This work was supported by the German Research Foundation (DFG RE 2740/3-1). We thank Frank Huethe and Thomas G&#252;nther for the in-house production of the custom-made tES set-up and DC-stimulator.

For research involving animals, the relevant (country-specific) approvals must be obtained before starting experiments. All animal experiments reported here are performed according to the EU directive 2010/63/EU, the updated German animal protection law (&#34;Tierschutzgesetz&#34;) of July 2013, and the updated German animal research regulations of August 2013. Animal protocols have been approved by the local authorities &#34;Commission for Animal Experimentation of the Regional Council of Freiburg&#34; and &#34;Commission for Animal Experimentation of the University Medical Center Freiburg&#34;.
1. Preparation of Instrumentation and Material for Surgery
<ol>
	<li>Make sure that the items listed in Figure 1 are available and already placed for surgery.</li>
	<li>Prepare a thin rectangular platinum plate (e.g., 10 mm x 6 mm x 0.15 mm), which will serve as the counter electrode placed subcutaneously on the chest, and punch two small holes in two opposite corners of the plate.</li>
	<li>Solder an insulated cable with a length of ~10 cm using a lead-free tin-solder to one of the corners (without a hole) of the platinum plate.</li>
	<li>Apply a small drop of histo-acrylic glue on the soldering joint for isolation.</li>
</ol>
2. Preparation of the Rodent for Surgery
<ol>
	<li>Assign a study number to the rodent and note this on the prepared surgery card.</li>
	<li>Weigh the rodent and note the weight on the surgery card. Calculate the dose of injection anesthetics (e.g., ketamine 80 mg/kg bodyweight plus xylazine 12 mg/kg bodyweight for rats).</li>
	<li>Induce anesthesia by intraperitoneal (i.p.) injection of the calculated amount of anesthetics. 
	NOTE: When using inhalation anesthesia instead (e.g., isoflurane), place the rodent in an induction chamber with continuous flow of ~4% in 1-2 L/min oxygen.</li>
	<li>Check depth of anesthesia by the toe pinch reflex starting 5 min post injection. If the toe pinch reflex is still present, reach prolongation and deepening of anesthesia by injection of 30% of the initial dose.
	<ol>
		<li>If at any time point in the experiment the toe pinch reflex returns, 30% of the initial dose of anesthesia should be injected.</li>
		<li>When using inhalation anesthesia, look for loss of the postural reflex of the rodent in the induction chamber and check the depth of anesthesia by the lack of a toe pinch reflex. If reflexes are still present, extend the duration in the anesthesia chamber. Throughout the whole experiment, adapt the percentage of isoflurane to the depth of anesthesia until reaching a maintenance concentration of ~1-1.5% isoflurane.</li>
		<li>When the frequency of breathing decreases and gasping occurs, lower the percentage; when the rodent regains the toe pinch reflex or shows spontaneous movement, increase the percentage of inhalation anesthetic.</li>
	</ol>
	</li>
	<li>As soon as reflexes are absent, place the rodent on the lab bench or hold it in hand. 
	NOTE: When using inhalation anesthesia, provide continued reduced isoflurane flow (now between 2-3%) by using a nozzle connected to the nebulizer.</li>
	<li>Remove the hair on the rat&#39;s head by shaving the area from ear to ear and from between the rostral eye level to just behind the ears with a clipper. Then remove the hair on the chest by shaving the area between the forelimbs from the xiphoid up to the clavicles. 
	NOTE: Keeping the skin under tension eases the shaving.</li>
	<li>Cover the eyes of the rat with a drop of eye ointment to protect the cornea.</li>
	<li>Mark the rat&#39;s ear according to the assigned study number. 
	NOTE: Depending on the length of the study, a tail mark might be sufficient, otherwise standardized earmarking is preferable.</li>
</ol>
3. Surgical Procedure: Chest Electrode Implantation
NOTE: This step can be skipped when the counter electrode is placed externally on the shaved chest with a vest.
<ol>
	<li>Place the rodent prone (on the chest) on the operation table. 
	NOTE: In case of inhalation anesthesia, keep the rat&#39;s snout placed in the anesthesia nozzle, further reducing the isoflurane concentration to 1.5-2%.</li>
	<li>Disinfect the shaved scalp with a disinfectant spray or with a swab soaked in antiseptic agent (e.g., ethanol 70%) and let air-dry. Repeat two times.</li>
	<li>Cut the skin with a scalpel in one line from the rostral eye level to the mid ear level. 
	NOTE: This allows for tunneling of the connecting cable from the implanted chest electrode toward the top of the head and is also the desired cut for the DCS electrode socket placement.</li>
