Direct effects of ethanol on neuronal differentiation: An in vitro analysis of viability and morphology.

The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development.