Recent molecular strategies demonstrated that the N-methyl-d-aspartate (NMDA) receptor is a major target site of anesthetic agents. In a previous article, the authors showed that knocking out the NMDA receptor epsilon1 subunit gene markedly reduced the hypnotic effect of ketamine in mice. In the current study, the authors examined the in vivo contribution of the NMDA receptor epsilon1 subunit to the action of other anesthetic drugs.
The authors determined the anesthetic effects of nitrous oxide on sevoflurane potency in NMDA receptor epsilon1 subunit knockout mice compared with those in wild-type mice. They then tested the hypnotic effect of gamma-aminobutyric acid-mediated agents, such as propofol, pentobarbital, diazepam, and midazolam, in knockout mice and wild-type mice.
The anesthetic action of sevoflurane itself was unaffected by the abrogation of the NMDA receptor epsilon1 subunit. Adding nitrous oxide reduced the required concentration of sevoflurane to induce anesthesia in wild-type mice, whereas this sparing effect was diminished in knockout mice. Furthermore, propofol, pentobarbital, diazepam, and midazolam also had markedly attenuated effects in knockout mice.
Although it has been demonstrated that knocking out the expression of receptors may induce changes in the composition of the subunits, the network circuitry, or both, the current findings show consistently that the NMDA receptor epsilon1 subunit mediates nitrous oxide but not sevoflurane anesthesia. Furthermore, the attenuated anesthetic impact of propofol, pentobarbital, diazepam, and midazolam as well as ketamine in knockout mice suggests that the NMDA receptor epsilon1 subunit could be indirectly involved in the hypnotic action of these drugs in vivo.
DURING the past few decades, a consensus has emerged that general anesthetic drugs may act on one or more superfamilies of ligand-gated ion channels that include γ-aminobutyric acid type A (GABAA), glycine, nicotinic acetylcholine, 5-hydroxytryptamine 3, and glutamate receptors.1,2Although anesthetic effects at the GABAAreceptors have received the most attention, various electrophysiologic studies suggest that nitrous oxide and ketamine minimally affect GABAAreceptors but inhibit N -methyl-d-aspartate (NMDA) receptors.3–7Recent genetic studies in Caenorhabditis elegans also showed that nitrous oxide acts by antagonizing NMDA receptors.8These results indicate that the molecular mechanisms of nitrous oxide and ketamine are different from volatile halogenated anesthetics and other intravenous anesthetics (γ-aminobutyric acid–mediated [GABAergic] agents).
N -methyl-d-aspartate receptor channels are formed by at least two families of subunits, i.e. , ε1–4(NR2A–D) and ζ1(NR1) superfamilies of the glutamate receptor channels.9In a previous study, we reported that knockout of the NMDA receptor ε1subunit gene markedly reduced the hypnotic effect of ketamine.10However, the in vivo contribution of the NMDA receptor ε1(NR2A) subunit to the effect of other anesthetic drugs remains unclear.
In the current study, we first examined the contribution of the NMDA receptor to the anesthetic effect of two gas anesthetics, sevoflurane and nitrous oxide, using NMDA receptor ε1subunit knockout mice. We then addressed the question of whether the NMDA receptor indirectly contributes to the effect of other categories of intravenous anesthetic agents that potentiate inhibitory synapses mainly through GABAAreceptors (GABAergic agents). The hypnotic effects of these GABAergic anesthetic drugs in knockout mice were compared to those in wild-type mice.
Materials and Methods
We used 8- to 10-week-old male C57BL/6 mice (CLEA Japan Inc., Tokyo, Japan) that were either wild-type (ε1+/+) or lacking the NMDA receptor ε1subunit gene (ε1−/−). Both ε1+/+ and ε1−/− mice with highly homogenous genetic backgrounds have been developed previously.11–13Although impaired hippocampal long-term potentiation and spatial learning have been observed in these knockout mice, no abnormal motor function has been observed under physiologic conditions.11,13–15No compensation by other NMDA receptor subunits in the brain and spinal cords has been observed.11,16The messenger RNA expression of the major GABAAreceptor subunits (α1, β1, β2, β3, and γ2) in the whole brain of NMDA receptor ε1subunit knockout mice was at the same level as that in wild-type mice by reverse transcriptase–polymerase chain reaction analysis (data not shown).
