Neurons release neurotransmitter in quantal units. Classically, quantal size (the number of neurotransmitter molecules released by a single vesicle during exocytosis) was thought to be invariant and changes in presynaptic efficacy were thought to occur as a result of changes in quantal frequency (the number of quanta released per stimulation); however, we have recently found that the quantal size of dopamine release from midbrain neurons can be altered by the psychostimulants cocaine and amphetamine. This suggests that changes in dopamine quantal size may contribute to psychostimulant-induced alterations in neuronal function and behavior. We now propose to explore one of the regulatory substrates of dopamine quantal size, the neuronal vesicular monoamine transporter VMAT2. We will determine if the VMAT2 transporter provides a regulatory mechanism to control dopamine quantal size and quantal frequency in cultured midbrain dopamine neurons and a heterologous system of VMAT2-expressing hippocampal neurons; we will then use microdialysis in the nucleus accumbens and striatum of freely moving wild-type and transgenic VMAT2-overexpressing mice to observe differences in basal, amphetamine- and depolarization-induced monoamine levels and locomotor behavior. The transfection of cultured midbrain dopamine neurons with a VMAT2-carrying adenoviral construct increases dopamine quantal size and frequency by 6-fold, over that provided by endogenous levels of VMAT2. This regulation is probably not due to feedback mechanisms driving tyrosine hydroxylase activity because we have also observed stimulation-dependent dopamine quanta in cultured non- catecholaminergic hippocampal neurons transfected with VMAT2 and preloaded with exogenous dopamine. We plan to extend these insights in the living brain to provide a basis for understanding the roles of presynaptic mechanisms in psychostimulant-induced reward, behavioral sensitization, drug reinforcement and addiction.