InteractiveFly: GeneBrief

ora transientless: Biological Overview | References


Gene name - ora transientless

Synonyms - hclA, outer rhabdomere absent

Cytological map position - 92A13-92A13

Function - channel

Keywords - histamine-gated chloride channel, eye, photoreceptor neurotransmitter, temperature-preference behavior, brain

Symbol - ort

FlyBase ID: FBgn0003011

Genetic map position - 3R: 15,485,470..15,489,401 [+]

Classification - Neurotransmitter-gated ion-channel ligand binding domain

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. This study identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, it was determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. It is proposed that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers (Gao, 2009).

Many animals respond differentially to light of different wavelengths: for example, most flying insects exhibit positive phototactic responses but prefer ultraviolet (UV) to visible light, whereas zebra fish are strongly phototactic to ultraviolet/blue and red light but weakly to green. Unlike true color vision, which distinguishes lights of different spectral compositions (hues) independently of their intensities, spectral preferences are strongly intensity-dependent and innate, probably reflecting each species' ecophysiological needs. Thus, water fleas (Daphnia magna) avoid harmful UV but are attracted to green light, which characterizes abundant food sources. Daylight is rich in UV, so flying insects' preference for UV over visible light is probably related to the so-called open-space response, the attraction towards open, bright gaps and away from dim, closed sites. The receptor mechanisms for spectral preference has been well studied in flying insects, especially in Drosophila. Two or more photoreceptor types with distinct spectral responses are required to detect different wavelengths of light, and mutant flies lacking UV-sensing photoreceptors exhibit aberrant preference for green light. However, the post-receptoral mechanisms of spectral preference are entirely unknown. Furthermore, it is not clear how spectral preference is related to true color vision. Color-mixing experiments suggest that color vision spectral preference are independent in honeybees. In Drosophila, however, spectral preference experiments have revealed that the phototactic response towards UV is significantly enhanced by the presence of visible light, suggesting a 'color' contrast effect in spectral preference behavior. Identifying and characterizing the neural circuits that process chromatic information is the first step to understanding the post-receptoral mechanisms of spectral preference and thus color vision (Gao, 2009).

With recent advances in genetic techniques that manipulate neuronal function, Drosophila has re-emerged as a model system for studying neural circuits and functions. In particular, the Gal4/UAS expression system combined with the temperature-sensitive allele of shibire makes it possible to examine the behavioral consequences of reversibly inactivating specific subsets of neurons. Such interventions allow direct comparisons between the connections of a neuron and its function, thereby establishing causality (Gao, 2009).

The Drosophila visual system comprises the compound eye and four successive optic neuropils (lamina, medulla, lobula and lobula plate. The compound eye itself has some 750 ommatidia, populated by two types of photoreceptors. The outer photoreceptors R1-R6, which are in many ways equivalent to vertebrate rod cells, express Rh1 opsin and respond to a broad spectrum of light, and are thus presumed to be achromatic. The inner photoreceptor neurons R7 and R8 have complex opsin expression patterns: R7s express one of two ultraviolet (UV)-sensitive opsins, Rh3 and Rh4, while beneath R7 the R8s coordinately express blue-sensitive Rh5 or green-sensitive Rh6 opsins. The achromatic R1-R6 channel mediates motion detection. R1-R6 innervate the lamina, where the achromatic channel input diverges to three or more pathways mediated by three types of lamina neurons, L1-L3. Their synaptic connections have been analyzed exhaustively at the electron microscopic (EM) level. Genetic dissection indicates that these three pathways serve different functions in motion detection and orientation. Much like vertebrate cones, R7 and R8 photoreceptors are thought to constitute chromatic channels that are functionally required for spectral preference behaviors. The axons of R7 and R8 penetrate the lamina and directly innervate the distal medulla, where until now their synaptic connections have been completely unknown (Gao, 2009).

The medulla, the largest and most heavily populated optic neuropil, is organized into strata (M1-M10) and columns, in a manner reminiscent of the mammalian cortex. All visual information converges upon the distal strata of the medulla: the axons of R7 and R8 directly innervate strata M6 and M3, respectively, while L1-L3 transmit information from the R1-R6 channel to multiple medulla strata (M1/5, M2, and M3, respectively). The R7, R8, and L1-L3, which view a single point in visual space innervate a single medulla column and there establish a retinotopic pixel. Previous Golgi studies have revealed about 60 morphologically distinct types of medulla neurons. Each arborizes in a stereotypic pattern within specific strata of the medulla, and projects an axon to a distinct stratum of the medulla, lobula or lobula plate. The distinct morphological forms of different types of medulla neurons reflect, at least in part, their diverse patterns of gene expression. Although it is widely presumed that the medulla incorporates key neural substrates for processing color and motion information, little is known about its synaptic circuits and their functions. EM analyses of synaptic circuits have not been possible because of the complexity of this neuropil, while electrophysiological investigations are technically challenging because of the small size of neurons (Gao, 2009).

This study investigated the chromatic visual circuits in the medulla. Using a combination of transgenic and histological approaches, the first-order interneurons in the medulla that receive direct synaptic inputs from the chromatic channels, R7 and R8 were identified. These neurons were subdivided based on their use of neurotransmitters and gene expression patterns. By systematically inactivating and restoring the activity of specific neuron subtypes, the neurons that are necessary and sufficient to drive a fly's phototactic preference to UV were identified (Gao, 2009).

Previous electrophysiological and histological studies have demonstrated that Drosophila photoreceptor neurons are histaminergic and that R7 and R8 photoreceptors provide the predominant histamine-immunoreactive input to the medulla. Two ionotropic histamine-gated channels, Ort (ora transientless; HisCl2) and HisCl1 have been identified. Mutants for ort exhibit defects in motion detection and their electroretinograms (ERGs), indicating that Ort is required to transmit R1-R6 input to the first-order interneurons (Gengs, 2002). To test whether Ort is required for visually guided behavior, flies' phototaxis towards either UV or green light in preference to dark was examined. This phototactic response is mediated primarily by the more sensitive, broad-spectrum photoreceptors, R1-R6, although R7 cells also contribute to UV, but not green, phototaxis under the light-adapted condition. Wild-type flies exhibit stronger phototaxis towards UV than towards green light by approximately one order of magnitude, and light-adaptation, when compared with dark-adaptation, reduces the sensitivity to UV and green light by approximately two orders of magnitude. In contrast, strong transallelic combination ort1/ortUS2515 mutant flies exhibit much weaker phototaxis towards either UV or green light (by about three and two orders of magnitude, respectively) as compared with wild-type. In negative geotaxis assays, ort mutants exhibit no apparent motor defects, suggesting that their reduced phototaxis was not a motor system defect but rather a visual deficit. In addition, the ort mutation affects UV phototaxis more severely than green phototaxis. It is speculated that Ort plays a role in relaying signals from UV-sensing R7s to their first-order interneurons, and that HisCl1 may participate in phototaxis, especially towards green light (Gao, 2009).

To assess whether Ort is required to transmit chromatic input mediated by R7 and R8, a quantitative spectral-preference assay was used. This spectral-preference assay tests the phototaxis towards UV in preference to green and depends on R7, but not significantly on R1-R6, function. This behavior depends on the circuit comparing UV and green light and likely reflects salience of UV and green lights rather than a simple linear summation of their phototactic responses. It was found that wild-type flies prefer short-wavelength UV to longer-wavelength green light in an intensity-dependent fashion. In contrast, homozygous null ort1 mutants and strong transallelic combination ort1/ortUS2515 mutants (as well as other allelic combinations, ortP306/ortUS2515 and ort1/ortP306) all exhibited reduced UV preference. Over five orders of magnitude in the ratio of UV/green intensities, the proportion of ort mutant flies that chose UV was significantly lower than that for wild-type flies. To quantify the UV preference, the isoluminance point, the UV/green intensity ratio at which flies found light of either wavelength equally 'attractive', was determined, and the negative logarithm of the intensity ratio was used as a measure of UV attractiveness. The UV attractiveness for ort mutants was significantly lower than that for wild-type flies (AttrUV/G=2.52±0.23) but higher than that for sevenless mutants (Gao, 2009).

Given that ort null mutants still exhibits phototaxis, whether the other histamine receptor, HisCl1, might contribute to UV preference, was examined. HisCl1134 null mutants were found to exhibit UV preference indistinguishable from the wild-type. In contrast, strong allelic combination HisCl1134 ort1/HisCl1134 ortP306 double-mutants shows weak phototaxis towards green light, while double-null HisCl1134 ort1 mutants, like the phototransduction mutant NorpA, fails to discriminate between wavelengths in the UV and green. It is concluded that Ort is essential for optimal UV preference while HisCl1 plays at most a minor and partially redundant role. It is noted that double-null HisCl1134 ort1 mutants are not entirely blind and still exhibit very weak fast phototaxis, suggesting that there might be residual synaptic transmission between photoreceptors and the first-order interneurons despite of the absence of these two known histamine receptors (Gao, 2009).

