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1 Neuropharmacology xxx (2009) 1 7 Contents lists available at ScienceDirect Neuropharmacology journal homepage: Effects of acute and repeated zolpidem treatment on pentylenetetrazole-induced seizure threshold and on locomotor activity: Comparison with diazepam Josipa Vlainić, Danka Peričić * Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, RuCer Bošković Institute, P.O. Box 180, Zagreb, Croatia article info abstract Article history: Received 31 July 2008 Received in revised form 20 March 2009 Accepted 23 March 2009 Keywords: Zolpidem Diazepam Pentylenetetrazole Seizure threshold Sedative activity Tolerance Zolpidem and diazepam are widely used drugs acting via benzodiazepine binding sites on GABA A receptors. While diazepam is non-selective, zolpidem has a high affinity for a1-, and no affinity for a5- containing receptors. Several studies suggested that behavioral effects of zolpidem might be more similar to classical benzodiazepines than previously thought. To compare the sedative and anticonvulsant properties of these drugs and to evaluate the importance of GABA A receptor subunits for development of tolerance during chronic treatment, we tested the effects of acute and repeated administration of zolpidem and diazepam on ambulatory locomotor activity (a measure of sedation) and on the threshold for myoclonic, clonic and tonic seizures in response to i.v. infusion of pentylenetetrazole (PTZ). Both drugs given acutely in doses 0.3, 1 and 3 mg/kg reduced locomotion, and in doses 1 and 3 mg/kg elevated the threshold for PTZ-induced seizures. The effects of zolpidem and diazepam on the tonic seizure threshold were greater than on myoclonus and clonic seizure threshold. Diazepam and zolpidem (3 mg/kg), given 18 or 42 h after repeated drug treatment (10 days, 5 mg/kg, twice daily), decreased the PTZ seizure threshold and increased the locomotor activity as compared to control mice, indicating development of tolerance to their anticonvulsant and sedative effects. After repeated treatment the PTZ seizure threshold was not different between the two drugs, while differences in sedation became larger than after the acute treatment. The results suggest that a5-containing GABA A receptors are not crucial for the development of sedative and anticonvulsant tolerance. Ó 2009 Published by Elsevier Ltd. 1. Introduction The imidazopyridine zolpidem is a widely used hypnotic drug known to produce its effects via benzodiazepine binding sites on the GABA A receptors (Depoortere et al., 1986; Sanger, 2004). Although zolpidem and benzodiazepines use the same binding sites, the pharmacological profile of zolpidem is different from that of classical benzodiazepines. Unlike benzodiazepines, zolpidem has very pronounced sedative and mild anxiolytic, anticonvulsant and myorelaxant properties (Depoortere et al., 1986; Sanger, 2004). Several groups of authors reported that unlike benzodiazepines, zolpidem does not produce either sedative (Perrault et al., 1992; Scharf et al., 1994; Elliot and White, 2000) or anticonvulsant tolerance (Perrault et al., 1992) during its prolonged administration. Differences in the behavioral properties of these positive allosteric modulators of GABA A receptor function were explained by selective affinity of zolpidem for a1-containing * Corresponding author. Tel.: þ ; fax: þ address: pericic@irb.hr (D. Peričić). GABA A receptors (Arbilla et al., 1986; Graham et al., 1996; Atack et al., 1999; Crestani et al., 2000). Furthermore, unlike classical benzodiazepines, which have a similar affinity and agonist efficacy for a1-3 and a5-containing GABA A receptors, zolpidem shows no affinity for receptors containing the a5 subunit (Pritchett and Seeburg, 1990; Graham et al., 1996; Atack et al., 1999). A recent study demonstrated that binding of zolpidem requires a phenylalanine residue (Phe77) in the g2 subunit(wulff et al., 2007). Crestani et al. (2000) suggested that the sedative action of zolpidem and its activity against pentylenetetrazole (PTZ)-induced tonic seizures are completely mediated by a1-containing GABA A receptors. Seizures induced by PTZ, a non-competitive antagonist of the GABA A receptors, are widely used in the screening of potential antiepileptic drugs. They are considered to be a model of petit mal or generalized absence seizures. PTZ seizures and human absence seizures share behavioral and EEG similarity and are pharmacologically characterized by high anticonvulsant efficacy of trimethadione, valproic acid, ethosuximide and benzodiazepines and by inefficacy of phenytoin and carbazepine (Löscher and Schmidt, 1988; Snead, 1992; White, 2003). In rodents, these seizures are /$ see front matter Ó 2009 Published by Elsevier Ltd. doi: /j.neuropharm

