Tail Clamp Responses in Stomatin Knockout Mice Compared with Mobility Assays in Caenorhabditis elegans  during Exposure to Diethyl Ether, Halothane, and Isoflurane

2006 ◽  
Vol 105 (3) ◽  
pp. 498-502 ◽  
Author(s):  
Margaret M. Sedensky ◽  
Melissa A. Pujazon ◽  
Phil G. Morgan
Keyword(s):  
Caenorhabditis Elegans ◽  
Diethyl Ether ◽  
Ganglion Cells ◽  
Volatile Anesthetic ◽  
Volatile Anesthetics ◽  
Mouse Strains ◽  
C Elegans ◽  
Anesthetic Action ◽  
Increased Sensitivity ◽  
Dorsal Root Ganglion Cells

Background The gene unc-1 plays a central role in determining volatile anesthetic sensitivity in Caenorhabditis elegans. Because different unc-1 alleles cause strikingly different phenotypes in different volatile anesthetics, the UNC-1 protein is a candidate to directly interact with volatile anesthetics. UNC-1 is a close homologue of the mammalian protein stomatin, for which a mouse knockout was recently constructed. Because the stomatin gene is expressed in dorsal root ganglion cells, the authors hypothesized that the knockout would have an effect on anesthetic sensitivity in mice similar to that seen in nematodes. Methods Mice were placed in semiclosed chambers and exposed to continuous flows of diethyl ether, halothane, or isoflurane in air. Using lack of response to tail clamp as an endpoint, the authors determined the EC50s for the knockout strain compared with the nonmutated parental strain. They compared the differences seen in the mouse strains with the differences seen in the nematode strains. Results Stomatin-deficient mice had a 12% increase in sensitivity to diethyl ether but no significant change in sensitivity to halothane or isoflurane compared with wild type. No defect in locomotion was noted in the mutant mouse. Conclusions Nematodes and mice with deletions of the stomatin gene both have increased sensitivity to diethyl ether. Neither nematodes nor mice with stomatin deficiencies have significantly altered sensitivity to isoflurane or halothane. The effects of stomatin deficiency cross phylogenetic boundaries and support the importance of this protein in anesthetic response and the use of C. elegans as a model for anesthetic action in mammals.

Behavioral Effects of Volatile Anesthetics in Caenorhabditis elegans

1996 ◽  
Vol 85 (4) ◽  
pp. 901-912 ◽  
Author(s):  
Michael C. Crowder ◽  
Laynie D. Shebester ◽  
Tim Schedl
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Time Course ◽  
Model Organism ◽  
Volatile Anesthetic ◽  
Volatile Anesthetics ◽  
Effective Concentration ◽  
Forward Genetics ◽  
C Elegans ◽  
Median Effective Concentration

Background The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic actions. It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans. Methods The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees C. Results The median effective concentration values for halothane inhibition of mating (0.30 vol%-0.21 mM), chemotaxis (0.34 vol%-0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations. Conclusions Volatile anesthetics selectively disrupt C. elegans behavior. The potency, time course, and recovery characteristics of halothane's effects on three behaviors are similar to its anesthetic properties in vertebrates. The affected nervous system molecules may express structural motifs similar to those on vertebrate anesthetic targets.

scholarly journals Goα Regulates Volatile Anesthetic Action in Caenorhabditis elegans

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 643-655 ◽  
Author(s):  
Bruno van Swinderen ◽  
Laura B Metz ◽  
Laynie D Shebester ◽  
Jane E Mendel ◽  
Paul W Sternberg ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Novel Mutation ◽  
Volatile Anesthetic ◽  
Loss Of Function ◽  
Wild Type ◽  
Α Subunit ◽  
C Elegans ◽  
Missense Mutant ◽  
Anesthetic Action ◽  
Mutant Phenotypes

Abstract To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the α-subunit of Go, have EC50s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goα, and presynaptic Goα-effectors are candidate VA molecular targets.

