PROJECT SUMMARY Prenatal nicotine exposure (PNE) remains a pressing public health concern. At least 10 percent of pregnant individuals in the US report smoking cigarettes (or e-Cigs) during gestation. PNE is linked to neurodevelopmental disorders such as attention-deficit hyperactivity disorder and autism spectrum disorders. Nicotine, via cholinergic nicotinic acetylcholine receptors (nAChRs), enhances cholinergic activity in the prefrontal cortex (PFC), dysregulating development and increasing the risk for PFC-linked neurodevelopmental disorders. PFC-linked neurodevelopmental disorders point at disruptions in synapse maturation leading to synaptic dysfunction. However, the mechanisms by which nAChR signaling, and nicotine exposure, influence synapse maturation are unknown. Our preliminary studies found that cholinergic denervation in the developing PFC impairs excitatory synapse maturation. Additionally, we found nicotine treatment promoted synapse maturation in the absence of glutamatergic activity. This proposal is composed of two aims which seek to test the hypothesis that postsynaptic nAChR signaling modulates excitatory synapse maturation in the developing PFC. The first aim uses a combination of innovative techniques including two-photon uncaging of glutamate, chemogenetics, electrophysiology, and pharmacology to determine if the up-regulation of endogenous cholinergic activity promotes synaptic maturation. The second aim uses two-photon, two-color uncaging of nicotine and glutamate, electrophysiology, and pharmacology to elucidate the synaptic and molecular mechanisms involved in the nicotinic signaling-mediated maturation of synapses. In sum, the studies outlined in this proposal will shed light to the cellular, synaptic, and molecular mechanisms involved in the nicotine-induced maturation of excitatory synapses. Results from this project will advance efforts towards mitigating the impact of neurodevelopmental disorders that result from PNE.