2 2 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) 1 7 characterized by 7 9 Hz bilaterally synchronous spike-wave discharges, which originate in thalamocortical circuitry (Snead, 1995). According to Löscher and Nolting (1991), one of the models that should be used in the laboratory evaluation of anticonvulsant drugs is the i.v. PTZ seizure threshold model to avoid underestimation of anticonvulsant activity of test compounds. The aim of this study was to determine and compare in the same experiment the acute doses of zolpidem and diazepam producing sedation and increasing the threshold for PTZ-induced myoclonic, clonic and tonic seizures. Our aim was also to compare the ability of zolpidem and diazepam, given in moderate doses, to induce sedative and anticonvulsant tolerance after prolonged treatment. If, as suggested by Van Rijnsoever et al. (2004), a5-gaba A receptors are required for the development of sedative tolerance to diazepam, then drugs lacking affinity for a5 receptor subtype (such as zolpidem) should not develop sedative tolerance. Sedative activity was assessed by measuring ambulatory locomotor activity. Anticonvulsant activity was evaluated by determination of threshold for myoclonic, clonic and tonic seizures following an i.v. infusion of PTZ. 2. Materials and methods 2.1. Animals Male CBA/HZgr mice (20 25 g) approximately 3 months old, raised in the breeding colony of the RuCer Bošković Institute, Zagreb, Croatia, were used. They were housed at a constant temperature (22 C) and under a light cycle of 12-h light/ 12-h darkness (lights on at 7:00 a.m.). Food and water were freely available. In order to improve the accuracy in calculating the dosage of drugs as well as PTZ (expressed per kg of body weight) required to induce seizures, the animals were deprived of food the night before the experiment. All animal care and experimental procedures were carried out in accordance with the National Institute of Health, Guide for the Care and Use of Laboratory Animals (NIH publication No ) revised 1996, and with the Croatian law on animal welfare Drugs Zolpidem hemitartrate (kindly provided by Pliva, Zagreb, Croatia), diazepam (Apaurin ampullae, Krka, Novo mesto, Slovenia) and pentylenetetrazole (PTZ, Sigma, St Louis, MO) were used. Zolpidem and PTZ were dissolved in, and diazepam was diluted with saline. Zolpidem and diazepam were administered i.p. in a volume of 1 ml per 100 g body weight. Control animals received i.p. injection of saline. In the acute experiment, doses of drugs were 0.3, 1 and 3 mg/kg. They were given 15 min before placing the animals in Ugo-Basile Activity Cage for measurement of ambulatory locomotor activity, i.e., 30 min before the PTZ seizure threshold determination started. For the acute experiment, the timing of zolpidem administration was chosen based on previous studies (e.g., Depoortere et al., 1986; Fahey et al., 2006). In the experiment designed to study the development of tolerance, mice were treated twice daily (at 8:00 a.m. and 5:00 p.m.) for 10 consecutive days with diazepam (5 mg/kg i.p.), zolpidem (5 mg/kg i.p.) or saline. On day 11 or 12, i.e., 18 or 42 h after termination of repeated treatment, diazepam treated mice received an additional i.p. injection of diazepam (3 mg/kg), and zolpidem treated mice an additional i.p. injection of zolpidem (3 mg/kg). Half of the animals in saline treated group were given for the first time diazepam (3 mg/kg i.p.), and the other half were given zolpidem (3 mg/kg i.p.). In the experiment performed 18 h after termination of repeated treatment, one part of saline treated mice were given an additional i.p. injection of saline. PTZ was given by constant i.v. infusion. A butterfly infusion needle (length 20 mm, gauge 27) was inserted into the tail vein and correct placement was verified by the appearance of blood in the infusion tubing. During the infusion the animal was held lightly by the tip of the tail. The concentration of PTZ was 4 mg/ml and the infusion rate