Agradecemos 4 evaluadores anónimos por su trabajo para mejorar el manuscrito. Agradecemos al Centro Nacional de Investigación Científica por su apoyo financiero a MBG y YG, y la Université de Bourgogne y el Ministerio francés de Investigación para MMS. La investigación en laboratorio YG es financiado por el Consejo Europeo de Investigación (ERC Starting Grant, GliSFCo-311403), la Agence Nationale de la Recherche (ANR-JCJC, GGCB-2010), el Conseil Régional de Bourgogne (Faber) y el CNRS.

1. Antes de empezar <ol><li> Utilizar una referencia de valores isogenized teniendo fenotipos conductuales estables y robustas. No existe una regla general para la elección de esta población, ya que todos los controles posibles pueden llevar heterogéneos alelos de fondo. </li><li> Utilice esta cepa de control para retrocruzar cada dos valores necesarios para los pasos posteriores. Este paso retrocruzamiento se representa típicamente por lo menos 5 cruces sucesivos de una sola hembra virgen (para permitir que sea posible entrecruzamiento entre cromosomas homólogos) a 2-3 machos de referencia isogénicas 5. Este paso es importante para homogeneizar el fondo genético entre las diferentes poblaciones de la mosca. </li><li> Mantener las poblaciones de Drosophila en una harina de maíz estándar (9%), levadura (10%), y el medio de agar (1,5%) complementado con antibióticos (0,4% de metil para-hidroxi-benzoato) en un ciclo de luz / oscuridad de 12 horas a 25 ° C. </li><li> Lograr experimentos quimiosensoriales en una habitación con temperatura controlada (25 º C) con luz roja lejana(Para eliminar la contribución de las señales visuales, y centrarse en las señales quimiosensoriales). Regularmente renovar el aire de la habitación a ventilar el área entre cada experimento. </li></ol> 2. Respuesta olfativa mediante un ensayo del laberinto <ol><li> Morir de hambre a las moscas de 16-18 horas a 25 ° C en tubos de vidrio que contienen una toalla de papel húmeda antes de la prueba. </li><li> Únase a un conector en forma de Y a dos viales de vidrio y un vial de plástico pequeña (carga vial). Utilice 1 ml puntas de pipeta que pasan a través de los tapones de espuma para unir el conector a los tres viales, y para obtener un laberinto Y cerrado herméticamente. Cortar los extremos estrechos de dos puntas de pipeta (~ 2 mm de diámetro, para evitar cualquier retorno de la marcha una vez que ha tomado su decisión) para formar dos viales &quot;atrapar&quot;, y un gran final de una punta de la pipeta para formar la &quot;carga&quot; tubo (Figura 1A). </li><li> Justo antes de la conexión de los viales &quot;atrapar&quot; (fig. 1B), coloque una ~ 6 mm de diámetro en el papel de filtrocada vial. Añadir 40 l de solución de odorante en una de papel de filtro, y 40 l de la correspondiente disolvente en el segundo papel de filtro. </li><li> Introducir diez 4-9 moscas de un día en el vial de &quot;carga&quot;. No utilice CO 2 anestesia durante esta transferencia, ya que tiene un fuerte efecto sobre el comportamiento 10. Más bien utilizar breve enfriamiento en hielo. La manipulación adecuada de las moscas anestesiadas es importante limitar el estrés en los temas tanto como sea posible. <ol><li> Lleve a cabo una serie de pruebas del laberinto a 25 ° C bajo luz roja lejana (el uso de bombillas LED para limitar la posible fuente de calor) para evitar los estímulos visuales tanto como sea posible. Tenga cuidado de alternar las orientaciones de los Y-laberintos (tubo odorante que contiene a la izquierda, oa la derecha, y el tubo de carga en el frente o en la espalda; Figura 1C). </li><li> Permita que transcurran varias horas para que las moscas entren en el vial que contiene la trampa odorizante o el disolvente. Cuente moscas después de 24 horas para aumentar la participatien un máximo de más de 80% y proporcionar el valor de índice olfativa máxima (Simonnet, comunicación personal). </li><li> Calcular el índice olfativa resultante usando la siguiente fórmula: (número en el tubo de olor - número en el tubo de disolvente) / número total de moscas cargados. </li></ol></li><li> Lave laberinto Y puesta a punto de la siguiente manera: remoje la desmantelado configuración en RBS 35 MD durante la noche. Enjuagar bien con agua del grifo. Por último, enjuagar con agua desionizada y dejar que se seque. </li></ol> 3. Análisis Estadístico de los Datos <ol><li> Lleve a cabo una prueba t, un ANOVA de una vía o un ANOVA de dos vías en función de los datos y variables. </li></ol>

