1Anesthesiology Research Laboratories, University of Pittsburgh, Pittsburgh, PA, USA. 2Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku, Finland. 3Department of Anaesthesia, Helsinki University Central Hospital, Helsinki, Finland, and Biomedical Research Center, Alko Group, Ltd, Helsinki, Finland
Presented in part to the 1995 annual meeting of the Society for Neuroscience (November 12 16, 1995, San Diego, CA).
Accepted May 30, 2000
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Abstract |
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Br J Anaesth 85; 2000: 75762
Keywords: anaesthetics volatile, halothane; anaesthetics volatile, desflurane; brain, GABA; rat
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Introduction |
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Abundant in vitro data demonstrating that structurally heterogeneous general anaesthetics enhance GABA-gated hyperpolarizing currents (reviewed by Tanelian et al.3) have led to the hypothesis that general anaesthetics act primarily via the GABAA receptor. One approach to testing this hypothesis is to identify experimental animals with a specific alteration in the genes encoding brain GABAA receptors and assay for changes in their anaesthetic requirement. Previous investigations have utilized GABAA receptor mutants arising spontaneously,4 due to ionizing radiation,5 or as a result of targeted gene disruption.68 In the case of a spontaneous mutation, a selectively outbred rat line highly susceptible to the motor ataxia produced by alcohol,9 benzodiazepines and barbiturates is available.10 The putative underlying mutation involves a single nucleotide substitution in the GABAA receptor 6 subunit resulting in an Arg100
Gln substitution.11 Given the susceptibility of this rat line, termed alcohol-nontolerant (ANT), to central depressants known to act via the GABAA receptor, we hypothesized that ANT rats would also be cross-sensitive to the actions of volatile general anaesthetics. To test this, anaesthetic requirements were determined in ANT and control, alcohol tolerant (AT) rats, using two volatile agents with widely differing potencies, as well as two separate endpoints reflecting obtundation and immobilization.
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Materials and methods |
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ANT and AT rats are outbred lines derived from crossbred Wistar, Sprague-Dawley, and Long-Evans rats,9 selected and bred for sensitivity (ANT) or resistance (AT) to ethanol. The selection procedure has been based on motor performance under the influence of a moderate, nonhypnotic dose of ethanol (2 g kg1, i.p.). The phenotype is rechecked every second generation. Stocks of AT and ANT rats are kept in stainless steel wire mesh cages on a 12-h light/dark cycle (light on at 6 a.m.) at the ambient temperature (20±2°C) and relative humidity (50±5%). For the present study, male rats of the F50 generation were housed four per cage and had continuous access to RMI(E)SQC pellet feed (SDS Ltd, Witham, Essex, UK) and tap water. The age of rats used in this study was 3.5 months (350430 g).
Six groups (three ANT, three AT) of four to six rats of either genotype (unknown to the observer) were studied during halothane and desflurane anaesthesia, in random order. Each group was placed within a Plexiglas chamber warmed by heating pads from below and an infrared lamp from above. The composition of the chambers atmosphere was monitored continuously on-line with Datex Capnomac UltimaTM and NormacTM devices (for oxygen/carbon dioxide, and volatile anaesthetic concentrations, respectively). Fresh oxygen flow was delivered via a Dameca oxygen vernitrol at rates of 1.58 litres min1. Volatile agents were added to the fresh gas stream by means of either a Fluotec 3TM (Cyprane Ltd, Keighley, UK) or an Ohmeda Tec 4TM vaporizer (Ohmeda, Inc., Steeton, West Yorkshire, UK). Carbon dioxide concentration was maintained at <1.0% atm by means of soda lime scattered on the floor of the chamber.
