We thank the Duke Physics Shop, especially Phil Lewis, for helping us build the behavioral apparatus and attachments and creating the drawing. This work is funded by the National Institutes of Health under award number R01GM100027.

1. Préparation de mouches à doser <ol><li> Culture vole sur la mélasse standards / media à la semoule de maïs incubateur réglé à 25 ° C et 65% d&#39;humidité. Prenez soin de ne pas surcharger les flacons. Par exemple, mettre 8 femmes et 6 hommes dans un flacon alimentaire étroite. Note: &quot;flacon alimentaire étroite» utilisée ici a un diamètre intérieur de 2,3 cm. Nous généralement distribué environ 10 ml de nourriture à la mouche dans chaque flacon. La recette de la nourriture à la mouche, nous avons utilisé est décrit ici: http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html. </li><li> 2 - 3 jours après les femelles eclosion, préparer des flacons avec de la pâte de levure fraîche comme le montre la figure 1E. Faire de la pâte de levure fraîche en mélangeant 6 g de levure active avec 10 ml d&#39;acide propionique 0,5%. Utiliser une spatule pour appliquer la pâte de levure sur la paroi latérale des flacons de produits alimentaires. Collecter 30 - 35 femelles éclos avec 20 - 25 mâles dans les flacons. <ol><li> En général, de recueillir les mâles des mêmes flacons où les femelles sont collectées(Afin de gagner du temps et de l&#39;effort). Toutefois, si l&#39;on se préoccupe de la fertilité des mâles, utilisez les mâles WT à la place. En outre, la pâte de levure est important pour stimuler la production d&#39;œufs donc le préparer frais tous les jours. </li></ol></li><li> Maintenir les hommes / femmes recueillies à 25 ° C, 65% d&#39;humidité, à moins que les mouches sont sensibles à la température. </li><li> Après ~ 4 - 5 jours, vérifiez les flacons pour voir si les femelles sont prêtes pour des expériences de ponte. Ils sont prêts lorsque la surface du milieu alimentaire est humide à partir de larves fouisseuses activement à la nourriture (voir la figure 1E - F). Remarque: Les femelles retiennent généralement pondre une fois que la surface de l&#39;aliment devient humide et grouillante de larves. Cette étape garantit que les femmes seraient prêtes à pondre d&#39;œufs quand ils rencontrent des substrats avec la texture désirée (par exemple., ~ 1% d&#39; agarose). </li></ol> 2. Chambre de la Construction, Assemblée, Assay Setup <ol><li> Avoir un atelier d&#39;usinage de construire des chambres de ponte acryliques (Figurer 1A - D). Le dessin technique de différentes pièces ont été montrés à la figure supplémentaire 1 -. 3 images haute résolution peuvent également être trouvés ici (http://www.rebeccayang.org/pdf/chamber%20design.pdf). </li><li> Insérez des feuilles de plastique dans le chargement ( en haut) morceau de la chambre comme le montre la figure 1C. Cela sert de surface de fond pendant le chargement vole dans les arènes de ponte individuels. </li><li> Anesthetize femelles sur un bloc de CO 2 et de les charger individuellement dans chaque arène de ponte. Autoriser ~ 30 min pour les mouches pour récupérer du CO 2 et de s&#39;acclimater à ce nouvel environnement. </li><li> Préparer les substrats d&#39;agarose. <ol><li> Pour plus de commodité, garder une bouteille de premade fondu 1% d&#39;agarose dans un bain d&#39;eau C 55 °. </li><li> Ajouter la quantité désirée de solution mère de saccharose (2 M) dans un tube conique de 50 ml et en le mélangeant avec une quantité appropriée d&#39;agarose. Par exemple, pour préparer le substrat mM, saccharose 150, plaCE 750 pi de solution 2 M de saccharose dans le tube, puis remplir le tube avec de l&#39;agarose à la marque 10 ml. </li><li> Préparer le substrat brut de la même manière mais en ajoutant de l&#39;eau distillée au lieu de la solution de saccharose. Remarque: La concentration finale d&#39;agarose dans ce protocole est légèrement inférieur à 1%. Dans notre expérience, la concentration exacte de l&#39;agarose n&#39;a pas d&#39;importance tant qu&#39;il est commandée pour être à l&#39;intérieur de ~ 0,9 à 1,1% et que les deux substrats sont de la même concentration d&#39;agarose. </li></ol></li><li> Prendre la pièce de la chambre et la pipette de 1000 ul de substrat d&#39; agarose substrat ( en bas) dans chaque creux comme on le voit sur ​​la figure 1D. </li><li> Laisser agarose se solidifier pendant ~ 30 min. </li><li> Une fois que les substrats d&#39;agarose et les mouches sont prêts, assembler les trois morceaux de la chambre de ponte et ensuite prendre les feuilles de plastique. </li><li> Placer les chambres dans les incubateurs de mouches. Note: Longueur des expériences de ponte peut varier selon expebesoins rimentales. Nous courons généralement l&#39;expérience O / N (14 - 16 h). En outre, on a observé aucun impact significatif de la synchronisation circadienne sur les préférences de ponte. </li><li> Anesthetize femelles par injection de CO 2 dans la chambre. Démonter la chambre, jeter les mouches anesthésiés dans la mouche morgue (ie., Un café vide peut rempli avec de l&#39; huile de maïs). Prenez des photos des résultats pour la tenue des dossiers (voir la figure 2). </li><li> Comptez le nombre d&#39;oeufs manuellement et calculer les indices de préférence pour l&#39;analyse. Calculer l&#39; indice de préférence en tant que (N a - N b) / (N a + N b) , où N et N a b représentent le nombre d&#39;oeufs sur le site d&#39; un site par rapport à b, respectivement. </li></ol>

