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. Matériel et réactifs créés interne (Voir liste des matières pour les autres) <ol><li> Préparer un Drosophila appareils d&#39;anesthésie froid pour effectuer des travaux à la mouche. <ol><li> Coupez une feuille de polyéthylène poreux pour couvrir une petite boîte en plastique (12,7 cm de long, 10,2 cm de large), typiquement le couvercle supérieur de la boîte de pointes de pipette (12,7 cm de long, 10,2 cm de large, 3,8 cm de profondeur). </li><li> Remplissez la boîte avec de la glace pilée, couvrir avec la feuille de polyéthylène poreux. </li></ol></li><li> Préparer un appareil T-labyrinthe; adapter l&#39;appareil décrit précédemment en détail 28-31. </li><li> Préparer un aspirateur à la mouche comme décrit 32. </li><li> Préparer un appareil de réponse d&#39;phototaxie vol pour déplacer dans et hors des flacons d&#39;essai sans contrainte: <ol><li> Utilisation d&#39;un appareil à contre-courant, comme décrit par 29,33. </li></ol></li></ol> NOTE: Le labyrinthe en T peut également être utilisé à d&#39;autres fins, tel que décrit par 28. <ol start="5"><li> Assurer homconditions d&#39;éclairage ogeneous: effectuer l&#39;expérience sur un banc recouvert d&#39;une couverture de banc blanc, et en face d&#39;un tableau blanc. NOTE: Pour ces expériences, les souches de mouches Drosophila melanogaster utilisés étaient les types sauvages: Canton-S, l&#39;Oregon et Samarkand vole de nos stocks de laboratoire 34; Elwood mouches ont été recueillies à l&#39;automne 2011 dans le quartier Elwood de Huntington, à Long Island, New York, USA. </li></ol> 2. Préparer les mouches avant l&#39;expérience 1-2 jours avant d&#39;effectuer l&#39;expérience: <ol><li> Maintenir les mouches sur le milieu de semoule de maïs-agar-mélasse-levure norme à 25 ° C sur un cycle de 12 heures de lumière / obscurité. </li><li> Recueillir les répondeurs mouches sous anesthésie froides 1-2 jours avant d&#39;effectuer l&#39;expérience <ol><li> Transférer les mouches des bouteilles dans lesquelles ils ont été soulevés dans 50 ml tubes Falcon en utilisant un entonnoir. </li><li> Placez le tube Falcon avec les mouches sous le niveau de la glace, dans unisolé seau à glace. </li><li> Placez l&#39;appareil d&#39;anesthésie froid avec de la glace pilée à -4 ° C pour se détendre. </li><li> Attendez 5 min pour les mouches d&#39;entrer dans le coma de refroidissement. </li><li> Transférer les mouches sur la feuille de polyethylene poreux de l&#39;appareil d&#39;anesthésie froid. </li><li> Recueillir les répondeurs mouches sur l&#39;appareil d&#39;anesthésie froid. A RT, sous un stéréomicroscope, séparer vieille 3-7 jours mâle de mouches femelles et maintenir ces mouches dans des groupes de 40. </li></ol></li><li> Recueillir vol d&#39;émetteur 1-2 jours avant d&#39;effectuer l&#39;expérience. <ol><li> Recueillir doucement Canton-S mixité vole de leurs bouteilles, en utilisant un aspirateur à bouche, le contrôle de leur nombre. </li><li> Maintenir ces mouches dans le groupe de 60 à 100, car ils seront les émetteurs. </li><li> Préparer autant d&#39;échantillons d&#39;émetteurs comme points de données nécessaires. </li></ol></li><li> Prévoyez au moins une nuit de récupération après la collecte des intervenants et des émetteurs, afin de minimiser tout effet de confusion de l&#39;anesthésie froideet la collecte bouche d&#39;aspiration sur le comportement. </li><li> Ouvrez le sac de flacons d&#39;essai, pour permettre l&#39;air en plastique odeur fade dans le sac pour être remplacé par de l&#39;air frais, au moins un jour avant l&#39;expérience. </li></ol> 2 h avant d&#39;effectuer l&#39;expérience: <ol start="6"><li> Se assurer que la température dans la salle où l&#39;expérience est réalisée est de l&#39;ordre de 23 à 25 ° C, même avec la lumière et de l&#39;humidité supérieure à 30%. </li><li> Transférez les intervenants et les mouches émetteurs de fruits dans des flacons de produits alimentaires frais pour se assurer que ces mouches ne sont pas affamés. </li><li> Laissez les mouches des fruits se adapter à l&#39;environnement pendant 2 heures, sur le banc sur lequel l&#39;expérience sera effectuée. </li></ol> 3. Effectuer l&#39;expérience évitement social <ol><li> Effectuez chaque expérience au même moment de la journée, dans une gamme de 3-4 heures dans l&#39;après-midi entre le temps Zeitgeber ZT5 et ZT9. </li><li> Placez le labyrinthe en T sur le pavé de martèlement, et serrer la pince à vis de sorte til est ascenseur stable. </li><li> Transfert vol d&#39;intervenants dans un flacon de test frais. Aligner cette fiole contenant vol d&#39;intervenants sur la partie supérieure de la T-labyrinthe. </li><li> Inclinez l&#39;appareil T-labyrinthe et appuyez sur le tapis battait si les mouches des fruits tombent dans l&#39;ascenseur. </li><li> Déplacez la partie supérieure de l&#39;ascenseur pour descendre de sorte que les mouches sont entre la partie supérieure et inférieure de la T-labyrinthe, mais attention à garder l&#39;ascenseur au-dessus du point de choix - pour empêcher les mouches de se échapper. </li><li> Obtenir