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. Author manuscript; available in PMC: 2014 Jun 5.
Published in final edited form as: Neuron. 2013 Jun 5;78(5):869–880. doi: 10.1016/j.neuron.2013.04.002

Analysis of NPR-1 reveals a circuit mechanism for behavioral quiescence in C. elegans

Seungwon Choi 1,2,3, Marios Chatzigeorgiou 4, Kelsey P Taylor 1,2,3, William R Schafer 4, Joshua M Kaplan 1,2,3,*
PMCID: PMC3683153  NIHMSID: NIHMS465731  PMID: 23764289

SUMMARY

Animals undergo periods of behavioral quiescence and arousal in response to environmental, circadian, or developmental cues. During larval molts, C. elegans undergoes a period of profound behavioral quiescence termed lethargus. Locomotion quiescence during lethargus was abolished in mutants lacking a neuropeptide receptor (NPR-1), and was reduced in mutants lacking NPR-1 ligands (FLP-18 and -21). Wild type strains are polymorphic for the npr-1 gene, and their lethargus behavior varies correspondingly. Locomotion quiescence and arousal were mediated by decreased and increased secretion of an arousal neuropeptide (PDF-1) from central neurons. PDF receptors (PDFR-1) expressed in peripheral mechanosensory neurons enhanced touch-evoked calcium transients. Thus, a central circuit stimulates arousal from lethargus by enhancing the sensitivity of peripheral mechanosensory neurons in the body. These results define a circuit mechanism controlling a developmentally programmed form of quiescence.

INTRODUCTION

Animals coordinately adjust their behaviors in response to changes in their environment and metabolic state. Co-regulated behaviors (often termed behavioral states) can persist for minutes to hours. Increased activity (or arousal) is associated with fear, stress, hunger, and exposure to sexual partners (Pfaff et al., 2008). Conversely, decreased activity (or quiescence) is associated with sleep and satiety (Cirelli, 2009).

Many aspects of behavior and metabolism exhibit rhythmic patterns with a periodicity of approximately 24 hours, which are generically referred to as circadian rhythms (Allada and Chung, 2010). Daily behavioral and metabolic rhythms are accompanied by a corresponding set of circadian changes in gene expression. Circadian rhythms are dictated by a cell autonomous clock that consists of a transcriptional feedback network that exhibits intrinsically oscillating activity. The period of this circadian clock is entrained by daily changes in light and temperature; although, daily rhythms persist even in constant conditions. Thus, circadian clocks provide a mechanism that allows animals to couple their behavior to anticipated changes in their environment.

Rhythmic changes in behavior and metabolism are also often coupled to developmental clocks. In the nematode C.elegans, molting exhibits a rhythmic pattern with a periodicity of 8-10 hours. This molting cycle is dictated by cell intrinsic developmental clock genes (termed heterochronic genes) (Moss, 2007). The periodicity of the molting cycle is dictated by rhythmic changes in the expression of a heterochronic gene (lin-42), which is homologous to the fly circadian gene PERIOD (Jeon et al., 1999; Monsalve et al., 2011). Thus, circadian and heterochronic clocks are mediated by similar biochemical mechanisms.

Although a great deal is known about the biochemical and genetic mechanisms controlling circadian and heterochronic timing, relatively little is known about how these clocks are coupled to changes in behavior, i.e. to their outputs. To address this question, we analyzed the rhythmic behaviors associated with the C. elegans molting cycle.

During each larval molt, C.elegans undergoes a prolonged period of profound behavioral quiescence, whereby locomotion and feeding behaviors are inactive for ~2 hours. This molt associated quiescence is termed lethargus behavior, and has been described for many wild type nematode species (Cassada and Russell, 1975). Lethargus has properties of a sleep-like state such as reduced sensory responsiveness and homeostatic rebound of quiescence following perturbation (Raizen et al., 2008). Several genes and molecular pathways involved in lethargus behavior have been identified (Monsalve et al., 2011; Raizen et al., 2008; Singh et al., 2011; Van Buskirk and Sternberg, 2007); however, a circuit mechanism controlling lethargus associated quiescence has not been defined.

Here we identify a central sensory circuit that dictates entry into and exit from locomotion quiescence during lethargus. Quiescence is associated with decreased activity in this central circuit, while arousal is associated with increased circuit activity. This central circuit regulates motility through the action of a neuropeptide (Pigment Dispersing Factor-1, PDF-1), which enhances the sensitivity of peripheral mechanosensory receptors in the body. These results provide a circuit mechanism controlling arousal and quiescence of locomotion in C. elegans.

RESULTS

Locomotion quiescence during lethargus is blocked in npr-1 mutants

Mutants lacking the neuropeptide receptor NPR-1 have heightened responsiveness to oxygen and pheromones, which results in altered foraging behavior and accelerated locomotion (Cheung et al., 2005; Gray et al., 2004; Macosko et al., 2009). Thus, NPR-1 is proposed to set the threshold for arousal of specific behaviors. Prompted by these results, we tested the idea that NPR-1 also regulates arousal from behavioral quiescence during lethargus. To analyze animals during the L4 to adult (L4/A) lethargus, we isolated a synchronous population of L4 animals and analyzed their behaviors during the subsequent molt. As in wild type animals, the pharyngeal pumping of npr-1 mutants was completely arrested during the L4/A lethargus (Fig. 1A). The duration of pharyngeal pumping quiescence was unaltered in npr-1 mutants, indicating that the duration of lethargus had not been altered (Fig. 1A). Pharyngeal pumping rate was also unaltered in npr-1 adults (Fig. 1B). To assess changes in locomotion during the L4/A lethargus, we analyzed the fraction of time animals undergo active motility (motile fraction) and locomotion velocity. Unlike wild type animals, npr-1 mutants exhibited fast and nearly continuous locomotion during the L4/A lethargus (Fig. 1C-E and Movies S1 and 2). The effects of npr-1 on locomotion persisted throughout the entire L4/A lethargus (as defined by pumping quiescence) (Fig. S1A-B). Inactivation of npr-1 had a significantly larger effect on locomotion during the L4/A lethargus (motile fraction, 17-fold increase; velocity, 50-fold increase) than in adults (motile fraction, 1.2-fold increase; velocity, 2-fold increase) (Fig. S1C-D). These results suggest that NPR-1 is required for locomotion quiescence during lethargus, but not for feeding quiescence.

