BRAIN DAT/5-HTT DYSREGULATION IN HUMAN COCAINE USERS

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DESCRIPTION: (Applicant's Abstract) Binding sites on the Dopamine Transporter (DAT) are markedly increased in striatum of human cocaine users versus matched controls, which is likely due to a complex post-translational mechanism. Alterations in cocaine binding sites, and accompanying changes in underlying DAT function, may significantly contribute to cocaine-induced clinical phenomenon such as binging, withdrawal, and craving symptoms and suggest that the DAT may represent an important regulatory focus for dopaminergic cells. A detailed knowledge of the molecular changes involved in DAT regulation may allow new pharmacotherapeutic manipulations of its function. SPECIFIC AIM #1 is to test the hypothesis that cocaine-altered DAT from human brain demonstrates changes in sensitivity to a number of critical parameters, including buffer, temperature, pH, ions, and to different ligands. SPECIFIC AIM #2 is to discover if either protein-protein interactions, detected by determining DAT apparent size, or increased expression of DAT mRNA splice-variants or monoamine transporter-analog mRNA species, detected by RNAase Protection assay or PCR cloning, contribute to the complex binding results found in human cocaine users. Because the norepinephrine transporter (NET is only subtly different from the DAT, understanding its regulation may shed light on the metamorphic potential of other monoamine transporters(MATs). SPECIFIC AIM #3 is to test the hypothesis that human brain NET is upregulated in response to blockade by cocaine exposure. The regulation of autoreceptors and transporters appear to be co-ordinated processes, perhaps involving direct interactions trans-membranally. Because autoreceptor regulation may compliment transporter regulation, SPECIFIC AIM #4 is to test the hypothesis that binding and mRNA levels for serotonin and dopamine autoreceptors are altered in cocaine users versus controls. SPECIFIC AIM #5 is to test the hypothesis that dopamine cells alter their metabolism during cocaine exposure because of cocaine's blockade of uptake. Successful therapeutic approaches targeted at dopaminergic function may need to take into account the adaptive possibilities available to dopamine neurons as they are perturbed. The human post mortem approach avoids complicating species differences, and allows correlative analyses with difficult to model human symptomatology and the possibility, because of the size of the human brain, for considerable inter-correlational analyses between interacting neuronal systems. As alterations are discovered in multiple neuronal systems (in particular serotonergic neurons which demonstrate a distinct human neuroanatomy compared to rodents), the likelihood of unique human responses further increases. For these reasons, continued examination of brain monoaminergic adaptations to cocaine exposure in human specimens now available, along with parallel development of validated cell-model systems, should prove valuable and informative.