It was reasoned that the first-order interneurons must express the histamine receptor Ort in order to respond to their inputs from histaminergic R7 and R8 terminals (see The histamine chloride channel Ort is expressed in subsets of lamina and medulla neurons). To identify these first-order interneurons, the ort promoter region was determined using comparative genomic sequence analysis (Odenwald, 2005; Yavatkar, 2008). In the ort locus, four blocks of non-coding sequence were found that are highly conserved among 12 species of Drosophila. The first three sequence blocks (designated C1-C3) are localized to the intergenic region and the first intron and are therefore likely to contain critical cis-elements. ort-promoter constructs driving Gal4 or LexA::VP16 were generated, designating these ortC1-3-Gal4 and ortC1-3-LexA::VP16. Both driver systems drove expression patterns in identical subsets of neurons in the lamina, medulla cortices and in the deep C and T neurons of the lobula complex, except that ortC1-3-Gal4 drove somewhat patchy expression with lower intensity. The fourth block of conserved sequences, located at 3'UTR, contains putative microRNA binding sites and, as examined in ortC1-4-Gal4, did not drive expression in additional cells, suggesting that it does not contain critical cis-elements. Overall, the expression patterns of these ort promoter constructs resembled previously published ort expression patterns (Witte, 2002) from in situ hybridization (Gao, 2009).

Comparative genomic sequence analysis was also performed for the HisCl1 locus and two blocks of highly conserved sequence (C1 and C2), located in the first introns of the HisCl1 gene and its neighboring gene (CG17360) were identified. A HisCl1-Gal4 construct was generated that included these conserved sequences. It was found that HisCl1-Gal4 drove strong expression in the lamina epithelial glia cells (as recently also reported by Pantazis (2008) and medulla cells that are not well characterized. This result is consistent with previous EM data that lamina epithelial glia enwrap each cartridge and are postsynaptic to R1-R6. Insofar as both the behavioral requirement and expression pattern indicate that Ort but not HisCl1 plays a critical role in the visual system, focus was placed on Ort in the following analyses (Gao, 2009).

Whether using the ort-promoter Gal4 drivers to express Ort is sufficient to rescue defects in the visual behavior ort mutants was examined. It was found that ortC1-4-Gal4-driven Ort expression restored a preference for UV (AttrUV/G=2.25±0.34) in ort mutants to the wild-type level. Since Ort, but not HisCl1, is also required in lamina neurons for normal ERG and motion detection responses (Gengs, 2002; Rister, 2007), the rescued flies were examined for these functions too. It was found that expressing Ort in ort mutants using ortC1-3-Gal4 restored, at least qualitatively, the 'on'- and 'off'-transients of the ERG, which report transmission in the lamina, as well as the optomotor behavior. These findings are consistent with the observation that ort-Gal4 drives reporter expression in lamina neurons L1-L3. In contrast, expressing Ort in lamina neurons L1 and L2 using an L1/L2-specific driver (L1L2-A-Gal4) rescued both the ERG, at least qualitatively, and optomotor defects, but not the UV preference of ort mutants. Thus, the actions of the ort-Gal4 drivers recapitulated the endogenous Ort expression pattern in the first-order interneurons of R1-R6 and R7 (Gao, 2009).

Next, whether the Ort-expressing neurons are required for UV reference and motion detection was examined. ortC1-4-Gal4 or ortC1-3-LexA::VP16 driving a temperature-sensitive allele of shibire, shits1, so as to block synaptic transmission in specific neurons was found to significantly reduce the UV attractiveness at non-permissive, but not permissive, temperatures. This reduction was smaller than that caused by inactivating the R7s alone. These results suggest that Ort-expressing neurons might mediate both UV and green phototaxis, presumably by relaying R7 and R8 channel signals, although the existence of an ort-independent UV-sensing pathway cannot be ruled out. Similarly, inactivating Ort-expressing neurons abolished the flies' ability to detect motion. Thus, it is concluded that Ort-expressing neurons are required for both spectral preference and motion detection (Gao, 2009).

To identify the Ort-expressing neurons that could be synaptic targets of the R7 and R8 channels, a single-cell mosaic technique based on the flip-out genetic method was employed. In this system, the ortC1-3-Gal4 flies that also carried the transgenes UAS>CD2,y+>CD8-GFP and hs-Flp were used. The flipase activity induced by brief heat-shock at the second- or third-instar larval stages excised the FLP-out cassette in small random populations of cells, thereby allowing Gal4 to drive the expression of the CD8-GFP marker. From over 1000 brain samples, 459 clones of transmedulla neurons, the projection neurons that arborize in the medulla and project axons to the lobula, were examined (see Ort is expressed in subsets of transmedulla neurons). To identify the exact medulla and lobula strata in which these processes extend, expression patterns of a series of known cell-adhesion molecules were screened, and three useful stratum-specific markers, FasIII, Connectin, and Capricious were found. In particular, anti-FasIII immunolabeled medulla and lobula strata of interest and, with MAb24B10 immunolabeling, was used primarily to identify the medulla and lobula strata. Based on the morphologies and stratum-specific locations of the arborization and axon terminals, four types of Ort-expressing projection neurons were assigned to types previously described from Golgi impregnation. These were Tm2, Tm5, Tm9, and Tm20. In addition, the ort-promoter driver labeled, albeit at a lower frequency, centrifugal neurons C2 and T2, and three types of medulla neurons with processes solely in the medulla, Dm8, other amacrine-like and also glia-like cells. All of these cells were identified multiple times in at least two independent ort-Gal4 lines, but given the sampling nature of the single-cell mosaic technique, the possibility cannot be excluded that some very rare Ort-expressing neurons were not detected. The amacrine-like and glia-like cells had not been previously described from Golgi impregnation, suggesting that there are even more classes of medulla cell types than those previously reported (Gao, 2009).

The Ort-expressing Tm neurons exhibit type-specific patterns of arborization and axon projection (see Ort-expressing Tm neurons receive multi-channel inputs in the medulla and are presynaptic at both the medulla and lobula). Tm5 neurons extend dendrite-like processes in medulla strata M3 and M6, where R8/L3 and R7 axons terminate, respectively, and they project axons to terminate in stratum Lo5 in the lobula. This pattern suggests that they relay information from the R7 and R8 or L3 channels to the lobula. The Tm5 neurons could be readily divided into three subtypes, Tm5a, b, and c, based on their unique dendritic patterns, the spread of their medulla arborization, and their gene expression patterns. Tm5a and Tm5b have medulla arborizations of different sizes and shapes; whereas Tm5c has dendritic processes in M1, in addition to strata M3 and M5, and the axon projects to both the Lo4 and Lo5 strata. The distinct morphology of Tm5c correlates with its unique expression of the vesicular glutamate transporter. Tm9 and Tm20 extend type-specific dendrite-like processes in strata M1-M3 and projected axons to distinct lobula strata. Tm20, like Tm5, projects to Lo5 while Tm9 projects to Lo1, suggesting that Tm9 and Tm20 relay information from R8 and (via lamina neurons) R1-R6, to different strata of the lobula. In medulla strata M1-M3, Tm2 extends dendrite-like processes which did not appear to make significant contacts with R7 or R8 terminals (Gao, 2009).

To determine if the Ort-expressing Tm neurons indeed receive synaptic input from photoreceptors, serial EM reconstructions were undertaken of Tm9, Tm2, and parts of a single Tm5 cell that resemble Tm5a, as well as the afferent input terminals that innervate the medulla, including R7, R8 and L1-L5 (see also Takemura, 2008). The fine dendritic arbor of Tm20 has so far eluded reconstruction. This study found that Tm9 received direct synaptic contacts from both R8 and L3 and the Tm5 received direct synaptic contacts from R7 and L3. Thus, Tm9 and Tm5 cells were postsynaptic to both the chromatic channels and an achromatic channel. Tm2, by contrast, received synaptic contacts from L2 and L4 but not, despite its Ort expression, R7 or R8. However, the possibility cannot be excluded that Tm2 responds to paracrine release of histamine from the R8 terminal, or an unidentified histamine input in the lobula (Gao, 2009).

