InteractiveFly: GeneBrief

ora transientless: Biological Overview | References

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 (ort^US6096) 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 ort^P306 and that underlie its (non-null) mutant phenotype (Gengs, 2002). Intriguingly, earlier recordings of the native receptor in LMCs from ort^P306 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 Hdc^P218 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 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 spineless^aristapedia, 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).

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: 20 May 2009

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