Audiogenic Seizure-susceptible Mice Research Articles

The riluzole derivative 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a mixed KCa2 activator and NaV blocker, is a potent novel anticonvulsant.

Inhibitors of voltage-gated sodium channels (Na(v)) have been used as anticonvulsants since the 1940s, while potassium channel activators have only been investigated more recently. We here describe the discovery of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a thioanalog of riluzole, as a potent, novel anticonvulsant, which combines the two mechanisms. SKA-19 is a use-dependent NaV channel blocker and an activator of small-conductance Ca(2+)-activated K(+) channels. SKA-19 reduces action potential firing and increases medium afterhyperpolarization in CA1 pyramidal neurons in hippocampal slices. SKA-19 is orally bioavailable and shows activity in a broad range of rodent seizure models. SKA-19 protects against maximal electroshock-induced seizures in both rats (ED50 1.6 mg/kg i.p.; 2.3 mg/kg p.o.) and mice (ED50 4.3 mg/kg p.o.), and is also effective in the 6-Hz model in mice (ED50 12.2 mg/kg), Frings audiogenic seizure-susceptible mice (ED50 2.2 mg/kg), and the hippocampal kindled rat model of complex partial seizures (ED50 5.5 mg/kg). Toxicity tests for abnormal neurological status revealed a therapeutic index (TD50/ED50) of 6-9 following intraperitoneal and of 33 following oral administration. SKA-19 further reduced acute pain in the formalin pain model and raised allodynic threshold in a sciatic nerve ligation model. The anticonvulsant profile of SKA-19 is comparable to riluzole, which similarly affects Na(V) and KCa2 channels, except that SKA-19 has a ~4-fold greater duration of action owing to more prolonged brain levels. Based on these findings we propose that compounds combining KCa2 channel-activating and Na(v) channel-blocking activity exert broad-spectrum anticonvulsant and analgesic effects.

Profile of the new pyrrolidone derivative seletracetam (ucb 44212) in animal models of epilepsy

Seletracetam is a pyrrolidone derivative with a one-log-unit higher affinity for the synaptic vesicle protein 2A (SV2A) than levetiracetam (Keppra®). This study explored its anticonvulsant properties in animal models of epilepsy. Seletracetam reduced both the amplitude and repetitive firing of population spikes induced by a high K+/low Ca2+ concentration fluid (HKLCF) in rat hippocampal slices. The reduction of HKLCF-induced increases in population spike amplitude was particularly pronounced, and occurred at ~10 times lower seletracetam concentrations than previously observed for levetiracetam. These invitro data suggest that desynchronisation of epileptiform activity may contribute significantly to the antiepileptic properties of seletracetam. Seletracetam also showed a potent anti-seizure activity in animal models mimicking partial-onset (kindled animals) and generalized epilepsy (audiogenic seizure susceptible mice and genetic absence epilepsy rats from Strasbourg (GAERS)). In amygdala-kindled rats, seletracetam increased the generalized seizure threshold current and decreased the duration of the after-discharge and the seizure severity observed at the after-discharge threshold current, and generally had a much more potent effect than previously observed for levetiracetam. Seletracetam showed no psychomimetic effects and a very high central nervous system (CNS) tolerability in both kindled and GAERS rats, markedly superior to that of levetiracetam and other antiepileptic drugs. These results suggest that seletracetam may represent an effective and very well tolerated broad-spectrum agent for the symptomatic treatment of epilepsy.

