Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Neuron. Author manuscript; available in PMC Mar 25, 2009.
Published in final edited form as:
Neuron. Sep 25, 2008; 59(6): 959–971.
doi:  10.1016/j.neuron.2008.07.038
PMCID: PMC2586605
HHMIMSID: HHMIMS74419

A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans

Summary

Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. Here we show that one olfactory neuron in Caenorhabditis elegans, AWCON, has the potential to direct both attraction and repulsion. Attraction, the typical AWCON behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWCON axons. gcy-28 mutants avoid AWCON–sensed odors; they have normal odor-evoked calcium responses in AWCON, but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWCON odor preferences. A behavioral switch in AWCON may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWCON. These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs.

Introduction

Animals of many species are born with innate odor and taste preferences that cause them to approach food-related odors and avoid toxic substances. Experiments in nematodes, fruit flies, and mice have demonstrated that innate preferences are encoded, at least in part, by dedicated sensory neurons. In each animal, certain sensory neurons are preferentially coupled to attraction or food acceptance, while others drive aversion (Marella et al., 2006; Mueller et al., 2005; Tobin et al., 2002; Troemel et al., 1997; Zhao et al., 2003). Downstream of sensory neurons, the anatomical or physiological pathways for behavioral preference are largely undefined. At one extreme, there could be a complete labeled-line segregation of sensory projections, as proposed in Drosophila, where sweet and bitter taste fibers project to different target regions in the brain (Thorne et al., 2004; Wang et al., 2004). The flexibility of animal behavior, however, suggests that even innate sensory pathways may be sensitive to modification.

Chemosensory preference can be examined in detail in the nematode C. elegans, whose compact nervous system with 302 neurons allows functions to be mapped to specific cell types. C. elegans is attracted to chemicals sensed by two pairs of olfactory neurons called AWC and AWA and a pair of gustatory neurons called ASE (reviewed in Bargmann, 2006). It avoids repulsive chemicals sensed by neurons called AWB, ASH and ADL. The sensory neurons detect environmental chemicals using G protein-coupled receptors (GPCRs) encoded by ~1700 chemoreceptor genes, as well as other receptors (Robertson and Thomas, 2006). Each sensory neuron expresses many chemoreceptor genes and detects many chemicals, an organization distinct from the one receptor-one neuron organization of the mammalian olfactory system. Sensory transduction downstream of C. elegans GPCRs is mediated either by cyclic guanosine monophosphate (cGMP) signaling through guanylate cyclases and cGMP-gated channels, or by signaling through transient receptor potential V (TRPV) channels, depending on the cell type (Bargmann, 2006).

In addition to the sensory properties defined by GPCRs, each sensory neuron is associated with a preferred behavioral output: attraction for AWC, AWA and ASE, and avoidance for AWB, ASH, and ADL. The strength of neuron-behavior coupling was demonstrated by expressing one receptor, the diacetyl-sensing GPCR ODR-10, in different sensory neurons (Troemel et al., 1997). Expression of ODR-10 in AWA or AWC neurons leads to attraction to diacetyl, whereas expression of ODR-10 in AWB leads to diacetyl avoidance (Sengupta et al., 1996; Troemel et al., 1997; Wes and Bargmann, 2001). The mechanisms by which AWA and AWC specify attraction and AWB specifies avoidance are not obvious, as these sensory neurons have largely overlapping synaptic targets (White et al., 1986).

Despite strong innate preferences, chemosensory behaviors of C. elegans can be altered by adaptation, sensitization, and associative learning. A striking change in behavior is caused by starving animals in the presence of NaCl, which is normally an attractive taste. Starvation/salt pairing for as little as ten minutes leads to salt avoidance, a reversal of normal behavior (Tomioka et al., 2006). In salt chemotaxis learning, an insulin signal from a downstream neuron activates an insulin receptor/PI 3-kinase signaling pathway within the ASER salt-sensing neuron to suppress its attractive activity (Tomioka et al., 2006). Mutation of the PI 3-kinase pathway eliminates salt chemotaxis learning, and constitutive activation by a mutation in the PTEN lipid phosphatase daf-18 suppresses salt attraction. The ASER sensory neuron has a normal NaCl-evoked sensory response in calcium imaging of PI 3-kinase/age-1 or PTEN/daf-18 mutants, indicating that the PI 3-kinase pathway acts downstream of ASER sensory transduction, probably at the level of synaptic modulation (Tomioka et al., 2006). While ASER accounts for the loss of attraction after conditioning, the cellular mechanism of avoidance is less clear. One possibility is that ASER itself can direct attraction or repulsion depending on prior sensory experience (Tomioka et al., 2006). Alternatively, other sensory neurons may drive avoidance of NaCl when ASER’s attractive role is silenced after conditioning. Several results support the second, multicellular model for avoidance. For example, if the ASH avoidance neurons are killed or inactivated, animals subjected to starvation/salt conditioning are no longer attracted to salt, but they do not avoid it (Hukema et al., 2006).

