Agradecemos a nuestros colegas en el Departamento de Biología de la Universidad de Friburgo para un debate fructífero. Damos las gracias a la existencia de Centro Bloomington de proporcionar las poblaciones de moscas. Este trabajo fue financiado por la Fundación Nacional de Ciencia de Suiza (PP00P3_123339) y la Fundación Velux para SGS

1. Cría de larvas <ol><li> Mantenga volar cepas o cruces genéticos en la cultura de masas a 25 ° C en medio de harina de maíz bajo un 12 hr luz-ciclo de 12 horas de oscuridad en un fly Incubadora equipada con luz y temporizador. </li><li> Diluir el respaldo de la levadura en agua para formar una pasta fluida (10 g de levadura respaldo diluido con 3-4 ml destilada H 2 O). Añadir una gota de la comida de harina de maíz y cubrir los viales. Deje secar durante al menos una hora para evitar las moscas adultas se pegue a la pasta de levadura. Poner una pequeña gota de levadura diluido en agua en la superficie de la comida puede mejorar la oviposición. Alternativamente panadero pasta de levadura, ácido acético al 20% (AcOH) (Sigma Aldrich, Suiza A6283-100 ML) se puede utilizar. Para esto, moje la punta de un Q-tip en la solución y se extendió sobre la superficie del alimento harina de maíz. </li><li> Ponga moscas adultas (por lo menos cuatro días de edad, pero no más de diez días) en viales de maíz alimento comida. Ponga suficientes adultos en cada vial con el fin de obtener larvas suficiente para repetir la preferenciaprobar varias veces y permitir la comparación estadística. Para cruces genéticos, por lo general utilizamos un mínimo de 20 hembras y 10.5 hombres por cada vial, pero algunas líneas necesitamos más mujeres para obtener una progenie larval suficiente. </li><li> Permitir que los adultos a poner huevos durante 12 horas y transferirlos a nuevos viales. Los adultos pueden ser transferidos por la mañana y la noche. Por lo general, transferimos los adultos de siete a diez días, y si es necesario tomar nuevas adultos. </li><li> Mantener las colecciones de huevo se toma cada 12 horas en la incubadora y hacer crecer las larvas a los 25 ° C, el ciclo de luz-oscuridad-12-hr 12-hr y 60% de humedad. Para la prueba de componentes circadianos de comportamiento visual larvas se mueven a la oscuridad constante (DD) 48 horas después de la recolección de huevos (que corresponden a dos ciclos de luz-oscuridad). Para ello, utilice una incubadora separada sin luz, pero mantener el resto de condiciones tales como la temperatura y humedad idénticas. Asegúrese de transferir los viales a la DD incubadora justo antes del cambio a la fase oscura. Antes de trasladar los frascos a Another incubadora puso los frascos en una caja de cartón o envolver los frascos con papel de aluminio para evitar la exposición a la luz durante el traslado (el embalaje en caja de cartón o embalaje tiene que ser hecho durante la &quot;luz-en fase de&quot; antes del cambio entre las incubadoras). Evitar que los animales de cualquier exposición a la luz después de este punto y hasta el inicio de los experimentos. </li><li> Después de 4 días (84 a 108 horas de la puesta de huevos) recoger temprano L3 (larvas de alimentación) en los puntos de tiempo a ensayar (ver 4. Prueba de preferencia de la Luz, punto. 4.4.). </li></ol> 2. Configuración de la prueba <ol><li> Lleve a cabo experimentos en una habitación oscura con una temperatura y humedad constantes. </li><li> Para mantener dos cuadrantes de la placa de Petri en la oscuridad, cubra la tapa con cinta negro y papel de aluminio. Con el fin de hacerlo, marcar el centro de la circunferencia de la tapa. También marcar cuatro puntos en el borde de la tapa con 90 ° de separación entre ellos. Para hacerlo más fácil, dibuja un cuadrado de 20 cm dividido en cuatro cuadrantes de 10 cm de longitud en unhoja de papel o con la ayuda de un ordenador. Cortar 10 cm cuadrados de papel de aluminio y cinta negro (Figura 1A). Cubrimos dos