An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-d-aspartate receptor function

Abstract 

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post–ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I_NMDA) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I_NMDA from CEE-treated cells were significantly larger than I_NMDA from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I_NMDA were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial–neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial–neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

Keywords: N-methyl-d-aspartate receptors, Cerebellar granule cells, Continuous ethanol exposure (CEE), Astrocytes, Confocal microscopy, Whole-cell patch clamp