Repeated anticonvulsant testing: contingent tolerance to diazepam and clobazam in kindled rats.

The acute anticonvulsant efficacy of diazepam (1.5 mg/kg, i.p.) was evaluated by repeated test injection in kindled rats subcutaneously implanted with diazepam-filled or empty silastic tubes for 3 weeks. Tolerance developed to acute test injections in both diazepam- and sham-implanted rats. Tolerance developed to a lesser extent in another group of diazepam-implanted rats which did not receive acute intermittent anticonvulsant tests. The hypothesis that contingent tolerance had developed to the anticonvulsant actions of benzodiazepines (diazepam, 1.5 mg/kg, i.p. and clobazam, 10 mg/kg, i.p.) in kindled rats given acute intermittent injections was investigated using a 'before-after' design. Significant contingent tolerance developed in rats which received intermittent benzodiazepine treatment before, but not after, amygdala stimulation. Tolerance developed to different extents depending on the seizure measure evaluated (forelimb clonus duration, amygdala afterdischarge duration, motor seizure latency and duration, and seizure stage). Contingent tolerance to both benzodiazepines developed at a similar rate. The findings suggest that contingent tolerance may contribute a sizeable component to the overall functional benzodiazepine tolerance measured in long-term anticonvulsant drug studies in kindled rats. Several questions regarding contingent tolerance phenomena are posed and the implications of these findings for studies using repeated anticonvulsant testing are discussed.     

Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats

While there is clinical evidence that progesterone has anticonvulsant activity in women with complex partial seizures, previous studies on the anticonvulsant effect of progesterone in experimental animal models are inconclusive. Moreover, the effect of progesterone on seizure parameters in fully amygdala-kindled rats which best resemble complex partial seizures has not been evaluated. Therefore, in the present work the anticonvulsant effect of progesterone at doses of 10, 30, 60 and 75 mg/kg in fully amygdala-kindled male rats was studied. Only at the high and sedative dose of 75 mg/kg, progesterone suppressed behavioral seizures and afterdischarges elicited 10 min after intraperitoneal (i.p.) administration. Pretreatment with the progesterone antagonist, 17β-hydroxy-11β-(4-dimethylaminophenyl)-17-(prop-1-ynyl)-estra-4,9-dien-3-one (RU 38486) at the dose of 3 mg/kg did not inhibit the anticonvulsant activity of progesterone, while pretreatment with the GABA_A receptor antagonist, bicuculline (2 mg/kg) blocked the anticonvulsant effect of progesterone. Neither RU 38486 nor bicuculline had any effect on the seizure parameters. These findings suggest that only at large and sedative doses, progesterone has some anticonvulsant activity in male amygdala-kindled rats which may be partly mediated via the GABA_A receptor complex interaction.     

Aminoglutethimide but not spironolactone enhances the anticonvulsant effect of some antiepileptics against amygdala-kindled seizures in rats

Summary. Aminoglutethimide (AGLD, an inhibitor of adrenal steroid synthesis) up to 5mg/kg and spironolactone (SPIR, a mineralocorticosteroid antagonist and a weak antiandrogen) up to 50mg/kg did not affect any seizure parameter in amygdala-kindled rats. AGLD (10mg/kg) significantly reduced seizure activity in rats of both gender. The combination of AGLD (5mg/kg) with phenobarbital (PB, applied at its subeffective dose of 15mg/kg) significantly shortened motor seizure and afterdischarge duration in amygdala-kindled seizures. The combined treatment of AGLD (5mg/kg) and clonazepam (CLO) at its subeffective dose of 0.01mg/kg caused significant reduction of the seizure severity, seizure duration and afterdischarge duration. Finally, AGLD (5mg/kg) proved ineffective upon the action of valproate (VPA) in this model of epilepsy. In contrast to AGLD, SPIR (50mg/kg) did not affect the action of PB, CLO or VPA against kindled seizures in rats. AGLD did not alter the free plasma levels and brain concentration of PB or CLO, so a pharmacokinetic interaction does not seem probable. Among a variety of chemoconvulsants, bicuculline and N-methyl-D-aspartic acid reversed the effects of AGLD/PB and AGLD/CLO combinations. Aminophylline, kainic acid, strychnine and the glucocorticosteroid (hydrocortisone) were ineffective in this respect. Our data confirm the hypothesis that AGLD-mediated events may play a role in seizure activity and can affect the anticonvulsant activity of some conventional antiepileptic drugs against kindled seizures. Moreover, extrapolation of obtained results to clinical practice may indicate that patients with complex partial seizures may be safely co-medicated with AGLD or SPIR without the risk of worsening of seizure control.     

