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><li> Prepare baja Na + fluido cefalorraquídeo artificial (ACSF): KCl 3 mM, 1 mM NaH 2 PO 4, sacarosa 230 mM, 26 mM NaHCO3, 0,8 mM CaCl 2, 8 mM MgCl2, 25 mM de glucosa, ácido ascórbico 0,4 mM, 1 mM Na-quinurenato, 2 mM Na-piruvato. Burbuja con un 95% de O2 y 5% de CO 2 (pH 7,4). Desde Na-quinurenato es a menudo difícil de disolver, asegúrese de comprar la que aparece en la tabla de materiales. </li><li> Preparar Solu K-gluconatoción: 130 mM K-gluconato, mM KCl 15, EGTA 0,05 mM, HEPES 20 mM, glucosa 25 mM, 1 mM Na-quinurenato, 2 mM Na-piruvato, se ajustó a pH 7,4 con KOH. </li><li> Preparar ACSF: NaCl 130 mM, KCl 2,5 mM, 2 mM CaCl2, 1 mM MgCl2, 1 mM NaH 2 PO 4, 26 mM NaHCO 3, glucosa 25 mM, ácido ascórbico 0,4 mM, 2 mM Na-piruvato. Burbuja con un 95% de O2 y 5% de CO 2 (pH 7,4). </li><li> Antes del comienzo de la disección, se disuelven 2% de agar en 80 ml de la solución de K-gluconato, y mantener caliente a 60 ° C. </li></ol></li><li> perfusión intracardiaca <ol><li> Realizar esta preparación en ratones hembras y machos, con edades de P2 a P11. </li><li> Anestesiar al ratón con una inyección intraperitoneal de 0,1 ml de 25 mM de sodio pentobarbital (50 mg / kg). </li><li> El uso de agujas o cinta, se debe inmovilizar el ratón sobre su espalda en una gran placa de Petri llena de silicio. Use un microscop diseccióne para el resto de la disección. </li><li> Sujetando la punta del esternón, levantar el pecho y cortar el diafragma utilizando unas tijeras finas. A continuación, abrir el pecho en ambos lados por el corte a través de las costillas para exponer el corazón. </li><li> Cortar la aurícula derecha antes de la punción del ventrículo izquierdo con una aguja 27G. </li><li> Perfundir con helado de baja ACSF Na +. Después de 30 seg, bajo Na + ACSF debe considerarse que fluye de la aurícula. bajas cantidades de sodio evitar que las células de adición y reducir la muerte celular durante la disección. </li><li> Mantener la celebración de la aguja en el corazón bajo el microscopio de disección hasta que el hígado se vuelve amarillo cuando se drena la sangre. </li></ol></li><li> Disección de la médula espinal <ol><li> Decapitar al animal y lo puso en su estómago. </li><li> Eliminar rápidamente la piel de la espalda (Figura 1A1). Hacer dos cortes a través de los hombros y bajando la caja torácica (Figura 1A2). A continuación, corte el cable como low como sea posible en la sección de caudal con el fin de aislar la columna vertebral con el inicio de las nervaduras de la parte inferior del animal. Da la vuelta al animal de nuevo y retirar las vísceras todavía unido a las costillas. </li><li> Transferir la columna vertebral, a otra, más pequeña placa de Petri llena de silicio y el uso de 4 pines de insectos para sostenerlo hacia arriba dorsal (Figura 1A). </li><li> Continuamente perfundir el animal con ACSF carbógeno-burbujeó (a aproximadamente 4 ° C) mientras se realiza una laminectomía del lado dorsal, y la exposición de la médula espinal desde el extremo rostral (Figura 1B). Para ello, inserte la punta de tijeras finas entre el hueso y la médula espinal y cortar el hueso poco a poco desde el extremo rostral, asegurándose de mantenerse alejado de la sustancia blanca. Alternan en cada lado durante el uso de pinzas para mantener lejos de la banda del hueso ya cortada (Figura 1B1). </li><li> El uso de las tijeras de primavera disponibles más pequeños y pinzas, levante la dura y cortaen ambos lados mientras sostiene la parte suelta de la duramadre con el fin de evitar daños en la médula espinal con las tijeras. Corte a lo largo del eje rostro-caudal. NOTA: La duramadre es una membrana continua semi transparente; a esta edad la piamadre es demasiado frágil y se