Introduction

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Classical benzodiazepines are in wide clinical use as hypnotics, tranquilizers, muscle relaxants, and anticonvulsants. These effects are caused exclusively by their interaction with the benzodiazepine site of GABA_A receptors. Based on the presence of more than a dozen subunit genes, the central nervous system contains a plethora of structurally diverse GABA_A receptors (Fritschy and Mohler, 1995; McKernan and Whiting, 1995; Barnard et al., 1998). The vast majority of GABA_A receptors are benzodiazepine-sensitive and can be grouped into 4 classes characterized by the type of  subunit being either 1, 2, 3 or 5. Diazepam and related classical benzodiazepines interact with equal affinity with all benzodiazepine-sensitive GABA_A receptors (Benke et al., 1996; Costa and Guidotti, 1996).

Recently, a promising strategy was developed to assign particular pharmacological effects of benzodiazepines to a specific GABA_A receptor subtype. This approach is based on a mutation-induced molecular switch by which the respective GABA_A receptor is rendered benzodiazepine-insensitive, as originally shown on recombinant receptors. When a conserved histidine residue in the benzodiazepine binding site of the respective  subunit is replaced by an arginine residue [1(H101R); 2(H101R); 3(H126R); 5(H105R)], the respective receptor is insensitive to diazepam but remains responsive to GABA (Wieland et al., 1992; Kleingoor et al., 1993; Benson et al., 1998). This molecular switch has recently been introduced into GABA_A receptors in vivo. A mutant mouse line was generated with a knock-in point mutation [1(H101R)] in which those benzodiazepine effects mediated via 1 GABA_A receptors were expected to be blunted (Rudolph et al., 1999). The behavioral analysis of this mutant mouse line demonstrated that the sedative, amnesic, and part of the anticonvulsant effects of diazepam are mediated by 1 GABA_A receptors. In contrast, the anxiolytic and myorelaxant effects of diazepam were unaltered in the 1 (H101R) mice compared with wild-type mice, suggesting that these effects are mediated by other GABA_A receptor subtypes (Rudolph et al., 1999).

To assign the contribution of 2 and 3 GABA_A receptors to the pharmacological spectrum of benzodiazepines, two further mouse lines were recently generated that contain the 2(H101R) and 3 (H126R) point mutations, respectively (Löw et al., 2000). The 2 GABA_A receptor is mainly expressed in the limbic system, whereas 3 GABA_A receptors are prominent in neurons of the reticular activating system of the brainstem. A detailed biochemical, autoradiographical, and immunohistochemical analysis demonstrated that the distribution and cellular location of the point-mutated receptors correspond to those of wild-type mice. However, their affinity for diazepam was reduced by a factor of at least 1000. An initial pharmacological analysis showed that the anxiolytic-like effect of diazepam is specifically mediated via 2 GABA_A receptors but not by 3 GABA_A receptors (Löw et al., 2000).

In the present investigation, an attempt is made to attribute the myorelaxant action of diazepam to 2 or 3 GABA_A receptors by comparing the diazepam-induced changes in muscle tone in the 2 and 3 mutant mouse strains compared with wild-type. The muscle tone was assessed in the horizontal wire test, in which the ability of the animals to grasp and hang on to a wire is measured. As a control that is independent of the benzodiazepine site, the myorelaxant activity of the GABA_B agonist baclofen was tested. The myorelaxant activity was differentiated from the diazepam-induced sedation by including measurements of the spontaneous locomotor activity of the mutant and wild-type mice.

	Materials and Methods

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Animals. Wild-type, 2(H101R), and 3(H126R) (five to six backcrosses to the 129/SvJ background) were generated as described previously (Löw et al., 2000). Female mice were raised in group-housed cages (8 to 10 mice per cage) under reversed light-dark cycle conditions (light on from 8:00 pm to 8:00 am) in the test room. Food and water were provided ad libitum. At the time of testing, body weight was 18 to 22 g.

Behavioral Procedures. The horizontal wire test was used to assess the drug effect on muscle tone (Bonetti et al., 1982). The number of mice unable to grasp the wire with both front paws and at least one hind paw within three trials was noted 30 min after oral administration of vehicle, diazepam (3-30 mg/kg), or baclofen (3-30 mg/kg). Another measure of muscle tone was obtained in the inverted screen test (Gasior et al., 1999). Mice were brought on a 22- × 9.5-cm wire mesh screen (0.9 cm screen mesh) placed 48 cm above the ground. The screen was inverted slowly by 180 degrees. Wild-type and 2(H101R) mice were able to move to the upper side of the screen three times. Thirty minutes after treatment with diazepam (20 mg/kg), the screen with the mice on the upper side was inverted and the latency to fall off the screen was noted (120-s observation period).

