Neurite guidance by non-neuronal cells in culture: Preferential outgrowth of peripheral neurites on glial as compared to nonglial cell surfaces

Abstract

Growing axons in the peripheral nervous system (PNS) encounter a variety of cellular and extracellular substrates. Since it is difficult to sort out the possible contributions of these diverse components of the extracellular environment to axonal guidance in vivo, I have developed an in vitro system to study neurite outgrowth on two classes of cells which may provide as substrates for growing axons during development or regeneration: glial cells, e.g., astrocytes and Schwann cells, and nonglial cells, e.g., fibroblasts. Although neurites from sympathetic and spinal sensory ganglia explants grew onto preformed monolayers of both glial and nonglial cells, glial cells were a markedly better substrate. On the glial cells the neurites extended at a rate of 25 to 30 μm/hr and traveled singly or in fine fascicles; their growth cones displayed long filopodia and migrated on the upper surface of the monolayer cells. Conditioned media experiments suggested that neurite outgrowth on glial cell monolayers was not mediated by soluble secreted factors. These results indicate that the glial cell surface is an attractive substrate for neurite outgrowth. In contrast, on nonglial cells the rate of outgrowth was only 10 to 15 μm/hr, large neurite fascicles were common, and the growth cones migrated beneath the monolayer cells in contact with the underlying artificial substrate. This location of the growth cone, coupled with the observation that conditioned medium from these cell promoted neurite outgrowth only when bound to artificial substrates, suggests that secreted substrate-associated components may be an important determinant of neurite outgrowth on nonglial cell monolayers. The surface of the nonglial cells thus appears to be either inhibitory or a poor substrate for neurite outgrowth. The behavior of neurites confronted with a choice of glial and nonglial cells was also tested. When neurites growing on astrocytes were confronted with fibroblasts, most of the neurites turned and stayed on the glial cells. The neurites which did cross to the nonglial portion of the monolayer slowed their growth rate and formed larger fascicles. In contrast, neurites growing on a fibroblast monolayer increased their growth rate and defasciculated when they encountered an adjacent astrocyte monolayer. Taken together, these experiments demonstrate that, although PNS neurites can grow both on glial and nonglial cell monolayers, glial cell surfaces are the preferred substrate. Furthermore, the results suggest that neurites are more adherent to glial as compared to nonglial cell surfaces, thus revealing an adhesive hierarchy between these two major cell classes encountered by growing axons during nerve development and regeneration.