cuadrantes opuestos pegando un cuadrado de papel de aluminio, como primera capa y cinta negro que lo cubre, tenga cuidado de cubrir también el borde de la tapa. En el pasado se utilizaron dos formas ligeramente diferentes de ensayos de este. Estamos aquí utilizamos el ensayo &quot;cuarto de plato&quot;, en la que la placa de Petri se divide en cuatro partes iguales 10. En el ensayo alternativo &quot;half-plate&quot; la placa de Petri se divide la mitad (la mitad expuesta a la luz, medio a oscuras) 1,7,8. Hasta hoy se ha publicado ninguna diferencia significativa entre los dos ensayos. </li><li> Pegamento una hoja de cuadrante, como la utilizada para el marcado de las tapas justo debajo de la fuente de luz sobre la mesa. Marque la circunferencia de una placa de Petri centrado en la intersección cuadrantes. Use las marcas como referencia para colocar la placa de la prueba siempre en la misma posición bajo la fuente de luz. </li><li> Instale unalámpara, ya sea una bombilla de luz blanca simple (Phillips, Softtone 5W) o una lámpara de LED (lámpara de LED, 80012 Blanco, Osram) por encima de la placa de Petri con la ayuda de un soporte de apoyo de hierro (Fisher Scientific, S47808). Ajustar la altura de una manera que la placa de prueba entera se ilumina homogéneamente. Recomendamos el uso de lámparas LED, ya que emiten longitudes de onda más específicas que las bombillas convencionales. Además LED emiten menos calor. </li><li> Ajuste la intensidad de la luz con la ayuda de un fotómetro (medidor PCE Ambiente EM882). Para lograr la intensidad de luz requerida de 350 a 760 lux, mueve la lámpara hacia arriba o hacia abajo según sea necesario. Al conectar la lámpara a una fuente de alimentación ajustable proporciona mayor flexibilidad para cambiar la intensidad sin mover la lámpara (Figura 1B). </li></ol> 3. Platos Preparación <ol><li> Hacer 200 ml de 2,5% de agar (Sigma-Aldrich, Suiza A5093-500G) con agua doble destilada (Millipore). </li><li> Coloque placas de Petri en una mesa (90 mm de diámetro;Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria) en hileras para permitir verter caliente agarosa. </li><li> Hervir la agarosa en un horno de microondas hasta que la solución es completamente transparente y fluida. Asegúrese de que la solución de líquido no contiene burbujas. PRECAUCIÓN: Transporte con cuidado a la mesa ya que la solución puede ser muy caliente! </li><li> Verter caliente de agarosa en las placas de Petri hasta que toda la superficie está cubierto homogéneamente con una capa delgada de la solución, aproximadamente dos o tres milímetros son suficientes. Ser rápido para evitar agarosa solidifica antes de recubrir todas las placas. Deje que los platos se enfríen. Almacenar las placas a temperatura ambiente y el uso de ellos sólo en el mismo día de la preparación. </li></ol> 4. Prueba de Preferencia Luz <ol><li> El trabajo en condiciones de luz roja en la sala de ensayo para evitar influencia de la luz antes de la prueba, ya Drosophila no es capaz de detectar longitudes de onda de luz roja. Utilice una bombilla de luz roja (Phillips, PFE712E * 8) montado en una lámpara para iluminar tque ponga a trabajar. </li><li> Mantenga la temperatura a través de todos los experimentos a 25 ° C. Control de temperatura ambiente por medio de un dispositivo de calefacción o de refrigeración si se requiere. </li><li> Tome un poco de comida de los viales criado durante ciclos de dos días bajo constante oscuridad (de la sección 1). Dado que las larvas son generalmente cavando en la superficie de la comida, tomar la capa superior (alrededor de 5 mm de profundidad) con ayuda de una espátula (Fisher Scientific, 14 de-373-25A). Extender la comida en el lado exterior de un plato de Petri tapa, añadir un poco de agua y mezclar suavemente con la espátula. </li><li> Recoger la alimentación de larvas L3 de la comida. Como se pondrá a prueba la preferencia la luz, elegir sólo a