Lack of cell density-dependent changes in gangliosides of rat primary culture neurons

Cell density-dependent changes in neuronal gangliosides, primarily relating to neurite outgrowth under dense to sparse conditions, were examined at cell seeding densities over an 8-fold range. During the first 24 h of incubation, the dissociated fetal rat neurons showed characteristic protrusion of neurites as a function of cell density. Ganglioside and protein contents per the same cell numbers were higher in dense cultures than sparse ones. However, the ganglioside pattern was essentially unchanged from dense to sparse culture, showing a predominance of GD3 and GT1b. The biosynthetic activity of gangliosides, as estimated by the incorporation of 3H-labeled N-acetyl-D-mannosamine, a precursor of sialic acid, was similar at various cell densities, with the labeling of b-series gangliosides predominating. The expression of neuronal gangliosides was monitored by indirect immunofluorescence using anti-GM1 antibody, but was found to be poor. A2B5 antigen, which was mainly identified as GT1b, appeared to be readily expressed on cell surfaces in sparse cultures. In contrast, the highly polysialylated form of the neural cell adhesion molecule (NCAM-H) was fully expressed on both the neurites and cell soma at various cell densities. The results suggest that the polysialic acids in NCAM have more important roles in neurite outgrowth than gangliosides, since the composition and synthesis of gangliosides are not affected by cell seeding density.