Synaptic failure is a cause of cognitive decline and memory dysfunction in Alzheimer’s disease (AD). Defective synaptic function and synaptic failure in AD is associated with the loss of dendritic spines and glutamatergic signaling. However, despite the importance of dendritic spine synapses in disease, the small size of spines and synapses has prevented us from understanding how pre- and post-synaptic molecules might serve as markers for the onset of AD and how to target these structures for AD intervention. Our proposal seeks to break down this barrier by using super-resolution imaging and state-of-the-art approaches to understand the nanomolecular organization of dendritic spines synapses, which begin to disappear before AD can be diagnosed. Our preliminary data indicate that AMPA and NMDA type glutamate receptors, which are both linked to AD, are organized using distinct rules in spines of different sizes, suggesting that individual synapses might have unique molecular signatures that can distinguish strong synapses from weak or weakening synapses. Here, we will test the hypothesis that changes in spine size determine the organization of AMPARs and NMDARs and that spine enlargement following cLTP will lead to the differential distribution of particular AMPAR and NMDAR subunits at synapses. We will take advantage of our approach combining morphological reconstructions of labeled spines and multi-color STED imaging. In Aim 1, we will determine the organization of glutamate receptors in discrete nanomodules of single and multi-module spines. We will use three-color STED imaging of PSD-95 and Bassoon, and either AMPAR or NMDAR subunits in EGFP labeled spines. We will examine how different subunits are organized relative to aligned PSD-95/Bassoon nanomodules and whether each nanomodule differs in their glutamate receptor make-up. In Aim 2, we will determine the organization of glutamate receptors in discrete nanomodules after induction of structural plasticity. Here, we will induce cLTP with glycine and combine live imaging of dendritic spine morphology with post-hoc three-color STED analysis of spine molecular organization. In the same spines that were imaged live, we will determine the number of nanomodules by staining for PSD-95 and Bassoon and assess the localization of AMPAR and NMDAR subunits in individual nanomodules of potentiated, unresponsive and control spines. These high risk/reward experiments will determine the molecular organization of glutamate receptors at synapses and will likely generate new avenues for investigation of synaptic failure and dysfunction in AD. Findings from our studies will drive new understanding of how changes in synaptic function are linked to AD and will likely yield transformative tools for the early diagnosis of memory loss that is associated with the onset of AD.