Name: Author Spotlight: Advancing Spinal Cord Stimulation - Erforschung der zellulären Reaktionen von Motoneuronen durch Patch-Clamp-Elektrophysiologie
Uploaded: 2023-09-08T14:15:00
Description: Watch this Scientific Journal Video about Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology at JoVE.com

Diese Studie wurde von der National Natural Science Foundation of China for Young Scholars (52207254 und 82301657) und dem China Postdoctoral Science Fund (2022M711833) finanziert.

Das Institutional Animal Care and Use Committee genehmigte alle Tierversuche und die Studien wurden in Übereinstimmung mit den einschlägigen Tierschutzbestimmungen durchgeführt.
1. Vorbereitung der Tiere
<ol><li>Tiere<ol><li>Haltungsinformationen: Männliche Sprague-Dawley-Ratten (postnatal 10-14 Tage, P10-P14) in einer spezifischen, pathogenfreien Umgebung. HINWEIS: Die Raumbedingungen wurden bei 20 °C ± 2 °C gehalten, Luftfeuchtigkeit: 50%-60%, mit einem 12-stünd...

Die Reaktionen von Motoneuronen auf die Stimulation des Rückenmarks verstehen

<ol>
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(116), e54525 (2016).</li><li>Sharples, S. A., Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+persistent+and+hyperpolarization-activated+inward+currents+shapes+the+differential+activation+of+motoneuron+subtypes+during+postnatal+development.">Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development.</a> Elife. 10, e71385 (2021).</li><li>Bhumbra, G. 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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=Na(V)1.1+is+essential+for+proprioceptive+signaling+and+motor+behaviors.">Na(V)1.1 is essential for proprioceptive signaling and motor behaviors.</a> Elife. 11, e79917 (2022).</li><li>Romer, S. H., Deardorff, A. S., Fyffe, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+rheostat:+Kv2.1+currents+maintain+or+suppress+repetitive+firing+in+motoneurons.">A molecular rheostat: Kv2.1 currents maintain or suppress repetitive firing in motoneurons.</a> The Journal of Physiology. 597 (14), 3769-3786 (2019).</li><li>Yao, X., 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=Structures+of+the+R-type+human+Ca(v)2.3+channel+reveal+conformational+crosstalk+of+the+intracellular+segments.">Structures of the R-type human Ca(v)2.3 channel reveal conformational crosstalk of the intracellular segments.</a> Nature Communications. 13 (1), 7358 (2022).</li><li>Bandres, M. F., Gomes, J., McPherson, J. 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Die durch SCS modulierten Bewegungsinformationen werden schließlich zu den Motoneuronen konvergiert. Daher könnte die Verwendung der Motoneuronen als Forschungsziel das Studiendesign vereinfachen und den Neuromodulationsmechanismus des SCS direkter aufdecken. Um gleichzeitig verschiedene Reizeigenschaften und zelluläre Reaktionen zu erfassen, ist eine Patch-Clamp eine gute Methode, um die elektrophysiologischen Eigenschaften auf Einzelzellebene zu untersuchen. Es gibt jedoch immer noch einige Schwierigkeiten, einschli...

