Increased seizure sensitivity, emotional defects and cognitive impairment in PHD finger protein 24 (Phf24)-null rats.

Phf24 is known as Gαi-interacting protein (GINIP) and is associated with the GABAB receptor. To study the function of Phf24 protein in the central nervous system (CNS), we have newly developed Phf24-null rats and investigated their behavioral phenotypes, especially changes in seizure sensitivity, emotional responses and cognitive functions. Phf24-null rats did not exhibit any spontaneous seizures. However, they showed a higher sensitivity to pentylenetetrazol (PTZ)- or pilocarpine-induced convulsive seizures. Phf24-null rats also showed an elevated susceptibility to kindling development with repeated PTZ treatments, suggesting that Phf24 acts as an inhibitory modulator in epileptogenesis. Although young Phf24-null rats showed normal gross behaviors, elevated spontaneous locomotor activity, especially in terms of the circadian dark period, emotional hyper-reactivity, reduced anxiety behaviors in the elevated plus-maze (EPM) test, and cognitive deficits in the Morris water maze test were explicitly observed at older age (20-week-old). The present results suggest that Phf24 is essential for proper functioning of the CNS, especially in preventing epileptogenesis and controlling emotional and cognitive functions.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

A Missense Mutation of the Gene Encoding Synaptic Vesicle Glycoprotein 2A (SV2A) Confers Seizure Susceptibility by Disrupting Amygdalar Synaptic GABA Release.

Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in the membranes of synaptic vesicles and modulates action potential-dependent neurotransmitter release. To explore the role of SV2A in the pathogenesis of epileptic disorders, we recently generated a novel rat model (Sv2a(L174Q) rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2a(L174Q) rats were hypersensitive to kindling development (Tokudome et al., 2016). Here, we further conducted behavioral and neurochemical studies to clarify the pathophysiological mechanisms underlying the seizure vulnerability in Sv2a(L174Q) rats. Sv2a(L174Q) rats were highly susceptible to pentylenetetrazole (PTZ)-induced seizures, yielding a significantly higher seizure scores and seizure incidence than the control animals. Brain mapping analysis of Fos expression, a biological marker of neural excitation, revealed that the seizure threshold level of PTZ region-specifically elevated Fos expression in the amygdala in Sv2a(L174Q) rats. In vivo microdialysis study showed that the Sv2a(L174Q) mutation preferentially reduced high K(+) (depolarization)-evoked GABA release, but not glutamate release, in the amygdala. In addition, specific control of GABA release by SV2A was supported by its predominant expression in GABAergic neurons, which were co-stained with antibodies against SV2A and glutamate decarboxylase 1. The present results suggest that dysfunction of SV2A by the missense mutation elevates seizure susceptibility in rats by preferentially disrupting synaptic GABA release in the amygdala, illustrating the crucial role of amygdalar SV2A-GABAergic system in epileptogenesis.

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.

New insight into the therapeutic role of the serotonergic system in Parkinson's disease.

Parkinson's disease (PD) is a common, late-onset neurodegenerative disorder that shows progressive extrapyramidal motor disorders (e.g., bradykinesia, resting tremors, muscle rigidity and postural instability) and various non-motor symptoms (e.g., cognitive impairment, mood disorders, autonomic dysfunction and sleep disorders). While dopaminergic agents such as L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine D2 agonists are widely used for the treatment of PD, there is still high clinical unmet need for novel medications that overcome the limitations of current therapies. Evidence is now accumulating that the serotonergic nervous system is involved in the pathophysiological basis of PD and can provide benefits in the treatment of PD through its diverse functions. Among 5-HT receptor subtypes, 5-HT1A, 5-HT2, 5-HT3 and 5-HT6 receptors play an important role in modulating extrapyramidal motor disorders. In addition, 5-HT1A, 5-HT2, 5-HT3, 5-HT4 and 5-HT6 receptors are implicated in modulation of cognitive impairment, mood disorders (e.g., depression and anxiety) and/or psychosis, which are frequently observed in patients with PD. Specifically, stimulation of 5-HT1A receptors seems to be effective for multiple PD symptoms including parkinsonism, L-DOPA-induced dyskinesia, cognitive impairment, mood disorders and neurodegeneration of dopamine neurons. Blockade of 5-HT2 receptors is also likely to improve parkinsonism, depressive mood and cognitive impairment. In addition, it was recently demonstrated that 5-HT2A inverse agonists can alleviate PD psychosis. All these findings emphasize the therapeutic roles of the serotonergic system in PD and stimulate new insight into novel treatments by modulating 5-HT1A and 5-HT2 receptors.

