• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neuron. Author manuscript; available in PMC Jan 4, 2008.
Published in final edited form as:
PMCID: PMC1855255
NIHMSID: NIHMS16253

C. elegans G Protein Regulator RGS-3 Controls Sensitivity to Sensory Stimuli

Summary

Signal transduction through heterotrimeric G proteins is critical for sensory response across species. Regulator of G protein signaling (RGS) proteins are negative regulators of signal transduction. Herein we describe a role for C. elegans RGS-3 in the regulation of sensory behaviors. rgs-3 mutant animals fail to respond to intense sensory stimuli, but respond normally to low concentrations of specific odorants. We find that loss of RGS-3 leads to aberrantly increased G protein-coupled calcium signaling, but decreased synaptic output, ultimately leading to behavioral defects. Thus, rgs-3 responses are restored by decreasing G protein-coupled signal transduction either genetically or by exogenous dopamine, by expressing a calcium binding protein to buffer calcium levels in sensory neurons, or by enhancing glutamatergic synaptic transmission from sensory neurons. Therefore, while RGS proteins generally act to down-regulate signaling, loss of a specific RGS protein in sensory neurons can lead to defective responses to external stimuli.

Keywords: chemosensation, olfaction, G protein, RGS, C. elegans, signal transduction, modulation, dopamine, neurotransmitter, sensory, behavior

Introduction

The nematode Caenorhabditis elegans detects environmental stimuli with specialized sensory neurons. In general, appropriate behavioral responses are generated when signals are relayed from the sensory neurons to downstream interneurons and motor neurons (White et al., 1986). C. elegans move towards odorants that indicate favorable conditions such as a food source (chemotaxis). Two pairs of head sensory neurons, AWA and AWC, detect volatile attractants including diacetyl (AWA) and isoamyl alcohol (AWC) (Bargmann et al., 1993). Several sensory neurons detect harmful stimuli that C. elegans avoids by initiating backwards locomotion. The well-characterized polymodal ASH sensory neurons detect a wide range of aversive stimuli, including high osmolarity, volatile odorants (e.g. octanol), soluble chemicals (e.g. quinine) and the mechanical stimulus of light touch to the nose (Bargmann et al., 1990; Kaplan and Horvitz, 1993; Troemel et al., 1995; Hilliard et al., 2004).

Like other metazoans, C. elegans use conserved G protein-coupled signal transduction pathways to mediate chemosensory responses to olfactory and gustatory stimuli (Prasad and Reed, 1999; Troemel, 1999). Signaling is initiated when a ligand binds to a seven-transmembrane G protein-coupled receptor (GPCR), inducing a conformational change that activates the associated heterotrimeric G proteins. Gα exchanges GDP for GTP and the active Gα-GTP and Gβγ subunits dissociate. Each can activate distinct signaling effectors to stimulate “second messenger” (e.g. cAMP) generating systems that ultimately mediate the cellular response to the bound ligand (Neer, 1995). Approximately 500 chemosensory GPCRs are encoded by the C. elegans genome (Troemel, 1999). Interestingly, 14 of the 21 C. elegans Gαs are expressed almost exclusively in a subset of sensory neurons (Zwaal et al., 1997; Roayaie et al., 1998; Jansen et al., 1999; Cuppen et al., 2003; Lans et al., 2004). While the AWC neurons utilize three stimulatory Gα proteins, ODR-3, GPA-3 and GPA-13, simultaneous loss of both ODR-3 and GPA-3 eliminates specific stimulus-evoked calcium transients in the ASH neurons (Zwaal et al., 1997; Roayaie et al., 1998; Jansen et al., 1999; Hilliard et al., 2004; Lans et al., 2004; Hilliard et al., 2005). It is not yet clear how signaling specificity is achieved and there is some functional redundancy between Gα proteins.

Although the AWC, AWA and ASH neurons all utilize G protein-coupled signaling pathways to mediate chemosensation, the second messengers and signaling components downstream of the G proteins differ (Coburn and Bargmann, 1996; Komatsu et al., 1996; Colbert et al., 1997; Birnby et al., 2000; L'Etoile and Bargmann, 2000; Tobin et al., 2002; Kahn-Kirby et al., 2004). Despite differences in signal transduction cascades, several lines of evidence suggest that the sensory neurons release glutamate in response to sensory stimuli. The ASH sensory neurons express the vesicular glutamate transporter EAT-4 (Lee et al., 1999; Bellocchio et al., 2000; Takamori et al., 2000) and synapse onto interneurons that express glutamate receptors (Hart et al., 1995; Maricq et al., 1995; Brockie et al., 2001). Mutations in eat-4 or glutamate receptors disrupt response to specific sensory stimuli. Specifically, EAT-4 is required for chemotaxis and all ASH-mediated avoidance behaviors (Berger et al., 1998; Hart et al., 1999; Nuttley et al., 2001; Hilliard et al., 2004). The glutamate gated ion channel GLR-1 is required for response to nose touch (Hart et al., 1995; Maricq et al., 1995), while two non-NMDA receptor subunits, GLR-1 and GLR-2, function together with the NMDA glutamate receptor subunit NMR-1 to mediate response to high osmolarity (Mellem et al., 2002). Finally, glutamate gated currents have been recorded in the AVA and AVD interneurons that are the direct synaptic targets of the ASH sensory neurons (Mellem et al., 2002).

Some C. elegans behaviors are modulated by feeding status and by the biogenic amines serotonin (5-HT) and dopamine (DA). The application of exogenous 5-HT mimics the effects of food for several behaviors (Horvitz et al., 1982; Avery and Horvitz, 1990; Ségalat et al., 1995; Sawin et al., 2000), suggesting that food increases endogenous 5-HT. Feeding status and 5-HT also modulate C. elegans sensory responses. Both enhance the ability of animals to respond to octanol and nose touch (Chao et al., 2004). 5-HT acts directly on the ASH sensory neurons (Chao et al., 2004; Hilliard et al., 2005) to increase stimulus-evoked calcium signaling (Hilliard et al., 2005). DA affects many of the same behaviors as 5-HT, but has distinct effects (Horvitz et al., 1982; Schafer and Kenyon, 1995; Ségalat et al., 1995; Weinshenker et al., 1995; Sawin et al., 2000; Chase et al., 2004). Although DA is involved in habituation to nonlocalized “tapping,” a mechanosensory stimulus (Sanyal et al., 2004), a role in C. elegans chemosensation has not been shown.

