Anxiogenic properties of an inverse agonist selective for alpha3 subunit-containing GABA A receptors

Abstract

Alpha3IA (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 GABA(A) receptors containing an alpha3 rather than an alpha1, alpha2 or alpha5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of alpha3IA are most probably mediated by the alpha3 subtype. Alpha3IA has good CNS penetration in rats and mice as measured using a [(3)H]Ro 15-1788 in vivo binding assay. At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. alpha1-containing receptors), alpha3IA (30 mg kg(-1) i.p.), like the nonselective partial inverse agonist N-methyl-beta-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, alpha3IA (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 GABA(A) receptors containing an alpha3 subunit is anxiogenic, and suggest that since alpha3-containing GABA(A) receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic.

Figure 1

Structure of α3IA.

Figure 2

Efficacy of α3IA measured by whole-cell patch clamping in human GABA_A receptors containing β3 and γ2 subunits plus either an α1, α2, α3 or α5 subunit stably transfected in L(tk⁻) mouse fibroblast cells. Efficacy was measured as the ability of the compound to modulate the current produced by a concentration of GABA corresponding to 20% of the maximal response (EC₂₀), with negative values indicating a reduction in the GABA-induced current. Values shown are mean±s.e.m. of recordings from four to seven individual cells (n=6, 5, 7 and 4 for α1, α2, α3 and α5 cells, respectively). The shaded areas represent the range of inverse agonist efficacies across subtypes for the nonselective partial inverse agonist FG 7142 (range of efficacies=−30 to −43%) or the nonselective full inverse agonist DMCM (range of efficacies=−53 to −71%; Chambers et al., 2003).

Figure 3

Occupancy of benzodiazepine binding sites by 10 or 30 mg kg⁻¹ α3IA (i.p. in 0.5% methyl cellulose suspension, pretreatment time=15 min) in the cerebellum and spinal cord, tissues enriched in GABA_A receptors containing α1 or α2/α3 subunits, respectively, of (a) mice and (b) rats. In both species, both doses of α3IA gave greater occupancy in the spinal cord relative to the cerebellum, indicating that the in vitro binding selectivity (affinity at α2 and α3>α1) is reflected in vivo. Values shown are mean±s.e.m. (n=5–6 and 7–8/group for mice and rats, respectively).

Figure 4

Effects of 10 or 30 mg kg⁻¹ α3IA or 30 mg kg⁻¹ FG 7142 relative to rats treated with vehicle (0.5% carboxymethylcellulose) on various parameters measured during a 5 min trial on the elevated plus maze. Parameters included: (a) percent time on the open arms; (b) percent time on the closed arms; (c) total arm entries; and (d) total distance traveled. Compared to vehicle-treated rats, both 30 mg kg⁻¹ α3IA and FG 7142 produced significant effects on each of these parameters. Values shown are mean±s.e.m. (n=18/group). ^*P<0.05 and ^**P<0.01 relative to vehicle group using an analysis of variance followed by Dunnett's post hoc t-tests.

Figure 5

Significant anxiogenic response of α3IA (30 mg kg⁻¹), shown here as a decrease in the time spent on the open arms, was blocked by the prototypic benzodiazepine antagonist Ro 15-1788 (flumazenil) (10 mg kg⁻¹ i.p.), the latter of which by itself had no effect on plus maze performance. Values shown are mean±s.e.m. (n=12/group). ^*P<0.05 relative to vehicle/vehicle group using an analysis of variance followed by Dunnett's post hoc t-tests.

Figure 6

(a) Average rate of chain-pulling during a 32 min response sensitivity trial expressed as a percentage of baseline responding prior to i.p. administration of either vehicle (0.5% methyl cellulose), 10 or 30 mg kg⁻¹ α3IA or 30 mg kg⁻¹ FG 7142 immediately prior to commencing the trial. Values shown are mean±s.e.m. (n=11–12/group). (b) The same data presented in panel (a) broken down into 4 min time bins. Each data point represents the mean (error bars omitted for clarity; n=11–12/group). ^*Significantly different from vehicle using an analysis of variance followed by Dunnett's post hoc t-tests.

Figure 7

Comparison of DOPAC concentrations in medial prefrontal cortex of rats receiving immobilisation stress or i.p. injections of vehicle (0.5% carboxymethylcellulose), 30 mg kg⁻¹ α3IA (15 min pretreatment) or 15 mg kg⁻¹ FG 7142 (30 min pretreatment). Values shown are mean±s.e.m. (n=9–11/group). ^*Statistically different from vehicle using analysis of variance followed by post hoc Dunnett's t-test.