Questo lavoro è stato sostenuto dal National Institutes of Health di Grant R01 GM54408 assegnato a LC Griffith. Vorremmo ringraziare Fred Wolf per il suo aiuto nel progettare la nostra camera di piazza e la creazione di nostri test, Frank Mello alla macchina negozio Brandeis University per costruire la nostra camera, e Dan Valente e Tim Lebestky per le conversazioni utili per quanto riguarda questioni di analisi.

Parte 1: alimentazione e gestione delle mosche <ol><li> Vola deve essere coltivata in flaconi contenenti lievito senza supporti standard. Avrete bisogno di un gran numero di mosche, così croci bisogno di essere impostato di conseguenza. Vola deve essere coltivate in una 12-ore di luce: buio ciclo a 25 ° C. </li><li> Le mosche devono essere raccolti subito dopo eclosion (1-3 giorni). Le mosche possono essere lavorato in questa fase utilizzando un diffusore di anidride carbonica, e deve essere ordinato in provette contenenti i media autoclavati o senza lievito. Noi usiamo solo i maschi, 10 memorizzati in una provetta. </li><li> Attendere almeno un giorno o due dopo l&#39;uso di anidride carbonica prima di eseguire gli esperimenti comportamentali. Corriamo vola nei nostri test comportamentali 3-5 giorni dopo Eclosion. </li><li> Test deve essere sempre effettuata all&#39;interno della stessa finestra 2-3 ore di tempo ogni giorno per evitare problemi di ritmo circadiano. </li></ol> Parte 2: Installazione del sistema di tracciamento in un ambiente controllato <ol><li> Tutti gli esperimenti sono fatti in una stanza ambiente che mantiene una temperatura costante di 25 º C con il 70% di umidità. </li><li> Sospendere una videocamera digitale sopra l&#39;area da registrare (telecamera rivolta verso il basso). Poiché il nostro software di monitoraggio è invece basato, sospendiamo la fotocamera su un light box. La fotocamera registra video direttamente su un computer Dell tramite una connessione firewire. Noi usiamo una fotocamera Sharp video digitale, collegato ad un computer Dell con Windows Moviemaker (versione Vista) per l&#39;acquisizione video. </li><li> Impulsi di aria sarà somministrato in questo protocollo, quindi una fonte di flusso d&#39;aria deve essere presente nella stanza. Utilizzando un tubo in gomma, collegare la sorgente d&#39;aria per un filtro a carbone per la filtrazione dell&#39;aria. </li><li> Utilizzando un tubo in gomma di più, collegare l&#39;altro lato del filtro a carbone per una beuta attraverso un tappo di gomma vuota, e riempire il pallone con circa un centimetro e mezzo d&#39;acqua. Questo umidificare l&#39;aria. </li><li> Il pallone deve anche avere un arma da fianco, che si collegherà a forma di Y valvola tramite tubi in gomma. </li><li> Un ramo della Y-valvola deve essere collegato ad un misuratore di portata. L&#39;altro ramo deve fornire aria alla camera di locomozione piazza. </li></ol> Parte 3: dare vola alla camera di locomozione <ol><li> La camera di locomozione piazza è basato sul design di Wolf et al. 2002 1, con l&#39;aggiunta di vassoi piccola goccia per evitare che le mosche di volare all&#39;interno della camera. La camera deve essere smontato, e tutti i componenti devono essere accuratamente pulito con etanolo al 70% e asciugato prima dell&#39;uso. </li><li> Anestesia immediatamente prima di effettuare un test comportamentale può potenzialmente compromettere le prestazioni. Per evitare questo, le mosche sono delicatamente bussato nella camera usando un imbuto con una presa di piccole dimensioni. Per fare una presa della misura giusta, allegare una punta blu pipetta (per P1000) alla fine di un imbuto. Usare le forbici per tagliare la fine del puntale per rendere l&#39;apertura abbastanza grande per una mosca a passare attraverso. </li><li> La parte superiore della nostra camera è un piccolo pezzo di plexiglass avvitato intorno ai bordi. Quando le viti vengono rimosse, i fori possono essere usati per fornire vola nella camera. Posizionare la parte superiore in plexiglass in modo che il foro della vite si trova direttamente sopra la camera interna al posto di una vite in cui si trova normalmente. Prendere una provetta contenente le mosche e metterlo a testa in giù sopra l&#39;imbuto, che dovrebbe poi essere inserito nel foro della vite. </li><li> Non spostare l&#39;imbuto, in quanto ciò potrebbe ferire o danneggiare le mosche. Invece, dolcemente botto tutta la camera, imbuto e tutti, fino a che tutte le mosche sono all&#39;interno della camera. Questo bussare dovrebbe