The glia of the adult Drosophila nervous system

Abstract

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.

Keywords: GAL4 lines; glial cell interaction; glial subtypes; morphology; multicolor mosaic.

Figure 1

Anatomy of the adult Drosophila central nervous system and its generic glial cell types. A.1: Schematic of the central nervous system (CNS). The cortical regions (dotted areas) contain all neuronal and most glial cell bodies, while the neuropile regions (grey areas) contain the synaptic connections. A.2: Magnified schematic of the central brain. The different neuropile regions are colored (light green), regions of particular interest in this study are highlighted (shade of gray). The planes of the most frequently used confocal sections (frontal, sagittal, coronal) are illustrated, the orientation of the brain is depicted as a coordinate system (A: anterior; P: posterior; D: dorsal; V: ventral; M: medial; L: lateral). B.1–6: Different generic glial cell subtypes are visualized using subtype‐specific drivers and a membrane‐tagged reporter (UAS‐mCD8GFP); 130–160 μm projections are shown. Note the color code for the different glial subtypes B.1: PNG (lavender; 85G01‐Gal4), B.2: SPG (dark red; 54C07‐Gal4), B.3: CG (mustard; 77A03‐Gal4), B.4: ALG (green; 86E01‐Gal4), B.5: EGN (light blue; 56F03‐Gal4), and B.6: EGT (dark blue; 75H03‐Gal4). Scale bar = 100 μm in B.1–6.

Figure 2

Morphology of PNG. A.1: Schematic showing the location of PNG (lavender) in the brain. A.2: PNG enclose the entire CNS and PNS; 85 μm projection. A.3–15: Individual cell morphologies of PNG in different regions of the CNS. A.3: In the central brain, PNG appear as long thin strips oriented along the dorso‐ventral axis; 61 μm projection. A.4,5: Higher magnification shows that PNGs interdigitate with neighboring cells through long thin extensions (arrows). A.6,7: 3D reconstruction of the cell boundary between two PNG showing small protrusions in the region of contact (arrow in A.6). As seen in cross section (A.7), PNGs retain their relative proximo‐distal position along the entire length of contact. A.8,9: In the optic lobe, PNG appear as long thin strips along the dorso‐ventral axis; 20 µm projection. A.10,11: In the ventral nerve cord, PNG show a more varied morphology, ranging from elongated to square shaped, with orientation mostly along the medio‐lateral axis; 43 µm projection. A.12,13: In the PNS, PNG enclose the peripheral nerves; 43 µm projection. A.14,15: 3D reconstructions show that PNGs form narrow strips along the long axis of the nerve, without covering its entire circumference. Scale bar = 100 µm in A.2; 50 µm in A.3, A.8, A.10; 10 µm in A.4, A.9, A.11–15; 5 µm in A.5; 2 µm in A.7; 1 µm in A.6.

Figure 3

Morphology of SPG. A.1: Schematic showing the localization of SPG (dark red) in the brain. A.2: SPG enclose the entire CNS and PNS; 85 μm projection. A.3–11: Individual cell morphologies of SPG in different regions of the CNS and PNS. A.3,4: In the central brain (A.3) and optic lobe (A.4), SPG are thin, very large square‐shaped cells; 38 μm projection. A.5,6 In higher magnification, many ring‐like membranous structures are apparent, which in cross section (A.6), reveal themselves as cap‐like basal protrusions (arrow) that cover individual neuronal cell bodies, which are also enveloped by CG, within the cortex. A.7: 3D reconstruction showing the overlap between two neighboring SPGs. A.8–11: Individual SPGs covering peripheral nerve; 102 µm projection. Cells have a square to oblong shape (red arrow in A.10) that extends along the long axis of the nerve; thinner nerves can be completely enveloped by a single cell (white arrow in A.10) forming a tube, as revealed by a cross section in A.11. A.9,10 3D reconstructions of A.8 at two different magnifications. Scale bar = 100 µm in A.2; 50 µm in A.3,4, A.8,9; 10 µm in A.5; 5 µm in A.6, A.10,11; 1 µm in A.7.

