Long-distance mechanism of neurotransmitter recycling mediated by glial network facilitates visual function in Drosophila

Abstract

Neurons rely on glia to recycle neurotransmitters such as glutamate and histamine for sustained signaling. Both mammalian and insect glia form intercellular gap-junction networks, but their functional significance underlying neurotransmitter recycling is unknown. Using the Drosophila visual system as a genetic model, here we show that a multicellular glial network transports neurotransmitter metabolites between perisynaptic glia and neuronal cell bodies to mediate long-distance recycling of neurotransmitter. In the first visual neuropil (lamina), which contains a multilayer glial network, photoreceptor axons release histamine to hyperpolarize secondary sensory neurons. Subsequently, the released histamine is taken up by perisynaptic epithelial glia and converted into inactive carcinine through conjugation with β-alanine for transport. In contrast to a previous assumption that epithelial glia deliver carcinine directly back to photoreceptor axons for histamine regeneration within the lamina, we detected both carcinine and β-alanine in the fly retina, where they are found in photoreceptor cell bodies and surrounding pigment glial cells. Downregulating Inx2 gap junctions within the laminar glial network causes β-alanine accumulation in retinal pigment cells and impairs carcinine synthesis, leading to reduced histamine levels and photoreceptor synaptic vesicles. Consequently, visual transmission is impaired and the fly is less responsive in a visual alert analysis compared with wild type. Our results suggest that a gap junction-dependent laminar and retinal glial network transports histamine metabolites between perisynaptic glia and photoreceptor cell bodies to mediate a novel, long-distance mechanism of neurotransmitter recycling, highlighting the importance of glial networks in the regulation of neuronal functions.

Fig. 1.

Carcinine and β-alanine are present in pigment cells and photoreceptor cell bodies of the retina. (A) Colabeling of histamine (a–b2), carcinine (c–d2), and β-alanine (e–f2) with chaoptin (blue) in the retina of WT flies. All three molecules are localized outside of chaoptin-labeled rhabdomeres, and their single-channel staining signals are shown in grayscale images b1, d1, and f1. Both lateral (a, c, and e) and cross-section (b, d, and f) views are presented. (B) Colabeling of histamine, carcinine, and β-alanine with ATPα (blue) in the retina of WT, tan¹, and ebony¹ mutants. All three molecules exist in both photoreceptor and pigment cells. Greyscale images show single-channel signals of respective molecules. Note that tan¹ mutants accumulate carcinine but have reduced β-alanine level in pigment cells, whereas ebony¹ mutants show the opposite. Both mutants have reduced histamine level in the retina. (C) Immunogold labeling of carcinine in cross eye sections of cn¹,bw¹ flies. Boxed areas in a1 and b1 are enlarged in a2, b2, and b3, which show the presence of immunogold particles (arrowheads) in both pigment (Pg, pink) and photoreceptor (Ph, gray) cells, but not in cone cells (Cn, green). (D) Quantification of immunogold particles in photoreceptor, pigment, and cone cells.

Fig. 2.

Gap-junction protein Inx2 is present in laminar glia. (A) Viabilities of different Inx2 KD, mutant, and rescue flies. (B) Immunolocalization of Inx2 (blue; a1) and its colabeling (a2) with a photoreceptor terminal marker discs-large (DLG) (red) in WT lamina. (C) Costaining of glial GFP (green; a1) and Inx2 (blue; a2) in the lamina of repo-Gal4>UAS-mCD8::GFP flies showing the localization of Inx2 in laminar glia. (D) Immunolocalization of CSP (green, axon marker; a1) and Inx2 (blue; a2) in the lamina of WT flies. The merged image (a3) shows localization of Inx2 in epithelial glia.

Fig. 3.

Inx2 KD in satellite glia leads to reduction of visual carcinine, and accumulation of β-alanine and reduction of histamine in the retina. (A) Colabeling of carcinine with ATPα (blue, a1 and b1, in cross-retina sections) or DLG [blue; a2 and b2, in both retina (RE) and lamina (LA)]. Arrows point to areas of reduced carcinine level in Inx2^RNAi flies. sfg, sg, eg, and mg indicate layers of surface, satellite, epithelial, and marginal glia. (B) Quantification of the staining intensity for carcinine in different laminar glia layers of WT, histamine metabolism mutants, and Inx2^RNAi flies. (C and D) Colabeling of β-alanine and histamine with ATPα (blue; a1 and b1) or DLG (blue; a2 and b2). Arrows point to areas where the respective molecule has changed level in Inx2^RNAi flies. (E) Quantification of staining intensities for β-alanine and histamine in laminar neuropil glia and the retina of WT and Inx2^RNAi flies (*P < 0.05, compared with WT, two-tailed t test, n = 10, for each quantification).

Fig. 4.

Satellite glial Inx2 is essential for fly visual synaptic transmission. (A) CSP staining signal is reduced in the lamina of Inx2^RNAi flies. (a1–b1 and a2–b2) Lateral and cross-views of lamina, respectively. (B) Quantification of laminar CSP immunostaining intensities. (C) EM images of photoreceptor terminals in the lamina. The red arrowhead-marked T-bar synapses in a1 and b1 are magnified in images a2 and b2, respectively. Black arrowheads point to synaptic vesicles. (D) Quantification of photoreceptor synaptic vesicles at the EM level (*P < 0.05, compared with WT, two-tailed t test, n = 10, for each quantification). (E) Inx2^RNAi flies lost both ON (black arrow) and OFF transients (white arrow) of ERG that represent histaminergic synaptic transmission activities. (F) Comparison of ON and OFF transient amplitudes among WT, Inx2^RNAi flies, and histamine metabolism mutants. Two independent Inx2 RNAi lines were tested (*P < 0.001, compared with WT, two-tailed t test, n = 20, for each quantification).

Fig. 5.

Inx2 KD in satellite glia alters fly visual alert response due to blockage in histamine recycling through a visual glia network. (A) Illustration of VAR behavior assay. In stationary condition of the block, fly exhibits antigeotaxis and climbs up for food in a vertical chamber. In moving condition of the block, wild-type fly freezes at a certain distance after seeing the block moving toward it. A visual defective fly, however, is unable to see the moving block from the same distance as WT and shows no response, delayed freezing, or brief pause response. (B) VARs of WT, norpA (blind), parent controls (mz0709-Gal4/+ and UAS-Inx2^RNAi/+), Inx2^RNAi flies, and histamine recycling-defective mutants. The number within colored bars represents percentage of flies showing the respective type of response. (C) Model showing that retinal pigment cells and the laminar glial network of Drosophila is actively involved in storage and recycling of histamine. After histamine is converted into carcinine by epithelial glia, carcinine moves through the laminar glial network to reach pigment (pig.) cells and enters the cell bodies of photoreceptor (ph.). β-alanine, however, moves in the opposite direction to reach epithelial glia.