Here we examine a switch in olfactory preference in C. elegans and provide evidence that a single olfactory neuron can switch between attractive and avoidance behaviors, in violation of the pure labeled-line model. At a behavioral level, alternative chemosensory preferences are associated with reversed biases in turning frequency during odor chemotaxis. At a molecular level, two components of the behavioral switch are axonal cGMP signaling and a DAG/PKC signaling pathway that affects synaptic transmission. Specific sensory experiences alter the balance between attractive and repulsive behavioral states. These results suggest that axonal signaling pathways can rapidly respecify the connections between sensory neurons and behavioral outputs.

Results

gcy-28 mutations switch AWCON odor preference from attraction to avoidance

The two AWC sensory neurons sense partly overlapping groups of odors. The AWCON neuron senses butanone, the contralateral AWCOFF neuron senses 2,3-pentanedione, and both AWC neurons sense benzaldehyde and isoamyl alcohol (Troemel et al., 1999; Wes and Bargmann, 2001). A screen for mutants defective in butanone chemotaxis yielded a notable mutant, gcy-28(ky713), that avoided butanone instead of approaching it (Fig. 1A, see Experimental Procedures). gcy-28(ky713) mutants avoided butanone across a range of concentrations that were attractive to wild-type animals (Fig. S1A). They showed reduced attraction, but not repulsion, to other odors sensed by AWC neurons, and were proficient in chemotaxis to odors sensed by AWA neurons (Fig. 1A). A deletion mutation in the gcy-28 gene, gcy-28(tm2411), had behavioral defects similar to those of gcy-28(ky713).

Figure 1
AWCON mediates odor avoidance instead of attraction in gcy-28 mutants

The butanone avoidance defect in gcy-28 mutants could be due to altered activity of AWCON, or due to altered function of other cells such as avoidance neurons. To distinguish between these possibilities, we examined mutants with defects in known cell types. ceh-36 mutants lack AWC neurons due to a mutation in an Otx-type homeobox gene (Koga and Ohshima, 2004; Lanjuin et al., 2003). gcy-28; ceh-36 mutants were not repelled by butanone, suggesting that AWC causes butanone avoidance in gcy-28 mutants (Fig. 1B). Consistent with a defect in AWC, mutations in the AWC signal transduction genes odr-1 and tax-4, which encode a ciliary guanylate cylase and a cyclic nucleotide-gated channel, respectively, also suppressed butanone avoidance of gcy-28 mutants (Fig. S1B, data not shown). gcy-28 butanone avoidance was not affected by a lim-4 mutation that inactivates AWB avoidance neurons and ADF neurons, by an osm-9 mutation that inactivates ASH and ADL avoidance neurons, or by a transgene that kills URX, AQR, and PQR oxygen-sensing neurons (Fig. S1C, S1D, and data not shown).

A marker for AWCON, str-2::GFP, was appropriately expressed in one AWC per animal in gcy-28 mutants, suggesting that both AWCON and AWCOFF were specified correctly (data not shown). To examine AWCON and AWCOFF function independently, we used mutants that eliminated each cell type. nsy-5 mutants have no AWCON cells but instead have two AWCOFF cells (Fig. 1C) (Chuang et al., 2007), and nsy-5 gcy-28 double mutants, like nsy-5 mutants, were neither attracted to nor repelled by butanone (Fig. 1D). nsy-1 mutants have two AWCON neurons and no AWCOFF neurons (Fig. 1C) (Sagasti et al., 2001), and gcy-28; nsy-1 double mutants showed enhanced avoidance of butanone compared to the gcy-28 single mutants (Fig. 1E). These results suggest that AWCON is responsible for butanone avoidance in gcy-28 mutants. To ask whether the gcy-28 defect was butanone-specific or more general to AWCON, the double mutants were examined for benzaldehyde chemotaxis. gcy-28; nsy-1 double mutants with two AWCON neurons avoided benzaldehyde (Fig. 1E). This result suggests that the AWCON neuron is systematically switched to an avoidance function in gcy-28 mutants.

The AWCOFF neuron is specifically required for chemotaxis to 2,3-pentanedione. nsy-5, nsy-5 gcy-28 and gcy-28; nsy-1 mutants all showed diminished attraction to 2,3-pentanedione, but not avoidance (Fig. 1D and E, Fig. S1E). Therefore, AWCOFF function is reduced but not reversed by the gcy-28 mutation. Reduced, but normal activity of AWCOFF is also suggested by the partial recovery of benzaldehyde chemotaxis in nsy-5 gcy-28 mutants with two AWCOFF neurons (Fig. 1D), in contrast with the benzaldehyde avoidance of gcy-28; nsy-1 mutants (Fig. 1E). Benzaldehyde chemotaxis in gcy-28 single mutants was variable, as might be expected from an interaction of antagonistic signals from (repulsive) AWCON and (weakly attractive) AWCOFF neurons.

