Name: functional analysis of the larval feeding circuit in drosophila
Uploaded: 2013-11-19T16:45:00
Description: Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Forfatterne ønsker å takke presidentens Forskningsfond fra Saint Louis University tildelt WSN

En. Vedlikehold av befolkningen Cages <ol><li> Oppretthold populasjons bur ved 25 ° C på en 12 timers lys-mørke-syklus. Så lenge kontroll-og forsøksgruppene er utsatt for de samme belysningsforhold, da denne teknikken kan utføres i en standard laboratorie-omgivelser. </li><li> Tillate kvinner å legge egg over natten på eplesaft-agar plater. </li><li> Samle nyklekkede larver ved å opprettholde platene med nylig avsatte egg ved 25 ° C i 24 timer. Plasser en liten porsjon av gjær i midten av platen for å tilt...

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Rev. 24, 341-353 (2000).</li><li>Schoofs, A., Niederegger, S., van Ooyen, A., Heinzel, H., Spie&#223;, 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+brain+can+eat:+Establishing+the+existence+of+a+central+pattern+generator+for+feeding+in+third+instar+larvae+of+Drosophila+virilis+and+Drosophila+melanogaster.">The brain can eat: Establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster.</a> J. 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Avvikende utvikling av serotonerge stomatogastric krets, som oppstår under sent embryogenese, vil påvirke sin modne funksjon. Endringer i neurite arkitekturen innervating tarmen kan være korrelert med den funksjonelle utgangen av kretsen, som mates hastighet (målt ved munn krok sammentrekninger i en gjæroppløsning) (figur 1). Bruken av UAS-Gal4 todelt system i Drosophila gjør det mulig å spesifikt målrette opp-eller ned-regulert ekspresjon av et bestemt transkript til et bestemt vev, e...

Drosophila er et ekstremt kraftig modellsystem for å studere neural kretsutvikling på grunn av rask generasjonstid, lav eksperimentelt kostnad og evne til å manipulere og kontrollere genetiske og miljømessige faktorer. Neurogenesis, neuronal Sti finne og synaptogenesis er bevart mellom mennesker og Drosophila, derfor mekanismene i å skape, vedlikeholde og modifisere nevrale kretser er bevart også. Klassiske neurotransmittere, slik som serotonin (5-hydroksytryptamin eller 5-HT) kan tjene som vekstfaktorer før bruk deres rolle som signalmolekyler i den modne nevrale kretsen 1-3 Tidligere studier har vist at opprørt nivåer av 5-HT under embryogenese endre tilkobling av modne nevroner fire. Andre har vist at ektopisk anvendelse av 5-HT til dyrkede neuroner Helisoma undertrykke neurite utvekst samt synaptogenesis 5-7. I Drosophila, er utviklings 5-HT-nivåer inverst relatert til åreknute antall og størrelse, så vel som graden av aborization, langs lengden av neurites rager til forutgående fra CNS 8.. Serotonerg neurotransmisjon har vist seg å modulere foringsoppførsel i forskjellige arter, inkludert Drosophila 8-9. Matekrets i Drosophila er en forholdsvis enkel krets som kan brukes som en modell for å korrelere den funksjonelle utgang (foring) med forandringer i utviklingen av de aksonale projeksjoner fra hjernen til den forutgående. Schoofs et al. har vist at Drosophila larve fôring er regulert av sentrale mønstergeneratorer som påvirker muskulaturen 10. Mens den spesifikke muskel anatomi er ikke helt forstått, har det vist seg at antennal nerve, maxillary nerve, og prothoracic tilbehøret nerve er ansvarlig for de muskuløse målene som er involvert ifôring atferd. De fleste data som involverer muskulaturen og nerve anatomi av virvelløse fôring er begrenset til Calliphora larver. Den tilførselshastighet på annen instar larver kan vurderes ved tilbaketrekning av cephalopharyngeal sclerites (munn kroker), og er reproduserbar og høy gjennomstrømning. De cephalopharyngeal platene er innervert av fibre fra sentrale 5-HT-neuroner via frontpartiet nerve. Den proventriculus, eller forutgående, er innervert av serotonerge fibre (recurrens nerven) som fasciculate i mellomtarmen og som er ansvarlig for sammentrekninger av forutgående (Figur 1) 11-12. Endringer i aksonal forgrening, og antallet og størrelsen av åreknuter langs neurite lengde, kan kvantifiseres ved hjelp av immunhistokjemiske teknikker. Manipulering av neuronal 5-HT under utvikling, enten direkte eller indirekte kan endre den funksjonelle resultatet av denne matekrets, som kan vurderes og korrelert med forandringer i morphology av neurite arkitektur.

