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>&lt;strong&gt;Durgs and solutions&lt;/strong&gt;</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?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>&lt;strong&gt;Surgical equipment&lt;/strong&gt;</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>&lt;strong&gt;Sugrgical materials&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</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 ...

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5.1K Views.  Poznań University of Physical Education. Dit protocol maakt gebruik van intracellulaire opnames van spinale motoneurons, om direct de invloed van trans-spinale directe stroom stimulatie op de functie van spinale netwerken te onderzoeken. Het grote voordeel van deze techniek is dat het mogelijk maakt intracellulaire opnames worden uitgevoerd op volledig volwassen zenuwstelsel. Het vergemakkelijken van de vertaling van de experimentele waarnemingen naar praktische toepassingen.Na bevestiging van een gebrek aan reactie op pedaal...

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 ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

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 ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

Understanding Motor Neuron Responses to Spinal Cord Stimulation

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Author Spotlight: Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

In Vivo Recording of Intracellular Signals from a Single Neuron

Source: Altwegg-Boussac, T., et al. <a href="https://app.jove.com/v/53576">Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of electrical signals from single brain neurons using a fine-tipped glass micropipette and a specialized setup. The micropipette is carefully inserted into the rat&#39;s brain and is buzzed to penetrate the neuron to record internal neuronal electrical signals.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Trans-Spinal Direct Current Stimulation of a Rat&#39;s Spinal Motoneuron

Source: B&#261;czyk, M., et al. <a href="https://app.jove.com/v/61439">In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation.</a> J. Vis. Exp. (2020).
This video describes the in vivo intracellular recording of rat motoneurons under trans-spinal direct current stimulation. The protocol describes how to measure membrane properties and record the rhythmic firing of motoneurons before, during, and after anodal or cathodal polarization of the spinal cord.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Stimulation and Modulation Techniques

In Vivo Measurement of the Compound Muscle Action Potential in a Rat

Source: Wild, B. M., et al. <a href="https://app.jove.com/v/56102">In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure for recording compound muscle action potential (CMAP) in rats by applying electrical stimuli to the ulnar nerve, adjusting electrodes for optimal signal, and increasing stimulation to assess nerve functionality. This method enables the in vivo investigation of the peripheral nerve functions in neurological disorders.

In Vivo Single-Fiber Recording of Sciatic Nerve C-Fibers in Rats

Source: Mao, H., et al. <a href="https://app.jove.com/v/59234">Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure.</a> J. Vis. Exp. (2019).
This video demonstrates a single-fiber recording of C-fiber nerve conduction in rats. An anesthetized rat is incised to expose the sciatic nerve, which provides sensory and motor supply to the lower limbs. The individual C-fibers are then exposed. Electrical stimulation is provided using stimulus electrodes implanted in the foot, and a response is recorded from a single nerve fiber.

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

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