DEVELOPMENTAL UP-REGULATION AND AGONIST-DEPENDENT DOWN-REGULATION OF GABA(A) RECEPTOR SUBUNIT MESSENGER-RNAS IN CHICK CORTICAL-NEURONS

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABA(A) receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively, The accumulation of the beta 4S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 mu M GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their spice variants (where present) were all reduced by 47-65% compared to untreated controls. However, GABA treatment for 4 days did not produce a significant change in the level of the alpha 1 subunit mRNA. The subunit specificity observed for developmental up-regulation and the lack of specificity in down-regulation suggests that mRNA down-regulation does not involve suppression of a developmental process. Comparisons with previous work indicate that the GABA-dependent reduction in the levels of subunit transcripts occurs after the loss of GABA(A) receptor binding sites [Hablitz et al., Brain Res., 501 (1989) 332-338] and subunit polypeptides [Calkin and Barnes, J. Biol. Chem., 269 (1994) 1548-1553]. Thus, for the down-regulation of GABA(A) receptors, it appears that translational or post-translational mechanisms may take precedence over the controlled synthesis or stability of subunit mRNAs.