Dynamic Structural Properties of Synapses

通过在培养中预嵌入游离海马神经元的纳米金标记，使用与CaMKII先前工作相似的策略，研究了PSD成分在活动期间的运动。高K+的去极化通常用于去极化和激活神经元培养物，但其他更接近生理刺激的方案，如NMDA的应用和在没有Mg2+的情况下用甘氨酸激活突触，正在测试中。现在，针对PSD几乎所有主要成分的抗体已经获得，并在免疫电子显微镜下进行了相容性测试，在分子水平上，活性诱导的PSD重组的总体图景开始出现。主要的PSD支架，由PSD-95和GKAPs组成，在短期活动中似乎相对稳定，而Shanks，尤其是Shank1，向PSD靠近。虽然我们之前描述了CaMKII在激活过程中向PSD移动，但我们现在发现另一个主要的调节蛋白SynGAP在刺激时离开PSD。这些刺激诱导的变化与PSD中AMPA受体标记的显著增加相一致，与CaMKII和SynGAP在调节PSD中AMPA受体运输的酶和结构作用相一致。

Movements of PSD components during activity were studied by pre-embedding Nanogold labeling of dissociated hippocampal neurons in culture, using strategies similar to those employed in previous work with CaMKII. Depolarization with high K+ is typically used to depolarize and activate neuronal cultures, but other protocols closer to physiological stimulation, such as application of NMDA and synaptic activation with glycine in the absence of Mg2+, are being tested. Now that antibodies for nearly all major components of the PSD have been obtained and tested for compatibility for immuno-electron microscopy, a general picture of activity-induced re-organization of the PSD at the molecular level begins to emerge. The main PSD scaffold, consisting of PSD-95 and GKAPs, appears to be relatively stable during short-term activity, while Shanks, especially Shank1, move closer to the PSD. While we have previously described movement of CaMKII towards the PSD during activation, we now show that another major regulatory protein, SynGAP, moves away from the PSD upon stimulation. These stimulation induced changes coincide with a marked increase of AMPA receptor labeling at the PSD, compatible with enzymatic as well as structural roles of CaMKII and SynGAP in regulating AMPA receptor trafficking at the PSD. EM tomography of isolated PSDs is being used to describe with more precision the re-localization of proteins at the PSD following activity. Two technical breakthroughs, the isolation of PSD fractions from hippocampal slice cultures, and a negative staining method compatibile with EM tomography, have been essential. PSDs isolated from depolarized hippocampal slices show elevated levels of CaMKII confirming that activity-induced changes at the PSD are preserved during isolation. CaMKII association domains appear as rings that are readily recognizable in tomograms, allowing accurate localization of individual CaMKII molecules within the PSD. Mapping the distribution of CaMKII will show exactly where in the PSD CaMKII is bound at rest, and after stimulation protocols with defined physiological consequences. This is an essential question to explore because CaMKII has multiple phosphorylation targets at multiple locations within the PSD. Localization of CaMKII after depolarization with high K+ will be followed by stimulation protocols known to induce long-term changes, such as chemLTP and chemLTD. Quantitative mass spectrometry protocols are in place to track parallel changes in protein composition and phosphorylation.