Agradecemos a Aylin Rodan para a leitura crítica do manuscrito, e os membros do laboratório para discussão útil. Esta pesquisa é apoiada pelo NIH, R01AA019526.

1. Criando a câmara de exposição <ol><li> Cortar um plug frasco pela metade utilizando uma lâmina de barbear. Alternativamente, use bolas de algodão. </li><li> Empurrar o meia ficha frasco, ou a bola de algodão, para o fundo de um frasco de comida para fazer uma superfície lisa que as moscas não vai ficar preso dentro </li><li> Se fazer câmaras de exposição múltipla, certifique-se que o plugue do frasco e alimentos ocupam o mesmo volume em cada câmara. A marca na parede do frasco pode facilitar este processo. </li></ol> 2. Expondo as moscas <ol><li> Colete 8 voa de um sexo, idade 1-5 dias, o dia antes da exposição em frascos de alimentos regulares, sem fermento. </li><li> Classificá-los em código, de modo que o experimentador é cego para o genótipo real / cepa utilizada. </li><li> Transferência dos oito voa para as câmaras de exposição tocando. Não anestesiar. </li><li> Adicionar uma pequena quantidade de alimentos corantes, por exemplo, ácido azul 9, a 200 etanol prova para torná-lo facilmente visível. </li><li> Brasão fundo do frasco de um plug, ou bola de algodão, com 0,5 ml de etanol. </li><li> Insira o plugue de etanol para o frasco de exposição. Garantir que o álcool é voltada para o frasco, e não em direção à parede do frasco. </li><li> Iniciar um temporizador de contagem acima. </li></ol> 3. Intoxicação registro induzida por etanol <ol><li> Cada minuto após o timer é iniciado, toque na câmara de exposição no banco de reservas para surpreender as moscas e observá-los por dez segundos. </li><li> Registrar o número de moscas estacionárias para cada minuto. Moscas são estacionários se eles não podem sair de costas, permanecem em um local, ou rapidamente vibrar as asas durante toda a dez segundos período de observação. As moscas últimos são considerados estacionários porque a grande maioria destas moscas são estacionários entre os períodos de movimento rápido das asas. Moscas não são estacionários se eles podem sair as costas e ir embora e / ou até a parede do frasco. </li><li> Um ensaio de exposição termina quando quatro ou mais moscas são registrados como estacionário. </li><li> Descartar moscas e deixar o ar câmara de exposição para fora para reutilização posterior. </li></ol> 4. Determinar o tempo de sedação 50% O tempo para sedação de 50% (ST50) é o tempo após o início da exposição ao etanol, quando quatro dos oito moscas permanecem estacionários. Isto é determinado da seguinte forma: Ou: no minuto primeira medição, quando exatamente quatro moscas são estacionárias. Ou: se menos de quatro moscas (Y1) são estacionários em um ponto do tempo (X minutos), e mais de quatro (Y2) no próximo ponto de medição do tempo (minutos X +1), então linearmente interpolar o ST50 utilizando a fórmula: ST50 = (4 - Y1) / (Y2 - Y1) + X 5. Repita a exposição para determinar a tolerância Expondo as moscas <ol><li> Determinar o ST50 (wt) para uma estirpe de controlo do tipo selvagem utilizado na experiência. </li><li> Expor os grupos experimentais de moscas para o etanol, como descrito acima. </li><li> Medir a sua ST50 (1), mas continuam a expor até chegar o tempo 2x ST50 (wt). </li><li> Transferência das moscas de volta em um frasco vazio de alimentos, e segure por 4 horas. </li><li> Re-expor as moscas, e determinar o ST50 (2) da exposição 2. </li></ol> Determinar a tolerância Calcule a tolerância em% como (ST50 (2) / ST50 (1) - 1) x100. 6. Resultados representativos: Mostrados aqui, são alguns resultados representativos obtivemos com 6-8 frascos de moscas por genótipo e dose. Se o seu erro padrão é substancialmente maior, então um problema sistemático provavelmente ocorreu. Certifique-se de que o etanol enfrenta nas câmaras de exposição, e que as câmaras são o mesmo volume. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Figura 1" /> Figura 1. Curva de resposta de dose do tipo selvagem voa expostos a aumento do percentual de etanol. Oito frascos de moscas foram expostos por dose e sexo. Machos e das fêmeas mostram o ST50 mesmo em cada dose (p&gt; 0,22, 2-way ANOVA), enquanto o ST50 em doses diminui significativamente com a dose crescente de etanol (p &lt;0,001). Note que voa de superlotado garrafas pode ser substancialmente menor do que os de garrafas saudável, e esta diferença de tamanho pode afetar o ST50. Garrafa de semente, cada uma com fêmeas 15-20 postura por 1-2 dias para obter a densidade larval ótimo por garrafa. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Figura 2" /> Figura 2. Expondo voa para tanto o etanol 90%, ou 100% reproduz o anteriormente descrito etanol resistência causada pelo branco rabbit1 mutation2 (p &lt;0,001, 2-way ANOVA). <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Figura 3" /> Figura 3. Repita a exposição ao etanol a 100%, espaçadas por um intervalo de recuperação de 4 horas, causa um aumento significativo na ST50 após a segunda exposição (p &lt;0,001, t emparelhado-Teste). Note que as moscas foram expostos por 24 minutos durante a primeira exposição (correspondente a duas vezes ve nd suas ST50). Exposição para um curto período de tempo (12 minutos) não causou um aumento significativo na ST50 sobre a re-exposição (dados não mostrados).

