The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

Todos os protocolos de mouse estavam de acordo com os institutos nacionais de saúde e orientações foram aprovadas pelo Comitê de animal Estudos da Escola de Medicina da Universidade de Washington (St. Louis, MO). 1. Preparando a Placa e Recintos Testing <ol><li> Limpar a superfície do vidro. </li><li> Segurar a sonda termopar filamento do tipo T à superfície no meio da placa de vidro com fita adesiva de laboratório. </li><li> Colocar o animal independente sobre a placa de vidro em uma única linha ao longo do meio da placa. </li><li> Passe a sonda termopar através do cerco animal centro e ligar para o registrador de dados. Vire o registrador de dados enquanto a desativação do recurso de auto-desligamento, e anexar o registrador de dados para o computador com o cabo fornecido. <ol><li> Se a gravação da temperatura da placa durante o experimento, abra o software registrador de dados para começar a gravar temperaturas placa. </li><li> Se necessário, ajustar o software de recabo a temperatura da placa de uma vez a cada segundo. </li><li> Começar a gravar temperaturas usando o software fornecido com o registrador de dados térmico. </li></ol></li><li> Separe o recinto com pastilhas pretas para evitar a interação visual entre camundongos. </li><li> Posição espelhos por baixo do vidro de modo que a parte de baixo das caixas é visível a partir de uma posição sentada confortável. </li></ol> 2. O aquecimento / resfriamento da placa de vidro <ol><li> Preencha caixas de alumínio com água aquecida, gelo molhado, ou gelo seco e posicioná-los de forma adequada sobre a placa de vidro (pacotes de folhas de alumínio preenchidos com gelo seco também pode ser usado para resfriar o vidro; Figura 1) 2. <ol><li> Para os testes, a 30 ° C, posicione as caixas de alumínio de aproximadamente 0,25 &#39;&#39; longe dos recintos de animais (Figura 2B) 2. <ol><li> Definir um circulador de água aquecida em ambos os lados da placa de vidro. Definir o circulador para 45 - 60 ° C, e nósenviar um e-lo para preencher as caixas de alumínio com um fluxo constante de água quente (Figura 1C) 2. </li><li> Posicionar os circuladores de tal modo que a água quente a partir das caixas de alumínio escoa directamente de volta para dentro do reservatório da bomba de circulação em cada um dos lados (Figura 1C) 2. </li></ol></li><li> Para os testes à temperatura ambiente, deixar as caixas vazias (Figura 2) 2. </li><li> Para os testes a 17 ° C, posicione as caixas de aproximadamente 0,25 &#39;&#39; longe dos recintos de animais de cada lado e encher com gelo molhado (Figura 2) 2. </li><li> Para os testes a 12 ° C, posicionar as caixas de aproximadamente 1,25 &#39;&#39; distância das caixas de cada lado e encher com gelo seco (Figura 2) 2. </li><li> Para os testes, a 5 ° C, posicionar as caixas de aproximadamente 0,25 &#39;&#39; distância das caixas de cada lado e encher com gelo seco (Figura 2) 2.<ol><li> Quando arrefecer o copo com gelo seco, verifique se não há ventilação suficiente para evitar o acúmulo de CO 2 no quarto. </li></ol></li></ol></li><li> Espera para o vidro para atingir a gama de temperatura desejada. </li><li> Adicionar os ratos para os compartimentos da placa. NOTA: Um gerador de ruído branco podem ser utilizados para diminuir as perturbações de ruído. </li><li> Aguarde até que os ratos para se aclimatar. NOTA: Em nosso serviço isso leva aproximadamente 2,5 horas, mas isso pode variar significativamente com base nas condições de manipulação e de alojamento dos animais. </li><li> Manter o vidro no intervalo de temperatura desejado, garantindo que as caixas são mantidas cheias de água aquecida, gelo molhado, ou gelo seco. NOTA: Com o nosso aparelho as caixas precisam ser recarregados com gelo aproximadamente a cada 90 min. NOTA: Para a 17 ° C condição, é útil para esvaziar a maior parte da água das caixas de alumínio