A cytomechanical investigation of neurite growth on different culture surfaces

Abstract

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity × (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to >200 μdynes, with some tendency for thresholds to cluster between 100 and 150 μdynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 μm/h/μdyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to us that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresh-olds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.