This article describes a method for studying synaptic transmission in Drosophila larval neuromuscular junctions using optogenetics and fluorescence imaging. The technique allows for the visualization of vesicle recycling and calcium dynamics during neuronal activity.
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.
For channelrhodopsin stimulation, attach a blue LED to a programmable stimulator using a coaxial cable and place the LED into the camera port. Focus the blue LED light beam onto the preparation with FM dye using the microscope Zoom function.
The most critical step at this point in the procedure is to make sure that the LED settings are kept constant, and that you illuminate the same part of each animal for all conditions being compared to ensure consistent stimulation strength.
Start the light stimulation, and track with the timer for the predetermined duration of the optogenetic stimulation period. When the timer stops, quickly remove the FM dye solution, and replace it with calcium-free saline to stop the SV cycling. For FM dye unloading, start the light stimulation and track with the timer for the predetermined duration of the optogenetic stimulation period. When the timer period ends, quickly remove the FM dye solution, and replace it with calcium-free saline to stop the SV cycling.
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