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., 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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 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 (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;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&#8221;x3&#8221;&#160; 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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Research

JoVE Journal

Neuroscience

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

8.5K 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

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálise de etanol durante a operante determinação auto-administração de etanol e etanol por cromatografia gasosa

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Neurociência

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Um ensaio para a permeabilidade do neuroepitélio Zebrafish Embryonic

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

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Sistema Modelo para Estudo Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

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.  ...

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 ...

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 ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Novas métricas para Caracterizar Embryonic Alongamento do nemátodo<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método eletrofisiológico para respostas gravação intracelular de tensão<em&gt; Drosophila</em&gt; Fotorreceptores e Interneurônios à luz Estímulos<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UMA<em&gt; Caenorhabditis elegans</em&gt; Ensaio de Aversão ao Cobre Baseado em Status Nutricional

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

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 ...