It was reasoned that Ort-expressing neurons might be divided into several groups based on their differential release of other neurotransmitters. To test this possibility, a series of promoter-Gal4 and enhancer trap lines driving the CD8 marker was used to label neurons with glutamatergic, cholinergic, GABAergic, serotonergic and dopaminergic phenotypes in the medulla. To determine whether these neurons also express Ort, and are thus likely to receive histaminergic input, the rCD2::GFP marker was expressed in the same animals using the ortC1-3-LexA::VP16 driver. By overlaying two expression patterns, it was found that many Ort-expressing neurons also expressed cholinergic or glutamatergic markers, while few did so for a GABAergic and none appeared to do so for serotonergic or dopaminergic phenotypes. In particular, it was found that a group of neurons labeled by both the vesicular glutamate transporter (vGlutOK371) and ort-Gal4 drivers extended processes in the M6 stratum where R7 axons terminate, suggesting that R7's target neurons might be glutamatergic (Gao, 2009).

To identify candidate R7 target neurons, a combinatorial gene expression system, the Split-Gal4 system, was employed to restrict Gal4 activity to glutamatergic Ort-expressing neurons. In this system, ort and vGlut promoters drive expression of the Gal4DBD (Gal4 DNA binding domain-leucine zipper) and dVP16AD (a codon-optimized VP16 trans-activation domain-leucine zipper), respectively. Thus, Gal4 activity was reconstituted only in the neurons that expressed both Ort and vGlut. A dVP16AD enhancer trap vector was used, and it was substituted for the Gal4 enhancer trap in the vGlut locus. The resulting hemidriver, vGlutOK371-dVP16AD, in combination with a general neuronal hemidriver, elav-Gal4DBD, drove expression in a pattern essentially identical to that driven by vGlutOK371-Gal4, indicating that the vGlutOK371-dVP16AD enhancer trap recapitulated the expression pattern of the vGlutOK371-Gal4 driver. The combination of the vGlutOK371-dVP16AD and ortC1-3-Gal4DBD hemidrivers (designated ortC1-3∩vGlut) gave rise to expression in a subset of Ort-expressing neurons in the optic lobe, namely those that express a glutamate phenotype and are thus likely to be glutamatergic. Single-cell mosaic analysis (using hs-Flp and UAS>CD2>mCD8GFP) revealed that the combinatorial ortC1-3∩vGlut driver was expressed in Dm8, Tm5c, and L1 neurons, as well as in the medulla glia-like cells. In contrast, cha∩ortC1-3, the combination of cha-Gal4DBD choline acetyltransferase-Gal4DBD) and ortC1-3-Gal4AD hemidrivers, drove expression in the Ort-expressing neurons that expressed a cholinergic phenotype, including L2, Tm2, Tm9, and Tm20. Notable among these findings, L1 and L2, paired lamina neurons that receive closely matched R1-R6 input in the lamina, express different neurotransmitter phenotypes (L1: glutamate; L2: acetylcholine) (Gao, 2009).

To determine whether glutamatergic Ort-expressing neurons confer UV preference in flies, whether expressing Ort in these neurons is sufficient to restore normal UV preference in ort mutants was examined. It was found that expressing Ort using the combinatorial ortC1-3∩vGlut driver restored normal UV preference in ort mutants. In contrast, expressing Ort in cholinergic Ort-expressing neurons using the cha∩ortC1-3 driver further reduced UV preference, suggesting that the cholinergic Ort-expressing neurons reduce UV attraction or, more likely, enhance green attraction. Although the cha∩ortC1-3 and ortC1-3∩vGlut drivers were expressed in specific subsets of Ort-expressing neurons in the optic lobe, they showed additional expression outside the visual system, and expressing shits1 with either driver caused non-specific motor defects at the non-permissive temperature. Although it was not possible to test whether the glutamatergic Ort-expressing neurons were required for UV preference, the rescue results indicated that the candidate glutamatergic Ort-expressing neurons, which included Dm8 and Tm5c, were involved in UV preference (Gao, 2009).

To distinguish whether Dm8 or Tm5c is required for UV preference, the ort promoter was dissected, and three promoter-Gal4 lines were generated, each of which contained one of the three highly conserved regions (C1-C3) of the ort promoter. It was found that the second and the third conserved regions (C2 and C3) gave rise to the expression in two different subsets of Ort-expressing neurons while C1 alone gave no detectable expression. Using single-cell analysis, it was found that ortC2-Gal4 drove expression in Dm8 and L1-L3 but not in any Tm neurons, while ortC3-Gal4 was expressed in L2, Tm2, Tm9, C2, and Mi1 neurons. All these neurons except Mi1 expressed Ort, suggesting that the C2 and C3 fragments of the ort promoter drives expression in distinct subsets of the Ort-expressing neurons, but that the combination of all conserved regions was required to suppress Ort expression in Mi1 (Gao, 2009).

Next, whether the ortC2 or ortC3 neuron subsets were sufficient and/or required for UV preference was examined. It was found that expressing Ort using the ortC2-Gal4 driver in ort mutants was sufficient to restore UV preference at least up to the wild-type level. Because the lamina neurons L1 and L2 are neither necessary nor sufficient for UV preference, this finding suggested that the Dm8 neurons alone are sufficient to drive a fly's normal preference for UV. Conversely, whether these neurons were required for UV preference was tested using shits1. It was found that flies carrying ortC2->shits1 exhibited strongly attenuated UV preference at the non-permissive, but not permissive, temperatures, indicating that the ortC2 subset is required for normal UV preference. In contrast, restoring the ortC3 subset activity further reduced UV preference, suggesting that the ortC3 subset inhibits UV sensing, or enhances green-sensing pathways. Moreover, blocking the activity of the ortC3 subset using shits1 did not confer a stronger UV preference, suggesting that the ortC3 subset is sufficient but likely not required for phototactic preference to green light (Gao, 2009).

The preceding evidence indicated that the two lines, ortC2 and ort∩vGlut, together identified the Dm8 neurons both functionally and anatomically as a substrate for UV preference. To test this possibility directly, an ortC2-Gal4DBD hemidriver was generated and combined with the vGlut-dVP16AD hemidriver. It was found that the combinatorial driver ortC2∩vGlut was expressed in most Dm8 neurons as well as in a small number of L1 neurons and glia-like cells. Restoring the expression of Ort in Dm8 in ort or HisCl1 ort double-null mutants completely restored normal UV preference. Conversely, flies carrying ortC2∩vGlut->shits1 exhibited reduced UV preference at the non-permissive, but not permissive, temperature. Thus, the Dm8 are necessary and sufficient for a fly's normal preference for UV (Gao, 2009).

Finally, using the single-cell mosaic method the morphology of the Dm8 neurons was examined (see Amacrine Dm8 neurons receive direct synaptic input from multiple R7 neurons). In stratum M6 the Dm8 neurons were found to extend web-like processes, which extensively overlap 13-16 R7 terminals. To determine whether Dm8 receives direct synaptic input from R7, an EM marker, HRP-CD2, was examined in the Dm8 neurons using the ortC2-Gal4 driver, and their synaptic structure was examined at the EM level. It was found that most R7 synapses are triads and that Dm8 contributes to at least one of the three postsynaptic elements in essentially all R7 synapses. Cumulatively, Dm8 contributes to ~38% (18 out of 47 identified) of the elements postsynaptic to R7s, suggesting that Dm8 is a major synaptic target for these photoreceptors. In addition, processes of three Dm8 neurons were reconstructed spanning seven medulla columns. It was found that Dm8 processes tiled the M6 stratum with partial overlapping so that each R7 terminal was presynaptic to one or two Dm8 neurons. Examining the presynaptic structures of the Dm8 neurons at EM and light microscopic levels, revealed that the Dm8 neurons were also presynaptic to small-field medulla neurons in stratum M6, including Tm5 and at a few contacts to a cell that resembles Tm9. In summary, the wide-field Dm8 neuron serves as a major target neuron for R7 input and provides output locally in stratum M6 to small-field projection neurons (Gao, 2009).

Before this study little was known about the synaptic target neurons of the R7 and R8 photoreceptors and the chromatic pathways their connection patterns subserve. This deficit reflected the inability until recently to penetrate the medulla's complexity. This study made use of prior knowledge of neurotransmitters and their receptors in the visual system to design corresponding promoter constructs that identify the first-order interneurons. These neurons were then labeled with genetically encoded markers and their morphology and synaptic connections were examined at the light and electron microscopic levels. Finally, promoter dissection and the Split-Gal4 system were combined with neurotransmitter hemidrivers to target particular neuron subtypes. It is envisioned that the same combinatorial approach can be applied to dissect other complex neural circuits (Gao, 2009).

