We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1. Equipos y Reactivos de creación propia (véase lista de los demás) <ol><li> Preparar un aparato de anestesia frío Drosophila para realizar el trabajo con mosca. <ol><li> Cortar una hoja de polietileno poroso para cubrir una pequeña caja de plástico (12,7 cm de largo, 10,2 cm de ancho), típicamente la cubierta superior de una caja de puntas de pipeta (12,7 cm de largo, 10,2 cm de ancho, 3,8 cm de profundidad). </li><li> Llene la caja con hielo picado, cubrir con la lámina de polietileno poroso. </li></ol></li><li> Preparar un aparato de laberinto en T; adaptar el aparato descrito anteriormente en detalles 28-31. </li><li> Preparar un aspirador de mosca como se describe 32. </li><li> Preparar un mecanismo de respuesta phototaxis para desplazar moscas dentro y fuera de viales de prueba sin estrés: <ol><li> Utilice un aparato de contracorriente, como se describe por 29,33. </li></ol></li></ol> NOTA: El laberinto en T también puede utilizarse para otros fines, como se describe por 28. <ol start="5"><li> Asegurar homcondiciones de iluminación ogeneous: realizar el experimento en un banco cubierto con una tapa banquillo blanco, y delante de una pizarra blanca. NOTA: Para estos experimentos, las cepas de moscas Drosophila melanogaster utilizados fueron tipos silvestres: Canton-S, Oregon y Samarcanda vuela desde nuestras reservas de laboratorio 34; Elwood moscas se recolectaron en el otoño de 2011 en el barrio de Elwood de Huntington, en Long Island, Nueva York, EE.UU.. </li></ol> 2. Preparación de las moscas antes de que el experimento 1-2 días antes de realizar el experimento: <ol><li> Mantener moscas en medio de harina de maíz-agar-melaza-levadura estándar a 25 ° C en un 12 horas de luz / oscuridad ciclo. </li><li> Recoger las moscas de respuesta bajo anestesia frías 1-2 días antes de realizar el experimento <ol><li> Transferir las moscas de las botellas en las que han sido planteadas en 50 ml tubos Falcon, usando un embudo. </li><li> Coloque el tubo Falcon con las moscas bajo el nivel del hielo, en uncubo de hielo aislado. </li><li> Coloque el aparato de anestesia fría con hielo picado a -4 ° C para relajarse. </li><li> Espere 5 minutos para que las moscas entren en un estado de coma escalofrío. </li><li> Transferencia de las moscas sobre la hoja de polietileno poroso del aparato de anestesia frío. </li><li> Recoge las moscas de respuesta sobre el aparato de anestesia frío. A temperatura ambiente, bajo un microscopio estereoscópico, separar 3-7 días de edad de sexo masculino de moscas hembras y mantener estas moscas en grupos de 40. </li></ol></li><li> Recoge las moscas emisor 1-2 días antes de realizar el experimento. <ol><li> Recoger suavemente Canton-S de ambos sexos vuela de sus botellas, utilizando un aspirador bucal, controlando su número. </li><li> Mantener estas moscas en el grupo de 60 a 100, ya que serán los emisores. </li><li> Preparar tantas muestras de emisores como puntos de datos necesarios. </li></ol></li><li> Deje al menos una noche de recuperación después de la recolección de los respondedores y emisores, para minimizar cualquier efecto de confusión de la anestesia fríoy la boca de recogida aspirador en el comportamiento. </li><li> Abra la bolsa de viales de prueba, para permitir el aire de plástico con olor a rancio en la bolsa para ser reemplazado por aire fresco, por lo menos 1 día antes del experimento. </li></ol> 2 hr antes de realizar el experimento: <ol start="6"><li> Asegúrese de que la temperatura de la habitación en la que se realiza el experimento es de alrededor de 23 a 25 ° C, incluso con la luz y la humedad por encima del 30%. </li><li> La transferencia de los respondedores y moscas de la fruta emisores en viales de alimentos frescos para asegurar que estas moscas no están hambrientos. </li><li> Deje que las moscas de la fruta se ajustan al medio ambiente durante 2 horas, en el banco en el que se llevará a cabo el experimento. </li></ol> 3. Realizar el Experimento Prevención Social <ol><li> Realizar cada experimento en el mismo momento del día, en un rango de 3 a 4 horas de la tarde entre el tiempo Zeitgeber ZT5 y ZT9. </li><li> Coloque el laberinto en T en el teclado de golpes, y apretar la pinza tornillo para tél ascensor es estable. </li><li> Transferir las moscas de respuesta en un vial de la prueba fresca. Ajustar este vial que contiene las moscas de respuesta sobre la parte superior de la T-laberinto. </li><li> Incline el aparato T-laberinto y golpecitos sobre el tapete golpeando por lo que las moscas de la fruta caen en el ascensor. </li><li> Mover la parte superior del elevador hacia abajo de modo que las moscas son entre la sección superior e inferior de la T-laberinto, pero prestar atención