<ol>
	<li>Perisse, E., Burke, C., Huetteroth, W., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking+revelations+and+saccharin+sweetness+in+the+study+of+Drosophila+olfactory+memory.">Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory.</a> Curr. Biol. 23 (17), 752-763 (2013).</li><li>Yamamoto, N. C., Koganezawa, M. <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+circuits+of+courtship+behaviour+in+Drosophila+males.">Genes and circuits of courtship behaviour in Drosophila males.</a> Nat. Rev. Neurosci. 14 (10), 681-692 (2013).</li><li>Zwarts, L., Versteven, M., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+neurobiology+of+aggression+in+Drosophila.">Genetics and neurobiology of aggression in Drosophila.</a> Fly. 6 (1), 35-48 (2012).</li><li>Joseph, R. M., Devineni, A. V., King, I. F., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviposition+preference+for+and+positional+avoidance+of+acetic+acid+provide+a+model+for+competing+behavioral+drives+in+Drosophila.">Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.</a> Proc. Natl. Acad. Sci. 106 (27), 11352-11357 (2009).</li><li>Miller, P. M., Saltz, J. B., Cochrane, V. A., Marcinkowski, C. M., Mobin, R., Turner, T. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+egg-laying+site+selection+a+system+to+study+simple+decision-making+processes.">Drosophila egg-laying site selection a system to study simple decision-making processes.</a> Science. 319 (5870), 1679-1683 (2008).</li><li>Kacsoh, B. Z., Lynch, Z. R., Mortimer, N. T., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+medicate+offspring+after+seeing+parasites.">Fruit flies medicate offspring after seeing parasites.</a> Science. 339 (6122), 947-950 (2013).</li><li>Wu, C. -. L., Fu, T. -. F., Chou, Y. -. Y., Yeh, S. -. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+neurons+on+the+internal+reproductive+tract+contribute+to+egg-laying+induced+acetic+acid+attraction+in+Drosophila.">Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying induced acetic acid attraction in Drosophila.</a> Cell Rep. 9 (2), 522-530 (2014).</li><li>Stern, U., Zhu, E. Y., He, R., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-duration+animal+tracking+in+difficult+lighting+conditions.">Long-duration animal tracking in difficult lighting conditions.</a> Sci. Rep. 5, 10432 (2015).</li><li>Zhu, E. Y., Guntur, A. R., He, R., Stern, U., Yang, C. 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The authors have nothing to disclose.

