| IntroductionVoltage-gated sodium channels(VGSCs)are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells,which consist of a complex of different glycosylated subunits,including a pore-forming a subunit of 260 kDa and fourβsubunits of 32-36 kDa(β1-β4 subunit).Theαsubunit forms the ion-selective pore,inner pore,voltage sensor and inactivation gate,including a transmembrane protein that consists of four homologous domains(â… -â…£),each with six transmembrane segments.It is generally agreed that both a andβsubunits play critical roles in modulating the activation and inactivation kinetics of sodium current.In addition,β1 subunit may function as cell adhesion molecules.Nine mammalian VGSC isoforms have already been identified.In the central nervous system(CNS),Nav1.1,Nav1.2,Nav1.3 and Nav1.6 are abundantly expressed, and Nav1.1 has been recognized to be highly detectable in the hippocampus, cerebellum,spinal cord,brainstem,neo-cortex,substantia nigra and caudate.Until now, many mutations in VGSC genes have been shown to be critically linked to specific epileptic syndromes.In generalized epilepsy with febrile seizures plus(GEFS+),an autosomal dominant epilepsy syndrome,mutations in the genes coding for VGSCâ… Î±-isoform(Nav1.1)or;β1 subunit(SCN1A,SCN1B)have been demonstrated.In human epilepsy,VGSCâ… a-isoform(Nav1.3)mRNA was found to be expressed at significantly higher levels in CA4 hilar cells in the epileptic hippocampus.It has also been reported that increased expression of neonatal Nay 1.3 mRNA was observed 4h after the induction of status epilepticus in neurons of CA1-CA3 and the dentate granule cell layer.Therefore,many previous studies have suggested that genetic abnormalities in the VGSC isoforms predominantly are likely to contribute to neuronal excitability in the CNS.The spontaneously epileptic rat(SER)is a double mutant obtained by mating heterozygous tremor rats(tm)(tm/+)and homozygous zitter rats(zi)(zi/zi).SER exhibits spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy.The mechanism underlying the epileptic seizures in SER was thought to include an abnormality of Ca channel function,an increase in extracellular glutamate concentrations,and enhanced levels of TVacetylaspartate because of lack of the aspartoacylase gene.However,so far,the effects of VGSC in SER have not yet been elucidated.We hypothesized that the etiopathogenisis of SER in genetic epilepsy might be involved in changes of VGSC.Thus,we investigated the expressions of VGSC in SER by researching VGSC Nav1.1,Nay1.3 andβ1 subunit at the mRNA and protein expression levels of SER,in hope that an insight in their roles in hippocampal excitability could be obtained.Materials and Methods1.MaterialsNormal Wistar rats and SERs at the age of 9-12 weeks were housed in individual cages under a controlled environment(12:12 h light/dark cycle,50-70%humidity, 24℃).Food and water were available ad libitum.For each experimental method,six SERs were in each experimental group and six Wistar rats were in each control group.The VGSCβ1 polyclonal antibody generated against the extracellular domain of VGSCβ1 subunit(KRRSETTAETFTEWTFR),was a gift from Doctor L.L.Isom, synthesized by the Protein and Carbohydrate Structure Core facility at the University of Michigan.The Nav1.1 and Nav1.3 antibodies were purchased from Sigma.2.MethodsRT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies) according to manufacturer's instructions(Takara).Real-time RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs(n=6)and control Wistar rats(n=6)using TRIzol(Life Technologies)according to manufacturer's instructions(Takara).A seven point standard curve for each gene was constructed using five fold serial dilutions of RT product.Immunofluorescence:The brains of SERs(n=6)and Wistar rats(n=6)were dissected out under anesthesia.Neonatal rat hippocampus was tested for Nav1.3 protein analysis and served as a positive control.Tissue sections were incubated with the primary antibody(anti-Nav1.1,1:25 dilution;anti-β1ex,1:100 dilution;anti-Nav1.3, 1:25 dilution).The sections were incubated at 30 min room temperature and overnight at 4℃,followed by incubation for 30 min in FITC-labeled goat anti-rabbit secondary antibody,and cell nuclei were labeled with Hoechst33258.After washes in PBS,the sections were mounted,viewed and photographed utilizing fluorescence microscopy.Immunohistochemistry:SERs(n=6)and Wistar rats(n=6)were perfused intracardially under anesthesia with normal saline,followed by 4%paraformaldehyde ice-cold fixative.Brains were dissected out and placed in 4%paraformaldehyde for 20 h at 4℃.The primary antibodies(anti-β1ex,1:100 dilution;anti-Nav1.1,1:25 dilution) were incubated for 30 min at room temperature and overnight at 4℃,and they were carried out using avidin-biotin peroxidase method and 3,3′-diaminobenzidine(DAB) as a chromogen.Control sections incubated without the primary antibody or with pre-immune sera were performed.The intensity,the cellular localization,and the frequency of immunoreactive