The β-alanine transporter BalaT is required for visual neurotransmission in Drosophila
Efficient clearance of neurotransmitters from the synaptic cleft is crucial for terminating a synaptic signal . These cleared neurotransmitters must then be recycled for sustained synaptic transmission ( i . e . , to maintain stable levels of transmitters ) ( Blakely and Edwards , 2012 ) . Neurons that utilize histamine as a neurotransmitter play important roles in a variety of biological processes , including cognition , sleep , and metabolism , and dysfunction of the histaminergic system in the brain has been linked to multiple neurological diseases ( Haas et al . , 2008; Panula and Nuutinen , 2013 ) . Histamine is particularly important in the Drosophila visual system ( Hardie , 1989 ) , and histamine recycling is the dominant pathway for maintaining adequate levels of histamine in adult photoreceptors ( Borycz et al . , 2002 ) . In this system , and in mammalian systems as well , the histamine recycling process involves both neurons and glial cells that surround the synapse ( Edwards and Meinertzhagen , 2010; Yoshikawa et al . , 2013 ) . In fact , a network of glia cells and pigment cells interconnected via gap junctions plays a key role in the long-distance recycling of histamine in the Drosophila visual system ( Chaturvedi et al . , 2014 ) . However , the mechanisms by which this cellular network recycles histamine and its metabolites are unknown . Once histamine is released by a photoreceptor into the optic lamina , it is quickly removed from the synaptic cleft by epithelia glial cells that surround the synapse . In these laminar glia , histamine is conjugated to β-alanine by an N-β-alanyl-dopamine synthase , called Ebony , to form the inactive metabolite , carcinine ( Borycz et al . , 2002; Richardt et al . , 2003 , 2002 ) . Recently , a carcinine transporter , CarT that is responsible for the uptake of carcinine from the synaptic cleft into photoreceptors has been identified ( Chaturvedi et al . , 2016; Stenesen et al . , 2015; Xu et al . , 2015 ) . The discovery that CarT retrieves carcinine directly from the laminar synaptic cleft indicates that carcinine is not transported from laminar glia to photoreceptor cell bodies through a long-distance trafficking mechanism involving the laminar glial and pigment cell network . Within photoreceptors , carcinine is hydrolyzed to generate histamine and β-alanine , a reaction catalyzed by an N-β-alanyl-dopamine hydrolase called Tan ( Borycz et al . , 2002; True et al . , 2005 ) . This regenerated histamine is then repackaged into synaptic vesicles for subsequent light-induced release , but it is unclear how β-alanine is returned to the laminar glial cells ( where it is needed to inactivate histamine ) . It is also unclear whether β-alanine recycling is required for visual transmission . The finding that high levels of β-alanine are detected in retinal pigment cells suggested that pigment cells may be critical for the transport and storage of β-alanine in this system ( Borycz et al . , 2012; Chaturvedi et al . , 2014 ) . To address these questions , we sought to determine whether there was a β-alanine transporter that was required for Drosophila vision transmission . Here , we identified BalaT , which is a plasma membrane transporter capable to transporting β-alanine into cells . Mutations in the balat gene disrupted photoreceptor synaptic transmission , phototaxis behaviors , and contents and retinal distribution of β-alanine . BalaT expression in retinal pigment cells completely rescued the defective visual transmission of balat mutants . Furthermore , the gap junction proteins Inx1 and Inx3 were required in retinal pigment cells for visual neurotransmission . We therefore provide evidence for a novel β-alanine recycling pathway , in which β-alanine that is released from photoreceptor cells is taken up and stored by retinal pigment cells , and then delivered to laminar glia via gap junctions , where histamine is inactivated by conjugation with β-alanine .
In Drosophila photoreceptors , carcinine is hydrolyzed to form histamine and β-alanine ( True et al . , 2005; Wagner et al . , 2007 ) . Histamine is then released as a neurotransmitter , taken up by laminar glial cells , and then conjugated with β-alanine to form carcinine . Although it is known that CarT transports carcinine into photoreceptors , it is unclear how β-alanine is delivered to laminar glia . To begin to understand this process , we sought to identify a β-alanine transporter in the fly visual system . We speculated that a transporter responsible for β-alanine uptake would likely be enriched in fly heads , so we examined previous RNA-seq data comparing mRNAs isolated from the head with mRNAs isolated from the body ( Xu and Wang , 2016 ) . Among ~600 putative transmembrane transporters encoded by the Drosophila genome , we identified 20 head-enriched genes ( Table 1 ) that represented potential β-alanine transporters ( Figure 1A ) . 10 . 7554/eLife . 29146 . 003Figure 1 . CG3790 is a retina-enriched β-alanine transporter . ( A ) Screening for a β-alanine transporter . HEK293T cells transiently expressing one of ~20 head-enriched potential transporters were exposed to [3H]-β-alanine , which was added to the ECF buffer ( final concentration 3 . 7 × 104 Bq ) . RFP ( red fluorescent protein ) was expressed as a negative control . ( B ) CG3790 transported β-alanine into S2 cells . Mouse GAT3 and RFP were used as positive and negative control , respectively . The results given are the mean values ± S . D . of four experiments . ( C–D ) CG3790 was expressed at high levels in the compound eye . ( C ) Relative RNA transcript levels ( RT-PCR experiments ) show that CG3790 expression is enriched in wild-type ( w1118 ) heads compared with wild-type bodies or GMR-hid heads . ( D ) Relative CG3790 transcript levels from wild-type ( wt ) bodies , wt heads , and GMR-hid heads ( gpdh served as an internal control ) . RNA levels were normalized to levels in wt bodies , which were set to 1 . Error bars indicate standard deviations ( SDs ) from three replicate experiments . Significant differences between candidates and control were determined using unpaired t-tests ( **p<0 . 01; ns , not significant ) . ( E ) S2 cells were transiently transfected with 3xFlag-tagged CG3790 , and then labeled with Flag antibody ( green ) and DAPI ( blue ) . Scale bar , 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00310 . 7554/eLife . 29146 . 004Figure 1—source data 1 . Relates to Figure 1A and B . [3H]-β-alanine uptake data for screening for β-alanine transporter in HEK 293 T cells and S2 cells ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00410 . 