essere fatto su un tappetino mouse per assorbire lo shock di questa sbattere. Quando le mosche sono all&#39;interno, togliere l&#39;imbuto, e riposizionare la parte superiore in plexiglass sopra i letti vite. Poi ri-avvitare la parte superiore in plexiglass in posizione. </li></ol> Parte 4: Esecuzione del saggio locomotore <ol><li> Una volta che le mosche vengono caricati correttamente, far loro ambientarsi nella camera per 30 minuti. Questo periodo di acclimatazione deve avvenire in ambiente controllato (25 ° C, 70% di umidità), e la camera deve essere posto in cima al light box (che dovrebbe essere acceso). Accendere le luci stanza off. </li><li> Dopo il periodo di acclimatazione, passare la Y-valvola nel sistema di flusso d&#39;aria in modo che il flusso d&#39;aria solo per il misuratore di portata. Accendere l&#39;aria, l&#39;aria e la rampa fino alla velocità desiderata (4,0-6,0 L / min). Generalmente usiamo 5,0-5,5 L / min, ma qualsiasi velocità in tale intervallo funzionerà. </li><li> Una volta che la velocità desiderata è raggiunta, l&#39;interruttore Y-valvola in modo che l&#39;aria fluisce verso la camera di locomozione. Tempo questo impulso d&#39;aria per 15 secondi, e poi girare bruscamente l&#39;aria spenta. Come si accende l&#39;aria spenta, accendere la fotocamera per registrare modalità. (Questa parte vuole pratica di fare tutto in una volta). </li><li> Interrompere la registrazione dopo il tempo desiderato e SAVE. Registriamo 30 prove secondo a 10 fotogrammi al secondo (almeno 8 prove per genotipo). </li></ol> Parte 5: Analisi dei Video <ol><li> Usiamo ilAnalisi dinamica del sistema di immagine (DIAS) 3.2 per il software di motion tracking 2. Per poter utilizzare questo software, che prima convertire i nostri video in un formato di file AVI con QuickTime 7.5.5. </li><li> Per tracciare le mosche in base al contrasto, si usa il &quot;AutoTrace da Threshold&quot; funzione. Siamo quindi utilizzare il &quot;Crea tracciato da traccia&quot; funzione di digitalizzare queste tracce. </li><li> Per emettere la velocità istantanea di ogni volo, si usa il &quot;Compute Parametri&quot; funzione di produrre un file di database (DIAS-specifico). Con questa funzione, abbiamo anche liscia i dati utilizzando un &quot;5,15,60,15,5&quot; Tukey Finestra levigante. </li><li> Una volta che questa informazione è fornito come un file di database, può essere aperto in Microsoft Excel. Noi usiamo uno script Matlab per compilare la velocità istantanea all&#39;interno di eccellere e di calcolare i parametri aggiuntivi necessari per comprendere la dinamica del movimento e la struttura incontro. </li></ol> Rappresentante dei risultati: La figura 1 mostra tracce rappresentante di organismi selvatici tipo Canton S (Figura 1, pannello a sinistra) e vola nullo per il gene dCASK cui sono stati generati dall&#39;incrocio di due grandi delezioni sovrapposte (Df (3R) X307 e Df (3R) X313) (Figura 1 , a destra del pannello). mosche nullo dCASK precedentemente dimostrato di avere problemi locomotori con Paradigma di Buridano 3, e nel nostro paradigma, rispetto al Canton S, mostrano notevolmente ridotta locomozione. Abbiamo sempre gestito organismi wild-type prima per assicurarsi che le condizioni e il comportamento sono all&#39;interno del range di normalità in un dato giorno. Abbiamo osservato deviazioni dalle risposte standard in meno del 5% dei giorni di test. Queste differenze di solito possono essere attribuiti a problemi con i parametri del nostro ambiente controllato. <img src="/files/ftp_upload/1096/figure_1.png" alt="" width="600" height="298" /> Figura 1. DIAS generati tracce di entrambe le mosche Canton S wild-type (sopra, a sinistra) e Locomotore-deficienti Null dCASK vola generato da cancellature sovrapposte (sopra, a destra). Le tracce rappresentano ciò che si vede nel video registrati. Entrambe le tracce controindicazione immagini 8-10 vola per 30 secondi nel test di monitoraggio video che segue un impulso d&#39;aria. <img src="/files/ftp_upload/1096/figure_2.png" alt="" width="600" height="438" /> Figura 2. Vola tipo selvaggio sono stati eseguiti nel test locomotore (sopra) in seguito ad un impulso di aria (barre viola) e senza un impulso d&#39;aria (blu). In tutti i parametri calcolati dal programma di analisi, non vi erano differenze significative tra le due condizioni (determinato con due code studenti t-test). Questo dimostra che in seguito ad un impulso di aria, vola locomote normalmente nel nostro setup.

