(430w) Micropatterned Substrates That Direct Neuronal Polarity

During development, young neurons sprout several processes (called neurites) that probe their environmental niche. Based on a combination of intracellular signaling molecules and extracellular cues, one neurite develops into the axon (the portion of the neuron that can transmit electrical and chemical signals) while the remaining neurites become dendrites. While this developmental program is tightly regulated in vivo, currently there are no methods to control neuronal polarity in vitro. We have designed a protocol to pattern various guidance cues on surfaces to guide the development of rat hippocampal neurons. Soft lithography was used to create microfluidic devices that generate patterned regions of guidance cues physically adsorbed to the culture substrate within a polypeptide matrix. This protocol is compatible with both small-molecule guidance cues as well as protein guidance cues such as the neurotrophins semaphorin 3a and brain-derived neurotrophic factor. Our patterned surfaces are able to specify axon initiation as well as guide the path-finding of the developing axon. This system is currently being used to study the signaling molecules that regulate neuronal polarity. For example, we have discovered the antagonistic role of two secondary messengers within the cell, cAMP and cGMP, on axon and dendrite development. Furthermore, the protocol is also being optimized as a method to create patterned arrays of neurons with specified polarity. By creating islands of cell-adhesive poly-L-lysine connected to patterns that promote axon and dendrite development, the location of the cell body as well as the axon can be specified. This is a first step towards guiding the formation of simple neuronal circuits in vitro. The ability to guide neuronal development on patterned substrates has potential application in bioMEMs devices, neural networks, biosensors, and scaffolds for tissue engineering of nerve grafts.