npr-1 action in the RMG hub-and-spoke circuit suppresses C9 avoidance

npr-1 animals show reduced avoidance of repulsive pheromones in accumulation assays (Macosko et al., 2009), consistent with their increased aggregation behaviors. As the aggregation behaviors are most prominent on food (de Bono and Bargmann, 1998; de Bono et al., 2002), we included food when comparing npr-1 and wild-type responses to C9. npr-1 mutants did not avoid 10 nM C9 in the drop test, although they avoided higher concentrations (Figure 2A, 2B). High-osmolarity glycerol avoidance was unaffected by npr-1 (Figure 2A). Silencing ADL synaptic output by TeTx expression in npr-1 mutants did not further affect their behavioral responses to 10 nM C9, but reduced their avoidance of 100 nM C9 (Figure 2B). These results suggest that ADL chemical synapses can drive C9 avoidance in npr-1 animals, but with reduced sensitivity compared to wild type.

Open in a separate window

Figure 2

NPR-1 acts in RMG to antagonize ADL chemical synapses.

A. npr-1 animals show decreased C9 avoidance. Avoidance assays in the presence of food. * indicates responses different from values indicated by brackets at P<0.05. n=40–100 animals each.

B. ADL chemical synapses can drive C9 avoidance in npr-1. Avoidance assays in the presence of food. * indicates responses different from wild type at P<0.05. n=40–100 animals each.

C. Heat shock-driven expression of npr-1 during the adult stage rescues the C9 avoidance defect of npr-1 animals. ** indicates responses different from the responses of the same animals before heat shock at P<0.01. Array(-) indicates animals from the hsp16.2::npr-1-expressing transgenic strain that have lost the extrachromosomal array, representing sibling controls for the array(+) transgenic animals. Behavioral assays were performed in the presence of food immediately after heat-shock at 33 C for 30 minutes. n=40–60 animals each.

D. Strengthening ADL chemical synapses enhances C9 avoidance in npr-1. Avoidance assays in the presence of food. * and ** indicate responses different from values indicated by brackets at P<0.05 and P<0.01, respectively. n=40–100 animals each. Also see Figure S2A.

E. (Left) Changes in G-CaMP fluorescence in response to C9 in ADL neurons of wild-type and npr-1(ad609) hermaphrodites. Wild-type and all experimental conditions (see Figure 4C and Experimental Procedures) were examined together on multiple days. n=12 neurons each. Shading around the lines represents SEM. (Right) Average peak percentage changes in fluorescence upon addition of C9. *** indicates responses different from wild-type at P<0.001. Also see Figures S2B and S2C. Error bar in all panels represent SEM.

Previous studies have indicated that the ADL neurons promote aggregation in npr-1 mutants, in apparent contradiction to their role in C9 avoidance in wild type (de Bono et al., 2002). A possible explanation of this paradox is provided by the proposed circuit for aggregation, which involves gap junctions between ADL and RMG neurons rather than ADL chemical synapses (Figure 1D) (White et al., 1986; Macosko et al., 2009). Aggregation through the RMG circuit is inhibited by npr-1 expression in RMG (Macosko et al., 2009). We hypothesized that this gap junction circuit might antagonize or inactivate C9 avoidance mediated by ADL chemical synapses. Indeed, the C9 avoidance defects in npr-1 animals were fully rescued by a transgene expressing an npr-1 cDNA in RMG (Figure 2A), indicating that NPR-1 acts in RMG to block C9 avoidance behaviors initiated by ADL.

To determine whether NPR-1 acts during development to affect connectivity, or in adults to regulate circuit function, we asked whether expression of NPR-1 during the adult stage could rescue C9 avoidance in npr-1 animals. An npr-1 cDNA expressed under a heat-shock promoter (Coates and de Bono, 2002) restored C9 avoidance fully after 30 minutes of heat shock (Figure 2C), suggesting that NPR-1 acts acutely to regulate neuronal responses and circuit output.

The model that npr-1 in RMG antagonizes ADL chemical synapses predicts that increased ADL synaptic function might restore avoidance. To test this prediction, we used an ADL-specific promoter to drive pkc-1(gf), a constitutively active protein kinase C isoform that enhances neuronal synaptic outputs (Okochi et al., 2005; Sieburth et al., 2007; Tsunozaki et al., 2008; Macosko et al., 2009). Expression of pkc-1(gf) in ADL enhanced C9 avoidance in npr-1 animals (Figure 2D), and blocking ADL chemical synapses with TeTx eliminated C9 avoidance in the pkc-1(gf) strain (Figure 2D). Expression of pkc-1(gf) in ADL neurons of wild-type animals had little effect on C9 avoidance (Figure S2A). These results suggest that strengthening ADL chemical synapses can override the effect of the npr-1 mutation.

