O trabalho foi financiado pelo NIH um prêmio Desenvolvimento Scientist Clínica K08NS002083, um Grant Instituto de Pesquisa Shervert Frazier, e uma Investigator Award NARSAD Young para Edgar A. Buttner.

1. Developmental Delay / Letalidade Ensaio: The Wild-Type (N2) e Dois Mutant (Mut1 e Mut2) Cepas são testadas em três concentrações de clozapina em 12 poços da placa <ol><li> No dia 1, 2 ml de derramar NGM médio de 10 em cada cavidade de uma placa de 12 poços (cada poço com um diâmetro de 2 cm) e deixa-se endurecer sobre o banco, à temperatura ambiente (RT) durante a noite. No mesmo dia, escolher uma colônia de bactérias Escherichia coli OP50, infectar uma garrafa de 50 ml de solução de LB e cultura-lo em um shaker 37 ° C a 220 rpm de rotação durante a noite. </li><li> No dia 2, transferir 20 ul de cultura bacteriana OP50 para o centro de cada NGM bem. Incubar as placas numa incubadora a 37 ° C, ou deixá-los à TA durante a noite para permitir que a camada de bactérias que crescem mais espessa. </li><li> Adicione uma solução de estoque 80 mM de clozapina por dissolução de clozapina em DMSO (dimetilsulfóxido) solvente. Em seguida, faça uma solução de trabalho de 80 mL com a solução estoque diluída em 1,7 mM de ácido acético based na Tabela 1. Nota: A solução de trabalho para uma droga em particular de interesse pode ser determinada fazendo uma série de provas preliminares de concentração para obter a concentração mais adequada para distinguir o tipo selvagem a partir dos mutantes. </li><li> Transferência de 80 uL de solução de trabalho clozapina na placa NGM semeado bem e agita-se a placa para permitir que a solução para distribuir uniformemente sobre a superfície. Esperar para a placa secar. Usar a placa para o ensaio, no mesmo dia ou colocá-lo em uma incubadora a 20 ° C até ser utilizado no dia seguinte. Nota: A solução de trabalho é uma suspensão do fármaco, devido à baixa solubilidade da clozapina, especialmente em concentrações mais elevadas. Portanto, vortex do tubo antes de se transferir a solução para a placa de droga, a fim de garantir que a concentração de fármaco é preciso. </li><li> Lave vermes de uma placa 3,5 centímetros contendo muitos adultos grávidas com tampão M9 e então colocá-los para baixo em um tubo de 15 mla 2.000 rpm durante 1 min. </li><li> Aspirar o sobrenadante. Adicionar 5 ml de solução de lixívia (NaOH: hipoclorito: DDH 2 O em 4:1:5) e perturbar os nematóides invertendo os tubos. </li><li> Observar os animais até a metade deles são dissolvidos em cerca de 5 min. Girar os ovos a 2.000 rpm por 1 min. </li><li> Aspirar o sobrenadante e adicionar 14 ml de tampão M9 rapidamente. </li><li> Girar os ovos a 2.000 rpm por 1 min e aspirar o sobrenadante, deixando cerca de 100 mL solução. Vortex para suspender os ovos. </li><li> Transferência de 2 ovos ul suspensa em M9 em uma placa NGM regulares para testar quantos ovos são transferidos. Ajustar o volume dos ovos em suspensão para assegurar que existem aproximadamente 30-35 ovos em cada transferência. Transferir 30-35 ovos de cada poço da placa de ensaio, e depois incubar numa incubadora a 20 ° C durante 24 hr. </li><li> No primeiro dia do ensaio, marcar o número de ovos eclodidos. Ajuste o número total de animais eclodidos para 25/well por Picking fora os vermes suplementares, se necessário. </li><li> Nos dias seguintes, observar os animais e pontuá-los a cada 24 horas. Estágio de desenvolvimento é pontuada com base no tamanho do animal, e a forma da vulva 11. O experimento dura 5-6 dias, com o efeito mais forte, tipicamente visto no terceiro ou no quarto dia. </li><li> Introduza os dados em um arquivo de Excel e calcular a percentagem de animais em cada fase de desenvolvimento para todos os dias do experimento. Gerar gráficos com a função &#39;100% Stacked Column &#39;no &#39;2-D coluna&#39; menu. </li></ol> 2. Pharyngeal Bombeamento Ensaio: Jovem Adulto Animais para o tipo selvagem e estirpes mutantes são pontuados em placas Clozapina Ensaio <ol><li> A placa de ensaio é feito seguindo o mesmo protocolo utilizado para o ensaio de atraso / letalidade desenvolvimento. </li><li> Escolha 50 animais L4 para cada estirpe de placas NGM semeadas 24 horas antes do ensaio e incubar os vermes, a 20 ° C. </li><li> Escolha 10 animais para uma assay placa também. Em seguida, escolher 10 animais para o próximo bem a cada 15-20 min. </li><li> Depois de os animais no primeiro poço foram expostas ao fármaco durante 30 minutos, iniciar o ensaio, observando bombeamento da faringe sob um microscópio de dissecção e marcar as bombas durante 20 seg 9. Uma vez bombeamento da faringe é marcado, pegar o verme para fora do prato. </li></ol>

