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First published online October 17, 2008
Journal of Experimental Biology 211, 3454-3466 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.021162
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Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain

J. Borycz1, J. A. Borycz1, A. Kubów1, V. Lloyd2,* and I. A. Meinertzhagen1,2,{dagger}

1 Department of Psychology, Life Sciences Centre, Dalhousie University, Halifax, NS, Canada B3H 4J1
2 Department of Biology, Life Sciences Centre, Dalhousie University, Halifax, NS, Canada B3H 4J1


Figure 1
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  		Fig. 1. Histamine, dopamine and 5-HT are reduced in the heads of white, brown and scarlet mutants relative to wild-type heads. (A) Histamine content, means of means from 10–13 samples for each genotype. (B) Dopamine content, means of means from 10 samples for each genotype. (C) 5-HT content, means of means from 10 samples for each genotype. All three mutants differ from Oregon-R wild-type for all three amines: *P<0.0005 (t-test).

 

Figure 2
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  		Fig. 2. Immunoreactivity to histamine is reduced in white, brown, scarlet, ebony and white; ebony relative to Oregon-R wild-type flies. Frozen 10 µm frontal sections immunolabeled with anti-CSP (B,E,H,K,N.Q) or anti-histamine (A,D,G,J,M,P), and the corresponding merged double label anti-CSP (green) and anti-histamine (magenta; C,F,I,L,O,R). (A–C) Oregon R wild-type; (D–F) white, (G–I) brown, (J–L) scarlet, (M–O) ebony and (P–R) white; ebony. Relative to the wild-type, the histamine immunosignal is reduced in the laminas of all mutants. The signal is similarly reduced for CSP, in the optic lobe and central brain in white, brown and largely lost in white; ebony mutants. Overlap between the expression patterns is complete only in the laminas of wild-type. Scale bar, 100 µm.

 

Figure 3
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  		Fig. 3. white, brown and scarlet mutants have organelle counts that mostly do not differ from wild-type controls. All values are mean ± s.d. (N=4 flies). (A,B) Sizes of R1–R6 profiles. Relative to wild-type controls, no differences were detected in profile sizes, either in their cross-sectional area (µm2; A) or membrane perimeter (µm; B). (C,D) The number of synapse profiles per micrometer of membrane perimeter, counted as either tetrad (C) or feedback (D). (E) Number of synaptic vesicle profiles per R1–R6 profile. These are significantly higher in Oregon-R wild-type than all mutant R1–R6 (*P<0.03, t-test). (F–H) Numbers of capitate projections (CP) in the same samples as C–E, seen as three profiles: single penetrating (F), shallow (G) and multiple headed (H). (For definition of types, see Materials and methods.) No significant differences were seen except in the number of multiple-headed penetrating invaginations, which was greater in the wild-type control than in either white or scarlet heads (*P<0.05, t-test, in H). Some counts showed wide variation, which we attribute to our methods for sampling organelle profiles at relatively low frequencies. By chance, the variation appeared larger in wild-type than mutant values (D,G,H).

 

Figure 4
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  		Fig. 4. Vesicle-enriched fractions from white, brown and scarlet mutants have reduced biogenic amines. (A) The pellet and supernatant fractions after centrifuging head homogenates of wild-type and mutant flies, calculated to show the average content per head in ng (histamine) or pg (dopamine, 5-HT). The pellet and supernatant fractions were significantly different (paired t-test, P<0.006), but the direction of the difference depended on the genotype. Values are means ± s.d. (B) Same data as in A, shown as the corresponding pellet:supernatant ratio for mutant and wild-type fly brain homogenates. These ratios are inverted for the mutant flies, compared with wild-type flies.

 

Figure 5
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  		Fig. 5. Western blot of supernatant (S) and pellet (P) fractions from head homogenates probed with anti-cysteine string protein (CSP; see Materials and methods). The antibody detects in the pellet fraction a single band at ~34 kDa, representing the combined isoforms of CSP. This band is not present in the supernatant.