ADL Ca²⁺ transients were slightly but significantly reduced in amplitude in npr-1 as compared to wild type animals (Figures 2E, S2B). Two results suggest that this small change in amplitude is due to indirect effects of RMG on ADL. First, ADL Ca²⁺ responses were rescued by expressing npr-1 under a promoter that is expressed in RMG (as well as a few other neurons) but not in ADL (Figure S2B). Second, the effect of npr-1 on ADL Ca²⁺ responses was reversed in animals mutant for unc-9, which encodes a gap junction subunit that is broadly expressed in muscles and neurons (Liu et al., 2006; Starich et al., 2009) (Figure S2C). This observation suggests that gap junctions are required for NPR-1 to affect ADL, as predicted by the hub-and-spoke model. However, unc-9 has stronger effects on ADL Ca²⁺ responses than npr-1 (Figure S2C), and acts at multiple sites, so it may have either direct or indirect effects on ADL. In summary, npr-1 has a strong effect on C9 avoidance behavior that is mediated by RMG, and an indirect effect on ADL Ca²⁺ responses. Our results suggest that npr-1 functions primarily by changing activity of the RMG gap junction circuit relative to ADL chemical synapses, and not solely by changing ADL sensory properties.

Reduced ADL C9 responses and ADL-ASK antagonism abolish C9 avoidance in wild-type males

Unlike wild-type hermaphrodites, wild-type C. elegans males accumulate in low concentrations of C9, a behavior that requires the ASK neurons and the male-specific CEM sensory neurons (Srinivasan et al., 2008). In agreement with this result, we found that wild-type males did not avoid either 10 nM or 100 nM C9 in the drop test, although they exhibited robust avoidance of high-osmolarity glycerol (Figure 3A).

Open in a separate window

Figure 3

ASK antagonism of ADL decreases C9 avoidance in males.

A. Wild-type males do not avoid C9. Avoidance assays were performed in the presence of food. n=20–70 animals each.

B. (Left) Changes in intracellular fluorescence in GCaMP3-expressing ADL neurons of wild-type hermaphrodites and males upon addition of 100 nM C9. n≥12 neurons each. Shading around the lines represents SEM. (Right) Average peak percentage change in fluorescence upon C9 addition. ** indicates responses different from wild-type at P<0.01. Also see Figures S3A and S3B.

C. ASK and RMG antagonize ADL in wild-type males. Avoidance of C9 in the presence of food. * and ** indicate responses different from wild type at P<0.05 and P<0.01, respectively. n=20–120 animals each. Also see Figure S3C.

D. C9 responses in wild-type male ADL neurons are unaffected by ASK ablation. Ca²⁺ transients upon C9 addition (red horizontal bar) in ADL in wild-type and ASK-ablated animals. n≥10 neurons each. Error bars in all panels represent SEM.

This sexually dimorphic behavioral response to C9 was accompanied by sexually dimorphic Ca²⁺ responses in ADL neurons. C9-induced Ca²⁺ transients in male ADL neurons were delayed by several seconds and reduced in amplitude compared to responses in hermaphrodites (Figures 3B, Figure S3A). Because slow changes in neuronal responses are often associated with neuropeptide signaling, we investigated ADL C9 responses in egl-3 and egl-21 mutants, which lack processed neuropeptides (Kass et al., 2001; Jacob and Kaplan, 2003). Both males and hermaphrodites showed sex-appropriate ADL Ca²⁺ transients in neuropeptide mutant backgrounds (Figure S3A), suggesting that classical neuropeptide signaling is not essential for sexual dimorphism. Thus altered male behaviors are associated with decreased and delayed pheromone signaling by the ADL neurons, which might or might not be intrinsic to ADL.

We next probed the roles of other sexually dimorphic neurons in C9 avoidance. The male-specific CEM sensory neurons are required for male accumulation at low C9 concentrations (Srinivasan et al., 2008), but were not central to C9 avoidance: sex-appropriate behaviors to C9 were observed both in males lacking CEM neurons (ceh-30(lf)), and in hermaphrodites with ectopic CEM neurons (ceh-30(gf)) (Schwartz and Horvitz, 2007) (Figure S3B). The ASK neurons are pheromone-sensing neurons that participate in the RMG gap junction circuit (Macosko et al., 2009a) (Figure 1D), and these neurons are functionally dimorphic between males and hermaphrodites (Srinivasan et al., 2008; Srinivasan et al., 2012). Males whose ASK neurons were killed with a mouse caspase gene (Kim et al., 2009) exhibited significant avoidance of 100 nM C9, unlike wild-type males (Figure 3C). Ablation of ASK had little effect on wild-type hermaphrodite C9 avoidance (Figure S3C). Thus, ASK effectively antagonizes ADL-mediated C9 avoidance in wild-type males, but not in wild-type hermaphrodites. ASK ablation did not affect C9-induced Ca²⁺ transients in male ADL neurons (Figure 3D), suggesting that ASK acts at a circuit level to suppress C9 avoidance.

