Gustatory receptor 5a
Recent studies have suggested that Drosophila taste receptors are encoded by a family of G protein-coupled receptor genes comprising at least 56 members. One of these genes, Gr5a, has been shown by genetic analysis to be required by the fly for behavioral and sensory responses to a sugar, trehalose. Gr5a is expressed in neurons of taste sensilla located on the labellum and legs. Expression is observed in most if not all labellar sensilla and suggests that many taste neurons express more than one receptor. It was demonstrate by heterologous expression in a Drosophila S2 cell line that Gr5a encodes a receptor tuned to trehalose. This is the first functional expression of an invertebrate taste receptor (Chyb, 2003).
To test the hypothesis that Gr5a plays a direct role in trehalose reception, it was asked whether it is expressed in taste neurons. Because previous attempts at in situ hybridization have proved unsuccessful with the great majority of Gr genes Gr5a promoter-GAL4 lines were generated. An 8.5-kb genomic region upstream of Gr5a was used to supply a promoter, and GAL4 was used to drive expression of both UAS-lacZ and UAS-GFP reporters. Wide expression was observed in taste neurons of the labellum as well as in four to six neurons in the tarsi of adult flies. No sexual dimorphism was observed in the expression pattern. Six independently derived lines were examined and all gave equivalent results (Chyb, 2003).
To examine Gr5a function at the cellular level, Gr5a cDNA was expressed in Drosophila S2 cells. This cell line was chosen for two reasons: (1) chemosensory receptors have been notoriously difficult to express in heterologous systems and it was predicted that use of a Drosophila cell line might improve the possibility of functional expression of a Drosophila receptor; (2) previous studies have documented Ca2+ release after activation of G protein-coupled receptors that couple to the endogenous Gq protein of S2 cells. In this system, ligand binding to the receptor results in the activation of the phosphoinositide (PI) pathway: hydrolysis of PIP2 by phospholipase C into InsP3 and 4,5-diacylglycerol, and release of Ca2+ from intracellular stores. The stimulus-activated change in [Ca2+]i can be monitored with Ca2+-sensitive fluorescent ratiometric indicators, such as fura 2 (Chyb, 2003).
Gr5a was transiently expressed in S2 cells: they were loaded with 100 µM fura 2, and 100 mM trehalose was applied via puffer pipette. Stimulation evoked Ca2+ release: cell response developed within ~5 s of ligand application and reached a peak intensity within ~15 s of application. Upon removal of the ligand, the level of intracellular calcium gradually returned to the baseline. These data provided initial evidence that Gr5a encodes a functional trehalose receptor when expressed in S2 cells. The results also suggested the possibility that Gr5a-encoded receptor protein couples to the endogenous phosphoinositide pathway. S2 cells were then cotransfected with Gr5a and promiscuous G proteins: Galpha15,Galpha16, or Galpha15 and Galpha16 together. These G proteins are known to couple a wide variety of G protein-coupled receptors to intracellular Ca2+ release. There was no significant increase in the response intensity to trehalose stimulation compared with the S2-Gr5a cells, consistent with the possibility that Gr5a couples efficiently to Gq (Chyb, 2003).
Therefore, Gr5a is expressed in all, or almost all, of the ~33 sensilla present on the labellum. Because the labellum responds to a variety of sugars, and because the sensilla each contain a single sugar-sensitive neuron, the broad expression observed is consistent with a model in which many, if not all, of the sugar-sensitive taste neurons express more than one receptor. This model is supported by the finding that mutation of Gr5a affects the physiological response of the sugar cell to trehalose, but not to sucrose, as if many of the sugar-sensitive cells contain both a trehalose receptor, Gr5a, and a sucrose receptor (Chyb, 2003).
The expression pattern of Gr5a is broader than that observed for previously described GAL4 lines established by using promoters of other Gr genes. The broad pattern is consistent with physiological data, which indicates that Gr5a is required for trehalose response in all L- and M-type sensilla. Most sensilla on the labellum respond to trehalose (Chyb, 2003).
The simplest interpretation of the results is that Gr5a functions as a homodimer, unlike the mammalian sweet receptors, which function as heterodimers. Furthermore, in contrast to the T1R2/T1R3 mammalian receptor that is rather broadly tuned to diverse sweet-tasting molecules such as sucrose, saccharin, dulcin, and acesulfame-K, the Gr5a receptor is tuned to trehalose and shows much less, if any, response to other sugars, such as sucrose, fructose, and glucose, which the fly encounters in its natural habitat (Chyb, 2003).
The relatively narrow tuning of Gr5a has implications for the mechanism of taste coding. If other Drosophila taste receptors are as specific as Gr5a, then an individual tastant is likely to be encoded largely by the activity of one or a small number of receptors, as opposed to the integrated activity of many receptors, each exhibiting a varying degree of response to a ligand. In the olfactory system of Drosophila, many individual odorants activate several distinct classes of receptor neurons, each expressing distinct odor receptors. This model of taste coding is also supported by the severe loss of trehalose response after mutation of a single receptor gene, Gr5a. Analysis of further Gr proteins will be required to determine whether the narrow tuning of Gr5a is representative of Gr receptors at large or of those that recognize tastants of particular metabolic significance to the fly, such as trehalose (Chyb, 2003).
The sense of taste allows animals to distinguish nutritious and toxic substances and elicits food acceptance or avoidance behaviors. In Drosophila, taste cells that contain the Gr5a receptor are necessary for acceptance behavior, and cells with the Gr66a receptor are necessary for avoidance. To determine the cellular substrates of taste behaviors, taste cell activity in vivo was monitored with the genetically encoded calcium indicator G-CaMP. These studies reveal that Gr5a cells selectively respond to sugars and Gr66a cells to bitter compounds. Flies are attracted to sugars and avoid bitter substances, suggesting that Gr5a cell activity is sufficient to mediate acceptance behavior and that Gr66a cell activation mediates avoidance. As a direct test of this hypothesis, different taste neurons were inducibly activated by expression of an exogenous ligand-gated ion channel, and it was found that cellular activity is sufficient to drive taste behaviors. These studies demonstrate that taste cells are tuned by taste category and are hardwired to taste behaviors (Marella, 2006).