flacons avec DSO (voir la section 5 pour générer DSO sans flacons qui étaient auparavant occupés par les mouches). <ol><li> Alors que les vol d&#39;intervenants se adaptent à ce nouvel environnement, placer vol d&#39;émetteur dans un flacon de test. </li><li> Placez un morceau de coton pour fermer le flacon de sorte que les mouches des fruits ne échappent pas. </li><li> Mécaniquement agiter cette fiole sur un mini-tourbillon comme suit: vortex pendant 15 secondes, retirer la cuvette du vortex pendant 5 sec. Répétez trois fois pour un total de 55 sec. </li><li> Remove vol d&#39;émetteur du flacon frais - en transférant dans un flacon alimentaire - et rapidement placer cette fiole remplie DSO dans l&#39;une des deux côtés de l&#39;appareil T-labyrinthe. </li><li> Alternez le côté de placement pour le flacon DSO pourvoir dans chaque nouvelle course. </li></ol></li><li> Placer un flacon d&#39;essai frais de l&#39;autre côté du labyrinthe en T. </li><li> Apportez l&#39;ascenseur complètement en bas, de sorte que les mouches des fruits peuvent choisir entre le flacon de test fraîche et le flacon de test avec DSO, et démarrer la minuterie. </li><li> Laissez les mouches choisir entre le flacon et le flacon frais DSO, pendant 1 min, à moins d&#39;indication contraire. </li><li> Après 1 min, déplacer l&#39;ascenseur jusqu&#39;à séparer les mouches dans le flacon frais, DSO flacon, et ceux qui sont coincés dans l&#39;ascenseur. </li><li> Comptez le nombre de mouches dans chaque flacon, et dans l&#39;ascenseur. </li><li> Répétez cette opération pour chaque génotype / état. </li></ol> 4. Analyser les données évitement social <ol><li> Comptez le nombre de mouches des fruits dans chaque flacon pour chaque genotype, et transférer les données vers un tableur. </li><li> L&#39;utilisation d&#39;un tableur, calculer l&#39;indice de performance (PI) pour chaque génotype en soustrayant le nombre de mouches dans le flacon DSO à partir du nombre de mouches dans le flacon DSO-libre, puis en divisant par le nombre total de mouches. </li><li> Comparez les moyens de le PiS l&#39;aide du logiciel d&#39;analyse statistique pour l&#39;analyse des résultats. <ol><li> Idéalement, préparer 2-3 répétitions internes pour chaque expérience, et trois répétitions indépendantes, effectuées à des jours différents, avec différents bouteilles ou traverse 30. </li><li> Utilisez la distribution des tests statistiques gaussiennes, que les données suivent une distribution normale. </li><li> Lorsque l&#39;essai d&#39;un effet (ce est à dire, la densité - Figure 2), utiliser un ANOVA à une voie, avec post-test de comparaison multiple (colonne par colonne), ou un t-test simple de comparer chaque résultat à un contrôle. </li><li> Lorsque expériences de groupe sont effectuées, tester les deux conditions et les sexes (les flacons vides, a souligné und mouches hors contrainte; et l&#39;effet de genre - Figure 1), en utilisant une ANOVA à deux voies. </li></ol></li></ol> 5. Générer flacons précédemment occupé par mouches atones - Effectuer Au lieu de 03.05 à 03.06 <ol><li> Utilisez phototaxie pour préparer flacons occupés par des mouches qui ne ont pas été sollicitées, comme témoin. </li><li> Transférez les mouches dans un flacon de test, et enclenchez dans l&#39;appareil à contre-courant, avec des flacons frais vides dans la position opposée. </li><li> Poser l&#39;appareil horizontalement avec le flacon distale directement en face d&#39;une lumière blanche froide fluorescente 15 W, et couvrir d&#39;un drap noir. </li><li> Laissez les mouches se déplacent doucement dans le flacon frais. Cela peut prendre plusieurs minutes. Une fois que la plupart des mouches marchent vers la lumière dans le flacon distale, démarrer le minuteur pendant 1 min. </li><li> Retirez délicatement le tissu noir, changer l&#39;emplacement de la lumière, de récupérer avec le drap noir, et permettent vol de quitter le flacon de test. </li><li> A cette époque, initiate l&#39;expérience de choix comme dans 3.2 à 3.5. Passer 3.6 et 3.7. </li><li> Déplacer le curseur de l&#39;appareil, afin de séparer les mouches qui a quitté le flacon de test à partir de l&#39;autre côté. </li><li> Utilisez le flacon de test libéré dans le labyrinthe en T tout de suite pour l&#39;expérimentation des choix, comme dans 3.7. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+decline+in+behavioral+performance+of+Drosophila+melanogaster+with+age.">Differential decline in behavioral performance of Drosophila melanogaster with age.</a> Mechanisms of Ageing and Development. , 127-647 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Ce protocole décrit une procédure détaillée pour le test d&#39;évitement social. Canton-S sera seulement d&#39;éviter un flacon dans lequel vole ont déjà été sollicitées mécaniquement, et que le sexe et le nombre d&#39;intervenants ne affecte pas que la performance d&#39;évitement social. Cependant, fond génétique des intervenants a une influence majeure. Voici plusieurs étapes critiques pour réaliser cette expérience avec succès: 1) transférer toujours les mouches 2 h av...