<ol>
	<li>Bainton, R. J., Tsai, L. T. -. Y., Singh, C. S., Moore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 10, 187-194 (2000).</li><li>Rothenfluh, A., Threlkeld, R. J., Bainton, R. J., Tsai, L. T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 127, 199-211 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 19, 2126-2132 (2009).</li><li>Scholz, H., Ramond, J., Singh, C. S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 28, 261-271 (2000).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 404, 394-398 (2000).</li></ol>

Não há conflitos de interesse declarados.

Nesta apresentação, nós esboçamos uma maneira simples para determinar a intoxicação induzida por etanol com base em ensaios anteriores descritos por Bainton et al. 2000 1, e Rothenfluh et al. 2006 2. Desde sedação induzida pelo álcool está correlacionado com a preferência de beber álcool 3, este ensaio é relevante para o estudo dos transtornos por uso de álcool. O ensaio descrito baseia-se em sobressalto induzida geotaxis negativo, e como a exposição de um álcool interfere com a resposta inata. Antes de iniciar a exposição, certifique-se todas as suas moscas mostram uma resposta normal a um tal de sobressalto, isto é, depois de tocar as moscas dentro do frasco de alimentos, todas devem subir de baixo para dentro de um tempo de observação de 10 segundos. Exemplos de moscas ter reduzido geotaxis negativos incluem mutantes bang-sensível apreensão 5, e com deficiência de locomoção moscas causada por degeneração dos neurônios dopaminérgicos 6. Mesmo que o ensaio descrito é relativamente simples, duas variáveis ​​merecem a maior atenção para garantir a reprodutibilidade dos resultados. Primeiro, todos os frascos de exposição deve ser o mesmo volume. Isto inclui assegurar que o fundo do frasco contém o mesmo volume de alimentos bola + frasco ficha / algodão, bem como inserir o plug-etanol contendo a mesma distância para cada frasco. Um frasco completamente vazio pode ser utilizado para este ensaio, mas leva a valores mais ST50, diminuindo a transmissão do ensaio. Este ensaio também é adequado para determinar a quantidade de moscas tolerância desenvolver após a exposição de etanol repetir. Uma série de variáveis ​​devem ser consideradas quando se faz exposições repetidas. Primeiro, o intervalo inter-exposição é aqui descrito como 4 horas. Esta variável pode ser alterado, mas recomendamos que não seja inferior a 2 horas, para garantir a liberação de etanol a partir da primeira exposição. Ver Scholz et al. 2000 4 para a cinética de desenvolvimento do etanol de tolerância e decadência. A segunda variável a considerar é a duração da primeira exposição. Isto, obviamente, tem que ser o mesmo para o controle e as moscas experimental. No entanto, isto não pode resultar na mesma quantidade de etanol entregue a estas duas estirpes voar. Moscas sedado absorvem mais etanol do que as moscas ativo 2, portanto, uma tensão que sedates anteriores podem receber uma dose mais elevada do que uma cepa mais tarde sedativo, e assim desenvolver mais tolerância devido a uma dose inicial mais elevado. É altamente advertir o leitor ao comparar o desenvolvimento de tolerância de estirpes que mostram diferenças na ST50 inicial (1). O ensaio aqui descrito é uma ferramenta útil em determinar facilmente reproduzível e moscas respostas comportamentais ao álcool.

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a simple way to measure ethanol sensitivity in flies

Baixas doses de etanol causa voa para tornar-se hiperativas, enquanto altas doses sedativas. A sensibilidade ao etanol induzida sedação de uma cepa voar dado é correlacionado com a preferência que a tensão s etanol, e, portanto, a sedação é uma medida altamente relevantes para o estudo da genética das respostas de álcool e beber. Nós demonstramos uma forma simples para expor voa para o etanol e medir seus efeitos inebriantes. O ensaio descrevemos pode determinar a sensibilidade aguda, assim como tolerância ao etanol induzida pela exposição repetida. Ele não requer uma instalação tecnicamente envolvidos, e pode, portanto, ser aplicada em qualquer laboratório com as ferramentas básicas da cultura voar.

Watch this Scientific Journal Video about A Simple Way to Measure Ethanol Sensitivity in Flies at JoVE.com

A Simple Way to Measure Ethanol Sensitivity in Flies

Um ensaio simples para medir os efeitos sedativos do etanol na<em&gt; Drosophila</em&gt; Moscas, com base na perda do reflexo de endireitamento, é descrito.

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 ...

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Neuroscience

23.1K visualizações.  University of Texas Southwestern Medical Center. Um ensaio simples para medir os efeitos sedativos do etanol na Drosophila Moscas, com base na perda do reflexo de endireitamento, é descrito.

Vídeo: A Simple Way to Measure Ethanol Sensitivity in Flies

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Hyponeophagia: Uma Medida de ansiedade no rato

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

Neurociência

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálise de etanol durante a operante determinação auto-administração de etanol e etanol por cromatografia gasosa

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

O Flybar: Administração álcool para Moscas

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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 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 Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Uma alternativa simples para injeção estereotáxica para Brain Knockdown específico de miRNA

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Uma maneira simples para medir alterações na Recompensa comportamento de busca Usando<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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 ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).