Signal transduction through G protein-coupled signaling pathways must be tightly controlled to allow animals to respond to changes in the environment. Regulator of G protein signaling (RGS) proteins bind to Gα subunits to stabilize their transition state for GTP hydrolysis, thus accelerating GTPase activity and dampening signaling (Ross and Wilkie, 2000; Hollinger and Hepler, 2002). Down-regulation of G protein-coupled signal transduction protects cells from overstimulation and allows cells to integrate information from multiple inputs and to respond to new stimuli. Sequencing of the human genome has revealed at least 37 human RGS proteins (Hollinger and Hepler, 2002; Siderovski and Willard, 2005) and puzzlingly, they are extremely promiscuous in their G protein targets when assayed in vitro. To date, relatively few mutants for RGS proteins have been generated to investigate the roles they play both in vivo and in the context of a whole organism (Ross and Wilkie, 2000; Hollinger and Hepler, 2002). Analysis of one mammalian RGS protein, RGS9, has revealed an important role in adaptation. Consistent with mouse knockout studies (Chen et al., 2000), loss-of-function mutations in the human retinal RGS9 (or its anchor protein R9AP) have been identified in patients with “bradyopsia” (slow vision) (Nishiguchi et al., 2004). These patients experience slow adaptation to bright light as well as difficulty seeing moving objects.

To further understand how loss of RGS function alters sensory response, we turned to C. elegans. The C. elegans genome contains thirteen predicted rgs genes and mutations or deletions exist for each (Bargmann, 1998; Dong et al., 2000; Hess et al., 2004). Although C. elegans chemosensation is mediated by G protein-coupled signaling, no RGS protein has previously been implicated in the regulation of sensory behaviors. Here, we present evidence that C. elegans RGS-3 regulates sensory response. We find that rgs-3 mutant animals are defective in behavioral responses to many sensory stimuli. These behavioral defects are due to increased signaling in sensory neurons, which in turns leads to diminished glutamatergic synaptic output.

Results

RGS-3 is Expressed in a Subset of Sensory Neurons

C. elegans rgs-3 (C29H12.3) encodes three predicted isoforms, each with two predicted RGS domains. The isoforms differ only in the length of their amino-termini (Figure 1A and Supplemental Figure 1). We note that C. elegans RGS-3 does not have obvious functional motifs outside the RGS domains. Based on neuron-specific mRNA pull-down studies, C. elegans rgs-3 is expressed in head sensory neurons (Kunitomo et al., 2005). To determine the precise cellular expression pattern of rgs-3, DNA encoding the Green Fluorescent Protein (GFP) was inserted in frame into an rgs-3 genomic clone, behind the first RGS domain, creating a translational fusion. RGS-3::GFP expression was observed only in nine pairs of sensory neurons (ASH, ADL, AWB, AWC, ASI, ASJ, ASK, PHA and PHB) (Figure 1B-C and Supplemental Figure 2). No other C. elegans rgs gene is expressed exclusively in sensory neurons, based on GFP reporter analysis (M.R.K., data not shown). RGS-3::GFP expression was seen in late-stage embryos, throughout development, and persisted through adulthood (data not shown). This expression pattern suggests a potential role in the sensory responses mediated by these neurons. Consistent with its neuronally restricted expression pattern, C. elegans RGS-3 is most similar to mammalian RGS8, a brain specific RGS (Saitoh et al., 1997).

Figure 1
C. elegans RGS-3 is Expressed in a Subset of Sensory Neurons.
  • (A) Protein structure of RGS-3. The rgs-3 locus encodes three predicted isoforms (A, B and C) that differ only in the length of their amino-termini. The vs19 deletion removes coding sequence
...

rgs-3(vs19) Disrupts Response to Sensory Stimuli

Using PCR-based screening techniques, we identified vs19 (Hess et al., 2004), a deletion allele of rgs-3 (Figure 1A). Although RGS proteins normally act to dampen G protein mediated signaling, rgs-3 mutant animals were not hypersensitive to environmental stimuli. Instead, rgs-3 animals were defective in their response to multiple stimuli detected by various sensory neurons (including octanol, quinine, high osmolarity and isoamyl alcohol). The volatile repellent 1-octanol is detected by the ASH, ADL and AWB sensory neurons when animals are tested in the standard assay format (Troemel et al., 1995; Chao et al., 2004). While wild-type animals responded to 100% octanol within a few seconds, rgs-3 animals responded poorly (Figure 2A). rgs-3 mutant animals were also defective in their response to the soluble repellent quinine (Figure 2B), which is detected primarily by the ASH neurons, with a small contribution by the ASK neurons (Hilliard et al., 2004). High osmolarity is also detected primarily by the ASH sensory neurons (Bargmann et al., 1990) and rgs-3 mutant animals were defective in their response to high osmolarity (Figure 2C). The behavioral defects of rgs-3 mutant animals were not specific to ASH-mediated avoidance behaviors. RGS-3 is also expressed in the AWC sensory neurons that detect the attractive odorant isoamyl alcohol; rgs-3 mutant animals were defective in their response to a 1:10 dilution of isoamyl alcohol (Figure 2D).

Figure 2
rgs-3(vs19) Disrupts Response to Sensory Stimuli.
  • (A-D) rgs-3(vs19) mutant animals are defective in response to (A) 100% octanol (B) 10mM quinine (C) 1M glycerol and (D) a 1:10 dilution of isoamyl alcohol. Expression of an rgs-3 genomic fragment significantly
...

To ensure that the behavioral defects observed in rgs-3(vs19) mutant animals were due to perturbation of RGS-3 function, a genomic construct containing the predicted rgs-3 gene was introduced into rgs-3 mutant animals. This construct was sufficient to restore all ASH and AWC-mediated behavioral responses (Figure 2A-D). Taken together, these results suggest that loss of RGS-3 function decreases sensory response, contrary to what might be expected for loss of a negative regulator of G protein signaling.

RGS-3 Functions in Adult Sensory Neurons

RGS-3 could function during neuronal development or in adult neurons to regulate response to sensory stimuli. To determine when RGS-3 function is required for sensory behaviors, full-length rgs-3 cDNA was placed under the control of a heat shock inducible promoter (Stringham et al., 1992) and introduced into rgs-3 animals. Induction of RGS-3 expression in adult animals significantly restored response to both 100% octanol (5.2 ± 0.9 s, Figure 2E) and 10mM quinine (95 ± 3%, not shown) when assayed 4 hours later. Without heat shock induction, no rescue was observed (100% octanol = 11.6 ± 0.8 s, 10mM quinine = 12.5 ± 3%). In addition, no changes in morphology, axonal projections, location or number of sensory neurons were observed by dye-filling in rgs-3 animals (data not shown). Combined, these results indicate that RGS-3 function is not required for development of the sensory neurons. Instead, expression of RGS-3 in adult sensory neurons, after the completion of cell fate determination and neuronal connectivity, is sufficient for normal sensory response.

Cell autonomous function of RGS-3 was confirmed in cell-specific rescue studies focused on the ASH sensory circuit. Since the ASH sensory neurons are the primary neurons that detect octanol, quinine and high osmolarity (Bargmann et al., 1990; Troemel et al., 1995; Hilliard et al., 2004), the osm-10 promoter (Hart et al., 1999) was used to express rgs-3 cDNA in the ASH neurons. The osm-10::rgs-3 transgene was sufficient to restore all ASH-mediated behavioral responses (Figure 2E and data not shown). Introduction of the osm-10 promoter construct alone had no effect (data not shown). Taken together, these results suggest that RGS-3 functions to regulate sensory behaviors in adult sensory neurons.