controlled by a microinfusion pump was 1.1 ml/min. As in the two previous studies (Fahey et al., 2006; Peričić et al., 2008), sedative and anticonvulsant effects of zolpidem against PTZ-induced seizures were studied in the same animal. Sedative activity was assessed by measuring the decreases in ambulatory locomotor activity Locomotor activity Prior to the experiment, the animals were brought from the adjacent animal room to the laboratory and allowed to acclimatize for approximately 1 h. The horizontal ambulatory activity in individual mice was registered by Ugo-Basile Activity Cage. The apparatus consists of an animal cage (with a transparent cover) and an electronic unit. The activity detection relies on horizontal sensors, designed for the assessment of the ambulatory activity. The movements the animal makes inside the cage interrupt one or more infrared beam/s. The beam interruptions are counted and recorded by the electronic unit. Data related to horizontal ambulatory activity are printed in digital form at pre-set intervals. The activity was recorded for 10 min, starting after placing the animal into the test cage. The locomotor measurements were performed between 08:00 and 13:00 h in a quiet room under normal laboratory lighting. The observer left the room after placing the animal in the apparatus Seizure threshold determination The animal was observed throughout the infusion, and the time between the start of PTZ infusion (4.4 mg/min) and the onset of seizures (latency) was recorded, mainly as previously described (Peričić et al., 2001, 2008). The first convulsive sign was myoclonus (a sudden involuntary muscle jerk, usually accompanied by a head twitch). Clonic seizures were characterized by whole-body clonus, including running and explosive jumps, while tonic seizures were characterized by extreme rigidity, with forelimbs and hindlimbs extended caudally. The infusion was stopped after tonic seizures. For each animal, the threshold dose of convulsant (mg/kg of body weight) required to elicit myoclonic, clonic and tonic seizures was calculated from the time of infusion (latency), infusion rate, concentration of convulsant and body weight. All experiments were carried out between 8:00 and 13:00 h Statistical analysis Statistical analysis and graphic presentation of results were carried out using SPSS version 13 and GraphPad Prism version 4.00 for Windows. Locomotor activity data were subjected to two-way, and the seizure threshold data to three-way ANOVA with seizure type as the repeated measure. For all significant effects, we reported the partial eta-squared (h p 2 ) values and the statistical power (Cohen, 1962). The partial eta squared is the proportion of the effect þ error variance that is attributable to the effect. It is computed as follows: partial h p 2 ¼ SS effect /(SS effect þ SS error ), where SS effect is the sum of squares for the effect, and SS error is the sum of squares for the error term associated with that effect. Partial eta-squared values can sum to more than 1 because each effect size estimate uses a different denominator. Statistical power is defined as 1 b, where b is the probability of correctly rejecting a false null hypothesis (Type II error). Type I error probability was set to Post hoc comparisons were conducted using Newman Keuls multiple comparison test. 3. Results 3.1. Acute effects of zolpidem and diazepam on the threshold for PTZ-induced myoclonic, clonic and tonic seizures As shown in Fig. 1A C, diazepam and zolpidem (0.3, 1 and 3 mg/kg) produced a dose-dependent increase of the threshold for PTZ-induced seizures. The three-way ANOVA (dose drug seizure type) with seizure type as repeated measure, revealed a significant (p ¼ 0.000) within-subject effect of seizure type (F 2,119 ¼ ), effect of seizure type by dose (F 6,119 ¼ 154.0) and by drug dose interaction (F 6,119 ¼ 6.24). The seizure type and the mentioned interactions explained 91.5, 79.5 and 13.6% of the within-subjects variance in PTZ-induced seizure threshold, as evidenced by their partial eta-squared values (h p 2 ¼ 0.915, and 0.136, respectively). The interaction drug seizure type was insignificant (F 2,119 ¼ 0.106). There was a significant (p ¼ 0.000) between-subject effect of drug (F 1,119 ¼ 105.3), of dose (F 3,119 ¼ 407.5) and drug dose interaction (F 3,119 ¼ 36.08), suggesting that differences in effects between the drugs were not consistent across different doses. The effects described above, accounted for 46.9, 91.1 and 47.6% of the variance in individual differences in PTZ-induced seizure threshold (h p 2 ¼ 0.469, and 0.476, respectively). The observed statistical power, computed using alpha ¼ 0.05, was in all cases 1.00, suggesting that there was a zero probability of committing Type II error. Post hoc analysis indicated that for all seizure types the seizure threshold obtained with 1 and 3 mg/kg was significantly different (p ¼ ) from that obtained in the saline treated group. The lowest dose of diazepam that produced a mild elevation (p ¼ 0.05)

