Anticonvulsant and antiepileptogenic effects of fluorofelbamate in experimental status epilepticus.

PURPOSE: To examine the seizure-protective properties of fluorofelbamate, a felbamate analog, on acute and chronic seizures in an experimental model of self-sustaining status epilepticus (SSSE). METHODS: SSSE was induced by stimulation of the perforant path for 30 min (PPS) through chronically implanted electrodes in free-running adult male Wistar rats. Fluorofelbamate was injected intravenously (i.v.) either 10 min, or 40 min after SSSE induction. Seizure and spike profiles were analyzed off-line. RESULTS: Fluorofelbamate injected during the early stages of SSSE (10 min after the end of PPS), shortened the duration of seizures in a dose-dependent manner. While a dose of 50 mg kg(-1) was ineffective, 100 and 200 mg kg(-1) reduced cumulative seizure time from 393 +/- 10 min to 15 +/- 8 min and 2.4 +/- 0.5 min respectively. Administration of fluorofelbamate (200 and 300 mg kg (-1)) at a late stage of SSSE, which is refractory to treatment with conventional anticonvulsants, also significantly attenuated seizures. Acute fluorofelbamate treatment (200 mg kg(-1) 10 min after PPS) significantly decreased the frequency of spontaneous seizures which follow SSSE after a 'latent' interval. Moreover, in contrast to control animals, fluorofelbamate-treated rats showed regression of spontaneous seizures, and an apparent remission of epilepsy within 2 months after SSSE. CONCLUSIONS: Acute treatment of SSSE with fluorofelbamate showed strong anticonvulsant effects even during the late stages of SSSE. In this model, it also displayed antiepileptogenic properties: it reduced the severity of chronic epilepsy after SSSE and lead to apparent remissions of that epilepsy.     

Anticonvulsant effects of agomelatine in mice

Agomelatine is a potent MT1 and MT2 melatonin receptor agonist and a 5-HT2C serotonin receptor antagonist. We analyzed whether agomelatine has anticonvulsant properties. The anticonvulsant activity of agomelatine (25, 50 or 75 mg/kg, i.p.) was evaluated in mouse models of pentylenetetrazole (PTZ-85 mg/kg, i.p.), pilocarpine (400mg/kg, i.p.), picrotoxin (7 mg/kg, i.p.), strychnine (75 mg/kg, i.p.) or electroshock-induced convulsions. In the PTZ-induced seizure model, agomelatine (at 25 or 50mg/kg) showed a significant increase in latency to convulsion, and agomelatine (at 50 or 75 mg/kg) also increased significantly time until death. In the pilocarpine-induced seizure model, only agomelatine in high doses (75 mg/kg) showed a significant increase in latency to convulsions and in time until death. In the strychnine-, electroshock- and picrotoxin-induced seizure models, agomelatine caused no significant alterations in latency to convulsions and in time until death when compared to controls. Our results suggest that agomelatine has anticonvulsant activity shown in PTZ- or pilocarpine-induced seizure models.     

Biochemical and behavioral effects of steroids on GABAA receptor function in long- and short-sleep mice.

The in vitro and in vivo effects of alphaxalone, a steroid anesthetic, and two physiological steroids, tetrahydrodeoxycorticosterone (THDOC) and pregnenolone sulfate (PS), on GABAA receptor function were evaluated in long-sleep (LS) and short-sleep (SS) mice. In vitro, both alphaxalone and THDOC enhanced GABAergic inhibition as measured by [3H]FNZ binding and GABA-stimulated 36Cl- flux. However, with the exception of alphaxalone potentiation of [3H]FNZ binding, which was greater in SS brain regions, LS and SS mice did not differ in their degree of enhancement. Pregnenolone sulfate produced mixed agonistic and antagonistic effects on GABAergic function, dependent upon brain region, with few differences between the lines of mice. In vivo effects of these steroids on sleep time indicated that, like other anesthetic agents, THDOC and alphaxalone induced longer sleep times in LS mice. Antagonism by PS of ethanol-induced sleep time was observed in LS mice only; however, this effect was dependent upon the dose of ethanol used and on the vehicle used to prepare the steroid. Pentobarbital-induced sleep time was not reduced by PS treatment in either line of mouse. These results demonstrate that few differences in sensitivity of the GABAergic receptor to these steroids exist between LS and SS mice. Thus, unlike the differences between LS and SS mice in GABAergic mediation of responses to ethanol and benzodiazepines, there is little genetic variability in subtypes of GABAA receptors capable of modulation by steroids in these lines of mice.     

Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes

Chick brain mRNA was isolated and injected into Xenopus oocytes. This led to the expression in the surface membrane of functional GABA-activated channels with properties reminiscent of vertebrate GABAA channels. The GABA-induced current was analyzed quantitatively under voltage-clamp conditions. Picrotoxin inhibited this current in a concentration-dependent manner with IC50 = 0.6 microM. The allosteric modulation of GABA currents by a number of drugs acting at the benzodiazepine binding site was characterized quantitatively. In the presence of the benzodiazepine receptor ligands diazepam and clorazepate, GABA responses were enhanced, and in the presence of the convulsant beta-carboline compound methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), they were depressed. Maximal stimulation of the response elicited by 10 microM GABA was 160% with diazepam and 90% with clorazepate, and maximal inhibition was 42% with DMCM, 30% with methyl beta-carboline-3-carboxylate (beta-CCM), 15% with ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate (Ro 15-1788), and 12% with ethyl beta-carboline-3-carboxylate (beta-CCE). Half-maximal stimulation was observed with 20 nM diazepam and 390 nM clorazepate, respectively, and half-maximal inhibition with 6 nM DMCM. beta-CCM had a similar effect to DMCM, whereas beta-CCE and Ro 15-1788 showed only small inhibition at low concentrations (less than 1 microM). All the tested carboline compounds and Ro 15-1788 showed a biphasic action and stimulated GABA current at concentrations higher than 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebellar GABAA and benzodiazepine receptor characteristics in young and aged mice

Age-related differences in GABAA and benzodiazepine receptors were investigated in cerebella of young (1-month-old), mature adult (3 months old), older (8 months old) and aged (20 months old) mice. In cerebellar membranes of aged mice, [3H]muscimol binding was significantly higher as compared to those from all three younger age groups. Binding was the same in these younger age groups. Scatchard analysis of binding isotherms in cerebellum of young and aged mice showed the presence of two components (with different affinities and binding capacities). In aged mice, a significantly lower KD of low affinity sites and a significantly higher Bmax of high affinity sites were noted as compared to those in young mice. However, [3H]flunitrazepam binding to benzodiazepine receptors in cerebellar membranes was the same in both age groups. These results suggested that GABAA receptor binding was increased during senescence in cerebella without altering benzodiazepine binding.     

Adenosine receptor ligands and dizocilpine-induced antinociception in mice

Interactions between adenosine receptor ligands and dizocilpine (uncompetitive NMDA receptor antagonist) was studied in antinociceptive, writhing test in mice. Minimal effective, antinociceptive doses of adenosine receptor agonists were: 0.1 mg/kg (NECA--A1/A2 agonist). Generally, these agonists did not potentiate the subthreshold dose of dizocilpine (0.05 mg/kg). Of all adenosine receptor antagonists used, only caffeine (A2 and A2 antagonists) reversed dizocilpine-induced (0.1 mg/kg) antinociception dose-dependently. These findings indicate that dizocilpin-induced antinociception in the writhing test is only partly influenced by adenosine receptor ligands.     

Anticonvulsant effects of acute and repeated fluoxetine treatment in unstressed and stressed mice

Comorbidity of epilepsy and depression is not rare. Stress can affect both depression and seizures. Therefore, it is important to know whether an antidepressant drug has pro- or anticonvulsant properties and whether these properties will be modified by stress. We tested the effects of the antidepressant drug fluoxetine on the seizure threshold for picrotoxin in unstressed and swim-stressed mice. The mice were, prior to exposure to swim stress and the intravenous infusion of picrotoxin (a non-competitive GABA(A) receptor antagonist), pretreated with fluoxetine (a selective serotonin reuptake inhibitor), either acutely or repeatedly (5 days), and the latency to the onset of two convulsant signs and death was registered. The convulsant signs were running/bouncing clonus and tonic hindlimb extension. As expected, swim stress enhanced the seizure threshold for picrotoxin. Fluoxetine (20 mg/kg ip) given acutely increased in unstressed and swim-stressed mice the dose of picrotoxin producing tonic hindlimb extension and in unstressed mice the dose of picrotoxin producing death. Neither 10 nor 20 mg/kg of fluoxetine affected doses of picrotoxin needed to produce running bouncing/clonus. Repeated treatment with fluoxetine (20 mg/kg ip) enhanced significantly in unstressed and swim-stressed mice doses of picrotoxin needed to produce tonic hindlimb extension and death, and in stressed mice also the dose of picrotoxin producing running/bouncing clonus. The results demonstrate that the antidepressant drug fluoxetine, given acutely or repeatedly, shows anticonvulsant properties against convulsions induced in unstressed and swim-stressed mice by antagonist of GABA(A) receptors, picrotoxin. Swim stress failed to modify the anticonvulsant properties of fluoxetine.     

