Name: measuring the effects of bacteria on c. elegans behavior using an egg retention assay
Uploaded: 2013-10-22T15:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay at JoVE.com

Os autores gostariam de agradecer a junho Middleton para o fornecimento de E. cepas faecalis e de orientação com a cultura de bactérias. C. elegans foram fornecidos pela CGC, que é financiado pelo NIH Escritório de Programas de Infra-estruturas de Investigação (P40 OD010440).

1) Elaboração de Nematóides Crescimento Mídia (NGM) <ol><li> Para fazer com que as placas 50, adicionar 1,5 g de NaCl, 8,5 g Agar ultrapura, e 1,25 g de peptona de um 1 Lflask. </li><li> Adicionar 487,5 ml de dH 2 O para o balão. Agite bem para misturar e cobrir abertura do frasco com um pedaço de papel alumínio. </li><li> Esterilizar a solução por tratamento em autoclave, em condições normalizadas (121 psi, 120 ° C, 20 min.) </li><li> Deixar a solução arrefecer até 45 ° C num banho de água. </li><li> Para a solução, adicionar o seguinte na ordem: 500 ul de colesterol a 5 mg / ml de solução (dissolvido em etanol), 500 ul de 1 M de CaCl2, 500 ul de 1 M de MgSO4, e 12,5 ml de tampão KPO4 (54,15 g KH 2 PO 4 e 17,8 g de K 2 HPO 4 em 500 ml de dH2O), tal como descrito em 18. </li><li> Usando pipetas esterilizadas, adicione 10 ml de solução de agar para cada 60 milímetros de poliestireno placa de Petri. Permitir que os meios de comunicação para solidificar à temperatura ambiente overnight. </li></ol> 2) Preparação de B Caldo E. Mídia coli <ol><li> Para fazer com que cinco culturas de 100 ml, adicionar 5,0 g de Bacto-triptona, 2,5 g de extracto de levedura, 5,0 g de NaCl para uma proveta. </li><li> Adicionar água destilada estéril para um volume final de 500 ml. Aquece-se a solução sobre uma placa quente, com agitação, até que os solutos se dissolveram. </li><li> Colocar 100 ml de caldo de B em cinco frascos de vidro (pelo menos, 200 ml de volume). </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Permitir B caldo de arrefecer até à temperatura ambiente antes do uso. </li></ol> 3) Sementeira C. elegans Placas de Manutenção <ol><li> Depois de permitir que as placas NGM a secar à temperatura ambiente durante pelo menos uma semana, inocular 100 ml de um caldo de cultura B com algumas colónias de E. coli (OP50). </li><li> Crescer E. coli, a cultura a 37 ° C durante a noite. </li><li> Pipeta uma ou duas gotas (aproximadamente 10081; l) OP50 de cultura para o centro de cada placa NGM. As placas são geralmente empilhados do lado da tampa para cima. Quando pipetagem (seeding), começam por pipetagem a cultura na placa, na parte inferior da pilha. Trabalhe seu caminho até cada pilha. Tente não mover as placas após a sementeira, porque é importante que a cultura não fique muito perto dos bordos da placa. </li><li> Permitir que as placas semeadas a secar à temperatura ambiente durante pelo menos uma semana antes da utilização. </li><li> Placas semeadas são armazenados (invertido), em recipientes de plástico vedados à temperatura ambiente. </li></ol> 4) A manutenção C. elegans Cepas <ol><li> Para manter bem alimentado C. estoques elegans, use um verme escolher para passar três L4s ou adultos grávidas para um novo, sem sementes NGM placa a cada 3-4 dias. </li><li> Placas de Loja (invertido), de preferência dentro de uma incubadora a 15 - 22 º C. Culturas em ensaios descritos foram armazenadas a 20 ° C. C. elegans desenvolvimento é dependente da temperatura. Como maintenance a temperatura aumenta, o tempo entre os estágios de desenvolvimento diminui. </li></ol> 5) Preparação de Tryptic Soy Agar (TSA), E. faecalis Meios de Cultura <ol><li> Para fazer 50 placas, meça 500 ml de água destilada estéril para um frasco e leve para ferver, agitando, numa placa de aquecimento. </li><li> Adicionar 20 g de pó de ágar TSA ao balão e permita que a solução para dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixar a solução arrefecer até 45 ° C num banho de água. </li><li> Usando pipetas esterilizadas, adicione 10 ml de solução de agar para cada 60 milímetros de poliestireno placa de Petri. Permitir que os meios de comunicação para solidificar à temperatura ambiente durante a noite. </li></ol> 6) Preparação de Tryptic Soy Broth (TSB), E. faecalis Meios de Cultura <ol><li> Para fazer com que os tubos 250 de cultura, medida a 500 ml de água destilada estéril para um frasco e quente numa placa de aquecimento com agitação. </li&gt; <li> Adicionar 15 g de pó de ágar TSB para o frasco e para permitir que o pó se dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixa-se arrefecer até à temperatura ambiente, em seguida, pipeta de 2 ml, em tubos de ensaio de 5 ml esterilizadas. </li></ol> 7) Preparação de Heart Infusion (BHI) Mídia Cérebro, com estreptomicina, para E. faecalis Cultura <ol><li> Medir 500 ml de água destilada estéril para um frasco e leve para ferver, agitando, numa placa de aquecimento. </li><li> Adicionar 26 g de meio BHI ao balão e permitir que a solução para dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixa-se arrefecer a 45 ° C num banho de água. </li><li> Utilizando uma técnica estéril, adicionar 0,5 mL de uma solução de estoque de 10 mg de estreptomicina / ml e agite para misturar. </li><li> Adicionar 10 ml da solução a cada 60 milímetros de Petri de poliestireno. </li><li&gt; Permitir mídia para solidificar à temperatura ambiente durante a noite. Placas de Loja (invertida) a 4 ° C até algumas horas antes do uso. </li></ol> 8) Mantendo E. faecalis Cepas <ol><li> Para manter E. estoques faecalis, use uma agulha esterilizada para raia colônias individuais em ágar placas de soja trípticos separados (TSA). </li><li> Crescer as culturas a 37 ° C durante a noite. Placas de Loja (invertida) em um saco ou caixa selada à temperatura ambiente durante várias semanas. </li></ol> 9) Preparar E. Placas faecalis para EIW Assay <ol><li> Inocular uma cultura ml 2 TSB com uma colônia de E. faecalis e incubar durante a noite a 37 ° C. </li><li> Pipetar 20 ul de E. faecalis cultura para o centro de uma placa BHI-estreptomicina preparada, e incubar durante a noite a 37 ° C. </li></ol> 10) Preparar E. Placas coli para EIW Assay (placas de controlo) <ol><li> Inocular um ml B-bro 100º cultura com uma colônia de E. coli OP50 e incubar durante a noite a 37 ° C </li><li> Pipetar 20 ul da cultura OP50 para o centro de uma placa NGM unseeded e permitir que as placas semeadas a secar à temperatura ambiente durante a noite. </li></ol> 11) Ovo em Ensaio Worm <ol><li> Escolha 15-20 encenou-L4 C. elegans (Figura 1) para o centro de uma camada de bactérias numa preparada BHI-Strep-E. placa faecalis. Tenha cuidado para não transferir um monte de OP50 a placa BHI. Escolha o mesmo número de L4s a um controle de placa NGM-OP50. </li><li> Incubar as placas durante 40 horas a 20 ° C. </li><li> Preparar a solução de água sanitária a 20%. Misture a água sanitária comercial (hipoclorito de sódio a 6,0%) com o volume adequado de água destilada. </li><li> Adicione 10 gotas mL de solução de água sanitária para dez locais distintos em uma tampa de plástico (de um worm ou placa bacteriana). </li><li> Usando uma picareta verme, transferir um verme em cada gota de água sanitária. Agite suavemente a ponta dopegar na gota de água sanitária para certificar-se o worm tem lavado o fim da escolha (confirmar vendo a gota sob um microscópio de dissecação). </li><li> Permitir que a cutícula para dissolver durante cerca de 10 min ou até que os vermes se abriu, expelindo os ovos (Figura 2). Tenha cuidado para não transferir os ovos a partir da placa. </li><li> Usando um microscópio de dissecação, a contagem dos ovos em cada gota de água sanitária. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Os passos mais importantes na realização com êxito neste ensaio são os seguintes: 1) usando stocks bem alimentados de C. elegans, 2) cultura de tipos individuais de bactérias na placa de ensaio, 3) identificação precisão encenado L4 vermes para a exposição a E. faecalis, 4), mantendo o tempo de exposição a E. faecalis consistentes em todos os ensaios e 5) branqueamento tempo não deve exceder 10 minutos para impedir a desintegração do ovo. Para este e...

