Summary: Over vertebrate evolution, the development of the myelin sheath has contributed to the expansion of the central nervous system and the emergence of complex brain function. Cumulative evidence indicates that the level of myelination and its positioning over the axon may be dependent on the class identity of myelinated neurons. A canonical example is the difference between L5 projection neurons, with extensive and uniform myelination, and the L2/3 callosal projection neurons, with lower and more diverse patterns of myelination, including “intermittent” profiles, where myelin tracts are separated by long unmyelinated regions rather than short nodes of Ranvier. Little is known about the mechanistic principles underlying cellular interaction between myelinating oligodendrocytes (OL) and axons of distinct neuronal classes in the CNS. Yet this knowledge is fundamental to understanding the cellular and developmental biology of myelination and regeneration. Focusing on the neocortex, we propose to answer fundamental questions regarding the mechanisms that control neuron-type specific myelination, and test hypotheses on how “attractive” and “repulsive” cues expressed by neuronal subtypes dynamically regulate their interactions with OLs. Here, we will 1) use molecular profiling of oligodendrocytes and cortical neuron subtypes across different cortical layers to map differences in their transcriptome, and use this data to generate a molecular interactome of candidates for genes mediating neuron- OL communication that may regulate neuron-subtype-specific myelination. We will 2) employ a screen to identify candidates able to induce or repress myelination (Aim 1). We will then 3) investigate membrane protein composition of myelinated and unmyelinated axonal segments of a specific neuronal class at subcellular resolution to understand the regulation of myelin positioning along the axon; and further 4) study whether long unmyelinated regions are differentially enriched for functionally-relevant structures such as synapses, gap junctions, and axonal branches (Aim 2). It has been reported that increased neuronal activity promotes myelination, which in turn stabilizes axon structure and neural circuit connectivity. Disrupted myelination can contribute to many debilitating neurological disorders, including multiple sclerosis and schizophrenia, and promoting oligodendrocyte differentiation and remyelination is an important therapeutic goal. We will investigate the molecular mechanisms that control cell-type specific adaptive remodeling of myelin and its regeneration after demyelination (Aim 3). In summary, the work proposed here aims to inform a conceptual framework for how different classes of neurons and oligodendrocytes interact to achieve differential myelination, mechanisms that will be critical in understanding the role of myelin in circuit function and dysfunction.