- Email: [email protected]
Control of anesthetic response in C. elegans
Toxicology Letters 100 – 101 (1998) 339 – 346
Control of anesthetic response in C. elegans Bernhard Kayser, Shantadurga Rajaram, Susan Thomas, Philip G. Morgan, Margaret M. Sedensky * Uni6ersity Hospitals of Cle6eland, Case Western Reser6e Uni6ersity, 11100 Euclid A6enue, Cle6eland, OH 44106 -5007, USA Accepted 7 May 1998
Abstract We describe the use of the animal model C. elegans to understand how the volatile anesthetics work at the molecular level. Mutations in several different genes can profoundly change the behavior of this animal under volatile anesthetics. Protein products of two of these genes are discussed. One gene is an integral membrane protein thought to regulate ion channels. The other is a subunit of the first protein complex of the electron transport chain. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C. elegans; Anesthetic response; Molecular level
1. Introduction Our laboratory uses the nematode Caenorhabditis elegans as an animal model to understand how the volatile anesthetics work. Over the years we have characterized the interactions of several different genes that control the animal’s behavior in gaseous anesthetics (Morgan and Cascorbi, 1985; Sedensky and Meneely, 1987; Morgan et al., 1988, 1990; Morgan and Sedensky, 1994) In addition, we now know the products of genes which, when altered, dramatically change the normal re* Corresponding author. Tel.: + 1 216 8447333; fax: +1 216 8443781; e-mail: [email protected]
sponse of the animal to volatile anesthetics. Since C. elegans brings numerous unique advantages to any attempts to understand behavior at the molecular level, it represents an extraordinary opportunity to probe the mechanism of anesthetic action. The basic premise that makes C. elegans an attractive model is that the phenomenon of general anesthesia is, of course, a whole animal occurrence. To find an anesthetic site of action in a whole animal is our fundamental goal. Given that an organism’s genes ultimately synthesize all the molecules that allow an animal to respond to volatile anesthetics, finding genes that encode an anesthetic site of action may be the easiest way to
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00204-5
340
B. Kayser et al. / Toxicology Letters 100–101 (1998) 339–346
avoid presuppositions as to the nature of an anesthetic site. This approach also avoids measuring superfluous or incidental effects of volatile anesthetics upon model systems. Changing the makeup of a gene, i.e. making a mutation, is the usual way to identify a gene amongst the many thousands that exist even in the simplest animals. Thus making a mutation in a gene that codes for an anesthetic site of action can lead to its identification and molecular characterization. In order for genetics to serve as a tool for a molecular understanding of a very complicated phenomenon like general anesthesia, a tractable genetic model system is needed.
2. C. elegans as a model C. elegans is a paragon of genetic usefulness. It is a nonparasitic soil nematode that is approximately 1 mm long (Fig. 1). In the laboratory it feeds on agar plates spread with Escherichia coli. The adult is usually a self-fertilizing herma-
phrodite that lays about 300 eggs, which grow to adulthood in about 31–32 days at 20°C. It consists of a cuticle, muscle, nerves, a gut, and a gonad. Males do occur, and can be easily propagated and used for genetic manipulations. The normal hermaphrodite always consists of exactly 959 somatic cells. Exactly 302 of these cells are neurons. Since the entire animal has been serially sectioned and studied under the electron microscope, every synapse is known. In addition, the embryonic lineage of every cell has been traced back to the single-celled zygote. The haploid DNA of this metazoan is only about 25 times that of E. coli; nearly all of its genome has been ordered relative to a map of known genetic mutations. Over 60% of its DNA has been sequenced, an enormous advantage in the molecular identification of genes and their products. Powerful tools exist to study the temporal and spatial expression of the gene products. Although the simplicity of this animal is an important aspect of its remarkable usefulness, it does possess complex behaviors that are mediated
Fig. 1. A micrograph of the nematode C. elegans. The adult hermaphrodite is pictured above. The male is depicted below. The hermaphrodite is approximately 1 mm long. Reprinted with permission from Sulston and Horvitz (1977).
B. Kayser et al. / Toxicology Letters 100–101 (1998) 339–346
by the same neurotransmitters that mediate behavior in more complicated animals. C. elegans possesses acetylcholine, GABA, dopamine, and serotonin. There is a large collection of well-studied mutants; for example there are over 100 known mutations designated as uncoordinated, with presumed derangements of neuromuscular function. These can be obtained through the mail from an archival national stock center. There also exists an extensive computerized data base that receives contributions from an international network of laboratories currently investigating diverse aspects of C. elegans’s development and behavior. For a full review of the organism’s salient features see Riddle et al. (1997).
