Este trabalho foi financiado pelo Instituto Nacional para a Saúde Mental, e da National Science Foundation. Agradecimentos especiais a membros do laboratório SIVE para muitas discussões úteis e críticas construtivas, e para Olivier Paugois para criação de peixes especialista.

1. Preparando-se para microinjeção <ol><li> Prepare agulhas microinjeção puxando tubos capilares utilizando Sutter extrator agulha instrumentos. </li><li> Agulha de microinjecção de carga com corante fluorescente (FITC-dextrano). </li><li> Montar a agulha no aparelho micromanipulador e microinjecção. </li><li> Cuidadosamente quebrar agulha microinjeção usando uma pinça para cerca de 2 m de largura, no entanto, isso vai variar dependendo da configuração do microinjetor. Para agulhas nossos microinjecção, esta corresponde à primeira região da agulha a partir da ponta, que não se curva. </li><li> Medir o tamanho da gota de óleo, ajustando o tempo de injecção e pressão, de modo que cada um proporciona uma injecção nl. Definições de exemplo para Harvard Apparatus microinjetor são: P = 1,4 psi equilíbrio, P = 1,4 psi fora, P = 22,9 psi injetar, P = 67,8 psi claras com um tempo de injeção de 0,4-0,7 seg. O diâmetro da nossa agulha usando essas configurações é de aproximadamente 2 m. No entanto, as definições serão microinjetor específico, e variamegundo a agulha de diâmetro. </li></ol> 2. Preparando os embriões <ol><li> Revestimento de dois pratos com agarose a 1% em água, para cada condição, furos puxão em agarose com uma ponta de pipeta de 1-200 uL, e remover tampões de agarose. Preencha pratos com a mídia de embriões. </li><li> Usando pinça, embriões dechorionate que são 18 hpf ou mais sob estereomicroscópio. Embriões são encenadas de acordo com Kimmel et al. 11. </li><li> Transferência de embriões dechorionated no prato agarose primeiro revestido. </li><li> Para anestesiar os embriões, adicionar tricaina (0,1 mg / ml) para o prato até embriões parar de se mover (feito de acordo com Westerfield 12). </li></ol> 3. Injetando os ventrículos cerebrais <ol><li> Embriões orientar para que você está olhando para o seu lado dorsal, colocando a cauda do embrião dentro do buraco. Se o micromanipulador está do lado direito, em seguida, deslocar o embrião de modo que o prosencéfalo é para a esquerda e para a direita rombencéfalo. </li><li> Posição da agulha ao widest ponto de ventrículo rombencéfalo. </li><li> Cuidadosamente placa telhado perfurar do ventrículo rombencéfalo tendo a certeza de não passar a profundidade do cérebro para a gema (Figura 1A). </li><li> Injectar 1-2 nl de corante fluorescente para os ventrículos, certificando-se que o corante enche todo o comprimento dos ventrículos do cérebro. </li><li> Transferir os embriões para o prato agarose segundo revestido cheio com meios de embriões e re-anestesiar como descrito em 2.4. </li><li> Começar imediatamente a imagem, como descrito na seção 4, a fim de obter uma imagem tempo zero. </li></ol> 4. Imagem <ol><li> Embriões orientar com sua cauda no buraco, como descrito em 3.1. </li><li> Use um microscópio de dissecação com luz fluorescente e transmitida tanto para obter uma imagem dorsal campo claro. Mantenha a ampliação constante entre imagiologia de embriões diferentes. Isto permite a comparação direta de análises realizadas utilizando Image J (5,2-6). </li><li> Sem mover o embrião microscópio, ou prato, dê umaimagem fluorescente correspondente. </li><li> Repetir para cada embrião em pontos de tempo desejados. </li></ol> 5. Quantificação de Movimento Dye <ol><li> Fundir de campo claro e as imagens fluorescentes em Photoshop como anteriormente descrito por Gutzman e Sive 10. </li><li> Medir a distância os movimentos frente de corante no software Image J disponível em <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> . </li><li> Arquivo mesclado