The wild-type and knockout mice were maintained in a pathogen-free room in a controlled environment (22 ± 2°C room temperature, light–dark cycle [12 h each], light on at 7:00). During the experiments, each mouse was housed in its own cage and had ad libitum access to food and water. All experiments were performed according to the Jichi Medical School Guide for Laboratory Animals (Tochigi, Japan).
Experiment 1: Assessment of the Anesthetic Effect of Sevoflurane and Nitrous Oxide in NMDA Receptor ε1Subunit Knockout Mice and Wild-type Mice
The total gas flow was 4 l/min administered in a volume %:volume % ratio of nitrous oxide:oxygen based on the required concentration of nitrous oxide. Sevoflurane (Maruishi Co., Ltd., Osaka, Japan) was added in each study. The concentrations of sevoflurane, nitrous oxide, and oxygen were continually measured using an infrared gas analyzer (Datex Ohmeda, Louisville, CO). Knockout and wild-type mice were randomly assigned to three groups: the 0% nitrous oxide group, the 50% nitrous oxide group, or the 75% nitrous oxide group (n = 18/group). In each part of the experiment, six mice were put into a large airtight acrylic chamber (40 × 40 × 40 cm) with a plastic glove tightly attached to one vertical side of the chamber. Two major anesthetic actions, hypnosis and inhibition of response to noxious stimulations, were studied. The hypnotic effect of the anesthetics was evaluated by the duration of the loss of righting reflex, which was defined as having occurred when the mouse did not right itself for at least 10 s after being placed on its back. Recovery from the loss of righting reflex was defined as having occurred when the mouse spontaneously righted itself.17In each group, the concentration of sevoflurane was increased by 0.1% at 30-min intervals until the mouse could not right itself. The inhibition of the response to noxious stimulation was measured as inhibition of movement in response to a tail pinch with an alligator clip.18If any movement to the stimulation was observed, the concentration of sevoflurane was increased by 0.1% until no movement was observed. Righting reflex ED50and response to noxious stimulation (median alveolar concentration [MAC]) were calculated for each mouse as the median value of the anesthetic concentrations that blocked the righting reflex and body movement to the tail clamp reflex, respectively.19
Experiment 2: Assessment of the Hypnotic Effect of Propofol, Pentobarbital, and Diazepam in NMDA Receptor ε1Subunit Knockout Mice and Wild-type Mice
Propofol (AstraZeneca Co., Ltd., Osaka, Japan), pentobarbital (Dainippon Co., Ltd., Tokyo, Japan), diazepam (Yamanouchi Co., Ltd., Tokyo, Japan), and midazolam (Yamanouchi Co., Ltd.) were diluted in saline. As described preciously, the hypnotic effect was evaluated as the duration of the loss of righting reflex after intraperitoneal injection of propofol (150 mg/kg), pentobarbital (50 mg/kg), diazepam (25 mg/kg), or midazolam (70 mg/kg). A preliminary experiment was run to determine the doses having a hypnotic effect in all tested mice and that lasted approximately 30–90 min in control wild-type mice.
In both experiment 1 and experiment 2, animals were kept warm on a plate heated to 38°C during anesthesia. The investigators were blinded to the experimental groups and continuously observed the behavior of the animals during anesthesia.
Statistical analyses were performed using the t test. Laboratory data are expressed as the mean ± SD. A value of P < 0.05 was considered statistically significant.
Experiment 1: Anesthetic Effect of Sevoflurane and Nitrous Oxide in NMDA Receptor ε1Subunit Knockout Mice and Wild-type Mice
We examined the contribution of the NMDA receptor to the anesthetic effect of sevoflurane and nitrous oxide on NMDA receptor ε1subunit knockout mice. Without nitrous oxide, there were no significant differences in the righting reflex ED50and MAC of sevoflurane between the knockout mice and wild-type mice (1.43 ± 0.11 and 1.42 ± 0.19%, 2.84 ± 0.19 and 2.83 ± 0.20%, respectively; figs. 1 and 2). The results implied that knocking out the NMDA receptor did not affect the anesthetic action of sevoflurane itself. Therefore, indirect measurements of the anesthetic effect of nitrous oxide by determining the righting reflex ED50and MAC of sevoflurane were validated in both knockout and wild-type mice.
