Worm Breeder's Gazette 15(1): 39 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Dept. of Anesthesiology, Washington University School of Medicine, St. Louis, MO.
Halothane is a volatile general anesthetic that disrupts coordinated movement in C. elegans at low, clinically-relevant concentrations (1) (0.2-0.5 volume % halothane), and abolishes gross movment at higher concentrations (3.5 vol. %). The nematicide drug aldicarb, an inhibitor of cholinesterase, paralyzes worms (>0.5 mM for N2) due to an excess of acetylcholine accumulating at the synapse. Mutations in pre-synaptic machinery can either confer resistance or hypersensitivity to aldicarb (a ric vs hic phenotype). Previous work has implicated several pre-synaptic components in halothane-induced anesthesia. We hypothesized that should halothane disrupt pre-synaptic machinery, then the reduction in acetylcholine release would confer resistance to aldicarb. Indeed, halothane makes worms ric. Halothane-induced resistance to aldicarb was measured by a simple movement assay. Worms (N = 20-30) were exposed to a seeded aldicarb plate for 4 hours, and were then picked into a 0.5 cm circle outlined on the same plate. An hour later, the percentage of animals having managed to crawl completely out of the circle was scored. Each assay was done in triplicate at various halothane concentrations within air-tight chambers,including a no-halothane control. Table 1 summarizes results for N2 and some mutant strains. Low, clinically-relevant, concentrations of halothane (0.2-0.5 vol. %) significantly reversed aldicarb-induced paralysis. Interestingly, higher concentrations of halothane (1-1.5 vol. %) do not show a significant effect in N2; the reversal effect peaks at 0.2-0.3 vol. % halothane and then gradually decreases to near control levels. Similar experiments performed with the acetylcholine agonist levamisole and halothane failed to show any reversal, suggesting that halothan acts pre-synaptically (data not shown). We tested several mutant lines for disruption of halothane-induced resistance to aldicarb, reasoning that by blocking this effect one might highlight clinically relevant molecular pathways targeted by volatile anesthetics. Aldicarb concentrations were optimized according to the ricness or hicness of each strain. Shown in Table 1 are results for some G protein and calcium channel mutant strains. egl-30 (n686) (a.k.a.Gqalpha) does not reverse at all, and neither do calcium channel mutations egl-19 (ad1013)lof and (n2368)gof. On the other hand, goa-1 (sy192) reverses significantly at low halothane concentrations, but continues to show robust reversals at higher concentrations as well. The same striking response was also seen for egl-10 (nIs51), which overexpresses the upstream negative regulator of goa-1 signalling. In conclusion, the anesthetic halothane can result in an organism "waking up." By understanding aldicarb's mechanism of action, some light is shed on the way volatile anesthetics work. The analysis of numerous mutants suggests that halothane has a pre-synaptic effect at clinically relevant concentrations, and that G proteins and calcium channels are involved . Table 1. (Crawling Indeces) ** significant movement compared to control Halothane Concentration Range (vol. %) Strains 0 (control) 0.2 - 0.5 1.0 - 1.5 [aldicarb] mM N2 0.16+/-0.04 0.52+/-0.04* 0.27+/-0.04 0.5 egl-30(n686) 0.14+/-0.02 0.15+/-0.03 0.04+/-0.01 0.8 egl-19(n2368) 0.15+/-0.03 0.20+/-0.03 0.05+/-0.02 0.5 egl-13(ad1013)0.20+/-0.03 0.23+/-0.03 0.11+/-0.02 0.5 goa-1(sy192) 0.10+/-0.02 0.62+/-0.06* 0.65+/-0.02* 0.1 egl-10(nIS51) 0.01+/-0.01 0.49+/-0.07* 0.63+/-0.03* 0.2 1. Crowder, C.M., Shebester, L.D. & Schedl, T (1996) Anesthesiology 85, 901-912.