7554/eLife . 29146 . 005Figure 1—source data 2 . Relates to Figure 1D . Relative CG3790 transcript levels from wild-type ( wt ) bodies , wt heads , and GMR-hid heads . RNA levels were normalized to levels in wt bodies , which were set to 1 ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00510 . 7554/eLife . 29146 . 006Figure 1—figure supplement 1 . CG3790 is a SLC22 family protein . Alignment of the Drosophila CG3790 amino acid sequence with mouse OCT3 ( mOCT3 ) and human OCT3 ( hOCT3 ) . Identical residues , found in at least two proteins , are enclosed in green boxes . CG3790 is 29% identical to mOCT3% and 30% identical to hOCT3 . The transmembrane domains are indicated by solid lines above the sequences . The running tally of amino acids is indicated to the right . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00610 . 7554/eLife . 29146 . 007Figure 1—figure supplement 2 . CG3790 is unable to transport histamine and GABA . ( A ) CG3790 did not transport GABA into S2 cells . Mouse GAT3 was used as a positive control , and RFP was used as a negative control . ( B ) CG3790 did not exhibit histamine transporting activity in S2 cells . Human OCT2 and RFP were used as positive and negative control , respectively . [3H]-GABA or [3H]-histamine was added to the ECF buffer ( final concentration 3 . 7 × 104 Bq ) . The results given are the mean values ± S . D . of three experiments . Significant differences were determined using unpaired t-tests ( ***p<0 . 001; ns , not significant ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00710 . 7554/eLife . 29146 . 008Figure 1—figure supplement 2—source data 1 . Relates to Figure 1—figure supplement 2A and B . [3H]-histamine uptake data in S2 cells transfected mock and RFP negative control or mGAT3 . [3H]-GABA uptake data in S2 cells transfected mock and RFP negative control or hOCT2 ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00810 . 7554/eLife . 29146 . 009Figure 1—figure supplement 3 . GMR-hid abolished expression of ninaE and pdh . ( A–B ) Wild-type heads contain more transcripts for ( A ) ninaE and ( B ) pdh than wild-type bodies and GMR-hid heads ( gpdh served as an internal control , and mRNA levels were normalized to wild-type body ) . Data is processed by logarithmic 2 and averaged from three replicate experiments . Error bars indicate SDs . Significant differences between candidates and control were determined using unpaired t-tests ( ***p<0 . 001 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 00910 . 7554/eLife . 29146 . 010Figure 1—figure supplement 3—source data 1 . Relates to Figure 1—figure supplement 3A and B . Relative ninaE and pdh transcript levels from wild-type ( wt ) bodies , wt heads , and GMR-hid heads . RNA levels were normalized to levels in wt bodies . Data is processed by logarithmic 2 ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01010 . 7554/eLife . 29146 . 011Table 1 . Description of 20 head-enriched genes . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 011CG numberNameDescription CG3790Organic anion transmembrane transporter activity CG18660Nckx30CSodium/potassium/calcium exchanger CG2893zydecoPotassium-dependent sodium/calcium exchanger CG7342Organic anion transmembrane transporter activity CG13610Orct2Organic cation transporter CG6331OrctOrganic anion transmembrane transporter activity CG6126Organic anion transmembrane transporter activity CG4360Nucleic acid binding CG3159Eaat2L-aspartate transmembrane transporter activity CG6723Sodium/solute symporter CG11010Ent3Nucleoside transmembrane transporter activity CG13743Amino acid transporter CG10804Neurotransmitter transporter activity CG4545SerTMonoamine transmembrane transporter activity CG5281EamA domain CG43066Neurotransmitter transporter activity CG7442SLC22ACholine transmembrane transporter activity CG7708Sodium/solute symporter CG13248Amino acid/polyamine transporter I CG42322Solute carrier family 35 member F3/F4 To determine if any of these candidate transporters could transport β-alanine , we used HEK293T cells to perform uptake assays . Each candidate was transiently expressed in HEK293T cell , and their ability to uptake [3H]-β-alanine was assessed . Only one candidate transporters , CG3790 , exhibited β-alanine uptake activity ( Figure 1A ) . The β-alanine content of CG3790-transfected cells was approximately 119 Bq/mg , which was 2 . 7-fold greater than measured for mock- or RFP-transfected controls ( 55 and 53 Bq/mg , respectively ) . CG3790 shares significant amino acid identity with the mammalian solute carrier family 22 ( SLC22 ) , which includes both mouse and human OCT3 ( Figure 1—figure supplement 1 ) . The β-alanine transporting activity of CG3790 was further confirmed in S2 cells ( Figure 1B ) . Mouse GAT3 ( GABA transporter type 3 ) , which is known to efficiently take up β-alanine , exhibited levels of β-alanine transporting activity that were similar to CG3790 , suggesting that CG3790 is a bona fide β-alanine transporter ( Christiansen et al . , 2007 ) ( Figure 1B ) . We next sought to determine whether CG3790 is a specific β-alanine transporter in Drosophila . Because a histamine transporter has not yet been identified , we first asked whether CG3790 is able to transport histamine . Histamine uptake assays revealed that CG3790 does not exhibit histamine uptake activity . As a control , the human Organic Cation Transporter ( OCT2 ) , which is known to take up histamine , exhibited high levels of histamine transport activity when expressed in S2 cells ( Figure 1—figure supplement 2A ) . Since mGAT3 is also a GABA transporter , we asked whether CG3790 is able to transport GABA . While mGAT3 efficiently transported GABA , CG3790 did not exhibit GABA transporting activity when expressed in S2 cells ( Figure 1—figure supplement 2B ) . Moreover , when we expressed Flag-tagged CG3790 in S2 cells the Flag signal localized exclusively to the plasma membrane ( Figure 1E ) . These data suggest that CG3790 encodes a plasma membrane β-alanine transporter . To further confirm that the CG3790 transporter functions in the fly head , we performed quantitative PCR comparing mRNAs isolated from wild-type heads , wild-type bodies , or heads from GMR-hid flies , which are devoid of eyes . As is seen with other eye-specific genes including ninaE ( neither inactivation nor afterpotential E ) , which is expressed exclusively in photoreceptor cells ( O'Tousa et al . , 1985 ) , and pdh ( pigment-cell-enriched dehydrogenase ) , which is expressed exclusively in retinal pigment cells ) ( Wang et al . , 2010 ) , CG3790 mRNA