Synaptic and extrasynaptic GABA transporters as targets for anti‐epileptic drugs

Inhibition of the GABA transporter subtype GAT1 by the clinically available anti-epileptic drug tiagabine has proven to be an effective strategy for the treatment of some patients with partial seizures. In 2005, the investigational drug EF1502 was described as possessing activity at both GAT1 and BGT-1. When combined with the GAT1 selective inhibitor tiagabine, EF1502 was found to possess a synergistic anti-convulsant action in the Frings audiogenic seizure-susceptible mouse model of reflex epilepsy. This effect was subsequently attributed to inhibition of BGT-1. In this study, the anti-convulsant effect of the GAT2/3 inhibitor SNAP-5114 was assessed in the Frings audiogenic seizure-susceptible mouse alone, and in combination with tiagabine and EF1502. The results showed that SNAP-5114 produced a synergistic anti-convulsant effect in combination with EF1502 but not when used in combination with tiagabine. These findings support anatomical evidence that GAT2/3 are most likely located at the synapse in close proximity to GAT1; whereas BGT-1 is located some distance away from the synapse and GAT1 and GAT2/3. Lastly, EF1502 and tiagabine were evaluated alone, and in combination, in the corneal kindled mouse model of partial epilepsy. The results of this evaluation provide further evidence in support of a role for BGT-1 in the control of seizure activity. In addition, they suggest that the combined inhibition of GAT1 and BGT-1 may afford some advantage over inhibiting either transporter alone.

Developing Novel Antiepileptic Drugs: Characterization of NAX 5055, a Systemically-Active Galanin Analog, in Epilepsy Models

The endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs, NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active after intravenous, intraperitoneal, and subcutaneous administration, and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved antiepileptic drugs.

Anti‐convulsive and anti‐epileptic properties of brivaracetam (ucb 34714), a high‐affinity ligand for the synaptic vesicle protein, SV2A

Screening of 12,000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure-susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure-susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index.

Brivaracetam: a rational drug discovery success story

Levetiracetam, the alpha-ethyl analogue of the nootropic piracetam, is a widely used antiepileptic drug (AED) that provides protection against partial seizures and is also effective in the treatment of primary generalized seizure syndromes including juvenile myoclonic epilepsy. Levetiracetam was discovered in 1992 through screening in audiogenic seizure susceptible mice and, 3 years later, was reported to exhibit saturable, stereospecific binding in brain to a approximately 90 kDa protein, later identified as the ubiquitous synaptic vesicle glycoprotein SV2A. A large-scale screening effort to optimize binding affinity identified the 4-n-propyl analogue, brivaracetam, as having greater potency and a broadened spectrum of activity in animal seizure models. Recent phase II clinical trials demonstrating that brivaracetam is efficacious and well tolerated in the treatment of partial onset seizures have validated the strategy of the discovery programme. Brivaracetam is among the first clinically effective AEDs to be discovered by optimization of pharmacodynamic activity at a molecular target.

First Demonstration of a Functional Role for Central Nervous System Betaine/γ-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K(i) of 4 and 5 muM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.

Correlation analysis between anticonvulsant ED 50 values of antiepileptic drugs in mice and rats and their therapeutic doses and plasma levels

Objective Although there are several animal models of epilepsy, the extrapolation of antiepileptic drug (AEDs) performance to epileptic patients from anticonvulsant activity results in animals is not straightforward. Consequently, the aim of this work was to perform a correlation analysis between therapeutic daily doses ( D) and average steady-state plasma concentrations ( C ss,av) of AEDs and their activity in common anticonvulsant animal models. Methods AED activity in anticonvulsant animal models was expressed as maximal electroshock seizure (MES) test ED 50 values in mice and rats and ED 50 values in audiogenic seizure-susceptible mice (AGS ED 50). Data were examined, by use of linear and logarithmic approaches, for an association between C ss,av (mg/L or μmol/L) and D (mg or mmol) for each AED in epileptic patients as the dependent variable ( Y) and its MES ED 50 in mice and rats and AGS ED 50 in mice (mg/kg or μmol/kg) as the independent variable ( X). Results Linear correlation analyses between C ss,av (mg/L) and ED 50 (mg/kg) for 11 AEDs gave the following correlation coefficients ( R 2): 0.68 (mice, MES); 0.73 (rat, MES); 0.64 (AGS). Switching the units from milligrams to micromoles improved the correlation significantly and gave the following R 2 values: 0.88 (mice, MES); 0.90 (rat, MES); 0.76 (AGS). The linear correlation between C ss,av and ED 50 was better than that between D and ED 50. Conclusions The results of this analysis suggest that the relationship between C ss,av and ED 50 is useful in predicting target concentration ranges in humans. The Y intercepts of the C ss,av-versus-ED 50 and D-versus- ED 50 plots were similar in all three animal models and ranged between 12 and 17 mg/L and between 570 and 890 mg, respectively, indicating that for all AEDs analyzed except valproic acid and ethosuximide, the therapeutic plasma concentration is in the range 10–20 mg/L.

