Name: Författare Spotlight: Främja ryggmärgsstimulering - Utforska de cellulära svaren hos motorneuroner genom 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

Denna studie finansierades av National Natural Science Foundation of China for Young Scholars (52207254 och 82301657) och China Postdoctoral Science Fund (2022M711833).

Institutional Animal Care and Use Committee godkände alla djurförsök och studierna utfördes i enlighet med gällande djurskyddsbestämmelser.
1. Förberedelse av djur
<ol><li>Djur<ol><li>Boendeinformation: Hushanar av Sprague-Dawley-råttor (postnatala 10-14 dagar, P10-P14) i en specifik patogenfri miljö. OBS: Rumsförhållandena bibehölls vid 20 °C ± 2 °C, luftfuktighet: 50%-60%, med en 12-timmars ljus/mörk cykel. Djuren hade fri tillgång till mat och vatten.<...

Förstå motorneuronernas svar på ryggmärgsstimulering

<ol>
	<li>Rowald, 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=Activity-dependent+spinal+cord+neuromodulation+rapidly+restores+trunk+and+leg+motor+functions+after+complete+paralysis.">Activity-dependent spinal cord neuromodulation rapidly restores trunk and leg motor functions after complete paralysis.</a> Nature Medicine. 28 (2), 260-271 (2022).</li><li>Kathe, C., 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=The+neurons+that+restore+walking+after+paralysis.">The neurons that restore walking after paralysis.</a> Nature. 611 (7936), 540-547 (2022).</li><li>Smith, S. J., Buchanan, J., Osses, L. R., Charlton, M. P., Augustine, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+distribution+of+calcium+signals+in+squid+presynaptic+terminals.">The spatial distribution of calcium signals in squid presynaptic terminals.</a> The Journal of Physiology. 472, 573-593 (1993).</li><li>Augustine, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+transmitter+release+at+the+squid+giant+synapse+by+presynaptic+delayed+rectifier+potassium+current.">Regulation of transmitter release at the squid giant synapse by presynaptic delayed rectifier potassium current.</a> The Journal of Physiology. 431, 343-364 (1990).</li><li>Llin&#225;s, R., McGuinness, T. L., Leonard, C. S., Sugimori, M., Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraterminal+injection+of+synapsin+I+or+calcium/calmodulin-dependent+protein+kinase+II+alters+neurotransmitter+release+at+the+squid+giant+synapse.">Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (9), 3035-3039 (1985).</li><li>Formento, E., 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=Electrical+spinal+cord+stimulation+must+preserve+proprioception+to+enable+locomotion+in+humans+with+spinal+cord+injury.">Electrical spinal cord stimulation must preserve proprioception to enable locomotion in humans with spinal cord injury.</a> Nature Neuroscience. 21 (12), 1728-1741 (2018).</li><li>Hari, K., 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=GABA+facilitates+spike+propagation+through+branch+points+of+sensory+axons+in+the+spinal+cord.">GABA facilitates spike propagation through branch points of sensory axons in the spinal cord.</a> Nature Neuroscience. 25 (10), 1288-1299 (2022).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annual Review Of Physiology. 46, 455-472 (1984).</li><li>Leroy, F., Lamotte d'Incamps, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+of+oblique+spinal+cord+slices+for+ventral+root+stimulation.">The preparation of oblique spinal cord slices for ventral root stimulation.</a> Journal of Visualized Experiments:JoVE. (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. S., Beato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+excitation+between+motoneurones+propagates+across+segments+and+is+purely+glutamatergic.">Recurrent excitation between motoneurones propagates across segments and is purely glutamatergic.</a> PLoS Biology. 16 (3), e2003586 (2018).</li><li>Leroy, F., Lamotte d'Incamps, B., Imhoff-Manuel, R. D., 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=Early+intrinsic+hyperexcitability+does+not+contribute+to+motoneuron+degeneration+in+amyotrophic+lateral+sclerosis.">Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis.</a> Elife. 3, 04046 (2014).</li><li>Tahir, R. A., Pabaney, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+hypothermia+and+ischemic+stroke:+A+literature+review.">Therapeutic hypothermia and ischemic stroke: A literature review.