Figure 1. NPR-1 and its ligands FLP-18 and FLP-21 regulate locomotion quiescence during lethargus.

Figure 1

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Pharyngeal pumping (A-B) and locomotion (C-G) was analyzed in the indicated genotypes. (A) The duration of feeding quiescence during the L4/A lethargus (defined by the absence of pharyngeal pumping) was unaltered in npr-1 mutants. (B) Adult pharyngeal pumping rate was also unaltered in npr-1 mutants. (C-G) Locomotion behavior of single worms during the L4/A lethargus was recorded for 30-75 seconds and velocity was measured (2 Hz sampling). Instantaneous locomotion velocity (C), average motile fraction (D,F), and average locomotion velocity (E,G) are plotted. (C-E) npr-1 null mutants had higher locomotion during the L4/A lethargus than WT (N2). (D,E) Wild type strains were polymorphic for L4/A locomotion, with 215V strains being more quiescent than 215F strains. (F,G) Mutations inactivating NPR-1 ligands, FLP-18 and FLP-21, decreased L4/A locomotion quiescence in animals expressing NPR-1(215F) receptors, but not in those expressing NPR-1(215V) receptors, i.e. npr-1(g320) mutants and N2 respectively. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly from npr-1(ky13) (D,E) or npr-1(g320) L4/A (F,G) are indicated (*, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant). See also Figure S1 and Movies S1 and S2.

Wild type strains are polymorphic for lethargus locomotion behavior

The npr-1 gene is polymorphic among wild type populations, with two frequent alleles observed (215V and 215F) (McGrath et al., 2009; Weber et al., 2010). These wild type alleles encode receptors that differ in their affinity for NPR-1 ligands (FLP-18 and FLP-21), with 215V exhibiting higher affinity (and lower EC50’s) than 215F receptors (Kubiak et al., 2003; Rogers et al., 2003). To determine if wild type strains are also polymorphic for lethargus behavior, we analyzed locomotion during the L4/A lethargus (Fig. 1D-E). All 215V containing strains exhibited similar levels of quiescence and were significantly more quiescent than 215F strains. The quiescence observed in 215F strains was more variable, with one strain (RC301) exhibiting L4/A locomotion similar to npr-1 null mutants while others (AB3 and CB4856) exhibited intermediate levels of quiescence. Thus, the extent of behavioral quiescence during lethargus is polymorphic among wild type strains. A strain carrying a 215F allele (g320) in the Bristol genetic background had significantly stronger quiescence than was observed in unrelated 215F wild type strains (e.g. CB4856 and RC301). These results suggest that variation in genes other than npr-1 also contributed to differences in the lethargus behaviors of wild type strains.

The NPR-1 ligands FLP-21 and FLP-18 regulate lethargus behavior

Two NPR-1 ligands have been identified, the neuropeptides FLP-18 and FLP-21 (Kubiak et al., 2003; Rogers et al., 2003). Both neuropeptides bind and activate NPR-1 receptors expressed in transfected cells; however, NPR-1 exhibits significantly stronger affinity for FLP-21. We found that mutations inactivating FLP-18 and FLP-21, and double mutants inactivating both ligands, had no effect on the L4/A locomotion behavior of worms expressing high affinity NPR-1(215V) receptors (Fig. 1F-G). By contrast, inactivating either FLP-18 or FLP-21 significantly decreased locomotion quiescence in a Bristol strain expressing low affinity NPR-1(215F) receptors, i.e. npr-1(g320) mutants (Fig. 1F-G). These results suggest that FLP-18 and FLP-21 function as endogenous NPR-1 ligands to regulate lethargus behavior in strains expressing NPR-1(215F) receptors.

The npr-1 lethargus defect is mediated by increased sensory activity

NPR-1’s effects on foraging are mediated by its expression in a sensory circuit in the head that is defined by gap junctions to the RMG interneuron (Fig. 2A) (Macosko et al., 2009). Hereafter, we refer to this circuit as the RMG circuit. In addition to the RMG circuit, NPR-1 is also expressed in GABAergic motor neurons in the ventral nerve cord (Coates and de Bono, 2002). We did two experiments to determine where NPR-1 functions to regulate motility during lethargus. First, an npr-1 transgene expressed in the RMG circuit (using the flp-21 promoter) (Fig. 2A) completely rescued the lethargus locomotion defect of npr-1 mutants, whereas a transgene expressed in GABAergic motor neurons (using the unc-30 promoter) had no rescuing activity (Fig. 2B-D). Second, the lethargus locomotion defect of npr-1 mutants was abolished by mutations inactivating ion channels required for sensory transduction, such as TAX-4/CNG and OSM-9/TRPV channels (Fig. 2E-G and S2A-B). A transgene expressing TAX-4 in the RMG circuit re-instated the L4/A quiescence defect in tax-4; npr-1 double mutants (Fig. 2F-G). These results suggest that the npr-1 defect in locomotion quiescence during lethargus was caused by heightened sensory activity in the RMG circuit.

Figure 2. The npr-1 lethargus defect is mediated by increased sensory activity.

Figure 2

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(A) A diagram illustrating the RMG circuit is shown. Sensory neurons (triangles) mediating the indicated aversive responses form direct gap junctions with the RMG interneuron (hexagon). Cells expressing NPR-1, TAX-4 CNG channels, PDF-1, and the flp-21 promoter (sensory rescue) are indicated (Barrios et al., 2012; de Bono et al., 2002; Janssen et al., 2009; Komatsu et al., 1996; Macosko et al., 2009; Rogers et al., 2003). ASI neurons are not directly connected to RMG but are also a potential source of PDF-1. This diagram is modified from that shown previously (Macosko et al., 2009). (B-G) Locomotion behavior of single worms during the L4/A lethargus was analyzed in the indicated genotypes. Instantaneous locomotion velocity (B,E), average motile fraction (C,F), and average locomotion velocity (D,G) are plotted. (B-D) The npr-1 L4/A locomotion quiescence defect was rescued by transgenes expressing NPR-1 in the RMG circuit (Sensory rescue, flp-21 promoter) but not by those expressed in GABAergic neurons (GABA rescue, unc-30 promoter), using the indicated promoters. (E-G) The npr-1 L4/A locomotion quiescence defect was suppressed in double mutants lacking TAX-4/CNG channels and was reinstated by transgenes expressing TAX-4 in the RMG circuit (Sensory rescue, flp-21 promoter). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (***, p <0.001). See also Figure S2.