	<li>Turn the rat to supine position, so that the chest is exposed.</li>
	<li>Disinfect the skin of the chest as described in step 3.2.</li>
	<li>Elevate the lateral skin of the right chest with a tissue forceps and cut a buttonhole with small scissors of about 0.5 cm medial from the right axilla. Then make a straight sagittal cut in cranial orientation with the scissors.</li>
	<li>Form a subcutaneous pouch by atraumatically disconnecting the skin from the left major pectoral muscle. Do so by repeatedly opening the small scissors (or by a saline soaked cotton swab).</li>
	<li>Turn the animal on its right side to tunnel the cable path from the left occipital corner of the opened head skin along the neck to exit into the pectoral pouch by penetrating the superficial fascia using homeostatic forceps.</li>
	<li>Carefully open the homeostatic forceps to grab the end of the electrode cable attached to the platinum electrode without allowing sharp wires to stray. Pull the cable through the tunnel until the electrode enters the pouch, oriented with the soldering point towards the rodents left hindlimb. Turn the rodent back to the prone position.</li>
	<li>Fix the platinum plate with a sterile synthetic braided non-absorbable suture to the pectoral fascia at the two opposing corner holes (4-5 knots are recommended for stability).</li>
	<li>Similarly attach the cable to the fascia by a loose knot, forming a slight loop before the entrance of the tissue tunnel.</li>
	<li>Close the skin with 3-4 cutaneous sutures depending on the size of the cut (the same suture material can be used as for the electrode and cable).</li>
</ol>
4. Surgical Procedure: Placement of the Epicranial tES Socket
<ol>
	<li>Place the animal in a stereotactic frame. 
	NOTE: If using inhalation anesthesia, lower the concentration of the anesthetic to a maintenance isoflurane flow of ~1.5-1%, adjusted to the toe pinch reflex and breathing pattern.</li>
	<li>Disinfect the shaved scalp as described in step 3.2.</li>
	<li>Cut the skin with a scalpel in one line from the rostral eye level to the mid ear level. 
	NOTE: If the chest electrode placement was performed, steps 4.2 and 4.3 have already been performed.</li>
	<li>Scrape off the periosteum (connective tissue on the skull) to the sides with the scalpel and thoroughly wipe off with cotton swaps. Fixate the connective tissue at the 4 corners of the cut with bulldog clamps and let them hang laterally to keep the surgery field open.</li>
	<li>Apply 0.9% saline to clean the bone surface and tissue with cotton swabs. Then clean the bone surface with 3% H2O2. Avoid contact with the tissue. Hereby the bone is cleaned more thoroughly and minor bleeding from the bone will be stopped. Also, residuals of the periosteum become visible. Remove these residuals with a cotton swab applying moderate pressure. 
	NOTE: Removal of the periosteum residuals will increase adhesion and durability of the tES socket glued onto the bone.
	<ol>
		<li>In case of unstoppable bleeding, use a bone drill and touch it for 1-3 s with slight pressure on the bone. This mechanical procedure will in most cases stop the bleeding without significant heating. Never use electrocautery on the bone; even brief application will result in brain tissue damage (electrocautery should solely be used for wound tissue bleeding).</li>
	</ol>
	</li>
	<li>As fixation screws will improve set-up adherence, choose a drill bit fitting the screw size. Place two burr holes on two different bone plates by pre-drilling with a hand drill and then by slight vertical pressure application with the bone drill. Avoid close proximity to the desired position of the tES socket, as it might hinder screwing in the electrode (e.g., for left primary motor cortical tES, choose right frontal and posterior parietal screw position).</li>
	<li>In case of an implanted counter electrode, burr a third hole located in the right posterior parietal bone for future fixation of the tunneled cable.</li>
	<li>Place the plastic screws in the burr holes and screw until the first friction is felt. Then perform three additional 180 &#176; screw turns. Check with forceps for stability of the screw and add one more turn if not tight enough. 
	NOTE: For adult rats this will ensure epidural placement of the screws without damaging the dura or brain (depending on the screw thread design, the turn number might vary) . The use of stainless steel screws should also be feasible, since even at DCS current densities above the neurodegeneration threshold, screw placement did not perturb lesion location or extent below the screws.</li>
