HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizuresusceptible El mice play an important role in the pathogenesis of seizures.     

Alteration of opioid receptors in seizure-susceptible El mouse brain

The distribution density of opioid receptors in the brain of El mice (seizure-susceptible strain) was examined to determine the relation between seizures and the opioid system. Saturation curves and Scatchard plots of [³H]2-d-alamine-5-d-leucine enkephalin binding revealed that the opioid delta receptor density in adult El mice during interictal periods was significantly increased in the cerebral cortex, hippocampus, and septal area. It was further shown that the concentration of such receptors in 25-day-old El mice that had no seizures was also significantly increased in the hippocampus and septal area, with no changes in apparent affinities, as compared with in the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). Such up-regulation of opioid receptors in the El mouse brain could result from deficits in endogenous opioid peptides, which could be associated with the pathogenesis of seizure diathesis in the El mouse.     

Metabolic profile of opioid peptides differs in the hippocampus and striatum of seizure-susceptible E1 mice

We previously suggested that a deficit of anticonvulsant endogenous methionine enkephalin, in the cerebral cortex, septal area, hippocampus, and striatum of seizure-susceptible E1 mice plays a role in the pathogenesis of seizures. To determine whether a hypofunction of enkephalinergic neuron may be due to metabolic abnormalities of opioid peptides in the E1 mouse brain, we measured methionine enkephalin-like immunoreactivity (ME-LI) of 50 fractions eluted by high performance liquid chromatography obtained from those four regions of the brain of E1 and seizure-nonsusceptible ddY mice (the mother strain of E1 mice). We observed the same ME-LI patterns of 50 fractions in the cerebral cortex and septal area in E1 and ddY mice, whereas exhibited differing ME-LI patterns in the hippocampus and striatum in the two stains. Different ME-LI patterns may imply the difference in the metabolic profile of opioid peptides. Thus, an abnormal metabolism of opioid peptides in the hippocampus and striatum of the E1 mouse may be involved in the pathogenesis of seizures.     

Differences in ME-LI and VIP-LI in discrete brain regions of seizure-naive and seizure-experienced El mice

In an attempt to elucidate the relationship between endogenous methionine-enkephalin (ME) and vasoactive intestinal polypeptide (VIP) with generalized seizures, we determined regional brain levels of ME-like and VIP-like immunoreactivity (ME-LI and VIP-LI) in El mice during and after seizures induced by repeated tossing stimulation. The levels of ME-LI in the striatum and hippocampus of seizure-naive El mice (El–) were lower than those of the control ddY mice, the mother strain of El mice. Conversely, the level of VIP-LI in the medulla oblongata and pons of El– was higher than that of ddY mice. The level of ME-LI in the striatum of seizure-experienced El mice (El+) killed 96 hours after three consecutive seizures was high, while levels of VIP-LI in the striatum and hypothalamus were low, in comparison to those of El– mice. A detailed time-course study revealed that seizures in El mice caused (1) significant decreases in levels of ME-LI in the striatum and hippocampus during seizures, (2) a significant decrease of VIP-LI content in the striatum 3 hours after seizures, and (3) a significant increase in hypothalamic VIP-LI 9 hours after seizures. These observations suggest that ME and VIP may play some role in El mouse seizures.     

Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis

Abstract: Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 ± 2.1 days (mean ± SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40–60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1–6), dynorphin-A (1–8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.     

Developmental and Regional Alteration of κ-Opioid Receptors in Seizure-Susceptible EL Mouse Brain

The binding of [³H]ethylketocyclazocine ([³H]EKC) under the suppression of and sites in the brain of EL mice (seizure-susceptible) was examined to determine the relationship between seizures and the dynorphinergic system. The density of -opioid receptors in the cerebrum of adult EL mice during interictal periods significantly increased, without changes in apparent affinities, compared with that of adult ddY mice (seizure-nonsusceptible; the mother strain of EL). Subsequently, the binding of 0.8 nM [³H]EKC in 8 brain regions was examined in the 2 strains. The [³H]EKC binding in 25-day-old EL mice that had no seizures significantly increased in the hippocampus and amygdala. At the age of 50 days, EL mice displayed abortive seizures, and the number of sites in EL mice was significantly greater in the hippocampus, amygdala and cerebral cortex. It was further shown that the binding of [³H]EKC in 150-day-old adult EL mice during interictal periods was markedly increased in the hippocampus, amygdala, cerebral cortex and striatum, compared with the corresponding regions in ddY mice. The up-regulation of receptors in the EL mouse brain may result from deficits in endogenous dynorphins, which could be involved in the pathogenesis of seizure diathesis and seizure manifestations in the EL mouse.     

