Wir danken 4 anonymen Gutachtern für ihre Arbeit, das Manuskript zu verbessern. Wir danken dem Centre National de la Recherche Scientifique für die finanzielle Unterstützung, um MBG und YG, und der Université de Bourgogne und der Französisch Ministerium für Forschung, um MMS. Research in YG Labor wird von der Europäischen Forschungsrates (ERC Starting Grant, GliSFCo-311403), der Agence Nationale de la Recherche (ANR-JCJC, GGCB-2010), des Conseil Régional de Bourgogne (Faber) und dem CNRS finanziert.

1. Vor dem Start <ol><li> Verwenden Sie ein isogenized Referenz Lager Lager stabil und robust Verhaltens Phänotypen. Es gibt keine allgemeine Regel für die Wahl dieses Lager, da alle potenziellen Kontrollen können heterogenen Hintergrund Allele tragen. </li><li> Verwenden Sie dieses Kontrollstamm, jede andere Lager, die für spätere Schritte Rückkreuzung. Diese Rückkreuzung Schritt wird in der Regel um mindestens 5 aufeinander folgenden Kreuzungen eines einzigen weiblichen Jungfrau vertreten zu 2-3 isogene Vergleichs Männer 5 (mögliche Crossing-over zwischen homologen Chromosomen zu ermöglichen). Dieser Schritt ist wichtig, um den genetischen Hintergrund der verschiedenen Flugbestände homogenisieren. </li><li> Pflegen Drosophila Aktien auf einem Standard-Maismehl (9%), Hefe (10%) und Agar-Medium (1,5%) mit einer Antibiotika-(0,4% Methyl-para-Hydroxy-Benzoat) in einem 12-Stunden Licht / Dunkel-Zyklus ergänzt bei 25 ° C. </li><li> Erreichen Sie chemosensory Experimente in einem temperierten Raum (25 ° C) unter viel rotes Licht(Um den Beitrag der visuellen Hinweisen zu beseitigen und die auf chemosensorischen Signale konzentrieren). Regelmäßig erneuern die Luft des Raumes, um den Bereich zwischen jedem Versuch zu lüften. </li></ol> 2. Olfaktorischen Reaktion mit einem Y-Labyrinth-Test <ol><li> Verhungern die Fliegen für 16-18 h bei 25 ° C in Glasröhrchen, die nassen Papiertuch vor der Prüfung. </li><li> Nehmen Sie an einer Y-Form-Stecker an zwei Glasflaschen und zu einem kleineren Kunststofffläschchen (Lade Fläschchen). Verwenden Sie 1 ml Pipettenspitzen, die durch die Schaumstopper geben, um den Anschluss an die drei Fläschchen zu verknüpfen und einen dicht verschlossenen Y-Labyrinth zu erhalten. Schneiden Sie die schmalen Enden von zwei Pipettenspitzen (~ 2 mm Durchmesser, um eine Rückkehr der Fliege zu vermeiden, sobald er seine Entscheidung gemacht), um zwei &quot;Falle&quot; Fläschchen zu bilden, und ein großes Ende einer Pipettenspitze, um den &quot;Laden&quot; zu bilden Rohr (1A). </li><li> Kurz vor dem Anschluss der &quot;Falle&quot; Fläschchen (1B), legen Sie ein ~ 6 mm Durchmesser Filterpapier injedes Fläschchen. 40 &amp;mgr; l Geruchsstoff-Lösung auf ein Filterpapier, und 40 ul des entsprechenden Lösungsmittels auf dem zweiten Filterpapier. </li><li> Führen zehn 4-9 Tage alten Fliegen in der &quot;loading&quot; Fläschchen. Verwenden Sie keine CO 2-Narkose während dieser Überweisung, da es einen starken Einfluss auf das Verhalten der 10 hat. Vielmehr verwenden kurzem Abkühlen auf Eis. Die richtige Handhabung der narkotisierten Fliegen ist wichtig, um Stress zu den Themen so weit wie möglich zu begrenzen. <ol><li> Führen Sie eine Reihe von Y-Labyrinth-Tests bei 25 ° C unter dunkelrotem Licht (mit LED-Lampen, um mögliche Wärmequelle zu beschränken) auf visuelle Reize zu vermeiden, so viel wie möglich. Achten Sie darauf, die Orientierungen der Y-Labyrinthe wechseln (Geruchsstoff enthalten Rohr auf der linken oder auf der rechten Seite, und Laderohr vorne oder hinten; Abbildung 1C). </li><li> Warten Sie einige Stunden für die Fliegen in der Falle Fläschchen mit dem Geruchsstoff oder Lösungsmittel ein. Graf Fliegen nach 24 Stunden, um die participati erhöhenauf bis zu mehr als 80% und bieten maximale Geruchsindexwert (Simmonet, persönliche Mitteilung). </li><li> Berechnen Sie die resultierende Geruchs Index nach folgender Formel: (Anzahl in den Geruch Rohr - Zahl in der Lösungsmittelrohr) / Gesamtzahl der geladenen Fliegen. </li></ol></li><li> Wash Y-Labyrinth Set-up wie folgt: einweichen abgebaut Set-up in RBS 35 MD Übernachtung. Gründlich mit Leitungswasser abspülen. Schließlich mit VE-Wasser spülen und trocknen lassen. </li></ol> 3. Statistische Analyse der Daten <ol><li> Führen t-Test, eine Einweg-ANOVA oder ein Zwei-Wege-ANOVA Abhängigkeit von Daten und Variablen. </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>