The vs19 Deletion Causes Loss of RGS-3 Function

Since the vs19 deletion does not remove the entire rgs-3 coding region, we confirmed that vs19 decreases RGS-3 function. Consistent with loss-of-function, vs19 is recessive and heterozygous animals (vs19/+) responded as well as wild-type animals to ASH and AWC detected stimuli (Figure 3 and data not shown). In addition, transgenes that encode wild-type RGS-3 restored all behavioral responses (Figure 2). Finally, vs19 does not create a dominant-negative form of RGS-3 (Supplemental Figure 3). These results suggest that the vs19 deletion creates a loss-of-function allele of rgs-3. If so, then rgs-3(vs19) homozygous animals should have behavioral defects equal to heterozygous rgs-3(vs19)/deficiency animals. maDf4 is a large chromosomal deficiency that deletes numerous genes on chromosome II, including rgs-3. Indeed, rgs-3(vs19)/maDf4 animals were indistinguishable from rgs-3(vs19) homozygous animals for all ASH-mediated behaviors (Figure 3 and data not shown). In addition, RNAi directed against rgs-3 in the ASH and ADL sensory neurons decreased response to 100% octanol (Figure 3). We conclude that vs19 is a loss-of-function allele.

Figure 3
rgs-3(vs19) is a Recessive Loss-of-Function Allele.

rgs-3 Animals Can Respond to Weak Sensory Stimuli

RGS proteins generally act to dampen signaling in other characterized systems (Ross and Wilkie, 2000; Hollinger and Hepler, 2002). But, instead of being hypersensitive (responding more robustly than wild-type animals), rgs-3 animals were defective in their response to many stimuli. However, the results described above assessed response to strong sensory stimuli (e.g. relatively high concentrations of odorants). To determine whether rgs-3 mutant animals were hypersensitive to weaker stimuli, we assessed their ability to respond to dilute stimulus concentrations. rgs-3 mutant animals did not respond better to more dilute quinine or glycerol (Figures 4A-B). However, for two odorants, rgs-3 mutant animals responded better to the diluted “weak” odorants than they did to the “strong” odorants. Although rgs-3 animals were defective in the ASH-mediated avoidance of 100% octanol, rgs-3 animals responded as well as wild-type animals to dilute octanol (Figure 4C). Similarly, when the attractive odorant isoamyl alcohol (detected by AWC) was diluted 1000-fold, rgs-3 animals responded as well as wild-type animals (Figure 4D). Thus, for both a repellant and an attractive chemosensory stimulus, rgs-3 animals failed to respond to intense stimuli, but responded as well as wild-type animals when the stimulus strength was decreased.

Figure 4
rgs-3 Mutant Animals Respond to Select Weak Sensory Stimuli
  • (A-B) rgs-3 animals do not respond better to more dilute concentrations (conc) of (A) quinine or (B) glycerol. n ≥ 20, p ≥ 0.30.
  • (C-D) rgs-3 animals respond like wild-type animals
...

This pattern of stimulus strength/response is not limited to chemosensory behaviors. C. elegans respond to the mechanosensory stimulus of light touch to the nose by initiating backwards locomotion (Kaplan and Horvitz, 1993). Response to nose touch is mediated primarily by the ASH neurons, with modest contributions from the FLP and OLQ sensory neurons (Kaplan and Horvitz, 1993). Nose touch is assayed by placing a hair on the agar surface in front of a freely moving animal. When the nose contacts the hair, the animal responds by initiating backwards locomotion. The strength of the nose touch stimulus is predetermined by the locomotion of the animal; directly decrementing stimulus strength is not possible in these behavioral assays. However, response to nose touch is modulated by feeding status. Wild-type animals have a diminished response to nose-touch when assayed 10-20 minutes after removal from food (bacterial lawn) due to decreased ASH sensory neuron signaling (Chao et al., 2004; Hilliard et al., 2005). Therefore, we compared the ability of rgs-3 animals to respond to nose touch both on food and off food, in effect manipulating stimulus strength (such that off food ≈ weaker stimulus). As shown in Figure 4E, rgs-3 animals responded to nose touch as well as wild-type animals when assayed on food. However, when assayed off food, rgs-3 animals responded twice as often as wild-type animals; loss of rgs-3 results in hypersensitivity to nose touch off food. Taken together, loss of RGS-3 function in sensory neurons leads to decreased response to several sensory stimuli. However, rgs-3 animals can respond to specific weaker stimuli.

Decreased Sensory Signaling Restores rgs-3 Response to Strong Stimuli

Since RGS proteins generally down-regulate G protein signaling, loss of RGS-3 function is expected to increase signaling in sensory neurons. If so, decreasing G protein-coupled signaling in sensory neurons might counteract RGS-3 loss-of-function and restore sensory response. GPA- 3 and ODR-3 are C. elegans Gα proteins that are required for primary sensory signal transduction (Zwaal et al., 1997; Roayaie et al., 1998; Jansen et al., 1999; Hilliard et al., 2004; Lans et al., 2004). ODR-3 Gα is a critical player in the sensory responses mediated by the ASH and AWC sensory neurons. GPA-3 Gα is also required for primary sensory signaling, but it plays a modest role in comparison to ODR-3 Gα. Underscoring the importance of both Gαs in primary signaling, loss of either ODR-3 Gαor GPA-3 Gα function decreases stimulus-evoked calcium influx into the ASH sensory neurons (Hilliard et al., 2005). To determine if rgs-3 behavioral defects are due to enhanced G protein-coupled signaling, we assayed rgs-3; odr-3 and rgs-3; gpa-3 double mutant animals for their ability to respond to strong stimuli. Loss of either ODR-3 Gα or GPA-3 Gα function significantly restored the ability of rgs-3 mutant animals to respond to 10mM quinine (Figures 5A-B). In addition, loss of GPA-3 Gα function restored response to 100% octanol (Figure 5C). Loss of neither ODR-3 Gα nor GPA-3 Gα restored response to high osmolarity or chemotaxis to isoamyl alcohol (data not shown). Since odr-3 mutant animals are defective in their response to octanol, high osmolarity and isoamyl alcohol (Roayaie et al., 1998), rgs-3; odr-3/+ animals were also tested; they remained defective in their response to these stimuli (data not shown). The ability to restore behavioral responses in rgs-3 animals by decreasing Gα signaling is consistent with loss of RGS-3 function causing an aberrant increase in sensory signaling.

Figure 5
Decreased Sensory Signaling Restores Behavioral Responses
  • (A-C) Decreased Gα function restores sensory response. Loss of either (A) ODR-3 Gα or (B) GPA-3 Gα function restores rgs-3 response to 10mM quinine.
  • (C) Loss of GPA-3 Gα
...

