Project Summary Navigating within an odor plume is a complex task due to unpredictable changes in odor concentration. The algorithms used by organisms to navigate the odor plume remain mysterious and how the brain solves this complex sensorimotor task key to escaping, mating and eating is unknown (1). The problem is challenging because it requires parallel monitoring of: 1) brain activity in multiple brain regions in the freely moving animal, 2) odor plume dynamics, 3) sniffing and 4) animal motion. While optical imaging is an attractive solution, imaging neural activity in multiple brain regions in the freely moving animal is currently impossible with either commercial or custom miniscopes (2). Members of our Odor Plume Neurophotonics (OPeN) interdisciplinary team developed a novel two photon fiber-coupled microscope for 3D imaging of brain activity in the freely moving mouse under funding from an NIH BRAIN U01 and an NSF NCS:FOUNDATIONS grant (3)(Bright, Gibson, Gopinath and Restrepo) and quantified realistic odor environments in the laboratory to explore algorithms used for odor-guided navigation under funding by an NSF Ideas Lab “Cracking the Olfactory Code” (Crimaldi)(4). Here, we leverage the extensive expertise of the team to crack the circuit basis for odor plume navigation. We will develop a multi-site miniature 3-photon fiber coupled microscope (3P-FCM) to record neuronal activity in four brain sites in two planes of view. In addition, OPeN will develop a portable photoionization (PID) sensor to detect the odorant concentration at the nostril as the animal navigates the odor plume. Finally, OPeN team members will develop a Bayesian analysis method to decode odor plume navigation from neural activity. The OPeN advisory board includes faculty from universities such as Cornell University and the University College of London, and will include two Directors of NSF NeuroNex Hubs. Understanding the circuit basis of odor plume navigation is impossible using current technology. This will be a major accomplishment involving systems neuroscience, neurophotonics and environmental engineering. The instruments and analysis methods developed by OPeN will be an important contribution to understanding brain function in complex environments. OPeN will foster training of graduate students and postdocs cracking the neural basis of odor plume navigation.