Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

Abstract

Source: Vasin, A., et al. Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction. J. Vis. Exp. (2017)

This video demonstrates the technique for recording synaptic currents from a synaptic bouton at the Drosophila larva's neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton, which is important for the study of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Protocol

1. Fabrication of Recording Electrodes

1. Pulling the glass electrodes
   1. Use the following protocol for the microelectrode puller (see Table of Materials):
      1. Line 1: Heat 510 Pull - Velocity 30 Time 250; Line 2: Heat 490 Pull - Velocity 30 Time 250.
         NOTE: Time units correspond to 0.5 ms per unit; the other units are relative. The value of the heat should be adjusted for every filament after the ramp test is performed.
   2. Use a microscope (35x magnification) to ensure that the inner diameter of the pulled electrode is in the range of 7 - 1....

Results

Figure 1. Final steps of micropipette fabrication. (A) Electrode tips after pulling and fire polishing. (B.1) The setup for tip bending. (B.2) The boxed area is shown enlarged on the right. The electrode and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microtorch 1WG61	Grainer	1WG61	Microtorch
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