Analyzing Synaptic Vesicle Recycling and Fluorescent Dye Uptake in Drosophila Larvae

Place a live Drosophila larval neuromuscular preparation in a recording chamber containing calcium-free saline. 

The larval neurons express a light-sensitive cation channel.

Switch to calcium-added saline containing a fluorescent dye.

The dye fluoresces weakly in the non-internalized state but strongly when internalized by the neurons.

Illuminate the larva with blue light.

This activates the channel, inducing cation influx and neuron depolarization, initiating an action potential.

The action potential reaches the synaptic terminal at the neuromuscular junction, allowing calcium influx and vesicle exocytosis.

Subsequently, the dye-bound membrane is internalized during vesicle recycling.

Wash with calcium-free saline to stop vesicle recycling and remove any non-internalized dye.

Visualize the fluorescence signal.

Next, introduce dye-free calcium-added saline and illuminate again.

This induces another action potential, calcium influx, and dye-containing vesicle exocytosis.

Visualize the signal again.

The dye release due to vesicle exocytosis reduces the fluorescence compared to the state during vesicle internalization.