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Anesth Analg 2004;99:1136-1140
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000131729.54986.30

ANESTHETIC PHARMACOLOGY

Reduced Sensitivity to Ketamine and Pentobarbital in Mice Lacking the N-Methyl-D-Aspartate Receptor GluR{epsilon}1 Subunit

Andrey B. Petrenko, MD*,{dagger}, Tomohiro Yamakura, MD PhD*, Naoshi Fujiwara, PhD{ddagger}, Ahmed R. Askalany, MD*, Hiroshi Baba, MD PhD*, and Kenji Sakimura, PhD{dagger}

*Department of Anesthesiology, Niigata University School of Medicine, Niigata, Japan; {dagger}Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata; and {ddagger}Department of Medical Technology, Niigata University School of Health Sciences, Niigata, Japan

Address correspondence and reprint requests to Tomohiro Yamakura, MD, PhD, Department of Anesthesiology, Niigata University School of Medicine, Asahimachi 1-757, Niigata 951-8510, Japan. Address e-mail to yamakura{at}med.niigata-u.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Ketamine is an IV anesthetic with N-methyl-D-aspartate receptor (NMDAR)-blocking properties. However, it is still unclear whether ketamine’s general anesthetic actions are mediated primarily via blockade of NMDAR. Functional NMDARs are composed by the assembly of a GluR{zeta}1 (NR1) subunit with GluR{epsilon} (GluR{epsilon}1–4; NR2A–D) subunits, which confer unique properties on native NMDARs. We hypothesized that animals deficient in GluR{epsilon}1, an abundant and ubiquitously postnatally expressed NMDAR subunit, might be resistant to the effects of ketamine. Here, we evaluated a righting reflex to determine the general anesthetic/hypnotic potency of ketamine administered intraperitoneally to GluR{epsilon}1 knockout mice and compared these results with those for wild-type mice. Mutant mice were more resistant to ketamine than control mice. Unexpectedly, mutant mice were also more resistant to pentobarbital, which is thought not to interact with NMDAR at clinically relevant concentrations. Although these data in no way eliminate the possibility of the involvement of the NMDAR GluR{epsilon}1 subunit in mediation of ketamine anesthesia/hypnosis, they suggest the difficulties with interpretation of altered anesthetic sensitivity in knockout animal models.

IMPLICATIONS: Mice deficient in the N-methyl-D-aspartate (NMDA) receptor GluR{epsilon}1 subunit showed reduced sensitivity to the anesthetic/hypnotic actions of ketamine. Sensitivity to pentobarbital, which, unlike ketamine, does not interact with NMDA receptors at clinically relevant concentrations, was also reduced. Our results illustrate the difficulties with interpreting altered anesthetic sensitivity in knockout animal models.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Ketamine is an IV anesthetic with N-methyl-D-aspartate receptor (NMDAR)-blocking effects at clinically relevant concentrations (1). However, it interacts with a wide range of molecular target sites in the central nervous system, and it is still unclear whether ketamine’s general anesthetic actions are mediated primarily via blockade of NMDAR (2,3).

Coexpression studies have demonstrated that formation of functional NMDAR channels requires a combination of the glycine-binding GluR{zeta}1 (NR1) subunit and at least one of the glutamate-binding GluR{epsilon} (GluR{epsilon}1–4; NR2A–D) subunits (4). The unique biophysical and pharmacological properties of the heteromeric GluR{zeta}1/GluR{epsilon} channels, such as affinity for agonists and antagonists and sensitivity to magnesium block, are conferred by the type of GluR{epsilon} subunit included in a heteromeric complex (5,6). Although the sensitivities to ketamine are only slightly variable among the four GluR{zeta}1/GluR{epsilon} channels (1), the relative contribution of each of four subunits of the GluR{epsilon} family to the anesthetic action of ketamine may be dissimilar because of their different expression patterns in the central nervous system (7,8). In fact, the GluR{epsilon}1 subunit is ubiquitously distributed in the adult mouse brain, whereas the GluR{epsilon}2 and GluR{epsilon}3 subunits are restricted to the forebrain and cerebellum, respectively. The GluR{epsilon}4 subunit is weakly expressed in the diencephalon and the brainstem. Similar to the brain, mature spinal cord shows differential distribution of GluR{epsilon} subunits. The GluR{epsilon}1 subunit extends to all laminae of the spinal cord except for lamina II, whereas the expression of GluR{epsilon}2 is lamina II specific. The GluR{epsilon}3 subunit is not found in the spinal cord, and the expression of GluR{epsilon}4 is insignificant.

