A Neuronal Signaling Pathway of CaMKII and Gqα Regulates Experience-Dependent Transcription of tph-1

Aversive training enhances the sensory-evoked calcium response of ADF neurons

Previously, we have shown that training with pathogenic bacteria, such as P. aeruginosa PA14, induces olfactory aversion to the pathogen (Zhang et al., 2005). Naive animals that are never exposed to the pathogen often prefer the smell of the pathogen, whereas trained animals that have ingested the pathogenic bacteria avoid the bacterial smell (Fig. 1A,B). This form of behavioral plasticity requires the activity of the C. elegans TPH-1 in ADF serotonergic neurons, because selective expression of tph-1 in the ADF neurons of the tph-1 null mutants rescues the learning defect of the mutants. Interestingly, aversive training increases tph-1 transcription in ADF, as evidenced by the increased signal of a tph-1::gfp reporter (Sze et al., 2000) in trained animals (Fig. 1C) (Zhang et al., 2005).

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Figure 1.

Aversive olfactory training enhances ADF neuronal activity to regulate tph-1 transcription. A, Standard and modified training procedure with P. aeruginosa PA14. B, Schematic for the aversive olfactory learning assay. C, Images of tph-1::gfp expression in naive or trained animals. Arrowheads indicate ADF; dashed lines outline the worms. D, FRET signals of YC3.60 in ADF neurons in response to medium conditioned with E. coli OP50 in naive and PA14-trained wild-type animals. E, Heat maps for the FRET signals shown in D. F, Time integral of the FRET signals in D during the last 20 s stimulus presentation. In D and F, ***p < 0.001; *p < 0.05, Student's t test. Error bars represent SEM. G, FRET signals of YC3.60 in ADF neurons in response to OP50-conditioned medium in naive and PAK-trained wild-type animals. H–J, FRET signals of YC3.60 in ADF neurons in response to OP50-conditioned medium in wild-type and itr-1(sa73) mutants (H) or in wild-type and unc-68(e540) mutants (I) or in wild-type and unc-2(e55) mutants (J). In D and G–J, solid lines indicate mean values and shading indicates SEM. K, Expression of a potassium channel encoded by twk-18(cn110) in ADF abolishes training-dependent increase in tph-1 transcription. A significant genotype × treatment interaction (**p < 0.01) was detected by two-way ANOVA after log transformation (p < 0.01 for both genotype and treatment; n ≥ 30 animals each sample). The data sets were tested as normal distribution by the Shapiro-Wilk test. Fluorescent signals were normalized by the signal of naive wild-type worms measured in parallel; error bars represent SEM.

To understand how tph-1 transcription in ADF neurons is regulated by training experience, we first examined the ADF neuronal activity using intracellular calcium imaging. We selectively expressed a genetically encoded calcium sensor, Cameleon YC3.60 (Nagai et al., 2004), in ADF using the srh-142 promoter and recorded the calcium response of transgenic animals in a microfluidic device (Chronis et al., 2007). We stimulated the transgenic animals with alternating fluid streams of buffer and the medium that was conditioned with the culture of E. coli OP50, the common laboratory bacterial food for C. elegans. We found that the ratio of YFP-to-CFP intensity increased when the stimulus switched from buffer to the conditioned medium, indicating increased intracellular calcium levels in response to the conditioned medium (Fig. 1D,E).

