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

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.

Striatal dopamine neurotransmission: Regulation of release and uptake

“…In Drosophila, reserpine prevents methamphetamine-induced loss of protons, while chloroquine, a non-VMAT substrate weak base, is not prevented by reserpine. The toxin MPP+ and its derivatives that are non-protonable VMAT substrates also collapse the pH in a reserpine-sensitive manner [85]. Together, these findings are consistent with a requirement for a role for VMAT uptake and net proton antiport in the amphetamine-mediated collapse of proton gradients of small synaptic vesicles, at least at lower drug concentrations.…”

“…Another challenge is that pHluorin probes are most sensitive near their pK which is near 7, so that a change from pH 5.0 to 5.5, typical of large dense core catecholamine secretory vesicles [82,83], would yield a very small change in signal. Nevertheless, this approach has been used to determine a synaptic vesicle pH of 5.6 in cultured DA neurons [84] and a pH of 5.8 in DA neurons in Drosophila brain [85]. As already noted, current data seem to indicate that in situ most synaptic vesicles have an undefineable pH value with no free proton, while a minority contain one or very few protons with a pH of ~ 4.3.…”

“…New work confirms that while amphetamines indeed collapse acidic gradients in DA synaptic vesicles in mammalian striatum and in Drosophila brain, the net reverse transport of protons through VMAT during vesicular amphetamine uptake plays a larger role in the collapse than does a “weak base” action, in which protons are buffered by intravesicular amphetamine [85,96]. In striatal brain slices, 20 µM para-chloroamphetamine can release a fluorescent antipsychotic compound from synaptic vesicles; however, this effect is blocked by a VMAT2 inhibitor, reserpine, indicating a requirement for VMAT mediated-proton efflux.…”

Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons

“…We used D. melanogaster to visualize the effects of increasing dVGLUT expression selectively in DA neurons on their overall morphology and organization in whole living brain (23). The tyrosine hydroxylase (TH) promoter was used to selectively label DA neurons with a soluble GFP marker distributed uniformly throughout the cells and visualized by multiphoton microscopy of intact living brain preparations from 3-day-old adult flies (24).…”

“…Isolated ex vivo whole adult fly brain preparations were microdissected as previously described, placed in a recording chamber (JG-23, Warner Instruments), and imaged under continuous perfusion with artificial hemolymph (in mM: 108 NaCl, 5 KCl, 2 CaCl 2 , 8.2 MgCl 2 , 1 NaH 2 PO 4 , 10 sucrose, 5 trehalose, 5 HEPES, 4 NaHCO 3 ; pH 7.5, 265 mOsm) as in earlier studies (23,63). Brains were imaged on an Ultima multiphoton laser scanning microscope (Bruker Corp.) using a 20× (1.0 NA) water immersion objective (Carl Zeiss Microscopy LLC).…”