Hcn1 is a tremorgenic genetic component in a rat model of essential tremor.

Genetic factors are thought to play a major role in the etiology of essential tremor (ET); however, few genetic changes that induce ET have been identified to date. In the present study, to find genes responsible for the development of ET, we employed a rat model system consisting of a tremulous mutant strain, TRM/Kyo (TRM), and its substrain TRMR/Kyo (TRMR). The TRM rat is homozygous for the tremor (tm) mutation and shows spontaneous tremors resembling human ET. The TRMR rat also carries a homozygous tm mutation but shows no tremor, leading us to hypothesize that TRM rats carry one or more genes implicated in the development of ET in addition to the tm mutation. We used a positional cloning approach and found a missense mutation (c. 1061 C>T, p. A354V) in the hyperpolarization-activated cyclic nucleotide-gated 1 channel (Hcn1) gene. The A354V HCN1 failed to conduct hyperpolarization-activated currents in vitro, implicating it as a loss-of-function mutation. Blocking HCN1 channels with ZD7288 in vivo evoked kinetic tremors in nontremulous TRMR rats. We also found neuronal activation of the inferior olive (IO) in both ZD7288-treated TRMR and non-treated TRM rats and a reduced incidence of tremor in the IO-lesioned TRM rats, suggesting a critical role of the IO in tremorgenesis. A rat strain carrying the A354V mutation alone on a genetic background identical to that of the TRM rats showed no tremor. Together, these data indicate that body tremors emerge when the two mutant loci, tm and Hcn1A354V, are combined in a rat model of ET. In this model, HCN1 channels play an important role in the tremorgenesis of ET. We propose that oligogenic, most probably digenic, inheritance is responsible for the genetic heterogeneity of ET.

Advances on genetic rat models of epilepsy.

Considering the suitability of laboratory rats in epilepsy research, we and other groups have been developing genetic models of epilepsy in this species. After epileptic rats or seizure-susceptible rats were sporadically found in outbred stocks, the epileptic traits were usually genetically-fixed by selective breeding. So far, the absence seizure models GAERS and WAG/Rij, audiogenic seizure models GEPR-3 and GEPR-9, generalized tonic-clonic seizure models IER, NER and WER, and Canavan-disease related epileptic models TRM and SER have been established. Dissection of the genetic bases including causative genes in these epileptic rat models would be a significant step toward understanding epileptogenesis. N-ethyl-N-nitrosourea (ENU) mutagenesis provides a systematic approach which allowed us to develop two novel epileptic rat models: heat-induced seizure susceptible (Hiss) rats with an Scn1a missense mutation and autosomal dominant lateral temporal epilepsy (ADLTE) model rats with an Lgi1 missense mutation. In addition, we have established episodic ataxia type 1 (EA1) model rats with a Kcna1 missense mutation derived from the ENU-induced rat mutant stock, and identified a Cacna1a missense mutation in a N-Methyl-N-nitrosourea (MNU)-induced mutant rat strain GRY, resulting in the discovery of episodic ataxia type 2 (EA2) model rats. Thus, epileptic rat models have been established on the two paths: 'phenotype to gene' and 'gene to phenotype'. In the near future, development of novel epileptic rat models will be extensively promoted by the use of sophisticated genome editing technologies.

Identification of QTLs involved in the development of amygdala kindling in the rat.

Amygdala kindling is useful for modeling human epilepsy development. It has been known that genetic factors are involved in the development of amygdala kindling. The purpose of this study was to identify the loci that are responsible for the development of amygdala kindling. To achieve this, rat strains from a LEXF/FXLE recombinant inbred (RI) strain panel were used. The phenotypes of amygdala kindling-related parameters for seven RI strains and parental LE/Stm and F344/Stm strains were determined. They included the afterdischarge threshold (ADT), the afterdischarge duration (ADD), and the kindling rate, an incidence of development of kindling. Quantitative trait loci (QTL) analysis was performed to identify linkage relationships between these phenotypes and 1,033 SNP markers. Although no significant differences in pre-kindling ADT and ADD were observed, a significant difference in the kindling rate was found for the LEXF/FXLE RI strain. Two QTLs for the amygdala kindling rate (Agkr1 and Agkr2) were identified on rat chromosome 2. These findings clearly prove the existence of genetic influences that are involved in kindling development and suggest that substantial genetic components contribute to the progression of partial seizures into generalized seizures.