This study identified four types of transmedulla neurons, Tm5a/b/c, Tm9, Tm20 and Tm2, that express Ort and are therefore qualified to receive direct input from R7 or R8. These Tm neurons arborize in the medulla and project axons to the lobula, suggesting that they relay spectral information from the medulla to the lobula. Supporting this interpretation, it was found that HA-syt, a presynaptic marker, is indeed localized to their terminals in the lobula. These data support previous suggestions that the lobula plays a key role in processing chromatic information for color vision. Lobula stratum 5 appears most critical for color vision because it receives all three subtypes of Tm5 neurons as well as Tm20. Moreover, it was observed that HA-syt also localized to the dendrite-like processes of all Tm neurons in the proximal medulla, suggesting the presence of presynaptic sites at this level, too. Especially, Tm5a, Tm5b, and Tm20 all extend processes with this presynaptic marker in medulla stratum M8, supporting a previous notion that this stratum might receive chromatic information (Gao, 2009).

All three subtypes of Tm5 neurons extend processes in medulla strata M6 and M3, suggesting that there they might be postsynaptic to R7 and to R8 or L3. Using serial EM, a Tm5 subtype was partially reconstructed that receives direct synaptic input from both the chromatic UV channel of R7 and the achromatic channel of L3. Serial EM also revealed that Tm9 receives inputs from the chromatic green/blue channel of R8 as well as the achromatic L3 channel. It is tempting to speculate that the Tm9 and Tm5 neurons function as color-opponent neurons by subtracting the L3-mediated luminance signal from the R7/R8 chromatic signal (see Medulla circuits in chromatic information processing). While the detailed neural mechanism must await electrophysiological studies, these anatomical data provide direct evidence that the achromatic and chromatic channels are not segregated. Instead they converge on the first/second-order interneurons, early in the visual pathway (Gao, 2009).

Using a quantitative spectral preference test, it was determined that in flies the Dm8 neurons are both necessary and sufficient to confer the animals' UV preference. Each Dm8 receives direct synaptic input from ~14 UV-sensing R7s. By pooling multiple R7 inputs, the Dm8 neurons may achieve high UV sensitivity at the cost of spatial resolution. Consistent with this notion, Dm8 is a main postsynaptic partner for R7 terminals: essentially all of R7's presynaptic sites contain at least one Dm8 postsynaptic element. The processes of Dm8 and their synapses with R7s are largely restricted to the medulla stratum M6. The stratum-specific arborization of Dm8 readily explains why R7 photoreceptors that fail to project axons to the M6 stratum are incapable of conferring UV preference (Gao, 2009).

Dm8 itself has no direct output to higher visual centers in the lobula; instead it is presynaptic to small-field projection neurons, such as Tm5 and possibly Tm9, in the medulla. Thus, Dm8 provides lateral connections linking projection neurons. The morphologies and connections of Dm8 are thus reminiscent of those made by horizontal and amacrine cells in the vertebrate retina. The vertebrate horizontal cells form reciprocal synapses with multiple cones, and in the case where the cones are of different spectral types, the horizontal cells can establish color opponency, as demonstrated in the goldfish retina. Dm8 in Drosophila receives inputs from both Rh3- and Rh4-expressing R7s, but does not provide feedback to photoreceptor terminals, suggesting that Dm8 is unlikely to contribute to color opponency, at least not in a way analogous to vertebrate horizontal cells. Vertebrate amacrine cells have diverse subtypes, which carry out very different functions, including correlating firing among ganglion cells, modulating center-surround balance of the ganglion cells and direction selectivity. The amacrine cells in vertebrate retina receive inputs from bipolar cells and provide the main synaptic input to ganglion cells. It is thus interesting to note that while direct synaptic connections from R7s to Tm5 projection neurons exists, the indirect information flow from R7, to Dm8, and then to Tm5, is both necessary and sufficient to confer UV preference, as suggested by inactivating and restoring experiments. It is hypothesized that the direct and indirect pathways function at different UV intensity levels: Dm8 pools multiple R7 inputs to detect low intensity UV in the presence of high-intensity visible light, while under high intensity UV, Tm5 receives direct input from R7 and mediates true color vision. Further studies using electrophysiology or functional imaging would be required to determine the neural mechanisms of Dm8 (Gao, 2009).

The spectral preference assay used in this study and others measure relative 'attractiveness' of UV and green light and therefore depends on the visual subsystems sensing UV and green light as well as the interactions between these subsystems. While in simple phototaxis assays, the broad-spectrum and most sensitive photoreceptors, R1-R6, dominate simple phototactic response to both UV and green light, they, as well as their first-order interneurons L1 and L2, appear to play an insignificant or redundant role in spectral preference. Thus, R8 alone, or together with R1-R6, provides the sensory input to promote green phototaxis and/or to antagonize UV attraction. The first-order interneurons that relay R8 input in this context have yet to be identified. While anatomical analysis revealed that Tm9 receives direct synaptic input from R8, the behavioral studies provided only weak and circumstantial evidence for its role in spectral preference. Expressing Ort using the cha∩ortC1-3 or ortC3-Gal4 driver significantly reduced UV preference in ort mutants, and Tm9 is covered by both drivers. Furthermore, inactivating Tm9 using the ortC3 driver and shits1 did not affect UV preference, suggesting that other neurons, such as Tm20, might function redundantly. Verification of these suggestions must await the isolation of Tm9-and Tm20-specific drivers, and the corresponding behavioral studies to assay the effects of perturbing activity in these neurons. It is worth noting that Ort-expressing neurons do not include any Dm8-like wide-field neurons for R8s, and restoring activity in the ortC3 neuron subset is sufficient to confer stronger green preference in ort mutants. It is thus tempting to speculate that Dm8 circuits evolved uniquely to meet the ecological need to detect dim UV against a background of ample visible light (Gao, 2009).

Distinct roles for two histamine receptors (hclA and hclB) at the Drosophila photoreceptor synapse

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC50 = 25 µM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states (~25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors are more sensitive to HA, with much smaller single-channel conductances. Null mutations of hclA (ortUS6096) abolish the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG 'on' transients in hclB mutants transients are approximately twofold enhanced, whereas intracellular recordings from their LMCs reveal altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, is exclusively localized to the glia cells, whereas HCLA expression is confirmed in the LMCs. The results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response (Pantazis, 2008).

This study compares the properties of recombinant HCLA and HCLB channels with those of the native channels, describes ERGs from null mutants in both genes, intracellular recordings from LMCs in wild-type and hclB mutants, and the expression profile of the hclB and hclA genes. As well as addressing the molecular identity of the native receptor on the LMCs, the results provide the first single-channel analysis of this new family of Cys-loop ligand-gated ion channel (LGIC) and reveal a novel role for the lamina glia in shaping the postsynaptic response (Pantazis, 2008).

Previous evidence had already clearly implicated HCLA as a subunit of the native channel (Gengs, 2002). It is required for synaptic transmission at the photoreceptor-LMC synapse and for orientation and motion vision (Rister, 2007). However, contradictory reports on the channels' properties and localization left the role of HCLB unclear. In particular, previous measurements of the HA dose-response (D-R) profiles of the two channels has led to widely discrepant EC50 values, none of which could be identified with the native receptor (Skingsley, 1995). The most conspicuous discrepancy concerned the EC50 value for HCLA, with the value of 166 µM reported by Gisselmann (2002) an order of magnitude larger than that found by Zheng (2002), despite using the same expression system (Xenopus oocytes). In this respect, it is interesting to note that the cDNA used by Gisselmann (2002) lacked a region of 5'UTR present in both the cDNA construct used in this study and that of Zheng (2002). This region includes 8 nt (-276 to -269) that are deleted in ortP306 and that underlie its (non-null) mutant phenotype (Gengs, 2002). Intriguingly, earlier recordings of the native receptor in LMCs from ortP306 yielded an EC50 of 190 µM (Gengs, 2002), representing a close match with Gisselmann's estimate. Although this may be coincidental, it may indicate the generation of an unrecognized splice variant under control of this region of the 5'UTR (Pantazis, 2008).

The present study examined the properties of HCLA and HCLB expressed in Drosophila S2 cells; HCLA homomers were an excellent match for the native receptor. Although the possibility cannot be excluded that channel properties are modified by the different cellular environments, recordings were made under very similar conditions to previous recordings of the native receptors from dissociated LMCs, and this equivalence is interpreted as strong evidence for the identity of the native receptor as HCLA homomers. The results from HCLB homomers and HCLA/B cotransfectants were broadly similar to previous results. Notably, all studies agree that HCLA/HCLB heteromers have distinctly higher Histamine sensitivity than either HCLA or HCLB homomers, as well as native LMC receptors, providing compelling evidence that these subunits can assemble into functional heteromers, but suggesting that they do not contribute to the native channels on the LMCs (Pantazis, 2008).

