Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

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.