The anticonvulsant effect of citalopram on El mice,and the levels of tryptophan and tyrosine and their metabolites in the brain

Serotonin (5-HT) plays an important role in the seizures of El mice since the seizure threshold of El mice correlates with the 5-HT concentration in the central nervous system. In this study, the anticonvulsant effect of a 5-HT reuptake blocker, citalopram, was evaluated behaviorally and biochemically. El mouse convulsions were inhibited by oral administration of citalopram for 2 weeks. Citalopram increased tryptophan and tyrosine amounts, and decreased the 5-HT, 5-hydroxy-indoleacetic acid, kynurenine, and dopamine amounts in the brain. These findings show that citalopram depresses monoaminergic metabolism. Given the known convulsant effect of kynurenine, it is suggested that its decrease by citalopram may involve attenuation of El mice seizures.     

Age- and seizure-related changes in noradrenaline and dopamine in several brain regions of epileptic El mice

Noradrenaline (NA) and dopamine (DA) levels in six brain regions of stimulated and nonstimulated El (El[s] and El[ns]) mice and their maternal ddY mice were determined at various ages and various times after a convulsion. The NA levels in the striatum and hippocampus of 12-week-old El[s] and El[ns] mice were lower than in ddY mice, and remained lower in 23-week-old El[s] mice, but not in El[ns] mice. DA levels were lower in the striatum of El[s] mice than in El[ns] and ddY mice at 16 and 23 weeks of age. NA levels decreased during seizure in the striatum and hippocampus of El[s] mice, and returned to preconvulsive levels 1 hr after convulsion in the striatum and 30 min in the hippocampus. DA levels in the striatum of El[s] mice decreased during convulsion and increased from 1 to 10 min after convulsion. These changes suggest that the NAergic systems in the striatum and hippocampus and the DAergic system in the striatum have important roles in relation to seizure susceptibility in El mice.     

Endogenous methionine enkephalin may play an anticonvulsant role in the seizure-susceptible El mouse

After the intracisternal injection of three protease inhibitors which prevent the degradation of methionine enkephalin (amastatin, Des-Pro²-bradykinin, and phosphoramidon) and a mixture of these protease inhibitors, we investigated the effect on convulsive seizures in the seizure-susceptible El mouse. We also measured the cerebral methionine enkephalin content by high-performance liquid chromatography coupled with radioimmunoassay. Protease inhibitors significantly decreased both the incidence of seizures and the seizure score in El mice in a dose-dependent manner. This anticonvulsant effect was reversed by naloxone (2 mg/kg, sc). The cerebral methionine enkephalin content increased significantly after the administration of protease inhibitors in comparison with saline injection. These findings suggest that it was not protease inhibitors but instead increase of endogenous methionine enkephalin that reduced the incidence of seizures and the seizure score in El mice. Together with our previous data, the present findings support our hypothesis that a deficit in anticonvulsant endogenous methionine enkephalin is involved in the pathogenesis of seizures in the El mouse.     

Effects of morphine andd-Ala2-d-Leu5-enkephalin in the seizure-susceptible El mouse

Opioid agonists were used to investigate the modulation of seizures in the seizure-susceptible El mouse. Morphine andd-Ala²-d-Leu⁵-enkephalin (DADLE) were injected subcutaneously or intracisternally as prototypic agonists for and opioid receptors. Systemic or intracisternal injection of both morphine and DADLE decreased the incidence of seizures and the seizure score in El mice in a dose-dependent manner. The anticonvulsant effects of morphine and DADLE were reversed by naloxone (2 mg/kg, s.c.). This implies that opioid agonists have anticonvulsant properties which are mediated by and opioid receptors. In conclusion, a deficit in endogenous opioid peptides, which act as anticonvulsants may play a significant role in the etiology or pathophysiology of seizures in the El mouse.     

Regional distribution of tyrosine,tryptophan, and their metabolites in the brain of epileptic El mice

Tyrosine, tryptophan, and their metabolites in the brain of ddY, non-stimulated El (El (–)), and stimulated El (El (+)) mice were measured using the three dimensional HPLC. The tryptophan content was lower in El (+) than ddY and El (–) mice. The 5-hydroxytrytophan content was much higher in both El groups. The serotonin content of El (+) was higher than that of ddY and El (–) mice. The kynurenine content was remarkably high in the El mice. The dopamine content was lower in El (–) than in ddY mice, whereas it was greater in El (+) than in El (–) mice. The norepinephrine showed higher levels in El (+) mice. These facts suggest that El mice posess congenital metabolic abnormalities of tryptophan and tyrosine and that kynurenine may play an important role as convulsant in El mice seizures along with changes in serotonin, dopamine, and norepinephrine that are inhibitory agents and responded to the repetitive convulsions.     

