Summary Dopamine is an important neuromodulator and pathologies in dopamine signaling are a hallmark of brain disease. Despite these roles, the organization and regulation of dopamine signaling are incompletely understood. The long-term goal of this project is to dissect the cell biology of axonal dopamine transmission. Spatial and temporal features of dopamine signaling are different from synaptic transmission. At conventional synapses, nanometer-scale synaptic structure enables robust receptor activation at sub-millisecond speeds and restricts communication to point-to-point contacts between select neurons. In contrast, dopamine is a volume transmitter that diffuses through the extracellular space after exocytosis and may influence many cells through G-protein coupled receptors. These properties suggest that dopamine transmission is slow and diffuse. Recent data from several laboratories, including some generated during the previous funding cycle, however, have revealed that dopamine transmission is highly dynamic and, in some cases, remarkably precise. Furthermore, dopamine release is powerfully and rapidly regulated by local cholinergic interneurons in the striatum. These findings suggest that the coding of dopamine volume transmission is more precise than previously thought. A major question that arises is how the architecture for dopamine transmission can support precise signaling. Our overarching model is that molecular machinery has evolved to support broad dopamine coding scales. We build on our previous findings that axonal dopamine exocytosis is executed with millisecond precision by sparse, sophisticated protein machinery typically present at synapses. In aim 1, we zoom in on the powerful local regulation and ask how cholinergic neurons trigger dopamine release. Based on preliminary data, we hypothesize that activity in cholinergic interneurons induces ectopic action potential firing in dopamine axons to trigger dopamine secretion. Our goal is to test this hypothesis and to understand the underlying mechanisms. Identification of an endogenous mechanism for ectopic axonal action potential initiation away from the dopamine neuron soma has important implications for dopamine neuron function. In aim 2, we dissect the organization of dopamine receptors relative to release sites. We build on recent work that identified markers for these sparse secretory sites. Our preliminary data reveal that dopamine receptors are clustered one to two micrometers away from release sites and suggest differences in D1 vs. D2 receptor distributions. We will systematically assess release-receptor organization in super-resolved 3D-images of large striatal volumes and will mechanistically dissect how it is set up. We propose that the organization is different from nanoscale synaptic structure and from the diffuse organization often associated with volume transmission, and may be suited to mediate distinct pathway activation by switches in dopamine neuron firing ...