To determine the molecular target site responsible for ketamine anesthesia/hypnosis, we used the loss of righting reflex (LORR) to examine the sensitivity to ketamine in mutant mice lacking the NMDAR GluR{epsilon}1 subunit, which is widely expressed postnatally in normal animals.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study was approved by the Committee for the Guidelines on Animal Experimentation of Niigata University. All procedures were performed on male adult (8–14 wk old) C57BL/6 and GluR{epsilon}1 subunit knockout mice, hereafter referred to as wild-type and mutant mice, respectively. Mutant mice were produced as described previously (9) and had more than 99.99% homogeneity with wild-type animals because of a series of backcrosses. They were generally smaller compared with age-matched wild-type controls (for animals used in this study—wild-type: n = 34, 25.3 ± 1.5 g; mutant: n = 28, 21.6 ± 1.3 g; P < 0.0001; unpaired Student’s t-test). The genotypes of mutant mice were confirmed by Southern blotting of genomic DNA (9) extracted from tail specimens. Mutant mice demonstrate increased spontaneous locomotor activity in a novel environment (10) and impairment of spatial, contextual, and latent learning (9–11). Nevertheless, they exhibit no alterations in responses to acute noxious stimuli (12,13), demonstrate a brisk righting reflex, and have no differences in gross motor abilities under control conditions. Animals were housed four to six per cage under a standard 12-h light/dark cycle; water and food were available ad libitum. The temperature of the testing room was kept at 24°C, and behavioral experiments were performed between 8:00 AM and noon. Each animal was not used for more than 2 drug injections, and at least 1 wk between each 2 treatments was allowed for mice to recover.

Ketamine hydrochloride was purchased from Sankyo Co. (Tokyo, Japan), and pentobarbital sodium was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Drugs were dissolved in 0.9% saline and injected intraperitoneally (IP) after an aspiration test. The drug doses were selected on the basis of previous reports (14,15).

Sensitivities to ketamine and pentobarbital were evaluated by a rating scale as previously described (14,16). Each animal was injected with the drug and placed in a 2-L glass beaker. At 2-min intervals, the beaker was tilted to an angle of approximately 45° with a horizontal plane 3 times to gently place animals on their backs, and the ability of mice to right themselves was noted as the anesthetic score according to the rating scale of Boast et al. (17), with minor modifications: a score of 0 indicated a normal righting reflex; +1 indicated that the mouse righted itself within 2 s on all 3 trials (slightly impaired righting reflex); +2 indicated that the latency to righting was >2 s but <10 s at the best response in 3 trials (moderately or severely impaired righting reflex); and +3 corresponded to the LORR (no righting within 10 s on all three trials). Total anesthetic scores (TASs) were the sums of all scores recorded after the drug injection. The time between the LORR (shown as a score of +3) and the time mice regained the ability to right themselves (shown as a score of +2) was considered the duration of LORR (minutes). The time required to return to a normal righting reflex (shown as a score of 0) was considered the recovery time (minutes). Mice that had one of these indexes >2 SD from the group mean were excluded from further analysis.

Cerebral levels of ketamine, norketamine (metabolite I), and pentobarbital were measured according to the protocols previously described (14,18), with some modifications. In brief, eight wild-type and seven mutant mice received IP injections of ketamine and pentobarbital 100 and 40 mg/kg, respectively. Animals were decapitated 10 min after injection, and the brains were quickly removed and weighed. Tissue homogenates were prepared by sonication of tissue samples in 20 volumes of 0.1 M perchloric acid and centrifuged at 28,000g for 20 min at 4°C. A 20-µL volume of the filtered supernatant was then injected into a high-performance liquid chromatography system (Shimadzu LC6A) equipped with Shimpack CLC-CN (for ketamine) and CLC-OSD (for pentobarbital) columns and an ultraviolet detector (210 nm). For ketamine assay, eluent buffer consisted of acetonitrile/water (1:9) containing 0.1 M NaClO4 (pH adjusted to 2.5). For pentobarbital assay, eluent buffer consisted of acetonitrile/methanol/water (1:3:6) containing 0.01 M NaPO4 (pH adjusted to 4.0).