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functional analysis of the larval feeding circuit in drosophila

Det serotonerge matekretsen i Drosophila melanogaster larver kan brukes til å undersøke nevronale substrater av kritisk betydning under utviklingen av kretsen. Ved hjelp av den funksjonelle utgangen av kretsen, foring, endringer i neuronal arkitekturen av stomatogastric system kan visualiseres. Mate problemet kan bli registrert ved å observere graden av tilbaketrekkingen av munn kroker, som mottar innervasjon fra hjernen. Bevegelsesatferd anvendes som en fysiologisk kontroll for foring, siden larver bruker sine munn kroker for å krysse over et agar-substrat. Endringer i fôring atferd kan være korrelert med den axonal arkitekturen av neurites innervating tarmen. Ved hjelp av immunohistokjemi er det mulig å visualisere og kvantifisere disse endringene. Feil håndtering av larvene under atferds paradigmer kan endre data som de er svært følsomme for manipulasjoner. Riktig avbildning av neurite arkitektur innervatingtarmen er kritisk for nøyaktig kvantifisering av antallet og størrelsen av åreknuter, så vel som omfanget av grennoder. Analyse av de kretser tillater bare for visualisering av neurite arkitektur eller atferdsmessige effekter, men gjør det mulig for denne modellen en å korrelere den funksjonelle utgangen av kretsen med svekkelser i neuronal arkitektur.

Det serotonerge matekretsen i Drosophila larven kan tjene som en ekstremt effektiv modell for å observere påvirkning av spesielle faktorer på nervesystemet utvikling. Ved kvantifisering mating hastighet, er det mulig å forbinde aksonal arkitekturen av foringskretsen med dets funksjonelle utgang (figur 1). Den lokomotoriske assay anvendes som en fysiologisk kontroll for retraksjon av munn kroker, siden larver bruker sine munn kroker til å drive seg selv på tvers av overflaten av agaren. De...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

Fôrings krets i Drosophila melanogaster larver serverer et enkelt men kraftig modell som gjør at endringer i fôring rate å være korrelert med endringer i stomatogastric nevrale kretser. Denne kretsen består av sentrale serotonerge neuroner som sender anslag til munningen kroker samt forutgående.

Funksjonell analyse av larve Fôring Circuit i Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

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Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Morfologisk analyse av Drosophila Larve perifer sensorisk Neuron dendritter og Axoner bruke Genetic Mosaikk

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisering av Proprioceptors i<em&gt; Drosophila</em&gt; Larver og pupper

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Hele Mount immunolabeling av olfactory Receptor nevroner i<em&gt; Drosophila</em&gt; Antenne

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live-Imaging av<em&gt; Drosophila</em&gt; Larve neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Hvorfor Kvantifisering Matters: Karakterisering av fenotyper på<em&gt; Drosophila</em&gt; Larve Nevromuskulær Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

fjerning av<em&gt; Drosophila</em&gt; Muskelvev fra Larve Fileter for Immunofluorescensanalyse av sensoriske nevroner og epidermal cellene

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Bruke Lineær Agarose kanaler å studere<em&gt; Drosophila</em&gt; Larve Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Fokal Macropatch opptak av Synaptic strøm fra Drosophila larver nevromuskulær krysset

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...