Morphometric and time lapse analyses of rapid-onset neurites stimulated by cycloheximide in NG108-15 cells

We previously reported that the laminin-induced outgrowth of neurites in the first 4 hr after plating ('rapid-onset neurites') is markedly stimulated by inhibitors of protein and RNA synthesis, including cycloheximide, puromycin and actinomycin D. Our original interpretation of this finding was that inhibiting protein synthesis led to metabolic changes in the cell which converged upon the intracellular pathway used by laminin to stimulate neurite formation. Alternatively, Preston et al. (1987, Proc. natn. Acad. Sci. U.S.A. 84, 5247-5251) suggested that cycloheximide acts by inhibiting neurite retraction. In order to resolve these conflicting interpretations, we decided to re-examine the effects of cycloheximide using time lapse analyses of NG108-15 cells grown on laminin, together with morphometric analyses of cultures fixed at 1.5 or 3 hr after plating. Cycloheximide was found to cause both an increase in neurite initiation within the first 1.5 hr after plating, and a decrease in the occurrence of neurite retractions between 1 and 3 hr. Investigators should be aware of the effects of biosynthetic inhibitors on neurite growth, since these agents are employed widely in studies of cultured neural cells, particularly those involving receptor metabolism and phenotypic differentiation.