Name: Yazar Spotlight: Omurilik Stimülasyonunu İlerletmek - Yama-Kelepçe Elektrofizyolojisi Yoluyla Motor Nöronların Hücresel Tepkilerini Keşfetmek
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

Bu çalışma, Genç Akademisyenler için Çin Ulusal Doğa Bilimleri Vakfı (52207254 ve 82301657) ve Çin Doktora Sonrası Bilim Fonu (2022M711833) tarafından finanse edilmiştir.

Kurumsal Hayvan Bakım ve Kullanım Komitesi tüm hayvan deneylerini onayladı ve çalışmalar ilgili hayvan refahı yönetmeliklerine uygun olarak yürütüldü.
1. Hayvanların hazırlanması
<ol><li>Hayvan<ol><li>Barınma bilgisi: Erkek Sprague-Dawley sıçanlarını (doğum sonrası 10-14 gün, P10-P14) belirli bir patojen içermeyen ortamda barındırın. NOT: Oda koşulları 20 °C ± 2 °C, nem: %50-60 arasında, 12 saatlik aydınlık/karanlık döngüsüyle korun...

Omurilik Stimülasyonuna Motor Nöron Yanıtlarını Anlama

<ol>
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(116), e54525 (2016).</li><li>Sharples, S. A., Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+persistent+and+hyperpolarization-activated+inward+currents+shapes+the+differential+activation+of+motoneuron+subtypes+during+postnatal+development.">Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development.</a> Elife. 10, e71385 (2021).</li><li>Bhumbra, G. 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. 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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. 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SCS tarafından modüle edilen hareket bilgisi sonunda motor nöronlara yakınsar. Bu nedenle, motor nöronları araştırma hedefi olarak almak, çalışma tasarımını basitleştirebilir ve SKS'nin nöromodülasyon mekanizmasını daha doğrudan ortaya çıkarabilir. Farklı uyaran özelliklerini ve hücresel tepkileri aynı anda kaydetmek için, bir yama kelepçesi, elektrofizyolojik özellikleri tek hücre ölçeğinde incelemek için iyi bir yöntemdir. Bununla birlikte, hücre canlılığının nasıl korunacağı...

Omurilik stimülasyonu (SCS), omurilik yaralanması (SCI) sonrası lokomotor fonksiyonu etkili bir şekilde geri yükleyebilir. Andreas Rowald ve ark. SCS'nin tek bir gün içinde alt ekstremite lokomotor ve gövde fonksiyonunu sağladığını bildirmiştir1. Lokomotor geri kazanım için SCS'nin biyolojik mekanizmasını keşfetmek, daha kesin bir SCS stratejisi geliştirmek için kritik ve trend olan bir araştırma alanıdır. Örneğin, Grégoire Courtine'in ekibi, omurilikteki uyarıcı Vsx2 internöron ve Hoxa10 nöronlarının SCS'ye yanıt veren anahtar nöronlar olduğunu ve hücreye özgü nöromodülasyonun SCI2'den sonra sıçan yürüme yeteneğini geri kazanmak için mümkün olduğunu gösterdi. Bununla birlikte, az sayıda çalışma, tek hücre ölçeğinde SCS'nin elektriksel mekanizmasına odaklanmaktadır. Klasik kalamar deneyi 3,4,5'te eşik üstü doğru akım uyaranının aksiyon potansiyellerini (AP'ler) ortaya çıkarabileceği iyi bilinmesine rağmen, SCS gibi darbeli alternatif elektriksel stimülasyonun motor sinyal üretimini nasıl etkilediği hala belirsizdir.
İntraspinal nöral devrelerin karmaşıklığı göz önüne alındığında, SKS'nin elektriksel mekanizmasının araştırılmasında hücre popülasyonu için uygun seçim önemlidir. SCS, propriyoseptif yol6'yı aktive ederek motor fonksiyonunu geri yüklese de, motor nöronlar, propriyosepsiyon bilgisi afferent girdisinin7 entegre edilmesinden türetilen motor komutu yürüten son birimdir. Bu nedenle, SCS ile motor nöronların elektriksel özelliklerini doğrudan incelemek, spinal motor modülasyonun altında yatan mantığı anlamamıza yardımcı olabilir.
Bildiğimiz gibi, yama kelepçesi, son derece yüksek uzay-zamansal çözünürlüğe sahip hücresel elektrofizyolojik kayıt için altın standart yöntemdir8. Bu nedenle, bu çalışma, motor nöronların SCS'ye elektriksel tepkilerini incelemek için bir yama kelepçesi kullanan bir yöntemi açıklamaktadır. Beyin yama kelepçesi9 ile karşılaştırıldığında, omurilik yama kelepçesi aşağıdaki nedenlerden dolayı daha zordur: (1) Omurilik, daha iyi hücre canlılığı elde etmek için çok ince mikromanipülasyon ve titiz buz gibi bakım gerektiren küçük hacimli vertebral kanal tarafından korunur. (2) Omurilik, kesme tepsisine sabitlenemeyecek kadar ince olduğundan, düşük erime noktalı agaroza daldırılmalı ve katılaşmadan sonra kesilmelidir.
Bu nedenle, bu yöntem, SCS'nin motor nöronlar üzerindeki elektriksel mekanizmasını sorunsuz bir şekilde incelemek ve gereksiz denemelerden ve hatalardan kaçınmak için omuriliğin diseke edilmesinde ve aynı zamanda hücre canlılığının korunmasında teknik ayrıntılar sağlar.

