El Mice Research Articles

Role of histaminergic neurons in development of epileptic seizures in EL mice

The EL mouse is an animal model for hereditary temporal lobe epilepsy. When the mice receive weekly vestibular stimulation, e.g., 30 “tosses”, 10–15 cm vertically, they start to convulse after 1–2 weeks. The aim of this study was to evaluate the role of the histaminergic neurons in the regulation of seizure development in the EL mice. The obtained results indicated that administration of either histidine, a substrate for histamine synthesis, or metoprine (2,4-diamino-5-(3,4-dichlorophnyl)-6-methyl-pyrimidine), an inhibitor of histamine N-methyltransferase (HNMT), retarded the onset of seizure episodes in the mice. The co-administration of histidine and metoprine caused a more marked delay in it. The histamine levels in the brain significantly increased in response to any of these treatments. The intraperitoneal injection of diphenhydramine, a H1-antagonist accelerated the initiation of seizure episodes in the mice, whereas thioperamide, a H3-antagonist caused a delay in the response. There were significant increases in the brain histamine levels upon injection of any of these drugs with concomitant rises in the activity of the histidine decarboxylase (HDC). These results, taken together, suggest that the histaminergic neurons play crucial roles in the development of seizures in the EL mice. They inhibit convulsion in a H1-dependent fashion, while the neurons enhance it in a H3-receptor-mediated way.

Nootropic Nefiracetam Inhibits Proconvulsant Action of Peripheral‐Type Benzodiazepines in Epileptic Mutant EL Mice

Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam >> oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

Genetic and phenotypic analysis of seizure susceptibility in PL/J mice.

Epilepsy is one of the most common but genetically complex neurological disorders in humans. Identifying animal models that recapitulate human epilepsies is important for pharmacological studies of anticonvulsants, dissection of molecular and biochemical pathogenesis of epilepsy, and discovery of epilepsy susceptibility genes. We discovered that the PL/J inbred mouse strain is susceptible to handling- and rhythmic tossing-induced seizure. The tonic-clonic and generalized seizures observed after induction were accompanied by abnormal EEGs, similar to seizures observed in EL and SWXL-4 mice. PL/J mice also had an extremely low threshold to electroconvulsive seizures compared to other strains and showed variable sensitivity to pentylenetetrazole-induced seizures. Gross neurostructural abnormalities were not found in PL/J mice. Crosses with the seizure-resistant C57BL/6 J strain revealed semidominant inheritance of the rhythmic tossing seizure trait with low penetrance. F2 progeny indicated that the genetic inheritance of seizure susceptibility in PL/J is non-Mendelian. We crossed DBA/2 J mice, which are resistant to rhythmic tossing seizure but susceptible to audiogenic seizures, to PL/J. We found that seizure penetrance in (DBA/2 J x PL/J)F1 mice was similar to the penetrance in (C57BL/6 J x PL/J)F1 mice but the severity and frequency of seizure were higher in (DBA/2 J x PL/J)F1 mice. The PL/J strain serves as an interesting new model for studying the genetics, neurobiology, and pharmacology of epilepsy.

Management of multifactorial idiopathic epilepsy in EL mice with caloric restriction and the ketogenic diet: role of glucose and ketone bodies

BackgroundThe high fat, low carbohydrate ketogenic diet (KD) was developed as an alternative to fasting for seizure management. While the mechanisms by which fasting and the KD inhibit seizures remain speculative, alterations in brain energy metabolism are likely involved. We previously showed that caloric restriction (CR) inhibits seizure susceptibility by reducing blood glucose in the epileptic EL mouse, a natural model for human multifactorial idiopathic epilepsy. In this study, we compared the antiepileptic and anticonvulsant efficacy of the KD with that of CR in adult EL mice with active epilepsy. EL mice that experienced at least 15 recurrent complex partial seizures were fed either a standard diet unrestricted (SD-UR) or restricted (SD-R), and either a KD unrestricted (KD-UR) or restricted (KD-R). All mice were fasted for 14 hrs prior to diet initiation. A new experimental design was used where each mouse in the diet-restricted groups served as its own control to achieve a 20–23% body weight reduction. Seizure susceptibility, body weights, and the levels of plasma glucose and β-hydroxybutyrate were measured once/week over a nine-week treatment period.ResultsBody weights and blood glucose levels remained high over the testing period in the SD-UR and the KD-UR groups, but were significantly (p < 0.001) reduced in the SD-R and KD-R groups. Plasma β-hydroxybutyrate levels were significantly (p < 0.001) increased in the SD-R and KD-R groups compared to their respective UR groups. Seizure susceptibility remained high in both UR-fed groups throughout the study, but was significantly reduced after three weeks in both R-fed groups.ConclusionsThe results indicate that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control is more associated with the amount than with the origin of dietary calories. Also, CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems.

