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Purpose: Previous quantitative trait loci (QTL) mapping studies from our laboratory identified a 6.6 Mb segment of distal chromosome 1 that contains a gene (or genes) having a strong influence on the difference in seizure susceptibility between C57BL/6 (B6) and DBA/2 (D2) mice. A gene transfer strategy involving a bacterial artificial chromosome (BAC) DNA construct that contains several candidate genes from the critical interval was used to test the hypothesis that a strain-specific variation in one (or more) of the genes is responsible for the QTL effect., Methods: Fertilized oocytes from a seizure-sensitive congenic strain (B6.D2-Mtv7a/Ty-27d) were injected with BAC DNA and three independent founder lines of BAC-transgenic mice were generated. Seizure susceptibility was quantified by measuring maximal electroshock seizure threshold (MEST) in transgenic mice and nontransgenic littermates., Results: Seizure testing documented significant MEST elevation in all three transgenic lines compared to littermate controls. Allele-specific RT-PCR analysis confirmed gene transcription from genome-integrated BAC DNA and copy-number-dependent phenotypic effects were observed., Conclusions: Results of this study suggest that the gene(s) responsible for the major chromosome 1 seizure QTL is found on BAC RPCI23-157J4 and demonstrate the utility of in vivo gene transfer for studying quantitative trait genes in mice. Further characterization of this transgenic model will provide new insight into mechanisms of seizure susceptibility.

Previous quantitative trait loci (QTL) mapping studies document that the distal region of mouse Chromosome (Chr) 1 contains a gene(s) that is in large part responsible for the difference in seizure susceptibility between C57BL/6 (B6) (relatively seizure-resistant) and DBA/2 (D2) (relatively seizure-sensitive) mice. We now confirm this seizure-related QTL ( Szs1) using reciprocal, interval-specific congenic strains and map it to a 6.6-Mb segment between Pbx1 and D1Mit150. Haplotype conservation between strains within this segment suggests that Szs1 may be localized more precisely to a 4.1-Mb critical interval between Fcgr3 and D1Mit150. We compared the coding region sequences of candidate genes between B6 and D2 mice using RT-PCR, amplification from genomic DNA, and database searching and discovered 12 brain-expressed genes with SNPs that predict a protein amino acid variation. Of these, the most compelling seizure susceptibility candidate is Kcnj10. A survey of the Kcnj10 SNP among other inbred mouse strains revealed a significant effect on seizure sensitivity such that most strains possessing a haplotype containing the B6 variant of Kcnj10 have higher seizure thresholds than those strains possessing the D2 variant. The unique role of inward-rectifying potassium ion channels in membrane physiology coupled with previous strong association between ion channel gene mutations and seizure phenotypes puts even greater focus on Kcnj10 in the present model. In summary, we confirmed a seizure-related QTL of large effect on mouse Chr 1 and mapped it to a finely delimited region. The critical interval contains several candidate genes, one of which, Kcnj10, exhibits a potentially important polymorphism with regard to fundamental aspects of seizure susceptibility.

Purpose: Our research program uses genetic linkage and association analysis to identify human seizure sensitivity and resistance alleles. Quantitative trait loci mapping in mice led to identification of genetic variation in the potassium ion channel gene Kcnj10, implicating it as a putative seizure susceptibility gene. The purpose of this work was to translate these animal model data to a human genetic association study., Methods: We used single stranded conformation polymorphism (SSCP) electrophoresis, DNA sequencing and database searching (NCBI) to identify variation in the human KCNJ10 gene. Restriction fragment length polymorphism (RFLP) analysis, SSCP and Pyrosequencing were used to genotype a single nucleotide polymorphism (SNP, dbSNP rs#1130183) in KCNJ10 in epilepsy patients (n = 407) and unrelated controls (n = 284). The epilepsy group was comprised of patients with refractory mesial temporal lobe epilepsy (n = 153), childhood absence (n = 84), juvenile myoclonic (n = 111) and idiopathic generalized epilepsy not otherwise specified (IGE-NOS, n = 59) and all were of European ancestry., Results: SNP rs#1130183 (C > T) alters amino acid 271 (of 379) from an arginine to a cysteine (R271C). The C allele (Arg) is common with conversion to the T allele (Cys) occurring twice as often in controls compared to epilepsy patients. Contingency analysis documented a statistically significant association between seizure resistance and allele frequency, Mantel-Haenszel chi square = 5.65, d.f. = 1, P = 0.017, odds ratio 0.52, 95% CI 0.33-0.82., Conclusion: The T allele of SNP rs#1130183 is associated with seizure resistance when common forms of focal and generalized epilepsy are analyzed as a group. These data suggest that this missense variation in KCNJ10 (or a nearby variation) is related to general seizure susceptibility in humans.

