Worm Breeder's Gazette 13(5): 35 (February 1, 1995)
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.
Biochemistry Department, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038. Several genes and drugs affect pharyngeal muscle excitation. The acetylcholine agonist arecoline and loss-of-function mutations in eat-6 (the Na,K-ATPase alpha subunit; WBG 13(2): 52) depolarize the muscles. An eat-11 mutation causes hypersensitivity to eat-6(lf) and arecoline. egl-30 mutations suppress the eat-11 hypersensitivity (WBG 12(5): 64). These genes could act directly in pharyngeal muscle, or they might act in the nervous system. To distinguish these possibilties, I designed the following experiment: kill the pharyngeal nervous system (PhNS) in mutant and wild-type worms, then compare them. If the phenotypes are still different, the gene has an effect outside the PhNS. This experiment, which eventually resulted in the senseless slaughter of thousands of pharyngeal neurons, became known as the Laser Experiment From Hell (LEFH). In practice all pharyngeal neurons except the essential neuron M4 were killed. As an objective measure of feeding, I determined whether each worm became a fertile adult, and if so the number of days from hatching to the production of the first progeny. By this test eat-6 (compare rows 2-4 to 1), eat-11 (row 9 vs 8), and egl-30 (row 10 vs 9) all act outside the PhNS. row Relevant [arecoline] number number days to genotype animals fertile fertility 1 + 0 mM 13 13 4.3 2 eat-6(ad601) 0 mM 11 5 8.4* 3 eat-6(ad792) 0 mM 2 1 16* 4 eat-6(ad467) 0 mM 13 4 7.3* 5 snt-1(md290) 0 mM 6 0 * 6 snt-1(ad596) 0 mM 14 10 10.5* 7 eat-11(ad541) 0 mM 11 11 5.4 8 + 5 mM 12 10 6.4 9 eat-11(ad541) 5 mM 10 0 * 10 egl-30(ad803) 5 mM 14 14 5.1^ eat-11(ad541) *Significantly different from wild-type at the same [arecoline], P < 1%. ^Significantly different from eat-11, P < 1%. The surprise was snt-1. snt-1 encodes the synaptic vesicle protein synaptotagmin, and within the pharynx antibodies were not reported to recognize anything other than neurons (Nonet et al, Cell 73: 1291). I expected that PhNS- snt-1 worms would be similar to PhNS- wild-type. In fact, they pumped little or not at all, and their growth was drastically slowed (rows 5 and 6 vs 1; md290 is a null, and ad596 a hypomorph). This result suggested that snt-1 has a site of action outside the PhNS capable of affecting pharyngeal muscle function. The snt-1 expression pattern, however, suggested that snt-1 does not act in pharyngeal muscle. Guessing that the depression of pumping in PhNS- snt-1 worms resulted from an inhibitory effect of cells outside the pharynx, I dissected pharynxes out of PhNS- snt-1(ad596) worms. In fact, the average firing rate increased from 1.1/min to 9.7/min, consistent with an extrapharyngeal inhibition. This experiment showed that the logic of the LEFH was flawed. Although it can exclude pharyngeal neurons as the sole cells affected by a gene, the LEFH cannot show that pharyngeal muscle is the site of action, since something outside the pharynx can also drastically affect pharyngeal muscle function. To get around this problem I killed the PhNS in mutant and wild-type, then dissected the pharynx out of the PhNS- worms that survived to adulthood. This experiment has been done only on wild-type, snt-1(ad596), and eat-6(ad467). (And if it is ever done again, it will not be me doing it.) The results are consistent with a nervous system site of action for snt-1, and support a muscle site of action for eat-6. After dissection 4 PhNS-eat-6 pharynxes hypercontracted, the characteristic eat-6 phenotype. 7 PhNS- snt-1 and 5 PhNS- wild-type didn't hypercontract, and weren't strikingly different from each other. There are two potential problems in interpreting the eat-6 results. First, since M4 was spared in the LEFH, it might in principle be the sole site of eat-6 action. However, I showed previously that eat-6 mutation suppresses the lethality of M4- worms (WBG 13(2): 53), implying a site of action outside M4. Second, it could be that eat-6 acts only in the PhNS, but that the mutant nervous system changes muscle physiology in a way that can't be reversed after removal of the PhNS. This is unlikely, because Wayne Davis has shown that the effects of a cold-sensitive eat-6 mutation are rapidly reversible. In conclusion, the LEFH strongly supports but doesn't prove the following two points: (1) A cell or cells outside the pharynx can profoundly inhibit pharyngeal muscle excitation. (2) eat-6 acts directly in pharyngeal muscle.