Name: the preparation of oblique spinal cord slices for ventral root stimulation
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Description: Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

Los autores agradecen a Marin Manuel y Olivia Goldman-Szwajkajzer por su ayuda en la toma de las fotografías. Los autores también agradecen a Arjun Masukar y Tobias Bock para la corrección del manuscrito. Ayudas económicas fueron proporcionados por la Agence Nationale pour la Recherche (Hyper-MND, ANR-2010-BLAN-1429/01), el NIH NINDS (R01NS077863), la Fundación Latran Thierry (OHEX Proyecto), la Asociación Francesa de miopatía ( la subvención número 16026) y Target ALS se agradece. Felix Leroy era el destinatario de un &quot;Contrato de Doctorado&quot; de la Escuela Normal Superior, Cachan.

Los experimentos se realizaron de acuerdo con las directivas europeas (86/609 / CEE y 2010 a 63-UE) y la legislación francesa, y fueron aprobados por el comité de ética de la Universidad de París Descartes. 1. Preparación de la rebanada de la médula espinal <ol><li> Preparar las siguientes soluciones diarias o un día de antelación. Si se mantiene durante toda la noche, la burbuja con un 95% de O2 y 5% de CO2 y mantener refrigerado en frascos bien cerrados. <ol...

<ol>
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rebanar oblicua de la médula espinal es importante ya que permite la estimulación unilateral de piscinas de neuronas motoras y las células de Renshaw en un solo segmento vertebral de una manera fiable, completa y específica. Además, se permite una identificación rápida, elegante y no ambigua de las motoneuronas grabados. A continuación, vamos a destacar las ventajas de esta técnica en comparación con otros métodos de preparación rebanada, y luego vamos a insistir en las trampas más comunes a evitar al reali...

Históricamente, las grabaciones motoneuronas utilizando Sharp-electrodo se llevaron a cabo in vivo en animales grandes tales como gatos o ratas 1 o en una médula espinal enteros aislados en ratones 2. La aparición de la técnica de grabación de patch-clamp durante la década de 1980, llamado por el acceso directo a la motoneurona somas como el sellado necesario para conseguir bajo control visual. Por lo tanto, la preparación de la rebanada de la médula espinal se ha logrado fácilmente desde principios de 1990 3. Sin embargo, rebanada preparación temprana a menudo no permitía la estimulación de las raíces ventrales. A lo mejor de nuestro conocimiento, sólo dos estudios han informado de la estimulación exitosa de las raíces ventrales en rodajas transversales, y ninguno se obtuvo a partir de ratones 4,5. En este artículo se presenta una técnica para lograr las rebanadas de la médula espinal viables de ratones recién nacidos (P2 - P11) en el que la piscina de motoneuronas conserva sus raíces ventrales saliendo axones. Respiraderoestimulación de la raíz ral desencadena potencial de acción antidrómica de nuevo en los somas de las neuronas motoras de la piscina que sale de la misma raíz ventral. También excita los objetivos colaterales de neuronas motoras, otras motoneuronas 6-10 y las células de Renshaw 11-13. Dado que sólo motoneuronas envían sus axones hacia abajo las raíces ventrales, se utiliza el registro de los potenciales de acción antidrómicos como una forma sencilla y definitiva a physiologicaly identificar motoneuronas 10. Además de utilizar criterios electrofisiológicos y morfológicos potencialmente no incluido o engañosas para confirmar la identidad de las neuronas motoras, estudios recientes sobre las neuronas motoras de la médula espinal también se basó en tinciones tedioso y requiere mucho tiempo post hoc 16. Tal identificación se realiza generalmente sólo en una muestra de las células grabadas. Otras estrategias de identificación se basan en las líneas de ratón en el que las motoneuronas expresan la fluorescencia endógena <sup&gt; 17-19. Sin embargo, el uso de marcadores codificados genéticamente puede ser difícil a una edad temprana, cuando la expresión del marcador sigue siendo variable o si el estudio ya se requiere el uso de una línea de ratones transgénicos. Alternativamente, la acción antidrómica potenciales grabaciones se pueden realizar de forma rutinaria en todos los ratones desde el inicio de la grabación de la célula. Los experimentadores que trabajan en los preparativos de la médula espinal intacta en el gato, la rata y el ratón, se han utilizado de forma fiable estas técnicas de identificación desde la década de 1950 1,2,20,21. En condiciones óptimas, hemos sido capaces de provocar potenciales de acción antidrómicos de virtualmente todas las neuronas motoras registradas. Además, la estimulación de la raíz ventral se puede utilizar para excitar de forma fiable otros motoneuronas 22,23 o sus objetivos. las células de Renshaw 10,24,25. Presentamos aquí las aplicaciones de la estimulación de la raíz ventral en forma de acción antidrómica posibles grabaciones de somas de las neuronas motoras, así como la excitación de las células de Renshaw.