Common Genetic Determinants of Halothane and Isoflurane Potencies in Caenorhabditis elegans 

1998 ◽  
Vol 89 (6) ◽  
pp. 1509-1517 ◽  
Author(s):  
Bruno van Swinderen ◽  
Alex Galifianakis ◽  
Michael C. Crowder
Keyword(s):  
Caenorhabditis Elegans ◽  
Recombinant Inbred ◽  
Recombinant Inbred Strain ◽  
Volatile Anesthetic ◽  
Inbred Strains ◽  
Volatile Anesthetics ◽  
Recombinant Inbred Strains ◽  
Genetic Determinants ◽  
Genetic Loci ◽  
C Elegans

Background Genetics provides a way to evaluate anesthetic action simultaneously at the molecular and behavioral levels. Results from strains that differ in anesthetic sensitivity have been mixed in their support of unitary theories of anesthesia. Here the authors use the previously demonstrated large variation of halothane sensitivities in Caenorhabditis elegans recombinant inbred strains to assess the similarities of the determinants of halothane action with those of another volatile anesthetic, isoflurane. Methods The recombinant inbred strains, constructed from two evolutionarily distinct C. elegans lineages, were phenotyped. A coordination assay on agar quantified the sensitivity to the volatile anesthetics; median effective concentrations (EC50s) were calculated by nonlinear regression of concentration-response data and were correlated between the drugs for those strains tested in common. Genetic loci were identified by statistical association between EC50s and chromosomal markers. Results The recombinant inbred strains varied dramatically in sensitivity to halothane and isoflurane, with a 10-fold range in EC50s. Heritability estimates for each drug were imprecise but altogether high (49-80%). Halothane and isoflurane EC50s were significantly correlated (r=0.71, P < 10(-9)). Genetic loci controlling sensitivity were found for both volatile anesthetics; the most significant determinant colocalized on chromosome V. A smaller recombinant inbred strain study of ethanol-induced immobility segregated different genetic effects that did not correlate with sensitivity to either halothane or isoflurane. Conclusions The genetic determinants driving the large variation in anesthetic sensitivity in these C. elegans recombinant inbred strains are very similar for halothane and isoflurane sensitivity.

Mitochondrial Complex I Function Affects Halothane Sensitivity in Caenorhabditis elegans 

2004 ◽  
Vol 101 (2) ◽  
pp. 365-372 ◽  
Author(s):  
Ernst-Bernhard Kayser ◽  
Phil G. Morgan ◽  
Margaret M. Sedensky
Keyword(s):  
Caenorhabditis Elegans ◽  
Oxidative Phosphorylation ◽  
Oxidative Damage ◽  
Adenosine Triphosphate ◽  
Complex I ◽  
Volatile Anesthetics ◽  
Mitochondrial Proteins ◽  
Wild Type ◽  
Complex Ii ◽  
C Elegans

Background : The gene gas-1 encodes a subunit of complex I of the mitochondrial electron transport chain in Caenorhabditis elegans. A mutation in gas-1 profoundly increases sensitivity of C. elegans to volatile anesthetics. It is unclear which aspects of mitochondrial function account for the hypersensitivity of the mutant. Methods : Oxidative phosphorylation was determined by measuring mitochondrial oxygen consumption using electron donors specific for either complex I or complex II. Adenosine triphosphate concentrations were determined by measuring luciferase activity. Oxidative damage to mitochondrial proteins was identified using specific antibodies. Results : Halothane inhibited oxidative phosphorylation in isolated wild-type mitochondria within a concentration range that immobilizes intact worms. At equal halothane concentrations, complex I activity but not complex II activity was lower in mitochondria from mutant (gas-1) animals than from wild-type (N2) animals. The halothane concentrations needed to immobilize 50% of N2 or gas-1 animals, respectively, did not reduce oxidative phosphorylation to identical rates in the two strains. In air, adenosine triphosphate concentrations were similar for N2 and gas-1 but were decreased in the presence of halothane only in gas-1 animals. Oxygen tension changed the sensitivity of both strains to halothane. When nematodes were raised in room air, oxidative damage to mitochondrial proteins was increased in the mutant animal compared with the wild type. Conclusions : Rates of oxidative phosphorylation and changes in adenosine triphosphate concentrations by themselves do not control anesthetic-induced immobility of wild-type C. elegans. However, they may contribute to the increased sensitivity to volatile anesthetics of the gas-1 mutant. Oxidative damage to proteins may be an important contributor to sensitivity to volatile anesthetics in C. elegans.

scholarly journals ACaenorhabditis elegansPheromone Antagonizes Volatile Anesthetic Action Through a Go-Coupled Pathway

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 109-119 ◽  
Author(s):  
Bruno van Swinderen ◽  
Laura B Metz ◽  
Laynie D Shebester ◽  
C Michael Crowder
Keyword(s):  
Sensory Neurons ◽  
Volatile Anesthetic ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Pheromone Activity ◽  
Dauer Formation ◽  
Anesthetic Action ◽  
Nervous System Function ◽  
Type C

AbstractVolatile anesthetics (VAs) disrupt nervous system function by an ill-defined mechanism with no known specific antagonists. During the course of characterizing the response of the nematode C. elegans to VAs, we discovered that a C. elegans pheromone antagonizes the VA halothane. Acute exposure to pheromone rendered wild-type C. elegans resistant to clinical concentrations of halothane, increasing the EC50 from 0.43 ± 0.03 to 0.90 ± 0.02. C. elegans mutants that disrupt the function of sensory neurons required for the action of the previously characterized dauer pheromone blocked pheromone-induced resistance (Pir) to halothane. Pheromone preparations from loss-of-function mutants of daf-22, a gene required for dauer pheromone production, lacked the halothane-resistance activity, suggesting that dauer and Pir pheromone are identical. However, the pathways for pheromone’s effects on dauer formation and VA action were not identical. Not all mutations that alter dauer formation affected the Pir phenotype. Further, mutations in genes not known to be involved in dauer formation completely blocked Pir, including those altering signaling through the G proteins Goα and Gqα. A model in which sensory neurons transduce the pheromone activity through antagonistic Go and Gq pathways, modulating VA action against neurotransmitter release machinery, is proposed.

Genetic Differences Affecting the Potency of Stereoisomers of Isoflurane

1996 ◽  
Vol 85 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Phil G. Morgan ◽  
Marianne F. Usiak ◽  
Margaret M. Sedensky
Keyword(s):  
Volatile Anesthetic ◽  
Genetic Differences ◽  
Wild Type ◽  
C Elegans ◽  
Mutant Strains ◽  
Anesthetic Action ◽  
Type C ◽  
Sites Of Action ◽  
Standard Techniques

Background In previous studies, researchers demonstrated the ability of a variety of organisms and in vitro sites of anesthetic action to distinguish between stereoisomers of isoflurane or halothane. However, it was not shown whether organisms with differing sensitivities to stereoisomers of one volatile anesthetic are able to distinguish between stereoisomers of another. In this study, the responses of mutants of Caenorbabditis elegans to stereoisomers of isoflurane were determined for comparison to previous results in halothane. Methods Mutant strains of C. elegans were isolated and grown by standard techniques. The EC50s (the effective concentrations of anesthetia at which 50% of the animals are immobilized for 10 s) of stereoisomers of isoflurane and the racemate were determined in wild type and mutant strains of C. elegans. Results Wild type C. elegans and strains with high EC50S of the racemate were more sensitive to the (+) isomer of isoflurane by approximately 30%. The racemate showed a EC50s similar to the less potent isomer, the (-) form. In the strains with low EC50s, one strain showed no ability to differentiate between the stereoisomers, whereas two showed a 60% difference between the (+) and (-) forms. Conclusions The ability to distinguish between stereoisomers of isoflurane is associated with genetic loci separate from those that distinguish between stereoisomers of halothane. These results are consistent with multiple sites of action for these anesthetics.

scholarly journals Volatile Anesthetic Preconditioning Present in the Invertebrate Caenorhabditis elegans 

2008 ◽  
Vol 108 (3) ◽  
pp. 426-433 ◽  
Author(s):  
Baosen Jia ◽  
C Michael Crowder
Keyword(s):  
Caenorhabditis Elegans ◽  
Cellular Response ◽  
Volatile Anesthetic ◽  
The Novel ◽  
Loss Of Function ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Anesthetic Preconditioning ◽  
Evolutionarily Conserved ◽  
Duration Of Protection

