NIH K08 EB003996

1. Poli-L-láctico (PLLA) Spinning Solución <ol><li> Disolver 0,4 g PLLA en 9 ml de cloroformo, agitando a fuego lento. </li><li> Añadir 1 ml de dimetilformamida a la solución, con lo que la concentración final de la solución al 4% PLLA (w / v) en cloroformo: DMF 9:1 (v / v). </li><li> Ponga la solución en una jeringa de vidrio o polipropileno, con una punta de metal 23ga contundente. </li></ol> 2. Spinning Preparación del substrato 1 <ol><li> Hacer una solución al 8% (w / v) de 85:15 PLGA (poli-láctico-co-glicólico) en cloroformo, agitando a fuego lento. </li><li> Capa limpia cubierta de vidrio se desliza en PLGA cubriendo la superficie de cada hoja de la cubierta con la solución de PLGA. Deje que el PLGA para secar a una capa delgada (aprox. 30 min). </li></ol> 3. Electrospinning 2 <ol><li> Asegure la cubierta de vidrio recubierta PLGA se desliza al colector con una cinta de carbón conductor. Para las fibras alineadas, el colector es una rueda con motor. Para las fibras de azar, el colector es una placa estacionaria. </li><li> Coloque la jeringa en la bomba con la punta de 20 cm de la rueda del colector. Conjunto de la bomba de aproximadamente 2 ml / hora y si se utiliza una rueda, ajustar el motor a 300-400 RPM. Si es posible, aplicar un sesgo -2 kV DC para el colector y 15 kV hasta la punta de metal. Ante la falta de acceso a una fuente de alimentación bipolar, baja del colector. </li><li> Las fibras se chorro de la punta de la jeringa y se acumulan en la rueda giratoria. Continúe girando hasta que la densidad deseada de las fibras se obtiene. Pase la punta de metal con una toalla de papel colocada en una barra no conductora periódicamente para evitar la obstrucción en la punta. </li></ol> 4. Moating 1 <ol><li> Girar a continuación, &#39;foso&#39; las muestras electrospun pipeteando una línea de PLGA en todo el perímetro de la hoja de la cubierta. Deje que el PLGA que se seque. Esto mantendrá la alineación de las fibras durante el cultivo. </li></ol> 5. Proteína de revestimiento <ol><li> Electrospun fibras y los controles de vidrio debe ser recubierto en proteínas antes de cultivo celular. Cubrir los sustratos en una solución de proteínas, por lo menos una hora y luego aspirar el exceso de solución y seguir aspirando hasta que los sustratos están secos. Posibles soluciones de proteínas son: PLL (poli-L-lisina) (100 mg / mL), laminina (25 mg / ml) o la fibronectina (50 mg / mL). Si PLL se utiliza, los sustratos deben ser enjuagados con agua estéril y seca dos veces después de la capa inicial. </li></ol> 6. Obtener embriones de rata E15 3 * Todos los experimentos se realizaron de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio aprobado por la Universidad de Michigan Comité de Uso y Cuidado de Animales. <ol><li> Preparación: Compruebe si el animal está embarazada. Descongele los 3 ml de tripsina, 3 ml de FBS y calentar un biberón de L15. Fuego pulir una pipeta Pasteur. Desinfectar todos los instrumentos quirúrgicos en el 70% de etanol durante 30 min. </li><li> Preparar y calentar el medio de placas (ver Tabla 1). </li><li> Eutanasia: Realizar una inyección IP de ketamina / xilazina. Espere 10-15 minutos y comprobar por falta de la córnea y el pedal de reflejos de retirada. Una vez que los reflejos están ausentes, realice una inyección de pentobarbital IC (FatalPlus). </li><li> La tienda de la piel del abdomen. Corte a través de la piel y las capas musculares para exponer la cavidad abdominal. Localizar el útero y separarlo en el cuello del útero y los ovarios. </li><li> La toma de embriones del útero con una tijera y colocarlos inmediatamente en caliente L15. (Todos los siguientes procedimientos se realizan bajo un microscopio de disección y los embriones y los cables de disección debe ser sumergido en agua tibia L15 en todo momento.) Decapitar a los embriones por el cierre de pinzas alrededor de la barbilla y debajo del hueso occipital del cráneo. </li></ol> 7. Disección de 3 <ol><li> Coloque el embrión de un lado. Proteger la médula espinal con un juego de pinzas, mientras que con el otro despojarnos de las extremidades y los órganos abdominales. </li><li> A su vez el embrión y mantenerlo fijo con un juego de pinzas. Recorte a lo largo del embrión, desde el cuello hasta la cola, hasta que toda la médula está expuesto. </li><li> Sujete el extremo del cable con cuidado y tire de ella sobre sí misma hacia la cola para retirar. El cable se tire libre con membrana y ganglios de la raíz dorsal (GRD) adjunto. </li><li> Si DRG explante o cultura disociada neurona sensorial se va a realizar, cortar la DRG de la médula y la transferencia a un plato fresco de tibia L15. <ol><li> Por DRG explante cultura, con el paso 10.2 </li><li> Para el cultivo disociado de neuronas sensoriales, vaya al paso 9 </li><li> Para el motor cultura neurona, el DRG se eliminará con la membrana en el paso 7.5 </li></ol></li><li> Quitar la membrana de la médula espinal, sujetándola en el extremo superior de la médula y el pelado hacia abajo, similar a la eliminación de una media de largo. Repita este procedimiento para todos los embriones y luego cortar los cables en pequeños (2-3 mm) piezas. </li></ol><pclass = &quot;jove_title&quot;&gt; 8. De las neuronas motoras (MN) Procesamiento de 5,6,7 <ol><li> Vierta los cables cortados y L15 en una forma cónica y giran a 1000 rpm durante 2-3 minutos para que sedimenten las piezas. </li><li> Retirar el sobrenadante y agregar 3 ml de tripsina a los cables de pellets. Incubar en un baño de agua a 37 ° C durante 20 min. </li><li> Añadir 3 ml de FBS cónica y giran a 1000 rpm durante 2-3 min para volver a pellet. </li><li> Retirar el sobrenadante y la capa de un incendio pulido pipeta Pasteur con FBS. </li><li> Añadir una pipeta Pasteur, lleno de L15 a la cónica y luego se tritura suavemente hasta que la solución es homogénea sin grumos visibles. Evitar las burbujas. </li><li> Prepare una solución de 9% de Optiprep en L15. Preparar un tubo con 3 ml de la solución Optiprep por cada dos embriones (si hay un número impar de embriones, redondear). </li><li> Caída de la solución homogeneizada en la solución Optiprep, dividiéndola en partes iguales entre todos los tubos. Girar los tubos a 2000 rpm durante 15 minutos. </li><li> Recoger cuidadosamente los 2 ml de cada tubo y la piscina en un Erlenmeyer de 50 ml. Llene el cono con L15 y giran a 1000 rpm durante 5 minutos para enjuagar las células de Optiprep. </li><li> Retirar el sobrenadante y resuspender las células en una pequeña cantidad de medios de chapado (250 a 500 l). Contar las células con azul de tripan exclusión para identificar las células vivas. Vaya al paso 10.1 </li></ol> 9. Neurona sensorial (SN) de elaboración 7 <ol><li> Vierta el DRG eliminado de la médula espinal y L15 en un tubo cónico y giran a 1000 RPM durante 2-3 min para precipitar las piezas. </li><li> Retirar el sobrenadante y agregar 3 ml de tripsina a la DRG granulado. Incubar en un baño de agua 37 ° C durante 10 min. </li><li> Añadir 3 ml de FBS cónica y giran a 1000 rpm durante 2-3 min para volver a pellet. </li><li> Retirar el sobrenadante y la capa de un incendio pulido pipeta Pasteur con FBS. </li><li> Añadir una pipeta Pasteur, lleno de L15 a la cónica y luego se tritura suavemente hasta que la solución es homogénea sin grumos visibles. Evitar las burbujas. </li><li> Girar el homogenado a 1000 rpm durante 5 min. Retirar el sobrenadante y resuspender las células en una pequeña cantidad de medios de comunicación placas SN (250-500 l). Contar las células con azul de tripan exclusión para identificar las células vivas. Continúe con el paso 10.1 </li></ol> 10. Revestimiento y Cultura <ol><li> Para disociar SN o la cultura MN: <ol><li> Cubrir los sustratos con unas gotas de los medios de comunicación a continuación, agregar un volumen apropiado de la suspensión celular. Las células disociadas se sembraron a una densidad baja (25-50 células / mm 2). Permitir que las células se adhieran por lo menos una hora antes de la inundación de los pozos con los medios de comunicación. </li></ol></li><li> Por DRG explante cultura: <ol><li> Cubrir los sustratos con unas gotas de los medios de comunicación a continuación, añadir DRG a los medios de comunicación. Organizar los GRD para que se distribuyen uniformemente a través del substrato (aproximadamente 4 DRG caben en una hoja de 22x22 mm cubierta). Deje que el GRD que se adhieran durante al menos 4 horas antes de la inundación de los pozos con los medios de comunicación. </li></ol></li><li> Culturas se puede mantener durante un máximo de 4 días sin cambiar los medios de comunicación. Durante más tiempo las culturas, la mitad de los cambios medios de comunicación se debe hacer con los medios de alimentación. Alimentar a los medios de comunicación es la misma composición que los medios de comunicación placas menos la L-glutamina. </li></ol> 11. Inmunocitoquímica 1,5,8 <ol><li> La fijación de las células: los medios de comunicación Aspirar y reemplazar con agua tibia 4% PFA (paraformaldehído) durante 30 minutos. A continuación, realizar 3 veces 5 min lavados con PBS 1x. </li><li> Bloqueo / permeabilización: Prepare bloque / perm solución (1,25% de suero albúmina bovina en PBS 1X, un 0,05% Triton X-100 y el 2,0% de suero normal de cabra). Aspirar PBS y aplicar bloque / permanente solución a las muestras durante 30 minutos. A continuación, realice 1X 5 min lavados con PBS 1x. </li><li> Anticuerpo primario: Preparar la solución de anticuerpo primario de trabajo (10% de suero normal de cabra, un 1,25% de albúmina sérica bovina, el 0,1% de azida Na, 0,05% de Triton X-100 y 10 ml con 1x PBS). Añadir la cantidad apropiada de anticuerpo primario (ver Tabla 2). Línea de varios platos con parafilm. Coloque un cordón de 200μl de