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.