Damos las gracias a N. Schwedhelm-Domeyer por su excelente asistencia técnica, C. y S. Hammer Papiol ayuda con los análisis estadísticos. Nuestro trabajo es financiado por la Sociedad Max Planck, la Deutsche Forschungsgemeinschaft, el Centro de Nanotecnología Microscopía y Fisiología Molecular del Cerebro (CNMPB), Göttingen, Alemania y por la GGNB Grupo Junior Mesada de la Universidad de Göttingen.

CGCs pueden producir o bien desde el primer día postnatal (P) 5 crías de ratón o de crías de rata P6. Seguimos un protocolo, descrito por Bilimoria y colegas, que utiliza un inhibidor mitótico para seleccionar para CGCs postmitotic 13. Declaración de Ética: Todos los experimentos con animales vivos se han realizado de acuerdo con el protocolo de los animales aprobado por el &quot;Verbraucherschutz und Lebensmittelsicherheit&quot; de la Baja Sajonia, Alemania. Ensayo in vitro: 1. Preparación de ADN plásmido, Medios, y tampones para el fosfato de calcio método de transfección <ol><li> Disolver el ADN plásmido en agua libre de endotoxina estériles; DMEM (glucosa alta); hacer CaCl2 2,5 M; hacer 2x HBSS (disolver 4 g NaCl, KCl 0,1775 g, 0,095 g de Na 2 HPO 4 • 7H 2 O, 0,675 g de glucosa y 2,5 g de HEPES en 250 ml de agua ultrapura y ajustar el pH a 7,05, 7,08 y 7,11). Nota: Cuando se preparala solución 2x ​​HBSS, pH de prueba que da mejores resultados en cuanto a la eficacia de transfección de determinada combinación de plásmidos. </li></ol> 2. La transfección de neuronas cultivadas <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig1highres.jpg" src="/files/ftp_upload/51070/51070fig1.jpg" /> Figura 1. Diagrama de flujo de los axones in vitro y el ensayo de crecimiento de las dendritas. Cultivadas CGCs (placa de 24 pocillos con cubreobjetos de vidrio), aislado de las crías de rata P6, se transfectan en DIV 0 o 1 con precipitado de ADN que contiene un marcador de transfección fluorescente (GFP por ejemplo). Después de la fijación y la inmunocitoquímica, las neuronas son imágenes en una forma ciega. Las imágenes se importan en ImageJ y se miden los procesos. Las mediciones se procesan entonces utilizando un programa estadístico. <ol><li> CGCs Semilla (20 x 10 6 por placa de 24 pocillos, BME, suero de ternera al 10%, 2 mM penicilina-Streptomycin-Glutamina (PSG), 25 mM KCl) en ácido-lavado, 12 mm cubreobjetos de vidrio nítrico polyornithine recubierto en una placa de 24 pocillos con 500 l de media por pozo. </li><li> En días in vitro (DIV) 0 (por lo menos 8 horas después de la siembra) o DIV 1, recoge medio de cultivo y mantener a 37 ° C. Lávese las neuronas dos veces con 500 l de DMEM precalentado y añadir 500 l de DMEM. </li><li> Colocar las neuronas en la incubadora (37 ° C, 5% de CO 2) durante 45 min. </li><li> Preparar 40 l de ADN precipitado para cada pozo mezclando: ADN (2-2,5 mg / pocillo, 10% del total de ADN debe ser un marcador de transfección por ejemplo, las buenas prácticas agrarias para visualizar las neuronas transfectadas), agua (hasta 18 l), añadir 2 l de CaCl2 2,5 M, mezclar bien y añadir 20 l de 2x HBSS. <ol><li> Incubar precipitado de ADN durante 5 min a TA. </li></ol></li><li> Añadir precipitado de ADN a cada pocillo e incubar las neuronas durante 18 min en la incubadora. </li><li> Retire la mezcla de DMEM / DNA y lavar dos veces con 500 neuronas l de DM precalentadoEM. </li><li> Añadir medio recogido desde el paso 2.2 de nuevo a las neuronas. Si las neuronas estarán en cultivo durante más de 3 días, suplemento de medios con glucosa 25 mM a DIV 3 para reponer la fuente de carbono. </li><li> Después de 1-5 días, las neuronas sometidas a inmunohistoquímica utilizando anticuerpos GFP. </li><li> Imagen por lo menos 30 neuronas individuales por condición en una forma ciega usando un microscopio de fluorescencia. </li></ol> 3. Mida los axones y dendritas con NeuronJ, un NIH ImageJ Plugin Importante: Asegúrese de que las imágenes se escalan correctluy utilizando píxeles adecuada: relación micras dependiendo de magnificación y resolución de imagen. <ol><li> Convertir imágenes a 8 bits con ImageJ: Abrir imagen, elija &#39;Imagen&#39; - Guardar Imagen &#39;&gt;&#39; Tipo &#39;- -&gt; &#39;8 bits&#39;&gt; &#39;. </li><li> Ejecute NeuronJ plugin y abrir la imagen de 8 bits. