We have developed a unique primary human culture model for neuronal cells to study the possible impact of EtOH on neuronal viability at different stages of differentiation (Fig. 1a). Primary human fetal neurons (PHFNs) and neurospheres (hNSPs) were cultured from matching human fetal donor brains (16–18 weeks of gestation) as described in materials and methods. In parallel to direct neuron cultures, hNSPs were also established from matching donor brains and maintained in the culture. In order to establish neural progenitors, neurospheres were dissociated and cultured as monolayers of neuronal progenitors (hNPCs) as described in materials and methods. Immunocytochemical verification revealed that young and mature neurons were stained for synaptophysin and Map2, hNSPs and hNPCs were stained for Nestin (Fig. 1b). PHFN cultures were followed for neuronal action potentials as the criteria for their full differentiation by multielectrode array (MEA) studies measuring local field potentials (LFP) at week 1, 3, and 4 post culturing. As shown in Fig. 1c, PHFNs started to show LFP activity at 3 weeks and reached the peak levels at 4 weeks post culturing. Neuronal cultures at
