Analysis of Molecular Assembly of Synaptic Molecules during Development

海马锥体神经元培养10 ~ 20 d后，兴奋性突触由树突轴向棘转变。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，我们生成了绿色荧光蛋白（GFP）标记的PSD蛋白，如PSD-95和PSD- zip45（也称为Homer Ic），以及突触前标记synaptophysin。第一组实验旨在揭示树突棘的形态发生、PSD蛋白的组装和突触囊泡在单个突触连接处的积累的时间过程。我们对活海马神经元进行了双波长延时荧光显微镜观察，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，发生在不到一个小时。在第二个研究项目中，我们分析了PSD-Zip45在培养神经元刺激后的动态再分布。与刺激后PSD-95的稳定性相反，PSD-Zip45在细胞内钙含量增加后，其分布发生了双向变化。这一结果表明存在通过神经元活动驱动psd - zip45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-Zip45的转基因小鼠，以实现探针分子在海马中的稳定表达。利用从转基因海马中分离出来的培养神经元，我们观察到PSD-95和PSD-Zip45簇在1周内的缓慢重塑。结果表明，存在camp依赖的信号系统，调节单个细胞内PSD簇的平均密度。综上所述，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蛋白在不同神经元活动下被差异激活的复杂信号系统对其分布的调控。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