Reversal of salt preference is directed by the insulin/PI3K and Gq/PKC signaling in Caenorhabditis elegans

Abstract

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the Gq signaling pathway composed of Gqα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.

Figure 1.—

Loss of a receptor-type guanylyl cyclase GCY-22 suppresses the chemotaxis defect of daf-18 mutants. (A) pe902, pe905, pe917, and pe922 were obtained in a suppressor screen for mutations that rescue the chemotaxis defect of daf-18(e1375) mutants. Naive animals were tested for NaCl chemotaxis. A deletion mutation gcy-22(tm2364) also suppresses the chemotaxis defect of the daf-18 mutants. (B) pe902, pe905, pe917, pe922, and tm2364 cause defects in attraction behavior to the ASER-sensed chemical ammonium chloride. (C) Genomic structure of gcy-22 receptor-type guanylyl cyclase. Solid boxes indicate predicted protein domains. Locations of the four suppressor mutations in the gcy-22 gene are depicted. Error bars represent standard error of the mean (SEM). (***) P < 0.001, (**) P < 0.01, Bonferroni t-test.

Figure 2.—

ASER promotes salt avoidance and ASEL promotes salt attraction in daf-18 mutants. (A) NaCl chemotaxis of wild-type animals, daf-18(mg198null), age-1(hx546rf), akt-1(ok525null), daf-18(mg198); age-1(m333null), and daf-18(mg198); akt-1(ok525null) mutants under naive conditions. On the new assay format (see Figure S1B), daf-18(mg198) mutants show salt avoidance. Mutations of age-1 and akt-1 suppress the salt avoidance of the daf-18 mutants. (B) Effect of the dysfunction of ASE neurons due to the che-1(p674) mutation. The che-1(p674) mutation suppresses the salt avoidance of daf-18(mg198) mutants. (C) Chemotaxis of daf-18(mg198) mutants with 2-ASEL (otIs204[ceh-36p∷lsy-6]) or 2-ASER (lsy-6(ot71)). ASER promotes aversion and ASEL promotes attraction in daf-18 mutants. (D) A model deduced from the analysis of the role of ASER in daf-18 mutants. ASER promotes salt avoidance when the PI 3-kinase signaling is activated. Error bars represent SEM. (***) P < 0.001, (*) P < 0.05, Bonferroni t-test. n.s., not significant.

Figure 3.—

The receptor-type guanylyl cyclase GCY-22 in ASER promotes salt avoidance. (A) Rescue experiments with expression of gcy-22 under its own promoter or ASER-specific gcy-5 promoter in the daf-18(e1375); gcy-22(pe917) mutant. (B) Suppression of the avoidance behavior of daf-18; 2-ASER mutants by the gcy-22(pe917) mutation. NaCl chemotaxis of wild-type animals, lsy-6(ot71), daf-18(mg198); lsy-6(ot71), lsy-6(ot71) gcy-22(pe917), and daf-18(mg198); lsy-6(ot71) gcy-22(pe917) mutants under naive conditions. Error bars represent SEM. (***) P < 0.001, (**) P < 0.01, Bonferroni t-test. n.s., not significant.

Figure 4.—

G_q/PKC signaling promotes salt attraction antagonistic to the insulin/PI 3-kinase signaling. (A) pe914 mutation causes an amino acid substitution in the linker I domain of the EGL-30 G_qα protein. (B) egl-30(pe914) was expressed under the ASER-specific gcy-5 promoter or under the ASEL-specific gcy-7 promoter. Expression of egl-30(pe914) in ASER switches the avoidance behaviors of daf-18(mg198) mutants and daf-18(mg198); lsy-6(ot71) mutants. (C) A gain-of-function form of nPKC TTX-4, TTX-4(A160E), was expressed in ASER under the gcy-5 promoter or in ASEL under the gcy-7 promoter. Expression of TTX-4(A160E) in ASER switches the avoidance behaviors of daf-18(mg198) mutants and daf-18(mg198); lsy-6(ot71) mutants. Error bars represent SEM. (***) P < 0.001, (*) P < 0.05, Bonferroni t-test. n.s., not significant.

Figure 5.—

Taste receptor neurons ASE promote salt avoidance and other sensory neurons are also required for salt avoidance. (A) Learning assays were performed with wild-type animals, che-1(p674) mutants, 2-ASEL animals (otIs204[ceh-36p∷lsy-6]), and 2-ASER animals (lsy-6(ot71)). che-1 mutants do not show salt chemotaxis plasticity, whereas 2-ASEL animals and 2-ASER animals show plasticity. (B) Learning assays were performed with the OH8585 strain (ASEL ablated) and OH8593 strain (ASER ablated). Both strains show impaired learned avoidance but still show plasticity. (C) Learning assays were performed with wild-type animals, gcy-22(pe917) mutants, lsy-6(ot71) animals (2-ASER), and lsy-6(ot71) gcy-22(pe917) mutants. ASER promotes learned salt avoidance depending on gcy-22. (D) dyf-11 was expressed under the dyf-11 promoter, in ASER under the gcy-5 promoter, or in ASEL under the gcy-7 promoter in the dyf-11(pe554) mutant background. The dyf-11 mutation causes dysfunction of sensory neurons. Selective rescue of ASER, ASEL, or both restore salt attraction after mock conditioning, but do not rescue the learned avoidance. (E) dyf-11 was expressed under the dyf-11 promoter or in ASER under the gcy-5 promoter in the dyf-11(pe554) mutant background or in the daf-18(mg198); dyf-11(pe554) mutant background. Expression of DYF-11 in ASER restores the attractive response in dyf-11 mutants. The daf-18 mutation downregulates the response of ASER but does not cause salt avoidance. Error bars represent SEM. (***) P < 0.001, (**)P < 0.01, (*) P < 0.05, Bonferroni t-test. n.s., not significant.

Figure 6.—

Constitutive activation of the G_q/diacylglycerol/PKC signaling fixes ASER to promote attraction behavior. (A) Animals were treated in a mock-conditioning liquid or a NaCl-conditioning liquid with or without the diacylglycerol analog PMA. Treatment with PMA disrupts starvation/NaCl learning and causes attraction behavior after the NaCl conditioning. (B) TTX-4(A160E) was expressed in ASER under the gcy-5 promoter or in ASEL under the gcy-7 promoter. Expression of TTX-4(A160E) in ASER causes attraction behavior even after NaCl conditioning. (C) ttx-4 mutants show defects in salt attraction but not in salt avoidance. (D) Schematic of molecular mechanisms in ASE neurons and AWC neurons that regulate gustatory and olfactory plasticity, respectively (see text). Guanylyl cyclases GCY-22, GCY-14, DAF-11, and ODR-1 are required for chemosensation. GCY-22 acts in ASER and GCY-14 acts in ASEL for sensing ions (Ortiz et al. 2009). In AWC, ODR-1 and DAF-11 are required for sensing odors (Birnby et al. 2000; L'etoile and Bargmann 2000). Error bars represent SEM. (***) P < 0.001, (**) P < 0.01, Bonferroni t-test.