	<li>Turn on the soldering iron and pre-heat for approximately 5 min. Wind the cable exiting the tissue tunnel occipitally around the right parietal screw and then cut it, leaving approximately 1 cm cable behind the winding. Carefully strip the insulation at the end of the cable with a scalpel.</li>
	<li>Fix the winded cable to the screw and bone with cyanoacrylic glue.</li>
	<li>Apply a small amount of the lead-free tin-solder to the connector and to the bare wires of the counter electrode cable and connect both by briefly pressing both pre-soldered parts together while touching the soldering tip until the tin-solder melts (about 2-3 s). Remove the soldering tip immediately to avoid excessive metal heating of the cable with subsequent tissue damage.</li>
	<li>Pick up the custom made tES electrode socket (Figure 1B, in red) with bent, serrated tip forceps and apply a thin layer of cyanoacrylic glue to the bottom rim of the socket. For placement above the motor cortex and using a 4 mm diameter socket, place the mid socket point at 2 mm anterior and 2 mm lateral from the bregma. For this position, the inner medial border of the socket should end directly at the sagittal suture and the caudal border should end at the height of bregma. Press the socket briefly onto the bone (most cyanoacrylic glues harden by pressure). 
	NOTE: Placing a light source directly above the socket can ease placing the socket.</li>
	<li>Ensure that the bone within the area of the socket is free of glue (by checking with light because the glue is reflective). In the case of glue spill, remove the socket, scrape the glue with the scalpel, and repeat step 4.12.</li>
	<li>After the socket is in place and the future stimulation area is free of glue, first seal the lateral border of the socket to the neighboring tissue with a small drop of cyanoacrylic glue to avoid a fluid bridge that could lead to shunting of current at this location. Do not apply too much glue as it may flow into the stimulation area (if this occurs, return to step 4.12). 
	NOTE: Keeping the stimulation area free of glue is crucial as a reduction of the stimulation area might dramatically increase current density (A/m&#178;).</li>
	<li>Cover all screws with cyanoacrylic glue.</li>
	<li>Mix the two-component dental acrylic cement in a small silicon tube or glass. As soon as it becomes viscous, apply it with a dental spatula to seal the remaining borders of the socket to the bone. Avoid any flow of dental acrylic cement into the stimulation area.</li>
	<li>Finally cover the whole skull, screws, counter electrode cable and the socket up to &#8531; of the socket with dental acrylic cement. Ensure that the cement has the correct viscosity: if too fluid, it will flow into the surrounding tissue; if too hard it is difficult to distribute it evenly.</li>
	<li>When all the bone is covered and the cement is hardened, remove the bulldog clamps; the skin should just touch the built-up cement so that suturing is not needed. (If the initial cut was chosen too long and the connective tissue or muscle is visible, apply a suture as described in step 3.12).</li>
	<li>Apply one layer of iodine with a cotton swab around the border of the cut skin and subcutaneously inject carprofen (5 mg/kg body weight dissolved in 5-7.5 mL of 0.9% saline for pain treatment and fluid replacement). 
	NOTE: If using inhalation anesthesia, turn it off now.</li>
	<li>Place the rodent in a warming box for recovery from anesthesia until the rodent is awake and postural stability is restored. 
	NOTE: Check the animal&#39;s weight development, wound state, and general well-being criteria daily according to the institution&#39;s recommendation.</li>
</ol>
5. Transcranial Electrical Stimulation Procedure
NOTE: As anesthesia affects tES effects, performing the stimulation in alert rodents whenever possible is recommended. Allow the rodent to recover for at least 5 days (healing of the head and chest wound) before starting experiments. Experiments can be performed at earlier time points after surgery when using an external counter electrode fixed with a vest, as the chest wound is most irritable; but animals need to be habituated to the electrode vest for several days and interference with behavioral tasks might occur.
<ol>
	<li>Fill the tES electrode socket half with 0.9% saline and remove air bubbles.</li>
	<li>Before cathodal tDCS sessions, always check chlorination, and if needed (such as a shiny silver surface), re-chlorinate the Ag/AgCl electrode. Before anodal tDCS sessions, remove possible excess AgCl deposits from previous stimulations with sandpaper to allow for good conductivity during stimulation. Screw in the tES electrode screw cap (Figure 1B, grey piece). 