Molecular Mechanism of Preventive Effect of Peony Root Extract on Neuron Damage

The molecular mechanism of the protective effects of peony root extract and its component substances on neuron damage induced by the cobalt focus epilepsy model and the EL mouse was investigated. Long-term administration of peony root extract for 30 days prior to metallic cobalt powder application to the cerebral cortex of mice resulted in increased expression of A20, an inhibitor gene of cell death. In the EL mouse, a hereditary epilepsy animal model with vulnerable neurons, increased expression of A20 was observed even without administration of peony root extract. Long-term administration of peony root extract to the EL mouse resulted in a marked increase of expression of A20. These results suggested that an increase in A20 expression is the main molecular mechanism of protective action of peony root extract on neuron damage.

Genetic mapping of the absence of third molars in EL mice to chromosome 3.

We noted the absence of all 4 third molars (M3) in Epilepsy-Like disorder (EL) mice, an animal model for the study of epilepsy. This study was conducted to identify the major candidate chromosome and to detect the region that included the candidate gene causing the absence of M3 in EL mice. Linkage analysis was performed on genetic crosses of EL mice and MSM (Mus musculus molossinus) strain mice, which had a normal complement of teeth. Genome-wide screening by individual genotyping of F(2) intercross mice identified mouse chromosome 3 as one of the candidate chromosomes. Based on high linkage scores in detailed genotyping of F(2) intercross and N(2) backcross mice, the candidate locus for the absence of M3 in EL mice was mapped on the middle of chromosome 3.

The significance of increase in striatal D 2 receptors in epileptic EL mice

The present study systematically and quantitatively analyzed the immunohistochemical distribution of various substances involved in synthesis, binding, and transport of dopamine in the forebrain of epileptic mice (EL mouse strain) using a brain mapping analyzer. A reduction in serum calcium levels decreases calcium/calmodulin-dependent-dopamine synthesis in the brain and subsequently increases susceptibility to epileptic convulsions and induces abnormal behavior in EL mice. The immunohistochemical levels of D 2 receptors in the medial area of the neostriatum were significantly higher in EL mice than in ddY mice (mother strain of EL mice), while there were no differences in the levels of tyrosine hydroxylase, calcium/calmodulin-dependent protein kinase II, calmodulin, D 1 receptors, and dopamine transporters. Together with our previous findings, the results suggest that the decrease in serum calcium levels and subsequent decrease in brain dopamine synthesis comprise the primary physiologic disorder in EL mice, and convulsions or increased D 2 receptors are secondarily-induced phenomena to improve or compensate for the principal disorder.

Down-regulation of GABA-transporter function by hippocampal translation products: its possible role in epilepsy

The genetically epileptic mouse strain (El) is used as a model for human temporal lobe epilepsy. To address the question of whether altered function of the neuronal GABA transporter GAT1 is involved in the pathology of epilepsy of El mice, we expressed in Xenopus oocytes cloned GAT1 of mouse brain by injection of complementary ribonucleic acid (cRNA) and co-injected messenger ribonucleic acid (mRNA) isolated from the hippocampus of non-epileptic control mother strain (ddY) mice and from El mice. GABA transporter activity was investigated by measurements of [ 3H]-GABA uptake as well as by steady-state and transient current measurements under voltage clamp. Co-injection of hippocampal mRNA into oocytes reduced GAT1-mediated transport. This effect was more pronounced for mRNA from ddY mice than for that from El mice that never experienced seizures, El(−), and being absent for mRNA from El mice that have had high seizure experience, El(+). The pronounced inhibition of GABA transport after injection of mRNA from the ddY strain results from reduced expression of functional GAT1, but to about one third also from a reduced GABA translocation rate. The reduced translocation can be attributed to a reduced forward rate of a step associated with extracellular Na + binding. If the results can be applied to the mouse brain, we may hypothesise that in ddY mice some GAT down-regulating factor translated from hippocampal mRNA may be involved to keep GAT1 activity low, and hence GABA concentration in synaptic cleft high. In El(−) mice such regulatory mechanism may be reduced or counteracted by another unknown factor present in El(−) brain. The repeated seizure experience in El(+) mice enhances this compensatory effect.