<ol>
	<li>Wolf, F. W., Rodan, A. R., Tsai, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Analysis+of+Ethanol-Induced+Locomotor+Stimulation+in+Drosophila.">High-Resolution Analysis of Ethanol-Induced Locomotor Stimulation in Drosophila.</a> J Neurosci. 22 (24), 11035-11044 (2002).</li><li>Soll, D. 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+use+of+computers+in+understanding+how+animal+cells+crawl.">The use of computers in understanding how animal cells crawl.</a> International review of cytology. 163, 43-104 (1995).</li><li>Martin, J. R., Ollo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+Drosophila+Ca2+/calmodulin-dependent+protein+kinase+(Caki)+is+localized+in+the+central+nervous+system+and+implicated+in+walking+speed.">A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed.</a> The EMBO journal. 15 (8), 1865-1876 (1996).</li><li>Levine, J. D., Funes, P., Dowse, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+Circadian+Clock+by+Social+Experience+in+Drosophila+melanogaster.">Resetting the Circadian Clock by Social Experience in Drosophila melanogaster.</a> Science. 298, 2010-2012 (2002).</li></ol>

Nella nostra impostazione, un impulso di aria moderatamente forte movimento si arresta temporaneamente volare. Quando l&#39;aria pulsata termina, le mosche vengono rilasciati da questo stato stazionario e locomote normalmente, come mostrato in figura 2. Perché questo impulso di aria sincronizza in modo efficace locomozione della popolazione, lo usiamo per cominciare il processo in modo da poter anche studiare l&#39;insorgenza del movimento a seguito di un momento di rottura stimolo. Il test può comunque essere fatto s...