After a 20-min equilibration period at a given anaesthetic concentration, rats were gently placed supine and observed for the ability to roll over spontaneously to the fully prone position within 30 s. Responses were scored in a quantal fashion. Immediately thereafter, the base of the tail of each rat in the group was clamped with a full-sized Kelly haemostat to the second ratchet position, and gross withdrawal within 5 s scored quantally. Withdrawal was defined as jerking of the head, twisting of the neck, or movement of an extremity; coughing, swallowing, chewing, and grimacing motions were not scored as positive responses, nor were increases in the respiratory rate.12 Rectal temperatures were then measured using a digital thermometer (Exacon MC8700, Denmark) with an accuracy of ±0.1°C. The rats were allowed to recover in an oxygen-rich atmosphere for at least 20 min before they were equilibrated with the next higher anaesthetic concentration and retested for both the obtundation and immobilization endpoints. For both agents, 913 different concentrations were tested in each group of rats.
After all experiments were completed, halothane and desflurane response data from the three ANT groups (n=17 rats) or three AT groups (n=16 rats) were pooled. The dose-response relationships were analysed by fitting to a logistic equation,13 which yielded estimates of the ED50 and slopes for each agent, as well as their respective standard errors. The ANT and AT group responses were then compared, using a method making the fewest assumptions about the distribution of errors.14 Briefly, the estimated variances for the AT and ANT groups were calculated from the standard errors; the sum of these then yielded the estimated variance of the difference in ED50 between groups. The ratio of this difference to its standard error was referred to a standard normal distribution.
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Results |
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Data obtained during halothane anaesthesia are summarized in Table 1 and Figures 1 and 2. These six experiments included three with AT rats (n=16) and three with ANT rats (n=17). For each genotype/endpoint combination, the arithmetic mean of the ED50 values obtained separately closely agreed with the ED50 obtained by a free fit of pooled data (for example, 0.98 vs 0.95% atm, respectively, for AT rats tested by loss-of-righting-reflex).
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Similarly, the slopes of the pooled tail clamp/withdrawal data for the AT vs ANT groups were not significantly different (P>0.10 for halothane; P>0.15 for desflurane). Halothane sensitivity in the AT line (1.72±0.09% atm) was again less than that previously reported for MAC in wild-type rats (approximately 11.2% atm), while the ANT value (1.1±0.08% atm) was similar to previous reports. Desflurane ED50 values for tail clamp for both AT (5.85±0.21% atm) and ANT (6.25±0.25% atm) lines were within the range of previous reports in rats (5.76.8% atm).16 17 Comparison of the ED50 values for these groups showed that for halothane, the ANT rats were significantly more sensitive than were AT rats with respect to the immobilization endpoint (P<0.0001), but ANT and AT rats had similar sensitivity to the immobilizing effects of desflurane (P>0.50).
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Discussion |
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We found that ANT rats were significantly more sensitive than AT rats to the obtunding effects of halothane and desflurane. ANT rats were also more sensitive to the immobilizing effects of halothane, but not desflurane. This inconsistent response to desflurane is unlikely to be anomalous, since our measurement of tail-clamp ED50 for desflurane in AT rats (5.85±0.21% atm) agreed closely with the minimum alveolar concentration (MAC) of desflurane in oxygen reported in normothermic Sprague-Dawley rats (5.72±0.40% atm).14 It is also unlikely that the immobilization assay result with desflurane was an artefact of the group sizes studied: a 20% difference between ED50 values would have been detectable in these groups by the statistical methods employed. Instead, this nonuniformity of response to the volatile agents more likely indicates that the immobilizing effect has different neuronal and neurochemical mechanisms than does the obtunding effect. Therefore, these data agree with the hypothesis that separate elements of the anaesthetic state are produced by independent mechanisms.18 The oil/gas partition coefficient of halothane is about 12 times greater than that of desflurane.19 The relatively greater lipid solubility of halothane allows for the possibility that halothane interacts with more hydrophobic regions of the membrane surrounding the GABAA or another receptor.20
ANT rats were first noted to display unusual cerebellar GABAA receptor binding pharmacology,21 which were subsequently proven to have a point mutation in the N-terminal extracellular portion of the 6 subunit.11 This
6 subtype is expressed almost exclusively in cerebellar granule cells,22 which are intrinsic to the cerebellar cortex and are the most ubiquitous neuron found in the mature mammalian nervous system. The role of cerebellum in motor coordination, posture maintenance, balance, and muscle tone is clear,23 but its role in conscious behaviours, including motor learning, is currently an area of intense research (reviewed by Glickstein).24 Classically, granule cells relay information between cerebellar input (mossy fibre) and output (Purkinje) neurons. The
6 subunit-containing GABAA receptor is among the most sensitive subtype to inhibition by GABA;25 thus, potentiation of GABA actions at granule cells by an anaesthetic could sharply reduce Purkinje cell output from the cerebellar cortex to the deep cerebellar nuclei, and subsequently to the brain. Indeed, attenuation of Purkinje output could explain, at least in part, the motor ataxia of intoxicated ANT rats, but might not be critical for deep anaesthesia.
