A growing number of diseases have been linked to synapse and circuit development in the brain, including autism, mental retardation, epilepsy, schizophrenia and depression. Therefore, it is important to understand the mechanisms of neuronal circuit and synapse formation. It has previously been shown that development of cortical circuits and sensory maps depends on NMDA receptors. We recently demonstrated that NMDA receptor activity bi-directionally controls presynaptic terminal formation during early postnatal development of neocortical neurons. The overall goal of this proposal is to understand how NMDA receptors regulate the formation of individual synapses. More specifically, the experiments of Aim 1 will test the novel hypothesis that NMDA receptors control the biochemical differentiation of excitatory presynaptic terminals in cerebral cortical neurons in a cell-autonomous manner. Because presynaptic terminal development could be regulated by either presynaptic or postsynaptic NMDA receptors, the experiments of Aims 1 and 2 of this proposal are designed to distinguish between these mechanisms. The existence and function of presynaptic NMDA receptors has been controversial. Therefore, the experiments of Aim 2 will systematically characterize the localization of NMDA receptors in the axon before, during and after synapse formation. This will constitute the first study of the mechanisms of delivery of NMDA receptors to presynaptic terminals and their trafficking within terminals. We expect that these experiments will also provide insight into novel strategies for selectively decreasing expression of NMDA receptors in either axons or dendrites. The experiments of Aim 3 will then investigate the function of presynaptic NMDA receptors during presynaptic terminal development. This will be directly compared to the relative contribution of postsynaptic NMDA receptors to presynaptic development. To achieve our Aims, quantitative, live time-lapse and fixed cell confocal imaging will be combined with novel genetic models and fluorescent reporters. Cell-autonomous control of presynaptic development by presynaptic NMDA receptors would provide neurons with a feed-back mechanism to ensure that functional presynaptic terminals are made and maintained, even at postsynaptically-silent synapses. Regulation of presynaptic development by trans-neuronal signaling from postsynaptic NMDARs would allow a neuron to tune its presynaptic output to match its postsynaptic input. Completion of the proposed project will not only improve our understanding of normal cortical development but may also establish new therapeutic targets for treatment of autism, intellectual disability, epilepsy or schizophrenia. In addition, this study will also shed light on the potential consequences of altered NMDA receptor activation during brain development, which is particularly relevant for infants and toddlers who either (1) have impaired sensory activity or (2) are exposed, either directly or through their mothers, to drugs that target NMDA receptors, including both FDA-approved therapeutics and drugs of abuse.