Specificity of monoclonal antibody N1 for cell surfaces of mouse central nervous system neurons

Abstract

Monoclonal antibody N1 reacts by indirect immunofluorescence with the cell surface of tetanus toxin-positive neurons from early postnatal mouse cerebellum. In freshly trypsinized single cell suspensions from early postnatal mouse cerebellum, 5-10% of all viable cells express N1 antigen on their surface. After 3-24 h of maintenance in vitro all N1 antigen-positive cells are tetanus toxin-positive. After culture periods of 3-4 days, most (approximately 90%) tetanus toxin-positive cells express N1 antigen on their surface. When horse serum-supplemented medium (HSSM) is used for cultivation, neurons begin to lose N1 antigen from their surface after about one week in vitro, until after two weeks in vitro, N1 antigen is no longer detectable, although some tetanus toxin-positive neurons can be shown to survive in culture. In defined medium, however, N1 antigen-positive neurons can still be detected after 34 days in vitro, the longest culture period examined so far. Complement-dependent immunocytolysis deletes all N1 antigen-positive and approximately 90% of all tetanus toxin-positive neurons from cultures. The remaining neurons reveal a morphology different from the one of the majority of small neurons, the granule cells. They have slightly larger cell bodies and several branched and unbranched cellular processes. Neonatal cerebellar cells show the same temporal sequence of appearance and disappearance of N1 antigen on most tetanus toxin-positive neurons in HSSM, and a persistence of N1 antigen on neurons in defined medium. N1 antigen becomes first detectable at embryonic day 17, and never becomes detectable in cell cultures derived from cerebella of younger mice. At all stages studied, N1 antigen expression is restricted to tetanus toxin-positive neurons, while it is absent from the cell surfaces of astrocytes, oligodendrocytes and fibroblasts. N1 antigen is also found in cultures derived from early postnatal mouse cerebrum, but is not detected in cultures derived from mouse retina, spinal cord, dorsal root ganglion, and embryonic telencephalon. It is also not detectable in cerebellar cultures from rabbit, rat, chicken and human. When N1 antibody is applied to fixed cultures where intracellular antigens are accessible, all cell types are labeled intracellularly, with astrocytes and fibroblasts revealing a fibrillary, vimentin-like staining pattern.