Este trabajo fue apoyado por subvenciones del NIH (F32 NS059120 a RLM y R01NS25217 y P30NS048154 a ABR).

Los autores declaran no tener intereses financieros en competencia.

Los métodos descritos aquí permiten la caracterización eléctrica y morfológica de las neuronas sensoriales y motoras de embriones de pez cebra después de la disección mínima de la médula espinal. Las neuronas permanecen sanos durante al menos 1 h, el límite de tiempo impuesto en estas grabaciones. Las neuronas han sido registrados utilizando la configuración de célula completa estándar, así como de parches nucleadas; el último método minimiza los problemas de espacio-clamp que se opone a que un estudio b...

<html> George Streisinger fue pionera en el uso de Danio rerio, conocido comúnmente como el pez cebra, como un sistema modelo para el análisis genético de desarrollo de los vertebrados 10. El modelo ofrece varias ventajas, incluyendo: (1) relativamente simple y barato cría de animales; (2) fertilización externa, lo que permite un fácil acceso a los embriones de las etapas de desarrollo más tempranas; y (3) un embrión transparente, permitiendo la observación directa y repetidas de células, tejidos y órganos, ya que forman. En las décadas siguientes, varios avances aumentaron aún más el poder del modelo de pez cebra. En particular, las pantallas adelante genéticos y los esfuerzos de secuenciación de todo el genoma jugaron un papel clave en la identificación de mutaciones y genes críticos para muchos procesos de desarrollo 11, 12, 13, 14,&quot;&gt; 15, métodos de clonación 16. Puerta de enlace han permitido la aplicación rutinaria de transgénicos se aproxima a 17, 18. Los recientes avances en la edición genoma, ejemplificada por la transcripción del tipo activador de (Talens) y agrupados repeticiones intercaladas regularmente cortos palindrómicas (CRISPR) -Cas9 nucleasas, permitir la introducción selectiva de mutaciones, así como knock-out y knock-in se aproxima a 19, 20, 21, 22. combinados, estos métodos hacen que el pez cebra un poderoso modelo para el estudio de los mecanismos genéticos que subyacen a los comportamientos específicos y varias enfermedades humanas 23, 24, 25, 26, 27. Este trabajo se centra en el desarrollola regulación mental y el papel de la actividad eléctrica en el desarrollo neuronal. La atención se centra en la médula espinal, para el que el modelo de pez cebra ofrece varias ventajas. En primer lugar, es relativamente fácil de acceder pez cebra en estadios embrionario y larval; Por lo tanto, se puede estudiar la función de la médula espinal durante las etapas de desarrollo que tienen menos neuronas y circuitos simples 28, 29. Por otra parte, la médula espinal pez cebra tiene un conjunto diverso de neuronas, similar a otros vertebrados, como se demuestra por los patrones característicos y distintivos de factores de transcripción 30, 31, 32, 33, 34, 35. La mayoría de los estudios en el pez cebra que tienen como objetivo descubrir los mecanismos que subyacen a la función de los circuitos de la médula espinal, especialmentelos que apoyan la locomoción, se centran comprensiblemente en estadios larvarios 36, 37, 38, 39, 40, 41, 42, 43. Sin embargo, muchas de las neuronas que forman las redes de locomotoras espinales iniciado su diferenciación en las etapas embrionarias tempranas, ~ 9-10 h después de la fertilización (HPF) 44, 45, 46, 47, 48, 49, 50, 51. En vista de esto, la comprensión de cómo surgen las propiedades morfológicas y eléctricas de las neuronas espinales y el cambio entre las etapas embrionario y larval es importante para un overacomprensión ll de la formación y la función de circuito locomotor. Los métodos de disección descritas aquí permiten grabaciones de patch clamp de neuronas de la médula y se han aplicado con éxito en las etapas embrionarias (~ 17-48 HPF) y estadios larvarios (~ 3-7 días después de la fertilización [dpf]). Este enfoque limita la cantidad de disección requerida para proporcionar acceso a las neuronas de interés. El protocolo se diferencia de la mayoría de los otros métodos publicados para la grabación de las neuronas espinales de pez cebra en que pegamento veterinario de sutura se utiliza, en lugar de un pasador de tungsteno bien, para unir el embrión o larva a la cámara de registro. La disponibilidad de dos enfoques diferentes (es decir, cola de sutura contra el pasador de tungsteno) para el montaje de los embriones de pez cebra o larvas para el análisis electrofisiológico proporciona a los investigadores con opciones alternativas para alcanzar sus objetivos experimentales específicas. En primer lugar, los procedimientos de acceso y grabación en una emergente<html> ulación de las neuronas sensoriales primarias, células Rohon-Barba, se describen. Los cuerpos celulares de estas neuronas se encuentran dentro de la médula espinal dorsal. Existen células Rohon-Barba en numerosas especies de vertebrados, se diferencian en el desarrollo temprano, y subyacen a la respuesta táctil embrionaria 6, 44, 47, 48. En segundo lugar, los procedimientos de acceso y registro de las neuronas motoras espinales se detallan. surgen de pez cebra neuronas motoras espinales durante dos oleadas de la neurogénesis. Surgen las neuronas motoras primarias nacidos antes-al final de la gastrulación (~ 9-16 hpf), con sólo 3-4 neuronas motoras primaria presentes por hemisegment 45, 46, 49. En contraste, la población nacida después de las neuronas motoras secundarias es más numeroso y se produce durante un período prolongado, a partir de ~ 14 HPFef &quot;&gt; 45, 50. génesis secundaria de neuronas motoras en los segmentos de mediados de tronco se ha completado todo por 51 HPF 