PWZ-029, a compound with moderate inverse agonist functional selectivity at GABA(A) receptors containing alpha5 subunits, improves passive, but not active, avoidance learning in rats

Abstract

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABA(A) receptors containing alpha1, alpha2, alpha3 or alpha5 subunits, and may have numerous experimental and clinical applications. The ability of non-selective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the alpha1 and/or alpha5 subunit-containing GABA(A) receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesized and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at alpha5-containing GABA(A) receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2'F) selective for GABA(A) receptors containing the alpha5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist ss-CCt exhibiting a preferential affinity for alpha1-subunit-containing receptors. These data suggest that moderate negative modulation at GABA(A) receptors containing the alpha5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined.

Figure 1

Concentration-effects curve for modulation of GABA elicited currents by PWZ-029 (a) on Xenopus oocytes expressing GABA_A receptor subtypes α₁β₃γ₂, α₂β₃γ₂, α₃β₃γ₂, and α₅β₃γ₂ (b). Concentrations of GABA that elicit 3% of the maximum GABA-triggered current of the respective cells were applied alone and with various concentrations of PWZ-029. Control currents represent responses in the absence of PWZ-029. Data points represent means ± SEM from 4 oocytes from ≥ 2 batches. 1µM PWZ-029 resulted in 114±4%, 105±8%, 118±5% and 80±4% of control current (at GABA EC₃) in α₁β₃γ₂, α₂β₃γ₂, α₃β₃γ₂, and α₅β₃γ₂ receptors, respectively. All these values except the one for α₂β₃γ₂ receptors were significantly different from that of the respective control currents (P<0.01, Student's t-test).

Figure 2

The effects of DMCM (0.2 mg/kg) and PWZ-029 (2, 5 and 10 mg/kg) on retention performance in a passive-avoidance task (*P<0.05 compared to solvent (SOL) group). Number of animals per treatment: 10.

Figure 3

The effects of DMCM (0.2 mg/kg) and PWZ-029 (2, 5 and 10 mg/kg) on acquisition (a) and retention (b) performance in an active-avoidance task (*P<0.05 compared to solvent (SOL) group). Number of animals per treatment: 8.

Figure 4

The effects of DMCM (2 mg/kg) and PWZ-020 (5,10 and 20 mg/kg) on distance travelled in the central (hatched bars) and peripheral (open bars) zone of the activity chamber during 30 min of recording (total activity corresponds to the height of the whole bar). *P<0.05 compared to solvent (SOL) group. Number of animals per treatment (Figure 4– Figure 5) was 10, except for PWZ-029 (8 rats).

Figure 5

The effects of DMCM (2 mg/kg) and PWZ-020 (5,10 and 20 mg/kg) on the distance travelled in 5-min intervals in the activity assay. *P<0.05 compared to solvent (SOL) group.

Figure 6

The influence of addition of antagonists (flumazenil – FLU and β-CCt – bCCt) and the agonist SH-053-R-CH3-2’F (SH-2’F-R) on the effect of PWZ-029, administered with solvent (SOL+PWZ-029), on distance travelled in the central (hatched bars) and peripheral (open bars) zone of the activity chamber during 30 min of recording (total activity corresponds to the height of the whole bar). *p<0.05 compared to solvent (SOL+SOL) group. +p<0.05 compared to SOL+PWZ-029 group, Student’s t-test. Number of animals per treatment, for SOL+SOL through SH-053-R-CH3-2’F, respectively, was: 7, 7, 7, ,6, 7, 8, 8, 8.

Figure 7

The effects of DMCM (1.25 mg/kg) and PWZ-029 (5,10 and 20 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 group was 8 *P<0.05 compared to solvent (SOL) group.