Effects of substrate geometry on growth cone behavior and axon branching

At the leading edge of a growing axon, the growth cone determines the path the axon takes and also plays a role in the formation of branches, decisions that are regulated by a complex array of chemical signals. Here, we used microfabrication technology to determine whether differences in substrate geometry, independent of changes in substrate chemistry, can modulate growth cone motility and branching, by patterning a polylysine grid of narrow (2 or 5 μm wide) intersecting lines. The shape of the intersections varied from circular nodes 15 μm in diameter to simple crossed lines (nodeless intersections). Time-lapse recordings of cultured hippocampal neurons showed that simple variations in substrate geometry changed growth cone shape, and altered the rate of growth and the probability of branching. When crossing onto a node intersection the growth cone paused, often for hours, and microtubules appeared to defasciculate. Once beyond the node, filopodia and lamellipodia persisted at that site, sometimes forming a collateral branch. At nodeless intersections, the growth cone passed through with minimal hesitation, often becoming divided into separate areas of motility that led to the growth of separate branches. When several lines intersected at a common point, growth cones sometimes split into several subdivisions, resulting in the emergence of as many as five branches. Such experiments revealed an intrinsic preference for branches to form at angles less than 90°. These data show that simple changes in the geometry of a chemically homogeneous substrate are detected by the growth cone and can regulate axonal growth and the formation of branches.