Continuous Exposure of Cultured Rat Cerebellar Macroneurons to Ethanol-Depressed NMDA and KCl-Stimulated Elevations of Intracellular Calcium

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Abstract
This series of experiments measured ethanol-induced changes in levels of free intracellular calcium. Cerebellar macroneurons, harvested from rat embryos on embryonic day 17, were cultured in the presence of 75 mM ethanol for 24, 48, or 96 hr. Intracellular calcium concentrations in control and ethanol-exposed neurons did not differ after 24 hr, but they were significantly elevated in the neurons exposed to ethanol for 48 or 96 hr. Similarly, increases in intracellular calcium elicited by stimulation with 50 microM NMDA were not significantly different in control and ethanol-exposed neurons after 24 hr. After 48 and 96 hr, however, NMDA-stimulated increases in intracellular calcium levels in control neurons were significantly greater than in the ethanol-exposed neurons. These results showed that, when calcium levels were elevated by prolonged exposure to ethanol, the neurons were significantly less responsive to NMDA stimulation. Increases in intracellular calcium elicited by stimulation with 30 mM KCI were not significantly different in the control and treated neurons after 24 and 48 hr of ethanol exposure. After 96 hr of exposure to ethanol, however, there was a significant increase in intracellular calcium levels in control neurons following KCI stimulation, but not in the ethanol-exposed neurons. The fact that neuronal responses to KCI stimulation were depressed only following 96 hr of exposure to ethanol makes it unlikely that voltage-regulated channels were the primary mediators of the ethanol-induced elevations in intracellular calcium in chronically exposed neurons.