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot1.jpg" width="500px" /></li><li> Utilice la opción &#39;agregar trazados&#39; para seguir el axón: click el botón izquierdo del ratón una vez al principio del axón y mover el ratón a lo largo del proceso. Doble click en la punta del axón si traza coincide con la forma axonal. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot2.jpg" width="500px" /> Nota: En caso de que la traza sugerido diferir de forma axonal, haga clic una vez en el proceso axonal anclar traza, haga doble click en la punta del axón. </li><li> Haz clic en &quot;trazados medida&quot;, elija &quot;Display mediciones rastreo &#39;opción y pulse&quot; Ejecutar &quot;. Mediciones Axon se visualizan todos de una nueva ventana. Para dendritas, elija la &quot;opción mediciones grupales Pantalla &#39;y pulse en&#39; Ejecutar &#39;. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot3.jpg" width="500px" /> Total de mediciones de dendritas se visualizan todos de una nueva ventana. Guardar como un archivo independiente que se puede abrir en cualquier programa de hoja de cálculo. </li><li> Como alternativa para el trazado manual, utilice el software de Fiji: Haga clic derecho en el the opción &#39;línea recta&#39;, seleccione &#39;línea a mano alzada », <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot4.jpg" width="500px" /> mantener presionado el botón izquierdo del ratón y trace manualmente el proceso, pulse la tecla &quot;Ctrl + M&quot; a la medida. </li><li> Calcule la media la longitud dendrítica / axonal por condición y el uso de la prueba estadística apropiada. En electroporación in vivo: </li></ol> 1. Equipo y Preparación de los reactivos <ol><li> Necesita 30 agujas G, espaciador (1-2 mm), jeringa, reductor de volumen muerto (DVR), electroporador y tweezertrodes, almohadilla térmica o una lámpara de calor infrarrojo, lámpara de cuello de cisne y el isoflurano. </li><li> Ponga DVR en la aguja, luego coloque la aguja a la jeringa, y, finalmente, poner espaciador en la aguja (Figura 2). <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig2highres.jpg" src="/files/ftp_upload/51070/51070fig2.jpg" /> Figura2. Preparación de la aguja. DVR se corta una punta de pipeta de 200 l y se coloca en la aguja para reducir el volumen muerto. Espaciador se deriva de una punta de 200 l de carga y se coloca en el extremo de la aguja para regular la profundidad de penetración en el cerebelo a aproximadamente 2 mm. Unidades de la regla: cm </li><li> Disolver ADN en PBS/0.03% Fast Green. Nota: Como un marcador de transfección, es ventajoso usar una proteína fluorescente que se encuentra bajo un promotor específico de neuronas (por ejemplo Synapsin) para visualizar las neuronas sólo. 25% de la cantidad total de plásmido debe ser el plásmido que codifica el marcador de transfección. </li><li> Hacer etanol al 70%. </li><li> Mezclar volúmenes iguales de PTU y, 30% de sacarosa disuelto en PBS. </li><li> Llene la jeringa con 4 l de ADN (4 g / l de ADN plásmido en PBS/0.03% Fast Green). </li></ol> 2. IVE de Ratas Diagrama de flujo del IVE: véase la Figura 3 <p class="jove_content" fo:keep-together.within-Page = &quot;always&quot;&gt; <img alt="Figura 3" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig3highres.jpg" src="/files/ftp_upload/51070/51070fig3.jpg" /> Figura 3. Diagrama de flujo de la electroporación in vivo. Crías de rata P4 se anestesian con isoflurano y el plásmido de ADN que codifica un marcador de transfección fluorescente (por ejemplo, GFP) se inyecta en el cerebelo, seguido por la exposición a 5 pulsos eléctricos. Cinco días más tarde, aislado de GFP-positivas cerebelo se seccionan y se somete a técnicas de inmunohistoquímica. Las imágenes se capturan utilizando un microscopio confocal y analizados utilizando el software Imaris. Los datos se procesan con un programa