A missense mutation of the gene encoding voltage-dependent sodium channel (Nav1.1) confers susceptibility to febrile seizures in rats.

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Na(v)1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic-clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Na(v)1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

Scn1a missense mutation causes limbic hyperexcitability and vulnerability to experimental febrile seizures.

Mutations of the voltage-gated sodium (Na(v)) channel subunit SCN1A have been implicated in the pathogenesis of human febrile seizures including generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy in infancy (SMEI). Hyperthermia-induced seizure-susceptible (Hiss) rats are the novel rat model carrying a missense mutation (N1417H) of Scn1a, which is located in the third pore-forming region of the Na(v)1.1 channel. Here, we conducted behavioral and neurochemical studies to clarify the functional relevance of the Scn1a mutation in vivo and the mechanism underlying the vulnerability to hyperthermic seizures. Hiss rats showed markedly high susceptibility to hyperthermic seizures (mainly generalized clonic seizures) which were synchronously associated with paroxysmal epileptiform discharges. Immunohistochemical analysis of brain Fos expression revealed that hyperthermic seizures induced a widespread elevation of Fos-immunoreactivity in the cerebral cortices including the motor area, piriform, and insular cortex. In the subcortical regions, hyperthermic seizures enhanced Fos expression region--specifically in the limbic and paralimbic regions (e.g., hippocampus, amygdala, and perirhinal-entorhinal cortex) without affecting other brain regions (e.g., basal ganglia, diencephalon, and lower brainstem), suggesting a primary involvement of limbic system in the induction of hyperthermic seizures. In addition, Hiss rats showed a significantly lower threshold than the control animals in inducing epileptiform discharges in response to local stimulation of the hippocampus (hippocampal afterdischarges). Furthermore, hyperthermic seizures in Hiss rats were significantly alleviated by the antiepileptic drugs, diazepam and sodium valproate, while phenytoin or ethosuximide were ineffective. The present findings support the notion that Hiss rats are useful as a novel rat model of febrile seizures and suggest that hyperexcitability of limbic neurons associated with Scn1a missense mutation plays a crucial role in the pathogenesis of febrile seizures.

Scn1a missense mutation impairs GABAA receptor-mediated synaptic transmission in the rat hippocampus.

Mutations of the Na(v)1.1 channel subunit SCN1A have been implicated in the pathogenesis of human febrile seizures (FS). We have recently developed hyperthermia-induced seizure-susceptible (Hiss) rat, a novel rat model of FS, which carries a missense mutation (N1417H) in Scn1a[1]. Here, we conducted electrophysiological studies to clarify the influences of the Scn1a mutation on the hippocampal synaptic transmission, specifically focusing on the GABAergic system. Hippocampal slices were prepared from Hiss or F344 (control) rats and maintained in artificial cerebrospinal fluid saturated with 95% O(2) and 5% CO(2)in vitro. Single neuron activity was recorded from CA1 pyramidal neurons and their responses to the test (unconditioned) or paired pulse (PP) stimulation of the Schaffer collateral/commissural fibers were evaluated. Hiss rats were first tested for pentylenetetrazole-induced seizures and confirmed to show high seizure susceptibility to the blockade of GAGA(A) receptors. The Scn1a mutation in Hiss rats did not directly affect spike generation (i.e., number of evoked spikes and firing threshold) of the CA1 pyramidal neurons elicited by the Schaffer collateral/commissural stimulation. However, GABA(A) receptor-mediated inhibition of pyramidal neurons by the PP stimulation was significantly disrupted in Hiss rats, yielding a significant increase in the number of PP-induced firings at PP intervals of 32-256ms. The present study shows that the Scn1a missense mutation preferentially impairs GABA(A) receptor-mediated synaptic transmission without directly altering the excitability of the pyramidal neurons in the hippocampus, which may be linked to the pathogenesis of FS.

Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling.

The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.

Anticataleptic 8-OH-DPAT preferentially counteracts with haloperidol-induced Fos expression in the dorsolateral striatum and the core region of the nucleus accumbens.

