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

The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR) in primary c ultured cerebellar granule cells. Several factors contributing to the inhib itory effects of ROH on NMDAR function were assessed using both whole-cell and perforated patch-clamp recordings. The NMDAR subunit composition was ex amined by Western blot analysis using NR2 subunit-specific antibodies and p harmacological 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 in creasing days in vitro (DIV). Although the NR2B subunit was detectable unti l 21 DIV, there was a significant decrease in ifenprodil sensitivity after 7 DIV. EtOH sensitivity did not change with an increasing DIV. A high conce ntration of glycine reversed EtOH inhibition of steady-state, but not peak, NMDA-induced current during whole-cell recordings. Significant glycine rev ersal of effects of a low concentration of EtOH on peak current was observe d under perforated patch-clamp conditions. A 30-s EtOH pretreatment signifi cantly enhanced ROH inhibition of NMDA-induced peak current. Collectively, these results indicate that EtOH sensitivity of the NMDAR in primary cultur ed cerebellar granule cells is not related to subunit composition nor ifenp rodil sensitivity, involves a kinetic interaction with glycine, and can be enhanced by a slowly developing transduction mechanism that occurs within t ens of seconds.