Ethanol Inhibits NMDA Receptor-Mediated Excitotoxicity in Rat Primary Neuronal Cultures

ABSTRACT

Excessive or prolonged stimulation of, N-methyl-D-aspartate (NMDA) receptors appears to play an important role in many neurodegenerative processes in brain through a process known as excitotoxicity. This study examined the effects of ethanol on NMDA receptormediated excitotoxicity in primary neuronal cultures obtained from embryonic rat whole brain. Neurotoxicity was quantitated by measuring the amount of lactate dehydrogenase released into the media during a 20-hr time period following NMDA washout. Exposure of 12-to 14-day-old cultures to NMDA in Mg²⁺-free HEPES buffer (pH 7.4) for a 25-min period resulted in a concentration-dependent toxicity (EC₅₀= 54 μM). Time-course experiments showed that exposure to NMDA for as little as 5 min was excitotoxic and reached a plateau after a 20-min exposure period. Preincubation of the cultures with ethanol (25 to 200 mm) resulted in a concentration-dependent inhibition of NMDA-mediated toxicity with approximately 38% inhibition produced by 25 mm ethanol and essentially complete inhibition at 200 mm ethanol (IC₅₀= 60 mm). Increasing the glycine concentration to 100 μM did not potentiate NMDA neurotoxicity or antagonize the neuroprotective effect of ethanol. NMDA-Mediated excitotoxicity was reduced by approximately 50% by the glycine antagonist 7-chlorokynurenate (50 μM). Ethanol (50 mm) reduced NMDA neurotoxicity similar to 7-chlorokynurenate, and the two together produced greater inhibition than either alone. These results show that intoxicating concentrations of ethanol can potently inhibit NMDA receptor-mediated excitotoxicity and may have important implications in terms of ethanols interactions with brain trauma, ischemia, and other neuropathologies associated with NMDA receptor-mediated neurotoxicity.