To identify the source of ADF calcium transients, we examined the effects of several mutations that disrupt either intracellular calcium release from endoplasmic reticulum or calcium influx from extracellular space. First, we imaged ADF neurons in itr-1(sa73) and unc-68(e540) mutant animals, which carried a reduction-of-function mutation in the inositol-1,4,5-trisphosphate receptor ITR-1 (Dal Santo et al., 1999) and a splicing-acceptor site mutation in the ryanodine receptor UNC-68 (Sakube et al., 1997), respectively. We quantified the sensory-evoked ADF calcium transients by calculating the time integral of the YFP/CFP ratio change during the 30 s exposure to the conditioned medium. We did not observe any obvious defect in ADF calcium responses to OP50-conditioned medium in these two mutants compared with wild-type animals (p > 0.05 for time integrals over 30 s stimulus exposure, Student's t test) (Fig. 1H,I). Because previous studies have indicated important roles of ITR-1 and UNC-68 in regulating intracellular calcium release (Maryon et al., 1996; Sakube et al., 1997; Clandinin et al., 1998; Baylis et al., 1999; Dal Santo et al., 1999; Busch et al., 2012), these results suggest that intracellular calcium stores are not likely to be the main source for the sensory-evoked calcium transients in ADF. Next, we tested ADF calcium response in unc-2(e55) animals, which carry a nonsense mutation in the C. elegans non-L-type voltage-gated calcium channel UNC-2 (Schafer and Kenyon, 1995; Estevez et al., 2004). We found that compared with wild-type animals, unc-2(e55) mutants displayed weaker ADF calcium transients in response to OP50-conditioned medium (Fig. 1J), and the integral of the calcium response during 30 s exposure of the stimulus is smaller in unc-2(e55) than in wild-type animals (p < 0.05, Student's t test). The partial defect of unc-2(e55) could be attributable to potential redundant roles played by other calcium channels. For example, the L-type voltage-gated calcium channel EGL-19 (Lee et al., 1997) has been shown to regulate sensory-evoked calcium transients in several types of sensory neurons (Suzuki et al., 2003; Hilliard et al., 2005; Frøkjaer-Jensen et al., 2006). However, we could not assess the effect of egl-19(ad1006) mutants because of weak expression of Cameleon YC3.60 in the ADF neurons of the egl-19 mutants. Both intracellular and extracellular sources of calcium contribute to the calcium responses of C. elegans neurons (Suzuki et al., 2003; Hilliard et al., 2005; Frøkjaer-Jensen et al., 2006; Busch et al., 2012; Hendricks et al., 2012). Our results together suggest that influx of extracellular calcium regulates the sensory-evoked calcium transients in ADF.

Next, we examined whether the aversive training alters ADF activity by comparing the calcium responses of ADF neurons in the naive and trained animals. We found that trained animals exhibited calcium transients for an extended amount of time, ranging from 10 to 30 s (Fig. 1D,E), in response to the conditioned medium. To quantify this change, we calculated the time integral of the YFP/CFP ratio change during the last 20 s exposure to the conditioned medium and found that PA14-trained animals displayed a significant increase compared with naive animals (Fig. 1F). In contrast, training with PAK, a nonpathogenic strain of P. aeruginosa, did not induce similar changes (p > 0.05 for time integrals of the calcium transients over the last 20 s stimulus presentation, Student's t test) (Fig. 1G), suggesting that the training-dependent enhancement in ADF activity is contingent on the aversive experience with the pathogenic bacterium PA14. Together, these results indicate that the sensory-evoked calcium response in ADF is enhanced in the PA14-trained animals, suggesting that aversive training activates ADF neurons.

Activating ADF neurons increases tph-1 transcription, whereas inhibiting ADF abolishes training-dependent induction of tph-1

Because aversive training increases both ADF neural activity and tph-1 transcription in these cells, we examined whether increased ADF activity can lead to upregulation of tph-1 transcription. First, we activated ADF neurons by selectively expressing a bacterially derived voltage-gated sodium channel, NaChBac (Ren et al., 2001), in ADF. Ectopic expression of NaChBac depolarizes and increases the excitability of several types of neurons in flies and mice (Nitabach et al., 2006; Kelsch et al., 2009). We found that compared with nontransgenic siblings (1.0 ± 0.06, intensity normalized by the signal in nontransgenic animals; n ≥ 49 animals), the transgenic animals that expressed NaChBac only in ADF neurons displayed significantly enhanced tph-1 expression in these neurons even in the absence of training (1.34 ± 0.08, intensity normalized by the signal in non-transgenic animals; p < 0.01, Student's t test; n ≥ 50 animals; mean ± SEM), thus supporting the hypothesis that increased ADF neuronal activity during aversive training induces tph-1 expression.

