Worm Breeder's Gazette 1(2): 17
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We have isolated several hundred levamisole (1-tetramisole)- resistant mutants and are characterizing them genetically and pharmacologically. There are at least 8 complementation groups including the four most frequent discovered by Brenner (tmr-1 to 4). The fifth most frequent maps right next to tmr-2 and tmr-3. Among the much rarer complementation groups are one corresponding to unc-50 (LG III) and two at least not strongly linked to dpy-5 (LG I). Mutants in seven of the complementation groups all have about the same mildly uncoordinated phenotype, extreme tetramisole-resistance, and a sensitivity to osmotic shock. The uncoordinated phenotype and resistance to levamisole can be phenocopied by high concentrations of mecamylamine, an acetylcholine blocking agent. Rare mutants resistant to mecamylamine can be isolated and their properties are being investigated viz. levamisole resistance. Revertants, ts mutants, etc. are being sought. We have developed a cut worm assay of body muscle contraction to compare the pharmacology of the wild type and our mutants . We have found that C. elegans cut in half will survive happily for hours in Ascaris Ringer's solution. The low sensitivity of the wild type to acetylcholine is greatly potentiated by esterase inhibitors (eserine, Aldicarb, Trichlorfon). Tetramisole and nicotinic agonists, nicotine and carbachol, cause rapid contraction by themselves. Muscarine, methacholine and carbamyl-B-methyl-choline, muscarinic agents, have little effect. Cholinergic blocking agents, curare, mecamylamine, and atrophine sulfate, and local anesthetics, tricaine and procaine, block reversibly the effects of cholinergic agonists. Only tricaine blocks contraction caused by ouabain. The high concentrations of drugs required (10+E-4 to 10+E-2 M) are probably due to impermeability caused by intervening tissue. Tetrodotoxin and bungarotoxin seem to have no effect. Other agents are under investigation. All uncoordinated levamisole-resistant mutants tested are very resistant to all acetylcholine agonists effective on the wild type. The mutants, however, retain sensitivity to ouabain, and are still paralyzed by cholinergic antagonists (we believe paralysis is an anesthetic side effect not directly related to blocking action). Strychnine, an inhibitor of inhibition, causes whole or cut wild type worms to convulse but has little effect (or causes rapid paralysis) on levamisole-resistant uncs. Whole levamisole-resistant uncs survive very much better than the wild type on plates containing the esterase- inhibitors Trichlorfon and Aldicarb but eventually die, too. We believe that levamisole is a nicotinic depolarizing blocking agent and that levamisole-resistant mutants are altered postsynaptically, possibly in the function of acetylcholine receptors. We are looking into bungarotoxin binding activity and the composition of plasma membrane proteins by 2-D gel electrophoresis in our mutants. Our work implies that nematode locomotory behavior depends mostly on myogenic activity with inhibitory regulation, cholinergic activity being largely dispensable. David Hirsh kindly sent us his Trichlorfon-resistant mutants to test in the cut worm assay. Mutants resistant to esterase inhibitors might be altered in the esterase itself or pre- or post-synaptically and be distinguished by testing with inhibitor alone, inhibitor plus acetylcholine, and a non-degradable acetylcholine analog, e.g. carbachol. Our preliminary impression is that all Trichlorfon mutants are altered presynaptically. The mutants do not seem to be as tight pharmacologically as levamisole-resistant uncs-a difficulty possibly circumventable by selecting for resistance at low temperature and screening for lethality at high temperature. We have not yet found any significant pharmacological abnormality for E933, resistant to esterase inhibitors lannate and aldicarb (Temik) .