Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines

Abstract

The GABA(A) receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that alpha2-rather than alpha3-containing GABA(A) receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an alpha3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an alpha3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders alpha2-containing receptors BZ insensitive (alpha2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of alpha3-containing GABA(A) receptors is sufficient to produce the anxiolytic effects of BZs and that alpha2 potentiation may not be necessary.

Figures

Figure 1.

Properties of TP003. A, Structure of TP003. B, Concentration-response curves representing potentiation by TP003 of whole-cell GABA currents evoked by an EC20 concentration on cells expressing human recombinant GABAA α1β3γ2 (n = 6), α2β3γ2 (n = 5), α3β3γ2 (n = 8), and α5β3γ2 (n = 7) receptors. The maximum potentiation produced by TP003 at the α3 subtype was 83%. The inset histogram shows the extent of potentiation of an EC20-equivalent concentration of GABA by 3 μm CDP at the α1, α2, α3, and α5 subtypes. Error bars indicate SEM. mod, Modulation.

Figure 2.

A, B, The effects of low doses (0.03, 0.1, and 0.3 mg/kg, p.o.) (A) and high doses (0.3, 1, and 3 mg/kg, p.o.) (B) of TP003 on the rat elevated plusmaze. The percentage of time spent on the open arms and the number of entries into the open arms are shown (mean ± SEM). CDP (5 mg/kg, i.p.) was included as the positive control; n = 17-18 per treatment group. *p < 0.05 compared with vehicle (0.5% methylcellulose). veh, Vehicle.

Figure 3.

The effects of TP003 on the mouse rotarod (mean ± SEM time in seconds on rotarod during a 2 min trial). Diazepam (Diaz; 10 mg/kg, p.o.) was included as the positive control; n = 8 per treatment group. *p < 0.05 compared with vehicle (veh).

Figure 4.

A, B, The effects of diazepam (Diaz; n = 10; B) and TP003 (n = 9; A) on response rates during the CS period of the conditioned emotional response paradigm (mean ± SEM percentage of pre-CS responding). *p < 0.05 compared with vehicle (veh).

Figure 5.

A, B, The effects of CDP (15 mg/kg, i.p.) and TP003 (1 mg/kg, i.p.) in the mouse SIH model on the mean temperature change (A) and activity (B); n = 6-8 per group. *p < 0.05 compared with vehicle of the same genotype before stress. Error bars indicate SEM. WT, Wild type; temp., temperature; Veh, vehicle.