Agradecemos Ciências da Vida e da Faculdade de Artes e Ciências da Universidade de Queen para financiar este trabalho. Assim, agradecemos o laboratório Chin-Sang para fornecer os reagentes necessários, equipamentos e suporte técnico. Agradecemos também QGEM 2011, especialmente Tony Ele, por sua contribuição para a discussão.

1. Preparação / lavar os Worms <ol><li> Sincronizar vermes adultos jovens 11. </li><li> Pipetar 2 ml da Basal S em uma placa de 5 cm de Chemotaxis encenado vermes que só limpou o gramado do OP50 E. coli. Incline a placa conforme necessário para garantir que os vermes são lavados da superfície da placa para o buffer. </li><li> Pipete 1 mL de solução de Basal verme-S para um tubo de microcentrífuga. </li><li> Centrifugar durante 10 segundos usando um PicoFuge em 6600 rpm. </li><li> Aspirar o Basal S, deixando a pelota de vermes não perturbadas. </li><li> Adicionar 1 ml de solução de S basal ao tubo de microcentrífuga, invertido várias vezes para lavar os vermes. </li><li> Repita os passos 1.4 a 1.6 mais três vezes. </li><li> Centrifugue durante 10 segundos a quinta vez, desta vez aspirando o sobrenadante para um volume final de ca. 100 ul. </li><li> Pipeta 2 ul dos vermes numa placa NGM para garantir que há entre 50 e 250 vermes em cada amostra de 2 ul. Ajustara concentração de vermes no basal S conforme necessário por ressuspensão dos vermes em um volume maior ou menor de S basal. </li><li> Usar os vermes no ensaio imediatamente após a lavagem dos vermes para até 1 hora. </li></ol> 2. Preparar as placas de teste <ol><li> Marcando o lado de baixo de uma placa de 5 cm, dividir o prato em quatro quadrantes iguais. Pode ser utilizado ou ágar Quimiotaxia NGM. Um mínimo de três ensaios são necessários por estirpe genética a ser testado. </li><li> Marque um círculo com um raio de 0,5 centímetros em torno da origem (figura 1). </li><li> Marcar um ponto em cada quadrante com qualquer um &quot;T&quot; para &quot;Test&quot; ou &quot;C&quot; para &quot;Control&quot;, garantindo que os sites estão equidistantes da origem e outro. Os pontos devem ser pelo menos 2 cm de distância da origem. Marcar o canto superior esquerdo e inferior direito quadrantes como quadrantes teste e no canto superior direito e inferior esquerdo quadrantes como controles (Figura 1). </li></ol> 3. A execução do ensaio </p&gt; <ol><li> Misturar a solução de ensaio através da combinação de volumes iguais de o composto de teste e 0,5 M azida (um anestésico usado para prender os vermes ao atingir um quadrante). Misturar a solução de controlo, combinando o solvente utilizado para diluir o composto de teste, com 0,5 M de azida. </li><li> Pipeta 2 ul da solução de verme (preparado em 1.8) a partir da pelota para a origem (onde as duas linhas se cruzam). </li><li> Imediatamente depois, pipeta 2 mL da solução de ensaio para os dois locais de &quot;T&quot;. Da mesma forma, pipetar a mesma quantidade da solução de controlo para os dois locais de &quot;C&quot;. </li><li> Uma vez que os vermes e odorante gotas foram absorvidas no agar, substituir as tampas e inverter as placas. </li><li> Após 60 min, coloque os vermes numa incubadora de 4 ° C. Apenas remover uma placa da incubadora, quando pode ser contado. Partindo vermes, à temperatura ambiente, pode permitir a mobilização de novo. </li><li> Anote o número de vermes em cada quadrante que cruzou completamente a circ interiorle. </li><li> Repita os passos 3.2 a 3.6, utilizando a solução de controlo em ambos os quadrantes de ensaio e de controlo. Isto servirá como a placa de controlo. Três dessas placas devem ser feitas e correr para servir como um controle adequado. </li></ol> 4. Interpretando os Scores / determinação do índice de quimiotaxia <ol><li> Calcular o índice de quimiotaxia usando a Equação 1. Isso vai render um índice quimiotático entre -1,0 e 1,0. </li></ol> (1) Quimiotaxia Index = (# Worms em ambos os quadrantes do teste - # Worms em ambos os quadrantes Controle) / (N º total de gols Worms) Uma pontuação de 1,0 indica atracção máxima em direcção ao alvo e representa 100% dos vermes que chegam nos quadrantes contendo o alvo química. Um índice de -1,0 evidência de repulsão máxima. Métodos de ensaio semelhantes foram já empregues 3 (Tabela 1). <ol start="2"><li> Indicar a média Chemotaxis Index (CI) e erro padrão da média (SEM). Realizar o t-teste de Student comparando os dados a partir das placas de ensaio e de controlo. </li></ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

Não temos nada a divulgar.