Analysis of single-channel properties provided additional strong evidence for the identity of the native receptor as an HCLA homomer. The single-channel conductance of both HCLB homomers and heteromeric receptors (~4 pS estimated by noise analysis) was an order of magnitude smaller than that of the native channels (Skingsley, 1995). This suggests a dominant conductance-limiting effect brought about by HCLB subunits to the receptor it is part of, be it an HCLB homomer or an HCLA/HCLB heteromer. In contrast, HCLA homomers had very similar properties to those of the native receptor. This extended to a close agreement of three distinct conductance states (25, 40, and 60 pS), with similar weighting and open times. An unusual feature common to both receptors was the disappearance of the largest conductance level at positive holding potentials. LMCs are not thought to depolarize above +20 mV, so the physiological significance of this feature, if any, is unclear. However, because this phenomenon has not been reported for other Cys-loop LGICs, it stands as further strong evidence for the identity of native receptors as HCLA homomers and an intriguing feature for future investigation (Pantazis, 2008).

The functional equivalence of their electrophysiological properties suggests that HCLA homomers are sufficient to account for the properties of the native receptor. The absence of ERG transients in the null hclA (ort) mutant also clearly showed that HCLA is required for synaptic transmission to the LMCs. In contrast, robust transients remained in null hclB (hisCl1134) mutants. Surprisingly however, the 'on' transients in ERGs from hisCl1134 mutants were actually enhanced compared with wild type, whereas recordings from LMCs revealed significantly slower responses that saturated over a smaller dynamic range. In principle, these phenotypes might be interpreted as evidence for a contribution of HCLB to the native channels. However, because the electrophysiological analysis did not support this, the cellular localization of the respective channels was investigated. Previous reports of HCLB localization are equivocal. Zheng (2002) reported that hclA and hclB transcripts were both predominantly expressed in eye tissue at comparable levels However, other studies using in situ hybridization failed to detect hclB RNA in any brain tissue, while confirming expression of hclA in the lamina (Gisselmann, 2002; Witte, 2002). More recently, HCLA, but not HCLB, was localized in the lamina by immunocytochemistry (Hong, 2006). The present study achieved higher resolution using a reporter gene strategy. Expression of hclA in the LMCs, and probably amacrine cells, was confirmed but it was found that, within the lamina, the hclB enhancer drives expression exclusively in glial cells. This conclusion is fully substantiated by an independent approach using mRNA tagging (Pantazis, 2008).

It has long been known that the lamina glia receive direct synaptic input from photoreceptors via the same tetradic synapses that innervate the LMCs, but the role of this glial synapse was unknown. The finding of altered LMC responses in hclB mutants now implies that the glia play a subtle but significant role in shaping the LMC response. Intracellular recordings have never been made from these glia, so it can only be speculated as to how this might be achieved. One possibility would be by contributing to extracellular field potentials, which are believed to be important for determining the effective transmembrane potential (and hence transmitter release) at the photoreceptor synapse. Another would be competition for transmitter binding between LMC and glia postsynaptic histamine receptors, which share the same synaptic cleft at the tetradic synapses (Pantazis, 2008).

Surprisingly, the 'on' transients in the hclB mutant ERG were substantially enhanced compared with control flies. A possible explanation for this unexpected phenotype derives from the fact that the ERG represents a low-pass filtered signal of the underlying neural responses. Because LMC responses in the hclB mutants had approximately twofold slower kinetics, they should suffer less attenuation in the resultant ERG. To estimate the extent of the low-pass filtering, the kinetics were compared of intracellular LMC responses and ERGs recorded with identical stimulation. At low intensities, at which the ERG is dominated by the LMC contribution, the wild-type ERG 'on' transient peaks ~50-60 ms after light onset, whereas the LMC response peaks much earlier (25-30 ms). By digitally filtering LMC responses, it was found that such a delay would be generated by an RC filter of ~20 Hz. When LMC responses from wild-type and hclB (hisCl1134) mutants were filtered in a similar manner, wild-type peak amplitudes were attenuated approximately twofold to threefold, whereas the slower mutant responses suffered only minor (~30%) attenuation. Potentially, this could fully account for the approximately twofold enhanced ERG transients in hclB mutants; however, other contributory factors cannot be excluded. For example, one would expect that the HCLB-mediated signal in the glia also contributes to the ERG; loss of this signal could in principle enhance the 'on' transients if the glia response was depolarizing (although this would require the chloride reversal potential in the glia to be positive to resting potential). In addition, the amplitude of extracellularly recorded responses is critically dependent on resistance barriers in the surrounding tissue. Because the glial cells form a sheath surrounding each cartridge in the lamina, they are likely to make a significant contribution to such resistance barriers, which might be expected to be increased in the absence of their only known synaptic conductance (HCLB) (Pantazis, 2008).

In conclusion, the loss of synaptic transients in ERGs of null hclA mutants indicates that HCLA is an essential component of the synaptic receptor, whereas the striking quantitative similarity between the properties of HCLA homomers and the native receptor strongly suggests their functional equivalence. Along with evidence showing lack of HCLB expression in the LMCs, it is consequently proposed that the native LMC receptor is composed of HCLA homomeric channels. It is further suggested that the hclB (hiscl1) phenotypes observed in the ERG and intracellular LMC recordings reflect a previously unrecognized contribution of the lamina glia to signaling at the photoreceptor synapse (Pantazis, 2008).

The role of carcinine in signaling at the Drosophila photoreceptor synapse

The Drosophila photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized Inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. By feeding wild-type flies carcinine, components of mutant ine ERGs can be duplicated. Finally, it was demonstrated that treatment with H3 receptor agonists (H3 receptor is a presynaptic G-protein-coupled autoreceptor, a metabotropic histamine receptor, that inhibits histamine release) or inverse agonists rescue several components of the mutant ine ERG phenotype. This sutdy provides pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. It is also speculated that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor (Gavin, 2007).

The findings of this study indicate that the presumed neurotransmitter transporter encoded by the ine gene in Drosophila transports the histamine metabolite carcinine. Using genetic epistasis this study shows that the oscillations observed in mutant ine ERGs require histidine decarboxylase activity and the carcinine-synthesizing enzyme Ebony, but not the carcinine-hydrolyzing enzyme Tan. Treating wild-type flies with carcinine can phenocopy components of the mutant ine ERG phenotype. Finally, by rescuing the ine2-associated phenotype with drugs that target the mammalian H3 receptor, pharmacological evidence is provided for the presence of a yet uncharacterized putative H3 receptor in Drosophila that may be responsible for the ERG oscillations observed in flies carrying mutations in the ine gene (Gavin, 2007).

Previous studies involving intracellular voltage recordings of ine mutants have led to the conclusion that the oscillations observed in ine mutant ERGs are the result of a defect occurring within the photoreceptor cell. These conclusions are supported by expressing ine specifically in photoreceptor cells and demonstrating a rescue of the ine2-associated oscillations. Neurotransmitter transporters are often able to function from either the presynaptic neuron or from neighboring glial cells, as shown at the neuromuscular junction in ine mutants. Glial cell-specific expression of the ine gene in ine2 flies results in a complete rescue of the ine mutant ERG phenotype. It was somewhat unexpected that ine expression in glial cells rescued the ine2 phenotypes, since glial cells have been shown to lack Tan protein and thus would be unable to convert carcinine back to a recycled pool of histamine. However, it is possible that glial cells do express trace amounts of the enzyme Tan to hydrolyze carcinine and generate a renewable source of histamine for photoreceptor cells, and it is also possible that the Inebriated protein is expressed in a non-autonomous manner and can be transported from glial cells to photoreceptors in the fly eye (Gavin, 2007).

The finding that an ERG recording can exhibit oscillations is somewhat surprising. An ERG does not record the electrical response of a single photoreceptor, but rather is a collective measure of the retinal photoresponse. Thus, if the mutant ine-associated ERG defects are indeed localized to the photoreceptor synapse, as the data suggest, then one would expect that different photoreceptors would be excited/inhibited at different timepoints, ultimately resulting in the oscillations simply canceling themselves out. The fact that oscillations are indeed observed, and appear to be due to a defect occurring at the photoreceptor synapse, implies the existence of an uncharacterized and complex synchronization of photoreceptor cell de-/repolarization (Gavin, 2007).

