Ethanol neuronotoxicity in the embryonic chick brain in ovo and in culture: Interaction of the neural cell adhesion molecule (NCAM)

Abstract

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.

Alcohol Exposure Alters the Expression Pattern of Neural Cell Adhesion Molecules During Brain Development

“…Because NCAM 180 and NCAM 140 are primarily located at the synapses and they are involved in synaptic stabilization (Persohn and Schachner, 1987), a reduction in the levels of these proteins might affect cell‐cell interactions, leading to a reduction in axonal growth and synaptic remodeling and stabilization. In fact, ethanol has been shown to alter neuronal growth patterns and neuritic branching in culture (Kentroty et al, 1995), and in vivo alcohol exposure affects synaptogenesis (Guerri, 1987; Inomata et al, 1987).…”

Murine Models for the Study of Fetal Alcohol Spectrum Disorders: An Overview

“…Several studies in animal models (zebrafish) and cell cultures show decreased levels of NCAM after ethanol exposure (135,136). In other studies, different patterns of NCAM expression were detected according to the developmental stage on which PAE occurs (133) or the NCAM isoform analyzed. For example, the highly sialylated form of NCAM is overexpressed after ethanol exposure but the NCAM 180 and NCAM 140 isoforms appear down-regulated in a rat model (137).…”

“…PAE affects the expression levels of synaptic proteins such as synapsin 1 and of other proteins of the pre-synaptic (GAP-43, synaptophysin, synaptotagmin) or post-synaptic machinery (MAP 2 and neurogranin). Moreover, ethanol interferes with the function of adhesion molecules such as NCAM (in chick embryo model) ( 133 ) and L1 (in mouse model) ( 134 ) involved in cell-cell interactions. During the neural processes of migration and morphogenesis, both proteins are involved in the organization and function of synaptic networks, which determine neuronal plasticity.…”

Ethanol neurotoxicity in culture: Selective loss of cholinergic neurons

“…Other reports have identified several conditions which lead to a shift in phenotypic expression within populations of pluripotential neuroblasts (Ramakers and Boer, 1991;Birgbauer and Fraser, 1994). Recently, we reported an alteration in embryonic neural cell adhesion molecule (NCAM) isoforms with ethanol exposure (Kentroti and Vernadakis, 1995b). This cell-adhesion molecule has been shown to influence greatly the cytoarchitecture of the developing central nervous system (Thiery et al, 1982), and may be involved in establishment of neuronal phenotypes.…”