solución de anticuerpos primarios en la parafina para cada sustrato. Invertir los sustratos, flotando a la par de la celda hacia abajo en la solución de anticuerpo primario de trabajo durante la noche (si la habitación tiene aire acondicionado o en seco sobre todo, un pedazo de papel saturado de agua se pueden agregar a una esquina del plato para evitar la evaporación de la solución de trabajo) . </li><li> Anticuerpo secundario: Retire los sustratos de la primaria (la solución de trabajo pueden ser recogidos, almacenados a 4 ° C y reutilizar) y llevar a cabo 2 veces 5 min lavados con PBS 1x. Aplicar 200 l de anticuerpos secundario diluido 1:200 en PBS (también se invierte en un plato forrado parafina) durante 4 horas. </li><li> Eliminar de secundaria a continuación, realizar 2 veces 5 min lavados con PBS 1x. Soportes de montaje en láminas de oro con prolongar la DAPI u otro contador nuclear adecuada mancha. </li></ol> 12. Los resultados representativos: La morfología típica de alineado y El azarfibras ectrospun se muestra en la Figura 1. Por lo general obtener la pureza de la MN&gt; 90% 7,8 neuronas con este protocolo y hemos mantenido DRG culturas durante todo el tiempo como una semana sin ningún tipo de crecimiento de fibroblastos significativo. La figura 2 muestra la apariencia típica de MN en fibras de vidrio y después de 24 horas de la cultura y la inmunotinción. DRG immunostained cultivadas durante tres días en fibras de vidrio y se muestra en la Figura 3. <img src="/files/ftp_upload/2389/2389fig1.jpg" alt="Figura 1" /> Figura 1. Alineación de la fibra es manipulado por la elección de los colectores. A.) Alineados fibras hiladas en un colector de rueda giratoria y B.) hilar fibras al azar en un colector de placa estacionaria. Las barras de escala = 20 micras. <img src="/files/ftp_upload/2389/2389fig2.jpg" alt="Figura 2" /> Figura 2. Representante neuronas motoras teñidas Tuj1 (verde) y DAPI (azul) después de 24 horas de la cultura en A. recubierto PLL) fibras y B) de cristal. Las barras de escala = 20 micras. <img src="/files/ftp_upload/2389/2389fig3.jpg" alt="Figura 3" /> Figura 3. Manchada Representante DRG para neurofilamentos (verde) después de 3 días de cultivo en el PLL recubiertas A.) de vidrio y B) las fibras. Las barras de escala = 200 micras. <table border="1"><tbody><tr><td> Solución de reserva: </td><td> MN medios de comunicación: </td><td> DRG / SN medios de comunicación: </td></tr><tr><td> 10 mg mL -1 albúmina </td><td> 12,5 l </td><td> - </td></tr><tr><td> 10 mg mL -1 apo-transferrina </td><td> 50 l </td><td> 40 ml </td></tr><tr><td> 50 mg mL -1 β-estradiol </td><td> 2,7 l </td><td> 2,2 l </td></tr><tr><td> 0,1 mg mL -1 biotina </td><td> 50 l </td><td> - </td></tr><tr><td> 16 mg mL -1 catalasa </td><td> L 8 </td><td> - </td></tr><tr><td> 15 mg mL -1 D-galactosa </td><td> 50 l </td><td> - </td></tr><tr><td> 50 mg mL -1 de hidrocortisona </td><td> 3,7 l </td><td> 2,9 l </td></tr><tr><td> 0,63 mg mL -1 de progesterona </td><td> 0,5 l </td><td> - </td></tr><tr><td> 16 mg mL -1 putrescina </td><td> 50 l </td><td> - </td></tr><tr><td> 50 mg mL -1 selenio </td><td> 3,4 l </td><td> 4,1 l </td></tr><tr><td> 2,5 mg mL -1 superóxido dismutasa </td><td> 50 l </td><td> - </td></tr><tr><td> * 500 ng ml -1 nervio del factor de crecimiento </td><td> - </td><td> 1 mL </td></tr><tr><td> * 100X PSN </td><td> 0,5 ml </td><td> 0,5 ml </td></tr><tr><td> * B27 </td><td> 1 mL </td><td> 1 mL </td></tr><tr><td> * 2 mM L-glutamina </td><td> 35 l </td><td> 35 l </td></tr><tr><td> * Neurobasal </td><td> a 50 ml </td><td> a 50 ml </td></tr></tbody></table> Tabla 1. Añadir asterisco (*) los componentes de los medios de comunicación justo antes del uso. El resto de componentes se pueden preparar como una solución de reserva y se mantiene a -20 ° C hasta el momento de 6,7. <table border="1"><tbody><tr><td> Primaria (concentración) </td><td> Las neuronas: </td><td> Glia: </td><td> Fibroblastos: </td></tr><tr><td> anti-β-tubulina (Tuj1) (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-neurofilamentos (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-S-100 (1:250) </td><td> X </td><td> ✓ </td><td> ✓ </td></tr><tr><td> anti-p75 NGFR (1:500) </td><td> ✓ </td><td> ✓ </td><td> X </td></tr></tbody></table> Tabla 2. La selección de anticuerpo primario depende de los objetivos del investigador. Los anteriores son varios anticuerpos y las concentraciones que se han utilizado con éxito en nuestro laboratorio. S-100 es especialmente útil para identificar las células de Schwann y / o detectar la contaminación fibroblastos, pero tenga en cuenta que se debe utilizar en combinación con NGFR p75 para distinguir entre células gliales y fibroblasts4. También hemos notado que NGFR p75 manchas neuritas muy ligeramente, mientras que neurofilamentos o Tuj1 son excelentes opciones si el objetivo es visualizar neuritas individual.

