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

Vorremmo ringraziare i membri del laboratorio Mari e il laboratorio Godenschwege, in particolare Aline Yonezawa, per i commenti e aiutare con questo protocollo. Questo lavoro è stato finanziato dall&#39;Istituto Nazionale per la Neurological Disorder and Stroke concessione R21NS06637 di FM e TAG, AB è stato finanziato dalla National Science il premio Foundation numero 082925, URM: Biologia Integrativa per i futuri ricercatori.

1. Elettrofisiologia Rig Set-up <ol><li> L&#39;attrezzatura necessaria per l&#39;impianto elettrofisiologia set-up è descritto in dettaglio da Augustin et al. in questo Journal 14. Si prega di fare riferimento a questo articolo per una spiegazione dettagliata degli apparati elettrofisiologici necessari. </li><li> Modificare il precedentemente descritto elettrofisiologia rig set-up 14 con l&#39;aggiunta di un micromanipolatore sesto, che detiene il nanoinjector. Per accedere facilmente a...

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Il nanoinjection / elettrofisiologia biotest qui presentata consente un rapido screening di composti nel sistema nervoso del moscerino della frutta. Si tratta di una nuova tecnica in vivo che richiede piccole quantità di un composto per provocare un effetto su una varietà di bersagli molecolari in un circuito ben caratterizzato neuronale. Questo metodo può essere utilizzato per testare la bioattività di diversi composti, da tossine sconosciuti agenti farmacologici disponibili in commercio. <p class="jov...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Registrazione elettrodi di vetro: vetro borosilicato capillari </td><td> Mondo Strumenti di precisione </td><td> 1B100F-4 </td><td> 1,0 millimetri OD, 0,58 millimetri ID </td></tr><tr><td> Stimolatore </td><td> Grass Instruments </td><td> Modello S48 </td><td></td></tr><tr><td> Amplificatore </td><td> Come Instruments Inc. </td><td> Modello 5A </td><td></td></tr><tr><td> Acquisizione dati Software: Digidata </td><td> Molecular Devices </td><td> Modello 1440A </td><td></td></tr><tr><td> Software di raccolta dati: PClamp </td><td> Molecular Devices </td><td> Versione 10 </td><td></td></tr><tr><td> Stereomicroscopio wesima fibra ottica microscopio illuminatore anulare </td><td> AmScope </td><td> SM-4T Modello HL 250-AR </td><td></td></tr><tr><td> Dissezione possibilità per il montaggio </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Kite &amp; Manual Micromanipolatore Tilting Base </td><td> Mondo Strumenti di precisione </td><td> Model # M3301 Kite: Model # KITE-M3-L </td><td></td></tr><tr><td> Drosophila melanogaster selvatica 10E genotipo (ceppo selvatico) </td><td> Bloomington magazzino centrale </td><td> Stock # 3892 </td><td></td></tr><tr><td> Vertical pipette puller </td><td> David Kopf Instruments </td><td> Modello 700c </td><td></td></tr><tr><td> Iniezione micropipette di vetro: vetro borosilicato capillari </td><td> Mondo Strumenti di precisione </td><td> Catalogo # 4878 </td&gt; <td> 1,14 millimetri OD, 0.5mm ID </td></tr><tr><td> Olio siliconico </td><td> Pescatore </td><td> Catalogo # S159-500 </td><td></td></tr><tr><td> Beyeler </td><td> Sutter Instrument Co. </td><td> KT Brown Type Model # BV-10 </td><td></td></tr><tr><td> Nanoliter2000 </td><td> Mondo Strumenti di precisione </td><td> Catalogo # B203XVY </td><td></td></tr><tr><td> Colorante alimentare blu </td><td> McCormick </td><td> N / A </td><td> Ingredienti: acqua, glicole propilenico, FD &amp; C Blue 1, e 0,1% Propylparaben (conservante). </td></tr><tr><td> Methyllycaconitine citrato (MLA) </td><td> Bioscience Tocris </td><td> Catalogo # 1029 </td><td></td></tr><tr><td> Bastoncini di cera plastica </td><td> Hygenic 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

