Name: an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans
Uploaded: 2015-04-09T13:00:00.000+00:00
Duration: 10 min 35 s
Description: Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

Estes estudos foram apoiados por bolsas de Institutos Nacionais de Saúde, Instituto Nacional de Alcoolismo e Abuso de Álcool: R01AA016837 (JCB) e P20AA017828 (AGD e JCB).

1. Passos para executar no dia anterior ao ensaio <ol><li> Escolha L4 vermes palco para meio de crescimento nematóide fresco (NGM) placas semeadas com um gramado de OP50 E. coli e cultura-los em 20 CO / N. Cada condição de ensaio requer 10 vermes; escolher um excesso de vermes para permitir O perda de vermes / N. <ol><li> Apenas animais de ensaio que são adultos do primeiro dia; muitos mutantes crescer a uma velocidade mais lenta do que o tipo selvagem. Ajuste o tempo de colheita para as estirpes que têm atrasos de desenvolvimento, de modo que todos os animais testados são adultos do primeiro dia. </li></ol></li></ol> 2. Passos a executar no dia do ensaio <ol><li> Preparação para o ensaio: <ol><li> Realizar ensaios em unseeded 60 x 15 milímetros placas NGM padrão. Seque todas as placas para ser usado em 37 ° C durante 2 horas, com tampas de folga. Para cada ensaio experimental, use quatro placas NGM secas; estes serão a 0 mm e 400 mm placas de ensaio etanol e suas placas de aclimatação de acompanhamento. NOTA:A utilização de placas NGM é importante aqui; diferenças na composição dos blocos, em particular, a sua osmolaridade, pode afectar fortemente a resposta à dose de etanol no comportamento, isto é devido, pelo menos em parte, a alterações na quantidade de etanol acumulada pelos animais 20. Agar NGM é de 160 mOsm. </li><li> Após a secagem, pesar cada uma das placas NGM unseeded para ser utilizado no ensaio e tomar nota do peso. Determinar o volume de meio em placas com base no peso de uma placa vazia. Para aproximar a conversão do peso do agar, para o volume, assumir que os meios de comunicação pesa o mesmo que um volume igual de água. NOTA: Os resultados mais consistentes foram encontrados com media NGM que desidratadas suficientemente que um volume original de 10 ml tem um volume de pós-secagem entre 8,3-8,9 ml. Uma alternativa para o tempo de secagem é de 2 horas para secar até que as placas atingem esta gama de volume para explicar as diferenças em incubadoras. </li><li> Derreta 4 anéis de cobre (diâmetro interno de 1.6 cm) para a superfície de cada uma das placas de agir como currais para os diferentes genótipos ou grupos de tratamento de worms.Grasp cada anel com uma pinça, e o calor de uma chama (em um forte chama de um queimador de Bunsen funciona bem) para cerca de 3 seg. Colocar imediatamente o anel em uma placa enquanto ainda mantém o anel com a pinça para impedi-lo de &quot;salto&quot;. <ol><li> Certifique-se de que o anel assenta plana contra a superfície do agar, pressionando suavemente com a pinça em vários pontos no anel. Ao colocar o anel, ter o cuidado de deixar espaço para mais três anéis. </li><li> Derreter os três anéis adicionais de cobre na superfície da placa, tendo o cuidado de colocá-los o mais próximo possível um do outro, de modo que todos os quatro estarão no campo de visão da câmara. NOTA: Fazendo uma boa vedação com o agar é essencial para manter os vermes nos anéis durante o ensaio. Anéis que pular na chapa não são susceptíveis de fazer a vedação correta com o agar epode scar o agar, que permite que os vermes de toca e interfere com a visualização dos vermes. </li><li> Para cada placa de ensaio preparar uma placa de acompanhamento &quot;aclimatação&quot;, que deve ser seco e não inoculada e não receberá qualquer etanol. Coloque quatro anéis de cobre sobre estas placas. </li></ol></li><li> Rotular o fundo das placas ao lado de cada anel com a estirpe sem-fim para ser usado no anel, tendo o cuidado para não escrever no campo de visão do próprio anel. Combinar as etiquetas na placa de ensaio com a sua placa de aclimatação que o acompanha. </li><li> Calcula-se a quantidade de etanol a 100% para adicionar a cada placa de ensaio de modo a que a concentração final é de 0 mM ou 400 mM de etanol (em peso por volume). Para cada experiência (n = 1), haverá um 0 mM e 400 uma placa de etanol mM; placas de aclimatação não receber etanol. Adicionar o etanol, pipetando-o em torno da superfície da placa. Use filme de laboratório para selar a placa e permitir que se equilibre no cimo da bancada durante 2 horas. </li><li> Quando tiver decorrido 1,5 horas, começa a etapa de aclimatação de ensaio, passo 2.2. </li></ol></li><li> Realizar ensaio locomoção: As placas de ensaio <ol><li> Transferir cuidadosamente 10 vermes de cada grupo experimental para o centro de um anel de cobre sobre a placa de aclimatação. Remova qualquer alimento que é visível no agar neste momento raspando suavemente com a pick worm. Variar a ordem