QUANTITATIVE CHANGES IN ALPHA-1 AND ALPHA-5 GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR SUBUNIT MESSENGER-RNAS AND PROTEINS AFTER A SINGLE TREATMENT OF CEREBELLAR GRANULE NEURONS WITH N-METHYL-D-ASPARTATE

It was previously demonstrated that daily administration of N-methyl-D -aspartate (NMDA) to primary cultures of cerebellar granule neurons fo r 5 days in vitro mediates an increase in the relative content of mRNA s encoding selected subunits of the gamma-aminobutyric acid (GABA)(A) receptor. This analysis was extended using a competitive polymerase ch ain reaction assay with internal standards to quantitate changes that occur in the absolute amounts of selected GABA(A) receptor subunit mRN As in cerebellar granule neurons in vitro after the single administrat ion of nontoxic doses of either NMDA or glutamate. For these studies, we focused on the alpha 1, alpha 5, and alpha 6 receptor subunit mRNAs and examined their absolute contents in cultures maintained in low KC l (12.5 mM), maintained in low KCl and treated with NMDA (10 mu M) for 24 hr, or maintained in high KCl (25 mM). The absolute amounts of eac h mRNA varied in these paradigms; whereas the alpha 1 and alpha 5 rece ptor subunit mRNAs increased in response to NMDA-selective glutamate r eceptor stimulation, the alpha 6 receptor subunit mRNA did not. The ti me course of the alpha 1 and alpha 5 mRNA increases, dose dependence, and effects of glutamate in the presence or absence of MK-801 were als o analyzed. Treatment of cultures maintained in 12.5 mM KCl with 5 mu M glutamate resulted in comparable changes in the alpha 1 and alpha 5 receptor subunit mRNA contents, and a somewhat smaller increase in the alpha 6 mRNA content was observed. Using corresponding GABA(A) recept or subunit-specific antibodies, it was shown that the observed mRNA ch anges are accompanied by increased expression of alpha 1- and alpha 5- like receptor subunit immunoreactivity. Collectively, these data demon strate that signal transduction mechanisms triggered by NMDA-selective glutamate receptor stimulation differentially modulate the levels of selected GABA(A) receptor subunit mRNAs and the corresponding proteins they encode.