<ol>
	<li>Donohoe, D. R., Jarvis, R. A., Weeks, K., Aamodt, E. J., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+adaptation+in+C.+elegans+produced+by+antipsychotic+drugs+requires+serotonin+and+is+associated+with+calcium+signaling+and+calcineurin+inhibition.">Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.</a> Neurosci. 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Res. 54, 361-372 (2006).</li><li>Karmacharya, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+effects+of+clozapine:+involvement+of+trace+amine+pathways+in+C.+elegans+and+M.+musculus.">Behavioral effects of clozapine: involvement of trace amine pathways in C. elegans and M. musculus.</a> Brain Res. 1393, 91-99 (2011).</li><li>Saur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans+Identifies+the+Nicotinic+Acetylcholine+Receptor+Subunit+ACR-7+as+an+Antipsychotic+Drug+Target.">A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target.</a> PLoS Genet. 9, (2013).</li><li>Avery, L., You, Y. 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Os autores declaram que não há conflito de interesse envolvidos.

Aqui, descrevemos métodos para testar os efeitos de APDs sobre o desenvolvimento e comportamento de C. elegans. DMSO ou o etanol é usado para dissolver a clozapina, uma vez que o fármaco é relativamente insolúvel em água. Porque os solventes foram relatados para afectar C. elegans biologia 12, grupos de controle de etanol-alone DMSO-alone ou são essenciais. A concentração mais elevada de DMSO utilizada nos ensaios é superior a 3%, o que não tem um efeito óbvio sobre C. elegan...

Caenorhabditis elegans é um organismo genético simples passíveis de telas genéticos frente e verso em grande escala e telas genéticos químicos. C. elegans é sensível a um amplo espectro de compostos bioactivos e, por conseguinte, tem sido usado com sucesso para definir os mecanismos de acção de uma variedade de tais compostos. Por exemplo, os compostos bioactivos estudada usando farmacogenética vermes incluem agonistas de receptor de acetilcolina (por exemplo, levamisole, nicotina, morantel, pirantel e), anestésicos (p.ex. halotano), cafeína, os inibidores da colinesterase (por exemplo, aldicarbe, Lannate, e TRICLORFOM), flúor, relacionadas-GABA compostos (por exemplo, GABA e muscimol), ivermectina, paraquat, ésteres de forbol e drogas relacionados com a serotonina (por exemplo, serotonina e imiprimine) 3. Além disso, C. elegans foi usado para telas de pequenas moléculas de grande escala, permitindo a descoberta de novos compostos bioactivoss e identificação de alvos genéticas novas 4. O C. elegans genoma inclui potencial antipsicótico (APD) alvos conservados em seres humanos, incluindo genes que codificam proteínas necessárias para a síntese de neurotransmissores e para a estrutura e função sináptica 5. Assim, C. elegans neurogenetics e métodos oferta neurobiologia para descobrir os mecanismos moleculares de ação dos APDs. Em nematóides, exposição DPA no início do desenvolvimento produz atraso do desenvolvimento, e em concentrações mais elevadas, a letalidade 2,6. Exposição APD durante a idade adulta produz fenótipos comportamentais. Por exemplo, a exposição a clozapina inibe locomoção e bombeamento da faringe e aumenta a postura de ovos 1,2,7. Atraso de desenvolvimento e letalidade induzida pela APD são fenótipos poderosas para telas genéticos químicos em larga escala. Estes fenótipos são complexa na medida em que eles provavelmente têm mais do que um basi celulares e genéticass. Portanto, tais telas genéticas são esperados para produzir uma variedade de alvos de drogas indirectos. No entanto, o nosso laboratório tem conduzido telas de genes candidatos e uma tela de RNAi de todo o genoma para supressores de atraso e a letalidade induzida por TPA de desenvolvimento e foi recuperado com sucesso genes que codificam provavelmente alvos directos, incluindo a dopamina, a insulina, e os receptores de acetilcolina nicotínicos 2,8. Telas genéticos baseados em comportamentos induzidos-APD no adulto também levaram à identificação de alvos APD novela, e agora estamos validando alvos das telas tanto de desenvolvimento e comportamentais em mamíferos 7. Assim, uma abordagem genética química invertebrados para descobrir os mecanismos moleculares de ação dos APDs parece ser viável 5,8. O C. elegans faringe é um órgão que inclui 20 neurônios, células musculares, 20 e 20 células acessórias, envolto por uma membrana basal. Semelhante ao coração dos mamíferos, a faringe é um autónomaª constantemente bombas alimentos a partir do ambiente externo 9. A inibição da taxa de bombeamento da faringe comprometendo a absorção de alimentos e, portanto, mutações ou drogas que inibem bombeamento da faringe causa atraso no desenvolvimento ou prender 9. APDs inibir a taxa de bombeamento da faringe, o que representa, em parte, por seus efeitos sobre o desenvolvimento e viabilidade 1,2. Aqui, usamos a clozapina APD atípico como um exemplo para demonstrar testes de drogas para o desenvolvimento de nematóides e da faringe de bombeamento.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

