Role of CaMKII in memory storage

项目摘要/摘要 活动依赖的突触修饰(LTP/LTD)是记忆机制的主要候选者。LTP 包括诱导、维持和表达过程。这项提议试图阐明分子 维护过程的基础，是印记的基础过程。 与维护有关的是“擦除测试”，即在LTP/Memory建立之后应用抑制物。如果 这会阻止LTP/记忆，并且在移除抑制器后效果仍然存在，则抑制器一定已经擦除了 维护流程。我们已经使用CaMKII(CN-肽)的抑制剂进行了擦除测试。我们 发现在LTP诱导后使用该肽可消除饱和的LTP。我们现在提议 两个实验在行为水平上测试了CaMKIIα在记忆维持中的作用。 询问显性-否定形式的CaMKIIα是否可以消除条件性位置回避。 第二个测试是“遮挡测试”。 激酶(CaMKIIα*)增强突触传递，阻断突触诱导的长时程增强。我们会像病毒一样 表达CaMKIIα*并测试两个预测：因为这最大限度地增加了所有突触的权重 应该破坏记忆功能，而且，在这些条件下学习应该是不可能的。 提供了支持这些预测的初步证据。这项提案中的其他实验旨在 了解储存印记的CaMKIIα复合体的性质。有很强的理由 怀疑维持LTP的实际上是CaMKIIα与NMDAR的复合体(可能也是致密的- 180)。蛋白质与CaMKII结合的研究一直依赖于体外工作，以前还没有 研究在实际LTP诱导过程中形成的络合物的方法。因此，有关这一事件的关键信息 缺乏复杂的阵型和持久性。我们已经开发并验证了一种新的光学方法 在电影院里。我们的初步证据表明，LTP诱导产生了CaMKIIα与 GluN2B在脊椎中，这种形成是突触特有的。我们将确定建筑群的持续时间 在诱导短期LTP(早期LTP)或同时产生早期和晚期LTP的条件下。我们会 还检查复合体的持续时间如何取决于增强(例如，BDNF)或防止(例如， 蛋白质合成抑制剂)晚期LTP。这一方法将被扩展到研究CaMKIIα与 致密蛋白-180。在确定了LTP背后的复合体的性质后，体内实验将是 进行测试以测试复合体内蛋白质相互作用的中断是否会扰乱 英格拉姆。

Project Summary/Abstract Activity-dependent synaptic modifications (LTP/LTD) are a major candidate for the mechanism of memory. LTP involves induction, maintenance, and expression processes. This proposal seeks to elucidate the molecular basis of the maintenance process, the process that underlies the engram. The critical test of any hypothesis regarding maintenance is the “erasure test” in which an inhibitor is applied after LTP/memory is established. If this blocks LTP/memory and the effect persists after the inhibitor is removed, the inhibitor must have erased a maintenance process. We have conducted the erasure test using an inhibitor of CaMKII (CN-peptide). We found that application of this peptide after LTP induction produced erasure of saturated LTP. We now propose two experiments that test the role of CaMKIIα in memory maintenance at the behavioral level. In the first, we ask whether a dominant-negative form of CaMKIIα can erase conditioned place avoidance. We present strong preliminary evidence that it does. The second test is the “occlusion test.” It has been shown that activated kinase (CaMKIIα*) enhances synaptic transmission that occludes synaptically induced LTP. We will virally express CaMKIIα* and test two predictions: that because this maximally increases all synaptic weights it should destroy memory function, and furthermore, that learning under these conditions should not be possible. Preliminary evidence supporting these predictions is presented. Other experiments in this proposal are aimed at understanding the nature of the CaMKIIα complex that stores the engram. There are strong reasons to suspect that what maintains LTP is actually the complex of CaMKIIα with NMDAR (and perhaps also densin- 180). Studies of the binding of proteins to CaMKII have relied on in vitro work, and there has been no previous method for studying the complexes formed during actual LTP induction. Thus, crucial information regarding the complex formation and persistence is lacking. We have developed and validated a novel optical method based on FLIM-FRET. Our preliminary evidence demonstrates that LTP induction produces complex of CaMKIIα with GluN2B in spines and that the formation is synapse specific. We will determine the duration of the complex under conditions that either induce short-lasting LTP (early LTP) or produce both early and late LTP. We will also examine how the duration of the complex depends on factors that enhance (e.g., BDNF) or prevent (e.g., protein synthesis inhibitors) late LTP. This approach will be extended to study the interaction of CaMKIIα with densin-180. Having identified properties of the complex that underlies LTP, in vivo experiments will be conducted to test whether disruption of protein interactions within the complex can disrupt maintenance of the engram.