Overview of a representative dopaminergic synapse and labeling by FFNs and APP+. (A) Dopamine is synthesized in the DA neuron and packaged into synaptic vesicles by the action of VMAT2 (blue ovals). Upon arrival of an action potential, DA is released via exocytosis into the synaptic cleft and extrasynaptic space where it interacts with pre- and postsynaptic dopamine receptors, and is transported in large part back into the presynaptic neuron via DAT. MSN = medium spiny neuron, D2 = dopamine receptor D2. (B) FFNs such as FFN102 are actively transported into dopaminergic axonal structures and stain both the cytosol and the synaptic vesicles. (C) Analogously, according to the proposed model, APP+ is transported via the same plasma membrane transporters, although APP+ then labels mitochondria, driven by the electrochemical potential across the mitochondrial inner membrane, as well as synaptic vesicles via VMAT2. The polar environment inside the cytosol and synaptic vesicle lumen likely leads to significant quenching of APP+ fluorescence. The vesicular signal is overwhelmed by the fluorescence from mitochondria, resulting in a nonreleasable fluorescent signal.