Project Summary/Abstract: The field of optogenetics — utilizing light to engage biological systems — is widely used for the dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, while many new opsins have been developed and are actively used, challenges still remain, and the current technology lacks a full toolbox for sub-cellular, spatiotemporal control of signaling — the predominant means for neuromodulator communication in the brain. Here we propose, an innovative effort combining neuroscience with structural biology and high-throughput pharmacology for the development of a series of cutting-edge novel Opto-GPCRs that will allow spatiotemporally precise and pathway-selective control of neuromodulator signaling in vitro and in freely moving animals. In four aims across five leading laboratories, we will develop and test these novel tools in vitro and in vivo. Specifically, we will work to 1) Develop and fully optimize OptoGPCR-v3.0 (Gi coupled) receptors for enhanced spectral multiplexing and altered sensitivity using structure-function analysis together with mutant-library HTS landing pad system; 2) Develop and fully optimize OptoGPCR-v3.0-Gq receptors for selective coupling to Gq signaling pathways using structure-guidance and the HTS landing pad system; 3) Utilize databases of less-explored naturally-occurring opsin-GPCRs to test, screen and further develop new optical tools with unique profiles; and 4) Assess the spectral compatibility for simultaneous use of OptoGPCR-v3.0 constructs in vivo, together with biosensors using photometry, 2p imaging and concurrent behavioral measures in vivo. Successful completion of the proposal will provide the wide neuroscience community with the long awaited capabilities of spatiotemporal manipulation of GPCR – neuromodulator signaling within neural circuits in vitro and in vivo, in awake freely behaving animals, and could be used for a wide variety of applications. This new technology will also further widen the field for unique optical approaches that allow discrete control and optodynamic simulation of neuromodulator function in brain tissue. We therefore believe that by the fulfilment of these goals we directly address the central purpose of this RFA-NS-21-027 call.

Project Narrative: The technology proposed here will lead to new techniques for spatial and temporal control of neuromodulator signaling in the brain. The light sensitive tools we will develop will have multi-functional abilities to allow for discrete control over time and space of critical receptor signaling pathways in the awake- behaving brain. These new optogenetic tools will allow neuroscientists and biologists to have unprecedented resolution in uncovering the basis for brain diseases, and have translational potential for delivery of new treatments for brain disorders.