PDF (Pigment Dispersing Factor) is required for the npr-1 lethargus defect

Neuropeptides play a pivotal role in sleep and wakefulness in other systems. For example, hypocretin/orexin regulates sleep, arousal, feeding, and metabolism in vertebrates (Sutcliffe and de Lecea, 2002). Thus, we tested if neuropeptides are required for the npr-1 lethargus defect. Consistent with this idea, the npr-1 lethargus quiescence defect was eliminated by mutations inactivating egl-3 PC2 and pkc-1 PKCε (Fig. 3A-C), which are required for pro-neuropeptide processing and dense core vesicle (DCV) exocytosis, respectively (Husson et al., 2006; Kass et al., 2001; Sieburth et al., 2007). These results suggest that the npr-1 lethargus defect was mediated by an endogenous neuropeptide.

Figure 3. PDF-1 and PDFR-1 mediate arousal from locomotion quiescence during lethargus.

Figure 3

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Locomotion behavior of single worms during the L4/A lethargus was analyzed in the indicated genotypes. Instantaneous locomotion velocity (A,D), average motile fraction (B,E), and average locomotion velocity (C,F) are plotted. The npr-1 L4/A locomotion quiescence defect was suppressed by mutations that block neuropeptide processing (egl-3 PC2 mutants, A-C) and dense core vesicle exocytosis (pkc-1 PKCε mutants, A-C), and by mutations inactivating PDF-1 and PDFR-1 (D-F). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (***, p <0.001). See also Figure S3.

In Drosophila, the neuropeptide Pigment Dispersing Factor (PDF) regulates circadian rhythms and promotes wakefulness (Parisky et al., 2008; Renn et al., 1999). Prompted by PDF’s role in Drosophila, we tested the idea that PDF mediates the lethargus quiescence defect in npr-1 mutants. C. elegans PDF peptides (PDF-1 and PDF-2) and their receptor (PDFR-1) were previously identified (Janssen et al., 2008; Janssen et al., 2009). PDF-1 is expressed in several classes of sensory neurons and interneurons, including ASK chemosensory neurons and RMG interneurons in the RMG circuit (Barrios et al., 2012; Janssen et al., 2009) (Fig. 2A). The locomotion rate and motile fraction of pdf-1;npr-1 and pdfr-1;npr-1 double mutants during the L4/A lethargus were significantly lower than in npr-1 single mutants (Fig. 3D-F). Inactivating PDF-1 and PDFR-1 had a much less dramatic effect on adult locomotion in pdf-1;npr-1 and pdfr-1;npr-1 double mutants (Fig. S3A). Thus, increased signaling by PDF-1 and PDFR-1 in npr-1 mutants was required for the increased motility during lethargus. The npr-1 foraging defect was unaltered in pdf-1;npr-1 and pdfr-1;npr-1 double mutants (Fig. S3B), indicating that PDF was not required for other npr-1 phenotypes. Inactivating PDF-2 had little effect on the locomotion of npr-1 mutants during lethargus (Fig. S3C-D), indicating the PDF-1 is the major form of PDF involved in lethargus behavior. Collectively, these results suggest that PDF-1 functioned as an arousal peptide in npr-1 mutants, preventing locomotion quiescence during lethargus. PDF-1’s effects on arousal were specific because knockdown of 14 other neuropeptides expressed in the RMG circuit had no effect on the npr-1 lethargus defect (Fig. S3E).

NPR-1 inhibits PDF-1 secretion during lethargus

If PDF-1 functions as an arousal peptide, PDF-1 expression or secretion should be inhibited during lethargus, when animals are quiescent. We did several experiments to test this idea. The abundance of pdf-1 and pdfr-1 mRNAs (assayed by quantitative PCR) was unaltered during the L4/A lethargus, whereas expression of mlt-10 (a gene required for molting) was significantly increased, as expected (Fig. S4A) (Frand et al., 2005). To assay PDF-1 secretion, we expressed YFP-tagged proPDF-1 with the pdf-1 promoter (Fig. 4A-B). During DCV maturation, the YFP linked to proPDF-1 is cleaved by proprotein convertases, and is subsequently secreted by DCV exocytosis. To assess the level of PDF-1 secretion, we analyzed PDF-1::YFP fluorescence in the endolysosomal compartment of coelomocytes, which are specialized scavenger cells that internalize proteins secreted into the body cavity (Fares and Greenwald, 2001; Sieburth et al., 2007). The PDF-1::YFP secretion reporter produced high levels of coelomocyte fluorescence in both L4 larvae and adults, whereas dramatically lower coelomocyte fluorescence was observed during the L4/A lethargus (Fig. 4A-B). Coelomocyte fluorescence produced by a second secretion probe (mCherry-tagged RIG-3 expressed in cholinergic motor neurons) (Babu et al., 2011) was unaltered during lethargus (Fig. S4B), indicating that secretion and coelomocyte function were not globally inhibited during lethargus.

Figure 4. NPR-1 inhibits PDF-1 secretion during lethargus.