<ol>
	<li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Kameda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+glial+progenitor+markers+p75NTR+and+S100+protein+in+the+developing+mouse+parathyroid+gland.">Expression of glial progenitor markers p75NTR and S100 protein in the developing mouse parathyroid gland.</a> Cell Tissue Res. 327, 15-23 (2007).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Vincent, A. M., Mobley, B. C., Hiller, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-I+prevents+glutamate-induced+motor+neuron+programmed+cell+death.">IGF-I prevents glutamate-induced motor neuron programmed cell death.</a> Neurobiol. Dis. 16, 407-416 (2004).</li><li>Russell, J. W., Windebank, A. J., Schenone, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+prevents+apoptosis+in+neurons+after+nerve+growth+factor+withdrawal.">Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.</a> J. Neurobiol. 36, 455-467 (1998).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

Este protocolo tiene varios pasos críticos. La primera consiste en la correcta producción de los sustratos de fibra electrospun. En medios líquidos, las fibras de PLLA, el cine y el foso PLGA PLGA se separará de la hoja de la cubierta de vidrio como una sola unidad. Si el PLGA se omite, las fibras de PLLA no seguirá siendo una hoja plana - que se acurrucaba en una maraña inutilizable. Así, la película de PLGA se incluye como un sustrato adecuado para mantener la alineación de la fibra durante el cultivo. El foso se añade después de girar tanto para garantizar que las fibras estén bien fijados a la película de PLGA y añadir rigidez estructural de la película. El foso que también sirve como un mango útil cuando los sustratos son manipulados durante la fijación, tinción y montaje. La trituración es el segundo paso crítico. Todo esfuerzo debe hacerse para evitar la formación de burbujas. Además, hemos obtenido un rendimiento mucho mayor cuando una pipeta pulida al fuego se utiliza para este paso. Para pulir el fuego, la luz de un mechero Bunsen y adjuntar una bombilla en la pipeta. Pase la punta de la pipeta rápidamente a través de la llama de 3.2 veces, mientras que continuamente apretar y soltar la bombilla - el flujo de aire a través de la pipeta le ayudará a evitar que la punta no cierran correctamente. Examine la punta de la pipeta para asegurarse el agujero sigue abierto y que los bordes ligeramente redondeados aparecen antes de su uso. Electrospinning es un proceso muy versátil; los parámetros electrospinning se pueden modificar para producir fibras con una variedad de morfologías. Tan et al. (2005) los detalles de un estudio sistemático de los parámetros que afectan el diámetro de las fibras de PLLA electrospun. Wang et al. (2009) presenta un estudio detallado de los parámetros que influyen en la alineación de las fibras de PLLA electrospun.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