Reasoning by analogy to the npr-1 circuit, we asked whether synaptic output of the RMG gap junction circuit antagonizes C9 avoidance in males. Indeed, expression of TeTx in the RMG neurons led to robust C9 avoidance behavior in wild-type males (Figure 3C). Expression of pkc-1(gf) in ADL also led to C9 avoidance, indicating that a strongly activated ADL neuron can drive repulsion in males (Figure 3C), as it can in npr-1 hermaphrodites (Figure 2D). These results suggest that ADL has a latent ability to drive C9 avoidance in males, but this activity is inhibited by ASK and RMG.

Male sexual identity and npr-1 have additive effects on C9 responses

Both males and npr-1 hermaphrodites have decreased C9 avoidance (compare Figures 2A and ​and3A),3A), and males also resemble npr-1 hermaphrodites in their avoidance of high oxygen, their rapid movement on food, and their propensity to aggregate (Figures S4A, S4B). Despite this similarity, behavioral analysis of npr-1 males suggest that npr-1 mutations and male sex have independent effects on C9 responses. First, in npr-1 males C9 failed to induce reversals as it did in npr-1 hermaphrodites and wild-type males, but instead suppressed spontaneous reversals (Figure 4A). Based on the biased random walk model for C. elegans chemotaxis, the suppression of reversals suggests that npr-1 males are attracted to C9 (Pierce-Shimomura et al., 1999; Luo et al., 2008). The suppression of reversals was eliminated by each of the genetic manipulations that increased C9 repulsion in wild-type males: killing ASK with the caspase transgene, reducing RMG synaptic output with TeTx, or enhancing ADL output with pkc-1(gf) (Figure 4B). Like other effects of npr-1, the effect on males was rescued by npr-1 expression in RMG neurons (Figure 4B), and was rapidly reversed after acute expression of npr-1 in adults (Figure S4C).

Open in a separate window

Figure 4

Masculinization and NPR-1 exert additive effects on a common circuit.

A. Spontaneous reversals are suppressed in the presence of C9 in npr-1 males. Avoidance assays were performed in the presence of food. n=30–120 animals each. Responses to 10 nM and 1 μM C9 are different from those of wild-type males at P<0.01 (see Figure 3A). Also see Figure S4.

B. C9-induced suppression of spontaneous reversals in npr-1 males is eliminated by circuit manipulations that enhance C9 avoidance. ** and *** indicate responses different from npr-1 at P<0.01 and P<0.001, respectively. n=40–120 animals each.

C. (Left) Intracellular Ca²⁺ dynamics in G-CaMP-expressing ADL neurons of wild-type and npr-1(ad609) mutant males in response to 100 nM C9. Only animals with positive responses were included in the averages at left: n=14 (of 17) neurons for wild-type males and n=4 (of 11) neurons for npr-1 males. Shading around the lines represents SEM. (Right) Scatter plot showing peak percentage changes in fluorescence in individual ADL neurons of animals of the indicated genotypes in response to a pulse of 100 nM C9. Data are included from Figure 2E for comparison. Dotted lines indicate the median. The percentage of ADL neurons that failed to exhibit significant changes in fluorescence were 14% in wild-type hermaphrodites (n=14); 14% in npr-1 hermaphrodites (n=14); 12% in wild-type males (n=17) and 64% in npr-1 males (n=11).

D. ASK responses to C9 are enhanced in npr-1 mutants. (Left) Intracellular Ca²⁺ dynamics in GCaMP3-expressing ASK neurons (Macosko et al., 2009b) of wild-type and npr-1(ad609) mutant males and hermaphrodites in response to 100 nM C9 (red horizontal bars). Only animals with quantifiable changes were included in these averages; n≥6 neurons for each. Shading around the lines represents SEM. (Right) Scatter plot showing peak percentage changes in fluorescence in individual ADL neurons of animals of the indicated genotypes in response to a pulse of 100 nM C9. Dotted lines indicate the median.

E. Inferred activities of ADL, ASK, and RMG neurons in animals of different sex and npr-1 genotype. High NPR-1 activity inhibits RMG. C9 responses are high in ADL and low in ASK in wild-type hermaphrodites; ADL drives C9 avoidance via its chemical synapses. In males, ASK C9 responses remain low and ADL C9 responses are decreased due to sexual dimorphism. ASK and the RMG circuit antagonize ADL output to further reduce ADL-driven C9 avoidance. In npr-1 animals, ASK C9 responses are increased in both males and hermaphrodites, and ADL C9 responses are significantly decreased in npr-1 males. ASK and RMG antagonize ADL chemical synapses and decrease avoidance (hermaphrodites) or promote attraction (males).

