Axons in vivo are guided by molecular signals acting as attractants and repellents, and possibly by physical constraints encountered in the extracellular environment. We analyzed the ability of primary sensory axons to extend and undergo guidance in three-dimensional (3-D) environments generated using photolithography. Confinement of neurons in fully enclosed square chambers decreased the percentage of neurons establishing axons as a function of chamber width. However, the ability to extend an axon in one or more directions allowed axons to form and extend similarly to those on two-dimensional (2-D) substrata. Live imaging of growth cones interacting with the walls of chambers or corridors revealed that growth cones respond to contact with a 3-D constraint by decreasing surface area, and circumvent constraints by repeated sampling of the constraint until an unobstructed path is encountered. Analysis of the ability of axons to turn around corners in corridors revealed that the angle of the corner and corridor width determined the frequency of turning. Finally, we show that the length of axons can be controlled through the use of 3-D constraints. These data demonstrate that 3-D constraints can be used to guide axons, and control the extent of axon formation and the length of axons.

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