Este trabajo fue apoyado por el Instituto Nacional de Salud Mental, y la National Science Foundation. Un agradecimiento especial a los miembros del laboratorio Sive para muchas discusiones útiles y críticas constructivas, ya Olivier Paugois para la cría de peces experto.

1. Preparación para la microinyección <ol><li> Preparar agujas de microinyección tirando tubos capilares utilizando Sutter extractor aguja instrumentos. </li><li> Carga aguja de microinyección con tinte fluorescente (FITC-dextrano). </li><li> Montar la aguja en un micromanipulador y un aparato de microinyección. </li><li> Corte cuidadosamente la aguja de microinyección con unas pinzas a unos 2 m de ancho, sin embargo, esto puede variar dependiendo de la configuración microinyector. Para nuestros agujas de microinyección, esto corresponde a la primera región de la aguja desde la punta que no se doble. </li><li> Medida de tamaño de gota en el aceite, el ajuste de tiempo de inyección y la presión, de modo que cada inyección ofrece 1 nl. Ejemplos de ajustes para Harvard Apparatus microinyector son: equilibrio P = 1,4 psi, P = 1,4 psi a cabo, P = 22,9 psi inyectar, P = 67,8 psi claras con un tiempo de inyección de .4 a 0.7 seg. El diámetro de la aguja con esta configuración es de aproximadamente 2 m. Sin embargo, los ajustes se microinyector específico, y variar unaegún el diámetro de la aguja. </li></ol> 2. Preparación de los embriones <ol><li> Capa 2 platos con agarosa al 1% en agua para cada condición, hacer agujeros en agarosa con una punta de pipeta 1-200 l, y quitar bloques de agarosa. Llenar los platos con los medios de embriones. </li><li> Utilizando pinzas, embriones dechorionate que son 18 hpf o más bajo un microscopio estereoscópico. Los embriones se organizaron de acuerdo a Kimmel et al. 11. </li><li> Transferencia de embriones dechorionated en el primer plato de agarosa recubierta. </li><li> Para anestesiar embriones, añadir tricaína (0,1 mg / ml) a la placa de embriones hasta que dejan de moverse (hecho de acuerdo con Westerfield 12). </li></ol> 3. La inyección de los ventrículos del cerebro <ol><li> Embriones Orient lo que está buscando en su lado dorsal poniendo la cola del embrión en el agujero. Si su micromanipulador está a la derecha, a continuación, mover el embrión, de manera que el cerebro anterior está a la izquierda y a la derecha rombencéfalo. </li><li> Posición de la aguja en widest punto de ventrículo rombencéfalo. </li><li> Con cuidado, perfore placa del techo del ventrículo cerebro posterior asegurándose de no pasar por la profundidad del cerebro en la yema (Figura 1A). </li><li> Inyectar 1-2 nl de colorante fluorescente en los ventrículos asegurándose de que el colorante se llena toda la longitud de los ventrículos del cerebro. </li><li> Transferencia de embriones al segundo plato de agarosa revestido lleno de Medios para embriones y volver a anestesiar, como se describe en 2.4. </li><li> Iniciar inmediatamente formación de imágenes, tal como se describe en la sección 4 con el fin de obtener una imagen de tiempo cero. </li></ol> 4. Proyección de imagen <ol><li> Embriones Oriente con su cola en el agujero como se describe en 3.1. </li><li> El uso de un microscopio de disección con luz transmitida y fluorescencia tanto para tomar una imagen de campo claro dorsal. Mantener la ampliación constante entre imágenes de embriones diferentes. Esto permite una comparación directa de los análisis realizados usando Image J (5,2-6). </li><li> Sin mover el embrión, un microscopio o un plato, tomar unaimagen fluorescente correspondiente. </li><li> Repita el procedimiento para cada embrión en momentos deseados. </li></ol> 5. Cuantificación del Movimiento Dye <ol><li> Combinar claro y las imágenes fluorescentes en Photoshop como se ha descrito previamente por Gutzman y Sive 10. </li><li> Mida la distancia que recorre la frente de colorante en el software Image J disponibles en <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> . </li><li> Abrir