Anti-depressant drugs such as fluoxetine (Prozac) increase levels of serotonin (5HT) in the brain, but little is known about how they affect the function of the nervous system. 5HT is implicated in a wide spectrum of brain functions and disorders. However, its precise role remains controversial and enigmatic. This is partly due to the previous inability to adequately isolate 5HT action in vivo. Recent developments in genetic tools and imaging techniques now offer an unprecedented opportunity to investigate such mechanisms in the living brain. This project aims to isolate 5HT transmission in the cerebral cortex and determine the precise cellular mechanisms underlying serotonergic neuromodulation of brain activity. Our objectives are to determine the action of 5HT in awake-behaving animals in three main ways: (1) on specific neuronal populations, at the network and single-cell level, by the local application of 5HT modulators (including fluoxetine); (2) when endogenously released, by controlling 5HT neuronal activity with optogenetic tools; and (3) on pathologic cortical network activity in an animal model of autism spectrum disorders. We will use the primary visual cortex as a model cortical area, investigating the role of 5HT in visually-evoked and activity-dependent cortical activation under normal conditions and during network dysfunction in autism spectrum disorders. In this project we will use state of the art in vivo two-photon calcium imaging and advanced genetic tools, including optogenetics. These cutting edge techniques will allow us to precisely activate 5HT neurons and measure the resulting effects on neuronal populations with single-cell resolution. The results will apply beyond the visual system to more broadly inform on cellular mechanisms of 5HT action in the central nervous system and its dysfunction in neurological disorders, leading to future advancements for therapeutic targeting.