Die Autoren haben nichts zu offenbaren.

Unsere Y-Labyrinth-Protokoll basiert auf einem zuvor beschriebenen Protokoll 9 basiert. Allerdings stellen wir zwei wesentliche Unterschiede. Erstens verwenden wir engen Pipettenspitzen, um die Fliegen von der Rückkehr zu verhindern, wenn sie sich entscheiden, in der Ampulle mit dem Lösungsmittel oder Lösungsmittel plus den Geruchsstoff geben. Diese schmalen Spitzen sind auch nützlich, um die Geruchsstoffdiffusion in der Y-Labyrinth begrenzen. Zweitens verwenden wir ein kleiner Laden Fläschchen, um die F...

Chemoreception durch Geschmack oder Geruchssinn ist ein Schlüssel sensorischen Modalität für das Überleben der Tiere. Es gibt dadurch wichtige Hinweise für die Erkennung einer Gefahr oder Nahrungsquellen notwendig, als auch für soziale Interaktionen. Es hilft auch Tieren eine Sex-Partner für ihre Fortpflanzung notwendig finden. Seit mehr als 20 Jahren intensiver Forschung, einschließlich der Nobelpreis ausgezeichneten Arbeiten von Richard Axel und Linda Buck im Jahr 2004 &quot;für ihre Entdeckungen der Geruchsrezeptoren und der Organisation des olfaktorischen Systems&quot;, wurde durchgeführt, um die molekularen und zellulären Grundlagen der offenbaren Riechen 1,2. Eines der beliebtesten Tiermodelle für Wissenschaftler Geruchswahrnehmung zu zerlegen ist D. melanogaster. Dieses Insekt teilt eine ähnliche zelluläre und molekulare Geruchscodierungsstrategie mit Säugetieren. Die wissenschaftliche Gemeinschaft nutzt diverse Verhaltensparadigmen, die Rolle der Geruchsstoffe in diesem Fruchtfliege studieren. Diese Tests umfassen multimodalen Assays wie courtship Tests, bei denen verschiedene Sinnesmodalitäten, einschließlich Geruchssinn, sind wichtig, um männliche Balz 3 entlocken. Andere Tests sind auch entwickelt worden, um die Rolle von Geruchsstoffen genauer anzugehen; Dazu gehören T-Labyrinthe, Y-Labyrinthe, trap-Assays, Vier-Felder-Arenen und Wind-Tunnel 4,5,6,7,8. In diesem Artikel stellen wir eine einfache modifizierte Y-Labyrinth-Test, die robust Riech Antworten bietet mit D. melanogaster. Unser Set-up verwendet End-Tipps im Gegensatz zu einer zuvor beschriebenen Verfahren 9. So hat unsere Y-Labyrinth zwei Vorteile. Erstens, eine Rückkehr in das System vermeidet es einmal die Fliege hat ihre Wahl getroffen. Zweitens schränkt sie den Austausch von Geruchsstoffen in allen Bereichen der Y-Labyrinth. Dieser letzte Vorteil ist wichtig, da Drosophila sind sehr empfindlich auf Luftstrom, der oft verwendet wird, um Geruchs Sättigung zu vermeiden. Zur Einstellung des experimentellen Aufbaus mit einem Luftstrom würde Zeit-und kostenintensiv sein. Daher sind unsere Y-Labyrinth-Test stellt eine efreichend und schnelle Möglichkeit, Riechleistung von Drosophila zu testen.

