Project Summary Neuromodulators control the state of an animal’s brain and thus behavior through the activation of G protein- coupled receptors (GPCRs). Although the role of neuromodulation has been appreciated for decades, the chemical logic of neuromodulation has remained elusive due to inadequate tools to study it. Recent advances in single-cell transcriptomics make it now clear that individual neurons co-express a huge diversity of neuromodulatory GPCRs, yet GPCRs transduce signaling primarily by coupling to only three classes of G- protein. This poses the question - does a neuron ‘know’ which of its many co-expressed receptors was activated? And if so, how? I hypothesize that individual neurons are able to distinguish this chemical diversity, even through shared signaling pathways, by utilizing spatiotemporal differences in GPCR signaling. To test this, I will first determine which receptors are functionally co-expressed in primary hippocampal neuron cultures, through a spatial-transcriptomic and functional screen. Then, I will compare response properties focusing on Gs-coupled receptors by taking advantage of a powerful palette of biosensors to interrogate their intracellular signaling with high spatial and temporal resolution. Finally, I will ask how these receptors are utilized in intact circuits by using spatial-transcriptomics and live imaging of cellular activity in acute brain slices. These findings will motivate my long term research goal of developing a subcellular understanding of neuromodulation, which is key to understanding neural circuits in both healthy and diseased states. In addition, this proposal includes a rigorous training plan and a team of collaborators that will bridge my research from molecular to systems neuroscience. It includes training in quantitative and computational methods that will both assist in my research aims and facilitate my development as a scientist. Through the excellence in research and postdoctoral support at UCSF, the training environment in the von Zastrow lab, and the planned activities listed in this proposal, this fellowship will prepare me for a career as an independent scientist invested in the priorities of the BRAIN Initiative.