Este trabalho foi financiado por uma bolsa NIH concedido a R. Brewster (1R01GM085290-01A1).

1.Microinjection <ol><li> Diluir plasmídeo codificando membrana-alvo proteína fluorescente verde (mGFP, cortesia de Richard Harland) a uma concentração de 40 ng / ml. DNA é preparada por linearização do plasmídeo (mGFP/PCS2 + cortesia, de Richard Harland) purificado usando o kit Qiagen Prep Maxi. Além do vermelho de fenol (diluído 1 / 10 do volume total) para a solução é o preferido para visualizar a solução. Mantenha a solução de DNA em gelo. </li><li> Sob microscópio estereoscópico, calibrar a agulha de injeção (90mm capilares de vidro de Narishinge Científico; Cat. NA-GD-1), alinhando a agulha em um slide graduado e batendo a parte da agulha onde o diâmetro é de 1 Hm usando uma navalha limpa blade (para obter uma ponta de tamanho apropriado). </li><li> A fim de calibrar a agulha para injetar um volume conhecido (2nl), conecte a agulha para o aparelho de microinjeção (PCI 100 microinjetor; Harvard Apparatus) e frente preencher a agulha com 0.5μl de uma solução contendo vermelho de fenol e água colocada em um pedaço de parafina. A solução pode ser dispensado através de um pedal. Ajuste a pressão para 7 KPa e variar o tempo (10mseg 30msec), de modo que é preciso dispensar 250 pedais 0.5μl da solução. </li><li> Colocar cuidadosamente a agulha calibrada em uma tira de cera em um prato de 100 milímetros umidificado Petri. Para umidificar o prato, coloque Kimwipes molhados em torno das bordas (isso evita a evaporação da solução na agulha). </li><li> Use uma micropipeta para carregar 0,5 1μl de DNA na extremidade da agulha (a fim de que não foi calibrado). </li><li> Uma vez que a solução atingiu a ponta da agulha, ligue a extremidade da agulha que foi usado para o carregamento a um aparelho de microinjeção. Ajustar as configurações no microinjetor para entregar cerca de 2 nl injecção / usando um pedal. </li><li> Em seguida, a posição de embriões zebrafish no meio de uma placa de Petri cheia de Medium Embrião (Stock 1,0 ml de Hank # 1, 0,1 de stock ml de Hank # 2, Stock 1,0 ml de Hank # 4, 95,9 ml DDH 2 O, Stock 1,0 ml de Hank # 5 , Stock 1,0 ml fresco Hank # 6, Use cerca de 10 gotas de NaOH 1 M pH 7,2). </li><li> Uma vez que os embriões se desenvolveram ao estágio desejado, gentilmente pega o córion com uma pinça fina e situar a agulha na área de segmentação injectável. Para a expressão ampla gama mGFP, injetar DNA na gema ou do citoplasma na fase 1CELL. Para restringir a expressão de mGFP a uma região menor, injetar DNA em 4 (ou mais) células estágio para o citoplasma de uma única célula. </li><li> Pressione o pedal do aparelho de microinjeção e entregar cerca de 2 nl, garantindo que a solução (visível devido à adição de vermelho de fenol) é adequadamente injetado. </li><li> Remova cuidadosamente a agulha do embrião. </li><li> Repita o procedimento para todos os embriões na placa de Petri. </li><li> Permitir que os embriões a desenvolver-se 28,5 ° C para o estágio desejado. </li><li> Embriões Dechorionate suavemente descascando o córion usando uma pinça fina (se os embriões são mais jovens do que 24hpf, dechorionating numa placa de Petri de vidro aumenta a taxa de sobrevivência de embriões). </li><li> Remover córion vazio da placa de Petri, utilizando uma pipeta de vidro. </li></ol> 2. Imagem mGFP marcado seções vibratome <ol><li> Use uma pipeta de vidro para o transporte de embriões em paraformaldeído (fixador de 4% em solução de 1X tampão fosfato (PBS). Fix embriões a 4 ° C durante a noite. </li><li> Lavar embriões em 1X PBS 3 vezes por 5 minutos cada. </li><li> Coloque os embriões em Placa de Petri e, usando uma pinça fina, retire a gema, sem danificar o embrião. Remover como gema tanto quanto possível. </li><li> Prepare 4% baixo-melt agarose em H 2 O. </li><li> Lugar agarose derretida no molde de corte de plástico. </li><li> Use uma pinça fina para levantar um embrião injetados a partir da placa de Petri e colocar e orientar na agarose. O embrião deve ser posicionado para uma extremidade do molde. </li><li> Uma vez que a agarose é endurecido, use uma ferramenta de superfície plana, como uma espátula, para remover o bloco de agarose do molde. </li><li> Corte a extremidade inferior do bloco de agarose com uma lâmina de barbear para que ele seja mesmo. </li><li> Adicionar uma gota de super-cola para o palco vibratome e colocar o bloco de agarose sobre ele (o lado do bloco que foi nivelado fora com a navalha deve enfrentar a cola). Para cortar vários embriões, montar várias agarose-embedded espécimes no palco. </li><li> Anexar o estágio vibratome ao vibratome (Vibratome, Inc.). </li><li> Definir os parâmetros do vibratome (configurações comuns: velocidade-3; espessura de 50 ìm). Se o corte vários embriões, alinhar