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Anesth Analg 2002;95:578-582
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Mice with Glycine Receptor Subunit Mutations Are Both Sensitive and Resistant to Volatile Anesthetics

Joseph J. Quinlan, MD*, Carolyn Ferguson, BS*, Katherine Jester, BS*, Leonard L. Firestone, MD*, and Gregg E. Homanics, PhD*{dagger}

Departments of *Anesthesiology and {dagger}Pharmacology, University of Pittsburgh, Pennsylvania

Address correspondence and reprint requests to Gregg E. Homanics, PhD, University of Pittsburgh, Department of Anesthesiology, W1356 Biomedical Science Tower, Pittsburgh, PA 15261. Address e-mail to homanicsge{at}anes.upmc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We used two mouse lines with glycine receptor mutations to determine whether glycine receptors might play an important role in anesthetic responses in vivo. Spastic (spA) mutants were slightly more sensitive (P = 0.02) to enflurane in the loss-of-righting reflex assay (50% effective concentration [EC50] = 1.17 ± 0.06 atm for controls versus 0.97 ± 0.06 atm for spA) but were also substantially more resistant (P = 0.01) to enflurane in the tail clamp assay (EC50 = 1.96 ± 0.10 atm for controls versus 2.58 ± 0.25 atm for spA). spA mice were also more sensitive to halothane (P < 0.001) in the loss-of-righting reflex assay (EC50 = 0.81 ± 0.03 atm for controls versus 0.57 ± 0.04 atm for spA), but the responses of mutant and control mice to tail clamp in the presence of halothane were similar. Spasmodic control and mutant mice did not differ in their responses to the two drugs. Sleep time was substantially longer in both mutant mouse lines after injection of three hypnotics (midazolam, pentobarbital, and ethanol). Our results suggest a complex involvement of glycinergic pathways in mediating anesthetic responses. Greater sensitivity to the hypnotic effect of enflurane, halothane, midazolam, pentobarbital, and ethanol in mutant mice with diminished glycinergic capacity suggests that glycinergic activity is inversely related to hypnosis, whereas resistance to enflurane in the tail clamp assay suggests that glycinergic activity potentiates the minimum alveolar anesthetic concentration response. Halothane seems to share some, but not all, of enflurane’s mechanisms, indicating that not all volatile anesthetics modulate glycinergic pathways equally.

IMPLICATIONS: We tested two mouse lines with glycine receptor mutations to determine whether glycine receptors might play an important role in anesthetic responses in vivo. Both sensitivity and resistance to common anesthetics were observed in mutant mice, depending on the behavioral end-point evaluated.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Pharmacogenetic approaches are useful in distinguishing plausible sites of anesthetic action. For example, mice bred for sensitivity to benzodiazepines (1) or those with gene deletions at certain {gamma}-aminobutyric acid (GABA)A receptor subunit loci (2) manifest altered responses to volatile anesthetics, supporting the hypothesis that the GABAA receptor is involved in mediating responses to these drugs. After GABAA receptors, glycine receptors are the second most abundant ligand-gated inhibitory ion channel in mammals and are thus another plausible target for anesthetic action. Glycine receptors consist of pentamers (formed by a mixture of {alpha} and ß subunits in adult rodents) that form transmembrane chloride channels. They are found in abundance in the spinal cord and medulla and at lower levels in the midbrain, thalamus, and hypothalamus, but they are essentially absent in more rostral structures. Volatile anesthetics and anesthetic alcohols potentiate glycine-gated currents at concentrations within the clinical range (3,4); however, there are no data bearing on the whole-animal relevance of these observations. Several mouse neurologic syndromes are caused by glycine subunit mutations that profoundly alter glycine receptor function. Spastic (spA) mice are caused by an intronic insertion of a LINE-1 transposable element in the ß subunit that causes premature termination of its expression (5). Glycine receptors function normally but are greatly reduced in number (6). Spasmodic (spD) mice result from a missense mutation in the {alpha}1 subunit, which decreases receptor sensitivity to glycine six-fold (7). Behaviorally, spA and spD mutants are similar but not identical (8). Both mutants are overtly normal if unperturbed. However, they exhibit a fine motor tremor when handled, clasp their hind legs together when picked up by the tail, and exhibit an exaggerated startle response. We used these two mutant mouse lines to determine whether glycine receptors might play an important role in anesthetic responses to enflurane, halothane, midazolam, pentobarbital, and ethanol in vivo.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Heterozygote breeding pairs of spA and spD mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Heterozygous breeding pairs of each line were interbred to produce wild type, heterozygous, and homozygous (mutant) animals. Adult mice were characterized as normal (wild type and heterozygous) or mutant (homozygous) by phenotype (homozygotes display tremor and hind leg clasping when lifted by the tail). Wild type and heterozygous animals are behaviorally indistinguishable (8). Within each assay, approximately equal numbers of males and females of each phenotype were used. All studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Anesthesia sensitivity was assessed using the end-points of loss-of-righting reflex (LORR) and tail clamp (TC) in a blinded manner, as previously described (2). For LORR, seven to 11 groups of mice were placed in individual wire mesh cages in a rotating carousel enclosed in a sealed acrylic chamber. Carbon dioxide tension and temperature were controlled to obviate effects on anesthetic requirement (9,10). Mice were equilibrated with a minimum of five concentrations of enflurane or halothane for 15 min. Anesthetic concentrations were confirmed by piezoelectric analysis (Siemens 120, Danvers, MA). A blinded observer scored the mice for LORR in a quantal fashion. The mice were then allowed to recover in air for 30 min before a new trial was begun. The TC assay was also performed in a similar chamber after a 15 min equilibration period with anesthetic. Six to 10 groups of mice were scored in a quantal fashion by a blinded observer for an organized motor withdrawal in response to clamping the tail before recovery for 30 min. All mice were between ~9–12 wk of age at the time of testing for responses to volatile anesthetics. Animal weights ranged from 19.3–25.2 g and 23.2–32.2 g for females and males, respectively. All mice were tested with both volatile drugs and were allowed at least 4 days to recover between exposures to different volatile anesthetics.

