Project Summary/Abstract The brain ECM constitutes about 10-20% of the brain’s volume and plays a critical role in diverse brain functions. In particular, the brain ECM regulates neuronal plasticity and behavior. Unlike collagen-based peripheral ECMs, the structure of brain ECM is highly flexible and dynamically regulated by experience. However, due to the technical limitations, the mechanism by which the brain’s ECM is organized and regulated is poorly understood. We have developed a novel live-cell probe that labels hyaluronan-based brain ECM, named H-Link. Sparse H-Link imaging shows that clustered ECM, such as perineuronal net (PNN), is not limited to a specific neuronal type (PV interneurons) but also elaborately coats excitatory neurons in vivo and in vitro. Furthermore, the application of H-Link to cultured neurons shows a striking specificity in ECM clustering among different types of neurons and reveals its organization process on the neuronal surface, providing an excellent platform for loss-of-function screens. Using this novel approach, we will identify cell-autonomous neuronal ECM organizers and reveal the homeostatic and activity-dependent remodeling process of the brain’s ECM. The tools and knowledge obtained from the proposed study will be a foundation for understanding the cell-type-specific role of the brain’s ECM in neuronal plasticity and behavior.