em que os grupos experimentais são colocados sobre as placas através de ensaios experimentais de modo a que as mesmas estirpes não são colocados nas placas na mesma ordem em todos os ensaios. NOTA: O objectivo é o de transferir os animais para a placa com quantidades mínimas de alimentos, porque se o alimento sobre a placa de aclimatação é transferido para a placa de ensaio os vermes vai agregar em redor dos alimentos e o efeito da droga sobre a locomoção vai ser obscurecida. </li><li> Tenha cuidado para não quebrar a superfície do agar com a escolha, pois isso permitirá que os vermes de toca e pode atrapalhar o ensaio. Incubar os vermes à TA durante 30 min. </li><li> Certifique-se que há um intervalo adequado entre as iniciações de cada placa. Um experimentador experiente pode mover-se 40 animais, 10 / anel para 4 anéis em &lt;1,5 min, mas qualquer intervalo de até 2 min é aceitável. O ensaio padrão tem gravar filmes em 10-12 min e 30-32 min de exposição tanto para 0 mM e 400 mM de etanol. </li><li> Iniciado a placa de aclimatação para a exposição 0 mM e a placa de aclimatação para a exposição de 400 mM a cerca de 2 min e 30 seg para além de permitir a poupança do primeiro arquivo de filme antes do segundo filme deve começar. </li><li> Após o período de aclimatação de 30 minutos, transferir os vermes das placas de aclimatação para as placas de ensaio. Transfira os vermes para as placas de ensaio (0 mm ou 400 mm) de etanol na mesma ordem em que foram adicionados à placa de aclimatação, mantendo o controle do tempo entre as conclusões de cada placa. Selar a placa com película de laboratório para minimizar a perda de etanol a vaporização. </li><li> Usi, um movimento de escavar com uma picareta verme fina gumes achatada para coletar vermes em cima da pick achatada. Executar a transferência de vermes a partir da placa não inoculada aclimatação à placa de ensaio, sem a utilização de bactérias, o que é comumente utilizados na transferência de vermes, uma vez que ajuda a colar o sem-fim para a retirada. NOTA: A velocidade em que os animais são transferidos para esta etapa é muito importante, porque as primeiras vermes sobre a placa será exposta a etanol mais tempo do que os últimos vermes adicionados à placa, e pontos de tempo anteriores podem apresentar alguns efeitos dependentes do tempo. Esta é a principal razão para a rotação que as estirpes são colocados sobre uma primeira placa através de experiências em replicado. </li></ol></li><li> Execute Locomotion Ensaio: As filmagens <ol><li> Use uma combinação de microscópio / câmera que permite a gravação simultânea de todos os quatro anéis no campo de visão (aproximadamente 42x42 mm 2 quadrado), como um objetivo 0,5x microscópio, ampliação de 0,8x e uma câmera com um 2048x2048 7,481; m CCD pixel. </li><li> Utilize uma iluminação uniforme em todo o campo de vista, que ajuda no reconhecimento de objecto no passo limiar de intensidade (ver abaixo). A &quot;x3&quot; backlight 3 funciona bem. </li><li> Imagem os vermes em uma placa posicionada do lado media para cima (tampa para baixo), o que gera contraste que se perde quando se utiliza a luz de fundo em comparação com um microscópio tradicional transmitida fonte de luz. </li><li> Prepare o software de análise de imagem para capturar filmes lapso de tempo dos vermes em movimento. Defina o software para capturar 12 bits imagens em escala de cinza a cada 1 s, como um filme de 2 min (120 frame). Para reduzir o tamanho do ficheiro de saída, mantendo a resolução de imagem suficiente, utilizar um modo de bins de 2x2 para capturar imagens 1024x1024 pixels. </li><li> Grave filmes. Começar a gravar um filme 120-frame da primeira placa (0 mM etanol) 10 min após o último vermes foram colocados nessa placa. Salve o arquivo de filme. </li><li> Grave a segunda placa (400 mM de etanol). Repita esse processo paraambas as placas com início 30 minutos após a última vermes foram colocados sobre a primeira placa (0 mM de etanol) para capturar os pontos 30-32 min de tempo para cada exposição. NOTA: Aguarde o tempo suficiente para salvar os arquivos de imagem depois de cada sessão de captura antes de começar a gravação do próximo prato a ser analisado. </li><li> Para futuro à prova de filmes arquivados e permitir que esses filmes sejam abertas e analisadas por outros programas de software de imagem de código aberto de domínio público, como ImageJ 28, converter uma cópia de cada filme para 8-bit 256 arquivos TIFF em escala de cinzento. NOTA: O software de análise de imagem descrito aqui usa um formato de arquivo proprietário. </li></ol></li><li> Análise de filmes usando software ImagePro. <ol><li> Uma vez capturado, analisar os filmes usando Imagem-Pro Plus software ou seu equivalente. NOTA: Uma variedade de outros softwares de seguimento de objectos está disponível que foi usado com sucesso para controlar múltiplos C. elegans