Nitrous oxide (N2O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N 2O and VAs may act by distinct mechanisms; N2O antagonizes the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in Caenorhabditis elegans, we tested whether the action of N 2 O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs. Distinct from the action of VAs, N 2O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N 2O, whereas a non-NMDA receptor-null mutant was normally sensitive. The N 2O-resistant nmr-1(null) mutant was not resistant to VAs. Likewise, VA-resistant mutants had wild-type sensitivity to N 2O. Thus, the behavioral effects of N 2O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N 2O is the NMDA receptor.

These data show that UNC-13, an evolutionarily conserved protein that promotes neurotransmitter release, is necessary for isoflurane sensitivity in C. elegans and suggest that its vertebrate homologs may be a component of the general anesthetic mechanism.

Xenon acts in C. elegans to alter locomotion through a mechanism requiring the non-NMDA glutamate receptor encoded by glr-1. Unlike for the action of nitrous oxide in C. elegans, the NMDA receptor encoded by nmr-1 is not essential for sensitivity to xenon.

Background Volatile general anesthetics inhibit neurotransmitter release by a mechanism not fully understood. Genetic evidence in C. 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 three-fold 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 levels 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 G□s – PKA – UNC-13-dependent pathway to regulate behaviour and isoflurane sensitivity. Conclusions Activation of neuronal adenylate cyclase antagonizes isoflurane inhibition of locomotion in C. elegans.

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.