Skidmore Facultad Donaciones para el Desarrollo financió la preparación y publicación de este manuscrito.

1. Montaje de los embriones (al término de técnica de visualización seleccionado, como la inmunocitoquímica, hibridación in situ, etc) <ol><li> Coloque los embriones de un plato Petri de 35 mm llena con tampón de elección (Figura 1A). La elección de tampón se basa en el procedimiento de etiquetado. Típicamente tampones basados ​​en PBS son adecuados. </li><li> Bajo un microscopio de disección, y el uso de fórceps, asegurar la cabeza con un par de pinzas mientras tira de la yema de distancia con el otro par (Figura 1B). Alternativamente, use un alfiler de insectos en un soporte de pasador de insectos para tirar suavemente la yema. </li><li> Usando el poker embrión (línea de pesca (0,41 mm de diámetro) pegado a cualquiera de un tubo capilar o una pipeta Pasteur) mover los embriones a una parte de la placa de Petri que no tiene una gran cantidad de restos de yema (figuras 1C-D). </li><li> Utilizando una pipeta Pasteur de vidrio, aspirar los embriones en tan poco líquido como sea posible, y gtemente pipetear ellos en un portaobjetos (Figura 1E). </li><li> Suavemente absorber y expulsar el exceso de líquido utilizando un laboratorio toallita. Evite el contacto con los embriones. </li><li> Añadir una gota de medio de montaje a los embriones. Para etiquetas fluorescentes, utilice un reactivo Antifade de elección. Para señales colorimétricas, 70% de glicerol se puede utilizar como un medio de montaje. </li><li> Usando el poker embrión, oriente los embriones en sus lados en filas (Figura 1F). </li><li> Añadir una &quot;pizca&quot; de vaselina o grasa de alto vacío a cada esquina de un cubreobjetos (Figura 1G). Esto evita el daño del embrión a partir de una compresión excesiva. </li><li> Coloque el cubreobjetos (lado vaselina hacia abajo) en la parte superior de los embriones (Figura 1H). </li><li> Golpear suavemente el cubreobjetos en cada esquina hasta que los medios de montaje (70% de glicerol o reactivo de montaje anti-fade) está tocando el cubreobjetos (Figura 1H). </li><li> Si es necesario, agregar material de montaje con la pipeta justo al lado del coverslip, usando una pipeta en el intervalo de 20-200 ml. Se mecha bajo. </li><li> El uso de un laboratorio de limpiar, limpiar completamente todo el cubreobjetos. Asegúrese de no aplicar presión en el cubreobjetos, ya que podría dañar los embriones. Esta área debe estar seca y no tienen vaselina o medio de montaje sobre el mismo. </li><li> Utilice el esmalte de uñas para sellar alrededor del borde del cubreobjetos (Figura 1I). </li></ol>

