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Anesth Analg 2006;103:117-120
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000220944.27963.b1

ANESTHETIC PHARMACOLOGY

Ethanol-Induced Hypnotic Tolerance Is Absent in N-Methyl-d-Aspartate Receptor {varepsilon}1 Subunit Knockout Mice

Yuki Sato, MD, PhD, Norimasa Seo, MD, PhD, and Eiji Kobayashi, MD, PhD

From the Department of Anesthesiology, Divisions of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Recent pharmacological studies suggest that N-methyl-d-aspartate (NMDA) receptors play an important role in neuroadaptive processes in the development of tolerance to addictive drugs, such as opioids, amphetamine, and cocaine. In the present study, we investigated the contribution of the NMDA receptor to ethanol-induced hypnotic tolerance using NMDA receptor {varepsilon}1 subunit knockout mice. Hypnotic sensitivity to a single injection of 3, 3.5, and 4 g/kg ethanol was not significantly different between wild-type mice and NMDA receptor {varepsilon}1 subunit knockout mice. In contrast, although wild-type mice displayed hypnotic tolerance after repeated administration of 4 g/kg ethanol for 4 consecutive days, no change in the duration of hypnosis was observed in knockout mice. No significant differences were observed in blood ethanol clearance between wild-type and knockout mice on day 4. Our results indicate {varepsilon}1 subunit containing the NMDA receptor might be involved in the development of ethanol-induced hypnotic tolerance.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ethanol is one of the oldest and most widely consumed drugs. It has many behavioral effects, some of which are shared with sedative, hypnotic, and anesthetic drugs (1). To mediate the behavioral, neurophysiological, and pathological effects of ethanol, both major inhibitory and excitatory amino acid transmitter systems, such as {gamma}-aminobutyric acid type A (GABAA) receptors and N-methyl-d-aspartate (NMDA) receptors, have been considered (2,3). Molecular analysis has revealed that ethanol shares a common site, within transmembrane domain 2 and transmembrane 3 of GABAA receptor {alpha}1, {alpha}2, and ß2 subunits, with volatile anesthetics such as halothane, enflurane, desflurane, and isoflurane (4–7). On the other hand, there is increasing evidence showing that NMDA receptors are important in neuroadaptive processes associated with the development of tolerance to addictive drugs, such as ethanol, opiate, amphetamine, and cocaine (8–11), which produce rewarding effects through an activation of the mesolimbic dopaminergic neuronal system in drug exposures (12,13). Pharmacological experiments have shown that the NMDA receptor antagonist, (+)MK-801, blocks the development of rapid and chronic tolerance to ethanol in motor-impairment, hypothermia, and sleep time tests (9,10,14,15).

Recently, genetically engineered animals have been used to investigate the mechanism of anesthetic actions at the whole animal level. We and others (16–18) have used NMDA receptor {varepsilon}1 subunit knockout mice (19,20) to study the involvement of NMDA receptors in the hypnotic effects of various anesthetic drugs in vivo. A reduced (but not absent) function of the NMDA receptor in this knockout mouse was implied by reduction of [3H]MK-801 binding and 45Ca+ uptake through the NMDA receptor (21,22). In previous studies we showed that whereas NMDA receptor {varepsilon}1 subunit gene knockout mice displayed markedly reduced hypnotic effects to ketamine, nitrous oxide, pentobarbital, propofol, diazepam, and midazolam, the anesthetic effects of sevoflurane did not differ from those in wild-type mice (16,18). Using the same knockout mice, Miyamoto et al. (22) have also shown that the development of methamphetamine and phencyclidine-induced sensitization and morphine-induced analgesic tolerance are all attenuated in these mice lacking the NMDA receptor {varepsilon}1 subunit.

In the present study, we first examined whether the hypnotic action by the single administration of ethanol in NMDA receptor {varepsilon}1 subunit knockout mice differs from that in wild-type mice. We then investigated the contribution of NMDA receptor to the development of ethanol-induced hypnotic tolerance. The sleep time produced by daily injection of ethanol in the NMDA receptor {varepsilon}1 subunit knockout mice was compared with that in wild-type mice observed for 4 consecutive days.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
All animal procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of Jichi Medical School. We used 8- to 10-wk-old male C57BL/6 mice that were wild-type (CLEA Japan Inc., Tokyo, Japan) ({varepsilon}1 +/+) or were lacking the NMDA receptor {varepsilon}1 subunit gene ({varepsilon}1 –/–) provided by Professor Masayoshi Mishina (Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan). Both {varepsilon}1 +/+ mice and {varepsilon}1 –/– mice with highly homogenous genetic backgrounds have been developed (19–21). Impairment of hippocampal long-term potentiation and contextual learning have been observed in these knockout mice (19,20), but no abnormal motor function has been observed under physiological conditions (21,23). These wild-type mice and knockout mice were maintained in a pathogen-free room under a controlled environment (temperature 22°C ± 2°C, 12-h light/ dark cycle, light on at 07:00). During the experiments, each mouse was housed in its own cage, and food and water were provided ad libitum.