transcripts were absent from fly bodies ( Figure 1C–D and Figure 1—figure supplement 3A–B ) . Ectopic expression of the pre-apoptotic gene hid via the glass multiple reporter ( GMR ) promoter results in eye ablation ( Grether et al . , 1995; Hay et al . , 1994 ) . As such , ninaE and pdh expression is greatly reduced in the heads of GMR-hid flies . Similarly , CG3790 transcript levels were also greatly reduced in the heads of GMR-hid flies , suggesting that CG3790 is primarily expressed in the fly compound eye ( Figure 1C–D and Figure 1—figure supplement 2A–B ) . These findings indicate that CG3790 encodes a retina-enriched plasma membrane β-alanine transporter . We therefore named this gene balat ( beta-alanine transporter ) . To study the function of BalaT in visual perception , we generated two null mutations in the balat gene by deleting ~900 bp and ~660 bp genomic fragments using the CRISPR-associated single-guide RNA system ( Cas9 ) ( Figure 2A ) . PCR amplification of the balat locus from genomic DNA isolated from wild-type , balat1 , and balat2 flies revealed a truncated balat locus in the mutant samples . Thus , the balat locus is disrupted in balat1 and balat2 flies ( Figure 2B ) . 10 . 7554/eLife . 29146 . 012Figure 2 . Mutations in balat disrupt photoreceptor cell synaptic transmission and visual behavior . ( A ) Schematic for balat knock-out by sgRNA targeting . The organization of the balat locus and the expected structures of balta1 and balat2 alleles are shown . Boxes represent exons with the coding region between ATG and TAG . The sgRNA1 and sgRNA2 primer pair was used to generate the balat1 allele; the sgRNA2 and sgRNA3 primer pair was used to generate the balat2 allele . Arrows indicate the primers used for genomic PCR . ( B ) PCR products obtained from balat1 and balat2 mutants show successful gene deletions . ( C ) ERG recordings from: wild-type ( w1118 ) , balat1 , balat2 , balat1;GMR-balaT , balat1;pdh-balaT , balat2;ninaE-balaT and w+; balat1 flies . Young flies ( <3 days after eclosion ) were dark adapted for 1 min and subsequently exposed to a 5 s pulse of orange light . ON and OFF transients are indicated by arrows . All flies contained the w1118 mutation except for the w+; balat1 flies . ( D ) Quantitative analysis of the amplitudes of ERG OFF transients shown in C . ( E ) Phototaxis behaviors of wt , balat1 balat1;GMR-balaT , balat1;pdh-balaT and balat2;ninaE-balaT flies . Significant differences between mutant and wild-type flies were determined using unpaired t-tests ( ***p<0 . 001; ns , not significant ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01210 . 7554/eLife . 29146 . 013Figure 2—source data 1 . Relates to Figure 2D and E . Quantitative analysis of the amplitudes of ERG OFF transients about wild-type ( w1118 ) , balat1 , balat2 , balat1;GMR-balaT , balat1;pdh-balaT , balat2;ninaE-balaT and w+; balat1 flies . Quantitative of phototaxis behaviors of wt , balat1 balat1;GMR-balaT , balat1;pdh-balaT and balat2;ninaE-balaT flies ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01310 . 7554/eLife . 29146 . 014Figure 2—figure supplement 1 . Quantification of the ERG ON transients . Percentage of flies of indicated genotypes that have ON transients were quantified . The ERG profiles from at least 7 flies of each genotype were scored . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 014 These balat mutations did not cause lethality or other visible phenotypes , so we performed electroretinogram ( ERG ) recordings to determine whether BalaT functions in phototransduction . Exposing wild-type flies to light results in two primary components in the ERG recording , including a sustained corneal negative response resulting from photoreceptor depolarization , and ‘on’ and ‘off’ transients resulting from postsynaptic neuronal activity in the optic lamina ( downstream of the photoreceptors ) ( Wang and Montell , 2007 ) ( Figure 2C ) . Mutations in genes that disrupt histamine recycling exhibit reduced ‘on’ and ‘off’ transients , reflecting defective synaptic transmission of photoreceptors ( Xu et al . , 2015 ) . Similarly , both balat1 and balat2 mutant flies lacked ERG ‘on’ and ‘off’ transients , indicating that BalaT is required for visual synaptic transmission ( Figure 2C and D , and Figure 2—figure supplement 1 ) . To confirm that the loss of ERG transients resulted from mutation of the balat locus , we generated a GMR-balat transgenic fly , which expresses BalaT in the compound eyes under control of the GMR promoter . The GMR-balat transgene completely rescued the ‘on’ and ‘off’ transients when crossed into the balat1 mutant flies ( Figure 2C ) . Mutations in the white gene , which encodes an ATP-binding cassette ( ABC ) transporter , has been reported to affect histamine levels ( Borycz et al . , 2008 ) . To make sure that the white mutation was not affecting ERG phenotypes exhibited by balat mutants , we crossed the balat mutation into a wild-type white ( w+ ) background and found that ‘on’ and ‘off’ transients were still absent in this w+; balat1 fly ( Figure 2C and D ) . Disrupting visual transmission results in blindness , which is reflected in the loss of phototaxis behavior ( Behnia and Desplan , 2015 ) . We next used this behavioral assay to assess the ability of balat mutant flies to see . Consistent with the loss of ‘on’ and ‘off’ transients , balat mutant flies exhibited impaired phototaxis , which was fully restored by the GMR-balat transgene ( Figure 2E ) . These results strongly support the conclusion that BalaT is involved in visual transmission and that the loss of BalaT severely disrupts vision . It is not currently known how β-alanine is transported out of the photoreceptor cells once generated ( along with histamine ) by the Tan hydrolase . Having established that BalaT functions as a β-alanine transporter in the retina , we next sought to determine which cells express balat , with the goal of determining the site of β-alanine transport during phototransduction . As we were unable to generate a high affinity antibody against BalaT , we used CRISPR/Cas9-based genome editing to introduce mCherry into the balat locus , downstream of the native balat promoter ( balat-mcherry ) ( Figure 3—figure supplement 1A ) . We identified balat-mcherry flies though PCR and RFP immunofluorescence ( Figure 3—figure supplement 1B–C ) . Importantly , homozygous balat-mcherry flies displayed the loss of ‘on’ and ‘off’ transients , as expected , indicating the insertion of mcherry into the balat locus ( Figure 3—figure supplement 1D ) . Photoreceptor cells and retinal pigment cells are the two major cell types in the compound eye . Staining with phalloidin and PDH labels the rhabdomere regions of photoreceptor cells and the retinal pigment cells , respectively . The mCherry signals in the balat-mcherry retina were predominantly detected in retinal pigment cells , as it overlapped with the PDH signal ( Figure 3A and B ) . No mCherry signaling was detected in wild-type flies ( Figure 3C and D ) . 