Differential Effects of Chronic Ethanol Treatment on N-Methyl-D-aspartate R1 Splice Variants in Fetal Cortical Neurons

Abstract

Functional N-methyl-D-aspartate receptors consisting of NR1 and NR2 subunits are an important site of action of ethanol. Chronic ethanol treatment increases the NR1 polypeptide levels in vivo and in vitro. Chronic ethanol treatment in vitro does not significantly alter the NR1 mRNA levels, even though under similar culture conditions ethanol (50 mM, 5 days) enhances the half-life of NR1 mRNA in fetal cortical neurons. To address this phenomenon, we determined by reverse transcription-polymerase chain reaction and Western blotting whether ethanol (50 mM, 5 days) has a splice variant-specific effect on the expression of the NR1 subunit in mouse fetal cortical neurons. This report analyzes for the first time the distribution of all NR1 splice variants in these neurons. Our data indicate the presence of NR1-3a,b and NR1-4a,b splice variants in cortical neurons. Chronic ethanol treatment significantly decreased the mRNA levels of exon 5-containing NR1 splice variants (NR1-3b and NR1-4b) (-E5/+E5 = 4.6 in untreated neurons and 6.1 in ethanol-treated neurons) and had no effect on the mRNA levels of NR1-3 (+E21/-E22) and NR1-4 (-E21/-E22) splice variants. At the polypeptide level, chronic ethanol treatment significantly reduced exon 5-containing splice variants (NR1-3b and NR1-4b). However, ethanol (50 mM, 5 days) induced a significant increase in polypeptide levels of NR1-4 (-E21/-E22), without any effect on NR1-3 (+E21/-E22) polypeptide levels. These results demonstrate that chronic ethanol treatment has a selective effect on the expression of NR1 splice variants at both the mRNA and polypeptide levels in mouse fetal cortical neurons.