Adrenalectomy and stress modulate GABAA receptor function in LS and SS mice.

The effects of manipulation of adrenal steroids by adrenalectomy (ADX) or stress on GABAA receptor function were characterized in long-sleep (LS) and short-sleep (SS) mice. 36Chloride flux was not altered in either line of mouse after ADX; however, exposure to a behavioral stressor resulted in a highly significant inhibition of ion channel activity measured in cortical membranes from both LS and SS mice. Adrenalectomy also had no effect on [3H]FNZ binding; whereas exposure to stress differentially altered benzodiazepine binding in LS and SS mice. In LS cortex both Bmax and Kd values increased, whereas in SS cerebellum, Bmax and Kd values were decreased after stress. In SS mice ADX did not affect GABA-enhancement of [3H]FNZ binding. In LS mice, however, ADX resulted in a potentiation of GABA-enhanced [3H]FNZ binding in cortex and an inhibition of enhancement in cerebellum. Corticosterone (CCS) replacement in ADX-LS mice returned enhancement values to those of sham-operated mice, indicating a role for basal levels of CCS in maintaining normal receptor coupling function in this line of mouse. These results suggest that GABAA receptor sensitivity is more labile under stressful conditions. Differential receptor responses to adrenal manipulation between LS and SS mice may be due to genetic variation in GABAA receptor subunit combinations in these lines of mice.     

Anticonvulsive and antiepileptogenic effects of levetiracetam in the audiogenic kindling model

Purpose: To study anticonvulsive and antiepileptogenic effects of singe levetiracetam (LEV) administration in the model of audiogenic kindling.
Methods: Rats of Krushinsky-Molodkina (KM) strain genetically susceptible to severe audiogenic seizures received one intraperitoneal injection of saline, low (6 mg/kg) or high (50 mg/kg) dose of LEV before or after audiogenic kindling. One hour postinjection, an audiogenic seizure was induced to assess anticonvulsive effect of LEV in nonkindled and kindled rats. To examine antiepileptogenic activity of LEV, nonkindled rats injected with the drug or saline were kindled with repeated sound stimulations. Audiogenic kindling development manifested in an appearance and progressive prolongation of an additional seizure phase, post-tonic–clonus. The latency and duration of audiogenic seizures and the duration of every seizure phase (running, tonic, post-tonic–clonic) were measured.
Results: One hour posttreatment, LEV dose-dependently lengthened the latency and reduced the duration of audiogenic seizures in both nonkindled and kindled rats. The seizure shortening resulted from selective suppression of tonic and kindled post-tonic–clonic phases. The dose of 50 mg/kg completely blocked tonic and clonic convulsions 1 h postinjection. The anticonvulsive effect of LEV was more pronounced in kindled than in nonkindled rats. Single LEV injection in the dose of 50 mg/kg prior audiogenic kindling significantly suppressed subsequent kindling progression indicating profound antiepileptogenic potency of the drug.
Conclusions: The present study shows that LEV exerts both short-lasting anticonvulsive effect on audiogenic seizures and very long-lasting antiepileptogenic effect on audiogenic kindling. Remarkably, a single injection of LEV is enough to significantly suppress kindling progression in KM rats.     

Anticonvulsant effects of 1,25-dihydroxyvitamin D in chemically induced seizures in mice

Here, we study the role of a neurosteroid hormone Vitamin D in epilepsy. To examine this problem, we used 1,25-dihydroxyvitamin D, an active form of Vitamin D, injected subcutaneously to NMRI mice (33 microg/20 microl) 40 min prior to seizures induced by systemic injection of pentylenenetrazole (PTZ, 70 mg/kg). Overall, compared to the vehicle-treated control animals (n=11 in each group), the Vitamin D-treated mice demonstrated reduced severity of PTZ-induced seizures (longer latency, shorter duration and lower mortality). In a separate experiment, we assessed the time-course of antiepileptic effects of 1,25-dihydroxyvitamin D. For this, we injected this compound (33 microg/20 microl) to NMRIx129S1 mice (n=11) 40 min, 3, 6, 12 and 24 h prior to seizures, showing that antiepileptic effects were short-term, almost disappearing 3h after administration. Our findings show that Vitamin D plays a direct anticonvulsant role in the brain and suggest that the Vitamin D endocrine system may represent a new target for the development of anticonvulsant drugs.     