In Drosophila, cells with the Gr5a taste receptor are necessary for sugar acceptance behaviors, and those with Gr66a are necessary for avoidance. These taste cells selectively recognize different taste modalities, such that there is functional segregation of taste qualities in the periphery and at the first relay in the brain. Moreover, activation of these different taste neurons is sufficient to elicit different taste behaviors. Thus, activity of the sensory neuron, rather than the receptor, is the arbiter of taste behavior. These studies argue that animals distinguish different tastes by activation of dedicated neural circuits that dictate behavioral outputs (Marella, 2006).
The patterns of sensory projections provide internal representations of the external world. For example, there is an odotopic map of olfactory projections in flies and mammals. Drosophila gustatory projections are segregated by taste organ such that there is an anterior-posterior map in the subesophageal ganglion of mouthpart, proboscis, and leg projections. Within the proboscis, two different populations of taste neurons can be defined by their expression of either the Gr5a receptor or the Gr66a receptor. Neurons with these different receptors show segregated projections in the brain, with Gr5a projections lateral and anterior to Gr66a projections. Genetic cell ablation experiments revealed that Gr5a cells are required for sugar acceptance behavior and Gr66a for avoidance of bitter compounds. These experiments suggest that in addition to the organotopic map of taste projections, there is also an anatomical map of different taste modalities (Marella, 2006).
This study directly demonstrates that there is functional segregation of different taste modalities in the fly brain. Taste responses were monitored in the living fly by expressing the calcium-sensitive indicator G-CaMP in different classes of taste neurons; Gr5a projections respond to a large number of sugars and Gr66a termini respond to several bitter compounds. Monitoring the responses of subsets of Gr66a cells to a panel of bitter compounds did not reveal striking differences in ligand recognition profiles. Although the possibility that different subsets of Gr5a or Gr66a cells show more selective responses cannot be rule out, clear spatial segregation of sugar and bitter responses was found in the SOG. This argues that there is a spatial activity map of different taste modalities in the fly brain that corresponds to the anatomical projections of Gr5a and Gr66a cells (Marella, 2006).
The character of the taste map in the fly brain is very different from the olfactory map. In the olfactory system, 70 receptors in flies and ~1000 in mammals are used to detect odors. Neurons generally express one receptor, and neurons with the same odorant receptor in the periphery form functional synapses at the same glomerulus in the first relay of the brain. Functional imaging experiments demonstrate that a given odor will activate multiple glomeruli, and one glomerulus will respond to multiple odors. This has led to a spatial model for odor coding in the brain in which the unique combination of activated glomeruli specifies a smell. An animal is thus able to distinguish thousands of different smells by the activation of thousands of different combinations of glomeruli. By contrast, in the fly taste system, sugars activate Gr5a taste projections and bitter compounds activate Gr66a projections. This suggests that there is not a combinatorial code for different tastes in the fly. Instead, the activation of segregated neural populations encodes different taste modalities. This simple map may allow the fly to distinguish sugars from bitter compounds, but may limit the ability to distinguish compounds within the same modality (Marella, 2006).
Sugars elicit food acceptance behavior, and bitter compounds elicit avoidance. The segregation of sugar and bitter responses in the fly brain suggests that activation of different classes of sensory neurons may be sufficient to generate different taste behaviors. This hypothesis was directly tested. Gr5a or Gr66a cells were inducibly activated by expression of a cationic ion channel, VR1E600K, in taste cells and application of its ligand, capsaicin, at the proboscis. G-CaMP imaging experiments demonstrated that taste cells show calcium increases in response to capsaicin. Behavioral studies showed altered taste preferences in flies containing the VR1E600K channel: flies with VR1E600K in Gr5a cells are attracted to capsaicin, and those with VR1E600K in Gr66a cells avoid it. This demonstrates that activation of different taste neurons is sufficient to generate different taste behaviors. Recent studies in C. elegans chemosensory neurons and mammalian gustatory cells demonstrate that exogenous activation of these cells is sufficient to generate acceptance and avoidance behaviors as well. The picture that is emerging from these studies is that the activity of selective sensory cells in the periphery generates behavioral programs through the activation of dedicated neural circuits (Marella, 2006).
G-CaMP imaging and behavioral studies have important implications for understanding how taste information is encoded in the periphery. Three different models have been suggested for how taste information is encoded in the brain: the labeled-line model, the population-coding model (or mixed-lines model), and the temporal-coding model. In the labeled-line model of taste coding, cells are dedicated to detecting different taste ligands, and this information remains segregated as it is relayed to the brain, such that different tastes are distinguished by the selective activation of nonoverlapping cells. In population-coding models, the comparative activity of many cell types rather than activation of one type conveys taste information. This model proposes that the ensemble activity encodes taste quality. In temporal-coding models, it is the precise pattern of action potentials that communicates taste quality (Marella, 2006).
The labeled-line model can be distinguished from the other models by the requirement for a neuron to have a unique identity in terms of recognition properties and behavior. The observation that neurons express subsets of receptors and selectively recognize different taste categories argues that taste neurons have different identities. Moreover, the finding that activation of an exogenous ion channel in discrete taste cell populations elicits specific behaviors argues that selective cell activation is sufficient to mediate behavior, under conditions that do not activate the entire taste cell population and are unlikely to mimic endogenous firing patterns. Taken together, these studies strongly favor the labeled-line model of taste coding in the periphery, although they cannot rule out a role for spike timing or ensemble encoding in fine-tuning the responses (Marella, 2006).
Seminal studies in the gustatory system of mammals strongly argue in favor of the labeled-line model of taste coding in the mammalian gustatory system in the periphery as well. Taste cells on the tongue selectively express either sugar, bitter, or amino acid receptors, such that different taste qualities are detected by different cells in the periphery. Activation of these different taste cells is sufficient to generate specific taste behaviors, with artificial activation of sugar cells eliciting acceptance behavior and artificial activation of bitter cells eliciting avoidance. The observation that cells are dedicated to detecting a specific taste modality and mediate a specific behavior suggests that there are labeled lines of taste information from peripheral detection to behavior. Thus, taste behaviors are hardwired to selective cell activation on the tongue in mammals and the proboscis in flies (Marella, 2006).
The advantage of having taste cell activation innately coupled to behavioral outputs via labeled lines is that the valence of a taste compound is dictated by the neural circuit and requires no previous association. The stereotypy of taste behaviors affords the opportunity to examine how neural connectivity elicits distinct behaviors. It is anticipated that live imaging of neural responses will be a powerful approach to dissect higher-order taste processing in the fly brain (Marella, 2006).
In Drosophila, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. This study proposes that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: (1) immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. (2) trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescues those impairments. (3) Expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depresses both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. It is suggested that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila (Ueno, 2006: full text of article).