<ol>
	<li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat. Rev. Neurosci. 5, 263-278 (2004).</li><li>Silbering, A. F., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionotropic+and+metabotropic+mechanisms+in+chemoreception:+'chance+or+design.">Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design.</a> EMBO Rep. 11, 173-179 (2010).</li><li>Ziegler, A. B., Berthelot-Grosjean, M., Grosjean, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+smell+of+love+in+Drosophila.">The smell of love in Drosophila.</a> Front. Physiol. 4, (2013).</li><li>Silbering, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+function+and+integrated+wiring+of+the+evolutionarily+distinct+Drosophila+olfactory+subsystems.">Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.</a> J. Neurosci. 31, 13357-13375 (2011).</li><li>Grosjean, Y., 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=An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila.">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478, 236-240 (2011).</li><li>Woodard, C., Huang, T., Sun, H., Helfand, S. L., Carlson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+olfactory+behavior+in+Drosophila:+a+new+screen+yields+the+ota+mutants.">Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants.</a> Genetics. 123, 315-326 (1989).</li><li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion.">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li><li>Budick, S. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-flight+responses+of+Drosophila+melanogaster+to+attractive+odors.">Free-flight responses of Drosophila melanogaster to attractive odors.</a> The Journal of Experimental Biology. 209, 3001-3017 (2006).</li><li>Martin, F., Charro, M. J., Alcorta, E. <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+the+cAMP+transduction+pathway+modify+olfaction+in+Drosophila.">Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila.</a> J. Comp. Physiol. A. 187, 359-370 (2001).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> Journal of Insect Physiology. 46, 439-442 (2000).</li><li>Ai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+the+Drosophila+olfactory+system.">Acid sensing by the Drosophila olfactory system.</a> Nature. 468, 691-695 (2010).</li><li>Becher, P. G., Bengtsson, M., Hansson, B. S., Witzgall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flying+the+fly:+long-range+flight+behavior+of+Drosophila+melanogaster+to+attractive+odors.">Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors.</a> J. Chem. Ecol. 36, 599-607 (2010).</li></ol>

Los autores no tienen nada que revelar.

Nuestro protocolo laberinto Y se basa en un protocolo previamente descrito 9. Sin embargo, se introducen dos grandes diferencias. En primer lugar, utilizamos puntas de pipeta estrechos para evitar que las moscas de regresar una vez que deciden entrar en el vial que contiene el disolvente o el disolvente más el odorante. Estos consejos estrechos también son útiles para limitar la difusión de aroma en el laberinto. En segundo lugar, se utiliza un vial de carga menor a la fuerza a las moscas para entrar en e...

Quimiorrecepción a través del gusto o el olfato es una modalidad sensorial clave para la supervivencia de los animales. Se da señales vitales necesarias para la detección de un peligro o de las fuentes de alimentos, así como para las interacciones sociales. También ayuda a los animales a encontrar un socio necesario el sexo para su reproducción. Durante más de 20 años, la investigación intensiva, incluyendo el premio Nobel de obra de Richard Axel y Linda Buck en 2004 &quot;por sus descubrimientos de los receptores odorantes y la organización del sistema olfativo&quot;, se ha llevado a cabo para revelar las bases moleculares y celulares de la olfacción 1,2. Uno de los modelos animales preferidos por los científicos para diseccionar la percepción olfativa es D. melanogaster. Este insecto comparte una estrategia de codificación de olor celular y molecular similar con mamíferos. La comunidad científica utiliza diversos paradigmas de comportamiento para estudiar el papel de los odorantes en esta mosca de la fruta. Estas pruebas incluyen ensayos multimodales como courtshipruebas p donde diversas modalidades sensoriales, incluyendo el olfato, son importantes para obtener el cortejo masculino 3. Otros ensayos también se han desarrollado para abordar el papel de los odorantes más específicamente; estos incluyen T-laberintos, Y-laberintos, ensayos de trampa, arenas de cuatro de campo y 4.5.6.7.8 viento túneles. En este artículo se presenta un ensayo del laberinto sencillo modificado, que proporciona respuestas olfativas robustas usando D. melanogaster. Nuestra puesta a punto utiliza-tips finales en contra de un método descrito anteriormente 9. Por lo tanto, nuestra laberinto Y tiene dos ventajas. En primer lugar, evita las subidas en el sistema una vez que la marcha ha hecho su elección. En segundo lugar, limita el intercambio de olores en todas las áreas de el laberinto. Esta última ventaja es importante, ya que de Drosophila son muy sensibles al flujo de aire que se utiliza a menudo para evitar la saturación odorante. Para ajustar el montaje experimental con un flujo de aire sería consume tiempo y costo. Por lo tanto, nuestro ensayo del laberinto representa un efciente y rápida para poner a prueba el rendimiento olfativo de Drosophila.