	CAUTION: Failure to re-chlorinate the electrode between cathodal tDCS sessions will lead to exhaustion of chlorination during stimulation and to toxic build-up by electrochemical reaction. This will induce tissue damage. Re-chlorination is not needed within a single session if stimulation duration is shorter than 20 min.</li>
	<li>Connect the cables to the two connectors on the head (for anodal stimulation, the anodal cable is connected to the connector on the screw cap, for cathodal stimulation, it is opposite). 
	NOTE: When using an externally placed counter electrode, cover the counter electrode with conductive gel and place on the rodent&#39;s chest. This is easiest if the electrode is pre-fixed in a small rodent vest, which the rodent can wear during stimulation.</li>
	<li>Place the rodent into the experimental cage, with the cables connected to a swivel above the cage that allows for free movement.</li>
	<li>Turn on the stimulator and adjust the stimulation parameters (stimulation intensity, duration, ramp up and down time).</li>
	<li>When not using a commercially available stimulation device with a safety shut down and disconnection alarm, include a meter in the circuit to check the constant current flow. 
	NOTE: With this set-up, stimulation can be applied during performance or training of behavioral tasks.</li>
	<li>Check for signs of stress or discomfort of the rodent during stimulation.</li>
	<li>After the end of the stimulation, disconnect the cables, unscrew the electrode cap on the head, and clean and dry the socket with a cotton swab. Return the rodent to the home environment or proceed with a behavioral procedure if desired.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-session+transcranial+direct+current+stimulation+(tDCS)+elicits+inflammatory+and+regenerative+processes+in+the+rat+brain.">Multi-session transcranial direct current stimulation (tDCS) elicits inflammatory and regenerative processes in the rat brain.</a> PloS one. 7 (8), e43776 (2012).</li><li>Liebetanz, D., Koch, R., Mayenfels, S., K&#246;nig, F., Paulus, W., 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=Safety+limits+of+cathodal+transcranial+direct+current+stimulation+in+rats.">Safety limits of cathodal transcranial direct current stimulation in rats.</a> Clinical Neurophysiol. 120 (6), 1161-1167 (2009).</li><li>Yoon, K. J., Oh, B. -. M., Kim, D. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+improvement+and+neuroplastic+effects+of+anodal+transcranial+direct+current+stimulation+(tDCS)+delivered+1+day+vs.+1+week+after+cerebral+ischemia+in+rats.">Functional improvement and neuroplastic effects of anodal transcranial direct current stimulation (tDCS) delivered 1 day vs. 1 week after cerebral ischemia in rats.</a> Brain Res. 1452, 61-72 (2012).</li><li>Spezia Adachi, L. N., 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=Exogenously+induced+brain+activation+regulates+neuronal+activity+by+top-down+modulation:+conceptualized+model+for+electrical+brain+stimulation.">Exogenously induced brain activation regulates neuronal activity by top-down modulation: conceptualized model for electrical brain stimulation.</a> Exp Brain Res. 233 (5), 1377-1389 (2015).</li><li>Jackson, M. 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=Safety+parameter+considerations+of+anodal+transcranial+Direct+Current+Stimulation+in+rats.">Safety parameter considerations of anodal transcranial Direct Current Stimulation in rats.</a> Brain, behavior, and immunity. , (2017).</li><li>Ordek, G., Groth, J. D., Sahin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+ketamine/xylazine+anesthesia+on+the+cerebral+and+cerebellar+cortical+activities+in+the+rat.">Differential effects of ketamine/xylazine anesthesia on the cerebral and cerebellar cortical activities in the rat.</a> J Neurophysiol. 109 (5), 1435-1443 (2013).</li><li>Sykes, 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=Differences+in+Motor+Evoked+Potentials+Induced+in+Rats+by+Transcranial+Magnetic+Stimulation+under+Two+Separate+Anesthetics:+Implications+for+Plasticity+Studies.">Differences in Motor Evoked Potentials Induced in Rats by Transcranial Magnetic Stimulation under Two Separate Anesthetics: Implications for Plasticity Studies.</a> Front Neural Circ. 10, 80 (2016).</li><li>Zhang, D. X., Levy, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine+blocks+the+induction+of+LTP+at+the+lateral+entorhinal+cortex-dentate+gyrus+synapses.">Ketamine blocks the induction of LTP at the lateral entorhinal cortex-dentate gyrus synapses.</a> Brain Res. 593 (1), 124-127 (1992).</li></ol>

