Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.
Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally-timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.
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. Support or Funding Information NSF Award #1845020 (PI Nelson)
Sleep is evolutionarily conserved and essential. Yet, it is disadvantageous as it renders animals vulnerable to predation and limits other essential behaviors, such as feeding and reproduction. Thus, animals must balance their sleep drive with behaviors that ensure their survival. How this is regulated at the cellular and molecular level is largely unknown. The genetically tractable nematode, Caenorhabditis elegans, is a powerful sleep model, possessing a compact and completely mapped nervous system of only 302 neurons. Stress‐induced sleep (SIS), analogous to sickness sleep of mammals, is a behavior that allows for a genetic dissection of these key questions. When C. elegans is exposed to noxious stimuli, avoidance responses are initiated, promoting their survival. However, if cellular damage is incurred SIS takes place, promoting recovery. Thus, SIS is an interesting model for identifying mechanisms underlying essential sleep/wake decisions. We conducted a forward genetic screen searching for sleep‐defective mutants, and isolated a strain containing a loss‐of‐function allele of the gene T10E10.3, which encodes a G‐protein coupled receptor (GPCR). Using CRISPR and cell‐specific rescue, we find that T10E10.3 acts to inhibit the sleep‐promoting RIS interneuron to promote arousal, while simultaneously signaling to multiple cells to promote sleep, including the ALA interneuron and a pharyngeal neuron called I6. Over‐expression of T10E10.3 results in a profound early sleep phenotype, suggesting an imbalance in the sleep/wake mechanisms. Our data supports a model in which a single GPCR can regulate the balance between wake/sleep by functioning in an antagonistic manner in unique cell types. We propose that this allows the worm to escape harmful stimuli early and then ensures proper levels of recovery sleep once a safe environment is reached.
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