Characterization of cytoskeletal and neuronal markers in micromass cultures of rat embryonic midbrain cells.

Micromass cultures of rat embryonic midbrain cells were characterized with regard to the immunolocalization of neuronal and cytoskeletal markers. Cells taken from gestational day-12 embryos and cultured for 5 days in vitro comprise at least two morphologically distinct cells types: fibroblast-like cells and neurons. Antibodies to the following markers yielded preferential staining of neuronal cells: A2B5 (GQ ganglioside), gamma-aminobutyric acid (GABA), microtubule-associated protein 2 (MAP2), MAP5, neuron-specific enolase (NSE), neural cell adhesion molecule (N-CAM), and tau. Antibodies to beta-tubulin, c-neu, MAP1, and neurofilament (NF-H) stained both neuronal and fibroblast-like cells. Antibodies to glial fibrillary acidic protein (GFAP) and vimentin failed to immunoreact with any cells in day-5 CNS cultures. SDS-PAGE and Western analysis were employed to determine the specificity of the antibodies and determine the electrophoretic profiles of the markers. We conclude that the pattern of neuronal differentiation in CNS micromass cultures exhibits certain similarities to that observed in vivo. In addition, certain markers identified in this study may be of potential utility as (1) biomarkers of chemically-induced developmental neurotoxicity, and (2) indicators of differential toxicity toward the diverse cell types that comprise the mammalian central nervous system.