Name: 저자 스포트라이트: Advancing Spinal Cord Stimulation - Exploring the cellular responses of motor neurons through patch-clamp electrophysiology (척수 자극의 발전 - 패치클램프 전기생리학을 통한 운동 뉴런의 세포 반응 탐구)
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

이 연구는 중국 국립 자연 과학 재단 젊은 학자(52207254 및 82301657)와 중국 박사후 과학 기금(2022M711833)의 지원을 받았습니다.

기관 동물 관리 및 사용 위원회는 모든 동물 실험을 승인했으며 연구는 관련 동물 복지 규정에 따라 수행되었습니다.
1. 동물 준비
<ol><li>마리<ol><li>주거 정보: 수컷 Sprague-Dawley 쥐(출생 후 10-14일, P10-P14)를 특정 병원체가 없는 환경에서 사육합니다. 알림: 실내 조건은 20°C ± 2°C, 습도: 50%-60%, 12시간 밝음/어두움 주기로 유지되었습니다. 동물들은 음식과 물을 자?...

척수 자극에 대한 운동 뉴런 반응의 이해

<ol>
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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. 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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. 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. 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SCS에 의해 조절된 운동 정보는 최종적으로 운동 뉴런으로 수렴됩니다. 따라서 운동 뉴런을 연구 목표로 삼으면 연구 설계를 단순화하고 SCS의 신경 조절 메커니즘을 보다 직접적으로 밝힐 수 있습니다. 다양한 자극 특성과 세포 반응을 동시에 기록하기 위해 패치 클램프는 단일 세포 규모에서 전기생리학적 특성을 연구하는 좋은 방법입니다. 그러나 세포 생존력을 유지하는 방법, 뼈 구조에서 척?...

척수 자극술(SCS)은 척수 손상(SCI) 후 운동 기능을 효과적으로 회복할 수 있습니다. 안드레아스 로왈드(Andreas Rowald) 등은 SCS가 단 하루 만에 하지 운동과 몸통 기능을 가능하게 한다고 보고했다1. 운동 회복을 위한 SCS의 생물학적 메커니즘을 탐구하는 것은 보다 정확한 SCS 전략을 개발하기 위한 중요하고 추세적인 연구 분야입니다. 예를 들어, 그레구아르 쿠르틴(Grégoire Courtine)의 연구팀은 척수의 흥분성 Vsx2 인터뉴런과 Hoxa10 뉴런이 SCS에 반응하는 핵심 뉴런이며, SCI2 이후 쥐의 보행 능력을 회복하기 위해 세포 특이적 신경조절이 가능하다는 것을 입증했습니다. 그러나 단일 셀 규모에서 SCS의 전기적 메커니즘에 초점을 맞춘 연구는 거의 없습니다. 고전적인 오징어 실험 3,4,5에서 초임계 직류 자극이 활동 전위(AP)를 유도할 수 있다는 것은 잘 알려져 있지만, SCS와 같은 펄스 교류 전기 자극이 모터 신호 생성에 어떤 영향을 미치는지는 여전히 불분명합니다.
척추 내 신경 회로의 복잡성을 감안할 때, 세포 집단에 대한 적절한 선택은 SCS의 전기적 메커니즘을 조사하는 데 중요합니다. SCS는 고유수용성 경로(proprioceptive pathway)6를 활성화시킴으로써 운동기능을 회복시키지만, 운동뉴런(motor neuron)은 고유수용감각 정보 구심성 입력(affrioception information afferent input)7의 통합으로부터 유도된 운동 명령을 실행하는 최종 단위이다. 따라서 SCS를 사용하여 운동 뉴런의 전기적 특성을 직접 연구하면 척추 운동 조절의 기본 논리를 이해하는 데 도움이 될 수 있습니다.
아시다시피, 패치 클램프는 매우 높은 시공간 분해능(spatiotemporal resolution)8을 가진 세포 전기생리학적 기록을 위한 황금 표준 방법입니다. 따라서 본 연구에서는 SCS에 대한 운동 뉴런의 전기적 반응을 연구하기 위해 패치 클램프를 사용하는 방법을 설명합니다. 뇌 패치-클램프(9)와 비교했을 때, 척수 패치-클램프는 다음과 같은 이유로 더 어렵다: (1) 척수는 아주 작은 부피의 척추관에 의해 보호되며, 이는 더 나은 세포 생존력을 얻기 위해 매우 미세한 미세 조작과 엄격한 얼음처럼 차가운 유지가 필요하다. (2) 척수가 너무 가늘어서 절단 트레이에 고정할 수 없기 때문에 저융점 아가로스에 담그고 응고 후 다듬어야 합니다.
따라서 이 방법은 척수를 해부하고 동시에 세포 생존력을 유지하는 데 있어 운동 뉴런에 대한 SCS의 전기적 메커니즘을 원활하게 연구하고 불필요한 시행착오를 피할 수 있는 기술적 세부 사항을 제공합니다.