Next, we examined whether the training-dependent increase in tph-1 transcription depends on ADF neuronal activity. We inhibited ADF neurons by selectively expressing an activated form of potassium channel twk-18(cn110) in ADF neurons. The expression of twk-18(cn110) in Xenopus oocytes generates a potassium channel that conducts significantly larger outward currents than the wild-type channels (Kunkel et al., 2000). We found that training-induced tph-1 transcription was abolished in the transgenic animals, whereas nontransgenic wild-type siblings displayed a normal level of induction (Fig. 1K). Together, these results support our hypothesis that activation of ADF neurons during the aversive experience leads to increased tph-1 transcription.

CaMKII/UNC-43 functions in ADF neurons to regulate ADF neuronal activity, tph-1 transcription, and aversive learning

Next, we sought the molecular mechanisms whereby tph-1 transcription in ADF neurons is upregulated by training experience. Because UNC-43, the only C. elegans homolog of CaMKII (Reiner et al., 1999; Rongo and Kaplan, 1999), regulates ADF tph-1 expression under normal conditions (Zhang et al., 2004), we examined whether UNC-43 also regulates training-dependent tph-1 expression in ADF.

First, we found that the training-dependent upregulation of tph-1 transcription in ADF neurons was completely abolished in the unc-43(n498n1186) null mutants, measured by the expression of the transcriptional reporter tph-1::gfp in both naive and trained animals (Fig. 2A). In addition, the unc-43(n498n1186) mutants failed to learn to avoid the smell of PA14 after training (Fig. 2C). UNC-43 is ubiquitously expressed in the nervous system (Reiner et al., 1999; Rongo and Kaplan, 1999). We found that selective expression of a wild-type unc-43 cDNA in the ADF neurons of unc-43(n498n1186) mutants fully restored the training-dependent upregulation of tph-1 expression in ADF and partially rescued the learning defects in the mutants (Fig. 2B,C). One potential reason for the partial rescue of learning is that UNC-43 also regulates the function of motor neurons and muscles (Reiner et al., 1999; Rongo and Kaplan, 1999), and the transgenic animals with UNC-43 activity restored only in ADF were still defective in locomotion. Thus, UNC-43 functions cell-autonomously in ADF neurons to regulate the training-dependent enhancement of tph-1 transcription and aversive olfactory learning.

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Figure 2.

UNC-43 functions cell-autonomously in ADF neurons to regulate training-dependent increase in ADF neuronal activity and tph-1 transcription, as well as aversive olfactory learning. A, The null mutant unc-43(n498n1186) is defective in training-dependent upregulation of tph-1::gfp expression in ADF neurons. B, Expression of unc-43 in the ADF neurons of unc-43(n498n1186) mutants fully rescues the defect in tph-1::gfp regulation. The fluorescent signals were normalized by the signal of naive wild-type worms (A) or naive rescued animals (B) measured in parallel. Significant genotype × treatment interactions (***p < 0.001) were detected by two-way ANOVA after log transformation (p < 0.001 for both genotype and treatment; n ≥ 59 animals each sample). The data sets were tested as normal distribution by the Shapiro-Wilk test. Error bars represent SEM. C, The null mutant unc-43(n498n1186) is defective in learning to avoid the smell of PA14 and expression of unc-43 in the ADF neurons of unc-43(n498n1186) mutants partially rescues the learning defect. **p < 0.01; *p < 0.05, Student's t test; n ≥ 6 assays. Error bars represent SEM. D, The gain-of-function mutant unc-43(n498) exhibits increased tph-1::gfp expression in ADF neurons in naive animals (***p < 0.001, Student's t test) and is defective in training-dependent increase in ADF tph-1 expression [a significant genotype × treatment interaction (***p < 0.001) was detected by two-way ANOVA after log transformation; p < 0.01 for genotype, p < 0.001 for treatment; n ≥ 57 each sample; error bars represent SEM; the data sets were tested as normal distribution by the Shapiro-Wilk test]. E, FRET signals of YC3.60 in ADF neurons in response to OP50-conditioned medium in unc-43(n498n1186) mutants and in the unc-43(n498n1186) mutant animals that selectively express wild-type UNC-43 in ADF under both naive and PA14 training conditions. F, Time integral of FRET signals in E during the last 20 s stimulus presentation. A significant genotype × treatment interaction (*p < 0.05) was detected by two-way ANOVA (p > 0.05 for genotype, p < 0.01 for treatment; n ≥ 13 each sample; the data sets were tested as normal distribution by the Shapiro-Wilk test; error bars represent SEM). G, FRET signals of YC3.60 in ADF neurons in response to OP50-conditioned medium in unc-43(n498n1186) mutants and in unc-43(n498n1186) mutants that selectively express the activated UNC-43(E108K) in ADF under both naive and PA14-training conditions. H, Time integral of the FRET signals in G and of the FRET signals in wild-type animals during the last 20 s stimulus presentation. A significant genotype × treatment interaction (*p < 0.05) between wild-type and unc-43(n498n1186) animals that selectively expressed UNC-43(E108K) in ADF was detected by two-way ANOVA (p < 0.01 for genotype, p > 0.05 for treatment; n ≥ 11 each sample; the data sets were tested as normal distribution by the Shapiro-Wilk test). Student's t test was used to compare wild-type animals (n = 17) and unc-43(n498n1186) mutants that selectively expressed UNC-43(E108K) in ADF under the naive condition (***p < 0.001). Error bars represent SEM.

