The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Our previous findings show that while pyramidal neurons in layer 2/3 of adult visual cortex show little, if any, change in branch tip length over time, GABAergic non-pyramidal interneurons display significant dendritic branch tip remodelling driven by visual experience in an input and circuit-specific manner. The fact that structural plasticity of interneurons is continuous through adulthood raises the intriguing possibility that local remodelling of inhibitory connections may underlie adult cortical plasticity. Yet, how experience alters inhibitory circuitry is unclear, and how modifications to inhibitory and excitatory circuits are locally coordinated remains unaddressed. In the previous funding period we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical layer 2/3 pyramidal neurons in vivo during normal and altered visual experience. We found that the rearrangements of inhibitory synapses and dendritic spines are locally clustered, mainly within 10 m of each other, and that this clustering is influenced by experience. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement a newly developed three-color labeling system to independently and simultaneously monitor the formation and disappearance of dendritic spines along with appearance or removal of the post-synaptic density in these spines, and the appearance and removal of inhibitory synapses along the same dendrites. Using spectrally resolved two-photon microscopy we will 1) monitor the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals; 2) monitor the effects of experience- dependent plasticity on coordination of inhibitory and excitatory synapse remodeling; 3) examine the specificity of afferent inputs to coordinated excitatory/inhibitory synaptic pairs. Further, 4) we will develop and implement spectrally resolved multifocal multiphoton microscopy to further enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.