Behavioral choice between conflicting alternatives is regulated by a receptor guanylyl cyclase, GCY-28, and a receptor tyrosine kinase, SCD-2, in AIA interneurons of Caenorhabditis elegans

Abstract

Animals facing conflicting sensory cues make a behavioral choice between competing alternatives through integration of the sensory cues. Here, we performed a genetic screen to identify genes important for the sensory integration of two conflicting cues, the attractive odorant diacetyl and the aversive stimulus Cu(2+), and found that the membrane-bound guanylyl cyclase GCY-28 and the receptor tyrosine kinase SCD-2 regulate the behavioral choice between these alternatives in Caenorhabditis elegans. The gcy-28 mutants and scd-2 mutants show an abnormal bias in the behavioral choice between the cues, although their responses to each individual cue are similar to those in wild-type animals. Mutants in a gene encoding a cyclic nucleotide gated ion channel, cng-1, also exhibit the defect in sensory integration. Molecular genetic analyses suggested that GCY-28 and SCD-2 regulate sensory integration in AIA interneurons, where the conflicting sensory cues may converge. Genetic ablation or hyperpolarization of AIA interneurons showed nearly the same phenotype as gcy-28 or scd-2 mutants in the sensory integration, although this did not affect the sensory response to each individual cue. In gcy-28 or scd-2 mutants, activation of AIA interneurons is sufficient to restore normal sensory integration. These results suggest that the activity of AIA interneurons regulates the behavioral choice between the alternatives. We propose that GCY-28 and SCD-2 regulate sensory integration by modulating the activity of AIA interneurons.

Figure 1.

gcy-28 mutants show behavioral defects in the interaction of the two sensory signals. A, Schematic diagram for an assay to test the interaction of two sensory signals, called the “interaction assay.” Worms must cross aversive Cu²⁺ barrier spread on the midline of the assay plate to reach the spot containing the attractive odorant diacetyl. Forty-five minutes after the start of the assay, the numbers of worms on the original side (A) and on the odorant side (B) were scored. The index was calculated as A/(A+B). B, Phenotypes of gcy-28 mutants in the interaction assay. Error bars in B–D indicate SEM. *p < 0.01. C, Chemotaxis toward various concentrations of diacetyl of gcy-28(qj4) was analyzed by the conventional chemotaxis assay. D, Avoidance of various concentrations of Cu²⁺ of gcy-28(qj4) was analyzed by the quadrant assay. In all points, the differences were not significant (p > 0.01). E, Gene structure and mutations of gcy-28. Three alternative splice forms (gcy-28.a, gcy-28.c, gcy-28.d) contain partly different 5′ exons encoding the extracellular domain. Black bars indicate deletions, and a filled triangle indicates a missense mutation. An open triangle indicates Mos1 insertion. ky713 missense mutation results in substitution of an amino acid in the conserved cyclase domain (Tsunozaki et al., 2008). The alleles tm3028, ky887, and tm2411 are deletions predicted to cause a frameshift. Mos1 transposon insertion (qj4) is located in the fourth exon (I:4499749..4499750) and would result in premature termination of GCY-28 protein at the extracellular domain. WT, Wild type.

Figure 2.

GCY-28 in the AIA interneurons is important for the interaction of two signals. A, The expression of the gcy-28.d::gfp fusion gene under control of the gcy-28.d promoter at the adult stage. The site of expression is indicated by an arrowhead. Scale bar, 10 μm. B, The phenotypes of gcy-28 mutant expressing gcy-28.d cDNA in AWC^ON+OFF neurons or AWC^ON were analyzed by the interaction assay. Worms carrying the transgene (+Ex, gray columns) and worms not carrying the transgene (−Ex, black columns) were reared on the same culture plate and subjected to the assay on the same assay plate to compare the indices. White columns indicate nontransgenic worms reared on separate culture plates and subjected to the assay on separate assay plates. C–E, The phenotypes of gcy-28 mutants expressing gcy-28.c cDNA by various promoters were analyzed by the interaction assay. The expression of H20 promoter: pan-neuron. The expression of ins-1 promoter: neurons including AIA. The expression of unc-86 promoter: several neurons including AIZ. The expression of ttx-3 promoter: AIY. The expression of odr-2 promoter: neurons including AIB. The expression of tax-4, odr-10, or osm-3 promoter: sensory neurons including ADL, ASE, ASH, AWA, and AWC. gcy-28(qj4) animals carrying phsp::gcy-28.c were heat shocked at young adult stage. F, The schematic diagram for the putative neural network for the interaction of two sensory signals, extracted from the complete circuit (White et al., 1986). Sensory neurons are shown as triangles and interneurons as hexagons. Arrows indicate chemical synapses, and lines with T-shaped ends indicate gap junctions. The AIA neurons expressing GCY-28.d are emphasized. G, gcy-28 mutants expressing gcy-28 cDNA in AIA interneurons by gcy-28.d promoter were analyzed in the interaction assay. Open bars show nontransgenic animals for comparison. B–E, G, +Ex and −Ex worms were compared. Error bars in B–E and G indicate SEM. *p < 0.01. WT, Wild type.

