Name: Autore in primo piano: Avanzamento della stimolazione del midollo spinale - Esplorazione delle risposte cellulari dei motoneuroni attraverso l'elettrofisiologia del patch-clamp
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

Questo studio è stato finanziato dalla National Natural Science Foundation of China for Young Scholars (52207254 e 82301657) e dal China Postdoctoral Science Fund (2022M711833).

Il Comitato Istituzionale per la Cura e l'Uso degli Animali ha approvato tutti gli esperimenti sugli animali e gli studi sono stati condotti in conformità con le normative vigenti in materia di benessere degli animali.
1. Preparazione degli animali
<ol><li>Animali<ol><li>Informazioni sull'alloggiamento: Ratti Sprague-Dawley maschi (10-14 giorni postnatali, P10-P14) in un ambiente specifico privo di agenti patogeni. NOTA: Le condizioni ambientali sono state mantenute a 20...

Comprendere le risposte dei motoneuroni alla stimolazione del midollo spinale

<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. 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Le informazioni di movimento modulate da SCS vengono infine condivise nei motoneuroni. Pertanto, prendere i motoneuroni come obiettivo di ricerca può semplificare il disegno dello studio e rivelare il meccanismo di neuromodulazione della SCS in modo più diretto. Per registrare simultaneamente diverse caratteristiche di stimolo e risposte cellulari, un patch-clamp è un buon metodo per studiare le caratteristiche elettrofisiologiche su scala di singola cellula. Tuttavia, ci sono ancora alcune difficoltà, tra cui come m...

La stimolazione del midollo spinale (SCS) può ripristinare efficacemente la funzione locomotoria dopo una lesione del midollo spinale (SCI). Andreas Rowald et al. hanno riportato che la SCS consente la funzione locomotoria e del tronco degli arti inferiori entro un solo giorno1. L'esplorazione del meccanismo biologico della SCS per il recupero locomotore è un campo di ricerca critico e di tendenza per lo sviluppo di una strategia SCS più precisa. Ad esempio, il team di Grégoire Courtine ha dimostrato che l'interneurone eccitatorio Vsx2 e i neuroni Hoxa10 nel midollo spinale sono i neuroni chiave per la risposta alla SCS e la neuromodulazione cellulo-specifica è fattibile per ripristinare la capacità di camminare del ratto dopo la lesione midollare2. Tuttavia, pochi studi si concentrano sul meccanismo elettrico della SCS su scala di una singola cellula. Sebbene sia ben noto che lo stimolo in corrente continua soprasoglia può suscitare i potenziali d'azione (AP) nel classico esperimento del calamaro 3,4,5, non è ancora chiaro come la stimolazione elettrica alternata pulsata, come la SCS, influenzi la generazione del segnale motorio.
Data la complessità dei circuiti neurali intraspinali, una selezione appropriata per la popolazione cellulare è importante per studiare il meccanismo elettrico della SCS. Sebbene la SCS ripristini la funzione motoria attivando la via propriocettiva6, i motoneuroni sono l'unità finale per eseguire il comando motorio, derivato dall'integrazione dell'input afferente alle informazioni sulla propriocezione7. Pertanto, studiare direttamente le caratteristiche elettriche dei motoneuroni con SCS può aiutarci a comprendere la logica sottostante alla modulazione motoria spinale.
Come sappiamo, il patch-clamp è il metodo gold standard per la registrazione elettrofisiologica cellulare con una risoluzione spazio-temporale estremamente elevata8. Pertanto, questo studio descrive un metodo che utilizza un patch clamp per studiare le risposte elettriche dei motoneuroni alla SCS. Rispetto al cerotto cerebrale9, il cerotto per il midollo spinale è più difficile per i seguenti motivi: (1) Il midollo spinale è protetto dal canale vertebrale con un volume minuscolo, che richiede una micromanipolazione molto fine e una rigorosa manutenzione ghiacciata per ottenere una migliore vitalità cellulare. (2) Poiché il midollo spinale è troppo sottile per essere fissato sul vassoio di taglio, deve essere immerso in agarosio a basso punto di fusione e tagliato dopo la solidificazione.
Quindi, questo metodo fornisce dettagli tecnici nella dissezione del midollo spinale e nel mantenimento della vitalità cellulare allo stesso tempo, in modo da studiare senza problemi il meccanismo elettrico della SCS sui motoneuroni ed evitare inutili prove ed errori.