Anticonvulsant activity, teratogenicity and pharmacokinetics of novel valproyltaurinamide derivatives in mice.

1 The purpose of this study was to synthesize novel valproyltaurine (VTA) derivatives including valproyltaurinamide (VTD), N-methyl-valproyltaurinamide (M-VTD), N,N-dimethyl-valproyltaurinamide (DM-VTD) and N-isopropyl-valproyltaurinamide (I-VTD) and evaluate their structure-pharmacokinetic-pharmacodynamic relationships with respect to anticonvulsant activity and teratogenic potential. However, their hepatotoxic potential could not be evaluated. The metabolism and pharmacokinetics of these derivatives in mice were also studied. 2 VTA lacked anticonvulsant activity, but VTD, DM-VTD and I-VTD possessed anticonvulsant activity in the Frings audiogenic seizure susceptible mice (ED(50) values of 52, 134 and 126 mg kg(-1), respectively). 3 VTA did not have any adverse effect on the reproductive outcome in the Swiss Vancouver/Fnn mice following a single i.p. injection of 600 mg kg(-1) on gestational day (GD) 8.5. VTD (600 mg kg(-1) at GD 8.5) produced an increase in embryolethality, but unlike valproic acid, it did not induce congenital malformations. DM-VTD and I-VTD (600 mg kg(-1) at GD 8.5) produced a significant increase in the incidence of gross malformations. The incidence of birth defects increased when the length of the alkyl substituent or the degree of N-alkylation increased. 4 In mice, N-alkylated VTDs underwent metabolic N-dealkylation to VTD. DM-VTD was first biotransformed to M-VTD and subsequently to VTD. I-VTD's fraction metabolized to VTD was 29%. The observed metabolic pathways suggest that active metabolites may contribute to the anticonvulsant activity of the N-alkylated VTDs and reactive intermediates may be formed during their metabolism. In mice, VTD had five to 10 times lower clearance (CL), and three times longer half-life than I-VTD and DM-VTD, making it a more attractive compound than DM-VTD and I-VTD for further development. VTD's extent of brain penetration was only half that observed for the N-alkylated taurinamides suggesting that it has a higher intrinsic activity that DM-VTD and I-VTD. 5 In conclusion, from this series of compounds, although VTD caused embryolethality, this compound emerged as the most promising new antiepileptic drug, having a preclinical spectrum characterized by the highest anticonvulsant potential, lowest potential for teratogenicity and favorable pharmacokinetics.

Characterization of the anticonvulsant profile and enantioselective pharmacokinetics of the chiral valproylamide propylisopropyl acetamide in rodents