Background Volatile anesthetics (VAs) have been found to induce a delayed protective response called preconditioning to subsequent hypoxic/ischemic injury. VA preconditioning has been primarily studied in canine and rodent heart. A more genetically tractable model of VA preconditioning would be extremely useful. Here, the authors report the development of the nematode Caenorhabditis elegans as a model of VA preconditioning. Methods Wild-type and mutant C. elegans were exposed to isoflurane, halothane, or air under otherwise identical conditions. After varying recovery periods, the animals were challenged with hypoxic, azide, or hyperthermic incubations. After recovery from these incubations, mortality was scored. Results Isoflurane- and halothane-preconditioned animals had significantly reduced mortality to all three types of injuries compared with air controls. Concentrations as low as 1 vol% isoflurane (0.64 mm) and halothane (0.71 mm) induced significant protection. The onset and duration of protection after anesthetic were 6 and 9 h, respectively. A mutation that blocks inhibition of neurotransmitter release by isoflurane did not attenuate the preconditioning effect. A loss-of-function mutation of the Apaf-1 homolog CED-4 blocked the preconditioning effect of isoflurane, but mutation of the downstream caspase CED-3 did not. Conclusions Volatile anesthetic preconditioning extends beyond the vertebrate subphylum. This markedly broadens the scope of VA preconditioning and suggests that its mechanisms are widespread across species and is a fundamental and evolutionarily conserved cellular response. C. elegans offers a means to dissect genetically the mechanism for VA preconditioning as illustrated by the novel finding of the requirement for the Apaf-1 homolog CED-4.

scholarly journals A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans

1999 ◽  
Vol 96 (5) ◽  
pp. 2479-2484 ◽  
Author(s):  
B. van Swinderen ◽  
O. Saifee ◽  
L. Shebester ◽  
R. Roberson ◽  
M. L. Nonet ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Volatile Anesthetic ◽  
Anesthetic Action

scholarly journals A Gain-of-function Mutation in Adenylate Cyclase Confers Isoflurane Resistance in Caenorhabditis elegans 

2011 ◽  
Vol 115 (6) ◽  
pp. 1162-1171 ◽  
Author(s):  
Owais Saifee ◽  
Laura B. Metz ◽  
Michael L. Nonet ◽  
C. Michael Crowder
Keyword(s):  
Caenorhabditis Elegans ◽  
Adenylate Cyclase ◽  
Neurotransmitter Release ◽  
Acetylcholinesterase Inhibitor ◽  
Volatile Anesthetic ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
General Anesthetics ◽  
Gain Of Function ◽  
C Elegans

Background Volatile general anesthetics inhibit neurotransmitter release by a mechanism not fully understood. Genetic evidence in Caenorhabditis elegans has shown that a major mechanism of action of volatile anesthetics acting at clinical concentrations in this animal is presynaptic inhibition of neurotransmission. To define additional components of this presynaptic volatile anesthetic mechanism, C. elegans mutants isolated as phenotypic suppressors of a mutation in syntaxin, an essential component of the neurotransmitter release machinery, were screened for anesthetic sensitivity phenotypes. Methods Sensitivity to isoflurane concentrations was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans acetylcholine release. Comparisons of isoflurane sensitivity (measured by the EC₅₀) were made by simultaneous curve-fitting and F test. Results Among the syntaxin suppressor mutants, js127 was the most isoflurane resistant, with an EC₅₀ more than 3-fold that of wild type. Genetic mapping, sequencing, and transformation phenocopy showed that js127 was an allele of acy-1, which encodes an adenylate cyclase expressed throughout the C. elegans nervous system and in muscle. js127 behaved as a gain-of-function mutation in acy-1 and had increased concentrations of cyclic adenosine monophosphate. Testing of single and double mutants along with selective tissue expression of the js127 mutation revealed that acy-1 acts in neurons within a Gαs-PKA-UNC-13-dependent pathway to regulate behavior and isoflurane sensitivity. Conclusions Activation of neuronal adenylate cyclase antagonizes isoflurane inhibition of locomotion in C. elegans.

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