el archivo resultante de la fusión en J de imagen y utilizar la herramienta de línea para dibujar una línea desde el prosencéfalo bisagra puntos para teñir delante en un ángulo de 10-20 ° de neuroepitelio (fig. 1A). Esta región fue elegido porque es el primer sitio y más notable de fuga del líquido del neuroepitelio de tipo salvaje. </li><li> Selecciona la herramienta de medición para calcular la longitud de la línea. </li><li> Repita el proceso para cada punto del tiempo. </li><li> Calcular la distancia neta el frente del colorante se movió a través del tiempo restando distancia en t = 0 a partir de otros puntos de tiempo. </li><li> Parcela engráfico. </li></ol> 6. Los resultados representativos Un ejemplo de los resultados obtenidos en un ensayo de permeabilidad neuroepitelial usando embriones de tipo salvaje se muestra en la figura 1B-D. Para distinguir con precisión la permeabilidad, es útil para poner a prueba los tintes con weightsto molecular diferente identificar un tamaño que es sólo ligeramente agujereado en embriones de tipo salvaje o de control (Figura 2). Esto permite la identificación de mutantes genéticos o condiciones ambientales que aumentan o disminuyen la permeabilidad (Figura 1D, verde y líneas rojas, respectivamente). Para el neuroepitelio pez cebra 24 HPF, 70 kDa dextrano FITC fugas lentamente durante 2 horas, mientras que 2.000 kDa no y 10 kDa casi inmediatamente se escapa. Por lo tanto 70 kDa es el peso ideal molecular para identificar las condiciones que aumentar y disminuir la permeabilidad neuroepitelial. Si la aguja no ve la luz ventricular, fluorescencia wenfermo aparecer fuera del cerebro en t = 0 (para un ejemplo, véase Gutzman y Sive, 2009 10). Estos embriones deben ser desechados ya que el medio de contraste inyectado no figuraba inicialmente en el cerebro y no hay ninguna conclusión clara sobre el movimiento del colorante y la permeabilidad de la neuropeithelium se pueden hacer. Por último, si los embriones tienen ventrículos pequeños o un-inflados ventrículos del cerebro, inyección usada de ventrículos con una solución salina se puede hacer antes de la inyección del colorante fluorescente. Esto infla los ventrículos de toma de visualización posterior de los ventrículos más fácil cuando se inyecta con el colorante fluorescente. Los controles apropiados debe ser realizado para determinar si la inyección de solución salina interrumpe el desarrollo normal del tubo neural. <img alt="Figura 1" src="/files/ftp_upload/4242/4242fig1.jpg" /> Figura 1. Evolución temporal de los diferentes tintes peso molecular. (A) Diagrama Experimental. Primero, Tinte fluorescente se inyecta en los ventrículos. X = posición de la aguja para la inyección. Siguiente imagen dorsales son capturados en el tiempo. Finalmente, la distancia recorrida por el frente del colorante desde el cerebro anterior se mide bisagra-punto (representado por una línea roja). (BC) Fusionada campo claro y fluorescencia imágenes dorsal a 22 HPF (t = 0 min, B) y 24 HPF (t = 120 min, C). Línea blanca indica la distancia de la parte frontal del cerebro anterior de tinte ventrículo. (D) hipotéticos datos de permeabilidad de la muestra. Azul = tipo salvaje o controles, rojo = muestra disminución de la permeabilidad con relación al control, y muestra verde = aumento de la permeabilidad con respecto al control. <img alt="Figura 2" src="/files/ftp_upload/4242/4242fig2.jpg" /> Figura 2. Medición de la permeabilidad neuroepitelial a diferentes colorantes peso molecular. (AE) y campo claro dorsal fusionada imágenes fluorescentes de 22 hpf embriones de tipo salvaje en t = 0 min después de la inyección con FITC-dExtran de los siguientes pesos moleculares: 10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D) y 2.000 kDa (E). (A&#39;-e &#39;) Igual que en el embrión (AE) en t = 120 min a 24 hpf. Anterior a la izquierda. F = cerebro anterior, cerebro medio = M, H = rombencéfalo. Asterisk = oreja.

