Damos las gracias a Aylin Rodan para la lectura crítica del manuscrito, y los miembros del laboratorio útil para el debate. Esta investigación es apoyada por el NIH, R01AA019526.

1. La creación de la cámara de exposición <ol><li> Cortar un tapón vial en la mitad de una hoja de afeitar. Como alternativa, utilice las bolas de algodón. </li><li> Empuje el tapón de media vial, o la bola de algodón, en el fondo de un frasco de comida para hacer una superficie lisa que las moscas no se queda bloqueado pulg </li><li> Si la toma de múltiples cámaras de la exposición, asegúrese de que el tapón del vial y los alimentos ocupan el mismo volumen en cada cámara. Una marca en el lado del vial puede facilitar esta tarea. </li></ol> 2. La exposición de las moscas <ol><li> Colecciona 8 moscas de un mismo sexo, la edad de 1-5 días, el día antes de la exposición en viales comida regular sin levadura. </li><li> Etiqueta en el código, de tal manera que el experimentador se desconocía el genotipo real / cepa utilizada. </li><li> La transferencia de los ocho moscas en las cámaras de la exposición tocando. No anestesiar. </li><li> Agregue una pequeña cantidad de colorantes en los alimentos, por ejemplo, ácido azul 9, a prueba de etanol 200 para que sea fácilmente visible. </li><li> Cubrir el fondo de un tapón de vial, o una bola de algodón, con 0,5 ml de etanol. </li><li> Inserte el enchufe del etanol en el vial de la exposición. Asegúrese de que el alcohol se enfrenta en el vial, y no hacia la pared del vial. </li><li> Iniciar un temporizador contando. </li></ol> 3. Registro de intoxicación inducida por el etanol <ol><li> Cada minuto después se inicia el temporizador, pulse en la cámara de exposición en el banco para asustar a las moscas y los observamos durante diez segundos. </li><li> Anote el número de moscas fijas por cada minuto. Las moscas son fijos si no pueden salir de sus espaldas, siendo en un solo lugar, o la velocidad de vibración de sus alas a lo largo de toda la observación de diez segundos de periodo. Las moscas últimos son considerados fijos porque la gran mayoría de estas moscas son estacionarias entre los períodos de rápido movimiento de las alas. Las moscas no son estacionarias, si se puede bajar la espalda y el pie y / o hasta la pared del vial. </li><li> Un ensayo de la exposición se termina cuando cuatro o más moscas se registran como estacionario. </li><li> Deseche las moscas y deja que el aire de la cámara a la exposición para su posterior reutilización. </li></ol> 4. Determinar el tiempo que el 50% de sedación El tiempo hasta el 50% de sedación (ST50) es el tiempo después del inicio de la exposición a etanol, cuando cuatro de los ocho moscas permanecen estacionarias. Esto se determina de la siguiente manera: O bien: el momento en la primera medición cuando exactamente cuatro moscas son estacionarias. O: si tiene menos de cuatro moscas (Y1) son estacionarias en un momento determinado (X minutos), y más de cuatro (Y2) en el punto de medir el tiempo siguiente (X +1 minutos), luego linealmente interpolar la ST50 con la fórmula: ST50 = (4 - Y1) / (Y2 - Y1) + X 5. Repita la exposición para determinar la tolerancia La exposición de las moscas <ol><li> Determinar el ST50 (en peso) para el control de la cepa de tipo salvaje utilizados en el experimento. </li><li> Exponer los grupos experimentales de las moscas de etanol como se describió anteriormente. </li><li> Medir su ST50 (1), pero continúan exponiendo hasta llegar a la hora 2x ST50 (en peso). </li><li> Transferencia de las moscas de nuevo en un frasco de comida vacías, y mantener durante 4 horas. </li><li> Volver a exponer las moscas, y determinar el ST50 (2) de la exposición 2. </li></ol> Determinar la tolerancia Calcular la tolerancia en% como (ST50 (2) / ST50 (1) - 1) x100. 6. Los resultados representativos: Se muestra aquí, son algunos de los resultados que obtuvimos con el representante de 6-8 viales de moscas por el genotipo y la dosis. Si el error estándar es sustancialmente mayor, entonces un problema sistemático que probablemente ocurrió. Asegúrese de que el etanol se enfrenta a las cámaras de la exposición, y que las cámaras tienen el mismo volumen. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Figura 1" /> Figura 1. Curva dosis-respuesta de tipo salvaje moscas expuestas a un porcentaje creciente de etanol. Ocho frascos de moscas fueron expuestas por dosis y el sexo. Machos y hembras muestran la ST50 mismo en cada dosis (p&gt; 0,22, 2-way ANOVA), mientras que el ST50 en todas las dosis se reduce significativamente con la dosis de etanol cada vez mayor (p &lt;0,001). Tenga en cuenta que las moscas de hacinamiento botellas pueden ser sustancialmente menores que los de las botellas de salud, y esta diferencia de tamaño puede afectar a la ST50. Semillas de cada botella con las hembras 15-20 por la que se durante 1-2 días para obtener la densidad óptima de las larvas por botella. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Figura 2" /> Figura 2. La exposición de moscas para el etanol del 90% o 100% reproduce el etanol se ha descrito anteriormente, la resistencia causada por el blanco rabbit1 mutation2 (p &lt;0,001, 2-way ANOVA). <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Figura 3" /> Figura 3. Repita la exposición a etanol al 100%, separadas por un intervalo de recuperación de 4 horas, provoca un aumento significativo en el ST50 después de la segunda exposición (p &lt;0,001, t pareada-Test). Tenga en cuenta que las moscas fueron expuestas durante 24 minutos durante la primera exposición (que corresponde al doble de su ve na ST50). La exposición de un corto período de tiempo (12 minutos) no causó un aumento significativo en ST50 en la re-exposición (datos no 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>

