Growth cone 3-D morphology is modified by distinct micropatterned adhesion substrates

The development, connectivity, and structural plasticity of neuronal networks largely depend on the directional growth of axonal growth cones (GCs). The morphology and 3-D profile of axons and GCs of primary hippocampal neurons, grown onto glass surfaces coated with poly-D-lysine (PDL) and micropatterned with stripes of the adhesion molecule L1 by using the indirect microcontact printing, were investigated. Neurons were fixed at early stages (one to seven days) of in vitro development prior to synapse formation, and analyzed by fluorescence and atomic force microscopy. The latter technique allowed us to investigate the 3-D morphology of the GCs, and detect their morphological rearrangements during axon outgrowth and during contact with the underlying substrate. We found that axons decreased their height-to-width ratio over development in culture, and that this value became particularly low when the axon and the GC proceeded onto a surface containing attracting cues such as L1 with respect to GCs growing onto a nonspecific adhesion substrate such as PDL. Along with this shape change of the axons, GCs lying onto L1 tracks displayed a flattened shape, ideal for sensing and progression, whereas GCs onto areas of nonspecific adhesion displayed more prominent shapes and steeper edges.