Visualizing Neurotransmission Using Fluorescent False Neurotransmitters

Begin with a brain slice rich in dopaminergic neurons.

Incubate the slice in aCSF containing fluorescent false neurotransmitters or FFNs.

FFNs are pH-sensitive markers that mimic dopamine and fluoresce more intensely in neutral than acidic environments.

These FFNs selectively enter dopaminergic neurons and get loaded into acidic synaptic vesicles, where the pH change reduces FFN fluorescence.

Transfer this slice to an imaging chamber, secure it, and continuously perfuse with aCSF to remove unbound FFNs.

Image the slice to confirm FFN accumulation as fluorescent puncta.

Next, position a stimulating electrode to deliver electrical stimulation.

The electrical pulses cause calcium to enter the neuron, prompting synaptic vesicle fusion with the neuronal membrane and the release of FFNs into the extracellular synaptic cleft.

In the neutral pH environment of the synaptic cleft, FFN fluorescence intensifies.

Image the slice again to observe diffuse fluorescence in the extracellular space, confirming FFN release from synaptic vesicles.