Este trabajo es apoyado por el NIH subvención NS065183 y los fondos de puesta en marcha de la Escuela de Medicina Robert Wood Johnson de Rutgers.

1. Preparación Rata Astrocyte la cultura para el medio condicionado y astrocitos-neuronas cocultivos. <ol><li> Prepare el tampón de disección (BSS, véase la Tabla 1 para la receta) y se almacena a 4 ° C hasta que esté listo para su uso. </li><li> Anestesie crías de ratas neonatales (P0-P2) con isoflurano en un vaso de 500 ml. </li><li> Cuando los cachorros son inmóviles, rocíe con etanol al 70% y decapitar. </li><li> Retire el cerebro. <ol><li> Sostenga firmemente la cabeza con un par de Dumont # 5 pinzas, y utilizar tijeras finas para hacer una incisión en la línea media a través de la piel y el cráneo. </li><li> Exponer el cerebro al reflejar el cráneo a los lados, y retire el cerebro en un recipiente lleno de frío BSS. </li></ol></li><li> Separar los hemisferios cerebrales desde el diencéfalo y el tronco cerebral bajo un microscopio de disección. Retire con cuidado todas las meninges al estabilizar el tejido con un par de pinzas y tirando suavemente las meninges con otro par de pinzas. </li><li> Recoge todos los hemisferios in una placa de 60 mm. Utilizando unas tijeras pequeñas, tejido picada lo más fino posible. A continuación, transferir el tejido picado a un tubo cónico de 50 ml. </li><li> Añadir 1,5 ml de 1% de DNasa I y 1,5 ml de 2,5% de tripsina y 12 ml de BSS (volumen total de 15 ml) a los cerebros. Incubar en un baño de agua en agitación durante 15 min a 37 ° C. Para asegurar una buena mezcla, el tubo se agita manualmente cada 5 minutos. </li><li> Transferir el sobrenadante a través de un filtro de 70 micras de células en un nuevo tubo cónico de 50 ml. Añadir 3 ml de FBS a este sobrenadante. </li><li> Para las piezas restantes, añadir 13,5 ml de BSS y 1,5 ml de 2,5% de tripsina y se incuba en un baño de agua agitando durante otros 15 min. </li><li> Transferir el sobrenadante restante a través del filtro celular y combinar con el sobrenadante de la Etapa 1.8. </li><li> Centrifugar a 1000 rpm durante 5 min para sedimentar las células. </li><li> Volver a suspender las pellas en 5 ml de medio gliales. El sobrenadante se puede centrifugar de nuevo para sedimentar las células restantes. </li><li> Contar las células utilizando un hemocitómetro. </li><li> Cel PlateLS a una densidad de 2 matraz de 1 x 10 7 por 150 cm. </li><li> Cambie medios de comunicación para medios gliales frescas del día después de la siembra. Después, las células alimentar dos veces por semana con los medios de comunicación de la glía. </li><li> Cuando las células han alcanzado&gt; 80% de confluencia (aproximadamente 10 días después de la siembra), congelar las células en 90% de suero de caballo y 10% de DMSO y mantener las existencias en nitrógeno líquido. Las células se congelaron a 2 x 10 6 por vial. Aproximadamente 5 viales se pueden congelar de cada matraz. </li><li> Acerca de 10-14 días antes de la creación de la cultura del hipocampo, las células gliales se siembran de las existencias congeladas. Por lo general, la placa de cinco placas de 6 pocillos, lo cual es suficiente para apoyar el crecimiento de 90 cubreobjetos de las neuronas del hipocampo (tres cubreobjetos de 15 mm redondo por pocillo). También PLACA un adicional de ocho a diez platos de 60 mm de las células gliales, que se utiliza para medios acondicionado N2.1 para la transfección. El día antes del cultivo neuronal, las células deben ser alimentados con medios NB27. Los astrocitos idealmente deben ser&gt; 90% de confluencia en el thes el punto. Encontramos que la congelación reduce en gran medida el número de microglia en las culturas astroglial. Dado que se observa una mayor neurotoxicidad