I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1. Crude Dauer Feromônio Produção <ol><li> Crescer OP50 E. coli a partir de uma única colónia O / N a 37 ° C, agitando, em 100 ml de caldo LB a 200 rpm. NOTA: Este cultura D / N pode ser feito com antecedência e guardadas a 4 ° C. </li><li> Crescer N2 C. elegans em junho 15-20 cm placas de Petri contendo ágar NGM (ver receita na Tabela 1) inoculadas com 50 ul de E. OP50 coli até que as bactérias está quase esgotada 19. </li><li> Faça 5x 250 ml de S mídia (ver receita na Tabela 1) em 1 L frascos Erlenmeyer de 19. NOTA: Esta solução pode ser feita com antecedência. </li><li> Crescer uma cultura de OP50 E. coli em 4 L de caldo LB S / N por meio da inoculação de 1 ml de OP50 em E. coli e agitação a 37 ° C durante 16 horas. </li><li> Lave os nematóides das placas (passo 1.2) com 1 ml de solução S basal (ver receita na Tabela 1) e nematóides de transferência de 4-5 placas em cada um dos quatro frascos de 1 L de Erlenmeyer com os meios de comunicação S preparada no passo 1.3 (4 5 placaspara cada frasco) 19. </li><li> Centrifuga-se o passo de OP50 1,4 a 650 xg durante 10 min e remover o sobrenadante. Ressuspender cada sedimento bacteriano em 10 ml de solução de S basal. Transferir quantidades iguais de bactérias em suspensão para cada frasco de nematóides. </li><li> Colocar os frascos num agitador orbital, à temperatura ambiente (20 - 25 ° C) e 100 - 150 rpm durante 4-5 dias, até que a solução começa a limpar, indicando uma exaustão de alimentos. </li><li> Uma vez que a comida está esgotado, crescer outra cultura O / N de OP50 E. coli em 4x 1 L de caldo de LB. Centrifuga-se as bactérias a 650 xg durante 10 min e adicionar uma pelete a partir de um balão para cada um dos frascos S media com nematóides. Devolver os frascos S de mídia para o agitador orbital, como no passo 1.7, por mais 3-4 dias até que o alimento está esgotado novamente. </li><li> Tomar um ~ alíquota de 1 ml de solução de cada frasco e estimar a concentração de nemátodos por sub-amostragem de 1 ul de cada alíquota de 1 ml e a contagem do número de nemátodos. NOTA: Opopulação do nematóide deve ser 50.000 - 75.000 nematóides / ml. A esta concentração, a maior parte dos nemátodes são dauers. </li><li> Descarte qualquer frascos contaminados. NOTA: Contaminação frequentemente se manifesta como hifas fúngicas visto durante a etapa 1.9. </li><li> Nemátodos de centrifugar a 4000 xg durante 10 min a 4 ° C. Desprezar o pelete nemátodo e reter o sobrenadante. Alternativamente, consulte a discussão para o uso desses animais para fazer um feromônio de baixa potência. </li><li> Filtrar o sobrenadante com # 1 filtro de papel Whatman. NOTA: uma garrafa térmica vai acelerar este processo. </li><li> Além disso filtrar o sobrenadante através de unidades de filtração de 0,45 um vácuo estéril. NOTA: O filtrado pode ser mantido O / N a 4 ° C ou prosseguir para 1,14. </li><li> Adicionar-se o filtrado para uma proveta de 2 L com uma barra de agitação. Coloque em um exaustor em uma placa de aquecimento mexa. Deixe ferver, mexendo e evaporar a cerca de 200 ml. Transferir a solução para um copo de 500 ml. Prosseguir a ebulição até cerca de 50 ml são lepés NOTA: Cuidados devem ser tomados durante este passo como é possível que a solução para transbordar. A cor dos finais de 50 ml é um