<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

Omurilik stimülasyonu (SCS), omurilik yaralanması (SCI) sonrası lokomotor fonksiyonu etkili bir şekilde geri yükleyebilir. Motor nöronlar, sensorimotor davranışları yürüten son birim olduğundan, motor nöronların elektriksel tepkilerini SCS ile doğrudan incelemek, spinal motor modülasyonunun altında yatan mantığı anlamamıza yardımcı olabilir. Farklı uyaran özelliklerini ve hücresel tepkileri aynı anda kaydetmek için, bir yama kelepçesi, elektrofizyolojik özellikleri tek hücre ölçeğinde incelemek için iyi bir yöntemdir. Bununla birlikte, hücre canlılığını korumak, omuriliği kemikli yapıdan hızlı bir şekilde ayırmak ve aksiyon potansiyellerini başarılı bir şekilde indüklemek için SCS'yi kullanmak da dahil olmak üzere bu hedefe ulaşmada hala bazı karmaşık zorluklar vardır. Burada, motor nöronların yüksek uzay-zamansal çözünürlükle SCS'ye elektriksel tepkilerini incelemek için yama-kelepçe kullanarak ayrıntılı bir protokol sunuyoruz, bu da araştırmacının omuriliği ayırma ve hücre canlılığını koruma becerilerini geliştirmelerine yardımcı olabilir.

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.

Yazar Spotlight: Omurilik Stimülasyonunu İlerletmek - Yama-Kelepçe Elektrofizyolojisi Yoluyla Motor Nöronların Hücresel Tepkilerini Keşfetmek 

Spinal Kord Stimülasyonu Sırasında Motor Nöronlarda Tüm Hücre Yama-Klemp Kaydı için Omurilik Diliminin Ex vivo Hazırlanması

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.

İnce işlem sırasında titiz düşük sıcaklık bakımı sayesinde (Ek Şekil 1, Ek Şekil 2 ve Şekil 1), hücre canlılığı sonraki elektrofizyolojik kayıtları gerçekleştirmek için yeterince iyiydi. Klinik senaryoyu mümkün olduğunca simüle etmek için, SCS katotunu ve anodu sırasıyla dorsal orta hat ve DREZ'in yakınına yerleştirmek için mikromanipülasyon kullandık (Şekil 2), bu da dorsal kornadaki nöral sinyali başlata...

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

Bu protokol, motor nöronların omurilik stimülasyonuna (SCS) elektriksel tepkilerini yüksek uzay-zamansal çözünürlükle incelemek için bir yama kelepçesi kullanan bir yöntemi açıklar ve bu da araştırmacıların omuriliği ayırma ve aynı anda hücre canlılığını sürdürme becerilerini geliştirmelerine yardımcı olabilir.

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

Omurilik stimülasyonu, hastaların yaralanmadan sonra çoğu hareket fonksiyonunu çözmesine yardımcı olabilir. Çoğu nöron, duyusal mod davranışlarını yürütmek içindir. Çoğu nöronun elektriksel tepkisini incelemek, spinal yük nöromodülasyonunun altında yatan mantığın anlaşılmasına yardımcı olabilir.Bir yama kelepçesi, son derece yüksek uzamsal zamansal çözünürlüğe sahip hücresel elektrofizyolojik kayıtlar için altın standart yöntemdir. Bu nedenle, bu çalışma, motor nöronların aort uyaran özelliklerini ve hücresel yanıtlarını tek hücre ölçeğinde eşzamanlı olarak kaydetmek için bir yama kelepçesi kullanan bir yöntemi açıklamaktadır. Beyin yama klempi ile karşılaştırıldığında, omurilik yama klempi daha zordur, çünkü omurilik küçük hacimli vertebral kanal tarafından iyi korunur.Bu nedenle, bu protokol, daha iyi hücre canlılığı elde etmek için omuriliğin hızlı bir şekilde mikro diseksiyonunda ve bakımın tersine çevrilmesinde teknik ayrıntılar sağlar. Yama klemp, sinaptik iletimlerin benzersiz bir şekilde anlaşılmasını ve aksiyon potansiyellerinin anlaşılmasını, omurilik stimülasyonu ile model aktivitelerinin kodlanmasını sağlar.

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.

Nörobilim

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.

Motor Nöronlarda Tüm Hücre Yama-Klemp Kaydı için Omurilik Diseksiyonu

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.

Motor Nöronlarda Tüm Hücre Yama-Kelepçe Kaydı için Omurilik Diliminin Hazırlanması

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.