La conception originale du compensateur de couple originaire avec Karl Goetz. La configuration particulière de cette présentation est dans une large mesure sur le prêt et a été initialement développé par Martin Heisenberg et Reinhard Wolf. Je suis particulièrement redevable à ces deux personnes pour leur soutien, encouragement et d&#39;expertise.

Fly moyennes La composition de la nourriture volée est critique pour l&#39;apprentissage (Guo et al, 1996.) <ul><li> Eau 1000 ml </li><li> 180 g de semoule de maïs </li><li> 10 g de soja </li><li> 18,5 g de levure </li><li> Agar 7,5 g </li><li> La mélasse 40 g </li><li> Sirop (betterave à sucre) 40 g </li><li> Nipagin 2,5 g </li></ul> Chaque flacon est fourni avec une noisette de frais, pâte de levure vivante et un morceau de papier filtre pour fournir une surface supplémentaire pour les mouches et les nymphes. Fly reproduction et de repos La procédure suivante est effectuée chaque jour, conduisant à justement mis en scène des animaux cultivés à la densité appropriée. Toutes les mouches nouvellement ecclosed depuis la dernière procédure le jour précédent sont collectées pour l&#39;élevage et des expériences. Le plus vieux flacons sans aucune pupes vivantes restant sont éliminés. Quatre mouches jours anciens sont ajoutés à un flacon de frais pour la ponte durant la nuit. La densité des mouches femelles devrait être d&#39;environ 20 pour chaque flacon, ajusté pour la taille du flacon et la souche utilisée. La densité idéale est celle qui est suffisamment élevée pour le milieu de volée pour liquéfier au cours des stades larvaires et suffisamment bas pour que toutes les larves ont pupated avant la ecclose premières mouches. Les mouches de ponte de la journée précédente sont enlevés et jetés. Volez la préparation Les mouches sont conservés sur le standard de semoule de maïs / mélasse moyennes comme décrit plus haut à 25 ° C et une humidité de 60% avec un régime light/12hr 12h sombre. Après avoir brièvement immobilisant 24-48h mouches anciennes par le froid-anesthésie, les mouches sont collés (superglue verre anti UV adhésif, 505127A, Pacer technologie, Cucamonga, en Californie., USA) avec la tête et le thorax à un crochet en cuivre en forme de triangle (diamètre de 0,05 mm ) le jour avant l&#39;expérience. Les animaux sont ensuite maintenues individuellement pendant la nuit dans de petites chambres humides contenant quelques grains de saccharose. Un appareil Le dispositif de base de la mise en place est le compensateur de couple (mètre de couple) (Götz, 1964). Il mesure le moment angulaire d&#39;une mouche autour de son axe vertical du corps, causées par des manoeuvres de vol prévu. La mouche, collé à l&#39;hameçon comme décrit ci-dessus, est joint à l&#39;appareil via un couple de serrage pour accomplir vol stationnaire au centre d&#39;un panorama cylindrique (aréna, diamètre 58mm), ce qui est homogène éclairées par l&#39;arrière. La source lumineuse est une 100W, 12V de tungstène-iode ampoule. Pour l&#39;éclairage vert et bleu de l&#39;arène, la lumière passe à travers des filtres monochromatiques large bande de gélatine Wratten Kodak (# 47 et # 99, respectivement). Les filtres peuvent être échangés par un solénoïde rapide au sein de 0.1s. Sinon, l&#39;arène est éclairé par &quot;jour&quot; en passant à travers un filtre bleu-vert (Rosco &quot;surfblue&quot; n ° 5433), ou sans filtre à tous. Le spectre de transmission du Rosco bleu-vert filtre utilisé dans cette étude est équivalente à celle d&#39;un filtre BG18 (Schott, Mayence) et constitue un intermédiaire entre le bleu de Kodak et de filtres verts (Brembs et Hempel de Ibarra, 2006; Liu et al ., 1999). L&#39;arène peut être tourné autour de la volée en utilisant un moteur électrique commandé par ordinateur. Dans un tel «simulateur de vol&quot; la situation, la vitesse angulaire de l&#39;arène est proportionnelle à, mais dirigée contre le couple de lacet de la mouche (coefficient de couplage K =- 11 ° / s • 10-10nm). Cela permet à la volée afin de stabiliser le panorama et de contrôler son orientation angulaire. Cette «direction de vol&quot; virtuels (ie, la position de l&#39;aréna) est enregistrée en continu via un potentiomètre circulaire (Novotechnik, A4102a306). Un convertisseur analogique-numérique de la carte (PCL812; Advantech Co.) alimente le poste arène et le signal de couple de lacet dans un ordinateur qui stocke les traces (fréquence d&#39;échantillonnage de 20Hz) pour une analyse ultérieure. La punition est obtenu par