PROJECT SUMMARY/ABSTRACT Myelin—the electrical insulator around neuronal axons—is essential in vertebrates for rapid nerve signaling, and its loss in diseases like multiple sclerosis and following injury causes severe disability in patients. Myelin’s traditional role as a passive electrical insulator has recently been reimagined as a dynamic process, in which oligodendrocytes build and remodel myelin sheaths in response to neuronal activity, and metabolically support the neurons they myelinate. These new discoveries open up many exciting new research questions, such as: how do oligodendrocytes regulate their morphology to tune conduction velocity or other neuronal functions, during development or in the adult central nervous system (CNS)? What roles do myelin dynamics play in higher-order brain functions and in neurodegenerative diseases beyond multiple sclerosis? A fundamental gap in current knowledge lies in understanding how oligodendrocyte cell biology is regulated in all of the contexts of development, dynamics, and disease. Bridging this knowledge gap requires building innovative new tools to break through the experimental limitations that have hindered our ability to study oligodendrocyte cell biology in vivo thus far. The goal of this proposal is to create a viral (AAV) toolkit for studying oligodendrocyte cell biology in vivo. This toolkit will allow for bright, sparse labeling and manipulation of single oligodendrocytes in the mouse CNS, which will empower studies to determine the mechanisms controlling oligodendrocyte morphology (Aim 1). In addition, this toolkit will allow subcellular targeting of reporters or perturbants to functionally-distinct regions within oligodendrocytes, including myelin sheaths (Aim 2). We propose that this viral cell biology toolkit will have a broad positive impact across the myelin and broader neuroscience fields, by: i) rapidly accelerating the time from idea to discovery, compared to traditional mouse genetics, ii) simplifying experimental design via “plug-and-play” modular construct architecture, iii) unlocking the ability to visualize and/or perturb single mouse oligodendrocytes in vivo, and iv) creating new tools to study functionally distinct subcellular structures in myelin. From this research, we will create and optimize a simple but powerful toolkit for studying myelin cell biology in vivo in the mouse CNS. It is our hope that these tools can be rapidly adopted by the field, and open exciting new frontiers in myelin research. We will share these tools as a free resource, thereby maximizing their potential impact to reveal new cell biological insights into myelin development, dynamics, and disease.