Benzodiazepine Binding Characteristics of Embryonic Rat Brain Neurons Grown in Culture

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Abstract
The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high-affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS-specific benzodiazepine clonazepam (Ki - 0.56 .+-. 0.08 nM) but was not affected by the peripheral-type receptor ligand Ro5-4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 .+-. 0.24 nM, and the Bmax was 952 .+-. 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose-dependently by .gamma.-aminobutyric acid (GABA), reaching 112 .+-. 7.5% above control values at 10-4 M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam > diazepam > .beta.-carboline-3-carboxylate ethyl ester > Ro15-1788 > CL218,872 >> Ro5-4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine-GABA-chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function.