This work was supported by the National Science Center grant No. 2017/25/B/NZ7/00373. Authors would like to recognize the work of Hanna Drzyma&#322;a-Celichowska and W&#322;odzimierz Mr&#243;wczy&#324;ski, who both contributed to the data gathering and analysis of the results presented in this paper.

All procedures connected to this protocol have been accepted by the appropriate authorities (e.g., Local Ethics Committee) and follow the national and international rules on animal welfare and management.
NOTE: Each participant involved in the procedure has to be properly trained in basic surgical procedures and has to have a valid license for performing animal experiments.
1. Anesthesia and premedication
<ol>
	<li>Anesthetize a rat with intraperitoneal injections of sodium pentobarbital (an initial dose of 60 mg&#183;kg-1 for 6-month old male Wistar rats weighing 400&#8210;550g). 
	NOTE: This protocol is not limited to the indicated strain, sex or age of rats. Also, alternative anesthesia such as ketamine-xylazine mix, alpha-chloralose or fentanyl+midazolam+medetomidine can be used if more suitable for different research goals or when required by the ethics committee.</li>
	<li>After approximately 5 min, check the depth of anesthesia by pinching the rat&#8217;s hind limb toe with blunt forceps. Proceed with the next steps of the protocol only when no reflex action is observed.</li>
	<li>Inject 0.05 mL of atropine subcutaneously in order to reduce mucus production after intubation.</li>
	<li>Inject subcutaneously 5 mL of phosphate buffer containing 4% glucose solution, NaHCO3 (1 %) and gelatin (14%). This buffer will be absorbed by the cutaneous vessels throughout an experiment and will help maintain fluid balance.</li>
	<li>Throughout the surgery, periodically check the animal for reflex actions and supplement anesthesia if required (10 mg&#183;kg-1&#183;h-1of sodium pentobarbital).</li>
</ol>
2. Surgery
<ol>
	<li>Prepare the animal for surgical treatment by shaving fur over the dorsal part of the left hindlimb, from the ankle to the hip, the backside, from tail to the high thoracic segments, the left side of the chest, and the ventral side of the neck area above the sternum</li>
	<li>Placement of the intravenous line
	<ol>
		<li>Place the rat on its back on a closed-loop heating pad (and secure it with limb fixations).</li>
		<li>Using a 21 blade, make a longitudinal cut through the skin from a sternum to a chin.</li>
		<li>Hold the skin with forceps and separate it from the underlying tissue.</li>
		<li>Using blunt dissection techniques expose the right jugular vein. Carefully dissect the vein from surrounding tissues.</li>
		<li>Locate the part of the vein without branching points, slip two 4-0 ligatures beneath it.</li>
		<li>Make one loose knot on the proximal end of the previously identified non-branching segment of the vein and one loose knot on the distal end of this segment of the vein. Clamp the vein proximal to the heart, and then ligate the distal part of the vein.</li>
		<li>Using iris scissors, make an incision between the clamp and distant ligature. Hold a flap of the vein, and introduce a pre-filled catheter to the point where it is blocked by the clamp.</li>
		<li>While holding the vein and the catheter together with forceps, remove the clamp and push the catheter several millimeters into the vein. Secure both ends of the catheter to the vein, and add an additional fixation point to the skin.</li>
	</ol>
	</li>
	<li>Introduction of the tracheal tube
	<ol>
		<li>Using blunt forceps separate the two mandibular glands covering the sternohyoid muscles. Separate sternohyoid muscles at the midline to expose the trachea.</li>
		<li>Slip three 4-0 ligatures beneath the trachea, then make two knots below the tracheal tube insertion point and one knot above.</li>
		<li>Locate the cricoid cartilage of the larynx and make an incision below the third tracheal cartilage.</li>
		<li>Insert a tracheal tube down the trachea and secure the tube in place with pre-prepared ligatures, then add an additional ligature to the skin.</li>
		<li>Place a small piece of cotton wool above the separated muscles, and suture the skin over the operated area.</li>
	</ol>
	</li>
	<li>Dissection of hind limb nerves
	<ol>
		<li>Using 21 blade, make a longitudinal cut on the posterior side of the left hind limb, from the Achilles tendon to the hip.</li>
		<li>Grab the skin with forceps, and using blunt dissection techniques separate the skin from underlying muscles on both sides of the incision.</li>
		<li>Locate the popliteal fossa at the back of the knee joint, which is covered by the biceps femoris muscle, and using scissors make a cut between the anterior and posterior part of this muscle.</li>
		<li>Moving upwards cut two heads of the biceps femoris all the way to the hip to expose the sciatic nerve. Cauterize as needed to prevent bleeding.</li>
		<li>Identify the sural, tibial and common peroneal branches of the sciatic nerve.</li>
		<li>Using scissors, separate the lateral from the medial head of the gastrocnemius muscle to expose the tibial nerve and its branches.</li>
		<li>Using 55 forceps grab the distal end of the sural nerve, cut it distally and dissect as far as possible.</li>
		<li>Repeat the procedure with the common peroneal nerve.</li>
