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

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.This work was financially supported by K08 DA031241 (Z.F.), Louis V. Gerstner, Jr, Scholars Program (Z.F.), Leon Levy Foundation (Z.F.), NARSAD Young Investigator Award (C.S.K.), K05 DA022413, (J.A.J.), P01 DA12408 (J.A.J.), Lieber Center for Schizophrenia Research and Treatment award (J.A.J.), R01 DA007418 (D. Sulzer), PO1 DA0 10154 (D. Su.), JPB and Parkinson's Disease Foundations (D. Su.), R01 MH086545 (D. Sames), G. Harold & Leila Y. Mathers Charitable Foundation (D. Sa.), NS075572 (B.D.M.), AG08702 (B.D.M.), the Dana Foundation (B.D.M.), the Gatsby Initiative in Brain Circuitry (B.D.M.), New York Presbyterian Seizure Disorders Fund (B.D.M.), R01 MH076900 (D.E.K.), R01 ES015747 (D.E.K.), P01 ES016732 (D.E.K.), Brain and behaviour Research Foundation (D.E.K.), UCLA Brain Research Institute (D.E.K.), UCLA Molecular Toxicology Program, Ruth L. Kirschstein T32 ES015457 (C.A.M.), Ruth L. Kirschstein GM07185 (A.C.), R01 NS075222 (E.M.), and the Intramural Research Program of NIDA (J.L.K.)