Characterization of benzodiazepine receptors in the cerebellum.

1. The goals of the work reported here were to further characterize benzodiazepine/GABA(A) (BDZR) receptor heterogeneity in the cerebellum and to measure the affinities and selectivities of structurally diverse benzodiazepines at each site identified. 2. Five chemical families were included in these studies. These were 1,4-benzodiazepines (flunitrazepam), imidazobenzodiazepines (RO15-1788 and RO15-4513 and RO16-6028), beta-carbolines (Abecarnil) and pyrazoloquinolines (CGS 8216, CGS 9895 and CGS 9896). 3. Saturation and competition binding assays were combined with powerful data analysis software developed in our laboratory. Among the capabilities of this software is the identification of multiple binding sites for a cold ligand using a non-selective labeled ligand that binds with equal, but high, affinity to all the binding sites 4. Saturation binding assays using either [3H]-RO15-1788 or [3H]-RO15-4513 revealed only one apparent binding site, with a higher affinity for RO15-4513 than for RO15-1788. However, using [3H]-RO15-4513 for the competition binding studies in the cerebellum, together with our data analysis software, led to the identification of two distinct binding sites with equal densities for the diverse benzodiazepines studied. 5. In rat cerebellum one of the sites identified corresponds to GABA(A) receptors exhibiting alpha1 subunit pharmacology and the other to GABA(A) receptors exhibiting alpha6 subunit pharmacology. In general, the diverse families of BDZR ligands studied had much lower affinities for the alpha6 containing receptors.