application de chaleur à partir d&#39;un laser infrarouge ajustable (825 nm, 150 mW), dirigé par derrière et au-dessus sur la tête de la mouche et le thorax. Le faisceau laser est pulsé (largeur d&#39;impulsion environ 200ms à ~ 4 Hz) et son intensité réduite pour assurer la survie de la mouche. Expériences Motif d&#39;apprentissage Pour le traditionnel modèle d&#39;apprentissage expérience (Dill et Heisenberg, 1995; Dill et al, 1993, 1995;. Liu et al, 2006;. Liu et al, 1998;. Liu et al, 1999;. Wolf et Heisenberg, 1991) , quatre noires, en forme de T schémas d&#39;orientation en alternance (ie, deux debout et deux inversée) sont régulièrement espacés sur le mur arène (Width Pattern ψ = 40 °, hauteur θ = 40 °, la largeur des barres = 14 °, comme on le voit partir de la position de la mouche). Un programme informatique divise les 360 ° de l&#39;arène en quadrants virtuel 4 ° 90, dont les centres sont désignés par les modèles. Les mouches de contrôler la position angulaire des modes avec son couple de lacet (situation simulateur de vol). Pendant la formation, la punition la chaleur se fait contigus avec l&#39;apparition de l&#39;une des orientations modèle dans le champ visuel frontal. Renforcement de chaque motifest toujours égalisé au sein des groupes. Lors du test, la chaleur est définitivement éteint et la préférence modèle de la mouche enregistrées. L&#39;apprentissage Couleur L&#39;apprentissage des couleurs est réalisée comme décrit précédemment (Brembs et Heisenberg, 2000; Brembs et Hempel de Ibarra, 2006; Brembs et Wiener, 2006; Wolf et Heisenberg, 1997). L&#39;arène est divisée en quadrants virtuels quatre ° 90, dont les centres sont notées par quatre bandes verticales identiques (largeur ψ = 14 °, hauteur θ = 40 °). La mouche est de contrôler la position angulaire des quatre bandes identiques avec son couple de lacet comme décrit pour les modèles en forme de T ci-dessus. La couleur de l&#39;éclairage de la scène entière est changé chaque fois que l&#39;une des frontières virtuelles quadrant passe un point en face de la volée. Pendant la formation, la punition la chaleur est subordonné à l&#39;une des deux couleurs. Pendant le test, la chaleur est définitivement éteint et préférences de couleur de la mouche enregistrées. Bien sûr, les couleurs peuvent être combinées avec des modèles pour le conditionnement composé (Brembs et Heisenberg, 2001). L&#39;apprentissage couple de lacet L&#39;apprentissage couple de lacet est réalisée comme décrit précédemment (Brembs et Heisenberg, 2000; Heisenberg et Wolf, 1993). Spontanée de la mouche plage de couple de lacet est divisé en une «gauche» et «droite» du domaine, correspondant approximativement à deux virages à gauche ou à droite. Il n&#39;y a pas des motifs sur le mur arène. Pendant la formation, la chaleur est appliquée à chaque couple de lacet de la mouche est dans un domaine et éteint quand le couple passe dans l&#39;autre. Dans les phases de test, la chaleur est définitivement éteint et le choix de la mouche des domaines couple de lacet est enregistrée. Composite de l&#39;apprentissage Composite de l&#39;apprentissage est une extension de l&#39;apprentissage couple de lacet, comme décrit précédemment (Brembs et Heisenberg, 2000). Fondamentalement, l&#39;apprentissage du couple de lacet et l&#39;apprentissage des couleurs sont combinées dans une expérience avec opérant équivalente (couple de lacet) et classiques (couleurs) prédicteurs. Pendant la formation, la mouche est chauffé à chaque couple de lacet de la mouche passe dans le domaine associé à la punition. Quand la volée commutateurs domaines couple de lacet, non seulement la température mais aussi la coloration arène est changé (du vert au bleu ou vice versa). Ainsi, le domaine du couple de lacet et de couleurs servent de prédicteurs équivalent de la chaleur. Dans les phases de test, la chaleur est définitivement éteinte et que la mouche choix des couleurs couple de lacet domaines / est enregistrée. Discussion Ce dispositif expérimental combine superbe contrôle sur les circonstances expérimentales avec un organisme modèle génétique de pointe. En utilisant les procédures décrites dans cette présentation, les fondements moléculaires et neurobiologiques d&#39;une variété de traits comportementaux peuvent être étudiés, y compris, mais sans s&#39;y limiter, les mécanismes de génération de comportements spontanés, conditionnement opérant et classique, la reconnaissance des formes, la vision des couleurs ou le contrôle bien sûr .

