Name: Autor destacado: Avance de la estimulación de la médula espinal: exploración de las respuestas celulares de las neuronas motoras a través de la electrofisiología de 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

Este estudio fue financiado por la Fundación Nacional de Ciencias Naturales de China para Jóvenes Académicos (52207254 y 82301657) y el Fondo de Ciencias Postdoctorales de China (2022M711833).

El Comité Institucional de Cuidado y Uso de Animales aprobó todos los experimentos con animales y los estudios se llevaron a cabo de acuerdo con las normas pertinentes de bienestar animal.
1. Preparación de los animales
<ol><li>Animales<ol><li>Información sobre el alojamiento: Aloje ratas macho Sprague-Dawley (Postnatal 10-14 días, P10-P14) en un ambiente específico libre de patógenos. NOTA: Las condiciones de la habitación se mantuvieron a 20 °C ± 2 °C, humeda...

Comprensión de las respuestas de las neuronas motoras a la estimulación de la médula espinal

<ol>
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(116), e54525 (2016).</li><li>Sharples, S. A., Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+persistent+and+hyperpolarization-activated+inward+currents+shapes+the+differential+activation+of+motoneuron+subtypes+during+postnatal+development.">Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development.</a> Elife. 10, e71385 (2021).</li><li>Bhumbra, G. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na(V)1.1+is+essential+for+proprioceptive+signaling+and+motor+behaviors.">Na(V)1.1 is essential for proprioceptive signaling and motor behaviors.</a> Elife. 11, e79917 (2022).</li><li>Romer, S. H., Deardorff, A. S., Fyffe, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+rheostat:+Kv2.1+currents+maintain+or+suppress+repetitive+firing+in+motoneurons.">A molecular rheostat: Kv2.1 currents maintain or suppress repetitive firing in motoneurons.</a> The Journal of Physiology. 597 (14), 3769-3786 (2019).</li><li>Yao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+R-type+human+Ca(v)2.3+channel+reveal+conformational+crosstalk+of+the+intracellular+segments.">Structures of the R-type human Ca(v)2.3 channel reveal conformational crosstalk of the intracellular segments.</a> Nature Communications. 13 (1), 7358 (2022).</li><li>Bandres, M. F., Gomes, J., McPherson, J. 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La información de movimiento modulada por el SCS finalmente converge a las neuronas motoras. Por lo tanto, tomar las neuronas motoras como objetivo de investigación puede simplificar el diseño del estudio y revelar el mecanismo de neuromodulación del SCS de manera más directa. Para registrar simultáneamente diversas características de estímulos y respuestas celulares, un patch-clamp es un buen método para estudiar las características electrofisiológicas a escala de una sola célula. Sin embargo, todavía exist...

La estimulación de la médula espinal (SCS, por sus siglas en inglés) puede restaurar eficazmente la función locomotora después de una lesión de la médula espinal (SCI, por sus siglas en inglés). Andreas Rowald et al. informaron que el SCS permite la función locomotora y troncal de las extremidades inferiores en un solo día1. La exploración del mecanismo biológico del SCS para la recuperación locomotora es un campo de investigación crítico y de tendencia para desarrollar una estrategia de SCS más precisa. Por ejemplo, el equipo de Grégoire Courtine demostró que la interneurona excitadora Vsx2 y las neuronas Hoxa10 en la médula espinal son las neuronas clave para la respuesta al SCS, y la neuromodulación específica de la célula es factible para restaurar la capacidad de caminar de la rata después de la LME2. Sin embargo, pocos estudios se centran en el mecanismo eléctrico del SCS a escala de una sola célula. Aunque es bien sabido que el estímulo de corriente continua supraumbral puede provocar los potenciales de acción (AP) en el experimento clásico del calamar 3,4,5, aún no está claro cómo la estimulación eléctrica alterna pulsada, como la SCS, afecta a la generación de señales motoras.
Dada la complejidad de los circuitos neuronales intraespinales, la selección adecuada de la población celular es importante para investigar el mecanismo eléctrico del SCS. Aunque el SCS restaura la función motora mediante la activación de la vía propioceptiva6, las neuronas motoras son la unidad final para ejecutar el comando motor, derivado de la integración de la información de la propiocepción de entrada aferente7. Por lo tanto, el estudio directo de las características eléctricas de las neuronas motoras con SCS puede ayudarnos a comprender la lógica subyacente de la modulación motora espinal.
Como sabemos, el patch-clamp es el método de referencia para el registro electrofisiológico celular con una resolución espacio-temporal extremadamente alta8. Por lo tanto, este estudio describe un método que utiliza una pinza de parche para estudiar las respuestas eléctricas de las neuronas motoras al SCS. En comparación con el parche cerebral9, el parche clamp de la médula espinal es más difícil debido a las siguientes razones: (1) La médula espinal está protegida por el canal vertebral con un volumen minúsculo, lo que requiere una micromanipulación muy fina y un riguroso mantenimiento en frío para obtener una mejor viabilidad celular. (2) Debido a que la médula espinal es demasiado delgada para asegurarla en la bandeja de corte, debe sumergirse en agarosa de bajo punto de fusión y recortarse después de la solidificación.
Por lo tanto, este método proporciona detalles técnicos para diseccionar la médula espinal y mantener la viabilidad celular al mismo tiempo para estudiar sin problemas el mecanismo eléctrico del SCS en las neuronas motoras y evitar pruebas y errores innecesarios.