de uma só vez 29 e talsoftware poderia razoavelmente substituir o descrito aqui como as etapas de identificação objeto são baseadas em princípios semelhantes. O método descrito refere-se a versões com software Image Pro Plus 6,0-6,3 e 7.0, as versões mais recentes de Imagem-Pro Plus software podem ter pequenas diferenças. </li><li> Analisar filmes nos segmentos 2-min (120 frame). Em primeiro lugar, aplica um filtro para as imagens para achatar o fundo e aumentar o contraste dos objectos de vermes. Selecione o filtro da seguinte forma: Menu&gt; Processos&gt; Filtros&gt; Acessórios&gt; Nivelar: Parâmetros: Fundo = escuro; Recurso Largura = 20 Pix. </li><li> Re-salvar os filmes após a etapa de filtragem, mas sempre manter o filme original em sua forma não filtrada. <ol><li> Analisar a locomoção dos animais em cada anel separadamente com uma região circular de interesse (ROI) que está colocado e dimensionado para se sobrepor com o anel de cobre. Identificar e acompanhar os vermes com o Menu&gt; Medida&gt; Track Objetos ... comando; Isso traz a Rastreamento Djanela Tabela ata. O botão Opções de controle permite faixas específicas a serem excluídos e limitar quaisquer artefatos experimentais. </li></ol></li><li> Sob a guia Auto Tracking, use os seguintes parâmetros: Pista Parâmetros: limite de velocidade (raio de busca) = 400 mm / frame, limite de Aceleração = Auto, comprimento total mínimo pista = 400 mm, tipo de movimento predominante = caótico; Objetos em parâmetros faixas: Permitir faixas parciais = yes, Min comprimento = 21 frames, Acompanhando profundidade previsão = 1 quadro. </li><li> Para iniciar o processo de rastreamento, clique no Encontrar Todos os rastreia automaticamente o botão de função para abrir a caixa de diálogo de Contagem / opções de tamanho e a caixa de diálogo Rastreamento. Selecione a opção Manual para a Seleção faixa de intensidade na caixa de diálogo Contagem / Size, que fornece o passo importante limiar que usa a intensidade da escala de cinzentos do worm pixels para destacar um objeto verme para análise e ignorar os mais leves de fundo pixels. <ol><li> Ajuste o sli limiar de intensidadeders (um para definir o limite superior, um para definir o limite inferior) para criar um intervalo inclusivo que destaca todos os objetos escuros. A gama de 0-1,500 em uma escala de 0-4,095 é um bom ponto de partida para a sintonia fina. </li><li> Aplicar um filtro de tamanho de excluir objectos que são maiores e menores do que um único objecto sem-fim, tal como o anel de cobre e quaisquer detritos nas placas. Definir dois parâmetros de tamanho para fazer isso que cobrir tamanhos para vermes individuais em uma variedade de posturas com a medida&gt; Selecionar Medidas item de menu na caixa de diálogo Opções de Contagem / Tamanho. (Intervalo Área: 28,000-120,000 mm 2 e gama Perimeter: 600-2,500 mm). </li><li> Se uma estirpe mutante é significativamente menor ou maior do que outros animais na chapa a ser analisado, ampliar a gama de configurações de filtro para reconhecimento de objetos para acomodar os diferentes tamanhos das cepas primeiro antes de rastreamento de animais, para que as configurações não precisam ser alteradas meados de análise. </li></ol> &lt;/ Li&gt; <li> Concluir o processo de rastreamento clicando em Continuar na caixa de diálogo Rastreamento. Visualmente comparar as faixas de saída com o progresso de cada verme no filme para garantir que cada verme é representada a menos que haja uma razão válida para excluí-lo com base nas definições de filtros automáticos. Exclua manualmente faixas que foram produzidos pela presença de objetos não-worm confirmaram que atenderam aos critérios de filtro de tamanho. </li><li> Use o software para calcular a velocidade de cada verme entre cada quadro (distância percorrida para o baricentro de um objeto por 1 segundo entre os quadros) e exibir a velocidade média para cada faixa e da velocidade média de todas as faixas para a população de vermes na anel de cobre. Considere esta média final para ser n = 1 em termos de ensaios experimentais. Exportar os dados para um programa de planilha para análises estatísticas e arquivamento de dados. </li><li> Uma vez que os dados foram registrados para o primeiro toque na placa, mova o ROI para o próximo anel erepita o processo de rastreamento, sem alterar qualquer um dos parâmetros. </li><li> Calcular a velocidade relativa de locomoção por meio de: </li><li> Velocidade relativa (%) = tratada (400 mm) Velocidade / não tratada (0 mm) Velocidade x 100. NOTA: Diferentes manipulações genéticas, muitas vezes altera a taxa de locomoção basal dos animais. A fim de determinar o efeito do etanol sobre a velocidade de locomoção dos animais e para ser capaz de comparar estes efeitos em diferentes genótipos ou condições, calcular uma velocidade relativa. </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

