Components of neurite outgrowth that determine neuronal cytoarchitecture: Influence of calcium and the growth substrate

The present study examined the cellular mechanisms underlying the generation of neuroarchitecture. Identified Helisoma neurons in isolated cell culture normally require factors present in brain-conditioned medium (CM) in order to display the different components of neurite outgrowth (sprouting, elongation, branching, and growth cone motility), which ultimately determine their overall architecture. We report here that cell calcium and cell-substrate can play quite specific roles in the regulation of these different components of neuronal outgrowth. CM-induced neurite outgrowth was inhibited by calcium ionophore A23187. In the absence of CM the calcium channel blocker La³⁺ (10 μM) reduced intracellular calcium levels and induced neurite sprouting and elongation; growth cone motility and branching were greatly reduced in the La³⁺-induced neurites. Neurons plated into an environment containing La³⁺ and a fibronectin substrate exhibited all of the components of neuronal outgrowth normally seen in response to CM. Fibronectin alone had little outgrowth-promoting activity. Neurite elongation rates and branching were increased by exposure to La³⁺ in neurons on either a CM or fibronectin substrate. The neurons growing on CM or fibronectin whose out-growth was accelerated by La³⁺ elaborated neuritic arbors that differed from those of neurons grown in response to CM; differences were seen in neurite length, area of outgrowth, branching frequency, and varicosity numbers. Taken together, these results indicate that 1) calcium and the growth substrate can exert specific effects on neurite sprouting, elongation, growth cone motility, and branching; 2) appropriate levels of activation of these two systems can elicit neurite out-growth that closely resembles that induced by endogenous growth factors; 3) both the differential expression of the separate components of outgrowth and the kinetics of outgrowth determine a neuron's morphology.