Characterization of febrile seizures and febrile seizure susceptibility in mouse inbred strains

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10- to 14-day-old mice and induced FS in all animals. Under the conditions used, seizure-induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.     

Enhanced Aspartate Release from Hippocampal Slices of Epileptic (El) Mice

The release of putative neurotransmitters [aspartate, glutamate, and gamma-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 microns thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatography with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice +/- SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 +/- 2.62 and 15.41 +/- 3.56), or GABA (17.28 +/- 2.90 and 12.73 +/- 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 +/- 0.69) than in the B6 mice (3.31 +/- 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.     

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.     

Genetic and environmental interactions determine seizure susceptibility in epileptic EL mice

Gene identification has progressed rapidly for monogenic epilepsies, but complex gene-environmental interactions have hindered progress in gene identification for multifactorial epilepsies. We analyzed the role of environmental risk factors in the inheritance of multifactorial idiopathic generalized epilepsy in the EL mouse. Seizure susceptibility was evaluated in the EL (E) and seizure-resistant ABP/LeJ (A) parental mouse strains and in their AEF1 and AEF2 hybrid offspring using a handling-induced seizure test. The seizure test was administered in three environments (environments I, II and III) that differed with respect to the number of seizure tests administered (one test or four tests) and the age of the mice when tested (young or old). The inheritance of seizure susceptibility appeared dominant after repetitive seizure testing in young or old mice, but recessive after a single test in old mice. Heritability was high (0.67-0.77) in each environment. Significant quantitative trait loci (QTL) that were associated with environments I and III (repetitive testing) were found on chromosomes 2 and 9 and colocalized with previously mapped El2 and El4, respectively. The El2 QTL found in environment I associated only with female susceptibility. A novel QTL, El-N, for age-dependent predisposition to seizures was found on proximal chromosome 9 only in environment II. The findings indicate that environmental risk factors determine the genetic architecture of seizure susceptibility in EL mice and suggest that QTL for complex epilepsies should be defined in terms of the environment in which they are expressed.     

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 forglial 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.     

Regional excitatory and inhibitory amino acid levels in epileptic El mouse brain

Inbred mutant El mice are highly susceptible to convulsive seizures upon tossing stimulation. The levels of excitatory (e.g. glutamate and aspartate) and inhibitory amino acids [e.g. -aminobutyrate (GABA)] were examined in discrete regions of stimulated El mice [El(+)] non-stimulated El mice [El(-)] and ddY mice, which do not have convulsive disposition. In comparison with ddY, a general increased levels of aspartate, glutamate, glutamine, and taurine were detected in brain regions of El(-). The levels of GABA and glycine were almost the same in ddY and El(-). Compared to El(+), the levels of aspartate, glutamate, glutamine, and GABA in El(-) were either the same or higher. In the case of taurine and glycine, the levels in El(-) were either the same or lower than El(+). Alanine is special in that El(-) have a higher level than El(+) in hippocampus but lower in cerebellum. Furthermore, while marked changes were registered in several brain regions, none of the amino acids investigated showed any significant differences in the hypothalamus of three different groups of mice.     

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 × PL/J)F1 mice was similar to the penetrance in (C57BL/6 J × PL/J)F1 mice but the severity and frequency of seizure were higher in (DBA/2 J × PL/J)F1 mice. The PL/J strain serves as an interesting new model for studying the genetics, neurobiology, and pharmacology of epilepsy.     

Use of chromosome substitution strains to identify seizure susceptibility loci in mice

Seizure susceptibility varies among inbred mouse strains. Chromosome substitution strains (CSS), in which a single chromosome from one inbred strain (donor) has been transferred onto a second strain (host) by repeated backcrossing, may be used to identify quantitative trait loci (QTLs) that contribute to seizure susceptibility. QTLs for susceptibility to pilocarpine-induced seizures, a model of temporal lobe epilepsy, have not been reported, and CSS have not previously been used to localize seizure susceptibility genes. We report QTLs identified using a B6 (host) × A/J (donor) CSS panel to localize genes involved in susceptibility to pilocarpine-induced seizures. Three hundred fifty-five adult male CSS mice, 58 B6, and 39 A/J were tested for susceptibility to pilocarpine-induced seizures. Highest stage reached and latency to each stage were recorded for all mice. B6 mice were resistant to seizures and slower to reach stages compared to A/J mice. The CSS for Chromosomes 10 and 18 progressed to the most severe stages, diverging dramatically from the B6 phenotype. Latencies to stages were also significantly shorter for CSS10 and CSS18 mice. CSS mapping suggests seizure susceptibility loci on mouse Chromosomes 10 and 18. This approach provides a framework for identifying potentially novel homologous candidate genes for human temporal lobe epilepsy.