Experimental study - The role of mint1, a novel synaptic protein, following epileptic seizures in mice

实验研究——新型突触蛋白 mint1 在小鼠癫痫发作后的作用

• 批准号: 13670678
• 负责人: NISHIMURA Hiroyuki
• 金额: $ 1.92万
• 依托单位: HYOGO COLLEGE OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670678/
• 关键词: synaptic protein; epilepsy; dentate gyrus; mossy fiber; granule cell layer; 歯状顆粒層細胞

Purpose : The aim of present study is to clarify the presynaptic function following epileptic seizures by estimating the change of mints in the brain.Methods : Adult male Sprague Dawley rate weighing 200-250 g were used. All animals were pretreatment with injections of atropine methylbromide 5 mg/kg intraperitoneally. Epileptic seizures were produced by intraperitoneal injection of pilocarpine hydrochloride 320-350 mg/kg. Seizure activity was monitored behaviorally, and after 3-4 hours of convulsive status epileptics, seizures were terminated with diazepam 10 mg/kg. Control rats were received saline only. Only animals that displayed continuous, convulsive seizure activity after pilocarpine treatment were used in the following experiments. Animals were perfusion-fixed with 4 % paraformaldehyde solution 2, 4, 6 hr, 1, 2, 3, 4, 7, 14 day 6 weeks after epileptic seizures (n=2-3 in each group). Serial coronal sections (20 μm in thickness) were obtained from the fixed brains with a vibratome … More and then subjected to immunocytochemistry for mint1, munc18, syp2. Immunostaining was performed with rabbit polyclonal antibody against rat mint1, munc18, syp2 as a primary antibody, followed by the ABC method. After reaction with respective primary antibodies, immunodetection was performed using the Vectastain ABC Elite Kit followed by reaction product visualization using 0.05 % diaminobenzidine.Results : In control rats (n=3) hippocampus, the immunoreactivity of mint1 was stained in the afferent fiber from the adjacent entorhinal cortex and mossy fiber from granule cell layer, in the molecular layer of the dentate gyres. The immunoreactivity of munc18 was uniformly stained in the hippocampus. Syp2 revealed in the mossy fiber and dentate gyros, In mossy fiber, the immunoreactivity of mint1 increased from 2 to 7 days after epileptic insult. On the other hand, there was no significant different of the immunoreactivity for munc18 and syp2 between control and epileptic mice in this area. In the dentate gyres, the immunoreactivity of mint1, munc18 and syp2 were greatly strained in the molecular layer. At the day 14, they marked distributed in the inner layer of molecular cell layer. These immunoreactivity continued until 6 weeks after initial epileptic status.Comments : The present study demonstrated that transient presynaptic dysfunction in the excitatory neurons to CA1 pyramidal neurons be prolonged since mint1, but not munc18 nor syp2 in the mossy fiber marked distributed until 7 days. These suggested that mint1 played the important role of neurotransmission from the entorhinal cortex to hippocampus following epileptic seizures. Less

目的：本研究的目的是通过估计大脑中的薄荷糖变化来阐明癫痫发作后突触前功能。方法：成年男性Sprague Dawley速率加权200-250 g。所有动物都是预处理，并注射了5 mg/kg腹膜内的阿托品甲基溴化物。癫痫发作是通过腹膜内注射盐酸盐酸320-350 mg/kg产生的。行为监测癫痫发作活性，并在3-4小时的抽搐状态癫痫发作后，用地西epa终止癫痫发作10 mg/kg。控制大鼠仅接受盐水。在以下实验中，仅使用毛果果处理后表现出连续的，抽搐的癫痫活性的动物。用4％多聚甲醛溶液2、4、6小时，1、2、3、4、7、14天6周（每组n = 2-3）灌注动物。从具有振动型的固定脑中获得了连续冠状切片（厚度为20μm）……更多，然后对MINT1，MUNC18，SYP2进行免疫细胞化学。用兔多克隆抗体对大鼠MINT1，MUNC18，SYP2作为原代抗体进行免疫染色，其次是ABC方法。与相对初级抗体反应后，使用Vectastain ABC Elite Kit进行免疫试验，然后使用0.05％二氨基苯胺进行反应产物可视化。反应：在对照大鼠（n = 3）海马中，MINT1的免疫反应性在邻接的Currorhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal Corterhinal interective中被染色。牙齿回旋的分子层。 Munc18的免疫反应性在海马中均匀染色。 Syp2在苔藓纤维和齿状陀螺中揭示，在苔藓纤维中，MINT1的免疫反应性从癫痫损伤后的2天增加到7天。另一方面，该区域对照小鼠和癫痫小鼠之间的Munc18和Syp2的免疫反应性没有显着不同。在牙齿回旋中，MINT1，MUNC18和SYP2的免疫反应性在分子层中极大地紧张。在第14天，它们标记在分子细胞层的内层中分布。这些免疫反应性一直持续到初始癫痫状态后的6周：本研究表明，由于MINT1，刺激神经元的兴奋神经元中的短暂性突触前功能障碍延长了MINT1，但在Mossy标记的纤维中分布直到7天，但在MOSSY FIBER中均未延长。这些表明MINT1在癫痫发作后从内嗅皮质到海马的神经递质发挥了重要作用。