Name: an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila
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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. Día Antes de Ensayo <ol><li> Recoger moscas en viales de alimentos frescos en grupos de 11 (solo sexo) bajo breve (1-5 min) CO 2. </li><li> Permitir que las moscas se recuperen O / N en viales de alimentos en un espacio con ambiente controlado (normalmente 25 ° C, humedad relativa del 60%, 12 horas de luz / oscuridad ciclo). </li><li> Preparar la solución de etanol (s) diluyendo puro (100%) de etanol en (≥18 MΩ) agua purificada hasta una concentración final (s) apropiado para el experimento planeado. Deje que la solución (s) para volver a RT O / N. Nota: La dilución de etanol es exotérmica. </li></ol> 2. Día de Ensayo <ol><li> Para cada vial de moscas a ensayar, preparar (i) un vial limpio y vacío de alimentos; es decir, las pruebas de vial, (ii) un nuevo tapón de acetato de celulosa, (iii) un tapón de silicona y (iv) 1 ml de solución de etanol (véase la Tabla 3). </li><li> Preparar sala de pruebas mediante el ajuste de la temperatura a 20-25 ° C y humedad relativa de 55-65%. </li><li> Haga que otro trabajadorasignar un código único a cada grupo de viales y registrar el código para más tarde. Colocar los viales codificados con moscas en sala de pruebas para aclimatarse durante unos minutos. </li><li> Etiquetar viales de ensayo vacías para que coincida con los códigos de los viales de la mosca de 2,3. </li><li> Construir un registro de las pruebas en papel mediante la introducción de los códigos en columnas (una columna / vial) en una hoja de cálculo similar a la Tabla 1. </li><li> Utilizando el registro de las pruebas como guía, organizar viales alimentos codificados con las moscas y los viales de prueba vacíos en juego matrices en la sala de pruebas. Nota: un número máximo razonable de viales para poner a prueba es de 24 (es decir, 6 juegos de 4 viales de cada uno). </li><li> Transferencia vuela desde viales de alimentos en pareados viales de ensayo vacío / etiquetados e inserte inmediatamente tapones de acetato de celulosa en viales de prueba hasta tapones de acetato de celulosa son 2 cm por debajo de la parte superior del vial. </li><li> De aquí en adelante, manejar cada fila de cuatro viales como un conjunto a intervalos escalonados min 1. </li><li> Para la evaluación de tiempo 0, sujete cada vial individual wITH pulgar y el dedo índice, toque suavemente sobre la mesa tres veces para golpear las moscas a la parte inferior del vial, espere 30 segundos y luego contar el número de moscas que son inmóviles / muerto. Anote el número de moscas inmóviles / muertas para cada vial en el tiempo 0 min en el registro de la prueba en papel. </li><li> Iniciar temporizador con cuenta de forma continua en el tiempo 0 y comenzar inmediatamente la adición de 1 ml de etanol a tapones de acetato de celulosa en los viales para la primera fila / juego de 4 viales. Añadir 1 ml de etanol para los tapones de acetato de celulosa en los viales a intervalos de 5 seg en el orden en que se pondrá a prueba. Añadir etanol para los tapones de acetato de celulosa en un movimiento circular de modo que el etanol se absorbe de manera uniforme a través de los tapones de acetato de celulosa. Cuando el etanol se ha añadido a todos los viales de prueba 4 en el conjunto, insertar un tapón de silicona en cada vial para sellarlo. </li><li> A veces 1, 2, 3, 4 y 5 minutos, añadir 1 ml de etanol para el segundo tercio cuarto, conjuntos, quinto y sexto de 4 viales, respectivamente. Continúe insertando tapones de silicona despuésadición de etanol a cada conjunto de 4 viales. </li><li> A la hora 6 minutos, probar la primera serie de 4 viales agarrando cada vial con el pulgar y el índice, golpeando suavemente sobre la mesa tres veces para golpear las moscas a la parte inferior del vial, espera 30 segundos y luego contar y registrar el número total de moscas que están sedados. Puntuación vuela como sedado si (i) se destacan en el suelo del vial, pero no caminan o (ii) se encuentran en la espalda con o sin batir sus alas. </li><li> Maneje cada vial dentro del conjunto a intervalos de 5 segundos con el horario en la Tabla 2. </li><li> A veces 7, 8, 9, 10 y 11 min, probar el segundo, tercero, cuarto, quinto y sexto juegos de viales, respectivamente, como se hizo para el primer set. </li><li> A la hora 12 min, probar la primera serie de 4 viales de nuevo como se describe en 2.12 y continuar probando la segunda tercera cuarta, conjuntos, quinto y sexto de viales a los 13, 14, 15, 16 y 17 minutos, respectivamente. </li><li> Continuar pruebas moscas como se describe en 2.12 hasta que todas las moscas son sedated. </li><li> Escriba el número total de moscas en cada vial en el registro de la prueba en papel. Censor moscas inmóviles / muertos en el tiempo 0 a partir del número total de moscas. </li><li> Calcular el porcentaje de moscas activas en cada punto de tiempo de evaluación y datos de la parcela como% moscas activos (eje y) en función del tiempo (eje x). Cuantificar la sedación etanol mediante la interpolación de Sedación Tiempo 50 valores (ST50, el tiempo a 50% sedación) de tercer orden curva polinómica o sigmoidal encaja o el cálculo del área bajo la curva. </li><li> Recopilar datos de viales decodificados y realizar análisis estadísticos (por ejemplo, un modelo lineal con Bonferroni test de comparación múltiple) según corresponda para el diseño experimental. </li></ol>