 

Figure 6
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  		Fig. 6. Immunoreactivity of the Drosophila visual system to White protein. Frontal 10 µm cryostat sections of fly heads immunolabeled with a polyclonal antibody that detects the extracellular loop of the White protein. (A) The wild-type shows a strong signal only in the lamina (La), with a much weaker signal in the photoreceptors (P). In the retina, signal is concentrated in the primary pigment cells (inset, top: arrows) and at in the basement membrane (inset, middle), probably in the base of secondary and tertiary pigment cells (Longley and Ready, 1995Go). In the lamina, White label is clear only in the epithelial glia (inset, bottom: asterisk) that surround the array of lamina cartridges. A ring of R1–R6 terminals is faintly visible within each cartridge, revealing weak photoreceptor expression. Terminals of the R7 and R8 photoreceptors are also faintly visible in the medulla (arrow), but label is otherwise very weak and diffuse in the deeper medulla (Me) neuropile, revealing no cellular expression site. (B–E) Matched wild-type and mutant visual systems immunolabeled in parallel, for comparison. (B) Wild-type. (C) white mutant. (D) brown mutant. (E) scarlet mutant. Relative to the wild-type, the immunosignal is essentially absent in white and brown, and greatly reduced in scarlet. Scale bars, in A, 50 µm and 10 µm (insets); and 100 µm (in D for B–D).

 

Figure 7
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  		Fig. 7. Histamine in the head is altered reciprocally by white, in double mutants with ebony and tan. In all single mutants, the histamine content is reduced, to 51% (white), 48% (ebony) and 10% (tan) of the wild-type value. Relative to these, double-mutant white, tan has significantly more histamine than the corresponding tan single mutant ({dagger}P<0.0005), which has 34% of the wild-type value; whereas white; ebony has less head histamine than either single mutant alone, reduced to 19% of the wild-type value, a reduction that is significant (*P<0.0005).

 

Figure 8
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  		Fig. 8. HPLC separation of head homogenates from flies that drank a 25% solution of [3H]histamine (37 MBq ml–1 and 858.4 GBq mmol l–1) in 4% aqueous glucose. (A) Wild-type, tan and double-mutant white, tan heads. (B) Wild-type, ebony and double-mutant white; ebony heads. Double-mutant white, tan has a significantly reduced carcinine (CA) peak compared with tan whereas double-mutant white; ebony rescues normal histamine (HA) uptake, which is otherwise significantly reduced in ebony. Both differences are indicated by arrows delimiting the magnitude of white's (w) added action. All data are plotted as 2-min bins, compiled from the emissions from two adjacent 1-min fractions. Retention time of carcinine is 12 min and retention time of histamine is 18 min. The tan peak with the shorter retention time (4 min) is an unknown metabolite. For a detailed comment see Borycz et al. (Borycz et al., 2000Go).

 

Figure 9
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  		Fig. 9. Histamine is reduced in the heads of four fly species with white eyes. These are: Sarcophaga spp., combined data from two species that compare S. bullata (wild-type) with S. barbata (mutant ivory, with white eyes); Calliphora erythrocephala (wild-type) with chalky (white eye); Musca domestica (wild-type and white-eyed); and Drosophila melanogaster, white null mutant and O-R wild-type.

 

Figure 10
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  		Fig. 10. White's proposed involvement in the ebony–tan histamine shuttle. Histamine (HA) is released from the photoreceptor terminal (R1–R6) into the synaptic cleft (1), where it activates postsynaptic histamine receptors on the dendrites of a lamina monopolar cell (LMC) target. Its action is terminated by glial uptake (2), which our evidence indicates may be partly white dependent. Histamine in the epithelial glia (EG) is then conjugated to β-alanine (β-A) to form carcinine (CA), regulated by Ebony, and the carcinine then extruded from the glia by an unknown transporter (3). Carcinine is next taken up from the cleft by the transporter Inebriated (4) (Gavin et al., 2007Go), into the R1–R6 terminal, where it is hydrolyzed under the action of Tan to liberate histamine and β-alanine. No clear evidence exists for a direct histamine reuptake mechanism at the R1–R6 terminal membrane (5).

 

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