The mammalian CaMKII isoform α_B is localized in the nucleus (Brocke et al., 1995). However, we did not observe any clear signal in the nuclei of transgenic animals that expressed a translational reporter unc-43::mcherry only in ADF neurons under either the naive or the trained condition (data not shown). The mammalian α_B-CaMKII possesses a nuclear localization signal (Brocke et al., 1995) that is absent in UNC-43. This notion is consistent with the imaging results. Therefore, UNC-43 likely functions through further downstream signaling events to regulate tph-1 transcription in ADF neurons.

Next, we investigated how UNC-43 functions cell-autonomously to regulate training-dependent tph-1 transcription in ADF neurons. First, we found that tph-1 transcription in the ADF neurons of a gain-of-function mutant, unc-43(n498) (Reiner et al., 1999; Rongo and Kaplan, 1999), was significantly elevated under the naive condition (Fig. 2D), consistent with previous studies (Zhang et al., 2004). In addition, this elevated tph-1 level was not further increased by training (Fig. 2D). The unc-43(n498) mutant exhibits opposing phenotypes to the unc-43 loss-of-function mutant in both locomotion and egg laying (Reiner et al., 1999; Rongo and Kaplan, 1999; Robatzek and Thomas, 2000), and the amino acid substitution E108K in unc-43(n498) results in constitutive activation of UNC-43 that is calcium independent (Umemura et al., 2005). These results suggest the possibility that activation of UNC-43 in ADF neurons during aversive training leads to increased tph-1 expression. To further test this possibility, we selectively expressed the constitutively active form UNC-43(E108K) in the ADF neurons of the unc-43(n498n1186) null mutants and found that this ectopic expression enhanced tph-1 transcription [1.84 ± 0.14, intensity normalized by the signals in unc-43(n498n1186) mutants that expressed wild-type UNC-43 only in ADF; n ≥ 37 animals; mean ± SEM] compared with transgenic animals that expressed the wild-type UNC-43 in the ADF neurons of the same mutants (1.0 ± 0.03, normalized intensity; n ≥ 37 animals; p < 0.001, Student's t test; mean ± SEM). This enhancement strongly mimicked the training effect. The transgenic animals that expressed ADF-specific UNC-43(E108K) appeared to exhibit higher expression of tph-1 in ADF neurons than the unc-43(n498) gain-of-function mutants (Fig. 2D). This difference may result from a higher expression level of UNC-43(E108K) in the ADF neurons of the transgenic animals and/or some unidentified defects in the unc-43(n498) mutant animals. Together, these findings support that activated UNC-43 in ADF neurons increases tph-1 transcription during aversive training.

