Tolerance to the anticonvulsant effects of carbamazepine, diazepam, and sodium valproate in kindled rats.

We assessed the development of tolerance to the anticonvulsant effects of carbamazepine (CBZ), diazepam (DZP), and sodium valproate (VPA) on convulsions elicited by amygdala stimulation in kindled rats in three similar experiments. In each experiment, amygdala-kindled rats were assigned to a drug group or to a corresponding vehicle control group. The rats in the three drug groups received a total of 10 bidaily (one every 48 h) IP injections of CBZ (70 mg/kg), DZP (2 mg/kg) or VPA (250 mg/kg) at a dose that initially blocked the forelimb clonus elicited by an amygdala stimulation (400 microA, 60 Hz, 1 s) administered 1 h after the injection. The rats in the three vehicle control groups were similarly treated except that they received injections of the saline vehicle. The drug tolerance test occurred 48 h after the final tolerance-development trial; the rats from each drug group and the corresponding vehicle control group received an injection of the appropriate drug followed 1 h later by the administration of a convulsive stimulation. The drug tolerance test revealed almost total tolerance in each of the three drug groups but no tolerance in any of the three vehicle control groups. Such large tolerance effects are inconsistent with the less dramatic effects reported in previous studies; possible reasons for this inconsistency were considered.     

Discriminative stimulus effects of the benzodiazepine receptor partial agonist bretazenil in pigeons and in rats

Bretazenil is a partial agonist at diazepam-sensitive (DS) GABA(A) receptors, and it also binds with high affinity to diazepaminsensitive (DI) GABA(A) receptors. A unique discriminative stimulus effect transduced by binding at DI benzodiazepine (BZ) receptors has been reported in pigeons, but has not been established in rats. Further, differences have been observed between rats and pigeons in results of drug discrimination experiments utilizing BZ receptor partial agonists. Therefore, to examine the discriminative stimulus effects of bretazenil and to explore the possibility of species differences in substitution profiles, pigeons and rats were trained to discriminate 0.3mg/kg bretazenil from vehicle. Flumazenil (0.03-1.0mg/kg) did not substitute for bretazenil in pigeons, despite full substitution of bretazenil for flumazenil in this species. Flumazenil (0.03-10.0mg/kg) also did not substitute for bretazenil in rats, despite the partial agonist effects of flumazenil in rats. Likewise, midazolam (0.3-1.0mg/kg) did not substitute for bretazenil in pigeons, despite the fact that bretazenil partially substitutes for midazolam in pigeons. However in rats, midazolam produced full, dose-dependent substitution (0.03-3.2mg/kg). Differences may result from different fractional receptor occupancy requirements for the mediation of discriminative stimulus effects through DS BZ receptors, and/or from a contribution of DI BZ receptor binding in pigeons.     

Tolerance to the anticonvulsant effects of phenobarbital, trimethadione, and clonazepam in kindled rats: cross tolerance to carbamazepine.

The kindled-convulsion model was used to assess the development of tolerance and cross tolerance to the anticonvulsant effects of antiepileptic drugs. In Experiment 1, tolerance developed to the anticonvulsant effects of bidaily (one every 48 h) IP injections of phenobarbital, trimethadione, and clonazepam on convulsions elicited 1 h after each injection in kindled rats by amygdala stimulation. In Experiment 2, kindled rats that were tolerant to the anticonvulsant effects of phenobarbital, trimethadione, or clonazepam received bidaily IP injections of carbamazepine, each followed 1 h later by a convulsive amygdala stimulation. There was a statistically significant transfer of tolerance from phenobarbital to carbamazepine, but not from either trimethadione or clonazepam to carbamazepine. Apparently, tolerance to anticonvulsant drugs is most likely to transfer between drugs that are effective against similar kinds of clinical and experimental seizures and have similar putative mechanisms of action.     

Discriminative stimulus properties of the benzodiazepine receptor partial agonist beta-carbolines abecarnil and ZK 95962: a comparison with chlordiazepoxide

Separate groups of rats were trained to discriminate one of three benzodiazepine receptor ligands from vehicle. The three ligands used, the benzodiazepine chlordiazepoxide, and the beta-carboline partial agonists ZK 95962 and abecarnil, have been reported to have different agonistic profiles. All three ligands formed specific benzodiazepine-receptor mediated discriminative stimuli antagonizable by at least one benzodiazepine antagonist. Different patterns of generalization were observed for each cue. As reported previously full and partial agonists substituted for chlordiazepoxide, whereas generalization to ZK 95962 was obtained more readily with partial agonists and antagonists with weak partial agonist activity. In contrast to the other two cues, the abecarnil discriminative stimulus was difficult to train and was unstable over time. Additionally, the abecarnil cue showed commonalities only with sedative or BZ1 receptor agonists. These results demonstrate qualitative differences between different benzodiazepine receptor ligands dependent on the intrinsic activity of the compound used.     

