Estos estudios fueron apoyados por becas de los Institutos Nacionales de Salud, Instituto Nacional de Alcoholismo y Abuso de Alcohol: R01AA016837 (JCB) y P20AA017828 (AGD y JCB).

1. Pasos para realizar en el día antes del ensayo <ol><li> Recogida L4 etapa gusanos a medio de crecimiento fresco nematodo placas (NGM) sembradas con un césped de OP50 E. coli, y la cultura de ellos a 20 CO / N. Cada condición de ensayo requiere 10 gusanos; recoger un exceso de gusanos para permitir la O / N pérdida de gusanos. <ol><li> Sólo los animales de ensayo que son los adultos de primer día; muchos mutantes crecen a una velocidad más lenta que la de tipo salvaje. Ajuste el tiempo de la cosecha para las cepas que tienen retrasos en el desarrollo a fin de que todos los animales analizados adultos de primer día. </li></ol></li></ol> 2. Los pasos para realizar en el día del ensayo <ol><li> Preparación para el ensayo: <ol><li> Realizar ensayos de 60 x 15 mm placas no cabezas de serie estándar NGM. Seque todas las planchas que se utilizarán a 37 ° C durante 2 horas, con tapas de descanso. Para cada ensayo experimental, utilizar cuatro NGM placas secas; Estos serán los 0 mM y mM placas de ensayo de etanol 400 y sus placas de aclimatación de acompañamiento. NOTA:El uso de placas de NGM es importante en este caso; las diferencias en la composición de las placas, en particular, su osmolaridad, pueden afectar fuertemente la respuesta a la dosis de etanol en el comportamiento, esto es debido, al menos en parte, a los cambios en la cantidad de etanol acumulado por los animales 20. Agar NGM es de 160 mOsm. </li><li> Después del secado, pesa cada una de las placas de NGM cabezas de serie para ser utilizado en el ensayo y anotar el peso. Determinar el volumen de los medios en las placas basadas en el peso de una placa de vacío. Para aproximar la conversión de peso del agar al volumen, se supone que los medios de comunicación pesa lo mismo que un volumen igual de agua. NOTA: Nuestros resultados más consistentes se han encontrado con los medios NGM que ha deshidratado suficientemente que un volumen original ml 10 tiene un volumen de post-secado entre 8.3 a 8.9 ml. Una alternativa para el tiempo de secado 2 hr es secar hasta que las placas llegan a este rango de volumen para tener en cuenta las diferencias en las incubadoras. </li><li> Derrita 4 anillos de cobre (diámetro interior de 1.6 cm) en la superficie de cada una de las placas para actuar como corrales para los diferentes genotipos o grupos de tratamiento de worms.Grasp cada anillo con fórceps, y el calor en una llama (una fuerte llama de un mechero Bunsen funciona bien) durante aproximadamente 3 seg. Inmediatamente coloque el anillo en un plato, mientras que mantiene el anillo con las pinzas para evitar que se &#39;salta&#39;. <ol><li> Asegúrese de que el anillo descansa plana contra la superficie del agar presionando hacia abajo suavemente con las pinzas en varios puntos en el anillo. Al colocar el anillo, tenga cuidado de dejar espacio para un período adicional de tres anillos. </li><li> Fundir los tres anillos adicionales de cobre en la superficie de la placa, teniendo cuidado de colocarlos lo más cerca posible, de manera que los cuatro estarán en el campo de visión de la cámara. NOTA: Hacer un buen sello con el agar es esencial para mantener los gusanos en los anillos durante el ensayo. Los anillos que se saltan alrededor en la placa son poco probable que el sello correcto con el agar ypuede dejar cicatrices en el agar, que permite a los gusanos de madriguera e interfiere con la visualización de los gusanos. </li><li> Para cada placa de ensayo preparar un plato de acompañamiento &quot;aclimatación&quot;, que debe ser seca y no cabeza de serie y no recibirán etanol. Coloque cuatro anillos de cobre en estas placas. </li></ol></li><li> Etiquetar el fondo de las placas junto a cada anillo con la cepa de gusano para ser utilizado en el anillo, teniendo cuidado de no escribir en el campo de visión del propio anillo. Coinciden con las etiquetas de la placa de ensayo con su placa de aclimatación acompaña. </li><li> Calcular la cantidad de etanol 100% a añadir a cada placa de ensayo de manera que la concentración final es de 0 mM o 400 mM de etanol (peso a volumen). Para cada experimento (n = 1), habrá un 0 mM y una placa de etanol 400 mM; placas de aclimatación no reciben etanol. Añadir el etanol, el pipeteado de que alrededor de la superficie de la placa. Utilice película de laboratorio para sellar la placa y deje que se equilibre en la mesa de laboratorio durante 2 horas. </li><li> Cuando ha transcurrido 1,5 horas, comenzar la etapa de aclimatación de ensayo, paso 2.2. </li></ol></li><li> Realizar el ensayo de locomoción: Las placas de ensayo <ol><li> Transferir cuidadosamente 10 gusanos de cada grupo experimental al centro de un anillo de cobre en la placa de aclimatación. Retire cualquier alimento que es visible en el agar en este punto, raspando suavemente con la selección gusano. Variar el orden en que los grupos experimentales se ponen en los platos entre los ensayos experimentales de modo que las mismas cepas