As a further test of AWCON function in gcy-28 mutants, we expressed the diacetyl receptor, ODR-10, in AWCON (Fig. 1F). In an odr-10 mutant, expression of odr-10 in AWCON restores attraction to diacetyl (Wes and Bargmann, 2001) (Fig. 1F). By contrast, in a gcy-28;odr-10 double mutant, odr-10 expression in AWCON causes animals to avoid diacetyl (Fig. 1F). Thus AWCON drives odor avoidance in gcy-28 mutants.

gcy-28 mutants reverse the turning bias in AWC-mediated chemotaxis

To better understand both normal chemotaxis and the behavioral basis of the olfactory defects in gcy-28 mutants, we analyzed olfactory chemotaxis using the framework of the biased random walk or “pirouette” model that was first developed for taste chemotaxis (Pierce-Shimomura et al., 1999). In the presence of attractive tastes, animals bias their turning frequency as a function of the concentration gradient, turning less when the concentration of an attractant is increasing and turning more when attractant concentration is decreasing (Pierce-Shimomura et al., 1999). This behavioral strategy results in eventual accumulation at an attractant source through a biased random walk, the mechanism of bacterial chemotaxis (Berg and Brown, 1972). Step changes in the concentration of the attractive odor isoamyl alcohol can regulate the turning rates of freely moving animals (Chalasani et al., 2007), or the turning of animals in liquid drops (Luo et al., 2008), but the turning response in odor gradients has not been examined.

Turning responses of C. elegans in odor gradients were observed by video microscopy of animals placed between a spot of odor and a spot of diluent (Fig. 2A and B). The frequency of turns was measured and classified as a function of the angle θ, where θ is the difference between the average direction of the animal’s motion in a five-second window before the turn and the direction of the odor source (Fig. 2C). This angle approximates the concentration change the animal had been experiencing, with θ < 90° corresponding to increasing odor and θ > 90° corresponding to decreasing odor. Wild-type animals showed net movement toward the odor source (Fig. 2D1), increased their turning frequency when heading down the odor gradient, and reduced their turning frequency when heading up the gradient (Fig. 2D2, Fig. S2A). In the absence of odor, animals showed neither net migration nor turning bias (Fig. 2E1 and E2). Wild-type animals had a similar turning bias in gradients of several attractive odors sensed by AWC neurons (Fig. 2G-I), and an opposite turning bias to the repulsive odor 2-nonanone, which is sensed by AWB neurons (Fig. S2C and D). These turning behaviors in odor gradients are consistent with a biased random walk model for positive and negative chemotaxis.

Figure 2
Regulated turning during chemotaxis to odors

A similar analysis with gcy-28 mutants revealed significant alterations of their turning bias. gcy-28 mutants showed net movement away from a butanone source (Fig. 2F1), increased their turning frequency when heading up the odor gradient, and reduced their turning frequency when heading down the gradient (Fig. 2F2, Fig. S2B). In gradients of 2,3-pentanedione or benzaldehyde, gcy-28 mutants showed little bias in turning frequency with respect to the odor gradient (Fig. 2H and I). Thus the regulated turning response of gcy-28 mutants to different odors matched their accumulation in chemotaxis assays, with a reversed bias to butanone and no bias to benzaldehyde and 2,3-pentanedione.

To determine whether the turning bias was generated by AWCON, we used a laser microbeam to kill the AWCON neuron in wild-type animals and gcy-28 mutants. Killing AWCON in wild-type animals abolished the net migration toward butanone (Fig. 3A)(Wes and Bargmann, 2001), and AWCON-ablated animals in a butanone gradient showed no turning bias going up or down the gradient (Fig. 3B). Killing AWCON in gcy-28 animals abolished butanone avoidance (Fig. 3C) and eliminated the butanone-evoked turning bias (Fig. 3D). Overall rates of turning were unchanged after killing AWCON; only odor regulation was altered. These results indicate that AWCON is necessary for butanone avoidance in gcy-28 mutants.

Figure 3
Ablation of AWCON abolishes turning bias in wild type and gcy-28 animals

gcy-28 encodes a receptor-type guanylate cyclase

gcy-28(ky713) was mapped to the left arm of chromosome I using standard methods, and determined to affect the predicted gene T01A4.1 (gcy-28) (Fig 4A, Supplementary Material). gcy-28 encodes several related receptor-type guanylate cyclases (rGCs), single-spanning transmembrane proteins with an extracellular ligand-binding domain and intracellular kinase-like and guanylate cyclase domains (Fig. 4B). Consistent with its sequence, the cyclase domain of gcy-28 has guanylate cyclase activity when expressed in heterologous cells (Baude et al., 1997). Three splice forms of gcy-28 were predicted from the genome sequence (T01A4.1a-c). We confirmed two of three forms by isolation of cDNAs (gcy-28.a and gcy-28.c, corresponding to T01A4.1a and T01A4.1c, respectively), and identified a fourth splice form, T01A4.1d (gcy-28.d), which fuses the upstream predicted gene T01A4.2 to T01A4.1 (Fig. 4A). The three confirmed gcy-28 isoforms share predicted intracellular domains, but have alternative exons encoding the N-terminal extracellular domain. The gcy-28(ky713) mutation is a C to G transversion predicted to substitute a conserved phenylalanine for a leucine in the cyclase domain (Fig. 4C).