We further tested this idea by perturbing a component of sensory signaling that acts downstream of the G proteins. OSM-9 is a TRPV channel required for primary signaling in the ASH neurons (Colbert et al., 1997; Tobin et al., 2002; Hilliard et al., 2004). OSM-9 acts downstream of stimulus-evoked G protein-coupled signaling and loss of OSM-9 function also decreases stimulus-evoked calcium influx into the ASH neurons (Kahn-Kirby et al., 2004; Hilliard et al., 2005). Similar to loss of Gα function, loss of OSM-9 restored rgs-3 response to 10mM quinine (Figure 5D). Combined, these results demonstrate that the behavioral defects of rgs-3 animals can be suppressed by mutations that decrease G protein-coupled signal transduction in sensory neurons.

Neuronal Synaptic Machinery is Intact in rgs-3 Animals

To determine whether loss of RGS-3 function perturbs signaling downstream of OSM-9/OCR-2 channels, including the synaptic release machinery, we assessed avoidance of the chili pepper irritant capsaicin. Normally, C. elegans do not respond to capsaicin because they lack the capsaicin receptor. However, when the rat TRPV1 capsaicin receptor is expressed in the ASH neurons, capsaicin elicits a robust avoidance response (Tobin et al., 2002). The response to capsaicin does not require ODR-3 Gα or the TRPV channels OSM-9 or OCR-2, but it is dependent upon the vesicular glutamate transporter EAT-4 (Tobin et al., 2002). Therefore, TRPV1 likely bypasses endogenous G protein signaling to directly depolarize ASH, activating calcium signaling and glutamatergic synaptic transmission (Tobin et al., 2002). rgs-3 and wild- type animals responded equally well to capsaicin across a wide range of concentrations (Figure 6A). This indicates that loss of RGS-3 function does not disrupt the efficacy of the synaptic machinery or the ability of the ASH neurons to communicate with their synaptic targets. This is also consistent with the ability of rgs-3 animals to respond to nose touch (Figure 4E) and to dilute odorants (Figures 4C-D). We conclude loss of RGS-3 function does not alter the inherent ability of the ASH neurons to signal to their synaptic targets.

Figure 6
A Calcium “Sponge” Restores Behavioral Responses
  • (A) Response to capsaicin is not altered. rgs-3 animals expressing the rat TRPV1 capsaicin receptor in the ASH neurons avoid both high and low concentrations of capsaicin as well as wild-type
...

Expression of a Calcium-Binding Protein in Sensory Neurons Restores Response to Strong Stimuli

The defects seen in rgs-3 animals may be attributed to increased stimulus-induced calcium signaling in sensory neurons. If so, wild-type animals with increased calcium signaling should also exhibit behavioral defects. To test this, we pre-exposed TRPV1 expressing animals to capsaicin to increase calcium signaling in ASH without activating G proteins. Animals were then assayed for response to chemosensory stimuli. Pre-exposure to 5mM capsaicin significantly diminished response to 10mM quinine (Figure 6B) and 100% octanol (not shown). If increased calcium signaling is responsible for the behavioral defects, expression of a calcium-binding protein could buffer the excess calcium and restore behavioral responses. The genetically encoded calcium indicator ”cameleon” consists of two fluorescent proteins (YFP and CFP) connected by a flexible linker composed of the calcium binding domain of calmodulin and the M13 calmodulin-binding peptide (Miyawaki et al., 1997). Each molecule of cameleon binds four calcium ions (Miyawaki et al., 1997), and it has previously been shown that cameleon expression can act to buffer cytosolic calcium (Miyawaki et al., 1999). Expression of cameleon in the ASH neurons (sra-6::YC2.12) of TRPV1 expressing animals restored chemosensory responses to animals that were pre-exposed to capsaicin (Figure 6B and data not shown), suggesting that cameleon can buffer excess calcium signaling in C. elegans sensory neurons.

We therefore asked if cameleon could also restore rgs-3 response to sensory stimuli. Expression of cameleon in the ASH neurons of rgs-3 animals significantly restored response to the ASH-detected stimuli 10mM quinine and 1M glycerol (Figures 6C-D). Mutation of defined glutamate residues in cameleon prevents calcium binding (Guerrero et al., 2005) and abolishes the ability of cameleon (sra-6::YC-EallQ) to restore rgs-3 response (Figures 6C-D). Combined, these results suggest that increased G protein-coupled signaling in the absence of RGS-3 function leads to increased stimulus-evoked calcium signaling in sensory neurons, which underlies the rgs-3 behavioral defects. Interestingly, the ability of rgs-3 animals to respond like wild-type animals to capsaicin (Figure 6A) suggests that basal calcium levels are not dramatically altered in rgs-3 sensory neurons. If loss of RGS-3 function resulted in elevated basal calcium levels, rgs-3 animals might have been more sensitive than wild-type animals to dilute concentrations of capsaicin.

Cameleon can be used to measure stimulus-evoked calcium fluxes in the ASH sensory neurons (Fukuto et al., 2004; Hilliard et al., 2005). Calcium binding induces a conformational change in cameleon that changes the relative fluorescence intensity of CFP and YFP (Miyawaki et al., 1997). When stimulus-evoked calcium fluxes were imaged in rgs-3 animals expressing cameleon in ASH, we saw no difference in the amplitude or duration of fluxes when compared to control animals (1M glycerol, 10mM quinine, data not shown). However, the fact that cameleon restores ASH-mediated behavioral responses in rgs-3 animals suggests that the behavioral defects of rgs-3 animals are indeed due to enhanced calcium signaling downstream of G proteins. Alterations in rgs-3 animals may not be observed via cameleon imaging because they are small or discretely localized away from the recording site at the soma.

Feeding Status and Biogenic Amines Modulate Sensory Response inrgs-3 Animals

If the behavioral defects of rgs-3 animals are the result of increased signaling in the sensory neurons, then neuromodulators that increase signaling in sensory neurons should exacerbate the rgs-3 behavioral defects. Serotonin (5-HT) enhances the behavioral response of wild-type animals to dilute octanol (Chao et al., 2004) and increases the calcium flux in ASH sensory neurons in response to nose touch (Hilliard et al., 2005). Unlike wild-type animals, the response of rgs-3 animals to 100% octanol was decreased in the presence of 5-HT; exogenous 5-HT exacerbated the rgs-3 octanol defect (Figure 7A).

Figure 7
Feeding Status and Biogenic Amines Modulate rgs-3 Chemosensory Behaviors
  • (A) 5-HT and food have opposing effects on rgs-3 response to 100% octanol. 5-HT exacerbates the behavioral defect (p < 0.001) while food restores response (p < 0.001).
...

Food mimics 5-HT in the modulation of several C. elegans behaviors (Horvitz et al., 1982; Avery and Horvitz, 1990; Ségalat et al., 1995; Sawin et al., 2000). In particular, both food and 5-HT enhance response to dilute octanol and nose touch (Chao et al., 2004). We therefore asked whether food mimicked 5-HT in its effects on rgs-3 sensory response. Surprisingly, although 5-HT exacerbated the rgs-3 defective response to octanol, food improved the response of rgs-3 animals to all ASH-detected stimuli (Figure 7A and data not shown). As 5-HT and food have opposing effects of rgs-3 behaviors, 5-HT probably does not account for the ability of food to restore rgs-3 sensory responses; another neuromodulator is likely responsible.

