Quantitative effects of laminin concentration on neurite outgrowth in vitro

Abstract

Recent studies indicate that mediation of neurite outgrowth by the glycoprotein laminin may be a significant factor in the outgrowth of neurites to their targets during embryogenesis. To further characterize the possible role of this extracellular matrix molecule during development, we have systematically measured several features of outgrowth by neonatal rat sympathetic neurons on different concentrations of laminin. Individual neurons, obtained by mechanical dissociation of superior cervical ganglia (SCG), were cultured at low density on laminin substrates ranging from 0.01 to 1.0 microgram/cm2. Outgrowth characteristics were subsequently analyzed for noninteracting cells in both fixed and live cultures. Data obtained from neurons fixed after 11 hr of culture showed approximately twofold increases in neurite initiation and outgrowth, and a twofold decrease in branching for a corresponding 100-fold increase in adsorbed laminin concentration. In time-lapse videomicroscopy observations, the root-mean square speed of growth cone movement increased from 60 to 90 microns/hr over the same range in concentration, while the persistence time remained constant at 0.10 hr. In general, neurite outgrowth parameters were relatively insensitive to changes in laminin concentration, supporting the idea that laminin is a permissive rather than an "instructive" substrate during development. Data obtained from fixed cultures were examined in terms of probability models to suggest possible mechanisms contributing to the dose-dependent effects observed.