L&#39;objectif de cette méthode est de quantifier facilement un nouvel aspect du comportement social simple dans Drosophila melanogaster, indépendant de la cour et de l&#39;agression. Les interactions sociales sont essentielles pour le bon développement et la santé des individus au sein d&#39;une société, ainsi que la fonctionnalité d&#39;un groupe social dans son ensemble. La grande complexité de ces interactions nécessite de grandes tailles d&#39;échantillon et un système qui permet une simplification du comportement que les bases génétiques et neuronales du comportement social sont encore mal compris. Drosophila melanogaster est un modèle génétique puissant qui peut être utilisé pour identifier la génétique et bases neurales des interactions sociales. En effet, D. melanogaster a un répertoire de comportements sociaux complexes et des mesures directes de socialisation ont déjà été fait 1-7. Cependant, la plupart de ces efforts ont été axés sur les comportements sociaux relativement complexes, comme agresinteractions sive 3,6, divers aspects de la cour 3,8-12, et comment l&#39;expérience sociale affecte d&#39;autres comportements tels que l&#39;apprentissage, ou rythme circadien 13-17. En outre, beaucoup de ces tests reposent sur l&#39;analyse des modèles d&#39;interaction complexes des groupes de mouches, par suivi vidéo et de logiciels pour analyser l&#39;abondance résultant des données. Ces analyses sont inestimables, et ouvrent de nouvelles perspectives importantes telles que la dynamique des interactions mouche mouche dans les groupes 7. Une limitation, cependant, est l&#39;inaccessibilité de ces tests à la communauté en général, et de la connaissance limitée des mécanismes sous-jacents reconnaissance des autres. En d&#39;autres termes, la base de l&#39;émission d&#39;un signal par un individu et sa reconnaissance par une autre est encore mal comprise 18. En revanche, vole également présenter un comportement simple, évitement social, où les individus se éloignent d&#39;un signal émis par les mouches stressées: D. melanogaster stress odorante ou DSO 19. Dans un dosage à haut débit, ce comportement peut être quantifiée comme l&#39;évitement d&#39;un signal de stress émis par d&#39;autres mouches, ou évitement social 19. Les mouches sont placées dans un appareil T-labyrinthe et ont le choix pour éviter un flacon contenant DSO. En utilisant ce dosage, CO 2 se est révélée être une composante de l&#39;ASM, et une partie du circuit neural nécessaire pour répondre à CO 2 a été disséqué 19. L&#39;essai d&#39;évitement sociale présentée ici est conceptuellement similaire aux tests comportementaux simples développées dans le laboratoire de Seymour Benzer qui ont permis à des générations de chercheurs pour disséquer des comportements complexes 20. Analyse des évitement social peut être réalisée en utilisant de manière rentable le test T-labyrinthe, permettant pour une étude plus généralisée du comportement social. Par exemple, l&#39;utilisation de ce test nous avons récemment démontré que les risques génétiques différents pour l&#39;autisme ont des effets contrastés b socialedosages ehavior. Mutants pour un gène candidat pour l&#39;autisme - neurobeachin 21,22 - les déficiences actuelles à la fois dans l&#39;espace social (décrits ailleurs 23) et évitement social 24. La signalisation dopaminergique anormale est également proposé de jouer un rôle dans l&#39;étiologie de l&#39;autisme chez les humains 25,26. Contrairement aux résultats obtenus avec neurobeachin, nous avons trouvé que la performance d&#39;évitement sociale était pas affectée par une augmentation ou une diminution du taux de la monoamine vésiculaire Drosophila Transporter (VMAT) dans des cellules dopaminergiques, bien que l&#39;espace social est directement corrélée à ces niveaux de VMAT 27. Les résultats contrastés obtenus avec neurobeachin et VMAT soulignent la possibilité d&#39;identifier diverses formes de comportement asocial, et donc les différents circuiteries neuronaux sous-jacents moduler la réponse à d&#39;autres.