Figure 4

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PDF-1 secretion (A,B) and the effect of forced secretion of PDF-1 on lethargus locomotion behavior (C,D) were analyzed in the indicated genotypes. (A,B) YFP-tagged PDF-1 was expressed with the pdf-1 promoter. Representative images (A) and summary data (B) are shown for coelomocyte fluorescence in L4, L4/A, young adult (0-2 eggs in uterus), and gravid adults of the indicated genotypes. PDF-1::YFP coelomocyte fluorescence was dramatically reduced during the L4/A lethargus of wild type animals, but not in npr-1 mutants. Decreased PDF-1::YFP coelomocyte fluorescence during lethargus was reinstated by transgenes expressing NPR-1 in the RMG circuit (Sensory rescue, flp-21 promoter). (C,D) Forced depolarization of PDF-1 expressing neurons decreased L4/A locomotion quiescence. PDF-1 and rat TRPV1 were ectopically expressed in ASH neurons (using the sra-6 promoter). Locomotion behavior of transgenic worms during the L4/A lethargus was analyzed with or without capsaicin treatment (6-7 hours). Average motile fraction (C), and average locomotion velocity (D) are plotted. Capsaicin treatment decreased L4/A quiescence in transgenic animals expressing both TRPV1 and PDF-1 in ASH neurons, but not in those expressing only TPRV1. The number of animals analyzed is indicated for each genotype. Scale bar indicates 10 μm. Values that differ significantly are indicated (*, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant). See also Figure S4.

If decreased PDF-1 secretion during lethargus is a cellular mechanism for inducing quiescence, we would expect that mutants retaining or lacking locomotion quiescence would exhibit reciprocal patterns of PDF-1 secretion during lethargus. We did several experiments to test this idea. In npr-1 mutants, which lack quiescence, the decrease in PDF-1::YFP coelomocyte fluorescence during the L4/A lethargus was eliminated (Fig. 4A-B), and was restored by a transgene expressing NPR-1 in the RMG circuit (Fig. 4B). 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 (Fig. 2F-G and S4C). By contrast, RIG-3 coelomocyte fluorescence was decreased in npr-1 mutants, in both L4 and L4/A animals (Fig. S4B). Consequently, the effects of NPR-1 and TAX-4 on PDF-1 coelomocyte fluorescence are unlikely to be caused by general changes in the stability of secreted proteins, nor 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 neurons, thereby increasing motility during lethargus.

The preceding results suggest that decreased and increased PDF-1 secretion during lethargus are correlated with and required for locomotion quiescence and arousal. To determine if increased PDF-1 secretion is sufficient to arouse locomotion, we constructed transgenic animals in which PDF-1 secretion can be pharmacologically induced (Fig. 4C-D). A prior study showed that capsaicin treatment depolarizes ASH neurons expressing rat TRPV1 channels (Tobin et al., 2002). When TRPV1 and PDF-1 were co-expressed in ASH neurons, capsaicin treatment significantly decreased locomotion quiescence during lethargus (Fig. 4C-D). This effect was not observed when only TRPV1 was expressed in ASH. These results suggest that forced secretion of PDF-1 during lethargus was sufficient to arouse locomotion behavior.

PDF-1 can function in ASK neurons to mediate arousal

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, and in ASK sensory neurons, which form direct gap junctions with RMG (Fig. 2A) (Barrios et al., 2012; Janssen et al., 2008). Transgenes expressing PDF-1 in ASK neurons re-instated the locomotion quiescence defect in pdf-1;npr-1 double mutants (Fig. S4D-E). 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 (Fig. 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 cell non-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 (Fig. S4D-E).

PDFR-1 acts in mechanosensory neurons to mediate arousal

How does enhanced PDF-1 secretion alter locomotion? The pdfr-1 promoter is expressed in mechanosensory neurons that sense vibration of the body wall (the touch neurons), in body wall muscles, and in a few other classes of neurons (Janssen et al., 2008). Transgenes expressing PDFR-1 in touch neurons or in body wall muscles both partially reinstated the lethargus locomotion quiescence defect in npr-1; pdfr-1 double mutants (Fig. 5A, C and D). These results suggest that PDFR-1 acts in both touch neurons and body wall muscles to promote arousal from locomotion quiescence during lethargus. The six touch neurons form gap junctions with the ventral cord command interneurons that control locomotion (Chalfie et al., 1985). Mutations that impair the mechanosensitivity of the touch neurons (termed Mec mutants) cause locomotion to become lethargic (Chalfie and Sulston, 1981). For these reasons, we focused our analysis on PDFR-1 function in touch neurons.

Figure 5. PDFR-1 receptors expressed in touch neurons and body muscles mediate locomotion arousal.

Figure 5

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Locomotion behavior of single worms during the L4/A lethargus was analyzed in the indicated genotypes. Instantaneous locomotion velocity (A,B), average motile fraction (C), and average locomotion velocity (D) are plotted. The npr-1 locomotion quiescence defect was partially reinstated in pdfr-1; npr-1 double mutants by transgenes expressing PDFR-1 in touch neurons (mec-3 promoter) and body muscles (myo-3 promoter) using the indicated promoters (A,C,D). Mutations disrupting touch neuron differentiation (mec-3 mutants) partially suppressed the npr-1 locomotion quiescence defect (B-D). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (**, p <0.01; ***, p <0.001).

Is the npr-1 lethargus defect mediated by increased activity of the touch neurons? We did several experiments to test this idea. First, we analyzed the lethargus behavior of mec-3; npr-1 double mutants. The MEC-3 transcription factor is required for differentiation of touch neurons; consequently, touch responses are disrupted in mec-3 mutants (Way and Chalfie, 1988). Mutations inactivating mec-3 partially suppressed the lethargus locomotion defect of npr-1 mutants (Fig. 5B-D). These results suggest that touch neuron function was required for NPR-1’s effect on motility during lethargus. Partial suppression of the lethargus defect in mec-3; npr-1 double mutants was expected because rescue experiments suggest that PDFR-1 function is required in both touch neurons and body muscles (Fig. 5A, C and D).