UNC-43 regulates training-dependent enhancement of ADF calcium responses

Having characterized the roles of UNC-43 and ADF neuronal activity in regulating tph-1 transcription in ADF, we examined whether UNC-43 regulates ADF activity. We performed calcium imaging on the ADF neurons of unc-43(n498n1186) null mutants (Fig. 2E,F). We found that loss of UNC-43 activity abolished training-induced increase in ADF calcium transients and that this defect was fully rescued by selective expression of wild-type UNC-43 in the ADF neurons (Fig. 2E,F). Conversely, ADF-specific expression of the activated UNC-43(E108K) isoform in the unc-43(n498n1186) null mutants is sufficient to increase ADF calcium transients, mimicking the training effect (Fig. 2G,H). Moreover, training did not further enhance ADF calcium response in these transgenic animals. Together, our results indicate that aversive training experience activates ADF serotonergic neurons via activated function of UNC-43 in ADF, which leads to upregulation of tph-1 transcription (Fig. 5E,F).

EGL-30 regulates increased tph-1 transcription during aversive training

Next, we investigated the molecular pathways in which UNC-43 acts to regulate tph-1 induction during training. Previously, it was shown that UNC-43 regulates locomotion and egg laying through a pathway of GOA-1 and EGL-30, the C. elegans Goα and Gqα, respectively (Miller et al., 1999; Robatzek and Thomas, 2000; Lee et al., 2004). To test whether UNC-43 signals in the same pathway to regulate tph-1 in ADF neurons during aversive learning, we analyzed the effect of mutations in the Goα/Gqα pathway on this process. Because mutants in this pathway are often deficient in locomotion, to ensure sufficient training we transferred trained animals from a standard training plate to a plate that was fully covered by a lawn of PA14 during the L4 larval stage and measured ADF tph-1 levels 12 h later (Fig. 1A).

First, we found that both egl-30(ad805) and egl-30(n686) (Brundage et al., 1996), two different mutations that disrupt EGL-30 function, abolished the training-dependent upregulation of tph-1 transcription in ADF (Fig. 3A,B). Surprisingly, the gain-of-function mutant egl-30(tg26) (Doi and Iwasaki, 2002; Bastiani et al., 2003) is similarly defective (Fig. 3C), suggesting that proper activity level of Gqα/EGL-30 is important in regulating tph-1 transcription. In contrast, a canonical mutant, goa-1(sa734), exhibited normal training-dependent induction of tph-1 despite an increased level of tph-1 expression under the naive condition (Fig. 3D). These results suggest that the functions of GOA-1 and EGL-30 in regulating training-dependent expression of tph-1 are different from their antagonistic roles in locomotion and egg laying and thus suggest a new mechanism of EGL-30 in regulating tph-1 transcription.

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Figure 3.

egl-30 regulates tph-1 transcriptional induction in ADF neurons during training. A–D, The egl-30(ad805) (A), egl-30(n686) (B), and egl-30(tg26) (C) mutants are defective in tph-1 transcriptional upregulation induced by aversive training; however, the goa-1(sa734) mutant animals (D) are normal in this training-dependent change. E, F, The egl-8(md1971) mutants (E) and unc-73(ce362) mutants (F) are not significantly defective in training-dependent tph-1 upregulation in ADF neurons. In A, B, D, and F, genotype × treatment interactions (***p < 0.001; **p < 0.01; n.s., p ≥ 0.05) were tested by two-way ANOVA after log transformation (p < 0.001 for both genotype and treatment; n ≥ 40 for each sample; the data sets were tested as normal distribution by the Shapiro-Wilk test). In C, a significant genotype × treatment interaction (**p < 0.01) was detected by the Scheirer-Ray-Hare nonparametric test (p < 0.001 for genotype and treatment; n ≥ 54 animals for each sample), because the data sets were tested as deviated from normal distribution by the Shapiro-Wilk test. In E, genotype × treatment interaction was tested by two-way ANOVA after log transformation (n.s., p > 0.05 for interaction, p = 0.902 for genotype, and p < 0.001 for treatment; n ≥ 40 for each sample; the data sets were tested as normal distribution by the Shapiro-Wilk test). For all, the worms were trained with a modified protocol as in Figure 1A. The fluorescent signals were normalized by the signal of naive wild-type worms measured in parallel. Error bars represent SEM.

