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

The present study was designed to compare the allosteric coupling between the Cl- channel of the GABA, 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 [H-3]flunitrazepam and two ligands with higher affinity for benzodiazepine BZ(1) sites relative to benzodiazepine BZ(2) 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-acetamide hemitartrate (zolpidem). Benzodiazepine BZ(1) 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 BZ(2) 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 similar to 150 nM) and benzodiazepine BZ(2L) (low affinity for zolpidem, K-d similar to 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 [H-3]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 [S-35]t-butylbicyclophosphorothionate ([S-35]TBPS) to the convulsant site of cortical GABA, 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 [S-35]TBPS binding in adult rats relative to neonates. The different affinities of CL 218,872 and zolpidem for benzodiazepine BZ(1) and BZ(2) receptors may account, at least in part, for the age-related changes in their inhibitory potencies. These results demonstrate that benzodiazepine BZ(2) sites mediate the modulation of [S-35]TBPS binding by benzodiazepine recognition site ligands in the cerebral cortex of newborn rats. Further, benzodiazepine BZ(2) sites may be involved in the inhibition of [S-35]TBPS binding by flunitrazepam, CL 218,872 and zolpidem in the cerebral cortex of adult rats.