Seizure-prone EL mice exhibit deficits in pup nursing and retrieval assessed using a novel method of maternal behavior phenotyping

The selectively bred EL mouse strain exhibits hyperreactivity to environmental disturbance reflected by handling-induced seizures and motor hyperactivity in an exploratory task relative to a non-seizure-prone control strain. One possible mechanism for the nongenomic transmission of an adult seizure-prone/hyperactive phenotype is the quality of parenting provided to immature offspring. In particular, the quality of maternal behavior has been implicated as an environmental determinant in rodent biochemical and behavioral development. A complication in testing this hypothesis is that human handling for husbandry and testing itself triggers seizures in seizure-prone EL mice. Thus, the present study evaluated potential EL versus control strain differences in maternal behavior using a novel apparatus for passive observation of undisturbed mice. Nonmaternal behaviors were also measured to control for any nonspecific differences in activity or exploration. EL dams were slower than DDY controls to initiate pup retrieval and spent less time nursing/crouching over pups than DDY mice. EL dams also exhibited a profile of sustained exploration and grooming over time relative to the profile of DDY controls. These results suggest that EL mothers exhibit an overabundance of motor activities that compete with crouching/nursing and retrieval behaviors required for viability of the litter.

Sexual dysfunction and sudden death in epileptic male EL mice: inheritance and prevention with the ketogenic diet.

The EL mouse is an animal model for multifactorial idiopathic epilepsy. Although EL mice have been studied extensively for >45 years, the etiology of male sudden death and its relation to seizures have not been defined. Here we investigated the cause of EL male sudden death and its relation to epilepsy. For histopathologic analysis, the terminally ill EL mice (n = 15) were killed, and the tissues were fixed. Blood chemical composition was compared between the terminally ill EL (n = 9) and the healthy age-matched EL (n = 17) and DDY (n = 11) males. To determine the effect of the ketogenic diet (KD) on sudden male death, young male EL mice (P30) were randomly separated into two groups that were fed ad libitum with either Agway lab chow (control n = 38) or with the KD (treated, n = 39) for 5 months. The genetic predisposition to sudden death was analyzed in the backcross generation (n = 106) of a cross between EL and the nonepileptic ABP strains. Sudden death coincided with the onset of seizures (70-80 days) and affected 94% of male EL mice by age 300 days. Urethral plugs were observed histologically in 13 of 15 longitudinally sectioned penises. Concentrations of blood urea nitrogen, creatinine, phosphorus, and calcium in the terminally ill mice were significantly elevated when compared with those of healthy animals. None of the mice treated with the KD experienced sudden death, whereas 15 (39%) of the untreated control mice died by age 5 months. The sudden death in male EL mice was inherited as an autosomal recessive sex-limited lethal trait. The cause of sudden death in male EL mice arises from abnormal ejaculation, which produces a urethral plug with consequent urinary retention and acute severe uremia. The coincident onset of seizures and sudden death in EL males suggests that a sexual dysfunction is associated with epilepsy in this model.

Ictogenesis and epileptogenesis in EL mice.