		<li>Using a blunt glass rod separate the tibial nerve from surrounding tissues, taking care not to damage the blood vessels, and cut it distally.</li>
		<li>Identify the medial gastrocnemius (MG) and the lateral gastrocnemius and soleus (LGS) nerves.</li>
		<li>Using 55 forceps, carefully dissect the MG and LGS nerves, disconnecting them from surrounding tissues, but maintaining their connection to the respective muscles.</li>
		<li>Place a saline-soaked piece of cotton wool under the exposed nerves.</li>
		<li>Close the skin over the operated area.</li>
	</ol>
	</li>
	<li>Laminectomy
	<ol>
		<li>Using 21 blade make a longitudinal incision from the sacrum up to the thoracic vertebrae.</li>
		<li>Separate the skin from underlying muscles.</li>
		<li>Cut the longissimus muscle on both sides of the thoracic and lumbar spinous processes.</li>
		<li>Using blunt-edged scalpel retract the muscles from the spinal column to expose the transverse processes of each vertebra.</li>
		<li>Using blunt tip scissors cut the tendons of muscles connected to the transverse processes along the exposed spinal column. Apply hemostatic agents if necessary.</li>
		<li>Identify the Th13 vertebra as the lowest thoracic segment with rib insertion and using fine rongeurs remove spinous processes and laminae from Th13 to L2 vertebrae to expose lumbar segments of the spinal cord. Remember not to damage the L3 spinous process which will be used as a fixation point for spine stabilization.</li>
		<li>Remove the Th12 spinous process and smooth the vertebra dorsal surface as much as possible.</li>
		<li>Using blunt dissection techniques separate the muscles from Th11 vertebra to create holder insertion points.</li>
		<li>Place thin saline-soaked cotton wool over the exposed spinal cord segments.</li>
		<li>Move the rat to the custom made metal frame with two parallel bars and two adjustable arms with clamps to support and stabilize the spine.</li>
	</ol>
	</li>
</ol>
3. Preparation for the recording and stimulation
<ol>
	<li>Vertebral column fixation and nerve arrangement
	<ol>
		<li>Place the rat in the custom-made frame on a heating pad, connected to the closed-loop heating system to maintain the animal body temperature at 37 &#177; 1&#176;C.</li>
		<li>Insert ECG electrodes under the skin and connect to an amplifier for heart rate monitoring.</li>
		<li>Using the skin flaps, form a deep pool over the exposed spinal cord.</li>
		<li>Using metal clamps, fix the vertebral column by putting clamps below Th12 transverse processes and at L3 spinous process.</li>
		<li>Make sure that the vertebral column is secured and arranged horizontally, and then apply dorso-ventral pressure on both sides of the column to retract the muscles.</li>
		<li>Fill the pool with warm (37 &#176;C) mineral oil and maintain it at this temperature.</li>
		<li>Thread a 4-0 ligature through the Achilles tendon, lift and stretch the operated left hind limb so that the ankle is leveled with the hip.</li>
		<li>Using the skin flaps make a deep pool over the exposed tibial, MG and LGS nerves.</li>
		<li>Fill the pool with warm (37 &#176;C) mineral oil.</li>
		<li>Place MG and LGS nerves on bipolar silver-wire stimulating electrodes and connect them to a square pulse stimulator. Use separate stimulation channels for each nerve.</li>
	</ol>
	</li>
	<li>Surface electrode placement
	<ol>
		<li>Place a silver ball electrode on the left caudal side of the exposed spinal cord, with a reference electrode inserted in the back muscles, and connect both electrodes to the differential DC amplifier. The surface ball electrode will be used to record afferent volleys from nerves.</li>
		<li>Using a constant-current stimulator, stimulate the MG and LGS nerves with square pulses of 0.1 ms duration, repeated at a frequency of 3 Hz, and observe afferent volleys.</li>
		<li>Determine the threshold (T) for nerve activation, stimulate each nerve at approximately 3&#183;T intensity, and record amplitude of afferent volley for each nerve.</li>
		<li>Move the surface electrode rostral and repeat the procedure to identify spinal segments at which amplitudes of the volleys are the highest for each nerve. After determining the maximum volley location, move the surface electrode to a safe distance from the spinal cord.</li>
	</ol>
	</li>
	<li>Muscle paralysis and forming a pneumothorax in order to reduce respiratory movements
	<ol>
		<li>Paralyze the rat intravenously with a neuromuscular blocker and connect the tracheal tube to an external ventilator in line with a rodent-compatible capnometer (Pancuronium bromide, at an initial dose of 0.4 mg&#183;kg-1, supplemented every 30 min in doses of 0.2 mg&#183;kg-1)</li>
		<li>Monitor the end-tidal CO2 concentration and maintain it at about 3&#8210;4% by adjusting ventilation parameters (frequency, air pressure, and flow volumes).</li>
		<li>Make a longitudinal incision in the skin between the 5th and 6th rib on a side of the recording.</li>
		<li>Using blunt tip scissors cut the overlying muscles to visualize intercostal space between the ribs.</li>
		<li>Using small sharp scissors, make a small incision in the intercostal muscles and in the pleura, then insert a tip of a blunt edge forceps into the opening, taking care not to press on the lungs.</li>
		<li>Allow forceps to expand or insert a small tube to keep the pneumothorax open throughout the experiment.</li>
