Vasopressin- and oxytocin-related neuropeptides are key regulators of animal physiology, including water balance and reproduction. Although these neuropeptides also modulate social behavior and cognition in mammals, the mechanism for influencing behavioral plasticity and the evolutionary origin of these effects are not well understood. Here, we present a functional vasopressin- and oxytocin-like signaling system in the nematode Caenorhabditis elegans. Through activation of its receptor NTR-1, a vasopressin/oxytocin-related neuropeptide, designated nematocin, facilitates the experience-driven modulation of salt chemotaxis, a type of gustatory associative learning in C. elegans. Our study suggests that vasopressin and oxytocin neuropeptides have ancient roles in modulating sensory processing in neural circuits that underlie behavioral plasticity.

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For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G proteincoupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα 16 construct is co-transfected. Following ligand binding, activation of the Gα 16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα 16 , which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

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