It was found that DGsalpha is localized not only in Gr5a-GRNs but also in non-Gr5a GRNs (~40 GRNs in a labelum). In labela, there are at least four types of GRNs sensitive to sugar, low concentrations of salt, bitter-substances/high concentrations of salt, water, and mechanosensory neurons. Then, two questions arise: (1) which GRN, other than Gr5a-GRNs, contains DGsalpha? (2) Is DGsalpha in unknown GRNs involved in the taste signaling of GRNs? The behavioral responses to bitter solutions were not different between heterozygous DGsalpha-null mutant and control flies, and the behavioral and electrophysiological responses to water were not different among all DGsalpha strains examined in this study. It is known that salt responses in larvae require amiloride-sensitive channels encoded by ppk11 and ppk19, and the low and high concentrations of salt responses do not require Ggamma1 in adult flies. These findings together with the current results suggest that DGsalpha in non-Gr5a GRNs serves for other signaling than taste or that the non-Gr5a GRNs containing DGsalpha are mechanosensory neurons. However, because the bitter and water responses in the homozygous DGsalphaR19 mutant were not studied, the possibility that DGsalpha is involved in bitter and/or water tastes cannot be rigorously excluded (Ueno, 2006).
It is suggested that, in Drosophila, the Gs-mediated cAMP transduction pathway is the main signaling route in sugar-sensitive GRNs. In contrast, the PLC/IP3 mediating pathway is involved in sugar-taste signaling in the fleshfly (Boettcherisca peregrina) and the guanosine-3',5'-cyclic monophosphate/nitric oxide pathway in the blowfly (Phormia regina). The cAMP pathway may be involved in sugar-taste perception in the frog, rat, and pig, whereas a recent study on T1R2/T1R3 gustatory sugar receptors of the mouse supports involvement of the PLC pathway. Additional comparative studies are necessary to elucidate the diversity of molecular mechanisms of sugar-taste signaling in various animals (Ueno, 2006).
Taste and olfaction are each tuned to a unique set of chemicals in the outside world, and their corresponding sensory spaces are mapped in different areas in the brain. This dichotomy matches categories of receptors detecting molecules either in the gaseous or in the liquid phase in terrestrial animals. However, in Drosophila olfactory and gustatory neurons express receptors which belong to the same family of 7-transmembrane domain proteins. Striking overlaps exist in their sequence structure and in their expression pattern, suggesting that there might be some functional commonalities between them. In this work, the assumption was tested that Drosophila olfactory receptor proteins are compatible with taste neurons by ectopically expressing an olfactory receptor (OR22a and OR83b) for which ligands are known. Using electrophysiological recordings, this study shows that the transformed taste neurons are excited by odor ligands as by their cognate tastants. The wiring of these neurons to the brain seems unchanged and no additional connections to the antennal lobe were detected. The odor ligands detected by the olfactory receptor acquire a new hedonic value, inducing appetitive or aversive behaviors depending on the categories of taste neurons in which they are expressed i.e. sugar- or bitter-sensing cells expressing either Gr5a or Gr66a receptors. Taste neurons expressing ectopic olfactory receptors can sense odors at close range either in the aerial phase or by contact, in a lipophilic phase. The responses of the transformed taste neurons to the odorant are similar to those obtained with tastants. The hedonic value attributed to tastants is directly linked to the taste neurons in which their receptors are expressed (Hiroi, 2008).
These experiments provide the first direct evidence that olfactory receptors are functional in true taste neurons of Drosophila. These neurons respond to odorants dissolved in paraffin oil upon contact, as if odorants were sapid molecules, and they can even respond to these molecules in air at close range. These observations indicate that the hedonic value that was associated with the detection of the odor is changed according to the identity of the GRNs expressing this receptor (Hiroi, 2008)
The results are consistent with and extend previous results published by Benton (2006) who expressed olfactory receptors in several classes of antennal neurons, including mechanosensory neurons of the Johnston organ and CO2-sensing neurons. Olfactory receptors like Or22a or Or43a need to be co-expressed with Or83b to be correctly addressed to the dendritic membranes and to induce functional responses to the proper odorant ligands. Benton expressed the olfactory receptor Or43a (with Or83b) in antennal neurons expressing Gr21a; these neurons respond to CO2 in the air and acquire the property of responding to cyclohexanol which is a ligand for Or43a. Although Gr21a and its partner Gr63a are classified as a taste receptors, these neurons should be considered as olfactory: (1) they are housed into sensilla ab1C which are lacking a terminal pore considered as characteristic to taste sensilla and (2) they project into the antennal lobe to the DM2-glomerulus while antennal taste sensilla in other insects project into the subesophageal ganglion. Nonetheless, these CO2-sensing sensilla express 'gustatory' receptors which are functional in the absence of Or83b. While Benton demonstrated that ectopic olfactory receptors are functional by population measurement using calcium imaging on the antennal lobe, this study used single-sensillum recordings that gives a greater temporal resolution. Lastly, this work extends Benton's work, by analyzing how the hedonic value of the odorants is changed after miss-expressing ORs into GRNs (Hiroi, 2008).
One important aspect of these experiments is that altered GRNs transduce odorants despite the obvious structural differences between olfactory and taste sensilla e.g. a single terminal pore for taste sensilla vs. a host of minute pores on the hair shaft for olfactory sensilla. The fact that volatile molecules can enter the terminal pore and stimulate taste neurons has received scant attention, except for reports showing that plant odors stimulate taste receptor neurons of tobacco hornworm larvae, Manduca sexta, the Colorado potato beetle, Leptinotarsa decemlineata (Say) and the blowfly. Further indications that taste sensilla may sense lipophilic molecules and odorants come from molecular studies that repeatedly report the presence of odorant-binding proteins in various taste sensilla of insects, which contribute to the transfer of chemicals from air to the sensillum lymph. While the tip-recording technique requires the use of lipophilic solvents that may damage the distal membrane of the taste cells, the technique used in this study should be suitable to record the responses of GRNs to other lipophilic compounds like cuticular pheromones or water-insoluble compounds from plants (Hiroi, 2008).
