Role of CaMKII in memory storage

 描述（由申请人提供）：活动依赖性突触修饰（LTP/LTD）是记忆机制的主要候选者。LTP涉及诱导、维持和表达过程。这一提议试图阐明存储记忆信息的维护过程。任何关于记忆维持的假设的关键检验是“擦除测试”，其中在LTP/记忆建立后应用抑制剂。如果这个能挡住 LTP/记忆，并且在去除抑制剂后效果仍然存在，则抑制剂必须清除维护过程。我们使用CaMKII抑制剂（CN-肽）进行了擦除试验。我们发现在LTP诱导后应用这种肽产生饱和LTP的擦除。此外，在LTP被擦除后，LTP可以被重新诱导，表明可塑性机制保持完整。本提案的总体目标是对CaMKII在维持LTP中的作用进行进一步的关键测试，并通过检查CaMKII在维持行为记忆中的作用来扩展这项工作。内存维护可能依赖于许多不同的进程。拟议工作的结果可以提供第一个强有力的证据，确定这些过程之一。目标1的目的是测试CaMKII假设的一个特定变体。生物化学实验表明，CaMKII的激活导致其与NMDAR结合。我们已经表明，CN-肽，一种肽，产生减少的复杂，逆转LTP。这些结果表明，CaMK Ⅱ/NMDAR复合物的形成过程中的LTP和负责的LTP的维持。然而，还没有以前的方法来监测LTP诱导期间和之后突触的复杂动力学。因此，缺乏关于复杂的形成和持久性的关键信息。我们已经开发和验证了一种新的光学方法的基础上FLIM-FRET。我们的初步证据表明，LTP的诱导产生复杂的形成，在脊柱和形成突触特异性。我们将确定复合物的持续时间的条件下，要么诱导短暂的LTP（早期LTP）或产生早期和晚期LTP。我们还将研究复杂的持续时间如何取决于增强的因素（例如，BDNF）或预防（例如，蛋白质合成抑制剂）晚期LTP。这些实验将提供一个强有力的测试，以证明复合物是否具有成为分子记忆所需的持久性。目标2的目标是在行为水平上进行擦除测试。我们将使用条件性位置厌恶，这是一种依赖于营地的记忆形式。在获得学习后，HSV病毒将用于将CaMKII（K42 M）的显性阴性形式递送至CA 1区，产生该突变激酶的瞬时（几天）表达。一周后将进行记忆力测试。我们的初步结果表明，这种形式的行为记忆可以通过这种程序消除。在其他实验中，我们将测试更具体的假设，即行为记忆依赖于激活的CaMKII与NMDAR（或CaMKII结合的其他PSD蛋白）的复合物。

 DESCRIPTION (provided by applicant): Activity-dependent synaptic modifications (LTP/LTD) are the leading candidate for the mechanism of memory. LTP involves induction, maintenance, and expression processes. This proposal seeks to elucidate the maintenance processes that store memory information. The critical test of any hypothesis regarding memory 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. Moreover, after LTP was erased, LTP could be reinduced, indicating that plasticity mechanisms remained intact. The overall goal of this proposal is to conduct further critical tests of the role of CaMKII in maintenance of LTP and to extend this work by examining the role of CaMKII in the maintenance of behavioral memory. Memory maintenance is likely to depend on many different processes. The results of the proposed work could provide the first strong evidence identifying one of these processes. The goal of Aim 1 is to test a specific variant of the CaMKII hypothesis. Biochemical experiments show that activation of CaMKII causes it to bind to the NMDAR. We have shown that CN-peptide, a peptide that produces a reduction in the complex, reverses LTP. These results suggest that the CaMKII/NMDAR complex forms during LTP and is responsible for the maintenance of LTP. However, there have been no previous methods for monitoring the complex dynamics at synapses during and after 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 formation 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. These experiments will provide a strong test of whether the complex has the persistence required to be a molecular memory. The goal of Aim 2 is to conduct the erasure test at the behavioral level. We will use conditioned place aversion, a hippocampal-dependent form of memory. After learning is achieved, HSV virus will be used to deliver a dominant-negative form of CaMKII (K42M) to the CA1 region, producing transient (several-day) expression of this mutant kinase. Memory retention will then be tested a week later. Our preliminary results indicate that this form of behavioral memory can be erased by this procedure. In additional experiments, we will test the more specific hypothesis that behavioral memory is dependent on the complex of activated CaMKII with the NMDAR (or other PSD proteins to which CaMKII binds).