Second, we measured touch-evoked calcium transients in the anterior touch neuron (ALM) of adult animals using the genetically encoded calcium indicator cameleon (Fig. 6A-B, and S5C-D). Cameleon expression in touch neurons did not disrupt NPR-1 and PDFR-1 effects on L4/A locomotion quiescence (Fig. S5A-B). Thus, calcium buffering by cameleon did not interfere with NPR-1 mediated regulation of touch cell function. PDF-1 secretion was increased in npr-1 adults (Fig. 4A-B); consequently, NPR-1’s effects on touch sensitivity should be evident in adults. Consistent with this idea, the magnitude of touch-evoked calcium transients in ALM was significantly increased in npr-1 mutant adults and this defect was rescued by transgenes expressing NPR-1 in the RMG circuit (Fig. 6A-B). The enhanced ALM touch-sensitivity exhibited by npr-1 adults was eliminated in pdfr-1; npr-1 double mutants (Fig. 6A-B) and was reinstated by transgenes expressing PDFR-1 in touch neurons but not by those expressed in body wall muscles (Fig. 6A-B). By contrast, in pdf-1; npr-1 double mutants, heightened ALM touch responsiveness was reduced but not eliminated (Fig. S5C-D). The residual effect of NPR-1 on ALM touch sensitivity in pdf-1; npr-1 double mutants was likely mediated by other PDFR-1 ligands (e.g. PDF-2). Collectively, these results suggest that increased PDF-1 secretion in npr-1 adults was associated with enhanced touch sensitivity.

Figure 6. PDFR-1 is required for enhanced ALM touch sensitivity in npr-1 mutant adults.

Figure 6

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Touch sensitivity and locomotion were analyzed in the indicated adult animals. (A,B) Touch-evoked calcium transients in ALM were analyzed using cameleon as a calcium indicator. Responses were analyzed in adult animals. Averaged responses (A), and the amplitudes of individual trials (B) are shown for each genotype. Each red trace represents the average percentage change in YFP/CFP fluorescence ratio. The black triangle indicates the time at which the mechanical stimulus was applied. Gray shading indicates SEM of the mean response. (A-B) Touch-evoked calcium transients in adult ALM neurons were significantly larger in npr-1 mutants. This defect was rescued by transgenes expressing NPR-1 in the RMG circuit (sensory rescue), and was suppressed by mutations inactivating PDF-1 and PDFR-1. Touch-evoked calcium transients in pdfr-1 mutants were not significantly different from wild type controls. Enhanced touch-evoked calcium transients in adult ALM neurons were reinstated in pdfr-1; npr-1 double mutants by transgenes expressing PDFR-1 in touch neurons but not by those expressed in body muscles. (C-E) Locomotion behavior of single adult worms was analyzed in the indicated genotypes. Instantaneous locomotion velocity (C), average motile fraction (D), and average locomotion velocity (E) are plotted. Both pdf-1 and pdfr-1 single mutants showed reduced locomotion in adult. The pdfr-1 adult locomotion defect was partially rescued by transgenes expressing PDFR-1 in touch neurons, but not in body wall muscles. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (***, p <0.001; **, p <0.01; ns, not significant). See also Figure S5.

Because PDF-1 and PDFR-1 enhanced touch sensitivity in npr-1 mutants, we would expect that pdf-1 and pdfr-1 single mutants would exhibit decreased touch sensitivity. Contrary to this idea, adult ALM touch responses were unchanged in either single mutant (Fig. 6A-B and S5C-D). These results do not exclude the idea that touch sensitivity was altered in these mutants. We may fail to detect differences in ALM responses for technical reasons. For example, an effect on touch sensitivity in single mutants may only be apparent at lower stimulus intensities, or upon repetitive stimulation. To further address this issue, we analyzed locomotion in the single mutants. Adult pdf-1 and pdfr-1 single mutants exhibited significantly slower locomotion and decreased motile fractions (Fig. 6C-E) (Meelkop et al., 2012), both of which could result from diminished touch sensitivity. Consistent with this idea, the decreased locomotion rate and motile fraction of pdfr-1 mutants was partially rescued by transgenes expressing PDFR-1 in touch neurons (Fig. 6D-E). These results support the idea that PDF-1 and PDFR-1’s effects on touch sensitivity are not restricted to npr-1 mutants.

To determine if NPR-1 also regulates touch sensitivity during lethargus, we analyzed ALM calcium transients during the L4/A lethargus (Fig. 7). A recent study reported that touch neuron calcium transients are significantly reduced during lethargus (Schwarz et al., 2011). Consistent with this prior study, we found that ALM touch-evoked calcium transients were significantly smaller during the L4/A lethargus; however, this effect was eliminated in npr-1 mutants (Fig. 7). The enhanced ALM touch responses during lethargus exhibited by npr-1 mutants was eliminated in pdfr-1; npr-1 double mutants (Fig. 7). Thus, NPR-1 inhibition of PDF signaling is required for inhibition of touch sensitivity during lethargus.

Figure 7. NPR-1 and PDFR-1 regulate ALM touch sensitivity during lethargus.

Figure 7

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Touch-evoked calcium transients in ALM were analyzed in L4, L4/A, and adults of the indicated genotypes. Averaged responses (A) and the amplitudes of individual trials (B) are shown for each genotype. Each red trace represents the average percentage change in YFP/CFP fluorescence ratio. The black triangle indicates the time at which the mechanical stimulus was applied. Gray shading indicates SEM of the mean response. (A-B) Touch-evoked ALM calcium transients were significantly reduced during L4/A lethargus, and this effect was abolished in npr-1 mutants. Enhanced touch-evoked calcium transients in npr-1 mutants were suppressed by inactivating PDFR-1. Values that differ significantly are indicated (*, p <0.05; **, p <0.01; ns, not significant).

DISCUSSION

We describe a circuit mechanism controlling arousal from a developmentally programmed form of behavioral quiescence in C. elegans. Increased RMG circuit activity in npr-1 mutants was accompanied by increased PDF-1 secretion and heightened peripheral sensitivity to touch, thereby increasing motility during lethargus. Below we discuss the significance of these results.

Related neuropeptides mediate quiescence and arousal/motivation in worms, flies, and rodents. Peptides homologous to NPY induce locomotion quiescence in C. elegans (FLP-18 and -21), inhibit locomotion and foraging on food in Drosophila (NPF) (Wu et al., 2003), and inhibit the arousing effects of hypocretin expressing neurons in mice (NPY) (Fu et al., 2004). By contrast, peptides homologous to PDF arouse locomotion in C. elegans (PDF-1), arouse circadian locomotor activity and decrease sleep duration in Drosophila (PDF) (Parisky et al., 2008; Renn et al., 1999), and regulate circadian behaviors and sleep in rodents (VIP) (Hu et al., 2011; Maywood et al., 2007). Thus, conserved molecular mechanisms are employed to regulate arousal and quiescence in developmentally programmed, metabolically driven, and circadian behavioral states.

