Decreases in Endogenous Opioid Peptides in the Rat Medullo-Coerulear Pathway after Chronic Morphine Treatment

Abstract

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 methionine5-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 leucine5- and methionine5-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 situhybridization 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.

Reward Processing by the Opioid System in the Brain

“…Decreased expression following chronic morphine was also described in other brain areas, like the HPT (PVN, 149), FrCx, (26), medulla oblongata (MO), and nucleus paragigantocellularis (PGi; Ref. 384). However, increased levels of Penk mRNA were detected in the HPC (26), whole cortex, and spinal cord (145).…”

Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies

“…For Western blot analysis, rat brains were sectioned and the DRN removed by punch dissection using a 2-mm trephine (Biomedical Research Instruments, Rockville, MD, USA). Protein was extracted in RIPA buffer with added soybean trypsin (25 μg/ml) and benzamidine (0.1 mM) as described previously (Van Bockstaele et al, 2000). Protein levels were quantified, 25 μg protein/lane was separated in a 4-20% Tris-glycine gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA).…”

Chronic Morphine Treatment Modulates the Extracellular Levels of Endogenous Enkephalins in Rat Brain Structures Involved in Opiate Dependence: A Microdialysis Study

“…Thus, increases or decreases in enkephalin immunoreactivity were reported to occur in brain tissues after chronic treatment with morphine, whereas in other studies, no change was observed (Van Bockstaele et al, 2000). These differences are very likely attributable to the following: (1) the various experimental protocols used in chronic opioid treatment and the regioselectivity of the effects (Tejwani and Rattan, 1997); and (2) the use of different techniques to measure the enkephalin levels, such as autoradiography , in situ hybridization (Lightman and Young, 1987;Tjon et al, 1997), or peptide extraction from tissues (Tejwani and Rattan, 1997).…”