The data are presented as mean ± SEM. For statistical analysis of differences in anesthetic sensitivity, we used the Mann-Whitney U-test. For statistical analysis of differences in brain concentrations of ketamine and its metabolite, we used unpaired Student’s t-tests. In all cases, P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The LORR assay was conducted to determine whether the anesthetic/hypnotic effects of ketamine (60, 80, and 100 mg/kg) were altered after GluR{epsilon}1 subunit gene knockout. IP injections of ketamine produced anesthetic effects/hypnosis in a dose-dependent manner in both groups of mice. As shown in Table 1, a smaller proportion of mutant mice demonstrated LORR at all doses tested. The duration of LORR was shorter in mutant mice and reached statistical significance only at a dose of 80 mg/kg. The ketamine-induced increase in TASs and recovery times was significantly attenuated in mutant mice compared with wild-type controls (Fig. 1, left).


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  		Table 1. Loss of Righting Reflex (LORR) After Intraperitoneal Administration of Ketamine and Pentobarbital to Wild-Type and Mutant Mice
 

Figure 1
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  		Figure 1. Duration of loss of righting reflex (LORR), total anesthetic scores (TASs), and recovery time after intraperitoneal administration of (A–C) ketamine 60, 80, and 100 mg/kg and (D–F) pentobarbital 30, 35, and 40 mg/kg to wild-type (white bars) and mutant (black bars) mice. The data are presented as mean ± SEM. *P < 0.05; **P < 0.001; ***P < 0.0001, wild-type versus mutant mice.

 
Mutant mice could be rendered less sensitive to ketamine because of its altered pharmacokinetics, especially taking into account body weight difference between the two groups of animals. Therefore, we next measured the concentrations of ketamine and its primary metabolite, norketamine, in brain tissue 10 min after IP administration of ketamine 100 mg/kg. Ten minutes corresponds to the declining part of the concentration curve of ketamine and to the peak of its metabolite (19). Ketamine concentrations that we obtained were similar to those previously reported (14), and there was no difference in the brain levels of ketamine and norketamine between wild-type and mutant mice (Table 2).


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  		Table 2. Whole-Brain Concentrations (Micrograms per Gram of Tissue) of Ketamine, Norketamine, and Pentobarbital
 
To investigate whether this reduction in anesthetic sensitivity was ketamine-specific, we studied the hypnotic effect of pentobarbital, a drug known to have no effect on NMDAR at clinically relevant concentrations. Surprisingly, fewer mutant mice lost the righting reflex after IP injection of pentobarbital (30, 35, and 40 mg/kg) compared with wild-type controls (Table 1); this effect was dose dependent and resembled that observed in ketamine experiments. As depicted in Figure 1 (right), the LORR was shorter and TASs and recovery times after pentobarbital administration were also less in mutant animals, thus displaying a tendency similar to ketamine trials.

Because there is a possibility that the resistance to pentobarbital is pharmacokinetic whereas that to ketamine is pharmacodynamic, we determined the concentrations of pentobarbital in brain tissue 10 min after its IP administration at a dose of 40 mg/kg. As Table 2 shows, both groups of animals displayed similar brain concentrations of pentobarbital.