A neurobiologia simples e ferramentas genéticas disponíveis em C. elegans fazer o sem-fim um excelente modelo para estudar as bases moleculares dos efeitos do etanol sobre o comportamento. Aqui, nós descrevemos um ensaio que foi utilizado para identificar vários mediadores moleculares e ambientais da resposta comportamental aguda para ethanol 8,10,20,24,25. Este método permite a diferenciação e análise simultânea de dois fenótipos diferentes comportamento de resposta etanol, sensibilidade i...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table><tbody><tr><td>C. elegans strains</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>60 x 15 mm Petri plates, triple vented</td><td>Greiner Bio-One</td><td>628161</td><td>Other plate brands will suffice.</td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>NaCl (3 g/L), agar (17 g/L), peptone (2.5 g/L), 1 ml cholesterol (5 mg/ml in ethanol), 1 ml (1 M) MgSO&lt;sub&gt;4&lt;/sub&gt;, 1 ml (1 M) CaCl&lt;sub&gt;2&lt;/sub&gt;, 25 ml (1 M) KPO&lt;sub&gt;4&lt;/sub&gt;, pH=6, 975 ml H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Forceps</td><td>Various</td><td></td><td>&lt;I&gt;e.g.&lt;/I&gt;, Fisher Scientific #10300</td></tr><tr><td>37 &amp;deg;C Incubator</td><td>Various</td><td></td><td>For drying agar</td></tr><tr><td>Digital balance</td><td>Various</td><td></td><td>For determining plate weights and agar volume</td></tr><tr><td>Copper rings</td><td>Plumbmaster</td><td>STK#35583 (48 cap thread gasket)</td><td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td></tr><tr><td>100% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>Parafilm M</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>CCD camera</td><td>QImaging</td><td>RET-4000R-F-M-12</td><td>This camera has a large field of view.</td></tr><tr><td>Stereomicroscope with C-mount and 0.5X objective</td><td>Leica</td><td>MZ6</td><td>Discontinued model, M60 is current equivalent.</td></tr><tr><td>Light source</td><td>Schott</td><td>A08923</td><td>3&amp;rdquo;x3&amp;rdquo;&amp;nbsp; backlight for even illumination across the field of view</td></tr><tr><td>Imaging and tracking software</td><td>Media Cybernetics</td><td>ImagePro-Plus v6.0-6.3</td><td>Newer versions of the software have tracking functions.</td></tr></tbody></table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