<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

척수 자극술(SCS)은 척수 손상(SCI) 후 운동 기능을 효과적으로 회복할 수 있습니다. 운동 뉴런은 감각 운동 행동을 실행하는 최종 단위이기 때문에 SCS로 운동 뉴런의 전기 반응을 직접 연구하면 척추 운동 조절의 기본 논리를 이해하는 데 도움이 될 수 있습니다. 다양한 자극 특성과 세포 반응을 동시에 기록하기 위해 패치 클램프는 단일 세포 규모에서 전기생리학적 특성을 연구하는 좋은 방법입니다. 그러나 이 목표를 달성하는 데에는 세포 생존력 유지, 뼈 구조에서 척수를 빠르게 분리, SCS를 사용하여 활동 전위를 성공적으로 유도하는 등 몇 가지 복잡한 어려움이 여전히 있습니다. 여기에서는 패치 클램프를 사용하여 높은 시공간 해상도로 SCS에 대한 운동 뉴런의 전기적 반응을 연구하는 상세한 프로토콜을 제시하며, 이를 통해 연구자가 척수 분리 및 세포 생존력 유지 기술을 향상시켜 운동 뉴런에 대한 SCS의 전기적 메커니즘을 원활하게 연구하고 불필요한 시행착오를 피할 수 있습니다.

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.

저자 스포트라이트: Advancing Spinal Cord Stimulation - Exploring the cellular responses of motor neurons through patch-clamp electrophysiology (척수 자극의 발전 - 패치클램프 전기생리학을 통한 운동 뉴런의 세포 반응 탐구) 

척수 자극 중 운동 뉴런의 전체 세포 패치-클램프 기록을 위한 척수 절편의 생체 외 제조

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.

미세 작동 중 엄격한 저온 유지 덕분에(보충 그림 1, 보충 그림 2 및 그림 1) 세포 생존율은 후속 전기생리학적 기록을 수행하기에 충분했습니다. 임상 시나리오를 최대한 시뮬레이션하기 위해 미세 조작을 사용하여 SCS 음극과 양극을 각각 등쪽 정중선과 DREZ 근처에 배치했으며(그림 2), 등쪽 뿔에서 신경 신호를 시작하여 운동 기둥의 배외측...

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

이 프로토콜은 패치 클램프를 사용하여 높은 시공간 분해능으로 척수 자극(SCS)에 대한 운동 뉴런의 전기적 반응을 연구하는 방법을 설명하며, 이를 통해 연구자들은 척수를 분리하고 세포 생존력을 동시에 유지하는 기술을 향상시킬 수 있습니다.

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

척수 자극은 환자가 부상 후 대부분의 운동 기능을 해결하는 데 도움이 될 수 있습니다. 대부분의 뉴런은 감각 모드 행동을 실행합니다. 대부분의 뉴런의 전기적 반응을 연구하는 것은 척추 부하 신경 조절의 기본 논리를 이해하는 데 도움이 될 수 있습니다.패치 클램프는 매우 높은 공간 시간 분해능을 가진 세포 전기생리학적 기록을 위한 황금 표준 방법입니다. 따라서 본 연구에서는 패치 클램프를 이용하여 운동 뉴런의 대동맥 자극 특성과 세포 반응을 단세포 규모로 동시에 기록하는 방법을 설명한다. 뇌 패치 클램프와 비교할 때 척수 패치 클램프는 척수가 작은 부피의 척추관에 의해 잘 보호되기 때문에 더 어렵습니다.따라서 이 프로토콜은 척수를 신속하게 미세 절개하고 더 나은 세포 생존력을 얻기 위해 역유지 관리에 대한 기술적 세부 정보를 제공합니다. 패치 클램프를 사용하면 시냅스 전달에 대한 고유한 이해와 활동 전위에 대한 이해, 척수 자극에 의한 코딩 패턴 활동을 이해할 수 있습니다.

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.

연구

신경과학

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.

운동 뉴런의 전체 세포 패치-클램프 기록을 위한 척수 절제

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.

운동 뉴런의 전체 세포 패치-클램프 기록을 위한 척수 절편 준비

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.