Gostaríamos de agradecer aos membros atuais e antigos do laboratório de ligação que ajudou a desenvolver os protocolos aqui descritos, particularmente Gin Fonte, que desenvolveu o procedimento original. Este trabalho foi suportado por concessões do NIH (AG12423) e Associação de Alzheimer (Prêmio Zenith) para CDL

1) Preparar os Suportes Nematóides placas de crescimento para ensaio de paralisia. Os ensaios são realizados paralisia no padrão Nematóides Crescimento Media (NGM) placas de rotina usado em C. elegans propagação e genética (Stiernagle, 2006). <ol><li> Autoclave a solução NGM contendo NaCl, Agar e peptona em um Erlenmeyer e adicionar as quantidades apropriadas de CaCl 2 estéril, Uracila, Colesterol, MgSO 4 e 1M 1M KPO 4. Alíquota de 10 mL de líquidos NGM em cada 60 mm x 15 mm placa de Petri. Permitir NGM para solidificar durante a noite a temperatura ambiente. </li><li> Se o teste para os efeitos de compostos / extratos, alíquota do extrato para cada placa e usar um difusor para distribuir uniformemente o extrato sobre a superfície do prato. Permitem extrair a secar durante a noite à temperatura ambiente. Volumes superiores a 1 mL vai exigir mais tempo para secar. </li><li> Ponto cada placa com 250 mL de E. coli OP5O cepa cultivadas em LB de mídia durante a noite para uma densidade óptica de 0,4-0,6. Permitir que as bactérias para secar durante a noite a temperatura ambiente. </li></ol> Variáveis ​​relevantes A idade de chapas tem um efeito significativo sobre o ensaio de paralisia, como placas mais velhas tendem a secar. Use placas derramado dentro de uma semana de ensaio de paralisia. Para resultados ideais, despeje placas 3-4 dias antes do início das populações verme síncrona [ver (2) abaixo]. Para qualquer experimento, apenas utilizar placas derramado a partir do mesmo lote. Alterando o tamanho do gramado (ou seja, manchado vs spread) e / ou o tipo de bactéria também vai alterar o comportamento dos vermes. Ensaios de paralisia pode ser realizada usando alternativa E. coli como fonte de alimento (por exemplo, a cepa HT115 utilizados na alimentação de experimentos RNAi), mas isso pode mudar a cinética de paralisia, exigindo controles internos adequados. 2) Preparação das Populações Idade-síncrona Worm Strain CL4176 (smg-1 (cc546 ts) I; dvIs27 [myo-3 / Aâ minigene + rol-6 gene marcador (su1006)] X é mais comumente usado para a dosagem Aâ induzida paralisia Esta estirpe e outros modelos transgênicos estão disponíveis. para pesquisadores acadêmicos para uma taxa nominal do Caenorhabditis Genetics Center (http://www.cbs.umn.edu/CGC/). <ol><li> Para maximizar a sincronia etária da população teste é preferível começar com uma geração sincronizado dos pais. Uma semana antes do início do ensaio paralisia da população teste, use um seletor de vermes de platina para transferir 20-30 adultos gravid para várias placas de 10 centímetros NGM espalhar com OP50 para um ovo de duas horas estava em 16 ° C (a temperatura permissivo para CL4176 ). Remove os adultos gravid e permitir que a progênie de crescer por 7 dias, época em que eles serão &quot;segundo dia&quot; adultos gravídico. </li><li> (Dia 1) Os adultos segundo dia gravid são usados ​​para preparar as populações em idade síncrona teste. Para cada condição experimental o objetivo é gerar placas triplicado contendo 50-75 idade síncrona worms. Usando um seletor de verme de platina, a transferência do dia 12/10 2 adultos gravid em 60 mm (10 mL de volume) placas NGM manchado com OP50. Permitirá que as minhocas para botar ovos a 16 ° C por 2 horas, em seguida, apanhar os adultos e permitir que os ovos para chocar e fazer crescer a 16 ° C. Neste ponto, é melhor ter alguém que não será marcar as placas para codificá-las, de modo que o artilheiro será cego às condições experimentais. </li></ol> Variáveis ​​relevantes O estágio de desenvolvimento embrionário, quando os ovos são colocados (~ o ​​estágio de 26 células para vermes tipo selvagem em condições ideais) é uma função da idade adulta e nutrição. Se os adultos gravid usado para a preparação da população de teste são mais velhas ou fome, ovos fase posterior serão estabelecidas, reduzindo a sincronia etária da população e da reprodutibilidade da cinética de paralisia. É essencial que mono-axênicas (ou seja, somente OP50 bactérias) culturas são utilizados (contaminantes microbianos podem afetar gravemente neste ensaio), e reprodutibilidade é maior se as placas têm triplicado número semelhante de worms. 