Il design originale del compensatore di coppia nasce con Karl Goetz. La configurazione particolare in questa presentazione è in gran parte in prestito ed è stato originariamente sviluppato da Martin Heisenberg e Reinhard Wolf. Sono particolarmente in debito con queste due persone per il loro continuo sostegno, incoraggiamento e competenza.

Vola media La composizione del cibo mosca è fondamentale per l&#39;apprendimento (Guo et al, 1996.) <ul><li> 1000 ml di acqua </li><li> Farina di mais 180 g </li><li> 10 g di semi di soia </li><li> 18,5 g di lievito </li><li> Agar 7,5 g </li><li> 40 g di melassa </li><li> Sciroppo (barbabietola da zucchero) 40 g </li><li> Nipagin 2,5 g </li></ul> Ogni flacone è fornito con una piccola quantità di fresco, pasta di lievito vivo e un pezzo di carta da filtro per fornire una superficie aggiuntiva per le mosche e le pupe. Vola l&#39;allevamento e la stadiazione La seguente procedura viene eseguita ogni giorno, portando a proprio messo in scena gli animali cresciuti alla densità appropriata. Tutte le mosche nuova ecclosed dopo l&#39;ultima procedura del giorno prima sono raccolti per l&#39;allevamento ed esperimenti. La più antica fiale senza pupe vivono rimanenti vengono scartati. Quattro mosche giorno di vita vengono aggiunti a una fiala fresca per la deposizione di uova durante la notte. La densità di mosche femmina dovrebbe essere di circa 20 per ogni fiala, corretto per la dimensione del flaconcino e il ceppo utilizzato. La densità ideale è quella che è abbastanza alto per il mezzo volano per liquefare durante gli stadi larvali e abbastanza basso in modo che tutte le larve hanno pupated prima della ecclose prime mosche. La deposizione delle uova d&#39;aria dal giorno precedente vengono rimossi ed eliminati. Vola preparazione Le mosche sono tenuti a standard di farina di mais / melassa media come descritto sopra a 25 ° C e 60% di umidità con un regime di 12 ore light/12hr buio. Dopo aver brevemente immobilizzare le mosche vecchio 24-48h dal freddo anestesia, le mosche sono incollato (colla UV vetro adesivo, 505127A, Pacer Tecnologia, Cucamonga, Ca., USA) con la testa e il torace di un triangolo a forma di gancio di rame (diametro 0,05 millimetri ) il giorno prima dell&#39;esperimento. Gli animali vengono poi tenuti individualmente durante la notte in piccole camere umido contenente alcuni chicchi di saccarosio. Apparato Il dispositivo centrale del set-up è il compensatore di coppia (torsiometro) (Götz, 1964). Esso misura il momento angolare di una mosca intorno al suo asse verticale del corpo, causata da manovre di volo previsto. La mosca, incollati al gancio come sopra descritto, è collegato al contatore di coppia tramite un morsetto per realizzare volo stazionario al centro di un panorama cilindrico (arena, diametro 58mm), che è omogeneamente illuminato da dietro. La sorgente luminosa è un 100W, 12V tungsteno-iodio bulbo. Per l&#39;illuminazione verde e blu dell&#39;arena, la luce viene fatta passare attraverso monocromatica a banda larga Kodak Wratten filtri gelatina (# 47 e # 99, rispettivamente). I filtri possono essere scambiati da un solenoide veloce all&#39;interno di 0.1s. In alternativa, l&#39;arena è illuminato con &quot;luce del giorno&quot;, passando attraverso un filtro blu-verde (Rosco &quot;surfblue&quot; No. 5433), nessun filtro o del tutto. Lo spettro di trasmissione del blu-verde filtro Rosco utilizzato in questo studio è equivalente a quella di un filtro BG18 (Schott, Mainz) e costituisce un intermedio tra il blu Kodak e filtri verdi (Brembs e Hempel de Ibarra, 2006; Liu et al ., 1999). L&#39;arena può essere ruotato attorno al volo usando un computer a controllo motore elettrico. In una tale situazione &quot;simulatore di volo&quot;, la velocità angolare del campo è proporzionale, ma contro una coppia di imbardata della mosca (accoppiamento fattore K =- 11 ° / s • 10-10Nm). Questo permette al volo per stabilizzare il panorama e per controllare il suo orientamento angolare. Questo virtuale &quot;direzione di volo&quot; (cioè, la posizione arena) è registrata in modo continuo tramite un potenziometro circolare (Novotechnik, A4102a306). Una scheda analogica a quella digitale (PCL812; Advantech Co.) alimenta la posizione arena e il segnale di coppia di imbardata in un computer che memorizza le tracce (frequenza di campionamento 20 Hz) per una successiva analisi. La punizione