<ol>
	<li>Harrington, M. J., 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=Comparative+analysis+of+neurulation:+first+impressions+do+not+count.">Comparative analysis of neurulation: first impressions do not count.</a> Mol. Reprod. Dev. 76, 954-965 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+vertebrate+neurulation+and+a+re-evaluation+of+teleost+neural+tube+formation.">Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation.</a> Mech. Dev. 121, 1189-1197 (2004).</li><li>Salehi, Z., Mashayekhi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cerebrospinal+fluid+on+neural+cell+survival+in+the+developing+chick+cerebral+cortex:+an+in+vivo+study.">The role of cerebrospinal fluid on neural cell survival in the developing chick cerebral cortex: an in vivo study.</a> Eur. J. Neurol. 13, 760-764 (2006).</li><li>Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryonic+brain+development+in+rats+requires+the+trophic+influence+of+cerebrospinal+fluid.">Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid.</a> Int. J. Dev. Neurosci. 27, 733-740 (2009).</li><li>Lehtinen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebrospinal+fluid+provides+a+proliferative+niche+for+neural+progenitor+cells.">The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.</a> Neuron. 69, 893-905 (2011).</li><li>Gato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cerebrospinal+fluid+regulates+neuroepithelial+survival,+proliferation,+and+neurogenesis+in+chick+embryos.">Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos.</a> Anat. Rec. A. Discov. Mol. Cell Evol. Biol. 284, 475-484 (2005).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Totally+tubular:+the+mystery+behind+function+and+origin+of+the+brain+ventricular+system.">Totally tubular: the mystery behind function and origin of the brain ventricular system.</a> Bioessays. 31, 446-458 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+neuroepithelial+barrier+by+Claudin5a+is+essential+for+zebrafish+brain+ventricular+lumen+expansion.">Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1425-1430 (2010).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+brain+ventricle+injection.">Zebrafish brain ventricle injection.</a> J. Vis. Exp. , (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M., Sprague, J., Doerry, E., Douglas, S., Grp, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Information+Network+(ZFIN):+a+resource+for+genetic,+genomic+and+developmental+research.">The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.</a> Nucleic Acids Research. 29, 87-90 (2001).</li></ol>

No hay conflictos de interés declarado.

Se demuestra la capacidad de cuantificar la permeabilidad del cerebro embrionario de pez cebra vivo tal como se determina por un medio de contraste inyectado de un peso molecular dado. Nuestra observación de que el neuroepitelio embrionario de pez cebra es diferencialmente permeable a los tintes de diferentes pesos moleculares sugiere que el colorante se está moviendo a través de la permeabilidad paracelular. Sin embargo, no podemos descartar la posibilidad de una contribución a la permeabilidad transcelular observa...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Dextrano, fluoresceína, aniónica, lisina corregible </td><td> Invitrogen </td><td> D7136, D7137, D1822, D1820, D1845 </td></tr><tr><td> Tricaína polvo </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Los tubos capilares </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

