Activation of Glial FGFRs Is Essential in Glial Migration, Proliferation, and Survival and in Glia-Neuron Signaling during Olfactory System Development

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

BACKGROUND:Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. METHODOLOGY/PRINCIPAL FINDINGS:We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. CONCLUSIONS/SIGNIFICANCE:We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment.

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/cne.2401

MβCD causes abnormal antennal lobe development.

<p>MβCD injection at early stage 3, animals allowed to develop to stage 14 (A–C) or 18 (D). Midline to the left. Brains were double labeled with WGA (A–D) and with Jacalin (A′–D′ plus insets). A: Control – ORN axons terminating in the male-specific macroglomerular complex (MGC, consisting of the Cumulus (C) and Toroids 1 & 2 (T1&T2)) label with WGA; axons terminating in the ordinary glomeruli (*) do not. A′: Jacalin-labeled AL neuron dendrites arborize in a glomerular pattern in both ordinary and MGC glomeruli. (CNP): coarse neuropil. A′ (inset): AL neuron dendrites in an untreated AL chronically deprived of ORN axon innervation have a diffuse, aglomerular arbor. B: 5 mg MβCD. Male-specific ORN axons retain some WGA labeling, but both MGC and ordinary glomerulus organization is perturbed and lobes elongate. Bright WGA labeling of the lateral and medial cell body clusters (LC, MC) is due to high WGA affinity for a nuclear membrane protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007222#pone.0007222-Gibson3" target="_blank">[40]</a>. B′: Jacalin labeling highlights the disordered arrangement and lower number of glomeruli in treated animals. B′ (inset): In another animal injected with 5 mg MβCD, glomerulus-like structures appear even in the normally glomerulus-free coarse neuropil. C,D: 7.5 mg MβCD. Bright WGA labeling of MGC axons is completely lost though an MGC-like structure (MGC*) is present in panel C. C′,D′: Lobular structure of neuropil is faintly visible (arrowheads), but organization of the lobe is deeply perturbed despite the presence of substantial antennal nerves (AN). OT: output tracts. SZ: sorting zone region of the AN. Scale bar in A applies to all panels.</p

MβCD treatment of cultured ORNs decreases EGFR labeling.

<p>Under control conditions (A_1–2), punctuate labeling for EGFRs (using antibody ab49966) appears along the length of the axons and in the growth cones (arrows), including the filopodia (arrowheads). ORNs exposed to 0.5 mM MβCD for 24 hours display no discernable changes in morphology or labeling for EGFRs (B_1–2).After 24-hr exposure to 1 mM MβCD (C_1–2), the punctuate labeling of filopodia and axons is reduced but labeling of flattened growth cones remains. Exposure to 1.5 mM MβCD (D_1–2) results in nearly complete absence of labeling of axons and filopodia; weak labeling of some growth cones persists. Scale bar in A₁ applies to all panels except D₂.</p

PD173074 treatment results in reduced glial numbers at later stages.

<p><b>A,B:</b> Syto 59 labeling of the same antennal lobes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033828#pone-0033828-g008" target="_blank">Figure 8A–D</a>. At lower magnification, Syto 59 labeling of cell nuclei illustrates the significant reduction in cell number in treated animals at later stages (stage 11 shown). The reduction occurs in regions normally occupied solely by glial cells. <b>Blocking glial FGFR activation leads to a decrease in proliferation. </b><b>C,D:</b> Control and PD173074-treated animals were allowed to develop to late stage 5, then dissected and their brains labeled with an antibody to phospho-histone H3, an indicator of mitosis (yellow). Syto 13 (blue) was used to visualize all glial nuclei. Projection depths = 30 µm in <b>A,B</b> and 10 µm in <b>C,D</b>.</p

WGA colocalizes with Triton-resistant Vybrant DiI.

<p>A: Explants of antennal sensory epithelium. Vybrant DiI (red) and WGA-Alexa 633 (green). B: Re-imaging after treatment with 0.5% Triton at 4°C shows WGA labeling only where Triton-resistant Vybrant DiI remains. C: Higher magnification reveals a population of Triton-resistant Vybrant DiI-labeled patches with no detectable WGA labeling.</p

MβCD treatment of cultured ORNs decreases MFas II labeling.