Sleep time (duration of the LORR) was determined after intraperitoneal injection of midazolam (35 mg/kg), pentobarbital (40 mg/kg), and ethanol (2.6 mg/g), as previously described (2). Animals still asleep 2 h after injections were assigned a sleep time of 120 min. Temperature was controlled in a manner similar to that for the other assays. Blood ethanol concentration on awakening was determined using an enzymatic assay (Sigma: Procedure number 333-UV) on serum collected from the retroorbital sinus. All mice were between ~12–15 wk of age at the time of testing for sleep time. Mice weights ranged from 20.8–25.5 g and 26.0–32.0 g for females and males, respectively. The mice that were tested for response to injectable drugs were a subset of those mice that had previously been tested for response to the volatile anesthetics. Mice were allowed at least 1 wk to recover between exposures to different injectable drugs.

Concentration-response data were fit to a logistic equation, yielding 50% effective concentration (EC50), slopes, and estimates of their respective SE (11). Control and mutant groups were compared statistically by referring the variance ratio to a standard normal distribution (12). Mean sleep times were compared using an unpaired t-test. Significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The slopes of the anesthetic response curves for LORR and TC in both spA and spD control when compared with their respective mutant mice were statistically similar, allowing valid comparisons of EC50 values (Table 1; Figs. 1 and 2). Anesthetic sensitivity of control spA and spD mice in the LORR and TC assays were comparable with each other and similar to previous reports (2,13,14), with the exception of the TC response of spA control mice to halothane, which was somewhat lower than previously reported (Table 1). spA mutants were slightly more sensitive than their normal controls to enflurane in the LORR assay (by approximately 17%; P = 0.02) but were also substantially more resistant to enflurane in the TC assay (by approximately 32%; P = 0.01) (Table 1; Fig. 1A). spA mutant mice were also more sensitive to halothane than their controls (see Fig. 1B) in the LORR assay (by approximately 30%; p < 0.001), but the responses of mutant and control mice to TC in the presence of halothane were similar (P = 0.16). spD control and mutant mice did not differ in their responses to the two drugs (see Fig. 2).


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  		Table 1. Table 1. Sensitivity of Glycine Mutant Mice to Volatile Anesthetics in Loss-of-Righting Reflex and Tail Clamp Assays
 


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  		Figure 1. Concentration-response relationship for (A) enflurane and (B) halothane in spastic (spA) versus control littermate mice for the loss-of-righting reflex (LORR) and tail clamp/withdrawal end-points. Curves were derived as described in the Methods section.

