| Objective Epilepsy is a multi-etiological brain dysfunction syndrome.With the continuous development of researches,it is generally believed that epilepsy is related to abnormal synchronous discharge of neuronal cells.In this study,based on the hypothesis of calcium in epilepsy,we used multi-channel fiber photometry system to simultaneously record the calcium activity of hippocampal CA1 and primary motor cortex M1 under anesthetized,free movement and intraperitioneal administrated of kainic acid(KA).The characteristics of calcium activities in two brain regions and their association with behavioral seizures were analyzed,and the effects of local uncaging Rubi-GABA,a photosensitive compound of GABA,in hippocampal CA1,on epileptiform calcium activity in primary motor cortex M1 and behavioral seizures were studied.Content 1.Simultaneously recording and characteristic analysis of calcium signals in hippocampal CA1 and primary motor cortex M1 under anesthetized;2.Simultaneously recording and characteristic analysis of calcium signals in hippocampal CA1 and primary motor cortex M1 in conscious free-movement state and acute epileptiform state;3.Effect of local uncaging Rubi-GABA in hippocampal CA1 on epileptiform calcium signal in primary motor cortex M1 and behavioral seizures.Methods 1.Subjects and groups: SPF level male C57BL/6 mice aged 8-12 weeks were selected in this study.To observe the epileptiform seizures induced by KA,the experiments were divided into saline control group and 10 mg/kg KA group.For uncaging of Rubi-GABA,the experiments were divided into Saline+light control group and Rubi-GABA +light experimental group.2.Induction and behavioral evaluation of acute epilepsy: intraperitoneal injection of 10 mg/kg KA.The behaviors of mice were recorded 10 min before KA and 1.5h after KA administration.According to the Racine grading rule,the behavior of mice with epilepsy-like seizures was graded.The epilepsy of grade III to V were considered as CMS.3.Calcium activities recording: Cal-520 AM was injected into hippocampal CA1 and primary motor cortex M1 after craniotomy,and recorded by optical fiber implanted.4.Uncage Rubi-GABA: Rubi-GABA was injected into hippocampal CA1 after craniotomy,and fibers were implanted above it for local photolysis.An optical fiber coupling circuit for local photolysis was constructed to adjust the output power to 15 m W.5.Calcium signal analysis and statistical methods: using multi-channel analysis software Op Signal based on MATLAB analyzed the experimental records of calcium signal,and the amplitude of calcium signal was expressed by ?F/F.The measurements of the amplitude,frequency and duration of calcium signals were performed by Clampfit 10.6.Paired t-test was performed by Graphpad Prism 7.Results 1.Calcium signals in hippocampal CA1 and primary motor cortex M1 were simultaneously recorded at different anesthesia concentrations and freely moving condition: a)Under the condition of 1.2% isoflurane/oxygen,the amplitude of calcium signal in CA1 region was 1.54±0.35%?F/F,and the frequency was 0.10±0.005 Hz,the amplitude of calcium signal in M1 region was 1.76 ±0.29%?F/F,and the frequency was 0.14±0.03Hz;b)Under the condition of 0.8% isoflurane/oxygen,the amplitude of calcium signal in CA1 region was 0.78±0.17%?F/F,the frequency was 0.20±0.02 Hz,and the amplitude of calcium signal in M1 region was 1.16 ±0.14 %?F/F,and the frequency was 0.24±0.06 Hz.c)Under the condition of freely moving,the amplitude of calcium signal in CA1 region was 0.92±0.20%?F/F,the frequency was 0.32±0.06 Hz,and the amplitude of calcium signal in M1 region was 1.05±0.17%?F/F,and the frequency was 0.35±0.04 Hz.2.Epileptiform calcium activities were simultaneously recorded in hippocampal CA1 and primary motor cortex M1 after KA administration: a)There were two types of calcium signals in hippocampal CA1,one was baseline elevated calcium wave with long duration,the other was baseline stable cluster high frequency pulse calcium wave.The first type could be divided into two sub-categories: one was calcium build-up conbined with CSD(cortical spreading depression)-like wave,which had the largest amplitude of 42.64±9.74%?F/F and the longest duration of 216.50±26.25s;the other was individual emergence,named non CSD-like calcium wave,and had the amplitude of 15.18±3.29%?F/F and the duration of 70.75±12.47 s.The amplitude of cluster high frequency pulsed calcium wave with stationary baseline was 9.83±2.64 %?F/F.b)Calcium signal in primary motor cortex M1 only appeared as elevated baseline and long-lasting calcium wave.For CSD-like calcium wave,the amplitude was 32.79±4.55%?F/F,the duration was 333.50±66.65s;and for non-CSD-like calcium wave,the amplitude of was 4.08±0.91%?F/F,the duration was 92.50±21.75 s.3.Effect of local uncaging Rubi-GABA in hippocampal CA1 on epileptiform calcium activity in M1: When the epileptiform calcium wave appeared in M1,the light was sustanined in the CA1 for 30 s.It was found that the epileptiform calcium wave in M1 showed ‘deformation' and the amplitude increased significantly.There was an aggravating effect on behavioral sezures associated with the appearance of calcium waves.Conclusion 1.Under anesthesia,calcium signals in hippocampal CA1 and primary motor cortex M1 had a certain synchronicity,and their amplitudes and frequency depend on the level of anesthesia.The higher the anesthesia concentration was,the lower the frequency became and the larger the amplitude was.There were statistical differences under different anesthesia levels.2.In the state of acute epileptiform seizures,the non CSD-like calcium waves in the two brain regions showed certain synchronization,while CSD-like calcium waves did not,but appeared in both brain regions respectively.Cluster high frequency pulsed calcium waves only appeared in hippocampal CA1.The emergence of different types of calcium signals had a certain correspondence with behavioral seizures.3.Local uncaging Rubi-GABA in hippocampal CA1 might aggravate its abnormal effect or be of no effect on epileptiform calcium activities in the primary motor cortex M1 and related behavioral seizures. |
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