Project Summary Uncovering the molecular properties of functionally-defined neural ensembles is essential for understanding how these networks give rise to circuit function and animal behavior. This proposal aims to develop and deploy new molecular technologies that will enable the sub-cellular tagging and enrichment of proteins in activated subgroups of neurons in the brain. Linking protein expression to neural function at a large and unbiased scale is currently not possible with existing technologies. Thus the research program proposed here will fill a critical unmet gap in the molecular toolbox for neuroscientists. While prior technologies have focused on gaining genetic access to the genome or transcriptome of activated neurons, the approach here will identify the actual proteins expressed in specific subcellular compartments of functionally-relevant neurons. This is a key distinction, as gene expression alone cannot reveal to the actual physical location and expression patterns of translated proteins – which are the ultimate molecules that carry out the specific biochemical functions of our cells. To enable this goal, new activity-dependent proximity labeling probes will be developed using protein engineering. These probes will be improved and optimized through high-throughput screens performed in cultured cells, and then adapted for use in mammalian neurons. Concurrently, the probes will be tested and characterized in the mouse brain to improve and benchmark their function. To demonstrate their utility, the activity-dependent probes will be used to tag the proteins that are present in neurons undergoing high neural activity in response to a behavioral drug experience in mice. 5-MeO-DMT is a hallucinogenic drug that in humans has been associated with therapeutic potential for treating neuropsychiatric diseases. Neurons activated by 5-MeO-DMT will be labeled by the activity-dependent probes, and their spatial distribution throughout the brain will be examined using the fluorescent read-out of the new molecular enzyme. In addition, the probes will also tag the proteins that are present in these activated neurons, allowing the enrichment and unbiased profiling of these molecules using liquid chromatography mass spectrometry (proteomics). The sub- cellular proteome of these neurons will provide essential biological insight into the mechanism of hallucinogenic drugs, in addition to providing potential downstream targets for new drug discovery and development. More broadly, the new probes will be distributed freely to the neuroscience community to enable the study of protein expression in functionally-relevant populations of neurons.