Role of GABAA α5-containing receptors in ethanol reward: The effects of targeted gene deletion, and a selective inverse agonist
Introduction
Ethanol achieves its effects on the central nervous system through diverse mechanisms, including interaction with several ligand-gated ion channels. A major contribution is played by GABAA receptors, at which ethanol acts to facilitate chloride flux, resulting in enhanced neuronal inhibition. Compounds acting as inverse agonists at the benzodiazepine (BZ) binding site of GABAA receptors (i.e. they reduce GABA-stimulated chloride flux, thereby reducing the membrane potential) may antagonize behaviorally sedative effects of ethanol (Bonetti et al., 1988, Dar, 1992, Suzdak et al., 1986, Wood et al., 1989), and it has also been suggested that such compounds may also reduce ethanol's euphoric and rewarding properties (June et al., 1994).
GABAA receptors are a family of oligomeric proteins that show considerable heterogeneity in their regional distribution and function. It has been suggested that receptors containing the α5 subunit isoform may be particularly important in mediating the rewarding properties of ethanol. This conclusion is predicated on three lines of pharmacological evidence. First, the imidazobenzodiazepine, RY 023 which has 67-fold higher affinity for and is an inverse agonist at recombinant GABAA receptors made up of β2, γ2 plus α5 subunits compared to the analogous receptors containing either an α1, α2 or α3 subunit (Liu et al., 1996, Liu et al., 1995), suppressed operant responding for ethanol reinforcers in the alcohol-preferring rat, when administered into dorsal hippocampal sites (June et al., 2001) rich in α5-containing receptors (Fritschy and Mohler, 1995, Pirker et al., 2000, Wisden et al., 1992). Higher doses of RY023 were required to reduce responding for saccharin, consistent with this latter effect being mediated by other GABAA receptor subtypes (June et al., 2001). Second, RY 024, which has an α5 binding selectivity (76-fold) comparable to RY 023 but has greater α5 inverse agonism (Liu et al., 1996, Liu et al., 1995), decreases ethanol self-administration in rats when given systemically(McKay et al., 2004). In keeping with a role for α5-containing receptors in ethanol reward, chronic alcohol administration increases expression of α5 mRNA in hippocampus (Charlton et al., 1997, Devaud et al., 1995). Thirdly, the prototypic imidazobenzodiazepine inverse agonist, Ro 15-4513, which has 10–20-fold higher affinity for α5-compared to α1-, α2-, α3-, α4- or α6-containing receptors (Hadingham et al., 1993, Lingford-Hughes et al., 2002, Liu et al., 1996, Smith et al., 2001), has long been known to antagonize the rewarding effects of ethanol in operant self-administration tests (Bao et al., 1992, Glowa et al., 1988, Rassnick et al., 1993, Samson et al., 1989, Samson et al., 1987). The ability of Ro 15-4513 to disrupt responding for ethanol might thus be related to its action at α5-containing receptors.
Nevertheless, the conclusions that can be reached with the hitherto available pharmacological tools are limited. Liu et al. (1996) suggest that both RY 023 and RY 024 can exert actions, presumably inverse agonism, at other GABAA subtypes, although it should be noted that to date only efficacy data for the α5 subtype has been presented (Liu et al., 1995). Similarly, the fact that Ro 15-4513 is proconvulsant (Lister and Nutt, 1988) and possesses inverse agonism at the α1, α2 and α3 subtypes (Benson et al., 1998, Hadingham et al., 1995, Kelly et al., 2002, Wafford et al., 1993) raises the possibility that the ability of Ro 15-4513 to disrupt ethanol reinforcement may represent actions at other receptor subtypes. An alternative approach to investigating the role of α5-containing subunits in ethanol's effects is to exploit the availability of mice in which the gene encoding subunits have been deleted (Collinson et al., 2002). If α5-containing receptors play an important role in mediating either the reinforcing effects of ethanol, or the ability of drugs like Ro 15-4513 to interfere with such reinforcing effects, then α5 knockout mice should show lower rates of responding for ethanol reward, and should be less sensitive than corresponding wildtype control mice to the disruptive effects of Ro 15-4513. To ensure that the α5 knockout mice were not generally different from wiltypes in the effects of ethanol, the pharmacological effects of ethanol on motor performance and operant performance were compared between the knockout and wildtype mice.
