Los autores desean agradecer a Bob Freeman para la formación y el laboratorio de Kirschner para la utilización de las instalaciones de rana, y los miembros del laboratorio Van Vactor para el apoyo. Damos las gracias a la Nikon Imaging Center de la Harvard Medical School de asistencia con microscopía de luz para las imágenes de la figura 1. Este trabajo fue financiado por el texto siguiente: NRSA NIH NIH K99 comunión y compañerismo con LAL, Ciencias Básicas Asociación financiación ( <a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a> ) a AEF, y NIH RO1 NS035909 para DVV

Nota: Se describen los pasos en las dos primeras secciones sólo en pocas palabras, como los protocolos excelentes con información detallada de haber sido publicada antes que se centran más concretamente en los siguientes pasos (por ejemplo, 8-12). Además, el protocolo general de colaboración con las neuronas espinales en Xenopus cultivo de células en vivo ha sido publicado previamente en un artículo de métodos detallados 8. Le recomendamos revisar ese artículo como complemento de este video, aunque aquí no proporcionan suficiente información para llevar a cabo con éxito y solucionar problemas de la disección del tubo neural y el protocolo de siembra en el paso 3. 1. Fertilización In Vitro de Xenopus huevos <ol><li> Obtener huevos de ranas hembra que fueron inyectados con gonadotropina coriónica (400 unidades / rana) 12-18 horas antes de la recolección de huevos. Recoger los huevos en 1X solución de Marc Ringer modificado (MMR) (0,1 M NaCl, 2,0 mM KCl, 1,0 mM MgSO 4, 2,0 mM CaCl 2, 5,0 mM de HEPES, pH 7,4 9). </li><li> Fertilizar los óvulos in vitro con testículos picada, como se describe anteriormente 9. </li><li> Después de al menos 20 min, eliminar embrión capa gelatinosa por incubación de embriones en 2% de cisteína en 1X MMR (se llevó a pH 7,8 con NaOH) durante 3-5 min. Lavar con 0,1 X MMR (o 0,1 X. MBS (Modificado salina de Barth, receta 1X: 88 mM NaCl, 1 mM de KCl, 0,7 mM CaCl 2, 1 mM MgSO 4, 2,5 mM NaHCO 3, 5 mM de HEPES, pH 7,8)) 3 embriones -5 horas y mantienen a temperatura ambiente hasta que las inyecciones, o si se desea, los embriones lugar a 14-18 ° C para retardar el desarrollo. </li></ol> 2. La microinyección de ARN Nota: mientras que nosotros utilizamos inyección de ARN aquí, estas técnicas no se limitan al ARN, y ADN, las proteínas o ácidos nucleicos modificados para la manipulación de genes también se pueden usar y se han descrito previamente 12. <ol><li> Antes del experimento, los ARN de etiquetado de las estructuras del citoesqueleto u otro are transcribe a partir de moldes de ADN linealizados utilizando el kit mMessage mMachine (Ambion). mRNAs requiere un 5 &#39;tapa y un 3&#39; cola de poliadenilación para promover la traducción y prevenir la degradación en los embriones. pCS2 + es un vector comúnmente usado para este propósito, y el kit de transcripción incluye un análogo de la tapa durante la reacción de síntesis. Se volvieron a suspender ARNm transcrito en nucleasa libre de ddH 2 0, a una concentración de valores entre 500 y 2.000 ng / l, seguido de almacenamiento del sistema a -80 ° C. Antes de la inyección, diluir ARN en ddH 2 0 a la concentración apropiada para inyección. Como sólo un pequeño volumen se inyectan en embriones, resuspendiendo en tampón no es necesario. Nuestra concentración final de inyección es típicamente 50-200 pg / nl, dependiendo de la construcción, y nosotros generalmente se inyecte hasta 4 nl por embrión, aunque muchos laboratorios tradicionalmente inyectar hasta 10 nl, es decir, aproximadamente 1% del volumen total de un embrión. ARN puede ser tóxico en altas dosis, y la dosis por géneros embrionesLLY varía de 10 pg a 1 ng, aunque hasta 5 ng puede ser tolerada en función del gen particular y pureza. Sin embargo, para la localización de imágenes de proteínas, el nivel más bajo posible se debe inyectar para evitar la sobre-expresión de artefactos. ARN para EB1-GFP en este protocolo se utiliza en cantidad final de 250 pg por embrión. </li><li> Preparar agujas de inyección tirando capilar con un