Supported by grants from CNRS and INSERM. PM acknowledges support from the Fondation pour la Recherche M&#233;dicale en France (Equipe FRM 2009). SC acknowledges support from the Fondation de France.

Los ratones utilizados en este trabajo fueron atendidos y manejados de acuerdo con las directrices del Consejo de la Unión Europea (86/609 / UE) para el uso de animales de laboratorio. 1 Preparación de soluciones requeridas <ol><li> Soluciones de archivo <ol><li> 10x poli-L-ornitina (OLP) Solución: Pesar 10 mg de bromhidrato de PLO (peso molecular = 30,000-70,000) y disolver en 70 ml de agua estéril. Se filtra la solución con 0,2 micras filtro de jeringa, alícuota, y se almacenan a -20 ° C. </li><li> 30% solución de glucosa: pesar 30 g de D (+) - Glucosa y disolver en agua estéril hasta un volumen total de 100 ml. Filtro, alícuota y almacena a 4 º C. </li><li> Medio de Eagle Modificado de 5x Dulbecco (DMEM) / mezcla de nutrientes F-12 de Ham: Pesar de 6 g de polvo de DMEM / F-12 de jamón y disolver en agua estéril hasta un volumen total de 100 ml. Filtro, alícuota y almacena a 4 º C. </li><li> 7,5% de bicarbonato de sodio (NaHCO3) Solutien: Pesar 7,5 g de NaHCO3 y se disuelven en agua estéril hasta un volumen total de 100 ml. Filtro, alícuota y almacena a 4 º C. </li><li> 1 M HEPES Solución: Pesar 23,8 g de HEPES y disolver en agua estéril hasta un volumen total de 100 ml. Filtro, alícuota y almacena a 4 º C. </li><li> 70% (v / v) de etanol: Diluir 70 ml de etanol absoluto con 30 ml de agua estéril. </li></ol></li><li> PBS con glucosa y antibióticos (PBS-GAB): añadir 10 ml de solución de glucosa al 30% y 5 ml de solución de penicilina-estreptomicina a 500 ml de fosfato de Dulbecco Buffered Saline. Almacenar a 4 ° C. </li><li> Fetal inactivado de suero bovino (IFB): Descongelar de suero bovino durante la noche a 4 ° C e inactivar a 56 ° C durante 30 min. Alícuota en 50 ml y se almacena a -20 ° C. </li><li> Medio de Cultivo: En agua estéril, se mezclan sucesivamente 40 ml de 5x DMEM / F12-Ham, 1 ml de HEPES 1 M, 2 ml de 200 mM de L-glutamina, 2 ml de penicilina-estreptomicina, 4 ml de 30% de glucosa unnd 3 ml de 7.5% de NaHCO3 a un volumen final de 180 ml. Filtrar y utilizar el mismo día. </li><li> Hormona Mix <ol><li> Preparar 180 ml de medio de cultivo. Disolver 200 mg de apo-transferrina en este medio. </li><li> Pesar 50 mg de insulina y disolver en 2 ml de HCl 0,1 M. Añadir lentamente 8 ml de agua estéril mientras se mezcla suavemente. Diluir esta solución en el medio de cultivo. </li><li> Pesar 19,3 mg de dihidrocloruro de putrescina y disolver en 10 ml de agua estéril. Añadir la solución de 10 ml a la mezcla de la hormona. </li><li> Preparar una solución de selenito de sodio 3 mM en agua estéril y una solución de progesterona 2 mM en etanol absoluto. Añadir 20 l de cada solución a la mezcla de la hormona. </li><li> Se filtra la mezcla de hormonas, alícuota de 10 ml y se almacena a -20 ° C. </li></ol></li></ol> 2 Preparación de las placas e instrumentos Cultura <ol><li> FBS Recubrimiento de las placas de cultivo: El día antes de la disección, preparar 180 ml de medio de cultivo. Añadir 10 ml deIFB a 90 ml de medio de cultivo. Añadir 300 l / pocillo de medio de cultivo / 10% IFB en precubiertas placas de 24 pocillos de poli-D-lisina. Incubar toda la noche a 37 ° C. Almacenar los residuos de medios (con y sin OFI) a 4 ° C. </li><li> Esterilizar los instrumentos y pipetas Pasteur. Reúna el equipo listado en la tabla de materiales, así como una campana de flujo laminar clase II y