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. 2002 Dec 15;22(24):10549-57.
doi: 10.1523/JNEUROSCI.22-24-10549.2002.

tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals

Janusz Borycz  1 , Jolanta A Borycz, Mohammed Loubani, Ian A Meinertzhagen
Affiliations

tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals

Janusz Borycz et al. J Neurosci. .

Abstract

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of beta-alanine to dopamine or hydrolysis (tan) of the beta-alanyl conjugate to liberate dopamine. beta-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, we show that both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. We demonstrate that when they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its beta-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas beta-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. Our findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. We suggest that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake.

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Figures

Fig. 1.
Fig. 1.
Distribution of histamine immunoreactivity in the lamina and distal medulla neuropiles of the optic lobe from wild-type (wt; A) and red-eye stocks ofebony1 (B) andtan1 (C)D. melanogaster. Confocal images of representative 10-μm-thick cryostat sections immunolabeled with antihistamine, revealing immunopositive labeling of photoreceptors, are shown. As shown in C, axons R1–R6 terminate in the lamina (La) or, for the long visual fibers, R7 and R8, in the distal medulla (Me), after crossing their positions in the external chiasma. Strong immunoreactivity is localized to the photoreceptor somata and their terminals (wt,ebony1), in neurons of the central brain (wt, ebony1), as well as in fenestration (arrowhead) and marginal (arrow) glia in the lamina (wt). Scale bar: C, 100 μm.
Fig. 2.
Fig. 2.
Histamine contents for theDrosophila head. Total head histamine fortan and three alleles of ebony, compared with the Oregon R wild-type contents of ∼2 ng.tan1 has ∼0.2 ng;ebonyexc, the ebonyexcision allele In(3R)eAFA has ∼0.7 ng; and ebony1 has ∼0.9 ng. Values are mean ± SEM for 10 samples per value. wt, Wild type.
Fig. 3.
Fig. 3.
Synaptic vesicle counts per R1–R6 photoreceptor terminal profile in Oregon R wild-type (wt),tan1, andebony1Drosophila. Counts (n) and their densities (N/μm2) in terminal cross sections are shown.
Fig. 4.
Fig. 4.
Distribution of 3H cpm in chromatographs obtained after the separation by HPLC of head extracts from tan1,ebony1, and wild-type Oregon R flies, after they had been permitted to drink [3H]histamine for 40 min. A clear 3H peak with the same retention time as carcinine (CA) appears in tan but not in the other two. Note the smaller overall 3H (HA) head content inebony1.
Fig. 5.
Fig. 5.
Distribution of 3H (cpm) in chromatographs obtained after the separation by HPLC of head extracts from wild-type Oregon R Drosophila, 20 min after microinjecting [3H]histamine individually into the head. Note the retention times for histamine (HA) and carcinine (CA).
Fig. 6.
Fig. 6.
Distribution of 3H (cpm) in chromatographs obtained after the separation by HPLC of head extracts from wild-type Sarcophaga microinjected into the head with [3H]histamine. A, At 5 min after injecting [3H]histamine (HA), 1 min fractions reveal a small peak at the same retention time as carcinine (CA), which is larger. B, After 30 min, two additional peaks (*) and a possible third (*?) with shorter retention times also appear.
Fig. 7.
Fig. 7.
The ratio between the peaks for histamine (HA) and carcinine (CA) at different times after injections into Sarcophaga. Each point is the mean, or the ratio between the indicated mean, of the corresponding fractions from at least two individually injected flies, from a batch carefully selected for similar age and weight. Values for [3H]carcinine and [3H]histamine from the two flies differed by <20%. Total [3H]histamine declines, presumably first by diluting into the hemolymph of the body, and then by excretion. The [3H]carcinine/[3H]histamine ratio is initially low but increases; [3H]carcinine does not accumulate, however, indicating that it is lost in parallel to [3H]histamine, presumably by back conversion, thereby establishing a dynamic equilibrium between histamine and its metabolite. The ratio between the major peak of the earlier3H retention peaks and the peak for [3H]histamine changes in a similar manner to, but is much larger than, the [3H]carcinine/[3H]histamine ratio (note different ordinate scales).
Fig. 8.
Fig. 8.
Distribution of 3H (cpm) in chromatographs obtained after the separation by HPLC of head extracts from black;tan double-mutant Drosophila, after they had been permitted to drink [3H]histamine (HA) for 40 min.A, Control double-mutant flies. B, Double mutants previously fed 5% β-alanine. CA, Carcinine.
Fig. 9.
Fig. 9.
Percentage changes relative to controls of3H (cpm) in chromatographs obtained after the separation by HPLC of head extracts from wild-type Sarcophaga, 30 min after they were injected with [3H]histamine (HA). Plotted are the ratios between control flies and flies that were pretreated with MAO inhibitors: pargyline at 10 μg, deprenyl at 0.5 μg, and clorgyline at 5 μg. The horizontal dashed line indicates control values.
Fig. 10.
Fig. 10.
Percentage changes relative to controls of3H (cpm) in Sarcophaga, 30 min after injecting [3H]histamine (HA).A, Flies pretreated with semicarbazide at concentrations between 0.5 and 2 μg/10 μl, relative to controls. B, Flies pretreated with hydroxylamine at concentrations between 0.05 and 0.25 μg/10 μl, relative to controls. The horizontal dashed lines indicate control values.
Fig. 11.
Fig. 11.
Tan and Ebony regulate the β-alanine conjugation and hydrolysis of dopamine to β-alanyl dopamine. In the lamina and possibly in the distal medulla, they also regulate the comparable conjugation of histamine to β-alanyl histamine (ebony) and its subsequent hydrolysis (tan) to yield free histamine. In both cases, conjugation requires β-alanine, which is also liberated during hydrolysis, along with the corresponding amine. Synthesis of β-alanine from aspartate or uracil by decarboxylation is under the control of the gene black.
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