3. Behavioral studies It initially appeared to us that C. elegans was a simple enough animal to study how volatile anesthetics work at the molecular level, yet complicated enough so that the data obtained may be relevant to more complex species. However, we first had to establish that the worms could be anesthetized. Nonmutated worms, denoted as N2, initially become hyperactive, as if avoiding a noxious stimulus, when placed in a gaseous anesthetic. Their usually sinuous movement across the agar plate becomes progressively uncoordinated until motion ceases altogether. At this endpoint the animals no longer withdraw from a tap to the snout. This flaccid paralysis is quickly reversed upon removing animals from the gas. We chose this reversible immobility as our endpoint with which to define ‘anesthesia’ in C. elegans, and proceeded to expose N2 to a range of gaseous agents whose oil – gas partition coefficients varied from 48 to 7230 (Morgan et al., 1988). We found that like every animal ever tested, N2 follows the Meyer – Overton rule: a log – log plot of EC50s versus oil – gas partition coefficients yielded a very good straight line fit with a slope of − 1. However, the absolute concentrations at which the worms become paralyzed is quite different from a mammalian MAC. For example, the EC50 of N2 in halothane is 3.3% at 20°C, which is scored after a
341
2 h exposure. Obviously 3% halothane is a toxic dose in humans, who possess both a circulatory and respiratory system that would ultimately collapse in the face of such an exposure. This necessitated a further look into our choice of endpoints. Specifically we tried to reproduce the results of Crowder et al. (1996), that implicate somewhat subtle behavioral changes of C. elegans after exposure to 4% halothane. We were unable to duplicate his results concerning toxicity. We have studied the effects of halothane exposure on male mating (Fig. 2). To try to reproduce exactly the results of Crowder we exposed N2 males to 16 h of 4% halothane and then tested them for mating. Since this represents paralysis and starvation for about 5% of their adult life, we performed age-matched controls that received no treatment, and also controls that were kept immobile in the refrigerator for 16 h. Anesthetized males, per the method of scoring of Crowder, were no different in this assay than age-matched controls, and much better at mating than those kept in the cold overnight. In addition, we mated young adult males and hermaphrodites after a 3 h exposure to 4% halothane, conditions which more closely simulate our method of generating dose– response curves. These males were tested for actual number of progeny produced. Once again, no difference was seen between males that had been anesthetized and those that had not. In addition, there was no effect on the number of recombinant progeny when the hermaphrodites had also been anesthetized. We have also tested C. elegans for its ability to move towards attractants after exposure to halothane (Fig. 3). After 3 h of exposure to 4% halothane and a 16 h recovery, N2 was scored for its coordination and its ability to move toward isoamyl alcohol (as per Crowder’s adaptation of the chemotaxis assay of Bargmann et al., 1993). There was no difference between animals exposed or not exposed to halothane in their coordination or ability to move to isoamyl alcohol. We also tested their ability to move toward a spot of food after exposure to halothane, and once again found no difference between controls and animals exposed to 4% halothane for 3 h. We are quite convinced that 4% halothane does not produce
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Fig. 2. Recovery of C. elegans after exposure to 4% halothane; effects on male mating. In 16 h experiments L4 males were exposed to 4% halothane at 20°C for 16 h. One set of age-matched controls was kept at 20°C and 0% halothane for 16 h, and another was kept at 4°C and 0% halothane for 16 h. Controls and anesthetized males were then mated to dpy-11 hermaphrodites as described by Crowder et al. (1996). Six sets of matings were performed per experimental condition. Mating was assayed as per Crowder and expressed on the y-axis as a percent compared to unanesthetized males kept at 20°C. Halothane concentration was measured at the beginning of the experiment via gas chromatography to be 4.1%, and 3.9% at the end of the exposure. In experiments assaying 3 h exposure to halothane, young adult worms were exposed to 4% halothane for 3 h at 20°C and allowed to recover for 16–18 h. Age-matched controls were kept at 20°C and 0% halothane for the entire pre-mating period. The following matings were then performed: (1) unanesthetized N2 males to unanesthetized dpy-11 hermaphrodites; (2) anesthetized N2 males to unanesthetized dpy-11 hermaphrodites; (3) unanesthetized N2 males to anesthetized dpy-11 hermaphrodites; (4) anesthetized N2 males to anesthetized dpy-11 hermaphrodites. Six matings were carried out per combination, each using three males to three hermaphrodites on a plate spotted with a 1 cm food source. Halothane concentration as checked by gas chromatography ranged from 3.8% to 4.1% at the end of the each experiment. Non-dumpy offspring were counted on each plate at 72 h after the beginning of mating. Recombinant offspring are expressed on the y-axis as a percent of mating (1), in which no worms were exposed to halothane. No differences were significant between worms exposed to 4% halothane vs. 0% halothane at 20°C for either the 16 or 3 h exposure.
immobility by toxic effects in these animals, which live in the wild at approximately 15°C amidst a verity of lipophilic compounds. They possess neither heart, lung, or liver. To try to place anthro-
pomorphic constraints upon the absolute concentration of volatile anesthetic that produces reversible immobility in C. elegans may be an endeavor fraught with misconceptions. Having established that N2 follows the Meyer– Overton rule when reversible immobility is used as an endpoint, we sought mutant animals that are changed in their sensitivity to volatile anesthetics. We have collected an array of behavioral changes that have arisen by single gene mutations. The first of these, unc-79 is extremely sensitive to halothane, with an EC50 only 1/3 that of N2. In fact it is very hypersensitive to all of the most lipid soluble volatile anesthetics that we tested (thiomethoxyflurane, methoxyflurane, chloroform, and halothane). However, it is slightly resistant to enflurane and flurothyl (hexafluorodiethylether), and no different from N2 in its behavior in isoflurane or fluroxene. It is mildly hypersensitive to diethyl ether. Clearly this animal’s behavior deviates from the Meyer–Overton rule (Morgan et al., 1988).