castanho escuro. Em qualquer altura durante o processo de cozimento, o calor pode ser desligado e o filtrado foi armazenado O / N à temperatura ambiente ou a 4 ° C. </li><li> Deixar arrefecer e centrifugar durante 5 minutos a 1000 x g. Verter o sobrenadante para uma proveta de 100 ml e descartar o sedimento. </li><li> Ferva o sobrenadante até uma crosta marrom. Retire do fogo e quebrar a crosta com uma espátula. Adicionar suficiente de etanol prova 200 para cobrir a casca. Cobrir o copo com Parafilm e deixe descansar O / N à temperatura ambiente. Deitar fora o etanol em um copo limpo e colocado para o lado. Adicionar etanol fresco para cobrir a casca. Repita extrações O / N 3 vezes. Reúnem-se os extratos. </li><li> Usar uma bomba de vácuo para secar os extractos de etanol. Se uma bomba de vácuo não está disponível, adicionar o extracto de etanol para um recipiente limpo e quente a 50 ° C com agitação numa hotte. NOTA: Uma vez que o etanol tenha evaporado um resíduo pegajoso de lcor marrom ight deve permanecer. Se o aquecimento é usado, deve ser tomado cuidado para não queimar a solução por meio de aquecimento por muito tempo. </li><li> Dissolve-se resíduo em 10 ml de água destilada estéril. Filtro de esterilizar a solução com um filtro de seringa de 0,22 um e armazenar a -20 ° C em alíquotas de 1 ml. NOTA: 1 ml de água é utilizada para dissolver o resíduo, para cada 100 ml de cultura inicial de nemátodos líquido. Por exemplo, se um dos frascos de cultura líquido é rejeitado devido a contaminação, em seguida, ajustar a quantidade de água utilizada para dissolver o resíduo. </li></ol> 2 Determinação do Feromônio Dose Response <ol><li> Prepare dauer feromona fazendo 6x 5 ml de meio de agar com NGM Noble, sem peptona, e suplementado com 50 ug / ml de sulfato de estreptomicina e uma gama de dauer feromona (ver receita na Tabela 1) 4, 19. Misturar os componentes num 13x100 mm tubo de cultura de vidro, ferver mais um bico de Bunsen, em seguida, despeje em 3 centímetros placas de Petri. </li><li> Pesar amtubo icrocentrifuge. Adicionar 1 ml de cultura O / N do E. coli OP50 para o tubo e centrifuga-se a 17.000 x g ~ em um tubo de microcentrífuga. Remover todo o sobrenadante. Pesa-se o tubo de novo e determinar o peso do sedimento de bactérias. Ressuspender o sedimento com tampão M9 (ver receita na Tabela 1) para uma concentração final de 4% (w / v). Aliquota de 10 ul da suspensão bacteriana sobre o centro de cada placa 4 de feromonas. Placas de loja O / N à temperatura ambiente. </li><li> Escolha 10-15 grávidas N2 do tipo selvagem hermafroditas, um dia após a muda até a idade adulta para as placas de feromônio 4. Armazenar as placas a 25 ° C durante 3-4 horas. </li><li> Escolha fora de todos os hermafroditas adultos. Registre o número de ovos postos por cada placa. Selar as placas com parafilme e regresso a 25 ° C durante 3 dias 17. </li><li> Contagem da percentagem de dauers (Figura 2). Nota: Para ambos os métodos, é importante notar que dauers (e, em menor extenda não dauers) vai subir as paredes e na tampa de placas de Petri. Por isso, é importante examinar todas as superfícies do prato para nematóides. </li><li> Ajustar os dados a uma curva de dose-resposta e calcular a concentração de EC90 de feromona (feromona suficiente para induzir 90% de animais para formar dauers) como