Caenorhabditis elegans é um organismo genético simples passíveis de grande escala para a frente e reverter telas genéticos e telas genéticos químicos. O C. elegans genoma inclui potencial antipsicótico (APD) alvos conservados em seres humanos, incluindo genes que codificam proteínas necessárias para a síntese de neurotransmissores e para a estrutura e função sináptica. Exposição APD produz atraso e / ou letalidade em nemátodos desenvolvimento de uma forma dependente da concentração. Estes fenótipos são causados, em parte, pela inibição induzida por TPA de bombeamento da faringe 1,2. Assim, o fenótipo de desenvolvimento tem uma base neuromuscular, tornando-o útil para estes estudos de neurolépticos. Aqui demonstramos procedimentos detalhados para testar efeitos APD sobre o desenvolvimento do nematóide e da faringe de bombeamento. Para o ensaio de desenvolvimento, os embriões são sincronizados colocado em meio de crescimento de nemátodos placas (NGM) contendo APD, e as fases de desenvolvimento de animals são, então, marcou diária. Para o ensaio de taxa de bombeamento da faringe, encenado animais adultos jovens são testados em placas NGM contendo APDs. O número de bombas da faringe por unidade de tempo é gravado, ea taxa de bombeamento é calculado. Estes ensaios podem ser utilizados para estudar muitos outros tipos de moléculas pequenas ou até mesmo moléculas grandes.

1. Atraso no desenvolvimento / resultado do ensaio de letalidade Um resultado típico para o ensaio de atraso / letalidade no desenvolvimento, é demonstrada nas Figuras 1a e 1b. Quando os animais do tipo selvagem no grupo de controle têm crescido para a fase adulta gravídico (Figura 1a), do tipo selvagem animais expostos à clozapina ainda estão nos estágios larvais jovens ou estão mortos (Figura 1b). Figura 1c mostra um ...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

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

Abordagens para testar os efeitos de drogas antipsicóticas (APDs) em Caenorhabditis elegans são demonstrados. Os ensaios estão descritos para testar efeitos de drogas sobre o desenvolvimento ea viabilidade e sobre a taxa de bombeamento da faringe. Estes métodos também são aplicáveis ​​para as experiências de farmacogenética com outras classes de drogas que APD.

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

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

methods-for-studying-mechanisms-action-antipsychotic-drugs

Research

JoVE Journal

Neuroscience

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

11.4K visualizações.  Harvard Medical School.  Abordagens para testar os efeitos de drogas antipsicóticas (APDs) em Caenorhabditis elegans são demonstrados. Os ensaios estão descritos para testar efeitos de drogas sobre o desenvolvimento ea viabilidade e sobre a taxa de bombeamento da faringe. Estes métodos também são aplicáveis ​​para as experiências de farmacogenética com outras classes de drogas que APD. 

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse No Utero Eletroporação: Transfecção Gênica Controlled Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neurociência

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers agarose para Imagem de cálcio a longo prazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

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

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

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

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

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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

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

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

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagem de cálcio Ratiometric de neurônios individuais se comportando Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Imagem de espinhas dendríticas em Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...