An in Vitro System for Studying Nematode Mechanosensory Neurons

The primary function of mechanosensory neurons is to detect and convey information about mechanical cues, including touch and self-generated changes in muscle length. This function depends on mechanosensitive ion channels, but also the interaction between mechanosensory neurons and their surrounding tissues. With its transparent body, easily manipulated genome, and hundreds of known mutants, the roundworm C. elegans is an ideal model animal for studying the biophysics of mechanosensory neurons in vivo. Calcium indicators, expressed under cell-specific promoters, enable imaging of neural impulses in response to mechanical cues. The elements of mechanosensitivity that are intrinsic to these neurons or dependent upon interactions with surrounding tissues are not known. We seek to address this question by developing an in vitro system that provides greater control over the neuron's environment while preserving the advantages of studying mechanosensation in C. elegans. We present a cell culture system consisting of a purified population of identified primary neurons from dissociated C. elegans embryos, grown on geometrically defined patterns of adhesion proteins that control neurite outgrowth and cell shape. Using a CRISPR/Cas-9 genetic approach, we introduced puromycin resistance under a cell-specific promoter allowing us to reduce the heterogeneity of the dissociation. In parallel, we used maskless photolithography to create shapes of protein on glass, surrounded by a bio-passivating layer, thereby restricting the suitable locations for cell adhesion. We have established that C. elegans neurons adhere to a variety of these patterns, they will extend neurites that follow the patterns, and in some cases bridge the gap between patterns across the bio-passivated region. This technological platform provides a springboard for further analyzing the mechanobiology and biophysical properties of cultured C. elegans neurons.