<p>A_1–4: In control conditions, a subset of ORN axons extending from explants are MFas II-positive, some strongly (arrowheads), some moderately (open arrowheads). Arrows indicate several unlabeled axons visible under brightfield optics. B_1–4: After 24-hr exposure to 1mM MβCD, more MFas II axons are moderately or only weakly labeled. C_1–4: At 2 mM MβCD, nearly all MFas II-positive axons are only faintly labeled. Rare axons that appear brightly labeled (C₃) were always less strongly labeled than those found in control or 1 mM dishes. No consistent changes were seen in axonal or growth cone morphology at the 1 mM dose; axon outgrowth was reduced at the 2 mg/ml dose.</p

Blocking activation of the FGFR blocks migration of neuropil glia.

<p><b>A,B:</b> Animals injected at stage 4 with DMSO or DMSO + PD173074 and examined at stage 7. <b>A:</b> Control animal injected with vehicle (DMSO) and labeled with the anti-pFGFR antibody (magenta) and Syto 13 (green) to show cell nuclei. Neuropil glial cells have migrated to surround glomeruli as in untreated animals. A more anterior view than those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033828#pone-0033828-g002" target="_blank">Fig. 2</a> was chosen to better illustrate the intense labeling of NP glial processes surrounding glomeruli. <b>B:</b> Animal injected with DMSO containing 0.5 mg PD173074. Labeling for activated FGFRs on glial cells is absent, and most neuropil-associated (NP) glia have failed to migrate to surround glomeruli. Sorting zone (SZ) glia have migrated normally despite their lack of labeling for activated FGFRs. <b>C:</b> A western blot of control and PD173074-treated antennal-lobe tissue from which neuronal cell bodies had been removed demonstrates a nearly complete absence of labeling for pFGFRs for the PD173074-treated lobes. <b>D:</b> Another animal treated with 0.5 mg PD173074 (beginning at stage 3), dissected at stage 7, and labeled with Syto 13. At increased gain, some NP glial processes can be seen to have extended into the neuropil (arrowheads) despite the absence of cell-body migration. <b>E:</b> A stage-6 antennal lobe from an animal chronically deprived of ORN innervation on one side (antennal anlagen removed at stage 1.) Although lack of ORN innervation resulted in lack of glial migration, glial cells did exhibit activated FGFRs (magenta, arrowheads). <b>E′:</b> Enlarged section from boxed area of panel <b>E</b> better illustrates the labeling of glial processes. <b>E″:</b> Opposite lobe, which was not deprived of ORN input, appears to have the same intensity of pFGFR labeling. <b>F′,F″:</b> Individual cells of deafferented (<b>F′</b>) and control (<b>F″</b>) lobes both display colocalization of pFGFR and DNA labels. LG, MG = lateral and medial group of AL neuron cell bodies. Projection depths = 10 µm in <b>A, B, D, E, E′</b>. <b>F′, F″</b> are single optical sections (40× objective).</p

MβCD treatment does not prevent correct regional targeting of the axons innervating an identified glomerulus (glomerulus X).

<p>Untreated (A–C) and MβCD-treated (D–F) animals. A,B: Stage 7. An antibody to human ankyrin B (yellow) labels ORN axons targeting a single glomerulus located dorso-posteriorly close to primary neurites of the medial cluster of AL neurons (outlined with dashed line). Neuronal cell bodies and glial nuclei labeled with Syto 13 (blue). C: Double labeling with the ankyrin B antibody (magenta) and the MFas II antibody (green) demonstrates that glomerulus X (arrow) is Fas II-negative. D–F: 7.5 mg MβCD, injected at early stage 3, brain dissected at stage 7. Ank B axons target a single glomerulus located near the primary neurites of the medial cluster of AL neurons although the shape of the glomerulus is variable and the pattern of fasciculation in the SZ is somewhat abnormal.</p