Developmental Regulation of Expression of GABAA Receptor α1 and α6 Subunits in Cultured Rat Cerebellar Granule Cells

Abstract

We have studied the postnatal development of GABAA receptor α1 and α6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor α1 and α6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-α1 subunit-specific antibodies, immunoreactive species with Mr 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the α1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da α1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3–5 DIV in P2 cultures. The GABAA receptor α6 subunit-like immunoreactivity (58,000 Da) was not detected until 5–7 DIV in P10 and 9–11 DIV in P2-derived cultures. The appearance of α6 subunit-like immunoreactivity was paralleled by an up-regulation of α1 subunit expression and a concomitant increase in diazepam-insensititive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of α6 and α1α6-subunit-containing GABAA 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 significantly affect the developmental profile, the level of GABAA receptor α6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor α6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors. Copyright © 1996 Elsevier Science Ltd