Decreases in endogenous opioid peptides in the rat medullo-coerulear pathway after chronic morphine treatment.

Several biochemical changes have been described in noradrenergic neurons of the locus coeruleus (LC) after chronic morphine treatment. Changes in neurochemical expression in opioid afferent projections to the LC may be equally important in modulating noradrenergic neurons during chronic opiate exposure. To test the hypothesis that opioid peptides in LC afferents are altered after chronic opiate administration, we exposed adult male rats to either morphine or placebo pellets for 5 d. Tissue sections through the LC were processed for peroxidase or gold-silver labeling of methionine(5)-enkephalin (met-ENK) and analyzed using light or electron microscopy, respectively. Light level densitometry and ultrastructural analysis showed that there was a significant decrease in immunolabeling for ENK in LC-afferent terminals of morphine-treated rats. Western immunoblot analysis confirmed that protein levels for both leucine(5)- and methionine(5)-ENK were significantly decreased in tissue samples containing the LC after chronic morphine treatment. To test whether decreases in ENK protein expression were mirrored by decreases in gene expression, Northern blot analysis of preproenkephalin (PPE) mRNA was conducted in tissue samples obtained through the medulla, a brainstem area that contains the major opioid afferents to the LC. PPE mRNA was reduced in samples obtained from morphine-treated rats. Finally, in situ hybridization experiments confirmed significant decreases in PPE mRNA expression in the nucleus paragigantocellularis, a region known to provide a robust opioid input to the LC. These data suggest that there is a decrease in the synthesis of the opioid peptide mRNA and protein in the medullo-coerulear pathway after chronic exposure to morphine. Such alterations in opioid peptide levels during opiate dependence may contribute to the observed hyperactivity of LC neurons during opiate withdrawal.