Genetic and molecular analysis of a rhythmic behavior in C. elegans: how neuropeptide signaling conveys temporal information

2013-10-11Rhythmic behaviors are those behaviors that occur at regular timing intervals and they are widely observed in animal kingdom. The time intervals are determined by pacemakers and the rhythmic behavioral outputs are generally performed by different tissues, including downstream neurons and muscles. However, how the timing information from the pacemaker is delivered to the downstream effectors remains unclear. ❧ To address this question, I study a simple rhythmic behavior in C. elegans: the enteric muscle contraction (Exp) in the defecation motor program. The pacemaker for the defecation motor program is the intestine and the timing is encoded by intestinal calcium oscillations, which drive rhythmical enteric muscle contraction about every 50 seconds. Using an in vivo calcium imaging approach, I find that the downstream GABAergic neurons (AVL and DVB) that innervate enteric muscles undergo rhythmic activation that happens immediately before Exp during each defecation cycle, suggesting the timing information from the intestine is transmitted step by step within the circuit controlling enteric muscle contraction. ❧ It has been hypothesized that an unknown neuropeptide signal may be released from the intestine to activate the downstream GABAergic neurons to cause rhythmic enteric muscle contraction. By combing forward genetic screens and whole genome sequencing, I identify a neuropeptide-like protein (NLP-40) as the timing signal from the intestine. First, nlp-40 is essential for enteric muscle contraction. Second, nlp-40 is exclusively expressed in the intestine. Third, NLP-40 is released from the intestine, which is mediated by SNT-2/synaptotagmin, the putative calcium sensor in the dense core vesicles (DCVs), suggesting calcium oscillations in the intestine may trigger rhythmic release of NLP-40. Fourth, using optogenetics and in vivo calcium imaging, I show that NLP-40 does not impact the integrity or development of the circuit that controls enteric muscle contraction. Instead, NLP-40 is both necessary and sufficient for the rhythmic calcium influx in the downstream GABAergic neurons. Fifth, I further demonstrate that NLP-40 triggers calcium influx in the GABAergic neurons by activating its receptor AEX/GPCR. ❧ I further delineate the molecular mechanism by which the NLP-40 signaling controls the activation of the GABAergic neurons. Previous studies show that NLP-40 signaling is dependent on cAMP. Here, I develop a genetic technique to either increase or inhibit PKA activity in a tissue specific manner in C. elegans. I demonstrate that PKA is the major target of cAMP in the NLP-40 signaling pathway in the GABAergic neurons to regulate calcium influx. Furthermore, I identify two voltage-gated calcium channels (VGCCs), UNC-2 and EGL-19 that partially mediate PKA-dependent calcium influx in the GABAergic neurons. ❧ In conclusion, I present evidence to uncover a mechanism by which neuropeptides could function as timing messengers to couple pacemakers to downstream neurons to coordinate the proper execution of rhythmic behaviors. Neuropeptides may encode timing information via synaptotagmin-dependent rhythmic release from pacemakers and they activate downstream neurons through binding to their receptors (GPCRs) which leads to PKA signaling cascade to trigger the calcium influx