Cannabinoid receptor ligands suppress memory-related effects of nicotine in the elevated plus maze test in mice

The purpose of the experiments was to examine the memory-related effects of nicotine using the mouse elevated plus maze. It has been shown that the acute doses of nicotine (0.1 and 0.5 mg/kg) significantly decreased the time of transfer latency (TL2) on the retention trial, indicating that nicotine improved memory processes. Similarly, acute doses of the CB1 cannabinoid receptor antagonist AM 251 (0.5, 1, 1.5 and 3 mg/kg) significantly decreased TL2 values. WIN55,212-2, a non-selective CB cannabinoid receptor agonist, at any dose tested (0.25, 0.5 and 1 mg/kg), did not provoke any effect in this model. Moreover, the acute injection of both WIN55,212-2 (0.25 and 0.5 mg/kg) and AM 251 (0.25 mg/kg), prior to injections of nicotine (0.1 and 0.5 mg/kg), significantly prevented nicotine-induced memory improvement. The results of this study provide clear evidence that the endogenous cannabinoid system participates in the responses induced by nicotine on memory-related behaviour in mice.     

Anticonvulsant effect of a ghrelin receptor agonist in 6Hz corneally kindled mice

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant‐induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild‐type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild‐type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV‐1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild‐type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV‐2959 in both models. This finding indicates that JMV‐1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.     

Differential effects of novel ligands for 5-HT receptor subtypes on nonopioid defensive analgesia in male mice

The effects of a number of 5-HT receptor ligands were examined on nonopioid defensive analgesia in male DBA/2 mice. MDL 73005EF (0.05-1.0 mg/kg), a selective 5-HT1A receptor agonist, potently and dose-dependently inhibited the analgesic consequences of social defeat. CGS 12066B (0.5-10.0 mg/kg) and MK-212 (0.3-10.0 mg/kg), selective agonists for 5-HT1B and 5-HT1C sites, respectively, failed to influence this particular form of adaptive pain inhibition. Two 5-HT2/1C receptor antagonists, ritanserin (0.05-10.0 mg/kg) and ICI 169.369 (0.3-10.0 mg/kg), were also devoid of specific effects upon defensive analgesia. Both ritanserin and ICI 169,369 were found to have intrinsic analgetic efficacy and to induce behavioural changes indicative of increased defensiveness. These data, together with previous findings, confirm the specific involvement of 5-HT1A receptor mechanisms in the analgesic consequences of social defeat in male mice. Results are discussed in relation to the role of anxiety in adaptive pain inhibition.     

Differential expression of two forms of GABAA receptor gamma 2-subunit in mice.

The gamma-aminobutyric acid type A (GABAA) receptor gamma 2-subunit is important for benzodiazepine action. In previous studies, two forms of gamma 2-subunit have been found which are generated by RNA alternative splicing (gamma 2L and gamma 2S) and differ by the presence or absence of an 8-amino acid insertion in the major intracellular loop between proposed transmembrane domains M3 and M4. We have used the RNase protection assay to study regulation of expression of the two forms of gamma 2-subunit in mouse brain, as judged from mRNA levels. We find that gamma 2L and gamma 2S are differentially expressed in brain regions and that gamma 2S is expressed at a fairly constant level during brain development while gamma 2L increases dramatically with maturation. Their differentially regulated expression suggests further that gamma 2L and gamma 2S form receptors with important functional differences.     

