【作者】 琚珏刚；

【导师】 李胜天；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 在神经系统中,N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受体是一类重要的离子通道型谷氨酸受体,其通道的开放可引起大量Ca2+流入胞内并激活下游的一系列信号通路。NMDA受体一方面在突触传递、突触可塑性以及学习与记忆等生理过程中起着举足轻重的作用,另一方面在诸如脑缺血、中风、癫痫、精神分裂症、抑郁症及一些神经退行性疾病的病理过程中亦是发挥着至关重要的作用。近年来的一系列研究表明:1)在生理状态下极少开放的突触外NMDA受体会在癫痫等病理状态下异常开放;2)星形胶质细胞分泌的谷氨酸可能是通过激活突触外NMDA受体来导致癫痫发作中的神经元同步放电的;3)突触外NMDA受体的激活能介导兴奋毒性,且可能导致癫痫发病过程中的神经元死亡。以上这些结果都提示突触外NMDA受体可能在癫痫的病理机制中起着重要的作用。但在癫痫发生阶段,目前很少有关于NMDA受体电生理学性质变化的研究,更无直接测试突触外NMDA受体的报告。因此,我们大胆假设突触外NMDA受体在癫痫发生过程中的异常激活介导了其病理机制,并在此研究了突触内、外NMDA受体介导的兴奋性突触后电流(NMDA receptors-mediated excitatory postsynaptic currents, NMDA-EPSCs)在癫痫发生早期过程中的变化。我们选取了6-8周的成年小鼠,通过氯化锂联合匹罗卡品的方法诱导了颞叶癫痫模型。在模型小鼠癫痫持续状态(status epilepticus, SE)发作之后的24小时,我们制备了急性海马切片,并利用膜片钳全细胞记录法研究了CA1区锥体神经元突触内NMDA受体所介导的EPSCs。我们随后利用了我们独自发展的一套实验方案来选择性地记录突触外NMDA受体介导的电流:在灌流NMDA受体拮抗剂MK-801 20分钟之后,给予低频刺激使得突触内NMDA受体先被激活,继而被MK-801不可逆地阻断;其后洗脱MK-801,30分钟后通过高频刺激来选择性地激活尚未被阻断的突触外的NMDA受体。我们对突触内、外NMDA受体介导电流的通道动力学指标进行了分析、统计,并与生理盐水对照组比较后发现:1)突触内NMDA-EPSCs的上升时间及衰减时间均无统计学差异;2)突触外NMDA-EPSCs的峰值、面积峰值比、以及上升时间亦无统计学差异;3)但突触外NMDA-EPSCs的衰减时程相对于对照组加快。以上结果表明在癫痫发生早期,突触内NMDA受体的通道动力学无显著改变,但突触外NMDA受体的通道性质却产生了变化,提示突触外NMDA受体的异常可能参与到了癫痫的发病机制之中。我们相信通过进一步地阐明突触外NMDA受体参与癫痫发生的分子机制,可为癫痫的治疗及预防提供合适的靶标作为参考。更多还原

【Abstract】 The N-methyl-D-aspartate (NMDA) receptor is a major type of ionotropic glutamate receptor, and its activation can induce a large amount of Ca2+ influx which mediates most of NMDA receptor activities through downstream signaling pathways. Many studies have shown that NMDA receptors play pivotal roles in the central nervous system under both physiological and pathological conditions. Recent reports have shown that: 1) extrasynaptic NMDA receptors can abnormally open under epileptic seizure conditions; 2) NMDA receptors can be activated by the elevated astrocytic glutamate, which may lead to the neuronal synchronous discharging during seizures; 3) the downstream cascades of extrasynaptic NMDA receptors can mediate excitotoxicity and promote neuronal death which can occur after status epilepticus (SE). These results suggest a key role for extrasynaptic NMDA receptors in the molecular mechanism of epilepsy. However, so far, there are few studies focusing on electrophysiological properties of NMDA receptors during epileptogenesis, even less is related to extrasynaptic NMDA receptor.Here, we hypothesize that the abnormal activation of extrasynaptic NMDA receptors participates in the epileptogenesis. We studied the changes of synaptic and extrasynaptic NMDA receptor-mediated currents at the early stage of epilepsy. Temporal lobe epileptic models are induced in six to eight week-old mice by pilocarpine (PILO) combined with lithium chloride. We prepared acute hippocampal slices at 24 hours after the onset of status epilepticus, and then recorded the synaptic NMDA receptors-mediated excitatory postsynaptic currents (NMDA-EPSCs) of pyramidal neurons in CA1 region by patch clamp whole-cell recording technique. Furthermore, we established a new protocol to record extrasynaptic NMDA receptor-mediated EPSCs. Firstly, synaptic NMDA-EPSCs were blocked by applying a 5 Hz stimulation in the presence of MK-801, an activity-dependent and irreversible NMDA receptor antagonist. Then, the MK-801 was washed out for 30 min, and the remaining extrasynaptic NMDA receptors, which were not blocked by MK-801, were finally activated by a 100 Hz stimulation.After analyzing the channel kinetic parameters of synaptic and extrasynaptic NMDA-EPSCs from both PILO- and saline-treated group, we found that: 1) there was no significant difference in both rise time and decay time of synaptic NMDA-EPSCs; 2) the peak amplitude, area to peak ratio, and rise time of extrasynaptic NMDA-EPSCs were not altered; 3) the decay of extrasynaptic NMDA-EPSCs, however, was faster in PILO-treated group than that of saline-treated mice. Taken together, these results suggest that the change in kinetics of extrasynaptic rather than synaptic NMDA receptors participates in epileptogenesis. Elucidating the extrasynaptic NMDA receptors-mediated molecular mechanisms in epileptogenesis will provide a potential cue for the effective treatment of epilepsy.更多还原