Several genes and drugs affect pharyngeal muscle excitation. Theacetylcholine agonist arecoline and loss-of-function mutations in
eat-6(the Na,K-ATPase alpha subunit; WBG 13(2): 52) depolarize the muscles. Aneat-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 inthe nervous system. To distinguish these possibilties, I designed thefollowing experiment: kill the pharyngeal nervous system (PhNS) in mutantand wild-type worms, then compare them. If the phenotypes are stilldifferent, the gene has an effect outside the PhNS. This experiment,which eventually resulted in the senseless slaughter of thousands ofpharyngeal neurons, became known as the Laser Experiment From Hell (LEFH).In practice all pharyngeal neurons except the essential neuron M4 werekilled. As an objective measure of feeding, I determined whether eachworm became a fertile adult, and if so the number of days from hatching tothe production of the first progeny. By this test
eat-6 (compare rows 2-4to 1),
eat-11 (row 9 vs 8), and
egl-30 (row 10 vs 9) all act outside thePhNS.The surprise was
snt-1.
snt-1 encodes the synaptic vesicle proteinsynaptotagmin, and within the pharynx antibodies were not reported torecognize anything other than neurons (Nonet et al, Cell 73: 1291). Iexpected that PhNS-
snt-1 worms would be similar to PhNS- wild-type. Infact, they pumped little or not at all, and their growth was drasticallyslowed (rows 5 and 6 vs 1;
md290 is a null, and
ad596 a hypomorph). Thisresult suggested that
snt-1 has a site of action outside the PhNS capableof affecting pharyngeal muscle function. The
snt-1 expression pattern,however, suggested that
snt-1 does not act in pharyngeal muscle. Guessingthat the depression of pumping in PhNS-
snt-1 worms resulted from aninhibitory effect of cells outside the pharynx, I dissected pharynxes outof PhNS-
snt-1(
ad596) worms. In fact, the average firing rate increasedfrom 1.1/min to 9.7/min, consistent with an extrapharyngeal inhibition.This experiment showed that the logic of the LEFH was flawed. Although itcan exclude pharyngeal neurons as the sole cells affected by a gene, theLEFH cannot show that pharyngeal muscle is the site of action, sincesomething outside the pharynx can also drastically affect pharyngealmuscle function. To get around this problem I killed the PhNS in mutantand wild-type, then dissected the pharynx out of the PhNS- worms thatsurvived 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 willnot be me doing it.) The results are consistent with a nervous systemsite of action for
snt-1, and support a muscle site of action for
eat-6. After dissection 4 PhNS-
eat-6 pharynxes hypercontracted, thecharacteristic
eat-6 phenotype. 7 PhNS-
snt-1 and 5 PhNS- wild-type didn'thypercontract, 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 ofeat-6 action. However, I showed previously that
eat-6 mutation suppressesthe lethality of M4- worms (WBG 13(2): 53), implying a site of actionoutside M4. Second, it could be that
eat-6 acts only in the PhNS, butthat the mutant nervous system changes muscle physiology in a way thatcan't be reversed after removal of the PhNS. This is unlikely, becauseWayne Davis has shown that the effects of a cold-sensitive
eat-6 mutationare rapidly reversible.In conclusion, the LEFH strongly supports but doesn't prove the followingtwo points: (1) A cell or cells outside the pharynx can profoundly inhibitpharyngeal muscle excitation. (2)
eat-6 acts directly in pharyngealmuscle.