Worm Breeder's Gazette 13(3): 76 (June 1, 1994)
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.
Defining the molecular targets that are affected by the widely used volatile anesthetics (VAs) to produce the stereotypical vertebrate behavioral response termed anesthesia has been complicated by their uniform lipophilicity, absence of drugs that block or reverse anesthesia, and lack of vertebrate strains with high level resistance to VAs. However, electrophysiologic techniques have been successful in demonstrating effects of VAs on several ligand- and voltagegated ion channels thus implicating them as potential targets. Which, if any, of these interactions results in anesthesia is unknown. Further, the VA concentration at which the channel properties are altered varies widely; most channels are not affected at clinically relevant anesthetic concentrations. Genetic studies have been started in order to bridge this molecular to behavioral gap, most notably the work by the lab of Phil Morgan and Margaret Sedensky with C. elegans. Morgan and Sedensky have shown that C. elegans is immobilized by all known VAs and have isolated alleles of the unc-79 and unc-80 genes that are immobilized at a 3-fold lower dose of the VA halothane when compared to N2 .In more recent screens, Morgan and Sedensky have found mutants that are hypersensitive to multiple VAs, not just halothane.
In order to further characterize the response of C. elegans to VAs and as a prelude to a search for other molecularly targets of VAs, we examined the VA sensitivity of other C. elegans behaviors besides locomotion. We set out to determine if other behaviors were affected in the vertebrate response range(for halothane approx. 0.1 - 1 vol% @ 20°C), if the response was rapid like that of vertebrates, and if the relative potency of the three VAs against each behavior mirrored the vertebrate rank order potency of halothane>isoflurane>enflurane. We measured the dose, time-course and order of potency for VA-induced abolition of mating, defecation, pumping, egg-laying and locomotion. For each assay, we examined at least 10 worms/VA concentration and constructed concentration/response curves fitted by logistic regression. The response endpoint varied for each behavior. For mating behavior, the endpoint was lack of cross progeny. For defecation, the endpoint was lack of expulsion of gut contents, and for pumping, a > 5-fold decrease in pumping rate. Worms not laying eggs in a two hour period were scored as defective. For locomotion, worms were scored for uncoordination at lower doses and lack of net movement at higher doses(the Morgan/Sedensky endpoint) The table below summarizes results.
Our results demonstrate that male-mating and coordination have halothane EC(50)'s that are in the clinical range. When gas phase concentrations are temperature-adjusted and converted to aqueous values, the EC(50) for male-mating of 0.29mM closely approximates the human EC(50)of 0.21mM. Although we could not directly measure the time-course of the male-mating response due to the constraints of the assay, we did observe mating behavior directly and found that within 5', males were no longer able to locate the female vulva, a behavioral defect that can be reproduced by ablation of specific neurons in the male tail(P. Sternberg, pers. comm.). When removed from anesthetic, 90 % of N2 males recover the ability to mate even after a 24° exposure to 1% halothane. Given the wide range of behavioral sensitivities, we believe that VAs may be affecting a different set of targets for each behavior, some targets perhaps homologous to those involved in the typical vertebrate anesthetic response and others potentially involved in anesthetic toxicity. We are in the process of determining the EC50 'sfor chemotaxis and mechanosensation and are beginning a search for mutants resistant to the halothane-induced mating defect.