Antiepileptogenic and anticonvulsant activity of interleukin-1 beta in amygdala-kindled rats

Ischaemic, excitotoxic and traumatic brain injuries have been associated with the occurrence of epileptic seizures. Microglia, the principal immune cells in the brain, produce a variety of proinflammatory and cytotoxic factors especially interleukin-1 (IL-1) early after an acute insult. We studied the effect of intracerebroventricularly administered IL-1beta on seizure acquisition and on fully kindled seizures in amygdala kindling model of epilepsy. IL-1beta (0.01 ng/rat) retarded acquisition of kindled behavioral seizures and growth of afterdischarges (AD). IL-1beta (0.01-10 ng/rat) also exhibited significant anticonvulsant effect on established kindled seizures and AD duration. This effect began 0.5 h after administration and was continued up to 72 h. Pretreatment of the kindled animals with nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, or cyclooxygenase inhibitor, piroxicam, reversed the anticonvulsant effect of IL-1beta at early time points. Although most of the previous studies indicate a proconvulsant or convulsant property of IL-1, our results support a protective and antiepileptogenic role of IL-1beta.     

The anticonvulsant effects of progesterone and 5alpha-dihydroprogesterone on amygdala-kindled seizures in rats

PURPOSE: Progesterone has been shown to be anticonvulsant in several animal seizure models. The purpose of the present study was to investigate the anticonvulsant actions of progesterone and its primary metabolite 5alpha-dihydroprogesterone in the amygdala kindling model. METHODS: Female Wistar rats were implanted in the right basolateral amygdala with a long-term, bipolar electrode. The subjects were kindled to 30 stage 5 seizures and stability tested. Multiple doses of progesterone and 5alpha-dihydroprogesterone were then tested for anticonvulsant activity against focal electrographic and generalized convulsive kindled seizures. The time course of progesterone's anticonvulsant action also was examined. RESULTS: Progesterone had a median effective dose (ED50) of 103 mg/kg against generalized convulsions at 15 min after injection. Subjects were not sedated at the time of seizure testing, although sedation developed later (40-60 min after injection). In time-course experiments, it was found that 120 mg/kg of progesterone caused complete suppression of the generalized convulsion from 20 to 160 min after injection. Suppression of the focal discharge also was seen in some animals between 20 and 160 min. 5alpha-dihydroprogesterone had an ED50 of 2.9 mg/kg against generalized kindled convulsions and an ED50 of 4.3 mg/kg against focal afterdischarge 15 min after injection. 5alpha-dihydroprogesterone did not produce sedation 15 min after injection, or at any later time interval. CONCLUSIONS: Progesterone is anticonvulsant only at high doses when tested against amygdala kindled seizures. 5alpha-dihydroprogesterone is considerably more potent than progesterone. At low, nonsedative doses, it was effective against both the kindled amygdala focal afterdischarge and the generalized convulsion.     