una incubadora de CO 2. </li></ol> 3. Disección de Ratón Mesencéfalo <ol><li> Sacrificar un ratón embarazada E13.5 (según las directrices institucionales). Limpiar el abdomen del ratón con etanol al 70% y abrir la pared del abdomen. Recoge los cuernos uterinos, abrirlas con unas tijeras delicadas, diseccionar cada embrión de los cuernos uterinos, y quitar las membranas amnióticas. Coloca los embriones en una placa de Petri de 100 mm que contiene PBS estéril-GAB y lavarlos por transferencia en 3 baños idénticos sucesivos utilizando pinzas. Para la disección, colocar los embriones en un 3 en un 60 mm placa de Petri estéril. </li><li> Move el plato final Petri bajo el microscopio estereoscópico. No decapitar a los embriones. Con unas tijeras Vannas, extirpar el cerebro. <ol><li> Retire con cuidado y deseche las regiones previsiones y cerebro posterior. Para el lado rostral, cortar cerca de la región talámica, a la corte lateral caudal en la región del istmo, retire el colículo superior. </li><li> Una vez que el cerebro medio ventral está aislado, retire con cuidado meninges utilizando fórceps ultrafinas. Recoge los segmentos disecados, sin las meninges, en un tubo de 13 ml estéril llena de PBS-GAB. </li></ol></li><li> Limpie el tubo que contiene la mesencephala a fondo con etanol al 70% y ponerla bajo el capó. </li></ol> Disociación 4. Cell <ol><li> Lave mesencephala 3x con PBS estéril-GAB. Permitir fragmentos cerebrales se asienten entre cada lavado y evitar la interrupción del tejido. Después del último lavado, eliminar cuidadosamente como cantidad posible de solución e incubar segmentos cerebrales en 3 ml de tripsina / EDTA durante 15 minutos a 37 ° C en unCO 2 incubadora. </li><li> Fuego-pula las pipetas Pasteur para maximizar la supervivencia de las neuronas como el final de una pipeta de vidrio no pulida es fuerte y puede dañar las células durante las etapas de disociación. Ligeramente reducir el diámetro de la extremidad (hasta 0,5 mm) de la pipeta Pasteur, mientras que el fuego-pulirlo. </li><li> Retire con cuidado tanto / EDTA solución tripsina como sea posible y añadir 10 ml de medio de cultivo / 10% IFB. Segmentos cerebrales Lave 3 veces con medio de cultivo / 10% IFB. </li><li> Usando la pipeta Pasteur pulida al fuego, comience disociación de mesencephala en 6 ml de medio de cultivo / 10% IFB. Triturar 10x (evitar las burbujas de aire), permiten que los trozos se asienten, a continuación, recoger el medio en un nuevo tubo estéril 13 ml. </li><li> Añadir 6 ml de medio de cultivo, repita el paso anterior una vez y recoger el medio que contiene células disociadas en el mismo tubo de 13 ml. </li><li> Centrifugar las células a 160 g durante 5 min. Desechar el sobrenadante y resuspender las células en medio de cultivo suplementado conMezcla de la hormona 10%. </li></ol> 5. Cell Plating <ol><li> Contar las células en suspensión en una célula de Malassez y ajustar el volumen del medio a una concentración de 600.000 células / ml. Alrededor de 1,7 millones de células se pueden obtener de un mesencéfalo ratón. </li><li> Retire que contenía FBS medio de revestimiento de placas de 24 pocillos y añadir en cada pocillo 1 ml de la suspensión celular (600.000 células / pocillo, el 2-4% de las células TH +). </li><li> Incubar la placa a 37 ° C y 5% CO 2/95% de aire. No es necesario cambio de medio. Las neuronas dopaminérgicas son maduros después de unos 5-7 días in vitro (expresión coherente del transportador de dopamina). Mantener las células en cultivo hasta 15 días sin reemplazo