Antagonism of the Ethanol-Like Discriminative Stimulus Effects of Ethanol, Pentobarbital, and Midazolam in Cynomolgus Monkeys Reveals Involvement of Specific GABAA Receptor Subtypes □S

The -aminobutyric acid (GABA)A receptors mediating the discriminative stimulus effects of ethanol were studied by comparing the potency of ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4Himidazol(1,5-a)benzodiazepine-3-carboxylate (Ro15-4513) and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)benzodiazepine-3-carboxylate (flumazenil, Ro15-1788) to antagonize ethanol, pentobarbital (PB), and midazolam substitution for ethanol. Ro15-4513 has high affinity for receptors containing 4/6 and 5 subunits and lower affinity for 1, 2, and 3 subunits. Flumazenil is nonselective for GABAA receptors containing 1, 2, 3, and 5 subunits and has low affinity for 4/6-containing receptors. Male (n 9) and female (n 8) cynomolgus monkeys (Macaca fascicularis) were trained to discriminate ethanol (1.0 or 2.0 g/kg i.g., 30-min pretreatment) from water. Ethanol, PB, and midazolam dose-dependently substituted for ethanol (80% ethanol-appropriate responding). Ro15-4513 (0.003–0.56 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam doseresponse functions rightward in a vast majority of monkeys tested (15/15, 16/17, and 11/12, respectively). In contrast, flumazenil (0.30–10.0 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in 9 of 16, 12 of 16, and 7 of 9 monkeys tested, respectively. In the monkeys showing antagonism with both Ro15-4513 and flumazenil, ethanol and PB substitution were antagonized more potently by Ro154513 than by flumazenil, whereas midazolam substitution was antagonized with similar potency. There were no sex or training dose differences, with the exception that flumazenil failed to antagonize ethanol substitution in males trained to discriminate 2.0 g/kg ethanol. GABAA receptors with high affinity for Ro15-4513 (i.e., containing 4/6 and 5 subunits) may be particularly important mediators of the multiple discriminative stimulus effects of ethanol through GABAA receptor systems. The GABAA ionotropic complex is a pentameric configuration of subunits forming a Cl -conducting channel. The pharmacologic response of GABAA receptors varies with subunit composition, which varies across brain regions (Sieghart, 1995). More than 60 years of research indicates that GABAA receptors mediate the discriminative stimulus effects of ethanol (Drug Comprehensive Bibliography of Drug Discrimination Research; www.dd-database.org). Benzodiazepines, barbiturates, ring-A-reduced 3 -hydroxy neuroactive steroids, and ethanol, which positively modulate GABAA receptors (Sieghart, 1995), produce ethanol-like discriminative stimulus effects (Grant et al., 2000). Research with subunit-preferring ligands further suggests that specific GABAA receptor subtypes mediate the discriminative stimulus effects of ethanol in non-human primates. In particular, 1 subunit-containing receptors seem to mediate discrimination of higher (i.e., 2.0 g/kg) doses of ethanol (Helms et al., 2008b), whereas 5but not 1-containing GABAA receptors mediate discrimination of lower (i.e., 1.0 g/kg) doses of ethanol (intravenous) in squirrel monkeys (Platt et al., 2005). Antagonism studies also suggest that GABAA receptors are involved in the discriminative stimulus effects of ethanol. The discriminative stimulus effects of ethanol are antagonized by Ro15-4513. In male CD-1 mice, Ro15-4513 (0.10–1.0 mg/kg) antagonized discrimination of 1.0 g/kg i.p. ethanol (Rees and Balster, 1988). In male rats, the discriminative stimulus effects of 1.0 and 1.5, but not 2.0 g/kg i.g. ethanol This work was supported by the National Institutes of Health [Grants AA10009, AA13860, AA17040]. This work was presented at the Annual Meeting of Experimental Biology; 2008 Apr 5–9; San Diego, CA (Helms et al., 2008a). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.156810. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: Ro15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)benzodiazepine-3-carboxylate; BEC, blood-ethanol concentration; Ro15-1788, flumazenil, ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)-benzodiazepine-3-carboxylate; FR, fixed ratio; PB, pentobarbital; ANOVA, analysis of variance. 