Behavioural effects of the benzodiazepine receptor partial agonist RO 16-6028 in mice

The imidazo-diazepinone RO 16-6028 is a benzodiazepine receptor partial agonist which exhibits some anticonflict effects in the two-chambered light/dark test without significantly affecting the behaviour of mice confronted with the staircase test. In addition, this drug slightly reduced locomotion and more markedly rearing in a free exploration procedure. These results indicate that RO 16-6028 appears to produce some anxiolytic and sedative properties like full agonists, but with weaker magnitude. This could be related to the benzodiazepine partial agonistic profile of the compound.     

Anxiolytic-like effects of PNU-101017, a partial agonist at the benzodiazepine receptor

PNU-101017 is a chemically novel ligand at the benzodiazepine recognition site of cloned GABA_A receptors. It was reported to potentiate GABA-mediated chloride current in cultured cells with a moderate intrinsic activity and a biphasic dose-response relationship. In this study, we confirmed that PNU-101017 has a partial agonist-like effect in the antagonism of metrazole-induced seizures in mice. It produced no sedation or ataxia, but did antagonize diazepam-induced motor deficit of mice in the rotarod test. PNU-101017 was weakly active in anti-conflict anxiolytic tests, but attenuated the plasma corticosteroid response to mild stress in rats. It also antagonized stress-induced elevation of cerebellar cGMP levels in mice. Like chlordiazepoxide, it increased drinking of saline solution in thirsty rats. PNU-101017 did not potentiate the CNS-depressant effects of ethanol, and produced no evidence of physical dependence when administered repeatedly. Agonists with low intrinsic activity at the benzodiazepine receptor, such as PNU-101017, should be further explored for therapeutic uses. Received: 18 June 1996/Final version: 22 November 1996     

RO 16-6028, a benzodiazepine receptor partial agonist, does not exhibit anticonvulsant tolerance in mice

The development of anticonvulsant tolerance with RO 16-6028, a benzodiazepine receptor partial agonist, was assessed in mice using an i.v. infusion of pentylenetetrazol as the convulsive stimulus. In contrast to other benzodiazepines tested previously in this seizure model the anticonvulsant protection afforded by RO 16-6028 did not change significantly during 10 days treatment (2 mg/kg b.i.d.). This result supports the hypothesis that partial agonists at the benzodiazepine receptor may induce less tolerance and/or dependence than full agonists.     

Contingent tolerance to the anticonvulsant effects of carbamazepine, diazepam, and sodium valproate in kindled rats.

The effect of convulsive stimulation during periods of drug exposure on the development of tolerance to the anticonvulsant effects of carbamazepine (CBZ), diazepam (DZP), or sodium valproate (VPA) was studied in three similar experiments. In each experiment, amygdala-kindled rats were assigned to one of three groups: one group received a drug injection (CBZ, 70 mg/kg, IP; DZP, 2 mg/kg, IP; VPA, 250 mg/kg, gavage) 1 h before each of a series of 10 bidaily (one every 48 h) convulsive stimulations, a second group received the same dose of the drug 1 h after each of the 10 stimulations, and a third group served as a vehicle control. The drug tolerance test occurred in each experiment 48 h after the 10th tolerance-development trial; every rat received the appropriate dose of CBZ, DZP, or VPA 1 h before being stimulated. In each experiment, only the rats from the drug-before-stimulation group displayed a significant amount of tolerance to the drug's anticonvulsant effect. Thus the development of tolerance to the anticonvulsant effects of CBZ, DZP, and VPA was not an inevitable consequence of drug exposure; the development of tolerance was contingent upon the occurrence of convulsive stimulation during the periods of drug exposure. These results support the idea that functional drug tolerance is an adaptation to a drug's effects on ongoing patterns of neural activity, rather than to drug exposure per se.     

Tolerance to the anticonvulsant effects of clobazam in mice.