Die Rückenmarkstimulation (SCS) kann die Bewegungsfunktion nach einer Rückenmarksverletzung effektiv wiederherstellen. Andreas Rowald et al. berichteten, dass SCS die Funktion des Bewegungsapparates und des Rumpfes der unteren Gliedmaßen innerhalb eines einzigen Tages ermöglicht1. Die Erforschung des biologischen Mechanismus von SCS für die Erholung des Bewegungsapparates ist ein kritisches Forschungsfeld für die Entwicklung einer präziseren SCS-Strategie. Das Team um Grégoire Courtine zeigte beispielsweise, dass exzitatorische Vsx2-Interneuronen und Hoxa10-Neuronen im Rückenmark die Schlüsselneuronen für die Reaktion auf SCS sind, und dass eine zellspezifische Neuromodulation möglich ist, um die Gehfähigkeit der Ratte nach einer Rückenmarksverletzung2 wiederherzustellen. Es gibt jedoch nur wenige Studien, die sich auf den elektrischen Mechanismus von SCS auf Einzelzellebene konzentrieren. Obwohl bekannt ist, dass der überschwellige Gleichstromreiz die Aktionspotentiale (APs) im klassischen Tintenfischexperiment 3,4,5 hervorrufen kann, ist noch unklar, wie sich die gepulste elektrische Wechselstimulation, wie z.B. SCS, auf die motorische Signalerzeugung auswirkt.
Angesichts der Komplexität intraspinaler neuronaler Schaltkreise ist eine geeignete Selektion für die Zellpopulation wichtig, um den elektrischen Mechanismus der SCS zu untersuchen. Obwohl SCS die motorische Funktion durch Aktivierung des propriozeptiven Pfades6 wiederherstellt, sind die Motoneuronen die letzte Einheit, die den motorischen Befehl ausführt, der von der Integration des afferenten Eingangs7 der Propriozeptionsinformation abgeleitet wird. Daher kann uns die direkte Untersuchung der elektrischen Eigenschaften von Motoneuronen mit SCS helfen, die zugrunde liegende Logik der spinalen motorischen Modulation zu verstehen.
Wie wir wissen, ist die Patch-Clamp-Methode die goldene Standardmethode für zellulär elektrophysiologische Aufzeichnungen mit extrem hoher raumzeitlicher Auflösung8. Daher beschreibt diese Studie eine Methode, bei der eine Patch-Klemme verwendet wird, um die elektrischen Reaktionen von Motoneuronen auf SCS zu untersuchen. Im Vergleich zur Gehirn-Patch-Klemme9 ist die Rückenmark-Patch-Klemme aus folgenden Gründen schwieriger: (1) Das Rückenmark wird durch den Wirbelkanal mit winzigem Volumen geschützt, was eine sehr feine Mikromanipulation und eine rigorose eiskalte Wartung erfordert, um eine bessere Zelllebensfähigkeit zu erhalten. (2) Da das Rückenmark zu dünn ist, um auf der Schneideschale befestigt zu werden, sollte es in Agarose mit niedrigem Schmelzpunkt getaucht und nach dem Erstarren gekürzt werden.
Daher liefert diese Methode technische Details bei der Dissektion des Rückenmarks und der gleichzeitigen Aufrechterhaltung der Zelllebensfähigkeit, um den elektrischen Mechanismus der SCS auf Motoneuronen reibungslos zu untersuchen und unnötige Versuche und Fehler zu vermeiden.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>Sigma</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide tweezers</td>
			<td>VETUS</td>
			<td>36A-SA</td>
			<td>Clip a slice</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>RWD Life Science</td>
			<td>S12006-10</td>
			<td>Cut the diaphragm</td>
		</tr>
		<tr>
			<td>Fluorescence Light Source</td>
			<td>Olympus&#160;</td>
			<td>U-HGLGPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-Gold</td>
			<td>Fluorochrome</td>
			<td>Fluorochrome</td>
			<td>Label the motor neuron</td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared CCD camera</td>
			<td>Dage-MTI</td>
			<td>IR-1000E</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator&#160;</td>
			<td>Sutter Instrument&#160;</td>
			<td>MP-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter instrument</td>
			<td>P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors&#160;</td>
			<td>Jinzhong</td>
			<td>wa1020</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>Microscope for anatomy</td>
			<td>Olympus&#160;</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-toothed tweezers</td>
			<td>RWD Life Science</td>
			<td>F11008-09</td>
			<td>Lift the cut vertebral body</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>V900182</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>LUMPLFLN60XW</td>
			<td>working distance 2 mm&#160;</td>
		</tr>
		<tr>
			<td>Osmometer&#160;</td>
			<td>Advanced&#160;</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier&#160;</td>
			<td>Axon&#160;</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp digitizer</td>
			<td>Axon&#160;</td>
			<td>Digidata 1550B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter&#160;</td>
			<td>Mettler Toledo&#160;</td>
			<td>FE28</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Anchor</td>
			<td>Multichannel system</td>
			<td>SHD-27H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinal cord stimulatior</td>
			<td>PINS</td>
			<td>T901</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezer</td>
			<td>RWD Life Science</td>
			<td>F13030-10</td>
			<td>Lift the xiphoid</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide band ultraviolet excitation filter</td>
			<td>Olympus&#160;</td>
			<td>U-MF2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo preparation of spinal cord slice for the whole-cell patch-clamp recording in motor neurons during spinal cord stimulation