En esta presentación, hemos delineado un método sencillo para determinar la intoxicación inducida por el etanol basado en los ensayos anteriores se ha descrito por Bainton et al. 2000 1, y Rothenfluh et al. 2006 2. Puesto que el alcohol sedación inducida se correlaciona con la preferencia de consumo de alcohol 3, este ensayo es relevante para el estudio de trastornos por consumo de alcohol. El ensayo se describe se basa en sobresalto inducida geotaxis negativo, y como una exposición al alcohol interfiere con la respuesta innata. Antes de iniciar la exposición, asegúrese de que todas las moscas de mostrar una respuesta normal a un sobresalto, es decir, después de tocar por las moscas en el frasco de alimentos, todos ellos deben moverse hacia arriba desde el fondo dentro de un tiempo de observación de 10 segundos. Ejemplos de las moscas de haber reducido geotaxis negativos incluyen bang-sensibles cinco mutantes convulsiones y deficiencia de locomoción-moscas causada por la degeneración de las neuronas dopaminérgicas 6. A pesar de que el ensayo descrito es relativamente simple, dos variables merecen la mayor atención para asegurar resultados reproducibles. En primer lugar, todos los viales de exposición debe ser el mismo volumen. Esto incluye asegurarse de que el fondo del frasco contiene el mismo volumen de alimentos + vial plug / bola de algodón, así como de insertar el etanol que contienen la misma distancia de cada vial. Un vial completamente vacío puede utilizarse para este ensayo, sino que lo conduce a los valores ya ST50, la disminución del rendimiento de la prueba. Este ensayo también es adecuado para determinar la cantidad de moscas de la tolerancia a desarrollar en la exposición repetida de etanol. Una serie de variables deben tenerse en cuenta al hacer exposiciones repetidas. En primer lugar, el intervalo entre la exposición es descrita aquí como 4 horas. Esta variable se puede cambiar, pero se recomienda que no sea inferior a 2 horas, para asegurar la eliminación de etanol a partir de la primera exposición. Ver Scholz et al. 2000 4 de la cinética de desarrollo de etanol de la tolerancia y la decadencia. La segunda variable a considerar es la duración de la primera exposición. Esto, obviamente, tiene que ser la misma para el control de las moscas y experimental. Sin embargo, esto no puede dar lugar a la misma cantidad de etanol entregado a estas dos cepas de volar. Moscas sedado absorber más etanol que las moscas activa 2, por lo tanto, una variedad que a principios de seda pueden recibir una dosis más alta que una cepa más sedante, y así desarrollar una mayor tolerancia, debido a una dosis inicial más alta. Le advierte al lector cuando se compara el desarrollo de la tolerancia de las cepas que muestran diferencias en los primeros ST50 (1). El ensayo se describe aquí es una herramienta útil en forma fácil y reproducible para determinar las respuestas de las moscas de la conducta con el alcohol.

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a simple way to measure ethanol sensitivity in flies

Las dosis bajas de etanol causa moscas se vuelven hiperactivos, mientras que altas dosis son sedantes. La sensibilidad a la sedación inducida por el etanol de una cepa de mosca dado se correlaciona con la preferencia de etanol que la cepa s, y por lo tanto, la sedación es una medida de gran importancia para estudiar la genética de las respuestas de alcohol y la bebida. Nos demuestran una manera sencilla de exponer a las moscas de etanol y medir sus efectos intoxicantes. El ensayo se describe se puede determinar la sensibilidad aguda, así como la tolerancia del etanol inducido por la exposición repetida. No requiere una instalación técnicamente involucrados, y por lo tanto se puede aplicar en cualquier laboratorio con las herramientas básicas de la cultura volar.

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

Una prueba sencilla para medir los efectos sedantes del alcohol en<em&gt; Drosophila</em&gt; Moscas, basado en la pérdida de los reflejos, se describe.

Una manera sencilla de medir la sensibilidad de etanol en las moscas

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 vistas.  University of Texas Southwestern Medical Center. Una prueba sencilla para medir los efectos sedantes del alcohol en Drosophila Moscas, basado en la pérdida de los reflejos, se describe.

Video: 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: una medida de la ansiedad en el ratón

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 ...

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Neurociencias

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.

Microdiálisis de etanol durante etanol operante determinación y la auto-administración de etanol por cromatografía de gases

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.

La barra estabilizadora: Administración de Alcohol 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.

Cerebro Slice Biotinilación: Una<em&gt; Ex Vivo</em&gt; Enfoque medir tráfico de proteínas específicas Región membrana plasmática en adultos las neuronas

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.

Un método simple y barato para la Determinación de la sensibilidad al frío y Adaptación en ratones

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.

Un ensayo de conducta Barato, escalable para medir etanol Sedación La sensibilidad y la tolerancia rápida en<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.

Una alternativa sencilla para inyección estereotáctica para el Cerebro Derribo específico de miARN

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.

Una manera sencilla de medir alteraciones en el comportamiento de búsqueda de recompensa Uso<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.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

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).