PROJECT SUMMARY Striatal dopamine serves a critical role in motivation, reward learning, and decision making. Aberrations in dopamine transmission are thought to underlie a variety of neuropsychiatric diseases and substance abuse disorders. Yet, despite dopamine’s prominent role in behavior and disease, mechanistic understanding of how transmission occurs at the subcellular level has remained limited due to methodological restrictions. Structural data shows that dopamine neurons form extensively arborized axons with sparse release sites (varicosities) that are often unassociated with postsynaptic receptors. Combined with early methods of measuring dopamine, these findings have led to the belief that dopamine signals primarily through ‘spillover’ transmission, characterized by slow changes (hundreds of milliseconds or more) in low concentrations of dopamine (<1 µM) over a broad radius (~10 µm). However, accumulating evidence has recently challenged this notion. Imaging of fluorescent dopamine sensors has revealed the presence of highly localized dopamine ‘hotspots.’ Further, electrophysiological recordings show that endogenous dopamine release rapidly modulates postsynaptic targets and is only precluded by concentrations far higher than those observed in spillover release. These data suggest that dopamine signaling can also occur through tightly coupled ‘synaptic’ transmission, characterized by rapid changes (milliseconds) in high concentrations of dopamine (10-100 µM) that are spatially restricted (~ 3 µm). The stark difference in spatiotemporal structure between spillover and synaptic transmission suggests that these two modes mediate distinct functions. Yet, little is known about how spillover and synaptic dopamine transmission are regulated or how these two forms of transmission inform healthy and diseased behaviors. This study will implement an innovative combination of synaptic electrophysiology and behavioral assays with a battery of perturbations to dopamine release to determine: (1) whether spillover and synaptic transmission occur through independent or intertwined mechanisms of release, and (2) whether these two forms of transmission serve distinct functions in reward behaviors. Together, these experiments will test the hypothesis that synaptic and spillover dopamine release are two independently regulated forms of transmission with distinct behavioral functions. By illuminating the microarchitecture of dopamine release, this work will lay a new framework for understanding how dopamine transmission shapes reward behaviors critical to both health and addiction.