Quimiotaxia, embora controladas por um conjunto complexo de mecanismos neuronais e celulares, pode ser facilmente e objectivamente quantificada usando ensaios de quimiotaxia. Para obter os melhores resultados a partir dos ensaios, devem ser tomadas determinadas etapas críticas. Em primeiro lugar, encenando os vermes é essencial para dar resultados experimentais consistentes. Worms em diferentes fases da vida se comportam de forma diferente 13; vermes estágio tão misturada pode distorcer os resultados expe...

Ward desenvolvido pela primeira vez o ensaio de quimiotaxia em 1973 5, e desde então tem tido aplicações de longo alcance. Neurobiology é um campo que beneficiou de utilizar uma variedade de ensaios de quimiotaxia. Adaptação olfativo, uma simples forma de aprendizagem e memória, foi demonstrada em C. elegans usando ensaios de quimiotaxia 6. Eles também têm sido usados ​​para mostrar que a C. elegans pode desenvolver-etanol tolerância resultado demonstra que não só a plasticidade comportamental dos vermes, mas que também mostra que os vermes podem ser muito útil no estudo da dependência do álcool em humanos 3. Os ensaios têm mesmo sido desenvolvidos para demonstrar a capacidade de C. elegans para armazenar curto e memória de longo prazo, mostrando que as associações são feitas pelos vermes entre chemoattractants e Alimentação (OP50) 7. Além disso, dada a extensa informação actualmente disponível sobre o C. elegans do genoma, o chemotaxiO comportamento de C. elegans foi alterada várias vezes através da indução de mutações de 1,8. Isto permite muitas possibilidades interessantes de engenharia, tais como o desenvolvimento de C. elegans como uma ferramenta de biorremediação. Assim, uma vez que o desenvolvimento inicial do ensaio de quimiotaxia, em 1973, tem sido frequentemente alterado e utilizado para elucidar mistérios numa variedade de áreas. Alguns ensaios teve como objetivo descobrir a rota específica tomada pelos vermes em direção a um alvo. O ensaio protótipo deste tipo foi desenvolvido por Ala 5. Três vermes foram colocados em agar derretido, durante 15 min. Os seus movimentos foram rastreados pelo cunho eles deixados como explorada a partir da periferia da placa superior de um gradiente de um atraente no centro da placa. Todos os vermes na placa foram presos utilizando clorofórmio no final de cada ensaio. Um descendente deste método de um único sem-fim colocado no meio da placa com o atractivo e a um controlet distâncias iguais e opostas a partir da origem 2. Pierce-Shimomura et al. desenvolvido um ensaio para observar a natureza exacta do movimento envolvidos na quimiotaxia 9. Vermes individuais foram colocados em placas de Petri de 9 centímetros, quer contendo uma concentração uniforme do atraidor ou um gradiente de forma radial, que culminou com a fonte do atraidor. Um programa de computador que reconhece o worm foi usado para registrar o comportamento observado. Uma câmara de vídeo acoplada a um microscópio trabalhadas em conjunto com a fase de ajustar a placa de Petri automaticamente como o ensaio correu para assegurar o verme permaneceu no campo de visão. A partir daí, a informação mais detalhada foi descoberto em relação à causa de pirueta indicadas por C. elegans. Outros ensaios, mais semelhantes ao descrito aqui, testou a resposta de uma grande população de vermes para compostos de teste. Dois quadrantes ensaios de quimiotaxia foramutilizada para explorar as funções que vários receptores, neurónios, e as moléculas de transdução de sinal quando reproduzido C. elegans foi exposto a vários compostos 1. Entre 20-50 vermes lavados foram colocados perto do centro da placa com um atraente e um controle nas extremidades polares, juntamente com o anestésico, azida de sódio (NaN3). Após 60 min, um índice quimiotáctico com valores a partir de -1,0 a +1,0 foi gerado com base na diferença entre o número de vermes foram afixadas no atractivo ou o controlo. Um índice quimiotáctico semelhante foi usada no ensaio relatado neste artigo, embora o ensaio anterior falhou para avaliar rigorosamente vermes não-móveis. Este ensaio foi posteriormente aplicada a testar os efeitos de ablação neuronal em quimiotaxia. Outra variação do ensaio acima mencionado foi realizada 100-200 vermes onde foram colocados no centro de uma placa contendo quatro quadrantes 3. Quadrantes adjacentes ou o teste, ou contidocontrolar a substância. Tal como nos ensaios anteriores, os vermes foram imobilizadas por acção de azida de sódio, antes de serem marcados. Um método semelhante é descrito aqui como uma forma de avaliar a resposta de C. elegans para vários compostos. No entanto, o método abaixo tem a vantagem de apenas avaliar vermes que passaram uma distância limite que separa móvel de vermes imóveis. Outros ensaios incorporaram diretrizes semelhantes para ignorar vermes imóveis. Frøkjær-Jensen et ai. desenvolvido um ensaio versátil que pode ser usado para testar os compostos solúveis tanto voláteis e água 10. A placa de Petri foi dividida em quatro quadrantes. Os quadrantes superior e inferior não contêm solventes. O quadrante esquerdo continha água, eo direito continha o atrativo. Ao testar odorantes voláteis, o analito foi colocado sobre a tampa da cápsula, ao longo do quadrante correcto, enquanto que os compostos solúveis em água, foram colocadas directamente no agar. Os métodos actualmente existentes para avaliar a resposta quimiotáctica de C. elegans estão constantemente a ser refinado para optimizar a sua facilidade de uso, a eficiência e precisão. Assim, enquanto que o ensaio aqui descrito tem a capacidade de avaliar um maior número de vermes (máximo rendimento:. Vermes 250 / hora por placa, um pouco maior do que a taxa de transferência demonstrada por Lee et al, 3), a força real deste método é o sucinta culminar de muitos dos atributos dos ensaios anteriores (Tabela 1).

