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：观察癫痫发作后大鼠脑内薄荷脑的变化，探讨癫痫发作后突触前功能的变化。所有动物均予阿托品甲基溴5 mg/kg腹腔注射。以盐酸匹罗卡品320~350 mg/kg腹腔注射致痫大鼠。对癫痫发作活动进行行为学监测，癫痫发作持续3~4h后，给予安定10 mg/kg终止癫痫发作。对照组大鼠只注射生理盐水。只有在匹罗卡品治疗后表现出持续的惊厥活动的动物才被用于以下实验。分别于癫痫发作后2、4、6h、1、2、3、4、7、14d灌流4%多聚甲醛溶液，每组2~3只。连续冠状切面(厚度20μm)取自固定的大脑，用振荡器…然后对mint1、Munc18、syp2进行免疫细胞化学染色。以兔抗大鼠mint1、Munc18、syp2多克隆抗体为一抗，采用ABC法进行免疫染色。结果：对照组(n=3)大鼠海马齿状回分子层，mint1免疫反应阳性染色位于邻近内嗅皮层的传入纤维和颗粒细胞层的苔藓纤维。Munc18的免疫反应在海马区呈均匀染色。Syp2表达于苔藓纤维和齿状回，在苔藓纤维中，mint1的免疫反应活性在癫痫发作后2~7d逐渐增强。而Munc18和syp2的免疫反应性在癫痫小鼠和正常小鼠之间无显著差异。在齿状回，mint1、Munc18和syp2的免疫反应主要集中在分子层。第14天，它们明显分布在分子细胞层的内层。这些免疫反应一直持续到癫痫发作后6周。注：本研究表明，兴奋性神经元对CA1锥体神经元的一过性突触前功能障碍从mint1开始延长，而苔藓纤维中Munc18和syp2的分布一直持续到7天。提示mint1在癫痫发作后内嗅皮层至海马区的神经传递中起重要作用。较少