<ol>
	<li>Gribble, S. L., Nikolaus, O. B., Dorsky, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+Dbx+genes+in+the+zebrafish+spinal.">Regulation and function of Dbx genes in the zebrafish spinal.</a> Dev. Dyn. 236 (12), 3472-3483 (2007).</li><li>Bonner, J., 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=Midline+crossing+is+not+required+for+subsequent+pathfinding+decisions+in+commissural+neurons.">Midline crossing is not required for subsequent pathfinding decisions in commissural neurons.</a> Neural Dev. 7 (1), 18 (2012).</li><li>Ross, A. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Wnt+signaling+using+Lithium+Chloride:+Inquiry-Based+Undergraduate+Laboratory+Exercises.">Activation of Wnt signaling using Lithium Chloride: Inquiry-Based Undergraduate Laboratory Exercises.</a> Zebrafish. , (2012).</li><li>Bonner, J., 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=Proliferation+and+patterning+are+mediated+independently+in+the+dorsal+spinal+cord+downstream+of+canonical+Wnt+signaling.">Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.</a> Dev. Biol. 313 (1), 398-407 (2008).</li><li>Gribble, S. L., 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=Tcf3+inhibits+spinal+cord+neurogenesis+by+regulating+sox4a+expression.">Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression.</a> Development. 136 (5), 781-789 (2009).</li><li>England, 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=Roles+of+Hedgehog+pathway+components+and+retinoic+acid+signalling+in+specifying+zebrafish+ventral+spinal+cord+neurons.">Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons.</a> Development. 138 (23), 5121-5134 (2011).</li><li>Challa, A. K., Beattie, C. E., Seeger, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+roundabout+orthologs+in+zebrafish.">Identification and characterization of roundabout orthologs in zebrafish.</a> Mech Dev. (1-2), 101-101 (2001).</li><li>Lee, J. S., Ray, R., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+three+zebrafish+roundabout+homologs+suggest+roles+in+axon+guidance+and+cell.">Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell.</a> 221 (2), 216-230 (2001).</li><li>Downes, G. B., Waterbury, J. A., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+labeling+of+identified+zebrafish+neurons.">Rapid in vivo labeling of identified zebrafish neurons.</a> Genesis. 34 (3), 196-202 (2002).</li><li>Kim, H., 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=Notch-regulated+oligodendrocyte+specification+from+radial+glia+in+the+spinal+cord+of+zebrafish+embryos.">Notch-regulated oligodendrocyte specification from radial glia in the spinal cord of zebrafish embryos.</a> Dev. Dyn. 237 (8), 2081-2089 (2008).</li><li>Sylvain, N. J., Brewster, D. L., Ali, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+exposed+to+alcohol+undergo+abnormal+development+of+motor+neurons+and+muscle+fibers.">Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.</a> Neurotoxicol. Teratol. 32 (4), 472-480 (2010).</li><li>de Soysa, T. Y., 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=Macondo+crude+oil+from+the+Deepwater+Horizon+oil+spill+disrupts+specific+developmental+processes+during+zebrafish+embryogenesis.">Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis.</a> BMC Biol. 10 (40), (2012).</li><li>Langenberg, T., Brand, M., Cooper, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+development+and+organogenesis+in+zebrafish+using+immobilized+embryonic+explants.">Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.</a> Dev Dyn. 228 (3), 464-474 (2003).</li><li>Deschene, E. R., Barresi, 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=Tissue+Targeted+Embryonic+Chimeras:+Zebrafish+Gastrula+Cell+Transplantation.">Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation.</a> J. Vis. Exp. (31), (2009).</li></ol>

No existen intereses económicos que compiten por cualquiera de autor.

Montaje lateral del pez cebra fija ayudas embriones visualización del desarrollo de la médula espinal. A través de la eliminación de la pelota yema de huevo, se reduce la distancia de trabajo requerido para imágenes microscópicas excepcional. Nuestros resultados representativos se limitan a la etapa 24 HPF, sin embargo, esta técnica se puede utilizar tan pronto como 18 HPF, aunque embriones anteriores son más difíciles de diseccionar debido al tamaño del embrión y la fragilidad del tejido. Esta técnica también es aplicable a los embriones de más de 24 HPF. Ayudado por la claridad óptica de la embriones de pez cebra, la capacidad de realizar pantallas adelante genéticos, genética inversa, y enfoques transgénicos, varios procesos de desarrollo pueden ser investigados. Esto incluye los índices mitóticos de progenitores neuronales con marcadores como BrdU y anti-fosfato histona marzo 4 a 6 y los patrones a través de la expresión de marcadores progenitoras neuronales y gliales en el pax, nkx, dbx, y familie oligs 1,4-6. Los reactivos que reportan determinación glial y neuronal (como la proteína glial fibrilar ácida (GFAP) y HuC / D) 1,4,5,10 se puede utilizar. Diferenciación subtipo neuronal puede ser analizada a través de la expresión de los islotes, vsx, engrailed y genes gata 1,4-6. Y, por último, el análisis de la orientación axón es posible a través de anticuerpos tales como anti-tubulina acetilada, 3A10 y ZNP-1 2,11,12. Aunque otros sistemas modelo de vertebrados (ratón y pollo) se utilizan para estudiar el desarrollo de la médula espinal, sólo el permiso de pez cebra visualización simultánea de todos los ejes de desarrollo en el embrión intacto. La limitación obvia de este enfoque es que los embriones se fijan, que excluye de imágenes en vivo. De hecho, la eliminación de (o daño a) los resultados de yema de huevo en la rápida muerte embrionaria, por lo que no es posible reducir la distancia de trabajo en embriones vivos utilizando esta técnica. Sin embargo, una gran ampliación de la médula coimágenes rd en embriones vivos es posible. Con el fin de preservar la yema durante de imágenes en vivo, los embriones se pueden colocar en las diapositivas de depresión, que proporcionan un espacio más grande entre la muestra y el cubreobjetos. Alternativamente, varios cubreobjetos de 22 mm x 22 mm se pueden pegar a cada diapositiva de un 3 en 1 x en portaobjetos de microscopio. Los cubreobjetos sirven como un &quot;puente&quot; para el cubreobjetos encima. Si los embriones son mayores de 18 HPF, tricaína se utiliza para anestesiar a los embriones para evitar el movimiento. Si bien se requiere una distancia de trabajo de 0,288 mm para los embriones deyolked en 24 HPF, cuando se trabaja con embriones vivos incrustados en agarosa, con yemas intactas, una lente microscópica con una distancia de trabajo de 3,3 mm es suficiente. Alternativamente, cultivos de explantes derivados a partir de embriones deyolked pueden ser visualizados utilizando una técnica de inmovilización coágulo de plasma 13. Varias poblaciones de células se pueden observar a través del uso de proteínas fluorescentes codificadas genéticamente, tales como GFP, mCherry, o el fotoconductortinte convertibles Kaede (por nombrar algunos). Además, las células marcadas con fluorescencia dentro de embriones quiméricos también se pueden ver con este enfoque 14. Una técnica de montaje consistente que es estable durante meses es una herramienta crítica para los biólogos celulares y de desarrollo. Esta técnica sencilla y reproducible, lo que permite una fácil comparación entre los diferentes ensayos experimentales. Además, la alineación de los embriones en filas permite fácilmente la identificación de los embriones específicos para su posterior análisis.

