(A) A primary antibody to histamine (magenta) labels photoreceptor cell terminals in the medulla (asterisks), an area in the proximal lamina (arrowheads) that contains the epithelial glia and axons of the outer photoreceptor cells, and a band just beneath the retina (arrows). The retina is weakly labeled in this section. (B) Co-labeling with anti-B₂ (green) shows co-localization (C) with histamine in the band beneath the retina. Bar: 40 microns.

Confocal images of whole adult w and dVMAT^Δ14 mutant brains were labeled with the DVMAT-B antiserum anti-B₁ (A,B). Labeling is visible in the distal lamina of the w control brain (A), but markedly reduced in the homozygous dVMAT^Δ14 mutant (B). Confocal images of wt and dVMAT^P1 homozygote head sections were labeled with anti-B₂ (C,D) (magenta) and anti-Ebony (C,D) (green). In the wt, control sections, DVMAT-B labeling is visible in the distal lamina (C). In the dVMAT^P1 mutant, however, no DVMAT-B expression is detected (D). These results demonstrate the specificity of both anti-B₁ and -B₂. Re, retina; La, lamina; Me, medulla. Bars: 50 microns.

(A) Electron micrograph of a tangential section through the proximal retina (Re) with seven rhabdomeres (arrowheads) visible in two complete cross-sections of ommatidia. The basement membrane, bm arrows in (A,B), separates the retina from the underlying lamina. Electron-dense gold particles lie just beneath the basement membrane. Beneath this band of labeling, an additional small profile quite distinct in appearance (double arrowhead) also expresses label, and may represent the profile of a serotonin-containing nerve terminal. (B) Higher magnification views of the labeled fenestrated glia and photoreceptor axons in the distal lamina. Gold particles (arrowhead) overlie the fenestrated glia. bm, basement membrane; pa, photoreceptor axons; t, tracheae; lc, glial nuclei. Bars: (A) 5 microns, (B) 2 microns.

(A) Photoreceptor cells R1–R6 terminate in the lamina where they innervate lamina target neurons. Groups of six R1–R6 terminals and lamina target neurons are organized into cartridges, the component modules of the lamina. The diagram illustrates the relationship between the photoreceptor cell axons (red), the basement membrane (blue) separating the retina and lamina, and the lamina target neurons (silver). Identified glial subtypes in the fly's lamina include the fenestrated (dark green), pseudocartridge (orange), satellite (light green), epithelial (yellow), and marginal glia (brown). (B) Possible functions of DVMAT-B in the fenestrated glia include: recycling metabolized histamine back to the photoreceptors (“Recycling”); preventing spillover of histamine into nearby cartridges (“Spillover”); and releasing a reserve pool of histamine into the lamina to regulate its concentration during periods of heavy release (“Reserve”).

A primary antibody to the protein Tan (A) (green) labels the photoreceptor cell bodies and their axons that extend into the lamina (La) and medulla (Me), seen in horizontal cryostat sections of the head. Co-labeling with anti-B₂ (B) (magenta) shows no overlap with Tan in merged images (C). Glia were labeled using repo-Gal4 to drive expression of the plasma membrane marker mCD8-GFP (D,G) (green). Some glial processes extend into the medulla (D,G) (small arrowheads). DVMAT-B was co-labeled using anti-B2 (E,H) (magenta). The merged images (F,I) show robust co-localization of DVMAT-B to profiles enclosed by glial cell membranes in the distal lamina, and additional, faint co-labeling of processes that extend distally into the retina (F,I) (large arrowheads). (G–I) Enlarged views of (D–F), to show the co-localization of the two signals. Bars: (A–F) 50 microns, (G–I) 10 microns.

(A–G) Confocal projection images of whole-mount preparations of retina and optic lobes in control w (A–D) and mutant dVMAT^Δ14 homozygotes (E–G). Tangential views are shown in (A,C,D,F,G); the surface of the lamina is shown in (B) and (E). In w tissues, a primary antiserum to histamine shows labeling of the retina (A) (arrowheads), the distal lamina (arrows) and nerve terminals in the medulla (asterisks). dVMAT^Δ14 mutants (E–G) show reduced labeling at the surface of the lamina (E) (arrows) and retina (F) (arrowheads) relative to the lamina (B) and retina (C) in w. Labeling of nerve terminals in the medulla (D,G) (asterisks) and an area near the optic chiasm (D,G) (small arrow) in the dVMAT mutant (G) is less prominently reduced relative to the w control (D). (H) Cryostat sections of a w head labeled with the anti-histamine antibody also show labeling of the lamina, including a band just beneath the retina (H) (large arrows) as well as processes in the medulla (asterisks). Note that labeling of the retina is less evident in these cryostat sections compared with that in the confocal stacks shown in (A–G). (I) dVMAT^Δ14 shows a decrease in labeling in the distal lamina relative to w—compare (H) and (I) (large arrows). Labeling in the medulla (asterisks) is less reduced in dVMAT^Δ14 and is strong in the proximal lamina (small arrow). Bars: (A) 50 microns, (B–G) 25 microns, (H,I) 50 microns.