no se ponen en los platos en el mismo orden entre los ensayos. NOTA: El objetivo es transferir los animales a la placa con cantidades mínimas de alimentos, porque si comida en el plato de aclimatación se transfiere a la placa de ensayo de los gusanos se agregarán alrededor de la comida y el efecto de la droga sobre la locomoción será oscurecido. </li><li> Tenga cuidado de no romper la superficie del agar con la selección, ya que esto permitirá que los gusanos madriguera y interrumpirá el ensayo. Incubar los gusanos a ta durante 30 min. </li><li> Asegúrese de que hay un intervalo adecuado entre las iniciaciones de cada placa. Un experimentador experimentado puede mover 40 animales, 10 / anillo de 4 anillos en &lt;1,5 min, pero cualquier intervalo de hasta 2 minutos es aceptable. El ensayo estándar tiene la grabación de películas en 10-12 minutos y 30-32 minutos de exposición para ambos 0 mM y etanol 400 mM. </li><li> Iniciar la placa de aclimatación para la exposición 0 mM y la placa de aclimatación para la exposición 400 mM aproximadamente 2 minutos y 30 segundos de separación para permitir el ahorro del primer archivo de la película antes de que debe comenzar la segunda película. </li><li> Después de que el período de aclimatación de 30 min, la transferencia de los gusanos de las placas de aclimatación a las placas de ensayo. La transferencia de los gusanos a las placas de ensayo (0 mm o etanol 400 mM) en el mismo orden en que se añadieron a la placa de aclimatación, el seguimiento de la sincronización entre las terminaciones de cada placa. Sellar la placa con película de laboratorio para minimizar la pérdida de etanol a la vaporización. </li><li> Use un movimiento de pala con un pico gusano delgado filo aplanado para recoger gusanos en la parte superior del pico aplanado. Realizar la transferencia de los gusanos de la placa de la aclimatación no cabeza de serie a la placa de ensayo y sin el uso de bacterias, que se utiliza comúnmente en la transferencia de los gusanos, ya que ayuda a pegar el gusano a la selección. NOTA: La velocidad a la que los animales se transfieren en este paso es muy importante porque los primeros gusanos en la placa estarán expuestos a etanol más largo que los últimos gusanos añadieron a la placa, y los puntos de tiempo anteriores puede mostrar algunos efectos dependientes del tiempo. Esta es la principal razón para hacer girar las cepas se colocan en un plato de primera a través de repeticiones experimentales. </li></ol></li><li> Realizar Locomotion Ensayo: El rodaje <ol><li> Use una combinación microscopio / cámara que permite la formación de imágenes simultánea de los cuatro anillos en el campo de visión (42x42 cuadrados aproximadamente 2 mm), tal como un objetivo de microscopio 0,5x, 0,8x de aumento y una cámara con un 7,4 2048x204881; m CCD pixel. </li><li> Utilice iluminación uniforme en todo el campo de visión, que ayuda en el reconocimiento de objetos en la etapa umbral de intensidad (véase más adelante). Una luz de fondo 3 &quot;x3&quot; funciona bien. </li><li> Imagen de los gusanos en una placa colocada de lado los medios de comunicación hacia arriba (de la tapa hacia abajo), lo que genera el contraste que se pierde cuando se utiliza la luz de fondo en comparación con un microscopio tradicional fuente de luz. </li><li> Preparar el software de análisis de imagen para capturar películas a intervalos de los gusanos en movimiento. Configure el software para capturar imágenes de 12 bits en escala de grises cada 1 seg, como a 2 min (120 marco) película. Para reducir el tamaño del archivo de salida, mientras que todavía conserva suficiente resolución de la imagen, usar un modo de binning 2x2 para capturar imágenes 1024x1024 píxeles. </li><li> Grabe las películas. Comienza la grabación de una película 120-marco de la primera placa (etanol 0 mM) 10 min después de los últimos gusanos se colocaron en ese plato. Guarde el archivo de película. </li><li> Registre la segunda placa (etanol 400 mM). Repita este proceso paraambas placas comenzando 30 minutos después de los últimos gusanos fueron colocados en la primera placa (etanol 0 mM) para capturar los puntos de tiempo de 30-32 min para cada exposición. NOTA: Deje tiempo suficiente para guardar los archivos de imagen después de cada sesión de captura antes de iniciar la grabación de la próxima placa para ser analizados. </li><li> Para prueba de futuro de las películas archivados y permitir que estas películas sean abiertos y analizados por otros programas de software de imágenes de código abierto de dominio público, como ImageJ 28, convertir una copia de cada película a 8 bits 256 archivos TIFF de escala de grises. NOTA: El software de análisis de imágenes se describe aquí utiliza un formato de archivo propietario. </li></ol></li><li> Análisis de películas usando software ImagePro. <ol><li> Una vez capturados, analizar las películas usando Plus software Image-Pro o su equivalente. NOTA: una variedad de otros software de seguimiento de objeto está disponible que ha sido utilizado con éxito para rastrear múltiples C. elegans de una sola vez 29 y talessoftware