Engineering connectivity by multiscale micropatterning of individual populations of neurons

Functional networks are the basis of information processing in the central nervous system. Essential for their formation are guided neuronal growth as well as controlled connectivity and information flow. The basis of neuronal development is generated by guiding cues and geometric constraints. To investigate the neuronal growth and connectivity of adjacent neuronal networks, two-dimensional protein patterns were created. A mixture of poly-L-lysine and laminin was transferred onto a silanized glass surface by microcontact printing. The structures were populated with dissociated primary cortical embryonic rat neurons. Triangular structures with diverse opening angles, height, and design were chosen as two-dimensional structures to allow network formation with constricted gateways. Neuronal development was observed by immunohistochemistry to pursue the influence of the chosen structures on the neuronal outgrowth. Neurons were stained for MAP2, while poly-L-lysine was FITC labeled. With this study we present an easy-to-use technique to engineer two-dimensional networks in vitro with defined gateways. The presented micropatterning method is used to generate daisy-chained neuronal networks with predefined connectivity. Signal propagation among geometrically constrained networks can easily be monitored by calcium-sensitive dyes, providing insights into network communication in vitro.