cuando microglia está presente en las culturas astrogliales, siempre utilizamos la cultura astroglial preparado a partir de material congelado por cocultivo con las neuronas. </li></ol> 2. Cultivos neuronales <ol><li> Limpie cubreobjetos de vidrio por primera enjuague en agua 2x milliQ. A continuación, se sumergen en ácido nítrico concentrado durante 24 horas, y se aclararon con agua MilliQ durante al menos cinco veces durante un total de 2 horas. Los cubreobjetos se secan y se esterilizan por cocción en un horno a 225 ° C durante 6 horas. </li><li> Transferencia cubreobjetos de 60 mm platos después de la esterilización. Coloque cuatro puntos de parafina estéril cerca del borde exterior de cada cubreobjetos para mantener las neuronas se separan a partir de células gliales durante cocultivo. </li><li> Cubreobjetos capa con 1 mg / ml de poli-L-lisina en tampón de borato 0,1 M durante la noche en 37 ° C incubadora. </li><li> El día siguiente, enjuague cubreobjetos dos veces en m estérililliQ agua durante al menos 15 minutos cada uno. A continuación, se incubaron durante la noche en medio de siembra en un 37 ° C incubadora. </li><li> La eutanasia a ratas preñadas (E18) por la anestesia isoflurano seguido de neumotórax, y la eutanasia a las ratas embrionarias por decapitación. Eliminar cerebros y diseccionar los hemisferios cerebrales, como se describe más arriba. </li><li> Diseccionar los hipocampos del hemisferio cerebral. Coloque todos los hipocampos en un tubo cónico de 15 ml, y digerir con tripsina al 0,25% (0,5 ml de 2,5% de tripsina, 4,5 ml de BSS) a 37 ℃ durante 15 min. </li><li> Hipocampos digeridos se enjuagan en BSS 3x durante 5 minutos cada uno. A continuación, se trituró con una pipeta Pasteur de vidrio, y la densidad celular se determina usando un hemocitómetro. Las neuronas se sembraron a continuación a una densidad de 2 x 10 5 células por placa de 60 mm. </li><li> Acerca de 2-4 horas después de la siembra, la transferencia cubreobjetos con las neuronas asociadas a las placas que contienen células astroglial, con las neuronas hacia abajo hacia la glía. En DIV3, añadir arabinósido de citosina a lacélulas a una concentración final de 5 mM para detener la proliferación de células gliales. </li></ol> 3. La transfección de calcio <ol><li> Medios N2.1 Condición de glia el día antes de la transfección. </li><li> En el día de la transfección, los medios de comunicación tomar N2.1 acondicionado fuera de la glía y transferir a un nuevo plato. Transferencia cubreobjetos con las neuronas en los medios de comunicación N2.1 acondicionado. Deje que equilibre en incubadora durante 10 a 30 min. </li><li> Para un conjunto de tubos estériles, combine 1-4 g de ADN, 12,5 l de 2 M de CaCl 2 y H 2 O estéril para un volumen total de 100 l. Para un segundo conjunto de tubos, añadir 100 ml de 2x HBS. </li><li> Añadir un volumen ⅛ de 2x HBS (12,5 l) a la vez para el tubo que contiene la mezcla de CaCl 2 / ADN, vórtice durante unos pocos segundos cada vez. Dejar que los tubos para sentarse durante 15 minutos. </li><li> Después de 15 min de incubación, añadir gota a gota la mezcla de transfección a las células. Incubar durante 1 a 1,5 horas. Una capa de precipitados como la arena debe ser visiblebajo el microscopio utilizando un objetivo de 10X. </li><li> Después de la incubación, enjuague cubreobjetos dos veces con tampón de lavado caliente HBS. </li><li> Cubreobjetos Volver a platos originales con glía, añadir ácido quinurénico a una concentración final de 0,5 mM. </li><li> Neuronas transfectadas se pueden obtener imágenes en vivo o procesadas para la inmunocitoquímica tan pronto como al día siguiente. Las neuronas pueden sobrevivir hasta tres semanas después de la transfección. </li></ol>