principios / alimentación de las larvas L3 es crucial ya que el phototaxis negativo está asegurada. Tarde / vagando larvas L3 o grande larva arrastrándose sobre la comida supuestamente ya cambiaron su photobehavior. La alimentación de las larvas L3 (fototácticas negativa) puede ser reconocido por sus espiráculos anteriores son abiertas y salían al exterior en forma de dedo. El espiráculo posteriors tienen tres aberturas cada una, y cuatro grupos de grandes pelos ramificados. Las glándulas salivales se extienden hasta el segundo segmento abdominal 11. Puesta en escena larva bajo un microscopio es conveniente mientras que no la luz blanca se debe utilizar. Una lámpara de luz roja es necesario iluminar las larvas si estadificación bajo microscopio estereoscópico se requiere antes de los experimentos. </li><li> Lávese las larvas brevemente en agua del grifo y recoger principios de amamantar larvas de tercer estadio (aún en luz roja). Antes de transferir las larvas a la placa de la prueba, tome las larvas con una brocha húmeda y absorba con cuidado el exceso de agua con una toalla de papel o papel de filtro. No seque larvas demasiado excesivamente ya que podría dañar el animal e influir en su comportamiento. </li><li> Utilizando el pincel mojado transferir cuidadosamente las larvas a las placas. Colocar un grupo de aproximadamente 30 larvas en el centro de la placa. Cubrir la placa con la tapa ya se prepara con los cuadrantes y fijar la placa bajo la fuente de luz de preparado para el experimento (véase la secciónción 2). </li><li> Encienda la lámpara de luz blanca e iniciar el temporizador. Deje que la larva se mueve libremente en la placa durante 5 min, a continuación, eliminar rápidamente la tapa y contar el número de larvas en la oscuridad y en los cuadrantes de luz. Marcar la posición de cada larva con un marcador puede facilitar el conteo. Alternativamente tomar una imagen de la placa y contar a posteriori. Las larvas que muestran preferencia la luz clara, como larvas arrastrándose en las paredes o excavando en agar debe ser considerado como preferencia neutro y sólo se incluye en el número total de larvas cuando se calcula el índice de preferencia de la luz. </li><li> Después de contar, deseche la placa con larvas y sustituirla por una nueva placa de ensayo para el siguiente experimento. Recoger las placas utilizadas en una bolsa de plástico autoclave para la manipulación y eliminación posterior. </li><li> Una vez que haya realizado un número suficiente de experimentos de un nuevo genotipo puede ser probada. 10-15 ensayos por genotipo son suficientes para el análisis. </li></ol> 5. Analysi Datas <ol><li> Para mayor comodidad transferir los datos a una hoja de datos como Excel (Microsoft) o de origen (Lab Origen) en un ordenador para su posterior análisis estadístico. Disponer en una columna el número de animales en la oscuridad, en una segunda columna el número de animales en los cuadrantes de luz y en el tercio del total de los animales en la placa (incluyendo larvas &quot;neutro&quot;). </li><li> Calcular el índice de preferencia (PREF) para la oscuridad para cada experimento usando la siguiente fórmula: PREF (oscuridad) = (número de larvas en la oscuridad - número de larvas en la luz) / número total de larvas </li><li> Comparar estadísticamente un conjunto de datos con un análisis adecuado para grupos. Aquí, utilizamos una prueba de Wilcoxon para comparar estadísticamente dos grupos. Una prueba de ANOVA con-comparación múltiple de Tukey post hoc de prueba se puede hacer, si supuesto de distribución normal se cumple en un grupo de comparación múltiple. </li><li> Hacer gráficos que muestran claramente las comparaciones entre líneas y puntos temporales a lo largo del ciclo de día. We Software origen uso (Lab Origen) para probar la significación estadística y generar gráficos adecuados, pero cualquier otro software de estadística puede ser útil. </li></ol>