<ol>
	<li>Rehm, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+disease+and+injury+and+economic+cost+attributable+to+alcohol+use+and+alcohol-use+disorders.">Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders.</a> Lancet. 373, 2223-2233 (2009).</li><li>Prescott, C. A., Kendler, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+contributions+to+alcohol+abuse+and+dependence+in+a+population-based+sample+of+male+twins.">Genetic and environmental contributions to alcohol abuse and dependence in a population-based sample of male twins.</a> The American journal of psychiatry. 156, 34-40 (1999).</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=The+evolution+of+Drosophila+melanogaster+as+a+model+for+alcohol+research.">The evolution of Drosophila melanogaster as a model for alcohol research.</a> Annual review of neuroscience. 36, 121-138 (2013).</li><li>Scholz, H., Mustard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+Models+of+Alcoholism.">Invertebrate Models of Alcoholism.</a> Current topics in behavioral neurosciences. 13, 433-457 (2011).</li><li>Rodan, A. R., Rothenfluh, A., Reilly, M. T., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Plasticity and Genetic Variation: Insights into the Neurobiology of Alcoholism. 91, (2010).</li><li>Schumann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+and+genetic+functional+studies+identify+autism+susceptibility+candidate+2+gene+(AUTS2)+in+the+regulation+of+alcohol+consumption.">Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 7119-7124 (2011).</li><li>Corl, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Happyhour,+a+Ste20+family+kinase,+implicates+EGFR+signaling+in+ethanol-induced+behaviors.">Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.</a> Cell. 137, 949-960 (2009).</li><li>Moore, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93, 997-1007 (1998).</li><li>Scholz, H., Franz, M., 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=The+hangover+gene+defines+a+stress+pathway+required+for+ethanol+tolerance+development.">The hangover gene defines a stress pathway required for ethanol tolerance development.</a> Nature. 436, 845-847 (2005).</li><li>Riley, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+dependence+is+associated+with+the+ZNF699+gene,+a+human+locus+related+to+Drosophila+hangover,+in+the+Irish+affected+sib+pair+study+of+alcohol+dependence+(IASPSAD)+sample.">Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish affected sib pair study of alcohol dependence (IASPSAD) sample.</a> Molecular psychiatry. 11, 1025-1031 (2006).</li><li>Morozova, T. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+sensitivity+in+Drosophila:+translational+potential+of+systems+genetics.">Alcohol sensitivity in Drosophila: translational potential of systems genetics.</a> Genetics. 183, 733-745 (2009).</li><li>Ogueta, M., Cibik, O., Eltrop, R., Schneider, A., Scholz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+Adh+function+on+ethanol+preference+and+tolerance+in+adult+Drosophila+melanogaster.">The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.</a> Chemical senses. 35, 813-822 (2010).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+GWASs+and+human+protein+interaction+networks+identifies+a+gene+subnetwork+underlying+alcohol+dependence.">Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence.</a> American journal of human genetics. 93, 1027-1034 (2013).</li><li>Lind, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomewide+association+study+of+nicotine+and+alcohol+dependence+in+Australian+and+Dutch+populations.">A genomewide association study of nicotine and alcohol dependence in Australian and Dutch populations.</a> Twin Res Hum Genet. 13, 10-29 (2010).</li><li>Schuckit, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+level+of+response+to+alcohol+as+a+predictor+of+future+alcoholism.">Low level of response to alcohol as a predictor of future alcoholism.</a> The American journal of psychiatry. 151, 184-189 (1994).