7-nitroindazole differentially affects the anticonvulsant activity of antiepileptic drugs against amygdala-kindled seizures in rats

PURPOSE: The objective of this study was to evaluate the interaction of the preferential brain nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), with conventional antiepileptic drugs (AEDs) against amygdala-kindled seizures in rats. METHODS: Experiments were performed on fully kindled rats. Adverse effects were evaluated with the rotorod test, which assesses motor coordination, and the passive-avoidance task, which assesses memory. Plasma levels of AEDs were measured by immunofluorescence. RESULTS: 7-NI (up to 100 mg/kg) failed to modify seizure parameters. However, it reduced the severity and duration of kindled seizures when coadministered with otherwise ineffective doses of carbamazepine (CBZ) (10-20 mg/kg) or phenobarbital (PB) (20 mg/kg). Combinations of 7-NI with valproate (VPA), diphenylhydantoin (DPH), or clonazepam (CLO) were not protective. L-Arginine (500 mg/kg) did not reverse the seizure-suppressing interactions between 7-NI and the conventional AEDs. The combinations of 7-NI and CBZ or PB did not impair performance in the rotorod test. Coadministration of 7-NI with CBZ did not affect long-term memory, and 7-NI given with PB didn't affect the mnemonic effect of PB. Finally, 7-NI did not affect the free plasma levels of CBZ or PB. CONCLUSIONS: Pharmacokinetic interactions do not seem to account for the anticonvulsant effects of 7-NI combined with CBZ or PB. Central nitric oxide (NO) is possibly not involved in the synergism between 7-NI and these AEDs.     

Lack of tolerance to the anxiolytic effect of diazepam and pentobarbital following chronic administration in perinatally undernourished rats

Adult female rats, undernourished at perinatal age, were evaluated for anxiolytic action in the plus-maze test after acute and chronic administration of diazepam (DZP) and pentobarbital (PTB). Deprived (D) rats chronically treated with vehicle showed an increased anxiety as compared with control (C) animals. A single intraperitoneal (i.p.) administration of DZP (1 mg/kg) or PTB (7.5 mg/kg) produced similar anticonflict effect in both C and D rats. Tolerance to the anxiolytic effect of DZP and PBT developed in C rats after a 15-day administration schedule, whereas no tolerance was observed in D animals. Drug disposition was not altered after chronic treatment either in C or in D rats. γ-aminobutyric acid (GABA)-mediated chloride uptake in microsacs of cerebral cortex of naive D rats was decreased as compared with naive C rats. After chronic DZP administration (1 mg/kg/day i.p. for 15 days), GABA-mediated ³⁶Cl⁻ influx in brain cortex microsacs of C rats did not change; however, GABA efficacy was increased in microsacs of D animals. In addition, chronic DZP treatment induced GABA-benzodiazepine uncoupling in brain cortex of C rats, but not in D animals, as assessed by chloride uptake in microsacs. Chronic PTB treatment (7.5 or 30 mg/kg/day i.p. for 15 days) did not modify GABA stimulation or GABA-PTB interaction in cortical microsacs of C or D rats.     

Time course for development of anticonvulsant tolerance and GABAergic subsensitivity after chronic diazepam

The time courses for development of neuronal and behavioral tolerance to diazepam (DZ) were estimated in rats continuously exposed to low levels of DZ for 3, 7, 14 or 21 days. Microiontophoretic sensitivity of dorsal raphe neurons to gamma-aminobutyric acid (GABA) was initially facilitated after short-term exposure to DZ released from implanted capsules for up to 3 days but returned to control levels by 7 days postimplantation and continued to decrease thereafter. GABAergic sensitivity remained depressed for a minimum of 5 days following removal of DZ capsules. To obtain a behavioral measure of tolerance, the anticonvulsant activity of DZ against bicuculline-induced seizures was also assessed. Rats studied 3 days after capsule implantation showed a significant elevation in seizure threshold. Seizure liability returned to control levels ca. 7 days after chronic treatment was initiated. These results indicate that tolerance to anticonvulsant efficacy against bicuculline seizures are temporally related to the onset of reduced GABA sensitivity on dorsal raphe neurons during prolonged exposure to DZ.     

