Neuronal cell culture is a cornerstone of neuroscience research and is used extensively in studies of brain and behavior. The in vitro environment offers researchers the opportunity to maintain neurons of interest under defined conditions and with strict control over experimental parameters. Although in vitro studies are easy to conduct and involve simply plating cells of interest into culture dishes, the cultured cells are distributed randomly on the substrate, and are mingled with their processes, which extend in arbitrary directions. The experimentalist must therefore expend valuable time and effort to search across the culture substrate to identify appropriate neurons and axons to study and to locate axons that are well separated from their cell bodies. Consequently, experimental throughput is limited, making the collection of large sample sizes difficult to achieve. Our long-term goal is to develop a high-throughput neuronal cell culture technology that maintains the advantages of low cost and ease of use of standard cell culture but provides investigators with precise control over the location of neuronal cell bodies and their axonal processes, including the patterning of large, high-density arrays of neurons and axons to enable substantially greater experimental throughput. We hypothesize that such high-throughput neurotechnology can be achieved using a novel and reliable method of cell patterning that relies on the deposition of high-resolution, cell adhesive geometries on top of a robust cell repellant background. In support of our long-term goal, we propose to answer three critical questions related to the utility of this novel patterning technology for the micropatterning of neurons and axons in culture. The proposed studies specifically address: 1) whether the neuropatterning process can be used to spatially segregate neurons and axons to form convenient experimental arrays;2) the performance of this technology in supporting key aspects of neuronal biology such as viability, differentiation, and functions such as axon growth;and 3) the ability of this patterning technology to immobilize and present a variety of bioactive molecules, thus greatly expanding its utility in many areas of neuroscience. We appreciate that ultimately additional studies must also be performed to more closely examine issues of manufacture and shelf life. However, the current studies will establish a knowledge base to support this future work. PUBLIC HEALTH RELEVANCE: Studies of neurons in culture often serve as an essential starting point for guiding broader research of the brain and behavior. Organizing cultures by precisely patterning the placement and orientation individual neurons to form an orderly array will greatly enhance experimental throughput and productivity. Towards this end, we are developing a robust technology to produce inexpensive, easy-to-use culture substrates to provide such precision micropatterning of neurons.