Este trabajo fue apoyado por una subvención del NIH otorgado a R. Brewster (1R01GM085290-01A1).

1.Microinjection <ol><li> Diluir plásmido que codifica la membrana dirigidos proteína verde fluorescente (mGFP, cortesía de Richard Harland) a una concentración de 40 ng / ml. El ADN es preparado por linearización del plásmido (mGFP/PCS2 +, cortesía de Richard Harland) purificado utilizando el kit Qiagen Maxi Prep. Además de rojo fenol (diluido volumen 1 / 10 en total) a la solución es preferible a visualizar la solución. Mantener la solución de ADN en el hielo. </li><li> Bajo un microscopio estereoscópico, calibrar la aguja de inyección (90 mm capilares de vidrio de Narishinge científica; Cat. N º NA-GD-1) por la alineación de la aguja sobre un portaobjetos de graduado y golpeando suavemente la parte de la aguja cuando el diámetro es 1μm con una navaja limpia hoja (para obtener una punta del tamaño adecuado). </li><li> Con el fin de calibrar la aguja para inyectar un volumen conocido (2NL), conectar la aguja con el aparato de microinyección (PCI 100 microinyector, Harvard Apparatus) y frente a llenar la aguja con 0.5μl de una solución de rojo fenol y agua colocado en una pieza de la parafina. La solución se puede prescindir de utilizar un pedal. Ajuste la presión a 7 kPa y variar el tiempo (10 ms 30msec) para que tenga 250 pedales para dispensar 0.5μl de la solución. </li><li> Coloque cuidadosamente la aguja calibrada en una tira de cera en un humidificado 100 mm placa de Petri. Para humedecer el plato, coloque Kimwipes húmeda alrededor de los bordes (esto evita la evaporación de la solución en la aguja). </li><li> Use una micropipeta para carga de 0.5 1μl del ADN en el extremo de la aguja (el extremo que no fue calibrado). </li><li> Una vez que la solución ha llegado a la punta de la aguja, conecte el extremo de la aguja que se usó para la carga a un aparato de microinyección. Ajustar la configuración de la microinyector para entregar aproximadamente 2 nl / inyección con un pedal. </li><li> A continuación, los embriones de la posición del pez cebra en el centro de una placa de Petri llena con un medio de embriones (Foto 1.0 ml de Hank # 1, 0,1 ml de Hank Foto # 2, de archivo de 1,0 ml de Hank # 4, 95,9 ml ddH2O, archivo 1,0 ml de Hank # 5 , 1,0 ml de archivo fresco de Hank º 6, el uso de 10 gotas de NaOH 1 M a pH 7,2). </li><li> Una vez que los embriones se han desarrollado hasta el punto deseado, suavemente agarre el corion con unas pinzas finas y situar la aguja en el área destinada a la inyección. Para la expresión de una amplia gama mGFP, inyectar ADN en el citoplasma de la yema de huevo o en la etapa de 1cell. Para limitar la expresión de mGFP a una región más pequeña, se inyecta el ADN en las cuatro (o más) etapa de las células en el citoplasma de una célula. </li><li> Presione el pedal del aparato de microinyección y entregar aproximadamente 2 nl, asegurando que la solución (visible debido a la adición de fenol rojo) está bien inyectada. </li><li> Retire con cuidado la aguja del embrión. </li><li> Repita este procedimiento para todos los embriones en la placa de Petri. </li><li> Permita que los embriones para desarrollar a 28,5 ° C a la etapa deseada. </li><li> Embriones Dechorionate suavemente quitando el corion con unas pinzas finas (si los embriones son más jóvenes que 24hpf, dechorionating en una placa de Petri de vidrio aumenta la tasa de supervivencia de los embriones). </li><li> Quitar corion vacío de la placa de Petri con una pipeta de vidrio. </li></ol> 2. Imágenes mGFP marcado con secciones vibratome <ol><li> Use una pipeta de vidrio para el transporte de los embriones en el fijador (4% de paraformaldehído en solución 1X tampón fosfato (PBS). Fix embriones a 4 ° C durante la noche. </li><li> Lavado de los embriones en 1X PBS tres veces durante 5 minutos cada uno. </li><li> Colocar los embriones en una placa Petri y, usando unas pinzas finas, eliminar la yema sin dañar el embrión. Retire como la yema de lo posible. </li><li> Prepare un 4% de agarosa de bajo punto de fusión de H 2 O. </li><li> Lugar agarosa fundida en el molde de plástico de seccionamiento. </li><li> Use unas pinzas finas para levantar un embrión inyectado desde la placa de Petri y colocar y orientar en la agarosa. El embrión debe ser colocado en un extremo del molde. </li><li> Una vez que la agarosa se ha endurecido, usar una herramienta de superficie plana, como una espátula, para quitar el bloqueo de agarosa del molde. </li><li> Corte el extremo inferior del bloque de agarosa con una cuchilla de afeitar para que sea uniforme. </li><li> Añadir una gota de pegamento a la etapa de vibratome y colocar el bloque de agarosa en el mismo (el lado del bloque que se estabilizó con la navaja debe enfrentarse a la cola). Para seccionar varios embriones, montar varios de agarosa incrustados de la etapa. </li><li> Conecte la etapa vibratome a la vibratome (Vibratome, Inc.). </li><li> Establezca los parámetros de la vibratome (conjunto de valores: 3 velocidades, 50 micras de espesor). Si el corte de múltiples embriones, alinear bloques de agarosa en el escenario y asegurarse