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).
Healthcare Relevance There is strong evidence that the decreased synaptic plasticity in aging and Alzheimer's involves reductions in CaMKII activation. Significant evidence now has been accumulated, suggesting that the kinase may also contribute to development of numerous pathological processes leading to neurological dysfunction and diseases such as epilepsy, neuropathic pain, addiction, and cell death during stroke [1]. Relevant to our specific hypothesis regarding the importance of the complex of CaMKII with NR2B, it is of interest that the memory deficits in aging rodents can be overcome by overexpression of NR2B [2]. This raises the possibility that, during aging, NR2B receptors may become saturated with CaMKII. This illustrates the importance of understanding the fundamental mechanisms by which memory is stored. Understanding these mechanisms is likely to have an impact on treating memory deficits.
