These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

1. Day Before Assay <ol><li> Recolha voa em frascos de alimentos frescos em grupos de 11 (do mesmo sexo), sob breve (1-5 min) CO 2. </li><li> Permitir moscas para recuperar S / N em frascos de alimentos num espaço de ambiente controlado (tipicamente 25 ° C, 60% de humidade relativa, 12 horas de luz / escuridão). </li><li> Preparar a solução de (s) diluindo etanol puro (100%) em etanol purificado (≥18 mohms) de água para a concentração (s) final adequada para a experiência planeada. Permitir que a solução (s) para voltar à TA O / N. Nota: Diluição de etanol é exotérmica. </li></ol> 2. Dia de Ensaio <ol><li> Para cada frasco de moscas para ser testado, preparar (i) um frasco vazio alimentos limpo; ou seja, testes de frasco, (ii) uma nova ficha de acetato de celulose, (iii) uma rolha de silicone e (iv) 1 ml de solução de etanol (ver Tabela 3). </li><li> Prepare a sala de ensaio, ajustando a temperatura de 20-25 ° C e humidade relativa de 55-65%. </li><li> Já um outro trabalhadoratribuir um código único para cada grupo de frascos e registrar o código para mais tarde. Coloque frascos codificados com moscas na sala de testes para se aclimatar para poucos min. </li><li> Rotular frascos vazios de teste para coincidir com códigos em frascos de voar a partir de 2.3. </li><li> Construir um log de ​​teste impressa, digitando os códigos em colunas (uma coluna / frasco) em uma planilha semelhante à Tabela 1. </li><li> Usando o log de teste como um guia, organizar frascos de alimentos codificados com moscas e frascos vazios de testes em matrizes correspondentes na sala de testes. Nota: Um número máximo razoável de frascos de testar é 24 (ou seja, 6 conjuntos de 4 frascos cada). </li><li> Transferência voa de frascos de alimentos em pareados / rotulados frascos de ensaio vazio e coloque imediatamente plugs de acetato de celulose em frascos de ensaio até plugs de acetato de celulose são 2 cm abaixo dos topos dos frascos. </li><li> A partir de agora, lidar com cada linha de quatro frascos como um conjunto em escalonados intervalos de 1 min. </li><li> Para a avaliação do tempo 0, segure cada frasco individualmente wom polegar eo indicador, bater suavemente sobre a mesa três vezes para bater as moscas para o fundo do frasco, esperar 30 segundos e depois contar o número de moscas que são imóveis / morto. Grave o número de moscas mortas / imóveis para cada frasco no tempo 0 min no log de teste impresso. </li><li> Comece contador de tempo de forma contínua no tempo 0 e começar imediatamente a adição de 1 ml de etanol para plugs de acetato de celulose nos frascos para a primeira linha / set de 4 frascos. Adicionar 1 ml de etanol para as velas de acetato de celulose nos frascos a intervalos de 5 seg na ordem em que irá ser testada. Adicionar etanol para os tampões de acetato de celulose em um movimento circular de modo que o etanol é absorvido uniformemente ao longo dos tampões de acetato de celulose. Quando o etanol foi adicionado a todos os 4 frascos de ensaio no conjunto, insira um plug silicone em cada frasco para selá-lo. </li><li> Às vezes 1, 2, 3, 4 e 5 min, adicionar 1 ml de etanol para o segundo, terceiro, quarto, conjuntos, quinto e sexto de 4 frascos, respectivamente. Continue inserir rolhas de silicone apósadição de etanol para cada conjunto de 4 frascos. </li><li> Em vez de 6 min, testar o primeiro conjunto de quatro frascos de agarrar cada frasco com o polegar eo dedo indicador, batendo suavemente na mesa três vezes para bater as moscas para o fundo do frasco, de espera de 30 segundos e, em seguida, a contagem e registo do número total de moscas que estão sedados. Score voa tão sedado se (i) estar no chão do frasco, mas não andam ou (ii) se situam em suas costas com ou sem bater as asas. </li><li> Lidar com cada frasco dentro do conjunto em intervalos de 5 seg usando o cronograma na Tabela 2. </li><li> Às vezes, 7, 8, 9, 10 e 11 min, testar as segunda, terceira, quarta, quinta e sexta séries de frascos, respectivamente, como é feito com o primeiro set. </li><li> Em tempo de 12 min, testar o primeiro conjunto de 4 frascos novamente como descrito em 2.12 e continuar testando o segundo terceiro, quarto sets, quinto e sexto de frascos em 13, 14, 15, 16 e 17 min, respectivamente. </li><li> Continuar ensaio moscas como descrito em 2,12 até que todas as moscas são sedated. </li><li> Digite o número total de moscas em cada frasco no log de testes cópia impressa. Censor moscas imóveis / mortas no tempo 0 a partir do número total de moscas. </li><li> Calcular a percentagem de moscas ativas em cada tempo de ponto de avaliação e dados de enredo como% moscas ativas (eixo y) em função do tempo (eixo-x). Quantificar sedação etanol interpolando Sedação Tempo 50 valores (ST50, tempo para 50% sedação) de terceira ordem curva polinomial ou sigmoidal encaixa ou área de cálculo sob a curva. </li><li> Compilar dados de frascos decodificados e realizar análises estatísticas (por exemplo, one-way ANOVA com Bonferroni teste de comparação múltipla) conforme apropriado para o projeto experimental. </li></ol>