Die Rückenmarkstimulation (SCS) kann die Bewegungsfunktion nach einer Rückenmarksverletzung effektiv wiederherstellen. Da die Motoneuronen die letzte Einheit sind, die sensomotorisches Verhalten ausführt, kann uns die direkte Untersuchung der elektrischen Reaktionen von Motoneuronen mit SCS helfen, die zugrunde liegende Logik der spinalen motorischen Modulation zu verstehen. Um gleichzeitig verschiedene Reizeigenschaften und zelluläre Reaktionen zu erfassen, ist eine Patch-Clamp eine gute Methode, um die elektrophysiologischen Eigenschaften auf Einzelzellebene zu untersuchen. Es gibt jedoch noch einige komplexe Schwierigkeiten, um dieses Ziel zu erreichen, einschließlich der Aufrechterhaltung der Zelllebensfähigkeit, der schnellen Trennung des Rückenmarks von der knöchernen Struktur und der Verwendung des SCS zur erfolgreichen Induktion von Aktionspotenzialen. Hier stellen wir ein detailliertes Protokoll vor, das Patch-Clamp verwendet, um die elektrischen Reaktionen von Motoneuronen auf SCS mit hoher räumlich-zeitlicher Auflösung zu untersuchen, was Forschern helfen kann, ihre Fähigkeiten bei der Trennung des Rückenmarks zu verbessern und gleichzeitig die Lebensfähigkeit der Zelle aufrechtzuerhalten, um den elektrischen Mechanismus von SCS auf Motoneuronen reibungslos zu untersuchen und unnötige Versuche und Fehler zu vermeiden.

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Andreas Rowald et al. reported that SCS enables lower limb locomotor and trunk function within a single day1. Exploring the biological mechanism of SCS for locomotor recovery is a critical and trending research field for developing a more precise SCS strategy. For example, Gr&#233;goire Courtine&#39;s team demonstrated that excitatory Vsx2 interneuron and Hoxa10 neurons in the spinal cord are the key neurons to response to SCS, and cell-specific neuromodulation is feasible to restore the rat walking ability after SCI2. However, few studies focus on the electrical mechanism of SCS at a single-cell scale. Although it is well-known that the suprathreshold direct current stimulus can elicit the action potentials (APs) in the classic squid experiment3,4,5, how the pulsed alternating electrical stimulation, such as SCS, affects the motor signal generation is still unclear.
Given the complexity of intraspinal neural circuits, appropriate selection for cell population is important for investigating the electrical mechanism of SCS. Although SCS restores motor function by activating the proprioceptive pathway6, the motor neurons are the final unit to execute the motor command, derived from integrating proprioception information afferent input7. Therefore, directly studying the electrical characteristics of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation.
As we know, patch-clamp is the golden-standard method for cellularly electrophysiological recording with extremely high spatiotemporal resolution8. Therefore, this study describes a method using a patch clamp to study the electrical responses of motor neurons to SCS. Compared with the brain patch-clamp9, the spinal cord patch-clamp is more difficult due to the following reasons: (1) The spinal cord is protected by the vertebral canal with tiny volume, which requires very fine micromanipulation and rigorous ice-cold maintenance to obtain better cell viability. (2) Because the spinal cord is too slender to be secured on the cutting tray, it should be immersed in low-melting point agarose and trimmed after solidification.
Hence, this method provides technical details in dissecting the spinal cord and maintaining the cell viability at the same time so as to smoothly study the electrical mechanism of SCS on motor neurons and avoid unnecessary trials and mistakes.

