DB Beitner-Johnson

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.

Previously, we have identified a number of morphine- and cyclic AMP-regulated phosphoproteins (MARPPs) in the rat locus coeruleus (LC) and other brain regions. We now show that one of these phosphoproteins, a 58 kDa protein designated MARPP-58, is tyrosine hydroxylase. First, MARPP-58 comigrates with immunolabeled, immunoprecipitated, and purified tyrosine hydroxylase on 1- and 2-dimensional electrophoresis. Second, MARPP-58, immunoprecipitated tyrosine hydroxylase, and purified tyrosine hydroxylase yield identical 1-dimensional phosphopeptide maps. Third, MARPP-58 exhibits a regional and subcellular distribution in brain consistent with tyrosine hydroxylase. Identification of MARPP-58 as tyrosine hydroxylase made it possible to determine whether increases in MARPP-58 phosphorylation induced by chronic morphine in the LC reported previously are associated with alterations in enzyme activity and expression in this brain region. We show that chronic treatment of rats with morphine increases levels of tyrosine hydroxylase activity, immunoreactivity, and mRNA in the LC. Induction of the enzyme by chronic morphine was blocked by concomitant treatment of rats with the opiate receptor antagonist naltrexone, indicating that morphine produces this effect through the activation of opiate receptors. Consistent with previous observations that the chronic morphine-induced change in MARPP-58 phosphorylation is specific to the LC, changes observed in enzyme activity, immunoreactivity, and mRNA were not observed in a number of other brain regions studied. The results indicate that chronic morphine regulates the expression of tyrosine hydroxylase specifically in the LC and suggest that such regulation reflects long-term adaptations of LC neurons to chronic morphine at the level of gene expression.