Clorazepate in dogs: tolerance to the anticonvulsant effect and signs of physical dependence

Dogs were treated with clorazepate, which is known to be completely metabolized to desmethyldiazepam. 2 mg/kg t.i.d. were given orally for 5-6 weeks, a dose regimen providing plasma concentrations of desmethyldiazepam in the range known to be therapeutic in man. The rate of development of tolerance to the anticonvulsant effect was followed by weekly determinations of the convulsive threshold for pentetrazole before, during and after cessation of treatment. The development of tolerance was not as clear and pronounced as that found after treatment with diazepam and clonazepam in earlier studies with dogs. The seizure threshold was elevated by a factor of 1.2-3.5 during the first 2 weeks of treatment; during the following weeks, tolerance developed in only 2 out of 6 dogs (decline of the pentetrazole threshold in spite of rising or unchanged plasma concentrations). 36 h after withdrawal of clorazepate, the convulsive threshold had fallen below the control values in all dogs, but 1 week later it had returned to the control level. One day after cessation of treatment, 2 out of 6 dogs showed withdrawal seizures, which, in 1 case, were lethal. This shows that severe withdrawal symptoms, even lethal seizures, may appear after abrupt discontinuation of chronic clorazepate treatment, in spite of the relatively low tolerance liability of clorazepate.     

Modification of amygdala-kindled postictal inhibition by pentylenetetrazol and diazepam

When fully amygdala-kindled rats are electrically stimulated in grouped trials with intertrial stimulation intervals of less than 60 min, significant residual inhibition can be demonstrated. When these grouped trials of stimulation are repeated daily, additional cumulative inhibition is seen. The present study examined the effect of daily pretreatment with three doses of pentylenetetrazol (5, 10, and 20 mg/kg) and two doses of diazepam (0.5 and 2 mg/kg) on daily, grouped trial electrical stimulations of fully amygdala-kindled rats. Little or no reduction was seen in postictal inhibition by pentylenetetrazol pretreatment including the highest dose tested in which prestimulation bursts of spiking were associated with short episodes of forelimb clonus. Pretreatment with the benzodiazepine receptor agonist, diazepam, resulted in a dose-dependent reduction in the first elicited seizure response each day compared with control trials. Subsequent daily seizure trials of diazepam-treated animals demonstrated a relatively constant degree of dose-dependent seizure suppression without evidence of further postictal inhibition. The neural substrate that governs grouped trial postictal inhibition of amygdala-kindled seizures appears to be resistent to modification by near-convulsant doses of pentylenetetrazol, an agent with a presumed GABA-mediated mechanism of action. Diazepam, an anticonvulsant with a presumed GABA-related or associated mechanism of action, suppressed seizures in a dose-dependent manner nearly equally across all trials.     

Dizocilpine does not prevent the development of tolerance to the anxiolytic effects of diazepam in rats

The non-competitive NMDA receptor antagonist, dizocilpine (0.25 mg/kg), has previously been shown to block the development of tolerance to the sedative effects of diazepam (2 mg/kg). Since there is considerable evidence that different mechanisms underlie the development of tolerance to the sedative and anxiolytic effects of diazepam, the present experiment examined whether dizocilpine would block the development of tolerance to diazepam's anxiolytic effects. Rats tested after an acute dose of diazepam (2 mg/kg) showed an anxiolytic effect, measured by an increase in the time spent in social interaction, whereas those tested after 21 days of treatment had scores equal to the control group. This development of tolerance was not blocked by concomitant administration of dizocilpine (0.25 mg/kg). Thus, these results provide further evidence that the mechanism underlying tolerance to the anxiolytic effects of diazepam is different from that underlying tolerance to the sedative effects.     

Development of long-term subsensitivity to GABA in dorsal raphe neurons of amygdala-kindled rats.