Tolerance to the anticonvulsant effects of clobazam has been studied in three murine models of epilepsy: pentylenetetrazole- and N-methyl-D,L-aspartic acid-induced seizures and audiogenic-induced seizures in the DBA/2 strain. Tolerance occurred most rapidly in the pentylenetetrazole model but the development of tolerance could be reduced by increasing the dose interval. Tolerance to the protective effects of clobazam occurred more readily to the first tonic seizure than to the full tonic clonic seizure. The development of tolerance could not be attributed to smaller concentrations of clobazam in brain or to increasing concentrations of the metabolite. Although slower to develop, tolerance to clobazam did occur in the N-methyl-D,L-aspartate model. However, tolerance to the protection from the full tonic clonic seizure in DBA/2 mice could not be detected, even when the dose of clobazam was reduced to the smallest dose that acutely protected most of the mice. In contrast, the protection given by clobazam to the induction of the wild-run in DBA/2 mice, did exhibit tolerance. Studies on the mechanism of tolerance to the anticonvulsant activity of benzodiazepines must take account of the seizure model used and the dose and interval between doses.     

Failure of the partial inverse benzodiazepine agonist Ro15-4513 to block the lethal effects of ethanol in rats

The partial inverse benzodiazepine agonist Ro15-4513 has been found to antagonize some of the behavioral and physiological effects of low to moderate doses of ethanol. In the present study, pretreating rats with Ro15-4513 (1, 3, 10, and 30 mg/kg) at doses equal to or greater than those used in prior investigations failed to block the lethal effects of intraperitoneal injections of ethanol at a dose of 5.4 g/kg. These results suggest that the lethal actions of ethanol may involve a mechanism that is not blocked by Ro15-4513, which is known to selectively antagonize ethanol-stimulated chloride uptake via the GABA-coupled chloride ion channel.     

The pharmacological properties of Y-23684, a benzodiazepine receptor partial agonist.

1. The pharmacological properties of a benzodiazepine receptor (BZR) partial agonist, Y-23684 were investigated in comparison with those of diazepam, a conventional BZR full agonist. 2. Y-23684 and diazepam showed high and selective affinity for the BZR with Ki values of 41 and 5.8 nM, respectively. 3. In contrast to diazepam, variability was noted in the anticonvulsive potency of Y-23684 depending on convulsants (bicuculline, pentylenetetrazol and maximal electrical shock). Y-23684 produced the most potent protective effect against bicuculline in rats and mice with ED50S of 1.3 and 1.2 mg kg-1, respectively. 4. In rat conflict models (Geller-Seifter and water-lick tests), Y-23684 produced an antipunishment action at doses 2-4 times lower than diazepam. In contrast to diazepam, Y-23684 did not affect unpunished responding up to 50 mg kg-1 in the Geller-Seifter test. 5. In other rat models of anxiety (social interaction and elevated plus-maze tests), Y-23684 was as efficacious as and ten fold more potent than diazepam. In a mouse model of anxiety (exploration (light/dark box) test), Y-23684 was as efficacious and two fold less potent as diazepam. In these paradigms, Y-23684 showed a selective anxiolytic profile over a wide dose-range without loss of efficacy and sedative action. 6. The impairment of motor coordination (rotarod) and potentiation of CNS depressants (ethanol and hexobarbitone) by Y-23684 was much weaker than that of diazepam. 7. These results suggest that Y-23684 would be a potent and selective anxiolytic agent in man with less side-effects than conventional BZ-anxiolytics.     

Lack of anticonvulsant tolerance and benzodiazepine receptor down regulation with imidazenil in rats.

1. Development of anticonvulsant tolerance and benzodiazepine (BZD) receptor down-regulation has been reported to occur upon chronic administration of conventional BZDs. We compared the effect of chronic treatment with imidazenil, a new BZD partial agonist, and diazepam in rats. 2. After acute administration, imidazenil was more potent though less effective than diazepam in protecting from bicuculline-induced seizure. The time-course analysis of two peak equieffective doses of imidazenil (2.5 mumol kg-1 p.o.) and diazepam (35 mumol kg-1, p.o.) showed a longer lasting action of the former drug. 3. The anticonvulsant efficacy of diazepam (35 mumol kg-1, p.o.) was reduced in rats given chronic diazepam (35 mumol kg-1 p.o., 3 times a day for 8-15 days). No tolerance to imidazenil (2.5 mumol kg-1, p.o.) was apparent after 130-day administration with imidazenil (2.5 mumol kg-1, p.o., 3 times a day). 4. Plasma levels of imidazenil and diazepam, assessed 30 min after administration, were not changed in chronically treated animals. 5. In rats made tolerant to diazepam, the maximum number of [3H]-flumazenil binding sites were reduced in both cerebral cortex (-36%) and cerebellum (-42%). No changes in [3H]-flumazenil binding were found in chronic imidazenil-treated rats. 6. Specific [3H]-flumazenil binding in vivo was decreased in the forebrain of chronic diazepam- but not of chronic imidazenil-treated animals. 7. These data indicate that imidazenil possesses a very low tolerance potential to its anticonvulsant activity and does not affect BZD receptor density even after prolonged administration.     