3) Indução Transgene e Ensaio Paralisia <ol><li> (Dia 3) placas upshift a 25 ° C 48 horas após o término do ovo síncrona leigos; worms devem estar no terceiro estágio larval (L3). As placas devem ser organizados, sem empilhamento de uma incubadora de 25 ° C para permitir que todas as placas para chegar a 25 ° C, ao mesmo tempo. </li><li> (Dia 4) Comece a marcar a paralisação de worms (linhagem CL4176) entre 18-20 horas após o início da troca de marcha. Neste ponto, todos os worms na população deve ter alcançado o quarto larval (L4) palco. Continuar a marcar em duas horas até que todos os incrementos de worms em cada uma das placas estão paralisados. Por razões desconhecidas, a paralisia não é nem mesmo todo o comprimento do corpo: a região da cabeça é a última parte do worm para cessar em movimento. Assim, wORMs que recentemente iniciou a paralisia não pode traduzir através da placa, mas pode mover a cabeça, abrindo assim as bactérias em torno de seu anterior e deixando um &quot;halo&quot; de bactérias limpo. Estes worms invariavelmente tornam-se completamente paralisado e, portanto, worms com halos são categorizados como paralisado. Alguns worms não terá halos, mas também não mostram movimento espontâneo, que são testados pela insistência com o selecionador de worm. Se um verme prodded não pode sofrer uma propagação de ondas de corpo inteiro em cima prodding, é também classificada como paralisado. Para marcar eficiente, é mais fácil para mover o worms paralisado para um sector sem mácula da placa de modo que não serão involuntariamente recodificados o período de pontuação próxima. (Isso também permite que mis-categorizado worms para demonstrar movimento, permitindo uma correção de pontuação). </li><li> Para gerar uma curva de paralisia, em cada momento a fração de worms em cada prato que não tenham sido paralisado é convertido em um percentual, ea média percentual &quot;unparalyzed&quot; é plotado contra o tempo desde o início upshift. (A justificativa para traçar desta forma é que a curva resultante é formalmente análogo a uma curva de sobrevivência e, portanto, podem ser analisados ​​usando estatísticas padrão não-paramétricos de sobrevivência.) </li></ol> Variáveis ​​relevantes O upshift de vermes myo-3/Aβ transgênicos deve ocorrer antes que eles atinjam o estágio L4 meados de paralisia para ser iniciada, como o mio-3 promotor é developmentally regulamentada, e reduziu significativamente a expressão em L4 tarde ou vermes adultos. Da mesma forma, a expressão deste transgene é bloqueado se tornar worms fome, por isso é essencial que os vermes estão bem alimentados durante o experimento. Nós nunca observamos um verme paralisado para recuperar sob quaisquer condições, e depois de 12-16 horas de troca de marcha, todos os vermes vão paralisar CL4176, mesmo que sejam downshifted a 16 ° C. Na linhagem CL4176, upregulation suficiente de expressão do transgene para causar paralisia pode ocorrer a temperaturas mais baixas (ie, 20-25 ° C), embora o tempo de paralisia será alterado. A taxa de paralisia depende da cepa específica transgénicos utilizados (temos construído com as duas cepas myo-3/Aβ taxas mais rápidas e mais lentas do que CL4176), assim com cepas alternativa a janela de pontuação pode precisar de ser ajustada. 4) Resultados Representante Mostrado na Figura 1 é um gráfico de uma curva de paralisia por CL4176, ambos não tratada e exposta a 6,27 mM cafeína (concentração final na placa NGM). Este tratamento resulta em um atraso altamente significante na paralisia de aproximadamente 4 horas, o que nós interpretamos como indicativo de supressão de toxicidade Aâ neste modelo. Mostrado na Figura 2 é um experimento independente assaying os efeitos da cafestol, um outro composto encontrado no café. Este enredo é típico para os tratamentos que não têm impacto toxicidade Aâ. Note que em ambos os experimentos cerca de metade dos tratados CL4176 populações estão paralisados ​​às 24 horas, e paralisia é completa por 28 hr. Estes são os prazos típicos para a paralisia do controle CL4176 populações. Desvios significativos deste momento são indicativos de alteração das condições ambientais ou de eliminação espontânea de cópias do transgene. (CL4176 contém uma matriz cromossomicamente integrado transgênica com várias cópias do transgene myo-3/Aβ. Embora esta matriz transgênicos é normalmente bastante estável, a propagação da estirpe a temperaturas&gt; 16 ° C irá selecionar para worms raro que sofreram uma eliminação espontânea de transgenes, resultando em variantes que terão reduzida expressão Aâ e resultados mais lento paralisia.) <img src="/files/ftp_upload/2252/2252fig1.jpg" alt="Figura 1" /> Figura 1. Curva de paralisia para a cepa CL4176 worms não tratada ou expostos à cafeína 6,27 mM (Sigma). Os tempos indicados são do início do upshift temperatura. Barras de erro = SEM <img src="/files/ftp_upload/2252/2252fig2.jpg" alt="Figura 2" /> Figura 2. Bares curva de paralisia por CL4176 worms expostos a 0,48 mM cafestol (Sigma). Error = SEM

