AbstractIn the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the insect CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments forms an internal diffusion barrier. We find that ensheathing glial cells are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous ßHeavy-Spectrin cytoskeleton. ßHeavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.