blocos de agarose no palco e assegurar que a lâmina atravessa o embrião todo, mas não por toda a largura dos blocos de agarose (de forma que as seções correspondentes ao embrião mesmo permanecerá em conjunto). </li><li> Despeje 1X PBS na fase vibratome. </li><li> Seção. </li><li> Uma vez que os embriões são sectioned, remova o bloco de agarose (s) a partir da fase vibratome. </li><li> Separe cuidadosamente cada seção cortando parte de trás do bloco de agarose (s). </li><li> Use uma pinça fina para selecionar seções desejado e coloque em um PBS 1X cheio de multi-prato bem (use poços diferentes para seções correspondentes aos embriões diferentes). </li><li> Repita para cada embrião. </li><li> Prosseguir com a rotulagem de anticorpos, embora as etapas 19 23, para melhorar a imagem mGFP (este passo pode não ser necessário se seções são gravadas dentro de um dia ou dois de preparação). </li><li> Tratar seções de 30 minutos em temperatura ambiente com solução de bloqueio em 1X PBS para bloquear ligações não específicas. </li><li> Seções Incubar com anticorpo primário (um coelho-GFP, Invitrogen Cat. A11122) diluído 1:200 @ na solução de bloqueio em um agitador por 5 horas em temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar quatro vezes (10 min, 15 min, 30 min e 60 min, cada) em 1% BSA/1X PBS / 1% de solução de DMSO. </li><li> Tratar seções com anticorpo secundário apropriado em solução de bloqueio por 5 horas em temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar quatro vezes (10 min, 15 min, 30 min e 60 min, cada) em 1% BSA/1X PBS / 1% de solução de DMSO. </li><li> Trate as seções com DAPI (0,1% em DAPI 50ml 1X PBS; Invitrogen Cat. D1306) por 30 minutos para corar os núcleos. </li><li> Lavar três vezes em 1X PBS por 5 minutos cada. </li><li> Montagem no slide. </li><li> Imagem utilizando microscopia confocal. </li></ol> 3. Ao vivo de imagens <ol><li> Dechorionate vivos, embriões injetados 30 minutos antes de chegar ao estágio desejado desenvolvimento. Coloque os embriões em solução sedativos (0,01% tricaina em Meio Embryo) por 15 min. </li><li> Coloque uma gota de agarose 1% em uma placa de cultura 35 milímetros de vidro de fundo (Mattek; Part No. P35G-o-20-C). </li><li> Permitir agarose para solidificar. </li><li> Tubo de calor capilar mais chama para fazer 3 furos em agarose (outros objetos, como dicas micropipeta pode ser usado para fazer os furos). Garantir que não há agarose residual no fundo buraco. </li><li> Encha o prato de Petri com meio de embriões. </li><li> Use uma pipeta de vidro para colocar o embrião no buraco. </li><li> Em seguida, oriente o embrião usando uma pinça fina para que a área a ser trabalhada (cabeça dorsal) está em contato com o vidro. </li><li> Cuidadosamente a placa de Petri de transporte para o estágio do microscópio (coloque a tampa sobre a placa de Petri para evitar a evaporação). </li><li> Enquanto imagem, certifique-se que os embriões estão em 28,5 ° C. </li></ol> 4. Resultados Descrevemos aqui uma abordagem direta às células de uma única imagem no tubo neural zebrafish usando a expressão de mosaico. Apesar da transparência óptica do embrião zebrafish, descobrimos que a visualização de células neurais é bastante reforçada em secções transversais obtidas por meio de vibratome. Estas seções são grossas (50 mm) permitindo a criação de imagens de extensão celular a partir de uma determinada célula em diferentes planos focais utilizando um microscópio confocal. Resultados utilizando esta metodologia ter sido publicado anteriormente 2, mas as imagens representativas das células do tubo neural de um embrião WT (Figura 1) e uma N-caderina (N-cad) mutantes (Figura 2) são fornecidos. Este último revela que as células em regiões lateral da placa neural em N-cad mutantes não orientar corretamente para a linha média (Figura 2). Em contraste com esta rotulagem mosaico, a imunocoloração neuroepitélio com um marcador geral da superfície celular, tais como beta-catenina não permite a morfologia das células individuais para ser visualizada (Figura 3). Imagem lapso de tempo de embriões vivos complementou a rotulagem mosaico de espécimes fixa, permitindo uma melhor compreensão da dinâmica celular que ocorrem durante a neurulação. Filme 1 revelaram que as células WT ativamente migrar para a linha média, estendendo saliências medialmente orientada membrana. <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="Figura 1" /> Figura 1. Expressão mGFP no tubo neural de um embrião zebrafish WT. <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="Figura 2" /> Figura 2. Expressão mGFP no tubo neural de um embrião de peixe-zebra N-caderina. <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="Figura 3" /> Figura 3. B-catenina coloração imuno da neuroepitélio.