Figure 4

Morphology of CG. A.1: Schematic showing the location of CG (mustard) in the brain (left), and the relationship between individual CG and neuronal cell bodies (right). A.2: CG fill the cortical regions of the brain without entering the neuropile regions; single section. A.3: CG cell bodies are found exclusively in the cortical regions of the brain (arrow); single section. B.1–4: Individual cell morphologies of CG in the central brain. Single section (B.1) and 3D reconstruction (B.2,3) of the same cell (arrow in B.1 points to glial nucleus), showing that a single CG cell envelopes 20–100 neuronal cell bodies. Neuronal cell bodies are wrapped individually, but two neighboring CG can contribute to wrapping one cell body (arrow in B.3). B.4: 3D reconstruction of an entire CG, with translucent membrane to reveal neuronal cell bodies within (filled arrow) and lamellipodial extensions (open arrow); 32 µm projection. C.1,2: Interaction of CG with neuronal cell bodies and fiber tracts; single section. CG support neuronal fibers (arrows) until they leave the cortical region. Scale = 50 µm in A.2,3; 10 µm in B.1,2, C.1,2; 10 µm in B.3,4.

Figure 5

Morphology of ALG. A.1: Schematic showing the location of ALG (green) in the brain. A.2: Location of neuronal cell bodies is always in the cortical regions (red arrow), while ALG cell bodies are frequently found in between neuropile regions (white arrow). A.3,4: Individual cell morphologies of ALG in the central brain. ALG show variable size and morphologies but cover largely non‐overlapping areas; single sections. A.5,6: Higher magnification views of ALG morphology; 3 μm projections. A.5: The glial mesh can be sparse (white arrow) or dense (red arrow), but individual cells cannot be distinguished when all cells are labeled. A.6: ALG project fine but long processes (arrow) from the neuropile into tract regions. B.1–5: Individual ALG cell morphologies in the central brain; filled arrows point to glial cell bodies, open arrows to processes. B.1: Single cell with low density of processes projecting into two neuropile (sub)‐regions; single section. B.2: Two neighboring cells, projecting into different neuropiles, each showing a markedly different density of processes; 2 µm projection. B.3,4: A single cell, projecting into two different neighboring neuropiles with different density of processes; B.3: 40 µm projection, B.4: single section. B.5: Single cell with high density processes; single section. C.1,2: Synaptic (C.1) and ALG (C.2) densities in neuropiles of the protocerebrum; single sections. C.3 The stainings shown in C.1,2, converted into structural density maps based on the distance between objects within a brain region and superimposed, show the anticorrelation of synaptic and glial densities. Regions with high synaptic density appear magenta, regions with high ALG density appear green (for description of algorithm see Materials and Methods; Supp. Info. Fig. S4). Scale bar = 50 µm in A.2; 10 µm in A.3–6, B.1,2,5, C.1–3; 5 µm in B.3,4.

Figure 6

Morphology of ensheathing glia. A.1: Schematic showing the location of ensheathing glia (EG) in the nervous system, comprising two subsets, EGN (light blue) and EGT (dark blue). A.2,3: Double labeling of EGN and EGT, visualizing the entire EG population of the nervous system, 122 µm projections; A.2: brain and neck commissure; A.3: ventral nerve cord and PNS. B.1–4: EGN; B.1: general expression pattern in the central brain; single section; B.2: EGN cell bodies (arrow) are mostly found in between neuropile regions in the central brain; single section. B.3,4: Individual EGN cell morphologies in the protocerebrum; 25 µm projections. The EGN take on many different shapes and sizes, as they ensheath and project complex and fine protrusions (arrow) into the neuropile regions. C.1–4 EGT; C.1: general expression pattern in the central brain; single section; C.2: EGT cell bodies (arrow) are mostly found in between neuropile regions in the central brain; single section. C.3,4: Individual EGT cell morphologies in giant fiber tract (C.3) and neck connective (C.4); single sections. EGT are extended along the long axis of the nerves; their ends are ragged but with their lateral neighbors they form a neatly contiguous sheath. Cross section shows that nerve tracts are completely ensheathed (C.5). Scale bar = 100 µm in A.2,3; 50 µm in B.1–3, C.1; 10 µm in B.4, C.3–5.