OR83b is an essential partner to OR22a and other odorant receptor proteins. These molecules form a dimer and adopt in vivo, a topology where their N-termini and most conserved loops are in the cytoplasm; this observation has been confirmed by another approach. This conformation suggested that signaling downstream of the ORs is non-canonical, a prediction that has been recently confirmed by two independent studies using in vitro heterologous expression systems. That OR receptors can induce spiking activities in taste neurons is therefore not surprising: these dimers form channels that when gated by an odorant, may generate current sufficient to induce a receptor potential and trigger the firing of action potentials. However, evidence is still missing about how these ORs are activated in vivo, especially considering that in addition to the odorant-gated channel activation, ORs may interact with more classical transduction pathways like cAMP or cGMP. From this perspective, Drosophila taste neurons represent a useful expression system to evaluate the specificity of olfactory receptors, since it provides cells fully equipped with compatible transduction pathways whose activities can be monitored by extracellular recording techniques or possibly by patch-clamp as done in fleshfly sugar-sensing GRNs (Hiroi, 2008).
Flies expressing olfactory receptors within subsets of taste neurons sharing the expression of the same GR should be particularly useful for understanding how the taste modalities are encoded at the periphery. Although the functional separation between sugar-sensing and bitter-sensing seems quite natural, it rests on chemical characteristics that may overlap. For example, NaCl was found to stimulate sugar-sensing cells at low concentration and bitter-sensing cells at high concentrations. Likewise, a number of artificial sweeteners are stimulating both sugar-sensing cells and bitter sensing-cells in humans and in flies. Because several Gr are co-expressed in Gr66a-GRNs and in Gr5a-GRNs, it is likely that more than one neuron detects the same molecule within a sensillum. The use of a heterologous receptor as a reporter gene for a given Gr has the advantage of activating only one cell without the confounding activity of the other cells (Hiroi, 2008).
While previous observations showed that impairing the expression of Gr5a or Gr66a in taste neurons changed the behavioral responses to sugars or to bitter substances and as well as the activities of the neurons projecting in the brain after 'ensemble' stimulations, the current experiments directly demonstrate that individual GRNs which express Gr5a and Gr66a are different and respond to sugar and to bitter compounds. This study indicates that taste sensory cells of insects encode broad qualities similar to those found in vertebrates (Hiroi, 2008).
How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. This study used the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. Food was shown to promote aggression in flies, and it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, it was shown that detection of sugar via Gr5a+ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs (Lim, 2014).
Because the Tre gene was isolated on the basis of its specific expression in taste sensory cells, the gene is likely to be expressed in taste cells. RT-PCR analyses on isolated labella and tarsi preparations have shown that the mRNA is expressed in the labella and tarsi of original EP(X) lines but is absent in DeltaTre and poxn flies with no taste sensory cells. In situ hybridization experiments have shown that the Tre mRNA is present in one of the taste sensory cells beneath a taste bristle. There were no signals in the labellum preparation of poxn and in the central brain. Thus, Tre seems to be specifically expressed in taste sensory cells (Ishimoto, 2000).
Fruit flies taste compounds with gustatory neurons on many parts of the body, suggesting that a fly detects both the location and quality of a food source. For example, activation of taste neurons on the legs causes proboscis extension or retraction, whereas activation of proboscis taste neurons causes food ingestion or rejection. Whether the features of taste location and taste quality are mapped in the fly brain was studied using molecular, genetic, and behavioral approaches. Projections were found to be segregated by the category of tastes that they recognize: neurons that recognize sugars project to a region different from those recognizing noxious substances. Transgenic axon labeling experiments also demonstrate that gustatory projections are segregated based on their location in the periphery. These studies reveal the gustatory map in the first relay of the fly brain and demonstrate that taste quality and position are represented in anatomical projection patterns (Wang, 2004).
Sixty-eight gustatory receptor (GR) genes have been identified in the sequenced Drosophila genome. These receptors are likely to recognize subsets of taste cues and therefore serve as molecular markers to distinguish neurons recognizing different taste stimuli. To determine whether there is a map of taste quality in the fly brain, the distribution of GRs in sensory neurons was examined. The potential number of tastes that a neuron may recognize was investigated and then the projections of different taste neurons in the brain were examined (Wang, 2004).
One of the difficulties in determining receptor expression patterns in the Drosophila taste system is that GR genes are expressed at very low levels. Most GR genes are not detectable by in situ hybridization experiments, and it has been necessary to generate transgenic flies in which GR promoters drive expression of reporters using the Gal4/UAS system to determine receptor expression. Two transgenic reporter systems were used to simultaneously detect the expression of different receptors. The Gal4/UAS system was used to label one set of neurons, using Gr-Gal4 to drive expression of UAS-CD2. Nine different GR promoters were used that have been reported to drive robust reporter expression in subsets of taste neurons (Dunipace, 2001; Scott, 2001; Chyb, 2003). To label the second set of GR-bearing neurons, transgenic flies were generated in which a GR promoter drives expression of multiple copies of GFP (e.g., Gr66a-GFP-IRES-GFP-IRES-GFP; for simplification, subsequently referred to as Gr-GFP) (Halfon, 2002). Although it is not known how much amplification multiple copies provide, this approach successfully allowed visualization of taste projections whereas direct promoter fusions to a single GFP did not. Transgenic flies for three different GR promoters (Gr32a, Gr47a, Gr66a) were generated and crossed to seven different Gr-Gal4, UAS-CD2 lines to generate a matrix of 21 double receptor combinations (Wang, 2004).
GRs are expressed in subsets of taste neurons, suggesting that one or a few receptors are expressed per cell. This hypothesis was tested by direct comparison of reporter expression for the matrix of three Gr-GFP by seven Gr-Gal4, UAS-CD2 receptor combinations. Focus was placed on the proboscis to compare reporter expression driven by different GR promoters. These studies revealed several surprising findings. (1) Many GR promoters drive reporter expression in partially overlapping cell populations. Gr66a-Gal4 drives expression in approximately 25 neurons per labial palp, in a single neuron in most or all sensilla. Of the six other GRs tested, five show expression in subsets of Gr66a-positive neurons. Of these five, four show largely overlapping expression with each other and one shows mostly non-overlapping expression. Therefore, Gr66a defines a population of gustatory neurons that express overlapping patterns of multiple receptors. (2) Some receptors are segregated into different cells. Gr5a-Gal4 drives reporter expression in approximately 30 neurons per labial palp, in one neuron in most sensilla. Gr5a is not expressed in Gr66a-positive cells. Thus, two non-overlapping neural populations can be identified by Gr66a and Gr5a. Together, these cells account for two of the four gustatory neurons in each taste sensillum (Wang, 2004).
