Finanziamenti forniti dalla National Science Foundation (MF).

Introduzione L&#39;OMT è un adattamento di un sistema di legare magnetica [1] progettato per incorporare un &quot;simulatore virtuale pennacchio&quot;. I seguenti protocolli spiegherà come legare correttamente le mosche (Parte 1) e fornire le strategie per creare i magneti (Parte 2) e la massa del flusso regolato sistema di erogazione degli odori (Part3). Le informazioni qui descritto è ottimizzato per questo particolare sistema e possono variare per i componenti con le altre specifiche tecniche. Parte 1: vola Tethering I passaggi per legare qui descritte sono simili a molti studi precedenti [2], ma adattati per l&#39;uso in OMT [3]. Tethering corretta è fondamentale per garantire solide prove sperimentali e ripetibili. L&#39;importanza di legare attento non può essere sopravvalutata. <ol><li> Raccogliere vola 4-6 giorni dopo l&#39;eclosion e farli morire di fame per 4-6 ore. Posizionare un kimwipe inumidito con acqua nel flacone senza cibo per prevenire l&#39;essiccamento. </li><li> Trasferimento di un lotto di mosche da una bottiglia di una piccola fiala e inserirlo in un blocco di ottone su un palco di raffreddamento Peltier fissato a circa 4 gradi Celsius. La temperatura corretta anestetizza le mosche entro circa 30 secondi. </li><li> Ordina le mosche sulla superficie fredda della fase di raffreddamento. Un Kimwipe umido garantisce un raffreddamento costante e assorbimento dell&#39;umidità dalla cuticola. Selezionare la più grande femmine per il tethering. </li><li> Utilizzando un pennello fine, spingere una mosca in posizione nel custom-built a forma di sarcofago per ospitare un unico selvatico moscerino della frutta femmina tipo. <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> Figura 1. <a href="https://jove.unicartagenaproxy.elogim.com/files/1063_2008_10_28_1/1063_Figure-01.png">Clicca qui per vedere la versione ingrandita della figura.</a> </li><li> Noi posizione un perno minutien (Multe Strumenti scienza) con una bacchetta magnetica montata su un micromanipolatore. Il perno è allineato in modo tale che l&#39;estremità smussata tocca il torace dorsale appena dietro la testa tra le due ali. Il perno deve essere perfettamente verticale quando viene visualizzato a testa alta verso gli occhi. Anche una piccola quantità di &#39;roll&#39; tra il volare e il pin non può essere tollerato. La vista laterale angolo tra il perno e l&#39;asse longitudinale del corpo deve essere di un naso-up angolo di 30 gradi rispetto all&#39;orizzonte (Fig. 1). Nota: Per un funzionamento ottimale nella pastoia magnete, il pin può essere necessario tagliati a lunghezze. Questo dipenderà interamente dalla distanza tra i magneti sospensione. </li><li> Una volta che il perno minutien è allineato, usare un sottile filo d&#39;acciaio per applicare una piccola goccia di colla UV-attivati ​​(Electrolite Corp) alla fine smussato del perno minutien. </li><li> Manipolare il perno delle gocce di colla in modo che il torace tocca al volo appena sopra la testa. Con la dimensione goccia corretta, la colla deve temprare e il flusso dal perno sul torace. Fare attenzione a non usare troppa colla. </li><li> Cura la colla con due 20 secondi scoppia di luce UV. </li><li> Posizionare il volano fisso da parte e ripetere. Fetta piazzette di Kimwipe e con una pinza dare uno a ciascuno volare legato a frenare volo. </li><li> Dopo un&#39;ora di recupero, le mosche legato sono pronti per gli esperimenti. </li><li> Incollaggio improprio può ostacolare la capacità di una mosca a svolgere nel campo magnetico. Ogni volo deve essere controllato per rotazione regolare all&#39;inizio di ogni esperimento per assicurare le mosche siano incollati. Questo può essere ottenuto dalla rotazione di un modello a strisce con un periodo sufficientemente ampio spazio per ottenere risposte optomotor forte [4]. Se il volo è incollato a l&#39;angolo di inclinazione sbagliata o inclinato sul pin con rispetto all&#39;asse di rollio, la mosca non possono (i) girare a tutti, (ii) hanno una gamma limitata di movimento, o (iii) ruotare a incoerente velocità. Nei casi di cui sopra al volo deve essere eliminata e sostituita con una nuova. Se questo risolve il problema era probabilmente un caso di incollaggio improprio. (Se la sostituzione al volo non risolve il problema, vedere la Parte 2.4) </li></ol> Parte 2: Allineare i magneti Allineando i magneti (terre rare-magnets.com) è necessario per la corretta volare per raggiungere una vasta liscia a 360 gradi di movimento. Attenersi alla seguente procedura per allineare correttamente i magneti. <ol><li> I magneti ad anello inferiore sono tenuti orizzontalmente da un collare in plastica e posto direttamente sopra una camera a vuoto chiaro (Fig. 1, descritta più avanti nella parte 3). Il magnete asta superiore è fissato in verticale di circa 3 / 4 &quot;sopra i magneti più basso. </li><li> Circa allineare il magnete superiore e inferiore visivamente posizionando il magnete superiore asta direttamente sopra il punto centrale dei magneti dell&#39;anello inferiore. E &#39;utile per fissare il magnete superiore ad un micromanipolatore (Siskiyou Inc.) per la regolazione scala fini. </li><li> Epossidica un V-gioiello del cuscinetto (parti piccole) sulla superficie inferiore del magnete superiore per ridurre al minimo l&#39;attrito di rotazione e standardizzare il posizionamento volare. </li><li> Mettere una mosca in campo e regolare la orizzontale e vertical posizione del magnete superiore fino al volo possa agevolmente e in costante rotazione di 360 gradi lungo l&#39;asse di imbardata. Ogni volo deve essere controllato per rotazione regolare all&#39;inizio di ogni esperimento per assicurare i magneti sono correttamente allineati e che singoli pin minutien non siano danneggiati (vedi Parte 1.11). Se i magneti non sono allineati correttamente al volo non possono (i) girare a tutti i (ii) hanno una gamma limitata di movimento (iii) ruotare a velocità incoerente. Se il problema è risolto sostituendo al volo con uno nuovo, è stato molto probabilmente dovuto ad un problema di incollaggio (vedi parte 1). Se questi problemi esistono per ogni volo, è probabile un problema con l&#39;allineamento dei magneti e di ulteriori aggiustamenti sarà richiesto. </li><li> Attenzione: durante il gioco con magneti in terre rare può essere divertente, sono molto pericolosi. L&#39;abuso si traduce in attrezzature danneggiate e / o minori lesioni personali. Prestare la massima attenzione a questi magneti allettante e facilmente sottovalutato. </li></ol> Parte 3: Consegna Odore <ol><li> Il sistema di erogazione odore è basato su quello descritto in precedenza [5] ed è la parte più difficile da ottimizzare (Fig. 1). Si richiede molta pazienza e tentativi ed errori. Seguire questa procedura per ottenere il monitoraggio stabile odore. </li><li> Il set-up iniziale del sistema odore richiede l&#39;esecuzione di stretta misura tubi in Teflon (parti piccole) dal gas multiplexer (Sable Systems International) per l&#39;acqua su misura / odore fiale. La presa di queste fiale si collega direttamente ai tubi porta il ipodermico utilizzando il tubo stesso in Teflon. </li><li> Una chiara camera a vuoto acrilico è posizionato sotto l&#39;arena per supportare i magneti. Usiamo un 4 millimetri tubo di vetro montato sotto il volano collegato alla camera a vuoto per portare il vuoto di apertura più vicino al volo e ridurre il diametro dell&#39;apertura. </li><li> Mettere una mosca legato nell&#39;arena con pinze. </li><li> Accendere un odore attraente (aceto di sidro di mele funziona bene), e verificare visivamente che la mosca tracce l&#39;odore. Se la mosca sembra avere alcuna preferenza per l&#39;odore, seguire i prossimi tre passi. Questo può richiedere vola diversi, e diverse prove. </li><li> Regolare la posizione del porto odore fino a quando il volo sembra seguire l&#39;odore. Abbiamo montato le porte odore di micromanipolatori (Siskiyou Inc.) e posizionati quattro millimetri anteriore dorsale e 3mm a testa della mosca. </li><li> Regolare la portata di massa fino a quando il volo sembra seguire l&#39;odore. Le nostre impostazioni sono 7ml/min (Sable Systems International MFC-4). </li><li> Regolare la portata del vuoto fino a quando il volo sembra seguire l&#39;odore. Abbiamo impostato la portata vuoto a 13l/min utilizzando un regolatore di flusso (Cole Parmer). Nota: Costruzione linee di alimentazione a vuoto possono variare nel tempo causando variazioni nella struttura pennacchio. Può essere utile per collegare una pompa esterna a vuoto per il regolatore di flusso per le condizioni di flusso costante di vuoto. </li><li> Una volta che la posizione del porto e le portate sono impostati, periodicamente passare da una fornitura di vapore acqueo e vapori odore utilizzando una commutabile tramite un multiplexer gas e verificare visivamente che la mosca tracce solo l&#39;odore, quando l&#39;odore è acceso. Occasionalmente, odore residuo all&#39;interno del porto odore può provocare monitoraggio odore è l&#39;odore è spento. Ciò può essere evitato senza rimuovere la porta odore posizionata correttamente compilando i tubi ipodermico con etanolo e raccolta alla fine consegna con un Kimwipe. Con aria compressa, soffiare ogni residuo di etanolo fuori e riprendere gli esperimenti. </li></ol> Rappresentante dei risultati: Quando questi passaggi sono seguiti correttamente due cose devono essere osservati. In primo luogo, sotto costante stimoli visivi a rotazione una mosca legato posto nel OMT dovrebbe girare senza problemi a 360 gradi lungo l&#39;asse di imbardata. &quot;Jerky&quot; giri (chiamati saccadi) sono caratteristici del comportamento spontaneo di svolta in assenza di costanti stimoli visivi. Se le mosche non possono ruotare apparentemente senza problemi, incollaggio improprio (vedi parte 1) e magneti allineati possano esserne la causa (cfr. parte 2). In secondo luogo, quando l&#39;odore è acceso, il volo dovrebbe dirigersi in direzione dell&#39;odore. Quando l&#39;odore è spento, la mosca dovrebbe saccade spontaneamente, senza preferenza per ogni singola posizione in campo. Se vola non attivamente traccia il pennacchio odore, la posizione del porto odore, la portata d&#39;aria e / o la portata di vuoto può avere bisogno di regolazione (cfr. parte 3).