Experiment 1: Hypnotic Sensitivity To Single Injection Of Ethanol In Wild-Type And NMDA Receptor {varepsilon}1 Subunit Knockout Mice
To determine whether acute sensitivity to ethanol differs between wild-type and NMDA receptor {varepsilon}1 subunit knockout mice, 3, 3.5, or 4 g/kg of 20% (volume/volume) ethanol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solution in saline (24,25) was injected IP into wild-type and NMDA receptor {varepsilon}1 subunit knockout mice (n = 12–13 in each case). The hypnotic effect was evaluated as the duration of loss of righting reflex (LORR) (16,18).

To confirm the hypnotic effects of volatile anesthetics, we also tested isoflurane (Dainippon CO, Ltd., Tokyo, Japan) in wild-type and NMDA receptor {varepsilon}1 subunit knockout mice (n = 15 in each case). The total gas flow was 4 L/min, and the concentrations of isoflurane and oxygen were continuously measured using an infrared gas analyzer (Datex Ohmeda, Louisville, CO). As descried previously (18), the concentration of isoflurane was increased in 0.1% steps at 30-min intervals until the mouse could not right itself. The righting reflex ED50 was calculated for each mouse as the median value of the anesthetic concentrations that blocked the righting reflex (18).

Experiment 2: Hypnotic Duration By The Repeatedly Administered Ethanol In Wild-Type And NMDA Receptor {varepsilon}1 Subunit Knockout Mice
To examine the role of NMDA receptors in ethanol-induced hypnotic tolerance, 4 g/kg of ethanol was injected intraperitoneally once a day in wild-type and NMDA receptor {varepsilon}1 subunit knockout mice (n = 9–10 in each case) for 4 consecutive days. The change of hypnotic effects was evaluated by the duration of the LORR over 4 days. To measure the blood ethanol concentrations on day 4, blood samples were obtained at 20, 120, and 240 min after administration of 4 g/kg ethanol, from wild-type and NMDA receptor {varepsilon}1 subunit knockout mice (n = 4 in each case). The blood concentration of ethanol was analyzed by an enzymatic method (26).

During anesthesia, the animals were kept warm on a plate heated to 38°C. The investigators were blinded to the experimental groups, and continuously monitored the behavior of the animals during anesthesia.

Statistical analyses were performed using one-way or two-way analysis of variance or Student's t-test. For multiple comparisons, Tukey's method was used as post hoc test. Laboratory data are expressed as mean ± sd. A 5% level of probability was considered significant.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In the first experiment, we tested the hypnotic effect of a single injection of ethanol in NMDA receptor {varepsilon}1 subunit knockout mice and wild-type mice. As shown in Figure 1, ethanol (3, 3.5, and 4 g/kg) produced LORR in a dose-dependent manner in both wild-type and knockout mice (F[2,74]) = 60.2; P < 0.001). The duration of LORR in knockout mice did not differ significantly from that in wild-type mice at each dose (F[1, 74] = 0.22; P = 0.64), whereas there have been several reports regarding inhibition of glutamate receptors by ethanol in several brain regions (27–29). Similarly, there were no significant differences for the ED50 of isoflurane for LORR between the knockout mice and wild-type mice (0.83 ± 0.11% and 0.83 ± 0.10%, respectively; P = 0.56), which is consistent with our previous results for sevoflurane (18). These results suggest that the hypnotic sensitivity to ethanol and volatile anesthetics may not be affected by the abrogation of NMDA receptor {varepsilon}1 subunits in whole animal levels. Together with our previous findings that the hypnotic effects of nitrous oxide, ketamine, pentobarbital, propofol, diazepam, and midazolam were attenuated in NMDA receptor knockout mice (18), the present data suggest that the molecular anesthetic mechanisms of ethanol and volatile anesthetics to exert hypnotic effects might differ from those of other anesthetics.


