Agradecemos a Homaira Nawabi, Frederic Moret e Isabelle Sanyas por su ayuda. Este trabajo cuenta con el apoyo de CNRS, Association Francaise contre les Myopathies (AFM), ANR YADDLE, Labex DevWeCan, Labex Cortex, ERC YODA to V.C.; C.D-B y A.J cuentan con el apoyo de las becas La Ligue contre le cancer y Labex DevWeCan, respectivamente.

1. Estrategia de clonación para etiquetar el receptor PlexinA1 con pHluorin
<ol><li>Elija un vector de expresión apropiado como columna vertebral(por ejemplo, el vector de expresión del receptor de ratón plexinA1, un regalo amable del Dr. Andreas Puschel11). Nota: Este vector plexinA1 fue diseñado para lograr la inserción eficiente del receptor ha- o VSV-tagged en la membrana plasmática.</li>
	<li>Amplificar por PCR la secuencia de codificación de pHluorin eclíptica utilizando el plásmido adecuado como plantilla(por ejemplo, receptor GABA A marcado con pHluorin, un regalo amable del Dr. Jacob2). Si es necesario, agregue un sitio de restricción al extremo 5' de la cartilla para facilitar el paso de clonación en la columna vertebral.</li>
	<li>Inserte la secuencia de pHluorin en el marco entre el péptido de señal y la secuencia de codificación del receptor utilizando sitios de restricción(por ejemplo, sitios de restricción NruI/Kpn2I como se describe en la Figura 1B). Nota: Debido a que el péptido de señal que asegura la correcta orientación de los receptores está escindida, el pHluorin debe colocarse después de él. Esto garantiza el reconocimiento del péptido señal y evita la escisión del pHluorin del receptor de interés.</li>
	<li>Secuenciar las construcciones obtenidas para asegurar que no se introdujera ninguna mutación por PCR.</li>
</ol>2. Caracterización Del Receptor De pHluorin-etiquetado In vitro En Células COS7
La capacidad de la proteína de fusión para llegar a la membrana plasmática y su pérdida reversible de fluorescencia a medida que se reduce el pH se puede confirmar mediante el siguiente procedimiento.
<ol><li>Día 1. Placa 1,5 x 105 células COS7 en un plato de fondo de cristal de 35 mm en 2 ml de medio de águila modificado de Dulbecco completo (DMEM - 10% suero fetal bovino - piruvato de sodio de 1 mM - 25 U/ml penicilina/estretomicina - 2,5 μg/ml Anfotericina B - pH 7,4).</li>
	<li>Día 2. Células transfectas: Nota: Las células deben ser 70-80% confluentes.<ol>
<li>Prepare 200 μl de NaCl 150 mM y agregue 3 μg de ADN, es decir, el vector que codifica el receptor marcado con pHluorin. Suavemente vórtice y girar hacia abajo brevemente. Nota: En la Figura 1Bse muestra un mapa del vector pHluorin-plexinA1 utilizado en estos experimentos.</li>
		<li>Añadir 10 μl de reactivo de transfección (o la cantidad adecuada del reactivo utilizado). Vórtice inmediatamente.</li>
		<li>Incubar 10 min en RT.</li>
		<li>Añadir 200 μl de la mezcla reactivo/ADN de transfección a las células.</li>
		<li>Mueva la placa suavemente para lograr la repartición de la mezcla y coloque las células de nuevo a la incubadora de 37 °C.</li>
	</ol>
</li>
	<li>Día 3. Retire el medio de transfección y reemplácelo por 2 ml de DMEM completo fresco.</li>
	<li>Día 4. Realizar imágenes de células vivas de cos7 pHluorin-plexinA1 células transfectadas:<ol>
<li>Preparar dos alícuotas de DMEM medio completo y ajustar el pH a 3,5 y a 9,5, respectivamente. Nota: Para una placa de 35 mm, se necesitan 1,5 ml de cada solución para realizar el experimento.</li>
		<li>Retirar el medio celular y sustituirlo por 1 ml de DMEM pH completo 7.4.</li>
		<li>Prepare una jeringa de 5 ml con un tipo apropiado de tubo para inyectar varios componentes directamente en el medio de cultivo celular sin abrir la cámara del microscopio.</li>
		<li>Utilice un módulo que permita el mantenimiento de una atmósfera de trabajo húmeda de 37 °C, 5% de CO2. Nota: Un enfoque alternativo para el uso de una cámara de CO2 es utilizar un medio tamponado con HEPES (generalmente en el rango de 10-25 mM según el tipo de célula).</li>
		<li>Coloque las células en la cámara y ajuste el tubo y la jeringa. Nota: El microscopio debe equilibrarse antes de comenzar a evitar la deriva mecánica durante la grabación.</li>
		<li>Abra el software de imágenes y seleccione el programa de adquisición multidimensional.</li>