cells were examined in different hippocampal regions (CA1,CA3 and dentate gyrus)of control rats and SERs.The expression levels of Nav1.1 andβ1 subunit were quantified by counting immunoreactive cells and measuring the intensity of staining using a computerized image analysis system.Western blot analysis:Western blot analysis was performed on samples of the whole hippocampus of adult SERs(n=6)and adult Wistar rats(n=6).For Nav1.3 protein analysis,six neonatal rats were served as positive controls.Samples were homogenized in lysis buffer containing 10 mM Tris(pH 8.0),150 mM NaCl,10% glycerol,1%NP-40,5 mM ethylenediamine tetra-acetic acid(EDTA)and protease inhibitor cocktail.Samples were incubated over night in TTBS:3%BSA,0.1%sodium azide,containing the primary antibodies(anti-β1ex,1:500 dilution;anti-Nav1.1,1:200 dilution;anti-Nav1.3,1:200 dilution).Immunoreactive bands were visualized using DAB and ECL kits.The levels of Nav1.1,Nav1.3 andβ1 protein were evaluated by measuring optical densities of the protein bands using Scion Image for Windows image-analysis software.Statistical analyses:Mean and standard deviation were calculated for all measurements in this study.Student's t-test was performed to determine statistical significance,which was evaluated at p＜0.05.All statistical analyses were conducted using SPSS for Windows version 12.0.Results1.The mRNA expressions of VGSC Nav1.1,Nav1.3 andβ1 subunit in SERs and control rats hippocampusThe data suggested that the mRNA expression of Nav1.1 in SERs hippocampus was significantly higher than that of control groups(1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001)by real-time RT-PCR;The mRNA expression level of Nav1.3 was abundantly increased compared with the control rats(0.55±0.15 in SERs vs 0.39±0.15 in control rats;p＜0.001);The mRNA expression ofβ1 subunit was also higher than that of control groups in hippocampus(0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05).The data by real-time RT-PCR were in agreement with the data by means of RT-PCR.On the whole,the mRNA expressions of VGSC Nav1.1,Nav1.3 andβ1 subunit were up-regulated in SERs hippocampus.2.The localization expressions of VGSC Nav1.1,Nav1.3 andβ1 subunit in SERs and control rats hippocampusNav1.1 andβ1 protein in SERs and control tissue were localized widely in the hippocampus by immunofluorescence.Under our investigation,Nav1.3 protein that was expressed in the neonatal(at the age of 3 days)hippocampus,was considered as a positive control.It was noting that the clear Nav1.3-immunoreactive cells were not found in control adult Wistar rats hippocampus.However,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.3.The protein expressions of VGSC Nav1.1 andβ1 subunit in SERs and control rats hippocampusBy means of immunohistochemistry,the protein expressions of Nav1.1 andβ1 subunit in the hippocampus of SERs were significantly higher than those in control rats. In addition,the increases in Nav1.1 andβ1 staining were clearly present throughout the hippocampus of SERs,including CA1,CA3 and dentate gyrus regions.Taken together, the data showed that the expressions at the protein level of Nav1.1 andβ1 subunit were up-regulated in SERs hippocampus.4.Additional tests that support the protein expressions of VGSC Nav1.1,Nav1.3 andβ1 subunit in SERs and control rats hippocampusThe protein expression of Nav1.1 in SERs hippocampus was significantly higher than that in control groups by western blot analysis(0.52±0.05 in SERs vs 0.30±0.04 in control rats;p＜0.001);The protein expression level of Nav1.3 was abundantly increased compared with the control rats(0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001).However,it seemed unchanged compared with neonatal rats(0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05).Taken together with the data of immunofluorescence,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.The protein expression ofβ1 subunit in SERs was higher than that of control rats in hippocampus(0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05).Altogether,the findings using western blot analysis further verified up-regulation expressions of VGSC Nav1.1,Nav1.3 andβ1 subunit at the protein level in SERs hippocampus.Conclusions1.The mRNA expressions of Nav1.1,Nav1.3 andβ1 subunit were first detected to be up-regulated in SERs hippocampus compared with control Wistar rats.2.Nav1.1 andβ1 subunit were first observed to be localized widely in SER hippocampus including CA1-CA3 and DG.3.The protein expressions of Nav1.1 andβ1 subunit were first found to be up-regulated in SERs hippocampus compared with control Wistar rats.4.Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.5.Sodium channel Nav1.1,Nav1.3 andβ1 subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER.6.The SER can be used as an epileptic animal model to screen effective specific VGSC isoforms for medicine and gene therapy.7.The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy. |
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