Methylphenidate (MPD) is a plasmalemmal dopamine (DA) transporter (DAT) inhibitor used to treat attention deficit hyperactivity disorder. In 2002, our laboratory reported a previously unidentified effect of this stimulant. Specifically, MPD administration in vivo rapidly and reversibly increases DA uptake via the vesicular monoamine transporter-2 (VMAT-2), a protein critical for vesicular DA sequestration, as assessed ex vivo in a non-synaptosomal membrane-associated (referred to herein as cytoplasmic) subcellular fraction prepared from treated rats. The impact of MPD on VMAT-2 is not restricted to vesicular trafficking. Preliminary data presented herein are the first to demonstrate that: 1) VMAT-2 co-fractionating with synaptosomal membranes after osmotic lysis (referred to throughout the proposal as VMAT-2M) display novel DA transport kinetics;and 2) MPD kinetically upregulates transport via the VMAT-2M, thus permitting increased vesicular DA accumulation. MPD enhances K+stimulated DA release as well. To better understand not only the functional role of VMAT-2M, but also the therapeutic and abuse properties of MPD, this proposal will test the hypothesis that MPD uniquely affects monoaminergic vesicular function and distribution by a mechanism that includes novel shifts in VMAT-2M-mediated DA transport kinetics. This will be accomplished by: 1) elucidating the unique features of the MPD-induced alterations in transport via the VMAT-2M;2) investigating mechanisms that regulate transport via the VMAT-2M per se, and after MPD treatment;and 3) demonstrating functional importance of MPD-induced alterations in DA transport via the VMAT-2M. Completion of the studies described in this proposal will accomplish two goals. First, the regulation and function of the VMAT-2M will be elucidated. This is important, given the large sequestration capacity and the unique kinetic profile of the VMAT-2M-associated vesicles and the role of these vesicular systems in regulating overall dopaminergic influences. Second, the impact of mechanistically dissimilar psychostimulants, particularly MPD, on the VMAT-2M will be explored. Taken together, these data will provide insight into mechanisms underlying the pharmacological, physiological and pathophysiological regulation of vesicular DA uptake and sequestration, and will thus advance understanding of a wide array of processes including those underlying addiction, and those contributing to the development of neurodegenerative diseases affecting dopaminergic systems. PUBLIC HEALTH RELEVANCE Methylphenidate (MPD) is a plasmalemmal dopamine (DA) transporter (DAT) inhibitor used to treat attention deficit hyperactivity disorder. Our laboratory reported previously that MPD also affects the function of the vesicular monoamine transporter-2 (VMAT-2). The goal of this project is to elucidate this effect, and thus advance understanding of a wide array of processes including those underlying addiction, and those contributing to the development of degenerative diseases affecting dopaminergic systems.