Este trabajo fue apoyado por los Institutos Canadienses de Investigación en Salud (CIHR) Otorgar a Wayne S. Sossin. Carole A. Farah es el beneficiario de una beca postdoctoral del Fonds de la Recherche en Santé du Québec (FRSQ) y una beca de Conrad Harrington. Los autores desean agradecer a Joanna Bugía, Hastings y Margaret Labban Margaret para recibir ayuda con la filmación del video y la piscina Madeline Richmond para ayudar con la descripción gráfica.

1. Preparación y mantenimiento de cultivos celulares en monocapa Sf9 <ol><li> Sf9 cultivo celular y el mantenimiento se lleva a cabo en una campana de cultivo de tejidos. </li><li> Sf9 células derivan de células de Spodoptera frugiperda ovario (Sf21 células) y se pueden comprar en las células congeladas en la gracia de los medios de Invitrogen. </li><li> Lugar 8 ml de medio de insectos de Grace, complementada (1X), a los cuales el 30% de suero fetal bovino (FBS) se ha añadido en un 75 cm 2 frasco de cultivo celular con el cuello inclinado. </li><li> Descongelar el tubo congelado de células Sf9 rápidamente en un baño de agua 37 ° C moviendo el tubo de ida y vuelta (1-2 minutos). </li><li> La transferencia de células Sf9 a los 75 cm 2 frasco de cultivo celular y la placa de roca suavemente con la mano para distribuir uniformemente las células. </li><li> Incubar 1-2 horas a 27 ° C hasta que las células se han unido. </li><li> Quitar medio viejo y reemplazarlo con 10 ml de medio de insectos freshGrace con FBS al 30%. </li><li> Se incuba a 27 ° C hasta que las células son confluentes. </li><li> Para mantener cultivos en monocapa, eliminar viejo medio del matraz confluente de células Sf9 y añadir 5 ml de medio de insectos de Grace con 10% de SFB. </li><li> Golpee ligeramente en el lado del frasco varias veces para separar las células Sf9. </li><li> Usando una pipeta de 10 ml y pipeta una, una pipeta de los medios arriba y hacia abajo una vez suavemente, pulverización pared frasco mientras pipeteo hacia abajo. Transferencia de las células a un tubo de 15 ml estéril poliestireno. </li><li> Añadir 2 ml de suspensión celular Sf9 a 8 ml de medio de insectos de Grace con FBS al 10% (dilución 1:5 para mantener el crecimiento en fase logarítmica) en los nuevos 75 cm 2 frasco de cultivo celular y la placa de roca suavemente con la mano. </li><li> Se incuba a 27 ° C hasta que las células son confluentes. </li></ol> 2. Expresión de la PKC marcado con fluorescencia en células Sf9 <ol><li> Para vivir de imágenes de experimento, la cultura Sf9 células en 35 mm MatTek cultura del vidrio disheswith inferior una superficie de vidrio de 14 mm y un espesor cubreobjetos de 0,16 a 0,19 mm. </li><li> PKC etiquetados con proteínas fluorescentes se expresan en células Sf9 con Cellfectin transfección II recomendación siguiente reactivo del fabricante con las modificaciones que se detallan a continuación. </li><li> Día 1: Antes de recubrimiento de las células, el tratamiento de cada plato con medio MatTek insectos de Grace con FBS al 10% por lo menos durante 30 minutos. </li><li> Contador de células Sf9 desde el paso 11 (arriba) con un hemocitómetro. </li><li> Placa de 0,05 x 10 6 células de cada cubreobjetos MatTek en un volumen total de 150 mL. Permiten a las células para conectar durante 30 minutos a temperatura ambiente. </li><li> Añadir 2 ml de Grace suplementado con FBS al 10% para cada plato 1 mL a la vez poco a poco para evitar desprendimiento de las células. </li><li> Se incuba a 27 ° C durante la noche. </li><li> A partir de ahora, utilice las recomendaciones del fabricante para la transfección de células Sf9 con el ADN del plásmido. </li><li> Día 2: transfectar células Sf9 con PKC marcado con