extractor de aguja 9, a un diámetro de la punta de ~ 0,2 micras. Con una Sutter P-87 extractor, utilizamos los siguientes parámetros: calor 644, 125 tracción, velocidad 70, hora 250, pero estos parámetros varían dependiendo de la máquina y debe ser reajustado después de cambiar el filamento (que usamos actualmente de 2,5 mm filamento caja cuadrada que es de 2,5 mm de ancho). Bajo el microscopio, romper punta de la aguja con una pinza en ángulo para generar una forma de pluma-como. Hay otros métodos para romper el llenado y agujas de inyección (por ejemplo 11,12), pero llenar la aguja con el ARN mediante la colocación de una gota pequeña (0,5 - 1 l) a la parte final de laaguja. Para la microinyección en embriones, hay un número de sistemas de inyección disponibles comercialmente, dos de las más comunes son el Pico-inyector (Medical Systems), y la Nanoject (Drummond Scientific) (ver 11 para más información). Usamos el Medical Systems PLI-100 Pico-inyector, que utiliza gas comprimido durante un período determinado de tiempo digitalmente con el fin de entregar volúmenes consistentes nanolitros. Volumen de inyección debe ser calibrado para cada micropipeta nueva usando un micrómetro, y el tiempo de inyección debe ser ajustada para alcanzar la cantidad deseada de ARN inyectado. </li><li> Place embriones fertilizados en la 1 - a etapa 4-células en un plato de plástico que contiene 5% de Ficoll en 0,1 X MMR. Sosteniendo el embrión en su lugar con pinzas, o colocar en una plataforma de soporte (una malla de plástico, con una rejilla de 1 mm ~, adherida a la parte inferior del plato con plastilina o cera dental), inyectar volumen deseado en blastómeros animales (ver paso 2,1 para más detalles sobre el volumen de inyección). Distribuir en varios locciones de todo embrión, al menos una inyección en cada uno de los blastómeros animales, para obtener una distribución más uniforme (por ejemplo, para un embrión en la fase 4-células, que normalmente inyectan 1-2 veces en cada blastómero, mientras que en una etapa de 2-células embrión, inyectamos 2-4 veces en cada uno de blastómeros). Una ventaja de esperar hasta la etapa de célula 2-4 es que se asegure de que el embrión haya comenzado a escindir normalmente. Sin embargo, si el tiempo es la esencia, se puede iniciar en la etapa 1-célula, con poca diferencia en el resultado final no sea más embriones que necesitan ser inyectados. Mayores embriones en la etapa se puede utilizar también, y esto es particularmente útil si los tipos específicos de células son objeto de inyección, pero como generalmente prefieren la expresión más amplia, se inyecta en los embriones más jóvenes para reducir la necesidad de inyecciones más numerosos. </li><li> Transferencia de embriones inyectados a un plato de plástico con MMR 0,1 X y les permiten desarrollar hasta la etapa 20-23 13. Incubar los embriones a los 14 a 22 ° C, dependiendo de SPE deseadaed de desarrollo. (Para la disección de los tubos neurales al día siguiente, utilice ~ 22 ° C. Para el día siguiente, utilice ~ 14 ° C) </li></ol> 3. Disección del tubo neural y Revestimiento <ol><li> Antes de realizar las disecciones, preparar las 14 placas de cultivo. En primer lugar, los cubreobjetos capa con suficiente solución de 200 mg / ml de poli-lisina en PBS para piscina sobre la superficie del cubreobjetos, de cualquiera de Mattek o Lab-Tek placas de cultivo, y se incuba durante una hora (alternativamente, se podría usar cubreobjetos de vidrio que se sientan suelta en una placa de Petri, para posterior colocación en portaobjetos de vidrio para la formación de imágenes y la inmunocitoquímica). Aspirar y lavar con un exceso de PBS 3 veces, y dejar secar. Entonces platos capa con 10 ug / ml de laminina en PBS (que suelen utilizar 500 l por cubreobjetos, suficiente para piscina sobre la superficie) durante una hora a 37 grados. Aspirar solución de laminina y lavar 3 veces con un exceso de PBS, teniendo cuidado de no dejar que las superficies recubiertas de laminina se exponen a la interfaz de aire. Sustitucie PBS con medios de cultivo (50% de Ringer, 49% 15 