Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

In this paper, two-photon microscopy is utlized in a scientific research context of important sociological impact. Specifically, the authors are investigating the effects of amphetamine, one of the most widely used and abused drugs, on the neuronal microenvironment by following in real time the dynamics of dopamine vesicles in the viable ex vivo Drosophila brain.

In a series of experiments, a fluorescent substrate of the vesicular monoamine transporter (VMAT) and a fluorescent biosensor of the intraluminal vesicular pH are used in order to directly monitor monoamine loading and release from synaptic vesicles, as well as changes in monoamine vesicle pH under amphetamine treatment in WT and transporter mutant Drosophila brains.

By combining different scientific approaches, such as genetic manipulation, optical monitoring through two-photon microscopy and pharmacology, the group was able to demonstrate for the first time the molecular mechanisms at the basis of amphetamine-mediated dopamine extracellular release, which determines the psychomotor stimulation and the behavioral effects already observed in mammals.

Clinically relevant amphetamine concentrations are able to alkalize dopamine vesicle pH and induce content release after its functional active transportation at both the plasma membrane (via DAT) and at the vesicular membrane (via VMAT) levels. Moreover, the mechanism of alkalization was also innovatively identified as an antiport, i.e. net export of H+ ions via VMAT for every amphetamine molecule transported into the vesicle.

In conclusion, the results provide a model for how pharmacologically relevant concentrations of amphetamines increase extracellular dopamine. The results also demonstrate the viability and utility of a novel experimental system for invesitgating the physiology of intact monoaminergic vesicles.