F. Integration of pheromone blend information in npr-1 hermaphrodites requires ASK. Assays were performed in the presence of food. *** indicates responses different from values indicated by brackets at P<0.001. n=60–80 animals each. Error bars in all panels represent SEM.

Additive effects of npr-1 and male sex were also observed in Ca²⁺ imaging. The majority of ADL neurons in npr-1 mutant males failed to modulate Ca²⁺ after C9 addition (Figure 4C, right panel). This reduction in ADL Ca²⁺ responses far exceeded that of wild-type males or npr-1 hermaphrodites, even considering only the small subset of npr-1 males that did modulate ADL Ca²⁺ in response to C9 (Figure 4C, left panel).

The strong reduction in ADL Ca²⁺ transients might explain the loss of C9 avoidance in npr-1 males, but would not predict the appearance of the new behavior of C9 attraction (strictly speaking, reversal suppression). Therefore, we sought another sensory neuron that enhances C9 attraction in npr-1 males. ASK was a plausible candidate to drive C9 attraction based on the behavioral analysis (Figure 4B), so we asked if its pheromone sensitivity was altered by npr-1. Indeed, ASK neurons showed much stronger C9-evoked Ca²⁺ transients in npr-1 males than in wild-type males (Figure 4D). A similar enhancement of ASK responses was present in npr-1 hermaphrodites, whose C9 avoidance is also antagonized by ASK (Figures 4D, S3C).

Together, these results indicate that npr-1 males have enhanced ASK C9 responses and decreased ADL C9 responses compared to wild-type males, and that these changes drive attraction to C9 through RMG chemical synapses. Circuit changes driving sexually dimorphic and NPR-1-dependent C9 pheromone responses are summarized in Figure 4E.

Pheromone blends are integrated by the RMG circuit

The results described above suggest that antagonism between repulsive signaling from ADL chemical synapses and attractive signaling mediated by ASK and the RMG gap junction circuit determine whether C9 is repulsive, neutral, or attractive. We considered what this might mean for the pheromone-dependent behaviors of npr-1 hermaphrodites, which are weakly attracted to mixtures of ascarosides, including C9 and C3, but not to either C3 or C9 alone (Srinivasan et al., 2008; Macosko et al., 2009). By analogy with the detection of pheromone blends in other animals (Slessor et al., 1988; Kaissling, 1996; Matsuura et al., 2010), synergistic attraction to ascaroside blends could result from cooperation of multiple pheromone-sensing neurons. Hermaphrodite ASK neurons detect C3 at nanomolar concentrations (Kim et al., 2009), and ASK pheromone responses are stronger in npr-1 than in wild type hermaphrodites (Macosko et al., 2009). If antagonism between ASK and ADL applies to pheromone blends, C3 detection by ASK should suppress repulsion to C9 detected by ADL. To address this hypothesis, the drop test assay was used to detect behavioral interactions between pheromones.

In agreement with the observation that C3 is not highly attractive or repulsive on its own (Macosko et al., 2009), C3 did not induce or suppress reversals in wild-type or npr-1 hermaphrodites (Figure 4F). However, C3 did modify the response to C9 in npr-1 hermaphrodites, suppressing their avoidance of 100 nM C9 almost to baseline levels (Figure 4F). No suppression was observed in wild-type hermaphrodites, indicating that the interaction depends on npr-1 and the gap junction circuit. Genetic ablation of the ASK neurons in npr-1 hermaphrodites abolished the interaction between C3 and C9 (Figure 4F). These results support the model that ASK suppresses ADL-mediated avoidance, and additionally are consistent with a circuit that can evaluate pheromone blends, so that the combination of C3 detected by ASK and C9 detected by ADL is less repulsive than C9 alone.

Behavioral assays

The drop test was performed essentially as previously described (Hilliard et al., 2002). “Fraction reversing” represents (fraction of animals reversing in 4s with pheromone) – (fraction reversing in 4s to buffer).

We are grateful to Eugene Kim and Andrew Gordus for assistance with ADL imaging experiments and analysis, the Caenorhabditis Genetics Center for strains and Eve Marder for discussion. This work was supported by the NSF (IOS 0542372 – P.S., DMR-0820492 – D.K. (MRSEC program), the HFSP (RGY0042-P.S.), the NIH (core grant P30 NS45713 to the Brandeis Biology Department; F31 DC011467 – D.M.Z., R00 GM87533 – R.A.B.), the DGIST MIREBrain and Convergence Science Center and Basic Science Research Program (2012009385) of the Ministry of Education, Science and Technology (K.K.), the Natural Sciences and Engineering Research Council of Canada (PGS-D3) and by the Brandeis National Committee (S.J.N.), a gift from the Jensam Foundation (C.I.B.), and the Howard Hughes Medical Institute (C.I.B.). C.I.B. is an Investigator of the Howard Hughes Medical Institute.