		<li>After the neuromuscular block, monitor anesthesia depth by checking ECG frequency, and supplement the anesthetic agent if the heart rate exceeds 400 bpm.&#160;Muscle paralysis and forming a pneumothorax in order to reduce respiratory movements, which will improve recording stability</li>
	</ol>
	</li>
	<li>Opening the dura and pia mater
	<ol>
		<li>Using #55 forceps, gently lift the dura mater, and cut it caudally from the L5 segment, rostrally up to the L4 segment.</li>
		<li>Using a pair of ultra-thin 5SF forceps make a small patch in the pia covering the dorsal column, between the blood vessels, exactly at the level of the maximum afferent volley from the MG or the LGS nerve.</li>
		<li>Use small pieces of saline-soaked and dried gel foam to block bleeding if necessary.</li>
	</ol>
	</li>
	<li>tsDCS electrode placement
	<ol>
		<li>Make a small incision in the skin on the ventral side of a rat abdomen at the rostro-caudal level corresponding to the location of L4-L5 spinal segments.</li>
		<li>Grab the exposed skin flap with a metal clip which will serve as a reference electrode.</li>
		<li>Place a saline-soaked sponge on the dorsal side of the Th12 vertebra. Make sure that the sponge size is equal to that of an active tsDCS electrode (circle-shaped stainless steel plate of 5 mm in diameter).</li>
		<li>Using a fine manipulator, press the sponge with an active tsDCS electrode to the bone and make sure that the entire surface of the electrode is pressed equally.</li>
		<li>Connect both reference and active tsDCS electrodes to a constant-current stimulator unit, capable of delivering a continuous flow of direct current.</li>
	</ol>
	</li>
	<li>Preparation of micropipettes
	<ol>
		<li>Using a microelectrode puller, prepare a microelectrode. 
		NOTE: Both filament and non-filament electrodes can be used, however, remember that the shank of the electrode must be long enough to reach the ventral horn while being thin enough not to compress the spinal cord while descending.
		<ol>
			<li>Adjust the puller setting so that the shank entering the spinal cord is approximately 3 mm long, while the tip of the electrode is no more than 1&#8210;2 &#181;m in diameter and microelectrode resistance is between 10 and 20 M&#937;.</li>
		</ol>
		</li>
		<li>Fill the microelectrodes with 2M potassium-citrate electrolyte.</li>
		<li>Mount the prepared microelectrode on the micromanipulator allowing 1&#8210;2 &#181;m stepping movement and stereotaxic calibration.</li>
		<li>Connect the microelectrode to the intracellular amplifier with the reference electrode placed in the back muscles.</li>
	</ol>
	</li>
	<li>&#160;After the neuromuscular block, monitor anesthesia depth by checking ECG frequency, and supplement the anesthetic agent so that the rat heart rate does not exceed 400 bpm.</li>
</ol>
4. Motoneuron tracking and penetration
<ol>
	<li>Place the afferent volley recording electrode back on the dorsal surface of the spinal cord, caudally to the location of the recording site, at the level of the L6 segment.</li>
	<li>Stimulate the MG and LGS nerves with electrical 0.1 ms pulses at a frequency of 3 Hz, and 3T intensity, to activate all the axons of alpha-motoneurons within a selected nerve.</li>
	<li>Drive the micropipette into a selected patch in the pia with a medio-lateral angle of 15&#8210;20&#176; (with a tip directed laterally).</li>
	<li>After descending below the surface, calibrate the microelectrode and compensate its capacitance and voltage offset, and continue penetration of the spinal cord when all parameters are stable. An antidromic field potential of the motoneuron pool will be visible at the microelectrode voltage trace while approaching a dedicated motor nucleus during stimulation of the respective nerve.</li>
	<li>Proceed penetration with the microelectrode at 1&#8210;2 &#181;m steps, and periodically use the buzz function of the intracellular amplifier to clear the electrode tip from any residue.</li>
	<li>Observe motoneuron penetration which will be characterized by a sudden hyperpolarization of the recorded voltage trace and appearance of an antidromic spike potential.</li>
</ol>
5. Recording motoneuron membrane and firing properties
<ol>
	<li>In a bridge mode of the intracellular amplifier, identify the motoneuron on the basis of the &#8220;all-or-nothing&#8221; appearance of the antidromic action potential by stimulating respective nerve branches. Record 20 subsequent traces for later averaging.</li>
	<li>&#160;Implement a strict inclusion criterion to ensure high-quality&#160;data: resting&#160;membrane potential of at least -50 mV in&#160;amplitude; action&#160;potential amplitudes greater than 50 mV, with a positive&#160;overshoot; membrane&#160;potential stable for at least 5 min prior to recording.</li>
	<li>In a discontinuous current-clamp mode (current switch rate mode 4&#8211;8 kHz) of the intracellular amplifier, evoke an orthodromic action potential in a motoneuron using 0.5 ms intracellular depolarizing current pulses. Repeat at least 20 times for offline averaging.</li>
	<li>Stimulate a motoneuron with 40 short pulses (100 ms) of hyperpolarizing current (1 nA) in order to calculate cell input resistance.</li>