Les chambres et les protocoles décrits ici ont plusieurs améliorations par rapport à des essais de ponte précédents. Tout d&#39;abord, ils augmentent le débit de dosage préférences des animaux isolés de manière significative. Chaque chambre peut doser 30 femmes célibataires et il faut moins d&#39;une heure à mettre en place. D&#39;autre part, ils augmentent la cohérence des préférences de ponte par rapport aux méthodes antérieures. La normalisation des dimensions de l&#39;arène, la taille des substrats...

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors1-3.
Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare and rank their options before "committing" to depositing an egg onto a given option4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.
In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Sucrose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water bath&#160;</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">15-462-6Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LifeCam Cinema webcam</td>
			<td style="width:104px;">Microsoft</td>
			<td style="width:119px;">H5D-00013</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:269px;">Red LEDs</td>
			<td style="width:104px;">Cree</td>
			<td style="width:119px;">C503B-RAN-CA0B0AA1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Egg-laying chambers</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Camera holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LED holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Fly vials (narrow)</td>
			<td style="width:104px;">Genesee</td>
			<td style="width:119px;">32-116BC</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput assay to examine egg-laying preferences of individual drosophila melanogaster

Récemment, la préférence de la drosophile ponte a émergé comme un modèle génétiquement traitable pour étudier les bases neurales des processus de prise de décisions simples. Lors de la sélection des sites pour déposer leurs œufs, les mouches femelles sont capables de classer l&#39;attractivité relative de leurs options et en choisissant les &quot;plus de deux produits.&quot; Cependant, la plupart des essais de préférence ponte ne sont pas pratiques si l&#39;on veut adopter une approche de dépistage génétique systématique à la recherche de la base de circuits qui sous-tend ce processus de prise de décision simple, comme ils sont et laborieux à mettre en place basée sur la population. Pour augmenter le débit de l&#39;étude des préférences de ponte des femelles simples, nous avons développé personnalisés chambres que chacun peut les préférences de ponte simultanément dosage allant jusqu&#39;à trente individu vole ainsi qu&#39;un protocole qui assure chaque femelle a un taux de ponte élevé (de sorte que leur préférence est facilement discernable et plus convaincante). Notre approche est simple à exécuteret produit des résultats très cohérents. En outre, ces chambres peuvent être équipées de différents accessoires pour permettre l&#39;enregistrement vidéo des animaux de ponte et de fournir de la lumière pour les études de optogénétique. Cet article fournit les plans pour la fabrication de ces chambres et la procédure pour la préparation des mouches à doser dans ces chambres.

Les chambres de ponte sont composés de plusieurs pièces: un morceau substrat ( en bas), un diviseur (milieu) pièce, une charge ( en haut) pièce, et 2 portes coulissantes (Figure 1A - D). Ces pièces sont utilisées pour indépendamment les mouches de configuration et des substrats avant des expériences de ponte. Figure 1F montre comment les flacons devraient regarder quand mouches femelles sont prêtes pour la ponte. Quand les mouches ont le choix e...

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

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

High Throughput Assay pour examiner les préférences de ponte de l&#39;individu<em&gt; Drosophila melanogaster</em

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.

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

high-throughput-assay-to-examine-egg-laying-preferences-individual

Research

JoVE Journal

Neuroscience

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

7.9K vues.  Duke University. 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.

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En électroporation in vivo de la rétine de souris en développement

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Recherche

Neurosciences

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 ...

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.

Méthodes de dosage à Drosophila Comportement

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

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 ...

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.

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 ...

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 ...

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 ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analyse Synaptic Modulation de<em&gt; Drosophila melanogaster</em&gt; Photorécepteurs après exposition à la lumière prolongée

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...