Anticonvulsant activity of the cyclooxygenase-2 (COX-2) inhibitor etoricoxib in pentylenetetrazole-kindled rats is associated with memory impairment

PurposeVarious selective and nonselective cyclooxygenase (COX) inhibitors are known to have effects on development and progression of seizures. In the present study, the effect of the selective COX-2 inhibitor etoricoxib on seizures, oxidative stress, and learning and memory was studied.MethodMale Wistar rats were kindled using subconvulsant dose of pentylenetetrazole (PTZ) (30 mg/kg, i.p.), on alternating days until animals were fully kindled. After a one-week PTZ-free period, kindled rats were challenged with PTZ 30 mg/kg, and the latency, duration, and severity of seizures were recorded. Etoricoxib was then administered intraperitoneally at 1 mg/kg and 10 mg/kg in kindled rats for nine days (days 6–14). On the ninth day of etoricoxib treatment, PTZ challenge (30 mg/kg) was given, and seizure parameters were noted. On day 15, behavioral assessment was carried out. The Morris water maze (MWM) apparatus and the passive avoidance (PA) apparatus were used for studying cognitive impairment. The rats were then sacrificed, and malondialdehyde (MDA) and glutathione (GSH), markers of oxidative stress, were estimated in the brain samples.ResultsEtoricoxib at lower dose (1 mg/kg) had an anticonvulsant effect which was reduced or reversed at higher dose (10 mg/kg). Etoricoxib also impaired the learning and memory in rats as tested by passive avoidance and Morris water maze tests.ConclusionThe results of the present study suggest that use of etoricoxib, especially at low dose, in patients with epilepsy may not be detrimental with regard to seizure control. However, attention should be paid to cognitive parameters.     

Autoradiography of opioid and ORL1 ligands in opioid receptor triple knockout mice

Three genes for the opioid receptors ( micro, delta and kappa) and a gene coding for a related receptor (ORL1) have been cloned but pharmacological studies suggest that further subtypes exist that remain poorly understood. To determine if there are other classically defined opioid binding sites we have carried out homogenate binding and section autoradiography with [3H]naloxone in mice that lack all three opioid genes and are hyperalgesic in a thermal nociceptive test. We have also examined [3H]bremazocine labelling in triple knockout brain and spinal cord as this ligand has been proposed to label novel kappa-receptors. No receptor labelling for either ligand was detected in the brains or spinal cord of knockout mice demonstrating that all binding is the product of the three known receptors and that there is no cross-labelling of the ORL1 receptor. Nociceptin (1 micro m) caused marked displacement of [3H]bremazocine in wild-type brains indicating that nociceptin at high concentrations can displace classical opioid binding. As a number of studies have proposed a close association between the classical opioid receptors and the ORL1 system we also hypothesized that loss of all of the classical opioid receptors might lead to compensatory changes in ORL1 receptors. Labelling of the ORL1 receptor with [3H]nociceptin showed region-dependent quantitative increases in triple knockout brains indicating a close relationship between the two systems in specific brain areas.     

Heterogeneity of GABAA receptor in goldfish retina

The possibility of GABA_A receptor heterogeneity in goldfish retina was studied with immunocytochemical and biochemical approaches: (1) immunoblotted membrane particulates of goldfish retina with mAb 62-3G1; (2) immunoprecipitation of the detergent-solubilized membrane proteins with mAb 62-3G1 followed by the receptor binding assay; (3) photoaffinity labeling of the membrane particulates with ³H-flunitrazepam (FNZ) and visualization of the labeled receptors by SDS-PAGE and fluorography; (4) dry autoradiography of ³H-muscimol and ³H-FNZ binding sites on frozen sections. Immunoblots showed that 62-3G1 reacted with 55–57.5 kDa Mr polypeptides, similar to the muscimol-binding subunit of the receptor complex in bovine brain; while ³H-FNZ photoaffinity labeled the 52.5 kDa and 41–43 kDa Mr polypeptides. Immunoprecipitated receptors bound only ³H-muscimol, not ³H-FNZ. An attempt to precipitate the ³H-FNZ photolabeled polypeptides failed. Dry autoradiography showed ³H-PNZ binding only in the inner plexiform layer (IPL); the binding was enhanced with γ-aminobutyric acid (GABA) and blocked by clonazepam. In contrast, ³H-muscimol was bound in both the outer plexiform layer (OPL) and IPL, similar to that observed with 62-3G1 immunocytochemistry. We suggest that there are two subtypes of GABA_A receptor in the goldfish retina: (1) GABA_A receptors that are not linked to a benzodiazepine (BZD) receptor are located in the OPL and at amacrine-to-amacrine and amacrine-to-ganglion cell synapses in the IPL and are recognized by 62-3G1; (2) GABA_A receptors that are linked to a BZD receptor are located only in the IPL, largely at amacrine-to-bipolar cell synapses and are not recognized by mAb 62-3G1. © 1994 Wiley-Liss, Inc.