Fig. 3. Recovery of C. elegans after 4% halothane exposure for 3 h: effects on mobility and chemotaxis. N2 worms were exposed to 4% halothane at 20°C and scored for mobility and chemotaxis per the method of Crowder et al. (1996). An additional experimental condition, chemotaxis to a food source, was also tested with E. coli as a substitute for the volatile attractants used by Crowder. Halothane concentrations were checked via gas chromatography and ranged between 3.9% and 4.5% in all cases. In mobility assays n =5, isoamyl alcohol chemotaxis, n =9, and food chemotaxis n =6, where n is the number of plates scored. For every experimental plate, a control plate of unanesthetized animals was scored simultaneously.
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Fig. 4. Percent change of unc-79 and unc-79:unc-1 in nine anesthetics compared to N2. Calculated as (unc-79 EC50 −N2 EC50)/(N2 EC50) or (unc-79:unc-1 EC50 − N2 EC50)/(N2 EC50). When no bar is seen, the response of the mutant is identical to N2. Abbreviations: TMOF, thiomethoxyflurane; MOF, methoxyflurane; CH, chloroform; H, halothane; E, enflurane; ISO, isoflurane; DE, diethylether; FLR, flurothyl; FLX, fluroxene.
Even in a simple animal ‘anesthesia’ is a complicated process, one that may be disrupted at any of a number of levels. A family of interacting genes would best uncover gene products that directly interact with volatile anesthetics (an actual anesthetic target). Therefore we sought suppressors of the gene unc-79, i.e. mutations that would cause an unc-79 animal to behave normally in volatile anesthetics. One of the best suppressors is the gene unc-1 (Morgan et al., 1990). unc-1 Is an X-linked gene that can restore the behavior of unc-79 in halothane exactly back to normal; the EC50 of a worm carrying both mutations unc-79:unc-1 is identical to that of N2. In fact it restores the hypersensitivity of unc-79 to chloroform, methoxyflurane, and thiomethoxy-flurane back to baseline, as well as restoring to normal unc-79 ’s resistance to enflurane and flurothyl. However it does not change unc-79 ’s sensitivity to diethyl ether (Fig. 4). unc-1 itself is sensitive to diethylether, but otherwise identical to N2 in its anesthetic response.
The fact that different mutations can specifically change C. elegans’s behavior to individual anesthetics is quite remarkable. It is consistent with the notion that there are multiple sites of action of volatile anesthetics, even in a simple animal. For example, there might be two sites (or two domains of a single molecule, or two different neural pathways, etc.) that are affected by mutations in unc-79. One of these is restored to normal by unc-1 and the other is not. However, there may be yet another site which governs the animal’s behavior in isoflurane, for example, that is not affected by mutations in either unc-79 or unc-1. By combining mutations within a single animal and observing the behavior of that animal in volatile anesthetics, it is possible to order them in a genetic pathway reflecting control of anesthetic sensitivity in C. elegans. Since the behavior of the unc-79:unc-1 animal is like that of unc-1 itself, the unc-79 gene product must somehow exert its effects through the unc-1 gene, i.e. unc-79 cannot behave like unc-79 without the normal unc-1 gene product.
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unc-1 Is an unusual gene in that there are many different versions of mutations in it (Park and Horvitz, 1986). There are both dominant and recessive types of unc-1 mutations, mutations that make the animal coil and mutations that make the animal kink, and mutations that only show their effects at particular temperatures. This makes it all the more compelling as a gene whose molecular characterization may yield some useful information as to its function. Another gene under intense investigation in our laboratory is gas-1 (Morgan and Sedensky, 1994). It was originally picked as part of a search for mutations that change C. elegans’s response to isoflurane, a gas for which we lacked hypersensitive mutants. gas-1 ( fc21 ) Moves well in air, but its EC50 in isoflurane is 1.3%, only 1/5 that of N2 (EC50 = 7.2%). However, unlike any other gene we have isolated, gas-1 is hypersensitive to every volatile anesthetic that we have tested. gas-1 is not suppressed by unc-1. In fact animals carrying gas-1 and any other mutation that changes anesthetic sensitivity always behaves like gas-1. In addition to its anesthetic sensitivity, this mutation causes the worms to lay fewer eggs than normal, develop more slowly than N2, and live about half as long as normal. In addition to these three genes, our laboratory has identified several other genes that can change anesthetic response in C. elegans. One of them, unc-80, behaves very much like unc-79 but maps to a completely different chromosome (Morgan et al., 1988). Another, fc34, is hypersensitive to all gasses but shrinks to about 1/3 its normal size when immobilized (Morgan and Sedensky, 1994). Another class of mutations suppresses the suppressor activity of unc-1 (Morgan and Sedensky, unpublished results). Overall we have accumulated an entire array of interacting genes that control anesthetic response, in an animal of 302 neurons.
4. Molecular characterization Our goal in accumulating a family of mutations that encompass a spectrum of behaviors in volatile anesthetics is to find all the genes that
control the anesthetic response in C. elegans. Clearly a complicated pathway exists even in this simple animal. However, this approach has allowed us to single out which genes to first characterize at the molecular level. It will also allow us to interpret our molecular data in light of a range of observed behaviors.
