Focal Macropatch optagelser af Synaptic strømninger fra Drosophila larve neuromuskulære Junction
Summary
Synaptic strømme kan registreres focally fra visualiseret synaptic boutons i Drosophila tredje instar larver neuromuskulære junction. Denne teknik gør det muligt for overvågning af aktiviteten af et enkelt synaptic bouton.
Abstract
Drosophila neuromuskulære junction (NMJ) er en fremragende modelsystem til at studere glutamatergic synaptisk transmission. Vi beskriver teknik af focal macropatch optagelser af synaptic strømninger fra visualiseret boutons i Drosophila larve NMJ. Denne teknik kræver tilpassede fabrikation af optager Mikropipetter, samt et sammensat mikroskop udstyret med en høj forstørrelse, langdistance vand fordybelse mål, differential interferens kontrast (DIC) optik og en fluorescerende vedhæftet fil. Optagelse elektrode er placeret på toppen af en markeret synaptic bouton visualiseret med DIC optik, epi-fluorescens eller begge. Fordelen ved denne teknik er, at det giver mulighed for overvågning af synaptic aktivitet af et begrænset antal sider om frigivelse. Optagelse elektroden har en diameter på flere mikron, og frigivelse sites placeret uden for den elektrode rand påvirker ikke væsentligt de indspillede strømninger. De indspillede synaptic strømninger har hurtig kinetik og kan løses let. Disse fordele er især vigtige for studier af mutant flyve linjer med forbedret spontan eller asynkron synaptic aktivitet.
Introduction
Drosophila er en fremragende modelsystem til at studere de molekylære mekanismer kontrollerende synaptisk transmission. Det neuromuskulære system i Drosophila er glutamatergic, og derfor Drosophila neuromuskulære junction (NMJ) kan bruges til at studere de bevarede funktioner i glutamatergic udgivelse. Siden Jan og Jans studie1, er de tredje instar larver bredt plejede at studere evoked og spontane synaptisk transmission af overvågning excitatoriske junction potentialer (EJPs) eller strømme (EJCs). EJPs registreres almindeligt intracellulært med en skarp glas mikro-elektrode, og de afspejler aktiviteten af det hele NMJ, herunder alle boutons at synapser på den givne muskelfiber.
Derimod kan aktivitet af et begrænset antal sider om frigivelse registreres focally ved at placere en mikropipette tip nær neuronal terminaler eller synaptic varicosities. Denne teknik blev oprindeligt ansat af Katz og Miledi2, og fokale ekstracellulære optagelser er blevet med held ansat på flere NMJ præparater, herunder frog3,4,5, mus6 , 7 , 8, krebsdyr9,10,11,12,13,14,15,16og Drosophila17,18,19,20,21,22,23. Denne tilgang blev yderligere udviklet af Dudel, der optimeret macropatch recoding elektroder24,25. I Dudels gennemførelse afstemt denne teknik nøje løs-patch-clamp metode26.
Drosophila larve NMJ har klart defineres synaptisk boutons, og transgene linjer med genetisk kodet neuronal fluorescerende tags (Se Tabel af materialer) er let tilgængelige. Disse fordele gjorde det muligt at optage EJCs og mEJCs fra en valgte synaptic bouton20,21,22. Her beskriver vi denne teknik i detaljer.
Protocol
Representative Results
Discussion
Drosophila repræsenterer en fordelagtig model organisme at studere synaptisk transmission. Flere optagelse konfigurationer er blevet anvendt på de larver NMJ, herunder intracellulære optagelser af synaptiske potentialer, optagelser af synaptic strømme med to elektrode spænding klemme33,34, og focal macropatch optagelser af synaptic strømninger beskrevet her. Sidstnævnte teknikken tillader en præcis kvantificering af synaptisk transmission på vis…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Støttet af NIH grant R01 MH 099557
Materials
| Sutter P-97 | Sutter instrument | P-97 | Microelectrode puller |
| Narishige MF-830 | Narishige | MF-830 | Microforge |
| WPI MF200 | WPI | MF200 | Microforge |
| Glass capilaries | WPI | B150-86-10 | Glass capilaries |
| Microtorch 1WG61 | Grainer | 1WG61 | Microtorch |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | SYLGARD 184 | Silicone for dissection plates preparation |
| Dissection pins | Amazon | B00J5PMPJA | Pins for larvae positioning |
| Tweezers | WPIINC | 500342 | Tweezers for placing pins, removing the guts and tracheas. |
| Scissors | WPIINC | 501778 | Scissors for cutting the cuticula of the larvae and nerves. |
| Olympus BX61WI | Olympus | BX61WI | Upright microscope |
| Olympus Lumplan FL N 60x | Olympus | UPLFLN 60X | Microscope objective 60X |
| Olympus UPlan FL N 10x | Olympus | Uplanfl N 10X | Microscope objective 10X |
| Narishige Micromanipulator | Narishige | MHW-3 | Three-axis Water Hydraulic Micromanipulator |
| npi Electronic GmbH ELC-03XS | npi Electronic GmbH | ELC-03XS | Electrophysiological amplifier |
| A.M.P.I Master 8 | A.M.P.I. | Master 8 | Electrical stimulator |
| A.M.P.I Iso-Flex | A.M.P.I. | Iso-Flex | Stimulus isolator |
| TMC antivibration table | TMC | 63-9090 | Antivibration table |
| TMC Faraday cage | TMC | 81-333-90 | Faraday cage |
| Digidata 1322A | Axon Instruments | Digidata 1322A | Digidata |
| Computer | Dell | Dell Dimension 5150 | Computer with Win XP OS |
| Electrode holder | WPI | MEH3SW | Electrode holder |
| Optical filter | Omega optical | XF 115-2 | Filter cube for Green Fluorescent Protein (GFP) detection |
| pCLAMP 8 | Axon Instruments | 8.0.0.81 | Software for signal recording |
| Quantan | In-house software | – | Software for signal processing |
| Canton-S (Wildtype) | Bloomington Stock Center | 64349 | Control fly line |
| cpx SH1 | Generous Gift of J.T. Littleton | – | Complexin knock-out fly line with increased spontaneous exocytosis |
| CD8-GFP | Bloomington Stock Center | 5137 | Fly line with neuronal fluorescent (GFP) Tag |
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