Worm Breeder's Gazette 15(1): 39 (October 1, 1997)

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.

Halothane, A General Anesthetic, Confers Resistance To Aldicarb.

Bruno van Swinderen, Russell Roberson, Mike Crowder

Dept. of Anesthesiology, Washington University School of Medicine, St. Louis, MO.

   Halothane is a volatile general anesthetic that disrupts coordinated
movement in C. elegans at low, clinically-relevant concentrations (1)
(0.2-0.5 volume % halothane), and abolishes gross movment at higher
concentrations (3.5 vol. %). The nematicide drug aldicarb, an inhibitor
of cholinesterase, paralyzes worms (>0.5 mM for N2) due to an excess of
acetylcholine accumulating at the synapse. Mutations in pre-synaptic
machinery can either confer resistance or hypersensitivity to aldicarb
(a ric vs hic phenotype). Previous work has implicated several
pre-synaptic components in halothane-induced anesthesia.  We
hypothesized that should halothane disrupt pre-synaptic machinery, then
the reduction in acetylcholine release would confer resistance to
aldicarb. Indeed, halothane makes worms ric.
   Halothane-induced resistance to aldicarb was measured by a simple
movement assay. Worms (N = 20-30) were exposed to a seeded aldicarb
plate for 4 hours, and were then picked into a 0.5 cm circle outlined
on the same plate. An hour later, the percentage of animals having
managed to crawl completely out of the circle was scored. Each assay
was done in triplicate at various halothane concentrations within
air-tight chambers,including a no-halothane control.
   Table 1 summarizes results for N2 and some mutant strains. Low,
clinically-relevant, concentrations of halothane (0.2-0.5 vol. %)
significantly reversed aldicarb-induced paralysis. Interestingly,
higher concentrations of halothane (1-1.5 vol. %) do not show a
significant effect in N2; the reversal effect peaks at 0.2-0.3 vol. %
halothane and then gradually decreases to near control levels. Similar
experiments performed with the acetylcholine agonist levamisole and
halothane failed to show any reversal, suggesting that halothan acts
pre-synaptically (data not shown).
   We tested several mutant lines for disruption of halothane-induced
resistance to aldicarb, reasoning that by blocking this effect one
might highlight clinically relevant molecular pathways targeted by
volatile anesthetics. Aldicarb concentrations were optimized according
to the ricness or hicness of each strain.  Shown in Table 1 are results
for some G protein and calcium channel  mutant strains. egl-30 (n686)
(a.k.a.Gqalpha) does not reverse at all, and neither do calcium channel
mutations egl-19 (ad1013)lof and (n2368)gof. On the other hand, goa-1
(sy192) reverses significantly at low halothane concentrations, but
continues to show robust reversals at higher concentrations as well.
The same striking response was also seen for egl-10 (nIs51), which
overexpresses the upstream negative regulator of goa-1 signalling.
   In conclusion,  the anesthetic halothane can result in an organism
"waking up." By understanding aldicarb's mechanism of action, some
light is shed on the way volatile anesthetics work. The analysis of
numerous mutants suggests that  halothane has a pre-synaptic effect at
clinically relevant concentrations, and that G proteins and calcium
channels are involved .

Table 1. (Crawling Indeces)   ** significant movement compared to
          Halothane Concentration Range (vol. %)

Strains     0 (control)      0.2 - 0.5      1.0 - 1.5   [aldicarb] mM

N2           0.16+/-0.04  0.52+/-0.04*   0.27+/-0.04     0.5
egl-30(n686)  0.14+/-0.02      0.15+/-0.03    0.04+/-0.01     0.8
egl-19(n2368) 0.15+/-0.03      0.20+/-0.03    0.05+/-0.02     0.5
egl-13(ad1013)0.20+/-0.03      0.23+/-0.03    0.11+/-0.02     0.5
goa-1(sy192)  0.10+/-0.02      0.62+/-0.06*   0.65+/-0.02*    0.1
egl-10(nIS51) 0.01+/-0.01      0.49+/-0.07*   0.63+/-0.03*    0.2

1.   Crowder, C.M., Shebester, L.D. & Schedl, T (1996) Anesthesiology
85, 901-912.