4.1. unc-1 The unc-1 mutation causes the hypersensitive mutant unc-79 to behave normally in the most lipid soluble volatile anesthetics. unc-1 is itself sensitive to diethylether. Through standard genetic mapping, we localized this gene to a very small region of the X chromosome. Because the entire genetic makeup of C. elegans is so wellstudied, we were able to correlate this small region to actual physical pieces of DNA that have been ordered relative to each other and to this region of the X chromosome. These pieces of DNA have been cloned, that is physically isolated from all other worm DNA and carried in a bacterium. Called cosmids, they were used to finally pinpoint the location of the unc-1 gene by a technique called mutant rescue (Fire, 1986). In mutant rescue, defective genes are restored to normal by the microinjection of DNA into the syncytial gonad of the C. elegans hermaphrodite. Subsequent offspring of the injected animal are able to take up and express the DNA injected into its parent. For example, we injected a small set of cosmids that we postulated would contain the unc-1 gene into an unc-1 hermaphrodite. From the injected unc-1 parent emerged normal appearing offspring, i.e. the mutant was ‘rescued’. By injecting subsets of the original mix of cosmids, and then fragments of cosmids, we were able to narrow unc-1 down to one of two predicted genes. Analyzing the intermediary molecules that DNA uses to make its protein product, we determined that the unc-1 gene codes for a protein called stomatin. This was confirmed by analyzing the DNA sequence of all the different versions of the unc-1 gene that are available. Changes in the DNA content of these various forms of unc-1 showed changes in different amino acids of the protein stomatin.
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In humans, stomatin is known to be an integral protein of the cell membrane (Stewart et al., 1992, 1993), thought to regulate cation conductance in red blood cells. Lack of this protein causes red blood cells to swell and lyse, presumably due to changes in membrane ion conductance. Human stomatin is though to activate and/or regulate an associated ion channel in a ball and chain model of receptor regulation. We are currently identifying the tissues in which the protein is expressed, as well as its pattern of expression in combination with other mutations like gas-1.
4.2. gas-1 gas-1 Was identified in a similar manner to unc-1, via fine genetic mapping, mutant rescue, and sequencing of DNA. It produces a protein that is homologous to one particular subunit of a large complex of proteins within the mitochondrial cell membrane. This complex, complex 1, is the first in the electron transport chain (Walker, 1992), gas-1 is similar to the 49 kDa subunit within it. Our version of gas-1, fc21, is a single base change in a very highly conserved region of this protein. It results in a very minor amino acid change of lysine to arginine at position 290 of this protein. However position 290 is always lysine in the 12 other species in which this protein has been sequenced, species ranging from bacteria to fungi to cows (Fearnley and Walker, 1992). Thus, although it appears most remarkable that a single base change in one subunit of 15 – 40 proteins within this complex can make an animal exquisitely sensitive to all volatile anesthetics, it also appears that this position is somehow crucial to its function. Mitochondrial function is obviously essential to many aspects of cellular physiology. Since a mutation in gas-1 overrides the effects of any other mutations we have isolated to date, we have postulated that it is part of a final common mechanism through which all volatile anesthetics act. Energy production, calcium homeostasis, and maintenance of membrane stability are critical roles of complex I (also known as NADH: ubiquinone oxidoreductase and NADH dehydrogenase). Any of these functions are candidates to
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mediate the effects of this mutation on behavior in volatile anesthetics. We are in the process of generating more mutations in this gene, and studying the pattern of expression of its protein. We also plan to study the metabolic effects and the oxidative state of the cell membranes of this mutant.
5. Summary We have characterized a genetic pathway that controls anesthetic sensitivity in the nematode C. elegans. This organism contains a fully-diagrammed nervous system, grows easily and cheaply in the laboratory, and is amenable to characterization of gene products at a molecular level. The endpoint of reversible immobility appears to have no toxic effects on the worms’ behavior in our hands. Multiple genes have been identified that profoundly alter the behavior of this animal in volatile anesthetics. Two genes that control anesthetic response have been cloned. One of these, stomatin, is postulated to activate associated ion channels. The other, a subunit of the first complex of the electron transport chain, is crucial to multiple aspects of intracellular physiology. Several avenues of future research are discussed. C. elegans is a very exploitable model in which to explore the mechanism of action of volatile anesthetics.
References Bargmann, C.I., Hartweig, E., Horvitz, H.R., 1993. Odorant selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515 – 527. Crowder, C.M., Shebester, L.D., Schedl, T., 1996. Behavioral effects of volatile anesthetics in C. elegans. Anesthesiology 85, 901 – 912. Fearnley, I.M., Walker, J.E., 1992. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochem. Biophys. Acta. 1140, 105 – 134. Fire, A., 1986. Integrative transformation of C.elegans. EMBO J. 5 (10), 2673 – 2680. Morgan, P.G., Cascorbi, H.F., 1985. Effect of anesthetics and a convulsant on normal and mutant Caenorhabditis elegans. Anesthesiology 62, 738 – 744.
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Morgan, P.G., Sedensky, M.M., Meneely, P.M., 1990. Multiple sites of action of volatile aesthetics in C. elegans. Proc. Natl. Acad. Sci. 87, 2965 – 2969. Morgan, P.G., Sedensky, M.M., 1994. Mutations conferring new patterns of sensitivity to volatile anesthetics in C. elegans. Anesthesiology 81 (4), 888–898. Morgan, P.G., Sedensky, M.M., Meneely, P.M., Cascorbi, H.F., 1988. The effect of two genes on anesthetic response in the nematode Caenorhabitidis elegans. Anesthesiology 69, 246 – 251. Park, E.C., Horvitz, H.R., 1986. Mutations with dominant effects on the behavior and morphology of the nematode Caenorhabditis elegans. Genetics 113, 821–852. Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R., 1997. C. elecans II. Cold Spring Harbor Laboratory Press, Oxford.