Extracellular recordings of taste responses from proboscis chemosensory bristles have suggested that all taste sensilla are equivalent and that each of the four taste neurons within a sensillum recognizes a different taste modality, with one neuron responding to sugars, two to salts, and one to water. However, more recent experiments suggest a greater diversity of responsiveness. Because Gr5a and Gr66a are expressed in different cells in a sensillum, it was wondered whether they might mark neurons recognizing different classes of tastes. Interestingly, Drosophila defective in Gr5a show reduced responses to the sugar trehalose both in behavioral and electrophysiological studies, and heterologous expression of Gr5a in tissue culture cells confers trehalose responsiveness, strongly arguing that the ligand for Gr5a is trehalose. Given that Gr5a marks a cell that responds to a sugar, it was hypothesized that Gr66a might mark a cell responding to a different taste category. This type of segregation has been demonstrated in the mammalian taste system, where taste cells that respond to sweet are different from those responding to bitter or umami tastants. The taste ligands that Gr5a and Gr66a cells recognize were examined using genetic cell ablation and behavioral studies. It was discovered that Gr66a cells participate in the recognition of bitter compounds (Wang, 2004).
How is taste quality represented in the brain? Because Drosophila taste receptor neurons need not only recognize different tastes but most likely also the gustatory source (e.g., proboscis, internal mouthparts, legs, and wings), gustatory projections were examined to determine whether taste quality or location is represented in sensory projection patterns (Wang, 2004).
The adult Drosophila brain contains approximately 100,000 neurons, with cell bodies in an outer shell surrounding the dense fibrous core. The primary gustatory relay is the subesophageal ganglion/tritocerebrum (SOG) located in the ventral region of the fly brain. It receives input from three peripheral nerves. Neurons from the proboscis labellum project through the labial nerve; mouthpart neurons project through the pharyngeal/accessory pharyngeal nerve, and neurons from thoracic ganglia project via the cervical connective. Early studies employing cobalt backfills provided evidence that mouthpart neurons project more anteriorly in the SOG than proboscis neurons, suggesting that there might be a map of different taste organs in the fly brain (Wang, 2004).
To examine whether taste neurons in different locations project to different brain regions, GR promoters that drive reporter expression in different peripheral tissues were exploited to follow gustatory projections from the proboscis, mouthparts, or leg. Brains of Gr-Gal4, UAS-GFP were stained by anti-GFP immunohistochemistry, and a series of 1 μm optical sections through the SOG was collapsed to produce a two-dimensional representation of projections. These studies reveal differences in projections for neurons in different peripheral tissues. For instance, Gr2a is expressed only in the mouthparts and these neurons exit the pharyngeal nerve and arborize anteriorly. Gr59b, however, is expressed only in proboscis neurons that arborize in a ringed web. Notably, some receptors are expressed both in the proboscis and mouthparts. Interestingly, their neural projections seem to be the composite of Gr2a and Gr59b projections (Wang, 2004).
Two color labeling approaches were used to examine whether projections are segregated by peripheral tissue. For example, differential labeling of Gr2a neural projections, expressed in the mouthparts, and Gr66a neurons expressed in the proboscis, mouthparts, and legs illustrate overlap of the mouthpart projections but not of proboscis projections. Similarly, when the projections of Gr59b, expressed only in the proboscis, and Gr66a are differentially labeled, there is overlap of projections in the ventral proboscis region but not the dorsal mouthparts region (Wang, 2004).
The different axonal patterns from mouth, proboscis, and leg are also seen in different optical sections through the SOG of Gr32a-Gal4, UAS-GFP flies, arguing that projections are segregated by peripheral tissue even if they contain the same receptor. To better resolve the projections of individual neurons with the same receptor, taste neurons were labeled using a genetic mosaic strategy that relies on postmitotic recombination to induce expression of reporters in single cells. The Gr32a receptor is expressed in proboscis, mouthpart, and leg neurons. Single Gr32a-positive neurons from each tissue were labeled and their arborizations were examined in the SOG. A single mouthpart neuron sends an axon that arborizes in a discrete arbor in the most anterior aspect of the SOG. However, a proboscis neuron with the same receptor sends an axon that shows diffuse branching in the medial SOG, a region different from mouthpart projections. Gr32a-positive leg neurons project through the thoracic ganglia and directly terminate in the most posterior part of the SOG (Wang, 2004).
Overall, these studies demonstrate that taste neurons in different tissues project to different locations in the SOG, with mouthpart projections more anterior than proboscis projections, which are more anterior than leg projections. The demonstration that neurons that express the same receptor in different parts of the body project to distinct locations argues that they elicit different spatial patterns of brain activity and provide a means for encoding different behaviors in response to the same tastant (Wang, 2004).
It was next asked whether neurons from the same peripheral tissue that recognize different tastes project to the same or a different brain region to evaluate if taste quality is encoded in sensory projection patterns. Two-color labeling strategy was used to differentially label projections from Gr5a neurons that recognize sugars and Gr66a neurons that recognize bitter compounds. Remarkably, the projections of proboscis neurons with these receptors are clearly segregated in the SOG: Gr5a projections are more lateral and anterior to Gr66a projections. The Gr5a projections are ipsilateral and resemble two hands holding onto the medial, ringed web of Gr66a projections. Interestingly, leg taste projections for Gr5a and Gr66a neurons are segregated: Gr66a neurons project to the SOG whereas Gr5a neurons project to thoracic ganglia (Wang, 2004).
By contrast, when receptors are contained in partially overlapping populations, there is no obvious segregation of projections. For example, Gr32a is contained in a small fraction of Gr66a-positive cells in the proboscis, yet Gr32a-positive fibers colocalize with Gr66a-positive fibers in all optical sections. Moreover, Gr32a and Gr47a are expressed in mostly non-overlapping subsets of Gr66a-positive proboscis neurons, and their projections overlap, showing that smaller populations of Gr66a-positive cells are not spatially segregated. The lack of segregation suggests that these cell types are not functionally distinct (Wang, 2004).
These studies demonstrate that receptors that are expressed in subsets of cells that recognize bitter substances do not show segregated projections. However, different projection patterns are clearly discernible for proboscis neurons that recognize bitter compounds versus those that recognize sugars. The segregated projections from Gr5a and Gr66a cells reveal that there is a spatial map of taste quality in the brain (Wang, 2004).