Expressional analysis of inwardly rectifying Kir4.1 channels in Noda epileptic rat (NER).

The inwardly rectifying potassium channel subunit Kir4.1 is expressed in brain astrocytes and involved in spatial K(+) buffering, regulating neural activity. To explore the pathophysiological alterations of Kir4.1 channels in epileptic disorders, we analyzed interictal expressional levels of Kir4.1 in the Noda epileptic rat (NER), a hereditary animal model for generalized tonic-clonic (GTC) seizures. Western blot analysis showed that Kir4.1 expression in NERs was significantly reduced in the occipito-temporal cortical region and thalamus. However, the expression of Kir5.1, another Kir subunit mediating spatial K(+) buffering, remained unaltered in any brain regions examined. Immunohistochemical analysis revealed that Kir4.1 was primarily expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes (somata) and foot processes clustered around neurons proved with anti-neuronal nuclear antigen (NeuN) antibody. In NERs, Kir4.1 expression in astrocytic processes was region-selectively diminished in the amygdaloid nuclei (i.e., medial amygdaloid nucleus and basomedial amygdaloid nucleus) while Kir4.1 expression in astrocytic somata was unchanged. Furthermore, the amygdala regions with reduced Kir4.1 expression showed a marked elevation of Fos protein expression following GTC seizures. The present results suggest that reduced activity of astrocytic Kir4.1 channels in the amygdala is involved in limbic hyperexcitability in NERs.

Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca²âº channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER).

Levetiracetam (LEV) is a widely used antiepileptic agent for partial refractory epilepsy in humans. LEV has unique antiepileptic effects in that it does not inhibit electroshock- or pentylenetetrazol-induced convulsion, but does inhibit seizures in kindling animal and spontaneously epileptic rat (SER: zi/zi, tm/tm) that shows both tonic convulsion and absence-like seizures. LEV also has unique characteristics in terms of its antiepileptic mechanism; it has no activity on Na⁺ and K⁺ channels or on glutamate and GABA(A) receptors. Recently, we found that LEV inhibits the depolarization shift and accompanying repetitive firing induced by mossy fiber stimulation in CA3 neurons of SER hippocampal slices. Therefore, this study was performed to determine whether LEV could inhibit the voltage-activated L-type Ca²âº current of hippocampal CA3 neurons obtained from SER and the non-epileptic Wistar rat. As previously reported, SER CA3 neurons were classified into type 1 and type 2 neurons. The application of LEV (100 µM) elevated the threshold for activation of the Ca²âº current, which was lowered in SER type 1 neurons and reduced the current size. Type 2 neurons of SER have a similar current-voltage relationship to Wistar rat neurons and the decay component of Ca²âº current during depolarization pulse in type 2 neurons was found to be smaller than that in Wistar rat neurons. LEV (100 µM) also reduced Ca²âº current in SER type 2 neurons. The effects of LEV were examined on such type 2 SER hippocampal CA3 neurons, compared with those on Wistar rat CA3 neurons. Application of LEV (10 µM) produced a significant decrease of amplitude of the Ca²âº current in SER neurons, although at this concentration of LEV there was no statistically significant decrease in the amplitude of Ca²âº current in Wistar rat neurons. Furthermore, LEV (100 nM-1 mM) reduced the Ca²âº current in a concentration-dependent manner in both SER and Wistar rat neurons, but the inhibition was much more potent in the former neurons than in the latter. Under the condition that the Ca²âº current had already been inhibited by LEV (10 µM), the addition of nifedipine (10 µM) did not cause further inhibition. Conversely, LEV had no effects on the current that had already been decreased by nifedipine (10 µM) given before LEV treatment (10 µM), indicating that LEV could act on the L-type Ca²âº channel. LEV elevated the threshold potential level for activation of the Ca²âº current and reduced the L-type Ca²âº current in type 1 neurons of SER, and the inhibitory action in type 2 neurons was much more potent than that in Wistar rat neurons, suggesting that these effects contribute, at least partly, to the antiepileptic action of LEV.

Region-specific elevation of D1 receptor-mediated neurotransmission in the nucleus accumbens of SHR, a rat model of attention deficit/hyperactivity disorder.