Author Spotlight: Advancing Spinal Cord Stimulation - Erforschung der zellulären Reaktionen von Motoneuronen durch Patch-Clamp-Elektrophysiologie 

Die Ex vivo Präparation eines Rückenmarksschnitts für die Ganzzell-Patch-Clamp-Aufnahme in Motoneuronen während der Rückenmarkstimulation

Spinal cord stimulation can help patients resolve most motion functions after injury. Most neurons are to execute sensory mode behaviors. Studying the electric response of most neurons can aid in understanding the underlying logic of spinal load neuromodulation.A patch clamp is the golden standard method for cellular electrophysiological recordings with the extremely high spatial temporal resolution. Therefore, this study describes a method using a patch clamp to simultaneously record the aortas stimulus characteristics and cellular responses of motor neurons at a single-cell scale. Compared with the brain patch clamp, the spinal cord patch clamp is more difficult, because the spinal cord is well protected by the vertebral canal with tiny volume.Therefore, this protocol provides technical details in quickly micro dissecting the spinal cord and reverse maintenance to obtain better cell viability. Patch clamp allows a unique understanding of the synaptic transmissions and the understanding of action potentials, coding pattern activities by the spinal cord stimulation.

Dank der rigorosen Tieftemperaturhaltung während des Feinbetriebs (ergänzende Abbildung 1, ergänzende Abbildung 2 und Abbildung 1) war die Lebensfähigkeit der Zellen gut genug, um nachfolgende elektrophysiologische Aufzeichnungen durchzuführen. Um das klinische Szenario so weit wie möglich zu simulieren, haben wir die SCS-Kathode und die Anode mittels Mikromanipulation in der Nähe der dorsalen Mittellinie bzw. der DREZ platziert (Abbildung 2</stro...

Watch this Scientific Journal Video about Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology at JoVE.com

Dieses Protokoll beschreibt eine Methode, bei der eine Patch-Clamp verwendet wird, um die elektrischen Reaktionen von Motoneuronen auf die Rückenmarkstimulation (SCS) mit hoher räumlich-zeitlicher Auflösung zu untersuchen, was Forschern helfen kann, ihre Fähigkeiten bei der Trennung des Rückenmarks und der gleichzeitigen Aufrechterhaltung der Zelllebensfähigkeit zu verbessern.

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

Die Rückenmarkstimulation kann Patienten helfen, die meisten Bewegungsfunktionen nach einer Verletzung aufzulösen. Die meisten Neuronen sollen sensorische Verhaltensweisen ausführen. Die Untersuchung der elektrischen Reaktion der meisten Neuronen kann dazu beitragen, die zugrunde liegende Logik der Neuromodulation der Wirbelsäulenbelastung zu verstehen.Eine Patch-Klemme ist die goldene Standardmethode für zelluläre elektrophysiologische Ableitungen mit der extrem hohen räumlich-zeitlichen Auflösung. Daher wird in dieser Studie eine Methode beschrieben, mit der eine Patch-Klemme verwendet wird, um gleichzeitig die Aorten-Stimuluseigenschaften und zellulären Reaktionen von Motoneuronen auf Einzelzellebene aufzuzeichnen. Im Vergleich zur Gehirn-Patch-Klemme ist die Rückenmark-Patch-Klemme schwieriger, da das Rückenmark durch den Wirbelkanal mit winzigem Volumen gut geschützt ist.Daher bietet dieses Protokoll technische Details für die schnelle Mikrodissektion des Rückenmarks und die umgekehrte Wartung, um eine bessere Zelllebensfähigkeit zu erreichen. Die Patch-Klemme ermöglicht ein einzigartiges Verständnis der synaptischen Übertragungen und das Verständnis von Aktionspotentialen, die durch die Rückenmarkstimulation kodierende Musteraktivitäten kodieren.

the-ex-vivo-preparation-spinal-cord-slice-for-whole-cell-patch-clamp

Research

JoVE Journal

Neuroscience

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.