de que la hoja pasa por todo el embrión, pero no a través de todo el ancho de los bloques de agarosa (por ejemplo, que las secciones correspondientes al embrión mismo estar juntos). </li><li> Vierta 1X PBS en la etapa de vibratome. </li><li> Sección. </li><li> Una vez que los embriones son sectioned, quitar el bloque de agarosa (s) de la etapa vibratome. </li><li> Separe cuidadosamente cada sección mediante la reducción de la parte trasera del bloque de agarosa (s). </li><li> Use unas pinzas finas para seleccionar las secciones que desee y coloque en un 1X PBS lleno de múltiples y plato (pozos de uso diferente para las secciones que corresponden a diferentes embriones). </li><li> Repita este procedimiento para cada embrión. </li><li> Continuar con el etiquetado de anticuerpos, aunque los pasos 19 23, para mejorar la imagen mGFP (este paso puede no ser necesario si las secciones son expuestas en un día o dos de preparación). </li><li> El tratamiento de las secciones durante 30 minutos a temperatura ambiente con solución de bloqueo en 1X PBS para bloquear la unión no específica. </li><li> Secciones se incuban con el anticuerpo primario (un conejo-GFP, Invitrogen Cat. No. A11122) diluido 1:200 @ en la solución de bloqueo en un agitador durante 5 horas a temperatura ambiente o durante la noche a 4 ° C. </li><li> Lavar cuatro veces (10 min, 15 min, 30 min y 60 min, cada uno) en el 1% BSA/1X PBS / 1% de solución de DMSO. </li><li> El tratamiento de las secciones con el anticuerpo adecuado secundaria en solución de bloqueo durante 5 horas a temperatura ambiente o durante la noche a 4 ° C. </li><li> Lavar cuatro veces (10 min, 15 min, 30 min y 60 min, cada uno) en el 1% BSA/1X PBS / 1% de solución de DMSO. </li><li> El tratamiento de las secciones con DAPI (0,1% en 50 ml de DAPI 1X PBS, Invitrogen Cat. No. D1306) durante 30 minutos para teñir los núcleos. </li><li> Lavar tres veces en PBS 1X durante 5 minutos cada uno. </li><li> Montaje de la diapositiva. </li><li> Imagen de microscopía confocal. </li></ol> 3. Imágenes en vivo <ol><li> Dechorionate embriones vivos, se inyecta 30 minutos antes de llegar a la etapa de desarrollo deseado. Colocar los embriones en la solución de sedantes (0,01% tricaína en medio de embriones) durante 15 minutos. </li><li> Coloque una gota de agarosa al 1% en una placa de cultivo de vidrio de 35 mm de fondo (MatTek; N º de P35G-o-20-C). </li><li> Permitir agarosa que se solidifique. </li><li> El calor del tubo capilar sobre la llama para hacer tres agujeros en agarosa (otros objetos como puntas de micropipeta se puede utilizar para hacer los agujeros). Asegúrese de que no hay residuos de agarosa en el fondo del agujero. </li><li> Llene la caja de Petri con medio de embriones. </li><li> Use una pipeta de vidrio para colocar el embrión en el agujero. </li><li> A continuación, orientar el embrión utilizando unas pinzas finas para que el área a explorar (la cabeza dorsal) está en contacto con el vidrio. </li><li> Cuidadosamente el transporte de la placa de Petri de la platina del microscopio (coloque la tapa sobre la placa de Petri para evitar la evaporación). </li><li> Mientras que imágenes, asegúrese de que los embriones son de 28,5 ° C. </li></ol> 4. Resultados Se describe aquí un acercamiento directo a las células de una sola imagen en el tubo neural del pez cebra con la expresión del mosaico. A pesar de la transparencia óptica del embrión de pez cebra, hemos encontrado que la visualización de las células neuronales es mucho mayor en las secciones transversales obtenidas mediante un vibratome. Estas secciones son de espesor (50 mm) que permite obtener imágenes de la extensión celular de una célula en diferentes planos focales con un microscopio confocal. Resultados utilizando esta metodología han sido publicados en otra 2, pero las imágenes representativas de las células en el tubo neural de un embrión de WT (Figura 1) y un N-cadherina (N-cad) mutante (Figura 2) se proporcionan. La última revela que las células en las regiones laterales de la placa neural en la N-cad mutantes no orientan adecuadamente hacia la línea media (Figura 2). En contraste con este documento de mosaico, el neuroepitelio inmunotinción con un marcador de superficie celular en general, tales como beta-catenina no permite la morfología de las células individuales para ser visualizado (Figura 3). Imagen de lapso de tiempo de embriones vivos complementa el etiquetado mosaico de muestras fijas, lo que permite una mejor comprensión de la dinámica celular que tienen lugar durante la neurulación. Película 1 reveló que las células WT activamente migrar hacia la línea media mediante la ampliación medial orientada protuberancias de la membrana. <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="Figura 1" /> Figura 1. MGFP expresión en el tubo neural de un embrión de pez cebra WT. <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="Figura 2" /> Figura 2. MGFP expresión en el tubo neural de un embrión de pez cebra N-cadherina. <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="Figura 3" /> Figura 3. B-catenina inmuno tinción de la neuroepithelium.