mostrado na Figura 3 Nota:. Nós normalmente utilizam um modelo de regressão logística para estimar a CE 90 , mas diferentes pacotes de software de estatística pode ter métodos alternativos que produzem estimativas similares. </li></ol> 3 Imagens ao vivo de Dauer IL2 Neurônios <ol><li> Prepare pratos Dauer feromônio como no protocolo 2, utilizando o valor de EC 90 determinou na etapa 2.6. </li><li> Induzir dauers usando os métodos no protocolo 2 com uma cepa carregando um IL2 repórter transgene integrado (myIs13 [P klp-6 :: GFP], myIs14 [P klp-6 :: GFP], myIs16 [P <em&gt; KLP-6 :: tdTomato] ou qIs56 [P lag-2 :: GFP]) 12, 20, 21. NOTA: Se q Is56 é usada, a expressão de GFP não será visível nos IL2Qs até várias horas para a muda Dauer e alguns dos eventos de remodelação iniciais não será visível. Além disso, enquanto o sinal de fluorescência a partir do desfasamento P-2 :: transgene GFP em última análise, é mais brilhante nos IL2s durante dauer que os P-6 klp repórteres fluorescentes, o desfasamento P-2 :: transgene gfp também fracamente expressa nos músculos da cabeça que pode mascarar os ramos IL2 finas. </li><li> Após a postura dos ovos, remover os hermafroditas adultos, selar as placas com parafilme e incubar durante 34 horas a 25 ° C 17. NOTA: O período de tempo para a muda Dauer varia ligeiramente entre os indivíduos. O período mais rápido de dendrito arborização começa 4-5 horas após o início da muda Dauer (39-44pós h a 25 ° C) 12, que estabelece-ovo. </li><li> O dia antes da imagem, formam uma solução de 10% (w / v) de agarose em tampão M9 com dauer feromona para a concentração EC90. Alíquota de aproximadamente 100 mL de agarose numa lâmina de microscópio e rapidamente, mas com cuidado, coloque outro slide na parte superior para criar uma superfície plana agarose. Após a agarose solidificou, enrole os slides em saran wrap e armazenar em uma câmara de umidade (caixa ponteira com papel-toalha molhada). </li><li> Após o período de incubação 34 h, examinar as placas de feromônio para nematóides que pararam de bombeamento da faringe. Lâminas separadas de tal forma que apenas uma superfície de uma lâmina de agarose tem nele. Escolher um ou mais nematóides numa almofada de agarose a 10% e 1 μ l de 0,1 micron esferas de poliestireno de 22. NOTA: Com uma população bem sincronizados, a maioria dos nematóides sobre a placa será entrar na muda de L2D de dauer. No entanto, pode haver variação entre indivíduos. Por isso, é importante observar características adicionais (oclusão do estoma, a remodelação da faringe) para estimar a quantidade de tempo que os animais tenham sido dentro da muda Dauer (ver resultados representativos, por exemplo). </li><li> Determinar a orientação (esquerda / direita, dorsal / ventral) do nematóide. Capturar imagens Z-stack de um dendrito IL2Q em 100x com a menor intensidade de fluorescência possível a cada 15 minutos ou quando necessário para o experimento específico. Continue a captura de imagens até que o nematóide se separou da cutícula L2D e imobilização é impossível ou se o animal se recupera de Dauer (faringe começa bombeamento). Quando não imaginando ativamente, coloque o slide em uma câmara de umidade. NOTA: Nós usamos um microscópio de epifluorescência com uma motorizada contraste fase e de interferência diferencial (DIC) óptica. </li></ol>