Figure 4
Sequence analysis of gcy-28

We subsequently obtained gcy-28(tm2411) (Shohei Mitani, National Bioresource Project, Japan), a deletion allele that results in a predicted frameshift upstream of the cyclase domain (Fig. 4A and B). The similar phenotypes of gcy-28(ky713) and gcy-28(tm2411) (Fig. 1A) suggest that strong loss-of-function mutations in gcy-28 cause AWC chemotaxis defects.

The genome sequence of C. elegans predicts 27 genes for rGCs and seven soluble GCs (Morton, 2004; Ortiz et al., 2006). Many of the nematode cyclases result from unique expansions within the nematode clade, but some are shared by nematodes and other invertebrates (Fitzpatrick et al., 2006). gcy-28 belongs to the natriuretic peptide receptor family of rGCs shared by both vertebrates and invertebrates (Fig. 4D). In mammals, natriuretic peptide signaling is important for proper regulation of blood pressure, cardiac myocyte growth, and bone development (Lopez et al., 1995; Oliver et al., 1997; Tamura et al., 2004; Tsuji and Kunieda, 2005). Although they are expressed in neurons in vertebrates, the role of natriuretic peptide receptors in the nervous system is largely unexplored.

GCY-28 can act in AWC to mediate chemotaxis

A reporter transgene driven by regions upstream of the gcy-28.c isoform has widespread expression in neurons and other tissues (Ortiz et al., 2006). We confirmed these results with additional reporters and found expression in almost all neurons, including AWC, as well as body wall muscle, gut, and hypodermis. The gcy-28.a upstream region was expressed in many, but not all, neurons, and the gcy-28.d upstream region was expressed in some neurons and in the gut.

A gcy-28.c cDNA expressed from its own promoter rescued the chemotaxis defects of gcy-28 mutants, but failed to rescue when it included the ky713 mutation (Fig. 5A). Heat-shock expression of gcy-28.c in adults resulted in behavioral rescue within two hours, indicating that the gene acts in mature neurons and not during nervous system development (Fig. 5B). The gcy-28.c isoform had high rescuing activity when expressed broadly (Fig. 5A-C) but incomplete rescuing activity when expressed only in AWC (Fig. 5D). By contrast, a gcy-28.d cDNA rescued chemotaxis when expressed under a pan-neuronal promoter (Fig. 5C), under the AWC-selective odr-3 promoter (Fig. 5D), or under the AWCON-selective str-2 promoter (Fig. 5E). The results with gcy-28.c suggest that gcy-28 may have roles in multiple neurons; nevertheless, the full rescue with gcy-28.d suggests that gcy-28 can function cell-autonomously in AWCON.

Figure 5
Spatial and temporal sites of GCY-28 action

GFP-tagged isoforms of GCY-28.c and GCY-28.d had different subcellular localization in AWC. GCY-28.d, the more active isoform, was enriched in the axonal process, whereas GCY-28.c was present at equivalent levels in dendrites and axons (Fig. 5F). Both isoforms were present in the cell body and excluded from sensory cilia, in contrast with the cilia-localized rGCs ODR-1 and DAF-11, which function in sensory transduction (Birnby et al., 2000; L’Etoile and Bargmann, 2000). The axonal localization of GCY-28 suggests that it does not mediate primary sensory transduction in AWC.

To ask directly whether sensory transduction was affected in gcy-28 mutants, we measured odor-evoked calcium transients in AWCON neurons expressing the genetically encoded calcium indicator G-CaMP. In both wild-type animals and gcy-28 mutants, removing animals from butanone resulted in a strong increase in AWCON calcium levels, reported as an increase in G-CaMP fluorescence (Fig. 5G). These results are consistent with prior studies that suggest a hyperpolarizing, or inhibitory, mode of sensory transduction in AWC (Chalasani et al., 2007). Wild type and gcy-28 showed similar AWCON responses across a range of butanone concentrations, and similar responses to other AWC-sensed odors (Fig. S3). In both wild-type and gcy-28 mutant animals, calcium levels in AWCON were unchanged or decreased upon odor addition (Fig. 5G, Fig. S3). Thus gcy-28 is not essential for the first steps of sensory transduction in AWCON.

gcy-28 interacts with axonal diacylglycerol and protein kinase C signaling

To establish a potential mechanism for cGMP effects on behavioral preference, we searched for mutations that resembled or suppressed gcy-28. A major effector of mammalian natriuretic peptide receptors is the cGMP-dependent kinase (PKG) (Potter et al., 2006). In C. elegans a PKG mutant, egl-4, has AWC chemotaxis and adaptation defects, as well as defects in other sensory behaviors (Daniels et al., 2000; Fujiwara et al., 2002; L’Etoile et al., 2002). However, close examination suggested that egl-4 is not the primary target of cGMP from GCY-28: egl-4 loss-of-function mutants had chemotaxis defects that were qualitatively different from gcy-28 mutants, and the double mutant had defects different from either single mutant (Fig. S4A). Moreover, a gain-of-function mutation in egl-4 did not suppress gcy-28 butanone avoidance or other chemotaxis defects (Fig. S4B).