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La prueba de preferencia luz descrita aprovecha la photobehavior innata larval. El ensayo es fácil de establecer, permite muchas repeticiones a bajo coste y ofrece información valiosa acerca de la detección de luz y de procesamiento. El paradigma experimental permite la cuantificación relativamente rápido de la cantidad de personas prefieren la luz o la oscuridad. Tal preferencia se puede mostrar como porcentajes de crudo o también como índice de preferencia (PREF). El PREF se expresa como la diferencia de los an...

Aquí se describe un ensayo de comportamiento basado en la preferencia larval para la oscuridad (o luz). Las larvas se reacciona con una respuesta fotonegativa fuerte y estereotipado durante las etapas de alimentación (L1 a L3 temprana) 1. El ensayo está destinado a evaluar el comportamiento fotofóbico de la larva y compara la preferencia claro u oscuro de un grupo de larvas mover libremente en una placa de Petri recubiertas con agar. Este ensayo de comportamiento no sólo proporciona información sobre la sensibilidad, la integración y temporal plasticidad del sistema visual, que proporciona más pistas sobre cómo sensibilidad a la luz y su proceso es controlado por el sistema circadiano. La larva de Drosophila ojo (también llamado órgano Bowlig; BO), es el principal órgano de percepción de la luz. Cada ojo se compone de 12 fotorreceptores (PR), ocho RP expresan la sensible al verde rhodopsin6 (RH6) y cuatro rupias expresan la sensible al azul rhodopsin5 (RH5) 2,3. Además de los RP del also clase IV neuronas multidendritic, que cubren la pared del cuerpo, las larvas se han identificado para responder a las intensidades de luz nocivos 4,5. También se sabe que las neuronas marcapasos situadas en el cerebro de larvas centro expresan la proteína sensible a la luz Cryptochrome (Cry) que actúa como sensor de luz azul intrínseca del reloj dentro del cerebro 6,7. Curiosamente photophobicity de animales de tipo salvaje se muestra un componente circadiano en diferentes puntos temporales durante el transcurso del día y de la noche cuando se prueba con este ensayo. Las respuestas a la luz de forrajeo larva L3 mostraron photophobicity fuerte al amanecer y al menor photophobicity al atardecer en las pruebas de preferencia de luz-oscuridad 7. Curiosamente sólo se requieren Th5-RP para evitar la luz, mientras Rh6-RP son prescindibles. Ambos, Th5-RP y Rh6-RP están involucrados en el restablecimiento del reloj molecular por la luz 8. La vía Cry debe coordinarse con las otras vías sensibles a la luz para organizar una salida de comportamiento apropiado en eltranscurso del día. La acetilcolina en RP juega un papel esencial en la conducta de evitación luz, así como el arrastre del reloj molecular. El bloqueo de la neurotransmisión acetilcolina de RP para las neuronas marcapasos circadiano reduce la respuesta fotofóbico en el ensayo de preferencia a la luz la oscuridad 8. Empleando el mismo ensayo, dos pares simétricos de neuronas se han identificado recientemente para cambiar la preferencia la luz del tercer estadio larval de Drosophila 9. Estos dos pares de neuronas pueden estar funcionando durante las etapas finales de las larvas, cuando los animales dejan la comida para encontrar presumiblemente un sitio pupariation apropiada. Sin embargo, la cuestión de cómo las vías visuales interactuar y controlar el comportamiento visual de las larvas de manera circadiana permanece en gran medida sin respuesta. El ensayo de preferencia la luz permite hacer comparaciones entre los puntos de tiempo circadiano, vuelan las líneas y estatales circadiano en diferentes calidades de luz. El ensayo es fácil de preparar y de bajo costo y ha sido de utilidad previamente in varios laboratorios para describir y estudiar el comportamiento de la luz procedente de la larva.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Sigma-Aldrich</td>
 <td>A5093-500G</td>
 <td>2.5%; Sigma-Aldrich, 9471 Buchs, Switzerland </td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Greiner Bio-One GmbH</td>
 <td>633180</td>
 <td>90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria</td>
 </tr>
 <tr>
 <td>LEDs Lamp</td>
 <td>OSARAM </td>
 <td>80012 White</td>
 <td>LED lamp, 80012 White</td>
 </tr>
 <tr>
 <td>Environment Meter</td>
 <td>PCE</td>
 <td>PCE EM882</td>
 <td>Lux, Temp, RH%</td>
 </tr>
 <tr>
 <td>Thermostatic cabinet</td>
 <td>Aqua Lytic (Liebherr)</td>
 <td>ET636-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Light timer</td>
 <td>Timer T</td>
 <td>6185.104</td>
 <td>230V/50HZ (check specifications for your country)</td>
 </tr>
 <tr>
 <td>Universal thermostat</td>
 <td>Conrad</td>
 <td>UT200</td>
 <td></td>
 </tr>
 <tr>
 <td>Humidifier</td>
 <td>Boneco</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Balck tape</td>
 <td>Tesa</td>
 <td></td>
 <td>5 cm </td>
 </tr>
 <tr>
 <td>Glue</td>
 <td>Uhu</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lncubator lamp</td>
 <td>Phillips</td>
 <td>Softtone</td>
 <td>5W</td>
 </tr>
 <tr>
 <td>Timer clock</td>
 <td>Ziliss</td>
 <td></td>
 <td>Ziliss, Switzerland</td>
 </tr>
 <tr>
 <td>Excel Software</td>
 <td>Microsoft</td>
 <td></td>
 <td>Excel</td>
 </tr>
 <tr>
 <td>Origin Software 8.5</td>
 <td>OriginLab</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Backer Yeast</td>
 <td>Migros Switzerland</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iron support stand 17X28CM</td>
 <td>Fisher Scientific</td>
 <td>S47808</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Sigma Aldrich</td>
 <td>A6283-100ML</td>
 <td>20% acetic acid dilluted in H2O</td>
 </tr>
 <tr>
 <td>Red light lamp</td>
 <td>Phillips</td>
 <td>PFE712E*8C</td>
 <td></td>
 </tr>
 <tr>
 <td>Spatula </td>
 <td>Fisher Scientific</td>
 <td>14-373-25A</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>EA</td>
 <td>EA PS 2042-06B</td>
 <td>Optional</td>
 </tr>
 <tr>
 <td>Aluminium foil</td>
 <td>Prix Coop</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heater</td>
 <td>GOON</td>
 <td>NSB200C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microwave Oven</td>
 <td>Intertronic</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Standard corn meal fly food</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Destilled water </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

light preference assay to study innate and circadian regulated photobehavior in drosophila larvae