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Sedensky, M.M., Meneely, P.M., 1987. Genetic analysis of halothane sensitivity in C. elegans. Science 236, 952 – 954. Stewart, G.W., Argent, A.C., Dash, B.C.J., 1993. Stomatin: a putative cation transport regulator in red cell membrane. Biochem. Biophys. Acta 1225, 15 – 25. Stewart, G.W., Hepworth, G.W., Jones, B.E., et al., 1992. Isolation of cDNA coding for a ubiquitous membrane protein deficient in high NA + , low K + , stomatocytic erythrocytes. Blood 79, 1593 – 1601. Sulston, J.E., Horvitz, H.R., 1977. Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56, 110 – 156. Walker, J.E., 1992. The NADH: ubiquinone oxidoreductase (complex 1) of respiratory chains. Q. Rev. Biophys. 5 (3), 253 – 324.
Control of anesthetic response in C. elegans Bernhard Kayser, Shantadurga Rajaram, Susan Thomas, Philip G. Morgan, Margaret M. Sedensky * Uni6ersity Hospitals of Cle6eland, Case Western Reser6e Uni6ersity, 11100 Euclid A6enue, Cle6eland, OH 44106 -5007, USA Accepted 7 May 1998
Abstract We describe the use of the animal model C. elegans to understand how the volatile anesthetics work at the molecular level. Mutations in several different genes can profoundly change the behavior of this animal under volatile anesthetics. Protein products of two of these genes are discussed. One gene is an integral membrane protein thought to regulate ion channels. The other is a subunit of the first protein complex of the electron transport chain. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C. elegans; Anesthetic response; Molecular level
1. Introduction Our laboratory uses the nematode Caenorhabditis elegans as an animal model to understand how the volatile anesthetics work. Over the years we have characterized the interactions of several different genes that control the animal’s behavior in gaseous anesthetics (Morgan and Cascorbi, 1985; Sedensky and Meneely, 1987; Morgan et al., 1988, 1990; Morgan and Sedensky, 1994) In addition, we now know the products of genes which, when altered, dramatically change the normal re* Corresponding author. Tel.: + 1 216 8447333; fax: +1 216 8443781; e-mail: [email protected]
sponse of the animal to volatile anesthetics. Since C. elegans brings numerous unique advantages to any attempts to understand behavior at the molecular level, it represents an extraordinary opportunity to probe the mechanism of anesthetic action. The basic premise that makes C. elegans an attractive model is that the phenomenon of general anesthesia is, of course, a whole animal occurrence. To find an anesthetic site of action in a whole animal is our fundamental goal. Given that an organism’s genes ultimately synthesize all the molecules that allow an animal to respond to volatile anesthetics, finding genes that encode an anesthetic site of action may be the easiest way to
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00204-5
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B. Kayser et al. / Toxicology Letters 100–101 (1998) 339–346
avoid presuppositions as to the nature of an anesthetic site. This approach also avoids measuring superfluous or incidental effects of volatile anesthetics upon model systems. Changing the makeup of a gene, i.e. making a mutation, is the usual way to identify a gene amongst the many thousands that exist even in the simplest animals. Thus making a mutation in a gene that codes for an anesthetic site of action can lead to its identification and molecular characterization. In order for genetics to serve as a tool for a molecular understanding of a very complicated phenomenon like general anesthesia, a tractable genetic model system is needed.
2. C. elegans as a model C. elegans is a paragon of genetic usefulness. It is a nonparasitic soil nematode that is approximately 1 mm long (Fig. 1). In the laboratory it feeds on agar plates spread with Escherichia coli. The adult is usually a self-fertilizing herma-
phrodite that lays about 300 eggs, which grow to adulthood in about 31–32 days at 20°C. It consists of a cuticle, muscle, nerves, a gut, and a gonad. Males do occur, and can be easily propagated and used for genetic manipulations. The normal hermaphrodite always consists of exactly 959 somatic cells. Exactly 302 of these cells are neurons. Since the entire animal has been serially sectioned and studied under the electron microscope, every synapse is known. In addition, the embryonic lineage of every cell has been traced back to the single-celled zygote. The haploid DNA of this metazoan is only about 25 times that of E. coli; nearly all of its genome has been ordered relative to a map of known genetic mutations. Over 60% of its DNA has been sequenced, an enormous advantage in the molecular identification of genes and their products. Powerful tools exist to study the temporal and spatial expression of the gene products. Although the simplicity of this animal is an important aspect of its remarkable usefulness, it does possess complex behaviors that are mediated
Fig. 1. A micrograph of the nematode C. elegans. The adult hermaphrodite is pictured above. The male is depicted below. The hermaphrodite is approximately 1 mm long. Reprinted with permission from Sulston and Horvitz (1977).