Previously we reported a long-term change in neuronal sensitivity to GABA following amygdala kindling. Dorsal raphe neurons of amygdala-kindled rats exhibited significant subsensitivity to GABA 4 weeks after the last fully generalized (Stage 5) seizure. We hypothesized that this alteration in GABA sensitivity might reflect neuronal changes corresponding to kindled seizure susceptibility and subsequent experiments have investigated this hypothesis. The progression towards neuronal subsensitivity to GABA during amygdala kindling can be correlated with the Stage to which an animal has been kindled. That is, when measured 4 weeks after the last kindled seizure, dorsal raphe neurons are supersensitive to GABA following a Stage 2 seizure, not different from controls following a Stage 3 seizure and subsensitive to GABA following a Stage 5 seizure. In addition, subsensitivity to GABA appears to be permanent in that it is still measurable 3 months after the last Stage 5 seizure. Thus, amygdala kindling produces long-term, perhaps permanent, changes in neuronal sensitivity to GABA and these changes reflect the Stage to which an animal has been kindled.     

Decreased reactivity to the anticonflict effect of diazepam in perinatally undernourished rats

Offspring of rats were submitted to a protein deprivation dietary treatment from the third week of gestation until 50 days of age, and later nutritionally rehabilitated for at least 90 days. In the punished lever-pressing conflict test, undernourished animals exhibited a decreased reactivity to the anticonflict effect of a 3 mg/kg dose of diazepam as compared to control animals. This decreased reactivity to diazepam in a shock-induced conflict test indicates that functional alterations in the GABAergic transmission might contribute to the state of hypersensitivity to stressful or aversive situations present in undernourished animals.     

Locomotor and convulsive responses to picrotoxin in amygdala-kindled rats

Amygdala-kindled rats and handled controls were tested for differences in locomotion and convulsive activity produced by picrotoxin, a specific synaptic antagonist of the putative neurotransmitter GABA. Compared with controls, the gross activity of kindled rats decreased more rapidly with dose (0.5 to 2.0 mg/kg, i.p.) and kindled rats were more likely to convulse with the higher doses (1.0 to 2.0 mg/kg). These results suggest a functional deficit in the GABA system of kindled rats as measured by locomotion and convulsive activity.     

Monoamine turnover in the brain of mice during development of tolerance to the anticonvulsant effect of clonazepam

Mice were treated for 14 days with clonazepam, 0.5 mg/kg i.p. twice daily, during which time partial tolerance to the anticonvulsant effect against pentetrazole developed. The development of tolerance was paralleled by a reduced turnover of noradrenaline in the whole brain, and of dopamine in the midbrain. The turnover of 5-HT was increased during the first week of treatment, but decreased thereafter. These changes in monoamine turnover, which are thought to be GABA-mediated, are consistent with an increased seizure susceptibility, and may contribute to the development of tolerance to the anticonvulsant effect of benzodiazepines.     

Glycine potentiates diazepam anticonvulsant activity in electroshock seizures of rats: possible sites of interaction in the brainstem

The effect of orally or intracerebrally injected glycine on the anticonvulsant actions of intraperitoneal diazepam was examined using a tonic-clonic electroshock seizure model in the rat. Orally administered glycine (1.125 g/kg) potentiated the anticonvulsant effect of diazepam (DZP) to convert tonic-clonic electroshock seizures to less severe subthreshold clonic seizures. Oral glycine by itself had no effect on the tonic-clonic seizure response. Bilateral substantia nigra (SN) microinjections of glycine (125 micrograms/site) failed to potentiate intraperitoneal DZP when compared with the most appropriate control, animals treated with DZP and intranigral saline. It was not possible to determine whether bilateral glycine microinjections into the inferior olivary nucleus (IO) potentiated anticonvulsant effects of DZP since glycine alone converted all tonic-clonic seizures to the clonic response. Finally, bilateral glycine microinjection alone into the nucleus reticularis pontis oralis (PNO) produced an anticonvulsant effect when compared to untreated control responses but did not potentiate the anticonvulsant actions of DZP. Although these results may indicate that the glycinergic potentiation of DZP involves a direct pharmacodynamic interaction between these two compounds at specific brain sites, these sites have not yet been demonstrated conclusively.     

