Effects of morphine withdrawal on μ-opioid receptor-stimulated guanylyl 5′-[γ-[35S]thio]-triphosphate autoradiography in rat brain

Abstinence from chronic morphine results in characteristic withdrawal symptoms in humans and experimental animals. Despite a large number of studies, the cellular mechanisms underlying opiate withdrawal symptoms are not clearly understood, in particular, the regulation of mu-opioid receptor function during this process. The present study investigated the It-opioid receptor-stimulated G-protein activity using guanylyl 5'-[gamma-[S-35]thio]-triphosphate ([S-35]-GTPgammaS) autoradiography. [S-35]-GTPgammaS binding was performed using coronal rat brain sections (20 mum) in the presence or absence of the mu-opioid selective agonist [D-Ala(2),N-MePhe(4)Gly(5)-ol] enkephalin (DAMGO). In experiment 1, rats (male, Sprague-Dawley) were injected every 12 h with increasing doses of morphine (5-100 mg/kg, s.c.) for 12 days; a separate group of rats which received saline injections served as control. Opiate withdrawal was induced by abstinence from morphine. Thirty-six hours after the last morphine injection, spontaneous withdrawal symptoms were assessed. Rats were then decapitated and brains rapidly removed. In experiment 2, withdrawal symptoms were precipitated with the opioid receptor antagonist naloxone (1 mg/kg). Brains were taken at 5, 10, 20 and 60 min after naloxone injection. In experiment 3, morphine dependence was induced by implantation of three morphine pellets (75 mg per pellet). After 7 days, withdrawal symptoms were precipitated by naloxone (1 mg/kg) and brains were removed 30 min after naloxone injection. [S-35]-GTPgammaS binding was measured in the locus coeruleus, nucleus parabrachialis, nucleus accumbens and central nucleus of amygdala. Although clear withdrawal symptoms were observed in all morphine-withdrawn rats, no significant changes in [S-35]-GTPgammaS binding were detected in animals undergoing withdrawal. The present lack of differences between morphine-withdrawn and control rats indicates that It-opioid receptor-stimulated G-protein activity is not modulated by chronic morphine administration and is not involved in the expression of opiate withdrawal. (C) 2002 Elsevier Science B.V. All rights reserved.