Analysis of Molecular Assembly of Synaptic Molecules during Development

在海马锥体神经元的培养中，兴奋性突触从树突轴向棘的过渡发生在10-20天的时间内。该培养体系为研究突触结构的形成和重塑提供了良好的模型。为了分析多种突触后密度（PSD）蛋白的动态特性，我们产生了绿色荧光蛋白（GFP）标记的PSD蛋白，例如PSD-95和PSD-Zip 45（也称为Homer Ic），以及突触前标记物突触素。第一组实验旨在揭示树突棘的形态发生、PSD蛋白的组装和单个突触连接处突触囊泡的积累的时间过程。我们进行了双波长，延时荧光显微镜的活海马神经元，并揭示了这三个事件之间的时间相关性。此外，突触发生是一个快速的过程，发生不到一个小时。在第二个研究项目中，我们分析了动态重分布 ...更多信息 在刺激培养的神经元后，PSD-Zip 45的分布。与PSD-95在刺激后的稳定性质相反，PSD-Zip 45在细胞内钙增加后双向改变其分布。该结果表明存在通过神经元活性驱动PSD-Zip 45特异性易位的分子机制。在第三组实验中，我们产生了表达GFP标记的PSD-95或PSD-Zip 45的转基因小鼠，以实现探针分子在海马中的稳定表达。使用从转基因海马分离的培养的神经元，我们表征了PSD-95和PSD-Zip 45簇的缓慢重塑>1周。结果表明，cAMP依赖性信号系统的存在下，调节PSD簇的平均密度在一个单一的细胞。总之，这三组实验揭示了PSD蛋白在活神经元中的新的分子动力学，以及PSD蛋白通过复杂的信号系统的分布的调节，该信号系统可以被各种神经元活动差异激活。少

Transition of excitatory synapses from dendritic shafts to spines takes place during the period of 10-20 days in culture of hippocampal pyramidal neurons. This culture system provides a good model for the study of formation and remodeling of synaptic structure. To analyze dynamic properties of multiple postsynaptic density (PSD) proteins, we generated green fluorescent protein (GFP)-tagged PSD proteins, such as PSD-95 and PSD-Zip45 (also known as Homer Ic), together with presynaptic marker synaptophysin. The first set of experiments aimed at revealing time-course of morphogenesis of dendritic spines, assembly of PSD proteins, and accumulation of synaptic vesicles at single synaptic junctions. We performed dual-wavelength, time-lapse fluorescence microscopy of living hippocampal neurons and revealed temporal correlation among these three events. Furthermore, synaptogenesis was a rapid process, which took place less than an hour. In the second research project we analyzed dynamic redistr … More ibution of PSD-Zip45 after stimulation of cultured neurons. In contrast to the stable nature of PSD-95 after stimulation, PSD-Zip45 changed its distribution bidirectionally after increase of the intracellular calcium. This result indicates the presence of molecular mechanism that drives PSD-Zip45-specific translocation by the neuronal activity. In the third set of experiments we generated transgenic mice expressing either GFP-tagged PSD-95 or PSD-Zip45 to achieve stable expression of probe molecules in the hippocampus. Using cultured neurons isolated from the transgenic hippocampus, we characterized slow remodeling of PSD-95 and PSD-Zip45 clusters for >1 week. The results indicate the presence of cAMP-dependent signaling system that regulates the average density of PSD clusters within a single cell. In summary, these three sets of experiments revealed novel molecular dynamics of PSD proteins in living neurons and regulation of the distribution of PSD proteins by complex signaling system that can be differentially activated by various neuronal activities. Less