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.

Forschung

Neurowissenschaften

Spinal Cord Dissection for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After exposing the heart of an anesthetized rat, cut the right atrium using fine scissors and inject 100 milliliters of chilled perfusing fluid at approximately two milliliters per second within one minute. Position the rat in the prone position. Cut the spine at the anterior superior iliac spine and the curvature shifting point of the thoracic column.Immediately place the isolated spine into the oxygenated ice cold perfusing solution to wash off residual blood and fat tissue. Then transfer the isolated spine to the anatomical tray with its dorsal side up and the rostral end near the operator. Fill the tray with 50 milliliters of continuously oxygenated ice cold cutting solution.Under a dissection microscope, cut the vertebral pedicles on both sides from the rostral end using micro scissors. Next, delicately sever the dura mater along its dorsal midline with micro scissors. Lift the rostral part of the spinal cord carefully and cut the nerve root, leaving about one millimeter reserved.After the spinal cord has been separated from the vertebral canal, secure it with the ventral side facing up using two insect pins. Trim any excessive nerve roots, leaving about one millimeter intact. Then separate the lumbar enlargement to a length of six to seven millimeters using a micro scissor.

Rückenmarksdissektion für die Ganzzell-Patch-Clamp-Aufnahme in Motoneuronen

Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After dissecting the spinal cord and separating the lumbar enlargement, place the lumbar enlargement on a 35 degree slope with the dorsal side up and the caudal side down. Absorb any excess water on the tissue surface using an absorbent filter paper. Carefully pour the molten agarose gel into the Petri dish containing the lumbar enlargement.Position the Petri dish in the ice water mixture to ensure rapid gel cooling. Shape the gel into a cube of 15 by 10 by 10 millimeters, and mount it onto the specimen disc using super glue. Adjust the vibratome settings to a thickness of 350 micrometers, a speed of 0.14 to 0.16 millimeters per second, an amplitude of one millimeter, and a vibration frequency of 85 hertz.Now, use the cover slide tweezers to clip a slice and place it into the incubation chamber, filled with continuously oxygenated artificial cerebrospinal fluid.

Präparation eines Rückenmarksschnitts für die Ganzzell-Patch-Clamp-Aufnahme in Motoneuronen

Patch-Clamp Recording to Study the Electrical Responses of Motor Neurons to Spinal Cord Stimulation

To begin, place the prepared spinal cord slice in the recording chamber. Then place the anode of the micro-manipulation system near the dorsal midline in the cathode close to the dorsal root entry zone for proper spinal cord stimulator positioning. Now switch to a 60X objective lens to identify a healthy neuron having a smooth and bright surface without visible nuclei.Slightly decrease the infrared intensity, turn on the fluorescent light source, and adjust the light filter to the wideband ultraviolet excitation option. Now, select an appropriate Fluoro-Gold-positive motor neuron and gradually lower the electrode until it is near the neuron. As the pipette contacts its surface, look for a small membrane indentation at the tip level and release the previously-applied positive pressure.Then apply negative pressure to the pipette using a syringe and wait until the resistance value rises to gigaohms on the software interface. Clamp the membrane potential at minus 70 millivolts. Press the fast capacitance compensation button on the amplifier's software interface, then apply a brief negative pressure to rupture the cell membrane and press the slow capacitance compensation button on the amplifier's software interface.Apply the spinal cord stimulation for one to two seconds keeping the amplitude between one and 10 milliamperes. Determine the motor threshold by gradually increasing the stimulation amplitude until the first action potential is observed. Distinguish delayed and immediate firing motor neurons using a five-second depolarizing current injection around rheobase in the current clamp mode.After turning off the spinal cord stimulation, continue recording the membrane potentials to capture the spontaneous action potentials firing. When the amplitude of spinal cord stimulation was raised, the membrane potential was increased, and the action potentials were triggered every 10 to 20 pulses. After the spinal cord stimulation was turned off, the neuron fired a series of spontaneous action potentials for a short period of time.Then, the resting membrane potential returned to minus 65 millivolts.