In EL mice, ictogenesis is established at age approximately 10 weeks, whereas epileptogenesis is induced through an experience of repetitive seizures during development. An "abnormal neural plasticity" has been suggested to be involved in these pathologic processes. It also is known that two isoforms of nitric oxide (NO) synthetase (nNOS and eNOS) are essential for the long-term potentiation (LTP), a plastic response of neurons. It appears, therefore, that these NO synthetases might play a major role in the establishment of abnormal neural plasticity. The purpose of the present study was to investigate ictogenesis and epileptogenesis by observing alterations of NO synthetases as well as immediate early gene (IEG) expressions and gamma-aminobutyric acid (GABA) distributions in the brain during development and with respect to seizure history. IEG (c-fos and zif) expression in EL mice were analyzed by in situ hybridization with 35S. Distribution of GABA concentrations and glutamic acid decarboxylase (GAD) activities in the parietal cortex of EL mice was quantitatively determined using ultramicroenzymatic chemistry (Lowry, 1978). Three isoforms of NOS were assayed by immunoblotting analysis for hippocampal tissues of EL mice and their control animals, DDY mice. DNA fragmentation was detected with the TUNEL method. In EL mouse brains, IEG expression was related to the seizure history, seizure threshold, and age. Even in the interictal period, the animals expressed IEG continuously when their seizure thresholds were very low. Among various IEG expression sites in the brain, hippocampal CA1 was the most remarkable. These IEG expression sites were almost identical to the brain regions of EL mice where GABA concentrations and GAD activities were altered. Unexpectedly, the eNOS content of EL was very small, although eNOS appears to be responsible for NO that mediates an increase in local cerebral blood flow during focal seizures. nNOS, iNOS, and to a lesser extent, eNOS were essential to establish both ictogenicity and epileptogenicity. DNA fragmentation was observed in the hippocampus of EL mice in the interictal period. Continuous IEG expression and abnormal GABAergic function are involved in the epileptogenesis of EL mice. Transiently expressed IEG, on the other hand, is associated with the ictogenesis. It is conceivable that an excess amount of iNOS (and subsequent increase in harmful antimicrobial NO) and a lesser amount of eNOS (and subsequent decrease in NO or endothelium-derived relaxing factor, EDRF) may work together to contribute to a focus complex and ictogenesis. Drugs that suppress iNOS and/or potentiate eNOS may be promising candidates for a new type of antiepileptic agent.

Selective suppression of hippocampal region hyperexcitability related to seizure susceptibility in epileptic El mice by the GABA-transporter inhibitor tiagabine

High seizure susceptibility in El mice is associated with disinhibition in the dentate gyrus (DG) and paired-pulse facilitation in the CA3 area in hippocampal slices [Brain Res. 745 (1997) 165; Brain Res. 779 (1998) 324]. A decrease in γ-aminobutyric acid (GABA)-mediated inhibition and an increase in excitatory inputs to the major neurons seem to be the responsible mechanisms, respectively, for these phenomena. In this study, we examined the effects of tiagabine, an inhibitor of GABA transporter, on hyperexcitation in vivo and in slice preparations. Tiagabine (0.3–0.5 mg/kg) suppressed the occurrence of seizures to about 20% of controls with an ED 50 value of about 0.17 mg/kg. In addition, perfusion of hippocampal slices with tiagabine (20 μM) counteracted the paired-pulse facilitation in the CA3 region over the entire range of interpulse intervals ( P<0.05, two-way ANOVA) and reduced the disinhibition in the DG measured at 10 and 20 ms during short interpulse intervals ( P<0.005, paired t-test). The CA1 region in the El mice, as well as in a non-epileptic parental strain of ddY mice did not respond to the drug. However, frequency potentiation of CA3 was enhanced in both strains ( P<0.05, paired t-test). Our results suggest that within the hippocampus the antiepileptic action of tiagabine is selectively suppressive for hyperexcitability of DG and CA3, which are responsible for seizure-susceptibility in El mice.

Foci identification of spike discharges in the EEGs of sleeping El mice based on the electric field model and wavelet decomposition of multi monopolar derivations

We report on spike discharges in the EEGs of sleeping El mice, which are considered to be homologous to the state seen in human epileptic patients. The discrete wavelet transform (DWT) was used to decompose the EEGs derived from seven electrodes, with the primary spike frequency detected in the 15.6–7.8 Hz detail. The synchronicity of the spikes was evaluated in 98 samples. We succeeded in detecting the foci of the spikes by applying the electric field model, in which the surface potential was expressed as a function of distance between the focus and each electrode based on Gauss’ theorem. The foci were obtained in 73 of 91 spikes so evaluated. In 69 of these 73 spikes, the calculated foci occurred within the brain, with none of the foci occurring at identical positions. The distribution occurred in relatively deeper brain regions, suggesting involvement of the paleocortex rather than the hippocampal system. The present study provides reasonable grounds for an explanation of the EEG using the electric field model supported by efficient foci detection of the spikes in a one-to-one correspondence. This paradigm will be applicable to all animal species including humans and will be helpful in the detection of the epileptic focus and in obtaining an insight into the mechanisms of spike genesis.

