Name: paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the drosophila melanogaster giant fiber system
Uploaded: 2012-04-15T14:00:00
Description: Watch this Scientific Journal Video about Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System at JoVE.com

Vi vil gerne takke medlemmerne af Mari laboratoriet og Godenschwege laboratoriet, især Aline Yonezawa, for kommentarer og hjælpe med denne protokol. Dette arbejde blev finansieret af det nationale institut for neurologiske sygdomme og slagtilfælde tilskud R21NS06637 til FM og TAG, AB blev finansieret af National Science Foundation prisen nummer 082925, URM: Integrative Biology for fremtidens forskere.

1. Elektrofysiologi Rig Set-up <ol><li> Det krævede udstyr til elektrofysiologi riggen opsætning er beskrevet i detaljer af Augustin et al. i denne Journal 14. Der henvises til denne artikel for en detaljeret forklaring af de elektrofysiologiske nødvendige apparater. </li><li> Rediger tidligere beskrevet elektrofysiologi rig set-up 14 ved at tilsætte 1/6 mikromanipulator, som holder nanoinjector. For nem adgang til dyrets hoved den skal placeres mellem de to stimulerende elektrode mi...

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Den nanoinjection / elektrofysiologi bioassay præsenteret her tillader en hurtig screening af forbindelser i nervesystem frugtflue. Dette er en hidtil ukendt in vivo teknik, der kræver små mængder af en forbindelse til at fremkalde en effekt på en række forskellige molekylære mål i en velkarakteriseret neuronal kredsløb. Denne fremgangsmåde kan anvendes til at teste den biologiske aktivitet af forskellige forbindelser, fra ukendte toksiner til kommercielt tilgængelige farmakologiske midler. <p cl...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Optagelse glaselektroder: borsilicatglas kapillærer </td><td> World Precision Instruments </td><td> 1B100F-4 </td><td> 1,0 OD, 0.58mm ID </td></tr><tr><td> Stimulator </td><td> Grass Instruments </td><td> Model S48 </td><td></td></tr><tr><td> Forstærker </td><td> Kom Instruments Inc. </td><td> Model 5A </td><td></td></tr><tr><td> Dataopsamlingssoftware: Digidata </td><td> Molecular Devices </td><td> Model 1440A </td><td></td></tr><tr><td> Dataindsamling software: pCLAMP </td><td> Molecular Devices </td><td> Version 10 </td><td></td></tr><tr><td> Stereomikroskop wed fiberoptisk mikroskop ring illuminator </td><td> AmScope </td><td> SM-4T Model HL250-AR </td><td></td></tr><tr><td> Dissektionsmikroskop for montering </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Kite Manuel mikromanipulator &amp; Vipbar Base </td><td> World Precision Instruments </td><td> Model # M3301 Kite: Model # KITE-M3-L </td><td></td></tr><tr><td> Drosophila melanogaster Wild 10E genotype (vildtype-stamme) </td><td> Bloomington Stock center </td><td> Stock # 3892 </td><td></td></tr><tr><td> Lodrette pipettedrev aftrækker </td><td> David Kopf Instruments </td><td> Model 700c </td><td></td></tr><tr><td> Injection glas mikropipetter: Borosilikat glas kapillærer </td><td> World Precision Instruments </td><td> Katalog # 4878 </td&gt; <td> 1,14 mm OD, 0,5 ID </td></tr><tr><td> Siliconeolie </td><td> Fisher </td><td> Katalog # S159-500 </td><td></td></tr><tr><td> Beveler </td><td> Sutter Instrument Co </td><td> KT Brown Type Model # BV-10 </td><td></td></tr><tr><td> Nanoliter2000 </td><td> World Precision Instruments </td><td> Katalog # B203XVY </td><td></td></tr><tr><td> Blå levnedsmiddelfarve </td><td> McCormick </td><td> N / A </td><td> Ingredienser: vand, propylenglycol, FD &amp; C Blue 1, og 0,1% propylparaben (konserveringsmiddel). </td></tr><tr><td> Methyllycaconitine citrat (MLA) </td><td> Tocris Bioscience </td><td> Katalog # 1029 </td><td></td></tr><tr><td> Plast voks sticks </td><td> Hygiejnisk Corporation (Akron Ohio USA) </td><td></td><td></td></tr></tbody&gt; </table>

paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the drosophila melanogaster giant fiber system