medio. </li></ol> Protocolo 6. inmunofluorescencia <ol><li> Limpieza de las Cubreobjetos <ol><li> El día antes de la disección, agregar 10 ml de HCl 1 M en una placa de Petri. Coloque en la superficie de las 12-15 cubreobjetos de vidrio líquido y esperar 15 min. </li><li> Hunde la cubiertacaer en el líquido y esperar 15 min. </li><li> Retire HCl y lavar 3 veces con agua. </li><li> Lavar los cubreobjetos con etanol puro rápidamente una vez, a continuación, añadir etanol puro al plato y esperar 30 min. </li><li> Bajo el capó, retire cubreobjetos limpios a partir de etanol y añadir 1 cubreobjetos por pocillo de una placa de cultivo celular de 12 pocillos usando fórceps esterilizados. </li><li> Lavado cubreobjetos dos veces con agua estéril (1 ml / pocillo). A continuación, lavar una vez con PBS estéril. </li></ol></li><li> Recubrimiento de los cubreobjetos <ol><li> Retirar PBS y añadir 500 l / pocillo de 2x OLP (diluido en PBS). Incubar durante 4 horas a 37 ° C en la incubadora de CO 2. </li><li> Eliminar la solución de la OLP y lavar 3 veces con PBS estéril. </li><li> Retire PBS y añadir 500 l / pocillo de 20% IFB / 1 g / ml laminina. Incubar toda la noche a 37 ° C en la incubadora de CO 2. </li></ol></li><li> Plating las células para inmunofluorescencia <ol><li> Retire medio de revestimiento de placas de 12 pocillos. </li><li> Añadir 900,000 células / pocillo en 2 ml de volumen y hacer crecer las células a 37 ° C en la incubadora. Las células se pueden mantener en cultivo de hasta 15 días sin reemplazo medio. </li></ol></li><li> La inmunotinción <ol><li> Retire medio de placas de 12 pocillos y lavar con 500 l / pocillo DMEM precalentado a 37 ° C. </li><li> Añadir 500 l / pocillo de DMEM precalentado a 37 ° C, a continuación, añadir con cuidado 160 l / pocillo de 8% de paraformaldehído (PFA, concentración final del 2%) y se incuba durante 10 min a 37 ° C. </li><li> Eliminar PFA y lavar 3 veces con PBS / 0,1 M de glicina durante 10 min. </li><li> Retirar PBS, añadir 300 l / pocillo de PBS / 0,05% de Triton X-100/20% de suero de cabra y se incuba durante 30 min a temperatura ambiente. </li><li> Retire medio, añadir 300 l / pocillo de anticuerpo primario diluido en PBS / 0,05% de Triton X-100/1% de suero de cabra y se incuba durante 2 horas a temperatura ambiente. Los anticuerpos primarios que se pueden utilizar para la detección de las neuronas dopaminérgicas y caracterización de la cultura se indican en la Tabla de Materiales. Mantenga1 bien sin anticuerpo primario para evaluar para el fondo de los diferentes anticuerpos secundarios. </li><li> Retire medio y lavar 3 veces con PBS / 0,2% de gelatina durante 10 minutos. </li><li> Retire medio, añadir 300 l / pocillo de anticuerpo secundario diluido en PBS / 0,05% de Triton X-100/1% de suero de cabra y se incuba durante 1 hora a temperatura ambiente, en la oscuridad. Secundaria anticuerpos utilizados se indican en la tabla de Materiales. </li><li> Retire medio y lavar 3 veces con PBS / 0,2% de gelatina durante 10 minutos. </li><li> Retire medio y lavar 3 veces con PBS. </li><li> Montar los cubreobjetos en el lado diapositivas celda de vidrio en una gota de VectaShield. Mantenga las diapositivas a temperatura ambiente durante la noche para permitir que el montaje medio seco, y luego almacenarlas a 4 ° C. </li><li> Adquirir imágenes utilizando un microscopio confocal o un microscopio de contraste de fase equipado para epifluorescencia. Imágenes representativas fueron adquiridas con un microscopio confocal Leica SP2 UV. </li></ol></li></ol>

