Name: Forfatter Spotlight: Fremme af rygmarvsstimulering - Udforskning af de cellulære reaktioner af motorneuroner gennem patch-clamp elektrofysiologi
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

Denne undersøgelse blev finansieret af National Natural Science Foundation of China for Young Scholars (52207254 og 82301657) og China Postdoctoral Science Fund (2022M711833).

Institutional Animal Care and Use Committee godkendte alle dyreforsøg, og undersøgelserne blev udført i overensstemmelse med relevante dyrevelfærdsbestemmelser.
1. Tilberedning af dyr
<ol><li>Dyr<ol><li>Boliginformation: Hushanrotter fra Sprague-Dawley (Postnatal 10-14 dage, P10-P14) i et specifikt patogenfrit miljø. BEMÆRK: Rumforholdene blev opretholdt ved 20 °C ± 2 °C, fugtighed: 50% -60%, med en 12-timers lys / mørk cyklus. Dyrene havde fri adgang til mad og...

Forståelse af motorneuronresponser på rygmarvsstimulering

<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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Bevægelsesinformationen moduleret af SCS konvergeres endelig til motorneuronerne. Derfor kan det at tage motorneuronerne som forskningsmål forenkle undersøgelsesdesignet og afsløre neuromodulationsmekanismen for SCS mere direkte. For samtidig at registrere forskellige stimulusegenskaber og cellulære reaktioner er en patch-clamp en god metode til at studere de elektrofysiologiske egenskaber på en enkeltcelleskala. Der er dog stadig nogle vanskeligheder, herunder hvordan man opretholder cellelevedygtighed, hvordan ma...

Rygmarvsstimulering (SCS) kan effektivt genoprette lokomotorisk funktion efter rygmarvsskade (SCI). Andreas Rowald et al. rapporterede, at SCS muliggør lokomotorisk og kropslig funktion i underekstremiteterne inden for en enkelt dag1. Udforskning af SCS' biologiske mekanisme til lokomotorisk genopretning er et kritisk og trendende forskningsfelt for udvikling af en mere præcis SCS-strategi. For eksempel demonstrerede Grégoire Courtines team, at excitatoriske Vsx2-interneuron og Hoxa10-neuroner i rygmarven er nøgleneuronerne til respons på SCS, og cellespecifik neuromodulation er mulig for at genoprette rottegangsevnen efter SCI2. Imidlertid fokuserer få undersøgelser på den elektriske mekanisme i SCS på en enkeltcelleskala. Selvom det er velkendt, at suprathreshold jævnstrømsstimulus kan fremkalde handlingspotentialerne (AP'er) i det klassiske blæksprutteeksperiment 3,4,5, er det stadig uklart, hvordan den pulserende vekslende elektriske stimulering, såsom SCS, påvirker motorsignalgenereringen.
I betragtning af kompleksiteten af intraspinale neurale kredsløb er passende selektion for cellepopulation vigtig for at undersøge SCS's elektriske mekanisme. Selvom SCS genopretter motorfunktionen ved at aktivere den proprioceptive vej6, er motorneuronerne den sidste enhed til at udføre motorkommandoen, afledt af integration af proprioceptionsinformation afferent input7. Derfor kan direkte undersøgelse af de elektriske egenskaber ved motorneuroner med SCS hjælpe os med at forstå den underliggende logik i spinalmotormodulation.
Som vi ved, er patch-clamp den gyldne standardmetode til cellulær elektrofysiologisk optagelse med ekstremt høj spatiotemporal opløsning8. Derfor beskriver denne undersøgelse en metode, der bruger en patchklemme til at studere motorneuronernes elektriske reaktioner på SCS. Sammenlignet med hjerneplaster9 er rygmarvsplasterklemmen vanskeligere af følgende årsager: (1) Rygmarven er beskyttet af rygsøjlen med lille volumen, hvilket kræver meget fin mikromanipulation og streng iskold vedligeholdelse for at opnå bedre cellelevedygtighed. (2) Da rygmarven er for slank til at blive fastgjort på skærebakken, bør den nedsænkes i agarose med lavt smeltepunkt og trimmes efter størkning.
Derfor giver denne metode tekniske detaljer ved dissekering af rygmarven og opretholdelse af cellelevedygtigheden på samme tid for jævnt at studere SCS's elektriske mekanisme på motorneuroner og undgå unødvendige forsøg og fejl.

