This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

NOTA: Los animales fueron mantenidos y manejados de acuerdo con las directrices institucionales y de todos los animales procedimientos fueron aprobados por el Bienestar Animal del Colegio Imperial y Órgano de Examen de Ética (AWERB) y el Ministerio del Interior y la Universidad de Harvard Institucional Cuidado de Animales y el empleo Comisión (IACUC), de conformidad con los reglamentos federales y estatales. 1. Reactivo y Configuración del equipo <ol><li> Thaw, alícuota y almacenar 1 mg / ml solución laminina a -80 ºC. Disolver 2 l en 1 ml de DMEM / F12, resultando en una concentración de recubrimiento de 1-2 g / cm 2. NOTA: Descongelar Laminin lentamente en hielo y se disuelve en frío DMEM / F12 y de inmediato añadir a los platos / cubreobjetos. </li><li> Diluir 75 ml de 0,01% de poli-L-ornitina en 425 ml de PBS y se almacena a 4 ºC. NOTA: La vida útil de la solución de trabajo es de hasta un mes. </li><li> Disolver 25% (p / v) de BSA en PBS, pH 7,4, y se almacena a 4 ° C durante un máximo de un year. </li><li> Preparar 50% de FBS (en HBSS) medios de desactivación. </li><li> Preparar medio completo mediante la adición de 50 U / ml de penicilina y estreptomicina, 1x suplemento N2, 5% (vol / vol) de FBS, 0,36% D - (+) - glucosa (peso / vol), 0,25% de BSA (peso / vol) a DMEM / F12 y almacenar a 4 ºC. NOTA: La vida útil es de hasta 10 días. </li><li> Coloque los cubreobjetos en ebullición 70% (vol / vol) de etanol durante al menos 30 min para eliminar los restos de grasa y autoclave. </li><li> Coloque los cubreobjetos en placas de 24 pocillos y añadir 500 l solución de poli-L-ornitina a cada pocillo. Incubar 1 hr bajo la campana de cultivo de tejido a TA y después, lavar 3 veces con 500 l de agua. Añadir solución de laminina 500 l a cada placa y se incuba O / N en la incubadora de cultivo de tejidos humidificado a 37 ºC. NOTA: Si bien no hay lavados son necesarias después del tratamiento laminina, lavar los poli-L-ornitina menos de tres veces resulta en la muerte de las células 24 h después del cultivo. </li><li> Pulir las puntas de pipetas de vidrio más de gas natural o antorcha fcojo. NOTA: Después de este paso, el diámetro de la punta debe ser aproximadamente la mitad de su tamaño normal. </li><li> Cortar las tapas de 1,5 ml tubos de microcentrífuga, utilizando una tijera autoclave y autoclave. </li></ol> 2. Disección de Embryonic ventral mesencéfalo <ol><li> Narcotizar oportuna embarazadas (E12.5) presas por inhalación de CO2 y luego practicar la eutanasia por dislocación cervical. </li><li> Pulverizar el abdomen con etanol al 70% y hacer una incisión abdominal hasta los sacos uterinos están expuestos. Utilizando unas pinzas cortan la fijación vaginal y eliminar útero. Coloque el útero en HBSS enfriado en hielo, en un 100 mm placa de Petri. </li><li> Retire embriones del útero y el saco amniótico, utilizando pinzas (Figura 1C). Coloque los embriones en fresco HBSS helada. NOTA: El rectángulo en la Figura 1D indica la ubicación del mesencéfalo ventral embrionario. </li><li> Coloca los embriones bajo un microscopio de disección (10 aumentos) y diseccionar el mesencéfaloarco cortando el cerebro en el istmo y la región frontera mesencefálico-diencefálica, utilizando el bioscissor y pinzas (Figura 1E, por favor refiérase a la Figura 1A para las líneas de incisión). </li><li> Después de sacar todo el cerebro medio, hacer un corte en el mesencéfalo mediodorsal (Figura 1F). </li><li> Eliminar meninges, utilizando dos pinzas (Figura 1H). NOTA: PASO CRÍTICO: Este paso es esencial para el rendimiento óptimo neuronal y la supervivencia. Dado que las condiciones de cultivo son óptimos para las células del cerebro, la mayoría de las células del tejido circundante mueren después de la siembra y la señal apoptótica partir de este tejido puede dañar la integridad de las culturas. </li><li> Acoplar el tejido del cerebro medio en la placa de Petri para observar la forma de mariposa y corte aproximadamente la mitad de cada ala, utilizando una cuchilla de afeitar esterilizada (Figura 1 H, I). NOTA: Figura 1J representa el mesencéfalo ventral aislado. </li><li> Transferir elpieza de mesencéfalo ventral en HBSS enfriado con hielo en un tubo cónico de 15 ml y proceder con el siguiente embrión. NOTA: Los pasos 2.1 a 2.8 no deben tomar más de 1 hora. Mantener los embriones más allá de este marco de tiempo en hielo disminuye significativamente la viabilidad celular. </li></ol> 3. La disociación de las células ventral mesencéfalo <ol><li> La transferencia de los trozos de cerebro medio ventral bajo una campana de flujo laminar. Retire la HBSS y añadir 1 ml precalentado (37 ºC) 0,05% de tripsina-EDTA en el tubo cónico (volúmenes suficientes para un máximo de 12 piezas de mesencéfalo ventral). Incubar el tejido a 37 ºC durante 5-10 minutos. </li><li> Eliminar tripsina-EDTA bajo el capó y se añade medio de la activación de-1 ml (el suero se desactivará tripsina) para el tejido. </li><li> Retire el medio de desactivación y lavar el tejido en 1 ml de medio completo dos veces. NOTA: evitar la eliminación de todo el medio del tubo y pipetear el tejido, para evitar desechar las células disociadas. </li><li> Añadir 1 ml de medio completo y triturato el tejido con una pipeta de vidrio pulida al fuego. Evitar la formación de burbujas y seguir triturando hasta que se logre la suspensión de células individuales. NOTA: Por lo general, 8-10 pasa por la punta restringido son necesarios para la disociación completa. </li><li> Centrifugar las células a 400 xg durante 5 min a temperatura ambiente y eliminar el medio. </li><li> Vuelva a suspender el sedimento en 1 ml de medio completo por lentamente la pipeta hacia arriba y hacia abajo para un máximo de cuatro veces. </li></ol> 4. Las células Chapado ventral mesencéfalo <ol><li> Mezclar 10 l de la suspensión celular con 90 l solución de azul de tripano y contar el número de células con un hemocitómetro. NOTA: La viabilidad de las células también se puede comprobar en esta etapa. </li><li> Coloque tapas de los tubos de microcentrífuga esterilizado en 100 mm placas de Petri y transferir los / laminina cubreobjetos recubiertos de poli-L-ornitina, utilizando fórceps, uno a la vez en la parte superior de las tapas. NOTA: No lave los cubreobjetos y evitar el secado de la laminina, ya que esto puede causar UNEVEculturas n y disminución de la supervivencia de las células. </li><li> Ajustar el volumen a 1500 células por 1 l mediante la adición de medio completo y añadir 100 l (un total de 150.000 células) de la suspensión celular en la parte superior de cada cubreobjetos (el volumen es óptimo para cubrir la superficie del cubreobjetos sin derrames). Cierre las placas de Petri y se incuba durante 1 hr ellos en una incubadora de cultivo de tejidos humidificado (37 ° C, 5% de CO 2). NOTA: PASO CRITICAL: Este paso es necesario para mejorar la unión y viabilidad de las células y para aumentar el número de cubreobjetos por embrión. Alternativamente, las células se pueden transferir directamente a los pozos (que contienen cubreobjetos), pero el número de células se debe aumentar a 350 mil por pozo (en lugar de 150.000). En otras palabras, utilizando este método, 8-10 cubreobjetos se pueden adquirir, mientras que la transferencia directa produce unos 3-4 cubreobjetos a causa de las células, que se unen a las placas (al lado o debajo de los cubreobjetos). El viabili promedioTy de los cultivos, utilizando este método fue de alrededor de 90%. </li></ol> 5. Crecimiento y Mantenimiento Cultura <ol><li> Después de 1 hora de incubación, transferir cuidadosamente el cubreobjetos incluyendo el medio en placas de 24 pocillos, que contiene 400 l de medio completo precalentado (a 37 ºC) (volumen total: 500 l). Se incuban las células O / N a 37 ºC. </li><li> Dentro de 24 horas después de la incubación de los pocillos, añadir suavemente medio completo 500 l en cada pocillo. NOTA: Las primeras horas es la etapa más crítica para la supervivencia de las neuronas dopaminérgicas y el número de células supervivientes no cambia significativamente más allá de este punto. </li><li> No agregue soportes adicionales a las culturas de las dos primeras semanas o hasta que el medio se convierte en amarilla. Si el cultivo durante más de dos semanas o cambios de color medio a amarillo, cambio de la mitad de los medios de comunicación (500 l) con frescos completas medios pre-calentado (normalmente cada dos semanas). NOTA: Las neuronas dopaminérgicas dentro de laculturas sobrevivirían durante más de 6 semanas bajo estas condiciones. </li></ol>

