Os autores gostariam de reconhecer que este trabalho foi apoiado pelas Ciências Naturais e Council of Canada (NSERC) concessão de Pesquisa de Engenharia.

<ol>
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Os autores não têm nada a revelar.

Este protocolo descreve o uso de vários tipos de microscopia de divisões celulares imagem durante meados embriogénese. Em particular, este protocolo destaca como a imagem da divisão de neuroblastos, células que podem facilitar epidérmica morfogênese. Comunicação célula-célula é importante para a formação de tecidos durante o desenvolvimento de metazoários e C. elegans é um excelente modelo para estudar a formação de tecido in vivo. Um evento que retrata muito bem a interação de teci...

Embora existam protocolos que descrevem como a divisão celular imagem no início do C. elegans embrião, este protocolo descreve como a divisão celular imagem dentro de um tecido em meados embriogênese. Um dos maiores desafios em imagem organismos durante o desenvolvimento tem sido a sua sensibilidade a fototoxicidade. No entanto, o aumento da acessibilidade a fiação microscópios confocal disco ou microscópios de campo varreram permitiu aplicações de imagem mais difundida. Ambos os sistemas utilizam lasers de estado sólido e de luz difusa, o que limita os níveis de UV que os organismos são expostos. No entanto, estandes Widefield ainda pode ser usado para geração de imagens in vivo, particularmente se eles são equipados com câmeras com alta sensibilidade (por exemplo EMCCD), controle de abertura e controle de luz (por exemplo, LEDs ou lâmpadas de mercúrio ajustável). Este protocolo descreve como usar um sistema confocal base ou um sistema de campo amplo para a divisão celular imagem dentro desenvolvimento C. elegans &lt;/ Em&gt; embriões. Como um exemplo, nós descrevemos como a imagem da divisão celular neuroblasto. Os neuroblastos podem facilitar epidérmico morfogénese fornecendo estímulos químicos ou mecânicos para as células epidérmicas sobrepostas, e fornecer um excelente exemplo da importância da comunicação intercelular para a formação dos tecidos. Caenorhabditis elegans é um organismo modelo ideal para estudos baseados microscopia devido à sua transparência e organização do tecido simples 1. Além disso, C. elegans é passível de métodos genéticos e RNAi, e uma vez que muitos dos seus genes têm homólogos humanos, ele pode ser usado para identificar os mecanismos conservadas para a formação de tecido de 2-5. Em C. elegans formação da epiderme ocorre durante o meio embriogênese, quando o embrião tem&gt; 300 células. Epidermal morfogénese consiste em várias fases principais durante os quais o embrião é colocado entre uma camada de células epidérmicas que se contraem e se estendem ao transformar o embryo de uma forma ovóide na forma alongada de um verme 6. Invólucro ventral descreve um desses eventos morfogenéticas, quando as células da epiderme ventral migrar em direcção à linha média ventral, para cobrir a superfície ventral do embrião (figura 1). Em primeiro lugar, dois pares de células anteriores localizado Leading Edge migrar para a linha média ventral, onde se aderem e se fundem com seus vizinhos contralateral 6. Esta é seguida pela migração de células de bolso posterior localizado, que formam cunha como formas criando uma bolsa ventral 6-7. O mecanismo que fecha a bolsa não é bem compreendida. Uma possibilidade é que uma estrutura contrátil actina miosina supracellular liga as células de bolso juntos em um cordão da bolsa como a moda, semelhante a cicatrização de feridas 8. Curiosamente, a migração de algumas das células de bolso é mediada por subconjuntos específicos de neuroblastos subjacentes 9 (precursores neuronais que são encontrados sob a epidermis, Figura 1B). Estudos anteriores mostraram que os neuroblastos regular a migração das células da epiderme ventral e invólucro ventral. VAB-1 (receptor de Efrina) e VAB-2 (ligando de Efrina) são altamente expressas em neuroblastos e facilitar a separação do anterior e posterior neuroblastos um do outro, e mutações em vab-1 ou vab-2 causa recinto ventral fenótipos 10-13. No entanto, as experiências mostraram que o promotor de salvamento também é necessário vab-1 nas células da epiderme ventral sobrepostas e receptores para outras pistas de orientação secretadas pelas neuroblastos são expressos nas células da epiderme ventral 9. Embora as mutações em qualquer um desses receptores causar fenótipos gabinete ventral, não está claro se os defeitos surgem devido a problemas no posicionamento neuroblasto ou devido à falha das células epidérmicas ventrais para responder a orientação cues 14. Alterando a divisão de neuroblastos without afetando sua capacidade de secretar pistas de orientação poderia lançar luz sobre o papel de neuroblastos e sua capacidade de fornecer entrada mecânica durante epidérmica morfogênese. Recentemente, verificou-se que um gene de divisão celular, ani-1 (anillin) é altamente expressa em neuroblastos (Figura 2A) e a sua exaustão faz com defeitos divisão neuroblastos. Curiosamente, estes embriões exibir fenótipos gabinete ventral (Fotopoulos, Wernike e Piękny, observações não publicadas). Anillin é necessário para a divisão celular e, particularmente, para a citocinese, a qual descreve o processo pelo qual uma célula mãe divide fisicamente em duas células filhas. Cytokinesis é impulsionada pela formação de um anel contrátil actomiosina, que precisa ser rigidamente controlado no espaço e no tempo para garantir que ele está corretamente juntamente com a segregação de cromátides irmãs. O regulador mestre de citocinese metazoan é RhoA (RHO-1 em C. elegans), uma pequena GTPase que é umctive em sua forma ligada a GTP. O GEF Ect2/ECT-2 ativa RhoA, após o qual RhoA-GTP interage com efetores downstream que formam o anel contrátil e mediam seu ingresso 15. Anillin é uma proteína do domínio de multi que se liga a RhoA, através do seu terminal C e a actina e miosina, através do seu terminal-N. Anillin é necessário para estabilizar a posição do anel contráctil, em células de mamíferos ou de Drosophila S2 16. Esgotamento Anillin provoca anéis contráteis a sofrer oscilações laterais e citocinese eventualmente falhar formando células multinucleadas 17-19. Curiosamente, apesar de C. elegans ani-1 Coordenadas actomiosina contratilidade no embrião inicial, isso não é essencial para a citocinese. No entanto, como descrito acima, é necessária uma ani-1 para neuroblastos citocinese durante meados de embriogénese (Fotopoulos, Wernike e Piękny, observações não publicadas). Falha Cytokinesis alteraria o número ea posição dos neuroblastos e poderia afetar o locção de pistas de orientação química, ou que poderiam alterar as propriedades mecânicas do tecido. Ambos os modelos destacam o papel não-autônomos de neuroblastos para o gabinete de ventral, ea importância da comunicação tecido-tecido durante o desenvolvimento embrionário. Este protocolo experimental descreve como a divisão celular durante a imagem C. elegans meados embriogênese usando microscopia de fluorescência. A maioria dos ensaios que estudam os mecanismos de divisão celular foram realizados em células individuais dentro de pratos de cultura (p. ex. HeLa ou células S2) ou em embriões precoces, com um número limitado de células (por exemplo, C. elegans embrião de uma célula, de Xenopus, ou Echinoderm embriões). No entanto, é importante também para estudar a divisão celular nos tecidos, uma vez que existem sinais externos que podem influenciar o tempo e de colocação do plano de divisão. Além disso, as células podem fornecer pistas químicas ou mecânicas para influenciar o desenvolvimento do tecido vizinhos, e é importante para entender como comunicação intercelular ajuda para formar tecidos durante o desenvolvimento.

