Modulation of [35S]TBPS binding by ligands with preferential affinity for benzodiazepine BZ1 sites in the cerebral cortex of newborn and adult rats

Abstract

The present study was designed to compare the allosteric coupling between the Cl⁻ channel of the GABA_A receptor and the different benzodiazepine recognition site subtypes (BZ sites) in the cerebral cortex of newborn (5-day-old) and adult rats (90-day-old). To this aim, we reexamined the heterogeneity of cortical GABA_A receptors in self- and cross-competition binding experiments using [³H]flunitrazepam and two ligands with higher affinity for benzodiazepine BZ₁ sites relative to benzodiazepine BZ₂ sites, the triazolopyridazine 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo [4,3-b] pyridazine (CL 218,872) and the imidazopyridine N,N,6-trimethyl-2-(4- methylphenyl)-imidazo[1,2-a-pyridine-3-acetemide hemitratrate (zolpidem). Benzodiazepine BZ₁ sites accounted for 52% of the total number of binding sites in adult rats, but were not detected in newborn rats. On the other hand, two classes of benzodiazepine BZ₂ sites with high and low affinity for zolpidem were present in newborn and adult rats. These sites were designated as benzodiazepine BZ_2H (high affinity for zolpidem, K_d ∼ 150 nM) and benzodiazepine BZ_2L (low affinity for zolpidem, K_d ∼ 3000 nM). High densities of benzodiazepine BZ_2H sites were measured in both newborn and adult rats (75% and 41% of the total number of [³H]flunitrazepam binding sites, respectively), whereas benzodiazepine BZ_2L sites accounted for 25% and 7% of the total number of cortical sites in neonates and adults, respectively. Flunitrazepam, CL 218,872 and zolpidem inhibited in a concentration-dependent manner the binding of $\text{[}{}^{35}\text{S}\text{]t-}\text{butylbicyclophosphorothionate}$ ([³⁵S]TBPS) to the convulsant site of cortical GABA_A receptors in newborn and adult rats. The IC₅₀ for flunitrazepam was about 3-fold greater in adults than in neonates. This rightward shift in the concentration-response curve may be due to a decrease with age in the intrinsic efficacy of flunitrazepam. In contrast, CL 218,872 and zolpidem were 4-fold more potent at inhibiting [³⁵S]TBPS binding in adult rats relative to neonates. The different affinities of CL 218,872 and zolpidem for benzodiazepine BZ₁ and BZ₂ receptors may account, at least in part, for the age-related changes in their inhibitory potencies. These results demonstrated that benzodiazepine BZ₂ sites mediate the modulation of [³⁵S]TBPS binding by benzodiazepine recognition site ligands in the cerebral cortex of newborn rats. Further, benzodiazepine BZ₂ sites may be involved in the inhibition of [³⁵S]TBPS binding by flunitrazepam, CL 218,872 and zolpidem in the cerebral cortex of adult rats.