Myelin formation and maintenance is vital for proper neuronal communication and its disruption is associated with numerous diseases of the central nervous system. Oligodendrocytes make myelin and are the only cells in the adult cerebral cortex that are continuously generated from a population of resident progenitors, called NG2 cells. Thus, protracted oligodendrocyte and myelin formation into adulthood constitutes a unique, understudied system for adult neuroplasticity, with broad implications for human cognition and disease. Understanding the process of oligodendrocyte generation is fundamental to dissect roles played by oligodendrocytes and myelination in nervous system function, plasticity, and disease. We have a rudimentary understanding of how new oligodendrocytes are generated in vivo. Reasons for this stem from inadequate tools for their dynamic investigation in the live brain. In light of these challenges, my long-term goals are to develop and apply optical and single cell molecular based approaches to dissect multicellular interactions in the intact developing and diseased nervous system, with a primary focus on the interface between the axon and oligodendrocyte. Realization of this goal has begun as we have now developed a range of novel complementary tools that allow unprecedented detailed investigation into the transformation of single progenitor cells into gap junction-coupled, mature myelinating oligodendrocytes in vivo. This proposal will implement and expand on these tools to ask several fundamental questions basic to our understanding of adult nervous system plasticity and response to injury. First, during the K99 phase, the in vivo dynamics of oligodendrocyte differentiation, gap junction coupling and internode assembly during initial myelin formation and after a demyelinating event will be determined. Next, a new method will be used to determine the developmental profile, longitudinal dynamics, and effects of demyelination on internode and Node of Ranvier assembly and distribution along extensive stretches of single axons. Finally, during the R00 phase, using a powerful combination of in vivo imaging and single cell molecular manipulation techniques learned during the K99 training period, the effects of myelin deposition on dynamic axonal structural plasticity will be tested. Overall the research portions of this proposal will uncover how functional internodes initially form, restructure throughout life, respond to oligodendrocyte death, and interact with the axon to influence its structural plasticity, all for the first time in the live brain. The aims set out in this proposal will provide the foundation for implementing these in vivo optical tools during the R00 phase. Furthermore this strategy will provide fundamental training in novel approaches for molecular design with unique intellectual, professional and academic guidance during the K99 phase under the mentorship of Dr. Jaime Grutzendler in collaboration with consultant Dr. Anthony Koleske and the vibrant neuroscience research environment at Yale University. The combination of learning a new set of molecular approaches and implementing our powerful in vivo imaging platform will ensure a unique skillset and perspective, critical components for a successful career as an independent investigator.