Mechanism of imipramine-induced seizures in amygdala-kindled rats

The present study was undertaken to get insight in the possible mechanisms of imipramine-induced seizures in amygdala-kindled rats. The intraperitoneal (i.p.) injection of imipramine produced potent behavioral and electroencephalogram (EEG) seizures in amygdala-kindled rats. Histidine (1500 mg/kg, i.p.) and histamine (10 and 20 microg, i.c.v.) significantly attenuated the seizures induced by imipramine (50 mg/kg, i.p.) in kindled rats. In addition, the inhibition of imipramine-induced seizures by histamine (20 microg, i.c.v.) was antagonized by an H1 antagonist, pyrilamine. An H3 antagonist, thioperamide (50 microg, i.c.v.), also significantly attenuated the imipramine-induced seizures in kindled rats. The i.p. injection of alpha-methyl-p-tyrosine at a dose of 250 mg/kg significantly diminished the seizures induced by imipramine. However, p-chlorophenylalanine and physostigmine did not affect the imipramine-induced seizures to any extent. These data give strong hints that the H1 receptor antagonistic properties and the brain noradrenaline activating effects of imipramine are centrally involved in imipramine-induced seizures, and central serotonergic and cholinergic neurotransmissions are not involved in the seizures induced by imipramine in amygdala-kindled rats.     

Lamotrigine treatment during amygdala-kindled seizure development fails to inhibit seizures and diminishes subsequent anticonvulsant efficacy

PURPOSE: Lamotrigine (LTG) is an anticonvulsant that is currently in use for the treatment of various seizure disorders and that shows promise in the treatment of affective illness. LTG is also effective in the suppression of amygdala-kindled seizures. Because many drugs show a differential efficacy profile as a function of the phase of kindling evolution, we evaluated LTG for its potential antiepileptogenic effects on the development of amygdala-kindled seizures. METHODS: In two separate studies, LTG (5 or 15 mg/kg versus vehicle) was administered before each daily amygdala stimulation (biphasic square wave pulses, 100 pulse pairs per second for a total of 0.5 second, 1-millisecond pulse width) at an intensity of 50 microA over the AD threshold. Seizure development was assessed, as well as the effect of this pretreatment on subsequent efficacy of LTG on completed kindled seizures. RESULTS: LTG at 5 mg/kg failed to block seizure development. At 15 mg/kg, LTG paradoxically enhanced seizure development and produced running fits in four of the nine animals tested. Animals previously treated with either dose of LTG during kindling development showed a diminished response to the anticonvulsant effects of LTG on fully kindled seizures compared with the vehicle-treated controls. CONCLUSIONS: Although LTG possesses potent anticonvulsant effects on completed amygdala-kindled seizures, it is either without effect (5 mg/kg) or facilitates (15 mg/kg) the initial phase of kindling development. In addition, exposure to LTG during kindled seizure development leads to a reduced subsequent response to the drug in fully kindled animals. These observations parallel those with carbamazepine and suggest that different stages of kindling (epileptogenesis versus fully manifest seizures) may have different underlying neural mechanisms that require distinct pharmacotherapies.     

Is the behavioural effect of diazepam in rats unique to negative secondary stimuli?

Summary Studies of the behavioural effects of benzodiazepines have focused in particular on situations with negative secondary stimuli, i.e., stimuli signalling negative primary events such as punishment or non-reward. The general result is that benzodiazepines attenuate behavioural reactions to this type of stimuli. The aim of the present study was to investigate if there are any differences between positive and negative secondary stimuli in this respect. Rats were treated with diazepam in a modified Skinner box with two levers. One of the levers always gave a small reward. A lamp being ON or OFF was used as a secondary stimulus indicating if the other lever would give a large or no reward. Pretreatment with diazepam (1.0 mg/kg) did not act differently on the response to the positive or negative secondary stimulus. The main effect was a general attenuation of optimal responding with a concomitant decrease in water intake. An alternative hypothesis stating that benzodiazepines alter the reactivity to secondary stimuli more in general is supported.