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3D Printed Microfluidics for Studying Sleep Pathways in Caenorhabditis elegans

Despite sleep being essential and highly conserved at the genetic level, a thorough understanding of the mechanisms of sleep regulation and its core function are lacking. Caenorhabditis elegans, a genetically tractable nematode worm, is a powerful model system for addressing these questions. Conserved neurochemistry and a compact 302‐celled nervous system allow for the identification and rapid characterization of novel sleep‐regulating genes of relevance to more complex animals. Orcokinin neuropeptides, predominantly found in ecdysozoan animals, have been implicated during the regulation of circadian rhythms and molting, however, a role during sleep regulation has not been described. In C. elegans the genes nlp‐14 and ‐15 code for peptides predicted to be orcokinin orthologs. We sought to determine if these peptides control a behavior called stress‐induced sleep (SIS), which occurs following incidents of noxious stimuli that damages their cells. Using a combination of loss‐of‐function and gain‐of‐function approaches, we find that nlp‐14 and nlp‐15 play a central role during SIS, regulating unique aspects of behavioral quiescence. To identify an orcokinin receptor, we have been conducting forward genetic screens to isolate mutants that suppress a strong gain‐of‐function sleep phenotype of nlp‐14. In the past, we employed a manual screening protocol that required an abundance of time and resulted in a high false‐positive rate. To improve upon this, we fabricated screening devices using 3D printing, which allows for higher throughput and selectivity. These microfluidic devices use gravity‐based sorting chambers which are both cheap and easily employed. In these chambers we place mutagenized animals, induced to fall asleep by nlp‐14 over‐expression, in a lower sorting chamber. Sleeping animals are trapped in the lower chamber, while suppressor mutants display gravitaxis behavior, the ability to swim against gravity, thus, swimming to an upper collection chamber. These rare suppressor mutants are then isolated for whole‐genome sequencing in order to reveal the essential genes involved in these sleep‐regulating pathways.