El sistema ventricular del cerebro se conserva entre los vertebrados, y se compone de una serie de cavidades interconectadas llamado ventrículos del cerebro, que se forman durante las primeras etapas del desarrollo del cerebro y se mantienen durante toda la vida del animal. El sistema ventricular del cerebro se encuentra en los vertebrados, y los ventrículos se desarrollan después de la formación del tubo neural, cuando el lumen central se llena con fluido cerebroespinal (CSF) 1,2. CSF es un fluido rico en proteínas que es esencial para el desarrollo normal del cerebro y la función 3-6. En el pez cebra, la inflación cerebro ventrículo comienza a aproximadamente 18 horas después de la fertilización (hpf), después de que el tubo neural se cierra. Varios procesos están relacionados con la formación del cerebro ventrículo, incluida la formación de un neuroepitelio, la formación de uniones estrechas que regula la permeabilidad y la producción de LCR. Hemos demostrado que la Na, K-ATPasa se requiere para la inflación ventrículo cerebral, afectando todos estos procesoses 7,8, mientras que claudin 5a es necesario para la formación de la unión estrecha 9. Además, se demostró que &quot;relajación&quot; del neuroepitelio embrionario, a través de la inhibición de la miosina, se asocia con la inflación cerebro ventrículo. Para investigar la regulación de la permeabilidad durante la inflación pez cebra ventrículo cerebral, hemos desarrollado un medio de contraste ventricular ensayo de retención. Este método utiliza la inyección ventrículo cerebral en un embrión de pez cebra de estar, una técnica desarrollada anteriormente en nuestro laboratorio 10, para etiquetar fluorescentemente el líquido cefalorraquídeo. Los embriones son entonces fotografiado con el tiempo a medida que el tinte fluorescente a través de los ventrículos cerebrales y el neuroepitelio. La distancia que el frente del colorante se aleja de la basal (no luminal) lado del neuroepitelio con el tiempo se cuantifica y es una medida de la permeabilidad neuroepitelial (Figura 1). Observamos que los colorantes de 70 kDa y más pequeñas se mueven a través del neuroepitelio y puede ser detected fuera del cerebro embrionario de pez cebra a las 24 HPF (Figura 2). Este ensayo de retención de colorante puede ser utilizado para analizar la permeabilidad neuroepitelial en una variedad de diferentes antecedentes genéticos, en diferentes momentos durante el desarrollo, y después de las perturbaciones ambientales. También puede ser útil en el examen acumulación patológica de CSF. En general, esta técnica permite a los investigadores a analizar la función y la regulación de la permeabilidad durante el desarrollo y la enfermedad.

Watch this Scientific Journal Video about An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium at JoVE.com

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Se describe una medición vivo animal entero cuantitativo para la permeabilidad del cerebro embrionario de pez cebra. La técnica analiza la capacidad de retener el líquido cefalorraquídeo y moléculas de diferentes pesos moleculares dentro del lumen del tubo neural y cuantifica su movimiento de salida de los ventrículos. Este método es útil para determinar las diferencias en la permeabilidad epitelial y maduración durante el desarrollo y la enfermedad.

Un ensayo para la permeabilidad del neuroepitelio embrionario de pez cebra

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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Research

JoVE Journal

Neuroscience

9.4K vistas.  Massachusetts Institute of Technology. Se describe una medición vivo animal entero cuantitativo para la permeabilidad del cerebro embrionario de pez cebra. La técnica analiza la capacidad de retener el líquido cefalorraquídeo y moléculas de diferentes pesos moleculares dentro del lumen del tubo neural y cuantifica su movimiento de salida de los ventrículos. Este método es útil para determinar las diferencias en la permeabilidad epitelial y maduración durante el desarrollo y la enfermedad.

Video: An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Investigación

Neurociencias

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imágenes de alta resolución en vivo del comportamiento de las células en el desarrollo de neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Drenaje Manual de los ventrículos del cerebro embrionario de pez cebra

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo de imágenes en vivo de las divisiones sola celda en ratón neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Las grabaciones de las células embrionarias de pez cebra Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un ensayo para medir los efectos del etanol sobre la velocidad de locomoción<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método electrofisiológico para registrar las respuestas de voltaje intracelular<em&gt; Drosophila</em&gt; Los fotorreceptores y interneuronas a la luz Estímulos<em&gt; En Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Un ensayo para la permeabilidad del neuroepitelio embrionario de pez cebra

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