Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

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Abstract
The specific bindings of [3H]flunitrazepam ([3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, altough their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p < 0.001). Taken together, our data suggest that the existence of several .gamma.-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-minetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol [THIP] in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.