Growth cone guidance and neuron morphology on micropatterned laminin surfaces

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Abstract
Neurite growth cones detect and respond to guidance cues in their local environment that determine stereo-typed pathways during development and regeneration. Micropatterns of laminin (which was found to adsorb preferentially to photolithographically defined hydro-phobic areas of micropatterns) were here used to model adhesive pathways that might influence neurite exten-sion. The responses of growth cones were determined by the degree of guidance of neurite extension and also by examining growth cone morphology. These parame-ters were found to be strongly dependent on the geom-etry of the patterned laminin, and on neuron type. Decreasing the spacing of multiple parallel tracks of laminin alternating with non-adhesive tracks, resulted in decreased guidance of chick embryo brain neurons. Single isolated 2 μm tracks strongly guided neurite extension whereas 2 μm tracks forming a 4 μm period multiple parallel pattern did not. Growth cones appear to be capable of bridging the narrow non-adhesive tracks, rendering them insensitive to the smaller period multiple parallel adhesive patterns. These observations suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by oriented extracellular matrix or certain axon fascicle structures, but could be guided by isolated adhesive tracks. Growth cone morphology became progressively simpler on progressively narrower single tracks. On narrow period multiple parallel tracks (which did not guide neurite extension) growth cones spanned a number of adhesive/non-adhesive tracks, and their mor-phology suggests that lamellipodial advance may be independent of the substratum by using filopodia as a scaffold. In addition to acting as guidance cues, laminin micropatterns also appeared to influence the production of primary neurites and their subsequent branching. On planar substrata, dorsal root ganglion neurons were multipolar, with highly branched neurite outgrowth whereas, on 25 μm tracks, neurite branching was reduced or absent, and neuron morphology was typi-cally bipolar. These observations indicate the precision with which growth cone advance may be controlled by substrata and suggest a role for patterned adhesiveness in neuronal morphological differentiation, but also high-light some of the limitations of growth cone sensitivity to substratum cues.