The lack of rescue of ine2-associated oscillations in flies carrying additional mutations in the postsynaptic histamine receptor gene ort, the finding that mutant ine oscillations were detected within single photoreceptor cells, and the observations that the mutant ine phenotype can be rescued when ine is expressed in photoreceptors, all combine to strongly suggest that the oscillation phenotype is likely a result of a defect occurring within the photoreceptor itself. In addition, by crossing ine2 animals with HdcP218 flies, it was demonstrated that the ine2-associated oscillations are dependent upon histamine synthesis. All of these results indicate that histamine is somehow contributing to the aberrant ERG witnessed in ine2 flies, and that histamine appears to be acting on the presynaptic photoreceptor cell to induce this oscillation phenotype. Further epistatic analyses also revealed that Ebony, but not Tan, activity is required for the generation of oscillations in ine2 ERGs. These genetic experiments are consistent with ine encoding either a carcinine importer found in the photoreceptor cell or a carcinine exporter found in glial cells. The homology of Inebriated with other known Na+/Cl- neurotransmitter transporters (which import neurotransmitter into cells) suggests that Inebriated protein is transporting carcinine into the photoreceptor, and not out of glial cells (Gavin, 2007).

While Ebony is known to act on multiple substrates, such as dopamine to generate β-alanyl-dopamine, the requirement of histamine synthesis for the maintenance of ine2-associated oscillations suggests that it is β-alanyl-histamine, or carcinine, that is somehow responsible for the oscillations observed in ine2 ERGs. It should be noted, however, that ebony mutations were not sufficient in rescuing the hyperpolarization response observed in mutant ine ERG traces. The origins of this hyperpolarization response are still unclear and further research will be required to elucidate its exact meaning. In tan mutants, one would predict that there would be a buildup of carcinine. However, this buildup does not give rise to an ERG recording similar to that of ine2. This is due most likely to the presence of functional Inebriated protein in tan mutant flies, which should effectively clear the carcinine from the synaptic cleft for degradation within the photoreceptor cell (Gavin, 2007).

By treating wild-type and ebony11 flies with carcinine and subsequently inducing components of the ine2-ERG phenotype, further evidence is provided suggesting that the sharp depolarization spike, the oscillations, and the hyperpolarization response all seen in ine2-ERGs are due to a buildup of carcinine within the photoreceptor synaptic cleft. While the oscillations observed in carcinine-treated wild-type flies do not mimic exactly the oscillations seen in ine2 ERG recordings, it is presumably difficult to replicate the carcinine and histamine balance occurring in the eyes of ine2 animals. Indeed, treatment of wild-type flies with higher (10%) or lower (1%) concentrations of carcinine were less effective in inducing the oscillations than the described 5% carcinine dose (Gavin, 2007).

It is possible that carcinine is being degraded or modified by the fly before the compound is able to exert its effects at the photoreceptor cell. In order to eliminate the activity of one enzyme known to be involved in carcinine metabolism, tan1 flies were treated with 5% carcinine overnight. Surprisingly, none of the tan1 flies treated with carcinine showed an aberrant ERG phenotype. It was surprising that carcinine treatment had a strong effect in flies of the ebony11, but not the tan1, background. While the results of these tan1 and ebony11 carcinine-treatment experiments are unexpected, one possible explanation may involve the regulation of carcinine clearance/degradation. The tan1 flies presumably suffer from a perpetual excess of carcinine even before exogenous carcinine treatment, and these flies, in order to reduce their sensitivity to this compound, may consequently decrease the levels of a putative carcinine receptor, increase their rate of carcinine degradation, or increase the levels of Inebriated protein for carcinine clearance. However, ebony11 flies are relatively 'naive' to the effects of carcinine, as their ability to synthesize this compound has been greatly diminished, and as a result these animals may have an increased level of the supposed carcinine receptor, a decrease in Inebriated receptor levels or a decrease in carcinine degradation, ultimately making them more sensitive to the effects of carcinine treatment (Gavin, 2007).

It remains to be seen whether or not all of the mutant ine-associated phenotypes, including increased neuronal excitability and increased sensitivity to osmotic stress, are due to the inability of these flies to transport carcinine. It is possible that the Inebriated protein transports other compounds that perhaps share the common feature of β-alanine conjugation. This might help explain why none of the more common neurotransmitters were taken up by ine-transfected Xenopus oocytes. In order to assist in confirming that Inebriated is indeed a carcinine neurotransmitter transporter, in vitro experiments, such as neurotransmitter uptake assays, will need to be performed. In addition, the ability of Inebriated protein to take up other β-alanyl-neurotransmitters/osmolytes also should be examined (Gavin, 2007).

The oscillations present within the photoreceptor response of ine2 ERGs appear as sharp depolarization/repolarization spikes, and this oscillation phenotype is dependent upon both histamine synthesis and Ebony activity, and is sensitive to drugs that target mammalian H3 receptors. It is perplexing that the synthesis of a single metabolite, carcinine, could be responsible for both the depolarization and repolarization spikes observed within ine mutant ERGs. It is speculated that these oscillations are the result of aberrant signaling involving both carcinine and histamine at a putative H3 receptor in Drosophila. H3 receptors are an unusual example of the G-protein coupled receptor family, in that they have partial constitutive activity, resulting in a constant small percentage of stimulated G-proteins that trigger a reduction of histamine synthesis and release as well as a decrease in extracellular calcium inflow. The presence of an H3 receptor agonist, such as histamine, causes an increase in activity of the associated G-protein and therefore a stronger inhibition of both histamine release and calcium inflow. Thus, synaptic histamine serves as a negative regulator for its own release and induces a slight repolarization of a stimulated presynaptic histaminergic neuron by inhibiting presynaptic calcium channels. An H3 receptor inverse agonist is believed to act by blocking the constitutive activity of the H3 receptor, resulting in the liberation from a histamine release checkpoint as well as the release of restrictions on calcium inflow. Recently, it has been shown that carcinine has the ability to act as an inverse agonist of presynaptic H3 receptors in mice. While significant further research is required to confirm this hypothesis, it is surmised that histamine and carcinine are exerting opposing effects on the polarization state of the histaminergic photoreceptor cell by activating or inhibiting presynaptic calcium channels via a putative Drosophila H3 receptor. While a recent search of the Drosophila genome did not uncover any direct homologs to vertebrate metabotropic histamine receptors, the CG7918 gene was listed as a possible candidate for encoding such a receptor, and this gene bears strong homology to genes encoding H3 receptors in mammals. In addition, the ine2-associated oscillations display sensitivity to mammalian H3 receptor agonists and inverse agonists, strengthening the possibility that an H3 receptor does exist in Drosophila. It is still unclear what the origins of the thioperamide-sensitive depolarization spikes are that are observed in ort5 ERGs. The presence of these thioperamide-sensitive spikes in ort5 ERG recordings implies the requirement of some postsynaptic retrograde signal for ERG stability, and this ort-dependent signal may be involved in the sensitization of the putative H3 receptor (Gavin, 2007).

It was unexpected that thioperamide treatment of wild-type flies resulted in the loss of on and off transients within their ERG traces. It is possible that histamine release was so extreme in the presence of the potent thioperamide that histamine levels were nearly depleted in the eye, resulting in the disruption of downstream signaling events. Indeed, treatment of mice with high concentrations of carcinine, which acts as an inverse agonist of H3 receptors similar to thioperamide, was shown to result in significantly lower overall levels of histamine within the brains of treated mice. This model of indirect histamine depletion has also been postulated to occur in ebony mutant flies. The absence of on and off transients in ebony mutant ERG recordings is attributed to the normal release of histamine by photoreceptor cells, but this histamine subsequently lacks the ability to be 'trapped' by β-alanine conjugation, ultimately resulting in histamine diffusing away from the eye. Interestingly, expression of pertussis toxin in photoreceptor and laminar neurons in Drosophila results in a similar loss of on and off transients in ERG traces, and this is believed to be the result of inactivation of an unknown G-protein coupled receptor found in photoreceptor cells that is unlikely to be rhodopsin. It is possible that pertussis toxin was acting within photoreceptor cells upon the putative H3 receptor in this study, resulting in a lack of negative feedback on histamine synthesis/release, eventually causing the exhaustion/depletion of histamine pools. Further research will be required to confirm or dismiss the presence of a histamine/carcinine-sensitive H3 receptor in Drosophila photoreceptor cells (Gavin, 2007).