<ol>
	<li>Bender, J. A., Dickinson, 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+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

Tethering mosche e suscitando monitoraggio stabile odore in questo campo è relativamente semplice. Si richiede pratica, pazienza e tentativi ed errori. Molte parti di questa arena può essere ulteriormente ottimizzato o sostituiti per produrre variazioni di potenziale sperimentale. Per esempio, le versioni più recenti della camera a vuoto sono molto più piccoli e permettere più spazio sotto l&#39;arena. Inoltre, la forza e il tipo di magneti in terre rare può essere variato leggermente l&#39;odore e le porte aggiuntive potrebbero esser...

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a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

E &#39;stato chiaro da molti anni che gli insetti utilizzano segnali visivi di stabilizzare la loro voce in un flusso di vento. Molti animali gli odori portata in pista dal vento. Come tale, la stabilizzazione visiva di bolina monitoraggio direttamente aiuti nel monitoraggio degli odori. Ma i segnali olfattivi influenzano direttamente il comportamento visivo di tracciamento indipendente di spunti vento? Inoltre, il diluvio recenti della ricerca sulla neurofisiologia e della genetica neurocomportamentale dell&#39;olfatto in Drosophila ha motivato test comportamentali sempre più tecnicamente sofisticate e quantitativi. Qui, abbiamo modificato un sistema magnetico tether originariamente ideato per gli esperimenti visione dotando l&#39;arena con pennacchi strette a flusso laminare odore. Una mosca è incollata ad un piccolo perno in acciaio e sospesa in un campo magnetico che permette di imbardata liberamente. Di piccolo diametro pennacchi odori degli alimenti sono diretti verso il basso sopra la testa al volo s, suscitando monitoraggio stabile da una mosca fame. Qui ci concentriamo sulla meccanica critica di tethering, allineando i magneti, definire il pennacchio odore, e confermando il monitoraggio stabile odore.

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Qui si descrivono come legare una mosca in un apparato olfattivo magnetico-legare (OMT). Noi descriviamo come allineare la magneti in terre rare e le porte degli odori, e come impostare le portate sia per la consegna stimolo e di aspirazione a vuoto per raggiungere il monitoraggio ottimale degli odori.

Un sistema magnetico Tether per Indagare visivo e olfattivo di controllo mediata Volo in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K Views.  University of California, Los Angeles. Qui si descrivono come legare una mosca in un apparato olfattivo magnetico-legare (OMT). Noi descriviamo come allineare la magneti in terre rare e le porte degli odori, e come impostare le portate sia per la consegna stimolo e di aspirazione a vuoto per raggiungere il monitoraggio ottimale degli odori.

Video: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

All&#39;inizio metamorfica Tecnologia Inserzione per Insect Volo monitoraggio del comportamento

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

Ricerca

Comportamento

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 ...

Biologia

Operant Learning of Drosophila at the Torque Meter

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 breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

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 ...

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Stimolazione Optogenetic del comportamento in Fuga<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Neuroscienze

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

Ad alta risoluzione quantificazione del comportamento Odor-guidata<em&gt; Drosophila melanogaster</em&gt; Utilizzo della<em&gt; Flywalk</em&gt; Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Un test comportamentale per Mechanosensation di cloni in marcm-based<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamenti olfattive analizzati da Computer di monitoraggio di<em&gt; Drosophila</em&gt; In un Olfattometro quattro quadranti

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Il comportamento larvale della drosofila in risposta alla stimolazione optogenetica dei neuroni olfattivi di rilevamento

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

Comportamento animale

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

Un sistema magnetico Tether per Indagare visivo e olfattivo di controllo mediata Volo in Drosophila

Riepilogo

Esplora Altri Video

Capitoli in questo video

All'inizio metamorfica Tecnologia Inserzione per Insect Volo monitoraggio del comportamento

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

L'apprendimento della Drosophila operante presso il misuratore di coppia

Visivamente monitoraggio Odore mediata durante il volo in Drosophila

Stimolazione Optogenetic del comportamento in Fuga

Ad alta risoluzione quantificazione del comportamento Odor-guidata

Un test comportamentale per Mechanosensation di cloni in marcm-based

Comportamenti olfattive analizzati da Computer di monitoraggio di

Il comportamento larvale della drosofila in risposta alla stimolazione optogenetica dei neuroni olfattivi di rilevamento

Comportamento animale