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The authors have nothing to disclose

Um exame de neuroplasticidade específicos de dauer e outras alterações morfológicas associadas à Dauer requer controlada e fiável a formação de dauers quer através de mutação genética (mutantes ou seja, daf-c) ou por exposição de animais de tipo selvagem de feromona. Enquanto a utilização de uma mutação genética para induzir dauers é conveniente, pode confundir os resultados. Assim, os dados coletados com mutações daf-C deve ser posteriormente confirmada com tipo selvagem animais ...

Plasticidade fenotípica, inclusive plasticidade do desenvolvimento e comportamental, é importante para as adaptações a condições ambientais adversas e é considerada uma força motriz na evolução 1. C. elegans é um organismo modelo frequentemente utilizado para estudos em desenvolvimento e neurobiologia. Sob condições ideais, C. elegans, desenvolvido em quatro fases larvares antes de entrar na fase adulta reprodutiva. No entanto, em condições de baixa disponibilidade de alimentos e altas densidades populacionais, C. elegans pode sofrer uma chave de desenvolvimento e entrar em um estágio Dauer longa vida e estresse resistente 2. Dauers exibir várias diferenças morfológicas e comportamentais de não-dauers que provavelmente medeiam esta resistência ao estresse. Os estímulos ambientais e vias genéticas que regulam a entrada Dauer têm sido amplamente descrito 3, 4, 5, 6, 7. Contudo, os mecanismos moleculares que controlam alterações fenotípicas individuais que ocorrem dformação Dauer urante são menos conhecidas. O exame de fenótipos específicos de Dauer requer a produção de animais dauer. Isto pode ser realizado de três maneiras: 1) colheita de uma cultura de C. fome elegans, 2) Utilização de formação dauer constitutiva (daf-c) mutantes, 3) indução da formação, através de feromonas dauer purificado. É relativamente simples para escolher dauers de uma placa com um padrão NGM C. população elegans que esgotou a sua bacteriana-abastecimento alimentar. Nas nossas mãos, uma placa NGM padrão originalmente semeada com 50 ul de OP50 Escherichia coli, deixou-se crescer durante 3 dias e em seguida inoculados com 3 - hermafroditas 4 adultos mantida a 25 ° C irá esgotar a fonte de alimento dentro de uma semana e produzem vários milhares dauers dentro de 3 dias adicionais (além de inúmeras não-dauers famintas). No entanto, este método não é satisfatório para os processos de exame que ocorrem durante a muda em dAuer. É difícil determinar qual dos vários milhares de animais em um prato típico faminto estão entrando em Dauer. Além disso, o uso de uma placa de fome oferece qualquer possibilidade de sincronização. A utilização de mutantes daf-c permite a sincronização e a geração segura de dauers. No entanto, não há qualquer garantia de que uma mutação daf-c em particular não irá afectar outros fenótipos específicos de Dauer sozinho ou em combinação com outras mutações. A presença de um feromônio dauer estava implícito pela primeira vez por Cassada e Russell e mais tarde demonstrado por Dourado e Riddle. A feromona dauer já foi caracterizada quimicamente 2, 8, 9, 10. A feromona purificado é constituído por uma mistura complexa de ascarosides, que em diferentes formas de regular a ambos os processos comportamentais e de desenvolvimento 9, 11. Utilização de um extracto bruto de feromonas dauer permite a indução controlada fiável do dauers sincronizados num fundo de tipo selvagem. </p&gt; O sistema nervoso e as células gliais associadas sofrer remodelação durante Dauer larva formação 12, 13, 14. Algumas dessas mudanças são irreversíveis, como a remodelação das células da glia durante Dauer 13, 14. Porém outros eventos de remodelação são reversíveis após um retorno à favorável condições ambientais. Por exemplo, um conjunto de quatro neurônios IL2Q sofrer remodelação neuronal rápida e reversível durante a formação Dauer incluindo: arborização dendrito, a transferência do único dendrítica de neurônios e axônios multidendritic remodelação 12. Os métodos aqui permitem a imagiologia in vivo de confiança de neuroplasticidade induzida pelo stress num modelo de organismo. Os métodos apresentados para a produção e teste de feromona dauer bruto são descritos anteriormente e compilada a partir de várias fontes de 4, 8, 15, 16, 17, 18.