a mantener el ascensor encima del punto de elección - para evitar que las moscas escapen. </li><li> Obtener los viales con la DSO (véase la Sección 5 para generar DSO libre viales que fueron ocupados previamente por las moscas). <ol><li> Mientras que las moscas de respuesta se están adaptando a este nuevo entorno, coloque las moscas de emisor en un vial de la prueba. </li><li> Coloque un pedazo de algodón para cerrar el vial por lo que las moscas de la fruta no se escapen. </li><li> Mecánicamente agitar este vial en un mini-vórtice de la siguiente manera: vórtice durante 15 segundos, retire el vial del vórtice durante 5 segundos. Repita 3 veces para un total de 55 seg. </li><li> Remove las moscas emisor del frasco fresco - mediante la transferencia en un vial de alimentos - y rápidamente colocar este vial lleno DSO en uno de los dos lados del aparato T-laberinto. </li><li> Alterne el lado de colocación para el vial DSO lleno en cada nueva ejecución. </li></ol></li><li> Colocar un vial de ensayo fresco en el otro lado de la T-laberinto. </li><li> Traiga el ascensor hasta el fondo, por lo que las moscas de la fruta pueden elegir entre el vial de la prueba fresca y el vial de la prueba con DSO, e iniciar el temporizador. </li><li> Deje que las moscas eligen entre el vial y el vial fresco DSO, durante 1 min, a menos que se indique lo contrario. </li><li> Después de 1 min, mover el ascensor hasta separar las moscas en el vial nuevo, DSO vial, y aquellos atrapados en el ascensor. </li><li> Cuente el número de moscas en cada vial, y en el ascensor. </li><li> Repita esto para cada genotipo / condición. </li></ol> 4. Análisis de los datos evitación social <ol><li> Cuente el número de moscas de la fruta en cada vial para cada genotipo, y transferir los datos a una hoja de cálculo. </li><li> Utilizando un programa de hoja de cálculo, calcular el Índice de Rendimiento (PI) para cada genotipo restando el número de moscas en el vial DSO partir del número de moscas en el vial DSO-libre, y luego dividiendo por el número total de moscas. </li><li> Comparación de las IP medios utilizando software de análisis estadístico para el análisis de los resultados. <ol><li> Lo ideal es preparar 2-3 repeticiones internas para cada experimento, y 3 repeticiones independientes, realizadas en días diferentes, con diferentes botellas o cruza 30. </li><li> Utilice pruebas estadísticas distribución gaussiana, como los datos siguen una distribución normal. </li><li> Al probar un efecto (es decir, la densidad de - la Figura 2), utilizar un ANOVA de una vía, con post-test para la comparación múltiple (columna por columna), o una simple prueba t para comparar cada resultado a un control. </li><li> Cuando se llevan a cabo experimentos de grupo, prueba ambas condiciones y géneros (viales vacíos, subrayó und moscas un-estresado; y el efecto de género - la Figura 1), utilizando un ANOVA de dos vías. </li></ol></li></ol> 5. Generar viales previamente ocupado por las moscas no acentuadas - Realizar En lugar de 03.05 a 03.06 <ol><li> Utilice phototaxis para preparar viales ocupados por las moscas que no han sido estresadas, como control. </li><li> Transferir las moscas en un vial de la prueba, y que encaje dentro del aparato en contracorriente, con viales vacíos frescas en la posición opuesta. </li><li> Coloque el aparato en posición horizontal con el vial distal directamente delante de una luz blanca fría 15 W fluorescentes, y cubrir con un paño negro. </li><li> Deje que las moscas se mueven suavemente en el vial fresco. Esto puede tardar varios minutos. Una vez que la mayoría de las moscas están caminando hacia la luz en el vial distal, iniciar el temporizador durante 1 min. </li><li> Retire suavemente el paño negro, cambiar la ubicación de la luz, recuperarse con el paño negro, y permitir que las moscas a abandonar el vial de la prueba. </li><li> En ese momento, initiate el experimento de elección como en 3.2 a 3.5. Salta 3.6 y 3.7. </li><li> Mueva el control deslizante del aparato, para separar las moscas que liberar a partir del vial de prueba desde el otro lado. </li><li> Utilice el vial de la prueba que dejó vacante en el laberinto en T de inmediato para el experimento de elección, como en 3.7. </li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo describe un procedimiento detallado para el ensayo de evitación social. Canton-S sólo evitará un vial en el que vuela han sido previamente tensionados mecánicamente, y que el sexo y el número de pacientes que respondieron no afecta que el rendimiento de evitación social. Sin embargo, los antecedentes genéticos de los respondedores tiene una gran influencia. Los siguientes son varios pasos críticos para llevar a cabo este experimento con éxito: 1) siempre transferir la...

