Axon Guidance and Growth Cone Collapse in Vitro

Abstract

Important information about the mechanisms of axon guidance in the developing and regenerating brain has been obtained from in vitro experiments. One particular system, the retinotectal projection of vertebrates, has been analyzed by several in vitro assays, three of which are described here in detail: (i) the stripe choice assay, (ii) the collapse assay, and (iii) the gradient assay. Each of these has revealed position-specific behavior of retinal axons in response to cell membranes derived from different regions of the optic tectum. The stripe choice assay tests the ability of growing axons to discriminate between two membrane substrates offered as alternating stripes. The gradient assay assesses whether growth cones can detect (and be guided by) smooth transitions from one substrate type to another. The collapse assay reveals instantaneous reactions of growth cones to inhibitory or repellent factors present in their environment. The protocols describe the preparation of retinal explants and tectal membranes, as well as the assays proper. Particular emphasis is placed on the gradient assay, which has not yet been described in detail. All of the approaches discussed here have in common that they are applicable to axon guiding components bound to cell membranes. By a few modifications, however, it should be possible to extend this type of investigation to a wider range of related questions, including cell migration and guidance. We do not consider the important aspect of chemotropic guidance of axons in response to diffusible factors.