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. 2000 Dec;131(7):1251–1254. doi: 10.1038/sj.bjp.0703717

Mechanism of action of the hypnotic zolpidem in vivo

Florence Crestani 1,*, James R Martin 2, Hanns Möhler 1, Uwe Rudolph 1
PMCID: PMC1572473  PMID: 11090095

Abstract

Zolpidem is a widely used hypnotic agent acting at the GABAA receptor benzodiazepine site. On recombinant receptors, zolpidem displays a high affinity to α1-GABAA receptors, an intermediate affinity to α2- and α3-GABAA receptors and fails to bind to α5-GABAA receptors. However, it is not known which receptor subtype is essential for mediating the sedative-hypnotic action in vivo. Studying α1(H101R) mice, which possess zolpidem-insensitive α1-GABAA receptors, we show that the sedative action of zolpidem is exclusively mediated by α1-GABAA receptors. Similarly, the activity of zolpidem against pentylenetetrazole-induced tonic convulsions is also completely mediated by α1-GABAA receptors. These results establish that the sedative-hypnotic and anticonvulsant activities of zolpidem are due to its action on α1-GABAA receptors and not on α2- or α3-GABAA receptors.

Keywords: GABAA receptor, zolpidem, benzodiazepines, knock-in mouse, targeted mutation

Introduction

The pharmacological profile of benzodiazepines and structurally unrelated ligands of the benzodiazepine site of GABAA-receptors is dominated by anxiolytic, sedative, myorelaxant and anticonvulsant properties. In the search for ligands with a selective pharmacological profile two lines of research have been followed. (1) Partial agonists were thought to provide anxioselective ligands based on the assumption that the receptor reserve for anxiolytic activity would be higher than that for other pharmacological actions. While this concept produced promising results in animal experiments no partial agonist has been introduced into clinical practice (Haefely et al., 1990; Stephens et al., 1993). (2) The second approach was based on the recognition that GABAA receptors are divided into subtypes which differ in their subunit composition and regional distribution in the brain (for review, see Möhler, 2000; Möhler et al., 2000). The triazolopyridazine CL218872 was the first ligand which displayed a differential affinity for the benzodiazepine site, since it displaced [3H]-diazepam with higher affinity from cerebellar than from hippocampal or cortical membranes (Lippa et al., 1979). The hypnotic zolpidem, an imidazopyridine, was the first subtype- selective ligand in clinical use. When tested on recombinant receptors, zolpidem displayed a high potency at α1-GABAA receptors (α1β2γ2, α1β3γ2: Ki=20 nM), medium potency at α2- and α3-GABAA receptors (α2β1γ2, α3β1γ2: Ki=400 nM) but failed to interact with receptors containing the α5 subunit (α5β3γ2, α5β2γ2: Ki⩾5000 nM) (Langer et al., 1992; Pritchett & Seeburg, 1990). Thus, it was postulated that the pronounced sedative-hypnotic effect of zolpidem (Depoortere et al., 1986) was mediated by its preferential interaction with α1-GABAA receptors. However, this view was in conflict with the characterization of the similarly α1-selective imidazopyridine alpidem as a selective anxiolytic (Zivkovic et al., 1990; Morselli, 1993). The extraordinary strong GABA shift for zolpidem – an increase in the affinity of zolpidem by a factor of 3 in the presence of 100 μM GABA in vitro – was considered as alternative explanation for the preferential sedative-hypnotic effect of zolpidem (Arbilla et al., 1985; Depoortere et al., 1986). Thus, it has remained unclear whether the sedative-hypnotic effect of zolpidem in vivo is exclusively due to its interaction with α1-GABAA receptors, or due to a combined interaction with α1-, α2- and α3-GABAA receptors or whether it is mainly determined by its failure to modulate the neuronal populations expressing α5-GABAA receptors. Therefore, in the present study, the subtype selectivity of the sedative effect of zolpidem was assessed in vivo. For this purpose, a mouse line was used in which the benzodiazepine binding site of α1-GABAA receptors was rendered drug-insensitive by the introduction of a histidine to arginine point mutation in position 101 [α1(H101R)] (Rudolph et al., 1999). In these animals, those pharmacological effects of zolpidem which are mediated via α1-GABAA receptors are expected to be absent, while potential drug effects mediated via α2- and α3-GABAA receptors would remain apparent. For comparison, we also included the classical non subtype-selective benzodiazepine diazepam in our analysis (see also Rudolph et al., 1999).

Methods

Animals

Wild type and α1(H101R) mice (third backcross of the 129/Ola chimeras to the 129/SvEv background) were generated as described in Rudolph et al. (1999). Male and female mice were raised in group-housed cages (8–10 mice per cage (either under normal 12 h day–night cycle conditions (light on at 06.00 h, motor activity test) or under reverse conditions (light on at 20.00 h, anticonvulsant test). The behavioural tests were performed between 08.00 and 15.00 h. Food and water were provided ad libitum. At the time of testing the body weight was about 18–22 g.

