Receptors for (3H)muscimol and for (3H)GABA in neurone-enriched cultures

Abstract The binding of ( 3 H)muscimol and ( 3 H)GABA to thoroughly-washed subcellular particles prepared from “neurone-enriched” and astroblast primary cultures of rat brain was examined in Na + -free, Tris-citrate medium using a centrifugation assay. Competition for binding sites by excess (10 −3 and 10 −5 M) unlabelled GABA provided estimates of “specific” binding. With 6.2 nM labelled ligand in the medium, about 37 f-moles ( 3 H)GABA/mg protein and about 220 f-moles ( 3 H)muscimol/mg protein were bound to particles of neurone-enriched cultures. Over a concentration range of 6.2–62 nM, ( 3 H)muscimol binding had K B ≈9 nM, B MAX ≈270 f-moles/mg protein. Neither ( 3 H)GABA nor ( 3 H)muscimol was bound to particles prepared from cultured astroblasts of neonatal rat brain or from C 6 glioma cells. ( 3 H)Muscimol binding to particles of neurone-enriched cultures was potently inhibited by muscimol itself, GABA, isoguvacine, and 3-aminopropanesulfonic acid, and less potently inhibited by bicuculline-methobromide. β-Alanine and β-proline also had some inhibitory effect, whereas (±) nipecotic acid, L-2,4-diaminobutyric acid and picrotoxin were relatively inactive ( IC 50 1 mM ). A similar binding of ( 3 H)GABA and ( 3 H)muscimol occurred to particles of a neurone-enriched culture of mouse brain. Electron-microscopic examination of neurone-enriched cultures did not reveal synaptic connections. Thus, GABA-receptors are present on neuronal membranes before the formation of most synapses. This binding of ( 3 H)muscimol and of ( 3 H)GABA that occurs to neuronal, but not to glial, membranes might serve as a useful “neuronal marker”.