</li><li>Schuckit, M. A., Smith, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+8-year+follow-up+of+450+sons+of+alcoholic+and+control+subjects.">An 8-year follow-up of 450 sons of alcoholic and control subjects.</a> Archives of general psychiatry. 53, 202-210 (1996).</li><li>Tabakoff, B., Cornell, N., Hoffman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+tolerance.">Alcohol tolerance.</a> Ann Emerg Med. 15, 1005-1012 (1986).</li><li>Chan, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+Influences+of+Drosophila+white/mini-white+on+Ethanol+Sensitivity+in+Two+Different+Behavioral+Assays.">Contrasting Influences of Drosophila white/mini-white on Ethanol Sensitivity in Two Different Behavioral Assays.</a> Alcohol Clin Exp Res. 38, 1582-1593 (2014).</li><li>Eddison, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=arouser+reveals+a+role+for+synapse+number+in+the+regulation+of+ethanol+sensitivity.">arouser reveals a role for synapse number in the regulation of ethanol sensitivity.</a> Neuron. 70, 979-990 (2011).</li><li>Wen, T., Parrish, C. A., Xu, D., Wu, Q., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuropeptide+F+and+its+receptor,+NPFR1,+define+a+signaling+pathway+that+acutely+modulates+alcohol+sensitivity.">Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 2141-2146 (2005).</li><li>Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+evoked+locomotor+behavior+in+Drosophila+reveals+a+role+for+integrins+in+ethanol+sensitivity+and+rapid+ethanol+tolerance.">An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.</a> Alcohol Clin Exp Res. 33, 1794-1805 (2009).</li><li>Bhandari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channels+modulate+acute+ethanol+behaviors+in+Drosophila,+Caenorhabditis+elegans+and+mice.">Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.</a> Genes, brain, and behavior. 11, 387-397 (2012).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, 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=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li><li>Chen, J., Wang, Y., Zhang, Y., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Bacchus+reveal+a+tyramine-dependent+nuclear+regulator+for+acute+ethanol+sensitivity+in+Drosophila.">Mutations in Bacchus reveal a tyramine-dependent nuclear regulator for acute ethanol sensitivity in Drosophila.</a> Neuropharmacology. 67, 25-31 (2013).</li><li>Lasek, A. W., Giorgetti, F., Berger, K. H., Tayor, 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=Lmo+genes+regulate+behavioral+responses+to+ethanol+in+Drosophila+melanogaster+and+the+mouse.">Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse.</a> Alcohol Clin Exp Res. 35, 1600-1606 (2011).</li><li>Maples, T., Rothenfluh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+way+to+measure+ethanol+sensitivity+in+flies.">A simple way to measure ethanol sensitivity in flies.</a> J Vis Exp. , e2541 (2011).</li><li>Rothenfluh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+behavioral+responses+to+ethanol+are+regulated+by+alternate+RhoGAP18B+isoforms.">Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.</a> Cell. 127, 199-211 (2006).</li><li>Rothenfluh, A., Troutwine, B. R., Ghezzi, A., Atkinson, N. S., Nohronha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+23.">Ch. 23.</a> Neurobiology of Alcohol Dependence. 23, 467-495 (2014).</li><li>Jones, M. A., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+age-related+impairment+in+locomotor+and+other+behaviors.">Drosophila as a model for age-related impairment in locomotor and other behaviors.</a> Experimental gerontology. 46, 320-325 (2010).</li><li>Martin, I., Grotewiel, 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=Oxidative+damage+and+age-related+functional+declines.">Oxidative damage and age-related functional declines.</a> Mechanisms of ageing and development. 127, 411-413 (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=Acute+ethanol+responses+in+Drosophila+are+sexually+dimorphic.">Acute ethanol responses in Drosophila are sexually dimorphic.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 21087-21092 (2012).</li></ol>

