Novel positive allosteric modulators of GABAA receptors: do subtle differences in activity at alpha1 plus alpha5 versus alpha2 plus alpha3 subunits account for dissimilarities in behavioral effects in rats?

Abstract

Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABA(A) receptors. This stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABA(A) receptors containing the alpha(1) subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently synthesized BZ site ligands: SH-053-2'N, SH-053-S-CH3-2'F, SH-053-R-CH3-2'F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABA(A) receptors containing the alpha(1) subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably alpha(1) receptor-mediated sedative effects of GABA(A) modulation were not fully eliminated with any of the ligands tested, only SH-053-2'N and SH-053-S-CH3-2'F, both dosed at 30 mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at alpha(2)- and alpha(3)-GABA(A) receptors, may have been partly connected with its preferential affinity at alpha(5)-GABA(A) receptors coupled with weak agonist activity at alpha(1)-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at alpha(1) GABA(A) receptors is needed for affecting spatial learning and memory impairments, while much weaker activity at alpha(1)- and alpha(5)-GABA(A) receptors is sufficient for eliciting sedation.

Figure 1

Concentration-effect curves for diazepam, zolpidem, SH-053-2'N, SH-053-S-CH3-2'F SH-053-R-CH3-2'F and JY-XHe-053 on α1β3γ2 (■), α2β3γ2 (▲), α3β3γ2 (◆), and α5β3γ2 (▼) GABA_A receptors, using an EC₃ GABA concentration. Data points represent means ±SEM from at least four oocytes from ≥ 2 batches. As explained in the Results section, the efficacy data for SH-053-2’N and JY-XHe-053 have been published in Rivas et al. (2009). A concentration of 100 nM of diazepam resulted in 246 ± 16%, 400 ± 22%, 461 ± 34%, and 322 ± 7% of control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. A concentration of 100 nM of zolpidem resulted in 180±14%, 132±4%, 121±3%, and non-significant changes relative to control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. A concentration of 100 nM of SH-053-2'N resulted in 113 ± 2%, 165 ± 2%, 149 ± 3%, and 130 ± 3% of control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. A concentration of 100 nM of SH-053-S-CH3-2'F resulted in non-significant changes relative to control current, 169 ± 5%, 138 ± 5%, and 218 ± 4% of control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. The effect of 1 μM of SH-053-S-CH3-2'F at α1β3γ2 receptors was significant relative to control (164 ± 6%). A concentration of 100 nM of SH-053-R-CH3-2'F resulted in 111 ± 2%, 124 ± 9%, 125 ± 8%, and 183 ± 7% of control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. A concentration of 100 nM of JY-XHe-053 resulted in 169 ± 10%, 307 ± 14%, 345 ± 26%, and 220 ± 2% of control current in α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 GABA_A receptors, respectively. All values given were significantly different from the respective control currents (p < 0.01, Student's t-test).

Fig. 2

The effects of diazepam (DZP 1.25 and 2.5 mg/kg) and SH-053-2’N 30 mg/kg (left graphs, a1 and b1), JY-XHe-053 (2.5, 5, 10, 20 and 40 mg/kg) (middle graphs, a2 and b2) and DZP 2 mg/kg and SH-053-R-CH3-2’F (10, 20 and 30 mg/kg) (right graphs, a3 and b3) on distance travelled in the central (hatched bars) and peripheral (open bars) zone of the activity chamber during 45 or 30 min of recording (total activity corresponds to the height of the whole bar) (upper graphs, a1, a2 and a3) as well as on distance travelled in 5-min intervals (lower graphs, b1, b2 and b3). *,** and ***, P<0.05; 0.01 and 0.001, respectively, compared to solvent (SOL) group in each of three experiments. Numbers of animals per treatment, for consecutive groups on each of panels, were 8, 8, 7, 8 (left); 8, 6, 6, 8, 8, 6 (middle) and 7, 5, 7, 8, 8 (right).

Fig. 3

The effects of diazepam (DZP 2.0 mg/kg) and SH-053-2’N (10, 20 and 30 mg/kg) on the a) closed arm entries, b) total arm entries and c) total distance travelled in the EPM. *P<0.05 compared to solvent (SOL) group. Number of animals per treatment (Fig. 3- 4, for SOL through SH-053-2’N 30 mg/kg, respectively): 6, 6, 5, 6, 6.

Fig. 4

The effects of diazepam (2.0 mg/kg) and SH-053-2’N (10, 20 and 30 mg/kg) on the a) distance travelled on open arms, b) percentage of entries in open arms and c) percentage of time spent on open arms of the EPM. *P<0.05 compared to solvent (SOL) group.

Fig. 5

The influence of JY-XHe-053 (0; 2.5; 5; 10; 20 and 40 mg/kg) on the a) distance travelled on open arms, b) percentage of entries in open arms and c) percentage of time spent on open arms of the EPM. Number of animals per treatment (for SOL through JY-XHe-053 40 mg/kg) was 7.

Fig. 6

The effects of a) zolpidem (ZOL 0.5, 1 and 2 mg/kg), b) SH-053-2’N, SH-053-S-CH3-2’F and SH-053-R-CH3-2’F (all dosed at 30 mg/kg) and c) JY-XHe-053 (5; 20 and 40 mg/kg), on latency to platform. *,** and ***, P<0.05; 0.01 and 0.001, respectively, compared to solvent (SOL) group in each of three experiments. In graph a): +, ++, and +++, P<0.05; 0.01 and 0.001, respectively compared to ZOL 0.5 group; #, P<0.05 compared to ZOL 1.0 group; ^ and ^^, P<0.05 and 0.01, ZOL 1 group compared to ZOL 0.5 group. In graph b): ++, P<0.01 compared to SH-053-S-CH3-2’F 30group. Number of animals per each treatment was 8 in a) and b), and 7 in c).

Fig. 7

The effects of SH-053-2’N, SH-053-S-CH3-2’F and SH-053-R-CH3-2’F, all dosed at 30 mg/kg, on a) path efficiency during 5-day acquisition sessions and b) number of entries to the zone of previous days’ position of platform in probe trial.

Fig. 8

The effects of b) zolpidem (ZOL 2), c) JY-XHe-053 30, d) SH-053-2’N 30, e) SH-053-RCH3-2’F and f) SH-053-S-CH3-2’F 30 (all doses in mg/kg) on the distance rats travelled in the NE quadrant and target region during 5-day acquisition trials in the water maze. The numbers inside the columns are the percent of the distance swam inside the target (NE) quadrant which was travelled in the target region. Control values (SOL) given in a) are taken from the experiment with ZOL. Numbers of animals are as in Figure 6.