C. eleganspharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes

Abstract

34Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-35 cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the 36 overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without 37 intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete 38 efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find 39 better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an 40 excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular 41 junctions show a high degree of molecular and functional conservation with the cholinergic 42 transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, 43 the anti-cholinesterase intoxication of the worm's body wall neuromuscular junction has been 44 unprecedented in understanding molecular determinants of cholinergic function in nematodes and 45 other organisms. We extend the use of the model organism's feeding behaviour as a tool to investigate 46 carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-47 dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the 48 acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay 49 allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole 50 organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-51 cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of 52 the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after 53 inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to 54 explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic 55

Cholinergic signalling at the body wall neuromuscular junction couples to distal inhibition of feeding inC. elegans

“…This assay has been extensively used to find molecular determinants at the NMJ of the body wall on the basis of resistance or hypersensitivity to paralysis of locomotion when nematodes are incubated on aldicarb-containing agar plates (26). We previously demonstrated that aldicarb and other cholinesterase inhibitors also cause a dose-dependent inhibition of the pharyngeal function (28). This inhibition was associated with the pharynx exhibiting hypercontraction observed by the opening of the lumen (28).…”

“…We previously demonstrated that aldicarb and other cholinesterase inhibitors also cause a dose-dependent inhibition of the pharyngeal function (28). This inhibition was associated with the pharynx exhibiting hypercontraction observed by the opening of the lumen (28). This implied that aldicarb intoxication in the context of the whole organism would result from drug-induced hyperactivation of the cholinergic transmission.…”

“…We screened the pharyngeal function of different mutant worms in the presence or absence of the cholinesterase inhibitor aldicarb under the conditions we previously optimized (28). The results of this screening are listed in Table 1.…”

“…However, this has highlighted a distinct non-pharyngeal modulation of drug-induced feeding inhibition. These investigations have been built on our previous observations indicating that aldicarb and other anti-cholinesterases cause a profound inhibition of the pharyngeal pumping (28). It was underpinned by a spastic paralysis of the radial muscles in the pharynx evidenced by an overt opening of the lumen (28).…”

“…These investigations have been built on our previous observations indicating that aldicarb and other anti-cholinesterases cause a profound inhibition of the pharyngeal pumping (28). It was underpinned by a spastic paralysis of the radial muscles in the pharynx evidenced by an overt opening of the lumen (28). The prediction of our initial investigations was explained by assuming that aldicarb-dependent inhibition of acetylcholinesterase leads to an excess of input to the pharyngeal muscle from the two cholinergic motor neurons, MC and M4 (18,23,59,60).…”