Mature male and female mice from six inbred stains were tested for susceptibility to behavioral seizures induced by a single injection of cocaine. Cocaine was injected ip over a range of doses (50-100 mg/kg) and behavior was monitored for 20 minutes. Seizure end points included latency to forelimb or hindlimb clonus, latency to clonic running seizure and latency to jumping bouncing seizure. A range of strain specific sensitivities was documented with A/J and SJL mice being most sensitive and C57BL/6J most resistant. DBA/2J, BALB/cByJ and NZW/LacJ strains exhibited intermediate sensitivity. EEG recordings were made in SJL, A/J and C57BL/6J mice revealing a close correspondence between electrical activity and behavior. Additionally, levels of cocaine determined in hippocampus and cortex were not different between sensitive and resistant strains. Additional studies of these murine strains may be useful for investigating genetic influences on cocaine-induced seizures.

DBA/2J (D2) and C57BL/6J (B6) mice exhibit differential sensitivity to seizures induced by various chemical and physical methods, with D2 mice being relatively sensitive and B6 mice relatively resistant. We conducted studies in mature D2, B6, F1, and F2 intercross mice to investigate behavioral seizure responses to pentylenetetrazol (PTZ) and to map the location of genes that influence this trait. Mice were injected with PTZ and observed for 45 min. Seizure parameters included latencies to focal clonus, generalized clonus, and maximal seizure. Latencies were used to calculate a seizure score that was used for quantitative mapping. F2 mice (n = 511) exhibited a wide range of latencies with two-thirds of the group expressing maximal seizure. Complementary statistical analyses identified loci on proximal (near D1Mit11) and distal chromosome 1 (near D1Mit17) as having the strongest and most significant effects in this model. Another locus of significant effect was detected on chromosome 5 (near D5Mit398). Suggestive evidence for additional PTZ seizure-related loci was detected on chromosomes 3, 4, and 6. Of the seizure-related loci identified in this study, those on chromosomes 1 (distal), 4, and 5 map close to loci previously identified in a similar F2 population tested with kainic acid. Results document that the complex genetic influences controlling seizure response in B6 and D2 mice are partially independent of the nature of the chemoconvulsant stimulus with a locus on distal chromosome 1 being of fundamental importance.

Agarose gel electrophoresis with ethidium bromide staining (AGE/EBS) is an efficient and reliable method for analyzing microsatellite polymorphisms. We report the use of AGE/EBS for analyzing DNA microsatellite polymorphisms in a preliminary quantitative trait loci (QTL) study of seizure susceptibility in which a candidate gene strategy was used to direct initial mapping efforts. F2 intercross progeny, derived from seizure-sensitive DBA/2J (D2) and seizure-resistant C57BL/6J (B6) inbred strains of mice, were tested for their sensitivity to the seizure-inducing effect of pentylenetetrazol (PTZ), a gamma-aminobutyric acid (GABA) receptor antagonist. A semi-automated method is described, in which DNA microsatellites were amplified by polymerase chain reaction (PCR) to yield products of 100-200 base pair (bp) in length. Alleles were separated on 3-6% MetaPhor agarose gels, stained with ethidium bromide, and visualized by ultraviolet (UV) illumination. Univariate analysis of genotype and phenotype data provides evidence for a seizure-related QTL on chromosome 5, near genes coding for the GABAA receptor subunits alpha 5 and gamma 3. Interestingly, this suggestive QTL derives from the more resistant B6 strain, but it nonetheless provides impetus for the characterization of possible strain differences in these two candidate genes. Overall, these results demonstrate that AGE/EBS can be useful for rapid screening of genomic regions of special interest in QTL mapping studies.

C57BL/6J (B6) and DBA/2J (D2) mice have been characterized previously as seizure-resistant and seizure-sensitive, respectively, a distinction based primarily upon a differential response to the convulsant effects of various drugs. In the present study, electroconvulsive shock (ECS) was used to assess maximal electroshock threshold (MET) in B6, D2 and hybrid mice. Results revealed that D2 mice have a significantly lower MET compared to B6 mice. There was also a significant gender effect for B6 and F2 mice with females exhibiting a lower MET compared to males. METs for F1 and F2 intercross mice were intermediate between the two parental strains. The difference in variance between F2 and F1 generation mice indicated that about three-quarters of the total variance is due to genetic influence. Taken together, results of this study suggest that the large difference in MET between B6 and D2 mice is a highly heritable trait which may yield to genetic dissection through use of quantitative trait locus mapping., (Copyright 1998 Elsevier Science B.V.)

