DEVELOPMENTAL REGULATION OF EXPRESSION OF GABA(A) RECEPTOR ALPHA-1 AND ALPHA-6 SUBUNITS IN CULTURED RAT CEREBELLAR GRANULE CELLS

We have studied the postnatal development of GABA(A) receptor alpha 1 and alpha 6 subunits expressed by primary cultures of cerebellar granu le cells originating from 2-day-old (postnatal day 2, P2) and 10-day-o ld (P10) rat neonates. At these ages, the granule cells are at distinc t stages of cerebellar development. In both cases, GABA(A) receptor al pha 1 and alpha 6 subunit-like immunoreactivities were detected, and d isplayed temporal expression profiles that were correlated with the ma turity of the cerebella from which the cultured granule cells were der ived. Using two different specificity anti-alpha 1 subunit-specific an tibodies, immunoreactive species with M(r) 53,000 Da and 54,000 Da wer e detected by immunoblotting. The lower 53,000-Da band co-migrated wit h the alpha 1 subunit-like immunoreactivity detected in GABA(A) recept ors purified from adult rat forebrain by benzodiazepine affinity chrom atography. This 53,000-Da alpha 1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3-5 DIV in P2 cultures. The GABA(A) receptor alpha 6 subunit-like immunoreactivit y (58,000 Da) was not detected until 5-7 DIV in P10 and 9-11 DIV in P2 -derived cultures. The appearance of alpha 6 subunit-like immunoreacti vity was paralleled by an up-regulation of alpha 1 subunit expression and a concomitant increase in diazepam-insensititive (DZ-IS) [H-3]Ro 1 5-4513 binding activity, a pharmacological characteristic of alpha 6 a nd alpha 1 alpha 6-subunit-containing GABA(A) receptors (Pollard et al . J. Biol. Chem., 270, 21,285-21,290, (1995)). Antagonism of both non- NMDA and NMDA subtypes of ionotropic glutamate receptors did not signi ficantly affect the developmental profile, the level of GABA(A) recept or alpha 6 subunit or the total DZ-IS or DZ-S [H-3]Ro 15-4513 binding activities expressed by these neurons. These results provide further e vidence that the expression of specific GABA(A) receptor subunit genes is subject to differential regulation. Furthermore, developmental exp ression of the GABA(A) receptor alpha 6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cu e that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors. Copyr ight (C) 1996 Elsevier Science Ltd.