Figure 7

Characterization of glia‐glia interactions. A.1: Schematic depicting glial subtypes and their relationship to the different neuronal compartments. A.2–4: Relationship between PNG and SPG on the surface of nervous system; 85 µm projections. A.3,4: Higher magnification shows that the PNG lie directly on top of the SPG. The surface glial sheath is ∼2–3 µm thick and continues across the CNS‐PNS boundary, as seen at the exit of peripheral nerves (arrow in A.4). B.1—3: Relationship between SPG (B.1) and CG (B.2), merge in (B.3); central brain; single section. SPG (B.1) form a contiguous cover over the entire cortical region. Occasionally, CG do not fully envelop neuronal cell bodies, in which cases SPG provide the necessary closure (arrow). C.1,2 Relationship between CG and EGN antennal lobe (C.1), mushroom body calyx (C.2); single section. The two subtypes abut in a very smooth fashion with little or no intermingling at the interface (white arrow). Occasionally, CG protrusions reach into neuropile regions, shown here a sub‐compartmental boundary within the antennal lobe (red arrow). D.1,2: ALG cell bodies (arrow) are embedded within the EGN layer; single section. E.1–5: Relationship between CG and ALG in cortical regions; E.1–3: single sections; E.4,5: 14 µm projections. ALG send fine processes into the CG region (arrows). F.1,2: Relationship between ALG and EGN, single sections. F.1: ALG processes (arrow) projecting into the EGN layer of a neuropile region in the protocerebrum. F.2: ALG cell bodies (arrows) are embedded in EGN sheath. Scale bar = 100 µm in A.1; 10 µm in C.2; 5 µm in A.2,3, B.1–3, C.1, D.1–5, E.1–4.

Figure 8

Equivalency of lamina‐specific glial subtypes with generic glial subtypes. A.1 Schematic of lamina and its interfaces. A.2 Generic and lamina‐specific nomenclature for glial subtypes. B.1–3 Equivalency of generic perineurial glia (B.1) and lamina fenestrated glia (B.2), merged in B.3; single section. B.4–6 Equivalency of generic perineurial glia (B.4) and lamina‐specific perineurial chalice glia (B.5), merged in B.6; single section. C.1–4 Equivalency of generic subperineurial glia (C.1) and lamina‐specific pseudo‐cartridge glia (C.2), merged in C.3; single section. C.4 Structure of subperineurial glia chalice; 84 µm projections. D.1–3 Equivalency of generic cortex glia (D.1) and lamina‐specific distal satellite glia (D.2), merged in D.3; single section. D.4–6 Equivalency of generic cortex glia (D.4) and lamina‐specific proximal satellite glia (D.5), merged in D.6; single section. E.1–3 Equivalency of generic astrocyte‐like glia (E.1) and lamina‐specific epithelial glia (E.2), merged in E.3; single section. F.1–3 Equivalency of generic ensheathing glia (F.1) and lamina‐specific marginal glia (F.2), merged in F.3; single section. Scale bar = 10 µm in all images.

