| Objective Fiber photometry is a new kind of signal detection technology,which is suitable to record the calcium activity of neurons in the deep brain of freely moving animals.At present,it is always used to study the relationship between one cerebral nuclei and its corresponding behavior under physiological conditions.In this research,the Ca2+ hypothesis of epileptogenesis was used as the theoretical basis,and the aim was to explore the calcium signals in the live,intact hippocampus(HPC)and entorhinal cortex(EC)before and after the acute epileptic seizures induced by kainic acid(KA)in mice.This study is expected to provide theoretical and methodological support for the application of fiber photometry on the research of epileptogenesis mechanism at the level of freely behaving animals.Content 1.The building of fiber photometry equipment and the optimizing of signal-noise ratio(SNR).2.Screening the method inducing seizure-like behavior of mice by KA for fiber photometry.3.Recording epileptiform calcium signals in CA3,CA1,dentate gyrus(DG)of HPC and EC in freely moving mice and exploring the relationship between epileptiform calcium signals and seizure-like behavior.Methods 1.Assembling and debugging fiber photometry equipment.Measuring the optical fiber coupling efficiency by optical power meter,the fluorescence recovery efficiency by optical phantom and SNR by anesthetized animal sample.2.Animals and groups: 2-month-old male C57BL/6 mice were used as experiment subject.To compare the different modes of administration,the mice were randomly divided into four groups: KA injectd by intracranial cannula group,saline injectd by intracranial cannula group,low-dose systemic administration group and high-dose systemic administration group.The animals used for calcium signal recording were randomly divided into four groups: CA3 group,CA1 group,DG group and EC group.3.Screening the method inducing seizure-like behavior of mice by KA: The mice in intracranial administration group were pre-implanted with cannula.After recovered from the operation for 3 to 5 days,the mice were injected 0.5 μl 2m M KA or 0.5μl saline by microinjection pump in awake.The mice in low-dose or high-dose systemic administration group were subcutaneously injected 15mg/kg and 20mg/kg KA,respectively.Video recordings were used to score behavioral seizures on modified Racine scale within 2h after KA injection.The mice whose seizure-like behavior reached stage-1 were considered being induced epileptic seizure.Convulsive motor seizures(CMS)means the seizures progressed to stage3-5.Assessing the different methods according to the proportion of animals that reached CMS and stage-5,the average number of CMS and the average duration of one CMS in every group.4.Calcium signals recording and acute epileptic seizures: After the craniotomy of mice,the calcium indicator OGB-1 AM was micro-injected into the targeted brain area and the fiber which connected to fiber photometry equipment was implanted above the indicator.When mice can freely behave,the calcium signals in the targeted brain area were recorded 30 min before and 2h after the KA administration.Animals were continuously monitored and the epileptiform calcium signals were analysed with seizures behavior at the same time.5.Data analysis: The calcium signals were analyzed using custom-made software based on Lab VIEW.△F/F was used to represent the amplitude of calcium signals.The number of CMS happened and the duration of CMS among KA injectd by intracranial cannula group,low-dose systemic administration group and high-dose systemic administration group were compared using one-way ANOVA.The amplitudes of calcium signals before and after the KA administration were compared using nonparametric tests for two related samples.Results 1.The optical fiber coupling efficiency of the own-built fiber photometry equipment is about 3%,the fluorescence collection efficiency is about 20%,and the minimum SNR is about 7.6d B,which satisfies the experimental requirement.2.KA induced epileptic seizure in intracranial KA administration group,low-dose and high-dose systemic administration group.100% of mice in intracranial KA administration group and high-dose systemic administration group reached CMS,while the percentage of low-dose systemic administration group was 75%.The proportion of animals that reached stage-5 is 100%,25%,40% respectively in intracranial KA administration group,low-dose and high-dose systemic administration group.The average number of CMS in intracranial KA administration group(9±2.14 times)was significantly more than that in low-dose(3±1.85 times)and high-dose(5±1.63 times)systemic administration group,and the average duration of one CMS in intracranial KA administration group(61±12.82s)was significantly longer than that in low-dose(31±8.80s)and high-dose(33±7.54s)systemic administration group.3.Compared with spontaneous irregular calcium signals,the epileptiform calcium signals recorded from freely moving mice after KA administration showed four different modes,including calcium build-up,large or small calcium wave,fast-spiking and slow-spiking.There were no specificity of brain area in epileptiform calcium signals,but the combinations of signals were different between areas.For example,the large calcium wave was always accompanied by the emergence of calcium build-up in hippocampal CA3 and CA1,while the small wave appeared before big calcium wave in DG area,and fast-spiking was synchronized with the small calcium wave in EC.The amplitude of epileptiform calcium signals after the KA administration in CA3(4.75±3.88%)is significantly larger than the amplitude of normal calcium signals(1.18±0.40%).Similar to CA3,The amplitudes of epileptiform calcium signals in CA1(13.88±9.78%),DG(6.36±3.56%),and EC(7.51±2.54%)were larger than the amplitude of normal calcium signals in correspongding brain location(1.59±0.19%,1.36±0.42% and 2.23±0.55%,respectively).4.The calcium build-up of hippocampal CA3 appeared at least 10 s in advance before the mice had obvious epileptic behavioral motions,and the small calcium wave accompanied by fast-spiking were synchronized with the seizure-like behavior of mice.Conclusion The fiber photometry can be effectively applied to record the epileptiform calcium signals in deep brain regions(the depth ≥ 4mm)of seizure-like freely behaving mice.The method of subcutaneous administration of KA at a dose of 20 mg/kg is suitable for inducing acute epileptic seizures in mice during calcium signals recording.The abnormalities of cell activity were expressed by different patterns of calcium signals in CA3 of HPC before mice showed obvious seizure-like motions induced by systemic administration of KA. |
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