<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

Detektieren von Signalen aus der Umgebung ist für Tiere ihr Überleben zu sichern. Zu diesem Zweck verwenden sie Umweltreize wie Sehen, Mechanorezeption, Hören und chemoperception durch Geschmack, über direkten Kontakt oder durch Geruchssinn, der die Antwort auf einen flüchtigen Molekül größere Reichweite handeln darstellt. Flüchtige chemische Moleküle sind sehr wichtige Signale für die meisten Tiere in der Erkennung von Gefahr-, Nahrungsquelle oder zwischen Individuen kommunizieren. Drosophila melanogaster ist einer der am häufigsten verwendeten biologischen Modellen für Wissenschaftler, die zellulären und molekularen Basis des Geruchssinns erkunden. Um olfaktorischen Fähigkeiten dieser kleinen Insekten zu markieren, beschreiben wir eine veränderte Wahl-Protokoll basiert auf der Y-Labyrinth-Test klassisch mit Mäusen verwendet. Mit Y-Labyrinthe erhaltenen Daten geben wertvolle Informationen, um besser zu verstehen, wie Tiere mit ihren sich ständig verändernden Umwelt umzugehen. Wir führen eine Schritt-für-Schritt-Protokoll zu studieren, dieWirkung von Geruchsstoffen auf Fly Sondierungs Antwort mit diesem Y-Labyrinth-Test.

1D zeigt zwei repräsentative Antworten mit diesem Y-Labyrinth-Test. Canton-S Männer stark zu vermeiden 10% Essigsäure in destilliertem Wasser verdünnt, während sie sich nicht signifikant von 10% Phenylessigsäure vermeiden. Diese Tests werden auf 10 Männer pro Wiederholung zusammen in den Ladefläschchen gegeben basiert. Dieses Protokoll kann manchmal zu großen Standardfehler des Mittelwerts führen. Bei Bedarf ist es möglich, diesen Nachteil, indem 20 Männer pro Wiederholungen statt nur 10. Ma...

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

Wir präsentieren eine Choice-Test, um den Einfluss von Geruchsstoffen auf Drosophila Verhalten mit einem Y-Labyrinth-Test offenbaren.

Testen<em&gt; Drosophila</em&gt; Riechen mit einem Y-Labyrinth-Test

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 Aufrufe.  UMR-6265 CNRS.  Wir präsentieren eine Choice-Test, um den Einfluss von Geruchsstoffen auf Drosophila Verhalten mit einem Y-Labyrinth-Test offenbaren. 

Serie: 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.

Gepaart Nanoinjection und Elektrophysiologie-Test zum Screening auf Bioaktivität der Verbindungen unter Verwendung des Drosophila melanogaster Riesen-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 ...

Forschung

Neurowissenschaften

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.

Methoden zum Test Drosophila Verhalten

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

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.

Ein Single-fly-Assay für Sammelverhalten in<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.

Eine einfache Verhaltenstest zur Prüfung von Visual Function in<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.

Live-Imaging von<em&gt; Drosophila</em&gt; Larven Neuroblasten

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.

Einfache Assay zur Quantifizierung von Sozialvermeidung in<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.

Ein Verhaltenstest für Mechanosensation der MARCM-basierte Klonen in<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.

Geschmackspräferenz Assay für Erwachsene<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).

Dynamische Quantitative Sensory Testing zu Charakterisieren zentralen Schmerzverarbeitung

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

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.

Die kapillare feeder Assay misst die Nahrungsaufnahme in<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 ...