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

Zachary Freyberg,Mark S Sonders,Jenny I Aguilar,Audrey Chen,Eugene V Mosharov,Takato Hiranita,Robin Freyberg,Zachary J Farino,David Sulzer,Kandatege Wimalasena,Brian D Mccabe,Yuchao Zhang,Jonathan L Katz,Caline S Karam,Hao Fei,Jonathan A Javitch,Gang Hu,Andrea B Pizzo,David E Krantz,Ciara A Martin,Jorge A Flores ,Ling‐Wei Hsin ,Theresa Kopajtic ,Dalibor Sameš ,Yi‐Ying Lin

doi:10.1038/ncomms10652

Abstract

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.

Highlights

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain
To validate VMAT2’s role in acute behavioural actions of amphetamines in rodents, we developed ( þ )-CYY477, a novel derivative of dihydrotetrabenazine that retains high VMAT2 affinity but has better selectivity (Supplementary Note 1). ( þ )CYY477 potently inhibited dopamine uptake by rat VMAT2 expressed in cultured cells (half-maximal inhibitory concentration (IC50) 1⁄4 26 nM; Supplementary Fig. 1a). ( þ )-CYY477 exhibited good selectivity for vesicular over plasma membrane monoamine transporters
That ( þ )-CYY477 selectively antagonized amphetamine and methamphetamine-induced behaviours but not those of cocaine, clearly illustrates that VMAT2 plays a critical role in the acute actions of amphetamines but not those of cocaine

Summary

Introduction

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Amphetamines are weak bases (pKa 8.8–9.9) and have been proposed to decrease the vesicular DpH by buffering luminal free protons, thereby depleting vesicles of stored dopamine and indirectly blocking substrate import This is the mechanism of action for weak bases such as chloroquine, which is lipophilic enough to diffuse across lipid bilayers (LogP 1⁄4 4.72)[11,12,13,14]. Amphetamines are less lipophilic (for example, LogP of amphetamine 1⁄4 1.41)[15], they have been hypothesized both to enter vesicles by diffusion as well as by VMAT-mediated transport[2] Evidence for these diverse mechanisms has come in large part from in vitro studies of isolated vesicles, cells and brain slices, but the actual relevance of these proposed mechanisms to amphetamines’ in vivo actions has not been ascertained

Methods

Results

Conclusion