 


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  		Figure 2. Concentration-response relationship for (A) enflurane and (B) halothane in spasmodic (spD) versus control littermate mice for the loss-of-righting reflex (LORR) and tail clamp/withdrawal end-points. Curves were derived as described in the Methods section.

 
There were substantial differences in sleep time between control and mutant mice from both lines with all three drugs tested. Sleep time in mutant mice was approximately 1.5- to four-fold longer in both lines of mutant mice compared with their controls (Table 2). spD mutant mice exhibited greater relative sensitivity to midazolam and pentobarbital compared with spA mice, but the reverse observation was made in the presence of ethanol. The difference in ethanol sensitivity was not because of a difference in ethanol pharmacokinetics. Both spD and spA mutant mice awoke at smaller ethanol blood concentrations than their control wild type mice, although this observation achieved statistical significance only in spD mice (Table 3).


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  		Table 2. Table 2. Sensitivity of Glycine Mutant Mice to Injected Anesthetics Using the Sleep Time Assay
 

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  		Table 3. Table 3. Blood Ethanol Concentrations on Awakening during the Sleep Time Assay
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The spA and spD lines of mice with mutant glycine receptors offer a unique opportunity to study the importance of glycine receptors in whole animal responses to anesthetics. Although the specific mutation is different in each line, the mutations’ effects in both lines are to substantially reduce glycinergic transmission in the nervous system. Thus, because anesthetics enhance glycine-gated currents, it follows that mutant mice should be relatively resistant to anesthetics if inhibitory glycinergic transmission were integral to anesthetic responses. Our results do not support this simple hypothesis; we found relative resistance, sensitivity, or no difference in anesthetic responses in mice with mutant glycine receptors compared with controls. The heterogeneity of the responses noted with respect to specific drug, assayed response, and specific mutation suggests that the role of glycinergic transmission in anesthesia is complex.

We cannot exclude the presence of additional mutations or compensatory alterations in other neurotransmitter systems in these animals. This complicates the interpretation of our results. In fact, spA mice have increased numbers of GABAA receptors in their spinal cord and brainstem (but not midbrain and cerebral cortex), and this has been hypothesized to be compensatory for the missing glycinergic inhibitory transmission (6). It is also possible that the apparent increase in sensitivity of the spA mutants on the LORR assay reflects motor deficiencies and not true differences in anesthetic sensitivity. Last, because we did not perform complete pharmacokinetic studies, we cannot exclude differences in adsorption or metabolism of injectable drugs as contributing to the observed differences in sleep time responses. However, we do not believe this is a confounding factor for the ethanol sleep time response. We measured blood ethanol concentrations at regaining of the righting reflex and found that not only was sleep time prolonged in the mutants, but they also regained the righting reflex at smaller blood ethanol concentrations than control animals.

Many but not all anesthetics seem to modulate glycine receptor function in vitro, but those that do act with an efficacy predicted by their anesthetic potency. Pentobarbitone (15), propofol (15), alphaxalone (3), trichloroethanol (15,16), normal alcohols from ethanol to dodecanol (17), and several volatile anesthetics (including methoxyflurane, sevoflurane, halothane, isoflurane, and enflurane) (4) all potentiate glycinergic currents in Xenopus oocytes at concentrations within the clinical range. Ketamine and the anesthetic neurosteroids seem to be inactive, and etomidate is far less effective than would be predicted (3,15). An experimental anesthetic cyclobutane was able to potentiate glycinergic currents, whereas a nonanesthetic cyclobutane was ineffective at concentrations that should have been effective based on its lipid solubility (3). In contrast to the findings at the GABAA receptor, the optical isomers of isoflurane did not differ in their effects at glycine receptors (4). Yet, two other findings suggest that anesthetic alcohols and volatile anesthetics interact with glycine receptors in a specific manner. Mutation of {alpha}267 from serine to glycine shifts the alkanol cutoff from dodecanol to butanol (17), whereas mutation of the same residue to tyrosine produces receptors that are completely insensitive to enflurane (18). Such specificity produced by point mutations strongly implicates a specific interaction between anesthetics and the receptor protein.

