Increased ambient glutamate concentration alters the expression of NMDA receptor subunits in cerebellar granule neurons.

Effects of prolonged (48 h) inhibition of glutamate reuptake on the relative abundance of mRNAs coding for N-methyl-D-aspartate (NMDA) receptor subunits, and the expression of corresponding proteins were investigated in primary cultures of rat cerebellar granule neurons. In cells exposed to the glutamate transport blocker, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), the expression of the C1 exon-positive NR1 mRNA variant was reduced by about 40% whereas, the expression of C1-negative mRNA was increased leading to significant reduction of the +C1/-C1 ratio. The expression of the N1-negative NR1 variants was slightly reduced following exposure to PDC, indicating that increased medium levels of glutamate changed the relative abundance of NR1 splice-variant expression but did not reduce the overall NR1 transcription. Expression of NR2A and NR2B mRNAs was 40-50% lower in PDC-treated cells as compared to control. Immunoblot experiments revealed that PDC exposure reduced the expression of NR1 and all NR2 proteins with NR2A and NR2B proteins being reduced to a greater extent than NR1. The overall decrease in NMDA receptor subunit protein expression was considerably more pronounced than the reduction of their corresponding mRNAs, suggesting involvement of a post-transcriptional regulation. Our data support the hypothesis that functional activity and number of NMDA receptors are regulated by strength of the glutamatergic input. Thus, reduced glutamate uptake resulting in increased concentration of ambient glutamate initiate a series of adaptive responses manifested as a gradual down-regulation of the functional activity and expression of NMDA receptors.