Kazumaru Wada

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Congenital malformations due to antiepileptic drugs

To identify the major risk factors for the increased incidence of congenital malformations in offspring of mothers being treated for epilepsy with antiepileptic drugs (AEDs) during pregnancy and, to determine the relative teratogenic risk of AEDs, we prospectively analyzed 983 offspring born in Japan, Italy, and Canada. The incidence of congenital malformations in offspring without drug exposure was 3.1%, versus an incidence with drug exposure of 9.0%. The highest incidence in offspring exposed to a single AED occurred with primidone (PRM; 14.3%), which was followed by valproate (VPA; 11.1%), phenytoin (PHT; 9.1%), carbamazepine (CBZ; 5.7%), and phenobarbital (PB; 5.1%). The VPA dose and level positively correlated with the incidence of malformations. This study first determined a cut-off value of VPA dose and level at 1000 mg/day and 70 microg/ml, respectively, to avoid the occurrence of malformations. The incidence of malformations increases as the number of drugs increases, and as the total daily dose increases. Specific combinations of AEDs such as VPA + CBZ and PHT + PRM + PB produced a higher incidence of congenital malformations. The incidence of malformations was not associated with any background factors studied except for the presence of malformations in siblings. These results indicate that the increased incidence of congenital malformations was caused primarily by AEDs, suggesting that malformations can be prevented by improvements in drug regimen, and by avoiding polypharmacy and high levels of VPA (more than 70 microg/ml) in the treatment of epileptic women of childbearimg age.

Determination of the effects of caffeine and carbamazepine on striatal dopamine release by in vivo microdialysis

The effects of carbamazepine and caffeine on adenosine receptor subtypes were determined using in vivo microdialysis in an attempt to elucidate their different psychotropic mechanisms of action. Adenosine and a selective adenosine A1 receptor agonist decreased the striatal extracellular dopamine level, whereas caffeine, carbamazepine and a selective adenosine A1 receptor antagonist increased it, but neither an adenosine A2 receptor agonist nor an antagonist affected it. Under conditions of adenosine A1 receptor blockade, adenosine, carbamazepine and a selective adenosine A2 receptor agonist increased the striatal extracellular dopamine level, whereas caffeine and a selective adenosine A2 receptor antagonist decreased it. These results suggest that adenosine A1 receptor stimulation reduces the striatal extracellular dopamine level, and that adenosine A2 receptor stimulation under conditions of adenosine A1 receptor blockade increases it. Therefore, caffeine is an antagonist of both adenosine A1 and A2 receptor subtypes, and carbamazepine is an adenosine A1 receptor antagonist as well as an adenosine A2 receptor agonist. These properties support the hypothesis that the central actions of both carbamazepine and caffeine result from effects on both adenosine A1 and A2 receptors.

Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode

1 Multiple components of hippocampal glutamate release were examined by study of Ca2+‐ and K+‐evoked hippocampal extracellular glutamate release using an in vivo microdialysis glutamate biosensor in urethane‐anaesthetized rats. In addition, the effects of the antiepileptic drugs, carbamazepine (CBZ) and zonisamide (ZNS) perfused through the probe on glutamate release were assessed. 2 Basal glutamate levels were below detection limits (∼0.1 μm). An increase in extracellular KCl (from 2.7 to 50 and 100 mm) increased extracellular hippocampal glutamate levels to 9.2±1.4 and 20.0±2.6 μm, respectively, calculated from the area under curve (AUC) for 60 min. 3 This KCl‐evoked glutamate release consisted of three components: an initial transient rise, a late gentle rise, and late multiple phasic transient rises. 4 An increase in or removal of extracellular CaCl2 levels respectively enhanced and reduced the 50 mm KCl‐evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 12.4±2.1 and 5.8±0.9 μm. 5 Perfusion with 100 μm CBZ or 1 mm ZNS inhibited both the 50 mm KCl‐evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 5.5±1.1 and to 5.8±1.3 μm, respectively, as well as the stimulatory effects of Ca2+ on KCl‐evoked hippocampal glutamate release. 6 These results suggest that both CBZ and ZNS may reduce epileptiform events by inhibiting excitatory glutamatergic transmission.

Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5‐HT) release and 5‐HT reuptake activity, hippocampal extracellular 5‐HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5‐HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5‐HT levels. Adenosine and A1 receptor agonist, 2‐chloro‐N6‐cyclopentyl‐adenosine (CCPA), decreased extracellular 5‐HT levels, whereas non‐selective antagonist, caffeine, and A1 antagonist, 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT) increased them. When 5‐HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5‐HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX), and A3 receptor agonist, N6–2‐(4‐aminophenyl)ethyladenosine (APNEA) did not affect 5‐HT levels; however, when A1 receptor was blocked by CPT, 5‐HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5‐HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5‐HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5‐HT reuptake activity might be modulated by hippocampal A3 receptor.

A novel mutation of KCNQ3 (c.925T→C) in a Japanese family with benign familial neonatal convulsions

At present, only one mutation of KCNQ3, a KCNQ potassium channel gene, has been identified as a cause of benign familial neonatal convulsions type 2 (BFNC2). We found a T to C substitution (c.925T→C) on one allele of affected individuals in a Japanese family with BFNC but not on 200 alleles from healthy subjects. c.925T→C replaced Trp309, a conserved residue within the P‐loop of the KCNQ potassium channel family that holds the channel pore open, with an Arg (W309R). We report c.925T→C as the second mutation of KCNQ3 responsible for BFNC2. Ann Neurol 2000;47:822–826

Biphasic effects of zonisamide on serotonergic system in rat hippocampus

To study the mechanisms of antiepileptic action of zonisamide (ZNS), we determined the effects of ZNS on extracellular, total levels and re-uptake activity of serotonin (5-HT) in rat striatum and hippocampus. After acute administrations, plasma ZNS concentrations associated with anticonvulsive action (effective concentrations) increased the total levels of 5-HT, its metabolise (5-hydroxyindoleacetic acid: 5-HIAA) and precursor (5-hydroxytryptophan: 5-HTP). After chronic administration of ZNS, effective plasma concentrations also increased the extracellular and total levels of 5-HT, 5-HIAA, and 5-HTP. On the other hand, after both acute and chronic administrations of ZNS, a supra-effective ZNS concentration either decreased or did not affect the total levels of these substances. Therefore, the stimulatory effects of ZNS on the 5-HT system were reduced by an increase in ZNS concentration to supra-effective concentrations. ZNS concentrations of 30-1000 microM did not affect hippocampal 5HT re uptake activity in vitro. These results suggest that ZNS has biphasic effects on the 5-HT system, in that effective concentrations of ZNS enhance and supra-effective concentrations of ZNS reduce the function of the 5-HT system. These biphasic effects of ZNS on the 5-HT system may be involved in the mechanisms of action of the antiepileptic and psychotropic effects, and side effects of ZNS.

Autosomal dominant epilepsy with febrile seizures plus with missense mutations of the (Na^+)-channel α1 subunit gene, SCN1A

Evidence that febrile seizures have a strong genetic predisposition has been well documented. In families of probands with multiple febrile convulsions, an autosomal dominant inheritance with reduced penetrance is suspected. Four candidate loci for febrile seizures have been suggested to date; FEB1 on 8q13-q21, FEB2 on 19p, FEB3 on 2q23-q24, and FEB4 on 5q14-15. A missense mutation was identified in the voltage-gated sodium (Na(+))-channel beta 1 subunit gene, SCN1B at chromosome 19p13.1 in generalized epilepsy with the febrile seizures plus type 1 (GEFS+1) family. Several missense mutations of the (Na(+))-channel alpha 1 subunit (Nav1.1) gene, SCN1A were also identified in GEFS+2 families at chromosome 2q23-q24.3. The aim of this report is precisely to describe the phenotypes of Japanese patients with novel SCN1A mutations and to reevaluate the entity of GEFS+. Four family members over three generations and one isolated (phenotypically sporadic) case with SCN1A mutations were clinically investigated. The common seizure type in these patients was febrile and afebrile generalized tonic-clonic seizures (FS+). In addition to FS+, partial epilepsy phenotypes were suspected in all affected family members and electroencephalographically confirmed in three patients of two families. GEFS+ is genetically and clinically heterogeneous, and associated with generalized epilepsy and partial epilepsy as well. The spectrum of GEFS+ should be expanded to include partial epilepsies and better to be termed autosomal dominant epilepsy with febrile seizures plus (ADEFS+).