spA mutants were either more (LORR) or less (TC) sensitive to the actions of enflurane, depending on which assay was used to measure anesthetic sensitivity. Sensitivity or resistance for an anesthetic for one, but not another, anesthetic end-point has been previously demonstrated (i.e., mice lacking the ß3 subunit of the GABAA receptor are resistant to enflurane and halothane in the TC assay but not the LORR assay) (2). However, we are unaware of any previous reports demonstrating sensitivity in one assay but resistance in another to the same anesthetic. This suggests a complex involvement of glycinergic pathways in mediating enflurane responses because of positive modulation of glycinergic activity that is involved in either negative feedback on some behaviors (LORR) or positive feedback of other behaviors (TC). Halothane seems to share some but not all of these mechanisms, indicating that not all volatile anesthetics modulate glycinergic pathways equally, and enflurane may have some unique characteristics at glycine receptors analogous to the finding with the {alpha}267 point mutation (4). The sensitivity of mutant mice to the hypnotic effects of the injected drugs suggests that glycinergic pathways are involved in a negative feedback on this behavior with these drugs. Taken together, this supports the hypothesis that individual elements of the anesthetic state are produced by separate mechanisms, and further, not all volatile anesthetics share the same mechanisms of action.


   Acknowledgments
 
Supported, in part, by the University of Pittsburgh Department of Anesthesiology and Critical Care Medicine and the National Institutes of Health (GM52035, GM47818, and AA10422).


   Footnotes
 
Presented, in part, at the American Society of Anesthesiologists Meeting, October 17, 2000, San Francisco, CA.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. McCrae AF, Gallaher EJ, Winter PM, Firestone LL. Volatile anesthetic requirements differ in mice selectively bred for sensitivity or resistance to diazepam: implications for the site of anesthesia. Anesth Analg 1993; 76: 1313–7.[ISI][Medline]
  2. Quinlan JJ, Homanics GE, Firestone LL. Anesthesia sensitivity in mice that lack the ß3 subunit of the {gamma}-aminobutyric acid type A receptor. Anesthesiology 1998; 88: 775–80.[ISI][Medline]
  3. Mascia MP, Machu TK, Harris RA. Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics. Br J Pharmacol 1996; 119: 1331–6.[ISI][Medline]
  4. Downie DL, Hall AC, Lieb WR, Franks NP. Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes. Br J Pharmacol 1996; 118: 493–502.[ISI][Medline]
  5. Mulhardt C, Fischer M, Gass P, et al. The spastic mouse: aberrant splicing of glycine receptor beta subunit mRNA caused by intronic insertion of L1 element. Neuron 1994; 13: 1003–15.[ISI][Medline]
  6. White WF, Heller AH. Glycine receptor alteration in the mutant mouse spastic. Nature 1982; 298: 655–7.[Medline]
  7. Ryan SG, Buckwalter MS, Lynch JW, et al. A missense mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor in the spasmodic mouse. Nat Genet 1994; 7: 131–5.[ISI][Medline]
  8. Simon ES. Phenotypic heterogeneity and disease course in three murine strains with mutations in genes encoding for {alpha}1 and ß glycine receptor subunits. Mov Disord 1997; 12: 221–8.[ISI][Medline]
  9. Eisele JH, Eger EI II, Muallem M. Narcotic properties of carbon dioxide in the dog. Anesthesiology 1967; 28: 856–65.[ISI][Medline]
  10. Eger EI II, Saidman LJ, Brandstater B. Temperature dependence of halothane and cyclopropane anesthesia in dogs: correlation with some theories of anesthetic action. Anesthesiology 1965; 26: 764–70.[ISI][Medline]
  11. Waud DR. On biological assays involving quantal responses. J Pharmacol Exp Ther 1972; 183: 577–607.[Abstract/Free Full Text]
  12. Alifimoff JK, Firestone LL, Miller KW. Anesthetic potencies of secondary alcohol enantiomers. Anesthesiology 1987; 66: 55–9.[ISI][Medline]
  13. Deady JE, Koblin DD, Eger EI II, et al. Anesthetic potencies and the unitary theory of narcosis. Anesth Analg 1981; 60: 380–4.[Abstract/Free Full Text]
  14. Mazze RI, Rice SA, Baden JM. Halothane, isoflurane, and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology 1985; 62: 339–41.[ISI][Medline]
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  16. Krasowski MD, Finn SE, Qing Y, Harrison NL. Trichloroethanol modulation of recombinant GABAA, glycine, and {rho}1 GABA receptors. J Pharmacol Exp Ther 1998; 284: 934–42.[Abstract/Free Full Text]
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Accepted for publication May 14, 2002.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press