razonablemente podría sustituir a la descrita aquí como los pasos de identificación de objetos se basan en principios similares. El método descrito se refiere a las versiones de Image-Pro Plus software 6,0-6,3 y 7.0, las versiones más recientes del software de Image-Pro Plus puede tener pequeñas diferencias. </li><li> Analizar películas en segmentos de 2 min (120 marco). En primer lugar, aplicar un filtro a las imágenes para aplanar el fondo y mejorar el contraste de los objetos de gusano. Seleccione el filtro de la siguiente manera: Menú&gt; Proceso&gt; Filtros&gt; Accesorio&gt; Acoplar: Parámetros: Antecedentes = oscuro; Característica Ancho = 20 Pix. </li><li> Vuelva a guardar las películas después de la etapa de filtración, pero siempre conservará la película original en su forma sin filtrar. <ol><li> Analizar la locomoción de los animales en cada anillo por separado con una región circular de interés (ROI) que se coloca y dimensiona para solaparse con el anillo de cobre. Identificar y seguir a los gusanos con el Menú&gt; Medida&gt; Pista Objetos ... comando; esto nos lleva a la Tracking Dventana Tabla ata. El botón Opciones de seguimiento permite pistas específicas que deben excluirse y limitar los artefactos experimentales. </li></ol></li><li> En la ficha de seguimiento automático, utilice los siguientes parámetros: Parámetros de pista: límite de velocidad (el radio de búsqueda) = 400 m / marco, límite de aceleración = Auto, longitud total de pistas mínima = 400 m, tipo de movimiento predominante = caótica; Los objetos en parámetros pistas: Permitir pistas parciales = yes, longitud Min = 21 marcos, Seguimiento profundidad predicción = 1 marco. </li><li> Para iniciar el proceso de seguimiento, haga clic en el Buscar sus temas de forma automática botón de función para abrir el / cuadro de diálogo Opciones en medidas Conde y el cuadro de diálogo de seguimiento. Seleccione la opción Manual para la Selección de rango de intensidad en el cuadro de diálogo Contador / tamaño, que proporciona el paso del umbral importante que utiliza la intensidad en escala de grises de los píxeles de gusano para resaltar un objeto gusano para el análisis y hacer caso omiso de los antecedentes más claros píxeles. <ol><li> Ajuste el sli umbral de intensidadDers (uno para establecer el límite superior, uno para establecer el límite inferior) para crear un rango inclusivo que resalta todos los objetos oscuros. Una gama de 0-1,500 en una escala de 0-4,095 es un buen punto de partida para la sintonía fina. </li><li> Aplicar un filtro de tamaño para excluir los objetos que son más grandes y más pequeño que un solo objeto gusano, tales como el anillo de cobre y cualquier residuo en los platos. Establecer dos parámetros de tamaño para hacer esto que cubren tamaños de gusanos individuales en una variedad de posturas con la Medida&gt; Seleccionar Mediciones elemento de menú en el cuadro de diálogo Opciones Conde / Tamaño. (Rango de Área: 28,000-120,000 m 2 y la gama Perímetro: 600-2,500 micras). </li><li> Si una cepa mutante es significativamente más pequeño o más grande que otros animales en la placa a analizar, ampliar la gama de los valores de filtro para el reconocimiento de objetos para dar cabida a los diferentes tamaños de las cepas primero antes de seguimiento de los animales para que los ajustes no necesitan ser cambiados mediados de análisis. </li></ol> &lt;/ Li&gt; <li> Completar el proceso de seguimiento, haga clic en Continuar en el cuadro de diálogo de seguimiento. Comparar visualmente las pistas de salida con el progreso de cada gusano en la película para asegurar que cada gusano se representa a menos que haya una razón válida para excluir que en base a la configuración de filtros automáticos. Elimine manualmente las pistas que fueron producidos por la presencia de objetos que no son gusanos confirmado que cumplían con los criterios de filtrado de tamaño. </li><li> Utilice el software para calcular la velocidad de cada gusano entre cada fotograma (distancia recorrida por el centro de gravedad de un objeto por 1 seg entre bastidores) y mostrar la velocidad promedio para cada pista y la velocidad promedio en todas las pistas para la población de gusanos en el anillo de cobre. Considere este promedio final sea n = 1 en términos de ensayos experimentales. Exportar los datos a una hoja de cálculo para el análisis estadístico y el archivado de datos. </li><li> Una vez que los datos han sido registrados en el primer anillo en la placa, mueva el retorno de la inversión para el siguiente anillo yrepita el proceso de seguimiento, sin cambiar ninguno de los parámetros. </li><li> Calcular la velocidad relativa de la locomoción a través de: </li><li> Velocidad relativa (%) = tratado (400 mM) Velocidad / no tratada (0 mm) Velocidad x 100. NOTA: Las diferentes manipulaciones genéticas menudo alteran la tasa de locomoción basal de los animales. Con el fin de determinar el efecto del etanol sobre la velocidad de locomoción de los animales y para poder comparar estos efectos a través de diferentes genotipos o condiciones, calcular una velocidad relativa. </li></ol></li></ol>