Glutamate and its receptors represent the major excitatory neurotransmission system in the mammalian brain and are considered important in the pathogenesis of many neurological diseases. The present study describes saturation binding experiments performed to measure the affinity (Kd) and density (Bmax) of kainate and AMPA receptors in striatum, cortex and hippocampus from mature DBA/2J (DBA) and C57BL/6J (C57) mice. Previous studies have documented that these two strains differ significantly in seizure susceptibility, with DBA mice exhibiting greater sensitivity in various convulsant tests compared to C57 mice. Non-linear regression analysis of binding data together with Student's t-test and ANOVA revealed significantly higher densities of kainate receptors in striatum and of AMPA receptors in cortex of DBA mice. C57 mice exhibited higher striatal [3H]AMPA binding. There were no significant differences between the mouse strains in binding sites prepared from hippocampus and no differences in affinity for either receptor in any brain region studied. The results support a role for kainate and AMPA receptors in seizure sensitivity, possibly by influencing glutamate transmission in specific pathways. It is unlikely, however, that these receptors account for the generation of seizures alone but rather cooperate with other glutamatergic and non-glutamatergic neurotransmitter systems.

Mature DBA/2J (D2) and C57BL/6J (B6) mice aged 9-10 weeks were studied to determine susceptibility to behavioral seizures induced by kainic acid (KA) and the possible influence exerted by differences in metabolism and blood-brain barrier (BBB) transport. Mice were observed for 4 h after subcutaneous (s.c.) KA injection. Behavioral seizure parameters included latency to first seizure (clonus), latency to tonic/clonic seizure, and latency to status epilepticus (SE). At a KA dose of 25 mg/kg, 80% of D2 mice exhibited tonic/clonic seizures, whereas all B6 mice remained seizure-free. At 30 mg/kg, tonic/clonic seizures were observed in 100% of D2 mice and 25% of B6 mice. Of D2 mice exhibiting at least one clonic seizure in response to KA at a dose of 25 mg/kg, 50% entered SE and eventually died. Administration of [3H]KA (6.6 x 10(6) dpm) at doses of 25 mg/kg (convulsive) or 11.1 micrograms (nonconvulsive) to mice of both strains resulted in similar levels of radioactivity in cortex, hippocampus, and cerebellum 30 and 60 min after injection. Bioconversion of [3H]KA to a radiolabeled brain metabolite in vivo could not be documented in mice from either strain. Results confirm previously reported differences between D2 and B6 mice in their relative susceptibility to seizures induced by systemic KA administration and suggest that these differences are not related to strain-specific variation in metabolism or BBB transport of KA. Further studies of these two strains of mice may be useful for investigating genetic influences upon seizure susceptibility.

This study reports comparative dose-response data for kainic acid (KA) induced seizures in juvenile (35-40 days old) and adult (70-90 days old) Wistar-Furth (WF), Fisher 344 (F344), Sprague-Dawley (SD) and Long-Evans Hooded (LEH) rats. Juvenile male WF (n = 51), F344 (n = 55), SD (n = 60), LEH (n = 50) and adult male WF (n = 48), F344 (n = 52), SD (n = 52), LEH (n = 53) rats were given KA 6, 8, 10, 12 or 14 mg/kg, sc. As previously demonstrated adult WF and F344 rats showed the greatest sensitivity and most reliable convulsant responses to kainic acid; SD and LEH rats were less sensitive and showed more variable convulsant responses. Regardless of strain, all juvenile rats exhibited greater sensitivity and less variable convulsant response to KA compared to adults. This was most evident in juvenile SD and LEH rats. Results suggest that while seizure sensitivity to KA decreases with age, genetic factors may regulate the expression of this resistance.

We describe a strain of rats (Wistar-Furth) that is highly susceptible to the neurotoxic effects of kainic acid (KA) and presents a reliable and quantifiable (with low within-group variability) animal model of status epilepticus. Wistar-Furth rats are more sensitive and demonstrate a less variable convulsant response than Sprague-Dawley and Long-Evans rats when tested for total time in seizure activity, latency to onset of first seizure, latency to status epilepticus, seizure severity scores, and percentage exhibiting behavioral seizures and status epilepticus. Results suggest that significant heterogeneity exists in the rodent population with regard to neuronal sensitivity to an excitotoxic amino acid and indicate that strain differences are an important consideration in studies using KA.

