Este trabalho foi financiado pelo Canadian Institutes of Health Research (CIHR) Grant para Wayne S. Sossin. Carole A. Farah é o destinatário de uma bolsa de pós-doutorado do Fonds de la Recherche en Santé du Québec (FRSQ) e um Conrad Harrington comunhão. Os autores gostariam de agradecer Joanna Bougie, Margaret Hastings e Margaret Labban para ajudar com a gravação de vídeo e Piscina Madeline Richmond para ajudar com a visão geral gráfica.

1. Preparação e manutenção de culturas monocamada de células Sf9 <ol><li> Sf9 cultura de células e manutenção é realizada em uma capa de cultura de tecidos. </li><li> Sf9 células são derivadas de células de ovário de Spodoptera frugiperda (Sf21 células) e podem ser comprados como células congeladas em meio Grace da Invitrogen. </li><li> Lugar de 8 mL de meio de insetos de Grace, completados, a que 30% de soro fetal bovino (FBS) foi adicionada em um balão de 75 centímetros cultura 2 celulares com o pescoço inclinado. (1X) </li><li> Descongelar o tubo congelado de células Sf9 rapidamente em banho-maria a 37 ° C, movendo o tubo de ida e volta (cerca de 1-2 minutos). </li><li> Transferência de células Sf9 para a 75 centímetros garrafa de cultura celular e duas placas de rocha suavemente com a mão para distribuir uniformemente as células. </li><li> Incubar de 1-2 horas a 27 ° C até que as células têm em anexo. </li><li> Remova o meio de idade e substituir com 10 mL de meio de insetos freshGrace com 30% FBS. </li><li> Incubar a 27 ° C até que as células são confluentes. </li><li> Para manter as culturas em monocamada, remova o meio de idade do frasco confluentes de células Sf9 e adicionar 5 mL de meio de insetos de Grace com 10% de FBS. </li><li> Toque levemente no lado do frasco várias vezes para retirar as células Sf9. </li><li> Usando uma pipeta 10 mL e uma pipeta, pipetar os meios de comunicação para cima e para baixo uma vez gentilmente, pulverização parede frasco enquanto pipetagem para baixo. Transferência das células para um tubo estéril 15 ml poliestireno. </li><li> Adicione 2 mL da suspensão de células Sf9 a 8 mL de meio de insetos de Grace com 10% FBS (1:5 diluição para manter o crescimento fase log) em novos 75 centímetros garrafa de cultura celular e duas placas de rocha suavemente com a mão. </li><li> Incubar a 27 ° C até que as células são confluentes. </li></ol> 2. Expressão de PKCs fluorescente Tagged em Células Sf9 <ol><li> Para a imagem live-experiência, cultura de células Sf9 em 35 milímetros de vidro de fundo Mattek cultura disheswith uma superfície de vidro de 14 mm e uma espessura de lamela 0,16-0,19 mm. </li><li> PKCs marcadas com proteínas fluorescentes são expressos em células Sf9 usando Cellfectin II recomendação de reagentes de transfecção seguintes do fabricante, com as modificações detalhadas abaixo. </li><li> Dia 1: Antes de revestimento das células, tratar cada prato Mattek com meio de insetos de Grace com FBS 10% para pelo menos 30 minutos. </li><li> Contagem de células Sf9 a partir do passo 11 (acima), utilizando um hemocitômetro. </li><li> Placa de 0,05 x 10 6 células em cada lamela de vidro Mattek em um volume total de 150 mL. Permitir que as células para anexar por 30 minutos em temperatura ambiente. </li><li> Adicionar 2 mL de Grace suplementado com 10% de SFB a cada mL prato 1 de cada vez lentamente para evitar separar as células. </li><li> Incubar a 27 ° C durante a noite. </li><li> Deste ponto em diante, use a recomendação do fabricante para a transfecção de células Sf9 com DNA plasmidial. </li><li> Dia 2: transfecção Sf9 células com PKCs fluorescente etiquetado usando Cellfectin reagente transfecção II. Temos muitas vezes co-expressas neste sistema PKCs marcados com Proteína Verde Fluorescente melhorada (eGFP) e proteína monomérica Vermelho Fluorescente (MRFP) (detalhes para a construção do plasmídeo foram previamente descritos 2,5). Expressão de uma proteína fluorescente de uma vez produz cerca de 70-80% expressar células 72 horas pós-transfecção. Co-expressão de duas proteínas fluorescentes diminui a eficiência de transfecção para cerca de 30% de co-células que expressam. Quando co-expressando eGFP marcadas PKC e MRFP marcadas PKC, use 2x mais DNA do plasmídeo para a proteína MRFP-marcadas para a expressão da proteína ideal. </li><li> Executar ao vivo de imagens de 48-72 horas pós-transfecção (para a expressão da proteína ideal, esperar 72 horas). </li></ol> 3. Expressão de PKCs fluorescente nos neurônios Tagged Aplysia Sensorial <ol><li> Preparação de culturas de células Aplysia neuronal tem sido descrita em um artigo de vídeo por Zhao e 4 colegas. </li><li> PKCs marcadas com proteínas fluorescentes são expressos em neurônios sensoriais Aplysia usando procedimento de microinjeção detalhadas abaixo. </li><li> Prepare uma solução estoque de 1% Verde Fast (FCF) em água destilada, filtro usando uma seringa e uma seringa de 28 milímetros filtro (0,20 m). Alíquotas e armazenar a -20 ° C. </li><li> Microinjeção dos neurônios sensoriais pode ser realizada no dia 1 ou 2 dias depois de isolar os neurônios mas nós achamos que esperar dois dias permite melhor adesão celular para a lamela. </li><li> No dia 2 após o isolamento