estadístico. <ol><li> Utilice crías de ratas P4 de la cepa albina (Wistar o Long Evans). </li><li> Anestesie cachorros (uno tras otro) con isoflurano en caja pequeña (por ejemplo, cajas de puntas de pipeta P1000) con 200 l de isoflurano (empapado en el tejido) durante 1-2 minutos hasta que las crías ya no está en movimiento. Tenga cuidado de que los cachorros no entrar en contacto with de liquido isoflurano. Vigilar estrechamente el tiempo como cachorros individuales responden de manera diferente a la anestesia. </li><li> Esterilizar parte posterior de la cabeza del cachorro con etanol al 70%. </li><li> Fijar la cabeza del cachorro entre el pulgar y el dedo índice y el uso de la lámpara de cuello de cisne para localizar cerebelo de cachorro albino. El seno transverso delimita de forma pronunciada el cerebro medio (colículo superior e inferior) de los hemisferios corticales (Figura 3). El cerebelo está ubicado adyacente al cerebro medio y aparece en un tono más oscuro. Use un marcador permanente para indicar el cerebelo con un punto. Importante: Mantenga las crías en posición fija! Nota: En caso de anestesia desaparecerá durante este procedimiento, exponga las crías al isoflurano antes de inyectar el ADN. </li><li> Insertar la aguja (Figura 3) e inyecte lentamente 3 l de ADN en el cerebelo. </li><li> Deje que la solución de ADN se difunden durante 30-60 seg. <ol><li> Coloque la cabeza del cachorro entre tweezertrodes modo que el polo negativo hace contacto con la parte posterior de la cabeza (cerebelola región r) y que hace contacto con el polo positivo el lado opuesto de la cabeza (Figura 3). </li><li> Asunto cachorro a 5 pulsos eléctricos. Ajuste de tensión con el peso de las crías para asegurar una buena eficiencia de la electroporación sin comprometer su supervivencia (Tabla 1). <table border="0"><tbody><tr><td> Peso </td><td> Voltaje </td><td> Pulso </td><td> Intervalo </td></tr><tr><td> 8-9 g </td><td> 160 V </td><td> 50 mseg </td><td> 950 ms </td></tr><tr><td> 9-10 g </td><td> 165 V </td><td> 50 mseg </td><td> 950 ms </td></tr><tr><td> &gt; 10 g </td><td> 170 V </td><td> 50 mseg </td><td> 950 ms </td></tr></tbody></table> Tabla 1. Electroporación de crías de rata P4. </li><li> Deje que los cachorros se recuperan en la almohadilla caliente o debajo de una lámpara de infrarrojos. Volver crías para represar. Importante: Asegúrese de que la fuente de calor no inflige quemaduras. </li></ol></li><li> Sacrificio cachorros 5 días después de la electroporación mediante la colocación de ellos en CO 2 seguido por decapitación. <ol><li> Aislar el cerebelo y la pantalla para las buenas prácticas agrarias-positivas cerebelo utilizando un microscopio de fluorescencia. </li></ol></li><li> Fijar el cerebelo en el 4% PFA O / N a 4 ° C, y luego incubar en sacarosa al 30% a 4 º C hasta su fregadero cerebelo al fondo del tubo. </li><li> Incrustar cerebelo en OCT/30% de sacarosa y cortar 40 micras secciones coronales utilizando un criostato. Nota: Sección cada cerebelo en una forma ciega. </li><li> Secciones reservamos el derecho de inmunohistoquímica utilizando el anticuerpo GFP. Contrateñir con tinte nuclear (DAPI o Hoechst 33258) y determinar la localización de al menos 200 neuronas transfectadas por animal. </li><li> Para un análisis en profundidad, subdividir el IGL en mitades, lo que resulta en una IGL superior frente al lavado de dinero y una IGL menor frente a la sustancia blanca y contar las neuronas positivas para GFP que residen en cada mitad. Nota: Número de neuronas positivas para GFP de cada sección en una forma ciega. </li&gt; </ol> 3. Medición Dendrita Largo, Adquirir las imágenes de la Sección de x, y, z Plane usa un microscopio confocal Nota: por ejemplo, usar 40 imágenes para una sección de 40 m con un z-STWp de 1 micra. <ol><li> Serie de imágenes Abrir en el software, Imaris, para generar una imagen en 3D de las dendritas. </li><li> Haga clic en el modo de &#39;Supera&#39; para ver la neurona en 3D. