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

目的：本研究的目的是通过测定癫痫发作后脑内薄荷糖的变化来阐明癫痫发作后突触前功能的变化。所有动物腹腔注射阿托品甲基溴5 mg/kg。腹腔注射盐酸毛果芸香碱320-350 mg/kg，造成大鼠癫痫发作。通过行为监测癫痫发作活动，在惊厥持续状态3-4小时后，用10 mg/kg地西泮终止癫痫发作。对照组大鼠仅接受生理盐水。在以下实验中仅使用在毛果芸香碱处理后显示出连续惊厥性癫痫发作活动的动物。癫痫发作后2、4、6小时，1、2、3、4、7、14天，6周用4%多聚甲醛溶液灌注固定动物（每组n=2-3）。用振动切片机对固定的脑组织进行连续冠状切片（厚度20 μm） ...更多信息 然后进行mint 1、munc 18、syp 2的免疫细胞化学。以兔抗大鼠mint 1、munc 18、syp 2的多克隆抗体为一抗，采用ABC法进行免疫组化染色。结果：在对照组大鼠（n=3）海马，mint 1免疫反应阳性表达于邻近内嗅皮质的传入纤维、颗粒细胞层的苔藓纤维和齿状回的分子层。munc 18在海马区呈均匀染色。在苔藓纤维和齿状回中，mint 1的免疫反应性在癫痫后2 ~ 7天逐渐增强。另一方面，正常小鼠和癫痫小鼠的munc 18和syp 2在该区域的免疫反应性无显著差异。在齿状回中，mint 1、munc 18和syp 2的免疫反应性在分子层中受到很大的限制。第14天，它们在分子细胞层的内层有明显的分布。这些免疫反应性持续到6周后，初始epileptic state.Comments：本研究表明，短暂的突触前功能障碍的兴奋性神经元CA 1锥体神经元被延长，因为薄荷1，但不是munc 18也不是syp 2的苔藓纤维显着分布，直到7天。提示mint 1在癫痫发作后内嗅皮层向海马的神经传递中起重要作用。少

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