50. neuronas motoras secundarias se considera que son la contrapartida de las neuronas motoras en amniotas 46. Curiosamente, las neuronas supraespinales, a través de la dopamina, regulan la locomoción en la larva y el motor secundario génesis neurona en el embrión y larva joven 50, 51. neuronas motoras primarias y secundarias comprenden cada uno varios subtipos diferentes. cada motor primario proyectos subtipo neurona un axón periférico que inerva un grupo muscular característica, lo que resulta en un estereotipo, la identificación de trayectoria axonal. Generalmente, las neuronas motoras secundarias siguen las vías axonales previamente establecidos por las neuronas motoras primarias. por lo tanto, con respecto a las trayectorias axonales, neuronas motoras primarias y secundarias son similares, con la excepción de que el espesor axonal y somata tamaño unare mayor para las neuronas motoras primarias 45. En tercer lugar, se discuten los métodos para la grabación de algunos tipos de interneuronas. Sin embargo, en estos casos, se requiere una cantidad limitada de la eliminación de otras células de la médula espinal, y por lo tanto la médula espinal es menos intacta que para las grabaciones de células Rohon-Barba o neuronas motoras.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vacuum filter/Storage bottle, 0.22 mm pore</td>
			<td>Corning</td>
			<td>431096</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.2 mm</td>
			<td>Whatman</td>
			<td>6780-2502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma</td>
			<td>A-5040</td>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt&#160;</td>
		</tr>
		<tr>
			<td>a-bugarotoxin</td>
			<td>Tocris</td>
			<td>11032-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>4368-28-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-549 hydrazine salt</td>
			<td>Molecular Probes</td>
			<td>A-10438</td>
			<td>fluorescent dye</td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>Corning</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Fisher</td>
			<td>12-550C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>silicone elastomer</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>30-0038</td>
			<td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Drummond Scientific</td>
			<td>1-000-1000-100</td>
			<td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Miniature barbed polypropylene fitting&#160;</td>
			<td>Cole-Palmer</td>
			<td>6365-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive&#160;</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter Instruments</td>
			<td>&#160;Model P-97&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized micromanipulator&#160;&#160;</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher</td>
			<td>14-169-1B</td>
			<td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Molecular Devices</td>
			<td>1-HC-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmaseal Three-Way Stopcocks&#160;</td>
			<td>Baxter</td>
			<td>K75</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Axon Instruments</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td>Zeiss</td>
			<td>Axioskop2 FS plus</td>
			<td></td>
		</tr>
		<tr>
			<td>40x/0.80w Achroplan objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition and analysis software&#160;</td>
			<td>Axon Instruments</td>
			<td colspan="2">PClamp 10 - Clampex and Clampfit&#160;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="2">Dissection and Recording Solutions(in mM)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">All solutions, except the intracellular, are stable for ~ 2 - 3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td>
		</tr>
		<tr>
			<td colspan="4">The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20&#176;C) in small aliquots that are individually thawed on the day of use.&#160;</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="1">Dissection/Ringer&#8217;s solution</td>
			<td></td>
			<td colspan="2">145 NaCl, 3 KCl, 1.8 CaCl2.2H2O, 10 HEPES; pH 7.4 (with NaOH)</td>
		</tr>
		<tr>
			<td colspan="2">Pipette (intracellular) recording solution</td>
			<td></td>
			<td colspan="2">135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td>
		</tr>
		<tr>
			<td colspan="2">Bath (extracellular) recording solution/voltage and current-clamp</td>
			<td></td>
			<td colspan="2">125 NaCl, 2 KCl, 10 CaCl2.2H2O, 5 HEPES; pH 7.4 (with NaOH).</td>
		</tr>
		<tr>
			<td colspan="2">Alexa-594 hydrazine salt stock solution.&#160;</td>
			<td></td>
			<td colspan="2">Prepare a 13.2 mM stock in ddH2O, aliquot (~ 100 &#181;l) and store at -20&#176;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&#160; with a centrifuge tube filter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immobilizing agents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">0.4 % ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td></td>
			<td colspan="2">Prepare a 0.4% stock solution in 0.2M Tris, pH9 (0.4 g Tricaine/100 ml 0.2 M Tris</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">Adjust pH to 7 with NaOH and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute the stock solution ~ 25 fold in embryo media</td>
		</tr>
		<tr>
			<td>250 &#956;M &#945;-bungarotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 250 &#956;M stock in ddH2O (1 mg/500 &#956;l), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td>
		</tr>
		<tr>
			<td>1 mM Tetrodotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 1 mM stock in ddH2O (1 mg/3 ml), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td>
		</tr>
	</tbody>
</table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

