The noradrenergic neurons of the locus coeruleus (LC) have been implicated in mediating physical opiate dependence and withdrawal. Over the last 10 years, the laboratories of Drs. Nestler, Aghajanian, Alreja, and Duman have carried out highly integrated biochemical and electrophysiological studies to investigate the molecular and cellular mechanisms underlying opiate tolerance, dependence, and withdrawal in these neurons and to relate these findings to behavioral manifestations of opiate withdrawal. One major finding is that chronic opiate administration up regulates the cAMP pathway in the LC at several major steps between receptor and physiological response, with increases seen for adenylyl cyclase, protein kinase A (PKA), and several substrates for the protein kinase, including tyrosine hydroxylase and CREB (cAMP-response element binding protein), a major cAMP-regulated transcription factor. Several lines of evidence now support the view that this upregulation of the cAMP pathway contributes to opiate tolerance, dependence, and withdrawal exhibited by LC neurons. The proposed studies will further characterize the role of the cAMP pathway in opiate action. One major aim is to identify the specific subtypes of adenylyl cyclase and specific subunits of PKA that are upregulated in the LC by morphine treatment and to investigate the molecular mechanisms by which this upregulation occurs. We have demonstrated upregulation of specific adenylyl cyclases and PKA subunits at the protein and mRNA levels, suggesting that these adaptations may occur, at least in part, at the level of gene expression. Indeed, preliminary investigations have provided direct evidence that some, but not all, of these morphine-induced adaptations are mediated via alterations in CREB. We will further pursue a role for CREB in opiate action by use of viral-mediated gene transfer where CREB or a specific inhibitor of CREB are over expressed in LC neurons. We will utilize novel transgenic mice, currently in development in the Transgenic Core, in which CREB or the CREB inhibitor are over expressed in the LC in an inducible fashion. A second major aim is to explore mechanisms for opiate tolerance in LC neurons. We will focus on a role for GRKs (G protein receptor kinases) and for RGS proteins (regulators of G protein signaling) as novel mechanisms of receptor-G protein uncoupling possibly related to tolerance. Together, the proposed molecular, cellular, and behavioral studies promise to advance our understanding of the long-term adaptations that chronic opiates induce in LC neurons.
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