	<li>Stimulate a motoneuron with 50 ms square-wave pulses at increasing amplitudes to determine the rheobase value as the minimum amplitude of depolarizing current required to elicit a single spike.</li>
	<li>Inject 500 ms square-wave pulses of depolarizing current, at increasing amplitudes in steps of 0.1&#8211;2 nA to evoke rhythmic discharges of motoneurons.</li>
</ol>
6. Trans-spinal direct current stimulation (tsDCS)
<ol>
	<li>While maintaining a stable penetration of the motoneuron, start the polarization procedure by trans-spinal application of direct current. Adjust the current intensity and application time to the experiment design (e.g., 0.1 mA for 15 min).</li>
	<li>Immediately after switching on the DC, observe the motoneuron membrane potential. Anodal polarization (the active electrode as an anode) should result in depolarization of the membrane potential, while cathodal polarization (the active electrode as a cathode) should evoke an opposite effect. Observe whether a change in the resting membrane potential in response to DC stimulation is constant, which ensures that electrical field intensity is not affected.</li>
	<li>During continuous current application, repeat steps 5.3&#8210;5.6 in 5 min intervals.</li>
	<li>Turn off the DC and continue to repeat steps 5.3&#8210;5.6 in 5 min intervals until recordings become unstable or inclusion criteria are compromised.</li>
	<li>Terminate the experiment and euthanizethe animal using intravenous administration of a lethal dose of pentobarbital sodium (180 mg&#183;kg-1).</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+properties+of+motoneurons+innervating+different+muscle+unit+types+in+rat+gastrocnemius.">Physiological properties of motoneurons innervating different muscle unit types in rat gastrocnemius.</a> Journal of Neurophysiology. 69 (4), 1160-1170 (1993).</li><li>Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-spinal+direct+current+stimulation+modifies+spinal+cord+excitability+through+synaptic+and+axonal+mechanisms.">Trans-spinal direct current stimulation modifies spinal cord excitability through synaptic and axonal mechanisms.</a> Physiological Reports. 2 (9), (2014).</li><li>Manuel, M., Iglesias, C., Donnet, M., Leroy, F., Heckman, C. J., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics,+high-frequency+oscillations,+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons.">Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 29 (36), 11246-11256 (2009).</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 Neurophysiology: Official Journal of the International Federation of Clinical Neurophysiology. 120 (6), 1161-1167 (2009).</li><li>B&#261;czyk, M., Jankowska, 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+effects+of+direct+current+are+reproduced+by+intermittent+depolarization+of+myelinated+nerve+fibers.">Long-term effects of direct current are reproduced by intermittent depolarization of myelinated nerve fibers.</a> Journal of Neurophysiology. 120 (3), 1173-1185 (2018).</li><li>B&#261;czyk, M., Drzyma&#322;a-Celichowska, H., Mr&#243;wczy&#324;ski, W., Krutki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motoneuron+firing+properties+are+modified+by+trans-spinal+direct+current+stimulation+in+rats.">Motoneuron firing properties are modified by trans-spinal direct current stimulation in rats.</a> Journal of Applied Physiology. 126 (5), 1232-1241 (2019).</li><li>B&#261;czyk, M., Drzyma&#322;a-Celichowska, H., Mr&#243;wczy&#324;ski, W., Krutki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lasting+modifications+of+motoneuron+firing+properties+by+trans-spinal+direct+current+stimulation+in+rats.">Long-lasting modifications of motoneuron firing properties by trans-spinal direct current stimulation in rats.</a> European Journal of Neuroscience. , (2019).</li><li>Miranda, P. C., Faria, P., Hallett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+the+ratio+of+injected+current+to+electrode+area+tell+us+about+current+density+in+the+brain+during+tDCS.">What does the ratio of injected current to electrode area tell us about current density in the brain during tDCS.</a> Clinical Neurophysiology: Official Journal of the International Federation of Clinical Neurophysiology. 120 (6), 1183-1187 (2009).</li><li>Rahman, 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=Cellular+effects+of+acute+direct+current+stimulation:+somatic+and+synaptic+terminal+effects.">Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.</a> The Journal of Physiology. 591 (10), 2563-2578 (2013).</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=Effects+of+uniform+extracellular+DC+electric+fields+on+excitability+in+rat+hippocampal+slices+in+vitro.">Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro.</a> The Journal of Physiology. 557, 175-190 (2004).</li><li>Jankowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+control+of+motor+outputs+by+intrinsic+and+externally+induced+electric+field+potentials.">Spinal control of motor outputs by intrinsic and externally induced electric field potentials.</a> Journal of Neurophysiology. 118 (2), 1221-1234 (2017).</li><li>Button, D. C., Gardiner, K., Marqueste, T., Gardiner, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-current+relationships+of+rat+hindlimb+alpha-motoneurones.">Frequency-current relationships of rat hindlimb alpha-motoneurones.</a> The Journal of Physiology. 573, 663-677 (2006).</li></ol>