1. Propylisopropyl acetamide (PID) is a new chiral amide derivative of valproic acid. The purpose of this study was to evaluate the anticonvulsant activity of PID in rodent models of partial, secondarily generalized and sound-induced generalized seizures which focus on different methods of seizure induction, both acute stimuli, and following short-term plastic changes as a result of kindling, and to assess enantioselectivity and enantiomer-enantiomer interactions in the pharmacokinetics and pharmacodynamics of racemic PID and its pure enantiomers in rodents. 2. Anticonvulsant activity of (S)-PID, (R)-PID and racemic PID was evaluated in the 6 Hz psychomotor seizure model in mice, in the hippocampal kindled rat, and in the Frings audiogenic seizure susceptible mouse. The pharmacokinetics of (S)-PID and (R)-PID was studied in mice and rats. 3. In mice (S)-PID, (R)-PID and racemic PID were effective in preventing the 6 Hz seizures with (R)-PID being significantly (P < 0.05) more potent (ED(50) values 11 mg kg(-1), 46 mg kg(-1) and 57 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively) than (S)-PID (ED(50) values 20 mg kg(-1), 73 mg kg(-1) and 81 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively). (S)-PID, (R)-PID and racemic PID also blocked generalized seizures in the Frings mice (ED(50) values 16 mg kg(-1), 20 mg kg(-1) and 19 mg kg(-1) respectively). 4. In the hippocampal kindled rat a dose of 40 mg kg(-1) of (R)- and (S)-PID prevented the secondarily generalized seizure, whereas racemic PID also blocked the expression of partial seizures following an i.p. dose of 40 mg kg(-1). Racemic PID also significantly increased the seizure threshold in this model. 5. Mechanistic studies showed that PID did not affect voltage-sensitive sodium channels or kainate-, GABA- or NMDA- evoked currents. 6. The pharmacokinetics of PID was enantioselective following i.p. administration of individual enantiomers to mice, with (R)-PID having lower clearance and longer half-life than (S)-PID. In rats and mice, no enantioselectivity in the pharmacokinetics of PID was observed following administration of the racemate, which may be due to enantiomer-enantiomer interaction. 7. This study demonstrated that PID has both enantioselective pharmacokinetics and pharmacodynamics. The better anticonvulsant potency of (R)-PID in comparison to (S)-PID may be due to its more favorable pharmacokinetic profile. The enhanced efficacy of the racemate over the individual enantiomers in the kindled rat may be explained by a pharmacokinetic enantiomer-enantiomer interaction in rats. This study also showed the importance of studying the pharmacokinetics and pharmacodynamics of chiral drugs following administration of the individual enantiomers as well as the racemic mixture.

Correlation between anticonvulsant activity and inhibitory action on glial gamma-aminobutyric acid uptake of the highly selective mouse gamma-aminobutyric acid transporter 1 inhibitor 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole and its N-alkylated analogs.

The inhibitory effect of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and its N-methylated (N-methyl-exo-THPO) and N-ethylated (N-ethyl-exo-THPO) analogs, derived from gamma-aminobutyric acid (GABA) and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) on GABA transport was investigated using cultured neocortical neurons (GABA-ergic) and astrocytes and cloned mouse GABA transporters GAT1-4 expressed in human embryonic kidney (HEK) 293 cells. Anticonvulsant activity was assessed after i.c.v. administration to Frings audiogenic seizure-susceptible mice. Anticonvulsant activity of the O-pivaloyloxymethyl prodrug of N-methyl-exo-THPO was assessed after i.p. administration. Results from these studies were compared with those obtained from similar studies with the novel anticonvulsant drug tiagabine, which acts via inhibition of GABA transport. exo-THPO and its N-alkyl analogs inhibited neuronal, astrocytic, and GAT1-mediated GABA transport but not GABA uptake mediated by GAT2-4. N-Methyl-exo-THPO was 8-fold more potent as an inhibitor of astrocytic versus neuronal GABA uptake. The IC(50) value for inhibition of GABA uptake by GAT1 closely reflected its IC(50) value for inhibition of neuronal uptake. Tiagabine was approximately 1000-fold more potent than exo-THPO and its alkyl derivatives as an inhibitor of GABA uptake in cultured neural cells and GAT1-expressing HEK 293 cells. exo-THPO, its alkylated analogs, and tiagabine displayed a time- and dose-dependent inhibition of audiogenic seizures after i.c.v. administration. N-Methyl-exo-THPO was the most potent anticonvulsant among the exo-THPO compounds tested and only slightly less potent than tiagabine. The findings suggest a correlation between anticonvulsant efficacy and selective inhibition of astroglial GABA uptake. Furthermore, results obtained with the N-methyl-exo-THPO prodrug demonstrate the feasibility of developing a glial-selective GABA uptake inhibitor with systemic bioavailability.

Acetoacetate, acetone, and dibenzylamine (a contaminant in l-(+)-beta-hydroxybutyrate) exhibit direct anticonvulsant actions in vivo.