Visualización de la médula espinal en el pez cebra se inhibe por un número de factores. Debido al espesor de los somitas suprayacentes y la ubicación interna de la médula espinal, se requiere una distancia considerablemente larga de trabajo para alta resolución celular. El balón ha yema (que todavía está presente durante las primeras etapas de la neurogénesis) aumenta aún más la distancia de trabajo necesario, y se daña fácilmente con la presión de un cubreobjetos. Además, los restos de yema dañado prohíbe la visualización clara de los tejidos. Aunque son posibles secciones transversales en el eje (DV)-dorso-ventral, que no permiten fácilmente la visualización simultánea en el eje antero-posterior (AP) 1. Para superar estos obstáculos, los embriones se disecan y se montaron en portaobjetos. Este procedimiento proporciona varias ventajas. En primer lugar, embriones de pez cebra se encuentran fácilmente con la cara lateral hacia arriba, lo que facilita la visualización eje AP. En segundo lugar, la eliminación de la yema de BALl disminuye la distancia de trabajo requerido, y limita los escombros. En tercer lugar, este procedimiento de montaje permite por tanto fluorescente y microscopía de campo claro. En cuarto lugar, los embriones montados son estables durante meses a 4 ° C, lo que permite la visualización espécimen prolongado. Por último, la progresión del desarrollo se da en que los segmentos anteriores son más maduros que los segmentos posteriores. Con el fin de asegurar que por etapas hemisegments espinales emparejados se comparan entre embriones, que recubre el tejido somite se utiliza como una guía. Por ejemplo, la décima somite más posterior se superpone a la décima hemisegment espinal, que es equivalente de desarrollo en embriones de etapa con ajuste. Este procedimiento de montaje de los embriones intactos permite una fácil identificación de los somitas. Utilizamos la genética y genes tecnologías knock-down para estudiar los mecanismos de guiado de los axones en la médula espinal en desarrollo. En particular, se ha determinado que se requieren robo2, ROBO3 y DCC para comisural Asce PrimariaNding (COPA) pathfinding axón. Usando el procedimiento de montaje descrito aquí, hemos sido capaces de examinar el crecimiento ventral, cruce la línea media, ancho de comisura, dorsal y anterior 2,3 de crecimiento. Este procedimiento de montaje también se puede aplicar a DV o AP patrón de la médula espinal. Hemos utilizado este procedimiento para determinar los papeles dispares de efectores aguas abajo de Wnt en DV patrón de la médula espinal. El uso de este montaje, hemos sido capaces de obtener la resolución de los marcadores de DV en el nivel de células individuales, y determinar los cambios de modelado minutos como consecuencia de la alteración de recepción de señales Wnt 4,5. Este procedimiento de montaje también permite el cálculo del índice mitótico a través de anti-fosfohistona 3 o etiquetado BrdU (proliferación de progenitores) 5 diferenciación.