aberto no Image J e use a ferramenta de linha para desenhar uma linha do prosencéfalo dobradiça ponto-a-tingir frente em um ângulo de 10-20 ° de neuroepitélio (Figura 1A). Esta região foi escolhida por ser o primeiro local e mais notável de corante vazar do neuroepitélio de tipo selvagem. </li><li> Selecione a ferramenta de medição para calcular o comprimento da linha. </li><li> Repetir para cada ponto do tempo. </li><li> Calcular a distância a frente de corante líquido movido ao longo do tempo, subtraindo a distância em t = 0 a partir de outros pontos de tempo. </li><li> Lote emgráfico. </li></ol> 6. Resultados representativos Um exemplo dos resultados obtidos num ensaio de permeabilidade utilizando embriões neuroepitelial do tipo selvagem é mostrada na Figura 1B, D-. Para diferenciar com precisão da permeabilidade, que é útil para testar os corantes com weightsto molecular diferente identificar um tamanho que é apenas ligeiramente permeável de tipo selvagem ou de embriões de controlo (Figura 2). Isto permite a identificação de mutantes genéticos ou condições ambientais que, ou aumento ou diminuição de permeabilidade (Figura 1D, verde e vermelho, respectivamente, de linhas). Para o neuroepitélio zebrafish 24 hpf, 70 kDa vazamentos FITC Dextran lentamente ao longo de 2 horas, enquanto 2.000 kDa não faz e 10 kDa quase imediatamente vaza. Portanto 70 kDa é o peso ideal molecular para identificar as condições que tanto aumentar e diminuir a permeabilidade neuroepitelial. Se a agulha falha do lúmen do ventrículo, a fluorescência wmal aparecem fora do cérebro em t = 0 (para um exemplo veja Gutzman e Sive, 2009 10). Estes embriões devem ser eliminados uma vez que a tinta não foi injectado inicialmente contido no interior do cérebro e nenhuma conclusão clara sobre o movimento do corante e permeabilidade da neuropeithelium podem ser feitas. Finalmente, se os embriões têm ventrículos pequenos ou ventrículos cerebrais un-inflados, pré-injecção dos ventrículos com uma solução salina pode ser feito antes da injecção do corante fluorescente. Isto faz a insuflação dos ventrículos tornando visualização posterior dos ventrículos mais fácil quando se injecta com o corante fluorescente. Controlos apropriados devem ser realizados para determinar se a injecção de uma solução salina normal, interrompe o desenvolvimento do tubo neural. <img alt="Figura 1" src="/files/ftp_upload/4242/4242fig1.jpg" /> Figura 1. Curso de tempo de diferentes corantes de peso molecular. (A) Diagrama Experimental. Primeiro, Corante fluorescente é injetado nos ventrículos. X = posição de agulha para injecção. Próxima imagens dorsais são capturados ao longo do tempo. Finalmente, a distância percorrida pela frente do corante a partir do prosencéfalo dobradiça ponto é medida (representada por uma linha vermelha). (BC) Incorporada campo claro e fluorescentes imagens dorsal a 22 hpf (t = 0 min, B) e 24 hpf (t = 120 min, C). Linha branca indica a distância da frente de corante do cérebro anterior do ventrículo. (D) de dados de permeabilidade hipotéticos amostra. Azul = tipo selvagem ou controles, vermelho = amostra com permeabilidade diminuiu em relação ao controle, e amostra verde = com permeabilidade aumentada em relação ao controle. <img alt="Figura 2" src="/files/ftp_upload/4242/4242fig2.jpg" /> Figura 2. Medição da permeabilidade de diferentes corantes neuroepitelial peso molecular. (AE) brightfield Dorsal resultante da fusão e as imagens fluorescentes de 22 HPF embriões de tipo selvagem em t = 0 min após a injecção, com FITC-dExtran dos pesos moleculares seguintes: 10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D) e de 2.000 kDa (E). (A&#39;-E &#39;) O mesmo que no embrião (AE) em t = 120 min a 24 hpf. Anterior para a esquerda. F = prosencéfalo, M = mesencéfalo, H = rombencéfalo. Asterisco = orelha.