<table >
	<tbody>
		<tr >
			<td >Na-kynurenate</td>
			<td >ABCAM</td>
			<td >ab120256</td>
			<td >dissolves better then other brands</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Sigma</td>
			<td >P3911</td>
			<td></td>
		</tr>
		<tr >
			<td >NaH2PO4</td>
			<td >Sigma</td>
			<td >P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >sucrose&#160;</td>
			<td >Sigma</td>
			<td >S9378</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3&#160;</td>
			<td >Sigma</td>
			<td >S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2&#160;</td>
			<td >G Biosciences</td>
			<td >R040</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2&#160;</td>
			<td >Quality Biological</td>
			<td >351-033-721</td>
			<td></td>
		</tr>
		<tr >
			<td >glucose&#160;</td>
			<td >Sigma</td>
			<td >G5767</td>
			<td></td>
		</tr>
		<tr >
			<td >ascorbic acid&#160;</td>
			<td >Sigma</td>
			<td >A5960</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-pyruvate&#160;</td>
			<td >Sigma</td>
			<td >P2250</td>
			<td></td>
		</tr>
		<tr >
			<td >K-gluconate&#160;</td>
			<td >Sigma</td>
			<td >P1847</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA&#160;</td>
			<td >Sigma</td>
			<td >E3889</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES&#160;</td>
			<td >Sigma</td>
			<td >H4034</td>
			<td></td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Sigma</td>
			<td >S9888</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Sigma</td>
			<td>A9799</td>
			<td></td>
		</tr>
		<tr >
			<td >QX-314</td>
			<td >Alomone</td>
			<td>Q150</td>
			<td></td>
		</tr>
		<tr >
			<td >Mg-ATP</td>
			<td>Sigma</td>
			<td >A9187</td>
			<td></td>
		</tr>
		<tr >
			<td >CsOH</td>
			<td>Sigma</td>
			<td >232041</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-GTP</td>
			<td>Sigma</td>
			<td >51120</td>
			<td></td>
		</tr>
		<tr >
			<td >gluconic acid</td>
			<td>Sigma</td>
			<td >G1951</td>
			<td></td>
		</tr>
		<tr >
			<td >Cesium hydroxide solution</td>
			<td>Sigma</td>
			<td >232041</td>
			<td></td>
		</tr>
		<tr >
			<td >KOH</td>
			<td>Sigma</td>
			<td >P5958</td>
			<td></td>
		</tr>
		<tr >
			<td >Vannas Spring Scissors - 2.5mm&#160;</td>
			<td >FST</td>
			<td>15000-08</td>
			<td>only use for cutting the dura, might get damaged if cutting bones</td>
		</tr>
		<tr >
			<td >Stimulator</td>
			<td>A-M Systems</td>
			<td colspan="2">Isolated Pulse Stimulator Model 2100</td>
		</tr>
		<tr >
			<td >Vibratome</td>
			<td >Campden</td>
			<td >Vibrating Microtome 7000 - Model 7000smz-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the preparation of oblique spinal cord slices for ventral root stimulation