<table><tbody><tr><td>N2 &lt;em&gt;C. elegans&lt;/em&gt; Bristol strain</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>PT2660 myIs13[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] III</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>PT2762 myIs14[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] V</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>JK2868 qIs56[P&lt;sub&gt;lag-2&lt;/sub&gt;::gfp] V</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>OP50 E. coli</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Tritech Research</td><td>60 mm T3308, 35 mm T3501</td><td></td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>3 g NaCl, 17 g agar, 2.5 g peptone in 975 ml H&lt;sub&gt;2&lt;/sub&gt;O autoclaved cooled to 55&amp;deg;C followed by 1 ml of 5 mg/ml cholesterol in ethanol, 25 ml of 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 1 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>S Media</td><td>Various</td><td></td><td>S media consists of S basal solution (1.46 g NaCl, 0.25g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.5 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.25 ml of 5 mg/ml cholesterol in ethanol and H&lt;sub&gt;2&lt;/sub&gt;O to 250 ml. Autoclaved.) To the S basal solution add 2.5 ml of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate&lt;sub&gt;, &lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml, autoclaved), 2.5 ml of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.2 g MnCl2&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O, 0.29 g ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.025 g CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Autoclaved and stored in the dark), 0.75 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, and 0.75 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;.</td></tr><tr><td>Dauer pheromone plates for dose-response assay</td><td>Various</td><td></td><td>Mix 0.085 g Noble agar, 0.5 ml of 0.513 M NaCl and 5 &amp;micro;l of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 ml culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H&lt;sub&gt;2&lt;/sub&gt;O to bring the volume to 4.725 ml.&amp;nbsp; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &amp;deg;C. Add 125 &amp;micro;l 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 50 &amp;micro;l 0.1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 50 &amp;micro;l 0.1 M MgSO&lt;sub&gt;4&lt;/sub&gt; and 50 &amp;micro;l of 5 mg/ml streptomycin sulfate to each tube.&amp;nbsp; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td></tr><tr><td>Microsphere beads 0.1 micron</td><td>Polysciences Inc.</td><td>00876-15</td><td></td></tr><tr><td>M9 buffer</td><td>Various</td><td></td><td>Mix 3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 liter. Sterilize by autoclaving</td></tr></tbody></table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

Os mecanismos que controlam a plasticidade fenotípica induzida por estresse em animais são muitas vezes complexas e difíceis de estudar in vivo. Um exemplo clássico de plasticidade induzida por estresse é a fase de Dauer C. elegans. Dauers estão numa fase de desenvolvimento larvar alternativa formada em condições de baixas concentrações de alimentos de bactérias e concentrações elevadas de uma feromona dauer. Dauers exibir extensa plasticidade do desenvolvimento e comportamental. Por exemplo, um conjunto de quatro quadrante interno-labial (IL2Q) neurônios passam por extensa remodelação reversível durante a formação Dauer. Utilizando a entrada de potenciais vias de regulação dauer bem conhecido, um método previamente estabelecido para a produção de dauer bruto feromona de culturas em larga escala de nematóides líquido é demonstrada. Com este método, uma concentração de 50.000 - 75.000 nemátodos / ml de líquido de cultura é suficiente para produzir uma altamente potente feromona dauer bruto. A potência de feromonas em bruto é determined por um bioensaio de resposta à dose. Por fim, os métodos utilizados para in vivo de lapso de tempo de imagem dos IL2Qs durante a formação Dauer são descritos.

A produção de resultados Dauer bruto feromônio em um líquido amarelo (Figura 1) que é tanto calor e frio estável. Alíquotas de feromona bruto são armazenadas a -20 ° C indefinidamente sem qualquer perda na actividade óbvia. Após a produção da feromona, um único bioensaio de dose-resposta é suficiente para uma estimativa da potência. No entanto, para a maioria das experiências, é necessário repetir o bioensaio de 2 - 3 vezes e ter uma média de cada experimento. Os resultados represent...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

In vivo de imagens de Dauer específica Neuronal Remodelação em C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

11.2K visualizações.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

Vídeo: In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos microfluídicos para<em&gt; In vivo</em&gt; Imaging de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Bioengenharia

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In vivo Calcium Imaging em C. elegans corpo parede músculos

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Bioquímica

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Na imagem cálcio vivo Neuronal em C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Neurociência

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers agarose para Imagem de cálcio a longo prazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biologia

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagem de cálcio Ratiometric de neurônios individuais se comportando Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Um chip microfluídico simples para crescimento a longo prazo e imagem de Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Avaliação da Dinâmica e Orientação em Caenorhabditis elegans Neurônios

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Quantificando níveis de alteração morfológica e degeneração do neurônio dopaminérgico em Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imagem Subcelular da Manipulação Neuronal de Cálcio In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

In vivo de imagens de Dauer específica Neuronal Remodelação em C. elegans

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