Electrospinning es una técnica para la producción de micro-fibras de escala nanométrica. Las fibras pueden ser electrospun con distintos grados de alineación, de muy bien alineados para completamente al azar. Además, las fibras se pueden girar de una variedad de materiales, incluyendo polímeros biodegradables tales como poli-L-láctico (PLLA). Estas características hacen que las fibras electrospun adecuado para una variedad de aplicaciones de andamios en ingeniería de tejidos. Nuestra atención se centra en el uso de fibras electrospun alineados para la regeneración nerviosa. Hemos demostrado previamente que las fibras alineadas electrospun PLLA directa el resultado de dos neuronas sensoriales primarias y el motor in vitro. Sostenemos que el uso de un sistema de células de cultivo primario es fundamental en la evaluación de los biomateriales a las neuronas modelo real en vivo tanto como sea posible. A continuación, describimos las técnicas utilizadas en nuestro laboratorio para electrospin andamios fibrosa y la cultura dorsal explantes de ganglios de la raíz, así como disociada neuronas sensoriales y motoras, en los andamios electrospun. Sin embargo, el electrospinning y / o técnicas de cultivo se presentan aquí son fácilmente adaptables para su uso en otras aplicaciones.

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Alineados fibras electrospun dirigir el crecimiento de las neuronas in vitro y son un componente potencial de andamios la regeneración nerviosa. Se describe un procedimiento para la preparación de sustratos electrospun fibra y el cultivo libre de suero de rata principal E15 sensorial (GRD) y las neuronas motoras. La visualización de las neuronas por inmunocitoquímica también se incluye.

La cultura de la motora primaria y las neuronas sensoriales en los medios de comunicación definidas en Electrospun poli-L-láctico andamios nanofibras

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

19.3K vistas.  University of Michigan. Alineados fibras electrospun dirigir el crecimiento de las neuronas in vitro y son un componente potencial de andamios la regeneración nerviosa. Se describe un procedimiento para la preparación de sustratos electrospun fibra y el cultivo libre de suero de rata principal E15 sensorial (GRD) y las neuronas motoras. La visualización de las neuronas por inmunocitoquímica también se incluye.

Video: The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

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 ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash fotólisis de compuestos enjaulados en los cilios de las neuronas sensoriales olfativas

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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

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

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

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo de dos fotones de imágenes de dependientes Experience-Los cambios moleculares en las neuronas corticales

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

El pez cebra<em&gt; In Situ</em&gt; Médula Espinal Preparación para Registros electrofisiológicos de Spinal Sensory y neuronas motoras

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...