<ol>
	<li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30, 6171-6177 (2010).</li><li>Zeitelhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+mammalian+neurons+via+nucleofection.">High-efficiency transfection of mammalian neurons via nucleofection.</a> Nat. Protoc. 2, 1692-1704 (2007).</li><li>Janas, J., Skowronski, J., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+delivery+of+RNAi+in+hippocampal+neurons.">Lentiviral delivery of RNAi in hippocampal neurons.</a> Method Enzymol. 406, 593-605 (2006).</li><li>Graham, F. L., vander Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52, 456-467 (1973).</li><li>Craig, A. M., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+cultured+neurons.">Transfecting cultured neurons.</a> Culturing Nerve Cells. , (1998).</li><li>Alavian, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13, 1224-1233 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+synaptic+function+by+VAC14,+a+protein+that+regulates+the+phosphoinositides+PI(3,5)P(2)+and+PI(5)P.">Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P.</a> EMBO J. 31, 3442-3456 (2012).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Curr. Protoc. Neurosci. Chapter 3, Unit 3 11 (2001).</li><li>Goslin, K. A. H., Banker, G., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Hippocampal+Neurons+in+Low-Density+Culture.">Rat Hippocampal Neurons in Low-Density Culture.</a> Culturing Nerve Cells. , (1998).</li><li>Kohrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+convenient,+and+effective+method+to+transiently+transfect+primary+hippocampal+neurons.">Fast, convenient, and effective method to transiently transfect primary hippocampal neurons.</a> J. Neurosci. Res. 58, 831-835 (1999).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Goetze, B., Grunewald, B., Baldassa, S., Kiebler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+formation+of+a+DNA/calcium+phosphate+coprecipitate:+application+for+transfection+of+mature+hippocampal+neurons.">Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.</a> J. Neurobiol. 60, 517-525 (2004).</li></ol>

No hay conflictos de interés declarado.

Hay varios parámetros clave que deben ser cuidadosamente controlada para transfecciones consistentemente exitosos 10,11. El parámetro más crítico para la transfección de fosfato de calcio es el valor de pH de 2x HBS, que en nuestras manos por lo general varía entre 7.10 hasta 7.15. Se recomienda hacer tres lotes de acciones con valores de pH en incrementos de 0.05 para dar cuenta de la diferencia entre los medidores de pH. Alternativamente, el kit de transfección de mamíferos Clontech proporciona 2x HBS que produce consistentemente buena eficiencia. Tenga en cuenta que el pH de la solución cambiará con el tiempo, incluso cuando se almacenan en el congelador. Generalmente Mantenemos alícuotas de 2x HBS a 4 ° C durante un máximo de un mes, ya -20 ° C durante un máximo de un año. El segundo parámetro es el pH del medio de cultivo. Para mantener este, medio N2.1 consistentes están condicionados de manera rutinaria en las culturas astrogliales noche a la mañana, pero no más de 24 horas. Además, tratamos de utilizar las culturas astrogliales ªa son similares en confluencia. El uso de los medios de comunicación glía acondicionado también ayuda a reducir la toxicidad a las neuronas durante la transfección. Los medios condicionados se equilibran en la incubadora antes de la transfección para mantener el pH constante. El tamaño y la densidad de los precipitados de fosfato de calcio es la clave para la transfección con éxito. Precipitados pequeños podrían conducir a una baja eficiencia de transfección, mientras que las grandes precipitados, aglutinadas por lo general conducen a citotoxicidad 10. Para asegurar la formación de fosfato de calcio precipitado coherente, utilizamos vórtice intermitente al mezclar el ADN / CaCl 2 y 2x HBS 11. Otro método comúnmente utilizado para la mezcla es a través de soplado de burbujas de aire utilizando una pipeta Pasteur 12. Hemos encontrado el vórtice intermitente para producir la formación de precipitado más consistente, especialmente para pequeños volúmenes de mezcla de transfección. Otras variables a considerar incluyen la calidad del ADN, y la edad de la neurons. Nos encontramos con que la transfección empezar a trabajar constantemente cuando las neuronas llegan DIV2, sin embargo, la eficiencia empieza a disminuir cuando las neuronas están más allá DIV10. Finalmente, la salud de los propios cultivos neuronales representa otro parámetro importante. Nos encontramos con el uso del sistema de co-cultivo que es la mejor manera de generar cultivos de hipocampo primaria saludables. Si las neuronas no son saludables, no van a sobrevivir mucho tiempo después de la transfección. Con el sistema de co-cultivo, las neuronas de baja densidad pueden sobrevivir por hasta 3 semanas después de la transfección, lo que facilita los estudios a largo plazo.