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			Petri dishes 35mm X 10mm
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			25373-041
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			12-541-B22X22-1.5
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			P36934
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			SZ61
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dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development

La médula espinal pez cebra es un modelo de investigación eficaz para la investigación del sistema nervioso por varias razones. En primer lugar, genética, transgénicos y de genes enfoques desmontables pueden ser utilizados para estudiar los mecanismos moleculares que subyacen desarrollo del sistema nervioso. En segundo lugar, las grandes garras de embriones de desarrollo sincronizados proporcionan grandes tamaños de muestras experimentales. En tercer lugar, la claridad óptica del embrión de pez cebra permite a los investigadores visualizar progenitoras, gliales, y poblaciones neuronales. Aunque los embriones de pez cebra son transparentes, espesor de la muestra puede impedir la visualización microscópica efectiva. Una razón para esto es el desarrollo en tándem de la médula espinal y el tejido somite suprayacente. Otra razón es la bola yema grande, que todavía está presente durante los períodos de la neurogénesis temprana. En este artículo, nos demuestran microdisección y la eliminación de la yema de embriones fijos, lo que permite la visualización microscópica, preservando el tejido circundante somito. Nosotros also demostrar montaje semipermanente de embriones de pez cebra. Esto permite la observación de neurodesarrollo en los ejes dorso-ventral y anterior-posterior, ya que conserva la tridimensionalidad del tejido.

Cuando elucidar los mecanismos que subyacen a diversos eventos de desarrollo tales como patrón celular, la diferenciación, y guía de axones, es importante ser capaz de visualizar las células en el contexto de su tejido. Defectos dorsoventral de modelado típicamente se presentan como un desplazamiento ventral o dorsal en el dominio de la expresión de factores de transcripción en el pax, nkx o familias dbx 4. A veces, los cambios pueden ser sutiles, que comprende sólo unos diámetros celulares 4. Análisis de corte transversal permite este tipo de análisis. Sin embargo, reactivos con las señales más débiles, o secciones transversales que no son perpendiculares al eje de AP pueden limitar la interpretación. Además, el análisis de la sección transversal no toma en cuenta fácilmente si los cambios persisten en el eje AP, y se basa en la disponibilidad de un criostato. Esta limitación se elude mediante vistas laterales de la médula espinal. Como se observa en la Figura 2B, Nkx6.1 ARNm es dISTRIBUIDO en aproximadamente la mitad ventral de la médula espinal en 24 horas después de la fecundación (HPF), dentro del dominio progenitora. Las células progenitoras son distinguibles de las células mitóticas mensaje debido a la presencia de la placa de piso, que se encuentra en la parte medial de la médula espinal. Visto con la óptica DIC, las células individuales son claramente distinguibles, y un límite definido entre la expresión y nonexpressing células es evidente. La salida del ciclo de células progenitoras se acompaña de cambios en la expresión génica. Por ejemplo, todas las neuronas post-mitóticas expresan HuC / D, que es detectable con 1,5 inmunocitoquímica. sondas de ARNm para los islotes, gata, y la familia VSX etiquetar las neuronas post-mitóticas específicos de posición coherente y número dentro de cada hemisegment médula espinal 6. Además, los receptores de orientación axón tales como los de la familia de robo se expresan en poblaciones restringidas de neuronas postmitóticas (Figura 2C) <sup&gt; 2,7,8. Montaje lateral de embriones permite conteos precisos de las neuronas post-mitóticas. Del mismo modo, los progenitores que continúan dividiendo pueden cuantificarse con anti-fosfato histona 3 inmunocitoquímica, así como el etiquetado BrdU. Además, la muerte celular se puede evaluar con TUNEL etiquetado 4-6. A las 24 HPF, los axones de las neuronas siguientes postmitóticas pueden visualizarse con varios métodos, y se distinguen en el único nivel celular: Dorsal Lateral Ascendente (Dola), comisurales Primaria ascendente (COPA), comisurales Secondary ascendente (COSA), ventral longitudinal Descendente (Veld), Kolmer-Agdur (KA), comisurales bifurcar / longitudinales (COB / L), circunferencial Ascendente (CIA), circunferencial Descendente (CID), y Unipolar comisural Descendente (CM subyacente), Rohon-Barba (RB), las neuronas motoras ( M) 9. Los axones se extienden desde estas neuronas en la parte dorsal, ventral, anterior y posterior direcciones. Ellos rama, cruzan la línea media, y salen tanto de la dorsaly ventral de la médula espinal. Cuando se combina con microscopía confocal de barrido láser, estos diversos comportamientos celulares son evidentes en los embriones montados lateralmente, usando inmunocitoquímica y proteínas fluorescentes codificadas genéticamente, tales como GFP 2. En la Figura 2D, se utilizó ZNP-1 inmunocitoquímica para etiquetar motoneuronas en desarrollo. Motoneuronas salen de la médula espinal ventral para inervar la musculatura que rodea el desarrollo. <img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50703/50703fig1highres.jpg" src="/files/ftp_upload/50703/50703fig1.jpg" width="500px" /> Figura 1. La disección y lateral de montaje de embriones de pez cebra. (A) Después del procesamiento para inmunofluorescencia, hibridación in situ, etc, los embriones se colocan en un plato de Petri de 35 mm llena de PBT (PBS con 0,5% de Triton X-100) o PTW (PBS con 0,1% de Tween-20). La yemabola (YB) es evidente. (B) La yema se separa por asegurar la cabeza del embrión seguida de disección cuidadosa. (C) Un póquer embrión (línea de pesca pegado a una pipeta Pasteur) se utiliza para embriones separados y escombros yema de (D). Embriones limpiados se pipetean sobre un portaobjetos de microscopio (E) y alineados (F). Jalea (G) Petróleo se aplica a las esquinas de la cubreobjetos. (H) El cubreobjetos se coloca suavemente en la parte superior de los embriones en medio de montaje. Polaco (I) Nail se utiliza para sellar los bordes del cubreobjetos. <a href="https://www.jove.com/files/ftp_upload/50703/50703fig1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande</a> . <img alt="Figura 2" fo:content-width="3.5in" fo:src="/files/ftp_upload/50703/50703fig2highres.jpg" src="/files/ftp_upload/50703/50703fig2.jpg" width="500px" /> &lt;<html> strong&gt; Figura 2. Análisis de los patrones de expresión de genes y axón pathfinding en montado lateralmente embriones de pez cebra. (A) Dibujo de un embrión de pez cebra en 24 HPF. En BD, aproximadamente cuatro hemisegments por encima de la extensión del saco vitelino se visualizan (área del recuadro). (B) Nkx6.1 se expresa en la médula espinal ventral, incluyendo la placa de piso (FP). (C) ROBO3 se expresa en las neuronas postmitóticas (PMN). La placa de suelo y progenitor zona no es evidente en este plano focal más lateral. En B, C, la médula espinal está delimitado por un soporte en la parte izquierda de la imagen. (D) microscopía confocal se utilizó para la imagen ZNP-1 de inmunofluorescencia, que las etiquetas de los axones motores que salen de la médula espinal ventral. En todas las imágenes, es anterior a la izquierda y dorsal es hacia arriba. A larga distancia de trabajo 40x, la inmersión en agua (NA 0.8) lente se utilizó (3,3 mm).oad/50703/50703fig2highres.jpg &quot;target =&quot; _blank &quot;&gt; Haga clic aquí para ver la imagen más grande.