In C. elegans, dopamine (DA) is also thought to be released upon exposure to food (Sawin et al., 2000) and DA contributes to specific food modulated behaviors (Schafer and Kenyon, 1995; Weinshenker et al., 1995; Sawin et al., 2000; Chase et al., 2004; Hills et al., 2004). To determine if DA modulates chemosensory sensitivity in C. elegans, we examined animals with decreased DA levels. cat-2 encodes a tyrosine hydroxylase enzyme specifically required for DA biosynthesis (Lints and Emmons, 1999). However, cat-2 mutant animals do synthesize ~40% DA as compared to wild-type animals (Sanyal et al., 2004), similar to the decreased DA levels observed in tyrosine hydroxylase deficient mice (Rios et al., 1999). We found that cat-2 animals were hypersensitive to dilute octanol and responded better than wild- type animals when they were assayed on food (Figure 7B). This suggests that dopamine normally dampens sensory responses on food.

We found that the ability of rgs-3 mutant animals to respond to strong stimuli on food is likely due to dopaminergic modulation. Exogenous DA substantially restored the ability of rgs-3 animals to respond to 100% octanol and 10mM quinine off food (Figures 7C-D). To confirm that DA is responsible for restoration of rgs-3 animals' response to strong sensory stimuli on food, rgs-3; cat-2 double mutant animals were examined. Loss of CAT-2 decreased the response to 100% octanol on food (Figure 7E). We note that rgs-3; cat-2 double mutants assayed on food were not as defective as rgs-3 animals assayed off food (compare Figures Figures2A2A and and7E).7E). This is likely due to the residual DA found in cat-2 animals (Sanyal et al., 2004). Alternatively, food may stimulate additional neuromodulatory pathways that function together with DA to restore rgs-3 response on food. We conclude that DA acts antagonistically to 5-HT and normally inhibits the sensory responses of wild-type animals on food. Hence, DA restores rgs-3 responses by decrementing signaling, potentially in sensory neurons.

Strengthening Glutamatergic Synaptic Transmission Restores ASH and AWC-mediated Behavioral Responses

The defective sensory responses of rgs-3 animals could result from either decreased or increased sensory neuron output. To determine which, we both decreased and increased glutamatergic signaling. GLR-1 and GLR-2 are glutamate gated ion channels that function in the interneurons downstream of the ASH sensory neurons (Hart et al., 1995; Maricq et al., 1995; Brockie et al., 2001; Mellem et al., 2002). If loss of RGS-3 results in enhanced glutamatergic synaptic transmission between the sensory neurons and the interneurons, then decrementing glutamate signaling to the interneurons could restore rgs-3 behavioral responses. However, loss of neither glr-1 nor glr-2 restored any rgs-3 behavioral responses (data not shown). While loss of either receptor may not have decremented synaptic signaling to the appropriate level to restore responses, these results suggest that loss of RGS-3 function does not result in enhanced glutamatergic synaptic transmission.

In fact, we found that increasing sensory neuron output restores rgs-3 response. In C. elegans, as in other organisms, glutamatergic signaling can be modulated by neuropeptides. Previous studies have shown that loss of the neuropeptide processing enzyme EGL-3 is sufficient to enhance glutamatergic signaling between the ASH sensory neurons and the interneurons (Kass et al., 2001; Mellem et al., 2002). To determine whether rgs-3 behavioral defects are due to diminished glutamatergic synaptic signaling, rgs-3; egl-3 double mutant animals were assayed for response to strong sensory stimuli. Loss of egl-3 significantly restored all ASH and AWC mediated behavioral responses to rgs-3 mutant animals (Figure 8 and data not shown). EGL-3 is expressed in many neurons, including sensory neurons and interneurons. Consistent with previous studies (Kass et al., 2001; Mellem et al., 2002), when EGL-3 was restored in the interneurons of rgs-3; egl-3 double mutants, animals were again defective in their sensory responses. Expression of EGL-3 in sensory neurons (including ASH) had no effect (Figure 8 and data not shown). Thus, enhancing glutamatergic signaling between the sensory neurons and the interneurons restores behavioral responses in rgs-3 animals. Combined with previous studies (Kass et al., 2001; Mellem et al., 2002), our analysis suggests that rgs-3 mutant animals are defective in their responses to sensory stimuli because loss of RGS-3 function in the sensory neurons ultimately leads to decreased synaptic transmission.

Figure 8
Loss of Neuropeptide Signaling Restores rgs-3 Behavioral Responses

Discussion

Loss of RGS-3 Function Increases Signaling in Sensory Neurons

To better understand the physiological consequences of misregulated G protein-coupled signaling, we have undertaken an analysis of C. elegans RGS-3. RGS proteins have classically been described as negative regulators of G proteins. Although mammalian RGS2 dampens cAMP production in olfactory epithelia by directly inhibiting specific adenylyl cyclase isoforms (Sinnarajah et al., 2001), our analysis of rgs-3 mutant animals suggests that the primary role of RGS-3 is regulation of G protein-coupled signaling. Loss of RGS-3 function alters behavioral response to sensory stimuli. rgs-3 animals are defective in their response to strong attractive (AWC-mediated) and aversive (ASH-mediated) stimuli. However, when the strength of either isoamyl alcohol (AWC) or octanol (ASH) is decreased, rgs-3 animals respond as well as wild- type animals. In addition, rgs-3 animals respond better than wild-type animals to the relatively weak stimulus (when assayed off food) of nose touch. This suggests that in the absence of RGS-3 function, there is actually “too much” signaling in response to strong stimuli, rendering animals unable to respond. Consistent with increased sensory signaling in the absence of RGS-3 function, decreasing signaling in C. elegans sensory neurons restores responses to strong stimuli. Loss of ODR-3 Gα, GPA-3 Gα or the OSM-9 TRPV channel restores ASH-mediated avoidance behaviors. Therefore, decreasing signaling at multiple points in the signaling cascade restores behavioral responses to rgs-3 mutant animals.

Cameleon proteins were designed to measure changes in intracellular calcium levels (Miyawaki et al., 1997). However, in some cellular contexts they also buffer cytosolic calcium (Miyawaki et al., 1999; Suzuki et al., 2003). While we cannot rule out the possibility that the M13 peptide of cameleon interacts with endogenous calmodulin (Palmer et al., 2006), it has previously been shown that cameleon expression does not interfere with endogenous calmodulin signaling at low concentrations (Miyawaki et al., 1999). The ability of cameleon, but not a calcium-insensitive cameleon, to restore behavioral responses in rgs-3 animals suggests that it is calcium binding that is critical for the rescue. Therefore, cameleon likely acts as a “sponge” to buffer cytosolic calcium in ASH.

