DEVELOPMENTAL EXPRESSION OF CHICK CORTICAL GABA(A) RECEPTOR ALPHA-1 SUBUNITS IN-VIVO AND IN-VITRO

In order to examine the expression of the GABA(A) receptor alpha 1 sub unit during chick cortical development in vivo and in vitro, we have u tilized a polyclonal antibody (RP4) directed against an alpha 1(331-38 1) fusion protein. This antibody exhibits a high titer for precipitati on of [H-3]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta 2- or beta 4-s ubunit fusion proteins, and a robust reaction with a single 51-kDa pol ypeptide on immunoblots of cortical membranes. This indicates monospec ificity of the RP4 antiserum for the GABA(A) receptor alpha(1) subunit . The alpha 1-subunit antibody also showed strong immunocytochemical r eactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum, The ontogeny of the alpha 1 subunit in chick cortex and in derived neuronal cultures was examined by quantit ative Western blotting. The level of the alpha 1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expressi on at day 4. Expression of the cortical GABA(A) receptor alpha 1 subun it increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alph a 1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-tran scriptase polymerase chain reaction (RT-PCR), showed a corresponding i ncline. These findings correlated well with previous developmental stu dies of GABA(A) receptor ligand binding sites both in vivo and in vitr o. The parallel increase of the alpha 1 subunit transcript and polypep tide with [H-3]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical o ntogeny. This was investigated further by quantitative immunoprecipita tion. At saturation, the RP4 antiserum consistently precipitated 50-65 % of the central [H-3]flunitrazepam binding sites in the developing co rtex from E12 through P7, despite a 5-fold increase in the binding lev el. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha 1 subunits remains relatively const ant. Furthermore, the alpha 1 polypeptide appears to be a major compon ent of GABA(A) receptor oligomers at all stages of cortical maturation . (C) 1997 Elsevier Science B.V.