Morphologic and neurotoxic effects of ethanol vary with timing of exposure in vitro☆

Abstract 

Results of investigations with animal models of fetal alcohol syndrome (FAS) seem to indicate that neuronal vulnerability to ethanol-induced cell death may be correlated with specific developmental events. In the present study, we sought to test this observation in a cell culture model of neuronal development in which morphogenesis as well as survival could be assessed. Using embryonic rat hippocampal pyramidal neurons in primary cultures, we compared the sensitivity of neurons to ethanol added, at 400 mg/dl, to the medium at different times relative to the development of axons and dendrites. Quantitative morphometric analysis was performed by using phase contrast at 12 h (0.5 day) and 24 h (1 day), or fluorescence microscopy after microtubule-associated protein-2 (MAP2) immunostaining at 6 and 14 days. Survival was assessed by counting the number of neurons per unit area of the substrate at 14 days. Addition of ethanol 1 day after plating, when most neurons had developed an axon, had no effect on survival up to 14 days in vitro, but resulted in significantly shorter, less branched dendrites than observed when ethanol was added 2 h after plating. Despite the shorter duration of ethanol exposure, the addition of ethanol on day 6, after rapid growth of dendrites and synapses had begun, resulted in loss of all but about one third of the neurons by 14 days. This supports the suggestion that increased neuronal vulnerability to the morphoregulatory effects of ethanol is correlated with the establishment of polarity, but that the sensitivity of neurons to the cytotoxic effects of ethanol occurs later, when dendrites and synapses are rapidly forming.

Keywords:  Hippocampus, Neuron development, Fetal alcohol syndrome, Dendrite, Alcohol, In vitro