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we used the knockout mouse model to investigate whether the anesthetic/hypnotic action of ketamine is mediated by the NMDAR GluR{epsilon}1 subunit. A previous report demonstrated that GluR{epsilon}1 mutant mice exhibit a reduction in hippocampal long-term potentiation (LTP) (9). This finding may be attributed to a hypofunction of NMDARs, as evidenced by alterations of [3H]MK-801 binding and 45Ca2+ uptake through the NMDARs (10). The observation that stronger stimulation restores normal LTP (11) and 45Ca2+ uptake (10) in mutant mice indicates that NMDAR function is not lost, but only reduced, after GluR{epsilon}1 subunit gene disruption in this setting. As demonstrated previously by in situ hybridization (9) and Western blot analysis (13), the compensation by other NMDAR subunits in the brain and spinal cord of mutant animals is unlikely to occur after GluR{epsilon}1 gene disruption. Thus, given the fact that altered ketamine pharmacokinetics as a possible cause of reduced ketamine effects in mutant mice was excluded in our experiments, the reduced anesthetic/hypnotic potency of ketamine in knockout mutant mice at first led us to the possibility that NMDAR GluR{epsilon}1 subunits may be involved in the anesthetic action of ketamine. However, mutant mice also displayed reduced sensitivity to the barbiturate pentobarbital, and this was not due to the altered pharmacokinetics of the drug. Because barbiturates markedly potentiate {gamma}-aminobutyric acid (GABAA) receptors but do not exert any effect on NMDARs at clinically relevant concentrations (20), the results of pentobarbital trials cast doubt on the suggestion that the reduction in ketamine sensitivity was specific to ketamine-NMDAR interaction. Thus, we could not clearly demonstrate the role of the NMDAR GluR{epsilon}1 subunit in ketamine anesthesia/hypnosis. In this regard, it would be interesting to cite two studies that used mice lacking the {alpha}-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid receptor (AMPAR) GluR2 subunit (15,21). AMPAR complexes that lack the GluR2 subunit are resistant to blockade by pentobarbital in vitro (22,23). However, GluR2-null mutant mice were more sensitive to the LORR induced by pentobarbital (15). Moreover, the sensitivity to volatile anesthetics, which do not inhibit AMPAR at clinically relevant concentrations, was also increased in these mutant mice (21). Thus, these data also illustrate difficulties in interpreting behavioral data in knockout animal models.

There are several possible explanations for how the GluR{epsilon}1 subunit gene knockout produced hyposensitivity to two anesthetics with different mechanisms of action. The reduced sensitivity to ketamine alone can be explained by the impairment of NMDAR function that has been documented in GluR{epsilon}1 knockout mice (9,10). At the same time, NMDA-stimulated [3H]GABA release was markedly diminished in striatal slices of GluR{epsilon}1 mutant mice (10), thus indicating a dys-function in GABAergic synaptic transmission. This dysfunction may explain the weaker effects of pentobarbital in this setting.

Alternatively, the reduced sensitivity to both anesthetics may have a common underling mechanism. In fact, GluR{epsilon}1 mutant mice exhibit hyperfunction of dopaminergic and serotoninergic neuronal systems in the frontal cortex and striatum (10). This is manifested as hyperlocomotion in whole-animal behavior. Conceivably, such functional changes in brain function inflicted by GluR{epsilon}1 subunit gene knockout, besides leading to hyperlocomotion, may also modify the ability of mutant mice to right themselves, not only after the administration of ketamine and pentobarbital, but also after the administration of any other sedative or anesthetic. However, our results with both anesthetics may not be straightforwardly explained even by the monoamine hypothesis, because these drugs were reported to exert diametrically opposite effects on the striatal monoaminergic system, with ketamine increasing and pentobarbital decreasing dopamine release in the nucleus accumbens of freely moving rats (24).

Animal models are indispensable to elucidating the mechanisms of anesthetic action in the brain, which shows a complexity in anatomy and richness in function that is not shared by any other organ. The generation of knockout mice offers an advantage of specific targeting of diverse molecules implicated in anesthetic mechanisms and relating them to changes in whole-animal behavior. However, our results show potential difficulties with interpretation of altered anesthetic sensitivity in global knockouts. The use of more sophisticated time- and region-specific transgenic tools will help to improve our understanding of the molecular basis of anesthesia.


   Acknowledgments
 
Supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.

The authors thank Rie Natsume for expert assistance in animal care.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication April 23, 2004.




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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