<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>

Não há conflitos de interesse declarados.

Nós demonstrar a capacidade de quantificar a permeabilidade do cérebro embrionário do peixe-zebra vivos, tal como determinado por um corante injectado de um determinado peso molecular. A nossa observação de que o neuroepitélio zebrafish embrionária é diferencialmente permeável aos corantes de diferentes pesos moleculares sugere que o corante está a avançar através da permeabilidade paracelular. No entanto, não podemos descartar a possibilidade de uma contribuição para a permeabilidade transcelular observa...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Dextrano, fluoresceína, Anionic, Lisina fixável </td><td> Invitrogen </td><td> D7136, D7137, D1822, D1820, D1845 </td></tr><tr><td> Tricaina pó </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Tubos capilares </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

O sistema ventricular do cérebro é conservada entre vertebrados e é composto de uma série de cavidades interconectadas chamado ventrículos cerebrais, os quais formam, durante as primeiras fases do desenvolvimento do cérebro e são mantidas durante toda a vida do animal. O sistema ventricular do cérebro é encontrado em vertebrados, e os ventrículos se desenvolver após a formação do tubo neural, em que o lúmen central, enche-se de líquido cefalorraquidiano (LCR) 1,2. CSF é um líquido rico em proteínas que são essenciais para o desenvolvimento do cérebro e função normais 3-6. No peixe-zebra, o cérebro de inflação ventrículo começa em cerca de 18 a fertilização pós hr (hpf), depois de o tubo neural está fechado. Vários processos estão associados com a formação de cérebro ventricular, incluindo a formação de um neuroepitélio, a formação da junção estanque, que regula a produção de permeabilidade e CSF. Nós mostramos que o Na, K-ATPase é necessária para a inflação do ventrículo cerebral, afectando todos estes processoses 7,8, ao passo 5a claudina é necessário para a formação da junção estanque 9. Além disso, mostramos que a &quot;relaxamento&quot; do neuroepitélio embrionário, através da inibição da miosina, está associada com a inflação do ventrículo cerebral. Para investigar a regulação da permeabilidade durante zebrafish inflação ventrículo cerebral, foi desenvolvido um ensaio ventricular retenção de corante. Este método utiliza a injecção ventrículo cerebral em um embrião do peixe-zebra vivos, uma técnica anteriormente desenvolvido no nosso laboratório 10, a fluorescência rotular o fluido cerebrospinal. Os embriões são então trabalhada ao longo do tempo à medida que o corante fluorescente, através dos ventrículos cerebrais e neuroepitélio. A distância da frente de corante se afasta do basal (não luminal) do lado do neuroepitélio ao longo do tempo é quantificado e é uma medida da permeabilidade neuroepitelial (Figura 1). Observamos que corantes 70 kDa e menores vão percorrer a neuroepitélio e pode ser detected fora do cérebro embrionário do peixe-zebra a 24 hpf (Figura 2). Este ensaio de retenção de corante pode ser usada para analisar a permeabilidade neuroepitelial em uma variedade de diferentes origens genéticas, em momentos diferentes durante o desenvolvimento, e após perturbações ambientais. Também pode ser útil para examinar a acumulação patológica de CSF. No geral, esta técnica permite aos pesquisadores analisar o papel e regulação da permeabilidade durante o desenvolvimento e doença.

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

Nós descrevemos uma medição animal vivo todo quantitativa para a permeabilidade do cérebro de zebrafish embrionário. A técnica de análise da capacidade de retenção de fluido cerebrospinal e moléculas de diferentes pesos moleculares no interior do lúmen do tubo neural e quantifica o seu movimento para fora dos ventrículos. Este método é útil para a determinação de diferenças de permeabilidade epitelial e maturação durante o desenvolvimento e na doença.

Um ensaio para a permeabilidade do neuroepitélio Zebrafish Embryonic

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

an-assay-for-permeability-of-the-zebrafish-embryonic-neuroepithelium

Research

JoVE Journal

Neuroscience

9.4K visualizações.  Massachusetts Institute of Technology. Nós descrevemos uma medição animal vivo todo quantitativa para a permeabilidade do cérebro de zebrafish embrionário. A técnica de análise da capacidade de retenção de fluido cerebrospinal e moléculas de diferentes pesos moleculares no interior do lúmen do tubo neural e quantifica o seu movimento para fora dos ventrículos. Este método é útil para a determinação de diferenças de permeabilidade epitelial e maturação durante o desenvolvimento e na doença....

Vídeo: An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Rotulagem e Células de imagem no rombencéfalo Zebrafish

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

Neurociência

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.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

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

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Potencial de membrana de neurônio motor biossensível em embriões vivos de peixe-zebra

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Biologia do Desenvolvimento

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.

Drenagem Manual dos Zebrafish ventrículos cerebrais embrionárias

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

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biologia

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Método de Electroporation para in vivo entrega de DNA plasmídeo no adulto zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Ensaio para a integridade da barreira hematoencefálica na Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Ablação e Regeneração a laser baseada em microscópio confocal em células interneuromast baseadas em zebrafish

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Ensaio de Captação de Anticorpos para Rastreamento de Endocitose Notch/Delta Durante a Divisão Assimétrica de Progenitores da Glia Radial de Zebrafish

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imagem e Reconstrução 3D de Estruturas cerebrovasculares em Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...