Nagisa Clinic

Hirakata, Japan

Nagisa Clinic

Hirakata, Japan
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Hanaya R.,Kagoshima University | Sasa M.,Nagisa Clinic | Sugata S.,Kagoshima University | Tokudome M.,Kagoshima University | And 3 more authors.
Brain Research | Year: 2010

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 Ca2+ 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. © 2010 Elsevier B.V. All rights reserved.


Ohno Y.,Osaka University of Pharmaceutical Sciences | Sofue N.,Osaka University of Pharmaceutical Sciences | Imaoku T.,Osaka University of Pharmaceutical Sciences | Morishita E.,Osaka University of Pharmaceutical Sciences | And 3 more authors.
Journal of Pharmacological Sciences | Year: 2010

To explore the role of the serotonergic system in modulating absence seizures, we examined the effects of 5-HT1A and 5-HT2 agonists on the incidence of spike-and-wave discharges (SWD) in Groogy (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-HT1A agonist (±)-8-hydroxy-2-(di-n-propylamino)-tetralin [(±)8-OH-DPAT] or the 5-HT2 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-HT1A antagonist) and ritanserin (5-HT2 antagonist), respectively. The present results suggest that the serotonergic system negatively regulates the incidence of absence seizures by stimulation of 5-HT1A and 5-HT2 receptors. ©2010 The Japanese Pharmacological Society.


Tokuda S.,Kyoto University | Tokuda S.,The Jackson Laboratory | Sofue N.,Osaka University of Pharmaceutical Sciences | Ohno Y.,Osaka University of Pharmaceutical Sciences | And 2 more authors.
Brain Research | Year: 2010

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 T1/2 of 2.7 h. These results revealed that LEV displays potent and relatively long-lasting inhibitory effects on absence seizures in GRY rats. © 2010 Elsevier B.V.


Ohno Y.,Osaka University of Pharmaceutical Sciences | Sofue N.,Osaka University of Pharmaceutical Sciences | Ishihara S.,Osaka University of Pharmaceutical Sciences | Mashimo T.,Kyoto University | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

Mutations of the Nav1.1 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% O2 and 5% CO2 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 GAGAA 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, GABAA 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 GABAA 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. © 2010 Elsevier Inc.


Tokudome K.,Osaka University of Pharmaceutical Sciences | Okumura T.,Osaka University of Pharmaceutical Sciences | Terada R.,Osaka University of Pharmaceutical Sciences | Shimizu S.,Osaka University of Pharmaceutical Sciences | And 7 more authors.
Frontiers in Pharmacology | Year: 2016

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 (Sv2aL174Q rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2aL174Q 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 Sv2aL174Q rats. Sv2aL174Q 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 Sv2aL174Q rats. In vivo microdialysis study showed that the Sv2aL174Q 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. © 2016 Tokudome, Okumura, Terada, Shimizu, Kunisawa, Mashimo, Serikawa, Sasa and Ohno.


Serikawa T.,Kyoto University | Serikawa T.,Osaka University of Pharmaceutical Sciences | Mashimo T.,Kyoto University | Kuramoto T.,Kyoto University | And 3 more authors.
Experimental Animals | Year: 2015

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 seizuresusceptible rats were sporadically found in outbred stocks, the epileptic traits were usually geneticallyfixed 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 Scn1amissense mutation and autosomal dominant lateral temporal epilepsy (ADLTE) model rats with an Lgi1missense mutation. In addition, we have established episodic ataxia type 1 (EA1) model rats with a Kcna1missense mutation derived from the ENUinduced rat mutant stock, and identified a Cacna1amissense 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. © 2015 Japanese association for Laboratory animal Science.


Ohno Y.,Osaka University of Pharmaceutical Sciences | Shimizu S.,Osaka University of Pharmaceutical Sciences | Tatara A.,Osaka University of Pharmaceutical Sciences | Imaoku T.,Osaka University of Pharmaceutical Sciences | And 5 more authors.
PLoS ONE | Year: 2015

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. © 2015 Ohno et al.


Ohno Y.,Osaka University of Pharmaceutical Sciences | Ishihara S.,Osaka University of Pharmaceutical Sciences | Terada R.,Osaka University of Pharmaceutical Sciences | Serikawa T.,Kyoto University | Sasa M.,Nagisa Clinic
Epilepsy Research | Year: 2010

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 (≈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. © 2010 Elsevier B.V.


Harada Y.,Osaka University of Pharmaceutical Sciences | Nagao Y.,Osaka University of Pharmaceutical Sciences | Shimizu S.,Osaka University of Pharmaceutical Sciences | Serikawa T.,Kyoto University | And 7 more authors.
Brain Research | Year: 2013

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. © 2013 Elsevier B.V. All rights reserved.


PubMed | Nagisa Clinic, Kyoto University and Osaka University of Pharmaceutical Sciences
Type: | Journal: Scientific reports | Year: 2016

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.

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