Watch this Scientific Journal Video about Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development at JoVE.com

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

Los procesos de desarrollo tales como la proliferación, el patrón, la diferenciación, y guía de axones se pueden modelar fácilmente en la médula espinal pez cebra. En este artículo se describe un procedimiento de montaje para los embriones de pez cebra, que optimiza la visualización de estos eventos.

La disección y de montaje lateral de embriones de pez cebra: Análisis para el Desarrollo de la médula espinal

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

dissection-lateral-mounting-zebrafish-embryos-analysis-spinal-cord

Research

JoVE Journal

Neuroscience

13.7K vistas.  Skidmore College.  Los procesos de desarrollo tales como la proliferación, el patrón, la diferenciación, y guía de axones se pueden modelar fácilmente en la médula espinal pez cebra. En este artículo se describe un procedimiento de montaje para los embriones de pez cebra, que optimiza la visualización de estos eventos. 

Video: Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Percusión fluido lateral: Modelo de Lesión Cerebral Traumática en ratones

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

La disección del ganglio y Cultura polluelo Statoacoustic y explantes médula espinal en geles de colágeno para ensayos neurite

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

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 ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Lesiones de la Médula Espinal completo y Protocolo Disección del cerebro por Wholemount posterior<em&gt; In Situ</em&gt; La hibridación en larvas lamprea de mar

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Análisis de Expresión diferencial de genes en electroporación de embriones de pollo de la médula espinal

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

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 ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Aislamiento de núcleos de adultos de la médula espinal para secuenciación masivamente paralela solo núcleo ARN

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

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

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