Ethanol neurotoxicity in culture: selective loss of cholinergic neurons

Studies from our laboratory have shown that in ovo exposure of chick embryos to ethanol decreases neuronal survival in culture, and shifts neurotransmitter phenotypic from cholinergic to catecholaminergic and GABAergic. In this study we attempted to determine if the shift from cholinergic expression is a result of selective loss of cholinergic neurons. Neuron-enriched primary cultures were prepared from 3-day-old whole chick embryos. Cells proliferating during the first 3 days in culture were labeled with BrdU, and one half of the cultures were exposed to 50 mM ethanol during the same time period. Selective survival in vitro of the cholinergic, catecholaminergic. GABAergic, glutamatergic, and somatostatinergic phenotypes was determined by counting cells double-stained for BrdU and either ChAT, TH, GAD, Glu, or SRIF. We found that ethanol exposure resulted in a significant reduction in neuronal survival within the cholinergic phenotype, both in the total number of ChAT+ cells and in the subpopulation born between 0-3 DIV. In addition, survival of glutamatergic neurons "born" between 0-3 days in vitro was significantly enhanced, while survival in catecholaminergic, GABAergic, and somatostatinergic phenotypes was also enhanced slightly. These results corroborate our earlier biochemical findings and suggest that the differential cholinotoxic effect of ethanol is due, at least in part, to enhancement of cell-death of cholinergic neuroblasts. This does not preclude the possibility that multipotent neuroblasts are also influenced to express alternative phenotypes, and analyses of these data, in fact, support this notion as well.