Abstract

During the development of the nervous system, neurons migrate and grow over great distances. During these processes, they are exposed to a multitude of signals determining their speed and direction. Currently, our understanding of neuronal development is, in large part, based on studies of biochemical signaling. Despite the fact that forces are involved in any kind of cell motion, mechanical aspects have so far rarely been considered. Here we used deformable cell culture substrates, traction force microscopy and calcium imaging to investigate how neurons respond to their mechanical environment. Axonal growth speed, directionality, and fasciculation, i.e., their tendency to grow in bundles, all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, axon bundles were repelled by stiff substrates. Calcium influxes through the activation of stretch-activated ion channels appear to be involved in neuronal mechanosensitivity. In vivo atomic force microscopy measurements revealed stiffness gradients in developing brain tissue, and showed that axons follow a soft pathway in vivo. Interference with brain stiffness and mechanosensitive ion channels suggest that mechanical signaling is involved in neuronal pathfinding in vivo and constitutes a formerly unknown control mechanism of axon growth.