Screening af forbindelser for aktivitet in vivo, kan anvendes som et første trin til at identificere kandidater, der kan udvikles til farmakologiske midler 1,2. Vi udviklet et hidtil ukendt nanoinjection / elektrofysiologi assay, som tillader påvisning af bioaktive modulatoriske virkninger af forbindelser på funktionen af en neuronal kredsløb, der medierer flugtrespons i Drosophila melanogaster 3,4. Vores in vivo assay, der anvender Drosophila Giant Fiber System (GFS, figur 1) tillader screening af forskellige typer af forbindelser, såsom små molekyler eller peptider, og kræver kun minimale mængder til at fremkalde en virkning. Desuden tilbyder Drosophila GFS en lang række potentielle molekylære mål på neuroner eller muskler. De Giant Fibers (GFS) synapse elektrisk (gap junctions) samt kemisk (kolinerge) på en ekstern Synapsing Interneuron (PSI) og Tergo trochanterale Muscle neuron (TTMn) 5 </sup&gt;. PSI til DLMn (Dorsal Longitudinal Muscle neuron) tilslutning er afhængig Dα7 nikotinacetylcholinreceptorer (nAChRs) 6. Endelig neuromuskulære junctions (NMJ) af TTMn og DLMn med spring (TTM) og fly muskler (DLM) er glutamaterge 7-12. Her vil vi vise, hvordan man kan injicere nanoliter mængder af et stof, mens få elektrofysiologiske intracellulære optagelser fra Giant Fiber System 13 og hvordan man kan overvåge virkningerne af forbindelsen på funktionen af dette kredsløb. Vi viser specificiteten af ​​assayet med methyllycaconitine citrat (MLA), en nAChR-antagonist, som forstyrrer PSI DLMn forbindelse, men ikke GF til TTMn forbindelse eller funktionen af ​​NMJ ved hop eller flugt muskler. Inden du begynder denne video er det vigtigt, at du nøje se og blive fortrolig med JOVE video med titlen &quot;Elektrofysiologiske Optagelser fra Giant Fiber Pathway af D. melanogaster &quot;fra Augustin et al 7, som videoen præsenteres her er tænkt som en udvidelse til den eksisterende teknik. Her bruger vi elektrofysiologiske optagelser metode og fokuserer i detaljer kun på tilsætning af de parrede nanoinjections og overvågning af teknik.

Watch this Scientific Journal Video about Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System at JoVE.com

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

En hurtig<em&gt; In vivo</em&gt; Assay til at teste for neuromodulatory forbindelser under anvendelse af Giant Fiber System (GFS) af<em&gt; Drosophila melanogaster</em&gt; Er beskrevet. Nanoinjections i hovedet af dyret sammen med elektrofysiologiske optagelser af GFS kan afsløre bioaktivitet af forbindelser på neuroner eller muskler.

Parret Nanoinjection og Elektrofysiologi Assay til screening for bioaktivitet af forbindelser ved hjælp af Drosophila melanogaster Giant fibersystem

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Mærkning af enkeltceller i centralnervesystemet hos<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Bestemmelse af Spontan motorisk aktivitet i<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Ligetil Assay til kvantificering af Social undgåelse i<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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

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

Høj opløsning Kvantificering af Lugt-styrede adfærd i<em&gt; Drosophila melanogaster</em&gt; Brug af<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

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

En Behavioral Assay for Mechanosensation af MARCM-baserede kloner i<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 ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay til at undersøge æglæggende præferencer af Individuel<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

En simpel måde at måle Ændringer i Reward-søger Behavior Brug<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Den kapillære Feeder-analysen Måler fødeindtagelse i<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...