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No conflict of interest declared.

Las neuronas dopaminérgicas en el mesencéfalo son la principal fuente de dopamina en el sistema nervioso central. Se dividen en tres grupos, sustancia negra pars compacta (SNpc), el área ventral tegmental (VTA) y el campo retrorubral (FRR) 10, 11. Las neuronas SNpc VTA y dan lugar a importantes vías dopaminérgicas, mesocorticales, mesolímbico y nigro-estriado, que participan en funciones como el control de las emociones, la motivación y el comportamiento motor. Desaparición de las neuronas en SNpc y a...

La pérdida de neuronas dopaminérgicas de la sustancia negra pars compacta conduce a los síntomas motores cardinal de la enfermedad de Parkinson (PD), el segundo trastorno neurodegenerativo más frecuente. La causa subyacente de la desaparición de esta población neuronal mesencephalic no se conoce. Para el estudio de las vías bioquímicas responsables del desarrollo y la modulación de las propiedades neurofisiológicas y la supervivencia de las neuronas Mesda, varios de cultivo de células y sistemas de modelos animales se han utilizado. Las líneas celulares inmortalizadas, incluyendo la línea celular de rata dopaminérgica 1RB 3 AN 27 (N27), el dopaminérgica línea celular de neuroblastoma humano SH-SY5Y, la línea celular híbrida de ratón dopaminérgica MN9D y mesencefálico humana LUHMES células se han utilizado para estudios mecanísticos bioquímicos y limitadas 1 -5. Para el estudio de la pérdida específica de neuronas Mesda, varios neurotoxina y basados ​​en modelos genéticos se han desarrollado 6-8. Culturas mesencéfalo ventral primaria, Proporcionar una herramienta indispensable para el estudio de las propiedades neuronales y sinápticas de las neuronas dopaminérgicas y las vías implicadas en la patogénesis de esta enfermedad común. Aquí presentamos un protocolo detallado para el aislamiento de las neuronas dopaminérgicas mesencefálicas, que contiene modificaciones resultantes en una mayor capacidad de supervivencia y el aumento de rendimiento de cubreobjetos por embrión. El uso del mesencéfalo E12.5 ratón prematuro (E14.5 en rata) mejora la supervivencia. A esta edad las neuronas no se han desarrollado todavía los axones, lo que deja las células intactas durante la disección y minimiza el estrés aumentando así significativamente la viabilidad. Además, la disección cuidadosa del mesencéfalo ventral, como se describe en la sección 2 de este protocolo, mejora aún más la capacidad de supervivencia. Para aumentar el número de cubreobjetos por embrión, una técnica de siembra alternativa se presenta en la sección 4 de este protocolo. Esto conduce a un rendimiento de hasta 10 cubreobjetos por embrión en comparación con 4 cubreobjetos bajocondiciones de chapado estándar reduciendo así la cantidad de animales por experimento. Las neuronas en la cultura de exposiciones consecuencia de los axones y dendritas, forman conexiones sinápticas y revelan la presencia de marcadores neuronales y sinápticas que hacen estas culturas adecuado para imágenes de células vivas, inmunocitoquímica y estudios electrofisiológicos. Además, el uso de cultivos neuronales facilita la manipulación genética y farmacológica. Excrecencia de neuritas a partir del día 2 in vitro permite estudios de desarrollo. Por otra parte, la supervivencia a largo plazo de las culturas (hasta seis semanas) hace que sean adecuadas para el estudio de la degeneración progresiva y lenta de estas neuronas.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:364px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified Eagle medium nutrient mixture F-12</td>
			<td>Invitrogen</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td>Invitrogen</td>
			<td>24020-117)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum, heat-inactivated (FBS)</td>
			<td>Invitrogen</td>
			<td>16140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td>17502-048)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose solution (45% (wt/vol) in water</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin BSA</td>
