Chronic ethanol modulates delta and mu-opioid receptor expression in rat CNS: immunohistochemical analysis with quantitiative confocal microscopy.

Ethanol consumption affects levels of endogenous opioids as well as opioid receptors in both animals and humans. We studied the expression of delta (delta) and mu (mu) opioid receptors (ORs) in brain sections of adult male Sprague-Dawley rats after 2 weeks of consuming ethanol in a liquid diet, with comparisons to sections from pair-fed control animals. Immunohistochemical staining for the ORs, using selective antibodies, and quantitation of confocal images, revealed increased expression of delta-ORs in hippocampal CA1 of the chronic ethanol-treated rats. In contrast, mu-ORs decreased in their expression after ethanol treatment in multiple brain areas, including cortex, hippocampus, midbrain colliculi, striatum and nucleus accumbens. The alterations in immunoreactive OR expression may be related to reduced functional coupling of the ORs to G-proteins, as found in prior studies in several brain regions, using the same chronic ethanol diet protocol. Changes in OR expression and functional coupling in the CNS may be factors in ongoing ethanol consumption and tolerance.

Chronic ethanol consumption reduces delta-and mu-opioid receptor-stimulated G-protein coupling in rat brain.

BACKGROUND: Ethanol consumption is thought to enhance the release of endogenous opioids acting at opioid receptors (ORs) in the central nervous system. Prior studies have shown that chronic ethanol consumption in alcohol-preferring rats uncouples mu-ORs from Gi proteins. The purpose of this study was to investigate the potential for uncoupling of the delta- and the mu-OR after chronic ethanol consumption in a nonpreferring rat strain. METHODS: We used radiohistochemical methods to study mu- and delta-OR-stimulated G-protein coupling in brain tissue of rats ingesting liquid diets containing 6.7% ethanol (v/v) for 16 days, as compared with 0% ethanol pair-fed control rats. Sections of brain from pair-fed and ethanol-treated rats were incubated with guanylyl 5'-[gamma-[35S]-thio]-triphosphate ([35S]-GTPgammaS) in the absence and presence of d-Pen2,d-Pen5 enkephalin (DPDPE), a delta-OR agonist, or Tyr-d-Ala-Gly-N(me)Phe-Gly-ol-enkephalin (DAMGO), a mu-OR agonist. RESULTS: DPDPE significantly stimulated [35S]-GTPgammaS binding in the hippocampal dentate gyrus (DG), CA1, cerebellum, and inferior colliculus of untreated pair-fed controls. By contrast, DPDPE-stimulated [35S]-GTPgammaS binding was reduced significantly in those brain regions in the ethanol-consuming group. DAMGO stimulated [35S]-GTPgammaS binding in cortex, caudate, nucleus accumbens, DG, CA1, and superior and inferior colliculi, whereas the DG, CA1, and colliculi showed a significant reduction of binding after chronic ethanol. Basal [35S]-GTPgammaS binding was not different between the two diet groups. CONCLUSIONS These data are the first to demonstrate functional uncoupling of delta-ORs from G proteins after chronic ethanol consumption. Uncoupling may result from modulation of receptors, possibly by internalization or phosphorylation. Alterations in functional coupling of both delta- and mu-ORs and subsequent effects may contribute to continued ethanol consumption.

Delta opioid receptor localization in the rat cerebellum.

Opioid receptors have been localized to a number of brain regions in rats as well as in other species. In situ hybridization has demonstrated the presence of mRNA for the delta receptor subtype in adult rat cerebellar cortex and in several deep nuclei, but there are no reports on localization of the delta receptor protein in cerebellar regions. In the present study, both quantitative immunohistochemistry and Western blots reveal the presence of delta receptors in the adult rat cerebellum, using a specific affinity-purified antibody. Purkinje cells and processes, as well as cells in the granule cell layer, were positively stained with the antibody. Quantitation of confocal microscopy images illustrated a lower relative level of delta receptor immunoreactivity in cerebellar cortical neurons as compared to neurons in hippocampal regions, striatum and cerebral cortex. Stimulation of delta receptors with a selective agonist, DPDPE, in frozen sections of rat brain, induced a significant increase in binding of [35S]-GTPgammaS in the cerebellar cortex as compared to basal binding levels, thereby demonstrating coupling of the receptor subtype to G-protein. Functional implications for the delta receptor in the cerebellum are discussed, particularly in light of evidence for the presence of a cerebellar opioid receptor for the endogenous opioid methionine enkephalin during early postnatal life.