Histamine-HisCl1 receptor axis regulates wake-promoting signals in Drosophila melanogaster

Histamine and its two receptors, Histamine-gated chloride channel subunit 1 (HisCl1) and Ora transientless (Ort), are known to control photoreception and temperature sensing in Drosophila. However, histamine signaling in the context of neural circuitry for sleep-wake behaviors has not yet been examined in detail. Mutant flies were obtained with compromised or enhanced histamine signaling, and their baseline sleep was tested. Hypomorphic mutations in histidine decarboxylase (HDC), an enzyme catalyzing the conversion from histidine to histamine, caused an increase in sleep duration. Interestingly, hisCl1 mutants but not ort mutants showed long-sleep phenotypes similar to those in hdc mutants. Increased sleep duration in hisCl1 mutants was rescued by overexpressing hisCl1 in circadian pacemaker neurons expressing a neuropeptide pigment dispersing factor (PDF). Consistently, RNA interference (RNAi)-mediated depletion of hisCl1 in PDF neurons was sufficient to mimic hisCl1 mutant phenotypes, suggesting that PDF neurons are crucial for sleep regulation by the histamine-HisCl1 signaling. Finally, either hisCl1 mutation or genetic ablation of PDF neurons dampened wake-promoting effects of elevated histamine signaling via direct histamine administration. Taken together, these data clearly demonstrate that the histamine-HisCl1 receptor axis can activate and maintain the wake state in Drosophila and that wake-activating signals may travel via the PDF neurons (Oh, 2013).

Using genetic and pharmacological methods to manipulate histamine signaling, this study shows that the HisCl1 receptor and its downstream signaling cascade regulate wake-evoking behavior in Drosophila, while Ort receptor does not show any sleep/wake regulatory function. Histamine promotes activity via the HisCl1 receptor. Reduced histamine in HDC mutants or loss of the HisCl1 receptor both show reduced activity and enhanced sleep. Additionally, the relevant signaling pathway downstream of the HisCl1 receptor may function in the PDF neurons. Finally, it was demonstrated that the histamine-HisCl1 receptor axis can activate and maintain wakefulness in PDF neurons (Oh, 2013).

These data show the complete functional segregation of the two histamine receptors for the first time. Ort receptor is expressed in large monopolar cells (LMC), postsynaptic to photoreceptors in the lamina and is a major target of photoreceptor synaptic transmission in Drosophila. In contrast to Ort, HisCl1 receptor is not expressed in postsynaptic neurons of photoreceptors. It is expressed in lamina glia and shapes the LMC postsynaptic response of Ort signaling. Both Ort and HisCl1 receptor are involved in temperature-preference behaviors, but the major independent function of HisCl1 receptor remains elusive. This study showed that sleep regulation is a novel and independent function of HisCl1 receptor. Additionally, this finding is an important clue in understanding the functional evolution of the two histamine receptors in Drosophila (Oh, 2013).

It is proposed that wake-activation by histamine signaling in Drosophila is similar to that found in mammals. hdc mutant flies have increased sleep durations compared to controls and a previous study showed that HDC-knockout mice have increased paradoxical sleep compared to controls. This suggests that the HDC enzyme has a common wake-promoting function in mammals and flies. However, the structures of histamine receptors differ between flies and mammals; the histamine receptors of Drosophila are histamine-gated chloride channels, whereas the mammalian histamine receptors belong to the rhodopsin-like G-protein-coupled receptor family. Currently, researchers are working to identify a metabotropic histamine receptor in Drosophila. Despite the structural differences of the mammalian and Drosophila receptors, they share a wake-activating function. This functional homology may be the result of evolution and gives us a hint to find out the metabotropic histamine receptors in Drosophila (Oh, 2013).

Surprisingly, functional conservations between flies and mammals are also found among the histamine receptor subtypes. The HisCl1 receptor has a wake-activating role, whereas the Ort receptor does not. This result parallels differences in the wake-activation roles of the H1 and H2 receptors in mammals: the H1 receptor can activate wakefulness, but the H2 receptor cannot. Thus, the data provide a more detailed understanding of the potential functional relationship between the HisCl1 and H1 receptors. A functional connection between the Ort receptor and the H2 receptor is also possible, since the two have little effect on sleep/wake regulation in their corresponding model systems. No auto-receptor of histamine has yet been found in Drosophila, suggesting that there may not be a Drosophila homolog for the mammalian H3 receptor. Further research should shed greater light on the evolutionary relationship between the histamine receptors of flies and mammals (Oh, 2013).

Histamine signaling modulates the maintenance of wakefulness and controls light sensing, and it is speculated that a number of interactions are possible between these two different pathways. Previous studies on light-perception mechanisms showed that histamine mutants exhibit light-sensing defects. However, this study found that the sleep duration was increased in histamine signaling mutants compared to wild-type flies in constant darkness. Thus, the perception of light in the context of evoking wakefulness is independent of vision-related light perception in Drosophila. Further research will be required to definitively establish the relationship between light perception and sleep regulation (Oh, 2013).

Previous studies revealed that the PDF neurons promote wakefulness in Drosophila. The current findings show that histamine signaling acts as a wake-promoting pathway in PDF neurons. The HisCl1 receptor is a chloride channel, which would be expected to inhibit the function of the neurons. However, since previous studies showed that chloride channels can activate the function of the neurons, hence the HisCl1 receptor might be an activator of the PDF neurons. The downstream signaling of histamine-HisCl1 receptor in PDF neurons should be further studied using genetic manipulation and electro-physiological methods (Oh, 2013).

Orexin is a neuropeptide that acts as an important wake-activating neurotransmitter in mammals, as shown by the demonstration that defects in orexin synthesis can cause narcoleptic symptoms in human and animals. Orexin neurons activate wakefulness in the lateral hypothalamic area and the feedback loop between orexin neurons and monoaminergic neurons such as histaminergic and serotonergic neurons (tuberomammillary nucleus, TMN, and dorsal raphe nucleus, DR) controls wakefulness in the hypothalamus and the brain stem. Histamine receptors are essential for wake-activation by orexin treatment, indicating that orexin and histamine signaling constitute an interactive wake-activating system in mammals. However, orexin has not been found in Drosophila. A previous study suggested that the PDF neuropeptide functions similar to those of orexin in Drosophila (Parisky, 2008), potentially explaining many aspects of the wake-activation cascade in Drosophila. Histamine and orexin have similar wake-activating function, but mammalian histamine mutants do not show narcoleptic symptoms. This study has shown that histamine and one of its receptors, HisCl1, constitute an important wake-evoking axis in Drosophila. Moreover, it was demonstrated that histamine-signaling mutants cannot maintain wakefulness during the daytime, which is similar to the phenotype of orexin mutants in mammals. Hence, it is proposed that, in Drosophila, histamine may have a function similar to that of the mammalian orexin. Further research is required to establish the functional relationship between wake activation of histamine signaling in Drosophila and wake-promoting function of orexin and histaminergic system in mammals (Oh, 2013).

Histamine and its receptors modulate temperature-preference behaviors in Drosophila

Temperature profoundly influences various life phenomena, and most animals have developed mechanisms to respond properly to environmental temperature fluctuations. To identify genes involved in sensing ambient temperature and in responding to its change, >27,000 independent P-element insertion mutants of Drosophila were screened. Defects were found in the genes encoding for proteins involved in histamine signaling [Histidine decarboxylase (Hdc), histamine-gated chloride channel subunit 1 (hisCl1) and ora transientless (ort)] cause abnormal temperature preferences. The abnormal preferences shown in these mutants were restored by genetic and pharmacological rescue and could be reproduced in wild type using the histamine receptor inhibitors cimetidine and hydroxyzine. Spatial expression of these genes was observed in various brain regions including pars intercerebralis, fan-shaped body, and circadian clock neurons but not in dTRPA1-expressing neurons, an essential element for thermotaxis. Histaminergic mutants showed reduced tolerance for high temperature and enhanced tolerance for cold temperature. Together, these results suggest that histamine signaling may have important roles in modulating temperature preference and in controlling tolerance of low and high temperature (Hong, 2006).

Animals are able to track suitable temperature for survival and maintain body temperature. Because of a large surface area-to-volume ratio, the body temperature of small poikilotherms like fruit flies is easily affected by the surrounding environment. To counter harmful effects of environmental temperature fluctuations, molecular and behavioral mechanisms are coordinated to maintain proper body temperature for survival (Hong, 2006).

Many studies on thermotaxis/temperature preference have been performed in model organisms. However, the molecular mechanisms mediating temperature preference are poorly understood. Caenorhabditis elegans is the most studied model system for temperature preference. Cell ablation studies and calcium imaging techniques were used to demonstrate that the neuronal circuitry is involved in thermotactic behavior, and genetic screens have further identified several thermotactic genes. In vertebrates, the preoptic area (POA) was discovered as a temperature-regulating center. In Drosophila, several temperature-sensing genes including dTRPA1, Painless, Hsp70, and Pyrexia have been characterized recently (Hong, 2006).