fluorescencia utilizando Cellfectin reactivo de transfección II. A menudo hemos co-expresados ​​en este sistema PKC etiquetados con aumento de proteína verde fluorescente (EGFP) y monomérico proteína fluorescente roja (mRFP) (los detalles de la construcción del plásmido se han descrito previamente 2,5). Expresión de una proteína fluorescente en un momento en los rendimientos de alrededor del 70-80% que expresan las células 72 horas después de la transfección. Co-expresión de dos proteínas fluorescentes disminuye la eficiencia de transfección de alrededor del 30% co-expresión de las células. Cuando se co-expresando EGFP de etiquetado de la PKC y mRFP etiquetados con PKC, use 2 veces más ADN plásmido para la proteína mRFP-etiquetados para la expresión óptima de proteínas. </li><li> Tocar en vivo de imágenes de 48-72 horas después de la transfección (para la expresión óptima de proteínas, esperar 72 horas). </li></ol> 3. Expresión de la PKC marcado con fluorescencia en las neuronas sensoriales Aplysia <ol><li> Preparación de los cultivos de células neuronales Aplysia ha sido descrito en un artículo de vídeo por Zhao y cuatro colegas. </li><li> PKC etiquetados con proteínas fluorescentes se expresan en las neuronas sensoriales Aplysia mediante el procedimiento de microinyección se detalla a continuación. </li><li> Prepare una solución de reserva del 1% Fast Green (FCF) en agua destilada, se filtra con una jeringa y un filtro de jeringa de 28 mm (0,20 m). Alícuota y almacenar a -20 ° C. </li><li> Microinyección de las neuronas sensoriales se pueden realizar en el día 1 o 2 días después de aislar a las neuronas, pero nos encontramos con que dos días de espera permite que la adherencia celular mejor que el cubreobjetos. </li><li> El día 2, después del aislamiento de las neuronas sensoriales, descongelar una alícuota de Fast Green y el filtro de nuevo con una jeringa y un filtro de jeringa de 28 mm (0,20 m). </li><li> Prepare una solución de ADN plásmido en agua destilada que contiene 0,5% Fast Green. Las concentraciones de ADN del plásmido de hasta 0,4 mg / l se puede utilizar, pero no se recomienda ir por encima de estos valores. </li><li> Filtrar el ADN del plásmido / Fast Green solución con una jeringa de 1 mL y un filtro de jeringa de 4 mm (0,20 m). </li><li> Centrifugar el ADN del plásmido / Fast Green solución a 16.110 xg durante 15 minutos usando un micrófonorocentrifuge. </li><li> Preparar electrodos fuerte para la microinyección de las neuronas con un extractor de microelectrodos (Sutter Flaming / Brown Micropipeta Puller, modelo P-97). El uso de un filamento de caja, tirar de microelectrodos de vidrio con 1 mm de diámetro exterior de 0,75 mm de diámetro interior y 100 mm de longitud con paredes delgadas capilares de vidrio con un filamento en el interior (World Precision Instruments TW100F-4). Para obtener más información acerca de tirar electrodos microinyección por favor refiérase a libros de cocina Sutter electrodo. </li><li> Llene cada electrodo con 2 l de ADN de plásmido / Verde solución rápida utilizando microloader punta de la pipeta (eppendorf CA32950-050). </li><li> La transferencia de la parte inferior de vidrio MatTek plato de cultivo que contienen cultivos Aplysia neuronal a la estación de microinyección. </li><li> La estación de microinyección consiste en lo siguiente: una de fabricación casera etapa de vidrio grande para sostener las placas de cultivo en una mesa de aislamiento de vibración (vibración cinética de estaciones de trabajo de aislamiento), equipo de música de alcance con iluminación halógena externa, un micromanipulador (Sutter Huxley Micromanipulador tipo de pared que permite el posicionamiento gruesas