0022-3565/09/3311-142–152$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 156810/3519664 JPET 331:142–152, 2009 Printed in U.S.A. 142 http://jpet.aspetjournals.org/content/suppl/2009/07/30/jpet.109.156810.DC1.html Supplemental material to this article can be found at: at A PE T Jornals on N ovem er 4, 2016 jpet.asjournals.org D ow nladed from were antagonized by Ro15-4513 (Gatto and Grant, 1997). However, in female rats and female C57BL/6cr mice, discrimination of 1.2 g/kg i.p. (Hiltunen and Järbe, 1988) and 1.0 g/kg i.p. ethanol (Middaugh et al., 1991) was not antagonized by Ro15-4513. Thus, sex and training dose may affect antagonism of the discriminative stimulus effects of ethanol by Ro15-4513, factors that were investigated in the current study in non-human primates. In contrast to antagonism by Ro15-4513, few acute effects of ethanol are blocked by Ro15-1788 (flumazenil). In rats, flumazenil does not block ethanol self-administration (Samson et al., 1989), consumption (McBride et al., 1988), sedative effects (Suzdak et al., 1986), or anticonflict effects (Glowa et al., 1988). However, flumazenil blocks tolerance to the ataxic effects of ethanol and behaviors associated with ethanol withdrawal in mice (seizures; Buck et al., 1991) and rats (anxiety; Knapp et al., 2004). Although it did not antagonize acute ethanol effects, flumazenil clearly antagonized the acute effects of other GABAA-positive modulators including benzodiazepines (Herling and Shannon, 1982; Rowlett and Woolverton, 1996, 1998; Paronis and Bergman, 1999; Lelas et al., 2000), and the neuroactive steroid allopregnanolone (Fernández-Guasti and Picazo, 1995), but not the barbiturate PB (Herling and Shannon, 1982; Woolverton and Nader, 1995). However, PB antagonized flumazenil discrimination in diazepam-dependent rhesus monkeys (McMahon et al., 2006), suggesting that barbiturates and flumazenil may act at common receptors in vivo. Flumazenil increased locomotion in female but not male rats (2.5–10.0 mg/kg; Holtman et al., 2003) and antagonized discrimination of high (15 and 20 mg/kg) but now low doses (2.5 and 3.0 mg/kg) of the benzodiazepine chlordiazepoxide (De Vry and Slangen, 1986). The current study therefore assessed sex and training dose effects on flumazenil antagonism of ethanol-like discriminative stimulus effects in cynomolgus monkeys. Flumazenil and Ro15-4513 act at common GABAA receptor subtypes as flumazenil blocks antagonism by Ro15-4513 (Suzdak et al., 1986) and displaces Ro15-4513 binding at 4 3 receptors (Wallner and Olsen, 2008). Studies in recombinant receptor expression systems suggest that flumazenil has very low affinity for “benzodiazepine-insensitive” GABAA receptors containing 4 and 6 subunits (Ki 90–107 nM), but equal affinity for “benzodiazepine-sensitive” GABAA receptors containing 1, 2, 3, or 5 subunits (Ki 0.40–1.5 nM). In contrast, Ro15-4513 has similar affinity for all subunit variants except 5, for which it has 10-fold greater affinity (Ki 0.50 nM) (Sieghart, 1995). Positive modulation of GABAA receptors by midazolam and PB occurs by binding to distinct receptor sites (Sieghart, 1995) and subunit configurations. Midazolam is inactive at benzodiazepine-insensitive GABAA receptors containing 4/6 subunits (Möhler et al., 2002). Pentobarbital has greater activity at human GABAA 6-containing compared with 1–3or 5-containing receptors, although positive modulation occurred at all receptor subtypes with similar potency (Xenopus oocytes: x 2 2s) (Thompson et al., 1996). Despite these differences, benzodiazepines and barbiturates substitute for the discriminative stimulus effects of ethanol (Grant et al., 2000). The current research is the first to investigate GABAA receptor mediation of ethanol-like discriminative stimulus effects by comparing the efficacy and potency of Ro15-4513 and flumazenil to antagonize the substitution of ethanol, PB, and midazolam in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol. Materials and Methods