10 . 7554/eLife . 29146 . 015Figure 3 . BalaT localizes to pigment cells . ( A–B ) Compound eyes from balat-mcherry flies that express mCherry ( red ) driven by the endogenous balat promoter were labeled for PDH ( green ) , mCherry ( red ) , and phalloidin ( blue ) . High-magnification images are shown in ( B ) . ( C–D ) mCherry signals were not detected in wild-type ( wt ) retinas . Scale bars represent 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01510 . 7554/eLife . 29146 . 016Figure 3—figure supplement 1 . Generation of balat-mcherry flies . ( A ) Schematics for generating balat-mcherry flies . The mcherry fluorescent protein , driven by an eye-specific 3XP3 promoter , was inserted into the balat genomic locus using CRISPR/Cas9-mediated homologous recombination . The 3XP3 promoter region was subsequently removed by Cre recombinase . PCR primers ( arrows , pF and pR ) were used to verify the balat-mcherry knock-in flies . ( B ) Images show flies with the 3XP3-mcherry reporter inserted into the balat locus ( balat-3XP3-mcherry ) , and the balat-mcherry knock-in flies after Cre/LoxP-mediated recombination . Scale bar represents 150 µm . ( C ) Genomic PCR products from wild-type ( w1118 ) , attP2 , balat-3XP3-mcherry , and balat-mcherry flies show successful gene targeting . ( D ) ERG recordings from wild-type ( w1118 ) , attP2 , balat-3XP3-mcherry , and balat-mcherry flies . The mcherry insertion caused the loss of ‘on’ and ‘off’ transients . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 016 Since BalaT localized to retinal pigment cells , we next asked whether BalaT is required in pigment cells by performing tissue-specific rescue experiments . We expressed BalaT specifically in photoreceptor cells using the ninaE promoter ( ninaE-balat ) or in retinal pigment cells using the pdh promoter ( pdh-balat ) . The pdh-balat transgene fully restored ‘on’ and ‘off’ transients and phototaxic behaviors when crossed into balat1 mutant flies , whereas expressing BalaT in photoreceptor cells failed to rescue these phenotypes ( Figure 2C–E , and Figure 2—figure supplement 1 ) . These results indicate that BalaT functions in pigment cells , and that pigment cells may play an important role in transporting β-alanine from photoreceptors to epithelia glial cells in the optic lamina . If β-alanine is transported into pigment cell by BalaT , it must then be transported from the pigment cells to the laminar glial cells , where it can be conjugated with histamine to form carcinine . It has recently been reported that a multicellular network involving laminar glia and pigment cells mediates long-distance recycling of histamine metabolites ( Chaturvedi et al . , 2014 ) . Since this transport relies on the gap junction protein Innexin 2 ( Inx2 ) in glia , it is possible that gap junctions between pigment cells and laminar glial cells are important for β-alanine recycling , and thus for maintaining visual synaptic transmission . We first asked whether disruption of Inx2 in the retina affects fly visual transmission . Knocking down Inx2 in the retina ( inx2RNAi: GMR-gal4/UAS-inx2RNAi ) did not affect ERG transients ( Figure 4A and C ) . However , there are seven Innexins , and Inx1 , Inx2 , and Inx3 are all expressed in the adult head . Knockdown of Inx1 or Inx3 in the compound eye also did not disrupt the ERG ‘on’ and ‘off’ transients ( Figure 4B and D ) . Because these Innexins may have redundant functions , we knocked down combinations of two retinal Innexins and measured the effect on fly visual transmission . Knockdown of Inx1/Inx2 or Inx2/Inx3 did not affect ERG transients , but ‘on’ and ‘off’ transients were largely reduce by Inx1/Inx3 double knockdown ( Figure 4E–4G ) . These results indicated that the gap junction proteins Inx1 and Inx3 are required in compound eyes for visual transmission . 10 . 7554/eLife . 29146 . 017Figure 4 . Specific knockdown of Inx1 and Inx3 in compound eyes blocks visual neurotransduction . ERG recordings of ( A ) control ( GMR-gal4 ) , ( B ) inx1RNAi ( GMR-gal4/ UAS-inx1RNAi ) , ( C ) inx2RNAi ( GMR-gal4/UAS-inx2RNAi ) , ( D ) inx3RNAi ( GMR-gal4/ UAS-inx3RNAi ) , ( E ) inx1RNAi + inx2RNAi ( GMR-gal4/UAS-inx1RNAi UAS-inx1RNAi ) , ( F ) inx2RNAi + inx3RNAi ( GMR-gal4/UAS-inx2RNAi UAS-inx3RNAi ) , and ( G ) inx1RNAi + inx3RNAi ( GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi ) are shown . Arrows indicate ON and OFF transients in ( A ) . Flies ( ~1 day after eclosion ) were dark adapted for 1 min and subsequently exposed to a 5 s pulse of orange light . ( H–I ) β-alanine was immunolabeled in horizontal sections of heads from ( H ) control: CG2198RNAi ( GMR-gal4/UAS- CG2198RNAi ) and ( I ) inx1RNAi + inx3RNAi ( GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi ) flies . Re , retina; La , lamina; Me , medulla; Lo , lobula . ( J ) Fluorescence intensity ratios of β-alanine signals between retina and lobula . Quantifications of all genotypes are averages of six replicate experiments . Significant differences between inx1RNAi + inx3RNAi and controls ( CG2198RNAi and CG9962RNAi ) were determined using unpaired t-tests ( ***p<0 . 001; ns: not significant ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01710 . 7554/eLife . 29146 . 018Figure 4—source data 1 . Relates to Figure 4J . Quantitative of fluorescence intensity ratios of β-alanine signals between retina and lobula of inx1RNAi + inx3RNAi and controls ( CG2198RNAi and CG9962RNAi ) ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01810 . 7554/eLife . 29146 . 019Figure 4—figure supplement 1 . Accumulation of β-alanine in heads of Inx1 and Inx3 double RNAi flies . Levels of β-alanine in heads of control and inx1RNAi + inx3RNAi ( GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi ) flies . Error bars indicate SDs , and significant differences between mutant and wt flies were determined using unpaired t-tests ( ***p<0 . 001 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 01910 . 7554/eLife . 29146 . 