To investigate whether ketone bodies are directly anticonvulsant. We tested the effects of acetoacetate (ACA), acetone, and both stereoisomers, D-(-)- and L-(+), of beta-hydroxybutyrate (BHB) on sensory-evoked seizures in Frings audiogenic seizure-susceptible mice. We found that these ketone bodies, with the exception of the D-(-)-isomer of BHB, were anticonvulsant in this model. Furthermore, with gas chromatography-mass spectrometry, we confirmed that the activity of L-(+)-BHB was due to dibenzylamine, a chemical contaminant. Our data indicate that the anticonvulsant efficacy of the ketogenic diet may be due in part to the direct actions of ACA and acetone.

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D-aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure.

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D-aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 209–216, 1998

Genetic Mapping of a Locus (mass1) Causing Audiogenic Seizures in Mice

Frings audiogenic seizure-susceptible mice are a model for sensory-evoked reflex seizures. Their seizure phenotype is characterized by wild running, loss of righting reflex, tonic flexion, and tonic extension in response to high-intensity sound stimulation. The Frings mice represent an inbred colony that has not been genetically characterized. This investigation studied the mode of inheritance for audiogenic seizures by crossing the Frings mouse with the seizure-resistant C57BL/6J mouse. Among the backcross progeny generated by crossing (Frings × C57BL/6J)F1 mice with the Frings strain, 391 of the 836 N2 progeny were audiogenic seizure susceptible, a finding consistent with monogenic inheritance. Genetic mapping and linkage analysis of hybrid mice using MIT microsatellite marker sequences localized the seizure gene, namedmass1for monogenic audiogenic seizure susceptible, to an approximately 3.6 cM interval in the middle of mouse chromosome 13. Linkage ofmass1to chromosome 13 is an important step in identifying the gene associated with a monogenic seizure disorder in mice, which may ultimately lead to a better understanding of the pathophysiology of human seizure disorders.

Interleukin-1α in the Brain Is Induced by Audiogenic Seizure

We examined the expression of the sleep-inducing cytokine interleukin-1α (IL-1α) in the brains of audiogenic seizure-susceptible mice subsequent to the induction of sound-induced seizure. Animal models of epilepsy often require lesioning or trauma that may nonspecifically alter IL-1α expression. To avoid this, we employed the Frings mouse strain; a model of auditory-evoked reflex epilepsy. Frings mice were exposed to a high-intensity sound stimulus to induce a tonic extension seizure, and the expression of IL-1α transcripts in different brain regions was measured thereafter. Compared to control animals, IL-1α transcripts were elevated 6 to 8 h postseizure in the hypothalamus, but not hippocampus, by a dexamethasone-sensitive pathway. Similar results were obtained from the genetically distinct DBA/2J audiogenic seizure-susceptible mouse strain. These findings demonstrate that the expression of IL-1α is altered following generalized seizure activity, induced by noninvasive sensory stimulation, in a brain-region-specific manner.

253 Effects of FK506 on the NMDA receptor-mediated potentials and synaptic plasticity in CA1 area of rat hippocampus

Although there are several animal models of epilepsy, the extrapolation of antiepileptic drug (AEDs) performance to epileptic patients from anticonvulsant activity results in animals is not straightforward. Consequently, the aim of this work was to perform a correlation analysis between therapeutic daily doses (D) and average steady-state plasma concentrations (Css,av) of AEDs and their activity in common anticonvulsant animal models.AED activity in anticonvulsant animal models was expressed as maximal electroshock seizure (MES) test ED50 values in mice and rats and ED50 values in audiogenic seizure-susceptible mice (AGS ED50). Data were examined, by use of linear and logarithmic approaches, for an association between Css,av (mg/L or μmol/L) and D (mg or mmol) for each AED in epileptic patients as the dependent variable (Y) and its MES ED50 in mice and rats and AGS ED50 in mice (mg/kg or μmol/kg) as the independent variable (X).Linear correlation analyses between Css,av (mg/L) and ED50 (mg/kg) for 11 AEDs gave the following correlation coefficients (R2): 0.68 (mice, MES); 0.73 (rat, MES); 0.64 (AGS). Switching the units from milligrams to micromoles improved the correlation significantly and gave the following R2 values: 0.88 (mice, MES); 0.90 (rat, MES); 0.76 (AGS). The linear correlation between Css,av and ED50 was better than that between D and ED50.The results of this analysis suggest that the relationship between Css,av and ED50 is useful in predicting target concentration ranges in humans. The Y intercepts of the Css,av-versus-ED50 and D-versus- ED50 plots were similar in all three animal models and ranged between 12 and 17 mg/L and between 570 and 890 mg, respectively, indicating that for all AEDs analyzed except valproic acid and ethosuximide, the therapeutic plasma concentration is in the range 10–20 mg/L.