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T., Silva, R. M., Burke, R. E., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitx3+is+required+for+development+of+substantia+nigra+dopaminergic+neurons.">Pitx3 is required for development of substantia nigra dopaminergic neurons.</a> Proc Natl Acad Sci U S A. 100, 4245-4250 (1073).</li><li>Ferri, A. 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The authors have nothing to disclose.

Este protocolo presenta los procedimientos y reactivos necesarios para preparar un cultivo primario de neuronas mesencefálicas desde el ratón embrionario y el procedimiento de inmunofluorescencia para detectar las neuronas dopaminérgicas. Los pasos críticos del procedimiento son la disección de los embriones y la disociación mecánica de los fragmentos del cerebro recogidos. Instrumentos de disección de alta calidad ayuda a dominar la técnica de disección. Neuronas DA constituyen una pequeña proporción de mes...

La dopamina, uno de los neurotransmisores cerebrales esenciales 1,2, se libera principalmente por dopaminérgicas del cerebro medio (DA), las neuronas. La mayoría de las neuronas DA residen en la parte ventral del mesencéfalo 2-6. Esquemáticamente, las neuronas dopaminérgicas del cerebro medio se pueden dividir en tres anatómica y funcionalmente distintos sistemas de proyección: mesostriatal, mesolímbico y vías mesocorticales 2,5. La vía nigroestriatal está implicado en el comportamiento motor, las vías mesolímbico juegan un papel importante en el refuerzo, la motivación y el aprendizaje, mientras que las vías dopaminérgicas que se proyectan hacia la corteza prefrontal están implicados en la cognición 2. Neuronas DA están implicados en varios trastornos neurológicos humanos tales como la esquizofrenia, déficit de atención, trastorno de hiperactividad, y la enfermedad de Parkinson (PD) 2,4. PD se caracteriza por una degeneración progresiva y selectiva de las neuronas DA de conexión sustancia negrapars compacta (SNC) a el cuerpo estriado. La pérdida de nigro-estriatal neuronas DA resultados en grave agotamiento de la dopamina en el cuerpo estriado que es responsable de los síntomas motores de la EP (bradicinesia, temblor de reposo, y la rigidez) 7. La causa inicial de la idiopática PD no se ha establecido y los tratamientos actuales sólo son sintomáticos, con el objetivo de restauración del nivel de dopamina en el cuerpo estriado. El fármaco más prescrito es la L-Dopa (Levodopa), el precursor natural de la dopamina. Aunque la administración de levodopa compensa la pérdida de dopamina durante un cierto tiempo, las complicaciones motoras se producen después de los tratamientos a largo plazo (discinesia y estados activado / desactivado) 8,9. La investigación sobre las neuronas dopaminérgicas y PD está en constante progresión y se están realizando intensos esfuerzos para desarrollar tratamientos basados ​​en el trasplante de células, la terapia génica, o agentes neuroprotectores 10,11. Sin embargo, un problema importante sigue siendo no dilucidado: ¿cuál es la causa de la extrema Vulnerability de neuronas DA? Parte de la respuesta se puede encontrar en la actividad de las neuronas dopaminérgicas. Una reducción en la actividad eléctrica y de la excitabilidad de las neuronas DA parece aumentar su propensión a degenerar 12. Sin embargo, la complejidad de la EP patogénesis requiere más estudios para identificar los mecanismos implicados en la degeneración de neuronas DA 13-15. Los cultivos primarios son especialmente relevantes para el estudio de las propiedades de las neuronas DA 16-19 y el de impugnar estas neuronas a varios tipos de estrés para la evaluación de agentes neuroprotectores 20-24. Se utilizan más a menudo modelos de cultivo de rata, como la disección de mesencéfalo de embriones de rata es más fácil, en comparación con el ratón, y cantidades más altas de las neuronas se pueden obtener en la rata. Sin embargo, la generación de modelos de ratones transgénicos de la enfermedad 25 ha aumentado considerablemente el interés de la comunidad neurocientífica para cultivos primarios de ratón 26-29. Aunque las culturas prepared de los animales recién nacidos puede ser utilizado, es mejor para prepararlos a partir de embriones en la etapa de post-mitótico (E13.5 para las neuronas de mesencéfalo), cuando las neuronas han conservado su capacidad de diferenciarse. El siguiente protocolo presenta mesencéfalo neuronas aisladas en cultivo primario a partir de embriones de ratón (E13.5), que son los más difíciles de preparar. En particular, se proporciona un protocolo de uso de medio de cultivo libre de suero para una mejor reproducibilidad. Los dos pasos más importantes en la preparación de la cultura (disección y disociación mecánica) se detallan cuidadosamente en el vídeo asociado.