Behavioural procedures

Motor activity was recorded for an hour in individual plexiglas chamber (20×40×31 cm) using a Digiscan Animal Activity Monitoring system (Omnitech Electronics Inc., Columbus, OH, U.S.A.) 5 min after oral administration of vehicle, diazepam (3–30 mg kg−1) or zolpidem (10–60 mg kg−1).

The anticonvulsant activity of diazepam (3, 10 and 30 mg kg−1 p.o.) and zolpidem (3, 10 and 30 mg kg−1 i.p.) were tested against pentylenetetrazole (PTZ) (Depoortere et al., 1986). This convulsant drug, when injected intraperitoneally at the dose of 120 mg kg−1 provokes myoclonic jerks associated with generalized tonic convulsions leading to death within a few seconds in all control mice. Diazepam and zolpidem were administered 30 min before PTZ. The number of mice developing the lethal tonic convulsion and/or myoclonic jerks within 10 min was noted.

Drugs

Diazepam and zolpidem were suspended in a 0.3% Tween 80/saline solution. Pentylenetetrazole (PTZ, Sigma, Buchs, Switzerland) was dissolved in saline. All drugs were administered in a volume of 5 ml kg−1.

Data analysis

Continuous random variables were analysed using two-way ANOVAs followed by Fisher's pair-wise comparisons when appropriate. Chi-Square analysis and Fisher's exact tests were used for dichotomic variables (Conover, 1999).

Results

Influence of diazepam on motor activity in wild type and α1(H101R) mice

Under vehicle treatment, wild type and α1(H101R) mice displayed a similar level of motor activity. When increasing doses of diazepam were administered, the motor activity was decreased in a dose-dependent manner in wild type mice. Wild type mice treated with 10 and 30 mg kg−1 of diazepam were less active in comparison with their vehicle-treated controls (P<0.01) (Figure 1A). However, as reported previously (Rudolph et al., 1999), the ability of diazepam to reduce motor activity was abolished in α1(H101R) mice (Figure 1A). Up to the highest dose tested all α1(H101R) mice showed the same amount of motor activity as the vehicle controls. ANOVA revealed a significant genotype-treatment interaction [F(3, 120)=4.90, P<0.003].

Figure 1.

Figure 1

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Effect of (A) diazepam (3–30 mg kg−1) and (B) zolpidem (10–60 mg kg−1) on motor activity in wild type and α1(H101R) mice. Motor activity was recorded for 1 h after drug administration. Results are expressed as mean counts ×103±s.e.mean. n=16 mice per group. V, vehicle; **P<0.01, Fisher's tests.

Effect of zolpidem on motor activity in wild type and α1(H101R) mice

In wild type mice, zolpidem depressed motor activity in a dose-dependent manner (P<0.01 versus vehicle) similar to diazepam (Figure 1B). However, in α1(H101R) mice zolpidem up to 60 mg kg−1 did not decrease motor activity. ANOVA showed a significant genotype-treatment interaction [F(3, 120)=3.27, P<0.02]. A separate analysis of the dose-response curve of zolpidem in α1(H101R) mice revealed no significant overall treatment effect on motor activity in α1(H101R) mice [ANOVA, F(3, 60)=1.91, n.s.]. Again, no genotype difference was observed in response to vehicle injection.

Anticonvulsant activity of diazepam in the pentylenetetrazole test in wild type and α1(H101R) mice

Wild type mice were dose-dependently protected by diazepam from the lethal tonic convulsion provoked by PTZ [χ2=21.79, P=0.001] (Figure 2A). Similarly, the number of wild type animals showing myoclonic jerks was dose-dependently decreased following diazepam treatment [χ2=31.11, P<0.001] (Figure 3A). At 30 mg kg−1 diazepam, all wild type mice were fully protected from both tonic and myoclonic convulsions. However, in α1(H101R) mice diazepam displayed a statistically significant activity against the lethal tonic convulsion only at the highest dose, 30 mg kg−1, at which 55% of the mice were protected [P<0.001 compared to vehicle, Fisher's exact test; χ2=7.53, P<0.056] (Figure 2A). Furthermore, diazepam displayed a dose-dependent partial activity against myoclonic jerks in α1(H101R) mice [χ2=12.08, P<0.01]. At the highest dose tested (30 mg kg−1), 37% of the mutant mice were protected from myoclonic jerks [P<0.05 versus vehicle] (Figure 3A). Thus, the anticonvulsant activity of diazepam in the PTZ convulsion test is partially due to its action on α1-GABAA receptors, in particular at low doses (10 mg kg−1), but GABAA receptors other than α1 (i.e. α2-, α3- and/or α5-GABAA receptors) clearly contribute to the full protective effect seen at the highest dose (30 mg kg−1) in wild type mice.

Figure 2.

Figure 2

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Effect of (A) diazepam (3–30 mg kg−1) and (B) zolpidem (3–30 mg kg−1) against the lethal tonic convulsion induced by pentylenetetrazole (120 mg kg−1) in wild type and α1(H101R) mice. Results are expressed as percentage of mice developing the tonic convulsion. n=10–11 per group. V, vehicle. *P<0.05, **P<0.01 and ***P<0.001, Fisher's exact tests.