Our model that loss of RGS-3 function increases sensory signaling but decreases sensory responses correlates well with the previous observation that overexpression of distinct Gα subunits can cause sensory defects. In particular, overexpression of ODR-3 Gα causes defects in chemotaxis toward attractive odorants (Roayaie et al., 1998). In addition, overexpression of a constitutively active (GTPase insensitive) ODR-3 Gα disrupts response to high osmolarity (Roayaie et al., 1998). Overexpression of constitutively active GPA-3 Gα also causes AWC- and ASH-mediated behavioral defects, but neuronal morphology is disrupted, making it difficult to determine whether the behavioral defects are the direct result of constitutively active GPA-3 Gα signaling (Zwaal et al., 1997; Jansen et al., 1999). A higher basal level of signaling might be obtained by overexpression of constitutively active Gα subunits than by loss of RGS-3 function, which more likely affects transient stimulus-evoked signaling events.

Loss of RGS-3 Function Decreases Synaptic Transmission

The strength of synaptic signaling was previously shown to be modulated by the neuropeptide processing enzyme EGL-3, which functions in the interneurons (Kass et al., 2001; Mellem et al., 2002). However, since glutamate-gated postsynaptic currents (in the AVA and AVD interneurons) are not altered in egl-3 mutants or in glr-1; egl-3 double mutants, loss of EGL-3 does not appear to alter the properties or levels of the postsynaptic glutamate receptors themselves (Kass et al., 2001; Mellem et al., 2002). Instead, loss of EGL-3 likely leads to an increase in the concentration of glutamate at ASH-interneuron synapses (Mellem et al., 2002). It was therefore postulated that a neuropeptide(s) processed by EGL-3 in the interneurons may normally function to either down-regulate glutamate release from the sensory neurons or to stimulate the uptake and removal of glutamate from the synaptic cleft (Mellem et al., 2002). Thus, in the absence of EGL-3, glutamatergic signaling from the sensory neurons to the interneurons is increased. As loss of EGL-3 restores sensory responses to rgs-3 mutants, we propose that loss of RGS-3 leads to decreased glutamatergic synaptic transmission.

There are striking parallels between the C. elegans rgs-3 sensory defects and loss of mammalian RGS9. In human patients with “bradyopsia,” loss of RGS9 causes temporary blindness upon exposure to bright light (Nishiguchi et al., 2004). This defect is reminiscent of the inability of C. elegans rgs-3 mutant animals to respond to strong chemosensory stimuli. Analogously, rgs-3 animals and patients lacking RGS9 have less difficulty responding to weaker stimuli (Nishiguchi et al., 2004). Electrophysiological studies also demonstrated that loss of mammalian RGS9 does not significantly alter initial retinal stimulus-evoked response, but instead disrupts recovery of photoreceptor cells (Chen et al., 2000; Nishiguchi et al., 2004). Similarly, blocking RGS4 activity in pancreatic acinar cells converts stimulus-induced calcium oscillations to a sustained response without affecting the amplitude of the calcium flux (Luo et al., 2001). Indeed, the ability of rgs-3 animals to respond better than wild-type animals to nose touch (off food) may be due to prolonged signaling through Gα in the absence of RGS-3.

How could increased sensory signaling in the absence of RGS-3 lead to decreased synaptic output in response to strong stimuli? Perhaps elevated basal Gα activity within the sensory neurons leads to a smaller maximal response amplitude achievable at saturating stimulus concentrations. This decreased level of Gα activation upon stimulus detection may be insufficient to trigger synaptic release. Although the Gα response amplitude to weaker stimuli should also be reduced, there could be compensatory mechanisms in place within the rgs-3 sensory neurons to allow response to weaker stimuli as a result of prolonged signaling through G proteins. Alternatively, many studies have demonstrated that the activity of calcium-permeable channels can be regulated by changing intracellular calcium concentrations (Taylor and Laude, 2002; Vazquez et al., 2004; Nijenhuis et al., 2005; Zhu, 2005). For example, in ASH neurons lacking RGS-3, increased (or prolonged) G protein activity, and thus increased calcium signaling, may cause C. elegans TRPV channels (e.g. OSM-9/OCR-2) or inositol 1,4,5-trisphosphate (IP3) receptors to be directly inhibited by excessive calcium-mediated feedback. This could result in decreased membrane depolarization and evoke insufficient synaptic release to elicit behavioral responses in rgs-3 animals.

Loss of RGS-3 Affects Sensory Signaling Uniquely

The mechanism underlying the rgs-3 behavioral defects stands in contrast to two previous reports where loss of negative regulators of signaling leads to defective behavioral responses. C. elegans grk-2 encodes a G protein-coupled receptor kinase (GRK) that likely negatively regulates signaling through G protein-coupled receptors. We previously showed that loss of GRK-2 function profoundly disrupts ASH, AWC and AWA-mediated sensory responses in C. elegans (Fukuto et al., 2004). In contrast to rgs-3 animals, grk-2 mutants are defective in their response to both “strong” and “weak” stimuli. While loss of RGS-3 function leads to enhanced signaling in sensory neurons, loss of GRK-2 leads to decreased signaling. Accordingly, loss of neither ODR-3 Gα (Fukuto et al., 2004) nor GPA-3 Gα (data not shown) function restores behavioral responses to grk-2 mutant animals. Instead, overexpression of ODR-3 Gα restores response to grk-2 animals (Fukuto et al., 2004). In addition, grk-2 animals have decreased stimulus evoked calcium fluxes in the ASH neurons and overexpression of cameleon does not restore response (Fukuto et al., 2004). Finally, feeding status does not affect grk-2 behavioral responses; grk-2 animals are defective when assayed either on or off food. Combined, these results indicate that there are key differences in the cellular response to loss of these two different negative regulators of G protein-coupled signaling.

TAX-6 is a calcineurin subunit that negatively regulates thermosensory, chemosensory and osmosensory signaling in C. elegans (Kuhara et al., 2002). Loss-of-function mutations in tax-6 lead to defects in AWC-mediated detection of odorants due to hyperadaptation to olfactory stimuli (Kuhara et al., 2002). Although the OSM-9 TRPV channel contributes to primary signal transduction in the ASH and AWA sensory neurons, it is required for adaptation to sensory stimuli detected by the AWC neurons (Colbert et al., 1997). Thus, loss of OSM-9 TRPV eliminates hyperadaptation due to loss of TAX-6 calcineurin and osm-9 tax-6 double mutant animals respond robustly to the AWC-detected odorant isoamyl alcohol (Kuhara et al., 2002). In contrast, loss of OSM-9 TRPV did not restore rgs-3 animals' response to isoamyl alcohol (data not shown). This suggests that if the rgs-3 behavioral defects are due to hyperadaptation, the hyperadaptation involves a different mechanism than that incurred by loss of TAX-6. Also in contrast to rgs-3 mutant animals, tax-6 mutants are hypersensitive to high osmolarity (Kuhara et al., 2002). Thus, while RGS-3 and TAX-6 both negatively regulate sensory signal transduction, their loss affects cellular signaling in a different way.

In many cellular contexts, RGS proteins negatively regulate G protein signaling. Yet, loss of specific RGS proteins results in defective sensory response from both vertebrate photoreceptors and C. elegans sensory neurons, despite distinct differences in their signal transduction cascades. This unique requirement for RGS proteins highlights their importance in the regulation of G protein-coupled signaling across species.

