Inositol 1,4,5-trisphosphate signalling regulates the avoidance response to nose touch in Caenorhabditis elegans

Abstract

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP(3)R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cbeta (EGL-8) and phospholipase Cgamma (PLC-3), which catalyse the production of IP(3), both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch-induced Ca(2+) transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP(3)R in two specific responses mediated by ASH.

Figure 1. Disruption of IP₃ signalling in the nervous system.

(A) Schematic diagram showing the IP₃ signalling cassette. Stimulation of a receptor (R) at the cell surface leads to the activation of phopholipase C (PLC), which catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ diffuses to the endoplasmic reticulum (ER), where it activates the IP₃ receptor (IP₃R), resulting in the release of Ca²⁺ into the cytoplasm. Expression of an IP₃ sponge [see (B)] should mop up free IP₃, thus interfering with its ability to activate IP₃Rs. (B) Strategy used to disrupt IP₃ signalling in the nervous system using IP₃ sponges. ITR-1, the C. elegans IP₃R subunit, consists of 3 functional regions, including an IP₃ binding domain. Overexpression of the binding domain allows it to act as an IP₃ sponge. Expression under the control of the unc-119 promoter leads to nervous system-wide expression of the IP₃ sponge. (C) Strategy used to express dsRNA, and thus disrupt itr-1 expression, in the nervous system. Forward and reverse copies of an itr-1 cDNA fragment are expressed under the control of the unc-119 promoter. A “linker” region allows the complementary RNA regions to form dsRNA.

Figure 2. IP₃ and itr-1 function in the aversive response to nose touch.

The reversal response (measured in worm lengths) of animals exposed to nose touch or light anterior body touch. (A) Animals expressing IP₃ sponge derivatives under the control of the unc-119 promoter. (B) Animals expressing dsRNA under the control of the unc-119 promoter. IR, inverted repeat. (C) Animals carrying the itr-1(sa73) loss-of-function allele. All three methods of disrupting ITR-1 function significantly disrupt the nose touch response in comparison to wt animals (P<0.001, Chi-squared test, in each case). The control sponge (A) and lacZ control IR (B) do not disrupt the response (P>0.05).

Figure 3. itr-1 functions in the avoidance response to a volatile repellent, benzaldehyde, but not in other ASH-mediated responses.

(A–I) The reversal response of animals (measured in worm lengths; see key, bottom right) exposed to a range of stimuli. (A–F) Reversal responses in animals expressing itr-1 or lacZ dsRNA (IR, inverted repeat) under the control of the unc-119 promoter and treated with: (A) Buffer alone (30 mM Tris [pH 7.5], 100 mM NaCl, 10 mM KCl), (B) SDS, (C) copper, (D) glycerol, (E) quinine, and (F) fructose, at the concentrations indicated, using a “dry drop” assay . (G–I) Reversal response to benzaldehyde (undiluted) of (G) animals expressing dsRNA under the control of the unc-119 promoter (IR, inverted repeat); (H) wild type and itr-1 loss-of-function animals; (I) animals expressing IP₃ sponge derivatives under the control of the unc-119 promoter. (J) Response to octanol, measured as the time taken to reverse, following administration of the octanol concentrations indicated, as a “smell-on-a-stick” . Animals in which itr-1 is knocked down in the nervous system, or which carry the itr-1(sa73) mutation are defective in the response to benzaldehyde [P<0.001, (G)] but not to other repellants (P>0.05). However, the IP₃ sponge failed to disrupt the response to benzaldehyde [P>0.05, (I)]. All P values are from Chi-squared tests.

Figure 4. PLCβ and PLCγ function, through the production of an IP₃ signal, in the aversive response to nose touch, but not to benzaldehyde.

(A) Reversal response of PLC deficient animals to nose touch. All showed a wild type response (>90% reversing >1 worm length) to anterior body touch (data not shown). (B) Reversal response of itr-1(sy290) gain-of-function animals to nose touch, following depletion of plc-3 or egl-8 by RNAi. CAT, E. coli chloramphenicol acetyltransferase. All showed a wild type response (>90% reversing >1 worm length) to anterior body touch (data not shown). (C) Reversal response of PLC deficient animals to benzaldehyde. All showed a wild type response (<5% reversing >1 worm length) to buffer alone (data not shown). Both plc-3 and egl-8 loss-of-function mutants exhibit defective responses to nose touch (P<0.001) compared to wt animals. RNAi of plc-3 or egl-8 similarly disrupted the response (P<0.001) compared to the CAT(RNAi) control animals. However, RNAi of plc-3 or egl-8 in an itr-1(sy290) background failed to disrupt the response to such an extent (plc-3, P<0.001; egl-8, P<0.05, when compared to RNAi of the same genes in wt animals). All P values are from Chi-squared tests.

Figure 5. itr-1 functions in ASH.

Reversal response to nose touch. (A) Animals expressing IP₃ sponge derivatives under the control of cell-specific promoters. CS, control sponge; SS, super sponge. (B) itr-1(sa73) loss-of-function animals expressing full-length itr-1 cDNA under the control of cell-specific promoters. pHP2 is the empty destination vector used in construction of the other plasmids. All showed a wild type response (>90% reversing >1 worm length) to anterior body touch (data not shown). When the IP₃ sponge is expressed under control of the sra-6 promoter, nose touch response is disrupted (P>0.001), while expression under control of the glr-1 promoter has no effect (P>0.05). Expression of the control sponge using either promoter has no effect (P>0.05). All P values are from Chi-squared tests.

Figure 6. egl-8 and plc-3 function in ASH.

Reversal response to nose touch. (A) plc-3(tm1340) loss-of-function animals expressing plc-3 genomic DNA under the control of cell-specific promoters. (B) egl-8(n488) loss-of-function animals expressing an egl-8 rescuing “minigene” under the control of cell-specific promoters. All showed a wild type response (>90% reversing >1 worm length) to anterior body touch (data not shown). Expression of plc-3 or egl-8 under control of the sra-6 promoter significantly rescues nose touch response in their respective mutants (P<0.001), while expression using the glr-1 promoter does not (P>0.05). All P values are from Chi-squared tests.

Figure 7. itr-1, egl-8, and plc-3 all function in nose touch–induced Ca²⁺ transients in ASH.

Ratio changes in cameleon-expressing ASH neurons, following nose touch. (A) Quantification of responses. Diamonds are individual observations; longer red lines are mean; error bars are s.e.m. (n = 20). (B) Representative responses, for wild type animals and the mutants indicated. Black bar indicates duration of stimulation. The CFP/YFP ratio decreases over the course of the recordings because YFP photobleaches faster than CFP; noise is relatively low in some animals, due to higher cameleon expression levels. Nose touch–evoked Ca²⁺ transients were significantly disrupted in itr-1, egl-8, and plc-3 loss-of-function animals (P<0.05, Mann-Whitney rank sum test).