The presence of GCY-28 protein in axons suggested a possible role in regulation of synaptic function. To investigate this possibility, we examined mutations that alter synaptic transmission. dgk-1 encodes a diacylglycerol (DAG) kinase that hydrolyzes DAG to phosphatidic acid (PA), and loss-of-function mutations in dgk-1 increase DAG signaling and enhance cholinergic neurotransmission at the C. elegans neuromuscular junction (Lackner et al., 1999; Nurrish et al., 1999). A mutation in dgk-1 strongly suppressed gcy-28 chemotaxis defects (Fig. 6A), and this suppression was partially rescued by expressing a dgk-1 cDNA in AWC (Fig. 6A). To ask whether this effect was due to increased DAG signaling or decreased PA signaling, we used a pharmacological agonist of DAG signaling, the synthetic β-phorbol ester phorbol 12-myristate 13-acetate (PMA) (Colon-Gonzalez and Kazanietz, 2006). Treating gcy-28 mutants with PMA for two hours rescued their chemotaxis defects (Fig. 6B). Thus increased DAG signaling suppresses gcy-28, perhaps by affecting synaptic release.

Figure 6
gcy-28 and DAG/PKC signaling interact to transform behavior

One of the targets of DAG signaling is protein kinase C (PKC); previous work has shown that AWC chemotaxis requires the function of the C. elegans PKC pkc-1/ttx-4 in AWC (Okochi et al., 2005). We found that the chemotaxis defect in pkc-1 is similar to that of gcy-28 (Fig. 6C): pkc-1 mutants avoided butanone, while showing little preference for benzaldehyde. Functional Ca2+ imaging of AWCON cells in pkc-1 mutants revealed normal butanone responses, suggesting that the pkc-1 defect, like the gcy-28 defect, is not in primary sensory transduction (Fig S5). gcy-28; pkc-1 double mutants avoided butanone as much as pkc-1 single mutants, suggesting that these two genes have related functions in AWC chemotaxis (Fig. 6C).

To further explore the similarity between gcy-28 and pkc-1, we asked whether activation of PKC-1 in AWC might rescue gcy-28 mutants. PKCs can be made constitutively active by an A160E mutation in the autoinhibitory pseudosubstrate motif (Dekker et al., 1993). Expression of PKC-1(A160E) in AWC fully rescued the chemotaxis defects of gcy-28 mutants (Fig. 6D). Thus synaptic DAG signaling through PKC-1 may act downstream of or in parallel to GCY-28 to regulate the behavioral preference encoded by AWCON.

AWC sensory neurons synapse onto three classes of interneurons called AIB, AIY, and AIA (White et al., 1986). To ask whether the predicted change in AWC synaptic function might be observed in target neurons, we monitored butanone-evoked calcium responses in the AIB interneurons using G-CaMP. In wild-type animals, AWC has an excitatory connection with AIB, so AIB, like AWC, is activated by odor removal and inhibited by odor addition (Chalasani et al., 2007). AIB had a small but significant increase in calcium after butanone removal in wild-type animals, and a weaker but similar increase in gcy-28 mutants (Fig. 6E). A difference between wild type and gcy-28 was observed upon butanone addition: wild-type AIB neurons were inhibited by butanone, but gcy-28 AIB neurons were not (Fig. 6E).

AWC olfactory preference changes with odor history

Certain odor conditioning protocols can lead to context-dependent, bidirectional modifications in AWC olfactory behavior. Odor conditioning in the absence of food causes adaptation, a reduction in chemotaxis (Colbert and Bargmann, 1995). Odor conditioning in the presence of food causes sensitization, or enhanced chemotaxis (Torayama et al., 2007). Previous studies have viewed these changes as increases or decreases in odor-specific AWC activity; we hypothesized that these context-dependent behaviors might, instead, affect the competing attractive and repulsive activities of AWC.

In the absence of food, conditioning wild-type animals with butanone for up to 90 minutes causes a butanone-specific reduction in chemotaxis (Colbert and Bargmann, 1995). We found that extending the conditioning to two hours led to butanone avoidance by wild-type animals (Fig. 7A). Long-term butanone conditioning also reduced benzaldehyde chemotaxis in wild-type animals, but did not affect chemotaxis to the AWCOFF odor 2,3-pentanedione, suggesting a broader reduction of AWCON activity (Fig. 7A). The AWCON behaviors of butanone-adapted wild-type animals thus resembled the AWCON behaviors of naïve gcy-28 mutants. In a gcy-28 mutant, butanone adaptation did not cause stronger avoidance of butanone or reduced attraction to benzaldehyde, but instead reduced butanone avoidance (Fig. 7A; see also Fig. S7). These results suggest that gcy-28 mutants occupy a behavioral state that wild-type animals enter after butanone adaptation.