If lethargus is a sleep-like state, as previously proposed (Raizen et al., 2008; Van Buskirk and Sternberg, 2007), one would expect that disrupting quiescence during lethargus would be deleterious. Contrary to this notion, the fertility and development of npr-1 mutants were not grossly altered, indicating that locomotion quiescence during lethargus is not essential for normal development or molting. These results do not exclude the idea that quiescence during lethargus has significant effects on health in native environments (where conditions are more variable).

How are arousal peptides functionally coupled to circadian and developmental cycles? VIP and PDF are expressed in central clock neurons: rat VIP in the suprachiasmatic nucleus (SCN) of the hypothalamus, fly PDF in LNv neurons, and worm PDF in the RMG circuit (Helfrich-Forster, 1995; Maywood et al., 2007). Rhythmic changes in pdf mRNA levels were not observed in the Drosophila circadian and C. elegans molting cycles (Janssen et al., 2009; Park and Hall, 1998). Instead, PDF-1 secretion was dramatically reduced during lethargus. Inhibition of PDF-1 secretion and inhibition of locomotion during lethargus were both abolished in npr-1 mutants. Thus, altered PDF-1 secretion provides a cellular mechanism for coupling changes in locomotor activity to the molting cycle.

How is PDF-1 secretion inhibited during lethargus? In npr-1 mutants, pheromone, and oxygen responses mediated by the RMG circuit are enhanced (Cheung et al., 2005; Gray et al., 2004; Macosko et al., 2009) and we observed a corresponding enhancement of PDF-1 secretion. Similarly, inactivation and restoration of TAX-4 CNG channel expression in the RMG circuit was accompanied by parallel changes in PDF-1 secretion. Based on these results, we propose that RMG circuit activity is diminished during lethargus, thereby inhibiting PDF-1 secretion. Consistent with this idea, forced depolarization of ASH neurons expressing PDF-1 was sufficient to arouse locomotion during lethargus.

How do central clock neurons engender rhythmic behaviors? A great deal is known about how the activity and expression profile of central clock neurons are regulated. Much less is known about how clock neurons dictate circadian behaviors. In C. elegans, responsiveness to several sensory cues is reduced during lethargus. In particular, touch sensitivity and touch-evoked calcium transients in the touch neurons are decreased during lethargus (Raizen et al., 2008; Schwarz et al., 2011; Singh et al., 2011). Our results provide a cellular mechanism for these effects. During lethargus, NPR-1 inhibited PDF-1 secretion from the RMG circuit, thereby decreasing touch neuron sensitivity. PDF-1’s effect on locomotion arousal was also mediated in part by activation of PDFR-1 receptors in body muscle. Interestingly, fly PDF and rodent VIP also have direct effects on muscle function (Talsma et al., 2012).

Although NPR-1, TAX-4, and PDF have profound effects on lethargus behavior, several results suggest that other signaling pathways must also contribute to both quiescence and arousal. For example, L4/A quiescence was restored in pdfr-1; npr-1 double mutants (Fig. 3D-F); consequently, changes in NPR-1 and PDF signaling are not absolutely required to induce locomotion quiescence or arousal. Similarly, the locomotion of pdfr-1 mutants during lethargus was significantly more quiescent than in adults. Thus, inactivating PDF signaling is unlikely to be the only mechanism producing L4/A quiescence. These results suggest that arousal and quiescence are behavioral states governed by multiple inputs, whose activities are integrated in the RMG circuit.

NPR-1 regulates several physiologically important traits. Inactivating NPR-1 alters sensitivity to environmental repellents (e.g. pheromones and oxygen), foraging behavior, innate immune responses, and lethargus behavior (Cheung et al., 2005; de Bono and Bargmann, 1998; Gray et al., 2004; Reddy et al., 2009; Styer et al., 2008) (Fig. 2A). Because NPR-1 sits at the nexus of multiple physiologically important traits, changes in NPR-1 activity and natural variation in the npr-1 gene provide a mechanism for coupling changes in behavioral quiescence to the demands of the local environment. Specifically, changes in NPR-1 signaling could allow isolated populations to optimize growth properties in environments with increased exposure to specific repellents or bacterial pathogens.

EXPERIMENTAL PROCEDURES

Strains

Strain maintenance and genetic manipulation were performed as described (Brenner, 1974). Animals were cultivated at 20°C on agar nematode growth media seeded with OP50 E.coli. Wild type reference strain was N2 Bristol. Strains used in this study are as follows:

Wild type strains

CB4555, TR389, AB3, CB4856, and RC301

Mutant strains and integrants

DA609 npr-1(ad609) X

KP6080 npr-1(g320) X

KP6048 npr-1(ky13) X

AX1410 flp-18(db99) X (Gift from Mario de Bono)

KP6077 flp-21(pk1601) V

PR678 tax-4(p678) III

KP3183 osm-9(ky10) IV

KP5966 egl-3(nr2090) V

KP5989 pkc-1(nj3) V

LSC27 pdf-1(tm1996) III

KP6340 pdfr-1(ok3425) III

KP6416 pdf-2(tm4393) X

CB1338 mec-3(e1338) IV

KP7044 flp-21(pk1601) V;flp-18(db99) X

KP7041 flp-18(db99) npr-1(g320) X

KP7042 flp-21(pk1601) V;npr-1(g320) X

KP7059 flp-21(pk1601) V;flp-18(db99) npr-1(g320) X

KP6060 tax-4(p678) III;npr-1(ky13) X

KP6841 osm-9(ky10) IV;npr-1(ky13) X

KP6054 egl-3(nr2090) V;npr-1(ky13) X

KP6682 pkc-1(nj3) V;npr-1(ky13) X

KP6100 pdf-1(tm1996) III;npr-1(ky13) X

KP6410 pdfr-1(ok3425) III;npr-1(ky13) X

KP6417 pdf-2(tm4393) npr-1(ky13) X

KP5364 nre-1(hd20) lin-15b(hd126) X

KP6050 npr-1(ky13) nre-1(hd20) lin15b(hd126) X

CX4978 kyIS200[sra-6p::VR1, elt-2p::NLS-gfp] (Gift from Cori Bargmann)

