The monoaminergic modulation of sensory-mediated aversive responses in Caenorhabditis elegans requires glutamatergic/peptidergic cotransmission

Abstract

Monoamines and neuropeptides interact to modulate behavioral plasticity in both vertebrates and invertebrates. In Caenorhabditis elegans behavioral state or "mood" is dependent on food availability and is translated by both monoaminergic and peptidergic signaling in the fine-tuning of most behaviors. In the present study, we have examined the interaction of monoamines and peptides on C. elegans aversive behavior mediated by a pair of polymodal, nociceptive, ASH sensory neurons. Food or serotonin sensitize the ASHs and stimulate aversive responses through a pathway requiring the release of nlp-3-encoded neuropeptides from the ASHs. Peptides encoded by nlp-3 appear to stimulate ASH-mediated aversive behavior through the neuropeptide receptor-17 (NPR-17) receptor. nlp-3- and npr-17-null animals exhibit identical phenotypes and animals overexpressing either nlp-3 or npr-17 exhibit elevated aversive responses off food that are absent when nlp-3 or npr-17 are overexpressed in npr-17- or nlp-3-null animals, respectively. ASH-mediated aversive responses are increased by activating either Galpha(q) or Galpha(s) in the ASHs, with Galpha(s) signaling specifically stimulating the release of nlp-3-encoded peptides. In contrast, octopamine appears to inhibit 5-HT stimulation by activating Galpha(o) signaling in the ASHs that, in turn, inhibits both Galpha(s) and Galpha(q) signaling and the release of nlp-3-encoded peptides. These results demonstrate that serotonin and octopamine reversibly modulate the activity of the ASHs, and highlight the utility of the C. elegans model for defining interactions between monoamines and peptides in individual neurons of complex sensory-mediated circuits.

Figure 1.

Monoamine-mediated G-protein signaling modulates ASH-mediated aversive responses to dilute octanol. Wild-type and G-protein signaling mutants were examined for aversive responses to dilute (30%) octanol in the presence and absence of exogenous 5-HT, OA or 5-HT and OA (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. Black, Off food; gray, +OA; white, +5-HT; hatched, +5-HT+OA.

Figure 2.

Monoamines modulate G-protein signaling in the ASH sensory neurons. Wild-type animals expressing RNAi transgenes were assayed for aversive responses to dilute (30%) octanol in the presence or absence of exogenous 5-HT, OA, or 5-HT and +OA (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. Black, Off food; gray, +OA; white, +5-HT; hatched, +5-HT+OA. ASH-selective promoter, sra-6p; ASI-selective promoter, gpa-4p.

Figure 3.

SER-5 signaling in ASH sensory neurons. Wild-type and mutant animals were examined for aversive responses to dilute (30%) octanol either in the presence or absence of exogenous 5-HT (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. ASH-selective promoter, sra-6p.

Figure 4.

Peptides encoded by nlp-3 are essential for the 5-HT-dependent stimulation of aversive responses to dilute octanol. Wild-type, mutant, and transgenic animals were examined for aversive responses to dilute (30%) octanol in the absence (top) or presence (bottom) of exogenous 5-HT (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. ASH-selective promoter, sra-6p.

Figure 5.

The release of peptides encoded by nlp-3 is essential for Gα_s stimulation of aversive responses mediated by the ASH sensory neurons. Animals were examined for aversive responses to dilute octanol (30%) in the absence of food or exogenous 5-HT (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. Black, Off food; white, +5-HT; gray, +OA. ASH-selective promoter, sra-6p.

Figure 6.

The RNAi knockdown of nlp-3 in the ASHs abolishes the food stimulation of aversive responses to dilute octanol. Aversive responses to dilute octanol were examined in wild-type animals in the presence of E. coli (OP50) after the promoter-selective RNAi knockdown of nlp-3 (ASHs, sra-6p::nlp-3; NSMs, ceh-2p::nlp-3; ADFs, srh-142p::nlp-3; AWBs, str-1p::nlp-3). Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions. Gray, +Food.

Figure 7.

5-HT stimulates ASH-mediated aversive responses in eat-4-null animals. Wild-type and mutant animals were examined for aversive responses to dilute octanol (30%) in the presence or absence of exogenous 5-HT (4 mm), as described in Materials and Methods. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from animals incubated in the absence of 5-HT. **Significantly different from eat-4-null animals incubated under identical conditions. Black, Off food; white, +5-HT. ASH-selective promoter, sra-6p.

Figure 8.

Octanol avoidance in eri-1 animals after the knockdown of predicted neuropeptide receptors by RNAi feeding. eri-1(kp3948) animals expressing global RNAi after bacterial feeding were examined for aversive responses to dilute octanol in the presence of 5-HT (4 mm). Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical conditions.

Figure 9.

npr-17 encodes a neuropeptide receptor that appears to be activated by nlp-3-encoded peptides. A, Putative neuropeptide receptor-null mutants were examined for 5-HT-dependent increases in aversive responses to dilute octanol (30%), as described in Materials and Methods. B, Putative neuropeptide receptor-null mutants overexpressing nlp-3p::nlp-3 were examined for aversive responses to dilute octanol. C, Wild-type and npr-17-null animals expressing npr-17p::npr-17 transgenes were examined for aversive responses to dilute octanol in the presence or absence of 5-HT. Black, Off food; white, +5-HT. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions.

Figure 10.

npr-17 is expressed in subsets of head and tail neurons. GFP fluorescence from a npr-17p::gfp transcriptional fusion. npr-17 transgene includes 5 kb upstream of the start ATG, as well as the first exon and intron of npr-17, fused to sequence encoding GFP. Merge of GFP fluorescence and DiD (a lipophilic dye) staining in the nerve ring.

Figure 11.

nlp-3- and npr-17-null animals exhibit identical phenotypes. Wild-type and mutant animals were examined for aversive responses to 100% octanol in the presence and absence of OA (4 mm) and spontaneous reversal immediately off food. Data are presented as mean ± SE and analyzed by two-tailed Student's t test. *p < 0.001, significantly different from wild-type animals under identical test conditions.

Figure 12.

Model of G-protein modulation of aversive responses mediated by the ASH sensory neurons. Green and red represent stimulation and/or inhibition, respectively, of aversive responses to dilute octanol.