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot5.jpg" width="500px" /></li><li> Seleccione &quot;Añadir nuevo filamento&quot; y haga clic en &quot;Cambiar la creación automática &#39;para iniciar el rastreo semiautomático. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot6.jpg" width="500px" /> Nota: Analizar cada imagen en 3D de una forma ciega </li><li> Seleccione la pestaña &#39;Draw&#39; y &#39;AutoPath&#39;. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot7.jpg" width="500px" /></li><li> Mueva el cursor del ratón sobre el ccuerpo ell y Shift + Clic derecho del ratón para seleccionar el cuerpo de la célula. Nota: cálculo automático de software puede requerir unos minutos. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot8.jpg" width="500px" /></li><li> Añadir caminos al filamento (dendrita) usando Shift + Clic izquierdo del ratón. Nota: Las rutas pueden ser visualizados en tiempo real. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot9.jpg" width="500px" /></li><li> Ir a la ventana de estadísticas de filamento y haga clic en, &quot;valores específicos &#39;&quot; detalladas &quot;y&quot; longitud de filamento dendrita (suma) &quot;por la suma de la longitud total de la dendrita. <img alt="pantalla" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot10.jpg" width="500px" /></li><li> Utilice las pruebas estadísticas apropiadas para analizar los datos. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII-NeuroD+Signaling+Pathway+Specifies+Dendritic+Morphogenesis.">CaMKII-NeuroD Signaling Pathway Specifies Dendritic Morphogenesis.</a> Neuron. 41, 229-241 (2004).</li><li>Yang, 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=A+Cdc20-APC+ubiquitin+signaling+pathway+regulates+presynaptic+differentiation.">A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation.</a> Science. 326, 575-578 (2009).</li><li>Litterman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+OBSL1-Cul7Fbxw8+ubiquitin+ligase+signaling+mechanism+regulates+Golgi+morphology+and+dendrite+patterning.">An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.</a> PLoS Biol. 9, (2011).</li><li>Kim, A. 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=A+centrosomal+Cdc20-APC+pathway+controls+dendrite+morphogenesis+in+postmitotic+neurons.">A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons.</a> Cell. 136, 322-336 (2009).</li><li>Shalizi, A., 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=A+calcium-regulated+MEF2+sumoylation+switch+controls+postsynaptic+differentiation.">A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.</a> Science. 311, 1012-1017 (2006).</li><li>Vadhvani, M., Schwedhelm-Domeyer, N., Mukherjee, C., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Centrosomal+E3+Ubiquitin+Ligase+FBXO31-SCF+Regulates+Neuronal+Morphogenesis+and+Migration.">The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration.</a> PLoS One. 8, (2013).</li><li>Puram, S. V., 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=A+CaMKIIbeta+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nat. Neurosci. , (2011).</li><li>Jia, Y., Zhou, J., Tai, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+promote+cerebellar+granule+neuron+survival.">TRPC channels promote cerebellar granule neuron survival.</a> Nat. Neurosci. 10, 559-567 (2007).</li><li>Huynh, M. A., 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=An+isoform-specific+SnoN1-FOXO1+repressor+complex+controls+neuronal+morphogenesis+and+positioning+in+the+mammalian+brain.">An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.</a> Neuron. 69, 930-944 (2011).</li><li>de la Torre-Ubieta, L., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+neuronal+polarity+and+morphogenesis+in+the+mammalian+brain.">Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain.</a> Neuron. 72, 22-40 (2011).</li><li>Calissano, P., 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=Recombinant+human+insulin-like+growth+factor+I+exerts+a+trophic+action+and+confers+glutamate+sensitivity+on+glutamate-resistant+cerebellar+granule+cells.">Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8752-8756 (1993).</li></ol>