Neuronas primarias son uno de los tipos de células más difíciles de transfectar ya que son postmitotic y son muy sensibles a los cambios micro-ambiental. Hay cuatro tipos de uso común de los métodos para la expresión de genes exógenos y los ARN corto horquilla (shRNAs) en estas células 1. Cada uno tiene sus propias ventajas y desventajas. Por ejemplo, electroporación se realiza generalmente en las neuronas recién aisladas 2, como las células deben ser transferidos en las cubetas para la transfección. Infección por el virus generalmente se puede lograr una eficiencia muy alta 3, pero es y arriesgado para los operadores más intensivos en mano de obra. Muchos reactivos de transfección mediadas por lípidos están disponibles comercialmente, con diversos grados de éxito en las neuronas y diferentes niveles de citotoxicidad. Transfección con fosfato de calcio representa un método conveniente y económico para la introducción de genes extraños en las neuronas. El método fue utilizado por primera vez para introducir ADN adenovirus en cel mamíferosls por Graham y Van Der Eb (1973) 4. La transfección se realizó mediante la mezcla de cloruro de calcio con el ADN recombinante en un tampón de fosfato. Esto permite la formación de fosfato de ADN / calcio precipitados que, cuando se deja caer poco a poco sobre una monocapa de células, se adhieren a la superficie celular, son absorbidos por endocitosis y finalmente entrar en el núcleo 5. Este proceso daría lugar a la expresión de genes extraños introducidos en la célula diana. Eficiencias típicas de calcio gama transfección con fosfato de entre 0,5-5% 6-8. Sin embargo, con la optimización cuidadosa y ejecución consistente del protocolo experimental, es posible llegar a una eficacia de transfección de casi el 50%. Aquí describimos nuestro protocolo detallado para la transfección de fosfato de calcio de las neuronas del hipocampo primarias, que se co-cultivaron con células astroglial en un formato sándwich 9.

<table border="0" cellpadding="0" cellspacing="0" width="879">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">Dumont #5 forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Fine scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td>straight / sharp 8.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Vannas-Tubinger spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15008-08</td>
			<td>angled / sharp / 8.5 cm / 5 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Standard pattern forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11000-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Student surgical scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">91401-14</td>
			<td>blunt / 14.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td>angled to side / 9 cm / 10 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Isoflurane</td>
			<td style="width:207px;">Webster Veterinary</td>
			<td style="width:119px;">14043-225-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Poly-L-lysine</td>
			<td>Sigma</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Sigma</td>
			<td>M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">100 mM Sodium pyruvate</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Glucose</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x HBSS</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">1 M HEPES, pH 7.3</td>
			<td>Gibco/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Gibco/Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Media</td>
			<td>Gibco/Invitrogen</td>
			<td>21130-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement (50x)</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">2.5% Trypsin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">DNase I</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Cytosine arabinoside</td>
			<td style="width:207px;">Calbiochem/EMD Millipore</td>
			<td align="right" style="width:119px;">251010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Kynurenic acid</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium phosphate transfection of primary hippocampal neurons

Precipitación con fosfato de calcio es un método conveniente y económico para la transfección de células cultivadas. Con la optimización, es posible utilizar este método en las células difíciles de transfectar tales como neuronas primarias. A continuación se describe el protocolo detallado para la transfección de fosfato de calcio de las neuronas del hipocampo cocultured con células astroglial.