020Figure 4—figure supplement 1—source data 1 . Relates to Figure 4—figure supplement 1 . Quantitative of levels of β-alanine in heads of control and inx1RNAi + inx3RNAi ( GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi ) flies ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 020 We next examined the in vivo levels of β-alanine , histamine , and carcinine in the heads of balat mutant flies via liquid chromatography-mass spectrometry ( LC-MS ) . For cart1 mutant flies , which cannot transport carcinine into photoreceptor cells , levels of histamine and β-alanine were significantly reduced ( Figure 5A and B ) , as has been reported ( Xu et al . , 2015 ) . Similarly , levels of β-alanine and histamine were significantly reduced in balat1 mutant flies ( Figure 5A and B ) . Reduction of histamine in balat1 mutant flies indicates defective histamine recycling and explains the disruption of photoreceptor synaptic transmission in balat1 mutants . Reductions in β-alanine may result from inadequate β-alanine storage and/or transport into pigment cells ( Borycz et al . , 2012 ) . These results therefore support the hypothesis that BalaT transports β-alanine into retinal pigment cells . 10 . 7554/eLife . 29146 . 021Figure 5 . Loss of BalaT affects the level and distribution of β-alanine in vivo . ( A–B ) Levels of ( A ) β-alanine and ( B ) histamine in heads of wild-type ( wt , w1118 ) , cart1 , and balat1 flies are shown . Error bars indicate SDs , and significant differences between mutant and wt flies were determined using unpaired t-tests ( *p<0 . 05; **p<0 . 01 ) . ( C–E ) β-alanine was immunolabeled in horizontal sections of heads from ( C ) wild-type ( w1118 ) , ( D ) balat1 , and ( E ) balat1;GMR-balat flies . Re , retina; La , lamina; Me , medulla; Lo , lobula . ( F ) Fluorescence intensity ratios of β-alanine labeling between retina and lobula . Significant differences between mutant and wild-type flies were determined using unpaired t-tests ( **p<0 . 01; ns: not significant ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 02110 . 7554/eLife . 29146 . 022Figure 5—source data 1 . Relates to Figure 5A and B . Quantitative of levels of β-alanine and histamine in heads of wild-type , cart1 , and balat1 flies ( . xlsx file ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 02210 . 7554/eLife . 29146 . 023Figure 5—figure supplement 1 . Distribution of β-alanine was unaffected in pyd1 and balck double mutant flies . β-alanine was immunolabeled in horizontal sections of heads from ( A ) wild type ( w1118 ) and ( B ) pyd1;b1 flies . Re , retina; La , lamina; Me , medulla; Lo , lobula . ( C ) Fluorescence intensity ratios of β-alanine signals between retina and lobula . Quantifications of both genotypes are averages of six replicate experiments . Significant difference between wild type and pyd1;b1 was determined using unpaired t-tests ( ns: not significant ) . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 023 If flies are not able to transport β-alanine into pigment cells for storage , β-alanine should be specifically decreased in the retina . To test this idea , we sectioned fly heads and labeled them with an antibody against β-alanine . In wild-type flies , levels of β-alanine were highest in the retina , consistent with previous analyses ( Borycz et al . , 2012; Chaturvedi et al . , 2014 ) ( Figure 5C and F ) . In contrast , levels of β-alanine were lower in balat1 sections ( for comparison , retinal signals were normalized to the β-alanine signal in the lobula ) ( Figure 5D and F ) . GMR-balat restored retinal β-alanine levels in balat1 mutants ( Figure 5E and F ) , and rescued photoreceptor synaptic transmission . Reduced levels of β-alanine in retina lacking BalaT support the hypothesis that BalaT transports β-alanine into retinal pigment cells . We next checked if reductions in β-alanine change its distribution . In pyd1 and black double mutants ( pyd1;b1 ) , two independent metabolic pathways for de novo β-alanine biosynthesis are blocked and thus β-alanine levels are largely reduced ( Borycz et al . , 2012 ) . However , in pyd1;b1 flies the pattern of β-alanine distribution is the same as seen in wild type , although immune intensity is reduced , as is the case with balat1 mutant flies ( Figure 5—figure supplement 1 ) . Therefore , pigment cells may serve as a reservoir of storedβ-alanine through a BalaT-dependent mechanism . Since Inx1/Inx3 double knockdown flies showed significant reductions in ‘on’ and ‘off’ transients , we asked whether Inx1/Inx3 is required for β-alanine trafficking . Of importance , β-alanine primarily accumulated in the retinal layer of Inx1/Inx3 double knock-down flies compared with control flies with either CG2198 or CG9962 RNAi ( Figure 4H–J ) . Meanwhile in vivo levels of β-alanine in heads of Inx1/Inx3 double knockdown flies are significantly higher than in control flies ( Figure 4—figure supplement 1 ) . These results further support our hypothesis that Inx1/Inx3 functions to transport β-alanine back into glia cell . Taken together , we have identified a previously uncharacterized β-alanine transporter , which we have named BalaT , and shown that BalaT is responsible for β-alanine transportation and storage in pigment cells .
Histamine is the primary neurotransmitter released by photoreceptors in flies , and the synthesis of histamine can occur de novo by photoreceptor cell-specific histidine decarboxylase ( Burg et al . , 1993; Hardie , 1987 ) . However , during light stimulation , flies depend primarily on histamine recycling to sustain tonic visual transmission . Histamine is inactivated when Ebony catalyzes its conjugation to β-alanine in laminar glial cells ( Borycz et al . , 2002; Richardt et al . , 2002 ) . In photoreceptors , CarT and Tan are involved in transporting carcinine and regenerating histamine , respectively ( Chaturvedi et al . , 2016; Stenesen et al . , 2015; Wagner et al . , 2007; Xu et al . , 2015 ) . Histamine is then loaded into synaptic vesicles , but fate of the β-alanine generated by Tan remains unknown . Here , we identify a new β-alanine transporter , BalaT , and provide evidence that BalaT is required for visual transmission . BalaT is a plasma membrane protein , and is sufficient to facilitate β-alanine uptake in cultured cells . BalaT is expressed predominantly by retinal pigment cells , and the specific expression of BalaT in these cells rescues photoreceptor synaptic transmission in balat1 mutant flies . The reduced levels and altered distribution of β-alanine in the heads of balat1 mutants support the hypothesis that BalaT functions to transport β-alanine . Therefore , we suggest that a novel β-alanine transporter , BalaT , functions in retinal pigment cells to help recycle β-alanine . Disrupting the synthesis of histamine and carcinine abolishes photoreceptor synaptic transmission ( Borycz et al . , 2002; Burg et al . , 1993 ) . The transport of β-alanine seems less important , as it is present at higher concentrations in Drosophila . Two common metabolic pathways have been shown to synthesize β-alanine: ( 1 ) aspartate decarboxylase , encoded by the black gene ( Hodgetts , 1972; Phillips et al . , 2005 ) , and ( 2 ) the uracil degradation pathway , which includes the enzymes dihydropyrimidine dehydrogenase ( Pyd1 ) , dihydropyrimidine amidohydrolase ( Pyd2 ) , and β-ureidopropionase ( Pyd3 ) ( Piskur et al . , 2007; Rawls , 2006 ) . The Black protein is enriched in lamina glia , together with the protein Ebony , and functions as the major enzyme in generating β-alanine ( Ziegler et al . , 2013 ) . Although levels of β-alanine are reduced in the heads of black mutants ( or pyd1 and black double mutants ) compared to wild type , these flies still exhibit ERG transients ( Borycz et al . , 2012; Ziegler et al . , 2013 ) . Therefore , an efficient β-alanine recycling systems has been proposed to compensate for the rapid loss of β-alanine that occurs in the conversion of histamine to carcinine ( Borycz et al . , 2012 ) . Here we show that mutations in balat disrupted transport and storage of β-alanine in the retina , resulting in dramatically reduced photoreceptor synaptic activity and visual behavior . Thus , we propose that Balat plays a key role in shuttling β-alanine from photoreceptor cells to glial cells . A similar recycling system has been demonstrated for histamine . Mutations in Hdc ( histidine decarboxylase ) result in the loss of visual transduction . Feeding these flies histamine fully restores the visual transduction , whereas supplying histamine fails to rescue the ERG phenotypes of ebony mutants or Hdc;ebony double mutant flies ( Melzig et al . , 1996 , 1998; Ziegler et al . , 2013 ) . In pyd1;black double mutants , β--alanine levels are still higher than histamine , which may be because of the uptake of exogenous β-alanine ( Borycz et al . , 2012 ) . Meanwhile , the β-alanine recycling pathway is able to sustain visual synaptic transmission in black mutant flies , even in the absence of in situ β-alanine generation . Therefore , β-alanine recycling is essential for maintaining histamine homeostasis and photoreceptor synaptic transmission . In addition , establishing and maintaining a β-alanine pool play key roles in sustaining visual synaptic transmission . The majority of pigment granules , which reduce the intensity of light exposure , are formed in retinal pigment cells of the Drosophila compound eye . Recently , it has been demonstrated that retinal pigment cells are functionally equivalent to cells within the mammalian retinal pigment epithelium ( RPE ) , in terms of chromophore cycling ( Travis et al . , 2007; Wang et al . , 2010 , 2012 ) . Just like chromophore ( a subunit of rhodopsin ) is sent back to pigment cells for regeneration , it has been suggested that the metabolism of β-alanine involves pigment cells ( Borycz et al . , 2012 ) . Several observations indicate that BalaT functions in retina pigment cells to help regenerate histamine . Most notably , BalaT is expressed in pigment cells and visual transmission is rescued in balat mutants by the expression of wild-type BalaT in pigment cells , but not photoreceptor neurons . Our work provides strong genetic evidence for the involvement of retinal pigment cells in histamine recycling , and suggests that BalaT is the key player responsible for the transport of β-alanine into pigment cell . It has been suggested that the transportation of histamine metabolites within the Drosophila visual system is mediated by a network of retinal pigment cells and laminar glia that are interconnected by gap junctions , and that this transport network is essential for fly vision ( Chaturvedi et al . , 2014 ) . However , histaminergic photoreceptor cells directly acquire carcinine from synaptic terminals within the optic lamina through a CarT-dependent trafficking pathway ( Xu et al . , 2015 ) . Here we provided evidence that the intercellular gap junctions between retinal pigment cells and laminar glial cells are involved in trafficking β-alanine , another histamine metabolite . Although the Inx2 gap junction protein is required in laminar glial cells for visual transmission , Inx2 was not required in retinal pigment cells . In contrast , downregulating the gap junction proteins Inx1 and Inx3 within the compound eye greatly impaired photoreceptor synaptic transmission . These data indicate that the Inx1 and Inx3 proteins are required in retinal pigment cells for maintaining fly vision . It has also been suggested that gap junctions may play a role in the formation of synapses between the retina and lamina in the Drosophila visual system ( Curtin et al . , 2002 ) . Therefore , an alternative explanation would be that Inx1 and Inx3 are required for the development of synapses between retinal and laminar neurons . Although electron microscopy studies in Drosophila have not revealed gap junctions between retinal pigment cells and laminar fenestrated glia , accumulation of β-alanine in the retinal layer of inx1/inx3 double mutant flies indicates defective β-alanine trafficking . BalaT belongs to the SLC22 family of transporters , which mediate sodium-independent transport of neurotransmitters , amino acids , and energy metabolites ( Koepsell , 2013 ) . We provide evidence that BalaT functions specifically in pigment cells as a β-alanine transporter , as BalaT mediated β-alanine transport in vitro , and is expressed predominately in retinal pigment cells . The reduced levels and altered distribution of β-alanine in the heads of balat null mutant flies supports this hypothesis . β-alanine is one of the two constituents of the naturally occurring dipeptides , carnosine and carcinine , and is considered rate-limiting for their synthesis . Both carnosine and carcinine are found in the retina where they exert protective effects . This is because they function as antioxidants , scavenging toxic activated oxygen species ( Marchette et al . , 2012; Pfister et al . , 2011 ) . There is evidence that several members of the SLC family of proteins that can transportβ-alanine are present in the retina , including taurine transporter ( Slc6a6/TauT ) ( Liu et al . , 1993 ) . This suggests a conserved function for β-alanine trafficking in mammals and Drosophila . Our current work , together with previous reports , provides evidence for the formulation of a more complete histamine recycling pathway , which is critical for sustaining photoreceptor synaptic transmission ( Figure 6 ) . In this pathway , carcinine is synthesized in epithelial glial cells within the optic lamina , and is transported back to photoreceptor cells via the synaptic cleft . Once in the photoreceptor cells it is hydrolyzed into histamine and β-alanine by Tan , and histamine is used as neurotransmitter . Meanwhile , β-alanine is released via an unknown mechanism , and is subsequently delivered to and stored within pigment cells by BalaT . Finally , β-alanine is transported back to epithelial glia through the network of retinal pigment cells and laminar glia for histamine inactivation and carcinine synthesis . 