We studied the effects of the 5-HT(1A/7) agonist 8-OH-DPAT on haloperidol-induced catalepsy and forebrain Fos expression in mice to clarify its mechanism in modulating extrapyramidal motor disorders. 8-OH-DPAT (0.1-1mg/kg, i.p.) markedly attenuated haloperidol-induced catalepsy in a dose-dependent manner with a potency greater than that of the antiparkinsonian agent trihexyphenidyl. The anticataleptic action of 8-OH-DPAT was completely antagonized by WAY-100135 (a selective 5-HT(1A) antagonist), but not by SB-269970 (a selective 5-HT(7) antagonist). Depletion of cerebral 5-HT by p-chlorophenylalanine (300mg/kg, i.p. for 3 days) did not attenuate, but rather potentiated the action of 8-OH-DPAT. Furthermore, the anticataleptic dose of 8-OH-DPAT showed a regionally specific reduction of haloperidol-induced Fos expression in the dorsolateral striatum (dlST) and the core region of the nucleus accumbens (AcC), without affecting that in the medial prefrontal cortex, the shell region of the nucleus accumbens or the lateral septal nucleus. These results suggest that 8-OH-DPAT alleviates antipsychotic-associated extrapyramidal motor disorders by stimulating the postsynaptic 5-HT(1A) receptors, which specifically counteracts the D(2) receptor blocking actions of antipsychotics in the dlST and AcC.

Evaluation of the antibradykinetic actions of 5-HT1A agonists using the mouse pole test.

To clarify the role and mechanism of the 5-HT1A receptor in modulating extrapyramidal motor disorders, we studied the actions of 5-HT1A agonists in the mouse pole test, a valid model of parkinsonian bradykinesia. Haloperidol markedly delayed pole-descending behavior of mice in the pole test, and this effect was alleviated by the antiparkinsonian agent trihexyphenidyl (a muscarinic antagonist). The selective 5-HT1A agonists, 8-hydroxydipropylaminotetraline (8-OH-DPAT) and tandospirone, significantly attenuated haloperidol-induced bradykinesia in a dose-dependent manner. The alleviation of haloperidol-induced bradykinesia by 8-OH-DPAT was completely antagonized by WAY-100135 (a selective 5-HT1A antagonist), but was unaffected by cerebral 5-HT depletion with p-chlorophenylalanine (PCPA) treatment (300 mg/kg, i.p. for 3 days). These results suggest that 5-HT1A agonists improve extrapyramidal motor disorders associated with antipsychotic treatments by stimulating the postsynaptic 5-HT1A receptor.

Synaptic vesicle glycoprotein 2A (SV2A) regulates kindling epileptogenesis via GABAergic neurotransmission.

Synaptic vesicle glycoprotein 2A (SV2A) is a prototype synaptic vesicle protein regulating action potential-dependent neurotransmitters release. SV2A also serves as a specific binding site for certain antiepileptics and is implicated in the treatment of epilepsy. Here, to elucidate the role of SV2A in modulating epileptogenesis, we generated a novel rat model (Sv2a(L174Q) rat) carrying a Sv2a-targeted missense mutation (L174Q) and analyzed its susceptibilities to kindling development. Although animals homozygous for the Sv2a(L174Q) mutation exhibited normal appearance and development, they are susceptible to pentylenetetrazole (PTZ) seizures. In addition, development of kindling associated with repeated PTZ treatments or focal stimulation of the amygdala was markedly facilitated by the Sv2a(L174Q) mutation. Neurochemical studies revealed that the Sv2a(L174Q) mutation specifically reduced depolarization-induced GABA, but not glutamate, release in the hippocampus without affecting basal release or the SV2A expression level in GABAergic neurons. In addition, the Sv2a(L174Q) mutation selectively reduced the synaptotagmin1 (Syt1) level among the exocytosis-related proteins examined. The present results demonstrate that dysfunction of SV2A due to the Sv2a(L174Q) mutation impairs the synaptic GABA release by reducing the Syt1 level and facilitates the kindling development, illustrating the crucial role of SV2A-GABA system in modulating kindling epileptogenesis.