através dos furos de drenagem antes de enchê-lo com gelo. Isto irá estabilizar a temperatura melhor, e prevento estouro NOTA: A quantidade exacta do gelo seco irá variar sazonal, mas, em geral, mantendo as caixas de mais do que ¼ cheio ao longo de todo o comprimento da caixa, será manter a temperatura constante. </li></ol> 3. Teste o Mice em temperaturas fixas <ol><li> Do lado de fora da sala de comportamento, encher um balde de gelo sobre meio cheio de gelo seco. </li><li> Usando um martelo ou malho, esmagar o gelo seco em um pó fino. NOTA: O excesso de balde irá tornar mais difícil para esmagar completamente o gelo seco em pó. </li><li> Usando uma lâmina de navalha ou tesoura, corte a parte de cima de uma seringa de 3 ml. </li><li> Usando uma agulha de 21 G, picar 3 furos em lados opostos da seringa (total de 6 furos). NOTA: Estes furos vai diminuir a pressão gerada por sublimação, enquanto comprime o gelo seco. A seringa de corte pode ser reutilizada para vários experimentos. </li><li> Pegue na seringa, o pó de gelo seco, e um cronômetro de mão na sala de comportamento. </li><li> Encha a câmara de seringa meio cheio de pó de gelo seco. Segurar a extremidade cortada da seringa de encontro a um objecto chato, e firmemente comprimir o pó usando o êmbolo. Seja cuidadoso; o êmbolo plástico pode entortar ou quebrar com a pressão. Se isso acontecer, substitua o êmbolo de uma seringa nova. </li><li> Estender a ponta do comprimido sedimento gelo seco além da extremidade da seringa. </li><li> Ratos de teste que são completamente em repouso. <ol><li> A 30 ° C, 23 ° C e 17 ° C, os ratinhos de teste, que têm todas as 4 patas sobre o vidro e não se deslocam, mas não totalmente 19 adormecido. </li><li> A 12 ° C e 5 ° C, os ratos de teste que são em 2 patas ou 4 patas e não em movimento ou de salto. </li></ol></li><li> Usando os espelhos para segmentação, gentil mas firmemente alisar o resplendor pellet contra a superfície de vidro debaixo da pata traseira do mouse (Figura 1A) 2. Comece a mão-timer. </li><li> Parar o temporizador e remover o sedimento quando o rato afasta-se do vidro arrefecida. NOTA: O movimento de retrocesso pode ser vertical ou horizontal. <ol><li> Se o mouse se move muito rapidamente a pata e, em seguida, retorna para a superfície de refrigeração, continuar a cronometragem e estimulando até que o rato faz um movimento permanente de distância. NOTA: O nosso laboratório usa um tempo máximo de 20 segundos de estímulo para os ratos, na maioria dos casos. </li></ol></li><li> Repita este procedimento até que o teste pelo menos três valores em cada pata de cada animal são coletados. Experimentações separadas testando as patas opostos no mesmo rato por, pelo menos, 7 min, e ensaios consecutivos separados em qualquer única pata por, pelo menos, 15 min. </li><li> Se for necessário, utilizam diferentes espessuras de vidro para gerar diferentes taxas de arrefecimento (Figura 3) 1. NOTA: A taxa de arrefecimento está inversamente correlacionada com a espessura do vidro. </li></ol> 4. Testando o Ratos Durante Fria Adaptação NOTA: Este é um protocolo alternativo que permite testar como o plat de vidroe esfria, ao invés de uma vez que a placa se estabilizou e os ratos estão totalmente adaptados ao ambiente frio. <ol><li> Siga as instruções listadas na Seção 1 para configurar o aparelho. </li><li> Siga as instruções indicadas no parágrafo 3 a tomar medidas de base na RT (Figura 7A) 2. </li><li> Pré-resfriar as caixas de alumínio com gelo seco. </li><li> Uma vez que as latências de retirada da linha de base foram medidos, posicione as caixas prï¿½arrefecida na placa aproximadamente 1,25 &#39;&#39; longe dos gabinetes de ambos os lados (Figura 7A, seta rotulado como &quot;gelo seco adicionado&quot;) 2. </li><li> Siga as instruções indicadas no parágrafo 3 a tomar medidas como a placa de vidro esfria, tomando medidas o mais rápido possível. </li></ol>