Figure 7
Olfactory plasticity is altered in gcy-28 mutants

Wild-type animals conditioned with butanone and food increase their chemotaxis to butanone (Fig. 7B). The sensitization response was intact in gcy-28 mutants; in fact, conditioned gcy-28 mutants were actually rescued, with levels of chemotaxis comparable to sensitized wild-type animals (Fig. 7B; see also Fig. S7). To determine if sensitization extended to all AWCON odors, animals were first conditioned with butanone and food, then tested for chemotaxis to benzaldehyde. Wild-type animals showed a small but consistent enhancement of benzaldehyde chemotaxis, and gcy-28 animals showed striking rescue of their benzaldehyde chemotaxis defect (Fig. 7B). Butanone-induced sensitization did not affect 2,3-pentanedione chemotaxis (Fig. 7B). The pattern of odor responses suggests that butanone-induced sensitization caused a systematic enhancement of AWCON-mediated chemotaxis in both wild-type and gcy-28 animals. The adaptation and sensitization experiments show that specific olfactory experiences can either mimic the gcy-28 mutation, or rescue it.

Its widespread expression in the nervous system suggests that GCY-28 may have a role in other C. elegans behaviors. Indeed, gcy-28 mutants had subtle defects in salt chemotaxis learning, and they also had strong defects in the regulation of thermotaxis by starvation (Fig. S6) (Mohri et al., 2005; Tomioka et al., 2006). Although neither of these changes is as dramatic as the effect on AWC, they suggest that gcy-28 signaling regulates multiple forms of sensory plasticity in C. elegans.

Discussion

A single-neuron switch between attraction and repulsion

Mutations in the receptor-type guanylate cyclase gcy-28 lead to an avoidance behavior in place of the attractive behavior normally directed by the AWCON neuron. The behavioral defect in gcy-28 mutants can be rapidly rescued by heat shock expression of gcy-28, by treatment with the DAG analog PMA, or, most strikingly, by conditioning animals briefly with butanone and food. These results suggest that AWCON neurons can switch rapidly between attractive and repulsive signaling modes.

A switch between attractive and repulsive signaling may contribute to wild-type behavior after odor adaptation or sensitization (Fig. 7C). The behavior of butanone-adapted animals resembles that of gcy-28 mutants, and an analogy between the adapted state and the gcy-28 mutant is suggested by the analysis of DAG signaling: genetic or pharmacological activation of DAG signaling in AWC neurons prevents adaptation in wild-type animals (Matsuki et al., 2006), and reverses the behavioral defects in gcy-28 mutants. As a potential receptor, GCY-28 could transmit an internal signal about food or another cue to AWC. We suggest that the repulsive drive after adaptation is associated with low gcy-28 activity and low DAG signaling, and the attractive drive from normal or sensitized AWC neurons is associated with high gcy-28 activity, DAG signaling, and active pkc-1.

Many aspects of AWCON olfactory neurons revealed by this and other studies parallel those of the ASER gustatory neurons. Based on calcium imaging, both AWCON and ASER are OFF-sensing cells whose activity rises after decreases in attractive odors or tastes (Chalasani et al., 2007; Suzuki et al., 2008). In their normal attractive modes, both AWCON and ASER promote turning behavior when active. Finally, both AWCON and ASER are subject to plasticity that can reverse the polarity of sensory behavior (Hukema et al., 2006; Tomioka et al., 2006). In each case, the behavioral change is observed despite a normal or near-normal sensory OFF-response. Moreover, both switches involve molecules that regulate lipid signaling and possibly synaptic function -- dgk-1and pkc-1 in AWCON, and Gqα/egl-30, PI 3-kinase/age-1, and PTEN/daf-18 in ASER (Tomioka et al., 2006).

For ASER, the behavioral switch after salt conditioning is regulated by a secreted insulin signal made by AIA interneurons (Tomioka et al., 2006). Several other neurons also contribute to salt plasticity, suggesting that conditioning affects multiple cell types. Although plasticity of the ASER neurons can fully explain the loss of attraction after conditioning, the switch to repulsion requires ASH avoidance neurons, ADF neurons, and URX, AQR, and PQR oxygen-avoidance neurons (Hukema et al., 2006). It is possible that ASER switches to an avoidance function, but it is not clear whether it does so. By contrast, our results show that repulsion directed by AWCON does not require any of the known avoidance neurons (AWB, ASH, ADL, URX, AQR, PQR) or ADF. Instead, AWCON appears to reverse its behavioral strategy directly, by changing its regulation of turning behavior during chemotaxis.

The gustatory ASE neurons and olfactory AWC neurons each have bilaterally asymmetric gene expression, and in each case the left-right pairs detect overlapping, but distinct, chemicals (Hobert et al., 2002). However, the functional asymmetry of these neurons is manifested differently in behavior. ASEL and ASER have reciprocal sensory and behavioral properties: unlike ASER, ASEL is an ON-sensing cell whose activity rises when attractive tastes appear, and ASEL inhibits turning when active (Suzuki et al., 2008). AWCON and AWCOFF are more similar; they are both OFF-sensing cells that stimulate turning when active (Chalasani et al., 2007). The AWCs differ, however, in their apparent sensitivity to the gcy-28 and pkc-1 signaling pathways, which reverse odor preference in AWCON but not in AWCOFF. One interesting possibility suggested by the behavioral analysis of gcy-28 turning behavior is that gcy-28 switches AWCON between an ASER-like behavioral output that stimulates turning, and an ASEL-like behavioral output that inhibits turning.