<ol>
	<li>Arya, U., Dwivedi, H., Subramaniam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

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O protocolo que temos descrito pode ser efetivamente usado para ensaio dos efeitos de compostos de toxicidade Aâ. Exemplos publicados incluem os efeitos dos constituintes Ginkgo biloba (Wu et al. 2006) e reserpina (Arya et al. 2009). Compostos que têm sido de proteção contra a toxicidade Aâ em outros sistemas também foram mostrados para ser protetor neste modelo (por exemplo, a memantina, nossos resultados não publicados). Uma lógica importante para o estabelecimento deste modelo é que as ferramentas bem desenvolvidas genética e molecular disponíveis em C. elegans pode ser usado para investigar os mecanismos de toxicidade Aâ. Assim, as mutações efeitos específicos sobre a toxicidade Aâ puderem ser analisadas (por exemplo, o efeito de reduzir insulin-like de sinalização através da introdução de uma daf-2 mutação de perda de função, Florez-McClure et al. 2007), e este modelo também pode ser usado para examinar os efeitos da sobre-expressão transgenes (Fonte et al. 2008). Recentemente, fez uso extensivo desse modelo para examinar os efeitos sobre a toxicidade de uma única substituições de aminoácidos na Aâ (dados não publicados). O modelo aqui descrito transgênicos ensaios o efeito de expressão Aâ aguda sobre a função das células musculares. No momento de células paralisia muscular e seus sarcômero estão intactos; o fenótipo paralisia não é o resultado de morte celular do músculo. No entanto, após a paralisia (~ 48 hr após upshift inicial) há uma ruptura da integridade muscular e eventual morte dos vermes. Nós não investigamos o efeito a jusante de expressão Aâ ou usado como um marcador de toxicidade. O modelo de expressão induzível Aâ descrito aqui não é apropriado para a investigação de tratamentos que modulam a β-amilóide formação de per se. Embora vermes transgênicos com forma constitutiva Aâ expressão depósitos de amilóide facilmente detectável (Fay et al 1998;.. Ligação et al 2001), worms transgênicos com a expressão induzível Aâ raramente têm depósitos de amilóide eo fenótipo paralisia aparece independente de amilóide (Drake et al. 2003). Nossos resultados são consistentes com a visão de que a toxicidade aguda de resultados Aâ induzida expressão da acumulação de Aâ oligomérico solúvel, não amilóide.

