PROJECT SUMMARY Functional brain development requires the precise coordination of neurochemicals that allow neurons to form synapses and communicate with one another. Improper synaptic maturation during critical developmental time windows often lead to cognitive disabilities, such as those observed in autism spectrum disorders, that can persist into adulthood. Studies in humans and rodents have linked disbalances of serotonin (5-HT) during prenatal and early postnatal development to increased rates of behavioral and cognitive deficits and irregular neuronal development. The prefrontal cortex (PFC) is a region of the brain that plays a key role in processing higher order cognitive functions that are disrupted when 5-HT levels are altered. During early PFC development, circuit formation is dependent on the functional maturation and stabilization of excitatory synapses, located on dendritic spines. Our preliminary studies identified a critical role for 5-HT in the development of dendritic spines within the PFC, indicating that both spine density and size are directly altered after manipulating 5-HT levels in early postnatal periods. This proposal is comprised of two aims which seek to test the hypothesis that 5-HT can potentiate and stabilize dendritic spines at a single synapse level. The first aim uses a combination of techniques including two-color, two-photon uncaging of 5-HT and glutamate neurotransmitters, electrophysiology, optogenetics, and pharmacology to determine whether 5-HT can induce functional long-term potentiation of individual dendritic spines. The second aim is designed to identify whether endogenous 5-HT signaling can stabilize nascent dendritic spines, which will be investigated by using optogenetics, two-photon 5-HT uncaging and mechanistic studies that include pharmacological approaches and fluorescence lifetime imaging. Together, these studies seek to establish the role of 5-HT dependent signaling in synapse maturation and stabilization at a cellular and molecular level. Results from this project will be essential knowledge towards better mitigating the cognitive disabilities that result from early 5-HT disbalances in the developing brain.