ETHANOL NEURONOTOXICITY IN THE EMBRYONIC CHICK BRAIN IN OVO AND IN CULTURE - INTERACTION OF THE NEURAL CELL-ADHESION MOLECULE (NCAM)

The present study was undertaken to investigate the involvement of NCA M in the neuroteratogenic effects of ethanol demonstrated by us and ot hers, In the first experiment we examined the effect of in-ovo ethanol exposure on expression of NCAM in various regions of the embryonic CN S throughout development, Chick embryos received ethanol(10 mg/50 mu l /day) or saline (control) at days 1-3 of development (E1-E3), were sac rificed at various embryonic ages and whole brain (WE), cerebral hemis pheres (CH) and cerebellum (CE) processed for SDS-polyacrylamide gel e lectrophoresis. The normal developmental profile of NCAM in the chick brain exhibited the same dynamics as previously reported by others. Wh en compared to age-matched control brains, an increase was observed in expression of high molecular weight forms of NCAM in cerebral hemisph eres between E8 and E10. These bands represented highly sialated (>180 kDa) forms of NCAM. In fact, the NCAM band from ethanol-treated embry os at E8 migrated at a higher molecular weight than did its control co unterpart, indicating an increase in sialic acid content. In contrast, no clear change was observed in NCAM expression in cerebellum from E1 0 through E20 as a result of ethanol exposure, In the second experimen t, we examined the involvement of NCAM in the alterations in neuronal growth patterns observed in ethanol-exposed cultures. Neuroblast-enric hed cultures derived from three-day-old whole chick embryos (E3WE) wer e maintained on poly-L-lysine pre-coated Petri dishes in DMEM+5% fetal bovine serum with or without 50 mM ethanol. Cultures were fixed at 3, 6 or 9 DIV and co-stained for NCAM and neurofilament (160 kDa). E3WE cultures exhibited intense NCAM immunoreactivity at 3 and 6 DIV decrea sing by 9 DIV, NCAM positive structures included all neuronal perikary a, neuritic processes and growth cones. Addition of 50mMethanol to the medium resulted in profound alterations in growth patterns of develop ing neurons which continued to exhibit intense NCAM staining. Ethanol- induced changes in the developmental profile of NCAM expression (i.e. increased sialation) in cerebral hemispheres correspond temporally wit h the shift in neuronal phenotype from cholinergic to catecholaminergi c and GABAergic which we have reported previously. Changes in the norm al pattern of cellular contact and interaction as a result of altered NCAM expression may influence establishment of neurotransmitter phenot ype. Findings from this study support the view that NCAM may be involv ed both directly and indirectly in shaping of the CNS during developme nt and we speculate that ethanol neuroembryotoxicity uncouples this re lationship,