Drugs. Diazepam (gift from F. Hoffmann-LaRoche, Basel, Switzerland) was suspended in a 0.3% Tween 80/saline solution. Baclofen (Sigma, Buchs, Switzerland) was dissolved in saline. The drugs were administered in a volume of 5 ml/kg by mouth.

Data Analysis. Continuous random variables were analyzed using two-way analysis of variance (ANOVA) followed by Dunnett's test, Newman-Keuls' test, or unpaired or paired t tests for post hoc mean comparisons when appropriate. ² Analysis and Fisher's exact tests were used for dichotomous variables (Conover, 1999). In addition, ANOVAs were performed on dichotomous variables after angular transformation.

	Results

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Myorelaxant Action of Diazepam in 2(H101R) and 3(H126R) Mice. To assess the potential involvement of the 2 and 3 GABA_A receptors in the muscle relaxant action of diazepam, 2(H101R) and 3(H126R) mice carrying diazepam-insensitive 2 and 3 receptors, respectively, were subjected to the horizontal wire test. Diazepam produced a dose-dependent impairment of the grasping reflex in wild-type mice (² = 47.11; P < 0.001). The percentage of mice that were unable to grasp the horizontal wire was significantly increased in response to 10 and 30 mg/kg of diazepam compared with vehicle (P < 0.001, Fisher's exact test) (Fig. 1 A). In contrast, diazepam up to 10 mg/kg did not affect the grasping reflex in 2(H101R) mice carrying a diazepam-insensitive 2 GABA_A receptor. Only at the highest dose (30 mg/kg) was the grasping reflex impaired in 33.3% of 2(H101R) mice (P < 0.01 versus vehicle) (² = 17.89, P < 0.001) (Fig. 1A). ANOVA revealed a significant genotype X treatment interaction [F(3,72) = 3.45, P < 0.05]. An independent measure of the muscle relaxant activity of diazepam in wild-type and 2(H101R) mice was obtained using the inverted screen test. The administration of 20 mg/kg diazepam was associated with a decreased latency to fall off the grid in wild-type mice (73.75 ± 15.20 s, n = 8; P < 0.05, paired t test) but not in 2(H101R) mice (111.07 ± 6.72; P < 0.05 versus wild-type). Repeated-measures ANOVA on the same subjects revealed a significant genotype X treatment interaction [F(1,20) = 6.70, P < 0.05]. In contrast, the GABA_B receptor agonist baclofen impaired the grasping reflex in the horizontal wire test dose-dependently to the same extent in wild-type (² = 19.29, P < 0.001) and 2(H101R) mice (² = 20.14, P < 0.001) (Fig. 1B), indicating that the 2(H101R) mice are responsive to other myorelaxants. Diazepam produced a similar impairment of grasping reflex in wild-type (² = 67.88, P < 0.001) and 3(H126R) mice (² = 43.77, P < 0.001) at doses up to 10 mg/kg. At the highest dose tested (30 mg/kg), a genotype difference was observed in that a significantly lower percentage of 3(H126R) mice (61.8%) showed impaired grasping reflex compared with wild-type mice (100%) (P < 0.001, Newman-Keuls' test). ANOVA revealed a significant genotype X treatment interaction [F(3,237) = 3.23, P < 0.05] (Fig. 1C).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of diazepam (3-30 mg/kg by mouth) (A,C) and baclofen (3-30 mg/kg p.o.) (B) in the horizontal wire test in wild-type, 2(H101R) (A,B), or 3(H126R) mice (C). Results are expressed as percentage of mice with impaired grasping reflex. n = 6 to 21 mice per group. V, vehicle. P < 0.05, P < 0.01, and P < 0.001, Fisher's exact tests.

Effect of Diazepam on Spontaneous Locomotor Activity in 2(H101R) and 3(H126R) Mice. To exclude the possibility that the differences in the myorelaxant action of diazepam observed in wild-type and mutant mice are caused by a differential sensitivity for the sedative action of the drug, the spontaneous locomotor activity was assessed in a familiar environment. Diazepam produced a dose-dependent decrease in locomotor activity similarly in wild-type and 2(H101R) mice [F(3,71) = 16.94, P < 0.001] (Fig. 2A). This depressant drug effect was significant from the dose of 3 mg/kg (P < 0.01 versus vehicle, Dunnett's post hoc mean comparisons) in the two genotypes. Similarly, diazepam at all doses tested depressed spontaneous locomotor activity in both wild-type and 3(H126R) mice [F(3,64) = 14.39, P < 0.001] (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of diazepam (3-30 mg/kg by mouth) on spontaneous locomotor activity in wild-type, 2(H101R) mice (A), and 3(H126R) mice (B). Locomotor activity was recorded for 1 h. Results were expressed as mean number of crossings ± S.E.M. n = 8 to 10 mice per group. V, vehicle. P < 0.01, Dunnett's post hoc tests.

