Ethanol-induced elevation of ceramide and PAR-4 activates caspase 3 in NCCs, which is prevented by betaine and citicoline. (a) Pregnant mice (three isocaloric controls, three treated) were intubated with 3 g/kg ethanol at days E8.5 and 9.5. Lipids were extracted from embryos at day E10.5 and analyzed by HPTLC. Lanes 1–5, vehicle-treated controls; lanes 6–10, ethanol-treated mice; lanes 11–13, ceramide standard (0.1, 0.2, 0.5 μg) (b) Immunoblot showing elevation of PAR-4 in embryos at day E10.5 after prenatal ethanol exposure at day E8.5 and E9.5. Lane 1, vehicle control; lane 2, embryo from ethanol-treated mouse. (c) Immunoblot showing elevation of PAR-4 in NCCs from explant culture exposed to ethanol (overnight). Lane 1, no ethanol; lane 2, 0.3% ethanol; lane 3, 0.5% ethanol. (d) Ethanol incubation (overnight) of NCCs leads to the activation of caspase 3, which is prevented by simultaneous incubation with citicoline (CDP-choline, 100 μM) or betaine (1 mM). For further quantitation by counting apoptotic cells, see Table 3. Lane 1, no ethanol; lane 2, 0.3% ethanol; lane 3, 0.5% ethanol; lane 4, 0.5% ethanol+citicoline; lane 5, 0.5% ethanol+betaine