Los autores declaran no tener intereses financieros en competencia.

Ventajas y limitaciones de la descrita in vitro e in vivo: CGCs cultivadas de ratón y ratas son igualmente aptas para los análisis morfológicos. Debido al tamaño más grande de un cerebelo de rata, el rendimiento de GCN de crías de rata que excede de crías de ratón 3-4x. Aparte de CGCs, las neuronas corticales y del hipocampo se pueden usar como sistema de cultivo así. El método de fosfato de calcio da como resultado un bajo (0,01-5%) eficiencia de transfección, q...

El cerebelo es un excelente sistema para estudiar los mecanismos de crecimiento de los axones y la migración. El cerebelo ha sido objeto de estudios anatómicos desde los albores de la neurociencia 1. Microscopía moderna y las técnicas inmunohistoquímicas han expandido significativamente y perfeccionado los descubrimientos iniciales por Santiago Ramón y Cajal 2-4. La genética del ratón de los estudios moleculares descubrieron los factores de crecimiento y de transcripción esenciales en el control del desarrollo del cerebelo, lo que condujo a una mayor comprensión de los acontecimientos cruciales requeridos para el cableado adecuado de los diferentes tipos de neuronas, incluyendo las neuronas granulares de cerebelo (CGCs) 5-7. El cerebelo es un derivado de rombómero 1 de la parte posterior del cerebro en desarrollo 8. El labio rómbica, que es parte del techo de la 4 ª ventrículo, da lugar a progenitores neuronales de gránulos cerebelosos, que constituirán la más numerosa población neuronal en elcerebelo adulto 9. A raíz de la migración rostral, se instalan en el esbozo del cerebelo. Aquí, la mitosis de precursores gránulo neuronas conduce a la dramática expansión de la capa granular externa (EGL), que tiene lugar después del nacimiento en roedores. Desde el EGL, las neuronas comienzan a migrar hacia el interior a través de la capa molecular (ML), más allá de la capa de células de Purkinje que tome en última instancia, la residencia en la capa granular interna (IGL 2). Durante este proceso migratorio, adquieren una forma bipolar con dos axones se extiende en el ML. Tras una posterior migración, el cuerpo de la célula migra lejos de los axones y los dos procesos se fusionan para formar un axón bifurcado, en forma de T 10. Posteriormente, estos axones fasciculada y se conocen como fibras paralelas. Habiéndose establecido en el IGL, CGCs crecen dendritas, que forman garras dendríticas para establecer sinapsis con las fibras musgosas. Para examinar los procesos fundamentales en el cerebelo en desarrollo, un combinado in vitro e in vivo approach permite obtener resultados fiables y conclusiones. CGCs no sólo son las más numerosas neuronas del cerebelo, sino de todo el cerebro y pueden ser cultivadas a gran pureza 11-13. En la cultura, esta población neuronal altamente homogénea se convierte rápidamente postmitotic y adquiere una morfología polar con axones fácilmente identificables y dendritas. CGCs cultivadas han demostrado ser de gran utilidad para estudiar diversos aspectos del desarrollo neuronal, incluyendo la proliferación de progenitores, la diferenciación y el desarrollo axonal dendrita, la migración neuronal, la apoptosis y las propiedades electrofisiológicas (14-19 y muchos otros). El empleo de la manipulación genética ha ampliado la versatilidad de CGCs cultivadas y permitió una mayor comprensión mecanicista de los hechos mencionados. Transfección de neuronas cultivadas utilizando fosfato de calcio de baja eficiencia o métodos lipofílicas seguidos por inmunocitoquímica con marcadores de polaridad o software soportado análisis faciliTates la evaluación de, por ejemplo la morfología de las neuronas individuales en un denso cultivo neuronal. Con este enfoque, el papel de las proteínas de interés en axón o la dendrita de crecimiento puede ser estudiado 20-25,26-28. Este sistema de cultivo sin embargo es menos útil para analizar la migración neuronal como la migración es muy limitada en cultivos de alta densidad y requeriría cocultivos. El análisis in vitro de axón y el crecimiento de dendritas también permite el examen de las proteínas interconectados de una vía de señalización que utilizan combinaciones de interferencia de ARN (i), sobre-expresión o pequeñas moléculas. Para establecer la relevancia de la proteína de interés en el axón y la regulación del crecimiento de dendritas o la migración neuronal, la electroporación in vivo técnica (IVE) permite el análisis en la corteza cerebelosa desarrollo. Debido al hecho de que el desarrollo del cerebelo en roedores se extiende camino en las dos primeras semanas después del parto, el cerebelo representa un accessibla estructura del cerebro le manipulaciones genéticas para examinar el desarrollo de los axones y dendritas, la migración neuronal, sinaptogénesis y apoptosis 20-24,29,30,26,27,31-34. Además, este sistema modelo es también útil para otros aspectos del desarrollo neuronal que requieren la corteza cerebelosa intacta como pathfinding axón, el cableado y la conectividad de las neuronas y de las interacciones neurona-glía Tomados en conjunto, este protocolo proporciona in vitro y en técnicas in vivo para hacer frente un enfoque complementario con respecto a la morfogénesis neuronal y la migración.