The authors have nothing to disclose.

This protocol describes typical materials and procedural steps for surgical realization of a permanent tES set-up, as well as for subsequent stimulation in alert rodents. During preparation of a rodent tES experiment, several methodological aspects (safety and tolerability of tES, outcome parameter) as well as conceptual aspects (comparability with human condition, anticipated effects of stimulation on a particular brain region) need to be taken into account. From a methodological point of view, the surgical set-up of th...

The transcranial administration of electrical currents to the brain (tES) has been used for decades to study brain function and to modify behavior. More recently, applying direct currents, or less frequently alternating currents (tACS and tRNS), noninvasively through the intact skull by use of two or more electrodes (anode(s) and cathode(s)) has gained scientific and clinical interest. In particular, tDCS has been used in more than 33,200 sessions in healthy subjects and patients with neuropsychiatric diseases and has emerged as a safe and easy, cost-effective bedside application, with possible therapeutic potential as well as long-lasting behavioral effects1. This clearly yielded the increased need and scientific interest in mechanistic studies, including safety aspects. This article focuses on the most commonly used form of stimulation, tDCS.
Across species, tDCS modulates cortical excitability and synaptic plasticity. Excitability changes have been reported as polarity-dependent alteration of spontaneous neuronal firing rate in rats and cats2,3,4, or as changes in motor evoked potential (MEP) amplitudes in humans and mice (both increased after anodal and decreased after cathodal tDCS: human5,6; mouse7). Anodal DCS increased synaptic efficacy of motor cortical or hippocampal synapses in vitro for several hours after stimulation or long term potentiation (LTP), when co-applied with a specific weak synaptic input or when given before a plasticity inducing stimulation8,9,10,11,12. In accordance, the benefits of stimulation on motor or cognitive training success are often revealed only if tDCS is co-applied with training8,13,14,15. While these previous findings are mainly attributed to functions of neurons, it should be noted that non-neuronal cells (glia) may also contribute to functional effects of tDCS. For instance, astrocytic intracellular calcium levels increased during anodal tDCS in alert mice16. Similarly, anodal tDCS at current densities below the threshold for neurodegeneration induced a dose dependent activation of microglia17. However, the modulation of neuron-glia interaction by tDCS needs further specific investigation.
Taken together, animal research clearly advanced our understanding of the modulatory effect of tDCS on excitability and plasticity. However, there is an &#34;inverse translational gap&#34; observable in the exponential increase in publications of human tDCS studies in contrast to the slow and minor increase in investigations of the underlying mechanisms of tES in in vitro and in vivo animal models. Additionally, rodent tES models are performed with high variability across research laboratories (ranging from transdermal to epicranial stimulation), and reported stimulation procedures are often not fully transparent hindering the comparability and replicability of basic research data as well as interpretation of results.
Here, we describe in detail the surgical implementation of a transcranial brain stimulation set-up targeting the primary motor cortex, which allows translation to the human tDCS condition while minimizing variability, and allows repeated stimulation without hindering behavior. A step-by-step protocol for subsequent tES in alert rats is provided. Methodological and conceptual aspects of safe application of tES in alert rodents are discussed.

