Regulation of synapse development, function and plasticity by the extracellular matrix of the central nervous system

化学突触是神经系统神经元之间交流的关键结构。这些单位由突触前和突触后组成，介导神经元之间快速而有效的信号传递，并被胶质细胞包围。后者影响突触的强度和可塑性，星形胶质细胞和神经元形成所谓的三部分突触。星形胶质细胞释放营养物质、神经营养因子、细胞因子、神经递质和糖蛋白以及细胞外基质(ECM)中的硫酸软骨素蛋白多糖(CSPGs)。在一个由SPP-1109神经胶质细胞和突触资助的项目中，实验室探索了神经细胞外基质对突触形成的功能。为此，开发了一种原代胚胎海马神经元培养系统，该系统允许在体外分析突触的形成。利用这个系统，我们已经能够证明CSPGs调节神经元表面的突触密度并影响海马神经元的微小兴奋性突触后电流(MEPSCs)的幅度。对一个缺失CSPG、NeuroCan和brevican以及糖蛋白tenascin-C和tenascin-R的四重基因敲除小鼠突变体的分析表明，神经ECM在培养的头两周内调节突触密度，是突触稳定和中期内形成被称为神经周网络(PNNS)的ECM超结构所必需的。四重敲除神经元表现出突触传递的缺陷，表现为mIPSCs和mEPSC频率降低。根据这些结果，实验室提出了指导本提案目标的三个假设。第一种假设假设细胞外基质环境调节与突触形成和功能相关的基因的表达。目前的转录组分析与这一建议是一致的，应该在不同的发育阶段进行。第二种假设认为，PNN结构在四头肌敲除组织中被修饰。这一假设可以通过免疫细胞化学得到证实，并将使用高分辨率标准辐射耗尽(STED)显微镜进行进一步阐述。第三种假说认为，四重突变神经元和PNN的遗传变化改变了神经元网络的活动模式。利用多电极阵列(MEA)技术，比较野生型和突变型神经元的网络活动，并将其提升到活体海马功能研究的水平。最近的遗传学和神经病理学研究表明，细胞外基质基因与神经精神疾病有关。四重基因敲除小鼠的模型提供了一个独特的机会，可以在神经元网络内的突触功能的背景下研究ECM和PNN结构的生物学效应，并发展与ECM变化在精神疾病领域的意义有关的概念。

Chemical synapses represent key structures for the communication between neurons of the nervous system. These units consist of a pre- and a post-synapse, mediate the rapid and efficient signal transmission between neurons, and are surrounded by glial cells. The latter affect synaptic strength and plasticity whereby astrocytes and neurons form the so-called tripartite synapse. Astrocytes release nutrients, neurotrophins, cytokines, neurotransmitters and glycoproteins and chondroitinsulfate proteoglycans (CSPGs) of the extracellular matrix (ECM). In a project funded within the SPP-1109 Neuroglia and Synapse the laboratory has explored functions of the neural ECM for synapse formation. To this end, a culture system for primary embryonic hippocampal neurons has been developed that allows for the analysis of synapse formation in vitro. Using this system, we have been able to demonstrate that CSPGs regulate synapse density on neuronal surfaces and influence the amplitude of miniature excitatory postsynaptic currents (mEPSCs) of hippocampal neurons. The analysis of a quadruple knockout mouse mutant that misses the CSPGs neurocan and brevican as well as the glycoproteins tenascin-C and tenascin-R revealed that the neural ECM regulates synapse density within the first two weeks of culture and is required for synapse stabilization and the formation of ECM superstructures designated as perineuronal nets (PNNs) in medium term. Quadruple knockout neurons displayed a deficit of synaptic transmission that manifested in reduced mIPSCS and mEPSC frequencies. Based on these results the laboratory has developed three hypotheses that direct the aims of the present proposal. The first hypothesis assumes that the ECM environment regulates the expression of genes that are relevant for synapse formation and function. Current transcriptome analyses are in agreement with this suggestion and shall be pursued at distinct developmental stages. The second hypothesis posits that PNN structures are modified in the quadruppel knockout tissue. This assumption could be confirmed by immunocytochemistry and will be elaborated further using high resolution standard emission depletion (STED) microscopy. The third hypothesis proposes that the genetic changes of the quadruple mutant neurons and PNNs modify the activity patterns of neuronal networks. Using the multi electrode array (MEA) technology the network activities of wild type and mutant neurons shall be compared and be carried to the level of investigations of hippocampal functions in vivo. Recent genetic and neuropathological investigations have suggested an association of ECM genes with neuropsychiatric diseases. The model of the quadruple knockout mouse offers a unique opportunity to investigate the biological effects of the ECM and PNN structures in the context of synaptic functions within neuronal networks and to develop concepts relating to the significance of ECM-changes in the realm of psychiatric diseases.