the temporal bones very carefully, mop up the moisture with ocular sticks. Test if the stimulation electrodes fit onto 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). Re-adjust the bulldogs and the retractors or shape the stimulation electrodes with scissors, if necessary.</li>
	<li>Fill the cavities of the stimulation electrodes with electroconductive gel and put a thin layer of glue onto the rim of the electrodes.</li>
	<li>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. Mop up with ocular sticks, if necessary. 
	Caution: In case any instability of the electrode is experienced, remove it. After cleaning the bone, repeat this step with a new stimulation electrode.</li>
	<li>Put 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.</li>
	<li>After the cement is completely hardened, repeat these steps on the contralateral side.</li>
	<li>Drill some holes all over the skull for anchoring screws. Drive miniature screws into the holes and put dental cement over them. Use a ~10% smaller diameter drill head compared to the screw diameter.</li>
	<li>Depending on the purpose of the experiments, either solder a connector to the ends of the cables, place it on the surface, and cover with cement, or continue implanting the recording electrodes and optical fibers, or prepare a cranial window over the skull. In this latter case, solder the connectors and anchor them to the construct only at the end. In case of the need of long-term simultaneous TES and LFP recordings, implant the aforementioned electrode triplets instead of single wires. 
	NOTE: This setup allows for the removal of common mode stimulation artefacts8. For the details of recording electrode implantation, see previous protocols14,15.</li>
	<li>Wash the exposed tissues abundantly with disinfectant. Inject 1 - 2% lidocaine (do not exceed a total dose of 7 mg/kg) subcutaneously.</li>
	<li>Debride wound edges and close them with simple interrupted sutures around the connector/implant. Disinfect the wound with povidone-iodine.</li>
	<li>Inject 5 mg/kg carprofene subcutaneously. Repeat this if necessary. 
	NOTE: Any other analgesics may be used matching the requirements set by the local ethical permission board.</li>
	<li>Suspend anesthesia, release the earbars and nosepiece, and put the animal in a postoperative recovery cage to regain consciousness.</li>
	<li>Ensure that the animal is under close supervision during the first postoperative hours. Continue to monitor the implant area periodically. 
	NOTE: The animal should not be left unattended until it has regained sufficient consciousness to maintain sternal recumbency. House the animal individually until full recovery from the procedure.</li>
</ol>

<ol>
	<li>Ozen, S., 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+electric+stimulation+entrains+cortical+neuronal+populations+in+rats.">Transcranial electric stimulation entrains cortical neuronal populations in rats.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (34), 11476-11485 (2010).</li><li>Ali, M. M., Sellers, K. K., Frohlich, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+alternating+current+stimulation+modulates+large-scale+cortical+network+activity+by+network+resonance.">Transcranial alternating current stimulation modulates large-scale cortical network activity by network resonance.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33 (27), 11262-11275 (2013).</li><li>Helfrich, R. 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 ...

chronic-transcranial-electrical-stimulation-intracortical-recording

Research

JoVE Journal

Neuroscience

8.6K Views.  University of Szeged. 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.

Video: Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...