A simple procedure for quantification of neurite outgrowth based on stereological principles

The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(pid/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.