El pez cebra, introducido por primera vez como un modelo de desarrollo, han ganado popularidad en muchos otros campos. La facilidad de la cría de un gran número de organismos que se desarrollan rápidamente, combinada con la claridad óptica embrionario, sirvió como atributos de peso iniciales de este modelo. Durante las últimas dos décadas, el éxito de este modelo ha sido impulsado además por su susceptibilidad a las pantallas de mutagénesis a gran escala y por la facilidad de transgénesis. Más recientemente, los enfoques de edición gen haber aumentado la potencia del modelo. Para los estudios del desarrollo neurológico, el embrión de pez cebra y larva proporcionan un modelo al que se pueden aplicar varios métodos. Aquí, nos centramos en los métodos que permiten el estudio de una propiedad esencial de las neuronas, excitabilidad eléctrica. Nuestra preparación para el estudio electrofisiológico de neuronas de la médula de pez cebra implica el uso de pegamento veterinario de sutura para asegurar la preparación de una cámara de registro. Los métodos alternativos para la grabacióna partir de embriones de pez cebra y larvas implicar la unión de la preparación a la cámara utilizando un alfiler fino de tungsteno 1, 2, 3, 4, 5. Un pasador de tungsteno es la más utilizada para montar la preparación en una orientación lateral, aunque se ha usado para montar larvas dorsal del lado hasta 4. El pegamento de sutura se ha usado para montar embriones y larvas en ambas orientaciones. Usando el pegamento, una disección mínima se puede realizar, permitiendo el acceso a las neuronas espinales sin el uso de un tratamiento enzimático, evitando así cualquier daño resultante. Sin embargo, para las larvas, es necesario aplicar un tratamiento enzimático breve para eliminar el tejido muscular que rodea la médula espinal. Los métodos descritos aquí se han utilizado para estudiar las propiedades eléctricas intrínsecas de las neuronas motoras, interneuronas, y las neuronas sensoriales en varios developmental etapas 6, 7, 8, 9.

Hemos registrado con éxito de las neuronas Rohon-barba en 17 hpf embriones a través de 7 dpf larvas (Figura 5A y 5B). Cuando se registraron células Rohon-barba, la preparación se monta en el lado dorsal hacia arriba. Dicho montaje permite la identificación inequívoca de células Rohon-Barba sobre la base de sus posiciones dorsal superficial y grandes tamaños soma. La identificación se confirmó adicionalmente por el potencial de membrana en repos...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

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

Este manuscrito describe métodos para registros electrofisiológicos de neuronas espinales de embriones de pez cebra y larvas. La preparación mantiene las neuronas in situ y a menudo implica la disección mínimo. Estos métodos permiten para el estudio electrofisiológico de una variedad de neuronas de la médula, de la adquisición inicial de la excitabilidad eléctrica a través de las primeras etapas larvales.

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 ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Research

JoVE Journal

Neuroscience

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

11.4K vistas.  University of Colorado Anschutz Medical Campus (UCAMC).  Este manuscrito describe métodos para registros electrofisiológicos de neuronas espinales de embriones de pez cebra y larvas. La preparación mantiene las neuronas in situ y a menudo implica la disección mínimo. Estos métodos permiten para el estudio electrofisiológico de una variedad de neuronas de la médula, de la adquisición inicial de la excitabilidad eléctrica a través de las primeras etapas larvales. 

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

La transección de la médula espinal en el pez cebra larval

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Un procedimiento para el implante Cámara Espinal para Longitudinal<em&gt; En Vivo</em&gt; Imágenes de la médula espinal de ratón

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Preparación de mezcla de culturas gliales primarios de ratón adulto espinal tejido del cordón

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Aislamiento y Cultura de Neuronas de la Médula Espinal de Ratones Neonatales

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imágenes de la actividad neuronal en la corteza somatosensorial primaria usando Thy1-ratones transgénicos GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

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

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 signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

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 ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Aislamiento y cultivo de las neuronas motoras oculomotoras, trocleas y espinales de Islmnprenatal :GFP Ratones transgénicos

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

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