【作者】 张智；

【导师】 徐天乐；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 长久以来,D型氨基酸被认为在高级进化的生物体中没有功能性作用,而只是通过整合进蛋白质多糖在低级进化的有机体中(如细菌中)发挥作用。然而,在过去的十年,随着胶质细胞和神经元的三突触结构的神经传递模型被大家认可,关于胶质细胞的高级功能无论是在健康的脑内还是在疾病模型上都得到了很大的关注。胶质细胞通过释放活性神经因子不仅可以倾听神经元活动而且可以和神经元对话。在此信号通路中,非典型的氨基酸D-丝氨酸(D-serine)的作用得到了广泛的关注。众所周知,D-丝氨酸是哺乳动物脑内NMDA型谷氨酸受体(NR1/NR2型)的辅助激动剂,在动物的大脑中一生都维持着很高的浓度:脑内的神经元和胶质细胞都含有D-丝氨酸并且具有特异的生物合成、胞外释放、摄取和降解途径。此外,选择性地清除D-丝氨酸可以降低NMDA受体(NMDAR)胞内信号和突触的长时程增强(LTP)。鉴于D-丝氨酸抗精神病的特征和其与NDMAR的作用对脑高级功能的意义,以前的研究都支持的观点是,D型氨基酸在不同的神经或精神医学上及其病理学条件下都是NMDAR的内源性激动剂。最近的研究报导了D-丝氨酸参与了LTP以及学习和记忆。并且,活动依赖的神经突触可塑性被认为是学习和记忆的细胞机制。因此,我们感兴趣的是,D-丝氨酸在突触可塑性和学习与记忆的高级脑功能中起到了什么调控作用呢?通过联合应用急性制备的海马脑片电生理记录、生物化学和行为学等方法,我们研究了以下几个问题:1,D-丝氨酸如何影响大鼠海马组织CA1区长时程可塑性?2,内源性的D-丝氨酸如何调控神经网络的长时程可塑性?3,D-丝氨酸如何影响动物的学习和记忆?实验结果如下:一)外源性的D-丝氨酸浓度依赖地影响海马CA1区的长时程抑制(LTD)和LTP在急性制备的海马脑片标本上,低浓度的D-丝氨酸(5微摩尔)提高了LTD的幅度而更高浓度的D-丝氨酸(100微摩尔)却不影响LTD的幅度,总体呈现“钟”形趋势。其对LTP作用亦如此。然而,在NMDAR的特异性拮抗剂7-chlorokynurenic或AP5都可以阻断D-丝氨酸对LTP和LTD的作用。这些结果表明,D-丝氨酸通过激活NMDAR影响LTP或LTD的幅度。二)胶质细胞通过感受突触前神经元活动释放相应浓度D-丝氨酸影响LTD应用不同的诱导强度产生的LTD幅度有显著的差别,我们的假设是不同诱导强度可以诱导不同水平的D-丝氨酸释放。应用D型氨基酸氧化酶特异性地清除生理条件下的D-丝氨酸后,LTD几乎完全被抑制;此外,特异性损伤胶质细胞,LTD和LTP均被阻断。然而,外源性D-丝氨酸的施加可以不同程度地修复胶质细胞破坏所损伤的LTD;有趣的是,增加诱导强度同样可以部分修复损伤的LTD;此外,这些条件下诱导的LTD均可被AP5所阻断。应用酶联免疫吸附实验检测(Enzyme-Linked Immunosorbent Assay)D-丝氨酸的水平发现,在不同的诱导强度下,D-丝氨酸的水平有明显的变化。这些数据表明胶质细胞可以通过感应神经元的活动而释放相应浓度的D-丝氨酸来调控长时程突触可塑性。三)D-丝氨酸影响大鼠的空间记忆提取并且呈剂量依赖性既然D-丝氨酸可以活动依赖地调控长时程突触可塑性,那么它在动物学习和记忆上的功能意义是什么?腹腔注射1000mg/kg D-丝氨酸明显提高了大鼠的水迷宫记忆提取水平;然而,腹腔注射100mg/kg或3000/mg/kg D-丝氨酸都不影响水迷宫的成绩。有趣地是,腹腔注射特异性损伤胶质细胞的药物sodimfluoroacetate明显抑制了大鼠的记忆能力,并且这种抑制效应可以被D-丝氨酸部分逆转。这些结果表明,D-丝氨酸不仅仅调控了长时程的突触可塑性并且影响着动物的记忆能力。总而言之,我们的研究表明,胶质细胞通过感应突触前神经元的活动模式,活动依赖地释放相应水平的D-丝氨酸从而浓度依赖地调控神经园长时程突触可塑性,这直接贡献于神经网络的平衡和记忆存储。更多还原

【Abstract】 Until the last decade,it was widely accepted that Damino acids had no functional role in higher organisms,but that they were restricted to lower organisms,such as bacteria, where they are integrated into the proteoglycans of the cell wall.Over the past decade, a growing body of evidence has emerged on the existence in the brain of a close bidirectional communication system between neurons and astrocytes,which is called tripartite synapse structure.Thus,the cases that glia not only listens but also talks to neurons through the release of neuroactive substances have been gained increasing attention as key players of higher functions in healthy brain,but also in diseases.In the signaling pathway,the nontypical amino acid,D-serine,is widely concerned.It has now been well established that D-serine,a coagonist for the N-methyl-Daspartate (NMDA)glutamate receptors(NR1/NR2 type),is maintained at a high concentration in mammalian brains for life.D-serine in the brain is contained in both the glia and neurons and has specific processes of biosynthesis,extracellular release,uptake,and degradation.Together with the anti-psychotic and anti-ataxic property of D-serine and the pivotal roles of the NMDA receptor in divergent higher brain functions,these observations support the view that the Damino acid may be involved as an endogenous modulator for the NMDA receptor in various neuropsychiatric functions and their pathological conditions.Recently reports show that D-serine participates not only in LTP but also in learning and memory.Generally,it is believed that activity-dependent synaptic plasticity serves as a cellular mechanism underlying learning and memory.Thus,we are interested that how D-serine regulates the higher brain functions,such as synaptic plasticity and learning and memory? Using acute prepared hippocampal slices,electrophysiological recording, biochemistry and behavioural testing methods,we investigated the following 3 questions:1.How dose D-serine affect hippocampal long-term synaptic plasticity? 