Caenorhabditis elegans, um microscópico, lombriga de vida livre, é um organismo modelo tradicionalmente usado para estudar os processos de desenvolvimento e de sinalização celular por causa de sua anatomia transparente, o desenvolvimento bem caracterizada, totalmente sequenciado o genoma, em tempo de geração curto, e homologia genética de seres humanos . Mais recentemente, C. elegans tornou-se um organismo modelo no campo da toxicologia ambiental e imunidade inata 10, 11. Esses vermes hermafroditas auto-fertilização se tornam sexualmente maduros dentro de dois a três dias de incubação do ovo. Durante o seu ciclo de vida, C. elegans passa por quatro estágios larvais (L1-L4), antes de atingir a idade adulta. Um isolado hermafrodita pode produzir, em média, 300 filhotes dentro de três dias de pico de fecundidade. Em reprodutivamente madura C. elegans hermafroditas, os ovos fertilizados são retidos no interior do útero durante várias horas antes de ser colocada. Onúmero normal de ovos armazenados no útero em qualquer momento (durante o pico da fecundidade) situa-se entre dez e quinze 12. O número de ovos no útero é uma função de ambos, a taxa de produção de ovos e a taxa de postura de ovos. Os ovos fertilizados são expulsos do útero pela contração da musculatura dezasseis vulvares dispostas em torno da abertura da vulva 13. Neurónios motores específicos do hermafrodita (do HSN) e neurónios motores VC sinapse para os músculos da vulva que afetam a contração muscular e, assim, postura comportamento 5,7,13,14. Expulsão dos ovos do útero ocorre devido à atividade coordenada de neurônios e músculos. Culturas de laboratório de C. elegans normalmente são levantadas em uma dieta de não patogênicas de Escherichia coli OP50. No ambiente natural, C. elegans entrar em contacto com uma variedade de fontes de alimentos, tais como as bactérias patogénicas, que podem ser potencialmente prejudiciais. Quando expostos a substâncias nocivas emambiente, C. elegans reter ovos até que o ambiente torna-se mais favorável. Presumivelmente, esta retenção de ovos é um esforço para proteger sua prole. Neste verme ovo em ensaio (EIW), C. elegans estão expostos às bactérias potencialmente patogénicas, Enterococcus faecalis, que é encontrada no meio ambiente. A exposição a formas patogénicas de E. faecalis pode causar infecção intestinal persistente e até mesmo a morte em C. elegans 15. A exposição a outras formas de bactérias patogénicas têm demonstrado afectar a retenção de ovo 16,17, no entanto, o efeito não foi quantificado. Além disso, o efeito de suavemente estirpes patogénicas de E. faecalis, as estirpes que não são imediatamente letal, sobre o comportamento de postura não foi estudado. EIW ensaios envolvem a contagem do número de ovos retidos no útero de C. elegans 4. Embora C. eleganssão transparentes, os ovos que se acumulam no útero pode ser difícil de quantificar num animal intacto. O ensaio EIW envolve branqueamento gravídico adulto C. elegans que foram expostos a bactérias, por um período fixo de tempo. A solução de água sanitária dissolve a cutícula externa deixando os ovos para trás. Os ovos são de refracção para os efeitos de branqueamento, devido à presença de uma casca de ovo. Após o branqueamento, é muito facilmente capaz de contar o número de ovos libertados do útero dos vermes após o branqueamento. O ensaio é descrito um método simples, barato e rápido para quantificar o número de óvulos no útero de uma só vez, e, assim, quantificar os efeitos de E. faecalis sobre retenção de ovo. Este ensaio pode ser utilizado para quantificar o efeito de outros tipos de bactérias, toxinas ou medicamentos ambientais sobre a retenção de ovo. Este ensaio tem também o potencial de ser usada como uma tela para patogenicidade.