Felbamate modulates the strychnine-insensitive glycine receptor

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a novel anticonvulsant substance whose mechanism of action is not clearly understood. The present investigation examined its ability to modulate the strychnine-insensitive glycine receptor associated with the N-methyl-D-aspartate (NMDA) receptor. Felbamate decreased the magnitude of glycine (100 microM)-enhanced NMDA (100 microM)-induced intracellular calcium ([Ca2+]i) transients in mouse cerebellar granule cells which had been loaded with the Ca(2+)-sensitive fluorescent probe indo-1 acetoxymethyl ester (indo-1/AM). This effect of felbamate was concentration dependent, with a maximal effect observed at 300 microM (65 +/- 4% of control). In the Frings audiogenic seizure-susceptible mouse model of reflex epilepsy, the glycine agonist D-serine (150 nmol, i.c.v.) completely blocked the anticonvulsant activity of a maximally effective dose of felbamate (19 mg/kg, i.p.). This effect of D-serine could be reversed by increasing the administered dose of felbamate to 29 mg/kg. Furthermore, administration of D-serine (300 nmol, i.c.v.) to felbamate-treated Frings mice produced a parallel right shift in felbamate's anticonvulsant dose-response curve (ED50s: 9.4 mg/kg for felbamate vs. 17.7 mg/kg for felbamate + D-serine). The results obtained in this investigation suggest that the ability of felbamate to modulate the strychnine-insensitive glycine receptor may be physiologically and behaviorally relevant to its anticonvulsant mechanism of action.

Altered GABAergic and glutamatergic transmission in audiogenic seizure-susceptible mice.

The C57BL/10 SPS/sps mouse mutant are audiogenic seizure-susceptible. The enzymatic activities of glutamate decarboxylase (GAD), GABA aminotransferase (GABA-T), alanine aminotransferase (ALA-T), aspartate aminotransferase (ASP-T), and glutamate dehydrogenase (GDH) of whole brain supernatant are significantly reduced in these epileptic mice. GABA uptake is decreased in cortex, midbrain, and pons medulla. Previous studies showed the presence of two sodium-dependent GLU uptake systems in normal (SPS/SP) mice. Glutamate Umax by System 1 is significantly decreased in these mice, whereas the Umax value for System 2 is significantly increased in the epileptic mice.

High affinity [ 3H]glutamate uptake systems in normal and audiogenic seizure-susceptible mice

Two high affinity sodium-dependent and PDC-sensitive glutamate (GLU) uptake systems are present in a whole brain synaptosomal preparation from adult C57BL/10 SPS/SPS normal mice. System 1 has an apparent K m of 3.65 μM while that of System 2 is 46.8 μM. Glutamate uptake in the normal mice increases gradually during development, displaying a striking peak at postnatal day 15, and decreases rapidly between PN 16 and PN 20 until it reaches adult levels. The developmental pattern of GLU uptake System 1 and System 2 in audiogenic seizure susceptible mice is similar to that described in normal mice. However, there are differences between GLU uptake system 1 and 2 during ontogenesis: (1) System 1 could not be detected until PN 15 while being markedly diminished in adulthood; and (2) GLU uptake by System 2 is increased in adults. In addition, the U max for System 2 is significantly greater than that of normal mice at PN 2 and PN 15.