In order to understand cocaine craving/relapse in humans it is critical to identify """"""""plastic"""""""" changes that occur in the CNS following repetitive drug administration. Repeated, intermittent treatment of rats with cocaine results in enhanced motor responsiveness, or """"""""behavioral sensitization"""""""". Sensitization is persistent and is an important form of long-lasting neuronal plasticity. The sensitivity and/or expression of the dopamine trans- porter (DAT) is persistently altered, particularly in the nucleus accumbens (NAc), after repeated exposure to cocaine, suggesting that DAT regulation in this brain region may be key to the enduring expression of sensi- tization. This competing renewal application focuses on this idea. To prove that changes in DAT are required, behavior and DAT activity must be measured concurrently. DAT activity can be altered via post-translational, as well as translational, mechanisms. Therefore, it is critical to also understand post-translational modifications by which cocaine could regulate DAT activity and expression. The proposed work will test the hypotheses that: (1) persistent changes in DAT sensitivity to cocaine in NAc contribute to the expression of behav- ioral sensitization and (2) protein kinase C (PKC) and Ca +/calmodulin-dependent protein kinase II (CaM kinase II) mediate cocaine-induced changes in DAT activity and/or expression.
Aim #1 will ad- dress the relationship between the persistent expression of behavioral sensitization and cocaine-induced changes in DAT activity using concurrent measures of locomotor activity and DA clearance, measured with in vivo electrochemical recording in NAc and caudate-putamen (CPu) of freely-moving rats. DAT expression will be determined using quantitative autoradiographic analysis of [ H]WIN 35,428 binding. Treatment regi- mens using repeated intermittent i.p. and i.v. injections, as well as continuous infusion, of cocaine will be compared.
Aim #2 will use in vitro brain slices containing NAc and CPu to address whether the persistent cocaine-induced changes hi DA clearance are unique to NAc, DAT and psychostimulants.
Aim #3 will focus on PKC and CaM kinase II as molecular mechanisms that regulate the activity and/or expression of human (h) DAT expressed in Xenopus oocytes. Measures of hDAT-mediated transport and reverse transport, as well as cocaine sensitivity, will be compared. By combining information about DAT at the systems level (behavioral measurements), cellular level (DA clearance studies), and molecular level (studies in expression systems), the relationship between cocaine behavioral sensitization and the transporter should be elucidated.
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