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	<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> Am. J. Psychiatry. 156, 34-40 (1999).</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=Genetics+of+the+risk+for+alcoholism.">Genetics of the risk for alcoholism.</a> Am. J. Addict. Article Review. 9, 103-112 (2000).</li><li>Heath, A. C., 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=Genetic+differences+in+alcohol+sensitivity+and+the+inheritance+of+alcoholism+risk.">Genetic differences in alcohol sensitivity and the inheritance of alcoholism risk.</a> Psychol. Med. 29, 1069-1081 (1999).</li><li>Rodriguez, L. A., Wilson, J. R., Nagoshi, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+psychomotor+sensitivity+to+alcohol+predict+subsequent+alcohol+use.">Does psychomotor sensitivity to alcohol predict subsequent alcohol use.</a> Alcohol. Clin. Exp. Res. 17, 155-161 (1993).</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> Arch. Gen. Psychiatry. 53, 202-210 (1996).</li><li>Kalu, N., 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=Heritability+of+level+of+response+and+association+with+recent+drinking+history+in+nonalcohol-dependent+drinkers.">Heritability of level of response and association with recent drinking history in nonalcohol-dependent drinkers.</a> Alcohol. Clin. Exp. Res. 36, 522-529 (2012).</li><li>Davis, S. J., Scott, L. L., Hu, K., Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+single+residue+in+the+BK+potassium+channel+required+for+activation+by+alcohol+and+intoxication+in.">Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in.</a> C. elegans. J. Neurosci. 34, 9562-9573 (2014).</li><li>Raabe, R. C., Mathies, L. D., Davies, A. G., Bettinger, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Omega-3+Fatty+Acid+Eicosapentaenoic+Acid+Is+Required+for+Normal+Alcohol+Response+Behaviors+in+C.+elegans.">The Omega-3 Fatty Acid Eicosapentaenoic Acid Is Required for Normal Alcohol Response Behaviors in C. elegans.</a> PLoS ONE. 9, e105999 (2014).</li><li>Topper, S. M., Aguilar, S. C., Topper, V. Y., Elbel, E., Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+disinhibition+of+behaviors+in+C.+elegans.">Alcohol disinhibition of behaviors in C. elegans.</a> PLoS ONE. 9, e92965 (2014).</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+Drosophila,Caenorhabditis+elegans+and+mice.">Chloride intracellular channels modulate acute ethanol behaviors Drosophila,Caenorhabditis elegans and mice.</a> Genes Brain Behav. 11, 387-397 (2012).</li><li>Davies, A. 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=A+central+role+of+the+BK+potassium+channel+in+behavioral+responses+to+ethanol+in+C.+elegans.">A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans.</a> Cell. 115, 655-666 (2003).</li><li>Davies, A. G., Bettinger, J. C., Thiele, T. R., Judy, M. E., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+the+npr-1+gene+modifies+ethanol+responses+of+wild+strains+of+C.+elegans.">Natural variation in the npr-1 gene modifies ethanol responses of wild strains of C. elegans.</a> Neuron. 42, 731-743 (2004).</li><li>Kapfhamer, D., 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=Loss+of+RAB-3/A+in+Caenorhabditis+elegans+and+the+mouse+affects+behavioral+response+to+ethanol.">Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol.</a> Genes Brain Behav. 7, 669-676 (2008).</li><li>Speca, D. 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=Conserved+role+of+unc-79+in+ethanol+responses+in+lightweight+mutant+mice.">Conserved role of unc-79 in ethanol responses in lightweight mutant mice.</a> PLoS Genet. 6, e1001057 (2010).</li><li>Thiele, T. E., Badia-Elder, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+neuropeptide+Y+in+alcohol+intake+control:+evidence+from+human+and+animal+research.">A role for neuropeptide Y in alcohol intake control: evidence from human and animal research.</a> Physiol. Behav. 79, 95-101 (2003).</li><li>Treistman, S. N., Martin, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Channels:+mediators+and+models+for+alcohol+tolerance.">BK Channels: mediators and models for alcohol tolerance.</a> Trends Neurosci. 32, 629-637 (2009).</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> Am. J. Hum. Genet. 93, 1027-1034 (2013).</li><li>Kendler, K. 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=Genomewide+association+analysis+of+symptoms+of+alcohol+dependence+in+the+molecular+genetics+of+schizophrenia+(MGS2)+control+sample.">Genomewide association analysis of symptoms of alcohol dependence in the molecular genetics of schizophrenia (MGS2) control sample.</a> Alcohol. Clin. Exp. Res. 35, 963-975 (2011).</li><li>Schuckit, M. 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=Autosomal+linkage+analysis+for+the+level+of+response+to+alcohol.">Autosomal linkage analysis for the level of response to alcohol.</a> Alcohol. Clin. Exp. Res. 29, 1976-1982 (2005).</li><li>Alaimo, J. T., 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+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., 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=The+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, 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=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. 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=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

