Mammalian and non-mammalian serotonergic neurons alike typically release specific neuropeptides, though it is not yet understood why or how serotonin and neuropeptide signals might function together. In the model system of the C. elegans egg-laying circuit, the Hermaphrodite Specific Neurons (HSNs) co-produce serotonin and NLP-3 neuropeptides to activate egg laying. Previous work1 has shown that these two signals synergistically enable activity of specific egg-laying muscles; however, because the receptor for NLP-3 has not been identified, it has remained difficult to understand how NLP-3 peptides mediate egg laying behavior alongside serotonin. Using an RNAi screen of 154 G protein coupled receptors that assessed their ability to mediate NLP-3 signaling, we identified a previously uncharacterized receptor, NPR-36, as a putative NLP-3 receptor in the egg-laying circuit. CRISPR knockouts of
npr-36 phenocopy the knockout of
nlp-3: firstly, in combination with
tph-1 (a mutation that prevents serotonin biosynthesis), both the
nlp-3 and
npr-36 knockouts severely disrupt egg laying behavior. Secondly,
npr-36 knockouts suppress the hyperactive egg-laying phenotype induced by transgenic overexpression of NLP-3 peptides, further evidence in favor of NPR-36 functioning as an NLP-3 receptor. Thirdly, the
nlp-3 npr-36 double knockouts show similar defects to those of either single knockout, consistent with the neuropeptide and receptor acting in the same genetic pathway. In ongoing work, we are using a CRISPR GFP knock-in at the endogenous
npr-36 promoter to identify the cells that express NPR-36. Concurrently, we are applying a range of concentrations of individual NLP-3 peptides to NPR-36-expressing cultured cells and measuring the downstream signaling this induces to determine if one or more of the five NLP-3 peptides activate NPR-36. Our goal is to understand how signals from serotonin and neuropeptides are integrated to activate a defined neural circuit. 1. Brewer et al. (2019) PloS Genet 15:
e1007896.