<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.

Le test d&#39;évitement social est un test robuste quantifier la capacité de Drosophila melanogaster de reconnaître un signal de stress (DSO) émis par d&#39;autres mouches, et d&#39;évaluer ainsi un aspect des interactions sociales. Le test est effectué en utilisant un appareil couramment utilisé dans divers essais de comportement connus comme un labyrinthe en T, qui présentent les mouches avec un choix entre deux options différentes - gauche ou à droite 19,28-31. Dans ce cas, l&#39;efficac...

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.

Essai simple pour la quantification de l&#39;évitement 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 vues.  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.

Vidéo: 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 paires et test d&#39;électrophysiologie à l&#39;écran de la bioactivité des composés en utilisant le Drosophila melanogaster Système de fibre géant

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 ...

Recherche

Neurosciences

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.

Stimulation optogenetic de comportement de fuite 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;

Détermination de l&#39;activité locomotrice spontanée,<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.

Conditions affectant espace social<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.

Haute résolution La quantification du comportement Odor-guidée dans<em&gt; Drosophila melanogaster</em&gt; Utilisation de la<em&gt; Flywalk</em&gt; Paradigm

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.

A Behavioral Essai pour mécanosensibilité de marcm-fondé clones dans<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.

High Throughput Assay pour examiner les préférences de ponte de l&#39;individu<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.

Foraging Protocole Chemin de longueur pour<em&gt; Drosophila melanogaster</em&gt; larves

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.

Capillaire Feeder Assay mesures prise alimentaire dans<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.

Développement de dosage pour une Quantification contenue élevée de Formation globale de protéine mutante Sod1 dans les cellules vivantes

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