Previous studies have implicated the Rho guanine nucleotide exchange factor (GEF) domain of UNC-73 (Williams et al., 2007) and the phospholipase Cβ EGL-8 as downstream effectors of EGL-30 in regulating locomotion and egg laying (Lackner et al., 1999; Miller et al., 1999). We found that a nonsense mutation in egl-8(md1971), which produces strong defects in locomotion and egg laying (Miller et al., 1999), displayed normal training-dependent induction of tph-1 expression in ADF neurons (Fig. 3E), suggesting that EGL-30 functions mostly independently of EGL-8 to enhance tph-1 transcription during aversive training. This result is consistent with a previous finding that EGL-8 acts as a minor downstream effector or in a parallel pathway of EGL-30 to regulate egg-laying behavior (Bastiani et al., 2003). Similarly, the unc-73(ce362) mutant that contains a mutation in the Rho GEF domain was also not significantly defective in tph-1 induction (Fig. 3F), implicating an unidentified or novel effector(s) of Gqα/EGL-30 in regulating training-dependent tph-1 transcription. Together, our results indicate that UNC-43 functions together with EGL-30 to modulate tph-1 expression in ADF neurons during aversive training.

EGL-30 mediates sensory response of AWB and AWC olfactory neurons to regulate tph-1 induction during aversive training

Next, we examined the mechanisms whereby EGL-30 regulates tph-1 transcription in ADF neurons during aversive training. egl-30 is known to express widely in the nervous system (Lackner et al., 1999). First, we confirmed the expression of egl-30 in ADF neurons (Fig. 4A–C) and the major olfactory sensory neurons AWB and AWC (Fig. 4D–F) by examining the coexpression of a transcriptional reporter, KP326 (Pegl-30::gfp) (Lackner et al., 1999), with mCherry reporters expressed in these neurons. Next, we found that, different from the cell-autonomous role of UNC-43, selective expression of a wild-type egl-30 cDNA in ADF neurons did not rescue the defect of egl-30(ad805) mutants in regulating training-dependent induction of tph-1 expression in ADF (Fig. 4G). This defect and the similar defect in the other egl-30 mutant, n686, required the activity of egl-30 in both AWB and AWC sensory neurons to rescue (Fig. 4H,I). AWB synapse onto ADF with multiple chemical synapses (White et al., 1986), but expression of egl-30 in AWB alone did not rescue (Fig. 4J). Together, our results reveal a small neuronal network in which unc-43 functions cell-autonomously in ADF neurons and egl-30 functions non-cell-autonomously in the upstream sensory neurons to produce activity-dependent upregulation of tph-1 transcription in ADF (Fig. 5E,F). Consistently, ADF-specific expression of the activated UNC-43(E108K) in egl-30(ad805) mutants significantly increases ADF tph-1 expression [Student's t test, p < 0.001 between transgenic animals (3.26 ± 0.18) and nontransgenic siblings (1 ± 0.08); tph-1 signals were normalized by the signals in nontransgenic animals; n ≥ 25 animals each group; mean ± SEM], further supporting the possibility that UNC-43 acts downstream of EGL-30 in regulating tph-1 expression in ADF.

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Figure 4.

EGL-30 functions in the AWB and AWC sensory neurons to regulate training-dependent induction of tph-1 transcription in ADF neurons. A–C, egl-30 is expressed in the ADF neurons, as shown by colocalization of the fluorescent signals generated by the Psrh-142::mcherry (A) and the Pegl-30::gfp (B) transcriptional reporters. D–F, egl-30 is expressed in the AWB and AWC neurons, as shown by colocalization of the fluorescent signals generated by the Podr-1::mcherry (D) and the Pegl-30::gfp (E) transcriptional reporters. G, Expression of egl-30 in ADF neurons does not rescue the mutant phenotype of egl-30(ad805) in tph-1 expression. Two-way ANOVA after log transformation, n.s. p > 0.05 for genotype × treatment interaction, p < 0.001 for genotype, p = 0.07 for treatment; n ≥ 28 for each group; the data sets were tested as normal distribution by the Shapiro-Wilk test. Error bars represent SEM. H, I, Expression of egl-30 in both AWB and AWC fully rescues the defect of egl-30(ad805) (H) and egl-30(n686) (I) in tph-1 regulation. A significant genotype × treatment interaction (***p < 0.001) was detected by the Scheirer-Ray-Hare nonparametric test (H) or by two-way ANOVA after log transformation (I), p < 0.001 for genotype and treatment (H, I); n ≥ 35 for each group; the data sets were tested as deviated from normal distribution (H) or normal distribution (I) by the Shapiro-Wilk test. Error bars represent SEM. J, Expression of egl-30 in AWB neurons alone does not rescue the mutant phenotype of egl-30(ad805) in tph-1 expression. The genotype × treatment interaction (n.s., p > 0.05) was tested by the Scheirer-Ray-Hare nonparametric test (p = 0.12 for genotype, p < 0.05 for treatment; n ≥ 40 for each sample), because the data sets were tested as deviated from normal distribution by the Shapiro-Wilk test. Error bars represent SEM.

