Similarly, tax-4; npr-1 double mutants exhibited locomotion quiescence and decreased PDF-1 secretion during lethargus, and both effects were reversed by a transgene expressing TAX-4 in the RMG circuit ( Figures 2F, 2G, and S4C). By contrast, RIG-3 coelomocyte fluorescence was decreased in npr-1 mutants in both L4 and L4/A animals ( Figure S4B). Consequently, the effects of NPR-1 and TAX-4 on PDF-1 coelomocyte fluorescence are unlikely to be caused either by general changes in the stability of secreted protein or by general changes in coelomocyte activity. Instead, these

results suggest that heightened RMG circuit activity in npr-1 mutants produced a corresponding increase in PDF-1 secretion from head sensory PLX3397 neurons, thereby increasing motility during lethargus. The preceding results suggest that decreased and increased PDF-1 secretion during lethargus are correlated with and required find more for locomotion quiescence and arousal.

To determine whether increased PDF-1 secretion is sufficient to arouse locomotion, we constructed transgenic animals in which PDF-1 secretion can be pharmacologically induced (Figures 4C and 4D). A prior study showed that capsaicin treatment depolarizes ASH neurons expressing rat TRPV1 channels (Tobin et al., 2002). When TRPV1 and PDF-1 were coexpressed in ASH neurons, capsaicin treatment significantly decreased locomotion quiescence during lethargus (Figures 4C and 4D). This effect was not observed when only TRPV1 was expressed in ASH. These results suggest that forced secretion of PDF-1 before during lethargus was sufficient to arouse locomotion behavior. Because RMG circuit activity controls PDF-1 secretion and locomotion arousal, a simple explanation for our data would be that PDF-1 is secreted by cells in the RMG circuit. Several results are consistent with this idea. The pdf-1 promoter is expressed in RMG interneurons, as well as in ASK sensory neurons, which

form direct gap junctions with RMG ( Figure 2A) ( Barrios et al., 2012; Janssen et al., 2008). Transgenes expressing PDF-1 in ASK neurons reinstated the locomotion quiescence defect in pdf-1; npr-1 double mutants ( Figures S4D and S4E). Similarly, coelomocyte fluorescence produced by PDF-1::YFP expressed in ASK neurons was decreased during lethargus in wild-type animals but not in npr-1 mutants ( Figure S4F). Thus, PDF-1 expression in ASK neurons was sufficient to reconstitute NPR-1’s effects on locomotion quiescence and PDF-1 secretion during lethargus. Because PDF-1 is secreted (and consequently acts in a non-cell-autonomous manner), PDF-1 secretion from other cells may also regulate lethargus behavior. Consistent with this idea, PDF-1 expression in ASI neurons also restored the L4/A quiescence defect in pdf-1; npr-1 double mutants ( Figures S4D and S4E).