		<li>Encuentre células COS7 transfectadas con el objetivo 40X y marque la posición en el software para cada una de ellas.</li>
		<li>Configure la pila Z para tener una adquisición de profundidad de 15 μm (el enfoque puede cambiar al agregar medios en la placa).</li>
		<li>Configure la exposición para el filtro GFP y la fase.</li>
		<li>Configurar el tiempo de adquisición. Nota: Para todo el experimento con 5 campos de interés, la adquisición cada 20 segundos durante 10 minutos debería ser suficiente.</li>
		<li>Iniciar la adquisición y tomar 5 imágenes de control en DMEM pH 7.4 medio.</li>
		<li>Pausar la adquisición, inyectar 1,25 ml de DMEM completo de pH 3,5 para lograr un pH de 5,5 en el medio de cultivo, marcar el evento en el software y reanudar la adquisición por 5 puntos de tiempo más. Nota: La fluorescencia verde debe desaparecer progresivamente.</li>
		<li>Pausar la adquisición, inyectar 1,2 ml de DMEM completo de pH 9,5 para lograr un pH 7,4 en el medio de cultivo, marcar el evento en el software y reanudar la adquisición por 5 puntos de tiempo más. Nota: La fluorescencia verde debe reaparecer en la membrana plasmática.</li>
		<li>Analizar imágenes. La Figura 2 muestra imágenes representativas obtenidas con dicho protocolo con el constructo pHluorin-plexinA1.
</li>
	</ol>
</li>
</ol>3. En ovo Electroporación De pHluorin-plexinA1 Constructo
<ol><li>Manipulación de huevos antes de la electroporación:<ol>
<li>Guarde los huevos fertilizados en una nevera a 14 °C hasta una semana antes de la incubación.</li>
		<li>Incubar los huevos a 38,5 °C (101,3 °F) en una incubadora con humedad saturada durante 50-52 horas hasta que los embriones alcancen el estadio HH1412. Nota: Los huevos deben colocarse horizontalmente durante la incubación para que el embrión esté correctamente posicionado para la electroporación, flotando en la parte superior de la yema. El estadio HH14 es adecuado para obtener la expresión de los plásmidos en neuronas diferenciadas de la médula espinal y en el ganglio de la raíz dorsal con una tasa de supervivencia adecuada.</li>
	</ol>
</li>
	<li>Embriones electropolados8-10:<ol>
<li>Preparar la electroporación:<ol>
<li>Preparar plásmidos de ADN libres de endotoxinas con una concentración superior a 2 μg/μl, para poder diluirlos como la concentración deseada.</li>
			<li>Tire de suficientes capilares de vidrio para inyectar las diferentes soluciones de ADN.</li>
			<li>Preparar PBS estéril (-Ca2+; -Mg2+)- 100 U/ml de penicilina/estreptomicina y equilibrar a 38,5 °C.</li>
			<li>Esterilizar la capucha, tijeras curvas y fórceps finos.</li>
			<li>Controle el espaciamiento del electrodo. Nota: Generalmente se utiliza un espacio de 4 mm entre los electrodos.</li>
		</ol>
</li>
		<li>Ventana el huevo13 (Figura 3A):<ol>
<li>Use tijeras curvas para perforar la cáscara en el lado romo del huevo.</li>
			<li>Retire 2 ml de albúmina con una aguja de 0,9 mm x 25 mm y una jeringa de 5 ml. Oriente la aguja verticalmente para evitar dañar el saco vitelino.</li>
			<li>Cubra la parte superior del huevo con cinta adhesiva para mantener la integridad de la cáscara.</li>
			<li>Usando tijeras curvas, perfore la cáscara en el centro de la cinta para igualar la presión al retirar 2 ml de albúmina del huevo. Luego, corte una ventana lo suficientemente grande como para visualizar el embrión y poder trabajar en él.</li>
			<li>Añadir ~2 ml de PBS caliente estéril (-Ca2+; -Mg2+)- 100 U/ml de penicilina/estreptomicina para evitar la deshidratación del embrión y hacerlo más accesible al manipulador.</li>
		</ol>
</li>
		<li>Inyectar ADN y electropórate el embrión<ol>
<li>Diluir el plásmido en PBS (-Ca2+; -Mg2+)a una concentración entre 0,5-2 μg/μl y añadir colorante verde rápido para alcanzar una concentración final del 0,025%. Cargue la mezcla de ADN en un capilar. Se recomienda el uso de un inyector. Nota: Compruebe que la resistencia capilar no es demasiado grande (lo que significa que podría haber dificultades al inyectar embriones) ni demasiado pequeña (lo que significa que el capilar podría ser demasiado grande y podría dañar el embrión). Además, la concentración de ácidos nucleicos superior a 2 μg/μl puede causar efectos inespecíficos y debe controlarse.</li>