3 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) of the threshold for myoclonic and clonic, but not tonic seizures, was 0.3 mg/kg Acute effects of zolpidem and diazepam on locomotor activity in mice To assess the sedative activity of zolpidem and diazepam, we tested the effects of these drugs on ambulatory locomotor activity. As shown in Fig. 2, both zolpidem and diazepam given in doses 0.3, 1 and 3 mg/kg reduced markedly the locomotor activity in mice, as quantified by recording interruptions of infrared beams during 10 min (counts/10 min). Two-way ANOVA (dose drug) revealed a significant main effect of dose (F 3,119 ¼ 321.3, p ¼ ) and drug (F 1, 119 ¼ 9.03, p ¼ ). The observed statistical power was and 0.846, respectively, and the partial eta-square values indicated that the effect of drug accounted only for a small (7%) and the effect of dose for a large proportion (89%) of the total variance in locomotor activity. A non-significant dose drug interaction (F 3, 119 ¼ 1.68) suggested similar differences between the two drugs across different doses Effects of repeated treatment with zolpidem and diazepam on the threshold for PTZ-induced myoclonic, clonic and tonic seizures in mice Challenge doses of both zolpidem and diazepam, given 18 h after repeated treatment (i.e., the 11th day from the start of the experiment), decreased the threshold for PTZ-induced myoclonic, clonic and tonic seizures (Fig. 3A C). The three-way ANOVA (treatment drug seizure type) with seizure type as repeated measure, revealed a significant main effect of treatment (F 1,30 ¼ 23.88, p ¼ 0.000) which accounted for 44.3% differences in the seizure threshold (observed power ¼ 0.997), confirming the development of tolerance to the anticonvulsant effects of drugs. There was also a significant effect of seizure type (F 2,30 ¼ , p ¼ 0.000, h p 2 ¼ 0.836, power ¼ 1.000) and a less convincing treatment seizure type (F 2,30 ¼ 6.99, p ¼ 0.013, h p 2 ¼ 0.189, power ¼ 0.725) interaction. There was no significant effect of either drug (F 1,30 ¼ 0.015), drug seizure type (F 2,30 ¼ 2.07) or drug treatment (F 1,30 ¼ 0.434) interaction. Post hoc analysis revealed significant differences (p ¼ ) between repeated (10 days) and acute treatment, and between the acute effects of zolpidem and diazepam on myoclonic (p ¼ 0.001) and clonic (p ¼ 0.05) seizure threshold. In the same experiment, the seizure threshold for PTZ-induced seizures in the group of mice which received saline after repeated saline, as evidenced in the legend to Fig. 3, was similar to that obtained in the acute experiment. Challenge doses of both zolpidem and diazepam, given 42 h after repeated drug treatment (i.e., the 12th day from the start of treatment), also, in comparison to acute doses of drugs given 42 h after repeated saline, decreased the threshold for PTZ-induced myoclonic, clonic and tonic seizures (Fig. 3D F). Three-way ANOVA with seizure type as repeated measure revealed a significant (p ¼ 0.000) effect of treatment (F 1,29 ¼ 29.04, h p 2 ¼ 0.500, observed power ¼ 0.999) indicating again a development of tolerance to the anticonvulsant effects of drugs. There was also a significant effect of Fig. 1. Acute effects of zolpidem and diazepam on the threshold for myoclonic (A), clonic (B) and tonic (C) seizures induced in mice by i.v. infusion of PTZ. Different doses of drugs were administered 30 min before the infusion of PTZ was started. To test the locomotor activity, the same animals spent 10 min (15 25 min following drug administration) in Ugo-Basile Activity Cage. Points represent means SEM from at least 8 animals per group. *p ¼ , **p ¼ versus control; j p ¼ 0.01, jj p ¼ versus the corresponding diazepam treated group (three-way ANOVA and Newman Keuls test).