L-media, 1% de suero bovino fetal, más 50 g / ml de penicilina / estreptomicina y gentamicina, pH 7,4 y se esterilizó filtro). (Media de Ringer, 115 mM NaCl, 2,5 mM KCl, 2 mM CaCl 2, 10 mM de HEPES, pH 7,4, 0,5 mM EDTA). Mientras que nuestro protocolo utiliza este medio, cabe señalar que se trata de un medio enriquecido común para cultivos más viejos ganglionares de la retina de Xenopus, donde las neuronas han reducido de energía reservados. Jóvenes culturas de la médula espinal va a sobrevivir y crecer durante más de 24 horas en los medios de comunicación Timbres puros, sin factores de crecimiento adicionales, y esto es de hecho una de las ventajas de utilizar este sistema. Opcional: añadir NT3 y el BDNF (a concentraciones finales de 25 ng / l cada uno) a los medios de cultivo para aumentar axón. </li><li> Debido a la variabilidad en la expresión de ARNm inyectado, los embriones pueden exhibir un mosaico de fluorescencia. Antes de realizar disecciones, embriones de pantalla para la presencia de fluorescencia, para identificar aquellos embriones que expulse la proteína de fusión de fluorescencia en el tubo neural. Colocar los embriones en la fase de 20-23 fluorescentes en un plato de plástico de agarosa recubierta lleno de medios de Steinberg (58 mM NaCl, 0,67 mM KCl, 0,44 mM Ca (NO 3) 2, 1,3 mM MgSO 4, 4,6 mM de Tris, pH 7,8 a continuación tratado en autoclave) 5. Para hacer el plato de agarosa recubiertas, cubrir el fondo del plato con plástico derretida 1% de agarosa en 0,1 X MMR y endurecer let. Esto proporciona una superficie suave para que no se produce daño a las pinzas finas y agujas de tungsteno utilizados durante las etapas posteriores de disección. La disección de los embriones más allá de la etapa 23 es posible, pero es más difícil ya que los tejidos se adhieren más estrechamente entre sí y por lo tanto requieren un tratamiento más largo con colagenasa. </li><li> Bajo un microscopio de disección, retirar la membrana vitelina con unas pinzas finas, y luego aislar toda la parte dorsal del embrión, haciendo una serie de incisiones. Mientras que un fórceps mantener el embrión en su lugar, utilizar la segunda para hacer una incisión en ellado del embrión para exponer el interior hueco. A continuación, utilice ambas pinzas para apretar a lo largo del tejido entre las mitades dorsal y ventral del embrión, cortando así el embrión en un medio para aislar la porción dorsal que contiene el tubo neural. </li><li> Coloque el explante dorsal en un tubo Eppendorf con 2 mg / ml de colagenasa en los medios de Steinberg durante 15-20 min en un agitador rotatorio, para aflojar los tejidos y, a continuación explante pipeta dorsal en un plástico recubierto con agarosa plato con solución fresca de Steinberg. Alternativamente, el explante se puede colocar en una placa de Petri que contiene la solución de colagenasa durante 15 min, donde la disección completa a continuación, podría llevarse a cabo. </li><li> El uso de un par de fórceps, suavemente diseccionar el tubo neural a partir de la epidermis dorsal y ventral de la notocorda. Deslice la punta de las pinzas entre la epidermis y el tejido subyacente y tire lentamente la epidermis, revelando el tubo neural debajo. A continuación, utilizar una pinza para sujetar el tejido y otro para deslizar la punta entre latubo neural y la notocorda. Por último, utilizar fórceps para extraer los somitas a cada lado del tubo. </li><li> Transferir el tubo neural a un plato de agarosa recubierta llena con medios de cultivo, tal como se describe en el paso 3,1. </li><li> Después de la recogida de varios tubos neurales, las corte en numerosas piezas (aproximadamente 20, si todo el tubo está aislado) utilizando agujas de tungsteno afiladas o fórceps, y después las piezas de la placa en placas de cultivo lleno de medio (preparado en el paso 3,1), extendiéndose los explantes uniformemente en filas. </li><li> Después de la siembra, no te muevas los platos porque esto perturbaría las células de fijación. Incubar los explantes bañados del tubo neural en torno a 20-22 º C. Por lo general dejan la placa de