Project Summary In the brain, neurons are connected by intermingled excitatory (E) and inhibitory (I) synapses. The correct E/I balance is essential for proper brain function. Recent in vivo studies demonstrate that excitatory and inhibitory synapses communicate with each other locally and inhibitory synapses could actively regulate the structure and function of excitatory synapses. In vivo optogenetic stimulation and suppression of inhibitory activity decreases and increases the stability of excitatory synapses, respectively. Our preliminary two-photon imaging and chemogenetic data further show that PFC interneuron activity may affect excitatory synaptic plasticity throughout brain development. These data strongly suggest that inhibitory synapses heterosynaptically regulate excitatory synapses. However, there is much yet unknown about the cellular and synaptic mechanisms by which inhibitory GABAergic synapses locally regulate excitatory glutamatergic synapse formation and elimination during brain development. Using advanced optical techniques, this R21 grant proposal addresses key questions of fundamental significance; 1) how do inhibitory synapses heterosynaptically regulate excitatory synapse structure and function throughout brain development in an age-dependent manner? And 2) what signaling mechanisms underlie the cross-talk between inhibitory and excitatory synapses during development and plasticity of neural circuits? Our work will provide a powerful experimental framework for understanding how the brain achieves and maintains E/I balance, and how this balance is altered in neurodevelopmental disorders arising from E/I imbalance such as autism and schizophrenia.