The patterns of Drosophila taste receptor expression resemble those of the mammalian taste system and the C. elegans chemosensory system, where multiple receptors are also expressed per cell. In the mammalian taste system, multiple bitter receptors are coexpressed in one population of cells on the tongue whereas receptors for sugars are expressed in a different population of taste cells, arguing that different sensory cells recognize different taste modalities. Remarkably, the concept of distinct sweet and bitter cells also applies to the fly (Wang, 2004).
This study identified two populations of proboscis neurons that show spatially segregated projection patterns in the SOG. These different patterns correspond to different taste categories: neurons that recognize bitter substances are mapped differently in the fly brain from those that recognize sugars, suggesting that there is a map of taste modalities or behaviors in the fly brain. In addition, several subpopulations of Gr66a-positive cells show convergent projections. Two different models could account for this convergence. (1) In the simplest model, convergence could imply similar function. For example, all neurons mediating avoidance behaviors might project to the same region and synapse on a second order neuron that conveys avoidance. (2) The apparent convergence could still yield segregated gustatory information if there is a molecular identity code such that second order neurons synapse exclusively with gustatory neurons containing the same receptors. This second model is akin to what is seen in the mammalian pheromone system and sensory-motor connectivity in the spinal cord. Future experiments examining synaptic connectivity will be essential to determine how gustatory information is transmitted to higher brain centers. Nevertheless, the observation that there is spatial segregation of Gr5a sugar cells and Gr66a bitter cells, but not of smaller populations of Gr66a-positive cells, suggests that the diversity of recognition afforded by 68 or so receptor genes may be simplified into only a few different taste categories in the fly brain (Wang, 2004).
Gustatory projections are also segregated according to the peripheral position of the neuron. Early studies employing cobalt backfills argue that mouthpart neurons project more anteriorly in the SOG than proboscis neurons. The results are consistent with, and extend, these observations. Using genetic mosaic approaches, single taste neurons were labeled, and it was found that projections from different organs are segregated even from neurons containing the same receptor. These studies argue that the same taste stimulus will produce different patterns of brain activity depending on the stimulus' location in the periphery and may mediate different behaviors, consistent with the observation that sugar on the leg causes proboscis extension whereas sugar on the ovipositor causes egg laying. An organotopic map of gustatory projections may provide a means for the fly to distinguish different taste locations (Wang, 2004).
This study examined the molecular and cellular basis of taste perception in the Drosophila larva through a comprehensive analysis of the expression patterns of all 68 Gustatory receptors (Grs). Gr-GAL4 lines representing each Gr are examined, and 39 show expression in taste organs of the larval head, including the terminal organ (TO), the dorsal organ (DO), and the pharyngeal organs. A receptor-to-neuron map is constructed. The map defines 10 neurons of the TO and DO, and it identifies 28 receptors that map to them. Each of these neurons expresses a unique subset of Gr-GAL4 drivers, except for two neurons that express the same complement. All of these neurons express at least two drivers, and one neuron expresses 17. Many of the receptors map to only one of these cells, but some map to as many as six. Conspicuously absent from the roster of Gr-GAL4 drivers expressed in larvae are those of the sugar receptor subfamily. Coexpression analysis suggests that most larval Grs act in bitter response and that there are distinct bitter-sensing neurons. A comprehensive analysis of central projections confirms that sensory information collected from different regions (e.g., the tip of the head vs the pharynx) is processed in different regions of the s ganglion, the primary taste center of the CNS. Together, the results provide an extensive view of the molecular and cellular organization of the larval taste system (Kwon, 2011).
Of the 67 Gr-GAL4 transgenes, 43 showed expression in the larva, of which 39 were expressed in the major taste organs of the head. The 39 Gr-GAL4 drivers are expressed in combinatorial fashion. Individual Gr-GAL4 drivers are expressed in up to 12 cells, in the case of Gr33a- and Gr66a-GAL4; approximately one-half, however, are expressed in only one cell (Kwon, 2011).
For some Gr-GAL4 drivers the observed pattern of expression may not be identical with that of the endogenous Gr gene. It was precisely with this concern in mind that a mean of 7.6 independent lines were analyzed for each of the 67 Gr drivers, and a rigorous, quantitative protocol was establised for identifying a representative line for each gene. In the absence of an effective in situ hybridization protocol, the approach used here seemed likely to be the most informative in providing a comprehensive systems-level view of larval taste reception (Kwon, 2011).
The Gr receptor-to-neuron map of the dorsal and terminal organs identified 10 neurons. Two neurons have cell bodies in the DOG and innervate the DO, two have cell bodies in the DOG and innervate the TO, and six have cell bodies in the TOG and innervate the TO (Kwon, 2011).
28 receptors were mapped to these 10 neurons. All of these neurons express at least two Gr-GAL4 drivers. Two receptors, Gr21a and Gr63a, are coreceptors for CO2; neither is sufficient to confer chemosensory function alone. It is conceivable that many other Grs may also require a coreceptor, which may explain the lack of neurons expressing a single Gr-GAL4. The number of receptors per neuron ranges up to 17, in the case of C1. This number is comparable with the maximum number of Gr-GAL4s observed in a labellar neuron (29), and much greater than the number of Ors observed in individual neurons of either the larval or adult olfactory system (Kwon, 2011).
Among the 10 identified cells, individual Gr-GAL4 drivers are expressed in as few as one cell and as many as six cells. Most of the drivers are expressed in only one of these 10 cells. The drivers expressed in six cells, Gr33a-GAL4 and Gr66a-GAL4, are expressed in all bitter neurons of the adult labellum. It is noted that Gr33a-GAL4 and Gr66a-GAL4 are the only drivers expressed in B1, arguing against the possibility that both of these receptors function exclusively as chaperones or as coreceptors that require another Gr for ligand specificity (Kwon, 2011).
There is little cellular redundancy. Only two neurons, A1 and A2, express the same complement of receptors. All other neurons contain a unique subset of the Gr repertoire. In this respect, the larval taste system differs from the adult taste system but is similar to the larval olfactory system, which also contains little if any cellular redundancy (Kwon, 2011).
Analysis of the central projections of all 39 Gr-GAL4 drivers provided evidence for a systematic difference among projection patterns between TO/DO neurons and pharyngeal neurons. These results support the conclusion that sensory information collected from the tip of the head is processed in different regions of the SOG than information collected in the pharynx, i.e., that evaluation of a potential food source before ingestion and the testing of food quality during ingestion are functionally partitioned in the brain. Similar inferences were drawn in an elegant study of a limited number of Gr-GAL4 transgenes (Colomb, 2007; Kwon, 2011 and references therein).
