Remapping of function after stroke relies on the rewiring of cortical circuitry. These structural modifications of circuits likely depend on nanoscale alteration in the molecular architecture of individual synapses that generate alternate connectivity and strengthen surviving contacts. NMDA- and AMPA-type glutamate receptors play critical roles in regulating structural and functional plasticity of synapses. Following a stroke, the signaling via AMPARsand NMDARs changes, likely due to changes in their nanoscale localization. The small size of synapses has prevented us from understanding how synaptic nano-architecture is altered by stroke and how nanoscale synaptic changes might lead to functional recovery, limiting our ability to target synapses for stroke intervention. Our proposal seeks to break down this barrier by combining state-of-the-art STEDsuper-resolution imaging with behavioral analyses of sensorimotor function in the mouse model of stroke. Our preliminary data indicate that AMPARsand NMDARs exhibit distinct organizational principles relative to spine size and presynaptic release sites, suggesting that the exact set of nanoscale rules governs synaptic transmission and plasticity. We will test the hypothesis that precise nanoscale remodeling of individual spine synapses underlies the functional recovery after stroke. In aim 1, we will determine how changes in pre- and post-synaptic nano-architecture on cortical pyramidal neurons are linked to functional recovery after stroke. By imaging the organization of scaffolding (PSD-95 and Bassoon) and functional (AMPARs, NMDARs, Munc-13) components of synapses in early and late phases of recovery after stroke, we will establish how changes in synaptic nano-architecture relate to the recovery of sensorimotor function in both young and old mice. In aim 2, we will determine the role of ephrin-B3 on the remodeling of spine nano-architecture on cortical pyramidal neurons during stroke recovery. Testing ephrin-B3 null mice in sensorimotor behavioral paradigms, we will determine whether ephrin-B3 is required for the functional recovery after stroke. Finally, using two-photon and STEDimaging of dendritic spines in the somatosensory cortex, we will determine whether ephrin-B3 regulates synaptic remodeling after stroke. Novel molecular insights gained from this research will improve our understanding of stroke pathology while providing mechanistic underpinnings into functional recovery.