Combined with nitrous oxide, the concentrations of sevoflurane needed to achieve a 1.0 righting reflex ED50and 1.0 MAC in knockout mice were significantly higher than in wild-type mice. Compared with the wild-type mice, the anesthetic effect of nitrous oxide was decreased in knockout mice. At 50% nitrous oxide, the differences in the values between knockout mice and wild-type mice were significant (1.16 ± 0.12 and 0.98 ± 0.12% [P = 0.01], 2.06 ± 0.17 and 1.84 ± 0.16% [P = 0.004], respectively). At 75% nitrous oxide, the differences were also significant (0.89 ± 0.06 and 0.82 ± 0.09% [P = 0.001], 1.64 ± 0.14 and 1.50 ± 0.1% [P = 0.003], respectively).
Experiment 2: Hypnotic Effect of Propofol, Pentobarbital, Diazepam, and Midazolam in NMDA Receptor ε1Subunit Knockout Mice and Wild-type Mice
In the first experiment, we found that the anesthetic action of sevoflurane, which mainly acts on the GABAAreceptor,20was not affected by abrogation of NMDA receptor ε1subunits. Therefore, in the second experiment, we examined whether the NMDA receptor contributes to the effect of the intravenous anesthetic drugs propofol, pentobarbital, diazepam, and midazolam, which potentiate inhibitory synapses mainly through GABAAreceptors (GABAergic agents).2
As shown in figure 3, the duration of the loss of the righting reflex resulting from the administration of propofol (150 mg/kg) was 32.7 ± 9.9 min in wild-type mice but only 13.9 ± 8.9 min in knockout mice (P = 0.003). Pentobarbital (50 mg/kg) induced loss of the righting reflex for 59.3 ± 22.8 min in wild-type mice but only for 17.9 ± 5.0 min in knockout mice (P < 0.001). Diazepam (25 mg/kg) was effective for 48.4 ± 3.6 min in wild-type mice but only for 12.6 ± 7.8 min in knockout mice (P < 0.001). Midazolam (70 mg/kg) was effective for 63.2 ± 10.0 min in wild-type mice but only for 41.2 ± 13.4 min in knockout mice (P = 0.01). Therefore, our data showed that the hypnotic effect of these GABAergic agents, propofol, pentobarbital, diazepam, and midazolam, as well as ketamine,10was significantly attenuated in knockout mice.
Research on general anesthesia has recently progressed rapidly at the molecular level. However, general anesthesia involves many direct and indirect neural pathways in the entire central nervous system.21–23When evaluating the functional effect of anesthetic agents in vivo , the balance of the neural networks mediating the inhibition of excitatory synapses and the potentiation of inhibitory synapses should be considered.2,24,25Gene-targeted mutant animals are therefore useful for evaluating the functional contributions of particular receptor subunits in research on general anesthetic activity. The NMDA receptor is considered to be a major target site of anesthetics, and our previous study demonstrated that the hypnotic effect of ketamine, which is a noncompetitive antagonist of the NMDA receptor, was markedly reduced in NMDA ε1subunit knockout mice.10The in vivo contribution of the NMDA receptor ε1subunit to the action of other anesthetic drugs is further interested.
In the current study, we first examined the contribution of the NMDA receptor to the anesthetic effect of two gas anesthetics, sevoflurane and nitrous oxide, using NMDA receptor ε1subunit knockout mice. Because nitrous oxide by itself does not provide an adequate level of anesthesia at one atmospheric pressure, sevoflurane was combined with nitrous oxide as a second potent inhalation anesthetic drug. The effects of these two inhalation anesthetic agents are considered to be additive. We found that knocking out the NMDA receptor ε1subunit did not affect the anesthetic action of sevoflurane itself and confirmed that adding nitrous oxide reduced the required concentration of sevoflurane for anesthesia in wild-type mice. This sparing effect was diminished in NMDA receptor ε1subunit knockout mice. Therefore, our findings suggest that nitrous oxide not sevoflurane induces general anesthesia partially through the NMDA receptors.
