Postnatal development of diazepam-insensitive [3H]RO 15-4513 binding sites

Abstract The postnatal development of the binding sites for an imidazobenzodiazepine, [ 3 H]Ro 15-4513, which labels all presently known GABA A receptor-associated benzodiazepine binding sites, was studied in the cerebellar, cerebrocortical and hippocampal tissues of Wistar rats. The binding sites in the hippocampal membranes were fairly similar at all ages studied (1–2, 7, 14, and 90 days), suggesting early development of the GABA A receptors. The density of the binding sites increased significantly with age in the cerebellar and cerebrocortical membranes, without any changes in the affinity for this ligand. As judged by displacement by a benzodiazepine agonist, diazepam, [ 3 H]Ro 15-4513 seemed to bind to a homogeneous pool of receptors in the cerebral cortex and hippocampus in all age groups, whereas in the cerebellum two pharmacologically distinct sites were observed. The benzodiazepine agonist-insensitive (“diazepam-insensitive”) binding first appeared at the time when the cerebellar granule cells started to mature, on the seventh postnatal day. As determined by autoradiography, such binding occurred selectively in the granule cell layer and was absent from the cerebellum of one- to two-day-old rats. The latter finding was confirmed by photoaffinity labelling experiments, in which diazepam inhibited all photolabelling in membrane homogenates from newborn rats. Diazepam-sensitive cerebellar binding, which increased steadily during development, was also localized in the molecular layer and deep nuclei. The time course of the development of the diazepam-insensitive binding obviously explains the previously observed inconsistencies in the number and location of the GABA A agonist and benzodiazepine binding sites in the cerebellum, as the ligands used in the earlier studies had no affinity for the unique binding sites of [ 3 H]Ro 15-4513.