KP6426 mec-3(e1338) IV;npr-1 (ky13) X

KP6693 nuIS472[pdf-1p::pdf-1::YFP, vha-6p::mCherry]

KP6744 tax-4(p678) III;nuIS472

KP6745 tax-4(p678) III;npr-1(ky13) X;nuIS472

KP6743 npr-1(ky13) X;nuIS472

AQ906 bzIS17[mec-4p::YC2.12]

KP6679 pdfr-1(ok3425) III;bzIS17

KP6680 pdfr-1(ok3425) III;npr-1(ky13) X;bzIS17

KP6681 npr-1(ky13) X;bzIS17

KP6699 pdf-1(tm1996) III;npr-1(ky13) X;bzIS17

KP6700 pdf-1(tm1996) III; bzIS17

Strains containing extrachromosomal arrays

CX9396 npr-1(ad609) X;kyEX1966[flp-21p::npr-1 SL2 GFP, ofm-1p::dsRed] (Gift from Cori Bargmann)

KP6053 npr-1(ad609) X;nuEX1520[unc-30p::npr-1::gfp, myo-2p::NLS-mCherry]

KP7144 tax-4(p678) III;npr-1(ky13) X;nuEX1601[flp-21p::tax-4, vha-6p::mCherry]

KP7141 npr-1(ky13) X;nuIS472;nuEX1607[flp-21p::npr-1, myo-2p::NLS-mCherry]

KP7143 tax-4(p678) III;npr-1(ky13) X;nuIS472;nuEX1612[flp-21p::tax-4, myo-2p::NLS-mCherry]

KP6819 nuEX1560[unc-17p::rig-3(-GPI)::mCherry]

KP6820 npr-1(ky13) X; nuEX1560

KP7053 kyIS200;nuEX1610[sra-6p::pdf-1::venus, myo-2p::NLS-mCherry]

KP6678 pdf-1(tm1996) III;npr-1(ky13) X;nuEX1547[sra-9p::pdf-1::venus]

KP6741 pdf-1(tm1996) III;npr-1(ky13) X;nuEX1552[str-3p::pdf-1::venus, vha-6p::mCherry]

KP6860 nuEX1611[sra-9p::pdf-1::venus, myo-2p::NLS-mCherry]

KP7146 npr-1(ky13);nuEX1611

KP6423 pdfr-1(ok3425) III;npr-1(ky13) X;nuEX1526[mec-3p::pdfr-1b, myo-2p::NLS-mCherry]

KP6594 pdfr-1(ok3425) III;npr-1(ky13) X;nuEX1534[myo-3p::pdfr-1a, vha-6p::mCherry]

KP6733 pdfr-1(ok3425) III;npr-1(ky13) X;bzIS17; nuEX1526 [mec-3p::pdfr-1b, myo-2p::NLS-mCherry]

KP6734 pdfr-1(ok3425) III;npr-1(ky13) X;bzIS17;nuEX1534[myo-3p::pdfr-1a, vha-6p::mCherry]

KP6736 npr-1(ad609) X;bzIS17; kyEX1966[flp-21p::npr-1 SL2 GFP, ofm-1p::dsRed]

KP6815 pdfr-1(ok3425) III;nuEX1526[mec-3p::pdfr-1b, myo-2p::NLS-mCherry]

KP6816 pdfr-1(ok3425) III;nuEX1534[myo-3p::pdfr-1a, vha-6p::mCherry]

Constructs

pdf-1 expression constructs (pdf-1p::pdf-1::YFP (KP#1861), sra-9p::pdf-1::YFP (KP#1923), str-3p::pdf-1::YFP (KP#1924), and sra-6p::pdf-1::YFP (KP#1925))

cDNAs corresponding to pdf-1 and YFP (VENUS) containing a stop codon were each amplified by PCR and ligated into pPD49.26 (Addgene) containing the pdf-1 (~5.4kb 5′ regulatory sequence), sra-9 (~3kb 5′ regulatory sequence: ASK expression), str-3 (~3kb 5′ regulatory sequence: ASI expression), and sra-6 (~3.8kb 5′ regulatory sequence: ASH expression) promoters.

npr-1 rescue constructs (unc-30p::npr-1::GFP (KP#1857) and flp-21p::npr-1 (KP#1921))

npr-1 cDNA (215V) was amplified by PCR and ligated into expression vectors (pPD49.26) containing the unc-30 promoter (~2.5kb 5′ regulatory sequence) and GFP at the 3′ end of MCSII or the flp-21 promoter (~4.1kb 5′ regulatory sequence).

tax-4 rescue construct (flp-21p::tax-4 (KP#1922))

tax-4 cDNA was amplified by PCR and ligated into expression vector (pPD49.26) containing the flp-21 promoter (~4.1kb 5′ regulatory sequence).

pdfr-1 rescue constructs (mec-3p::pdfr-1b (KP#1863) and myo-3p::pdfr-1a (KP#1866))

pdfr-1 cDNAs were amplified by PCR and ligated into expression vectors (pPD49.26) containing the mec-3 promoter (3.4kb upstream of the start codon of mec-3 genomic region) or myo-3 promoter (~2.4kb 5′ regulatory sequence).

Transgenes and germline transformation

Transgenic strains were generated by microinjection of various plasmids with coinjection markers (myo-2p::NLS-mCherry (KP#1480) and vha-6p::mcherry (KP#1874)). Injection concentration was 40 - 50 ng/μl for all the expression constructs and 10 ng/μl for coinjection markers. The empty vector pBluescript was used to bring the final DNA concentration to 100 ng/μl. Integration of transgenes was obtained by UV irradiation of strains carrying extrachromosomal arrays. All the integrants were outcrossed to wild type strains (N2 bristol) 10 times.