Figure 3.

Figure 3

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Effect of (A) diazepam (3–30 mg kg−1) and (B) zolpidem (3–30 mg kg−1) against the myoclonic jerks induced by pentylenetetrazole (120 mg kg−1) in wild type and α1(H101R) mice. Results are expressed as percentage of mice developing myoclonic jerks. n=10–11 per group. V, vehicle. *P<0.05, **P<0.01 and ***P<0.001, Fisher's exact tests.

Effectiveness of zolpidem in the pentylenetetrazole test in wild type and α1(H101R) mice

In wild type mice zolpidem was protective against the lethal tonic convulsion in a dose-dependent manner [χ2=29.45, P<0.001] (Figure 2B). This effect was not observed in α1(H101R) mice even at the highest dose tested (30 mg kg−1) [χ2=3.07, n.s.]. This result suggests that the protective effect of zolpidem against the lethal tonic convulsion is mediated via α1-GABAA receptors. In contrast, myoclonic jerks, which were assessed irrespective of whether they were followed by tonic convulsions or not, failed to be suppressed by zolpidem in the wild type mice even at the highest dose tested (30 mg kg−1) [χ2=6.31, n.s.] (Figure 3B). This was the more surprising as α1-GABAA receptors are occupied by zolpidem under these experimental conditions (Figure 1B). In α1(H101R) mice, zolpidem was similarly ineffective in suppressing myoclonic jerks [χ2=3.07, n.s.].

Discussion

Classical benzodiazepine hypnotics interact with comparable affinity with all drug-sensitive GABAA-receptor subtypes (Kupfer & Reynolds, 1997; Benke et al., 1996). In contrast, the imidazopyridine hypnotic zolpidem is a ligand with a preferential affinity for α1-GABAA receptor subtype as determined by radioligand binding studies in vitro (Arbilla et al., 1985; Depoortere et al., 1986; Bartholini, 1993). However, it had not been clarified whether its interactions with α1-GABAA receptors is indeed the basis for its sedative-hypnotic activity. To assess the pharmacological relevance of the α1-GABAA-receptor subtype for the effectiveness of zolpidem in vivo an exquisite tool has become available through the recent development of a mouse line containing a knock-in point mutation in the benzodiazepine binding site. The α1(H101R) point mutation renders the benzodiazepine binding site of α1-GABAA receptors insensitive to classical benzodiazepines in vivo (Rudolph et al., 1999; McKernan et al., 2000). Similarly, binding of zolpidem to the point-mutated α1-GABAA receptor is virtually abolished as determined after immunoprecipitation of α1-GABAA receptors from α1(H101R) mice. Zolpidem displayed a Ki value of 13 nM at wild type α1-GABAA receptors and a Ki value of >10 μM at α1(H101R) receptors (Rudolph et al., 1999). Mice with a point mutation (H101R) in the α1 subunit are therefore suitable to test the contribution of α1-GABAA receptors to the pharmacology of zolpidem in vivo.

The sedative effect of zolpidem was found to be entirely mediated by α1-GABAA receptors at least up to a dose of 60 mg kg−1 as shown by the failure of zolpidem to significantly reduce motor activity in α1(H101R) mice (Figure 1B). The result is in line with the finding that the sedative effect of diazepam is likewise mediated via α1-GABAA-receptors (Figure 1A) (Rudolph et al., 1999). Thus, the neuronal populations which express α1-GABAA receptors mediate the sedative action of agonists acting at the benzodiazepine site irrespective of their chemical structure.

Apart from its sedative-hypnotic activity zolpidem had been characterized as a weak anticonvulsant active against the lethal PTZ-induced tonic convulsion (Depoortere et al., 1986). In the present study zolpidem was effective in reducing the PTZ-induced tonic convulsions in wild type mice with a potency comparable to that of diazepam. This anticonvulsant effect was abolished in α1(H101R) mice, suggesting that the protection by zolpidem against tonic convulsions is likewise mediated via α1-GABAA receptors (Figure 2B). However, even at the highest dose tested zolpidem failed to show anticonvulsant effectiveness against myoclonic seizures not only in α1(H101R) mice but also in wild type mice (Figures 2B and 3B). This occurred despite the fact that α1-GABAA receptors in wild type mice were occupied under the drug concentrations used. This failure by zolpidem to display activity against myoclonic jerks may be due to its reduced affinity to α2- and α3-GABAA receptors and/or its lack of interaction with α5-GABAA receptors.

In summary, the present results clearly show that the sedative effect of zolpidem is mediated via α1-GABAA receptors in vivo. This conclusion is of relevance for the development of future selective hypnotics acting at the benzodiazepine site.

Acknowledgments

We thank Daniel Blaser, Humberto Pochetti and Guido Schmid for animal care. This work was supported by a grant from the Swiss National Science Foundation.

Abbreviations

GABA

γ-aminobutyric acid

PTZ

pentylenetetrazole

ANOVA

analysis of variance

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