<table border="0" cellpadding="0" cellspacing="0" width="1291">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">DMEM 4.5g/L Glucose with L-Glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">0.05% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td style="width:139px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 200 mM Solution</td>
			<td>Life Technologies</td>
			<td>25030123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D0547</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Laminin - 1 mg/mL in Tris buffered NaCl</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Ornithine hydrobromide</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin from porcine pancreas</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">I5523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apo-Transferrin human</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Putrescine dihydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sodium selenite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">H4034&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G7126</td>
			<td>Stock solution 1M in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G9391</td>
			<td>Stock solution 2% (w/v) in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% in water</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:139px;">RT 15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">106329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D(+)-Glucose, Monohydrate</td>
			<td>Merck Millipore</td>
			<td>4074-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Hydrochloric acid - c(HCl) = 1 mol/l (1 N) Titripur</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">109057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile water - Aqua B. Braun</td>
			<td style="width:176px;">Braun</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Ethanol absolute NORMAPUR analytical reagent</td>
			<td>VWR</td>
			<td style="width:139px;">20821.321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile Petri Dishes</td>
			<td>VWR</td>
			<td>82050-566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td>
			<td>VWR</td>
			<td>612-2297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Counting chamber Malassez</td>
			<td>VWR</td>
			<td style="width:139px;">631-0975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &#181;m, 37 mm</td>
			<td>PALL Life science</td>
			<td style="width:139px;">4525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td>14002-14</td>
			<td>To open the abdominal wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors - Straight, pointed, delicate, 10 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>4877A</td>
			<td>To open the uterine wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Curved, usual, serrated jaws 1 mm</td>
			<td style="width:176px;">MORIA</td>
			<td>2183</td>
			<td>To manipulate embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Vannas Scissors - Curved, pointed, 7 mm blades</td>
			<td style="width:176px;">MORIA</td>
			<td>MC50</td>
			<td>To take out the mesencephalon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Forceps - Curved, delicate, 13 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>9987</td>
			<td>To remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">356414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Microscope Slides, Ground edges 90&#186;</td>
			<td>MENZEL-GL&#196;SER</td>
			<td style="width:139px;">AG00008032E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision cover glasses thickness No. 1.5H circular 18 mm &#216;</td>
			<td>MARIENFELD</td>
			<td>117580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td>
			<td>Chemicon Millipore</td>
			<td>AB5622</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td>
			<td>Chemicon Millipore</td>
			<td>MAB5406</td>
			<td>1/400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&#160;</td>
			<td>Chemicon Millipore</td>
			<td>MAB369</td>
			<td>1/500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-5-HT Antibody</td>
			<td style="width:176px;"></td>
			<td style="width:139px;"></td>
			<td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum, New Zealand Origin</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">A-31556</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11001</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11007</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Stereomicroscope</td>
			<td>Carl Zeiss microscopy</td>
			<td>Stemi-2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Bunsen Burner FIREBOY</td>
			<td style="width:176px;">VWR</td>
			<td style="width:139px;">451-0136</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of mouse dopaminergic neurons