Increased expression of mitochondrial respiratory enzymes in the brain of epilepsy-prone, naive EL mice

The present studies demonstrate that expression of both type 5 and type 6 subunits of NADH dehydrogenase and the type 1 subunit of cytochrome oxidase is enhanced significantly in the brains of naive, epilepsy-prone EL mice. In contrast, no apparent change in expression occurred with type 1 and type 2 subunits of NADH dehydrogenase. When expression of type 5 and 6 subunits of NADH dehydrogenase was determined at 24 h after a single series of vestibular stimulation, significant down-regulation was detected. The expression of subunit 2 of NADH dehydrogenase augmented gradually after vestibular stimulation. The increased expression of these mitochondrial respiratory enzymes may reflect enhanced demand for energy due to inherent, spontaneous neuronal hyperactivity in the brains of EL mice.

Hippocampal neurons and glia in epileptic EL mice.

Reactive changes in hippocampal astrocytes are frequently encountered in association with temporal lobe epilepsy in humans and with drug or kindling-induced seizures in animal models. These reactive changes generally involve increases in astrocyte size and number and often occur together with neuronal loss and synaptic rearrangements. In addition to producing astrocytic changes, seizure activity can also produce reactive changes in microglia, the resident macrophages of brain. In this study, we examined the effects of recurrent seizure activity on hippocampal neurons and glia in the epileptic EL mouse, a natural model of human multifactorial idiopathic epilepsy and complex partial seizures. Timm staining was used to evaluate infrapyramidal mossy fiber organization and the optical dissector method was used to count Nissl-stained neurons in hippocampus of adult (about one year of age) EL mice and nonepileptic C57BL/6J (B6) and DDY mice. Immunostaining for glial fibrillary acidic protein (GFAP) and Iba1, an actin cross-linking molecule restricted to macrophages and microglia, was used to evaluate astrocytes and microglia, respectively. The EL mice experienced about 25-30 complex partial seizures with secondary generalization during routine weekly cage changing. No significant differences were found among the mouse strains for Timm staining scores or for neuronal counts in the CA1 and CA3 pyramidal fields or in the hilus. However, the number of GFAP-positive astrocytes was significantly elevated in the stratum radiatum and hilus of EL mice, while microglia appeared hyper-ramified and were more intensely stained in EL mice than in the B6 or DDY mice in the hilus, parietal cortex, and pyriform cortex. The results indicate that recurrent seizure activity in EL mice is associated with abnormalities in hippocampal astrocytes and brain microglia, but is not associated with obvious neuronal loss or mossy fiber synaptic rearrangements. The EL mouse can be a useful model for evaluating neuron-glia interactions related to idiopathic epilepsy.

Mapping of a Gene causing congenital Absence of the third Molars in EL mice to Chromosome 3

Mapping of a Gene causing congenital Absence of the third Molars in EL mice to Chromosome 3

Caloric restriction inhibits seizure susceptibility in epileptic EL mice by reducing blood glucose.