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PicoFuge</td>
 <td>Stratagene</td>
 <td>400552</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P1000 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P10 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M Sodium Azide</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl2, 1 mM MgSO4]</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol/Distilled Water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Test Compounds (eg. 0.5% diacetyl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Bio-Rad</td>
 <td>166-0600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>Amresco</td>
 <td>CA97062-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>VWR</td>
 <td>CA12001-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4OH</td>
 <td>BDH</td>
 <td>CABDH8641-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Tween 20</td>
 <td>Bio-Rad</td>
 <td>170-6531</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

c. elegans chemotaxis assay

Muitos organismos usam a quimiotaxia para buscar fontes de alimentos, evitar substâncias nocivas, e encontrar companheiros. Caenorhabditis elegans tem comportamento quimiotaxia impressionante. A premissa subjacente testar a resposta dos vermes a um odorante é colocá-los em uma área e observar o movimento evocada em resposta a um odorante. Mesmo com os diversos ensaios disponíveis, optimizando verme localização inicial em relação a ambas as áreas de teste e de controlo, enquanto minimiza a interacção de vermes com o outro, mantendo ao mesmo tempo uma amostra significativa, continua a ser um trabalho em progresso 1-10. O método descrito aqui pretende abordar estas questões através da modificação do ensaio desenvolvido por Bargmann et al. 1. A placa de Petri é dividido em quatro quadrantes, dois quadrantes opostos marcados &quot;teste&quot; e dois são designados como &quot;Controle&quot;. Anestésico é colocado em todos os testes e os locais de controlo. Os vermes são colocados no centro da placa com uma circle marcados em torno da origem para assegurar que os vermes não-móveis serão ignorados. Utilizando um sistema de quatro quadrantes, em vez de um ou 2 dois elimina uma polarização no movimento dos vermes, como eles são equidistantes a partir de amostras de ensaio e de controlo, independentemente de que lado da origem começaram. Isso contorna o problema de vermes sendo forçados a viajar através de um conjunto de outros vermes para responder a um odorante, o que pode atrasar vermes ou forçá-los a tomar um caminho mais tortuoso, produzindo uma interpretação errada do seu caminho pretendido. Este método também apresenta vantagens práticas por ter um tamanho de amostra maior e permitindo que o pesquisador para executar o ensaio autônoma e marcar os vermes uma vez que o tempo estipulado expirou.

Comparando-se o tipo selvagem (N2) C. elegans para o ODR-10 (KY10) mutante. Usamos diacetil, um conhecido C. elegans quimioatractor, para comparar os vermes de tipo selvagem para o de um mutante que carece do receptor para diacetil 1,12. Por tipo selvagem (N2), o índice quimiotáctico vermes era de 0,100 ± 0,066 para o etanol, e 0,839 ± 0,031-0,5% de diacetil. Como esperado, diacetil provoca uma resposta significativa quimioatratante de vermes d...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

Um método para avaliar quantitativamente a resposta quimiotáctica de<em&gt; Caenorhabditis elegans</em&gt; É descrito. Um índice quimiotáctico (Cl) foi utilizada como uma forma de avaliar precisamente a resposta de minhocas, para determinados objectivos, e servir como uma plataforma de comparação entre estirpes e os compostos de interesse.

C. elegans Chemotaxis Assay

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

Research

JoVE Journal

Biology

45.0K visualizações.  Queen's University. Um método para avaliar quantitativamente a resposta quimiotáctica de Caenorhabditis elegans É descrito. Um índice quimiotáctico (Cl) foi utilizada como uma forma de avaliar precisamente a resposta de minhocas, para determinados objectivos, e servir como uma plataforma de comparação entre estirpes e os compostos de interesse.

Vídeo: C. elegans Chemotaxis Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biologia

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicação de um elegans dopamina C. Ensaio degeneração dos neurônios de Validação do Potencial Doença de Parkinson Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Paradigmas para Caracterização farmacológica de C. elegans Mutants Transmissão Sináptica

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecação e gravação a partir da junção neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Geração de transgênicos C. elegans Por biobalística Transformação

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Um Método para o cultivo embrionário<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Anticorpo de coloração em<em&gt; C. Elegans</em&gt; Usando o &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

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

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

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

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

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

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