Tolerance to anxiolytic- and antidepressant-like effects of a partial agonist of glycineB receptors.

The present study examined effects of acute and repeated administration of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist of glycineB receptors, in the conflict drinking test and the forced swim test in rats. Diazepam and imipramine were used, respectively, as reference drugs in those tests. In the conflict drinking test, acute administration of ACPC (200 mg/kg) increased fivefold the number of punished licks. A three- and fivefold increase in the number of punished licks was observed in rats treated repeatedly with ACPC (200 mg/kg daily; 14 days) and challenged with the same dose of the drug 24 h or 4 days later, respectively. A single injection of ACPC (400 mg/kg) reduced by 40% the immobility time in the forced swim test. In rats treated repeatedly with ACPC (400 mg/kg daily; 14 days) and challenged with the same dose 24 h or 4 days later, the drug either produced no significant effect or reduced the immobility time by 50%, respectively. On the other hand, no changes in anxiolytic- and antidepressant-like effects of chronically administered diazepam (10 mg/kg daily; 14 days) and imipramine (30 mg/kg daily; 14 days), respectively, were observed. The above results indicate that tolerance develops to the anxiolytic- and, particularly, to the antidepressant-like activity of ACPC.     

Chronic exposure to a benzodiazepine partial agonist does not alter GABAA receptor function in cultured neurons.

The benzodiazepine partial agonist Ro16-6028 does not lead to GABAA receptor downregulation in vivo. To assess effects of this compound in vitro, cultured neurons were exposed to Ro16-6028, 1 and 10 microM. GABA-dependent chloride uptake was unaffected at either dose of Ro16-6028 from 2 to 10 days, in contrast to decreased function observed with clonazepam, 1 microM, at 10 days, Ro16-6028 exposure did not alter GABA-independent chloride uptake, total neuronal protein, or cellular protein synthesis.     

The anticonvulsant effects of diazepam and phenobarbital in prekindled and kindled seizures in rats

The effects of various doses of diazepam (0.5–4 mg/kg) and phenobarbital (7.5–60 mg/kg) were determined on prekindled and kindled amygdaloid seizures in the same rats. Diazepam was ineffective against the prekindled focal seizures, but demonstrated profound and statistically significant control of the kindled seizures. In the kindled state, diazepam reduced the afterdischarge duration and seizure rank score to prekindled levels. Only the largest sedating dose of phenobarbital produced a reduction of both prekindled afterdischarge duration and seizure rank score. Against the kindled seizure, phenobarbital showed a marked and statistically significant increase in effectiveness in all but the smallest dose tested. The afterdischarge duration of kindled seizures was reduced to prekindled levels by 15–60 mg/kg of phenobarbital, while seizure rank score was reduced to prekindled levels by 30 and 60 mg/kg phenobarbital. The effects of two doses of diazepam (0.5 and 2.5 mg/kg) and phenobarbital (7.5 and 30 mg/kg) were tested against prekindled and kindled pentylenetetrazol (PTZ)-induced seizures. Preliminary work with 3 doses of pentylenetetrazol (30, 40 and 60 mg/kg) demonstrated that repeated doses of 30 mg/kg readily kindled seizures without the significant mortality seen with larger doses. Both diazepam and phenobarbital were less effective against seizures kindled with 30 mg/kg pentylenetetrazol compared to prekindled seizures. The comparative lack of effect that was seen with diazepam and phenobarbital against the pentylenetetrazol kindled seizure at doses associated with control of the kindled amygdaloid seizure may reflect an underlying difference in the pathogenesis of kindling between these seizure models. Further, the lack of suppression of the prekindled amygdaloid afterdischarge duration by large doses of diazepam, in contrast to large doses of phenobarbital, may also reflect differences between the mechanisms of action of these two drugs. This paradigm provides a model for testing the effectiveness of anticonvulsants during the progressive development of various epileptogenic seizures.     

Effects of gender and gonadectomy on responses to chronic benzodiazepine receptor agonist exposure in rats.