</a> Surgical Neurology International. 7, S381-S386 (2016).</li><li>Lu, Y., 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=Management+of+intractable+pain+in+patients+with+implanted+spinal+cord+stimulation+devices+during+the+COVID-19+pandemic+using+a+remote+and+wireless+programming+system.">Management of intractable pain in patients with implanted spinal cord stimulation devices during the COVID-19 pandemic using a remote and wireless programming system.</a> Frontiers in Neuroscience. 14, 594696 (2020).</li><li>Yao, Q., 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=Wireless+epidural+electrical+stimulation+in+combination+with+serotonin+agonists+improves+intraspinal+metabolism+in+spinal+cord+injury+rats.">Wireless epidural electrical stimulation in combination with serotonin agonists improves intraspinal metabolism in spinal cord injury rats.</a> Neuromodulation. 24 (3), 416-426 (2021).</li><li>Arlotti, M., Rahman, A., Minhas, P., Bikson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+terminal+polarization+induced+by+weak+uniform+dc+electric+fields:+a+modeling+study.">Axon terminal polarization induced by weak uniform dc electric fields: a modeling study.</a> 2012 Annual International Conference of the IEEE Engineering in Medicine and Biology Society. , 4575-4578 (2012).</li><li>Espino, C. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+multimodal+neural+transmission+suggests+that+adult+spinal+networks+maintain+an+intrinsic+state+of+readiness+to+execute+sensorimotor+behaviors.">Spontaneous multimodal neural transmission suggests that adult spinal networks maintain an intrinsic state of readiness to execute sensorimotor behaviors.</a> Journal Of Neuroscience. 41 (38), 7978-7990 (2021).</li><li>Manuel, M., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intracellular+recording+of+a+lumbar+motoneuron+and+the+force+produced+by+its+motor+unit+in+the+adult+mouse+in+vivo.">Simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo.</a> Journal of Visualized Experiments: JoVE. (70), e4312 (2012).</li><li>Luo, X., Wang, S., Rutkove, S. B., Sanchez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhomogeneous+volume+conduction+effects+affecting+needle+electromyography:+an+analytical+and+simulation+study.">Nonhomogeneous volume conduction effects affecting needle electromyography: an analytical and simulation study.</a> Physiological Measurement. 42 (11), (2021).</li><li>Barra, B., 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=Epidural+electrical+stimulation+of+the+cervical+dorsal+roots+restores+voluntary+upper+limb+control+in+paralyzed+monkeys.">Epidural electrical stimulation of the cervical dorsal roots restores voluntary upper limb control in paralyzed monkeys.</a> Nature Neuroscience. 25 (7), 924-934 (2022).</li><li>Powell, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidural+stimulation+of+the+cervical+spinal+cord+for+post-stroke+upper-limb+paresis.">Epidural stimulation of the cervical spinal cord for post-stroke upper-limb paresis.</a> Nature Medicine. 29 (3), 689-699 (2023).</li><li>Wenger, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+neuromodulation+therapies+engaging+muscle+synergies+improve+motor+control+after+spinal+cord+injury.">Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.</a> Nature Medicine. 22 (2), 138-145 (2016).</li><li>&#214;zyurt, M. G., Ojeda-Alonso, J., Beato, M., Nascimento, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+longitudinal+lumbar+spinal+cord+preparations+to+study+sensory+and+recurrent+motor+microcircuits+of+juvenile+mice.">In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice.</a> Journal of Neurophysiology. 128 (3), 711-726 (2022).</li><li>Moraud, E. 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=Mechanisms+underlying+the+neuromodulation+of+spinal+circuits+for+correcting+gait+and+balance+deficits+after+spinal+cord+injury.">Mechanisms underlying the neuromodulation of spinal circuits for correcting gait and balance deficits after spinal cord injury.</a> Neuron. 89 (4), 814-828 (2016).</li><li>Capogrosso, 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=A+computational+model+for+epidural+electrical+stimulation+of+spinal+sensorimotor+circuits.">A computational model for epidural electrical stimulation of spinal sensorimotor circuits.</a> Journal of Neuroscience. 33 (49), 19326-19340 (2013).</li></ol>

