Controlling growth cone behavior through substrate patterning
Smirnov, Michael Sergey
Urbach, Jeffrey S
Geller, Herbert M
Throughout the process of development, billions of neuronal axons are responsible for navigating through the nervous system and synapsing onto their appropriate targets. To establish their individual paths, neurons are guided by a complex and dynamic map of biochemical, topographic, and mechanical signals. At the tip of each neurite lies the growth cone - a dynamic structure responsible for interpreting extracellular cues and steering neuronal growth in the appropriate direction. Growth cones in vivo exhibit various morphologies and behavioral changes, though the underlying mechanisms of these changes remain widely unknown. Through the use of substrate patterning techniques, we examined the relationship between growth cone morphology and behavior with the purpose of optimizing guidance in engineered regenerative systems.We investigated multiple substrate patterning techniques in order to achieve easily observable manipulations to growth cone morphology. We found that common micro-contact printing and microfluidic techniques are poor at creating isolated regions of overlapping biochemical cues. We also found that established protein patterning methods for soft substrates are ill-suited for use on surfaces with nervous system-relevant elasticity. Using laser ablation, we designed micron-scale patterns capable of confining dissociated mouse cerebellar granule neuron growth cones to channels of different widths. Growth cone dynamics in these channels were observed using time lapse microscopy. Growth cone area was decreased in channels between 1.5 and 6 µm as compared to that in 12 µm and unpatterned substrates. Growth cone aspect ratio was also affected as narrower channels forced growth cones into a narrow, elongated shape. There was no difference in the overall rate of growth cone advance in uniform channels between 1.5 and 12 µm as compared to growth on unpatterned substrates. The percentage of time growth cones advanced, paused, and retracted was also similar. However, growth cones responded to changes in confinement: growth cones in narrow lanes rapidly sped up when encountering a wide region and then slowed down as they entered another narrow region. Our results suggest that the rate of neurite extension is not affected by the degree of confinement, but does respond to changes in confinement.
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