Composti di screening per l&#39;attività in vivo può essere utilizzato come un primo passo per identificare candidati che possono essere sviluppati in 1,2 agenti farmacologici. Abbiamo sviluppato un nanoinjection / elettrofisiologia nuovo metodo di analisi che permette il rilevamento di effetti modulatori di composti bioattivi sulla funzione di un circuito neuronale che media la risposta di fuga in Drosophila melanogaster 3,4. Il saggio in vivo, che utilizza la fibra Drosophila Giant System (GFS, Figura 1) permette lo screening dei vari tipi di composti, come piccole molecole o peptidi, e richiede quantità minime per provocare un effetto. Inoltre, il GFS Drosophila offre una grande varietà di potenziali bersagli molecolari sui neuroni e muscoli. Le fibre giganti (GFS) sinapsi elettricamente (Gap Junction) e chimicamente (colinergica) su una sinapsi Interneuron Peripheral (PSI) e il Trochanteral Tergo Muscle neurone (TTMn) 5 </sup&gt;. Il PSI di DLMn (dorsale neurone muscolare longitudinale) la connessione dipende Dα7 recettori nicotinici dell&#39;acetilcolina (nAChR) 6. Infine, la giunzioni neuromuscolari (NMJ) del TTMn e la DLMn con il salto (TTM) e dei muscoli di volo (DLM) sono glutamatergica 7-12. Qui, si dimostra come iniettare quantità nanolitri di un composto, ottenendo registrazioni elettrofisiologiche intracellulari dal sistema fibra Giant 13 e come controllare gli effetti del composto sulla funzione di questo circuito. Mostriamo la specificità del test con citrato methyllycaconitine (MLA), un antagonista nAChR, che interrompe il PSI di DLMn connessione, ma non il GF a TTMn connessione o la funzione del NMJ al salto o dei muscoli del volo. Prima di iniziare questo video, è fondamentale che si guarda con attenzione e familiarizzare con il video intitolato &quot;JOVE registrazioni elettrofisiologiche dal sentiero fibra gigante di D. Melanogaster &quot;da Augustin et al 7, come il video presentato qui è inteso come un ampliamento di questa tecnica esistente. Qui si utilizza il metodo elettrofisiologico registrazioni e focalizzazione in dettaglio solo l&#39;aggiunta delle nanoinjections accoppiati e la tecnica di monitoraggio.

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

Un rapido<em&gt; In vivo</em&gt; Test per verificare i composti neuromodulatori che utilizzano il sistema di Giant Fiber (GFS) di<em&gt; Drosophila melanogaster</em&gt; Descritto. Nanoinjections nella testa dell&#39;animale insieme registrazioni elettrofisiologiche di GFS può rivelare la bioattività dei composti sui neuroni e muscoli.

Nanoinjection associato e test Elettrofisiologia allo schermo per la bioattività dei composti con il Drosophila melanogaster Sistema Fiber Giant

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 ...

Research

JoVE Journal

Neuroscience

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

Le registrazioni elettrofisiologiche dal Percorso di Fiber Gigante D. melanogaster</em

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

Ricerca

Neuroscienze

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

Monte Preparazione tutto il Adulti Drosophila Cordone nervoso ventrale per iniezione Giant Dye Fibra

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

Etichettatura di singole cellule nel sistema nervoso centrale di<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 ...

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Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determinazione del locomotore attività spontanea in<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

Assay Semplice per la quantificazione della Social Prevenzione in<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

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

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

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 ...

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 test per esaminare Preferenze deposizione delle uova dei singoli<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

Un modo semplice per misurare alterazioni in ricompensa il comportamento di ricerca Utilizzando<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

Il capillare alimentatore Assay Misure assunzione di cibo in<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 ...