The repeated use of various drugs of abuse produces long term alterations in behavior and brain function that contribute to the compulsive drug-seeking that characterizes addiction. Research conducted in the Integrative Neuroscience Section seeks to identify the neural substrates that underlie these adaptive responses as well as those endogenous systems within the brain that oppose their development and long-term expression. The long-term goal of these studies is to identify effective targets for the treatment of drug and alcohol addiction and biological factors that may predispose compulsive drug-seeking in certain individuals. Our studies have continued to focus on endogenous opioid sytems since we have demonstrated important and contrasting roles of the various opioid receptor types (mu-,delta-,kappa-) in regulating circuits in the brain upon which drugs of abuse act to affect behavior. Building on our previous demonstration that kappa-opioid receptors regulate the activity of the dopamine transporter, a membrane protein upon which cocaine and other psychostimulants act to exert their pharmacological effects, we have used immunofluorescence confocal microscopy and live cell imaging to examine the cellular mechanisms mediating the interaction of kappa-opioid receptors with the dopamine transporter. In collaborative studies with Vanderbilt University we have developed an imaging technique which permits the real-time quantification of drug-induced alteration in transporter function in single cells. Using a high affinity fluorescent dopamine transporter substrate, ASP-+, and cells co-transfected with the dopamine transporter and the kappa-opioid receptor, we have shown that kappa-opioid receptor activation increases uptake. This effect is concentration dependent and reversed by a selective kappa-opioid receptor antagonist. Examination of the trafficking of a fluorescently-tagged dopamine transporter in HEK-293 and Neuro 2a cells has shown that this increase is associated with decreased transporter internalization. On-going studies are examining whether these effects result from a physical interaction of these two proteins or are secondary to alterations in signaling cascades that we have previously shown to regulate dopamine transporter activity and cell surface expression. Evidence that systemic administration of kappa-opioid receptor agonists regulates transporter trafficking in the nucleus accumbens, a brain region implicated in mediating the rewarding effects of cocaine, has also been obtained using recently developed biotinylation techniques. In-vivo studies examining the role of endogenous kappa-opioid receptor systems in mediating individual differences in responsiveness to cocaine have also been completed. Using pharmacological and gene inactivation techniques, we have shown that hypofunction of this opioid receptor system results in enhanced responsiveness to the behavioral, neurochemical and cellular effects of cocaine. Furthermore, inactivation of this system not only increases basal dopamine release in reward circuits within the brain but also triggers short- and long-term alterations in dopamine transporter function. Studies examining the mechanism of action of dynorphin, the postulated endogeous ligand for the kappa-opioid receptor have revealed that this peptide can also interact with NMDA receptors and modulate their function. Since the expression and release of dynorphin is increased following the repeated use of cocaine, ampethamine, and methamphetamine, we are examining the role of opioid- versus non-opioid receptor mediated effects of this endogenous peptide in modulating the behavioral and neurochemical effects resulting from the repeated use of these agents.