B. Kayser et al. / Toxicology Letters 100–101 (1998) 339–346
by the same neurotransmitters that mediate behavior in more complicated animals. C. elegans possesses acetylcholine, GABA, dopamine, and serotonin. There is a large collection of well-studied mutants; for example there are over 100 known mutations designated as uncoordinated, with presumed derangements of neuromuscular function. These can be obtained through the mail from an archival national stock center. There also exists an extensive computerized data base that receives contributions from an international network of laboratories currently investigating diverse aspects of C. elegans’s development and behavior. For a full review of the organism’s salient features see Riddle et al. (1997).
3. Behavioral studies It initially appeared to us that C. elegans was a simple enough animal to study how volatile anesthetics work at the molecular level, yet complicated enough so that the data obtained may be relevant to more complex species. However, we first had to establish that the worms could be anesthetized. Nonmutated worms, denoted as N2, initially become hyperactive, as if avoiding a noxious stimulus, when placed in a gaseous anesthetic. Their usually sinuous movement across the agar plate becomes progressively uncoordinated until motion ceases altogether. At this endpoint the animals no longer withdraw from a tap to the snout. This flaccid paralysis is quickly reversed upon removing animals from the gas. We chose this reversible immobility as our endpoint with which to define ‘anesthesia’ in C. elegans, and proceeded to expose N2 to a range of gaseous agents whose oil – gas partition coefficients varied from 48 to 7230 (Morgan et al., 1988). We found that like every animal ever tested, N2 follows the Meyer – Overton rule: a log – log plot of EC50s versus oil – gas partition coefficients yielded a very good straight line fit with a slope of − 1. However, the absolute concentrations at which the worms become paralyzed is quite different from a mammalian MAC. For example, the EC50 of N2 in halothane is 3.3% at 20°C, which is scored after a
341
2 h exposure. Obviously 3% halothane is a toxic dose in humans, who possess both a circulatory and respiratory system that would ultimately collapse in the face of such an exposure. This necessitated a further look into our choice of endpoints. Specifically we tried to reproduce the results of Crowder et al. (1996), that implicate somewhat subtle behavioral changes of C. elegans after exposure to 4% halothane. We were unable to duplicate his results concerning toxicity. We have studied the effects of halothane exposure on male mating (Fig. 2). To try to reproduce exactly the results of Crowder we exposed N2 males to 16 h of 4% halothane and then tested them for mating. Since this represents paralysis and starvation for about 5% of their adult life, we performed age-matched controls that received no treatment, and also controls that were kept immobile in the refrigerator for 16 h. Anesthetized males, per the method of scoring of Crowder, were no different in this assay than age-matched controls, and much better at mating than those kept in the cold overnight. In addition, we mated young adult males and hermaphrodites after a 3 h exposure to 4% halothane, conditions which more closely simulate our method of generating dose– response curves. These males were tested for actual number of progeny produced. Once again, no difference was seen between males that had been anesthetized and those that had not. In addition, there was no effect on the number of recombinant progeny when the hermaphrodites had also been anesthetized. We have also tested C. elegans for its ability to move towards attractants after exposure to halothane (Fig. 3). After 3 h of exposure to 4% halothane and a 16 h recovery, N2 was scored for its coordination and its ability to move toward isoamyl alcohol (as per Crowder’s adaptation of the chemotaxis assay of Bargmann et al., 1993). There was no difference between animals exposed or not exposed to halothane in their coordination or ability to move to isoamyl alcohol. We also tested their ability to move toward a spot of food after exposure to halothane, and once again found no difference between controls and animals exposed to 4% halothane for 3 h. We are quite convinced that 4% halothane does not produce
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Fig. 2. Recovery of C. elegans after exposure to 4% halothane; effects on male mating. In 16 h experiments L4 males were exposed to 4% halothane at 20°C for 16 h. One set of age-matched controls was kept at 20°C and 0% halothane for 16 h, and another was kept at 4°C and 0% halothane for 16 h. Controls and anesthetized males were then mated to dpy-11 hermaphrodites as described by Crowder et al. (1996). Six sets of matings were performed per experimental condition. Mating was assayed as per Crowder and expressed on the y-axis as a percent compared to unanesthetized males kept at 20°C. Halothane concentration was measured at the beginning of the experiment via gas chromatography to be 4.1%, and 3.9% at the end of the exposure. In experiments assaying 3 h exposure to halothane, young adult worms were exposed to 4% halothane for 3 h at 20°C and allowed to recover for 16–18 h. Age-matched controls were kept at 20°C and 0% halothane for the entire pre-mating period. The following matings were then performed: (1) unanesthetized N2 males to unanesthetized dpy-11 hermaphrodites; (2) anesthetized N2 males to unanesthetized dpy-11 hermaphrodites; (3) unanesthetized N2 males to anesthetized dpy-11 hermaphrodites; (4) anesthetized N2 males to anesthetized dpy-11 hermaphrodites. Six matings were carried out per combination, each using three males to three hermaphrodites on a plate spotted with a 1 cm food source. Halothane concentration as checked by gas chromatography ranged from 3.8% to 4.1% at the end of the each experiment. Non-dumpy offspring were counted on each plate at 72 h after the beginning of mating. Recombinant offspring are expressed on the y-axis as a percent of mating (1), in which no worms were exposed to halothane. No differences were significant between worms exposed to 4% halothane vs. 0% halothane at 20°C for either the 16 or 3 h exposure.
