Picture from 1999.....We are interested in understanding the cellular and molecular mechanisms controlling behavior and anesthetic-induced disruption of behavior in the nematode Caenorhabditis elegans. Despite intense and long-term interest, the mechanisms underlying general anesthesia are unknown. Genetics is a relatively untapped but powerful approach to the problem. C. elegans is a small non-parasitic worm with a well-characterized nervous system, composed of 302 neurons whose synaptic connections have been entirely mapped. While simple, its nervous system directs a number of complex behaviors. We have shown that volatile general anesthetics alter each C. elegans behavior in a dose-dependent fashion. C. elegans mating, chemotaxis, and coordination are abolished at anesthetic concentrations similar to those that anesthetize humans. Through both classical and quantitative genetic techniques, we have identified single gene mutations and quantitative trait loci that markedly alter sensitivity to volatile anesthetics. We have found that mutations in the presynaptic SNARE proteins syntaxin, SNAP-25, VAMP, an N-type Ca++ channel, and some G proteins drastically alter the sensitivity of C. elegans to anesthetics. In the end, we should know what are the mechanisms of anesthesia in C. elegans and, perhaps, will learn how anesthetics produce unconsciousness, amnesia, and loss of sensation in the vertebrate nervous system.
For the past three and a half years, I have been studying suppressors of syntaxin reduction-of-function mutations. One of these suppressor mutatons was a gain-of-function in Gq-alpha, and such a mutation conferred reistance to volatile anesthetics. We confirmed this discovery by testing previously discovered gain-of-function Gq-apha mutants. Furthermore, we examined the effects of drugs which activate the pathway upstream and downstream of Gq. Finally, we tested several double mutants to confirm a previously predicted pathway.
Abstract for 2001 International C. elegans Meeting
Gain-of-function mutations in egl-30 produce resistance to volatile anesthetics
Ammar Hawasli1, Stephen Hunt1, Owais Saifee2, Mike Nonet2, C. Michael
Crowder1,3
1 Department of Anesthesiology, Washington University
2 Department of Anatomy and Neurobiology, Washington University
3 Department of Molecular Biology and Pharmacology, Washington University
Because mutations in the neuronal syntaxin gene unc-64 alter volatile anesthetic
(VA) sensitivity, we have screened for unc-64(rf) suppressors in hopes of identifying
regulators of VA action. For different reasons, Owais Saifee in Mike Nonet's lab performed
a similar screen. A total of 38,000 genomes were screened by the two labs, and 21
suppressor mutations were
isolated. The suppressors were outcrossed from unc-64(rf) and fell into two phenotypic
classes loopy and jerky that cosegregate with unc-64(rf) suppression. Two semidominant
loopy suppressors mapped by their behavioral and VA resistant phenotypes to chromosome IL
near egl-30. egl-30 was sequenced in the mutants, and one of the alleles js126 was found
to
have a missense mutation in the coding sequence. We did not find an egl-30 mutation in the
other strain. egl-30(tg26), kindly given to us by K. Iwasaki, was also loopy and resistant
to VAs. Testing of strains transformed with a constitutively active egl-30 array (thanks
to Carol Bastiani - Sternberg lab) were also resistant to VAs confirming that egl-30(gf)
could
indeed produce VA resistance. Furthermore, we treated N2 with phorbol-ester, which
activates diacyl glycerol-binding proteins and effectively enhances EGL-30 signaling;
treated worms were resistant to VAs. Finally, we found that egl-30(js126), egl-30(tg26),
the egl-30(gf) array-containing strains, and phorbol ester treated strains were all
aldicarb hypersensitive.
Aldicarb hypersensitivity suggests that presynaptic release of acetylcholine is increased.
Thus, consistent with our previous results for goa-1(rf) and unc-64(rf) that suggested a
presynaptic mechanism of VA action, egl-30 regulates VA sensitivity, presumably by
controlling transmitter release. Currently, we are constructing double mutants between
various VA
resistant and hypersensitive strains to place egl-30 in the VA sensitivity pathway.
Abstract for a Howard Hughes Medical Institute Symposium
SUPPRESSORS OF SYNTAXIN PARTIAL-LOSS OF FUNCTION SHOW ANESTHETIC RESISTANCE IN
CAENORHABDITUS ELEGANS.
Ammar Hawasli1, Stephen Hunt1, Christine Liu1, CM Crowder1,2. Department of
Anesthesiology, Washington Univ. School of Medicine1; Department of Molecular
Biology/Pharmacology, Washington Univ. School of Medicine2
Although anesthetics were first developed and clinically introduced over a century ago,
the underlying mechanisms behind anesthetics and anesthetic sensitivity are still
unresolved. By taking a genetic approach to uncovering anesthetic mechanisms, scientists
may be able to bridge the mysterious gap between molecular effects and behavioral effects
of anesthetics. Scientists have chosen to study "anesthesia genes" in the fruit
fly Dresophila melanogaster and the nematode Caenorhabditus elegans due to their powerful
genetics.
Previous studies have shown that mutants of the syntaxin gene in C. elegans drastically
alter anesthetic sensitivity. Syntaxin is a membrane-spanning protein located in the
cellular membrane of presynaptic nerve terminals. As vesicles undergo exocytosis, syntaxin
plays a fundamental role in synaptic transmitter release from the presynaptic nerve
terminal. Anesthetics in C. elegans reduce transmitter release by a syntaxin
mediated mechanism. In C. elegans, a partial loss or reduction of function in the syntaxin
gene, unc-64, leads to viable and fertile but rather uncoordinated and abnormal adults.
For example, unc-64(e246) reduction of function strain is very uncoordinated and immobile
in comparison with the wild type strain.
In order to identify proteins that may function with syntaxin to regulate transmitter
release and anesthetic action, Stephen Hunt mutagenized a population of unc-64(e246)
mutants and screened their progeny for suppressors of the reduction of function mutation.
I took two of these mutants and out-crossed them with wild-type animals several times to
remove the unc-64(e246) mutation from the background. The resulting strains both have a
"loopy" hyperactive phenotype. In anesthetic locamotion assays, both suppressor
strains are very resistant to isoflurane; the suppressors’ isoflurane EC50 values
(the anesthetic concentration at which the effect of locomotion is half-maximal) are 2.5
and 2.9 times greater than the wild-type value. Other behavioral anesthetic assays also
confirm that both strains appear very resistant.
The loopy phenotypes in both strains are semi-dominant, with one strain being much less
dominant than the other. Through a recessive mapping technique, I mapped one suppressor
mutation onto the first chromosome. The other suppressor may likely be on the first or
fifth chromosome; however, further mapping is necessary.
To fully map each suppressor, more fine-tuned techniques, such as three-factor mapping,
will be necessary. Fine mapping will serve as a prelude to identification of the mutated
gene. Furthermore, it will be essential to formally determine if the loopy phenotype
corresponds to the unc-64(e246) suppression and to the anesthetic resistant phenotypes.
Howard Hughes Fellows Link:
http://www.nslc.wustl.edu/Research/HHMI/99fellows/ammar_hawasli.html
Liu C, Hawasli A, Metz LB, Hunt S, van Swinderen B, Crowder CM. A Truncated Neuronal Syntaxin
Antagonizes Volatile Anesthetics in C. elegans. Alcoholism: Clinical and Experimental Research. In
Press
Hawasli A, Saifee O, Hunt SF, Nonet ML, Crowder CM. Volatile anesthetic resistance by enhancing Gqa
in Caenorhabditis elegans. Manuscript in preparation
Picture from 1999.....