deshaga durante la disección y corte en rodajas (Figura 1B2). </li><li> Una vez que la duramadre es suprimido el uso de una copa roma o punta de plástico para empujar suavemente el cable en el lado izquierdo de la ranura formada por la columna vertebral mitad cortar, y cortar las raíces ventral y dorsal en el lado derecho, comenzando desde el lado rostral , más lejos de donde entran en el cable (unos pocos mm, al menos, la figura 1B2). </li><li> Repetir la operación en el lado izquierdo, siempre pasando de rostral a caudal. Si zurdo, comience desde el lado izquierdo y luego moverse hacia el lado derecho. </li></ol></li><li> Incorporación en agar <ol><li> Deslizar el cable fuera de la columna vertebral. Use un alfiler de insectos más pequeños de precisarel cordón con la superficie dorsal y quitar delicadamente cualquier pieza de membrana todavía unido a ella (Figura 1C1). </li><li> Una vez limpia, recortar ambos extremos (Figura 1C2). Inserte un alfiler de insectos doblada en la parte anterior de la cuerda para manipular el cable y tenga en cuenta su orientación (flecha en la figura 1C2). A continuación, la transferencia de la médula espinal a una solución de K-gluconato enfriado con hielo. NOTA: Esta solución imita la composición intracelular de la CSF y evitará que las células mueran de choque osmótico una vez que las motoneuronas se cortarán 26. </li><li> Una vez que la médula espinal se encuentra dentro de la solución intracelular, tomar el vaso de precipitados con el agar salir del baño seco y enfriarlo en una mezcla de hielo y agua. </li><li> Mantener la agitación mientras que la medición de la temperatura. Cuando la temperatura alcanza los 38 ° C sumergir la médula espinal, sujetándolo por el pasador de insectos y colocarlo lado rostral hacia abajo. Asegúrese de que la médula espinal es como straight posible, lejos de las paredes, parte caudal ligeramente hacia arriba (Figura 1D1). </li><li> Deje el vaso de precipitados en la mezcla de hielo y agua para permitir que el agar se solidifique lo más rápidamente posible. Asegúrese de que se mantiene en su lugar y que el cable es lo más recto posible (Figura 1D1). </li></ol></li><li> El rebanar <ol><li> Después de la solidificación, cortar el bloque de agar que contiene la médula espinal de una manera tal que la base del bloque es en un ángulo de 35 ° con la parte lumbar de la médula (flecha en la Figura 1D2). La superficie dorsal debe estar mirando hacia fuera de la base (Figura 1D2). NOTA: Este es un paso crítico en el procedimiento, para mantener la continuidad de las piscinas de las motoneuronas a las raíces ventrales de la que salen. </li><li> Pegar el bloque en la cámara de la vibratome utilizando pegamento de cianoacrilato. Sumergirla en una solución de K-gluconato y añadir solución de K-gluconato congelada para mantener slushedel baño de enfriado (por debajo de 2 ° C). </li><li> Cortar 350 - 400 micras rodajas gruesas de la región lumbar (identificable por la curvatura y el diámetro más grande). En su orientación correcta (véase 1.4.1.), Utilice la cuchilla para cortar desde el dorsal a la superficie ventral y hacer rodajas de forma continua más rostral. Típicamente, hay 4-5 rebanadas adecuados con raíces ventrales que se extienden de 2 mm o más. Utilice los siguientes parámetros: 10 ° de ángulo de referencia, la frecuencia de vibración de 70 Hz y 10 mm / min de velocidad de corte. </li></ol></li><li> Incubación <ol><li> Transferir las rebanadas de ACSF a 34 ° C. Después de aproximadamente 30 min, enfriar las rodajas a RT y comenzar la sesión de grabación. </li></ol></li></ol><img alt="Figura 1" src="/files/ftp_upload/54525/54525fig1.jpg" /> La figura 1. La disección A1:. La eliminación de la piel de la espalda para exponer la columna dorsal A2:El