dos neurônios sensoriais, descongelar uma alíquota do Verde Fast e filtrá-la novamente usando uma seringa e uma seringa de 28 milímetros filtro (0,20 m). </li><li> Prepare uma solução de DNA plasmidial em água destilada contendo 0,5% Verde Fast. Concentrações de DNA plasmídeo tão alto quanto 0,4 g / mL pode ser usado, mas não é recomendável ir acima desses valores. </li><li> Filtrar o DNA plasmidial / solução Verde rápida usando uma seringa de 1 mL e um filtro de seringa de 4 milímetros (0,20 m). </li><li> Centrifugar o DNA plasmidial / solução Verde rápida a 16.110 xg por 15 minutos, utilizando um microfonerocentrifuge. </li><li> Prepare eletrodos afiada para microinjeção de neurônios utilizando um extrator de microeletrodos (Sutter Flaming / Brown Micropipeta Puller, modelo P-97). Utilizando um filamento de caixa, puxe microeletrodos de vidro com 1 mm de diâmetro externo, 0,75 mm de diâmetro interno e 100 mm de comprimento com parede fina capilares de vidro com um filamento dentro (World Precision Instruments TW100F-4). Para mais informações sobre puxando eletrodos microinjeção consulte cookbook eletrodo Sutter. </li><li> Preencha cada eletrodo com 2 mL de DNA plasmídeo / solução rápida usando Verde microloader pipeta ponta (eppendorf CA32950-050). </li><li> Transferir o vidro Mattek placa de cultura de fundo contendo culturas Aplysia neuronal para a estação de microinjeção. </li><li> A estação de microinjeção consiste no seguinte: um home-made estágio copo grande para manter os pratos da cultura em uma mesa de isolamento de vibração (Kinetic Sistemas de estação de trabalho de isolamento de vibrações), um aparelho de som com escopo de iluminação halógena externo, um micromanipulador (Sutter Huxley micromanipulador tipo de parede que permite o posicionamento tanto grosseiro e ultra-fino nos três eixos), um suporte de microeletrodos conectado a uma bomba pneumática (Instrumentos de Precisão Mundial PV820 Pneumatic Pico-Bomba). A entrada de pressão da bomba pneumática é conectado a um tanque de nitrogênio comprimido. </li><li> Inserir um microeletrodo no suporte de microeletrodos. </li><li> Visualização sob um estereomicroscópio, introduza a ponta da micropipeta no núcleo da célula. </li><li> Entregar pulsos de pressão curto de nitrogênio (10 a 100 ms de duração, 20 lb / pol 2) até o núcleo torna-se uniformely verde. </li><li> Incubar as células durante 4 horas à temperatura ambiente e manter a 4 ° C até o uso. Para a translocação da PKC ideal, tocar ao vivo de imagens experimento 20-24 horas após a injecção. </li></ol> 4. Visualização da translocação da PKC em células Sf9 Vivendo e em neurônios sensoriais Aplysia <ol><li> Para geração de imagens, nós usamos um microscópio invertido Zeiss LSM510 confocal com um 200 Axiovert. </li><li> Protocolo de imagens é a mesma para células Sf9 e para os neurônios sensoriais Aplysia, exceto para o seguinte: Sf9 células são gravadas com uma objetiva de imersão X63 óleo enquanto neurônios sensoriais Aplysia são gravadas com um objetivo de imersão em óleo x40. Sf9 células são gravadas em uma câmara com temperatura controlada mantida a 26-27 º C enquanto que os neurônios sensoriais Aplysia são gravadas em temperatura ambiente mantida em torno de 18-20 º C. </li><li> Nós usamos um laser de argônio 30 mW (excitação em 488nm), com saída do laser de 50% a PKC imagem marcada com eGFP proteína e um laser HeNe (excitação em 543nm) para PKC imagem marcada com a proteína MRFP. A Argon e linhas de laser HeNe são atenuadas a 4% e de saída da transmissão de 50%, respectivamente, antes da imagem e do pinhole é ajustado para um. </li><li> É importante ter a placa de cultura estabilizado no palco de imagem e para evitar o movimento ou vibração. </li><li> Retire 1 mL de meios de cultura do prato com cuidado usando uma pipeta 10 mL deixando um volume de mL total no prato. </li><li> Tire fotos na seção intermediária das células onde o núcleo pode ser visto. </li><li> Configurar uma série temporal de 10-20 imagens confocal de tirar uma foto a cada 30 segundos. </li><li> Iniciar a série de tempo. </li><li> Depois de 2 fotos são tiradas (após o ponto de tempo de 30 segundos), adicione 1 mL da droga (2X concentração) suavemente gota a gota, em cima das células utilizando uma pipeta P1000. A droga é adicionada continuamente por cerca de 30 segundos. </li><li> Algumas drogas induzem uma rápida translocação, que pode ser visível imediatamente após o tratamento medicamentoso (no ponto de tempo de 60 segundos), enquanto outros induzem uma translocação lenta que só é ideal 5-10 minutos pós-tratamento (Filme 1 e Filme 2). </li><li> Se o medicamento precisa ser lavado, use 2 x 10 mL pipetas para fazer a lavagem, pipetagem do tampão de lavagem suavemente com uma pipeta de um lado do prato e pipetagem até a mídia do outro lado. </li><li> Se a lavagem tem sido eficaz e se o efeito da droga é reversível, PKC reverte para o citosol dentro de 30-60 segundos. </li><li> Salve o filme como um arquivo de LSM. </li><li> Use software NIH Image J para abrir os arquivos LSM e quantificar as imagens pré e pós tratamento medicamentoso. Quantificação tem sido descrito em outros lugares 1. </li></ol>