			<li>Perfore el saco vitelino y el tubo neural en el lado caudal con el capilar cargado. Ingrese al tubo neural con un ángulo poco profundo y llene el lumen desde la cola hasta la cabeza con la mezcla de ADN (Figura 3B). Nota: El verde rápido permite controlar la precisión de la inyección.</li>
			<li>Coloque rápidamente los electrodos de platino de 4 mm a cada lado del tubo neural en el nivel que desea electroborrar y aplique 3 pulsos a 31 V durante 50 milisegundos con intervalos de 500 milisegundos(Figura 3C). Nota: Evite colocar los electrodos en el corazón o en grandes vasos embrionarios adicionales para evitar dañar el embrión en desarrollo. Las burbujas deben formarse en los electrodos.</li>
			<li>Con la aguja, retire 2 ml de albúmina para disminuir el nivel en la cáscara.</li>
			<li>Selle la ventana y el lado romo herméticamente con cinta adhesiva.</li>
			<li>Volver a colocar los huevos a 38,5 °C en la incubadora hasta que alcancen la etapa deseada.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. Incrustación de embriones y criosección
<ol><li>48 horas después de la electroporación, cosechar cuidadosamente embriones electroporados (etapa HH24). Cortar la cinta y la mitad de la membrana corioallantoidea. Para evitar que los embriones se hundan en la yema, coloque un colador debajo del embrión y corte la segunda mitad de la membrana corioallantoidea.</li>
	<li>Transfiera el embrión a un plato de disección lleno de PBS helado.</li>
	<li>Compruebe la eficiencia de la electroporación buscando fluorescencia en el tubo neural con un microscopio estéreo de fluorescencia. Nota : La coelectroporación de un plásmido control que codifica la RFP puede ayudar a visualizar el área electroporada.</li>
	<li>Diseccione embriones usando un microscalpel para seleccionar el área electroporada de la médula espinal.</li>
	<li>Transferir embriones disecados a una placa de 24 pozos y fijar en pH 7,4 4% Paraformadehído (PFA) - Tampón Fosfato Salino (PBS), O/N a 4 °C. Nota: La etapa de fijación es crucial para permitir la estabilización de pHluorin en su conformación "viva" y así poder utilizar pHluorin en tejido fijo/permeabilizado. Aunque la fijación retarda drásticamente el cambio dependiente del pH de la fluorescencia, se debe tener en cuenta que los cambios conformacionales /protonación todavía pueden ocurrir después de la fijación. Así, el siguiente protocolo (incrustación, criosecciones y observación) debe realizarse dentro de los 3 días posteriores a la etapa de fijación, con todos los tampones a pH 7. Si no se requiere fijación, se recomienda realizar la observación en secciones de tejido vivo.</li>
	<li>Eliminar el 4% de PFA y lavar los embriones en pH 7,4 PBS.</li>
	<li>Incubar embriones en PBS-15% sacarosa y conservar a 4 °C hasta que los embriones se hundan.</li>
	<li>Incubar embriones fijos en pH 7.4 7.5% gelatina- 15% sacarosa durante 45 min a 37 °C para que los embriones estén completamente incrustados.</li>
	<li>Coloque los moldes de incrustación sobre hielo y agregue 400 μl de pH 7.4 7.5% gelatina- 15% sacarosa para lograr una base sólida de 2 mm.</li>
	<li>Aspirar el embrión incrustado con una punta cortada y colocar el embrión sobre la base de gelatina sólida.</li>
	<li>Cubrir con pH 7,4 gelatina al 7,5%, sacarosa al 15% y colocar el embrión con fórceps antes de que la gelatina se solidifique.</li>
	<li>Una vez que la gelatina sea sólida, prepare un baño de isopropanol de -40 °C (use hielo seco o nitrógeno líquido) y congele el bloque de gelatina durante 5 min.</li>
	<li>Mantenga los bloques congelados a -80 °C.</li>
	<li>Coloque el bloque congelado a -20 °C durante 1 hora.</li>
	<li>Retire el molde y fije el bloque en un mandril con un medio de polietilenglicol.</li>
	<li>Una vez que el bloque esté bien fijado, coloque el mandril en el sistema de criostato. Nota: Use portaobjetos recubiertos para evitar la pérdida de tejido durante la tinción.</li>
	<li>Realizar criosecciones seriales (generalmente se realizan criosecciones de 20 μm).</li>
	<li>Deje secar las criosecciones durante 15 min en rt. Nota: las criosecciones deben protegerse de la exposición innecesaria a la luz para evitar el blanqueo de la fluorescencia de GFP.</li>