corte de los hombros y las costillas para liberar el dorsal columna B1:. La columna vertebral fijada en la placa de Petri de silicio-llenado (lado dorsal, de lado caudal izquierda) B2:. Lo mismo con la médula espinal expuesta y diseccionado C1:. Aislado de la médula espinal . (lado izquierdo rostral) C2: la médula espinal listo para ser incorporado (lado ventral hacia arriba, lado izquierdo rostral). Tenga en cuenta el pasador de insectos más pequeños en la parte rostral D1:.. De la médula espinal en el vaso de precipitados de agar (lado rostral Dow, lado ventral hacia la parte inferior) D2: bloque de agar Cortar con la médula espinal incrustado. Tenga en cuenta el ángulo de 35 ° las formas de la médula espinal con la base del bloque y la ampliación lumbar (Flecha). Las barras de escala 1 cm. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> 2. Colocar la rebanada en la Cámara NOTA: Ventral raíces son de tamaño variable. <ol><li> Prepare una caja de varias pipetas con diámetros de punta que van desde 40 hasta 170 micras con antelación. Para preparar las pipetas de aspiración, preparar muchas pipetas con una larga puesta a punto. Usando un cuchillo de diamante, hacer un corte en diferentes posiciones. Entonces bajo un microscopio de disección, se rompen por golpear la punta con unas pinzas. </li><li> Retire la cámara del microscopio grabación y colocarlo bajo un microscopio de disección. </li><li> Seleccionar un segmento que contiene una raíz ventral de longitud suficiente (2 mm o más) para ser montado en un electrodo de estimulación de succión. Seleccione la orientación adecuada de la rebanada con las raíces ventrales hacia arriba (figura 2A) y cortar delicadamente el agar alrededor de las raíces ventrales, dejando el resto del agar alrededor de la rebanada (Figura 2B). NOTA: Debido a que el agar es más firme que la rodaja, esto permitirá que los hilos de anclaje de la rebanada para descansar en el agar en lugar de en la rebanada y por lo tanto la unanchor no dañará el tejido. Asegúrese de que los hilos de anclaje de la rebanada están por encima de la piscina de las neuronas motoras (se muestra en rojo en la figura 2C). </li><li> Montar la cámara de nuevo en el microscopio y perfundir continuamente la cámara de registro con ACSF a una velocidad de 1-2 ml / min, a temperatura ambiente. Usando una pipeta de vidrio lleno de ACSF y conectada a una jeringa, chupar uno de la raíz ventral (flecha en la Figura 2C). Con el fin de lograr una buena estimulación de la raíz ventral, la punta de la pipeta tiene que ser apretado alrededor de la raíz ventral. Un polo debe estar en el electrodo de estimulación y el otro en el baño (o conectado a la referencia de electrodo de conexión de sujeción). </li><li> Lograr la grabación de patch-clamp del tipo de célula deseado y registrar el efecto de la estimulación de la raíz ventral como se describe previsouly 10. <ol><li> Aquí, utilizar un amplificador para la adquisición de datos. Filtrar registros de células completas a las 3 kHz. Digitalizar a 10 kHz. Compensar la resistencia puente en curremodo nt-clamp. </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/54525/54525fig2.jpg" /> Figura 2. ventral Root Preparación R: Lumbar rebanada de la médula espinal incrustado en agar con la raíz ventral hacia arriba. B: Lumbar rebanada de la médula espinal con las raíces ventrales liberados del agar C:. Rebanada de la médula espinal lumbar con un electrodo de estimulación firmemente colocado alrededor de las raíces ventrales (flecha) . Tenga en cuenta la ubicación de las células de Renshaw que expresan fluorescencia roja en el 28 chrna2-Cre ratón cruzado con el reportero del ratón R26 Tom 17. Las barras de escala 1 mm. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