<table border="1"><tbody><tr><td> Reagentes </td><td> Quantity/2L </td><td> Empresa / Catálogo # </td><td> Comentários </td></tr><tr><td> NaCl </td><td> 6g </td><td> Sigma-Aldrich / S9888 </td><td></td></tr><tr><td> Agar </td><td> 40g </td><td> Sigma-Aldrich / A7002 </td><td></td></tr><tr><td> Peptona </td><td> 5g </td><td> Sigma-Aldrich / P0556 </td><td></td></tr><tr><td> 2 O DDH </td><td> 2L </td><td></td><td></td></tr><tr><td> CaCl 2 1M </td><td> 2mL </td><td></td><td> 147.02mg/mL em DDH 2 O </td></tr><tr><td> Uracil </td><td> 2mL </td><td></td><td> 2mg/ml em DDH 2 O </td></tr><tr><td> Colesterol </td><td> 2mL </td><td></td><td> 5mg/mL em etanol (não autoclave) </td></tr><tr><td> 1M MgSO 4 </td><td> 2mL </td><td></td><td> 246.5mg/mL em DDH 2 O </td></tr><tr><td> 1M KPO 4 </td><td> 50mL </td><td></td><td> 98mg KH 2 PO 4. ML, 48mg K2HPO4/mL em DDH 2 O, pH </td></tr><tr><td> Erlenmeyer </td><td></td><td> VWR </td><td></td></tr></tbody></table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

Acúmulo do peptídeo β-amilóide (Aâ) é acreditado geralmente para ser central para a indução da doença de Alzheimer, mas o mecanismo relevante (s) de toxicidade ainda não estão claros. Aâ também é depositado por via intramuscular em Miosite Inclusão Corpo, uma miopatia severa humana. A intensamente estudada nemátodo Caenorhabditis elegans worm pode ser projetado para expressar transgenicamente Aâ humana. Dependendo do tecido ou o momento de expressão Aâ, worms transgênicos podem ter fenótipos facilmente mensuráveis, que servem como uma leitura de toxicidade Aâ. Por exemplo, worms transgênicos com pan-neuronal expressão Aâ têm defeitos é aprendizagem associativa (Dosanjh et al 2009.), Enquanto worms transgênicos com constitutiva músculo-específicos expressão mostram uma progressiva, dependente da idade fenótipo paralisia (Link, 1995; Cohen et al . 2006). Um C. particularmente útil elegans modelo emprega uma mutação sensível à temperatura no sistema de vigilância mRNA para engenheiro de temperatura induzida por expressão de um transgene muscular Aâ, resultando em um fenótipo paralisia reprodutíveis sobre upshift temperatura (Link et al. 2003). Tratamentos que a toxicidade Aâ contra neste modelo [por exemplo, a expressão de um transgene de proteção (Hassan et al. 2009) ou exposição a extratos de Ginkgo biloba (Wu et al. 2006)] reproducibly alterar a taxa de paralisia induzida por upshift temperatura desses transgênicos worms. Aqui nós descrevemos nosso protocolo para medir a taxa de paralisia neste C. transgênicos elegans modelo, com particular atenção às variáveis ​​experimentais que podem influenciar esta medida.

Watch this Scientific Journal Video about Assaying ÃŽÂ²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

O verme nematóide intensamente estudada<em&gt; Caenorhabditis elegans</em&gt; Pode ser transgenicamente projetadas para expressar o peptídeo β-amilóide humana (Aâ). Expressão induzida de Aâ em<em&gt; C. elegans</emMuscular&gt; leva a uma rápida paralisia fenótipo, reproduzível que pode ser usado para monitorar tratamentos que modulam a toxicidade Aâ.

Assaying Toxicidade β-amilóide utilizando um transgênico C. elegans Modelo

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

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Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

25.5K visualizações.  University of Colorado. O verme nematóide intensamente estudada Caenorhabditis elegans Pode ser transgenicamente projetadas para expressar o peptídeo β-amilóide humana (Aâ). Expressão induzida de Aâ em C. elegans leva a uma rápida paralisia fenótipo, reproduzível que pode ser usado para monitorar tratamentos que modulam a toxicidade Aâ.

Vídeo: Assaying β-amyloid Toxicity using a Transgenic C. elegans Model

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Aprender Butanona Positivo, de curto prazo e de longo prazo Ensaios Memória Associativa

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Neurociência

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo Laser axotomia em C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Quantificação automatizada de fluorescência em Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Acompanhamento e Medição do Comportamento

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

A Leitura Molecular de adaptação a longo prazo olfativa em<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Ensaio danos no DNA em neurônios do hipocampo Usando o Ensaio Cometa

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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, ...

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 ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizando neuroblasto Cytokinesis Durante<em&gt; C. elegans</em&gt; Embriogênese

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

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

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

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 ...