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		<th>Comment</th>
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<tr>
<td>Square Chamber</td>
<td>Tool</td>
<td>Machine Shop</td>
<td>N/A</td>
<td>Design from Wolf et al. 20021</td>
</tr>
<tr>
<td>Digital Camera</td>
<td>Camera</td>
<td>Sharp</td>
<td>ViewcamZ VL-23</td>
<td>&nbsp;</td>
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<td>Flowmeter</td>
<td>Tool</td>
<td>Cole Parmer</td>
<td>SY-32003-12</td>
<td>&nbsp;</td>
<tr>
<td>Light Box</td>
<td>Tool</td>
<td>DNASTAR, Inc.</td>
<td>Seq-Easy</td>
<td>&nbsp;</td>
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<td>Charcoal Filter</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>09-744-37</td>
<td>&nbsp;</td>
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<td>DIAS 3.2</td>
<td>Software</td>
<td>Soll technologies</td>
<td>N/A</td>
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high-resolution video tracking of locomotion in adult drosophila melanogaster

Vola fornire un importante modello per lo studio del comportamento complesso a causa della pletora di strumenti genetici a disposizione dei ricercatori in questo campo. Studiare il comportamento motorio in<em&gt; Drosophila melanogaster</em&gt; Si basa sulla capacità di essere in grado di quantificare i cambiamenti in atto durante o in risposta a un determinato compito. Per questo motivo, ad alta risoluzione sistema di monitoraggio video, come quello descritto in questo articolo, è un valido strumento per la misurazione locomozione in tempo reale. Il nostro protocollo prevede l&#39;utilizzo di un impulso d&#39;aria iniziale per rompere il momento vola, seguito da un periodo di trenta riprese secondo in una camera quadrata. Un programma di monitoraggio viene poi utilizzato per calcolare la velocità istantanea di ogni volo all&#39;interno della camera con incrementi di 10 msec. Il software di analisi compila questi dati, e le uscite una serie di parametri quali la velocità media, velocità massima, il tempo trascorso in movimento, accelerazione, ecc Questo protocollo discuterà corretta alimentazione e gestione delle mosche per le attività comportamentale, la gestione vola senza anestesia o immobilizzazione , la creazione di un ambiente controllato e l&#39;esecuzione del test, dall&#39;inizio alla fine.

Watch this Scientific Journal Video about High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster at JoVE.com

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Lo studio del comportamento motorio complesso in<em&gt; Drosophila melanogaster</em&gt; Dipende dalla capacità di quantificare i cambiamenti in movimento una mosca dato. Questo articolo viene illustrato come eseguire questa operazione utilizzando ad alta risoluzione sistema di tracciamento.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

high-resolution-video-tracking-locomotion-adult-drosophila

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JoVE Journal

Biology

13.4K Views.  Brandeis. Lo studio del comportamento motorio complesso in Drosophila melanogaster Dipende dalla capacità di quantificare i cambiamenti in movimento una mosca dato. Questo articolo viene illustrato come eseguire questa operazione utilizzando ad alta risoluzione sistema di tracciamento.

Video: High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Ricerca

Biologia

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Ad alta risoluzione misurazione del comportamento Odor-Driven in Larve Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Visivamente monitoraggio Odore mediata durante il volo in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolamento di Drosophila melanogaster Testicoli

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

L&#39;insulina per iniezione ed estrazione emolinfa per misurare la sensibilità all&#39;insulina nei adulti Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Misura della durata della vita in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging attraverso il caso pupa di<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Mantenimento di un<em&gt; Drosophila melanogaster</em&gt; Cage Popolazione

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Determinazione di zona ottico della sezione trasversale del muscolo dei muscoli adulto volo indiretto di Drosophila Melanogaster

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

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Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

Ad alta risoluzione misurazione del comportamento Odor-Driven in Larve Drosophila

Visivamente monitoraggio Odore mediata durante il volo in Drosophila

Dissezione di Oenocytes da adulti Drosophila melanogaster

Isolamento di Drosophila melanogaster Testicoli

L'insulina per iniezione ed estrazione emolinfa per misurare la sensibilità all'insulina nei adulti Drosophila melanogaster

Misura della durata della vita in

Imaging attraverso il caso pupa di

Mantenimento di un

Determinazione di zona ottico della sezione trasversale del muscolo dei muscoli adulto volo indiretto di Drosophila Melanogaster