Drug addiction is a neurological disorder characterized by dysregulation of brain circuits which regulate motivation and reward signaling. Drugs of abuse increase dopamine (DA) concentrations in neural structures involved with reward processing;psychostimulant drugs do so by acting on dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN), resulting in increased dopamine concentrations in their targets (respectively, the nucleus accumbens and dorsal striatum). The psychostimulant amphetamine (AMPH) acts at monoamine transporters, including the plasma membrane dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT2). DAT is responsible for DA reuptake from the extracellular space into the presynaptic terminal. VMAT2 transports cytosolic DA into synaptic vesicles. AMPH binds to DAT causing a reversal of DA transport and a net efflux of DA from the cytosol to the extracellular space. The actions of AMPH at VMAT2 are less-understood and a subject of controversy;however, it is accepted that binding of AMPH to VMAT2 results in efflux of DA from synaptic vesicles into the cytosol. Recent findings demonstrate that DAT and VMAT2 are physically coupled via the synaptic vesicle membrane protein synaptogyrin-3 (SYGR3). The formation of this DAT-SYGR3-VMAT2 complex is required for normal uptake of DA through DAT, and depends not only on the presence of SYGR3, but also on the presence of functional VMAT2. The hypotheses directing this work is that the action of AMPH on DAT and VMAT2 may also depend on SYGR3-mediated coupling, and that exposure of neurons to AMPH may result in alterations in SYGR3- mediated coupling of DAT and VMAT2. These hypotheses will be tested in primary cultures of dopaminergic neurons, as well as in adult rats in vivo, using an adeno-associated viral vector (AAV) containing a short hairpin RNA (shRNA) designed to knock down SYGR3. We will use this virus to determine the effects of SYGR3 removal on AMPH-induced reverse transport of DA using molecular, cellular, and behavioral techniques. Experiments are designed so that, regardless of the outcome, we will have an increased understanding of the extent to which the molecular mechanisms of AMPH action require and/or interfere with normal interactions between proteins regulating DA homeostasis;furthermore, these experiments may increase our understanding of the role of VMAT2 in generating AMPH-induced DA efflux.
Addiction to amphetamine and related stimulant drugs is a devastating behavioral disorder, and a major threat to public health. The broad objective of this research is to determine whether recently-discovered protein- protein interactions (between synaptic vesicle proteins and the dopamine transporter) are required for amphetamine to cause release of dopamine through the dopamine transporter, a major mechanism by which amphetamine exerts its psychomotor effects. This research will increase our understanding of the molecular mechanisms by which amphetamine works, and lay the groundwork for future studies aimed at designing interventions to treat addiction to amphetamine and related drugs of abuse.