Registros electrofisiológicos de láminas de médula espinal han demostrado ser una técnica valiosa para investigar una amplia gama de preguntas, de celular a las propiedades de red. Mostramos cómo preparar rodajas oblicuas viables de la médula espinal de ratones jóvenes (P2 - P11). En esta preparación, las motoneuronas conservan sus axones que sale de las raíces ventrales de la médula espinal. La estimulación de estos axones provoca back-propagan los potenciales de acción que invaden el somas de las neuronas motoras y emocionantes colaterales el motoneuronas en la médula espinal. Grabación de potenciales de acción antidrómicos es una forma inmediata, definitiva y elegante para caracterizar la identidad de las neuronas motoras, que supera a otros métodos de identificación. Además, la estimulación de las neuronas motoras colaterales es una manera simple y fiable para excitar los objetivos colaterales de las neuronas motoras en la médula espinal, tales como otros motoneuronas o Renshaw células. En este protocolo, se presentan grabaciones antidrómicos de la motoneurona somas, así como excitación de la célula de Renshaw, resultante de la estimulación de la raíz ventral.

La confirmación de la identidad Motoneuron base de los potenciales de acción antidrómica La orientación de la célula Motoneuronas se encuentran en el cuerno ventral (visible en rojo en la figura 2C). Comience desde el haz de axones que forman la raíz ventral y subir hasta que el haz se dispersa completamente y u...

Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Mostramos cómo preparar rodajas oblicuas de la médula espinal en ratones jóvenes. Esta preparación permite la estimulación de las raíces ventrales.

La Preparación de oblicua de la médula espinal ventral de segmentos para Root Estimulación