In addition, and as a complement to the knockout mice studies, we have used a novel compound, α5IA-II, which preferentially modulates the functions of α5-containing GABAA receptors (21). This differs from the α5 binding-selective compounds RY 023, RY 024 and Ro 15-4513 in that α5IA-II possesses subtype selective efficacy. In other words, whilst α5IA-II binds with comparable affinity to the α1, α2, α3 and α5 GABAA subtypes, it has much greater inverse agonist efficacy at the α5 compared to α1, α2 or α3 subtypes and consequently it's in vivo effects will be exerted primarily via α5-containing GABAA receptors (see Table 1 and Fig. 1 for summary).
Section snippets
Animals
Wildtype and α5−/− mice (Collinson et al., 2002) were bred from wildtype and homozygous α5−/− mice in a mixed 50% C57BL6 and 50% 129SvEv genetic background, constructed and selectively bred by Dr Thomas Rosahl, Merck Sharp and Dohme Neuroscience Centre, Terlings Park Essex, UK. Female animals of the F3 generation only were used for this study. On arrival at the University of Sussex, the animals were housed in pairs and maintained on a 12-h light/dark cycle with lights off at 19:00; temperature
Self-administration
Both genotypes learned to lever press on an FR4 schedule to obtain 0.01 ml of 10% sucrose/10% ethanol mixture, obtaining approximately 50 reinforcers in a 60 min session. There were no differences between genotypes at any stage of training (data not shown). Fig. 2A shows that Ro 15-4513 administration reduced the number of reinforcers delivered during the operant session, in a dose-related fashion (F3, 36 = 6.50; p = 0.001). A significant dose by genotype interaction (F3, 36 = 5.55; p < 0.01) indicated
Discussion
Although several reports indicate that drugs acting at the benzodiazepine site of α5-containing GABAA receptors to reduce chloride flux (inverse agonists) reduce self-administration of ethanol (Bao et al., 1992, Glowa et al., 1988, June et al., 2001, McKay et al., 2004, Rassnick et al., 1993, Samson et al., 1989, Samson et al., 1987) in the present experiments we found no differences between female mice in which the α5 subunit had been deleted, and wildtype mice in the self-administration of
Acknowledgements
This work was supported by UK Medical Research Council Grant G9806260. JP held a Marie-Curie Training Award.
References (33)
- et al.
Effects of Ro 15-4513 and ethanol on operant behavior of male and female C57BL/6 mice
Alcohol
(1992) - et al.
Pharmacology of recombinant gamma-aminobutyric acid A receptors rendered diazepam-insensitive by point-mutated alpha-subunits
FEBS Lett.
(1998) - et al.
gamma-Aminobutyric acid A receptor subunit mutant mice: new perspectives on alcohol actions
Biochem. Pharmacol.
(2004) - et al.
Ro 15-4513: partial inverse agonism at the BZR and interaction with ethanol
Pharmacol. Biochem. Behav.
(1988) Selective antagonism of acute ethanol-induced motor disturbances by centrally administered Ro 15-4513 in mice
Pharmacol. Biochem. Behav.
(1992)- et al.
Ethanol and the GABA receptor complex: studies with the partial inverse benzodiazepine receptor agonist Ro 15-4513
Pharmacol. Biochem. Behav.
(1988) - et al.
Expression and pharmacology of human GABAA receptors containing gamma 3 subunits
Eur. J. Pharmacol.
(1995) - et al.
GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain
Neuroscience
(2000) - et al.
Oral ethanol reinforcement in the rat: effect of the partial inverse benzodiazepine agonist RO15-4513
Pharmacol. Biochem. Behav.
(1987) - et al.
Antagonism of ethanol-reinforced behavior by the benzodiazepine inverse agonists Ro15-4513 and FG 7142: relation to sucrose reinforcement
Pharmacol. Biochem. Behav.
(1989)
The intrinsic and interactive effects of RO 15-4513 and ethanol on locomotor activity, body temperature, and blood glucose concentration
Life Sci.
An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties
J. Med. Chem.
Chronic ethanol administration regulates the expression of GABAA receptor alpha 1 and alpha 5 subunits in the ventral tegmental area and hippocampus
J. Neurochem.
Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor
J. Neurosci.
Chronic ethanol consumption differentially alters the expression of gamma-aminobutyric acid A receptor subunit mRNAs in rat cerebral cortex: competitive, quantitative reverse transcriptase-polymerase chain reaction analysis
Mol. Pharmacol.
GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits
J. Comp. Neurol.
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Present address: Department of Pharmacology, Masaryk University, Brno, Czech Republic.