Research Of Epileptiform Calcium Activity And Inhibition Of Uncaging Rubi-GABA On Seizure Events Based On Fiber Photometry

Objective Epilepsy is a multi-etiological brain dysfunction syndrome characterized by synchronously repeated spontaneous neuronal cell discharges.In recent years,with the rapid development of calcium imaging technology,basic research on epilepsy based on the calcium overload hypothesis is emerging.In this study based on the calcium hypothesis of epileptogenesis,epilepsy-related calcium activity induced by 4-AP(4-aminopyriding)or KA(Kainic acid)administration were recorded by fiber photometry,a collection method for recording neuronal calcium transients in freely behaving animal,the correlation between epileptic-calcium signal and behavioral episodes were analysed,and effect of local photolytic inhibitory neurotransmitter GABA complex(Rubi-GABA)on behavioral seizures and seizure-related epilepsy like calcium signal were studied.Content 1.Record and character analysis of epilepsy-like calcium signals induced by topical 4-AP administration,and correlation study between abnormal calcium signals in primary motor cortex and behavioral episodes.2.Study the effect of local photolysis of hippocampal CA1 of GABA cage compound Rubi-GABA on acute epileptiform seizures and abnormal calcium signals induced by KA systemic administration.Methods 1.Subjects and groups: 2-month-old male C57BL/6 mice were selected as the study subjects;for the 4-AP topical administration part,the experiment was divided into three groups of ACSF group,1 m M 4-AP group and 10 m M 4-AP group;for photolysis inhibition Rubi-GABA inhibition seizure experimental part,the experiment was divided into KA group,KA+Saline group and KA + Rubi-GABA group.2.Seizure induction methods and behavioral scoring criteria for acute epileptic seizures: 1)Local induction: The mice were subjected to craniotomy.A cranial window with a diameter of approximately 0.5 mm was opened in M1,and 1 m M or 10 m M was added dropwise to the surface of the cranial window.4-AP solution;2)Systemic systemic administration: A 20 mg/kg dose of KA solution was injected subcutaneously.Record behavioral video of mice before and after administration of epileptic drugs,according to the improved Racine's rules to assess the extent of mice's seizures.Grade III and above behavioral episodes were regarded as convulsive motor seizures.3.Calcium signal recording: After the craniotomy of experimental animals,calcium indicator(Oregon Green BAPTA-1 AM,OGB-1 AM)was loaded on target brain regions M1,CA1 and EC(Entorhinal cortec,EC)by multicell bolus loading(MCBL)methods,and optical fiber was implanted into the same area.4.Photodissociation of Rubi-GABA: For the photodissociation Rubi-GABA experimental part,cannula was pre-filled into CA1 for drug administration and fiber implantation.A fiber-coupled optical path for photolysis of Rubi-GABA was set up with the light intensity to 10 m W.5.Calcium signal analysis and statistical processing methods: Calcium signal analysis software written based on Lab VIEW was used to read and analyze the calcium signal obtained from the experiment.Its amplitude was expressed by ?F/F.The non-parametric t-test of paired samples was performed on the calcium signal before and after KA injection together with diverse isoflurane level;The one-way ANOVA was performed on other groups.Results 1.Epileptiform-calcium signal induced by 4-AP local administration: a)Compared with rhythmic spontaneous calcium activity in the anesthetic state,local 4-AP administration induced abnormal calcium wave in M1,and amplitude statistics of characteristic calcium signal in control group,1 m M 4-AP group and 10 m M 4-AP group were 2.87 ± 0.08%?3.93 ± 0.22%?10.02 ± 0.62%,respectively.b)Compared with spontaneous calcium activity in freely behaving conditions,4-AP induced abnormal calcium waves in M1 with high-frequency oscillations,and amplitude statistics of characteristic calcium signals in control,1 m M 4-AP,and 10 m M 4-AP groups were 2.82 ± 0.24%,4.18 ± 0.38%,and 6.77 ± 0.47%,respectively.2.Inhibition of epileptic seizures by local photolysis of Rubi-GABA: a)Systemic administration of KA(20 mg/kg,sc)induced acute epileptic seizures in mice,and calcium signal recordings in hippocampal CA1 appeared as three types: KA-I type calcium waves(KA-wave1),KA-II type calcium wave(KA-wave2)and KA-slow flash.b)Local photolysis of Rubi-GABA in hippocampal CA1 could alleviate KA-induced acute epileptic seizures,including seizure progression from V to grade II,and suppression of epileptic-like calcium signals.Conclusion 1.Local injection of 4-AP induces epileptoid-calcium activity in M1 that is significantly different from spontaneous activity.The higher the 4-AP concentration,the greater the amplitude of epileptic-like calcium signal and the stronger the synchronization of abnormal excitation of nerve cells.2.KA subcutaneous injection(20mg/kg)can induce mice to produce acute epileptic seizures.Before the onset of behavior,abnormal hippocampal CA1 has formed epileptiform-calcium activity,manifested as abnormal synchronization calcium waves with high-frequency pulse-like signals.3.Local photolysis of Rubi-GABA in hippocampal CA1 can improve KA-induced acute epileptiform seizures and epileptiform-calcium activity in freely behaving mice.