Using Enhancer-directed expression within AAVs to target subtypes within the four major neuromodulatory populations

Grant Summary Neuromodulatory systems—comprising noradrenergic, serotonergic, cholinergic, and dopaminergic neurons—play a critical role in regulating mood, cognition, motor control, and physiological processes. These systems are central to understanding how the brain encodes reward, action selection, and memory, and they remain key therapeutic targets for neuropsychiatric and neurodegenerative disorders. Recent advances in high-throughput single-cell and spatial genomics have revealed a remarkable diversity among neuromodulatory cell types, with dozens to hundreds of distinct subpopulations. Despite this, tools capable of targeting these subpopulations with precision remain limited, hindering progress in both basic and translational neuroscience. This project seeks to address this gap by developing a comprehensive pipeline to nominate, validate, and disseminate cell-type-specific enhancers for neuromodulatory systems. Building upon prior successes in enhancer discovery for cortical interneurons and pyramidal neurons, we will employ advanced spatial multiomic profiling, computational prediction, and high-throughput AAV-based enhancer testing to generate a robust set of validated enhancers. Specifically, we will: 1) Nominate candidate enhancers by integrating publicly available data and performing spatial multiomic profiling on sorted cholinergic, dopaminergic, serotonergic, and noradrenergic cell types. 2) Quantitatively validate enhancer activity using cutting-edge techniques, including smFISH, Slide-Tag molecular profiling, and functional assays such as optogenetic and chemogenetic manipulation, neuronal activity monitoring, and CRISPR-based gene editing. 3) Disseminate validated tools through collaboration with the Allen Institute, Addgene, and other platforms, ensuring wide accessibility and standardization across the neuroscience community. The proposed research will produce at least 60 highly specific and validated enhancers targeting distinct neuromodulatory subpopulations, each characterized for their activity, specificity, and functional applications. These tools will be invaluable for studying neuromodulatory circuits in health and disease and for advancing therapeutic interventions targeting these systems. By providing the neuroscience community with these transformative tools, this project will facilitate unprecedented insights into the molecular and cellular underpinnings of brain function and dysfunction, addressing pressing challenges in the fields of psychiatry and neurology. The successful completion of this work will not only enable basic scientific discoveries but also lay the groundwork for the development of precision therapies for neuropsychiatric and neurodegenerative diseases. Project Number: 1U01MH142288-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: GORDON FISHELL (+3 co-PIs) | Institution: BROAD INSTITUTE, INC., CAMBRIDGE, MA | Award Amount: $3,312,425 | Activity Code: U01 | Study Section: Special Emphasis Panel[ZRG1 BN-H (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11261262