células en el banquillo a temperatura ambiente durante la noche. Uno de los beneficios de Xenopus laevis neuronas es que una incubadora especial para el cultivo celular que regula los niveles de CO 2 o la temperatura no es necesario. Neuritas y conos de crecimiento pueden ser observadas y fotografiadas a temperatura ambiente 12-24 Horas después de la siembra, aunque excrecencia puede acelerarse con la adición de factores de crecimiento. En general, la expresión de un ARN o producto de plásmido tendrá como resultado la expresión variable entre las células, y por lo tanto puede haber una gama de niveles de expresión de fluorescencia entre los conos de crecimiento. Hemos observado la expresión de ARN de proteínas fluorescentes para persisten más allá de 48 h después de la siembra, aunque esto depende de la construcción particular. </li></ol> 4. Los resultados representativos Después de seguir el protocolo, que se resume en la Figura 1A, sano, correctamente, disecados y se cultivaron explantes neuronales enviará numerosos axones o neuritas en todas direcciones sobre sustrato de laminina / poli-lisina sustrato, que se muestra en la Figura 1B. Los conos de crecimiento en las puntas de los axones pueden ser reflejados por cualquiera de óptica DIC, como se ve en la Figura 1C, a la dinámica de la imagen motilidad general, o alta resolución de la microscopía de fluorescencia para examinar la LOcalization de fluorescentemente etiquetado proteínas del citoesqueleto, por ejemplo, EB1-GFP se muestra en la Figura 1D. <img alt="Figura 1" src="/files/ftp_upload/4232/4232fig1.jpg" /> Figura 1. Resumen del protocolo experimental y los resultados esperados. A) Protocolo de diagrama de flujo. Ver película para detalles con respecto a la disección en el día 3. El tubo neural entera debe ser cortado en piezas de 20 del mismo tamaño y distribuyen de manera uniforme en hileras sobre el cubreobjetos. En esta caricatura, tijeras se representan para representar a cortar el tejido con unas pinzas finas. CG gonadotropina coriónica B) Imagen DIC de los axones o neuritas que crecen fuera de explante (explante en la esquina superior izquierda). C) Ampliación de imagen superior del cono de crecimiento en el extremo de un axón en crecimiento. D) Imagen de microscopía de fluorescencia de EB1-GFP en cono de crecimiento. Barra de nivel 10 micras. <a href="http://www.jove.com/files/ftp_upload/4232/4232fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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Xenopus laevis explantes neuronales enviar neuritas de una manera muy robusta por 24 hr después de la siembra en la laminina / poli-lisina sustrato, si las condiciones son apropiadas. Con este sustrato, los conos de crecimiento son muy móviles y pueden alcanzar longitudes de los axones de hasta 1 mm, que se extiende en todas las direcciones hacia el exterior a partir del explante, aunque longitudes típicas son 100 micras o más. Si neuritas no crecen hacia fuera, hay un número limitado de razones para que e...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Gonadotropina Coriónica </td><td> Argent Chemical Laboratories </td><td> C-HCG-ON-10 </td></tr><tr><td> Cisteína </td><td> Sigma-Aldrich </td><td> 52-90-4 </td></tr><tr><td> mMessage mMachine kit </td><td> Ambion </td><td> AM1340 </td></tr><tr><td> Capilar Agujas Borosil 1,2 mm (diámetro exterior) x 0,9 mm (ID) </td><td> FHC </td><td> 30-31-0 </td></tr><tr><td> Ficoll </td><td> Sigma </td><td> F2637 </td></tr><tr><td> Dumont # 5 Biologie Inox Pinzas </td><td> Herramientas Artes Ciencias </td><td> 11252-20 </td></tr><tr><td> La colagenasa </td><td> Sigma-Aldrich </td><td> 9001-12-1 </td></tr><tr><td> Mattek platos </td><td> Mat Tek Corporation </td><td> P35G-1.5-14-C </td></tr><tr><td> L-15 </td><td> Invitrogen </td><td> 21083-027 </td></tr><tr><td> Poli-L-Lysinordeste </td><td> Sigma </td><td> P-1399 </td></tr><tr><td> La laminina </td><td> Sigma </td><td> L2020 </td></tr><tr><td> NT3 </td><td> Sigma </td><td> N1905 </td></tr><tr><td> BDNF </td><td> Sigma </td><td> B3795 </td></tr></tbody></table>