Experimental Procedures

Strains

Strains were maintained under standard conditions (Brenner, 1974). The strains used in this study include: N2 Bristol wild-type, HA865 Ce-grk-2(rt97) III, LX242 rgs-3(vs19) II, GE24 pha-1(e2123) III, CB1467 him-5(e1467) V, CB128 dpy-10(e128) II, CX3390 odr-3(n1605) V, NL335 gpa-3(pk35) V, CX10 osm-9(ky10) IV, KP4 glr-1(n2461) III, VM1854 glr-2(ak10) III, CB1112 cat-2(e1112) II, PD4792 mIs11 IV, MT1541 egl-3(n729) V, HA1738 maDf4/mIn1[dpy-10(e128) mIs14] II, CX4978 kyIs200 X, and HA1203 rtIs25 X. Also see Supplemental Data.

Supplementary Material

Supplementary Data

Acknowledgements

We thank David Clapham, Hidetoshi Komatsu, Michael Chao, Judy Pepper and members of the Hart, van den Heuvel, Schafer and Koelle labs for valuable discussions. We also thank Mike Boxem, Massimo Hilliard and Rex Kerr for critical feedback on this manuscript. We are grateful to Cori Bargmann, Josh Kaplan, Yuji Kohara, Andrew Fire, Gert Jansen, Jane Mendel, Atsushi Miyawaki, Roger Tsien and the Caenorhabditis Genetics Center for reagents. This work was supported by the NIH (#GM057918) (A.C.H.), American Cancer Society (#PF-03-130-01-MBC) (D.M.F.), American Psychological Association Diversity Program in Neuroscience (#T32MH18882) (R.H.), Human Frontier Science Program Organization (#LT00726/2004-C) (G.H.), NIDA (#R01DA016445-01) and HFSP (W.R.S.) and NIH (#NS036918) (M.R.K.).

