背景:神经细胞的电活动以细胞膜离子通道的活动为基础。神经元异常放电是癫痫的基本特征,其基础是细胞膜离子通道的激活与离子的跨膜运动,然而马桑内酯致痫机制中是否存在钙激活钾通道的激活还不清楚。目的:以大鼠海马锥体神经元为靶细胞,了解马桑内酯在其致痫机制中对神经元钙激活钾通道的作用。设计:非随机对照实验。单位:四川大学华西医院神经内科和泸州医学院心肌电教研室。材料:实验于2000-05/12在四川省泸州医学院完成。选择出生24h之内Wistar乳鼠100只。方法:Wistar乳鼠在麻醉状态下和无菌条件下分离出海马组织,以培养第7～10天,生长良好、形态典型的锥体神经元进行膜片钳试验。将培养皿随机分成9组:①正常对照组,19皿,加入DMEM培养基,给以不同的钳制电压,以后加入四乙基胺。②10-8,10-7,10-6mol/L钙浓度组;加入含不同浓度钙离子的DMEM培养基,以后加入四乙基胺,每一浓度8皿,共24皿;马桑内酯0,0.25,0.5,1.0,2.0mL/L致痫组,加入不同浓度含马桑内酯的DMEM培养基,以后加入四乙基胺。每一浓度26皿,共130皿。运用膜片钳制技术贴附式和内面向外式方法记录神经元单通道电活动,并分析通道活动的开放概率、平均开放时间、平均关闭时间、电流幅值等。主要观察指标:①观察并记录正常,不同钳制电压、不同钙离子浓度对锥体神经元钙激活钾通道的激活作用和四乙基胺的影响。②观察并记录致痫剂马桑内酯对锥体神经元细胞膜钙激活钾通道的激活作用及四乙基胺的影响。结果:①在钳制电压为0mV时,锥体神经元钙激活钾通道有少量的随机开放,具有明显的电压依赖性,通道电导值为(122.79±21.68)pS,可被钾通道阻断剂四乙基胺所阻断。②在内面向外式膜片下,钙激活钾通道表现出钙离子的浓度依赖性。当钙浓度为10-8,10-7,10-6mol/L时,平均开放概率分别为0.022±0.006,0.040±0.007,0.142±0.049(P<0.01)。③在细胞贴附式膜片下,浴液中游离钙离子浓度10-8mol/L,膜电位在20mV时,发现马桑内酯能明显增加钙激活钾通道的开放概率。④与马桑内酯0mL/L比较,马桑内酯1.0mL/L能增加钙激活钾通道平均开放时间(1.867±0.210,6.900±0.120,P<0.01),减少平均关闭时间(78.505±7.192,6.233±0.854,P<0.01)。结论:在马桑内酯诱导的癫痫发病中,钙激活钾通道活化可能起重要的负反馈调节作用。 Background: The electrical activity of nerve cells is based on the activity of cell membrane ion channels. The abnormal discharge of neurons is the basic feature of epilepsy, which is based on the activation of ion channels in the plasma membrane and the transmembrane movement of ions. However, it is unclear whether the activation of calcium-activated potassium channels is involved in the mechanism of epilepsy induced by coriaria lactone. OBJECTIVE: To investigate the effect of coriaria lactone on calcium-activated potassium channels in neurons in rat hippocampal pyramidal neurons. Design: Non-randomized controlled trials. Unit: Department of Neurology, West China Hospital of Sichuan University and Department of Myocardial Electrophysiology, Luzhou Medical College. MATERIALS: The experiment was completed at Luzhou Medical College, Sichuan Province from May 2000 to December 12. Select 100 Wistar pups within 24 hours of birth. METHODS: Wistar rats were isolated under anesthesia and under aseptic conditions to obtain hippocampal tissue. The 7th to 10th day of culture was performed. The well-developed and typical pyramidal neurons were subjected to patch clamp test. The culture dishes were randomly divided into 9 groups: 1 normal control group, 19 dishes, DMEM medium was added, different clamping voltages were given, and tetraethylamine was added later. 210-8,10-7,10-6mol/L calcium concentration group; adding DMEM medium containing different concentrations of calcium ions, followed by tetraethylamine, 8 dishes per concentration, 24 dishes in total; Coriaria lactone 0 , 0.25, 0.5, 1.0, and 2.0 mL/L epileptogenesis groups. Different concentrations of DMEM medium containing coriaria lactone were added followed by tetraethylamine. Each concentration of 26 dishes, a total of 130 dishes. The single-channel electrical activity of neurons was recorded using the patch-clamp technique and the inner-out-side method. The open probability, average opening time, average closing time, and current amplitude of the channel activities were analyzed. MAIN OUTCOME MEASURES: 1 Observe and record normal, different clamp voltage, different calcium ion concentration on the activation of calcium-activated potassium channels in pyramidal neurons and the effect of tetraethylamine. 2 Observe and record the activation of epileptic agent coriaria lactone on calcium-activated potassium channels in pyramidal neurons and the effect of tetraethylamine. RESULTS: 1 When the clamped voltage was 0mV, the calcium-activated potassium channels of pyramidal neurons had a small amount of random opening, with obvious voltage dependence. The conductance value of the channel was (122.79±21.68) pS, which could be blocked by potassium channels. IV. Ethylamine is blocked. 2 The calcium-activated potassium channels exhibit calcium ion concentration dependence under the inner-facing outer membrane. When the calcium concentration was 10-8, 10-7, and 10-6 mol/L, the average open probability was 0.022±0.006, 0.040±0.007, and 0.142±0.049, respectively (P<0.01). 3 In the cell-attached patch, the free calcium ion concentration in the bath was 10-8 mol/L, and when the membrane potential was 20 mV, it was found that coriaria lactone significantly increased the opening probability of the calcium-activated potassium channel. 4 Compared with 0 mL/L of coriaria lactone, coriaria lactone 1.0 mL/L increased the average opening time of calcium-activated potassium channels (1.867±0.210, 6.900±0.120, P<0.01), and reduced the average closure time (78.505±7.192). , 6.233±0.854, P<0.01). CONCLUSION: Activation of calcium-activated potassium channels may play an important role in negative feedback regulation in the pathogenesis of coriaria lactone-induced epilepsy.