In Vitro Assays for Molecules That Inhibit Growth Cone Motility during Neural Development and Regeneration

Abstract

There is increasing evidence that molecules that inhibit growth cone motility are involved in the guidance of axons to their appropriate targets during neural development and contribute to the suppression of axon regeneration in the mammalian CNS. Two tissue culture phenomena have been used to detect and monitor these molecules: inhibition of neurite outgrowth and growth cone collapse. In neurite outgrowth assays the inhibitory material is used as a culture substratum. It can be presented to neurons either as a continuous layer or in a form that growing axons will encounter, such as an explant or a stripe. Tissue explants or sections, monolayer cultures of cells, membrane fractions, and purified or partially purified material have all been used. In the growth cone collapse assay, the growth cones of axons extending on a permissive substratum are treated with liposomes incorporating the putative inhibitory material. This method is particularly useful for testing the inhibitory effects of membrane-derived molecules. The relevance of results obtained with these in vitro assays to axon growth phenomena in vivo must always be established. Their principal value lies in the provision of a means of monitoring biochemical purification procedures aimed at identifying and characterizing molecules that inhibit nerve growth.