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

En conclusión, las técnicas de etiquetado descritos aquí permiten el análisis de células individuales de los procesos morfogenéticos en el embrión de pez cebra. El énfasis principal de este protocolo es sobre los métodos para que las células de imágenes etiquetadas en el tubo neural con mGFP bajo el control de un promotor en todas partes. Para otras aplicaciones de este ensayo de expresión transitoria lectores deben referirse a un documento reciente de Andersen et al. 3.

A correction was made to <a href="http://www.jove.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

Soluciones <ul><li> Foto de Hank # 1: 8,0 g de NaCl, 0,4 g de KCl en 100 ml dd H 2 O </li><li> Foto de Hank # 2: 0,358 g de Na 2 HPO 4 anhidro, 0,60 g KH 2 PO 4 en 100 ml ddH2O </li><li> Foto de Hank # 4: 0,72 g de CaCl2 en 50 ml ddH2O </li><li> Foto de Hank # 5: 1,23 g MgSO4 x7H 2 O en 50 ml dd H 2 O </li><li> 1 X tampón fosfato: 0,8% NaCl, KCl 0,02%, 0,02 M PO4, pH 7,3 </li><li> Solución de bloqueo: 2% de suero normal de cabra, 1% de albúmina sérica bovina (BSA), dimethysulfoxide 1% (DMSO) </li></ul>

labeling and imaging cells in the zebrafish hindbrain

La clave para entender los procesos morfogenéticos que forma el embrión de vertebrados temprana es la capacidad de las células de la imagen en alta resolución. En embriones de pez cebra, la inyección de los resultados de ADN plásmido en la expresión del mosaico, lo que permite la visualización de células individuales o pequeños grupos de células<sup&gt; 1</sup&gt;. Se describe cómo la inyección de ADN que codifica plásmido membrana dirigidos proteína verde fluorescente (mGFP) bajo el control de un promotor en todas partes se puede utilizar para que las células sometidas a imágenes neurulación. Central de este protocolo es la metodología de las células de imágenes etiquetadas en alta resolución en las secciones y también en tiempo real. Este protocolo implica la inyección de ADN en embriones de pez cebra mGFP jóvenes. Los embriones son luego procesadas para seccionar vibratome, el etiquetado de anticuerpos y la imagen con un microscopio confocal. Por otra parte, los embriones vivos que expresan mGFP se pueden crear imágenes con lapso de tiempo de microscopía confocal. Hemos utilizado anteriormente este método sencillo para analizar los comportamientos celulares que conducen a la formación del tubo neural en la región posterior del cerebro de embriones de pez cebra<sup&gt; 2</sup&gt;. Los preparativos fijo permitido para la visualización sin precedentes de formas de organización celular y en el tubo neural, mientras que imágenes en vivo complementa este enfoque permite una mejor comprensión de la dinámica celular que tienen lugar durante la neurulación.

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

La clave para entender los procesos morfogenéticos que forma el embrión temprano es la capacidad de las células de la imagen en alta resolución. Se describe aquí una técnica para el etiquetado de las células individuales o pequeños grupos de células en embriones de pez cebra con toda la membrana dirigidos proteína verde fluorescente.

Etiquetado y las Pilas de imágenes en la parte posterior del cerebro de pez cebra

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

Research

JoVE Journal

Neuroscience

11.2K vistas.  University of Maryland, Baltimore County. La clave para entender los procesos morfogenéticos que forma el embrión temprano es la capacidad de las células de la imagen en alta resolución. Se describe aquí una técnica para el etiquetado de las células individuales o pequeños grupos de células en embriones de pez cebra con toda la membrana dirigidos proteína verde fluorescente.

Video: Labeling and Imaging Cells in the Zebrafish Hindbrain

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 ...

Investigación

Neurociencias

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 ...

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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

El marcaje de células individuales en el Sistema Nervioso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etiquetado retrógrada de células ganglionares retinianas en adultos de pez cebra con tintes fluorescentes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

El cerebelo de ratón como modelo para el estudio de la neurogénesis embrionaria

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Etiquetado e imágenes de placas amiloideas en el tejido cerebral utilizando la curcumina de polifenoles naturales

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Imágenes in vivo de tejido cerebral completamente activo en larvas y juveniles de pez cebra despiertos mediante la extirpación del cráneo y la piel

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...