A neuronal signaling pathway of CaMKII and Gqα regulates experience-dependent transcription of tph-1

Abstract

Dynamic serotonin biosynthesis is important for serotonin function; however, the mechanisms that underlie experience-dependent transcriptional regulation of the rate-limiting serotonin biosynthetic enzyme tryptophan hydroxylase (TPH) are poorly understood. Here, we characterize the molecular and cellular mechanisms that regulate increased transcription of Caenorhabditis elegans tph-1 in a pair of serotonergic neurons ADF during an aversive experience with pathogenic bacteria, a common environmental peril for worms. Training with pathogenic bacteria induces a learned aversion to the smell of the pathogen, a behavioral plasticity that depends on the serotonin signal from ADF neurons. We demonstrate that pathogen training increases ADF neuronal activity. While activating ADF increases tph-1 transcription, inhibiting ADF activity abolishes the training effect on tph-1, demonstrating the dependence of tph-1 transcriptional regulation on ADF neural activity. At the molecular level, the C. elegans homolog of CaMKII, UNC-43, functions cell-autonomously in ADF neurons to generate training-dependent enhancement in neuronal activity and tph-1 transcription, and this cell-autonomous function of UNC-43 is required for learning. Furthermore, selective expression of an activated form of UNC-43 in ADF neurons is sufficient to increase ADF activity and tph-1 transcription, mimicking the training effect. Upstream of ADF, the Gqα protein EGL-30 facilitates training-dependent induction of tph-1 by functional regulation of olfactory sensory neurons, which underscores the importance of sensory experience. Together, our work elucidates the molecular and cellular mechanisms whereby experience modulates tph-1 transcription.

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.

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.

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.

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.

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.