Figure 9

Glial cell morphologies and glia‐neuron interactions in the lamina. A.1,2: Schematics illustrating the glial (A.1) and neuronal (A.2) components of a lamina column. All generic glial subtypes are present but show lamina‐specific morphologies, inspiring the distinct nomenclature for lamina glia. B.1–6: Individual cell morphologies of L‐SPG; B.1,2: 5 µm projection; B.3,4: single sections; B.5,6: 3 µm projections. B.1,2: L‐SPG are large oblong cells extended along the a‐p axis, with each cell enveloping multiple incoming retinula fibers. B.3.6 Fine protrusions of the L‐SPG ensheath incoming retinula fibers on the distal lamina surface (arrows in B.3,4). B.7: Schematic of relationship between L‐SPG and retinula fibers. C.1–5: Individual cell morphologies of lamina distal cortex glia (L‐dCG); C.1: 42 µm projections; C.2: 6 µm projection; C.4,5: single sections. C.1: L‐dCG are uniformly large and oblong in shape along the a‐p axis, with each cell enveloping multiple cartridges. C.2: L‐dCG form honey comb‐like structures that house lamina neuron cell bodies. Note that two L‐dCG may jointly form one honey comb. C.3: L‐dCG ensheath neuronal cell bodies as well as tracts. More than one neuronal cell body can be found within one L‐dCG pocket (arrow). C.4,5: L‐dCG cells partially lie on top of each other (arrow in C.5). C.6 Schematic of relationship between L‐dCG, retinula fibers, and lamina neuron cell bodies. D.1–5: Individual cell morphologies of lamina proximal cortex glia (L‐pCG); D.1: 4 µm projection; D.2: 6 µm projections; D.3: 12.5 µm projection; D.4: single section. D.1: L‐pCG are smaller and show more variable orientation along the a‐p axis, as compared with their distal counterparts. D.2,3: Higher magnification shows that L‐pCG are thin and compact/dense cells. D.4: L‐pCG lie at the interface between lamina cortex and neuropile. On their distal surface, they form pockets (red arrow) for neuronal cell bodies, on their proximal surface, and they send protrusions into the neuropile (white arrow). D.5: Schematic of relationship between L‐pCG, retinula fibers and lamina neuron cell bodies. E.1–8: Individual cell morphologies of lamina L‐ALG; E.1–7: single sections E.8: 4 µm projection. E.1: L‐ALG form a thin lattice. E.2,3: Single lamina cartridge, showing that photoreceptor projections (E.2) are in close contact with L‐ALG lattice (E.3). E.4–6: Distal to proximal sections through single L‐ALG, showing that the main branches of the glial cell are maintained but smaller processes appear and disappear. E.7,8: L‐ALG mosaic revealing a mesh‐like structure extending through the lamina neuropile. E.9: Schematic of relationship between single L‐ALG and lamina columns. F.1–5: Individual cell morphologies of lamina ensheathing glia (L‐EG); F.1: 30 µm projection; F.2: 3 µm projection; F.3: 17 µm projection, F.4,5: single sections. F.1: L‐EG are narrow cells oriented along the dorso‐ventral axis, ensheathing multiple lamina cartridges. F.2,5: L‐EG send many fine protrusions deep into the lamina neuropile as well as shorter processes medially into the outer chiasm. F.3,4: Cross‐sections of single marginal cell at its base (F.3) and tip (F.4). F.11: Schematic of relationship between single L‐EG and lamina columns. Scale bar = 10 µm in B.1,2, C.1,2, D.1, E.1, F.1; 5 µm in B.3–5, D.2–5, E.2,3,7,8, F.2–5.

Figure 10

Glial cell morphologies in the medulla and interactions with neurons. A.1: Schematic showing the location of ALG and EGN cell bodies. Cell bodies are located at the distal and proximal border of the medulla, as well as the serpentine layer, which forms the border between distal and proximal medulla. A.2: Schematic depicting the main features of ALG and EGN. A medulla column is magnified and illustrates the relationship between photoreceptor projections and different glial subtypes. B.1–7: Individual cell morphologies of ALG; B.1: 3 µm projection; B.2: 30 µm projection; B.3–6: 9–18 µm projections; B.7,8: single sections. B.1–6: ALG with cell bodies in the distal medulla cortex occupy the distal medulla, forming irregular columnar structures (white arrows in B.1) and sending fine processes toward the outer chiasm (red arrows in B.1,4). ALG with cell bodies in the proximal medulla cortex occupy the proximal medulla, forming irregular square‐like structures (“chandelier glia,” red arrow B.2). ALG with cell bodies around the serpentine layer branch out into both distal and proximal medulla (green arrows B.1,2). B.7,8: ALG send long processes into the outer chiasm (red open arrows). B.8. These ALG processes lie within the sheath of chiasm glia next to neuronal tracts. C.1–5: Individual EGN cell morphologies; C.1–3: 3–5 µm projections; C.4: 8 µm projection; C.5: 11 µm projection. The EGN in the distal medulla are organized in highly columnar structures (C.1,2) and show a characteristic branching pattern in layers M3, M6, and M7. The fine processes in the proximal medulla (red open arrow in C.2) belong to the inner chiasm glia. The EGN in the serpentine layer send columnar branches into both distal and proximal medulla (white arrow C.4). The EGN of the inner chiasm send fine protrusions into the proximal medulla (C.5). D.1–5: EGN accompany photoreceptor projections, which terminate in medulla layers M3 and M6. D.1: 3 µm projection; D.2–4: Cross sections (3 µm projections) through M3 (D.2), in between (D.3) and M6 (D.4), showing differences in the density of the glial ensheathment. D.5: Schematic illustrating the association of individual EGN with medulla columns. D.6–8: EGN partially envelop (arrows) the many tracheal branches that invade the medulla. D.7,8: 3D reconstruction of a substack of D.6. Scale bar = 50 µm in B.1,2, C.1; 5 µm in B.3–8, C.2–5, D.1–4, D.6,7; 2 µm in D.8.

