Native extracellular matrix induces a well-organized bipolar outgrowth pattern with neurite extension and retraction in cultured neurons

Abstract

Cultured anterior pagoda (AP) neurons from the leech develop characteristic outgrowth patterns that depend on the molecular composition of the substrate. This article analyzes how native substrates from the central nervous system (CNS), such as the extracellular matrix (ECM) inside the capsules that enwrap the ganglia, determine the outgrowth patterns of AP neurons. When plated on the internal side of ganglion capsules, the remaining primary portion (stump) of AP neurons sprouted two main branches in opposite directions with bifurcations. This T-shaped pattern was distinctive for AP neurons and was different from the patterns of the same cell type plated on the external side of the capsule or on leech laminin extracts, in which they generated multiple neurites and branching points. AP neurons plated on tritonized CNS homogenates reproduced the outgrowth pattern displayed on ganglion capsules, in terms of the number of primary neurites, their length, their orientation, and the number of branch points. The development of the T-shaped outgrowth pattern of AP neurons on ganglion capsules and CNS homogenates started by the sprouting of one branch that later bifurcated, followed by a second branch in the opposite direction after a lag of several hours. Extension of the second branch and retraction of secondary neurites of the first were synchronous and contributed to refine the T-shaped pattern. These results suggest that during development or regeneration of the CNS, particular sets of ECM proteins have multiple effects regulating the number, direction, extension, and retraction of neurites.