4 4 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) Discussion Fig. 2. Acute effects of zolpidem and diazepam on ambulatory locomotor activity (LMA) in mice. Zolpidem and diazepam were administered i.p. 15 min before placing the animals in the apparatus (Ugo-Basile Activity Cage) equipped with infrared sensors. The horizontal locomotor activity was measured for 10 min and quantified by interruptions of infrared beams, which were counted and recorded automatically (counts/10 min). Points represent means SEM from at least 8 animals per group. *p ¼ versus control saline treated group (two-way ANOVA and Newman Keuls test). seizure type (F 2,29 ¼ , h p 2 ¼ 0.856, observed power ¼ 1.000) and treatment seizure type interaction (F 2,29 ¼ 12.89, p ¼ 0.000, h p 2 ¼ 0.308, observed power ¼ 0.996) on the threshold for PTZinduced seizures. There was no significant effect of drug (F 1,29 ¼ 1.69), neither interaction between drug and treatment (F 1,29 ¼ 1.31) nor between drug and seizure type (F 2,29 ¼ 0.687). Post hoc comparisons revealed that repeated diazepam in comparison with acute diazepam and repeated zolpidem in comparison with acute zolpidem produced a significant reduction of the threshold for all seizure types (p ¼ 0.05 or p ¼ 0.001). The anticonvulsant effects of acute diazepam against PTZ-induced myoclonic and clonic seizures were significantly (p ¼ 0.001) greater than the effects of zolpidem. After repeated treatment, this difference in anticonvulsant potency between diazepam and zolpidem was lost Effects of repeated treatment with zolpidem and diazepam on locomotor activity in mice The ambulatory locomotor activity of mice treated with diazepam acutely or repeatedly (10 days) was increased in comparison to mice treated with zolpidem, suggesting a higher level of sedation in zolpidem treated animals. In addition, mice repeatedly treated with diazepam and zolpidem had an increased locomotor activity as compared to mice treated acutely with these drugs, suggesting a development of tolerance to sedative effects of drugs (Fig. 4A and B). Two-way ANOVA confirmed (statistical power ¼ 1.000) a highly significant (p ¼ 0.001) effect of drug: A (F 1,30 ¼ 61.63, h p 2 ¼ 0.673); B (F 1,29 ¼ 78.81, h p 2 ¼ 0.731) and treatment: A (F 1,30 ¼ 11.72, h p 2 ¼ 0.281, power ¼ 0.912); B (F 1,29 ¼ 20.85, h p 2 ¼ 0.418, power ¼ 0.993), a nonsignificant drug treatment interaction for A (F 1,30 ¼ 1.83) and a significant interaction (p ¼ 0.005) for B (F 1,29 ¼ 9.15, h p 2 ¼ 0.240, power ¼ 0.877), indicating that in the latter case the differences between the two drugs were dependent on the treatment. It is generally accepted that unlike classical benzodiazepines, the imidazopyridine zolpidem has very pronounced sedative and mild anxiolytic and anticonvulsant properties (Depoortere et al., 1986; Sanger, 2004). Several studies (Perrault et al., 1992; Scharf et al., 1994; Elliot and White, 2000) reported a lack of tolerance to zolpidem upon repeated administration. This study demonstrated that zolpidem given acutely is slightly more effective than diazepam in producing sedation and less potent as a convulsant. Although both drugs given acutely were more potent against tonic than against myoclonic and clonic seizures, this difference was dependent on the dose and it was greater after zolpidem than after diazepam administration. After repeated treatment, mice developed tolerance to the anticonvulsant and sedative effects of both drugs so that differences in their anticonvulsant activity were lost, while differences in the level of sedation were potentiated. Diazepam is known to be non-selective for GABA A receptor subtypes, while zolpidem has the highest affinity to a1- and no affinity to a5-containing GABA A receptors (Arbilla et al., 1986; Pritchett and Seeburg, 1990; Graham et al., 1996; Atack et al., 1999). GABA A receptors containing a1 subunit are supposed to be responsible for the sedative activity of zolpidem and benzodiazepines (Perrault et al., 1988; Rudolph et al., 1999; McKernan et al., 2000; Kralic et al., 2002) and for the anticonvulsant effect of zolpidem against PTZ-induced tonic seizures (Crestani et al., 2000). Myoclonic, clonic and tonic seizures have different neuronal substrates. Myoclonic seizures originate in thalamocortical circuitry; clonic seizures have been shown to arise from the cerebral cortex and forebrain, whereas tonic seizures originate in the brain stem (cf. André et al., 1998). Immunocytochemical studies demonstrated that the mentioned brain regions exhibit different distribution of GABA A receptor subtypes (Pirker et al., 2000). While brain stem regions (medulla, pontine and cranial nerve nuclei) exhibit strong subunit a1-, b2- and g2-immunoreactivities, a2-imunoreactivity was preferentially located in forebrain areas and a3- especially in reticular thalamic nucleus and inner layers of the cerebral cortex. The strongest a5-subunit immunoreactivity was present in forebrain regions such as