Figure 11

Glial cell morphologies in the lobula complex. A.1–3: Schematics of lobula complex, consisting of lobula and lobula plate, and neighboring medulla. A.1: Main neuronal connections; transneurons connect medulla and lobula complex. Lobula and lobula plate contain large dendritic arborizations of visual PNs. A.2: Location of ALG and EGN cell bodies. The cell bodies of large inner chiasm glia (XGI) are located between proximal medulla and lobula complex. A.3: Main morphologic features of ALG and EGN. B.1–4: Individual ALG cell morphologies; 1 µm sections. In both lobula and lobula plate, ALG take on various shapes and sizes and can send branches into both lobula and lobula plate; location of cell bodies marked by white arrows (B.1–4). ALG entering the lobula from the anterior margin show a (loosely) columnar organization (B.4). C.1–6 Individual EGN cell morphologies; C.1: 12.5 µm projection; C.2: 10 µm projection; C.3; 10 µm projection; C.4–6 single sections. C.1 EGN have column‐like processes perpendicular to the margin, as well as tangential processes parallel to the margin. C.2,3: EGN form a complex three‐dimensional network of branches. C.4–6 EGN enclose the large dendritic arborizations of lobula plate tangential cells. C.6 Cross sections of single tubes reveal that neuronal processes are completely enclosed (white and red arrows). Scale bar = 10 µm in all images.

Figure 12

The glia of the outer and inner chiasm. A.1: Schematic of the optic lobes and their chiasms. The outer chiasm connects lamina and distal medulla, the inner chiasm connects proximal medulla, lobula and lobula plate. A.2: Labeling of chiasm glia (XG) in outer (XGO) and inner (XGI) chiasm; 1 µm section. B.1,2: XG and EGN are distinct populations in outer chiasm; B.1: 15 µm projection; B.2: 4 µm projection. C.1–5: Individual XG cell morphologies in outer chiasm; C.1: 30 µm projection; C: 2,3: single sections. Neuronal tracts are enveloped by XGO (arrows in C.2,3). C.4,5: 3D reconstruction of C.1, view from medial medulla into chiasm (C.4), view onto lamina portion of chiasm (C.5). Different fibers crossing the chiasm at perpendicular angles can be ensheathed by a single XGO cell. D.1–4 Individual XG cell morphologies in inner chiasm; D.1: 93 µm projection; D.2: 60 µm projection. D.1: A single XGI cell may extend its sheath‐like processes along the entire coronal plane of the inner chiasm. D.2: XGI send many long protrusions (open arrows) into all three neighboring neuropiles. Most XGI cell bodies lie outside the chiasm (filled arrow). D.3,4: 3D reconstruction of D.1, view of the dorso‐ventral axis showing the multilayered structure of the chiasm (D.3); D.4: cross section through D.3, XGI envelop neuronal tracts. Scale bar = 10 µm in all images.

Figure 13

Characterization of glia in the antennal lobe. A.1: Schematic of the olfactory system and its major neuronal connections by PNs that connect the antennal lobe with higher brain centers and by mushroom body intrinsic KC. A.2: Schematic of antennal lobe and its glial subtypes, ALG and EGN. A.3,4: Location of glial cell bodies; single sections. ALG (A.3) and EGN (A.4) cell bodies are found surrounding the antennal lobe neuropile but not inside. B.1–4: ALG in the antennal lobe. B.1,2: General expression pattern; B.1: single section; B.2: 2 µm projection. ALG show a low density of processes throughout the antennal lobe (B.1), high magnification reveals thick branches, fine processes, and occasional thickenings. B 3,4: Single ALG cell morphology; B.3 single section; B.4: 3D reconstruction of cell in B.3. Individual ALG cells penetrate multiple glomeruli with their processes without any anatomical registration. C.1–8: EGN in the antennal lobe. C.1–6: General expression pattern; C.1,6: 3.5 µm projection; C.3,5: single sections. C.1: EGN processes cover the surface of the antennal lobe. C.2: 3D reconstruction of C.1: showing that the glial sheath is nearly contiguous, with small holes remaining for nerve entries (white arrow). C.3: EGN processes projecting between the glomeruli. C.4: 3D reconstruction of C.3 showing that, in contrast to the antennal sheath, the glial cover of individual glomeruli is not continuous. C.5: Neuronal tracts enter the antennal lobe along the glomerular boundaries and are in contact with glial processes (white arrow). C.6: High magnification of EGN at glomerular boundaries reveals tube‐like morphologies. C.7,8: Single EGN cell morphologies; C.7: single section; C.8: 12.5 µm projection. C.7: Neighboring EGN cells cover distinct areas but partially interdigitate in regions of contact (white arrow). C.8: Individual EGN are not confined to single glomeruli. From the main glial processes along the glomerular boundaries smaller branches project perpendicularly into different glomeruli. Scale bar = 10 µm in A.3,4, B.1,3, and C.1–5; 5 µm in B.2, C.7,8.