<table><tbody><tr><td>Agar, ultrapure</td><td>Affymetrix</td><td>10906</td><td></td></tr><tr><td>Bacto Peptone</td><td>Becton Dickinson</td><td>211677</td><td></td></tr><tr><td>Bacto Tryptone</td><td>Becton Dickinson</td><td>211705</td><td></td></tr><tr><td>Brain Heart Infusion dehydrated medium</td><td>Carolina Biological Supply</td><td>781781</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;, N2 strain</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;C. elegans&lt;/em&gt;</td><td>Tritech Research Inc.</td><td>T3308</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;E. faecalis&lt;/em&gt;</td><td>Fisher Scientific-Fisherbrand</td><td>875713</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;(OP50)</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt; strains</td><td></td><td></td><td>provided by J. Middleton. All isolates were confirmed as enterococci</td></tr><tr><td colspan="4">by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td></tr><tr><td colspan="4"> &lt;p&gt;all strains grew at 44.5 &amp;ordm;C and were catalase negative and hydrolyzed esculin. A simplified&amp;nbsp;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;dichotomous key based on pigmentation and fermentation reactions for six sugars&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;(arabinose, mannitol, methyl-&amp;alpha;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;presumptive identification of all &lt;em&gt;E&lt;/em&gt;. &lt;em&gt;faecalis&lt;/em&gt; strains (Efs lacks pigmentation and is arabinose, MGP&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;were confirmed using the API 20 STREP system (Biomerieux).&lt;/span&gt;&lt;/p&gt; </td></tr><tr><td>Microscope</td><td>Motic</td><td>SMZ 168B</td><td>any microscope with transmitted illumination and 50X magnification should be sufficient</td></tr><tr><td>Streptomycin sulfate</td><td>Fisher BioReagents</td><td>BP910-50</td><td></td></tr><tr><td>Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td><td>Becton Dickinson</td><td>236950</td><td></td></tr><tr><td>Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td><td>Becton Dickinson</td><td>211768</td><td></td></tr><tr><td>Yeast extract</td><td>Acros</td><td>61180-1000</td><td></td></tr></tbody></table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

