Involvement Of Acid Sensing Ion Channel 3 In Epileptic Seizure And Its Mechanism

Backgrouds(1)Epilepsy is a common and disabling brain disorder characterized by recurrent unprovoked seizures.It results from abnormal discharge of highly synchronized neurons and affects approximately 50 million people worldwide.Twenty to thirty percent of patients remain refractory to treatment and require lifelong therapy,which has a serious impact on the patients' physical and mental health,working life and other aspects.Therefore,epilepsy has become a public health issue of concern to the whole society,and it is urgent to study its onset,occurrence,and development mechanisms as well as the intervention targets based on new findings.(2)The fact that seizures can reduce brain pH through C02 accumulation,lactic acid production and other mechanisms has drawn much attention to ASICs.The ASICs,which belong to the degenerin/epithelial sodium channel superfamily,are proton-gated,cation-selective,amiloride-sensitive channels.However,Previous research on the role of ASICs in epileptic seizures is puzzling and controversial.Particularly,acid sensing ion channel 3(ASIC3)is the most sensitive to extracellular pH and has the characteristic ability to generate a biphasic current,but evidence concerning the function of ASIC3 in epilepsy also remains scant and elusive.Therefore,it is meaningul and necessary to further explore the role of ASIC3 in epileptic seizure.Objective(1)To explore the expression changes of ASIC3 in epileptic seizure animal model and in an in vitro Mg2+-free epileptiform discharge cell model.(2)To provide new ideas and theoretical basis for anti-seizure drug development by studying the effects of ASIC3 on the behaviors of epileptic seizure animals and the associated molecular mechanismsMethods(1)ASIC3 expression in the epileptic seizure animals:we adopted Western blot,RT-PCR and immunohistochemistry(IHC)repectively to study the ASIC3 expression in the hippocampus of lithium chloride-pilocarpine induced seizure animal models.(2)ASIC3 expression in the Mg2+-free epileptiform discharge cell model:immunofluorescence technique was used to investigate the purity of primary cultured hippocampal neurons.The spontaneous epileptifrom discharge of primary neurons induced by Mg2+-free medium was verified by patch clamp technique.Western blot was used to detect the changes of ASIC3 protein expression after Mg2+-free treatment.(3)The impact of ASIC3 inhibition on the behaviours of epileptic seizure rats:APETx2 was intracerebroventricularly microinjected into animal brains and Racine score grading system was used to evaluate the seizure onset latency,the maximum seizure severity,the incidence of GTCS and the seizure severity over time.(4)The mechanism of the impact of ASIC3 inhibiton on epileptic seizure:Western blot was adopted to evaluate the membrane expression of subunits of NMDA receptors and AMPA receptors in seizure animals pretreated with APETx2.Co-immunoprecipitation(Co-IP)was used to study whether there were interactions between NMDA receptors,AMPA receptors and ASIC3.Western blot was also adopted to detect the changes of CaMK ?and CREB downstream of NMDA receptors.The spontaneous and evoked discharges of action potentials of primary neurons in Mg2+-free medium pretreated with APETx2 were recorded by whole-cell patch clamp.The NMDA receptor mediated mEPSC and eEPSC were also recorded in brain slice of seizure models pretreated with APETx2.Results(1)ASIC3 protein expression began to increase 2 hours after seizure onset,reached a peak at 48 hours and then slightly decreased at 72 hours but still higher than the control.The relative mRNA expression of ASIC3 in the seizure group was higher than in the control at all time points.IHC showed that the mean OD value of ASIC3 in the hippocampus of the rats 24 hours after seizure was higher than that in the control.(2)Immunofluorescence staining with MAP-2 antibody proved the high purity of primary cultured hippocampal neurons.The control neurons displayed infrequent spontaneous action potentials,while neurons after a treatment with Mg2+-free solution for 3 hours demonstrated characteristic epileptiform discharges.The ASIC3 protein level in Mg2+-free group was higher than in the control group.(3)APETx2 significantly shortened the latency to seizure of level 4 and enhanced the maximum Racine score compared with the sham group.More than half the rats in the APETx2 groups developed GTCS,whereas those in the sham group were less likely to have GTCS.The APETx2-treated groups had more severe seizures than the sham group except at the 45-minute time point.(4)The membrane expression of NR1,NR2A,NR2B and GluRl was significantly increased 2 hours after seizure onset.The membrane expression of these NMDARs but not GluRl was even higher in the APETx2 group than in the seizure group.The expression profile of NMDAR subunits 24 hours after seizure onset was similar to that at the 2-hour time point except that,NR1 in the APETx2 group remained statistically unchanged compared with the seizure and the PBS group.Co-IP experiments verified the interaction between NMDARs and ASIC3,but not AMPARs and ASIC3.AP5,a selective NMDAR antagonist,abolishes the aggravated epileptic seizures caused by ASIC3 inhibition.The elevated changes of NMDARs were accompanied by activation of the CaMK? and CREB signaling pathway.The primary neurons in Mg2+-free medium treated with APETx2 have more spontaneous and evoked discharge of action potentials compared with those in Mg2+-free medium alone.NMDAR mediated mEPSC and eEPSC in acute hippocampal slices were significantly increased in APETx2 treated seizure group compared with those observed in seizure group.Conclusions(1)The level of ASIC3 is elevated in pilocarpine-induced seizure model and in vitro Mg2+-free epileptiform discharge model.(2)ASIC3 blockade increases seizure severity and enhances the excitability of primary cultured hippocampal neurons in Mg2+-free medium.(3)The aggravated epileptic seizure behavioral changes were accompanied by upregulation of NMDA receptors and subsequent activation of the CaMKII and CREB signaling pathway,which were accompanied by increased NMDAR component of mEPSC and eEPSC.(4)ASIC3 functions as part of an endogenous anticonvulsant system and may emerge as a new target for prevention and control of seizure.