Kindling-associated SV2A expression in hilar GABAergic interneurons of the mouse dentate gyrus.

Immunohistochemical studies were performed to analyze the expressional changes in hippocampal synaptic vesicle protein 2A (SV2A) following pentylenetetrazole (PTZ) kindling. Repeated treatments of mice with sub-convulsive PTZ (40 mg/kg, i.p.) for 15 days progressively enhanced seizure susceptibility and induced clonic convulsions in most animals examined. Topographical analysis of hippocampal SV2A-immunoreactivity revealed that SV2A was densely expressed in the hilar region of the dentate gyrus, stratum lucidum of the CA3 field and around the periphery of CA3 pyramidal neurons. PTZ kindling region-specifically increased SV2A expression in the dentate hilus without affecting that in the stratum lucidum or the pyramidal cell layer of the CA3 field. Confocal laser microscopic analysis using PTZ-kindled mice illustrated that most SV2A was co-expressed with glutamic acid decarboxylase 67 in the cell bodies and dendrites of hilar interneurons. However, SV2A-immunoreactivity was negligibly observed in the hilar glutamatergic nerve terminals (mossy fibers) probed with the anti-vesicular glutamate transporter 1 antibody. The present study suggests that SV2A specifically regulates hilar GABAergic neurotransmission in the kindled hippocampus probably as a compensatory or prophylactic mechanism against kindling epileptogenesis.

Antiepileptogenic and anticonvulsive actions of levetiracetam in a pentylenetetrazole kindling model.

Levetiracetam (LEV) is a unique antiepileptic drug that preferentially interacts with synaptic vesicle protein 2A (SV2A). To evaluate the antiepileptogenic action of LEV, we studied its effects on the development and acquisition of pentylenetetrazole (PTZ) kindling and compared them to those of sodium valproate (VPA). Anticonvulsive actions of LEV in PTZ-kindled animals were also determined. LEV did not affect PTZ seizures in naïve animals even at high doses (approximately 300 mg/kg, i.p.). However, combined treatment of LEV (30 and 100 mg/kg, i.p.) with PTZ significantly suppressed the development and acquisition of PTZ kindling. In addition, LEV at relatively low doses (3-30 mg/kg, i.p.) inhibited PTZ-evoked seizures in fully kindled animals. In contrast to LEV, VPA at sub-anticonvulsive doses (30 and 100 mg/kg, i.p.) failed to prevent the development of PTZ kindling and its anticonvulsive potency was similar in PTZ-kindled and naïve mice. The present study shows that LEV contrasts VPA by preventing the development of PTZ kindling and inhibiting seizures selectively in kindled animals.

Regional expression of Fos-like immunoreactivity following seizures in Noda epileptic rat (NER).

Noda epileptic rat (NER) is a genetic rat model of epilepsy that exhibit spontaneous generalized tonic-clonic (GTC) seizures with paroxysmal discharges. We analyzed the regional expression of Fos-like immunoreactivity (Fos-IR) following GTC seizures in NER to clarify the brain regions involved in the seizure generation. GTC seizures in NER elicited a marked increase in Fos expression in the piriform cortex, perirhinal-entorhinal cortex, insular cortex and other cortices including the motor cortex. In the limbic regions, Fos-IR was highest in the amygdalar nuclei (e.g., basomedial amygdaloid nucleus), followed by the cingulate cortex and hippocampus (i.e., dentate gyrus and CA3). As compared to the above forebrain regions, NER either with or without GTC seizures exhibited only marginal Fos expression in the basal ganglia (e.g., accumbens, striatum and globus pallidus), diencephalon (e.g., thalamus and hypothalamus) and lower brain stem structures (e.g., pons-medulla oblongata). These results suggest that GTC seizures in NER are of forebrain origin and are evoked primarily by activation of the limbic and/or cortical seizure circuits.