Conspicuously absent from the list of Gr-GAL4 drivers expressed in the larval taste system are those representing the eight members of the sugar receptor subfamily (Gr5a, Gr61a, Gr64a-f). The founding member of this family, Gr5a, mediates response to the sugar trehalose, and two other members of the subfamily have been shown to encode sugar receptors as well. No GFP expression for these genes was observed in cells of the taste organs or in neural fibers in the brain or ventral ganglion. Most of these Gr-GAL4 transgenes drive expression in the adult, but it is acknowledged that these transgenes may not faithfully reflect expression in the larva (Kwon, 2011).
Given that Drosophila larvae respond to sugars, as do larvae of other insect species, how do they detect them without members of the sugar receptor subfamily? Other Grs, including the recently identified fructose receptor Gr43a, may underlie sugar detection in the larva. It is noted that Gr59e-GAL4 and Gr59f-GAL4 are coexpressed in a cell that does not express the bitter cell markers Gr33a-GAL4 or Gr66a-GAL4. Sugar reception may also be mediated by other kinds of receptors, such as those of the TRPA family (Kwon, 2011).
In adult Drosophila, Gr33a-GAL4 and Gr66a-GAL4 are coexpressed with other Gr-GAL4s in bitter neurons; the simplest interpretation of expression and functional analysis is that multiple bitter receptors are coexpressed (Kwon, 2011).
In the larva, it ws found that most larval Gr-GAL4s are coexpressed with Gr33a- and Gr66a-GAL4, suggesting the possibility that most larval Grs act in bitter response. It is noted that, of the 17 Gr-GAL4s coexpressed in the C1 neuron, 15 are coexpressed in a bitter neuron of the labellum. It was also establish that there are distinct molecular classes of Gr33a-GAL4, Gr66a-GAL4-expressing neurons. The simplest interpretation of these results is that there are distinct bitter-sensing neurons in larvae (Kwon, 2011).
Larvae must determine whether to accept or reject a food source, and in principle this determination could be made by a simple binary decision-making circuit. However, the existence of six Gr33a-GAL4, Gr66a-GAL4-expressing neurons expressing distinct subsets of Gr-GAL4s suggests a greater level of complexity in the processing of gustatory information. One possibility is that C1, which expresses the largest subset of drivers among the TO/DO neurons, may activate an aversive behavior in response to many of the bitter compounds that the larva encounters, while C2, C3, C4, or B2 either potentiates the response or activates a different motor program in response to chemical cues of particular biological significance or exceptional toxicity. The existence of heterogenous bitter-sensing cells, some more specialized than others, is a common theme in insect larvae. In particular, many species contain a taste cell that responds physiologically to many aversive compounds and whose activity deters feeding. C1 could be such a cell, and its coexpression of many receptors may provide the molecular basis of a broad response spectrum (Kwon, 2011).
It is striking that the number of TO/DO neurons that express Gr-GAL4s is small compared with the total number of TO/DO neurons. Gr-GAL4 expression was mapped to only 10 cells in the TO/DO (although Gr2a-GAL4 and Gr28a-GAL4 were each expressed in two TO neurons that were not mapped). The DOG and TOG contain 36-37 and 32 sensory neurons, respectively, among which 21 in the DOG are olfactory. Thus, of the nonolfactory cells, on the order of 20%-30% express Gr-GAL4 drivers. It will be interesting to determine how many of the other DOG/TOG cells express other chemoreceptor genes, such as Ppk, Trp, or IR genes, and how many of the other neurons have mechanosensory, thermoreceptive, hygrosensory, or other sensory functions (Kwon, 2011).
The role of Gr genes in the larval pharyngeal organs is unknown. In adult pharyngeal sensilla, the TRPA1 channel, which detects irritating compounds, regulates proboscis extension. It is possible that Grs expressed in larval pharyngeal organs may also play a role in modulating feeding behavior. Of the 24 Gr-GAL4 drivers expressed in the larval pharyngeal organs, 9 are coexpressed with Gr33a-GAL4 and Gr66a-GAL4 in the TO/DO; it seems plausible that they may monitor ingested food for the presence of aversive compounds (Kwon, 2011).
In summary, this study has analyzed essential features of the molecular and cellular organization of a numerically simple taste system in a genetic model organism. Ten gustatory receptor neurons were described and evidence was provided that they express Grs in combinatorial fashion, with most of these neurons and receptors acting in the perception of bitter compounds. The results lay a foundation for a molecular and genetic analysis of how these receptors and neurons, and the downstream circuitry, underlie a critical decision: whether to accept or reject a food source (Kwon, 2011).
Functional evidence is provided that one of the gustatory receptor genes, Gr5a, encodes a taste receptor required for response to the sugar trehalose. In two different mutants that carry deletions in Gr5a, electrophysiological and behavioral responses to trehalose are diminished but the response to sucrose is unaffected. Transgenic rescue experiments show that Gr5a confers response to trehalose. The results correlate a particular taste ligand with a Gr receptor and indicate a role for G protein-mediated signaling in the transduction of sweet taste in Drosophila (Dahanukar, 2001).
To test the hypothesis that Gr genes encode taste receptors, it was asked whether any Gr genes reside at loci implicated in taste perception. The trehalose response locus, whose alleles confer different levels of response to the disaccharide trehalose, has been mapped to cytogenetic region 5A on the X chromosome. One Gr gene, Gr5a, is also located in region 5A and is thus tightly linked to this locus. Gr5a is a member of a small subfamily of eight Gr genes. The intron-exon structure of Gr5a was determined by 5' and 3' RACE and RT-PCR experiments. The experiments also show expression of Gr5a in the proboscis and the legs, both of which contain sensilla that respond to trehalose. The 5' end of Gr5a lies less than 900 base pairs from CG3171, which has previously been reported to encode the trehalose receptor and has been named Tre1 (Ishimoto, 2000; Dahanukar, 2001).
To investigate the possible role of Gr5a in trehalose reception, two strains of flies, each with a deletion in the Gr5a-Tre1 genomic region, were used. The two deletion lines, DeltaEP(X)-19 and DeltaEP(X)-5, henceforth referred to as Delta19 and Delta5, were generated by imprecise excision of a P element that lies in the region between the two genes in strain EP(X)0496, henceforth referred to as 496. Sequence analysis determined that in Delta19, the proximal endpoint of the deletion lies within the second exon of Gr5a (3' to the codon specifying Ala 62), and in Delta5, the proximal endpoint lies in the first intron (which lies 3' to the codon specifying Arg 36) (CG15779 in Gadfly). Thus, in each deletion strain, the translation initiation codon of Gr5a is removed. Flies that are either homozygous or hemizygous for these deletions are viable (Dahanukar, 2001).