<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 estimulación de la médula espinal (SCS, por sus siglas en inglés) puede restaurar eficazmente la función locomotora después de una lesión de la médula espinal (SCI, por sus siglas en inglés). Debido a que las neuronas motoras son la unidad final para ejecutar los comportamientos sensoriomotores, el estudio directo de las respuestas eléctricas de las neuronas motoras con SCS puede ayudarnos a comprender la lógica subyacente de la modulación motora espinal. Para registrar simultáneamente diversas características de estímulos y respuestas celulares, un patch-clamp es un buen método para estudiar las características electrofisiológicas a escala de una sola célula. Sin embargo, todavía existen algunas dificultades complejas para lograr este objetivo, incluido el mantenimiento de la viabilidad celular, la separación rápida de la médula espinal de la estructura ósea y el uso del SCS para inducir con éxito potenciales de acción. Aquí, presentamos un protocolo detallado que utiliza patch-clamp para estudiar las respuestas eléctricas de las neuronas motoras al SCS con alta resolución espacio-temporal, lo que puede ayudar a los investigadores a mejorar sus habilidades para separar la médula espinal y mantener la viabilidad celular al mismo tiempo para estudiar sin problemas el mecanismo eléctrico del SCS en la neurona motora y evitar pruebas y errores innecesarios.

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.

Autor destacado: Avance de la estimulación de la médula espinal: exploración de las respuestas celulares de las neuronas motoras a través de la electrofisiología de patch-clamp 

La preparación ex vivo del corte de la médula espinal para el registro de patch-clamp de células enteras en neuronas motoras durante la estimulación de la médula espinal

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.

Gracias al riguroso mantenimiento a baja temperatura durante la operación fina (Figura suplementaria 1, Figura suplementaria 2 y Figura 1), la viabilidad de la celda fue lo suficientemente buena como para realizar registros electrofisiológicos posteriores. Para simular al máximo el escenario clínico, utilizamos la micromanipulación para colocar el cátodo y el ánodo del SCS cerca de la línea media dorsal y el DREZ, respectivamente (Figura 2</stron...

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

Este protocolo describe un método que utiliza una pinza de parche para estudiar las respuestas eléctricas de las neuronas motoras a la estimulación de la médula espinal (SCS) con alta resolución espacio-temporal, lo que puede ayudar a los investigadores a mejorar sus habilidades para separar la médula espinal y mantener la viabilidad celular simultáneamente.

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

La estimulación de la médula espinal puede ayudar a los pacientes a resolver la mayoría de las funciones de movimiento después de una lesión. La mayoría de las neuronas deben ejecutar comportamientos de modo sensorial. El estudio de la respuesta eléctrica de la mayoría de las neuronas puede ayudar a comprender la lógica subyacente de la neuromodulación de la carga espinal.Una pinza de parche es el método estándar de oro para los registros electrofisiológicos celulares con una resolución espacio-temporal extremadamente alta. Por lo tanto, este estudio describe un método que utiliza una pinza de parche para registrar simultáneamente las características del estímulo de la aorta y las respuestas celulares de las neuronas motoras a escala de una sola célula. En comparación con la pinza de parche cerebral, la pinza de parche de la médula espinal es más difícil, porque la médula espinal está bien protegida por el canal vertebral con un volumen pequeño.Por lo tanto, este protocolo proporciona detalles técnicos en la microdisección rápida de la médula espinal y el mantenimiento inverso para obtener una mejor viabilidad celular. La pinza de parche permite una comprensión única de las transmisiones sinápticas y la comprensión de los potenciales de acción, codificando las actividades del patrón por la estimulación de la médula espinal.

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.

Investigación

Neurociencias

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.

Disección de la médula espinal para el registro de patch-clamp de células enteras en neuronas motoras

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.

Preparación del corte de la médula espinal para el registro de patch-clamp de células enteras en neuronas motoras

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.