Lethargus locomotion and behavior analysis

Well-fed late L4 animals were transferred to full lawn OP50 bacterial plates. After 1 hour, locomotion of animals in lethargus (determined by absence of pharyngeal pumping) was recorded on a Zeiss Discovery Stereomicroscope using Axiovision software. Locomotion was recorded at 2 Hz for 30-75 seconds. Centroid velocity of each animal was analyzed at each frame using object-tracking software in Axiovision. Motile fraction of each animal was calculated by dividing the number of frames with positive velocity value with total number of frames. Speed of each animal was calculated by averaging the velocity value at each frame. For long-term lethargus locomotion analysis (Fig. S1A-B), 1 min-long video was recorded every 20 minutes for each animal after the transfer to full lawn OP50 bacterial plates, and motile fraction was calculated for each time point. For the forced secretion of PDF-1 (Fig. 4C-D), early L4 animals were transferred to NGM plates containing 50 μM capsaicin (with food) and treated with capsaicin for 6-7 hours. Duration of L4/A pumping quiescence was calculated by summating the time period from the cessation to the resumption of pharyngeal pumping. Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparison and two-tailed Student’s t test for pairwise comparison.

Adult locomotion and behavior analysis

Locomotion of adult animals was analyzed with the same setup as lethargus locomotion analysis described above, except that well-fed adult animals were monitored within 5-10 minutes after the transfer to full lawn OP50 bacterial plates. Pharyngeal pumping rate of adult animals was calculated by counting the number of pharyngeal pumping for 10 seconds under the Leica MS5 routine stereomicroscope. Foraging behavior was analyzed as described (de Bono and Bargmann, 1998). Briefly, approximately 150 well-fed adult animals were placed on NGM plates seeded with 200 μl OP50 E.coli 2 days before the assay. After 3 hours, images were taken for each genotype. Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparison and two-tailed Student’s t test for pairwise comparison.

RNAi feeding screen

A small-scale RNAi feeding screen was performed as described (Kamath et al., 2003). The screen was performed in the neuronal RNAi hypersensitive mutant background (nre-1 lin-15b)(Schmitz et al., 2007). 15 neuropeptide genes known to be expressed in RMG circuit were selected for the screen (Li and Kim, 2008). After 5 days of RNAi treatment (2 generation) at 20°C, well-fed late L4 animals were transferred to full lawn OP50 bacterial plates. After 1 hour, animals in lethargus (determined by absence of pharyngeal pumping) were scored for their motility. Statistical significance was determined using chi-square test.

Quantitative PCR

Total RNA was purified from synchronized animals in L4/A lethargus (determined by absence of pharyngeal pumping) and synchronized young adult animals (4-5 hours after L4/A lethargus) using standard protocol. 6 biological replicates of wild type (N2 Bristol) and npr-1(ky13) samples were collected on 3 different days. 2 μg of total RNA was used to synthesize cDNA using RETROscript (Ambion). Real-time PCR was performed using iTaq SYBR Green Supermix with ROX (BioRad) and a 7500 Fast Real-Time PCR System (Applied Biosystems). Statistical significance was determined using two-tailed Student’s t test.

Fluorescence microscopy and image analysis

Quantitative imaging of coelomocyte fluorescence was performed using a Zeiss Axioskop equipped with an Olympus PlanAPO 100× (NA=1.4) objective and a CoolSNAP HQ CCD camera (Photometrics). Worms were immobilized with 30 mg/ml BDM (Sigma). The anterior coelomocytes were imaged in L4, L4/A lethargus (determined by absence of pharyngeal pumping), young adult (0-2 eggs), and gravid adult animals. Image stacks were captured and maximum intensity projections were obtained using Metamorph 7.1 software (Universal Imaging). YFP fluorescence was normalized to the absolute mean fluorescence of 0.5 mm FluoSphere beads (Molecular Probes). Statistical significance was determined using one-way ANOVA with Tukey test.

Calcium imaging and analysis

To image touch-evoked calcium transients in the ALM cell body, we used a transgenic line (bzIs17) that expresses the calcium-sensitive protein cameleon in touch neurons (using the mec-4 promoter). Calcium imaging was performed on a Zeiss Axioskop 2 upright compound microscope equipped with a Dual View Beam Splitter and a Uniblitz Shutter. Images were recorded at 10 Hz using an iXon EM camera (Andor Technology) and captured using IQ1.9 software (Andor Technology). Individual worms were glued using Dermabond topical skin adhesive glue to pads composed of 2% agarose in extracellular saline (145mM NaCl, 5mM KCl, 1mM CaCl2, 5mM MgCl2, 20mM D-glucose, 10mM HEPES buffer, pH7.2). Gentle touch stimuli were delivered using a M-111.1DG micromanipulator. The micromanipulator was used to drive a pulled glass microcapillary with a 15μm diameter rounded tip against the side of the glued worm. The tip was positioned adjacent to the body wall and was driven forward to cause a 10μm (adults, Fig. 6) or 20μm (L4/A lethargus, Fig. 7) deflection of the cuticle. Optical and mechanical stimuli were synchronized by flashing a white LED on the sample a second before the stimulus was delivered. Analysis was done using a custom written Matlab (Mathworks) program. A rectangular region of interest (ROI) was drawn surrounding the cell body and for every frame the ROI was shifted according to the new position of the center of mass. Fluorescence intensity, F, was computed as the difference between the sum of pixel intensities and the faintest 10% pixels (background) within the ROI. Statistical significance was determined using one-way ANOVA with Tukey test.

Supplementary Material

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ACKNOWLEDGEMENTS

We thank the following for strains, advice, reagents, and comments on the manuscript: Cori Bargmann for NPR-1 sensory rescue and rat TRPV1 transgenes, Mario de Bono for flp-18 mutants, the Caenorhabditis Genetics Center (CGC) and S. Mitani for strains, and members of the Kaplan lab for comments on the manuscript. This work was supported by a Kwanjeong Educational Foundation Predoctoral Fellowship (S.C.), by research grants to J.K. (NIH DK80215), and by the Medical Research Council (W.S.).

Footnotes

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