Spontaneously hypertensive rats (SHR) are widely used as a rat model of attention deficit/hyperactivity disorder (AD/HD). Here, we conducted neurochemical and behavioral studies in SHR to clarify the topographical alterations in neurotransmissions linked to their behavioral abnormalities. In the open-field test, juvenile SHR showed a significant hyperactivity in ambulation and rearing as compared with Wistar Kyoto rats (WKY). Brain mapping analysis of Fos-immunoreactivity (IR) revealed that SHR showed a marked increase in Fos expression in the core part (AcC) of the nucleus accumbens (NAc). Small to moderate increases were also observed in the shell part of the NAc and some regions of the cerebral cortex (e.g., parietal association cortex). These changes in Fos expression were region-specific and the Fos-IR levels in other brain regions (e.g., hippocampus, amygdala, striatum, thalamus and hypothalamus) were unaltered. In addition, treatment of SHR with the selective D1 antagonist SCH-23390 significantly reversed both behavioral hyperactivity and elevated Fos expression in the AcC and cerebral cortex. The present study suggests that D1 receptor-mediated neurotransmission in the AcC is region-specifically elevated in SHR, which could be responsible for behavioral hyperactivity.

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.

Modulation of abnormal synaptic transmission in hippocampal CA3 neurons of spontaneously epileptic rats (SERs) by levetiracetam.

Levetiracetam (LEV) inhibits partial refractory epilepsy in human, and both convulsive and absence-like seizures in the spontaneously epileptic rat (SER). Two-thirds of hippocampal CA3 neurons in SER show a long-lasting depolarization shift, with accompanying repetitive firing upon mossy fiber stimulation. This abnormal excitability is probably attributable to abnormalities in the L-type Ca(2+) channels. We performed electrophysiological studies to elucidate the mechanism underlying the antiepileptic effects of LEV via intracellular recording from the hippocampal CA3 neurons in slice preparations of SER and non-epileptic Wistar rats. LEV (100 µM) inhibited the depolarization shift with repetitive firing by mossy fiber stimulation (MFS), without affecting the first spike in SER CA3 neurons. At a higher dose (1mM), LEV suppressed the first spike in all SER neurons (including the CA3 neurons which showed only a single action potential by MFS), while the single action potential of Wistar rat CA3 neurons remained unaffected. SER CA3 neurons with MFS-induced abnormal firing exhibited a higher number of repetitive spikes when a depolarization pulse was applied in the SER CA3 neurons. LEV (100 µM, 1mM) reduced the repetitive firing induced by a depolarization pulse applied without affecting Ca(2+) spike in SER neurons. LEV is known not to bind glutamate and gamma-aminobutyric acid (GABA) receptors. These findings suggest that the therapeutic concentration of LEV inhibits abnormal firing of the CA3 neurons by modulating abnormal synaptic transmission and abnormal Na(+) channels in SER.

Neuroprotective effect of levetiracetam on hippocampal sclerosis-like change in spontaneously epileptic rats.

The spontaneously epileptic rat (SER) begins to exhibit both tonic convulsions and absence seizures from 6 weeks of age and SERs have stable seizures after 10 weeks of age. Low-dose administrations of levetiracetam (LEV) for 4- to 5-weeks-old SERs which did not show spontaneous seizures reduced both seizures 5 weeks after termination of administration. The hippocampus of SER exhibited decreased CA3 neurons, sprouting of mossy fibers, and hyperexpression of the brain-derived neurotrophic factor (BDNF). We attempted prophylactic LEV administrations in preseizure-manifesting SERs to evaluate if such a treatment regimen would protect the hippocampal sclerosis-like changes observed in SERs. The osmotic mini-pump administered LEV dissolved in saline to 4-weeks-old SERs for 4 weeks at 2.5 µl/h. LEV was administered at 420 mg/ml for 4 weeks in Group A. In Group B, LEV was given at 420 mg/ml for the first 2 weeks followed by doubling the dosage (840 mg/ml) in the following 2 weeks. LEV administrations in preseizure-manifesting SERs reduced the decrease of CA3 neurons and mossy fibers sprouting at 10-11 weeks of age in both group A and B. LEV attenuated BDNF expression in inner molecular layers of the dentate gyrus, striatum radiatum, and CA3 in 10- to 11- and 14- to 15-weeks-old SERs. In group B, LEV decreased BDNF expression in hilus and CA1 of 10- to 11- weeks-old SER. The present results suggest that prophylactic treatment with LEV in preseizure-manifesting SERs inhibits hippocampal sclerosis-like neuronal degeneration and/or regeneration.

