Name: chronic transcranial electrical stimulation and intracortical recording in rats
Uploaded: 2018-05-11T13:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats at JoVE.com

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.7K 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

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Behavior

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

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 ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Source: Haumesser, J. K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

Stimulation and Modulation Techniques

Implanting an On-Head Electrode System in a Rat Model for Real-Time Seizure Detection

Source: Koz&#225;k, G., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56669">Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the method of implanting an on-head electrode system in an anesthetized rat for real-time seizure detection. The procedure includes placing stimulation and recording electrodes, securing them with dental cement, and connecting them to a head-mounted amplifier.

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

Source: Kang, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56447">Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent.</a> J. Vis. Exp. (2017).
This video demonstrates the procedure for concurrent recordings of co-localized electroencephalography (EEG) and local field potentials (LFPs) in an anesthetized rat. The process involves preparing the rat and attaching stimulating electrodes to its whisker pads. An EEG spider electrode is placed on the rat&#39;s skull for surface EEG measurements. At the same time, a multi-channel microelectrode is inserted into a specific brain region through a burr hole in the skull to record neural activity from a localized brain area.

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Summary

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Chapters in this video

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Transcranial Electrical Brain Stimulation in Alert Rodents

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Implanting an On-Head Electrode System in a Rat Model for Real-Time Seizure Detection

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat