Of Mice and Nematodes

WE still do not know how inhaled anesthetics work, but significant advances have been made in recent decades, as researchers have shifted from primarily biophysical models (i.e. , lipid perturbations) to systematic neurobiologic approaches. The goal of this enterprise, progressing in dozens of laboratories around the world, is to identify molecular targets of general anesthetics and to understand how they mediate the cellular, tissue, and behavioral effects of these drugs. In this issue of Anesthesiology, Sedensky et al.  1describe anesthetic sensitivity studies on genetically modified animals from two different phyla that suggest remarkably similar roles for one potential target, stomatin.

Stomatin is so named because a deficiency of this membrane protein in humans causes hereditary stomatocytosis, a hemolytic disorder where erythrocytes seem to have pale central areas with the shape of a smile or a fish mouth. The protein is also found in sensory neurons, where it is thought to play a role in mechanical sensation. A group of researchers at the University of California at San Francisco Medical School, wishing to create a laboratory animal model for the hematologic disease, created a knockout mouse lacking the gene for murine stomatin.2Independently, Sedensky and Morgan's research team at Case Western Reserve Medical School (Cleveland, Ohio) mapped mutated genes in Caenorhabditis elegans  nematodes that caused abnormal sensitivity to volatile anesthetics. A number of these mutations were in the unc-1  gene, which encodes a homologue of stomatin, resonantly named UNC-1.3Mutations that reduce UNC-1 expression or activity result in immobilized worms at lower than normal partial pressures of diethyl ether, without altering sensitivity to halogenated agents. In their new article, Sedensky and Morgan report that stomatin knockout mice also display increased sensitivity to the immobilizing effects of diethyl ether (i.e. , decreased minimum alveolar concentration), but not to other volatiles. Testing whether patients with hereditary stomatocytosis also have increased sensitivity to diethyl ether is theoretically possible but ethically indefensible.

Molecular genetic techniques have proven to be powerful tools for linking putative molecular targets to anesthetic effects. In classic forward genetic studies, researchers induce random mutations in animal genes and screen viable mutants for altered anesthetic sensitivity (phenotype). Interesting mutants are analyzed by mapping the mutated gene and defining its sequence, which encodes a protein that may be an anesthetic target. Small animals with short generation times, such as C. elegans  or fruit flies (Drosophila ) are ideal for forward genetics, although translating anesthetic effects in humans to behavioral screens in worms and flies is challenging. Sedensky and Morgan also identified another C. elegans  gene that affects anesthetic sensitivity; gas-1 alters mitochondrial function and may provide insight into human mitochondrial myopathies.4C. Michael Crowder, M.D., Ph.D. (Associate Professor, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO) has also exploited C. elegans , identifying a number of genes that alter anesthetic sensitivity through changes in G proteins5and excitatory neurotransmitter release.6 

Another approach, sometimes called reverse genetics, has been used in studies of mice. In reverse genetics, a putative target is identified based on thorough molecular studies, and then the gene for that target is selectively targeted for deletion (knockout) or mutation (knock-in). Techniques also exist for triggering knockouts after normal development (conditional knockout) and for restricting the expression of altered genes to specific regions of the nervous system. The resulting transgenic animals are then characterized for the phenotype of interest, in this case altered anesthetic sensitivity. Anesthetic-sensitive neuronal ion channels, including γ-aminobutyric acid receptor type A (GABA^A) receptors, glutamate receptors, and background-leak potassium channels have been studied using these techniques. One particularly successful case was based on the finding that single amino acid mutations in GABA^Areceptor β²and β³subunits largely eliminate the effects of etomidate and propofol on these neurotransmitter receptors. Transgenic mice containing these mutations were found to be resistant to etomidate and propofol anesthesia, and further behavioral testing revealed specific roles for β²- versus β³-containing receptors in different anesthetic-sensitive behaviors.7,8Knockout mice also reveal that GABA^Areceptors9and background potassium channels10mediate some of the effects of volatile anesthetics. Based on molecular studies showing that nitrous oxide and xenon inhibit mammalian glutamate receptors, reverse genetic approaches were also used in C. elegans . Crowder and his coworkers studied nematodes lacking the homolog of the major N -methyl-d-aspartate–sensitive glutamate receptor subunit and found that the effects of nitrous oxide were ablated,11whereas a different glutamate receptor was required for xenon anesthesia.12 

Can we expect that forward genetics in nematodes and reverse genetics in mice will converge on a conserved set of anesthetic targets that will also be relevant to humans? So far, stomatin seems to be unique in this regard. This is not too surprising, because worms express fewer genes, have a much simpler nervous system, and have a very limited repertoire of “behaviors” in comparison with vertebrates. Moreover, although mice and humans are in the same phylum and class, genetic changes in these two mammals are not always expressed in the same way. Stomatin itself illustrates this issue, because stomatin knockout mice do not develop hemolytic anemia.2 

Nonetheless, studies using simple animal models such as C. elegans  will continue to provide information about general anesthetic mechanisms in a variety of ways. First, we have only begun to identify the proteins that are important anesthetic targets. With complete genome maps available, forward genetic screening in simple animals is a much more efficient method of identifying novel candidate targets. The alternative is functionally testing the anesthetic sensitivity of every plausible gene product that is identified. Second, lack of convergence between animal models can also be informative. For example, propofol does not anesthetize C. elegans  (C. Michael Crowder, M.D., Ph.D., electronic personal communication, April 2006). Because C. elegans  GABA^Areceptors are insensitive to propofol,13this observation bolsters the conclusion that the anesthetic acts selectively in vertebrates via  specific GABA^Areceptor subtypes. Third, there are transgenic experiments that are easier to perform in worms, because their small size and simple nervous systems allow them to survive mutations that are lethal in mammals. The C. elegans N -methyl-d-aspartate receptor knockout study with nitrous oxide is an example. Knocking out the most common N -methyl-d-aspartate receptor subunit (NMDAR1) in mice is lethal, so testing the role of this subunit in mice may require finding a nonlethal mutation that selectively alters nitrous oxide sensitivity.

Molecular genetic experiments in nematodes and mice are in many ways complementary, because the advantages of each experimental model compensates for the weaknesses of the other. Our understanding of anesthetic mechanisms has already been tremendously enriched by experiments in both mice and worms, and future molecular genetic research on anesthetic targets in a variety of animal models should be encouraged.

Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts. saforman@partners.org