Factors that enhance ethanol inhibition of N-methyl-D-aspartate receptors in cerebellar granule cells

Abstract

The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR) in primary cultured cerebellar granule cells. Several factors contributing to the inhibitory effects of EtOH on NMDAR function were assessed using both whole-cell and perforated patch-clamp recordings. The NMDAR subunit composition was examined by Western blot analysis using NR2 subunit-specific antibodies and pharmacological manipulation with the NR2B-specific antagonist infenprodil. Western blot analysis indicated that NMDAR subunit composition changed from a combination of NR2A and NR2B containing NMDARs to primarily NR2A with increasing days in vitro (DIV). Although the NR2B subunit was detectable until 21 DIV, there was a significant decrease in ifenprodil sensitivity after 7 DIV. EtOH sensitivity did not change with an increasing DIV. A high concentration of glycine reversed EtOH inhibition of steady-state, but not peak, NMDA-induced current during whole-cell recordings. Significant glycine reversal of effects of a low concentration of EtOH on peak current was observed under perforated patch-clamp conditions. A 30-s EtOH pretreatment significantly enhanced EtOH inhibition of NMDA-induced peak current. Collectively, these results indicate that EtOH sensitivity of the NMDAR in primary cultured cerebellar granule cells is not related to subunit composition nor ifenprodil sensitivity, involves a kinetic interaction with glycine, and can be enhanced by a slowly developing transduction mechanism that occurs within tens of seconds.