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 ønsker å takke medlemmene av Mari lab og Godenschwege lab, særlig Aline Yonezawa, for kommentarer og hjelp med denne protokollen. Dette arbeidet ble finansiert av National Institute for nevrologiske lidelser og Stroke stipend R21NS06637 til FM og TAG; AB ble finansiert av National Science Foundation award nummer 082925, URM: Integrative Biology for fremtidige forskere.

1. Elektrofysiologi Rig Set-up <ol><li> Den nødvendige utstyret for elektrofysiologi riggen Oppsettet er beskrevet i detalj av Augustin et al. i denne Journal 14. Vennligst referer til denne artikkelen for en detaljert forklaring av elektrofysiologiske apparater som trengs. </li><li> Endre tidligere beskrevet elektrofysiologi rigg set-up 14 ved å legge en sjettedel micromanipulator, som holder nanoinjector. For enkel tilgang til hodet på dyret det skal plasseres mellom de to stimulere...

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Den nanoinjection / elektrofysiologi bioassay presenteres her åpner for en rask screening av forbindelser i nervesystemet av bananflue. Dette er en roman in vivo teknikk som krever små mengder av et sammensatt for å lokke fram en effekt på en rekke molekylære mål i en godt karakterisert neuronal krets. Denne metoden kan brukes til å teste bioaktivitet av ulike forbindelser, fra ukjente giftstoffer til kommersielt tilgjengelige farmakologiske agenter. Her demonstrerte vi funks...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Opptak glass elektroder: borsilikatglass capillaries </td><td> Verden presisjonsinstrumenter </td><td> 1B100F-4 </td><td> 1.0mm OD, 0.58mm ID </td></tr><tr><td> Stimulator </td><td> Grass Instrumenter </td><td> Model S48 </td><td></td></tr><tr><td> Forsterker </td><td> Komme Instruments Inc. </td><td> Model 5A </td><td></td></tr><tr><td> Datainnsamling Programvare: Digidata </td><td> Molekylære enheter </td><td> Model 1440A </td><td></td></tr><tr><td> Datainnsamling programvare: pCLAMP </td><td> Molekylære enheter </td><td> Versjon 10 </td><td></td></tr><tr><td> Stereomikroskop wed fiberoptisk mikroskop ring illuminator </td><td> AmScope </td><td> SM-4T Modell HL250-AR </td><td></td></tr><tr><td> Dissekere muligheter for montering </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Kite Manuell Micromanipulator &amp; Skråstille Base </td><td> Verden presisjonsinstrumenter </td><td> Model # M3301 Kite: Model # KITE-M3-L </td><td></td></tr><tr><td> Drosophila melanogaster Wild 10E genotype (villtype stamme) </td><td> Bloomington Stock sentrum </td><td> Stock # 3892 </td><td></td></tr><tr><td> Vertikal pipette avtrekker </td><td> David Kopf Instrumenter </td><td> Modell 700C </td><td></td></tr><tr><td> Injection glass mikropipetter: borsilikatglass capillaries </td><td> Verden presisjonsinstrumenter </td><td> Katalog # 4878 </td&gt; <td> 1.14mm OD, 0.5mm ID </td></tr><tr><td> Silicon olje </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 Modell # BV-10 </td><td></td></tr><tr><td> Nanoliter2000 </td><td> Verden presisjonsinstrumenter </td><td> Katalog # B203XVY </td><td></td></tr><tr><td> Blå konditorfarge </td><td> McCormick </td><td> N / A </td><td> Ingredienser: Vann, Propylene Glycol, FD &amp; C 1 Blå, og 0,1% Propylparaben (konserveringsmiddel). </td></tr><tr><td> Methyllycaconitine citrat (MLA) </td><td> Tocris Biovitenskap </td><td> Katalog # 1029 </td><td></td></tr><tr><td> Plast voks pinner </td><td> Hygienisk 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 forbindelser for in vivo aktivitet kan brukes som et første skritt for å identifisere kandidater som kan utvikles til farmakologisk agenter 1,2. Vi utviklet en roman nanoinjection / elektrofysiologi analysen som tillater påvisning av bioaktive regulerende effekt av forbindelser på funksjonen til en neuronal krets som medierer flukten respons i Drosophila melanogaster 3,4. Vår in vivo-forsøk, som bruker Drosophila Giant Fiber System (GFS, figur 1) tillater screening av ulike typer forbindelser, for eksempel små molekyler eller peptider, og krever bare minimale mengder å lokke fram en effekt. I tillegg tilbyr Drosophila GFS et stort utvalg av potensielle molekylære mål på nerveceller og muskler. The Giant fiber (GFS) synapse elektrisk (Gap veikryss) samt kjemisk (kolinerge) på en ytre Synapsing Interneuron (PSI) og Tergo Trochanteral Muscle nervecellen (TTMn) 5 </sup&gt;. PSI til DLMn (Dorsal Longitudinal Muscle nevron) tilkoblingen er avhengig Dα7 nikotin acetylkolinreseptorer (nAChRs) 6. Til slutt, den nevromuskulære knutepunktene (NMJ) av TTMn og DLMn med hopp (TTM) og fly muskler (DLM) er glutamatergic 7-12. Her viser vi hvordan du injiserer nanoliter mengder av et stoff, mens skaffe elektrofysiologiske intracellulære opptak fra Giant Fiber System 13, og hvordan å overvåke virkningene av det sammensatte på funksjonen til denne kretsen. Vi viser spesifisitet av analysen med methyllycaconitine citrat (MLA), en nAChR antagonist, som forstyrrer PSI til DLMn tilkobling, men ikke GF å TTMn tilkobling eller funksjonen av NMJ på hopp eller fly muskler. Før du begynner denne videoen er det viktig at du nøye se og bli kjent med Jove video med tittelen &quot;Elektrofysiologiske Opptak fra Giant Fiber Pathway av D. melanogaster &quot;fra Augustin et al 7, som video som presenteres her er ment som en utvidelse til denne eksisterende teknikken. Her bruker vi elektrofysiologisk innspillinger metode og fokus på detaljer bare om tilsetting av de sammenkoblede nanoinjections og overvåking teknikk.

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 rask<em&gt; In vivo</em&gt; Analysen for å teste for neuromodulatory forbindelser ved hjelp av Giant Fiber System (GFS) av<em&gt; Drosophila melanogaster</em&gt; Er beskrevet. Nanoinjections i hodet på dyret sammen med elektrofysiologiske opptak av GFS kan avsløre bioaktivitet av forbindelser på nevroner og muskler.

Sammenkoblede Nanoinjection og Elektrofysiologi Assay til skjerm for bioaktivitet av forbindelser ved hjelp av Drosophila melanogaster Giant Fiber System

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

Merking av enkeltceller i Central Nervous System av<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

Fastsettelse av Spontan Locomotor 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

Enkel analyse for Kvantifisering av Social Avhold 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

Høyoppløselig Kvantifisering av Lukt styrt Atferd<em&gt; Drosophila melanogaster</em&gt; Bruke<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 analyse for Mechanosensation av MARCM baserte Clones 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

Høy gjennomstrømning analyse for å undersøke Eggleggingen Preferences av Individuell<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 enkel måte å måle endringer i Reward-søkende atferd med<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

Kapillær mater analysen Måler matinntak 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 ...