Guiding neuronal development with in situ microfabrication

We report the ability to modify microscopic 3D topographies within dissociated cultures, providing a means to alter the development of neurons as they extend neurites and establish interconnections. In this approach, multiphoton excitation is used to focally excite noncytotoxic photosensitizers that promote protein crosslinking, such as BSA, into matrices having feature sizes ≥250 nm. Barriers, growth lanes, and pinning structures comprised of crosslinked proteins are fabricated under conditions that do not compromise the viability of neurons both on short time scales and over periods of days. In addition, the ability to fabricate functional microstructures from crosslinked avidin enables submicrometer localization of controllable quantities of biotinylated ligands, such as indicators and biological effectors. Feasibility is demonstrated for using in situ microfabrication to guide the contact position of cortical neurons with micrometer accuracy, opening the possibility for engineering well defined sets of synaptic interactions.