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

Os dados representativos (Figura 1) de vários genótipos diferentes e seus controles pareados são apresentados 8,24; dados foram escolhidos especificamente que as diferenças de destaque nos animais testados. O grau de efeito de 10 min de exposição é considerada a sensibilidade inicial de uma estirpe, que é mostrada nos eixos esquerda na Figura 1B-G. Estirpes mutantes com uma maior velocidade relativa do que o controlo em 10 minutos são considerados como sendo resisten...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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

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

Um ensaio para medir os efeitos do etanol sobre a velocidade de Locomotion<em&gt; Caenorhabditis elegans</em

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

8.6K visualizações.  Virginia Commonwealth University. C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. 

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

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Uma maneira simples para medir a sensibilidade de etanol no Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Neurociência

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

Medindo a sensibilidade de Caenorhabditis para o Receptor acetilcolina Agonista Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

Genética

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Métodos para o estudo dos mecanismos de ação dos antipsicóticos Drogas em<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Método de alta duração para medir o tempo de sedação de álcool de melanogaster individual drosophila

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Comportamento

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Análise comparativa de métodos experimentais para quantificar a atividade animal em Caenorhabditis elegans Modelos de Doença Mitocondrial

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Um Ensaio Behavioral Barato, escalável para medição Ethanol Sedação sensibilidade e tolerância rápida em<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biologia

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medição dos Efeitos de bactérias em<em&gt; C. Elegans</em&gt; Comportamento Usando um Retenção Ensaio Egg

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Touch Habituation Assay: A Method to Study Non-Associative Learning in C. elegans

Source: Mersha, M. D., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/56771/long-term-behavioral-reproductive-consequences-embryonic-exposure-to">Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants.</a> J. Vis. Exp. (2018).
This video introduces the touch habituation assay, a C. elegans behavioral method of assessing mechanosensation and quantifying time to habituation.

Ensaio de Habituação ao Toque: Um Método para Estudar a Aprendizagem Não Associativa em C. elegans

Source: Mersha, M. D., et al. Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants. J. Vis. Exp. (2018).
This ...

JoVE Encyclopedia of Experiments

Caenorhabditis elegans (nematódeo)

Electrotaxis Assay: A Method to Observe Locomotion in C. elegans

Source: Tong, J,&#160;et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/50226/microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis">Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.</a>&#160;J. Vis. Exp.&#160;(2013).
Researchers can trigger on-demand movement in C. elegans by applying an electric field that worms sense and respond to.&#160; This video introduces electrotactic behavior and shows a sample protocol that is done in a microfluidic device.

Ensaio de eletrotaxia: um método para observar a locomoção em C. elegans

Source: Tong, J,&#160;et al. Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.&#160;J. ...

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