hippocampus, olfactory bulb and hypothalamus. Fradley et al. (2007), who examined the ability of diazepam to reduce PTZ-induced and maximal electroshock-induced seizures in mice containing point mutations in single or multiple alpha subunits, suggested that GABA A receptors containing a5 subunit do not mediate the anticonvulsant effects of diazepam. Their results also suggested that a2-containing receptors play a larger role in mediating the anticonvulsant properties of this drug than those containing a1, although the efficacy at different subtypes of GABA A receptors may act synergistically. Based on the experiments demonstrating that b-cct, a selective antagonist of BZ 1 receptors i.e., of a1-containing GABA A receptors, failed to affect the effect of diazepam and zolpidem on seizures produced by PTZ, Griebel et al. (1999) suggested that BZ 2 receptors may be primarily involved in the convulsant action of PTZ. Hence, a possible predominant involvement of a2-containing GABA A receptors in the convulsant action of PTZ (Griebel et al., 1999) and a different affinity of zolpidem and diazepam for different subtypes of GABA A receptors (primarily those containing a1 and a2 subunits), may explain the greater potency of zolpidem against tonic than against myoclonic and clonic seizures as well as dosedependent differences in the potency of zolpidem and diazepam against different PTZ-induced seizure types. It was already known that zolpidem has anticonvulsant activity against convulsions induced by isoniazid, PTZ and maximal

5 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) Fig. 3. Chronic effects of zolpidem and diazepam challenged with 3 mg/kg i.p. on the 11th (A C) or 12th (D F) day from the start of the repeated treatment (5 mg/kg i.p. twice daily for 10 days) on the threshold for myoclonic (A, D), clonic (B, E) and tonic (C, F) seizures induced in mice by i.v. infusion of PTZ. Drugs were administered 30 min before the infusion of PTZ was started. To test the locomotor activity, the same animals spent 10 min (15 25 min following drug administration) in Ugo-Basile Activity Cage. Points represent means SEM from 7 to 10 animals per group. *p ¼ , **p ¼ versus the corresponding group receiving test dose of drugs after repeated saline; j p ¼ 0.05, jj p ¼ versus the corresponding diazepam treated group (three-way ANOVA and Newman Keuls test). In the control group (N ¼ 10), which received saline on the 11th day from the start of repeated treatment with saline, the threshold for myoclonic ( ), clonic ( ) and tonic ( ) seizures was similar to that obtained in the acute experiment. electroshock (Depoortere et al., 1986; Sanger et al., 1996), but early studies reported that sedation with zolpidem occurs at doses 10 times lower than those inducing anticonvulsant effect (Depoortere et al., 1986). In the present work, a significant reduction of locomotor activity i.e., a sedative effect of zolpidem, but of diazepam as well, was seen already after 0.3 mg/kg, a dose that failed to increase the threshold for PTZ-induced seizures. The same dose of diazepam had a weak although significant anticonvulsant effect

6 6 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) 1 7 Fig. 4. Chronic effects of zolpidem and diazepam challenged with 3 mg/kg i.p. on the 11th (A) or 12th (B) day from the start of repeated treatment (5 mg/kg i.p. twice daily for 10 days) on ambulatory locomotor activity (LMA) in mice. Drugs were administered 15 min before placing the animals in the apparatus (Ugo-Basile). The horizontal locomotor activity was measured for 10 min. Points represent means SEM from 7 to 10 animals per group. *p ¼ , **p ¼ versus the corresponding group receiving test dose of drugs after repeated saline; jj p ¼ versus the corresponding diazepam treated group (two-way ANOVA and Newman Keuls test). In the control group (N ¼ 10), which received saline on the 11th day from the start of repeated treatment with saline, the locomotor activity count (556 23) was slightly greater than that obtained in the acute experiment. against myoclonic and clonic seizures, while 1 mg/kg was active also against tonic seizures. The lowest dose of zolpidem that produced a significant enhancement of the PTZ threshold for myoclonic, clonic and tonic seizures was 1 mg/kg. Previous studies failed to show the anticonvulsant effectiveness of zolpidem against PTZ-induced myoclonic seizures (Crestani et al., 2000; Fradley et al., 2007). The results of this study showing that zolpidem (1 mg/kg) enhanced the threshold for PTZ-induced myoclonic seizures, suggest that the determination of the seizure threshold in response to i.v. infusion of PTZ is a more sensitive method than the use of a lethal dose of PTZ. This is in accordance with the results published previously (Löscher and Nolting, 1991). As expected, mice treated repeatedly with diazepam developed tolerance to its anticonvulsant and sedative activity, as evidenced by lower seizure thresholds for PTZ-induced convulsions