Rörelseinformationen som moduleras av SCS konvergerar slutligen till de motoriska nervcellerna. Att ta de motoriska nervcellerna som forskningsmål kan därför förenkla studiedesignen och avslöja neuromoduleringsmekanismen för SCS mer direkt. För att samtidigt registrera olika stimulusegenskaper och cellulära svar är en patch-clamp en bra metod för att studera de elektrofysiologiska egenskaperna på encellsskala. Det finns dock fortfarande vissa svårigheter, bland annat hur man upprätthåller cellviabiliteten,...

Ryggmärgsstimulering (SCS) kan effektivt återställa rörelsefunktionen efter ryggmärgsskada. Andreas Rowald et al. rapporterade att SCS möjliggör rörelse- och bålfunktion i nedre extremiteter inom en enda dag1. Att utforska den biologiska mekanismen för SCS för motorisk återhämtning är ett kritiskt och trendigt forskningsområde för att utveckla en mer exakt SCS-strategi. Till exempel visade Grégoire Courtines team att excitatoriska Vsx2-interneuron och Hoxa10-neuroner i ryggmärgen är de viktigaste neuronerna för att svara på SCS, och cellspecifik neuromodulering är möjlig för att återställa råttans gångförmåga efter SCI2. Få studier fokuserar dock på den elektriska mekanismen för SCS på encellsskala. Även om det är välkänt att supratröskeln likströmsstimulus kan framkalla aktionspotentialerna (AP) i det klassiska bläckfiskexperimentet 3,4,5, är det fortfarande oklart hur den pulserande växelströmsstimuleringen, såsom SCS, påverkar motorsignalgenereringen.
Med tanke på komplexiteten hos intraspinala neurala kretsar är lämpligt urval för cellpopulationen viktigt för att undersöka den elektriska mekanismen för SCS. Även om SCS återställer motorisk funktion genom att aktivera den proprioceptiva vägen6, är de motoriska nervcellerna den slutliga enheten för att utföra det motoriska kommandot, härlett från att integrera proprioceptionsinformation afferent input7. Att direkt studera de elektriska egenskaperna hos motorneuroner med SCS kan därför hjälpa oss att förstå den underliggande logiken för spinal motorisk modulering.
Som vi vet är patch-clamp den gyllene standardmetoden för cellulärt elektrofysiologisk inspelning med extremt hög spatiotemporal upplösning8. Därför beskriver denna studie en metod som använder en patch clamp för att studera de elektriska svaren hos motorneuroner på SCS. Jämfört med hjärnklämma9 är ryggmärgsklämman svårare på grund av följande skäl: (1) Ryggmärgen skyddas av kotkanalen med liten volym, vilket kräver mycket fin mikromanipulation och rigoröst iskallt underhåll för att få bättre cellviabilitet. (2) Eftersom ryggmärgen är för smal för att fästas på skärbrickan, bör den nedsänkas i agaros med låg smältpunkt och trimmas efter stelning.
Därför ger denna metod tekniska detaljer för att dissekera ryggmärgen och samtidigt upprätthålla cellviabiliteten för att smidigt studera den elektriska mekanismen för SCS på motorneuroner och undvika onödiga försök och misstag.

<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

Ryggmärgsstimulering (SCS) kan effektivt återställa rörelsefunktionen efter ryggmärgsskada. Eftersom de motoriska nervcellerna är den sista enheten för att utföra sensomotoriska beteenden, kan direkta studier av de elektriska svaren hos motorneuroner med SCS hjälpa oss att förstå den underliggande logiken för ryggradsmotorisk modulering. För att samtidigt registrera olika stimulusegenskaper och cellulära svar är en patch-clamp en bra metod för att studera de elektrofysiologiska egenskaperna på encellsskala. Det finns dock fortfarande några komplexa svårigheter för att uppnå detta mål, inklusive att upprätthålla cellviabiliteten, snabbt separera ryggmärgen från den beniga strukturen och använda SCS för att framgångsrikt inducera aktionspotentialer. Här presenterar vi ett detaljerat protokoll med hjälp av patch-clamp för att studera de elektriska svaren hos motorneuroner på SCS med hög spatiotemporal upplösning, vilket kan hjälpa forskare att förbättra sina färdigheter i att separera ryggmärgen och upprätthålla cellviabiliteten samtidigt som de smidigt studerar den elektriska mekanismen för SCS på motorneuron och undviker onödiga försök och misstag.

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.

Författare Spotlight: Främja ryggmärgsstimulering - Utforska de cellulära svaren hos motorneuroner genom patch-clamp elektrofysiologi 

Ex vivo-beredning av ryggmärgsskiva för helcellspatch-clamp-registrering i motorneuroner under ryggmärgsstimulering

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.

Tack vare det rigorösa underhållet vid låg temperatur under finoperationen (kompletterande figur 1, kompletterande figur 2 och figur 1) var cellviabiliteten tillräckligt god för att utföra efterföljande elektrofysiologiska registreringar. För att simulera det kliniska scenariot så mycket som möjligt använde vi mikromanipulation för att placera SCS-katoden och anoden nära den dorsala mittlinjen respektive DREZ (Figur 2), vilket kunde...

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

Detta protokoll beskriver en metod som använder en patch-clamp för att studera de motoriska nervcellernas elektriska svar på ryggmärgsstimulering (SCS) med hög spatiotemporal upplösning, vilket kan hjälpa forskare att förbättra sina färdigheter i att separera ryggmärgen och upprätthålla cellviabilitet samtidigt.

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

Ryggmärgsstimulering kan hjälpa patienter att lösa de flesta rörelsefunktioner efter skada. De flesta neuroner är till för att utföra sensoriska beteenden. Att studera den elektriska responsen hos de flesta neuroner kan hjälpa till att förstå den underliggande logiken för neuromodulering av ryggradsbelastning.En patch clamp är den gyllene standardmetoden för cellulära elektrofysiologiska inspelningar med den extremt höga rumsliga tidsupplösningen. Därför beskriver denna studie en metod som använder en patchklämma för att samtidigt registrera aortas stimulusegenskaper och cellulära svar hos motorneuroner på encellsskala. Jämfört med hjärnklämman är ryggmärgsklämman svårare, eftersom ryggmärgen är väl skyddad av kotkanalen med liten volym.Därför ger detta protokoll tekniska detaljer för att snabbt mikrodissekera ryggmärgen och omvänt underhåll för att få bättre cellviabilitet. Patch clamp möjliggör en unik förståelse av de synaptiska överföringarna och förståelsen av aktionspotentialer, kodningsmönsteraktiviteter genom ryggmärgsstimulering.

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.

Ryggmärgsdissektion för helcellsinspelning av patch-clamp 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.

Förberedelse av ryggmärgsskiva för helcellsinspelning av patch-clamp 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.