è ottenuta applicando calore da un laser a infrarossi regolabile (825 nm, 150 mW), diretto da dietro e soprattutto sulla testa della mosca e del torace. Il raggio laser è pulsato (circa 200ms a larghezza d&#39;impulso ~ 4 Hz) e la sua intensità ridotta per assicurare la sopravvivenza della mosca. Esperimenti Modello di apprendimento Per il tradizionale modello di apprendimento-esperimento (Dill e Heisenberg, 1995; Dill et al, 1993, 1995;. Liu et al, 2006;. Liu et al, 1998;. Liu et al, 1999;. Wolf e Heisenberg, 1991) , quattro nero, a forma di T modelli di orientamento alternato (cioè due in piedi e due capovolto) sono distribuiti uniformemente sulla parete arena (modello larghezza ψ = 40 °, altezza θ = 40 °, larghezza delle barre = 14 °, come si è visto dalla posizione della mosca). Un programma per elaboratore divide il 360 ° dell&#39;arena virtuale in 4 quadranti 90 °, i cui centri sono indicati con i modelli. Le mosche controllare la posizione angolare dei modelli con la sua coppia di imbardata (simulatore di volo situazione). Durante l&#39;addestramento, la punizione di calore è fatto contiguo con la comparsa di uno degli orientamenti modello nel campo visivo frontale. Rinforzo di ciascun modelloè sempre pareggiato all&#39;interno dei gruppi. Durante il test, il calore è sempre spento e la preferenza modello della mosca registrato. Colore di apprendimento Apprendimento del colore viene eseguita come descritto in precedenza (Brembs e Heisenberg, 2000; Brembs e Hempel de Ibarra, 2006; Brembs e Wiener, 2006; Wolf e Heisenberg, 1997). L&#39;arena è divisa in quattro quadranti virtuali 90 °, i cui centri sono indicati con quattro strisce verticali uguali (larghezza ψ = 14 °, altezza θ = 40 °). La mosca è il controllo della posizione angolare dei quattro strisce uguali con la sua coppia di imbardata, come descritto per il T-forma di modelli di cui sopra. Il colore di illuminazione del campo tutto è cambiato ogni volta che una delle frontiere quadrante virtuale passa un punto di fronte al volo. Durante l&#39;addestramento, la punizione di calore è subordinata su uno dei due colori. Durante il test, il calore è sempre spento e la preferenza del colore della mosca registrato. Naturalmente, i colori possono essere combinati con i modelli per il condizionamento composto (Brembs e Heisenberg, 2001). Yaw coppia di apprendimento Apprendimento della coppia di imbardata viene eseguita come descritto in precedenza (Brembs e Heisenberg, 2000; Heisenberg e Wolf, 1993). La mosca spontanea gamma di coppia di imbardata è diviso in una &quot;sinistra&quot; e del dominio &quot;destra&quot;, che corrisponde circa a uno gira a sinistra oa destra. Non ci sono modelli a parete arena. Durante l&#39;addestramento, il calore viene applicata ogni volta che una coppia di imbardata della mosca è in un dominio e spento quando la coppia passa nell&#39;altro. Nelle fasi di test, il calore è sempre spento e la scelta della mosca di imbardata domini coppia viene registrato. Apprendimento composito Apprendimento composito è un&#39;estensione di apprendimento della coppia di imbardata, come descritto in precedenza (Brembs e Heisenberg, 2000). Fondamentalmente, l&#39;apprendimento della coppia di imbardata e l&#39;apprendimento dei colori sono combinati in un esperimento con operante equivalente (coppia di imbardata) e classica (colori) predittori. Durante l&#39;addestramento, la mosca è riscaldato ogni volta che una coppia di imbardata della mosca passa nel dominio associato con la punizione. Ogni volta che il volo passa domini coppia di imbardata, non solo la temperatura ma anche colorazione arena è cambiato (da verde a blu o viceversa). Così, il dominio della coppia di imbardata e di colore servire come predittori equivalente di calore. Nelle fasi di test, il calore viene definitivamente spenta e solo la scelta della mosca di colori coppia di imbardata domini / viene registrato. Discussione Questa configurazione sperimentale abbina il controllo superbo su circostanze sperimentale con un organismo avanzato modello genetico. Utilizzando le procedure descritte in questa presentazione, le basi molecolari e neurobiologiche di una serie di tratti comportamentali possono essere indagati, tra cui, ma non limitati a, i meccanismi di generazione comportamento spontaneo, condizionamento operante e classica, pattern recognition, visione a colori o in corso di controllo .

