GLYCINE DOES NOT REVERSE THE INHIBITORY ACTIONS OF ETHANOL ON NMDA RECEPTOR FUNCTIONS IN CEREBELLAR GRANULE CELLS

The effects of ethanol and/or glycine on NMDA-induced enhancement of c ytoplasmic free Ca2+ concentrations ([Ca2+](i)), Ca-45(2+) influx, 4-b -[H-3]phorbol-12,13-dibutyrate ([H-3]PDBu) binding, and neuronal necro sis in cultured rat cortical and cerebellar granule neurons were exami ned. Using microfluorimetric techniques in combination with rapid perf usion of single brain neurons, we found that glycine (10 mu M) was a n ecessary co-agonist for NMDA-induced depolarization in cerebellar gran ule cells. In contrast, depolarization with NMDA in cortical cells was observed even without the addition of exogenous glycine as well as in the absence or presence of 1 mM MgCl2. Ethanol (50 mM) inhibited the effects of NMDA in some, but not all, neurons indicative of the existe nce of ethanol-sensitive and ethanol-insensitive cortical and cerebell ar granule neurons. In studies performed in monolayers of cortical and cerebellar granule cells, we observed that the presence of glycine (1 0 mu M) was a necessary prerequisite to unmask inhibitory actions of e thanol on Ca-45(2+) influx induced by NMDA. In another set of experime nts, we noted that NMDA-induced stimulation of [H-3]PDBu binding to mo nolayers of intact cerebellar granule cells was inhibited by ethanol ( 50 mM). Finally, we report that ethanol caused a concentration-depende nt inhibition of NMDA-induced necrotic cell death, assessed by measuri ng the ability of cerebellar granule cells to transform 4,-5-dimethylt hiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan. In n one of the four assays used to demonstrate the inhibitory effects of e thanol on NMDA receptor activity, the ethanol-induced inhibition was r eversed by glycine (up to 100 mu M). Thus, in contrast to earlier repo rts, our data suggest that ethanol and glycine produce their effects b y acting at different regulatory sites within the NMDA receptor system in brain neurons.