<ol>
	<li>Brooks, C. M., Downman, C. B., Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After-potentials+and+excitability+of+spinal+motoneurones+following+antidromic+activation.">After-potentials and excitability of spinal motoneurones following antidromic activation.</a> J Neurophysiol. 13 (1), 9-38 (1950).</li><li>Bories, C., Amendola, J., Lamotte d'Incamps, B., Durand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+electrophysiological+abnormalities+in+lumbar+motoneurons+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Early electrophysiological abnormalities in lumbar motoneurons in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Eur J Neurosci. 25 (2), 451-459 (2007).</li><li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+currents+in+visually+identified+motoneurones+of+neonatal+rat+spinal+cord.">Membrane currents in visually identified motoneurones of neonatal rat spinal cord.</a> J Physiol. 423, 27-46 (1990).</li><li>Hori, N., Tan, Y., Strominger, N. L., Carpenter, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+activity+of+rat+spinal+cord+motoneurons+in+slices.">Intracellular activity of rat spinal cord motoneurons in slices.</a> J Neurosci Methods. 112 (2), 185-191 (2001).</li><li>Arai, Y., Mentis, G. Z., Wu, J. Y., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventrolateral+origin+of+each+cycle+of+rhythmic+activity+generated+by+the+spinal+cord+of+the+chick+embryo.">Ventrolateral origin of each cycle of rhythmic activity generated by the spinal cord of the chick embryo.</a> PLoS One. 2 (5), e417 (2007).</li><li>Cullheim, S., Lipsenthal, L., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+monosynaptic+contacts+between+type-identified+alpha-motoneurons+in+the+cat.">Direct monosynaptic contacts between type-identified alpha-motoneurons in the cat.</a> Brain Res. 308 (1), 196-199 (1984).</li><li>Cullheim, S., Kellerth, J. O., Conradi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+direct+synaptic+interconnections+between+cat+spinal+alpha-motoneurons+via+the+recurrent+axon+collaterals:+a+morphological+study+using+intracellular+injection+of+horseradish+peroxidase.">Evidence for direct synaptic interconnections between cat spinal alpha-motoneurons via the recurrent axon collaterals: a morphological study using intracellular injection of horseradish peroxidase.</a> Brain Res. 132 (1), 1-10 (1977).</li><li>Gogan, P., Gueritaud, J. P., Horcholle-Bossavit, G., Tyc-Dumont, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+excitatory+interactions+between+spinal+motoneurones+of+the+cat.">Direct excitatory interactions between spinal motoneurones of the cat.</a> J Physiol. 272 (3), 755-767 (1977).</li><li>Ichinose, T., Miyata, Y. <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+of+motoneurons+in+the+isolated+spinal+cord+of+newborn+rats+detected+by+whole-cell+recording.">Recurrent excitation of motoneurons in the isolated spinal cord of newborn rats detected by whole-cell recording.</a> Neurosci Res. 31 (3), 179-187 (1998).</li><li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+excitatory+postsynaptic+ionotropic+receptors+coactivated+at+the+motoneuron-Renshaw+cell+synapse.">Four excitatory postsynaptic ionotropic receptors coactivated at the motoneuron-Renshaw cell synapse.</a> J Neurosci. 28 (52), 14121-14131 (2008).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+centripetal+impulses+in+axons+of+spinal+ventral+roots.">Central effects of centripetal impulses in axons of spinal ventral roots.</a> J Neurophysiol. 9, 191-204 (1946).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+nerve+impulses+in+the+gray+matter+as+a+mechanism+in+central+inhibition.">Interaction of nerve impulses in the gray matter as a mechanism in central inhibition.</a> Fed Proc. 5 (1 Pt 2), 86 (1946).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+interaction+of+nerve+impulses+in+the+gray+matter+and+on+the+nature+of+central+inhibition).">Observations on interaction of nerve impulses in the gray matter and on the nature of central inhibition).</a> Am J Physiol. 146, 443-448 (1946).</li><li>Pambo-Pambo, A., Durand, J., Gueritaud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+excitability+changes+in+lumbar+motoneurons+of+transgenic+SOD1G85R+and+SOD1G(93A-Low)+mice.">Early excitability changes in lumbar motoneurons of transgenic SOD1G85R and SOD1G(93A-Low) mice.</a> J Neurophysiol. 102 (6), 3627-3642 (2009).</li><li>Quinlan, K. A., Schuster, J. E., Fu, R., Siddique, T., 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=Altered+postnatal+maturation+of+electrical+properties+in+spinal+motoneurons+in+a+mouse+model+of+amyotrophic+lateral+sclerosis.">Altered postnatal maturation of electrical properties in spinal motoneurons in a mouse model of amyotrophic lateral sclerosis.</a> J Physiol. 589 (Pt 9), 2245-2260 (2011).</li><li>Martin, E., Cazenave, W., Cattaert, D., Branchereau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+alteration+of+motoneuronal+morphology+induces+hyperexcitability+in+the+mouse+model+of+amyotrophic+lateral+sclerosis.">Embryonic alteration of motoneuronal morphology induces hyperexcitability in the mouse model of amyotrophic lateral sclerosis.</a> Neurobiol Dis. 54, 116-126 (2013).</li><li>Hadzipasic, 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=Selective+degeneration+of+a+physiological+subtype+of+spinal+motor+neuron+in+mice+with+SOD1-linked+ALS.">Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS.</a> Proc Natl Acad Sci U S A. 111 (47), 16883-16888 (2014).</li><li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li><li>Tallini, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenic+mice+express+enhanced+green+fluorescent+protein+in+central+and+peripheral+cholinergic+neurons.">BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.</a> Physiol Genomics. 27 (3), 391-397 (2006).</li><li>Manuel, 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=Fast+kinetics,+high-frequency+oscillations,+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons.">Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons.</a> J Neurosci. 29 (36), 11246-11256 (2009).