y ultra fino en los tres ejes), el titular de microelectrodos conectados a una bomba neumática (Instrumentos del Mundo de precisión neumática PV820 Pico-bomba). La entrada de la presión de la bomba neumática está conectada a un tanque de nitrógeno comprimido. </li><li> Inserte un microelectrodo en el soporte de microelectrodos. </li><li> Viendo bajo un microscopio estereoscópico, inserte la punta de la micropipeta en el núcleo de la célula. </li><li> Entregar pulsos cortos de presión de nitrógeno (10 a 100 ms de duración, 20 lb / in 2) hasta que el núcleo se convierte de manera uniforme verde. </li><li> Se incuban las células durante 4 horas a temperatura ambiente y mantenga a 4 ° C hasta su uso. Para la translocación de PKC óptima, presentación en vivo de imágenes de experimento 20 a 24 horas post-inyección. </li></ol> 4. Visualización de la translocación de PKC en células Sf9 vida y en las neuronas sensoriales Aplysia <ol><li> Para las imágenes, se utiliza un microscopio confocal Zeiss LSM510 invertido con un Axiovert 200. </li><li> Imagen del protocolo es el mismo para las células Sf9 y de las neuronas sensoriales Aplysia excepción de los siguientes: Sf9 células son imágenes con un objetivo de aceite de inmersión x63 mientras que las neuronas sensoriales Aplysia son imágenes con un objetivo de inmersión en aceite de x40. Sf9 células son expuestas en una cámara de temperatura controlada mantenida a 26-27 º C mientras que las neuronas sensoriales Aplysia son expuestas a temperatura ambiente mantiene alrededor de 18-20 º C. </li><li> Nosotros usamos un 30 mW láser de argón (excitación a 488 nm) con la potencia del láser 50% de la PKC imagen etiquetados con la proteína EGFP y un láser de HeNe (excitación a 543nm) para PKC imagen etiquetados con proteínas mRFP. El argón y láser de HeNe líneas se atenúan y el 4% y el 50% de transmisión de salida, respectivamente, antes de la imagen y el agujero se ajusta a uno. </li><li> Es importante contar con la placa de cultivo estabilizado en el escenario de imágenes y para evitar el movimiento o vibración. </li><li> Retire 1 ml de medio de cultivo en el plato suavemente con una pipeta de 10 mL dejando un volumen total de ml en el plato. </li><li> Tomar fotos en la sección central de las células donde se puede ver el núcleo. </li><li> Establecer una serie de tiempo de 10-20 imágenes confocal de tomar una foto cada 30 segundos. </li><li> Iniciar la serie de tiempo. </li><li> Después de dos fotografías se toman (después del punto de tiempo de 30 segundos), agregar 1 ml de la droga (2X concentración) con suavidad gota a gota en la parte superior de las células con una pipeta P1000. El fármaco se añade de forma continua durante unos 30 segundos. </li><li> Algunos fármacos inducen un desplazamiento rápido que puede ser visible inmediatamente después del tratamiento de drogas siguientes (en el punto de tiempo de 60 segundos), mientras que otras inducen un desplazamiento lento, que está a sólo unos 50-10 óptima del tratamiento (Película 1 y Película 2). </li><li> Si el medicamento tiene que ser lavado, el uso de 2 x 10 ml pipetas para hacer el lavado, pipetear el tampón de lavado suavemente con una pipeta en un lado del plato y la pipeta hasta los medios de comunicación en el otro lado. </li><li> Si el lavado ha sido eficaz y si el efecto de la droga es reversible, PKC vuelve al citosol dentro de 30-60 segundos. </li><li> Guarde la película como un archivo de LSM. </li><li> Uso de imágenes NIH J software para abrir los archivos de LSM y cuantificar las imágenes pre y post tratamiento de drogas. La cuantificación se ha descrito en otra parte 1. </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

