PROJECT SUMMARY Brain circuits are dynamic networks of neurons that process information in the form of electrical and chemical signals to form memories and shape behaviors. To investigate how brain circuits instantiate fundamental computations underlying behaviors, we need to map their wiring diagrams coupled with functional analysis at cellular resolution. However, the electrical (voltage) and chemical (e.g. neuropeptides) signals are not directly visible, and current circuit tracing tools are insufficient for meaningful functional analysis. Using protein engineering this proposal aims to develop a toolbox of genetically-encoded fluorescent reporters and tracers specifically tailored to study neural circuits. At the electrical level, voltage sensors can image the precise timing of action potentials and subthreshold voltage not detectable by other means. However, even the latest voltage sensors do not perform well with high-resolution microscopes that use 2-photon illumination for imaging deep in the brain. To overcome these limitations, we are taking a two-pronged approach by evolving amino acids at the mechanistic heart of voltage sensor proteins and by using spectroscopy to aid our protein engineering efforts. We believe directed evolution will improve voltage sensitivity and 2-photon functionality >10 fold, enabling us to image currently invisible signals, like synaptic potentials, deep inside the brain. At the chemical level, neuropeptides are highly expressed in almost all cortical neurons, but their role and impact in animals can only be inferred because current detection methods, like microdialysis, are invasive and lack spatiotemporal resolution. We are using phage display to evolve nanobodies capable of recognizing neuropeptides and coupling their conformational changes to fluorescence changes from reporter molecules. These sensors will provide visualization of neuropeptide release at cellular resolution throughout an animal’s brain during behavior paradigms that mimic human health and disease states. At the cellular connectivity level, current tools for circuit- mapping, like rabies virus, exhibit substantial neurotoxicity, prohibiting meaningful functional analyses. We are engineering proteins with a natural propensity to assemble into structures capable of delivering a genetic payload to specific cells to produce more effective and less toxic tools to map and manipulate brain circuits. Effective and robust tools to map the brain will bridge functional and structural analysis and finally allow long- term studies of neural networks based on their connectivity. Overall, the optogenetic tools developed in this proposal will translate the chemical and electrical signals between neural circuits into fluorescence that can be easily measured. Consequently, they can be used to unravel the functional basis and causes of neuronal disorders at a level of detail that has not been accessible to date and empower us to develop novel treatments.

PROJECT NARRATIVE Brain cells communicate using electrical and chemical signals and measuring these is crucial to understanding brain function. However, there is no natural contrast mechanism that allows us "to see" these messages. This project aims to illuminate brain communication through developing novel classes of genetically-encoded fluorescent reporters and tracers to study the brain and unravel the functional basis of neural disorders at a level of detail that has not been accessible to date empowering us to develop novel treatments.