<table border="0" cellpadding="0" cellspacing="0" width="835">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Drosophila Polystyrene tube</td>
			<td>VWR europe</td>
			<td>734-2255</td>
			<td>30 x 25 mm&#160; 
			Y-maze</td>
		</tr>
		<tr>
			<td>Drosophila Borosilicate tube</td>
			<td>Dijon verre</td>
			<td></td>
			<td>95 X 25 mm 
			Y-maze</td>
		</tr>
		<tr>
			<td>Foam stopper</td>
			<td>Dutscher</td>
			<td>999038</td>
			<td> 
			Y-maze</td>
		</tr>
		<tr>
			<td>Y-shaped connector</td>
			<td>Europrix</td>
			<td>11020605</td>
			<td> 
			Y-maze</td>
		</tr>
		<tr>
			<td>100-1000&#181;l pipet tips</td>
			<td>Corning</td>
			<td>4868</td>
			<td>Join the following pipet tips to the Y-shaped connector. Cut 2 pipet tips at 65 mm from the wide end, and connect the narrow end (with a &#8764;2 mm opening) to 2 test vials. These openings will limit the U-turns once the flies enter the tubes containing the odors. Cut 1 pipet tip at 35 mm from the wide end, and connect it to the loading vial. 
			Y-maze</td>
		</tr>
		<tr>
			<td>Far-Red LED Bulb</td>
			<td>Rubin-Lacaque</td>
			<td>0RB180238</td>
			<td>625-630 nm</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>45725</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>P16621</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>Sensient Flavors Strasbourg</td>
			<td>1018880464</td>
			<td></td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>eurogerm</td>
			<td>Farine de ma&#239;s</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Kalys</td>
			<td>HP-697-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl hydroxy 4 benzoate</td>
			<td>VWR international</td>
			<td>25605293</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing drosophila olfaction with a y-maze assay

La detección de las señales del medio ambiente es esencial para los animales para asegurar su supervivencia. Para ello, utilizan señales ambientales tales como la visión, mecanorrecepción, el oído y chemoperception a través del gusto, por contacto directo oa través del olfato, el cual representa la respuesta a una molécula volátil que actúa en un mayor alcance. Moléculas químicas volátiles son señales muy importantes para la mayoría de los animales en la detección de peligro, una fuente de alimento, o para la comunicación entre individuos. Drosophila melanogaster es uno de los modelos biológicos más comunes para los científicos a explorar la base celular y molecular del olfato. Con el fin de resaltar las habilidades olfativas de este pequeño insecto, se describe un protocolo elección modificado basada en la prueba del laberinto en clásicamente utilizado con ratones. Los datos obtenidos con Y-laberintos dan información valiosa para comprender mejor cómo tratan los animales con su entorno permanentemente cambiante. Introducimos un protocolo paso a paso para estudiar laimpacto de los olores en mosca respuesta exploratorio utilizando este ensayo del laberinto.

Figura 1D muestra dos respuestas representativas utilizando este ensayo del laberinto. Cantón-S machos evitan fuertemente 10% de ácido acético diluido en agua destilada, mientras que no evitan significativamente ácido fenilacético 10%. Estos ensayos se basan en 10 varones por réplica colocados juntos en los viales de carga. Este protocolo a veces puede conducir a grandes errores estándar de la media. Si es necesario, es posible reducir este inconveniente mediante el uso de 20 hombres por repetici...

Watch this Scientific Journal Video about Testing Drosophila Olfaction with a Y-maze Assay at JoVE.com

Testing Drosophila Olfaction with a Y-maze Assay

Se presenta una prueba de elección para revelar la influencia de sustancias odoríferas en el comportamiento de Drosophila utilizando un ensayo del laberinto.

Pruebas<em&gt; Drosophila</em&gt; El olfato con un ensayo del laberinto

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

testing-drosophila-olfaction-with-a-y-maze-assay

Research

JoVE Journal

Neuroscience

Testing Drosophila Olfaction with a Y-maze Assay

14.8K vistas.  UMR-6265 CNRS.  Se presenta una prueba de elección para revelar la influencia de sustancias odoríferas en el comportamiento de Drosophila utilizando un ensayo del laberinto. 

Video: Testing Drosophila Olfaction with a Y-maze Assay

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

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 ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Microinyección insulina hipocampo y<em&gt; En vivo</em&gt; Microdiálisis durante la prueba de memoria espacial

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

Un ensayo de una sola mosca por el comportamiento de forrajeo en<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Un ensayo de comportamiento simple para Pruebas de función visual en<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imágenes en vivo de<em&gt; Drosophila</em&gt; larvales neuroblastos

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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

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

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

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 ...

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 ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).