Biphasic effects of carbamazepine on the dopaminergic system in rat striatum and hippocampus

To clarify the effects of carbamazepine (CBZ) on dopamine (DA) release and their metabolism, the extracellular and total levels of DA, its metabolites (DOPAC and HVA) and precursor, 3,4-dihydroxyphenylalanine (DOPA) in the striatum and hippocampus were studied. DA re-uptake and DOPA accumulation in the striatum and hippocampus, and monoamine oxidase (MAO) activities were also determined. After acute and chronic administrations of CBZ, the plasma concentration of CBZ associated with therapeutic activity increased the extracellular and total levels of all substances determined, whereas supratherapeutic concentration of CBZ decreased extracellular and total levels of all substances. Neither therapeutic nor supratherapeutic concentrations of CBZ affected MAO-A nor -B activities, nor DA re-uptake. DOPA accumulation caused by NSD1015 was inhibited by therapeutic and supratherapeutic concentrations of CBZ. These results suggest that a therapeutic concentration of CBZ enhances DA turnover, whereas a supratherapeutic concentration of CBZ inhibits DA turnover. These effects of CBZ on dopaminergic systems may be, at least partially, involved in the mechanisms of action of CBZ.

Effects of carbamazepine on hippocampal serotonergic system

To establish the mechanism of action of the antiepileptic and psychotropic effects of carbamazepine (CBZ), its effects on serotonin (5-HT) transmission, metabolism and re-uptake activity in the rat hippocampus were studied. After acute and chronic administrations of 25 mg/kg CBZ, the plasma concentration of CBZ was found to be within the therapeutic range, whereas both acute and chronic administrations of 50 and 100 mg/kg CBZ resulted in a supratherapeutic plasma concentration. Acute administration of the therapeutic dose of CBZ resulted in an increase in the hippocampal extracellular and total level of 5-HT, its metabolite, 5-hydroxydoleacetic acid (5-HIAA) and its precursor, 5-hydroxytryptophan (5-HTP). The acute administration of 50 mg/kg CBZ resulted in an increase in the hippocampal levels of extracellular 5-HT and 5-HIAA as well as in the total levels of 5-HTP, whereas hippocampal levels of extracellular 5-HTP, total 5-HT and 5-HIAA remained unaffected. CBZ at a dose of 100 mg/kg decreased the levels of all of these substances. After chronic administration, 25 mg/kg/day CBZ increased hippocampal total levels of 5-HT, 5-HTP and 5-HIAA, whereas 100 mg/kg/day CBZ decreased all of these total levels. CBZ at a dose of 50 mg/kg/day decreased total levels of 5-HT, however neither total levels of 5-HIAA nor 5-HTP were affected. Both therapeutic and supratherapeutic plasma concentrations of CBZ inhibited 5-HTP accumulation, and did not affect 5-HT re-uptake activity in vitro. These results suggest that a therapeutic concentration of CBZ enhances 5-HT turnover and transmission, whereas a supratherapeutic concentration of CBZ inhibits 5-HT turnover and transmission without affecting 5-HT re-uptake activity. These effects of CBZ on serotonergic systems may be, at least partially, involved in the mechanisms of action of CBZ.