</li><li>Obeidat, A. Z., Nardelli, P., Powers, R. K., Cope, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+motoneuron+firing+by+recurrent+inhibition+in+the+adult+rat+in+vivo.">Modulation of motoneuron firing by recurrent inhibition in the adult rat in vivo.</a> J Neurophysiol. 112 (9), 2302-2315 (2014).</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, (2014).</li><li>Leroy, F., Lamotte d'Incamps, B., 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=Potassium+currents+dynamically+set+the+recruitment+and+firing+properties+of+F-type+motoneurons+in+neonatal+mice.">Potassium currents dynamically set the recruitment and firing properties of F-type motoneurons in neonatal mice.</a> J Neurophysiol. 114 (3), 1963-1973 (2015).</li><li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+composition+and+kinetics+of+the+Renshaw+cell+heteromeric+nicotinic+receptors.">Subunit composition and kinetics of the Renshaw cell heteromeric nicotinic receptors.</a> Biochem Pharmacol. 86 (8), 1114-1121 (2013).</li><li>Lamotte d'Incamps, B., Krejci, E., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+shaping+the+slow+nicotinic+synaptic+current+at+the+motoneuron-renshaw+cell+synapse.">Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.</a> J Neurosci. 32 (24), 8413-8423 (2012).</li><li>Dugue, G. P., Dumoulin, A., Triller, A., Dieudonne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+use+of+co-released+inhibitory+transmitters+at+central+synapses.">Target-dependent use of co-released inhibitory transmitters at central synapses.</a> J Neurosci. 25 (28), 6490-6498 (2005).</li><li>Mentis, G. Z., Siembab, V. C., Zerda, R., O'Donovan, M. J., Alvarez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+afferent+synapses+on+developing+and+adult+Renshaw+cells.">Primary afferent synapses on developing and adult Renshaw cells.</a> J Neurosci. 26 (51), 13297-13310 (2006).</li><li>Perry, S., 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=Firing+properties+of+Renshaw+cells+defined+by+Chrna2+are+modulated+by+hyperpolarizing+and+small+conductance+ion+currents+Ih+and+ISK.">Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.</a> Eur J Neurosci. 41 (7), 889-900 (2015).</li><li>Thurbon, D., Luscher, H. R., Hofstetter, T., Redman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+electrical+properties+of+ventral+horn+neurons+in+rat+spinal+cord+slices.">Passive electrical properties of ventral horn neurons in rat spinal cord slices.</a> J Neurophysiol. 79 (5), 2485-2502 (1998).</li><li>Zengel, J. E., Reid, S. A., Sypert, G. W., Munson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electrical+properties+and+prediction+of+motor-unit+type+of+medial+gastrocnemius+motoneurons+in+the+cat.">Membrane electrical properties and prediction of motor-unit type of medial gastrocnemius motoneurons in the cat.</a> J Neurophysiol. 53 (5), 1323-1344 (1985).</li><li>Cooper, S., Sherington, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gower's+tract+and+spinal+border+cells.">Gower's tract and spinal border cells.</a> Brain. 63, 123-124 (1940).</li><li>Morin, F., Schwartz, H. G., O'Leary, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+the+spinothalamic+and+related+tracts.">Experimental study of the spinothalamic and related tracts.</a> Acta Psychiatr Neurol Scand. 26 (3-4), 371-396 (1951).</li><li>Sengul, G., Fu, Y., Yu, Y., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+projections+to+the+cerebellum+in+the+mouse.">Spinal cord projections to the cerebellum in the mouse.</a> Brain Struct Funct. 220 (5), 2997-3009 (2015).</li><li>Russier, M., Carlier, E., Ankri, N., Fronzaroli, L., Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-,+T-,+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons.">A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.</a> J Physiol. 549 (Pt 1), 21-36 (2003).</li><li>Dourado, M., Sargent, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord).">Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord).</a> J Neurophysiol. 87 (6), 3117-3125 (2002).</li><li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse.">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> J Neurophysiol. 107 (2), 728-741 (2012).</li><li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx.">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> J Neurosci. 5 (6), 1483-1489 (1985).</li><li>Olney, J. W., Price, M. T., Samson, L., Labruyere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+specific+ions+in+glutamate+neurotoxicity.">The role of specific ions in glutamate neurotoxicity.</a> Neurosci Lett. 65 (1), 65-71 (1986).</li></ol>

The authors have nothing to disclose.

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>
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			<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-preparation-oblique-spinal-cord-slices-for-ventral-root

Research

JoVE Journal

Neuroscience

9.4K vistas.  Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes.  Mostramos cómo preparar rodajas oblicuas de la médula espinal en ratones jóvenes. Esta preparación permite la estimulación de las raíces ventrales. 

Video: The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

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

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

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

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

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

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

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 ...

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 ...

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 ...

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

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 result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

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, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

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

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

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 ...

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 ...

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

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