Authors have no conflict of interest to disclose.

If performed correctly, the surgical part of the described protocol should be completed within approximately three hours. One should take particular care in maintaining stable physiological conditions of an animal during the surgery, in particular body temperature and depth of anesthesia. Apart from obvious ethical considerations, a lack of proper anesthesia can result in excessive limb movements during nerve dissection or laminectomy and lead to damage to the preparation or a premature experiment termination. Upon paral...

Trans-spinal direct current stimulation (tsDCS) is gaining recognition as a potent method to modify spinal circuit excitability in health and disease1,2,3. In this technique, a constant current is passed between an active electrode located above selected spinal segments, with a reference electrode located either ventrally or more rostrally4. Several studies have already confirmed that tsDCS can be used in managing certain pathological conditions, such as neuropathic pain5, spasticity6, spinal cord injury7 or to facilitate rehabilitation8. Researchers&#160;suggest that tsDCS evokes alterations in the ion distribution between the intracellular and the extracellular space across the cell membrane, and this can either facilitate or inhibit neuronal activity depending on the current orientation9,10,11. However, until recently, a direct confirmation of this influence on motoneurons was lacking.
Here, we describe a detailed protocol to conduct in vivo intracellular recording of electrical potentials from lumbar spinal motoneurons in the anesthetized rat with simultaneous application of tsDCS, in order to observe changes in motoneuron membrane and firing properties in response to anodal or cathodal polarization of the spinal neuronal network. Intracellular recordings open several areas of investigation of neuron properties, unavailable for previously used extracellular techniques9,12. For example, it is possible to precisely measure motoneuron membrane voltage response to direct current flow induced by tsDCS, to indicate voltage threshold for spike generation, or to analyze action potential parameters. Moreover, this technique allows us to determine motoneuron passive membrane properties, such as input resistance, and to observe the relationship between intracellular stimulation current and frequency of rhythmic firing of motoneurons. Antidromic identification of recorded motoneuron, based on the stimulation of functionally identified nerves (i.e., nerves providing efferents to flexors or extensors) allows us to additionally identify types of innervated motor units (fast versus slow), which gives an opportunity to test whether polarization differently influences individual elements of the mature spinal neuronal system. Due to extensive surgery preceding the recording and high requirements on stability and reliability of recordings, this technique is highly challenging but allows direct and long-term assessment of electrophysiological characteristics of one motoneuron: before, during and after application of tsDCS, which is crucial to determine both its acute actions and persistent effects13. As a motoneuron directly activates extrafusal muscle fibers14 and takes part in feedback control of a muscle contraction and developed force15,16 any observed influence of tsDCS on the motor unit or muscle contractile properties may be linked to modulations of motoneuron excitability or firing characteristics.