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
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			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
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			<td height="21" style="height:21px;">Agar</td>
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			<td>For making bacteria LB agar plates</td>
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			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
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			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

Este protocolo descreve a utilização de microscopia de fluorescência para a imagem a divisão das células dentro desenvolvendo Caenorhabditis elegans embriões. Em particular, este protocolo se concentra em como a imagem dividindo neuroblastos, que são encontrados debaixo das células da epiderme e podem ser importantes para epidérmica morfogênese. Formação de tecido é crucial para o desenvolvimento metazoan e depende de estímulos externos a partir de tecidos vizinhos. C. elegans é um excelente organismo modelo para estudar a morfogênese do tecido in vivo devido à sua transparência e organização simples, fazendo com que seus tecidos fáceis de estudar via microscopia. Invólucro ventral é o processo em que a superfície ventral do embrião é coberto por uma única camada de células epiteliais. Este evento é pensado para ser facilitado pelas neuroblastos subjacentes, que fornecem sinais de orientação química de mediar a migração das células epiteliais sobre jacentes. No entanto, os neuroblastos são altamente proliferativo e também podem agircomo um substrato mecânico para as células da epiderme ventral. Estudos utilizando este protocolo experimental pode descobrir a importância da comunicação intercelular durante a formação do tecido, e pode ser utilizada para revelar o papel dos genes envolvidos na divisão celular em tecidos em desenvolvimento.

Este protocolo experimental descreve como a divisão celular imagem em C. elegans embriões durante meados embriogênese. Em particular, ela descreve como neuroblastos de imagem, o que pode facilitar epidérmica morfogênese. Epidermal morfogênese ocorre devido a uma combinação de mudanças no formato de células epidérmicas, migração e adesão, mas também depende de estímulos químicos ou mecânicos dos neuroblastos subjacentes (Figura 1B). Os neuroblastos secretam pistas de orientaç?...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Este protocolo descreve como as células em divisão imagem dentro de um tecido em Caenorhabditis elegans embriões. Embora vários protocolos descrevem como a divisão celular imagem no embrião precoce, este protocolo descreve como imagem divisão celular dentro de um tecido em desenvolvimento em meados embriogénese tardia.

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

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

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.8K visualizações.  Concordia University.  Este protocolo descreve como as células em divisão imagem dentro de um tecido em Caenorhabditis elegans embriões. Embora vários protocolos descrevem como a divisão celular imagem no embrião precoce, este protocolo descreve como imagem divisão celular dentro de um tecido em desenvolvimento em meados embriogénese tardia. 

Vídeo: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

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

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

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

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

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

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

Neurociência

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

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

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

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

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

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

In vivo Laser Axotomy in C. elegans

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

In vivo Laser Axotomy in C. elegans

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

In vivo Laser axotomia em C. elegans

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

Automated Quantification of Synaptic Fluorescence in C. elegans

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

Automated Quantification of Synaptic Fluorescence in C. elegans

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

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

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

C. elegans Tracking and Behavioral Measurement

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

C. elegans Tracking and Behavioral Measurement

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

C. elegans Acompanhamento e Medição do Comportamento

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

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

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

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

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

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

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

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Microinjeção insulina hipocampal e<em&gt; Em vivo</em&gt; Microdiálise Durante um teste de memória espacial

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Na imagem cálcio vivo Neuronal em C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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

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

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

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

In vivo de imagens de Dauer específica Neuronal Remodelação em C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

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 ...