Effect of Chronic Ethanol and Withdrawal on the m-Opioid Receptor- and 5-Hydroxytryptamine1A Receptor-Stimulated Binding of [S]Guanosine-59-O-(3-thio)triphosphate in the Fawn-Hooded Rat Brain: A Quantitative Autoradiography Study

Previous studies have shown that chronic ethanol influences the density of central m-opioid receptors and serotonin1A (5-hydroxytryptamine1A) receptors. To determine whether the functional coupling of these two receptors to G proteins in the rat brain, particularly in mesocorticolimbic regions, is affected by ethanol, receptor-mediated [S]guanosine-59O-(3-thio)-triphosphate ([S]GTPgS) binding stimulated by [D-Ala,N-MePhe,Gly-ol]-enkephalin (DAMGO) or L694,247 was used. By quantitative autoradiography, receptor-mediated [S]GTPgS binding activated by the two agonists was mapped throughout brain sections at the level of the nucleus accumbens and hippocampus from groups of alcohol-preferring Fawn-Hooded (FH) rats after different ethanol consumption paradigms. Significant DAMGO (m-opioid receptor agonist)-stimulated binding of [S]GTPgS was obtained in the striatum, nucleus accumbens, and lateral septum, whereas L694,247 (5-hydroxytryptamine1A/1B/1D receptor agonist)-stimulated binding of [S]GTPgS was observed in the lateral septum, amygdala, and cingulate cortex. Chronic ethanol self-administration significantly reduced DAMGOstimulated [S]GTPgS binding in the nucleus accumbens (219%), lateral septum (215%), and striatum (223%), which recovered toward control levels after ethanol withdrawal. However, chronic ethanol, as well as ethanol withdrawal, failed to produce any significant alteration in L694,247-stimulated [S]GTPgS binding in all tested brain regions. The region-specific and receptor-specific alteration of agoniststimulated [S]GTPgS binding suggests that the change of functional coupling of m-opioid receptors to G proteins induced by chronic ethanol drinking may have a pathophysiological role in the consequences of ethanol consumption. Opioidergic and serotonergic neurotransmissions in the central nervous system have been shown to play significant roles in alcohol abuse or alcoholism (Gianoulakis, 1993; LeMarquand et al., 1994; Nevo and Hamon, 1995a). A number of studies have indicated a link between m-opioid receptors and serotonin1A [5-hydroxytryptamine1A (5-HT1A)] receptors to alcohol preference, tolerance, and dependence (Wong et al., 1990; Hyytia, 1993; McBride et al., 1998). In addition, both m-opioid receptors and 5-HT1A receptors belong to a superfamily of seven-transmembrane-domain (heptahelical) receptors that couple to the same subgroup of G protein, an inhibitory GTP-binding regulatory protein (Gi/ Go). This subfamily of G proteins (Gi/Go) is sensitive to pertussis toxin and able to mediate a variety of effector systems, such as inhibition of adenylyl cyclase, reduction in calcium channel conductance, and activation of potassium channels when activated by either endogenous or extracellular signals (i.e., hormones, neurotransmitter, or agonists; Aghajanian and Wang, 1986; Hescheler et al., 1987; Harrington et al., 1992). Theoretically, ethanol is assumed to interfere with the process of signal transduction by acting at any part of the cascade chain from the membrane receptor to G protein and thus to the effector. In fact, ethanol was found to influence the signal transduction cascade at the level of receptor Received for publication October 7, 1999. 1 This work was part of the Ph.D. thesis of F.C. and was supported by a Monash Graduate Scholarship, Australia, and funds to A.J.L. from Australian Brewers’ Foundation and the National Health and Medical Research Council, Australia. A.J.L. is an R. D. Wright Fellow of the National Health and Medical Research Council, Australia. ABBREVIATIONS: FH, Fawn-Hooded; GTPgS, guanosine-59-O-(3-thio)triphosphate; G protein, guanine nucleotide regulatory protein; Gi/Go and Gs, adenylyl cyclase inhibitory and stimulating G proteins; Gia/Goa and Gsa, a-subunit of Gi/Go and Gs proteins, respectively; DAMGO, [D-Ala,N-MePhe,Gly-ol]-enkephalin; 5-HT, hydroxytryptamine (serotonin); L694,247, 2-[5-[3-(4-methylsulfonylamino)benzyl-1,2,4-oxadiazol-5yl]-1H-indol-3-yl]ethanamine; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin. 0022-3565/00/2931-0159$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 1 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:159–165, 2000 159 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from through, for example, both m-opioid receptor and 5-HT1A receptors, even though their associated cellular functions were not monitored at the same time (Nevo et al., 1995b; Winkler et al., 1998), or at the level of G proteins such as Gs and Gi/Go (Wand et al., 1993; Ozawa et al., 1994) and at the level of effector such as adenylyl cyclase (Tabakoff and Hoffman, 1979; Wand et al., 1993). However, whether the functional coupling of these two receptors to their G proteins is affected by ethanol has not been investigated. The advent of an assay based on measurement of agonist-stimulated [S]guanosine-59-O-(3-thio)triphosphate ([S]GTPgS) binding allows the coupling efficiency between specific receptors and their G proteins to be monitored by radioligand binding in membranes (Traynor and Nahorski, 1995) or by quantitative autoradiography on brain slices (Sim et al., 1995). The Fawn-Hooded (FH) rat, a strain of inbred rat with high alcohol preference, consumes large amounts of ethanol in a two-bottle free-choice situation (Rezvani et al., 1990; Chen et al., 1998). In addition, behavioral studies demonstrated that compounds such as opioid receptor antagonists and 5-HT1A receptor agonists were able to reduce the ethanol consumption in FH rats (Rezvani et al., 1991; Cowen et al., 1999). Therefore, it is hypothesized that the function of both m-opioid receptors and 5-HT1A receptors in mesocorticolimbic structures may be affected by ethanol consumption in FH rats. To test this hypothesis, brain sections at the level of the nucleus accumbens and the hippocampus from FH rats under a paradigm of ethanol self-administration with or without 48-h withdrawal were analyzed by means of quantitative in vitro autoradiography. Specifically, a comparative study of m-opioid receptorand 5-HT1A receptor-mediated [S]GTPgS binding was mapped throughout selected brain regions from the different experimental groups. Experimental Procedures All experiments were performed in accordance with the Prevention of Cruelty to Animals Acts 1986 under the guidelines of the Code of Practice for the Care and Use of Animals for Experimental Pur-