<table>
	<tbody>
		<tr>
			<td>Durgs and solutions</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Atropinum sulfuricum</td>
			<td>Polfa Warszawa</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>346351</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Merck</td>
			<td>106329</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pancuronium Jelfa</td>
			<td>PharmaSwiss/Valeant</td>
			<td>-</td>
			<td>Neuromuscular blocker</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Biowet Pu&#322;awy Sp. z o.o</td>
			<td>-</td>
			<td>Main anesthetic agent</td>
		</tr>
		<tr>
			<td>Pottasium citrate</td>
			<td>Chempur</td>
			<td>6100-05-06</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tetraspan</td>
			<td>Braun</td>
			<td>-</td>
			<td>HES solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical equipment</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>21 Blade</td>
			<td>FST</td>
			<td>10021-00</td>
			<td>Scalpel blade</td>
		</tr>
		<tr>
			<td>Cauterizer</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Chest Tubes</td>
			<td>Mila</td>
			<td>CT1215</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dumont #4 Forceps</td>
			<td>FST</td>
			<td>11241-30</td>
			<td>Muscle forceps</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Dura forceps</td>
		</tr>
		<tr>
			<td>Dumont #5F Forceps</td>
			<td>FST</td>
			<td>11255-20</td>
			<td>Nerve forceps</td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>FST</td>
			<td>11252-00</td>
			<td>Pia forceps</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>Blunt forceps</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11053-10</td>
			<td>Skin forceps</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13013-14</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>FST</td>
			<td>16021-14</td>
			<td>For laminectomy</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15000-08</td>
			<td>Vein scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15002-08</td>
			<td>Dura scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14184-09</td>
			<td>For trachea cut</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>104075-11</td>
			<td>Muscle scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14002-13</td>
			<td>Skin scissors</td>
		</tr>
		<tr>
			<td>Tracheal tube</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Vein catheter</td>
			<td>Vygon</td>
			<td>1261.201</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vessel cannulation forceps</td>
			<td>FST</td>
			<td>18403-11</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vessel clamp</td>
			<td>FST</td>
			<td>18320-11</td>
			<td>For vein clamping</td>
		</tr>
		<tr>
			<td>Vessel Dilating Probe</td>
			<td>FST</td>
			<td>10160-13</td>
			<td>For vein dissection</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sugrgical materials</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gel foam</td>
			<td>Pfizer</td>
			<td>GTIN 00300090315085</td>
			<td>Hemostatic agent</td>
		</tr>
		<tr>
			<td>Silk suture 4.0</td>
			<td>FST</td>
			<td>18020-40</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Silk suture 6.0</td>
			<td>FST</td>
			<td>18020-60</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Axoclamp 2B</td>
			<td>Molecular devices</td>
			<td>discontinued</td>
			<td>Intracellular amplifier/ new model Axoclamp 900A</td>
		</tr>
		<tr>
			<td>CapStar-100 End-tidal CO2 Monitor</td>
			<td>CWE</td>
			<td>11-10000</td>
			<td>Gas analyzer</td>
		</tr>
		<tr>
			<td>Grass S-88</td>
			<td>A-M Systems</td>
			<td>discontinued</td>
			<td>Constant current stimulator</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket Systems with Flexible Probe</td>
			<td>Harvard Apparatus</td>
			<td>507222F</td>
			<td>Heating system</td>
		</tr>
		<tr>
			<td>ISO-DAM8A</td>
			<td>WPI</td>
			<td>74020</td>
			<td>Extracellular amplifier</td>
		</tr>
		<tr>
			<td>Microdrive</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement IVM/Scientifica</td>
		</tr>
		<tr>
			<td>P-1000 Microelectrode puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>Microelectrode puller</td>
		</tr>
		<tr>
			<td>SAR-830/AP Small Animal Ventilator</td>
			<td>CWE</td>
			<td>12-02100</td>
			<td>Respirator</td>
		</tr>
		<tr>
			<td>Support frame</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement lab standard base 51601/Stoelting</td>
		</tr>
		<tr>
			<td>Spinal clamps</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement Rat spinal adaptor 51695/Stoelting</td>
		</tr>
		<tr>
			<td>TP-1 DC stimulator</td>
			<td>WiNUE</td>
			<td>-</td>
			<td>tsDCS stimulator</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1B150-4 glass capillaries</td>
			<td>WPI</td>
			<td>1B150-4</td>
			<td>For microelectrodes production</td>
		</tr>
		<tr>
			<td>Cotton wool</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>flexible tubing</td>
			<td>-</td>
			<td>-</td>
			<td>For respirator and CO2 analyzer connection</td>
		</tr>
		<tr>
			<td>MicroFil</td>
			<td>WPI</td>
			<td>MF28G67-5</td>
			<td>For filling micropipettes</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>-</td>
			<td>-</td>
			<td>For nerve electrodes</td>
		</tr>
	</tbody>
</table>