immobility by toxic effects in these animals, which live in the wild at approximately 15°C amidst a verity of lipophilic compounds. They possess neither heart, lung, or liver. To try to place anthro-
pomorphic constraints upon the absolute concentration of volatile anesthetic that produces reversible immobility in C. elegans may be an endeavor fraught with misconceptions. Having established that N2 follows the Meyer– Overton rule when reversible immobility is used as an endpoint, we sought mutant animals that are changed in their sensitivity to volatile anesthetics. We have collected an array of behavioral changes that have arisen by single gene mutations. The first of these, unc-79 is extremely sensitive to halothane, with an EC50 only 1/3 that of N2. In fact it is very hypersensitive to all of the most lipid soluble volatile anesthetics that we tested (thiomethoxyflurane, methoxyflurane, chloroform, and halothane). However, it is slightly resistant to enflurane and flurothyl (hexafluorodiethylether), and no different from N2 in its behavior in isoflurane or fluroxene. It is mildly hypersensitive to diethyl ether. Clearly this animal’s behavior deviates from the Meyer–Overton rule (Morgan et al., 1988).
Fig. 3. Recovery of C. elegans after 4% halothane exposure for 3 h: effects on mobility and chemotaxis. N2 worms were exposed to 4% halothane at 20°C and scored for mobility and chemotaxis per the method of Crowder et al. (1996). An additional experimental condition, chemotaxis to a food source, was also tested with E. coli as a substitute for the volatile attractants used by Crowder. Halothane concentrations were checked via gas chromatography and ranged between 3.9% and 4.5% in all cases. In mobility assays n =5, isoamyl alcohol chemotaxis, n =9, and food chemotaxis n =6, where n is the number of plates scored. For every experimental plate, a control plate of unanesthetized animals was scored simultaneously.
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Fig. 4. Percent change of unc-79 and unc-79:unc-1 in nine anesthetics compared to N2. Calculated as (unc-79 EC50 −N2 EC50)/(N2 EC50) or (unc-79:unc-1 EC50 − N2 EC50)/(N2 EC50). When no bar is seen, the response of the mutant is identical to N2. Abbreviations: TMOF, thiomethoxyflurane; MOF, methoxyflurane; CH, chloroform; H, halothane; E, enflurane; ISO, isoflurane; DE, diethylether; FLR, flurothyl; FLX, fluroxene.
Even in a simple animal ‘anesthesia’ is a complicated process, one that may be disrupted at any of a number of levels. A family of interacting genes would best uncover gene products that directly interact with volatile anesthetics (an actual anesthetic target). Therefore we sought suppressors of the gene unc-79, i.e. mutations that would cause an unc-79 animal to behave normally in volatile anesthetics. One of the best suppressors is the gene unc-1 (Morgan et al., 1990). unc-1 Is an X-linked gene that can restore the behavior of unc-79 in halothane exactly back to normal; the EC50 of a worm carrying both mutations unc-79:unc-1 is identical to that of N2. In fact it restores the hypersensitivity of unc-79 to chloroform, methoxyflurane, and thiomethoxy-flurane back to baseline, as well as restoring to normal unc-79 ’s resistance to enflurane and flurothyl. However it does not change unc-79 ’s sensitivity to diethyl ether (Fig. 4). unc-1 itself is sensitive to diethylether, but otherwise identical to N2 in its anesthetic response.
The fact that different mutations can specifically change C. elegans’s behavior to individual anesthetics is quite remarkable. It is consistent with the notion that there are multiple sites of action of volatile anesthetics, even in a simple animal. For example, there might be two sites (or two domains of a single molecule, or two different neural pathways, etc.) that are affected by mutations in unc-79. One of these is restored to normal by unc-1 and the other is not. However, there may be yet another site which governs the animal’s behavior in isoflurane, for example, that is not affected by mutations in either unc-79 or unc-1. By combining mutations within a single animal and observing the behavior of that animal in volatile anesthetics, it is possible to order them in a genetic pathway reflecting control of anesthetic sensitivity in C. elegans. Since the behavior of the unc-79:unc-1 animal is like that of unc-1 itself, the unc-79 gene product must somehow exert its effects through the unc-1 gene, i.e. unc-79 cannot behave like unc-79 without the normal unc-1 gene product.
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unc-1 Is an unusual gene in that there are many different versions of mutations in it (Park and Horvitz, 1986). There are both dominant and recessive types of unc-1 mutations, mutations that make the animal coil and mutations that make the animal kink, and mutations that only show their effects at particular temperatures. This makes it all the more compelling as a gene whose molecular characterization may yield some useful information as to its function. Another gene under intense investigation in our laboratory is gas-1 (Morgan and Sedensky, 1994). It was originally picked as part of a search for mutations that change C. elegans’s response to isoflurane, a gas for which we lacked hypersensitive mutants. gas-1 ( fc21 ) Moves well in air, but its EC50 in isoflurane is 1.3%, only 1/5 that of N2 (EC50 = 7.2%). However, unlike any other gene we have isolated, gas-1 is hypersensitive to every volatile anesthetic that we have tested. gas-1 is not suppressed by unc-1. In fact animals carrying gas-1 and any other mutation that changes anesthetic sensitivity always behaves like gas-1. In addition to its anesthetic sensitivity, this mutation causes the worms to lay fewer eggs than normal, develop more slowly than N2, and live about half as long as normal. In addition to these three genes, our laboratory has identified several other genes that can change anesthetic response in C. elegans. One of them, unc-80, behaves very much like unc-79 but maps to a completely different chromosome (Morgan et al., 1988). Another, fc34, is hypersensitive to all gasses but shrinks to about 1/3 its normal size when immobilized (Morgan and Sedensky, 1994). Another class of mutations suppresses the suppressor activity of unc-1 (Morgan and Sedensky, unpublished results). Overall we have accumulated an entire array of interacting genes that control anesthetic response, in an animal of 302 neurons.