Gonadal steroid hormones or their derivatives have been shown to modulate the GABA receptor complex and GABA-mediated responses in a manner similar to the benzodiazepines. The present study examines if hormonal status modulates the development of tolerance and/or the neural adaptations in GABAA receptors associated with chronic benzodiazepine exposure. Anticonvulsant effects of diazepam were compared in groups of male, female, orchidectomized, and ovariectomized rats following acute (3 day) and chronic (3 week) exposure to diazepam-filled silastic implants. Results indicated that hormonal status did not significantly modify either the neural levels of drug resulting from the diazepam implants or the diazepam-induced increases in bicuculline seizure thresholds following acute (3 day) exposure. Unlike males and gonad-intact females, ovariectomized rats continued to display elevated seizure threshold values due to the diazepam released from the implants even after chronic diazepam exposure. This suggests that the tolerance to benzodiazepine actions observed in male and intact female rats was prevented by ovariectomy. Analysis of the anticonvulsant effects of additional challenge doses of diazepam in chronic diazepam-treated rats paradoxically suggested that benzodiazepine tolerance developed in all hormone groups. The discrepancies between these two tests of anticonvulsant tolerance may be related to the divergent neural GABAA receptor adaptations seen between hormone groups. Ovariectomized rats displayed a reduction in GABA IC50 values for [3H]bicuculline-thiocyanate binding in cortex following chronic diazepam exposure that was not observed in males or intact females. These results suggest that the diminution of ovarian steroid hormones may modulate the neural GABAergic changes associated with the development of tolerance to benzodiazepine actions during chronic agonist exposure.     

Tolerance to the anticonvulsant action of benzodiazepines. Relationship to decreased receptor density

Tolerance to the anticonvulsant action of benzodiazepines was studied in rats that had been treated for 4 weeks with 100-150 mg/kg per day of flurazepam. Previous studies had shown that this treatment produced tolerance to motor impairment induced by benzodiazepines and also down-regulation of benzodiazepine receptors in brain, which was seen as a reduced number of binding sites with no change in binding affinity. In the present study, seizures were produced using pentylenetetrazol (PTZ). In rats that had been chronically treated with flurazepam, pretreatment with diazepam was significantly less effective in blocking pentylenetetrazol-induced seizures, thus indicating tolerance. This tolerance could not be explained by a change in sensitivity to pentylenetetrazol resulting from chronic treatment, nor by any differences in levels of active drug in the brain following doses of diazepam. Residual amounts of flurazepam and its active metabolites may have artifactually reduced the apparent degree of tolerance measured 12 hr after the end of chronic treatment, but not at later times. Tolerance to the antipentylenetetrazol action of diazepam was evident up to the fourth day following chronic treatment with flurazepam, but tolerance had largely disappeared a week after chronic treatment. The duration of tolerance was much longer than that reported for tolerance to motor impairment induced by benzodiazepines, and for down-regulation of receptors. These results suggest that different mechanisms or different neural systems must mediate tolerance to these different actions of benzodiazepines. Furthermore, an adaptive reduction in the number of benzodiazepine receptors does not seem to be a likely mechanism for tolerance to the anticonvulsant action of these drugs.     

Discriminative stimulus properties of RU 33965, a benzodiazepine receptor weak partial inverse agonist.

Rats were trained to discriminate the low-efficacy benzodiazepine receptor inverse agonist RU 33965 from vehicle in a two-lever discrimination task on a fixed ratio (FR) 20 schedule. Consistent discrimination was obtained at 0.5 mg/kg PO RU 33965. Both leptazol and stronger inverse agonists (FG7142, S-135, RU 34000) substituted for the cue. The weak inverse agonists/antagonists RU 33094, RU 34030, Ro 15-1788, and ZK 93426 also substituted for the cue with the latter two compounds being particularly potent. The agonist and partial agonists diazepam, RU 33203, and RU 39419 did not substitute for the RU 33965 cue but RU 39419 antagonised it. The full agonists diazepam and loprazolam only consistently antagonised the cue when given IP 5 min pretest. These data suggest that the RU 33965 cue results from its weak inverse agonist activity at benzodiazepine receptors, but kinetic factors must be considered when interpreting drug effects in discrimination studies.     

Effects of the delta-opioid receptor agonist SNC80 on learning relative to its antidepressant-like effects in rats

Delta-opioid receptor agonists produce decreases in immobility in the forced swim test, suggesting that these compounds have antidepressant-like activity. There is also the possibility that these compounds decrease immobility in the forced swim test by disrupting learning processes that occur during the swim, or with successive swim exposures, thus falsely identifying them as having "antidepressant" potential. This study investigated the effects of the delta-opioid receptor agonist, SNC80, on responding in a repeated-acquisition procedure and in the forced swim test in rats, and the effects were compared directly to those of scopolamine, a compound known to disrupt memory and learning. SNC80 disrupted acquisition of a response sequence (learning) and produced a significant antidepressant-like effect in the forced swim test. Scopolamine, however, produced larger decrements in learning without producing behavioral changes consistent with an antidepressant-like profile of action. These results suggest that SNC80 produces antidepressant-like activity through a mechanism independent of its disruptive effects on learning.