</ol>5. Análisis microscópico de criosecciones
<ol><li>Rehidratar criosecciones en pH 7,4 PBS a RT durante 10 min. Nota: Si es necesario, los núcleos se pueden teñir con Hoechst.</li>
	<li>Utilizar una solución de Hoechst de 0,5 μg/ml en PBS e incubar criosecciones durante 15 min.</li>
	<li>Enjuague los portaobjetos 3x con pH 7.4 PBS durante 5 min.</li>
	<li>Proceda al montaje por deslizamiento. Se puede utilizar una solución de montaje de alcohol polivinílico de pH 7.4 (o más básico) que endurece la O/N: coloque cuidadosamente el cubrebocas para evitar la formación de burbujas de aire entre la corredera y la cubierta.</li>
	<li>Deje que el medio de montaje se endurezca O/N a 4 °C en la oscuridad.</li>
	<li>Utilice un microscopio confocal invertido para visualizar con precisión la proteína de fusión pHluorin-plexinA1: realice z-stack a la resolución óptima de agujeros de alfiler y óptico y utilice lentes 20X (NA 0.75) o 40X (NA 1.3). Nota: pHluorin es detectable con los mismos parámetros utilizados para detectar GFP(es decir, pico de emisión a 509 nm). Los ajustes del filtro de excitación y detección de longitud de onda se definen de forma óptima mediante el software de imágenes. Hoechst se detecta entre 425-460 nm (la excitación está a 405 nm), GFP o pHluorin se detecta entre 485-545 nm (la excitación está a 473 nm) y la RFP se detecta entre 575-675 nm (la excitación está a 559 nm).</li>
</ol>Las imágenes representativas de la expresión de pHluorin-plexinA1 y eGFP en la médula espinal del embrión de polluelo se muestran en la Figura 4.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+regulating+the+abundance+and+localization+of+synaptobrevin+in+the+plasma+membrane.">Factors regulating the abundance and localization of synaptobrevin in the plasma membrane.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11399-11404 (2006).</li><li>Bozza, T., McGann, J. P., Mombaerts, P., Wachowiak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+neuronal+activity+by+targeted+expression+of+a+genetically+encoded+probe+in+the+mouse.">In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse.</a> Neuron. 42, 9-21 (2004).</li><li>Sankaranarayanan, S., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurements+of+vesicle-SNARE+recycling+in+synapses+of+the+central+nervous+system.">Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.</a> Nat. Cell. Biol. 2, 197-204 (2000).</li><li>Stark, D. A., Kasemeier-Kulesa, J. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+cell+labeling+for+cell+tracing+in+avian+development.">Photoactivation cell labeling for cell tracing in avian development.</a> CSH Protoc.. 2008, (2008).</li><li>Hildick, K. L., Gonzalez-Gonzalez, I. M., Jaskolski, F., Henley, 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=Lateral+diffusion+and+exocytosis+of+membrane+proteins+in+cultured+neurons+assessed+using+fluorescence+recovery+and+fluorescence-loss+photobleaching.">Lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching.</a> J. Vis. Exp. (60), (2012).</li><li>Hanson, G. T., 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=Green+fluorescent+protein+variants+as+ratiometric+dual+emission+pH+sensors.+1.+Structural+characterization+and+preliminary+application.">Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application.</a> Biochemistry. 41, 15477-15488 (2002).</li><li>Rose, T., Schoenenberger, P., Jezek, K., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+refinement+of+vesicle+cycling+at+schaffer+collateral+synapses.">Developmental refinement of vesicle cycling at schaffer collateral synapses.</a> Neuron. 77, 1109-1121 (2013).</li><li>Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHTomato,+a+red,+genetically+encoded+indicator+that+enables+multiplex+interrogation+of+synaptic+activity.">pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity.</a> Nat. Neurosci. 15, 1047-1053 (2012).</li><li>de Wit, J., Toonen, R. F., Verhage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+local+retention+of+secretory+vesicle+cargo+in+cortical+neurons.">Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.</a> J. Neurosci. 29, 23-37 (2009).</li></ol>