10 . 7554/eLife . 29146 . 024Figure 6 . A model of the histamine recycling pathway . Histamine is initially synthesized by histidine decarboxylase ( Hdc ) in photoreceptor cells ( PR ) . Upon light stimulation , PRs release histamine into the synaptic cleft . Released histamine is quickly taken up by an unknown histamine transporter into epithelial glial cells that express Ebony . In these glia , histamine is conjugated to β-alanine , which inactivates histamine and generates carcinine . Carcinine is released into the synaptic cleft and subsequently internalized , via CarT , by the PRs . After carcinine is hydrolyzed to histamine and β-alanine by Tan hydrolase in the PR , histamine is re-packaged into synaptic vesicles , whereas β-alanine is delivered to and subsequently internalized , via the BalaT transporter , by retinal pigment cells ( PCs ) . PCs can store β-alanine or deliver β-alanine to the laminar glia cells through a gap junction network involving Inx1 and Inx3 , which are expressed in PCs . In glial cells , β-alanine is conjugated to histamine , and the cycle repeats . Moreover , β-alanine can be synthesized by Black , which is an aspartate decarboxylase that is expressed together with Ebony in optic lobe glia . DOI: http://dx . doi . org/10 . 7554/eLife . 29146 . 024
The pyd1; b1 ( su ( r ) rC; b1 ) , UAS-inx1RNAi , UAS-inx2RNAi , and UAS-inx3RNAi flies were provided by the Bloomington Drosophila Stock Center . The nos-Cas9 flies were obtained from Dr . J . Ni at Tsinghua University , Beijing , China . The cart1 mutant flies were maintained in the lab of Dr . T . Wang ( Xu et al . , 2015 ) . The balat1 and balat2 mutations were generated using the Cas9/sgRNA system ( Xu et al . , 2015 ) . Briefly , two pairs of guide RNAs targeting the balat locus were designed ( sgRNA1: ATATAGTGCGCTATCTTGAG , sgRNA2: GTGTCTACGTGGGACTGAGT , sgRNA3: GAGGCCGGAACACCGGTTTT ) and cloned into the U6b-sgRNA-short vector . The sgRNA1 and sgRNA2 plasmids were used to generate the balat1 mutant flies , whereas sgRNA2 and sgRNA3 were used to generate the balat2 mutant flies . The plasmids were injected into the embryos of nos-Cas9 flies , and deletions were identified by PCR using the following primers: forward primer 5’-CGCCACCAGTTCCTGGAC-3’ and reverse primer 5’-CCAGATGTAAGAGACGCAGTG-3’ . The balat-mcherry knock-in flies were generated as shown in Figure S3 . Briefly , sgDNA sequence was designed and cloned into the U6b-sgRNA-short vector . Two fragments from the balat locus ( −1327 to −38 and +380 to+1660 , where + 1 represents the transcription start site ) were subcloned into a pDM19-mcherry vector such that they were separated by the 3XP3-mcherry marker . The two plasmids were co-injected into the embryos of nos-Cas9 flies , and mCherry-positive progeny were identified . By crossing these flies with hs-Cre flies , the 3XP3 promoter region was deleted . The balat-mcherry flies were finally confirmed by PCR using the following primers: forward primer 5’-AATTAATTAATGCGCACGAGAGGCC-3’ and reverse primer 5’-TTCCACGATGGTGTAGTCCTCGTTG-3’ . 20 head-enriched potential transporters and the balat cDNA sequences were amplified from cDNA clones obtained from DGRC ( Drosophila Genomics Resource Center , Bloomington , IN , USA ) . The mouse GAT3 cDNA sequences were amplified from mouse liver cDNA obtained from mRNA . Their entire CDS sequences were subcloned into the pCDNA3 vector ( Invitrogen , Carlsbad , USA ) for expression in HEK293T cells and PIB vector ( Invitrogen , Carlsbad , USA ) for expression in S2 cells . To construct PninaE-balat , Ppdh-balat , and PGMR-balat , the entire coding region of balat was amplified from cDNA clones and subcloned into the pninaE-attB , ppdh-attB and pGMR-attB vectors ( Xu et al . , 2015 ) . These constructs were injected into M ( vas-int . Dm ) ZH-2A;M ( 3xP3-RFP . attP ) ZH-86Fb embryos , and transformants were identified on the basis of eye color . The 3xP3-RFP . attP locus was removed by crossing with P ( Crey ) flies . HEK 293 T cells was obtained from ATCC ( RRID:CVCL_0063 ) and S2 cells was obtained from Drosophila Genomics Resource Center ( RRID:CVCL_Z232 ) , which have been tested for contamination of bacteria , yeast , mycoplasma and virus and has been characterized by isozyme and karyotype analysis . ERGs were recorded as described ( Xu et al . , 2015 ) . Briefly , two glass microelectrodes were filled with Ringer’s solution and placed on the surfaces of the compound eye and thorax ( one each surface ) . The source light intensity was ~2000 lux , and the light color was orange ( source light was filtered using a FSR-OG550 filter ) . ERG signals were amplified with a Warner electrometer IE-210 , and recorded with a MacLab/4 s A/D converter and the clampelx 10 . 2 program ( Warner Instruments , Hamden , USA ) . Alanine , β-[3-3H ( N ) ] ( 30–60 Ci/mM , American radiolabeled chemicals , Saint Louis , USA ) uptake was measured as described ( Tomi et al . , 2008 ) . Briefly , pcDNA3/RFP/BalaT or mock-transfected HEK293T cells were cultured in 12-well plates ( BD-Falcon ) and washed with 1 mL extracellular fluid ( ECF ) buffer consisting of 120 mM NaCl , 25 mM NaHCO3 , 3 mM KCl , 1 . 4 mM CaCl2 , 1 . 2 mM MgSO4 , 0 . 4 mM K2HPO4 , 10 mM D-glucose , and 10 mM Hepes ( pH 7 . 