In vitro, some anaesthetics (propofol and trichloroethanol) have significantly lower efficacy, but not apparent affinity, at 6 subunit-containing GABAA recombinant receptors expressed in cell lines, as compared with those containing the
1 subunit.26 However, pentobarbital potentiates GABA-gated currents more significantly in
6-containing cells than in those containing
1.27 The significance of these findings in vivo remains to be determined. Studies of mice with
5 and
3 GABAA receptor gene deletions suggest that these subtypes are not essential for the halothane response.5 In contrast, targeted gene disruption of the ß3 subunit of the GABAA receptor increases halothane and enflurane MAC but not the ED50 for loss of righting.8
Despite extensive characterization of the ANT 6 GABAA receptor genotype, there is still a possibility that other mutations coexist in ANT rats that affect the central nervous system.28 Additional but unidentified mutations could, in principle, have become enriched in the selected population along with the defective
6 gene and be the actual basis for the drug-response phenotype reported here. The role of possible unidentified mutations could be assessed in crossbreeding experiments in which AT rats are mated with ANT, siblings of the first generation are mated again, and anaesthetic sensitivity is determined in this resulting generation of rats. The degree of dominance of the
6 mutation should determine the distribution of anaesthetic sensitivity in rats of the second generation. However, such experiments are costly and require large numbers of animals. Additional unidentified mutations may be excluded by using targeted disruption, or site-specific alteration, of the gene in question. Such an approach was recently used to establish that the GABAA
2 receptor subunit is essential to benzodiazepine agonist action in vivo; targeted disruption of the subtype gene by homologous recombination resulted in mice for whom diazepam was behaviourally inactive.7 Mice completely lacking the
6 subunit, created using similar techniques, do not differ from wild type mice in their sensitivity to either halothane or enflurane in the loss-of-righting reflex assay or the tail clamp assay.6 These mice also exhibit normal responses to ethanol and pentobarbital6 29 but their response to the motor ataxic effects of diazepam was enhanced.29 The dramatic difference in the pharmacological profile of rats with an intact but altered
6 subunit and null allele mice completely lacking any
6 subunits suggests that other undocumented mutations in the ANT line may be responsible for at least some of their abnormal behavioural responses, but the difference could also be explained by substitution of other subunits for the missing
6 subunits in the null allele mice.6 An alternative explanation is that the
6 null allele is pharmacologically and behaviorally very different from the gain of function mutation that is present in the ANT rat line.
In conclusion, we have documented that the obtunding effects of halothane and desflurane are markedly enhanced in ANT rats compared to AT controls. The immobilizing effects of halothane, but not desflurane, were also enhanced in the ANT rat line. Since ANT rats are known to contain a point mutation in the 6 subunit of the GABAA receptor that segregates with the ANT phenotype, it is tempting to speculate that the enhanced behavioral sensitivity to the volatile agents is also due to this mutation. However, it is equally plausible that an additional mutation(s) is the basis for the observed phenotype.
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Acknowledgements |
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Footnotes |
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References |
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