Caloric restriction (CR) involves underfeeding and has long been recognized as a dietary therapy that improves health and increases longevity. In contrast to severe fasting or starvation, CR reduces total food intake without causing nutritional deficiencies. Although fasting has been recognized as an effective antiseizure therapy since the time of the ancient Greeks, the mechanism by which fasting inhibits seizures remains obscure. The influence of CR on seizure susceptibility was investigated at both juvenile (30 days) and adult (70 days) ages in the EL mouse, a genetic model of multifactorial idiopathic epilepsy. The juvenile EL mice were separated into two groups and fed standard lab chow either ad libitum (control, n=18) or with a 15% CR diet (treated, n=17). The adult EL mice were separated into three groups; control (n=15), 15% CR (n=6), and 30% CR (n=3). Body weights, seizure susceptibility, and the levels of blood glucose and ketones (beta-hydroxybutyrate) were measured over a 10-week treatment period. Simple linear regression and multiple logistic regression were used to analyze the relations among seizures, glucose, and ketones. CR delayed the onset and reduced the incidence of seizures at both juvenile and adult ages and was devoid of adverse side effects. Furthermore, mild CR (15%) had a greater antiepileptogenic effect than the well-established high-fat ketogenic diet in the juvenile mice. The CR-induced changes in blood glucose levels were predictive of both blood ketone levels and seizure susceptibility. We propose that CR may reduce seizure susceptibility in EL mice by reducing brain glycolytic energy. Our preclinical findings suggest that CR may be an effective antiseizure dietary therapy for human seizure disorders.

Expression of vesl-1S/homer-1a, a gene associated with long-term potentiation, in the brain of the epileptic EI mouse

The El mouse is an animal model for hereditary epilepsy. It undergoes a convulsive seizure lasting 10–30 s after vestibular stimulation, such as ‘tossing-up’. To better characterize the effects of seizure activity in the El mouse, we examined the expression profile of vesl-1S/homer-1a mRNA in the brain. It has been shown that this mRNA is expressed after long-term potentiation or seizures induced by electrical or pharmacological stimulations. The basal level of vesl-1S mRNA was very low even in seizure-susceptible El mice. The El mouse hippocampus, but not that of ddY mice, showed a remarkable increase in vesl-1S mRNA expression in the dentate granule cell layer after seizure induced by ‘tossing-up’. The mRNA remained for about 3 h after the seizure and disappeared after 8 h.

Influences of caffeine to nitric oxide production and zonisamide concentration in the brain of seizure-susceptible EL mice.

To investigate changes of nitric oxide (NO) productions and zonisamide (ZNS) concentrations in the brain of seizure-susceptible EL mice given caffeine orally, mice were given caffeine (600 microg/mL) solution ad libitum as a drinking fluid for 1-3 weeks. Nitric oxide production in the brain was determined by measuring levels of nitrite plus nitrate (NOx). The brain NOx levels of mice treated with caffeine for 3 weeks were significantly higher than the control. Seizures in mice treated with caffeine for 2 and 3 weeks were not suppressed by ZNS at a dose of 75 mg/kg. Serum ZNS concentrations of mice with caffeine intake for 1-3 weeks were higher than in untreated mice. Conversely, brain ZNS concentrations of mice with caffeine intake for the same periods were significantly lower than in untreated mice. These results suggested that caffeine influenced brain NO production and ZNS concentrations in the seizure susceptibility of EL mice.

Alterations of nitric oxide and monoamines in the brain of the EL mouse treated with phenobarbital and zonisamide.

The effects of phenobarbital (PB; doses, 5, 10, and 25 mg/kg, intraperitoneally (i.p.)) and zonisamide (ZNS; doses, 30, 75, and 150 mg/kg, i.p.) on nitric oxide (NO) production, and those of coadministration of PB (5 mg/kg, i.p.) and ZNS (75 mg/kg, i.p.) on monoamines in the brain of the seizure-susceptible EL mouse were investigated. Nitric oxide production was obtained by measuring the combined level of nitrite plus nitrate (NOx). Zonisamide and PB dose-dependently suppressed the seizure of the EL mouse, and coadministration of PB (5 mg/kg) and ZNS (75 mg/kg) induced a greater degree of seizure suppression than treatment with ZNS or PB alone. Although PB (5 mg/kg) had no effect on brain NOx levels, ZNS (150 mg/kg) and coadministration of ZNS (75 mg/kg) and PB (5 mg/kg) decreased NOx levels significantly. Phenobarbital (5 mg/kg) did not influence monoamines, while coadministration of PB (5 mg/kg) and ZNS (75 mg/kg) decreased dihydroxyphenylacetic acid and increased 5-HT concentrations. The effect of the coadministration of two drugs on monoamines were similar to that of ZNS alone. These results suggest that one of the anticonvulsant effects of coadministration of PB and ZNS may be caused by changes in NOx levels.