The authors have nothing to disclose.

Ensayos sencillos que cuantifican reproducible fenotipos significativos son de gran valor para el análisis del comportamiento. El trabajo que se describe aquí aborda varios aspectos prácticos de un ensayo para medir la sensibilidad sedación etanol y rápida tolerancia en Drosophila. Aunque no es un tema central de este trabajo, análisis de comportamiento son facilitadas por el mantenimiento del medio ambiente y los antecedentes genéticos constante para los sujetos de prueba en un estudio. Además, por lo ...

Trastorno por consumo de alcohol (AUD) es un enorme problema de salud en todo el mundo (revisado en 1). Aunque los mecanismos que impulsan el desarrollo de AUD son complejos, estos trastornos tienen un componente genético importante (por ejemplo, 2). La gran heredabilidad de la AUD y las respuestas conductuales conservadas en etanol a través de muchas especies (revisado en 3,4) han generado un gran interés en el uso de organismos modelo genético para investigar la participación de genes específicos en los comportamientos relacionados con etanol hacia una mejor comprensión de las bases moleculares de AUD. La mosca de la fruta, Drosophila melanogaster, se ha convertido en un organismo líder en modelo para explorar los mecanismos moleculares genética de los comportamientos relacionados con etanol (revisado en 3,4). Los estudios en moscas han puesto de relieve los roles de varias vías de señalización en las respuestas conductuales a etanol (revisado en 5). Curiosamente, algunos de los genes y las vías que influir respons conductualeses a etanol en las moscas también se han implicado en los comportamientos relacionados con etanol de roedores y / o AUD humano (por ejemplo, 6-14). La conservación de los mecanismos de los comportamientos relacionados con la conducción de etanol a través de especies, junto con la suite de herramientas genéticas disponibles en el sistema modelo de Drosophila, ponen de relieve la utilidad del modelo de mosca de la fruta para la investigación de la genética de las respuestas de comportamiento a etanol. Sensibilidad 15,16 y la tolerancia (revisado en 17) a etanol en los seres humanos está relacionada con el desarrollo de AUD. Ambas de estas respuestas de comportamiento a etanol puede ser modelado en las moscas a través de una variedad de ensayos de laboratorio (revisado en 3,4). Todos los ensayos de moscas que se sabe que los autores se basan en cualquiera de tiempo-dependiente inducida por etanol sedación / falta de coordinación o de recuperación en función del tiempo de sedación etanol. En un artículo anterior de nuestro grupo sobre la genética de la sensibilidad de etanol y rtolerancia apid en Drosophila, se usó un ensayo de comportamiento basados ​​en etanol sedación inducida por vapor de moscas-18. Prueba en este ensayo se inició mediante la transferencia de adulto vivo vuela sin anestesia para vaciar viales de alimentos, atrapando las moscas en los viales con un tapón de acetato de celulosa, la adición de etanol a la parte superior (es decir, el lado no-fly) de la bujía de acetato de celulosa, y sellar el vial que contiene moscas, tapón de acetato de celulosa y el etanol con un tapón de silicona (véase el esquema de la Figura S3, referencia 18). Viales de múltiples que representan los diferentes grupos de moscas fueron evaluados en paralelo, aumentando el rendimiento de este ensayo. Los viales se les dio un código anónimo y experimentadores estaban cegados al grupo de tratamiento para evitar el sesgo no intencional en la evaluación de la sedación. En un experimento estándar, moscas en viales se golpeó suavemente a intervalos de 6 min y, después de una recuperación de 30 segundos, se contó y converte el número de moscas sedados en cada viald para ciento moscas activos. Las moscas absorben vapor de etanol a partir de la clavija de acetato de celulosa de una manera dependiente del tiempo, causando aumentos progresivos en etanol interno 18 y sedación (referencia cf 18 y la Figura 1A y 1B en este informe). Sedación en este ensayo se define operacionalmente como moscas (i) de pie en la ausencia de caminar o (ii) que se encuentra en la espalda con o sin batir sus alas. Aquí, este ensayo sedación etanol se describe en detalle, se proporciona una mayor optimización operativa relevante para su uso, y el ensayo se utiliza para tratar la contribución de opciones de administración de suplementos de alimentos sobre la sensibilidad a la sedación mosca.