Cuando los diferentes parámetros de la transfección se optimizan y controlan cuidadosamente de experimento a experimento, es posible obtener eficiencias de transfección de hasta el 50%. La Figura 1 muestra un campo de neuronas que están transfectadas con GFP en DIV4. El campo contiene un total de 28 neuronas, entre los cuales 16 fueron transfectadas. Esto representa un rendimiento de más del 50%. Un muestreo de otros campos de la misma cubreobjetos muestra la eficiencia global es de alrededor de 50% (datos no mostrados). Con el sistema de cocultivo astroglial, las neuronas permanecen sanos y desarrollarse normalmente después de la transfección. Figura 2 muestra una neurona del hipocampo en DIV15, 10 días después de la transfección con una construcción de PSD-95-GFP. La neurona ha desarrollado numerosas espinas dendríticas maduras en forma de hongo, lo que indica que las neuronas son saludables. La alta eficiencia de transfección y mínima toxicidad de este protocolo hace que sea una herramienta valiosa para el estudio de las funciones de genes en hipopótamo primarianeuronas Campal. Finalmente, este protocolo puede ser potencialmente adaptada para transfectar cultivos de otros tipos de neuronas en el cerebro, así como otros tipos de células difíciles de transfectar. <img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50808/50808fig1highres.jpg" src="/files/ftp_upload/50808/50808fig1.jpg" width="600px" /> Figura 1. Las neuronas del hipocampo transfectadas con GFP en DIV4, mostrando una alta eficiencia de transfección. 16 de 28 neuronas en el campo son positivas. <a href="https://www.jove.com/files/ftp_upload/50808/50808fig1highres.jpg" target="_blank">Haz clic aquí para ver la imagen más grande</a> . <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50808/50808fig2highres.jpg" src="/files/ftp_upload/50808/50808fig2.jpg" width="600px" /> Figura 2. Neurona del hipocampo transfectadas con el PSD-95-GFP en DIV15, mostrando numerosas espinas dendríticas maduras en forma de hongo. <a href="https://www.jove.com/files/ftp_upload/50808/50808fig2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande</a> . Tabla 1. Buffer / Recetas Medios. <table border="1"><tbody><tr><td> Reactivo </td><td> Componentes </td></tr><tr><td> Glial Medios </td><td> 425 ml de MEM 5 ml GlutaMAX 5 ml de piruvato de sodio, 100 mM 15 ml de 20% de glucosa 25 ml de FBS (5% final) Suero de 25 ml de ternera (5% final) 5 ml de penicilina / estreptomicina </td></tr><tr><td> 0,1 M tampón borato </td><td> 2,48 g de ácido bórico 3,9 g de tetraborato de sodio (bórax) 800 ml de H2O milliQ pH con NaOH a 8,5 Se esteriliza con filtro </td></tr><tr><td> Plating Medios </td><td> 425 ml de MEM 5 ml GlutaMAX 5 ml de piruvato de sodio, 100 mM 15 ml de 20% glucose FBS 50 ml </td></tr><tr><td> NB27 Medios </td><td> 485 ml Neurobasal Medios 5 ml GlutaMAX 2 ml de suplemento B27 (50x) </td></tr><tr><td> Disección Buffer (BSS) </td><td> 50 ml de HBSS 10x 5 ml de HEPES 1 M, pH 7,3 5 ml de penicilina / estreptomicina 440 ml de H2O </td></tr><tr><td> N2.1 Medios </td><td> 425 ml de MEM 5 ml GlutaMAX 5 ml de piruvato de sodio, 100 mM 15 ml de 20% de glucosa 50 ml de ovoalbúmina, 1% en MEM 5 ml de suplemento de N2 </td></tr><tr><td> 2x HEPES Buffered Saline (HBS), estéril </td><td> NaCl 274 mM 9,5 mM KCl 15 mM de glucosa 42 mM HEPES 1,4 mM Na 2 HPO 4 Preparar lotes de 3 pH diferentes (7,05, 7,10, 7,15) </td></tr><tr><td> Tampón de lavado (HEPES Buffered Saline) </td><td> 50 ml de HBSS 10x 5 ml de HEPES 1 M, pH 7,3 445 ml de H2O</td></tr><tr><td> 50 mM de ácido quinurénico </td><td> Disolver 0,473 g de ácido quinurénico en 1x PBS. Añadir gotas de NaOH conseguirlo en la solución. A continuación, utilice HCl para ajustar de nuevo el pH a 7,0 </td></tr></tbody></table> Tabla 2. Línea de tiempo para la preparación de la cultura. <table border="1"><tbody><tr><td> Día </td><td> Acción </td></tr><tr><td> Antes de iniciar </td><td> Preparar la cultura de astrocitos y congelar las células hacia abajo. Un lote de la cultura general de los astrocitos puede soportar varios meses de cultivos de hipocampo. </td></tr><tr><td> Semana 1 Lunes </td><td> Descongele los astrocitos. </td></tr><tr><td> Semana 1 Martes </td><td> Alimente astrocitos. </td></tr><tr><td> Semana 1 Viernes </td><td> Alimente astrocitos. </td></tr><tr><td> Semana 2 Lunes </td><td> Alimente astrocitos. Enjuague cubreobjetos en H 2 O, y luego sumergirse en ácido nítrico durante 24 horas. </td></tr><tr><td> Semana 2 Martes </td><td> RinSE cubreobjetos 5x. Esterilizar seco cubreobjetos. </td></tr><tr><td> Semana 2 Miércoles </td><td> Coloque los puntos de parafina en cubreobjetos. Escudo cubreobjetos con poli-L-lisina. </td></tr><tr><td> Semana 2 Jueves </td><td> Enjuague cubreobjetos recubiertos en H2O estéril, luego incubar cubreobjetos en chapado de medios durante la noche a 37 ° C. Cambie los medios en los astrocitos a NB27. </td></tr><tr><td> Semana 2 Viernes </td><td> Disección y galjanoplastia del hipocampo. </td></tr></tbody></table>