Homotaurine and taurine are two powerful inhibitory aminoacids with anticonvulsant properties against various experimental models of focal epilepsy. This study reports on their effects in the feline model of corticoreticular epilepsy induced by parenteral administration of large amounts of penicillin. Both aminoacids, but particularly homotaurine, remarkably potentiate epileptiform discharges in cats. Brainstem transection at the precollicular level does not modify the activation, thus ruling out the intervention of mesoromboencephalic structures in the observed effect. The opposing action of these two amino acids on focal epilepsy as compared to corticoreticular epilepsy suggests that the two types of epileptiform activity stem from very different pathophysiological mechanisms. Homotaurine is a powerful GABA agonist that exerts a central action upon parenteral administration. Other GABA analogs such as muscimol, imidazole acetic acid, and gamma-hydroxybutyrate have been reported to potentiate experimental models of spike and wave epilepsy. Thus, the activating effects of homotaurine in this epilepsy model are in keeping with the demonstrated GABAmimetic properties of the compound.

Generalized corticoreticular epilepsy was established in adult cats by parenteral penicillin, and electroencephalographic monitoring was carried out. Ketamine HCl was injected intravenously in doses of 2.5 to 20 mg per kilogram. If doses of penicillin were inadequate to establish typical spike-wave activity, ketamine induced the spike-wave pattern typical of much higher doses of penicillin. At doses of penicillin that established the spike-wave pattern, ketamine potentiated the spike-wave activity and sometimes induced spike-and-wave status. These findings suggest caution in the clinical use of ketamine in patients with corticoreticular epilepsy. Because analogous effects have been observed upon administration of GABA-mimetic agents, GABA systems may play a role in ketamine anesthesia and corticoreticular epilepsy. Precollicular brain transections failed to modify ketamine effects, excluding a possible influence of mesencephalic centers on the observed potentiation.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg s.c. for 5 days) to mice resulted in complete abolishment of strychnine seizure and of the tonic phase of the maximal electroshock response. Bicuculline and picrotoxin convulsions were not significantly affected by MPTP treatment. The severity of the pentylenetetrazole seizures was mildly, but significantly affected in the protective way. MPTP depleted neostriatal dopamine and its metabolites, together with hippocampal norepinephrine. No nigral neuronal loss was detected histologically. Strychnine seizures and the tonic phase of the maximal electroshock response are thought to depend mostly on hindbrain (bulbo-spinal) structures. Thus, these experiments suggest that a caudally projecting system originates from the substantia nigra, pars compacta, and/or locus coeruleus, controlling seizures that involve bulbo-spinal centers. While neostriatal dopamine depletion offers a good index of seizure resistance, its role in the protection from seizures remains to be established.

Direct GABA agonists that suppress spikes induced by penicillin in cats failed to do so in rats. Phenytoin and large doses of THIP increased the rate of spiking activity of the penicillin focus. Only progabide caused marked, initial, short-lasting suppression and a modest reduction of frequency of spikes for 1 hr. Homotaurine (3APS) reduced the amplitude and changed the morphology of the contralateral "mirror" spike. Antagonism of penicillin-induced spikes in rats is considered to be an unsuitable parameter for the screening of anticonvulsant agents.

Intracerebral injection of L-acetylcarnitine in rats induced interictal and ictal epileptic phenomena with immediate onset, lasting up to 4 h. Pretreatment with systemic atropine prevents all epileptiform phenomena. Local injection of muscimol and THIP abolish ictal events, but not single spikes. L-carnitine induced only ictal discharges with a latency of 40-90 min. Acetylcarnitine epileptogenic properties are probably related to muscarinic agonism. The transition from interictal to ictal events may involve failure of GABAergic mechanisms.

Pretreatment of rats with homotaurine (3 aminopropanesulfonic acid; 3APS), a synthetic gamma-aminobutyric acid (GABA) analog, protected from the convulsant and cytotoxic action of systemically injected kainic acid (KA). Wet dog shaking (WDS) behavior was significantly reduced. Taurine, an inhibitory non-GABA-mimetic amino acid, and muscimol (another direct GABA-agonist) reduced the number of seizures and lesions in the brain but were less effective than homotaurine. Progabide (a GABA-agonist) did not modify kainic acid effects. The neurotoxicity of kainic acid could have been due to repetitive convulsive activity. Activation of GABA-mediated inhibition is an effective, but not the determinant means of preventing KA-induced abnormalities.