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Figure 5.

EGL-30 mediates the sensory function of the AWB and AWC olfactory neurons to regulate training-dependent increase in tph-1 transcription in ADF neurons. A, The resistance of egl-30(n686) mutants to PA14 is not significantly rescued by the expression of egl-30 in AWB and AWC neurons (n = 3 assays, 3 plates per assay; no significant difference among the egl-30(n686) groups; the difference between wild-type and all other genotypes is significant; log-rank test with Bonferroni correction; error bars represent SEM). B, C, GCaMP signals of AWB neurons in wild-type and egl-30(ad805) mutant animals (B) and in transgenic animals that express egl-30 selectively in AWB and nontransgenic siblings (C). D, GCaMP2 signals of AWC neurons in wild-type and egl-30(ad805) mutant animals. In B–D, solid lines indicate mean, and shading indicates SEM. *p < 0.05, Student's t test. E, F, Working model that describes the role of CaMKII/UNC-43 and Gqα/EGL-30 signaling pathway in regulating tph-1 transcription in ADF neurons in the naive (E) and trained (F) animals.

Next, we investigated the function of egl-30 in AWB and AWC sensory neurons. First, we found that expression of egl-30 in AWB and AWC neurons did not significantly alter locomotory speed (transgenic animals, 0.13 ± 0.04 pixels s⁻¹; nontransgenic siblings, 0.27 ± 0.13 pixels s⁻¹; Student's t test, p > 0.05; n = 5 animals each group; mean ± SEM), suggesting that egl-30 function in these neurons is not essential for locomotion. Using slow-killing assays (Kawli et al., 2010) to measure resistance to PA14 infection, we found that the activity of egl-30 in AWB and AWC also did not significantly change the susceptibility of the egl-30(n686) mutants (Fig. 5A), consistent with the previous finding on the role of intestinal EGL-30 in regulating pathogen resistance (Kawli et al., 2010).

Finally, we examined whether egl-30 mediates the sensory response of AWB and AWC, the major sensory neurons that detect repellents and attractants, respectively (Bargmann et al., 1993). The sensory ability of these two pairs of neurons is essential for worms to learn to avoid the smell of pathogenic bacteria (Ha et al., 2010), and both AWB and AWC cells are activated by the removal of medium conditioned by the worm food E. coli OP50 (Chalasani et al., 2007; Ha et al., 2010). Using intracellular calcium imaging and transgenic animals that selectively expressed the calcium-sensitive fluorescent protein GCaMP (Nakai et al., 2001) or GCaMP2.0 (Tallini et al., 2006), we found that compared with wild-type animals, egl-30(ad805) mutants displayed a significantly weaker calcium response in AWB neurons after the removal of OP50-conditioned medium (Fig. 5B). This defect was fully rescued by selective expression of egl-30 cDNA in AWB neurons (Fig. 5C). Similarly, egl-30(ad805) mutants showed a mild defect in the calcium response of AWC neurons (Fig. 5D). Together, these results indicate that EGL-30 functions in AWB and AWC neurons to regulate olfactory sensory response, suggesting an important role of sensory experience in the upregulation of tph-1 transcription during aversive training.