<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.

Questa configurazione sperimentale abbina il controllo superbo su circostanze sperimentale con un organismo avanzato modello genetico. Utilizzando le procedure descritte in questa presentazione, le basi molecolari e neurobiologiche di una serie di tratti comportamentali possono essere indagati, tra cui, ma non limitati a, i meccanismi di generazione comportamento spontaneo, condizionamento operante e classica, pattern recognition, visione a colori o in corso di controllo .

operant learning of drosophila at the torque meter

Per gli esperimenti al contatore di coppia, le mosche sono conservati su supporto standard di volare a 25 ° C e 60% di umidità con un regime di 12 ore light/12hr buio. Un regime di allevamento standard assicura adeguata densità larvale e di pari età coorti. Freddo anestetizzati mosche sono incollati con la testa e il torace di un triangolo a forma di gancio il giorno prima l&#39;esperimento. In allegato al metro coppia tramite un morsetto, manovre di volo destinato al volo sono misurati come il momento angolare attorno al suo asse verticale del corpo. La mosca è posto al centro di un panorama cilindrico per realizzare volo stazionario. Un convertitore analogico digitale a carta alimenta il segnale di coppia di imbardata in un computer che memorizza la traccia per un&#39;analisi successiva. Il computer controlla anche una varietà di stimoli che può essere tenuta sotto controllo della mosca chiudendo il circuito di feedback tra questi stimoli e la traccia della coppia di imbardata. La punizione è ottenuta applicando calore da un laser a infrarossi regolabile.

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

Misurazione della coppia di imbardata Drosophila legato con il misuratore di coppia consente il controllo squisito neuroscienziato della situazione stimolo degli animali sperimentali. Insieme con gli strumenti genetico unico disponibile nel moscerino della frutta, questo paradigma è utilizzato per una vasta gamma di ricerca neurobiologica.

L&#39;apprendimento della Drosophila operante presso il misuratore di coppia

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

Research

JoVE Journal

Biology

13.4K Views.  Free University of Berlin. Misurazione della coppia di imbardata Drosophila legato con il misuratore di coppia consente il controllo squisito neuroscienziato della situazione stimolo degli animali sperimentali. Insieme con gli strumenti genetico unico disponibile nel moscerino della frutta, questo paradigma è utilizzato per una vasta gamma di ricerca neurobiologica.

Video: 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.

Caricamento terminazioni nervose Drosophila con gli indicatori di calcio

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 ...

Ricerca

Biologia

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 stimolazione mediata dei potenziali Synaptic a Drosophila Giunzioni neuromuscolari

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.

Etichette fluorescenti di Drosophila Cuore Strutture

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.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

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.

Confronto quantitativo di Cis-Regulatory Element (CRE) Attività in transgenici 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.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 costrutti omologhi ricombinazione<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.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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.

Dissezione e Montaggio di<em&gt; Drosophila</em&gt; pupa Dischi Eye

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