Los autores no tienen nada que revelar.

Este protocolo proporciona un procedimiento paso a paso para seguir la dinámica de un receptor de guía de axones tanto en cultivo celular como en el contexto de desarrollo de la médula espinal del embrión de polluelo.
Para diseñar una proteína etiquetada con pHluorin de novo, es necesario considerar dos puntos con respecto a la estrategia de clonación. En primer lugar, la etiqueta de pHluorin debe exponerse a la luz de los endosomas ácidos y, en consecuencia, al compartimento ...

Durante su navegación, los axones integran múltiples señales ambientales que los guían hacia su objetivo. Estas señales activan los receptores de guía en la superficie de los terminales de los axones, los conos de crecimiento, que a su vez inician una vía de señalización adecuada. Por lo tanto, la regulación temporal y espacial de la distribución de la superficie celular de los receptores es fundamental para establecer la sensibilidad del cono de crecimiento1. En este contexto, el cruce de la línea media por axones comisurales es un excelente modelo para investigar la regulación de los niveles de superficie celular del receptor. En la médula espinal en desarrollo, los axones commissurales son inicialmente atraídos hacia la placa del piso ventral donde cruzan la línea media. Después del cruce, pierden su capacidad de respuesta a los atrayentes de la placa del piso y ganan respuesta a los repelentes de la placa del piso para que puedan salir de la placa del piso y navegar hacia su destino final en el lado contralateral del sistema nervioso2,3. La regulación de la disponibilidad del receptor en la superficie del cono de crecimiento es uno de los mecanismos subyacentes al cambio de capacidad de respuesta a las señales de la línea media4,5. Por lo tanto, la monitorización selectiva de los receptores presentes en la membrana plasmática de los conos de crecimiento es de primordial importancia. Se describe aquí un método basado en la propiedad de fluorescencia dependiente del pH de una proteína fluorescente verde (GFP) variante para visualizar específicamente los receptores de guía axón que se dirigen a la membrana plasmática in vitro e in vivo,en el desarrollo de la médula espinal polluelo.
Rothman y sus colegas diseñados por mutaciones puntuales variantes sensibles al pH de GFP, incluida la eclíptica pHluorin6. La pHluorgina eclíptica tiene la propiedad de ser no fluorescente cuando se expone al pH ácido (<6), mientras que es fluorescente a pH neutro. Esto permite distinguir los receptores no fluorescentes localizados en compartimentos ácidos intracelulares(es decir, endosomas, vesículas de tráfico) de los receptores fluorescentes incorporados a la membrana plasmática y, por lo tanto, expuestos al pH neutro extracelular7. Aprovechamos esto para monitorizar la localización de la membrana plasmática de plexinA1, un receptor de guía axón que media la respuesta del cono de crecimiento a la semaforina repelente de línea media 3B5 (Figura 1A). Se describe aquí la caracterización in vitro de una construcción de pHluorin-plexinA1, junto con en ovo electroporación8-10 de esta construcción en la médula espinal polluelo en desarrollo seguido por el análisis microscópico de criosecciones que permiten seguir la dinámica del receptor de orientación axón in vivo con resoluciones espaciales y temporales.

