Experiments were performed to characterize diazepam-insensitive [3H]Ro 15-4513 binding sites in discrete regions of rodent brain and cultured rat cerebellar granule cells. Scatchard analysis of [3H]Ro 15-4513 binding in the presence of 10 microM diazepam revealed that diazepam-insensitive binding sites in the rat brain were most abundant in the cerebellum, followed by the hippocampus, cerebral cortex and olfactory bulb. Diazepam-insensitive sites represented approximately 80% of the total [3H]Ro 15-4513 binding sites in the membranes of cultured rat cerebellar granule cells. The Bmax values for total [3H]Ro 15-4513 and [35S]TBPS are almost identical, and 5-6 times larger than that for [3H]diazepam in this preparation. Although some annelated [1,5-a]benzodiazepine analogues such as Ro 15-4513, ro 16-6028, flumazenil and Ro 15-3505, and an imidazothieno-diazepine, Ro 19-4603, showed high affinity for cortical and cerebellar diazepam-insensitive sites, all the annelated benzodiazepine compounds tested showed higher affinity for cerebellar diazepam-insensitive sites than cortical ones. In contrast, a pyrazoloquinoline compound, CGS 8216, and beta-carboline analogues such as beta-carboline-3-carboxylate ethyl ester (beta-CCE) and beta-carboline-3-carboxylate methyl ester (beta-CCM) exhibited higher affinity for cortical than cerebellar sites. These results suggest that diazepam-insensitive sites are heterogeneous in brain areas with respect to ligand specificity.

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