Glia play crucial roles in the development and homeostasis of the nervous system. While the GLIA in the Drosophila embryo have been well characterized, their study in the adult nervous system has been limited. Here, we present a detailed description of the glia in the adult nervous system, based on the analysis of some 500 glial drivers we identified within a collection of synthetic GAL4 lines. We find that glia make up ∼10% of the cells in the nervous system and envelop all compartments of neurons (soma, dendrites, axons) as well as the nervous system as a whole. Our morphological analysis suggests a set of simple rules governing the morphogenesis of glia and their interactions with other cells. All glial subtypes minimize contact with their glial neighbors but maximize their contact with neurons and adapt their macromorphology and micromorphology to the neuronal entities they envelop. Finally, glial cells show no obvious spatial organization or registration with neuronal entities. Our detailed description of all glial subtypes and their regional specializations, together with the powerful genetic toolkit we provide, will facilitate the functional analysis of glia in the mature nervous system. GLIA 2017 GLIA 2017;65:606–638

How does the sensory environment shape circuit organization in higher brain centers? Here we have addressed the dependence on activity of a defined circuit within the mushroom body of adult Drosophila. This is a brain region receiving olfactory information and involved in long-term associative memory formation. The main mushroom body input region, named the calyx, undergoes volumetric changes correlated with alterations of experience. However, the underlying modifications at the cellular level remained unclear. Within the calyx, the clawed dendritic endings of mushroom body Kenyon cells form microglomeruli, distinct synaptic complexes with the presynaptic boutons of olfactory projection neurons. We developed tools for high-resolution imaging of pre- and postsynaptic compartments of defined calycal microglomeruli. Here we show that preventing firing of action potentials or synaptic transmission in a small, identified fraction of projection neurons causes alterations in the size, number, and active zone density of the microglomeruli formed by these neurons. These data provide clear evidence for activity-dependent organization of a circuit within the adult brain of the fly.

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Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

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