Nitrous oxide is an excellent analgesic agent and also can produce hypnosis.4Like other NMDA antagonists, such as MK801, phencyclidine, and ketamine, nitrous oxide protects against the excitotoxic action of NMDA in the in vivo rat brain and inhibits ionic current induced by NMDA in cultured rat hippocampal neurons.3,5Sevoflurane, a halogenated ether, produces hypnosis and unconsciousness and depresses the spinal reflexes yet confers little analgesia. Recent analysis of the recombinant receptors expressed in Xenopus oocytes demonstrated that nitrous oxide but not halogenated inhalation anesthetic drugs such as isoflurane blocks the current response of NR1a/NR2A and that ketamine inhibits the NR1a/NR2A–D channels to a similar extent.26,27Therefore, the pharmacologic profile of nitrous oxide anesthesia is considered to be similar to that of ketamine,4which is consistent with our results obtained from NMDA receptor ε1subunit knockout mice.10
In the second experiment, we tested the GABAergic agents propofol, pentobarbital, diazepam, and midazolam in knockout mice to determine whether the NMDA receptor is involved in their hypnotic effect. We initially hypothesized that the hypnotic effects of these anesthetics in knockout mice do not differ from those in wild-type mice, because recent molecular and gene targeting observations using various GABAAreceptor subunit knockout/knock-in mice suggest that propofol and pentobarbital potentiate the GABAAreceptor β1or β3subunit, benzodiazepine potentiates the GABAAreceptor α1or γ2subunit, and midazolam potentiates the GABAAreceptor β3subunit to achieve their anesthetic effects.25,28–34To the contrary, our results showed that these anesthetic agents as well as ketamine have a markedly reduced hypnotic effect on NMDA receptor ε1subunit knockout mice.10
To date, little is known about the in vivo functional contribution of the NMDA receptor ε1subunits to the GABAergic anesthetic effect. Because our previous findings showed no significant difference in hepatic metabolism and the pharmacokinetics of ketamine between wild-type and knockout mice10and because NMDA receptor ε1subunit disruption did not influence the anesthetic effect of sevoflurane in the first experiment of this study, our current findings about GABAergic agents suggest that the NMDA receptor may be involved indirectly in the action of anesthetic drugs that mainly mediate GABAAreceptors to exert hypnotic activity. We believe that this result most likely occurs through dynamic neural network interactions in whole animal bodies. These findings also support the hypothesis of Flohr et al. 35that the action of anesthetic agents that primary interact with other targets such as the GABA synapse can eventually be explained as an indirect effect on the NMDA receptor mediated synapse.
Although a genetically modified animal is a powerful tool to investigate targeted gene function, there are some limitations in our experimental model, and a number of confounders exist in such studies. In knockout studies, it is always possible that the lack of a certain receptor subtype may be compensated for by up-regulating of other subtypes or down-regulating of other receptors.36Although no abnormal motor function has been observed during physiologic conditions in the NMDA receptor ε1knockout mice, it was recently suggested that disruption of the ε1subunit results in enhanced dopaminergic and serotonergic neuronal activities.13Therefore, careful consideration must be made before definitive conclusions are drawn.37
In conclusion, we demonstrated that the sensitivity to nitrous oxide anesthesia was significantly decreased in NMDA receptor ε1subunit knockout mice compared with wild-type mice. Our findings suggest that the anesthetic action of nitrous oxide but not sevoflurane is partially mediated by NMDA receptors in vivo . Furthermore, our current data showed a clear reduction in the hypnotic effects of propofol, pentobarbital, diazepam, and midazolam in NMDA ε1subunit knockout mice, suggesting that the NMDA receptor might be involved indirectly in the hypnotic action of these GABAergic anesthetic drugs in vivo through dynamic neural network interactions in whole animal bodies.
The authors thank Jason Waechter, M.D. (Exchange Researcher, University of British Colombia, Vancouver, Canada), for his assistance in preparing the manuscript.