The G protein regulators EGL-10 and EAT-16, the Giα GOA-1 and the G(q)α EGL-30 modulate the response of the C. elegans ASH polymodal nociceptive sensory neurons to repellents

Abstract

Background: Polymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation.

Results: We discovered that the regulator of G protein signalling (RGS) protein, EGL-10, is required for appropriate avoidance responses to noxious stimuli sensed by ASH. As it does for other behaviours in which it is also involved, egl-10 interacts genetically with the G(o)/(i)α protein GOA-1, the G(q)α protein EGL-30 and the RGS EAT-16. Genetic, behavioural and Ca²(+) imaging analyses of ASH neurons in live animals demonstrate that, within ASH, EGL-10 and GOA-1 act downstream of stimulus-evoked signal transduction and of the main transduction channel OSM-9. EGL-30 instead appears to act upstream by regulating Ca²(+) transients in response to aversive stimuli. Analysis of the delay in the avoidance response, of the frequency of spontaneous inversions and of the genetic interaction with the diacylglycerol kinase gene, dgk-1, indicate that EGL-10 and GOA-1 do not affect signal transduction and neuronal depolarization in response to aversive stimuli but act in ASH to modulate downstream transmission of the signal.

Conclusions: The ASH polymodal nociceptive sensory neurons can be modulated not only in their capacity to detect stimuli but also in the efficiency with which they respond to them. The Gα and RGS molecules studied in this work are conserved in evolution and, for each of them, mammalian orthologs can be identified. The discovery of their role in the modulation of signal transduction and signal transmission of nociceptors may help us to understand how pain is generated and how its control can go astray (such as chronic pain) and may suggest new pain control therapies.

Figure 1

egl-10 is involved in avoidance of aversive stimuli. N2 is the wild-type control, and egl-10 is egl-10(md176). For each genetic background, ≥50 animals were tested in at least three independent assays: in (a) and (b), each animal was subjected to three trials; in (c), each animal was subjected to 30 trials. The avoidance index is the number of positive responses divided by the total number of trials. In (B), avoidance is expressed as the time in seconds that the animal took to respond. In all panels, each bar represents the mean ± SEM. *Difference from N2, P < 0.01.

Figure 2

Avoidance responses of single and double mutants. N2 is the wild-type control, egl-10 is egl-10(md176), egl-30 is egl-30(n686), eat-16 is eat-16(ad702), goa-1 is goa-1(n363) and dgk-1 is dgk-1(nu62). The transgene psra-6::PTX is described in the text, in the Results section and represents an ASH-specific goa-1 loss of function. For each genetic background, ≥50 animals were tested in at least three independent assays, and each animal was subjected to three trials. The avoidance index is the number of positive responses divided by the total number of trials. In all panels, each bar represents the mean ± SEM. In (b), different concentrations of glycerol were used to detect the hypersensitivity of the wild type and of the eat-16, goa-1 and dgk-1 mutants. *Difference from N2, P < 0.01; **difference from egl-10(md176), P < 0.01; ***difference from egl-30(n686), P < 0.01.

Figure 3

egl-10, goa-1 and eat-16 function in ASH to affect avoidance. N2 is the wild-type control, egl-10 is egl-10(md176), eat-16 is eat-16(ad702) and goa-1 is goa-1(n363). The transgenes psra-6::egl-10, psra-6::eat-16, podr-10::egl-10 and psra-6::PTX are described in the text, in the Results and in the Methods sections. psra-6::PTX represents an ASH-specific goa-1 loss of function. For each genetic background, ≥50 animals were tested in at least three independent assays, and each animal was subjected to three trials. The avoidance index is the number of positive responses divided by the total number of trials. In all panels, each bar represents the mean ± SEM. *Difference from egl-10(md176), P < 0.01; **difference from egl-10(md176); eat-16(ad702), P < 0.01. For each genetic background, only the results obtained with animals from one transgenic line are represented in the figure. The results from the other lines obtained and tested are reported in Additional file 2.

Figure 4

egl-10, eat-16 and goa-1, but not egl-30 mutant animals show normal stimulus-evoked Ca²⁺ transients in ASH neurons. Ca²⁺ transients in ASH neurons. All animals carry the psra-6::G-CaMP transgene driving expression of the G-CaMP Ca²⁺ sensor in ASH. Genetic background is wild type for control, egl-10(md176) for egl-10, egl-30(n686) for egl-30, eat-16(ad702) for eat-16 and goa-1(n363) for goa-1. ASH Ca²⁺ transients are reported in response to (a) high osmotic strength stimulus, (b) quinine and (c) copper. For each repellent and for each genotype, at least 20 animals were tested. Three individual imaging trials were recorded for each animal. In the graph panels, the time courses of the change in green fluorescent protein (GFP) fluorescence intensity after stimulus delivery are shown relative to averaged prestimulus (10 seconds) baseline (ΔF/F); the grey bands represent the SEM for each time point (0.2 second). The black horizontal bars indicate the time and duration of the stimulus. In the histogram panels, the means ± SEM of the maximum ΔF/F of all the imaging trials for a given stimulus and genotype are reported. *Difference from control, P < 0.01.

Figure 5

egl-10 and eat-16 have opposite effects on the frequency of spontaneous reversals. N2 is the wild-type control, egl-10 is egl-10(md176) and eat-16 is eat-16(ad702). For each genetic background, ≥50 animals were tested in at least three independent assays. Numbers of spontaneous inversions per minute are represented. Each bar represents the mean ± SEM. *Difference from N2, P < 0.01; **difference from egl-10(md176), P < 0.01.

Figure 6

A model for the ASH modulatory pathways. The model is discussed in the text, in the Discussion section. The molecules in the ovals are those studied in this paper. Only the main players are depicted. The question marks indicate that only the type of molecule (GPCR) is known, while its precise identity is not. DAG, diacylglycerol; PA, phosphatidic acid. Arrows indicate proven activities or pathways. Stimuli and molecules upstream of the two modulatory pathways: food, dopamine, octopamine, serotonin, DOP-3, OCTR-1 and SER-5 have been identified by Wragg et al. [10], Harris et al. [11] and Ezak and Ferkey [35] as modulators of the response to dilute octanol.