Dendritic spines are the remarkable, specialized membrane compartments on neurons that house the postsynaptic, receiving end of most excitatory, glutamatergic synapses in the brain. They are highly plastic and change during development, learning, and in disease. This proposal relies on multi-laser 2-photon imaging and photostimulation approaches to generate and evaluate new connections on genetically targeted striatal neurons, dissecting interacting variables of sex, age, and neuromodulatory state. The proposal builds on preliminary observations of sex differences in spinogenesis, which may interact with other convergent signaling cascades to control dendritic spine formation and sensitivity to therapeutic agents. Further, the earliest stages of nascent synapses in striatal neurons will be defined functionally and ultrastructurally, using newly developed molecular tools and imaging approaches to assist this objective. The proposed work would yield valuable insights into new spine and synapse genesis, early-stage function, and stability, impacting basic research relevant to synaptic development and rules that guide plasticity. The resulting platform will help to drive technical innovation that would allow researchers to design therapies to augment reconstruction of neuronal architecture or deconstruct aberrant connectivity.