4 ) at 37°C . Uptake was initiated by applying 200 μL ECF buffer containing 7400 Bq [3H] β-alanine at 37°C . After 10 min , uptake was terminated by removing the solution , and cells were washed with 1 mL ice-cold ECF buffer . The cells were then solubilized in 1 N NaOH and subsequently neutralized . An aliquot was taken to measure radioactivity and protein content using a liquid scintillation counter and a DC protein assay kit ( Bio-rad , USA ) , respectively , with bovine serum albumin as a standard . Histamine , histamine [ring , Methylenes-3H ( N ) ] dihydrochloride , ( 10–40 Ci/mM , American radiolabeled chemicals , Saint Louis , USA ) , and GABA , 4-amino-n-[2 , 3-3H]butyric acid ( [3H]GABA , 30–60 Ci/mmol , PerkinElmer , Waltham , Massachusetts , USA ) uptake assays were initiated by applying 200 μL ECF buffer containing 7400 Bq radiolabeled histamine or radiolabeled GABA . The mixture was incubated for 30 min . The continuous procedures are similar to the β-alanine transport assay . Fly head sections ( 10 μm thick ) were prepared from adults that were frozen in OCT medium ( Tissue-Tek , Torrance , USA ) . Immunolabeling was performed on cryosections sections with rabbit anti-β-alanine ( 1:200 , Abcam , USA; RRID:AB_305476 ) , rat anti-RFP ( 1:200 , Chromotek , Germany; RRID:AB_2336064 ) , rabbit anti-PDH ( 1:200; RRID:AB_2570065 ) ( Wang et al . , 2010 ) as primary antibodies . Cells transfected with BalaT-3XFLAG were incubated with mouse monoclonal anti-Flag M2 antibody ( 1:200 , Sigma-Aldrich , USA; RRID:AB_439685 ) and . Goat anti-rabbit lgG conjugated to Alexa 488 ( 1:500 , Invitrogen , USA; RRID:AB_143165 ) , goat anti-rat lgG conjugated to Alexa 568 ( 1:500 , Invitrogen , USA; RRID:AB_2534121 ) , and goat anti-mouse lgG conjugated to Alexa 647 ( 1:500 , Jackson ImmunoResearch , USA; RRID:AB_2535805 ) were used as secondary antibodies . Phalloidin conjugated to Alexa 650 ( 1:500 , Thermo scientific , Germany; RRID:AB_2532159 ) and DAPI ( 1:1000 , Life Technologies , USA; RRID:AB_2307445 ) was used to indicate photoreceptor cells and nucleus respectively . The images were recorded with a Nikon A1-R confocal microscope . Flies were dark adapted for 15 min before phototaxis assay , as described ( Xu et al . , 2015 ) . Phototaxis index was calculated by dividing the total number of flies by the number of flies that walked above the mark . At least three groups of flies were collected for each genotype and three repeats were made for each group . Each group contained ≥20 flies . Results were expressed as the mean of the mean values for the three groups . Total RNA was prepared from the heads of 3-day-old flies using Trizol reagent ( Invitrogen , Carlsbad , USA ) , followed by TURBO DNA-free DNase treatment ( Ambion , Austin , USA ) . Total cDNA was synthesized using an iScript cDNA synthesis kit ( Bio-Rad Laboratories , USA ) . iQ SYBR green supermix was used for real-time PCR ( Bio-Rad Laboratories , USA ) . Three different samples were collected from each genotype . The primers used for qPCR were as follows: ninaE-fwd , 5’-ACCTGACCTCGTGCGGTATTG-3’ ninaE-rev , 5’-GGAGCGGAGGGACTTGACATT-3’ gpdh-fwd , 5’-GCGTCACCTGAAGATCCCATG-3’ gpdh-rev , 5’-CTTGCCATACTTCTTGTCCGT-3’ pdh-fwd , 5’-GCTTGGCGAACGAAAAGTACT-3’ pdh-rev , 5’-GTCACTCGTTTCCGGGAAGAT-3’ balat-fwd , 5’- AGACATCACACTGCTGCTCTAC -3’ balat-rev , 5’- CCTCCTCCAAAGTCTGTGGAAG -3’ The Dionex Ultimate 3000 UPLC system was coupled to a TSQ Quantiva Ultra triple-quadrupole mass spectrometer ( Thermo Fisher , CA ) , equipped with a heated electrospray ionization ( HESI ) probe in negative ion mode . Extracts were separated by a Fusion-RP C18 column ( 2 × 100 mm , 2 . 5 μm , phenomenex ) . Data acquired in selected reaction monitoring ( SRM ) for histamine , carcinine , and β-alanine with transitions of 112/95 . 2 , 183/95 , and 90/72 , respectively . Both precursor and fragment ions were collected with resolution of 0 . 7 FWHM . The source parameters are as follows: spray voltage: 3000 V; ion transfer tube temperature: 350°C; vaporizer temperature: 300°C; sheath gas flow rate: 40 Arb; auxiliary gas flow rate: 20 Arb; CID gas: 2 . 0 mTorr . Data analysis and quantification were performed using the software Xcalibur 3 . 0 . 63 ( Thermo Fisher , CA ) . Each sample contained 50 Drosophila heads , and the mean values from five samples were calculated .
The recycling of neurotransmitters is essential for sustained synaptic transmission . In Drosophila , histamine recycling is required for visual synaptic transmission . Synaptic histamine is rapidly taken up by laminar glia , and is converted to carcinine . After delivered back to photoreceptors , carcinine is hydrolyzed to release histamine and β-alanine . This histamine is repackaged into synaptic vesicles , but it is unclear how the β-alanine is returned to the laminar glial cells . Here , we identified a new β-alanine transporter , which we named BalaT ( Beta-alanine Transporter ) . Null balat mutants exhibited lower levels of β-alanine , as well as less β-alanine accumulation in the retina . Moreover , BalaT is expressed and required in retinal pigment cells for maintaining visual synaptic transmission and phototaxis behavior . These results provide the first genetic evidence that retinal pigment cells play a critical role in visual neurotransmission , and suggest that a BalaT-dependent β-alanine trafficking pathway is required for histamine homeostasis and visual neurotransmission .
Neurons transmit information around the body in the form of electrical signals , but these signals cannot cross the gaps between neurons . To send a message to its neighbor , a neuron releases a molecule known as a neurotransmitter into the gap between the cells . The neurotransmitter binds to proteins on the recipient neuron and triggers new electrical signals inside that cell . When the message has been received , the neurotransmitter molecules are returned to the first neuron so that they can be reused . This recycling is particularly important in the visual system , where neurons communicate via rapid-fire signaling . In fruit flies , for example , light-sensitive neurons in the eye known as photoreceptors release a neurotransmitter called histamine when they detect light . Supporting cells called laminar glia take up any leftover histamine and combine it with another molecule known as β-alanine to form a larger molecule . The photoreceptors absorb this larger molecule and break it back down into histamine and β-alanine . However , it is not clear how the β-alanine returns to the glia to allow this cycle to continue . Many molecules rely on so-called “transporter” proteins to help them move into or out of cells . To identify transporters that might help to move β-alanine , Han , Xiong et al . prepared a list of fruit fly genes that encode transporter proteins found inside the insect’s head . Testing the resulting proteins in a cultured cell system revealed that one of them was able to transport β-alanine . This protein , named BalaT , is found in another type of support cell called retinal pigment cells . Mutant flies that cannot produce BalaT are blind because their photoreceptors have problems in transmitting information to other neurons . Han , Xiong et al . propose that BalaT transports β-alanine from photoreceptors to retinal pigment cells , which then pass β-alanine on to the laminar glia . Follow-up studies are required to find out exactly how the laminar glia take up histamine .