El objetivo de este método es cuantificar fácilmente un nuevo aspecto de la conducta social sencilla en Drosophila melanogaster, independiente desde el cortejo y la agresión. Las interacciones sociales son cruciales para el buen desarrollo y la salud de los individuos dentro de una sociedad, así como la funcionalidad de un grupo social en su conjunto. La alta complejidad de estas interacciones requiere grandes tamaños de muestra y un sistema que permite la simplificación del comportamiento como las bases genéticas y neuronales del comportamiento social son aún poco conocidos. Drosophila melanogaster es un modelo genético de gran alcance que se puede utilizar para identificar la genética y bases neurales de las interacciones sociales. De hecho, D. melanogaster tiene un repertorio de comportamientos sociales complejos y algunas mediciones directas de socialización ya se han hecho 1-7. Sin embargo, la mayoría de estos esfuerzos se han centrado en los comportamientos sociales relativamente complejas, como los aggresinteracciones sive 3,6, diversos aspectos de cortejo 3,8-12, y cómo la experiencia social afecta a otros comportamientos tales como el aprendizaje, o ritmo circadiano 13-17. Además, muchos de estos ensayos se basan en el análisis de los patrones de interacción complejas de grupos de moscas, mediante el seguimiento de vídeo y software de computadora para analizar la abundancia de datos resultante. Estos análisis son muy valiosos, y dar lugar a nuevas e importantes ideas tales como la dinámica de las interacciones con mosca mosca en grupos de 7. Una limitación, sin embargo, es la falta de acceso a estos ensayos a la comunidad en general, y el limitado conocimiento de los mecanismos subyacentes reconocimiento de los demás. En otras palabras, la base de la emisión de una señal por un individuo y su reconocimiento por otro aún es poco conocido 18. Por el contrario, también vuela exhibir un comportamiento simple, evitación social, donde las personas se alejan de una señal emitida por las moscas estresados: el D. melanogaster Estrés Odorante o DSO 19. En un ensayo de alto rendimiento, este comportamiento se puede cuantificar como la evitación de una señal de tensión emitida por otras moscas, o evitación social 19. Las moscas se colocan en un aparato de laberinto en T y se les da la opción de evitar un vial que contiene DSO. Utilizando este ensayo, CO 2 ha demostrado ser un componente de la DSO, y parte de la circuitería neural necesaria para responder a CO 2 se disecó 19. El ensayo de evitación social que aquí se presenta es conceptualmente similar a los ensayos de comportamiento simples desarrollados en el laboratorio de Seymour Benzer que permitieron a generaciones de investigadores para diseccionar conductas complejas 20. Análisis de evitación social puede llevarse a cabo de forma rentable utilizando el ensayo de laberinto en T, lo que permite un estudio más generalizado de comportamiento social. Por ejemplo, el uso de este ensayo hemos demostrado que los diferentes riesgos genéticos para el autismo tienen efectos contrastantes en b socialesensayos ehavior. Los mutantes de un gen candidato para el autismo - neurobeachin 21,22 - deficiencias presentes tanto en el espacio social (describen en otra parte 23) y evitación social 24. También se propone la señalización dopaminérgica anormal para jugar un papel en la etiología del autismo en los seres humanos 25,26. En contraste con los resultados obtenidos con neurobeachin, se encontró que el rendimiento evitación social no se vio afectada por el aumento o disminución de los niveles de la Drosophila vesicular de monoamina Transporter (VMAT) en células dopaminérgicas, aunque el espacio social se correlacionó directamente a estos niveles de VMAT 27. Los resultados obtenidos con contrastantes neurobeachin y VMAT subrayan la posibilidad de identificar diversas formas de comportamiento asocial, y por lo tanto los diferentes trazados de circuito neuronales subyacentes modulación de la respuesta a otros.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Porous Polyethylene, 12&#34; x 12&#34; Sheet</td>
			<td>Flystuff - Genesee</td>
			<td>46-100</td>
			<td>http://www.flystuff.com/ProductInfo.php?productID=46-100 
			Porous Plastic sheet for the cold anesthesia box</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortex</td>
			<td>Fisher</td>
			<td>14-955-151&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail 
			For mechanical agitation of the flies - any vortex would work.</td>
		</tr>
		<tr>
			<td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14mL;&#160; 17 x 100mm</td>
			<td>Fisher</td>
			<td>14-959-8 &#160;&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalogId=29104&#38;productId=2771811&#38;distype=0&#38;highlightProductsItemsFlag=Y&#38;fromSearch=1&#38;searchType=PROD&#38;hasPromo=0 
			These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td>
		</tr>
		<tr>
			<td>cotton balls</td>
			<td></td>
			<td></td>
			<td>to close the vials after the experiment.</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>white bench cover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td>large black cloth</td>
			<td></td>
			<td></td>
			<td>to cover the counter-current apparatus in phototaxis response.</td>
		</tr>
		<tr>
			<td>cool-white light&#160;</td>
			<td>Home Depot</td>
			<td>1000516563</td>
			<td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423 
			any similar linear light with fluorescent light bulb cool-white at 13-15W would work</td>
		</tr>
	</tbody>
</table>

