Thermal avoidance in Caenorhabditis elegans: an approach to the study of nociception

Abstract

Upon perception of a noxious stimulus, an organism executes defense mechanisms, such as escape responses. The molecular basis of these mechanisms is poorly understood. In this paper we show that upon exposure to noxious temperature, Caenorhabditis elegans reacts by a withdrawal reflex. To analyze this thermal avoidance behavior, we developed a laser-based assay to quantify the response. The escape reflex can be observed in 98% of the adult animals, but is not executed in animals in diapause. The thermal avoidance response differs significantly from the thermotaxis behavior that is based on the perception of physiological temperature. It involves different neurons and is influenced by mutations in distinct genes. As in mammals, the strength of the thermal avoidance response is increased by application of capsaicin, the pungent ingredient in chili peppers. We find that thermal avoidance is strongly reduced in mutants affecting the neural transmission modulated by glutamate and neuropeptides as well as in mutants affecting the structure and function of sensory neurons. We suggest that the study of this nociceptive behavior in C. elegans can be used to understand the genetic and molecular basis of thermal nociception.

Figure 1

Tav response of wild-type animals at different developmental stages. (a) The behavior of animals in a nonstarved population. Twelve hundred animals (L4 or adult animals) were tested over 15 days, and the Tav response was scored according to the behavioral classes defined in Materials and Methods (solid bars, class I; darkly shaded bars, class II; lightly shaded bars, class III; open bars, class IV). (b) Dauer animals do not respond to noxious heat. Two hundred and sixty-seven animals in the dauer stage were tested on 2 days. (c) In comparison, 122 animals of the alternative L3 non-dauer stage. (d) After exit from the dauer stage, animals exhibit a normal Tav response again. Two hundred and five adult animals recovered from dauer were tested on 2 days. Bars represent the percentage of animals within a particular response class. Error bars represent SE.

Figure 2

Capsaicin-induced hyperalgesia is blocked by the competitive inhibitor capsazepine. Wild-type and osm-9(ky10) mutant animals were tested in liquid culture after the addition of the chemicals, as indicated. Bars represent the percentage of animals that responded (solid bars) or did not respond (open bars) in the Tav assays. Control, addition of 4% ethanol in M9; +cap, 100 μM capsaicin; +cap+czp, preincubation in 100 μM capsazepine, followed by addition of 100 μM capsaicin; +czp, 100 μM capsazepine. Shading of the bars is as in Fig. 1; for statistical data, see text.

Figure 3

Thermoreceptive regions/neurons in C. elegans. (a) The schematic drawing shows an adult animal with the anterior/tip of the nose to the left and the posterior/tail to the right. Somata of neurons are darkly shaded. The solid areas are the somata and axonal processes of AFD, AIY, and AIZ neurons that mediate thermotaxis response (34). The table below the drawing shows the percentage of wild-type (wt) and vab-3(e648) animals that responded to the heat stimulus in the indicated body region. The size of the laser is indicated to the left. (b) Neural circuit for thermotaxis (modified after ref. 34) showing the connectivity of AFD, AIY, and AIZ. Both ablation of the sensory neuron AFD or the interneuron AIY and mutations eliminating their functions lead to a cryophilic phenotype, whereas ablation or mutations abolishing the function of AIZ lead to a thermophilic phenotype (34). Neither mutant affected the thermal avoidance response.