Modulation of synaptic transmission may modify sensory preference

The similar chemotaxis phenotypes of gcy-28 and pkc-1 mutants and the genetic interactions between gcy-28 and the DAG/PKC pathway suggest that these two signaling pathways regulate a common process in AWCON. The most likely possibilities are excitability or synaptic release. The most active GCY-28 isoform resides in axons, and while subject to the potential artifacts of GFP-tagged proteins, its localization suggests an interaction with the synaptic machinery. Synaptic transmission in vertebrate neurons can be regulated by cGMP-regulated protein kinases, cGMP-gated channels, and cGMP-regulated phosphodiesterases (Herring et al., 2001; Murphy and Isaacson, 2003; Richard and Bourque, 1996; Savchenko et al., 1997; Yu et al., 2006). DAG and phorbol esters modulate synaptic transmission by activating PKCs and Munc13/UNC-13 (Betz et al., 1998; Lackner et al., 1999; Shapira et al., 1987); in C. elegans, genetic or pharmacological manipulations that increase DAG enhance synaptic transmission, and PKC-1 regulates neuropeptide release (Nurrish et al., 1999; Lackner et al., 1999; Sieburth et al., 2007). The DAG/PKC synaptic signaling pathway is implicated in thermal behavioral plasticity, where DAG signaling affects the synaptic output of AFD thermosensory neurons (Okochi et al., 2005; Biron et al., 2006).

The altered butanone-evoked calcium signals in AIB are consistent with a change in synaptic function in gcy-28 mutants. It is interesting that the defect appears mainly upon odor addition, which normally suppresses AIB activity, because it suggests that suppression and activation of AIB could each convey unique information. Electrophysiology in Ascaris has demonstrated that nematode motor neurons have graded neurotransmission; they release tonic transmitter at rest, increase release upon depolarization, and decrease it upon hyperpolarization (Davis and Stretton, 1989). If AWCON has similar synaptic properties, the altered AIB response in gcy-28 could result from changes in tonic AWCON transmission, changes in hyperpolarization suppression of release, or an altered resting potential. These results should be interpreted with caution; the effects of gcy-28 on AIB are subtle, and the cellular site of action has not been determined, so they could be direct or indirect.

There are several mechanisms by which gcy-28 could alter turning behavior. AWC neurons use glutamate as a transmitter and also express neuropeptides, either of which could be regulated by gcy-28 (Chalasani et al., 2007; Nathoo et al., 2001). gcy-28 could redistribute the weights of AWC connections with its functionally distinct synaptic partners, AIA, AIB, and AIY (White et al., 1986; Chalasani et al., 2007). gcy-28 could also alter the quality of AWC connection with individual synaptic partners: AWC normally activates the AIB target neuron and inhibits the AIY target neuron, but can activate AIY in certain receptor mutants (Chalasani et al., 2007). The biased random walk strategy for chemotaxis is intrinsically probabilistic and variable. Through relatively simple changes in synaptic weights or dynamics, the probabilistic circuit may have more flexibility to change its properties than deterministic circuits with fixed behavioral outputs.

GCY-28 is the sole C. elegans member of the natriuretic peptide receptor family, corresponding to vertebrate guanylate cyclase A (GC-A) and guanylate cyclase B (GC-B)(Potter et al., 2006). The natriuretic peptides do not have obvious homologs in the C. elegans genome; if there is a peptide ligand for GCY-28, it may have a distinct structure. Both GC-A and GC-B are expressed in the vertebrate brain, and in the rat olfactory bulb (Gutkowska et al., 1997; Potter et al., 2006); it will be interesting to ask whether they have a role in olfactory behavior.

Context and experience modify sensory preferences in all animals. In C. elegans, we find that modulatory pathways acting in single sensory neurons can transform behaviors, allowing behavioral changes to emerge from changes in peripheral neurons. C. elegans has a small nervous system, but more complex animals also have anatomical pathways that support peripheral modulation. The mammalian olfactory bulb receives central innervation from adrenergic, serotonergic, and cholinergic fibers that could act as early as the presynaptic terminal of olfactory sensory neurons (Gomez et al., 2005). Our results suggest that modulatory information at this first synapse might generate qualitative differences in olfactory signaling, not just increases or decreases in sensory activity. Rapid behavioral switching provides a new view of the functional plasticity that can be generated within a fixed neuroanatomy.

Experimental Procedures

Standard techniques were used for nematode culture and molecular biology. A complete strain list, description of gcy-28 cloning, detailed molecular biology, and sequence analysis methods are in Supplementary Material.

Behavioral assays

Population chemotaxis assays were performed on assay agar (1.6 % agar, 1 mM MgSO4, 1 mM MgCl2, 5 mM phosphate buffer, pH 6.0) in 10 cm square plates, which are better for detecting avoidance behavior than round assay plates (Troemel et al., 1997). Unless otherwise indicated, odor dilutions in ethanol were 2-butanone 1:1,000; 2,3-pentanedione 1:10,000; benzaldehyde 1:200; diacetyl 1:1,000; 2-methylpyrazine 1:1,000; 2-nonanone 1:10. 2 μl of diluted odor was placed on one side of the plate, and 2 μl of ethanol at the other side, with azide to anaesthetize animals that reached odor or ethanol sources. Washed adult animals were placed in the center of the plate, and the distribution of animals counted after 1-2 hours. A score of 1.0 represents perfect attraction, -1.0 perfect repulsion, and 0 random behavior. All data points are averages of ≥ 4 assays, with ≥ 50 animals each, repeated on at least two different days.