Las neuronas dopaminérgicas representan menos del 1% del número total de neuronas en el cerebro. Esta baja cantidad de neuronas regula importantes funciones cerebrales como el control motor, la motivación y la memoria de trabajo. Las neuronas dopaminérgicas nigroestriatal selectivamente degeneran en la enfermedad de Parkinson (PD). Esta pérdida neuronal progresiva está inequívocamente asociada con los síntomas motores de la patología (bradicinesia, temblor en reposo, rigidez muscular y). El principal agente responsable de la degeneración de neuronas dopaminérgicas es aún desconocido. Sin embargo, estas neuronas parecen ser muy vulnerables en diversas condiciones. Los cultivos primarios constituyen uno de los modelos más relevantes para investigar las propiedades y características de las neuronas dopaminérgicas. Estas culturas se pueden enviar a varios agentes de estrés que imitan patología PD y para compuestos neuroprotectores para detener o ralentizar la degeneración neuronal. Los numerosos modelos de ratones transgénicos de PD que han sido generATED durante la última década se incrementó aún más el interés de los investigadores por cultivos de neuronas dopaminérgicas. En este caso, el protocolo de vídeo se centra en la delicada disección de cerebro de ratón embrionarias. La escisión precisa del mesencéfalo ventral es crucial para obtener cultivos neuronales suficientemente ricos en células dopaminérgicas para permitir estudios posteriores. Este protocolo puede ser realizado con ratones transgénicos embrionarias y es adecuado para la tinción de inmunofluorescencia, PCR cuantitativa, la cuantificación segundo mensajero, o la evaluación de la muerte / supervivencia neuronal.

Un diagrama de flujo que ilustra los pasos de cultivo mesencéfalo se muestra en la Figura 1. Brevemente, después de la recolección de embriones E13.5 de un ratón suizo embarazada, mesencéfalo ventral se diseca de todo el embrión. Los fragmentos cerebrales aisladas se someten sucesivamente a la digestión enzimática y disociación mecánica. Las células disociadas se sedimentan por centrifugación, se resuspendieron en medio de cultivo y se sembraron en placas de 12 o 24 pocillos pre-revestido. L...

Watch this Scientific Journal Video about Primary Culture of Mouse Dopaminergic Neurons at JoVE.com

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultivo primario de ratón neuronas dopaminérgicas

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

primary-culture-of-mouse-dopaminergic-neurons

Research

JoVE Journal

Neuroscience

37.2K vistas.  Institut de Génomique Fonctionnelle, Montpellier. Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Video: Primary Culture of Mouse Dopaminergic Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection y cultivos primarios de neuronas del hipocampo de embriones de ratón y corticales

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Investigación

Neurociencias

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultivo de neuronas de rata primaria cortical y la glía

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Disección y Cultura de ratón dopaminérgico estriatal en explantes y tridimensionales ensayos matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Aislamiento, cultivo y mantenimiento a largo plazo de Primaria mesencefálico neuronas dopaminérgicas a partir de cerebros de embriones de roedores

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imágenes en vivo de la proteína GLUT4 trata de ratón las neuronas hipotalámica primaria utilizando microscopía de deconvolución

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

Cultura celular primaria de las neuronas purificadas GABAérgicas o glutamatérgicas establecidas a través de la clasificación celular activada por fluorescencia

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Etiquetado balístico de las neuronas piramidales en las rebanadas cerebrales y en el cultivo celular primario

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.