Histamine is a biogenic amine synthesized from L-histidine via a single decarboxylation step by histidine decarboxylase (hdc). In invertebrates, histamine has various roles in neurotransmission in the brain, such as olfaction in crustaceans and photoreception in various arthropods, as well as in mechanoreception. The histaminergic system in the vertebrate CNS projects its neurites to most regions in the brain and plays a key role in the regulation of basic body functions. Until recently, all histamine receptors identified in vertebrates were merely known as G-protein-coupled receptors (termed H1, H2, H3, and H4). Immunohistochemical studies indicated the presence of histamine in a variety of neuron types in the brain and optic lobes, as well as in the ganglia of the ventral nerve cord of several insect species. In arthropods, it was reported that histamine increases chloride conductance (Claiborne, 1984; Hardie, 1989; McClintock, 1989; Gisselmann, 2002) and that its receptors are members of the ligand-gated chloride channel family (Hong, 2006).

This study identified novel genes involved in temperature-preference regulation: histidine decarboxylase (hdc) and two histamine receptors, ora transientless (ort) and histamine-gated chloride channel subunit 1 (hisCl1). Drosophila strains with mutations in these genes showed abnormal temperature preferences. Furthermore, it was found that these genes are essential in determining critical thermal limits (insects enter a state of coma when they go beyond this temperature limit). Moreover, expression of these genes was detected in various regions of the brain, further supporting their critical roles. Collectively, these results implicate that the histaminergic system participates in regulating physiological responses to cold temperature (or thermal threshold to low and high temperature) as well as modulating temperature preference (Hong, 2006).

The histaminergic mutants showed abnormal temperature preferences, which were rescued by restoring corresponding gene functions. This finding indicates that the histamine-signaling genes function by controlling preference for temperature. However, preference profile changes in these histaminergic mutants were weaker than previously reported thermosense mutants such as spinelessaristapedia, bizarre, and dTRPA1(RNAi) (Sayeed, 1996; Rosenzweig, 2005). Furthermore, the histamine-signaling genes were not coexpressed with dTRPA1, suggesting that histamine, as an essential element for thermotaxis, might be insufficient to function as a central regulator to control temperature preference but finely modulates the temperature preference in Drosophila (Hong, 2006).

This is the first report clearly showing spatial expression patterns of histamine receptors. Compared with previous reports, some unique features were observed. (1) Gisselmann (2002) and Zheng (2002) showed that Ort and HisCl1 could be assembled into functional homomultimers or heteromultimers. They reported that the histamine binding affinity of the heteromultimeric receptors is higher than that of homomultimers. Because neurons with both receptors can respond to low levels of histamine, it is possible they have a more critical role in controlling various biological functions than the singly expressing ones. Accordingly, newly identified neurons and brain regions, expressing both receptors (such as FB, OF, dorsolateral neurons, and pars intercerebralis), may have critical roles in determining temperature preference. Consistent with this hypothesis, it was found that defects in the central complex cause abnormal temperature preferences. (2) Another interesting finding is that Ort is used as major postsynaptic receptors in visual processing, whereas HisCl1 is not. In contrast to Ort, HisCl1 was not expressed in the postsynaptic neurons of photoreceptors. For this reason, the hisCl1 mutant with defects in visual functions might not be recognized in the previous experiments for screening visual defective mutants (Gengs, 2002). (3) Finally, hisCl1 is expressed in circadian clock neurons. Recently, several reports showed that changes in environmental temperature could directly control the circadian clock. In addition, it has been suggested that clock neurons can affect temperature preference in Drosophila. These results imply that thermal behavior-circadian rhythm in Drosophila might be interlocked like the mammalian POA/anterior hypothalamus (an important site for thermoregulation) and suprachiasmatic nucleus (a clock for timing circadian rhythms) (Hong, 2006).

The histaminergic mutants showed changes in tolerance to low and high temperatures as well as temperature preference. Tolerance to low and high temperature is affected by critical thermal limits. To survive in the range of the thermal limit, critical thermal limits must be regulated according to changes in preferring temperature or accommodated temperature. From this point of view, changes in thermal limits accompanying temperature preference in the histaminergic mutants are quite probable. Drosophila showed an increase in cold tolerance and a decrease in hot tolerance when there are defects in histamine function. This coincides with previous reports that resetting the lower thermal limit may come at the expense of a corresponding decrease in the upper thermal limit (Hong, 2006).

This study demonstrates that histamine plays important roles in temperature preference and tolerance to low and high temperature, roles that extend beyond its well accepted activity in visual reception, mechanosensory reception, and sleep. This study provides new insight into the mechanism of temperature sensing or thermotactic decisions in Drosophila. Regulation of histamine secretion may help to control physiological responses such as rapid cold hardening or seasonal acclimation that occur according to changes in temperature and light. Given the potential relationship between temperature and the circadian clock, this work could provide clues for additional insight into sleep and arousal. Finally, this study will help pave the way toward a better understating of the molecular mechanisms and neural circuits of temperature preference as well as the critical thermal limit (Hong, 2006).

Retrograde signaling from the brain to the retina modulates the termination of the light response in Drosophila

A critical factor in visual function is the speed with which photoreceptors (PRs) return to the resting state when light intensity dims. Several elements subserve this process, many of which promote the termination of the phototransduction cascade. Although the known elements are intrinsic to PRs, this study found that prompt restoration to the resting state of the Drosophila electroretinogram can require effective communication between the retina and the underlying brain. The requirement is seen more dramatically with long than with short light pulses, distinguishing the phenomenon from gross disruption of the termination machinery. The speed of recovery is affected by mutations (in the Hdc and ort genes) that prevent PRs from transmitting visual information to the brain. It is also affected by manipulation (using either drugs like neostigmine or genetic tools to inactivate neurotransmitter release) of cholinergic signals that arise in the brain. Intracellular recordings support the hypothesis that PRs are the target of this communication. It is inferred that signaling from the retina to the optic lobe prompts a feedback signal to retinal PRs. Although the mechanism of this retrograde signaling remains to be discerned, the phenomenon establishes a previously unappreciated mode of control of the temporal responsiveness of a primary sensory neuron (Rajaram, 2005).

Two cDNAs coding for histamine-gated ion channels in D. melanogaster

Histamine, a neurotransmitter and neuroregulatory compound in diverse species, serves as the neurotransmitter of photoreceptors in insects and other arthropods by directly activating a chloride channel. By systematic expression screening of novel putative ligand-gated anion channels predicted from the Drosophila genome project, two cDNAs (DM-HisCl-alpha1 and -alpha2) coding for putative histamine-gated chloride channels were identified by functional expression in Xenopus laevis oocytes. DM-HisCl-alpha1 mRNA localizes in the lamina region of the Drosophila eye, supporting the idea that DM-HisCl-alpha1 may be a neurotransmitter receptor for histamine in the visual system (Gisselmann, 2002).

Identification of two novel Drosophila melanogaster histamine-gated chloride channel subunits expressed in the eye

Histamine has been shown to play a role in arthropod vision; it is the major neurotransmitter of arthropod photoreceptors. Histamine-gated chloride channels have been identified in insect optic lobes. This study reports the first isolation of cDNA clones encoding histamine-gated chloride channel subunits from the fruit fly Drosophila melanogaster. The encoded proteins, HisCl1 and HisCl2, share 60% amino acid identity with each other. The closest structural homologue is the human glycine alpha3 receptor, which shares 45% and 43% amino acid identity respectively. Northern hybridization analysis suggested that hisCl1 and hisCl2 mRNAs are predominantly expressed in the insect eye. Oocytes injected with in vitro transcribed RNA, encoding either HisCl1 or HisCl2, produced substantial chloride currents in response to histamine but not in response to GABA, glycine, and glutamate. The histamine sensitivity was similar to that observed in insect laminar neurons. Histamine-activated currents were not blocked by picrotoxinin, fipronil, strychnine, or the H2 antagonist cimetidine. Co-injection of both hisCl1 and hisCl2 RNAs resulted in expression of a histamine-gated chloride channel with increased sensitivity to histamine, demonstrating coassembly of the subunits. The insecticide ivermectin reversibly activated homomeric HisCl1 channels and, more potently, HisCl1 and HisCl2 heteromeric channels (Zheng, 2002).

The target of Drosophila photoreceptor synaptic transmission is a histamine-gated chloride channel encoded by ort (hclA)

By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), ort was identified as a candidate gene encoding a histamine receptor subunit on L1/L2. Evidence is provided that the ort gene corresponds to CG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using cloned hclA cDNA and by showing that several ort mutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions in hclA transcript levels. Other workers have shown that hclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. This study showed unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclA and ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo (Gengs, 2002; full text of article).


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date revised: 10 October 2014

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