Loss of CarT affects the histamine, β-alanine, and carcinine contents <i>in vivo</i>.

<p>(A-C) Head histamine, β-alanine, and carcinine contents in the three genotypes indicated. (A-B) The <i>tan</i>^<i>1</i> and <i>cart</i>^<i>1</i> mutants had significantly less histamine and β-alanine than wild-type flies (<i>w</i>^<i>1118</i>). (C) The <i>tan</i>^<i>1</i> mutants had nearly three times as much carcinine as wild-type flies, and <i>cart</i>^<i>1</i> flies only showed a 35% increase in carcinine content. Error bars indicate SD; significant differences between mutant and wt flies were determined using unpaired t-tests (*p < 0.05; ***p < 0.001). (D) Model of the pathway for histamine recycling. After a light stimulus, the photoreceptor cells (PR) release histamine, synthesized by histidine decarboxylase (Hdc), into the synaptic cleft to activate histamine-gated chloride channels (HisClA) on postsynaptic neurons (LMC). The released histamine is quickly removed by an unknown histamine transporter in epithelial glial cells that express Ebony, and is then deactivated by conjugation to β-alanine. The histamine metabolite carcinine is then transported out of epithelial glial cells (Glia) by a second unknown transporter, and back to photoreceptors by means of the CarT transporter at the photoreceptor cell terminals, where carcinine is then hydrolyzed back into histamine by Tan ready to be pumped into synaptic vesicles in preparation for further release.</p

CG9317 is photoreceptor cell-enriched carcinine transporter.

<p>(A-B) Photoreceptor cells express CG9317 at a high level. (A) qPCR experiments show that <i>CG9317</i> expression is enriched in wild-type (wt: <i>w</i>^<i>1118</i>) heads compared with <i>gl</i>^<i>3</i> heads or wild-type bodies. (B) The ratio of <i>CG9317</i> transcript levels versus <i>gpdh</i> transcript levels was determined using quantitative PCR. The mRNA level was normalized to the wild-type body, relative to which the <i>CG9317</i> transcript levels were increased about 43 fold and 5 fold in the heads of wild-type and <i>gl</i>^<i>3</i> mutant flies respectively. Error bars indicate the SD. (C-F) S2 cells transiently expressed (C) mCherry, (D) Ine-mCherry, (E) CG3790-mCherry or (F) CG9317-mCherry. Carcinine was added to the culture medium at a final concentration of 20μm. Cells were labeled with rabbit anti-carcinine (green) and DAPI (blue). The mCherry (red) signal was observed directly. Scale bar, 25μm.</p

Rescue of <i>cart</i>^<i>1</i> phenotype by expressing human OCT2 in photoreceptors.

<p>(A) OCT2 was able to transport carcinine. S2 cells transiently expressed OCT2-mCherry in a culture medium to which carcinine was added at a final concentration of 20μM or 100 μM. Cells were immunolabeled with anti-carcinine (green), and the mCherry (red) signal was observed directly. The carcinine signal is stronger in the presence of 100μM carcinine. (B-D) ERG recordings from (B) wt: <i>w</i>^<i>1118</i>, (C) <i>cart</i>^<i>1</i>, and (D) <i>cart</i>^<i>1</i>; <i>PninaE-oct2</i> flies showed restored “on” and “off” transients after photoreceptor cell-specific expression of human OCT2 in <i>cart</i>^<i>1</i> flies. (E) Quantification of phototactic behaviors of wt, <i>cart</i>^<i>1</i>, and <i>cart</i>^<i>1</i>; <i>PninaE-oct2</i> (<i>cart</i>^<i>1</i>; <i>oct2</i>) flies. Error bars represent SD. Significant differences between mutant and wt flies were determined using unpaired <i>t</i>-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).</p

CarT is required for synaptic transmission in photoreceptor cells.

<p>(A-E) ERG paradigm that elicits “on” and “off” transients (arrows) in (A) wt (<i>w</i>^<i>1118</i>) flies but not in (B) <i>e</i>^<i>1</i>, (C) <i>tan</i>^<i>1</i>, (D) <i>ine</i>^{<i>MI05077</i>} or (E) <i>cart</i>^<i>1</i> mutant flies. Flies (~1 d after eclosion) were dark adapted for 1 min and subsequently exposed to a 5s pulse of orange light. (A’-E’) “Off” transients were observed in the wt mosaic, <i>e</i>^<i>1</i> and <i>ine</i>^{<i>MI05077</i>} mosaic eyes but not in <i>tan</i>^<i>1</i> or <i>cart</i>^<i>1</i> mosaic eyes. The genotypes are as follows: (A’: wt mosaic) <i>ey-flp; FRT40A/GMR-hid CL FRT40A</i>. (B’: <i>e</i>^<i>1</i> mosaic) <i>ey-flp; FRT82B e</i>^<i>1</i><i>/FRT82B GMR-hid CL</i>. (C’: <i>tan</i>^<i>1</i> mosaic) <i>tan</i>^<i>1</i><i>FRT19A/GMR-hid FRT19A; ey-flp</i>. (D’: <i>ine</i>^{<i>MI05077</i>} mosaic) <i>ey-flp; ine</i>^{<i>MI05077</i>}<i>FRT40A /GMR-hid CL FRT40A</i>. (E’: <i>cart</i>^<i>1</i> mosaic mosaic) <i>ey-flp; cart</i>^<i>1</i><i>FRT40A /GMR-hid CL FRT40A</i>. (F) Expression of CarT in photoreceptor cells (<i>cart</i>^<i>1</i><i>; PninaE-cart</i>), but not in pigment cells (<i>cart</i>^<i>1</i><i>; PrdhB-cart</i>), restored the “on” and “off” transients in <i>cart</i>^<i>1</i> flies. (G) Quantification of phototaxis of flies with the indicated genotypes. Error bars represent SD. Significant differences between mutant and wt flies were determined using unpaired <i>t</i>-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).</p

CarT is localized to terminals of photoreceptor neurons.

