AGONIST ADMINISTRATION IN OVO DOWN-REGULATES CEREBELLAR GABA(A) RECEPTORS IN THE CHICK-EMBRYO

Chick embryos with an undeveloped blood-brain barrier were used to exa mine the down-regulation of GABA(A) receptors in vivo. The GABA(A) rec eptor agonist isoguvacine (5 mu mol) was applied to the vascularized c horioallantoic membrane of 8 day embryos. This treatment was repeated on embryonic days 11, 14, and 17, and the embryos were sacrificed on d ay 18 (stage 42). Isoguvacine administration reduced the clonazepam-di splaceable binding of [H-3]flunitrazepam to washed cerebellar membrane s by 34.0 +/- 3.0% compared to vehicle-treated controls. Binding reduc tions of lower magnitude were found in membranes from the cerebrum and optic lobes. Administration of isoguvacine had no significant effect on the wet weights of whole embryos or cerebella, the yield of cerebel lar membranes, or the binding of [H-3]N-methylscopolamine. The reducti on of [H-3]flunitrazepam binding to cerebellar membranes was dose-depe ndent, allowing a half saturation value of 8 mu M isoguvacine to be es timated. Scatchard analysis showed that the B-max for [H-3]flunitrazep am binding was reduced by 28.3 +/- 6.7% compared to controls, without a change in the K-d. Embryonic exposure to isoguvacine also caused a r eduction of 43.6 +/- 6.0% in the binding of the GABA(A) receptor chann el ligand [S-35]t-butylbicyclophosphorothionate to washed cerebellar m embranes. Taken together, these results indicate that isoguvacine indu ces a down-regulation of the receptor subunits in vivo. However, measu rements of cerebellar GABA(A) receptor mRNAs for the alpha 1, beta 2L, beta 2S, beta 4, gamma 1, gamma 2L, and gamma 2S subunits by reverse transcriptase - polymerase chain reaction (RT-PCR) revealed no signifi cant alterations by isoguvacine administration. The data suggest that translational or post-translational mechanisms, rather than those modu lating the synthesis or stability of subunit mRNAs, take precedence in establishing GABA(A) receptor down-regulation.