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

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Nós descrevemos uma técnica para translocação imagem de PKCs fluorescente etiquetado em tempo real em Sf9 células e nos neurônios sensoriais Aplysia. Sf9 células fornecer um sistema simples de translocação imagem PKC desde cultura e transfecting deles é bem simples. Em contraste, a microinjeção de neurônios sensoriais Aplysia leva algum tempo para dominar, até alguns meses. Dificuldades relacionados a essa técnica incluem o entupimento do eletrodo. Filtragem da solução de injeção e c...

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

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

Proteína quinase Cs (PKCs) são serina treonina quinases que desempenham um papel central na regulação de uma ampla variedade de processos celulares como crescimento celular e aprendizagem e memória. Há quatro famílias conhecidas de isoformas da PKC em vertebrados: PKCs clássica (α, βI, βII e γ), tipo romance que PKCs (ε e η), novo tipo II PKCs (δ e θ), e PKCs atípicos (ζ e ι ). O PKCs clássica são ativadas por Ca 2 + e diacylclycerol (DAG), enquanto o PKCs romance são ativadas por DAG, mas são Ca 2 +-independente. O PKCs atípicos são ativadas por Ca 2 + nem nem DAG. Em Aplysia californica, o nosso sistema modelo para estudar a formação da memória, há três isoformas específicas do sistema nervoso PKC um de cada classe principal, ou seja, o convencional PKC Apl I, o novo tipo I PKC Apl II ea PKC atípica Apl III. PKCs são lipídios cinases ativadas e, portanto, a ativação de PKCs clássica e romance em resposta a sinais extracelulares tem sido freqüentemente correlacionada com a translocação da PKC do citoplasma para a membrana plasmática. Portanto, visualizando translocação da PKC em tempo real em células vivas tornou-se uma ferramenta valiosa para a elucidação das vias de transdução de sinal que levam à ativação da PKC. Por exemplo, esta técnica permitiu-nos estabelecer que diferentes isoformas da PKC translocate sob diferentes condições de mediar tipos distintos de plasticidade sináptica e que a serotonina ativação (5HT) da PKC Apl II requer a produção de ambos os DAG e ácido fosfatídico (PA) para translocação 02/01. Importante, a capacidade de visualizar o mesmo neurônio repetidamente nos tem permitido, por exemplo, para medir a dessensibilização da resposta PKC no requintado detalhe 3. Neste vídeo, demonstramos cada etapa de preparação de culturas de células Sf9, culturas de neurônios sensoriais Aplysia foram descritas em outro artigo de vídeo 4, expressando PKCs fluorescente etiquetado em Sf9 células e nos neurônios sensoriais Aplysia e live-imaging de translocação da PKC em resposta a ativadores diferentes, utilizando microscopia de varredura a laser.

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

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

Neste vídeo, demonstramos a visualização da translocação da PKC em células vivas usando PKCs fluorescente etiquetado.

Live-imagem de translocação da PKC em células Sf9 e em neurônios sensoriais Aplysia

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

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Research

JoVE Journal

Neuroscience

11.9K visualizações.  McGill University. Neste vídeo, demonstramos a visualização da translocação da PKC em células vivas usando PKCs fluorescente etiquetado.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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 ...

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

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

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

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

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

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

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

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

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

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 ...

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

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

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

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

Lentivírus mediada Manipulação Genética e Visualização de Olfactory neurônios sensoriais In vivo</em

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

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

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

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

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

Extracelularmente Identificar os neurônios motores de um pool do motor muscular em<em&gt; Aplysia californica</em

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

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

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

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

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

Isolamento de neurônios sensoriais de<em&gt; Aplysia californica</em&gt; Para Remendo Recordings Grampo de Correntes glutamatérgicos

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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

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

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

Um método para alvejar e Isolar Airway-inervação neurônios sensoriais em Ratos

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

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

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 ...