La luz actúa como señal ambiental para controlar el comportamiento de los animales en los distintos niveles. El sistema nervioso de Drosophila larval se utiliza como un modelo único para responder preguntas básicas sobre cómo se procesa y se comparte entre los comportamientos rápidos y circadiano información de la luz. Larvas de Drosophila mostrar una conducta de evitación estereotipo cuando se expone a la luz. Para investigar las conductas dependientes de la luz pruebas de preferencia de luz-oscuridad comparativamente simples que se pueden aplicar. En los vertebrados y artrópodos, los caminos de los nervios implicados en la detección y procesamiento de entradas visuales se superponen parcialmente con las de procesamiento de información fótica circadiano. El fascinante cuestión de cómo el sistema de detección de luz y el sistema circadiano interactúan para mantener las salidas de comportamiento coordinados sigue siendo en gran medida inexplorada. Drosophila es un modelo de impacto biológica de acercarse a estas preguntas, debido a un pequeño número de neuronas en el cerebro y la disponibilidad de herramientas genéticas para MANIPUL neuronalación. El ensayo de preferencia de luz-oscuridad presentado permite la investigación de una serie de comportamientos visuales, incluyendo control circadiano de phototaxis.

Siguiendo el protocolo descrito anteriormente, hemos probado la preferencia de luz-oscuridad a principios del tercer estadio larval de tipo salvaje Canton-S vuela en dos tiempos circadianos diferentes CT0 y CT12. Los adultos fueron criados de 12 horas de luz-oscuridad de 12 horas y se fueron a poner huevos durante 12 horas. Las larvas crecen los dos primeros días bajo el mismo régimen de luz-oscuridad. Ya que queríamos probar la modulación circadiana en condiciones constantes (funcionamiento libre del reloj circadia...

Watch this Scientific Journal Video about Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae at JoVE.com

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Aquí se describe una prueba de preferencia de luz-oscuridad de Drosophila larva. Este ensayo proporciona información acerca de la regulación circadiana innata y de la luz photobehavior detección y tratamiento.

Ensayo Preferencia luz para estudiar innata y circadiano Photobehavior Regulado de<em&gt; Drosophila</em&gt; Las larvas

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

light-preference-assay-to-study-innate-circadian-regulated

Research

JoVE Journal

Neuroscience

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

12.8K vistas.  University of Fribourg. Aquí se describe una prueba de preferencia de luz-oscuridad de Drosophila larva. Este ensayo proporciona información acerca de la regulación circadiana innata y de la luz photobehavior detección y tratamiento.

Video: Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Láser microdisección de captura de Drosophila Neuronas periféricas

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Investigación

Neurociencias

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Ensayo Actividad locomotora para el Estudio de los ritmos circadianos y del sueño en los parámetros Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Medición de las respuestas de luz circadianos y aguda en ratones con actividad rueda para correr

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Métodos de ensayo a Drosophila Comportamiento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Métodos locales y globales de evaluación de nocicepción térmica en<em&gt; Drosophila</em&gt; Las larvas

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualización de los propioceptores<em&gt; Drosophila</em&gt; Las larvas y pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Apetitivo aprendizaje asociativo olfativa en<em&gt; Drosophila</em&gt; Las larvas

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

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, ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Ensayo Preferencia gusto por adulto<em&gt; Drosophila</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Ensayo novedoso para la nocicepción en frío<em&gt; Drosophila</em&gt; Las larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Ensayo Preferencia luz para estudiar innata y circadiano Photobehavior Regulado de

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Capítulos en este video

Láser microdisección de captura de Drosophila Neuronas periféricas

Ensayo Actividad locomotora para el Estudio de los ritmos circadianos y del sueño en los parámetros Drosophila

Medición de las respuestas de luz circadianos y aguda en ratones con actividad rueda para correr

Métodos de ensayo a Drosophila Comportamiento

Métodos locales y globales de evaluación de nocicepción térmica en

Visualización de los propioceptores

Apetitivo aprendizaje asociativo olfativa en

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

Ensayo Preferencia gusto por adulto

Ensayo novedoso para la nocicepción en frío