Ontogeny of the GABA receptor complex in chick brain: studies in vivo and in vitro

The ontogeny of the gamma-aminobutyric acid-A (GABAA) receptor complex in the chick brain was studied by specific binding of [3H]muscimol, [3H]flunitrazepam (Flu) and [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes. During development in ovo, the specific binding of muscimol and flunitrazepam increased from day 8 and reached 50% of adult levels of day 20, while a comparable level of TBPS binding was achieved by day 17. The increases in TBPS and Flu binding were reflected in Bmax rather than Kd changes. In embryonic brain, only a low-affinity site for muscimol (Kd = 23 nM) was observed while an additional high-affinity site (Kd = 0.4 nM), as well as the low-affinity site, was found in adult tissue. Similar studies were carried out with cultures of cerebral neurons prepared from 8-day embryos. The level of specific binding of muscimol, Flu and TBPS increased in culture, achieved one half of the maximum level by days 4-5, maximal levels by day 10 and decreased slowly thereafter. The maximal levels in culture corresponded, respectively, to 27%, 67% and 57% of these found in the 18-day embryo. The binding of Flu to membranes from neurons, embryos and adults was enhanced by addition of GABA while TBPS binding was inhibited. The EC50 and IC50 values for these effects corresponded to those for gating of chloride channels. These findings indicate a coordinated expression of receptors for GABA, benzodiazepines and convulsant/TBPS during neuronal maturation both in vivo and in vitro. The schedule for this postsynaptic ontogeny is very similar to that for presynaptic markers of GABAergic neurons (see companion paper).