<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

Rygmarvsstimulering (SCS) kan effektivt genoprette lokomotorisk funktion efter rygmarvsskade (SCI). Fordi motorneuronerne er den sidste enhed til at udføre sensorimotorisk adfærd, kan direkte undersøgelse af de elektriske reaktioner fra motorneuroner med SCS hjælpe os med at forstå den underliggende logik i spinalmotormodulation. For samtidig at registrere forskellige stimulusegenskaber og cellulære reaktioner er en patch-clamp en god metode til at studere de elektrofysiologiske egenskaber på en enkeltcelleskala. Der er dog stadig nogle komplekse vanskeligheder med at nå dette mål, herunder opretholdelse af cellelevedygtighed, hurtig adskillelse af rygmarven fra knoglestrukturen og brug af SCS til succesfuldt at fremkalde handlingspotentialer. Her præsenterer vi en detaljeret protokol ved hjælp af patch-clamp til at studere de elektriske reaktioner fra motorneuroner til SCS med høj rumlig tidsmæssig opløsning, hvilket kan hjælpe forskere med at forbedre deres færdigheder i at adskille rygmarven og opretholde cellelevedygtigheden på samme tid for jævnt at studere SCS's elektriske mekanisme på motorneuron og undgå unødvendige forsøg og fejl.

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.

Forfatter Spotlight: Fremme af rygmarvsstimulering - Udforskning af de cellulære reaktioner af motorneuroner gennem patch-clamp elektrofysiologi 

ex vivo-forberedelse af rygmarvsskive til helcelle-patch-clamp-optagelse i motorneuroner under rygmarvsstimulering

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.

Takket være den strenge vedligeholdelse ved lave temperaturer under den fine operation (supplerende figur 1, supplerende figur 2 og figur 1) var cellelevedygtigheden god nok til at udføre efterfølgende elektrofysiologiske registreringer. For at simulere det kliniske scenarie så meget som muligt brugte vi mikromanipulation til at placere SCS-katoden og anoden nær henholdsvis dorsal midterlinje og DREZ (figur 2), som kunne initiere neuralt si...

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

Denne protokol beskriver en metode, der bruger en patch-clamp til at studere de elektriske reaktioner fra motorneuroner til rygmarvsstimulering (SCS) med høj rumlig tidsmæssig opløsning, hvilket kan hjælpe forskere med at forbedre deres færdigheder i at adskille rygmarven og opretholde cellelevedygtighed samtidigt.

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

Rygmarvsstimulering kan hjælpe patienter med at løse de fleste bevægelsesfunktioner efter skade. De fleste neuroner skal udføre sensorisk tilstandsadfærd. At studere det elektriske respons hos de fleste neuroner kan hjælpe med at forstå den underliggende logik i spinal load neuromodulation.En patchklemme er den gyldne standardmetode til cellulære elektrofysiologiske optagelser med den ekstremt høje rumlige tidsopløsning. Derfor beskriver denne undersøgelse en metode, der anvender en patchklemme til samtidig at registrere aortas-stimuluskarakteristika og cellulære reaktioner fra motorneuroner på en enkeltcelleskala. Sammenlignet med hjerneplasterklemmen er rygmarvsplasterklemmen vanskeligere, fordi rygmarven er godt beskyttet af rygsøjlen med lille volumen.Derfor giver denne protokol tekniske detaljer i hurtig mikrodissekering af rygmarven og omvendt vedligeholdelse for at opnå bedre cellelevedygtighed. Patch clamp giver en unik forståelse af de synaptiske transmissioner og forståelsen af handlingspotentialer, kodning af mønsteraktiviteter ved rygmarvsstimulering.

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.

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.

Rygmarvsdissektion til optagelse af hele celleplasterklemmen i motorneuroner

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.

Forberedelse af rygmarvsskive til helcelleplaster-klemmeoptagelse i motorneuroner

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.