Role of CaMKII in memory storage

 描述(申请人提供)：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。这项提议试图阐明存储记忆信息的维护过程。对任何关于记忆维持的假说的关键检验是“擦除试验”，即在LTP/Memory建立之后应用抑制物。如果这阻止了 LTP/Memory，并且在移除抑制剂后效果仍然存在，则该抑制剂一定已经擦除了维护过程。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诱导后使用该肽可以消除饱和的LTP。此外，在LTP被清除后，LTP可以被重新诱导，表明塑性机制保持不变。这项建议的总体目标是对CaMKII在LTP维持中的作用进行进一步的关键测试，并通过研究CaMKII在行为记忆维持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过程。拟议工作的结果可能提供确定其中一个进程的第一个强有力的证据。目标1的目标是测试CaMKII假说的一个特定变体。生化实验表明，CaMKII的激活使其与NMDAR结合。我们已经证明了CN-肽，一种在复合体中产生还原的肽，逆转了LTP。这些结果表明CaMKII/NMDAR复合体在LTP过程中形成，并负责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，关于复杂的形成和持久性的关键信息是缺乏的。我们开发并验证了一种基于Flim-FRET的新型光学方法。我们的初步证据表明，LTP诱导在脊椎中产生复杂的结构，并且这种结构是突触特异性的。我们将确定在诱导短期LTP(早期LTP)或产生早期和晚期LTP的条件下复合体的持续时间。我们还将研究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的因素。这些实验将提供一个强有力的测试，以确定该复合体是否具有成为分子记忆所需的持久性。目标2的目标是在行为级别进行删除测试。我们将使用条件性位置厌恶，这是一种依赖于海马体的记忆形式。在学习完成后，HSV病毒将被用来将显性-阴性形式的CaMKII(K42M)传递到CA1区域，产生这种突变激酶的瞬时(几天)表达。记忆保持将在一周后进行测试。我们的初步结果表明，这种形式的行为记忆可以通过这个过程来消除。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)的复合体。

 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).