Figure 123
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  		Figure 1. Duration of loss of righting reflex (LORR) after single IP administration of 3, 3.5, and 4 g/kg ethanol in wild-type (closed square) and NMDA receptor {varepsilon}1 subunit knockout mice (open squares) (n = 9–10 in each). Two-way analysis of variance showed there was no significant difference in the sensitivity to ethanol in wild-type mice and knockout mice (F[1,74]) = 0.22; P = 0.64).

 

On the other hand, the contribution of NMDA receptors to neuroadaptive processes associated with ethanol tolerance has received growing attention (30). Features of the development of drug tolerance might involve learning or experience-dependent neuroplasticity, as modeled by long-term potentiation or long-term depression (30). Lately, it has been proposed that internalization of the NR2A ({varepsilon}1) subunit in the presence of ethanol contributes to the changes in synaptic plasticity, which might lead to ethanol-associated tolerance, dependence, withdrawal, and intoxication (31), whereas alterations in GABAA receptor function might also play an important role (32). We therefore investigated the effect of repeated administration of ethanol for 4 consecutive days on the sleep time in NMDA receptor {varepsilon}1 subunit knockout mice and compared it with that in wild-type mice.

As shown in Figure 2, there was a statistically significant difference between the two groups of mice (F[1,74] = 57.1; P < 0.001). In wild-type mice, the duration of LORR gradually decreased with the repeated administration of ethanol (F[3,36] = 4.37, P = 0.007). Compared with the hypnotic duration on day 1, the righting reflex was lost for a significantly shorter time on days 3 (P < 0.01) and 4 (P < 0.01). This indicates the development of hypnotic tolerance to ethanol in wild-type mice. In contrast, the sleep time in NMDA receptor knockout mice was not altered by the daily injection of ethanol (F[3, 37]) = 1.23, P = 0.31) (Fig. 2). The righting reflex was lost for a significantly shorter time in wild-type mice than in knockout mice on day 2 (P = 0.003), day 3 (P < 0.001), and day 4 (P < 0.001). However, as shown in Figure 3, wild-type mice and knockout mice displayed similar blood ethanol clearance on day 4 after repeated administration of ethanol; no significant difference was observed between the two groups (F[1, 23]) = 0.11; P = 0.75). Therefore, the observed behavioral difference between the two groups of mice was not a result of changes in ethanol metabolism. These findings are also supported by our previous data showing that cytochrome P450 activity in the liver of knockout mice is not significantly different from that in wild-type mice (16).


Figure 223
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  		Figure 2. Duration of loss of righting reflex (LORR) after the repeated administration of ethanol in wild-type (closed circles) and NMDA receptor {varepsilon}1 subunit knockout mice (open squares). One-way ANOVA showed that the duration of LORR was significantly reduced in wild-type mice by the repeated administration of ethanol (F[3,36]) = 4.37, P = 0.007) on day 3 and 4 (P < 0.01 for both compared with day 1), however, the sleep time was not altered by daily injection of ethanol in NMDA receptor knockout mice (F[3,37]) = 1.23, P = 0.31).

 

Figure 323
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  		Figure 3. Mean blood ethanol concentration after IP injection of ethanol on the 4th day of repeated administration of ethanol, in wild-type (closed circles) and NMDA receptor {varepsilon}1 subunit knockout mice (open squares). Two-way ANOVA showed no significant difference was observed between the two groups (F[1,23]) = 0.11, P = 0.75).

 

In conclusion, we found that, although hypnotic sensitivity to the single injection of ethanol in NMDA receptor {varepsilon}1 subunit knockout mice did not differ from that in wild-type mice, hypnotic tolerance to ethanol was absent in knockout mice. These results suggest that {varepsilon}1 subunit containing-NMDA receptors might be involved in the development of ethanol-induced hypnotic tolerance.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Professor Masayoshi Mishina (Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan) for providing us NMDA receptor {varepsilon}1 subunit knockout mice.


   Footnotes
 
Accepted for publication March 1, 2006.

Supported, in part, by "21st Century Center of Excellence (COE)" program of Japan's Ministry of Education, Tokyo, Japan.

Address correspondence and reprints to Eiji Kobayashi, MD, PhD, Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, 3311-1, Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Address e-mail to eijikoba{at}jichi.ac.jp.


   REFERENCES
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 Introduction
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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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