<p>(A) CarT expression was enriched in the lamina and medulla neuropils by a mCherry-tagged transgene labeled with anti-mCherry. <i>Pcart-cart-mcherry</i> flies expressing mCherry-tagged CarT driven by the <i>cart</i> promoter were used. Cryosections of fly heads were labeled with anti-mCherry (red), anti-Chaoptin (24B10, expressed in photoreceptors) (blue) and anti-Ebony (green, expressed in lamina epithelial glia). La, lamina; Me, medulla; Re, retina. (B) Cross sections of the lamina of <i>Pcart-cart-mcherry</i> flies immunolabeled with anti-mCherry (red) and anti-Ebony (green) antibodies showed a complementary pattern. (C) Cross sections of the lamina of <i>Pcart-cart-mcherry</i> flies labeled with anti-mCherry (red) and anti-Tan (green) showed an overlapping pattern. (D) Longitudinal sections of the medulla of <i>Pcart-cart-mcherry</i> flies labeled with anti-mCherry (red) and anti-Tan (green) showed an overlap in the pattern of R7/R8 labeling.</p

Mutations in <i>cart</i> eliminate “on” and “off” transients in ERG recordings.

<p>(A) Schemes for <i>cart</i> deletion by sgRNA targeting. The organization of the <i>cart</i> locus and the expected structure of the deletion alleles (<i>cart</i>^<i>1</i> and <i>cart</i>^<i>2</i>) are shown. Boxes represent exons, and deep gray boxes represent the coding region. The sgRNA1 and sgRNA2 primer pair was used to generate the <i>cart</i>^<i>1</i> mutation. The sgRNA1 and sgRNA3 primer pair was used to generate the <i>cart</i>^<i>2</i> mutation. The positions of the DNA primers used for PCR (arrows) are indicated. (B) PCR products obtained from successful <i>cart</i> deletion mutants. The agarose gel electrophoresis of PCR products using the primers indicated in (A) (pF and pR) is shown and genomic DNA templates prepared from wt (<i>w</i>^<i>1118</i>), <i>cart</i>^<i>1</i> and <i>cart</i>^<i>2</i> flies. (C) ERG recordings from wt (<i>w</i>^<i>1118</i>), <i>cart</i>^<i>1</i>, <i>cart</i>^<i>2</i> and <i>cart</i>^<i>1</i>; <i>Pcart-cart</i> flies. Flies (~1 d after eclosion) were dark adapted for 1 min and subsequently exposed to a 5s pulse of orange light. (D) Phototaxis assays revealed a difference in behavior between wt and <i>cart</i> mutant flies. Error bars: SD; significant differences between mutant and wt flies were determined using unpaired <i>t</i>-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).</p

Histamine is reduced in <i>cart</i>^<i>1</i> mutant photoreceptor neurons.

<p>Histamine was immunolabeled in horizontal sections of heads from (A-B) wild type (wt: <i>w</i>^<i>1118</i>) and (C-D) <i>cart</i>^<i>1</i> mutant flies. (A) Strong signals in the lamina neuropile (La) and R7/R8 terminals in the distal medulla (Me) were detected in control <i>w</i>^<i>1118</i>. Additional immunolabel also appeared from cells in the lobula (Lo) (arrowhead). (B) The enlarged image of the wt head section in (A). Arrowheads in the lamina and medulla identified histamine positive photoreceptor terminals. (C) Loss of photoreceptor histamine signals and strong signals in lamina marginal glia were detected in <i>cart</i>^<i>1</i> mutant. (D) The enlarged image of the <i>cart</i>^<i>1</i> head section in (C) showing labeled marginal glia (arrow) but no photoreceptor signals. Scale bars: 50μm</p

Transverse frozen sections from a series taken through the right half of the thorax of <i>Philaenus</i> at the planes indicated in <b>Figure 1D</b> and viewed from their anterior surfaces.

<p>Dorsal is to the top and ventral to the bottom of each image. A. Superimposed images of the same sections taken with UV and with bright field illumination. Intense blue fluorescence occurs in the pleural arch (energy store) within the thorax. Weaker blue fluorescence is present in the exoskeleton. B. The same sections in which antibody labelling is superimposed on the bright field image. The immuno-signal is restricted to the fluorescent regions with a much weaker signal in parts of the exoskeleton.</p

Combined fluorescence and antibody staining of the pleural arches (energy stores) in transverse sections of the thorax of <i>Delphacodes</i>.

<p>Combined images of bright field and UV fluorescence are shown on the left and of UV fluorescence and antibody labelling on the right.</p

Preadsorption controls to show the specificity of the antibody.

<p>A. Transverse section of the thorax of <i>Philaenus</i> illuminated with bright field and UV light. Blue fluorescence is restricted to the pleural arch (energy store). The large muscles that power depression of the hind legs in jumping occupy most of the volume of the thorax. B. Incubation in the antibody after preadsorption with the antigen now fails to label the pleural arch. Only some weak immunolabelling is still present in the exoskeleton.</p