neural explant cultures from xenopus laevis

El complejo proceso de orientación axón está impulsado principalmente por el cono de crecimiento, que es la estructura dinámica móviles en la punta del axón en crecimiento. Durante axón resultado, el cono de crecimiento debe integrar múltiples fuentes de información de guía señal para modular su citoesqueleto con el fin de propulsar el cono de crecimiento hacia adelante y precisa navegar a encontrar sus objetivos específicos 1. Cómo esta integración se produce a nivel del citoesqueleto todavía está emergiendo, y el examen de proteína del citoesqueleto y la dinámica efectoras dentro del cono de crecimiento puede permitir la elucidación de estos mecanismos. Xenopus laevis conos de crecimiento son lo suficientemente grandes (10-30 micras de diámetro) para realizar alta resolución de imagen en directo de la dinámica del citoesqueleto (por ejemplo, 2-4) y son fáciles de aislar y manipular en un ambiente de laboratorio en comparación con otros vertebrados. La rana es un sistema modelo clásico de desarrollo de los estudios de neurobiología e importantes reflexiones tempranas en mic cono de crecimientodinámica rotubule se encontraron inicialmente utilizando este sistema de 5-7. En este método 8, se recogen los huevos y fertilizados in vitro, inyectados con ARN que codifica las proteínas de fusión etiquetadas fluorescentemente citoesqueleto o de otras construcciones para manipular la expresión génica, y luego se dejó desarrollar a la etapa del tubo neural. Tubos neurales están aislados por la disección y luego cultivadas son, y los conos de crecimiento de neuritas outgrowing fotografiado está. En este artículo, se describe cómo llevar a cabo este método, el objetivo de las cuales es para los conos de crecimiento de cultivo de Xenopus laevis para su posterior análisis de imágenes de alta resolución. Si bien damos el ejemplo de proteína de fusión + TIP EB1-GFP, este método se puede aplicar a cualquier número de proteínas para dilucidar sus comportamientos en el cono de crecimiento.

Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Neural Explant Cultures from Xenopus laevis

Cultivo de explantes neuronales de disección<em&gt; Xenopus laevis</em&gt; Embriones que expresan proteínas fluorescentes de fusión permite la obtención de imágenes de cono de crecimiento dinámica del citoesqueleto.

Los cultivos neuronales de explantes de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

neural-explant-cultures-from-xenopus-laevis

Research

JoVE Journal

Neuroscience

Neural Explant Cultures from Xenopus laevis

12.4K vistas.  Harvard Medical School. Cultivo de explantes neuronales de disección Xenopus laevis Embriones que expresan proteínas fluorescentes de fusión permite la obtención de imágenes de cono de crecimiento dinámica del citoesqueleto.

Video: Neural Explant Cultures from Xenopus laevis

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivación de las Culturas enriquecido oligodendrocitos y neuronas oligodendrocitos / myelinating Co-culturas de Post-natal tejidos murinos

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Investigación

Neurociencias

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Disección, Cultura y Análisis de<em&gt; Xenopus laevis</em&gt; Tejido de la retina embrionaria

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultánea pre-y post-sináptica de registro electrofisiológico<em&gt; Xenopus</em&gt; Músculo Nervio-Co-culturas

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Un ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Un ensayo de comportamiento simple para Pruebas de función visual en<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Grabación de la actividad neuronal inducida por temperatura a través de la supervisión de cambios de calcio en el bulbo olfatorio de<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Derivación de las leptomeninges de explantes de culturas postmortem Humanos Los donantes de cerebro

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Grabaciones simultáneas de potenciales de campo Local Cortical, Electrocardiograma, electromiograma y respirar a ritmo de una rata libremente móvil

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...