2. How dose endogenous D-serine regulate the homeostasis of neural network? 3.How dose D-serine affect animal learning and memory?1.Exogenous D-serine affects hippocampal CA1 regional LTD and LTP in a concentration-dependent mannerIn slices incubated in 5μM D-serine,the magnitude of LTD was enhanced significantly compared with that in the control group.However,in slices incubated in 100μM D-serine,the expression of LTD was not affected.According to these results,we wondered whether D-serine regulated LTD in a concentration-dependent manner.Thus,LTD was induced in the slices incubated by D-serine at different concentrations(3μM,5μM,10μM,50μM,100μM). The magnitudes of LTD had a bell-shape:5μM D-serine enhanced the magnitude of LTD to maximum;with the concentration of D-serine increasing,LTD returned to control level in the slices incubated in 100μM D-serine.Having established these data,we further revealed that the LTD was NMDAR dependent,being blocked by AP5 or 7-chlorokynurenic,both are special antagonists of NMDAR.It is the case to LTP.The data presented so far indicate that D-serine regulates long-term synaptic plasticity via NMDAR activation in a concentration-dependent manner.2.Gila release D-serine through sensing presynaptic activities to regulate LTDSince different magnitudes of LTD were induced by different stimulus protocol,we hypothesized that different stimulus intensities during LFS induced the release of corresponding D-serine.After Damino acid oxidase(DAAO,the enzyme which degrades D-serine)eliminated the physiological D-serine,LTD induction was blocked. However,exogenous D-serine rescued the DAAO-inhibited LTD to different degrees. Furthermore,a strong stimulus intensity during rescued the glial impairment-inhibited LTD.in addition,the LTD induced under these condition was blocked by AP5. Interestingly,Enzyme-Linked Immunosorbent Assay(ELISA)showed that the D-serine level altered significantly after LFS compared with that during baseline recording.3.D-serine affected memory in dose-dependent mannerIt is widely believed that long-term synaptic plasticity serves as a cellular mechanism underlying learning and memory[1-3].Thus,we investigated the effects of glial cell impairment and exogenous D-serine on spatial memory retrieval in Morris water maze.Intraperitoneal injection with D-serine(1000 mg/kg) increased memory retrieval significantly compared with the control group while injection with a lower(100 mg/kg)or higher(3000 mg/kg)dose of D-serine did not affect memory retrieval.In contrast,injection with NaFAC(3 mg/kg)impaired memory retrieval and D-serine(1000 mg/kg)restored it partially(Fig.4B).This result provides strong evidence that glial cells are crucial to memory retrieval through releasing D-serine.In brief,these findings suggest that the patterns of presynaptic activity can be sensed by glia leading to corresponding concentrations of D-serine release.And then,as a crucial neuromodulator,D-serine may modify activity-dependent long-term synaptic plasticity and thus maintain neural network homeostasis and memory storage.更多还原