This article details a method for visualizing synaptic vesicle endocytosis at the neuromuscular junction in Drosophila larvae. By using fluorescent membrane dye and a suction microelectrode, researchers can stimulate nerve fibers and observe vesicle dynamics.
Begin with Drosophila larvae, with exposed muscle and nerve fibers immersed in a solution containing vesicles of fluorescent membrane dye, or FM dye.
The FM dye binds to the lipids in the cell membrane, forming a fluorescent coating over it.
Using a micromanipulator unit, position the suction microelectrode tip under a microscope.
Focus on the nerve fibers and the microelectrode.
Use negative pressure to suck a loop of the nerve fiber into the microelectrode.
Apply current pulses to stimulate the nerve fiber.
This triggers the neuromuscular junction, where a motor neuron connects to a muscle fiber, to initiate synaptic vesicle endocytosis.
During this process, the cell engulfs the FM dye-coated membrane to form stained endocytic vesicles, ideal for visualizing presynaptic stages of neurotransmission.
Wash off the FM dye and replace the external solution.
Apply current pulses to unload the FM dye-stained vesicles out of the cell.
For electrical stimulation, prepare a suction pipette using the microelectrode puller to obtain the required taper and tip size. Afterward, fire-polish the microelectrode tip with a microforge to create a tight fit when sucking up a single motor nerve.
Slide the suction pipette onto the electrode holder on a micromanipulator, which is attached to a long, flexible plastic tube and a syringe. Next, put the preparation with FM dye on the microscope stage and raise the stage until the larva and suction pipette are in focus.
Then, suck up a loop of cut motor nerve, innervating the selected hemi-segment with negative air pressure generated by the syringe into the suction electrode. Subsequently, stimulate the motor nerve using the selected parameters to drive SV endocytosis and FM1-43 dye uptake.
The most critical step at this point in the procedure is to suck up a single motor axon into the suction electrode with a tight fit. This may take some trial and error. Once an adequate suction electrode is found, use the same one for all conditions being compared to ensure consistent stimulation strength.
For FM dye unloading, suck the same motor nerve into the same electrode, and then stimulate the motor nerve to activate SV exocytosis and FM1-43 dye release.
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