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The authors have nothing to disclose.

Ensaios simples que reproducibly quantificada fenótipos significativas são de grande valor para a análise do comportamento. O trabalho descrito aqui aborda vários aspectos práticos de um ensaio para medir a sensibilidade sedação etanol e tolerância rápida em Drosophila. Apesar de não ser um foco deste trabalho, análises de comportamento são facilitadas pela manutenção do ambiente e genético constante fundo para indivíduos de teste dentro de um estudo. Além disso, comparações devem tipicamente...

Transtorno de uso de álcool (AUD) é um enorme problema de saúde mundial (revisto em 1). Embora os mecanismos que conduzem o desenvolvimento de R são complexas, estas perturbações têm um importante componente genético (por exemplo, 2). O grande herdabilidade de AUD e as respostas comportamentais conservados em etanol através de muitas espécies (revisto em 3,4) têm gerado grande interesse na utilização de organismos-modelo genéticos para investigar o envolvimento de genes específicos em comportamentos relacionados com o álcool para uma melhor compreensão das bases moleculares de AUD. A mosca da fruta, Drosophila melanogaster, surgiu como um organismo modelo de liderança para explorar mecanismos genético-moleculares de comportamentos relacionados com o etanol (revisto em 3,4). Estudos em moscas têm destacado papel para várias vias de sinalização em respostas comportamentais ao etanol (revisto em 5). Curiosamente, alguns dos genes e vias que influenciam respons comportamentaises para etanol em moscas também têm sido implicados em comportamentos relacionados com etanol roedores e / ou R humano (por exemplo, 6-14). A conservação dos mecanismos de condução comportamentos relacionados com o etanol em toda a espécie, juntamente com o conjunto de ferramentas genéticas disponíveis no sistema modelo de Drosophila, ressaltam a utilidade do modelo mosca da fruta para investigar a genética de respostas comportamentais ao etanol. Sensibilidade e tolerância 15,16 (revisto em 17) ao etanol em seres humanos está associada com o desenvolvimento de AUD. Ambas as respostas comportamentais ao etanol pode ser modelado em moscas através de uma variedade de ensaios laboratoriais (revisto em 3,4). Todos os ensaios de mosca conhecidos para os autores são baseados quer dependente do tempo induzida pelo etanol sedação / ou descoordenação recuperação dependente do tempo a partir de etanol sedação. Em um artigo anterior do nosso grupo sobre a genética da sensibilidade etanol e rtolerância APID em Drosophila, um ensaio comportamental, baseada em etanol sedação induzida por vapor de moscas foi utilizado 18. Testes neste ensaio foi iniciado através da transferência de adulto vivo voa sem anestesia para esvaziar frascos alimentares, aprisionando as moscas em frascos com um tampão de acetato de celulose, adição de etanol para a parte superior (ou seja, o lado não-fly) do tampão de acetato de celulose, e selando o frasco contendo moscas, plugue de acetato de celulose e de etanol com uma rolha de silicone (ver esquema na figura S3, Referência 18). Vários frascos que representam diferentes grupos de moscas foram avaliados em paralelo, aumenta o rendimento do presente ensaio. Os frascos foram dadas um código anônimo e experimentadores ficaram cegos para grupo de tratamento para evitar viés não intencional na avaliação da sedação. Numa experiência normal, moscas em frascos foram bate-se suavemente a intervalos de 6 min e, após uma recuperação de 30 seg, o número de moscas sedados em cada frasco foi contado e Converted para moscas por cento ativos. Moscas absorvida vapor de etanol a partir do tampão de acetato de celulose de uma maneira dependente do tempo, causando aumentos progressivos em etanol interna 18 e sedação (cf referência 18 e Figura 1A e 1B neste relatório). Sedação neste ensaio foi definida operacionalmente como moscas (i) que está na ausência de curta ou (ii) que encontra-se em suas costas com ou sem bater as asas. Aqui, este ensaio de sedação etanol é descrita em detalhe, uma maior optimização operacional relevante para usá-lo é fornecido, e o ensaio é utilizado para tratar a contribuição de opções de suplementação de alimentos sobre a sensibilidade mosca sedação.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>food vials</td>
			<td>VWR</td>
			<td>89092-772</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>Flugs</td>
			<td>Genesee/flystuff.com</td>
			<td>49-102</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>silicone stopper</td>
			<td>Fisher Scientific</td>
			<td>09-704-1l</td>
			<td>#4</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Pharmaco-Aaper</td>
			<td>111000200</td>
			<td>200 proof</td>
		</tr>
	</tbody>
</table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