Watch this Scientific Journal Video about Calcium Phosphate Transfection of Primary Hippocampal Neurons at JoVE.com

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Fosfato de Calcio La transfección de Primaria neuronas del hipocampo

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

calcium-phosphate-transfection-of-primary-hippocampal-neurons

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

25.1K vistas.  Rutgers University.  Precipitación con fosfato de calcio es un método conveniente y económico para la transfección de células cultivadas. Con la optimización, es posible utilizar este método en las células difíciles de transfectar tales como neuronas primarias. A continuación se describe el protocolo detallado para la transfección de fosfato de calcio de las neuronas del hipocampo cocultured con células astroglial. 

Video: Calcium Phosphate Transfection of Primary Hippocampal 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

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 ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

La transfección de anticuerpos en neuronas como una herramienta para estudiar la patogénesis de enfermedades

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imaging las espinas dendríticas de las neuronas del hipocampo de rata primaria utilizando Estructurado Iluminación Microscopía

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

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 ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescencia y bioluminiscencia de imágenes de subcelular Ca<sup&gt; 2 +</sup&gt; Envejecido en las neuronas del hipocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Baja densidad primaria del hipocampo Neurona Cultura

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Medición de endocitosis vesícula sináptica en neuronas Hippocampal cultivadas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagen radiométrica calcio de neuronas individuales en comportamiento Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...