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

The overall goal of this procedure is to prepare oblique slices of the spinal cord that allow for the stimulation of the ventral roots. The main advantage of this technique is that it allows motoneural identifications for ventral-root stimulation and to study the ventral cells enervated by motoneural axon collaterals. We first had the idea for this method when needing a reliable way to stimulate ventral cells.Individuals new to this method will struggle because of the difficulty to properly execute the different steps of the dissection. Begin by perfusing a P2 to P11 mouse with low-sodium ACSF. After decapitating the mouse and removing the skin on the back, make two cuts through the shoulders and down the rib cage.Then, cut the spinal cord as low as possible in the caudal section to isolate the vertebral column and ribs from the lower part of the animal. Flip the vertebral column and remove the viscera still attached to the ribs. Transfer the vertebral column to another smaller, silicon-filled Petri dish.Add cold ACSF and bubble with carbogen and use four insect pins to hold the tissue dorsal-side up. Then perform a laminectomy of the dorsal side by first inserting the tip of fine scissors between the bone and the spinal cord and cutting the bone from the rostral end, making sure to stay away from the white matter. Alternate the cuts on each side while using tweezers to hold the band of bone already cut away.Next, use small tweezers to lift the dura and small scissors to cut along the rostral caudal access on both sides. Removal of the dura is a critical step. It is well worth spending a few extra minutes to do this thoroughly.Once the dura is removed, use a blunt glass tip to gently push cord towards the left side of the groove formed by half-cut spinal column. Then cut the ventral and dorsal roots on the bright side as far as possible from where they enter the cord. Repeat the operation on the left side, always going from rostral to caudal.After dissection, slip the spinal cord out of the vertebral column. Then use a small insect pin to secure the cord for the dorsal surface-up. Delicately remove any pieces of attached membrane.Once cleaned, trim both ends of the cord and insert a bent insect pin into the anterior part of the cord to mark its orientation. Then transfer the spinal cord to ice-cold intracellular solution. Next, cool the previously prepared molten agar on a mixture of ice and water.Stir the agar while measuring the temperature. When the temperature of the agar reaches 38 degrees Celsius, hold the spinal cord by the insect pin and immerse it in the agar, rostral-side down. Make sure the spinal cord is as straight as possible, away from the walls, with the caudal part slightly upward.Leave the beaker in the mixture of ice and water to allow the agar to solidify around the cord as quickly as possible. After solidification, cut the agar block containing the spinal cord in such a way that the base of the block is at a 35-degree angle with the lumbar part of the cord. The dorsal surface should be facing away from the base.Glue the block into the chamber of the vibratome, using cyanoacrylate glue. It is critical to mount the block correctly to maintain the continuity of the motor pools with the ventral roots from which they exit. Immerse the block in potassium gluconate solution.Set the vibratome parameters. Next, cut 350 400 micron-thick slices of the lumbar region, which is identifiable by its curvature and larger diameter. Transfer the suitable slices to ACSF at 34 degrees Celsius.Typically, there are four to five suitable slices with ventral roots extending two millimeters or more. After approximately 30 minutes at 34 degrees Celsius, cool the slices to room temperature, then begin the recording session. Prepare a box of various pipettes with tip diameters ranging from 40 to 170 microns in advance.To prepare the section pipettes, select pipettes with a long tapure. Then, using a diamond knife, make cuts at different positions and under a dissecting microscope, break each pipette by hitting the tip with tweezers. Next, remove the chamber from the recording microscope and place it under a dissecting microscope.Select a slice that contains a ventral root of sufficient length to be mounted on a suction-stimulation electrode. Mount the slice, orienting it with the ventral roots upward, then delicately cut the agar around the ventral roots while leaving the rest of the agar around the slice and place the grid to hold the sample down. Mount the chamber back onto the microscope and continuously perfuse the recording chamber with ACSF at a rate of one to two milliliters per minute at room temperature.Using a glass pipette filled with ACSF and connected to a syringe, suck one of the ventral roots. In order to achieve good stimulation of the ventral root, the pipette tip needs to be tight around the ventral root. Achieve patch-clamp recording of the desired cell type and record the effect of the ventral root stimulation as described previously.This image shows the results from voltage-clamp recording. The recordings of the upper panels are taken from different motoneurons than the ones in the bottom panels. The upper panel shows the antidromic response of a motoneuron to a one-volt stimulation in red, and a five-volt stimulation in black.The lower trace shows the orthodromic action potential response to a 20-volt stimulation in red, and a 30-volt stimulation in black. Black dots indicate stimulus timing. The double arrow heads emphasize the difference in latencies between ortho-and antidromic spikes.Here, current clamp recordings are shown. The upper panel shows the antidromic response to 10-volt and 15-volt stimulations in red and black, respectively. The lower panel shows the orthodromic response to 25-and 40-volt stimulations in red and black, respectively.Again, the stimulus timing and differences in latencies are indicated. This image of the Renshaw cell pool was acquired using an oblique condenser and a 20x objective. Note the motoneuron axons merging into the ventral root as shown by the arrow.This image of the same region was taken using a 40x objective. Putative Renshaw cells are indicated with arrow heads. Shown here is a voltage-clamp recording of a Renshaw cell following stimulation of the ventral root, indicated by the black dot.QX-314 in the intracellular solution prevented the cell from firing and glycine and gaba responses were blocked by 3 micromolar gabazine and one micromolar strychnine, respectively. The red trace is the average of the gray ones. Once mastered, this technique can be performed in 20 or 30 minutes.While attempting this procedure, it's important to remember to keep the preparation in cold medium at all time. After watching this video, you should have a good understanding of how to prepare oblique slices of spinal cord suitable for ventral-root stimulation. Ventral-root stimulation can be used to easily confirm the motoneural identity, and more importantly, to reliably stimulate the ventral cells.

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Investigación

Neurociencias

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Preparación Todo el montaje del Adulto Drosophila Cordón nervioso ventral para inyección gigante tinte de fibra

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Preparación de las rebanadas parasagitales para la Investigación de dorsal-ventral Organización de los roedores de corteza entorrinal medial

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Extrusión hidráulica de la médula espinal y aislamiento de los ganglios de la raíz dorsal en roedores

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

El pez cebra<em&gt; In Situ</em&gt; Médula Espinal Preparación para Registros electrofisiológicos de Spinal Sensory y neuronas motoras

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Preparación de láminas agudas de la médula espinal para celulares Patch-clamp de grabación en las neuronas de la sustancia Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Preparación de rítmicamente activa en Vitro Neonatal roedor del médula oblonga-medulares y rebanada fina

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Entrega intra-arterial de células madre neuronales a la rata y el cerebro del ratón: aplicación a la isquemia cerebral

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

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

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

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