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The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

Medir a capacidade de resposta fria em roedores é importante para melhorar a compreensão dos potenciais mecanismos de sensibilidade frio em seres humanos, tanto em condições normais e patológicas. The Cold Plantar Assay (CPA), originalmente desenvolvido há vários anos um, é projetado para gerar, respostas inequívocas murino comportamentais reprodutíveis a um estímulo frio entregue na RT. Mais recentes melhorias deste ensaio permitiu a medida reprodutível de sensibilidade frio a uma vasta gama de temperaturas de 2. Ambas as versões também são projetados para ser relativamente alto rendimento, e barato de usar. Uma grande quantidade de progresso tem sido feito na compreensão dos mecanismos de sensibilidade frio usando outros métodos comportamentais. Um método é o teste de evaporação de acetona, enxugando ou que envolve a pulverização acetona na pata do rato e medindo a quantidade de tempo que o rato gasta sacudindo a 3,4 pata. Infelizmente,as respostas a acetona evaporação são confundidos pela sensação molhada e o cheiro da acetona. Além disso, o estímulo frio que é aplicado no teste de acetona evaporação pode variar de acordo com a quantidade de acetona aplicada, e é difícil de quantificar. Finalmente, os ratinhos lesionados têm respostas mínimas para acetona a linha de base, o que torna impossível de medir a analgesia na ausência de hipersensibilidade com este método. Outro ensaio clássico para respostas frias é o ensaio da ponta da cauda, ​​onde a latência para retirada é medida após a cauda está imerso em 5,6 água fria. Enquanto as respostas comportamentais neste ensaio são inequívocos e o ensaio mede as respostas a uma temperatura específica, os animais devem ser imobilizados durante os testes, o que pode alterar a capacidade de resposta fria através bem descritos mecanismos analgésicos estresse induzido por 7. Outra ferramenta utilizada é o teste de placa fria, que mede o comportamentorespostas dos ratos depois de serem colocados sobre uma placa arrefecida com Peltier 8-10. Enquanto esta ferramenta fornece informações sobre as respostas de animais em temperaturas específicas, ele também tem sido usado de forma inconsistente; diferentes grupos foram medidos diferentes tipos de resposta incluindo o número de saltos 8,11, a latência a primeira resposta 8,11- 13, e o número de pata elevadores 11,13,14 com resultados muito diferentes. O teste de placa de frio é também relativamente baixa taxa de transferência uma vez que apenas um animal pode ser testada ao mesmo tempo, e necessitam de um dispositivo de Peltier caros e frágeis. O teste de preferência temperatura 2 placa é um derivado normalmente usado para o teste de placa fria, que mede a quantidade relativa de tempo em que os animais passam em 2 placas conectadas de diferentes temperaturas 9,15- 17. Outro ensaio utilizado é semelhante ao ensaio de gradiente térmico, em que a quantidade de tempo que os ratos passam em diferentes zonas de temperaturavariando entre 5 ° C e 45 ° C em uma placa de metal é medida a longo 16. Enquanto estes ensaios permitir a comparação das temperaturas, não está claro se o comportamento representa aversão temperatura ou a preferência de temperatura. Finalmente, o ensaio de placa fria dinâmica tem sido usado para medir como ratos responder às mudanças nas temperaturas ambiente 18. Este método envolve a colocação de ratos em um dispositivo Peltier RT e aumentando-a para 1 ° C enquanto mede o quanto os ratos saltar ou lamber as patas em diferentes temperaturas de placas. Enquanto isto testa como ratos se adaptar a um ambiente de arrefecimento, ele não fornece uma maneira de testar como os ratos responder a um estímulo frio na definição de uma temperatura ambiente mais fresco. Além disso, requer equipamentos caros para realizar e não fornece uma maneira de se aclimatar ratos para o equipamento de teste antes de medir a sua sensibilidade frio. Para complementar estes ensaios, o CPA testa a ACCLIMAted respostas a um estímulo frio bem definida em uma variedade de faixas de temperatura, ou durante o processo de adaptação a temperaturas ambientes frios. Pode testar-se a 14 ratinhos de cada vez com o nosso aparelho actual, com o potencial de ser barata aumentada a escala para ensaios de elevado rendimento.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

As respostas comportamentais induzidos de ratinhos a partir de 30 ° C, 23 ° C, 17 ° C, e 12 ° C são altamente reprodutível (Figura 4A) 20. A fim de medir o estímulo frio sendo gerado sob a pata traseira, os ratos foram anestesiados com uma mistura de cetamina / xilazina / acepromazina e cocktail suas patas foram assegurados no vidro na parte superior de um termopar de tipo T filamento (Figura 4B) 20. O vidro foi arrefecida ou aquecida para a faixa de teste d...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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Neuroscience

14.6K visualizações.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Vídeo: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

O Vermicelli e Testes Handling Capellini: Simple medidas quantitativas da função forepaw dexterous em ratos e camundongos

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Neurociência

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Uma maneira simples para medir a sensibilidade de etanol no Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

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 ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero Eletroporação e Explantes hemisfério inteiro: um método simples Experimental de Estudos do Desenvolvimento Cortical Precoce

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Um Ensaio Behavioral Barato, escalável para medição Ethanol Sedação sensibilidade e tolerância rápida em<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Um método para alvejar e Isolar Airway-inervação neurônios sensoriais em Ratos

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Um de uma etapa simples protocolo de dissecação para Whole mount-Preparação do Adulto<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Romance Ensaio para Fria nocicepção em<em&gt; Drosophila</em&gt; Larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Cultivo In Vivo-gosto murino astrócitos usando o protocolo AWESAM rápida, simples e barato

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...