and an increased locomotor activity. Our results demonstrated that repeated zolpidem also induces both sedative and anticonvulsant tolerance. Mice which received diazepam (3 mg/kg) after repeated (10 days) i.p. injections of saline had a lower seizure threshold and a greater locomotor activity than mice receiving only one acute injection of diazepam. Because repeated i.p. injections are stressful for the animals, these results appear to be in line with those of Weizman et al. (1989), showing a decrease of benzodiazepine binding sites and the reduction in clonazepam s anticonvulsant potency after repeated swim stress. Irrespective of whether the testing was done 42 or 18 h after the last of 20 repeated drug injections (i.e., whether the treatment was discontinued or not), decreases of the threshold for both diazepam and zolpidem were greater for tonic than for clonic and myoclonic seizures. The effect of diazepam against tonic seizures appeared to be more pronounced when mice were tested 42 than 18 h after repeated treatment. In both experiments, a significant treatment seizure type interaction, but not the effect of drug or drug treatment and drug seizure type interaction, suggested that the treatment effect changes with the seizure type. In contrast to this, testing of locomotor activity 18 or 42 h after termination of repeated treatment indicated that both treatment and drug had a significant effect on the development of tolerance to the sedative effects of drugs. It appears that mice withdrawn from diazepam for 42 h developed a greater level of tolerance to its sedative effects than mice receiving a test dose of diazepam in continuation of repeated treatment. The animals which were drug free for 42 h did not show visible withdrawal symptoms. A significant drug treatment interaction, obtained when analyzing this experiment, indicated that mice did not develop the same level of tolerance to sedative effects of zolpidem and diazepam. As reviewed by Löscher and Schmidt (2006) there are two major types of tolerance, pharmacokinetic or metabolic and pharmacodynamic or functional tolerance. While the first type of tolerance is due to changes in the pharmacokinetics of drugs, the other is due to a reduced sensitivity of target tissue. Previous studies suggested that long-term treatment with diazepam (Rundfeldt et al., 1995; Auta et al., 2008) or zolpidem (Thénot et al., 1988; Holt et al., 1997) did not change the pharmacokinetics of drugs. Thus, we can presume that both drugs produce functional tolerance. The mechanisms involved in the development of tolerance and dependence following chronic treatment with benzodiazepines are not clear (Costa et al., 2002; Wafford, 2005), although different mechanisms such as allosteric uncoupling of GABA and benzodiazepine binding sites (Gallager et al., 1984; Friedman et al., 1996) have been suggested. Van Rijnsoever et al. (2004), who demonstrated that mice with point mutation in a5 subunits fail to manifest sedative tolerance, proposed that diazepam binding to a5-gaba A receptors is a key mechanism underlying the diminution of its sedative efficacy with chronic treatment. However, in this study, zolpidem, which does not have an affinity for receptors containing the a5 subunit (Pritchett and Seeburg, 1990; Graham et al., 1996), produced sedative tolerance. The results of a recent study suggested the important role of the a1, but not of the a5 subunit in the development of tolerance to the anticonvulsant actions of positive allosteric modulators of benzodiazepine binding sites (Auta et al., 2008). These authors demonstrated that long-term treatment with imidazenil, which has a high affinity and intrinsic efficacy for a5, and low for a1-containing GABA A receptors, failed to induce anticonvulsant tolerance. In conclusion, our results demonstrate that in the acute study the lowest doses of zolpidem and diazepam producing sedation were 0.3, while those elevating the threshold for PTZ-induced tonic

7 J. Vlainić, D. Peričić / Neuropharmacology xxx (2009) seizures were 1 mg/kg. Both diazepam and zolpidem were more effective against PTZ-induced tonic than against myoclonic and clonic seizures. Mice treated repeatedly with zolpidem, like those treated with diazepam, developed tolerance to its sedative and anticonvulsant effects. Because zolpidem does not have an affinity for a5-containing GABA A receptors, the results suggest that prolonged stimulation of this receptor subtype is not essential for the development of sedative and anticonvulsant tolerance. After repeated treatment zolpidem and diazepam are approximately equipotent anticonvulsants, at least against PTZ-induced convulsions in mice, but zolpidem remains more potent in producing sedation. Acknowledgements This study was supported by the Croatian Ministry of Science, Education and Sports (Project: Stress, GABA A receptors and mechanisms of action of neuropsychoactive drugs). 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