4. Molecular characterization Our goal in accumulating a family of mutations that encompass a spectrum of behaviors in volatile anesthetics is to find all the genes that
control the anesthetic response in C. elegans. Clearly a complicated pathway exists even in this simple animal. However, this approach has allowed us to single out which genes to first characterize at the molecular level. It will also allow us to interpret our molecular data in light of a range of observed behaviors.
4.1. unc-1 The unc-1 mutation causes the hypersensitive mutant unc-79 to behave normally in the most lipid soluble volatile anesthetics. unc-1 is itself sensitive to diethylether. Through standard genetic mapping, we localized this gene to a very small region of the X chromosome. Because the entire genetic makeup of C. elegans is so wellstudied, we were able to correlate this small region to actual physical pieces of DNA that have been ordered relative to each other and to this region of the X chromosome. These pieces of DNA have been cloned, that is physically isolated from all other worm DNA and carried in a bacterium. Called cosmids, they were used to finally pinpoint the location of the unc-1 gene by a technique called mutant rescue (Fire, 1986). In mutant rescue, defective genes are restored to normal by the microinjection of DNA into the syncytial gonad of the C. elegans hermaphrodite. Subsequent offspring of the injected animal are able to take up and express the DNA injected into its parent. For example, we injected a small set of cosmids that we postulated would contain the unc-1 gene into an unc-1 hermaphrodite. From the injected unc-1 parent emerged normal appearing offspring, i.e. the mutant was ‘rescued’. By injecting subsets of the original mix of cosmids, and then fragments of cosmids, we were able to narrow unc-1 down to one of two predicted genes. Analyzing the intermediary molecules that DNA uses to make its protein product, we determined that the unc-1 gene codes for a protein called stomatin. This was confirmed by analyzing the DNA sequence of all the different versions of the unc-1 gene that are available. Changes in the DNA content of these various forms of unc-1 showed changes in different amino acids of the protein stomatin.
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In humans, stomatin is known to be an integral protein of the cell membrane (Stewart et al., 1992, 1993), thought to regulate cation conductance in red blood cells. Lack of this protein causes red blood cells to swell and lyse, presumably due to changes in membrane ion conductance. Human stomatin is though to activate and/or regulate an associated ion channel in a ball and chain model of receptor regulation. We are currently identifying the tissues in which the protein is expressed, as well as its pattern of expression in combination with other mutations like gas-1.
4.2. gas-1 gas-1 Was identified in a similar manner to unc-1, via fine genetic mapping, mutant rescue, and sequencing of DNA. It produces a protein that is homologous to one particular subunit of a large complex of proteins within the mitochondrial cell membrane. This complex, complex 1, is the first in the electron transport chain (Walker, 1992), gas-1 is similar to the 49 kDa subunit within it. Our version of gas-1, fc21, is a single base change in a very highly conserved region of this protein. It results in a very minor amino acid change of lysine to arginine at position 290 of this protein. However position 290 is always lysine in the 12 other species in which this protein has been sequenced, species ranging from bacteria to fungi to cows (Fearnley and Walker, 1992). Thus, although it appears most remarkable that a single base change in one subunit of 15 – 40 proteins within this complex can make an animal exquisitely sensitive to all volatile anesthetics, it also appears that this position is somehow crucial to its function. Mitochondrial function is obviously essential to many aspects of cellular physiology. Since a mutation in gas-1 overrides the effects of any other mutations we have isolated to date, we have postulated that it is part of a final common mechanism through which all volatile anesthetics act. Energy production, calcium homeostasis, and maintenance of membrane stability are critical roles of complex I (also known as NADH: ubiquinone oxidoreductase and NADH dehydrogenase). Any of these functions are candidates to
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mediate the effects of this mutation on behavior in volatile anesthetics. We are in the process of generating more mutations in this gene, and studying the pattern of expression of its protein. We also plan to study the metabolic effects and the oxidative state of the cell membranes of this mutant.
5. Summary We have characterized a genetic pathway that controls anesthetic sensitivity in the nematode C. elegans. This organism contains a fully-diagrammed nervous system, grows easily and cheaply in the laboratory, and is amenable to characterization of gene products at a molecular level. The endpoint of reversible immobility appears to have no toxic effects on the worms’ behavior in our hands. Multiple genes have been identified that profoundly alter the behavior of this animal in volatile anesthetics. Two genes that control anesthetic response have been cloned. One of these, stomatin, is postulated to activate associated ion channels. The other, a subunit of the first complex of the electron transport chain, is crucial to multiple aspects of intracellular physiology. Several avenues of future research are discussed. C. elegans is a very exploitable model in which to explore the mechanism of action of volatile anesthetics.
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