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.

Inhibitory effects of levetiracetam on absence seizures in a novel absence-like epilepsy animal model, Groggy rat.

Levetiracetam (LEV) is known to inhibit convulsive seizures and is clinically used for treating both partial and generalized seizures. The study was performed to determine whether LEV possesses an inhibitory effect on absence seizures in a novel genetic animal model of absence epilepsy, Groggy (GRY) rats. Single injections of LEV at doses ranging from 20 to 160 mg/kg i.p. markedly inhibited absence seizures in GRY rats. The anti-absence action of LEV was potent and the cumulative duration of spike and wave discharges (SWD) in GRY rats was almost completely suppressed even at 20 mg/kg (i.p.). When the time-course of the inhibitory action of LEV (80 mg/kg i.p.) was examined up to 24 h after the treatment, the appearance of SWD was suppressed for over 6 h after injection of LEV in contrast to the action of sodium valproate (200 mg/kg i.p.) which had a very short effect (< 2 h). The maximum level of blood concentration of LEV was attained within 2 h after administration, and the drug disappeared from the blood in 24 h with T(¹/2) of 2.7 h. These results revealed that LEV displays potent and relatively long-lasting inhibitory effects on absence seizures in GRY rats.

Serotonergic modulation of absence-like seizures in groggy rats: a novel rat model of absence epilepsy.

To explore the role of the serotonergic system in modulating absence seizures, we examined the effects of 5-HT(1A) and 5-HT(2) agonists on the incidence of spike-and-wave discharges (SWD) in Groggy (GRY) rats, a novel rat model of absence-like epilepsy. GRY rats exhibited spontaneous absence-like seizures characterized by the incidence of sudden immobile posture and synchronously-associated SWD. The total duration of SWD in GRY rats was about 300 - 400 s/15-min observation period under the control conditions. However, the incidence of SWD was markedly reduced either by the 5-HT(1A) agonist (±)-8-hydroxy-2-(di-n-propylamino)-tetralin [(±)8-OH-DPAT] or the 5-HT(2) agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI]. The 5-HT reuptake inhibitors, fluoxetine and clomipramine, also inhibited the SWD generation. In addition, the inhibitory effects of (±)8-OH-DPAT and (±)DOI were reversed by WAY-100135 (5-HT(1A) antagonist) and ritanserin (5-HT(2) antagonist), respectively. The present results suggest that the serotonergic system negatively regulates the incidence of absence seizures by stimulation of 5-HT(1A) and 5-HT(2) receptors.

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.

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.

Hippocampal cell loss and propagation of abnormal discharges accompanied with the expression of tonic convulsion in the spontaneously epileptic rat.

Spontaneously epileptic rats (SER) are double mutants with both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 neurons in SER display a long-lasting depolarizing shift accompanied by repetitive firing (attributed to abnormalities of the Ca(2+) channels) with a single stimulation of the mossy fibers. In the present investigation, we examined if the seizure discharges of SER were correlated with the hippocampal abnormality of SER using electrophysiological and histological methods. In CA1 neurons of seizure-susceptible mature SER, higher-voltage (<8-11 V) stimulations induced a long depolarization shift (in 25% of neurons) with repetitive firing (in 12.5% of neurons). However, the tremor rat, one of the parent strains of SER, did not exhibit such abnormal firing in the CA3 region of the hippocampus. The number of CA3 neurons in SER was significantly (p<0.01) lower than that in tremor rats and Wistar rats, although no significant difference was established in the hilus. Sprouting of mossy fiber was observed in the dentate of mature SER; however, negligible staining was spotted in the dentate of both mature tremor and Wistar rats. Interestingly, expression of the brain-derived neurotrophic factor was higher in the hilus, CA3, and granular cell layer of dentate gyrus in SER than normal Wistar rats. The expression levels of TUNEL, bax, and Caspase-3 did not show significant changes between the SER and Wistar rats. SER exhibited hippocampal sclerosis-like changes which did not have enough potential for epileptogenesis. Repetitive tonic seizures and vulnerable CA3 neurons of SER could be involved in the induction of sclerosis-like changes in the hippocampus.