Anxiogenic properties of an inverse agonist selective for α3 subunit‐containing GABAA receptors

α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype. α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo binding assay. At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. α1-containing receptors), α3IA (30 mg kg−1 i.p.), like the nonselective partial inverse agonist N-methyl-β-carboline-3-carboxamide (FG 7142), not only caused behavioural disruption in an operant, chain-pulling assay but was also anxiogenic in the elevated plus maze, an anxiogenic-like effect that could be blocked with the benzodiazepine antagonist Ro 15-1788 (flumazenil). Neurochemically, α3IA (30 mg kg−1 i.p.) as well as FG 7142 (15 mg kg−1 i.p.) increased the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid in rat medial prefrontal cortex by 74 and 68%, respectively, relative to vehicle-treated animals, a response that mimicked that seen following immobilisation stress. Taken together, these data demonstrate that an inverse agonist selective for GABAA receptors containing an α3 subunit is anxiogenic, and suggest that since α3-containing GABAA receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic. Keywords: GABAA receptor, anxiety, inverse agonist, benzodiazepine, anxiogenic, 3,4-dihydroxyphenylacetic acid Introduction The GABAA receptor is generally considered to be a pentamer comprising subunits of members of the GABAA receptor family (α1–6, β1–3, γ1–3, δ, ɛ, θ and π), with the majority of native receptors containing two α, two β and a single γ subunit (Sieghart & Sperk, 2002). In addition to the agonist (GABA) recognition site, the GABAA receptor also contains binding sites for a number of pharmacologically relevant substances such as neurosteroids, barbiturates, ethanol, convulsants, anaesthetics and benzodiazepines (Korpi et al., 2002). In light of the clinical use of benzodiazepines based upon their anxiolytic, sedative, myorelaxant, cognition impairing and anticonvulsant properties, the binding site for these compounds has been the focus of considerable attention. Detailed analyses of recombinant GABAA receptors have established that the benzodiazepine binding site occurs at the interface of the α and γ subunits (Sieghart & Sperk, 2002). Since the predominant γ subunit occurring in native GABAA receptors is the γ2, then the benzodiazepine site pharmacology of GABAA receptors in the brain is dictated primarily by the α subunit present (McKernan & Whiting, 1996). The influence of the α subunit on benzodiazepine binding site pharmacology is best illustrated by the fact that GABAA receptors containing either an α4 or α6 subunit have essentially no affinity for classical benzodiazepines such as diazepam or lorazepam, a difference that can be solely attributed to the presence of an arginine residue in α4 and α6 subunits, which in α1, α2, α3 and α5 subunits is histidine (Wieland et al., 1992). Thus, the benzodiazepine binding site is associated with GABAA receptors containing a β and γ2 subunit in conjunction with either an α1, α2, α3 or α5 subunit, a receptor population accounting for roughly three-quarters of the total brain GABAA receptor population (McKernan & Whiting, 1996; Sieghart & Sperk, 2002). The clinically used ‘classical' benzodiazepines (e.g. diazepam, lorazepam, flunitrazepam, alprazolam) are nonselective full agonists in that they potentiate the effects of GABA at α1-, α2-, α3- and α5-containing GABAA receptors as a result of increasing the number of channel opening events when GABA is bound (Sieghart & Sperk, 2002). This causes an increased chloride ion flux into the cell, resulting in a hyperpolarisation of the resting membrane potential, the behavioural manifestations of which are anxiolysis, sedation, myorelaxation, cognitive impairment and anticonvulsant activity (Korpi et al., 2002). On the other hand, nonselective inverse agonists, such as N-methyl-β-carboline-3-carboxamide (FG 7142) or methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), have the opposite effects in that they decrease the number of GABA-induced channel opening events, resulting in depolarisation and increased neuronal excitability. The opposite effects of benzodiazepine site agonists and inverse agonists are reflected at the behavioural level, since inverse agonists are anxiogenic, increase vigilance and are either convulsant in their own right or enhance the efficacy of a convulsant compound (i.e. are proconvulsant) (Haefely et al., 1993). Between the extremes of full agonism or full inverse agonism (e.g. DMCM) lie a spectrum of efficacies, which include partial agonists such as bretazenil or imidazenil, partial inverse agonists such as FG 7142 and antagonists, the prototypic example of which is Ro 15-1788 (flumazenil) (Haefely et al., 1993). With respect to Ro 15-1788, it is important to note that this compound has no effect on GABA-induced channel opening effects and therefore does not affect the resting membrane potential nor does it have marked effects in vivo (Haefely, 1988). Recent molecular genetic approaches have begun to define which of the specific pharmacological features of benzodiazepines are associated with particular (i.e. α1-, α2-, α3- or α5- containing) subtypes of GABAA receptor (Rudolph & Mohler, 2004). For example, mice in which the α1-containing GABAA receptors are rendered insensitive to diazepam are less sensitive to the sedative effects of diazepam, establishing the role of α1-containing GABAA receptors in mediating the sedative properties of nonselective benzodiazepines (Rudolph et al., 1999; Crestani et al., 2000a; McKernan et al., 2000). Pharmacological confirmation that α1-containing GABAA receptors play a key role in mediating the sedative properties of nonselective benzodiazepines comes from observations that the α1 binding selective imidazopyridine zolpidem is hypnotic (Crestani et al., 2000a), whereas a compound lacking α1 efficacy, L-838417, has a much reduced sedation liability (McKernan et al., 2000). While GABAA receptors containing an α1 subunit are associated with sedation and anticonvulsant activity and those containing α5 are associated with certain cognitive processes (Rudolph et al., 1999; Collinson et al., 2002; Crestani et al., 2002), the role of α3-containing GABAA receptors is less well defined. Thus, the α3 subtype does not appear to be associated with diazepam-induced changes in either sleep architecture (Kopp et al., 2003), motor performance, sedation or anticonvulsant activity (Low et al., 2000). Moreover, although a comparison of transgenic mice containing diazepam-insensitive α2 or α3 GABAA populations suggests that the α3 subtype does not mediate the anxiolytic effects of diazepam, whereas α2 does (Low et al., 2000), the interpretation of these behavioural data is confounded by methodological issues (Crestani et al., 2000b; Reynolds et al., 2001). Clearly, it would be useful to resolve the relative anxiolytic contributions of the α2 and α3 subtypes pharmacologically, but as yet there are no compounds reported to have subtype selective agonism for α2- versus α3- or α3- versus α2-containing GABAA receptors (Cooke & Hamilton, 2002). However, in the present study, we describe the properties of 6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one (α3IA), which has α3 subtype selective inverse agonism. Thus, α3IA possesses a degree of binding and inverse agonist efficacy selectivity for α3-containing receptors such that its in vivo effects are presumably mediated primarily through this GABAA receptor subtype. In rats, this compound not only disrupted behaviour in the chain-pulling assay but was also anxiogenic in the elevated plus maze and produced changes in the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the medial prefrontal cortex consistent with a stress response. These data implicate α3-containing GABAA receptors in mediating at least part of the anxiogenic effects of nonselective inverse agonists and suggest that a compound with agonist efficacy at the α3 subtype may be anxiolytic.