<table><tbody><tr><td>Softasept N</td><td>B. Braun Melsungen AG,&lt;br/&gt; Melsungen, Deutschland</td><td>3887138</td><td>antiseptic agent</td></tr><tr><td>Ethanol 70 %</td><td>Carl Roth GmbH &amp;amp; Co. KG, Karlsruhe, Deutschland</td><td>T913.1</td><td></td></tr><tr><td>arched tip forceps</td><td>FST Fine science tools, Heidelberg, Deutschland</td><td>11071-10</td><td></td></tr><tr><td>Iris Forceps, 10cm, Straight, Serrated</td><td>World Precision Instruments, Inc, Sarasota, FL, USA, Inc, Sarasota, FL, USA</td><td>15914</td><td></td></tr><tr><td>Scalpel Handle #3, 13cm</td><td>World Precision Instruments, Inc, Sarasota, FL, USA, Inc, Sarasota, FL, USA</td><td>500236</td><td></td></tr><tr><td>Standard Scalpel Blade #10</td><td>World Precision Instruments, Inc, Sarasota, FL, USA, Inc, Sarasota, FL, USA</td><td>500239</td><td></td></tr><tr><td>Zelletten cellulose swabs</td><td>Lohmann und Rauscher, Neuwied, Deutschland</td><td>13349</td><td>5 x 4 cm&amp;nbsp;</td></tr><tr><td>Isoflurane</td><td>AbbVie Deutschland GmbH &amp;amp; Co</td><td>N01AB06</td><td></td></tr><tr><td>Iris Scissors, 11.5cm, Straight</td><td>World Precision Instruments, Inc, Sarasota, FL, USA, Inc, Sarasota, FL, USA</td><td>501758</td><td>small scissors</td></tr><tr><td>cotton swab/cotton buds</td><td>Carl Roth GmbH &amp;amp; Co. KG, Karlsruhe, Deutschland</td><td>EH12.1</td><td>Rotilabo</td></tr><tr><td>Kelly Hemostatic Forceps, 14cm, Straight</td><td>World Precision Instruments, Inc, Sarasota, FL, USA, Inc, Sarasota, FL, USA</td><td>501241</td><td>surgical clamp</td></tr><tr><td>electrode plate (platinum)</td><td>custom made</td><td>Wissenschaftliche Werkstatt Neurozentrum Uniklinik Freiburg, Deutschland</td><td>10x6 mm, 0.15 mm thickness</td></tr><tr><td>insulated copper strands (~1 mm diameter)</td><td>Reichelt elektronik GmbH &amp;amp; Co. KG, Sande, Germany</td><td>LITZE BL</td><td>electrode cable</td></tr><tr><td>Weller EC 2002 M soldering station</td><td>Weller Tools GmbH, Besigheim, Germany</td><td>EC2002M1D</td><td></td></tr><tr><td>Iso-Core EL 0,5 mm</td><td>FELDER GMBH L&amp;ouml;ttechnik, Oberhausen, Deutschland</td><td>20970510</td><td>lead free solder</td></tr><tr><td>MERSILENE Polyester Fiber Suture</td><td>Johnson &amp;amp; Johnson Medical GmbH, Ethicon Deutschland, Norderstedt, Germany</td><td>R871H</td><td>nonabsorbable braided suture, 4-0</td></tr><tr><td>Histoacryl</td><td>B. Braun Melsungen AG,&lt;br/&gt; Melsungen, Deutschland</td><td>9381104</td><td>cyanoacrylate</td></tr><tr><td>Ketamin 10%</td><td>Medistar GmbH, Germany</td><td>n/a</td><td>anesthetics</td></tr><tr><td>Rompun 2% (Xylazine)</td><td>Bayer GmbH, Germany</td><td>n/a</td><td>anesthetics</td></tr></tbody></table>

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.

The described implementation of a set-up for reliable repeated tES in alert rodents can be easily integrated into mechanistic experiments, dose-response studies, or experiments including behavioral tasks. To date, the comparability of data from animal studies using (noninvasive) tES is hindered by the variability of the tES stimulation set-ups between laboratories and by differences in stimulation parameters (e.g., various current densities applied at exorbitant high levels compa...

Watch this Scientific Journal Video about Transcranial Electrical Brain Stimulation in Alert Rodents at JoVE.com

Transcranial Electrical Brain Stimulation in Alert Rodents

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 ...

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Research

JoVE Journal

Neuroscience

10.6K Views.  Albert-Ludwigs-University Freiburg. Dit protocol beschrijft een chirurgische set-up voor een permanente epicranial-elektrode-aansluiting en een geïmplanteerde borst-elektrode in knaagdieren. Door het plaatsen van een tweede elektrode in het stopcontact, kunnen verschillende soorten elektrische hersenstimulatie transcranial worden afgeleverd bij het locomotorisch stelsel in alert dieren via de intacte schedel.

Video: Transcranial Electrical Brain Stimulation in Alert Rodents

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Behavior

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 ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

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.

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 ...

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 ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

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 ...

Transcranial Electrical Brain Stimulation in Alert Rodents

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