Figure 3.

gcy-28 and cng-1 regulate the interaction of two sensory signals in the same genetic pathway. A, Phenotypes of cng-1 mutants in the interaction assay. Error bars in A–D and F indicate SEM. *p < 0.01. B, Chemotaxis toward various concentrations of diacetyl of cng-1 was analyzed by the conventional chemotaxis assay. C, Avoidance of various concentrations of Cu²⁺ of cng-1 was analyzed by the quadrant assay. D, The genetic interaction between cng-1 and gcy-28 in the interaction assay (15 mm copper acetate was used as Cu²⁺ barrier). E, Confocal views of wild-type animals expressing GFP under the control of the cng-1 promoter at the adult stage. The expression of GFP was colocalized with monomeric red fluorescent protein expressed in AIA by ins-1 promoter (bottom, arrow). Scale bars, 10 μm. F, RNAi knockdown of cng-1 in AIA interneurons of wild-type animals; their behavior was analyzed in the interaction assay. Worms carrying the transgene (+Ex) and worms not carrying the transgene (−Ex) were compared. WT, Wild type.

Figure 4.

SCD-2 in AIA interneurons is important for the interaction of two sensory signals. A, The genetic interaction between hen-1 and scd-2 in the interaction assay. Error bars in A–C, E, and F indicate SEM. B, Chemotaxis toward various concentrations of diacetyl of scd-2 was analyzed by the conventional chemotaxis assay. C, Avoidance of various concentrations of Cu²⁺ of scd-2 was analyzed by the quadrant assay. D, Confocal view of wild-type animal expressing the functional scd-2::gfp fusion gene under the control of its own promoter at the adult stage. The colocalization of GFP with monomeric red fluorescent protein expressed in AIA by ins-1 promoter is indicated by arrows. Scale bar, 10 μm. E, The phenotype of scd-2 mutants expressing scd-2 cDNA by gcy-28.d promoter was analyzed by the interaction assay. Worms carrying the transgene (+Ex) and worms not carrying the transgene (−Ex) were compared. *p < 0.01. F, Analysis of gcy-28;scd-2 double mutants in the interaction assay. *p < 0.01. WT, Wild type.

Figure 5.

The AIA interneurons regulate sensory integration. A, The functions of AIA interneurons were inhibited by the expression of MEC-4(d) or constitutively active UNC-103 in wild-type animals; their behavior was analyzed in the interaction assay. B, Chemotaxis toward various concentrations of diacetyl of worms expressing MEC-4(d) or UNC-103(gf) in AIA was analyzed by the conventional chemotaxis assay. C, Avoidance of various concentrations of Cu²⁺ of worms expressing MEC-4(d) or UNC-103(gf) in AIA was analyzed by the quadrant assay. All points showed no significant differences from wild type (p > 0.01). D, E, The expression of constitutively active PKC-1 in AIA interneurons could rescue the defects of gcy-28 mutants, scd-2 mutants, and cng-1 mutants in the interaction assay. F, The effect of the expression of constitutively active PKC-1 in AIA interneurons on the phenotype of wild-type worms in the interaction assay (100 mm copper acetate was used as Cu²⁺ barrier). A–F, Worms carrying the transgene (+Ex) and worms not carrying the transgene (−Ex) were compared. Error bars in A–F indicate SEM. *p < 0.01. WT, Wild type.

Figure 6.

AIA interneurons receive excitatory and inhibitory inputs from distinct sensory neurons. A, Calcium responses of AWA sensory neurons to diacetyl measured by the Ca²⁺-sensitive fluorescent indicator Cameleon in wild-type animals. The black line indicates the average trace, and the shaded areas around the trace represent SEM (n = 3 animals). The black bar indicates the presence of diacetyl. B, The effect of mutations in glutamate-gated chloride channel GLC-3 on the behavior of wild-type animals and gcy-28 mutants in the interaction assay (100 mm copper acetate was used as Cu²⁺ barrier). Worms carrying the transgene (+Ex) and worms not carrying the transgene (−Ex) were compared. Error bars indicate SEM. *p < 0.01. C, A neuronal circuit model for the interaction of two sensory signals. Sensory neurons are shown as triangles, and interneurons, as hexagons. Arrows indicate excitatory connections, and lines with T-shaped ends indicate inhibitory connections. The activity of AIA interneurons would be regulated by the balance between excitatory inputs from AWA through gap junction and inhibitory inputs from ADL/ASH through GLC-3. Activated AIA interneurons would increase the relative strength of the signals for diacetyl by inhibiting the signals for Cu²⁺. GCY-28 and SCD-2 may enhance the inhibitory synaptic outputs from AIA.