<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>

Não há conflitos de interesse declarados.

Em conclusão, as técnicas descritas aqui rotulagem permitem uma análise única célula de processos morfogenéticos no embrião do zebrafish. A ênfase principal deste protocolo é sobre os métodos de células imagem rotulada no tubo neural usando mGFP sob o controle de um promotor onipresente. Para aplicações adicionais leitores deste ensaio transiente expressão deve se referir a um artigo recente 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

Soluções <ul><li> Ações de Hank # 1: 8,0 g NaCl, 0,4 g KCl em 100 ml dd H 2 O </li><li> Ações de Hank # 2: 0,358 g Na 2 HPO 4 anidro, 0,60 g KH 2 PO 4 em 100 ml 2 O DDH </li><li> Ações de Hank # 4: 0,72 g CaCl 2 em 50 ml DDH 2 O </li><li> Ações de Hank # 5: 1,23 g MgSO 4 x7H 2 O em 50 ml dd H 2 O </li><li> Um tampão fosfato X: 0,8% NaCl, KCl 0,02%, 0,02 M PO4, pH 7,3 </li><li> Solução de bloqueio: soro de cabra 2% normal, 1% albumina sérica bovina (BSA) dimethysulfoxide, 1% (DMSO) </li></ul>

labeling and imaging cells in the zebrafish hindbrain

Chave para a compreensão dos processos morfogenéticos que forma o embrião de vertebrados precoce é a capacidade de células de imagem em alta resolução. Em embriões de peixe-zebra, a injeção de DNA plasmídeo resultados na expressão de mosaico, permitindo a visualização de células isoladas ou pequenos aglomerados de células<sup&gt; 1</sup&gt;. Descrevemos como injeção de codificação de DNA plasmídeo membrana-alvo Proteína Fluorescente Verde (mGFP) sob o controle de um promotor onipresente pode ser usado para as células de imagem passando neurulação. Central a este protocolo é a metodologia para as células de imagens rotuladas em alta resolução em seções e também em tempo real. Este protocolo envolve a injeção de DNA em embriões de peixe-zebra mGFP jovens. Embriões são então processadas para o seccionamento vibratome, rotulagem de anticorpos e de imagem com um microscópio confocal. Alternativamente, embriões vivos expressando mGFP pode ser fotografada usando lapso de tempo de microscopia confocal. Nós já usou esta abordagem simples para analisar os comportamentos celulares que levam a formação do tubo neural na região rombencéfalo de embriões zebrafish<sup&gt; 2</sup&gt;. Os preparativos fixo permitido para visualização sem precedentes de formas e organização das células no tubo neural, enquanto imagens ao vivo complementada esta abordagem, possibilitando uma melhor compreensão da dinâmica celular que ocorrem durante a neurulação.

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

Chave para a compreensão dos processos morfogenéticos que forma o embrião precoce é a capacidade de células de imagem em alta resolução. Descrevemos aqui uma técnica para a rotulagem células isoladas ou pequenos aglomerados de células em embriões de peixe-zebra inteira com membrana-alvo Proteína Fluorescente Verde.

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 ...

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

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Neuroscience

11.2K visualizações.  University of Maryland, Baltimore County. Chave para a compreensão dos processos morfogenéticos que forma o embrião precoce é a capacidade de células de imagem em alta resolução. Descrevemos aqui uma técnica para a rotulagem células isoladas ou pequenos aglomerados de células em embriões de peixe-zebra inteira com membrana-alvo Proteína Fluorescente Verde.

Vídeo: 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.

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 ...

Neurociência

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

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As 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.

Eletroporação do rombencéfalo rastrear Trajetórias axonal e Metas Synaptic no embrião de galinha

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.

Marcação de células individuais no Sistema Nervoso 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.

Marcação retrógrada de células ganglionares da retina em Zebrafish Adulto com corantes 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.

O rombencéfalo Mouse como um modelo para o estudo embrionário neurogênese

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.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

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.

Identificando marcadores de superfície celular da haste neural primária e células progenitoras por rotulagem metabólica de 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 ...

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.

Rotulagem e imagem de placas amilóides no tecido cerebral usando a curcumina polifenóis naturais

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.

In vivo Imagem de Tecido Cerebral Totalmente Ativo em Larvas de Zebrafish Acordados e Juvenis por Remoção de Crânio e Pele

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