Functions of neuronal membrane-associated proteins for neuronal connection and synapase formation.

1.大脑的许多部分都有层叠结构，每一层都接受来自特定神经元组的传入，然后形成生理上相关的突触。在鸡的顶盖中，可以区分出16层，视网膜的输入只在其浅层的3层(视网膜前板；RRL)中形成树枝和突触。免疫球蛋白超家族细胞黏附分子SC1/JC7/DM-GRGRAP/BEN在RRL深层表达，胚胎14~16天达高峰，以后逐渐减少。建立了一套共培养系统来分析板层特异性和视网膜神经元的终末行为。当应用N-钙粘附素抗体时，观察到更多的神经突起超过RRL边界，并减少了终末分枝。SC1抗体对N-钙粘附素的过度表达有相似的影响，但作用较小，但不影响树枝形成。Tho…更多的结果表明，一些具有空间限制性表达模式的细胞表面分子可能调控雏鸡视网膜顶盖神经元板层特异性连接的发育，也可能适用于其他层状脑部和/或其他物种。我们开发了一种新生小鼠的海马片培养系统，其器官型结构至少保持了三周。为了获得外源基因的神经元特异性表达，我们从CaMKII基因启动子控制下的Cre重组酶转基因系制备了切片培养物，分别感染带有启动子的腺病毒和带有loxP-Stop-loxP盒的EGFP。腺病毒之所以被选择，是因为它们对哺乳动物培养的细胞毒性很低。使用双光子激光显微镜可以实现对活神经元中突触结构的高分辨率观察。我们还分析了PSD-95-GFP和GFP-Zip45的动力学，表明它们的行为方式与我们之前报道的解离培养相似。为了研究N-钙粘蛋白在突触形成和可塑性改变中的作用，我们目前正在准备几种反义寡核苷酸抑制N-钙粘蛋白基因表达的系统，以及直接抑制其与抑制性多肽或蛋白质的黏附活性的系统。较少

1. Many parts of brain have laminated structures, and each lamina receives afferents from specific sets of neuron groups, then make physiologically relevant synapses. In the chick tectum, sixteen layers can be distinguished, and retinal inputs make arborizations and synapses only in the three of those lamina (retinorecipient laminae; RRL) in its superficial region. N-cadherin is concentrated specifically in RRL, and remained in adult, while SC1/JC7/DM-GRASP/BEN, an immunoglobulin superfamily cell adhesion molecule appeared to be expressed in deeper two RRL with its peak at embryonic day 14-16, then gradually decreased. A coculture system was developed to analyze the lamina-specificity and retinal neuron terminal behavior. When antibodies against N-cadherin were applied, more overshooting of neurites beyond RRL border, and reduced terminal arborization were observed. SC1 antibodies had similar but less effects on the overshooting to N-cadherin, however arborization was not affected. Tho … More se results suggest that some of cell-surface molecules with spatially-restricted expression patterns may regulate the development of lamina-specific retinotectal neuronal connectivity in the chick, and may also be applicable to other laminated brain portions and/or other species.2. We developed an hippocampal slice culture system from neonatal mice of which organotypic structures are well maintained for at least three weeks. In order to obtain neuron-specific expression of exogenous genes, we prepared the slice cultures from a transgenic line with Cre recombinase under control of CaMKII gene promoter, the the cultures are infected with adenoviruses bearing a promoter and EGFP with loxP-STOP-loxP cassette in between. Adenoviruses are chosen, because of their low cytotoxicity to mammalian cultures. High resolutional observation of synaptic structures in living neurons can be achieved using a two-photon laser microscope. We also analyzed dynamics of PSD-95-GFP and GFP-Zip45, showing that they behave in similar way to that of dissociation cultures which we previously reported. To study the function of N-cadherin in synapse formation and plastic change, we are now preparing several systems for suppressing N-cadherin gene expression with anti-sense oligonucleotides as well as direct inhibition of its adhesion activity with inhibitory peptides or proteins. Less