<ol>
	<li>Guo, A., 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=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., 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=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., 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=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

Ce dispositif expérimental combine superbe contrôle sur les circonstances expérimentales avec un organisme modèle génétique de pointe. En utilisant les procédures décrites dans cette présentation, les fondements moléculaires et neurobiologiques d&#39;une variété de traits comportementaux peuvent être étudiés, y compris, mais sans s&#39;y limiter, les mécanismes de génération de comportements spontanés, conditionnement opérant et classique, la reconnaissance des formes, la vision des couleurs ou le contrôle bien sûr .

operant learning of drosophila at the torque meter

Pour les expériences sur le compteur de couple, les mouches sont conservés sur support volée standard à 25 ° C et une humidité de 60% avec un régime light/12hr 12h sombre. Un régime d&#39;élevage standardisées assure bonne densité des larves et appariés selon l&#39;âge des cohortes. Cold-anesthésiés mouches sont collés avec la tête et le thorax à un crochet en forme de triangle de la journée avant l&#39;expérience. Rattaché à l&#39;indicateur de couple par l&#39;intermédiaire d&#39;une pince, des manœuvres de la mouche vol prévu sont mesurés comme le moment angulaire autour de son axe vertical du corps. La mouche est placé au centre d&#39;un panorama cylindrique pour accomplir le vol stationnaire. Une carte de l&#39;analogique au convertisseur numérique alimente le signal de couple de lacet dans un ordinateur qui stocke la trace pour une analyse ultérieure. L&#39;ordinateur contrôle également une variété de stimuli qui peut être mise sous contrôle de la mouche en fermant la boucle de rétroaction entre ces stimuli et la trace du couple de lacet. La punition est obtenu par application de chaleur à partir d&#39;un laser infrarouge ajustable.

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

Mesure du couple de lacet de la drosophile attachés avec le compteur de couple permet le contrôle neuroscientifique exquis de la situation de relance de l&#39;animal expérimental. Ensemble, avec les outils de génétique unique disponible dans la mouche des fruits, ce paradigme est utilisé pour une grande variété de recherches en neurobiologie.

L&#39;apprentissage opérant de la drosophile à la couplemètre

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

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Biology

13.4K vues.  Free University of Berlin. Mesure du couple de lacet de la drosophile attachés avec le compteur de couple permet le contrôle neuroscientifique exquis de la situation de relance de l'animal expérimental. Ensemble, avec les outils de génétique unique disponible dans la mouche des fruits, ce paradigme est utilisé pour une grande variété de recherches en neurobiologie.

Vidéo: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Terminaux de chargement nerveuse chez la drosophile avec des indicateurs de calcium

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Recherche

Biologie

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 stimulation médiée potentiels synaptiques au Drosophile Jonctions neuromusculaires

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marquage fluorescent Drosophile</emOuvrages d&#39;art> Coeur

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

L&#39;imagerie in vivo chez la drosophile larves Intact au sub-cellulaire Résolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparaison quantitative des Cis-Regulatory Element (CRE) Activités dans transgéniques Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering homologues constructions de recombinaison<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culture de cellules humaines épithéliales nasales à l&#39;interface Air Liquide

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissection et de montage de<em&gt; Drosophila</em&gt; pupes disques yeux

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Étudier mitochondrial Structure et fonction<em&gt; Drosophila</em&gt; ovaires

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...