Figure 14

Characterization of glia in the mushroom body. A.1: Schematic of the olfactory projections in the mushroom body. A.2: Schematic of the mushroom body neuropile and the focal planes represented in the panels below. B.1–7: ALG. B.1–3,6,7: 5 µm projections; B.4,5: single sections. B.1–3: General expression pattern revealing low structural density of ALG projections in the calyx. B.2,3: Higher magnification showing that ALG processes are highly branched and have occasional thickenings. B.4,5: In the peduncle, ALG processes are rare even in the proximity of synapses (B.4), but emerge in the transition from peduncle to heel (B.5). B.6,7: In the mushroom body lobes, the structural density of ALG is low. C.1–7: EGN in the mushroom body. C.1–6: General expression pattern; C.1: 5 μm projection; C.2–5: single sections; C.6: 10 μm projection. C.1: EGN processes permeate the calyx. C.2–5: Cross‐sections through different portions of calyx and peduncle. EGN subcompartmentalize the calyx by enwrapping individual subpopulations of KC (white arrows). C.6: β, β′, and γ lobes are separated by sheaths of EGN (white arrow); even within the individual lobes, EGN provide further subcompartmentalization (red arrow). C.7: Individual EGN cell morphologies in the lobes; 10 μm projection. Both sheath‐like structures and fine protrusions are visible (white and red arrow). Scale bar = 10 µm in B.1–3, C.1–7, D.1,6,7, E.1–7; 5 µm in D.2–5.

Figure 15

Characterization of the glia‐neuron interactions in the olfactory system. A.1,2: Schematic of the olfactory system and its major neuronal projections. The focal planes of the different panels are shown in A.2. B.1–3: Spatial relationship between the cell bodies of PN and CG in the antennal lobe cortex; B.1: 2 µm projection; B.2,3: single sections. Both the neuronal cell body and the neuronal processes (arrows) that project from the cell body to the neuropile region are ensheathed by CG extensions. C.1–3: PN axons within the antenno‐cerebral tract are ensheathed by EGN; 2.5 µm projections. The tract is completely enclosed. D.1,2: EGN at glomerular boundaries in the antennal lobe; D.1: 1.5 µm projection; D.2: horizontal projection of D.1. D.1: The glomeruli of the antennal lobe are ensheathed by a discontinuous layer (arrow) of EGN. D.2, EGN extensions accompany neural processes until they enter the neuropiles (arrow). D.3,4: ALG; D.3 2 µm projection; D.4 Magnification of D.3. The bushy postsynaptic compartment of PNs are intertwined with ALG protrusions, but not all postsynaptic regions are directly in contact with the glia. E.1–7: Spatial relationships of EGN and ALG with pre‐ (E.1–3) and post‐ (E.4–7) synaptic compartments in the calyx. E.1,2: 2.5 µm projections; E.3: horizontal projection of E.1. EGN processes contact PN extensions until they diverge into different terminal regions. E.4,6: single sections; E:5: 1.5 µm projection. Both ALG and EGN processes are found in proximity to postsynaptic regions, but do not closely associate with them in a stereotypic manner. E.7: 7 Association of ALG with PN presynaptic boutons in calyx; 1.5 µm projections. ALG processes loosely intermingle with sites of synaptic connections but lack specific association with the presynaptic compartment. Scale bar = 10 µm in B.1, C.1, D.1,3, E.1,4; 5 µm in B.2,3, C.2,3, D.2,4, E.2,3,5–7.