straightforward assay for quantification of social avoidance in drosophila melanogaster

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

El ensayo de evitación social es una prueba robusta cuantificar la capacidad de Drosophila melanogaster para reconocer una señal de estrés (DSO) emitida por otras moscas, y la evaluación de este modo uno de los aspectos de las interacciones sociales. El ensayo se realizó a través de un aparato de uso común en diversos ensayos de comportamiento conocido como un laberinto en T, que presentan las moscas con una elección entre dos opciones diferentes - izquierda o derecha 19,28-31. En este caso, ...

Watch this Scientific Journal Video about Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster at JoVE.com

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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

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

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

straightforward-assay-for-quantification-social-avoidance-drosophila

Research

JoVE Journal

Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

11.1K vistas.  Yale University. Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Video: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection parejas y Ensayo de electrofisiología para la detección de bioactividad de compuestos utilizando el Drosophila melanogaster Sistema de fibra gigante

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Investigación

Neurociencias

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenético estimulación de comportamiento de escape en<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Determinación de la actividad locomotora espontánea en<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

Condiciones que afectan Espacio Social en<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

De alta resolución de cuantificación del comportamiento guiado Olor en<em&gt; Drosophila melanogaster</em&gt; Uso de la<em&gt; Flywalk</em&gt; Paradigma

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Un ensayo de conducta para Mechanosensation de clones en MARCM-basa<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Ensayo de Alto Rendimiento para el Examen de Preferencias de la puesta de huevos individual<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Forrajeo Protocolo Ruta de longitud de<em&gt; Drosophila melanogaster</em&gt; Las larvas

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

El alimentador de ensayo capilar para analizar el consumo de alimentos en<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...