in vivo intracellular recording of type-identified rat spinal motoneurons during trans-spinal direct current stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

Parameters of action potentials and several membrane properties can be calculated on the basis of intracellular recordings when stable conditions of cell penetration are ensured. Figure 1A presents a typical orthodromic action potential evoked by intracellular stimulation, which meets all criteria for data inclusion (the resting membrane potential of at least -50 mV, and spike amplitude higher than 50 mV, with a positive overshoot). Action potential parameters, such as the spike amplitude, t...

Watch this Scientific Journal Video about In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation at JoVE.com

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

in-vivo-intracellular-recording-type-identified-rat-spinal

Research

JoVE Journal

Neuroscience

5.1K Views.  Poznań University of Physical Education. This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Video: In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

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

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

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

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while maintaining clinical standards. The current consensus regarding the efficacy of tDCS is that multiple treatment sessions are needed to observe targeted behavioral reductions in symptom burden. However, the requirement for patients to travel to clinic daily for stimulation sessions presents a major obstacle for potential participants, due to work or family obligations or limited ability to travel. This study presents a protocol that directly overcomes these obstacles by eliminating the need to travel to clinic for daily sessions.
This is an updated protocol for remotely supervised self-administration of tDCS for daily treatment sessions paired with a program of computer-based cognitive training for use in clinical trials. Participants only need to attend clinic twice, for a baseline and study-end visit. At baseline, participants are trained and provided with a study stimulation device, and a small laptop computer. Participants then complete the remainder of their stimulation sessions at home while they are monitored via videoconferencing software.
Participants complete computerized cognitive remediation during stimulation sessions, which may serve a therapeutic role or as a &#34;placeholder&#34; for other computer-based activity. Computers are enabled for real-time monitoring and remote control by study staff.
Outcome measures that assess feasibility and tolerance are administered remotely with the aid of visual analogue scales that are presented onscreen. Following completion of all RS-tDCS sessions, participants return to clinic for a study end visit in which all study equipment is returned.
Results support the safety, feasibility, and scalability of the RS-tDCS protocol for use in clinical trials. Across 46 patients, 748 RS-tDCS sessions have been completed. This protocol serves as a model for use in future clinical trials involving tDCS.