C. elegans comportamento de postura de ovos é afetado por estímulos ambientais, como a osmolaridade 1 e vibração 2. Na ausência total de alimentos C. elegans também deixará postura de ovos e manter ovos fertilizados em sua 3 útero. No entanto, o efeito de diferentes fontes de alimentos, especialmente bactérias patogénicas e, particularmente, Enterococcus faecalis, sobre o comportamento de postura não está bem caracterizado. O ensaio in ovo sem-fim (EIW) é uma ferramenta útil para quantificar os efeitos de diferentes tipos de bactérias, neste caso E. faecalis, sobre o comportamento de postura. EIW ensaios envolvem a contagem do número de ovos retidos no útero de C. elegans 4. O ensaio EIW envolve branqueamento encenado, grávida adulto C. elegans para remover a cutícula e separar os ovos retidos do animal. Antes do branqueamento, os vermes são expostos a bactérias (ou qualquer tipo de sinalização ambiental) Por um período fixo de tempo. Após o branqueamento, é muito facilmente capaz de contar o número de ovos retidos no interior do útero dos vermes. Neste ensaio, a um aumento na retenção quantificável de ovos após E. exposição faecalis pode ser facilmente medido. O ensaio EIW é um ensaio comportamental que poderia ser utilizado para rastrear para bactérias potencialmente patogénicos ou a presença de toxinas ambientais. Além disso, o ensaio EIW pode ser uma ferramenta para o rastreio de fármacos que afectam a sinalização de neurotransmissores, pois o comportamento de postura é modulada por neurotransmissores, tais como serotonina e acetilcolina 5-9.

Este ensaio permite a quantificação do número de ovos retidos no interior C. elegans após exposição a E. faecalis. L4 encenado vermes (caracterizados por espaço aberto transparente acima de sua vulva, Figura 1) foram expostos a E. faecalis até a idade adulta. Após 40 horas de exposição a E. faecalis, branqueamento foi realizado. À medida que a cutícula desintegrado na gotícula de lixívia, os ovos se tornou mais evidente (Figura 2). O n?...

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Medindo os efeitos das bactérias no comportamento de C. Elegans usando um ensaio de retenção de ovos | Biologia | Vídeo

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

Um ensaio ovo-in-worm (EIW) é um método útil para quantificar o comportamento de postura. Alterações na postura de ovos pode ser uma resposta comportamental do organismo modelo<em&gt; Caenorhabditis elegans</em&gt; A substâncias ambientais potencialmente prejudiciais, tais como os produzidos por bactérias patogénicas.

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 ...

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

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17.0K visualizações.  Fairleigh Dickinson University. Um ensaio ovo-in-worm (EIW) é um método útil para quantificar o comportamento de postura. Alterações na postura de ovos pode ser uma resposta comportamental do organismo modelo Caenorhabditis elegans A substâncias ambientais potencialmente prejudiciais, tais como os produzidos por bactérias patogénicas.

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

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

O nemátode Caenorhabditis Elegans - um modelo versátil na Vivo para estudar interações do anfitrião-micróbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genética

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

Análise automatizada de um ensaio de preferência quimiossensorial baseado na população de um nematodo

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Comportamento

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

Pesquisando métodos de baixo custo para medir a expectativa de vida e a saúde em Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

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.

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 ...

Neurociência

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 ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Medir o efeito de produtos químicos sobre o crescimento e a reprodução de Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicina

C. elegans Chemotaxis Assay

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

C. elegans Chemotaxis Assay

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

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

Biologia

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Inativação Metabólica Bacteriana em Caenorhabditis elegans Usando Paraformaldeído

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

Autor em destaque: Avançando na pesquisa de Caenorhabditis elegans usando bactérias tratadas com paraformaldeído 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

Metodologia para Inativação Metabolicamente de Bactérias para Pesquisa de Caenorhabditis elegans

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

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