Project Summary Neuromodulators play an important role in our life by dynamically adapting the brain to an ever-changing environment. By simultaneously activating multiple receptor classes that are distributed amongst distinct components of neural circuits, neuromodulators can effectively rewire the brain in a reversible manner. However, the involvement of specific neuromodulators in distinct cognitive processes has been difficult to establish. Neuropeptides, in particular, are a unique and mysterious class of neuromodulator that are released from neurons that also release classic neurotransmitters such as glutamate or GABA. The behavioral contexts and neural activity patterns that drive neuropeptide release have been difficult to establish experimentally due to a lack of spatiotemporally precise tools for monitoring neuropeptide levels in the brain. Our current understanding is based largely on studies that rely on microdialysis for detection and pharmacology to interfere with or mimic neuropeptide signaling in behaving animals, leaving the following fundamental questions unanswered: When and where are neuropeptides released in the brain during distinct behaviors? Once released, how far do they spread from release sites, and how long do they remain to influence neural activity? To address these and related questions, we propose to engineer Nanobody-Evolved Optical Neuropeptide Sensors (NEONS) that will report the presence of neuropeptide in the extracellular space. NEONS will readily interface with contemporary imaging technology to provide highly sensitive, spatially and temporally precise monitoring of neuropeptide release in the brain. Our approach relies on the ability to evolve nanobodies in the laboratory that bind to neuropeptides with high affinity. Uniquely, our strategy is to engineer two nanobodies that form a ternary complex in the presence of neuropeptide and to translate complex formation into an optical signal using fluorescent proteins. In Specific Aim 1 we will implement a novel, powerful method for directed evolution of nanobodies developed in the Chang Laboratory called GAIN recombination. In Specific Aim 2 we will engineer these dual-binding affinity elements into optical sensors by fusing them with fluorescent proteins that function as FRET pairs and by tethering the nanobodies to circularly permutated GFP. The resulting NEONS will be optimized and characterized in brain tissue in Specific Aim 3 using fluorescence microscopy. NEONS promise to enable large-scale, potentially whole brain imaging of neuropeptidergic transmission in behaving animals. Such unbiased experiments will reveal otherwise invisible roles for neuropeptides in specific brain regions and will motivate further studies that dissect the mechanisms by neuropeptides transform circuit function to regulate behavior.