Transtorno de uso de álcool (AUD) é um desafio de saúde grave. Apesar de uma grande componente hereditário para AUD, alguns genes têm sido implicados de forma inequívoca na sua etiologia. A mosca da fruta, Drosophila melanogaster, é um modelo poderoso para explorar mecanismos genético-moleculares subjacentes comportamentos relacionados com o álcool e, portanto, é uma grande promessa para identificar e compreender a função dos genes que influenciam AUD. A utilização do modelo de Drosophila para estes tipos de estudos depende da disponibilidade de ensaios que medem a forma fiável respostas comportamentais ao etanol. Este relatório descreve um ensaio adequado para avaliar a sensibilidade de etanol e tolerância rápida em moscas. Sensibilidade etanol medido neste ensaio é influenciado pelo volume e concentração do etanol usado, uma variedade de manipulações genéticas previamente relatados, e também o período de tempo em que as moscas são alojados sem alimentos imediatamente antes do teste. Em contraste, s etanolensitivity medido neste ensaio não é afectado pela força de manipulação mosca, sexo das moscas, e a suplementação do meio de crescimento com antibióticos ou com levedura viva. Três métodos diferentes para quantificar a sensibilidade de etanol encontram-se descritos, todos levando a resultados essencialmente indistinguíveis sensibilidade etanol. A natureza escalável deste ensaio, combinada com a sua simplicidade global de set-up e custo relativamente baixo, tornam adequado para a análise genética de pequena escala e grande sensibilidade de etanol e tolerância rápida em Drosophila.

Os dados em bruto a partir de etanol neste ensaio de sedação são os números de moscas que são sedados como uma função do tempo de exposição ao vapor de etanol. Os dados em bruto são convertidos para as moscas por cento ativos como uma função do tempo (dados primários, as Figuras 1A, B, D - F). Sensibilidade para sedação de etanol a partir dos dados primários podem ser quantificados como sedação 50 Tempo (ST50), o tempo necessário para que 50% das moscas de se tornar s...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

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 ...

an-inexpensive-scalable-behavioral-assay-for-measuring-ethanol

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Neuroscience

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

10.2K visualizações.  Virginia Commonwealth University. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combinando Behavioral Endocrinologia e Economia Experimental: Decisão da testosterona e Social Fazendo

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

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 ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Medindo Mudanças na Tátil sensibilidade na pata traseira de ratos utilizando um aparelho eletrônico von Frey

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

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.

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 ...

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 ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Clones em um ensaio comportamental para Mechanosensation de MarcM baseada em<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método eletrofisiológico para respostas gravação intracelular de tensão<em&gt; Drosophila</em&gt; Fotorreceptores e Interneurônios à luz Estímulos<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Medir e alterando Acasalamento Drive em Male<em&gt; Drosophila melanogaster</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.