Footnotes

Supplemental Data

For more information on rgs-3 sequences, expression pattern, vs19, behavioral assays, transgenic strains, plasmid construction and calcium imaging, see Supplemental Data.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • Avery L, Horvitz HR. Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans. J Exp Zool. 1990;253:263–270. [PubMed]
  • Bargmann CI. Neurobiology of the Caenorhabditis elegans genome. Science. 1998;282:2028–2033. [PubMed]
  • Bargmann CI, Hartwieg E, Horvitz HR. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 1993;74:515–527. [PubMed]
  • Bargmann CI, Thomas JH, Horvitz HR. Chemosensory cell function in the behavior and development of Caenorhabditis elegans. Cold Spring Harb Symp Quant Biol. 1990;55:529–538. [PubMed]
  • Bellocchio EE, Reimer RJ, Fremeau RT, Jr., Edwards RH. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science. 2000;289:957–960. [PubMed]
  • Berger AJ, Hart AC, Kaplan JM. Gαs-induced neurodegeneration in Caenorhabditis elegans. J Neurosci. 1998;18:2871–2880. [PubMed]
  • Birnby DA, Link EM, Vowels JJ, Tian H, Colacurcio PL, Thomas JH. A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory behaviors in Caenorhabditis elegans. Genetics. 2000;155:85–104. [PMC free article] [PubMed]
  • Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. [PMC free article] [PubMed]
  • Brockie PJ, Madsen DM, Zheng Y, Mellem J, Maricq AV. Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J Neurosci. 2001;21:1510–1522. [PubMed]
  • Chao MY, Komatsu H, Fukuto HS, Dionne HM, Hart AC. Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit. Proc Natl Acad Sci U S A. 2004;101:15512–15517. [PMC free article] [PubMed]
  • Chase DL, Pepper JS, Koelle MR. Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans. Nat Neurosci. 2004;7:1096–1103. [PubMed]
  • Chen CK, Burns ME, He W, Wensel TG, Baylor DA, Simon MI. Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature. 2000;403:557–560. [PubMed]
  • Coburn CM, Bargmann CI. A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron. 1996;17:695–706. [PubMed]
  • Colbert HA, Smith TL, Bargmann CI. OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci. 1997;17:8259–8269. [PubMed]
  • Cuppen E, van der Linden AM, Jansen G, Plasterk RH. Proteins interacting with Caenorhabditis elegans Gα subunits. Comparative and Functional Genomics. 2003;4:479–491. [PMC free article] [PubMed]
  • Dong MQ, Chase D, Patikoglou GA, Koelle MR. Multiple RGS proteins alter neural G protein signaling to allow C. elegans to rapidly change behavior when fed. Genes Dev. 2000;14:2003–2014. [PMC free article] [PubMed]
  • Fukuto HS, Ferkey DM, Apicella AJ, Lans H, Sharmeen T, Chen W, Lefkowitz RJ, Jansen G, Schafer WR, Hart AC. G protein-coupled receptor kinase function is essential for chemosensation in C. elegans. Neuron. 2004;42:581–593. [PubMed]
  • Guerrero G, Reiff DF, Agarwal G, Ball RW, Borst A, Goodman CS, Isacoff EY. Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nat Neurosci. 2005;8:1188–1196. [PMC free article] [PubMed]
  • Hart AC, Kass J, Shapiro JE, Kaplan JM. Distinct signaling pathways mediate touch and osmosensory responses in a polymodal sensory neuron. J Neurosci. 1999;19:1952–1958. [PubMed]
  • Hart AC, Sims S, Kaplan JM. Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature. 1995;378:82–85. [PubMed]
  • Hess HA, Roper JC, Grill SW, Koelle MR. RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans. Cell. 2004;119:209–218. [PubMed]
  • Hilliard MA, Apicella AJ, Kerr R, Suzuki H, Bazzicalupo P, Schafer WR. In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J. 2005;24:63–72. [PMC free article] [PubMed]
  • Hilliard MA, Bergamasco C, Arbucci S, Plasterk RH, Bazzicalupo P. Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J. 2004;23:1101–1111. [PMC free article] [PubMed]
  • Hills T, Brockie PJ, Maricq AV. Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J Neurosci. 2004;24:1217–1225. [PubMed]
  • Hollinger S, Hepler JR. Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev. 2002;54:527–559. [PubMed]
  • Horvitz HR, Chalfie M, Trent C, Sulston JE, Evans PD. Serotonin and octopamine in the nematode Caenorhabditis elegans. Science. 1982;216:1012–1014. [PubMed]
  • Jansen G, Thijssen KL, Werner P, van der Horst M, Hazendonk E, Plasterk RH. The complete family of genes encoding G proteins of Caenorhabditis elegans. Nat Genet. 1999;21:414–419. [PubMed]
  • Kahn-Kirby AH, Dantzker JL, Apicella AJ, Schafer WR, Browse J, Bargmann CI, Watts JL. Specific polyunsaturated fatty acids drive TRPV-dependent sensory signaling in vivo. Cell. 2004;119:889–900. [PubMed]
  • Kaplan JM, Horvitz HR. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1993;90:2227–2231. [PMC free article] [PubMed]
  • Kass J, Jacob TC, Kim P, Kaplan JM. The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans. J Neurosci. 2001;21:9265–9272. [PubMed]
  • Komatsu H, Mori I, Rhee JS, Akaike N, Ohshima Y. Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans. Neuron. 1996;17:707–718. [PubMed]
  • Kuhara A, Inada H, Katsura I, Mori I. Negative regulation and gain control of sensory neurons by the C. elegans calcineurin TAX-6. Neuron. 2002;33:751–763. [PubMed]
  • Kunitomo H, Uesugi H, Kohara Y, Iino Y. Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pull-down of poly(A) tails. Genome Biol. 2005;6:R17. [PMC free article] [PubMed]
  • L'Etoile ND, Bargmann CI. Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1. Neuron. 2000;25:575–586. [PubMed]
  • Lans H, Rademakers S, Jansen G. A network of stimulatory and inhibitory Gα-subunits regulates olfaction in Caenorhabditis elegans. Genetics. 2004;167:1677–1687. [PMC free article] [PubMed]
  • Lee RY, Sawin ER, Chalfie M, Horvitz HR, Avery L. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in Caenorhabditis elegans. J Neurosci. 1999;19:159–167. [PMC free article] [PubMed]
  • Lints R, Emmons SW. Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene. Development. 1999;126:5819–5831. [PubMed]
  • Luo X, Popov S, Bera AK, Wilkie TM, Muallem S. RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations. Mol Cell. 2001;7:651–660. [PubMed]
  • Maricq AV, Peckol E, Driscoll M, Bargmann CI. Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature. 1995;378:78–81. [PubMed]
  • Mellem JE, Brockie PJ, Zheng Y, Madsen DM, Maricq AV. Decoding of polymodal sensory stimuli by postsynaptic glutamate receptors in C. elegans. Neuron. 2002;36:933–944. [PubMed]
  • Miyawaki A, Griesbeck O, Heim R, Tsien RY. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A. 1999;96:2135–2140. [PMC free article] [PubMed]
  • Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997;388:882–887. [PubMed]
  • Neer EJ. Heterotrimeric G proteins: organizers of transmembrane signals. Cell. 1995;80:249–257. [PubMed]
  • Nijenhuis T, Hoenderop JG, Bindels RJ. TRPV5 and TRPV6 in Ca2+ (re)absorption: regulating Ca2+ entry at the gate. Pflugers Arch. 2005;451:181–192. [PubMed]
  • Nishiguchi KM, Sandberg MA, Kooijman AC, Martemyanov KA, Pott JW, Hagstrom SA, Arshavsky VY, Berson EL, Dryja TP. Defects in RGS9 or its anchor protein R9AP in patients with slow photoreceptor deactivation. Nature. 2004;427:75–78. [PubMed]
  • Nuttley WM, Harbinder S, van der Kooy D. Regulation of distinct attractive and aversive mechanisms mediating benzaldehyde chemotaxis in Caenorhabditis elegans. Learn Mem. 2001;8:170–181. [PMC free article] [PubMed]
  • Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, Baker D, Tsien RY. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol. 2006;13:521–530. [PubMed]
  • Prasad BC, Reed RR. Chemosensation: molecular mechanisms in worms and mammals. Trends Genet. 1999;15:150–153. [PubMed]
  • Rios M, Habecker B, Sasaoka T, Eisenhofer G, Tian H, Landis S, Chikaraishi D, Roffler-Tarlov S. Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine hydroxylase. J Neurosci. 1999;19:3519–3526. [PubMed]
  • Roayaie K, Crump JG, Sagasti A, Bargmann CI. The Gα protein ODR-3 mediates olfactory and nociceptive function and controls cilium morphogenesis in C. elegans olfactory neurons. Neuron. 1998;20:55–67. [PubMed]
  • Ross EM, Wilkie TM. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem. 2000;69:795–827. [PubMed]
  • Saitoh O, Kubo Y, Miyatani Y, Asano T, Nakata H. RGS8 accelerates G-protein-mediated modulation of K+ currents. Nature. 1997;390:525–529. [PubMed]
  • Sanyal S, Wintle RF, Kindt KS, Nuttley WM, Arvan R, Fitzmaurice P, Bigras E, Merz DC, Hebert TE, van der Kooy D, et al. Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans. Embo J. 2004;23:473–482. [PMC free article] [PubMed]
  • Sawin ER, Ranganathan R, Horvitz HR. C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway. Neuron. 2000;26:619–631. [PubMed]
  • Schafer WR, Kenyon CJ. A calcium-channel homologue required for adaptation to dopamine and serotonin in Caenorhabditis elegans. Nature. 1995;375:73–78. [PubMed]
  • Ségalat L, Elkes DA, Kaplan JM. Modulation of serotonin-controlled behaviors by Go in Caenorhabditis elegans. Science. 1995;267:1648–1651. [PubMed]
  • Siderovski DP, Willard FS. The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits. Int J Biol Sci. 2005;1:51–66. [PMC free article] [PubMed]
  • Sinnarajah S, Dessauer CW, Srikumar D, Chen J, Yuen J, Yilma S, Dennis JC, Morrison EE, Vodyanoy V, Kehrl JH. RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III. Nature. 2001;409:1051–1055. [PubMed]
  • Stringham EG, Dixon DK, Jones D, Candido EP. Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans. Mol Biol Cell. 1992;3:221–233. [PMC free article] [PubMed]
  • Suzuki H, Kerr R, Bianchi L, Frokjaer-Jensen C, Slone D, Xue J, Gerstbrein B, Driscoll M, Schafer WR. In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation. Neuron. 2003;39:1005–1017. [PubMed]
  • Takamori S, Rhee JS, Rosenmund C, Jahn R. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature. 2000;407:189–194. [PubMed]
  • Taylor CW, Laude AJ. IP3 receptors and their regulation by calmodulin and cytosolic Ca2+ Cell Calcium. 2002;32:321–334. [PubMed]
  • Tobin D, Madsen D, Kahn-Kirby A, Peckol E, Moulder G, Barstead R, Maricq A, Bargmann C. Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron. 2002;35:307–318. [PubMed]
  • Troemel ER. Chemosensory signaling in C. elegans. Bioessays. 1999;21:1011–1020. [PubMed]
  • Troemel ER, Chou JH, Dwyer ND, Colbert HA, Bargmann CI. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell. 1995;83:207–218. [PubMed]
  • Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney JW., Jr. The mammalian TRPC cation channels. Biochim Biophys Acta. 2004;1742:21–36. [PubMed]
  • Weinshenker D, Garriga G, Thomas JH. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci. 1995;15:6975–6985. [PubMed]
  • White JG, Southgate E, Thomson JN, Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil Trans R Soc Lond B. 1986:1–340. [PubMed]
  • Zhu MX. Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels. Pflugers Arch. 2005;451:105–115. [PubMed]
  • Zwaal RR, Mendel JE, Sternberg PW, Plasterk RH. Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone. Genetics. 1997;145:715–727. [PMC free article] [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...