For tracking assays (Fig. 2, ,3),3), about 50 animals were picked onto an NGM plate without bacteria, washed with S-basal and assay buffer, and placed on round 10 cm plates with assay agar. Excess liquid was wicked off, and recording was started after a spot of odor and diluent were placed on the agar surface. Unlike standard chemotaxis assays, no sodium azide was added. Tracking assays were performed under a dissecting scope (Stemi 2000; Zeiss, Thornwood, NY) with a custom-modified base that illuminates a wide field (Serco Technical Services, Livermore, CA). The field was captured by a digital camera (MacroFire; Optronics, Goleta, CA) with 1240×1240 pixel resolution at 2 frames per second. Captured movies were analyzed using MATLAB (MathWorks, Natick, MA) scripts (Ramot et al., 2008; http://wormsense.stanford.edu/). Tracks were segmented into turns and runs, essentially as described (Pierce-Shimomura et al., 1999). Runs were analyzed in 5-second bins, and an average bearing relative to the odor was obtained for each bin. Absolute angles were binned in 30° intervals.

PMA treatment was performed as described (Okochi et al., 2005). Briefly, adult animals were washed onto culture plates with 1 μg/ml PMA (PMA+) or DMSO solvent (PMA-) in the agar, incubated at room temperature for two hours, and then tested for chemotaxis in drug-free assay plates.

Heat shock treatment was performed as described (L’Etoile et al., 2002). Animals were incubated at 33°C for two hours, and then incubated for another two hours at 20°C before testing for chemotaxis.

Butanone sensitization assays were performed as described, with some modifications (Torayama et al., 2007). Animals on their NGM growth plate were exposed to butanone vapor by spotting 12 μL of butanone on agar plugs on the plate lid and sealing the plate with parafilm. After 90 minutes animals were washed and tested for chemotaxis on square plates. The butanone dilution used for chemotaxis was 1:1000. Adaptation assays were performed essentially as described (Colbert and Bargmann, 1995). Animals were washed and placed on 3% assay agar plates. 20 μL butanone was placed on agar plugs on the plate lid and the plates were sealed with parafilm. After two hours animals were washed off and tested for chemotaxis on square plates. Controls were treated identically except that butanone was omitted from the conditioning plate.

Laser ablations were performed on L1 animals as described (Bargmann and Avery, 1995). The AWCON cell was identified by expression of str-2::GFP in the integrated strain CX6343. About 20 animals were ablated for each tracking assay, and some animals were tested twice on two different days.

Calcium imaging

Calcium imaging was performed as described (Chalasani et al., 2007; Chronis et al., 2007). For AWCON imaging, the strain CX10281 expresses the calcium indicator G-CaMP2.0 (Tallini et al., 2006) in AWCON under the str-2 promoter. gcy-28(tm2411) and pkc-1(nj1) were crossed into CX10281 to generate the strains CX10223 and CX10784, respectively. For AIB imaging, the strain CX7469 expressing G-CaMP1.0 in AIB neurons (Chalasani et al., 2007) was crossed with gcy-28(tm2411) to generate the strain CX8994. Animals were washed in buffer without food for ~20 minutes prior to imaging, a protocol designed to mimic the washes before chemotaxis assays. This brief washing step enhances the reliability of chemotaxis and of AWC calcium imaging. Imaging was conducted in a polydimethylsiloxane (PDMS) chamber in which an animal’s nose was exposed to a stream of buffer that could be switched between odor-containing and odor-free solutions using an electronically gated valve. The standard stimulus protocol consisted of a 5-minute step pulse of the indicated dilution of odor in S-basal (without cholesterol) followed by odor removal. G-CaMP fluorescence intensity was measured for 10 seconds before and 50 seconds after the onset or offset of the odor stimulus; the same animals were imaged for odor onset and offset. All G-CaMP strains had the appropriate olfactory behaviors for their respective genetic backgrounds.

Supplementary Material

01

Acknowledgments

We thank the late David Garbers, Norio Suzuki, Isao Katsura, Loren Looger, the National Bioresource Project, and the Caenorhabditis Genetic Center (CGC) for strains and reagents; Daniel Ramot, Miriam Goodman, Jennifer Garrison, Navin Pokala, Carl Procko, and Shai Shaham for methods and advice; and members of the Bargmann lab for comments and discussions. MT designed and conducted genetic and behavioral experiments; MT and CHS conducted imaging experiments; MT and CIB interpreted results and wrote the paper. This work was funded by NIDCD and by the G. Harold and Leila Y. Mathers Charitable Foundation. CIB is an Investigator of the Howard Hughes Medical Institute.

Footnotes

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