This manuscript provides an updated remote supervision protocol that enables participation in transcranial direct current stimulation (tDCS) clinical trials while receiving treatment sessions from home. The protocol has been successfully piloted in both patients with multiple sclerosis and Parkinson&#39;s disease.

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...

The Combined Use of Transcranial Direct Current Stimulation and Robotic Therapy for the Upper Limb

Neurologic disorders such as stroke and cerebral palsy are leading causes of long-term disability and can lead to severe incapacity and restriction of daily activities due to lower and upper limb impairments. Intensive physical and occupational therapy are still considered main treatments, but new adjunct therapies to standard rehabilitation that may optimize functional outcomes are being studied.
Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that polarizes underlying brain regions through the application of weak direct currents through electrodes on the scalp, modulating cortical excitability. Increased interest in this technique can be attributed to its low cost, ease of use, and effects on human neural plasticity. Recent research has been performed to determine the clinical potential of tDCS in diverse conditions such as depression, Parkinson&#39;s disease, and motor rehabilitation after stroke. tDCS helps enhance brain plasticity and seems to be a promising technique in rehabilitation programs.
A number of robotic devices have been developed to assist in the rehabilitation of upper limb function after stroke. The rehabilitation of motor deficits is often a long process requiring multidisciplinary approaches for a patient to achieve maximum independence. These devices do not intend to replace manual rehabilitation therapy; instead, they were designed as an additional tool to rehabilitation programs, allowing immediate perception of results and tracking of improvements, thus helping patients to stay motivated.
Both tDSC and robot-assisted therapy are promising add-ons to stroke rehabilitation and target the modulation of brain plasticity, with several reports describing their use to be associated with conventional therapy and the improvement of therapeutic outcomes. However, more recently, some small clinical trials have been developed that describe the associated use of tDCS and robot-assisted therapy in stroke rehabilitation. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

The combined use of transcranial direct current stimulation and robotic therapy as an add-on for conventional rehabilitation therapy may result in improved therapeutic outcomes due to modulation of brain plasticity. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

Neurologic disorders such as stroke and cerebral palsy are leading causes of long-term disability and can lead to severe incapacity and restriction of ...

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 ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through ...

Transcranial Direct Current Stimulation (tDCS) for Memory Enhancement

Memory enhancement is one of the great challenges in cognitive neuroscience and neurorehabilitation. Among various techniques used for memory enhancement, transcranial direct current stimulation (tDCS) is emerging as an especially promising tool for improvement of memory functions in a non-invasive manner. Here, we present a tDCS protocol that can be applied for memory enhancement in healthy-participant studies as well as in aging and dementia research. The protocol uses weak constant anodal current to stimulate cortical targets within cortico-hippocampal functional network engaged in memory processes. The target electrode is placed either on the posterior parietal cortex (PPC) or the dorsolateral prefrontal cortex (DLPFC), while the return electrode is placed extracranially (i.e., on the contralateral cheek). In addition, we outline a more advanced method of oscillatory tDCS, mimicking a natural brain rhythm to promote hippocampus-dependent memory functions, which can be applied in a personalized and non-personalized manner. We present illustrative results of associative and working memory improvement following single tDCS sessions (20 minutes) in which the described electrode montages were used with current intensities between 1.5 mA and 1.8 mA. Finally, we discuss crucial steps in the protocol and methodological decisions that must be made when designing a tDCS study on memory.

Transcranial Direct Current Stimulation tDCS for Memory Enhancement

A protocol for memory enhancement by using transcranial direct current stimulation (tDCS) targeting dorsolateral prefrontal and posterior parietal cortices, as core cortical nodes within hippocampo-cortical network, is presented. The protocol has been well evaluated in healthy-participant studies and is applicable to aging and dementia research as well.

Memory enhancement is one of the great challenges in cognitive neuroscience and neurorehabilitation. Among various techniques used for memory enhancement, ...