The present study was undertaken to investigate the involvement of NCAM in the neuroteratogenic effects of ethanol demonstrated by us and others. In the first experiment we examined the effect of in-ovo ethanol exposure on expression of NCAM in various regions of the embryonic CNS throughout development. Chick embryos received ethanol (10 mg/50 microliters/day) or saline (control) at days 1-3 of development (E1-E3), were sacrificed at various embryonic ages and whole brain (WB), cerebral hemispheres (CH) and cerebellum (CE) processed for SDS-polyacrylamide gel electrophoresis. The normal developmental profile of NCAM in the chick brain exhibited the same dynamics as previously reported by others. When compared to age-matched control brains, an increase was observed in expression of high molecular weight forms of NCAM in cerebral hemispheres between E8 and E10. These bands represented highly sialated (> 180 kDa) forms of NCAM. In fact, the NCAM hand from ethanol-treated embryos at E8 migrated at a higher molecular weight than did its control counterpart, indicating an increase in sialic acid content. In contrast, no clear change was observed in NCAM expression in cerebellum from E10 through E20 as a result of ethanol exposure. In the second experiment, we examined the involvement of NCAM in the alterations in neuronal growth patterns observed in ethanol-exposed cultures. Neuroblast-enriched cultures derived from three-day-old whole chick embryos (E3WE) were 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 decreasing by 9 DIV.NCAM positive structures included all neuronal perikarya, neuritic processes and growth cones. Addition of 50 mM ethanol to the medium resulted in profound alterations in growth patterns of developing 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 with the shift in neuronal phenotype from cholinergic to catecholaminergic and GABAergic which we have reported previously. Changes in the normal pattern of cellular contact and interaction as a result of altered NCAM expression may influence establishment of neurotransmitter phenotype. Findings from this study support the view that NCAM may be involved both directly and indirectly in shaping of the CNS during development and we speculate that ethanol neuroembryotoxicity uncouples this relationship.

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In this study, we examined the effects of ethanol administered to chick embryos, on the maturation of astrocytes, using glutamine synthetase (GS) activity as an astrocyte marker. Ethanol (50 mM) was administered in ovo via the air sac, embryos were sacrificed at various days of embryonic development and GS activity was determined in cerebral hemispheres and cerebellum. We found that in both cerebral hemispheres and cerebellum, GS activity was higher in the ethanol-treated embryos, as compared to controls, during the embryonic periods, E6 to E10 in the cerebral hemispheres and E10 to E14 in the cerebellum. These periods are characterized by increased neuronal differentiation in these CNS areas. The increase in GS activity in the ethanol-treated embryos is speculated to reflect either a transient reactive gliosis and/or an enhancement in the differentiation of radial glia, immature glia, to more mature astrocytes.

The goal of this study was to examine the responsiveness of an immortalized catecholaminergic neuronal line, 2N27, to various growth factors and identify those which promote catecholaminergic expression. 2N27 is a newly established neural cell line derived from fetal rat mesencephalic tissue and, thus, contains tyrosine hydroxylase (TH), a reliable marker for catecholaminergic neurons. Using TH activity as a biochemical index, we examined the responsiveness to both recognized trophic factors (NGF, TGF-beta and basic- and acidic-FGF) as well as novel, glia-derived factors present in conditioned media from several glial sources. The glial cells included MACH, a normal cell line derived from aged mouse cerebral hemispheres NBCC, normal glia derived from newborn mouse cerebral hemispheres; and C-6 glioma cells, 2B clone, passage 72, predominately astrocytes. Cells were cultured in the presence of added factors from 0 to 3 days in vitro (DIV) and were harvested on day 4. We found that 2N27 neural cells responded differentially to growth factors. No change was observed in TH activity in response to NGF, TH activity even decreased in response to b-FGF ad TGF-beta addition to the culture medium. However, a dose dependent increase in TH activity was observed following treatment with a-FGF and the increase to a-FGF was associated to an increase in cell proliferation as compared to TH increase by cAMP associated to differentiation. However, the 2N27 cells responded with a marked increase in TH when cultured in the glial cell conditioned media. We conclude that immortal cells require a variety of microenvironmental signals to maintain their phenotype.