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

Trastorno por consumo de alcohol (AUD) es un grave problema de salud. A pesar de un gran componente hereditario AUD, algunos genes se han implicado de forma inequívoca en su etiología. La mosca de la fruta, Drosophila melanogaster, es un poderoso modelo para explorar los mecanismos moleculares que subyacen a los comportamientos genéticos relacionados con el alcohol y por lo tanto representa una gran promesa para la identificación y la comprensión de la función de los genes que influyen en AUD. El uso del modelo de Drosophila para estos tipos de estudios depende de la disponibilidad de ensayos que miden de forma fiable respuestas de comportamiento a etanol. Este informe describe un ensayo adecuado para evaluar la sensibilidad de etanol y rápida tolerancia en las moscas. Sensibilidad etanol medido en este ensayo está influenciada por el volumen y la concentración de etanol utilizado, una variedad de manipulaciones genéticas se informó anteriormente, y también la longitud de tiempo que las moscas se alojan sin alimentos inmediatamente antes de la prueba. En contraste, el etanol sensitivity medido en este ensayo no se ve afectada por el vigor de la manipulación de la mosca, el sexo de las moscas, y la suplementación de medio de crecimiento con antibióticos o levaduras vivas. Tres métodos diferentes para cuantificar la sensibilidad de etanol se describen, todo lo cual lleva a los resultados de sensibilidad etanol esencialmente indistinguibles. La naturaleza escalable de este ensayo, junto con su simplicidad general de puesta a punto y relativamente de bajo costo, lo hacen adecuado para el análisis genético pequeña y gran escala de sensibilidad etanol y rápida tolerancia en Drosophila.

Los datos brutos de este ensayo sedación etanol son los números de moscas que son sedados como una función de vapor de etanol tiempo de exposición. Los datos en bruto se convierten a las moscas por ciento activos como una función del tiempo (datos primarios, las figuras 1A, B, D - F). Sensibilidad a la sedación etanol a partir de los datos primarios se puede cuantificar como la sedación Tiempo 50 (ST50), el tiempo requerido para el 50% de las moscas para convertirse en sedado o A...

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.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

10.2K vistas.  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.

Video: 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.

La combinación del comportamiento de Endocrinología Experimental y Economía: Decisión de la testosterona y sociales que

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

Investigación

Neurociencias

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.

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 ...

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.

Un método general para la evaluación de incubación de sacarosa en ratas Deseo

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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 ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Método de alto rendimiento para medir el tiempo de sedación del alcohol de la Drosophila melanogaster individual

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Comportamiento

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Flypub para estudiar la desinhibición conductual inducida por etanol y la sensibilización

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Ensayo sencillo para cuantificación de Prevención Social en<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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.

Un ensayo para medir los efectos del etanol sobre la velocidad de locomoción<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 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 ...

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

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