			<td>Sigma</td>
			<td>A9430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% (wt/vol)</td>
			<td>Invitrogen</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA) cell culture tested</td>
			<td>Sigma&#160;</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Tyrosine hydroxylase (Th) antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td>P60101-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-ornithine, 0.01% solution</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td>
			<td>Millipore</td>
			<td>MAB3418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Synapsin-1 antibody</td>
			<td>Millipore</td>
			<td>AB1543P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-mouse IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue solution (0.4% (wt/vol)</td>
			<td>Biowhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted phase contrast microscope</td>
			<td>Carl Zeiss</td>
			<td>Axiovert 40 C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>May-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover Slip Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11251-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two Dumont #45 Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11245-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blade Holder/Breaker Flat Grip - 11cm</td>
			<td>Fine Scientific Tools</td>
			<td>10052-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Iris Scissors - Straight 11.5cm</td>
			<td>Fine Scientific Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber optic halogen illuminator</td>
			<td>Nikon</td>
			<td>MKII</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Proscitech</td>
			<td>SVZ2NIOU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m sterile filter units</td>
			<td>Nalgene</td>
			<td>NL-CE-156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100x20mm Petri dishes</td>
			<td>BD Biosciences</td>
			<td>351005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Cover Slip #1 Thickness German Glass 12mm</td>
			<td>Bellco Glass</td>
			<td>1943-10012&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La inmunohistoquímica contra la tirosina hidroxilasa (TH) muestra que entre 0,5-1% de las células en cultivo son dopaminérgica. Proyecciones neuronales aparecen dentro de 2 horas después de la siembra y antes del primer día, los axones y dendritas son distinguibles (Figura 2), utilizando proteína 2 (Map2) anticuerpos asociada a los microtúbulos (Figura 3) tirosina hidroxilasa (TH) y. Las neuronas a sobrevivir durante más de seis semanas y muestran una amplia extensión. La relac...

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

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

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 ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Research

JoVE Journal

Neuroscience

13.8K vistas.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’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.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Aislamiento y cultivo de neuronas del hipocampo de ratones prenatales

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

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 ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Aislamiento y cultivo de neuronas sensoriales disociadas De embriones de pollo

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Un método simplificado para determinar la densidad ultra-baja, a largo plazo del hipocampo primaria Neurona Cultura

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Aislamiento y Cultura de Neuronas de la Médula Espinal de Ratones Neonatales

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generación y Mantenimiento a Largo Plazo de Nervios<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Aislamiento y extracción de ARN de las neuronas, macrófagos y Microglia de cerebros de larvas de pez cebra

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...