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 d evelopment and plasticity. Determination of neurite outgrowth in cell cultu re is a widely used approach to investigate these phenomena. This is genera lly done by a time consuming tracing of individual neurites and their branc hes. We have used stereological principles to determine the length of neuri tes. The total neuritic length per cell was estimated by counting the numbe r of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional comp uter-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cel l by means of the equation L = (pi d/2)I describing the relationship betwee n 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 intersectio ns correlated statistically significantly with data obtained using a conven tional tracing technique. However, information was acquired more efficientl y using the stereological approach. Thus, using the described set-up, the s tereological procedure was approximately five times less time consuming tha n the conventional method based on neurite tracing. The study shows that st ereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons an d cell lines. (C) 2000 Elsevier Science B.V. All rights reserved.