	Discussion

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Apart from baclofen, classical benzodiazepines represent the main group of drugs that are widely used to reduce the heightened muscle tone that accompanies various neurological diseases and injuries of the brain or spinal cord as well as states of anxiety. However, their clinical use as myorelaxants is limited by the lack of selectivity. The reduction in muscle stiffness is frequently associated with drowsiness and sedation. Until recently, it was not possible to dissociate, on the molecular level, the effects of diazepam on motor control systems from its various other actions. However, in a recent study using 1(H101R) mice , we were able to attribute the sedative effect of diazepam to the 1 GABA_A receptors, whereas the reduction in muscle tone was associated with other GABA_A receptor subtypes (Rudolph et al., 1999). To identify the molecular substrate of the myorelaxant property of diazepam, we have recently generated two novel lines of mice that contain the point mutations 2(H101R) and 3(H126R) (Löw et al., 2000). These two novel animal models are exquisite tools to investigate the specific role of the 2 or 3 GABA_A receptor subtypes in mediating the myorelaxant effect of diazepam.

The reduction in muscle tone produced by diazepam was found to be almost exclusively mediated by 2 GABA_A receptors, at least up to the dose of 10 mg/kg by mouth. This is demonstrated by the failure of diazepam to impair the grasping reflex in 2(H101R) mice (Fig. 1A) but not in 3(H126R) mice (Fig. 1C).

The molecular target for the muscle relaxant effect is clearly distinct from that mediating sedation, because the 2(H101R) mice showed a marked decrease in spontaneous locomotor activity in response to the respective dose of diazepam (10 mg/kg) (Fig. 2A). The failure of 2(H101R) mice to display diazepam-induced myorelaxation is not attributable to a principal inability to respond in the horizontal wire test because the 2(H101R) mice were responsive to the muscle relaxant baclofen, indicating that the polysynaptic spinal reflex transmission was not impaired in the 2(H101R) mice (Fig. 1B). The attribution of the myorelaxant effect of diazepam to 2 GABA_A receptors is in line with the highly specific expression of the 2 GABA_A receptor in the spinal cord, notably in the superficial layer of the dorsal horn and in motor neurons (Bohlhalter et al., 1996).

The anxiolytic effect of diazepam has recently been shown to be mediated via 2 GABA_A receptors, which are located mainly in limbic areas. However, the two 2 GABA_A receptor mediated effects are apparent at different dose ranges. Low doses (1-2 mg/kg) are sufficient for anxiolysis (Löw et al., 2000), whereas myorelaxation is evident in the horizontal wire test only at doses of 10 mg/kg (Fig. 1A).

The 3 GABA_A receptors seem to contribute to the muscle relaxant effect at high doses of diazepam as shown by the significantly reduced percentage of 3(H126R) mice with impaired grasping reflex in response to 30 mg/kg of diazepam (Fig. 1C). This is consistent with the widespread expression of the 3 subunit in the spinal cord and notably its colocalization with the 2 subunit on primary afferent terminals, which are the targets of GABA-ergic presynaptic inhibition (Bohlhalter et al. 1996). Again, this partial loss of drug effect was restricted to the control of the muscle tone. The spontaneous locomotor activity of the 3(H126R) mice was similarly affected by diazepam as in wild-type mice (Fig. 2B). We did not use doses of diazepam higher than 30 mg/kg because they produce a significant degree of sedation, which would nonspecifically affect the performance in tests used to assess muscle tone.

It is noteworthy that in 2(H101R) mice, a residual myorelaxation was observed only at the highest dose of diazepam tested (30 mg/kg) (Fig. 1A). This effect may be attributable to 3 GABA_A receptor-dependent impairment of the grasping reflex at high doses of diazepam (Fig. 1C).

The -carboline abecarnil is an anxiolytic compound that is markedly less myorelaxant than diazepam, although both types of benzodiazepine actions are thought to be mediated via 2 GABA_A receptors. This differential modulation might be explained by the partial agonistic activity of abecarnil at 2 GABA_A receptors (Pribilla et al., 1993; Turner et al., 1993), which appears to be sufficient to induce anxiolytic but not muscle relaxant activity.

In summary, the present results demonstrate that the myorelaxant effect of diazepam is largely mediated via 2 GABA_A receptors. The respective 2 GABA_A receptors are presumably those expressed on motor neurons and in the superficial layer of the dorsal horn although supraspinal 2 GABA_A receptors may also be involved. In response to high doses of diazepam, the 3 GABA_A receptors may additionally contribute to the muscle relaxant action. These results are of relevance for the development of future selective myorelaxants acting at the benzodiazepine binding site.