The process of network formation depends on the development of neuronal polarity with a specified axon as well as several dendrites and requires precise regulation of microtubule and actin dynamics. Microtubule-associated proteins (MAP) control the dynamic properties of microtubules, which differ in their complement of MAPs. For example, MAP2 is found mostly in dendrites and tau is found mainly in axons. Micropatterned substrates provide a suitable environment to study neuronal polarity because they allow for the distinction of axons and dendrites on the surface due to the separation of cells on cell-adhesive areas. Neurons were plated on day 30 of differentiation onto star-shaped micropatterned substrates and either immunostained 10 days later (day 40 of differentiation) or 70 days later (day 100 of differentiation). Using antibodies against tau (to label axons; Figure 4B, arrow with open arrowhead), and MAP2 (to label dendrites; Figure 4B, arrow with closed arrowhead), we could visualize the formation of individual connections to neighboring cell bodies. Culturing neurons until day 100 of differentiation on star-shaped micropatterned substrates resulted in a progressively more organized and denser neuronal network, further demonstrating the utility of micropatterns in studies of neuronal maturation and network development.