Hemos descrito una técnica de translocación de PKC imagen marcado con fluorescencia en tiempo real en células Sf9 y en las neuronas sensoriales Aplysia. Sf9 proporcionar un sistema simple a la translocación de PKC imagen desde el cultivo y la transfección de ellos es bastante sencillo. Por el contrario, la microinyección de las neuronas sensoriales Aplysia toma algún tiempo para dominar, hasta unos pocos meses. Dificultades relacionadas con esta técnica son la obstrucción de los electrodos. Fi...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
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live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

La proteína quinasa C (PKC) son serina treonina cinasas que juegan un papel central en la regulación de una amplia variedad de procesos celulares, tales como el crecimiento celular y el aprendizaje y la memoria. Hay cuatro familias conocidas de las isoformas de PKC en los vertebrados: PKC clásicas (α, βI, βII y γ), el tipo de novela que PKC (ε y η), la novela de tipo II PKC (δ y θ), y PKC atípicas (ζ y ι ). La PKC clásicas son activados por Ca 2 + y diacylclycerol (DAG), mientras que las PKC nuevas son activadas por DAG, pero son Ca 2 +-independiente. Las PKC atípicas son activados por Ca 2 + ni ni DAG. En Aplysia californica, nuestro sistema modelo para estudiar la formación de la memoria, hay tres isoformas específicas del sistema nervioso PKC uno de cada clase principal, a saber, la PKC convencionales APL I, el tipo de novela que PKC Apl II y de la PKC atípicas APL III. PKC son lípido quinasas activadas y por lo tanto la activación de la PKC clásicas y nuevas en respuesta a señales extracelulares con frecuencia se ha correlacionado con translocación de PKC desde el citoplasma a la membrana plasmática. Por lo tanto, la visualización de la translocación de la PKC en tiempo real en células vivas se ha convertido en una herramienta invaluable para dilucidar las vías de transducción de señales que conducen a la activación de la PKC. Por ejemplo, esta técnica ha permitido que nos permite establecer que las diferentes isoformas de PKC trasladar bajo diferentes condiciones de mediar en distintos tipos de plasticidad sináptica y que la serotonina (5-HT) activación de la PKC Apl II requiere la producción de los DAG y ácido fosfatídico (PA) para translocación 01/02. Es importante destacar que la capacidad de visualizar la misma neurona en repetidas ocasiones nos ha permitido, por ejemplo, para medir la desensibilización de la respuesta de la PKC en exquisito detalle 3. En este vídeo, que muestran cada paso de la preparación de Sf9 cultivos celulares, cultivos de neuronas sensoriales Aplysia se han descrito en otro artículo de vídeo 4, expresando PKC marcado con fluorescencia en células Sf9 y en las neuronas sensoriales Aplysia y en vivo de imágenes de la translocación de PKC en respuesta a activadores diferentes utilizando microscopía de barrido láser.

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

En este vídeo, se demuestra la visualización de la translocación de la PKC en células vivas mediante PKC marcado con fluorescencia.

En vivo de imágenes de la translocación de PKC en células Sf9 y en las neuronas sensoriales Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K vistas.  McGill University. En este vídeo, se demuestra la visualización de la translocación de la PKC en células vivas mediante PKC marcado con fluorescencia.

Video: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse de imágenes en vivo de la relación clonal con células progenitoras neurales en el cerebro anterior El desarrollo de pez cebra

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus mediada por la manipulación genética y la visualización de las neuronas sensoriales olfativas In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracelularmente Identificar las neuronas motoras de un músculo en la piscina Motor<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

El aislamiento de las neuronas sensoriales de<em&gt; Aplysia californica</em&gt; Se Patch clamp grabaciones de Corrientes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Un método para detectar y aislar las vías aéreas que inervan las neuronas sensoriales en ratones

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

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 ...