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			<td height="25" style="height:25px;width:160px;">CaCl2</td>
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			<td height="22" style="height:22px;width:160px;">Tissue-Tek OCT</td>
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			<td height="44" style="height:44px;width:160px;">Material Name</td>
			<td style="width:125px;">Company</td>
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			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:88px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">ImageJ</td>
			<td style="width:125px;">NIH</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
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			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
			<td style="width:125px;">Bitplane, Inc.</td>
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			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
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			<td height="22" style="height:22px;width:160px;">MS Excel</td>
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genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration

Eventos de desarrollo en el cerebro, incluyendo la morfogénesis neuronal y la migración son altamente orquestados procesos. In vitro e in vivo análisis permite una caracterización en profundidad para identificar las vías que participan en estos eventos. Neuronas granulares de cerebelo (CGCs) que se derivan de la cerebelo en desarrollo son un sistema modelo ideal que permite análisis morfológicos. Aquí se describe un método de cómo manipular genéticamente CGCs y cómo estudiar axono y dendritogénesis de las neuronas individuales. Con este método los efectos de la interferencia de ARN, la sobreexpresión o moléculas pequeñas se pueden comparar para controlar las neuronas. Además, la corteza cerebelosa roedor es un sistema de fácil acceso en vivo debido a su desarrollo postnatal predominante. También presentamos una técnica de electroporación en vivo para manipular genéticamente el cerebelo en desarrollo y describir posterior del cerebelo se analizan para evaluar la morfología neuronal unnd migración.

Para analizar la morfología de las CGCs en respuesta a diferentes condiciones de cultivo, transfectadas las neuronas en DIV 0 como se describió anteriormente. Después de la transfección, colocamos un conjunto de neuronas en un medio completo (BME, suero de ternera al 10%, 2 mM PSG, 25 mM KCl) y otro conjunto en medio mínimo que contiene insulina (BME, glucosa 25 mM, PSG 2, 10 g / ml de insulina). Hemos sometido las neuronas para inmunocitoquímica utilizando el anticuerpo GFP en DIV 1, 2 y 3, seguido por la medici?...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Morfogénesis neuronal y la migración son eventos cruciales que subyacen el desarrollo adecuado del cerebro. Aquí se describen los métodos para manipular genéticamente las neuronas granulares de cerebelo en cultivo y el cerebelo en desarrollo para la evaluación de la morfología y las características migratorias de las neuronas.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

genetic-manipulation-cerebellar-granule-neurons-vitro-vivo-to-study

Research

JoVE Journal

Neuroscience

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

13.6K vistas.  Max Planck Institute of Experimental Medicine.  Morfogénesis neuronal y la migración son eventos cruciales que subyacen el desarrollo adecuado del cerebro. Aquí se describen los métodos para manipular genéticamente las neuronas granulares de cerebelo en cultivo y el cerebelo en desarrollo para la evaluación de la morfología y las características migratorias de las neuronas. 

Video: Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatizado de Análisis de la morfología neuronal Sholl digitalizados en múltiples escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Investigación

Neurociencias

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus mediada por la manipulación genética y la visualización de las neuronas sensoriales olfativas In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection de roedores neuroblastos para el Estudio de la Migración Neuroblast<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

El sistema olfativo como un modelo para estudiar los patrones de crecimiento axonal y Morfología<em&gt; En Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging de Postnatal cerebelosa gránulo migración celular utilizando confocal Macroscopía

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analizando dendríticas morfología en las columnas y capas

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Evaluación de la Viabilidad Neuronal Utilizando Colorante de Fluoresceína-Ioduro de Propidio Doble Coloración en Gránulos Cerebelosos Cultura de Neuronas

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...