La neurobiología simple y herramientas genéticas disponibles en C. elegans hacer el gusano un excelente modelo en el que el estudio de las bases moleculares de los efectos del etanol sobre el comportamiento. Aquí se describe un ensayo que se ha utilizado para identificar varios mediadores moleculares y ambientales de la respuesta de comportamiento aguda a etanol 8,10,20,24,25. Este método permite la diferenciación y el examen simultáneo de dos fenotipos comportamiento de respuesta etanol difere...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

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.

Los datos representativos (Figura 1) de varios genotipos diferentes y sus controles emparejados se presentan 8,24; datos fueron escogidos específicamente que las diferencias de relieve en los animales ensayados. El grado de efecto en 10 minutos de exposición se considera la sensibilidad inicial de una cepa, que se muestra en los ejes izquierda en la figura 1B-G. Las cepas mutantes con una velocidad relativa más grande que el control en 10 min se considera que son resistent...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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 ...

an-assay-for-measuring-effects-ethanol-on-locomotion-speed

Research

JoVE Journal

Neuroscience

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

8.5K vistas.  Virginia Commonwealth University. 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. 

Video: An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

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.  ...

Investigación

Neurociencias

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Un ensayo para la permeabilidad del neuroepitelio embrionario de pez cebra

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

La<em&gt; Caenorhabditis elegans</em&gt; Sistema Modelo para el Estudio Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Métodos para el estudio de los mecanismos de acción de los fármacos antipsicóticos en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

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 ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microcámaras de agarosa para Imagen de calcio a largo plazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Las métricas para caracterizar nuevos embrionarias Alargamiento del nematodo<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

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

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

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

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

Método electrofisiológico para registrar las respuestas de voltaje intracelular<em&gt; Drosophila</em&gt; Los fotorreceptores y interneuronas a la luz Estímulos<em&gt; En Vivo</em

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UN<em&gt; Caenorhabditis elegans</em&gt; Ensayo de aversión al cobre basado en el estado nutricional

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagen radiométrica calcio de neuronas individuales en comportamiento Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...