<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

La stimolazione del midollo spinale (SCS) può ripristinare efficacemente la funzione locomotoria dopo una lesione del midollo spinale (SCI). Poiché i motoneuroni sono l'unità finale per eseguire i comportamenti sensomotori, studiare direttamente le risposte elettriche dei motoneuroni con SCS può aiutarci a comprendere la logica sottostante della modulazione motoria spinale. Per registrare simultaneamente diverse caratteristiche di stimolo e risposte cellulari, un patch-clamp è un buon metodo per studiare le caratteristiche elettrofisiologiche su scala di singola cellula. Tuttavia, ci sono ancora alcune difficoltà complesse nel raggiungere questo obiettivo, tra cui il mantenimento della vitalità cellulare, la rapida separazione del midollo spinale dalla struttura ossea e l'utilizzo dell'SCS per indurre con successo potenziali d'azione. Qui, presentiamo un protocollo dettagliato che utilizza patch-clamp per studiare le risposte elettriche dei motoneuroni a SCS con un'elevata risoluzione spaziotemporale, che può aiutare i ricercatori a migliorare le loro capacità di separare il midollo spinale e mantenere la vitalità cellulare allo stesso tempo per studiare senza problemi il meccanismo elettrico di SCS sul motoneurone ed evitare inutili tentativi ed errori.

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.

Autore in primo piano: Avanzamento della stimolazione del midollo spinale - Esplorazione delle risposte cellulari dei motoneuroni attraverso l'elettrofisiologia del patch-clamp 

La preparazione ex vivo di una fetta di midollo spinale per la registrazione del patch-clamp a cellula intera nei motoneuroni durante la stimolazione del midollo spinale

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.

Grazie al rigoroso mantenimento a bassa temperatura durante l'operazione fine (Figura 1 supplementare, Figura 2 supplementare e Figura 1), la vitalità cellulare è stata sufficiente per eseguire successive registrazioni elettrofisiologiche. Per simulare il più possibile lo scenario clinico, abbiamo utilizzato la micromanipolazione per posizionare il catodo e l'anodo SCS vicino alla linea mediana dorsale e DREZ, rispettivamente (Figura 2), che ...

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

Questo protocollo descrive un metodo che utilizza un patch-clamp per studiare le risposte elettriche dei motoneuroni alla stimolazione del midollo spinale (SCS) con un'elevata risoluzione spazio-temporale, che può aiutare i ricercatori a migliorare le loro capacità di separare il midollo spinale e mantenere contemporaneamente la vitalità cellulare.

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

La stimolazione del midollo spinale può aiutare i pazienti a risolvere la maggior parte delle funzioni motorie dopo una lesione. La maggior parte dei neuroni deve eseguire comportamenti sensoriali. Lo studio della risposta elettrica della maggior parte dei neuroni può aiutare a comprendere la logica sottostante alla neuromodulazione del carico spinale.Un patch clamp è il metodo standard di riferimento per le registrazioni elettrofisiologiche cellulari con una risoluzione temporale spaziale estremamente elevata. Pertanto, questo studio descrive un metodo che utilizza un patch clamp per registrare simultaneamente le caratteristiche dello stimolo dell'aorta e le risposte cellulari dei motoneuroni su scala di singola cellula. Rispetto al cerotto encefalico, il cerotto per midollo spinale è più difficile, perché il midollo spinale è ben protetto dal canale vertebrale con un volume ridotto.Pertanto, questo protocollo fornisce dettagli tecnici per la microdissezione rapida del midollo spinale e la manutenzione inversa per ottenere una migliore vitalità cellulare. Il patch clamp consente una comprensione unica delle trasmissioni sinaptiche e la comprensione dei potenziali d'azione, codificando le attività del pattern da parte della stimolazione del midollo spinale.

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.

Ricerca

Neuroscienze

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.

Dissezione del midollo spinale per la registrazione del patch-clamp dell'intera cellula nei motoneuroni

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.

Preparazione di una fetta di midollo spinale per la registrazione del patch-clamp a cellula intera nei motoneuroni

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.