(A) Projected confocal image of whole w adult brain labeled with the antibody against the N-terminus of DVMAT (anti-N) that recognizes both DVMAT-A and -B. The entire surface of the lamina is labeled with anti-N in a honeycomb pattern (white arrows). In the central brain, cell bodies (asterisk) and a large number of processes are labeled, consistent with the previously described expression of DVMAT-A. Additional punctate labeling in the lamina (B) (arrowheads) is likely to represent previously described serotonergic varicosities, more easily seen in a single optical section of the lamina (B) (arrowheads) and in a tangential section through the lamina (C) (La, see arrowheads) and distal medulla (C) (Me). Labeling of the lamina surface is also apparent (C) (arrows) in the tangential view of the lamina and distal medulla. (D–F) Cryostat sections of w adult brains labeled for Repo (green) and DVMAT-A+B (magenta). Repo label in glial cell nuclei of the optic lobe and central brain does not co-localize with the label for DVMAT-A+B, but the DVMAT-A+B label in the distal lamina appears to co-localize with glial cell nuclei (D) (arrow) (overlap shown in F). Bars: (A) 50 microns, (B,C) 25 microns, (D–F) 50 microns.

(A) Histamine content is decreased by 30% in the homozygous dVMAT^P1 allele (P/P) and 47% in homozygotes of the dVMAT^Δ14 allele (Δ14) compared with the control line w¹¹¹⁸Cs₁₀ (w; +/+). Additional controls include a precise excision of the P in dVMAT^P1 (p.e.) and dVMAT^P1 heterozygotes (P/+); one-way ANOVA p<0.0001, with Bonferroni post-hoc test used to compare all data. Differences in head histamine from w;+/+ are indicated as **, p<0.01 and ***, p<0.001. Bars show the mean±SEM of independent trials measuring 3–4 heads/trial of randomly mixed sexes, with the number of trials (n) indicated in the bars. (B) Genetic rescue of dVMAT-B. All lines are in a genetic background containing the transgene repo-Gal4/+, and the genotype of the line used as a baseline control is dVMAT^P1/+; repo-Gal4/+ (indicated as “Base. P/+; Repo/+”). In this background, histamine content is reduced 45% by rendering the dVMAT^P1 allele homozygous (dVMAT^P1/dVMAT^P1; repo-Gal4/+, indicated as “Mut. P/P; Repo/+”. Rescue was performed using repo-Gal4 with two separate UAS-dVMAT-B transgenes, UAS-dVMAT-B1 and -B2, indicated as “Res.1 P/P; Repo/B1” and “Res.2 P/P; Repo/B2”, respectively. Additional controls include dVMAT^P1 heterozygotes with repo-Gal4 and either UAS-dVMAT-B1 or -B2, (indicated as “Con.1 P/+; Repo/B1”, and “Con.2 P/+; Repo/B2”). One-way ANOVA (p<0.0006, with Dunnett's multiple comparison tests) shows that “Mut.” head histamine content differs from all other lines (p<0.05: *). Bars show mean±SEM of independent trials measuring 4 heads, of randomly mixed sexes, with the number of trials indicated in the bars.

(A) dVMAT-A and -B share a common translational start site (indicated as “A, B start”) and a common N-terminus, but diverge at their C-termini. Coding exons in the dVMAT gene that are common to both dVMAT-A and-B are shown as gray boxes, introns as black lines. To generate alternative carboxy termini, the indicated genomic sequence (magenta box) is spliced out from dVMAT-A and retained in dVMAT-B. The in situ probe for dVMAT-B contains the first 260 nucleotides of this sequence. The P element insert in the dVMAT mutant allele dVMAT^P1 (black arrowhead) disrupts the coding sequence of both dVMAT-A and -B. (B,C) In situ hybridization of head sections shows transcription of the dVMAT-B gene in a layer between the retina and lamina (B). Magnified view of (B) shown in (C). (D) Cartoons showing the predicted topology of DVMAT with lumenal domains above and cytoplasmic domains below the parallel gray lines representing the vesicle membrane. Open circles indicate domains shared by DVMAT-A and -B. Filled, magenta circles indicate the C-terminal domain specific for DVMAT-B. The P element insertion site in the last exon of the dVMAT^P1 (marked with “P” and a black arrowhead) functionally deletes transmembrane domains 11 and 12 and the C-terminus (shaded gray). The imprecise excision allele dVMAT^Δ14 results in an insertion of 51 base pairs, and 17 amino acids in-frame with the original downstream codons (marked as “Δ14” with a blue arrowhead, with blue circles indicating the inserted residues). (E) Western blot using the N-terminus antibody directed against both DVMAT-A and -B splice variants shows an absence of DVMAT protein in dVMAT^P1 homozygotes (P/P), and dramatically reduced levels in dVMAT^Δ14 (Δ14/Δ14), compared with heterozygous controls (P/CyO and Δ14/CyO). The plasma membrane associated protein Late Bloomer (indicated as “lb”) was used as a loading control. Re, retina; La, lamina; Me, medulla. Bars: (B) 50 microns, (C) 20 microns.