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

Durante el desarrollo, los receptores de guía de axones juegan un papel crucial en la regulación de la sensibilidad de los axones a las señales atractivas y repulsivas. De hecho, la activación de los receptores de guía es el primer paso de los mecanismos de señalización que permiten que las puntas de los axones, los conos de crecimiento, respondan a los ligandos. Como tal, la modulación de su disponibilidad en la superficie celular es uno de los mecanismos que participan en el establecimiento de la sensibilidad del cono de crecimiento. Describimos aquí un método para visualizar exacto la dinámica espacio-temporal de la superficie de la célula de un receptor de la dirección del axón in vitro e in vivo en la médula espinal del polluelo que se desarrolla. Aprovechamos la propiedad de fluorescencia dependiente del pH de una variante de proteína fluorescente verde (GFP) para detectar específicamente la fracción del receptor de guía de axones que se dirige a la membrana plasmática. Primero describimos la validación in vitro de tales construcciones dependientes del pH y detallamos aún más su uso in vivo,en la cuerda espinal del polluelo, para evaluar la dinámica espacio-temporal del receptor de guía de axones de interés.

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg"> Figura 1. A. Esquema de las propiedades de fluorescencia pHluorin-plexinA1 en un contexto celular. La PHluorin es no fluorescente en compartimentos intracelulares donde el pH es ácido (<6) como en vesículas de tráfico o en endosomas y es fluorescente cuando se expone al medio extracelular donde el pH es n...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Se describe aquí el uso de una variante de proteína fluorescente verde sensible al pH, pHluorin, para estudiar la dinámica espacio-temporal de los receptores de guía de axones que se trafficking en la superficie celular. El receptor marcado con pHluorin se expresa tanto en cultivo celular como in vivo,utilizando la electroporación del embrión de pollito.

Uso De pHluorin Para Evaluar La Dinámica De Los Receptores De Orientación Axón En El Cultivo Celular Y En El Embrión De Polluelo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

use-phluorin-to-assess-dynamics-axon-guidance-receptors-cell-culture

Research

JoVE Journal

Neuroscience

8.2K vistas.  University of Lyon. Se describe aquí el uso de una variante de proteína fluorescente verde sensible al pH, pHluorin, para estudiar la dinámica espacio-temporal de los receptores de guía de axones que se trafficking en la superficie celular. El receptor marcado con pHluorin se expresa tanto en cultivo celular como in vivo,utilizando la electroporación del embrión de pollito.

Video: Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualización de los propioceptores<em&gt; Drosophila</em&gt; Las larvas y pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Investigación

Neurociencias

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

La electroporación del Rombencéfalo trazar trayectorias axonal y metas sinápticas en el embrión de pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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

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

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

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

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo el montaje de imágenes de embriones de ratón Proyecciones sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagen Ca<sup&gt; 2 +</sup&gt; Dinámica de Terminales cono fotorreceptor axón de la retina del ratón

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

El uso de condicionamiento de clásico de Eyeblink rastro para evaluar disfunción hipocampal en un modelo de rata de trastornos del espectro alcohólico Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Uso De Las Neuronas Del Hipocampo Cultivadas Primarias Para Estudiar El Ensamblaje De Los Segmentos Iniciales Axón

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Aislamiento y cultura de chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...