It was next asked whether the deletion mutants lacking Gr5a were defective in trehalose response, using two independent assays. First, electrophysiological response was measured to sugars in individual labellar taste hairs of the large (L) or medium (M) class, using the tip-recording method. L and M sensilla each have four chemosensory neurons, classified according to the stimuli that elicit a response from them: a sugar cell, two salt cells and a water cell. The sugar cell responds to a number of different sugars, including disaccharides such as sucrose and trehalose, which is present in yeast (an important food source of Drosophila). Individual sensilla were stimulated by placing an electrode containing the sugar over the tip of the sensillum. Action potentials are elicited in both the sugar and the water cell, but the activities of the two cells can be distinguished by their different amplitudes. Dose-response curves for sucrose and trehalose in the various strains reveal that sucrose response is the same for all strains across a broad range of concentrations. In the same cells, however, the trehalose responses of Delta5 and Delta19 were drastically reduced compared to that of the parental strain 496 (Dahanukar, 2001).
Next the behavioral response to trehalose was tested using the two-choice preference test. This protocol compares the consumption of two sugars offered simultaneously to populations of flies. In the first control experiment, the preference for different concentrations of sucrose versus a standard concentration of 2 mM sucrose was tested. Flies of all strains tested preferred 3 mM (10-2.5 M) sucrose to 2 mM sucrose, with a preference index (PI) of 0.9, and they preferred 2 mM sucrose to 1 mM sucrose. Consistent with the physiological data, there was no difference in the response to sucrose between strain 496 and the strains that are mutant for Gr5a. To investigate trehalose response, several concentrations of trehalose were tested against 2 mM sucrose. The PI50 value, the concentration of trehalose at which flies consume as much trehalose as sucrose, was 11 mM for 496, but 77 mM for Delta5 and 76 mM for Delta19. Thus, in this behavioral protocol both mutants were severely defective in their response to trehalose (Dahanukar, 2001).
To determine whether loss of Gr5a expression is responsible for the reduced response to trehalose in the deletion mutants, a 10-kb genomic DNA rescue construct was engineered that includes both the Gr5a and the Tre1 coding regions. To assess the contributions of each gene separately, derivatives of this 10-kb construct were generated that include a stop codon near the N-terminus of either Gr5a or Tre1. Transgenic flies were generated containing these constructs and crossed into Delta5 and Delta19 genetic backgrounds. The resulting flies were tested for rescue of the trehalose response defect using the physiological and behavioral assays described above (Dahanukar, 2001).
The physiological response of the sugar cell to trehalose was rescued by the construct with wild-type copies of both Tre1 and Gr5a. Although rescue occurs in both the Tre1+Gr5a+ and the Tre1-Gr5a+ transgenic flies, it does not occur in the Tre1+Gr5a- transgenic flies, indicating that rescue required a wild-type Gr5a but not a wild-type Tre1. None of the transgenes affected response to sucrose (Dahanukar, 2001).
Next, the behavioral phenotype of the transgenic flies was tested. The preference for trehalose was measured at a 10-1.5 M (31.6 mM) concentration, which gave the maximal difference in PI between the parental and deletion strains. Rescue again occurred, and the pattern of rescue was consistent with the physiological data: it was dependent on Gr5a and not on Tre1 in both the Delta19 and Delta5 flies (Dahanukar, 2001).
Thus, Gr5a, a member of a large family of candidate taste receptors, corresponds to the Tre locus, a genetic locus that affects response to the sugar trehalose. The most compelling evidence that Gr5a is responsible for the trehalose sensitivity phenotype is that deletion mutations affecting Tre function can be rescued by a transgenic construct containing a wild-type copy of Gr5a, but not by a construct containing a mutant copy of Gr5a. Rescue has been shown both by measurements of single-cell electrophysiology and by behavioral assays. The equivalence of Gr5a and the Tre locus is also consistent with mapping data indicating that all reported Tre deletions remove portions of Gr5a (Dahanukar, 2001).
The simplest interpretation of these results is that Gr5a encodes a taste receptor for trehalose. The absence of a functional Gr5a affects the physiological response of a taste neuron to trehalose, but not the response of the same neuron to another disaccharide sugar, sucrose. Thus, the specificity of Gr5a function is consistent with that expected of a taste receptor, and inconsistent with that expected of a GPCR playing a general role in taste neuron development or function. Trehalose reception could also be mediated in part by additional receptors; however, the severely reduced trehalose response in flies lacking Gr5a and the rescue data suggest that the response to trehalose is mediated primarily by Gr5a (Dahanukar, 2001).
A previous report identified the product of Tre1 as the trehalose receptor (Ishimoto, 2000). That report used a heat shock-inducible Tre1 cDNA construct to rescue the phenotype of a deletion mutant similar to Delta5. Although the results do not formally exclude a role for Tre1 in trehalose response, neither do they support such a role. In considering the differing conclusions of these two studies, it is noted that although Tre1 mutants were shown to be abnormal in both a two-choice preference test and a proboscis extension test, rescue was described only in the two-choice preference test and only for a single pair of concentrations (2 mM sucrose and 80 mM trehalose). Moreover, although some limited data are reported to indicate a physiological phenotype for a Tre1 mutant, rescue of the peripheral physiological defect by the hs-Tre1 transgene has not been shown. It is noted that Tre1 is expressed in embryonic EST collections, suggesting that it is expressed in early Drosophila development. Tre1 is also expressed ubiquitously in adult tissue, according to a paper published while the current manuscript was under review (Ueno, 2001). Polymorphisms in the sequence of Gr5a, but not Tre1, correlate with the trehalose phenotype (Dahanukar, 2001).
The sugar cells in all wild-type L- or M-class sensilla from which recording were taken responded to both sucrose and trehalose. In recordings from Gr5a deletion mutants, no sensilla of these types were found whose sugar cells respond at wild-type levels to trehalose. Though not exhaustive, these data suggest that Gr5a is expressed in the sugar cells of all the L and M sensilla on the labellum. These two sensillar classes together constitute ~30 of the ~66 sensilla on the labellum; thus, Gr5a seems to be expressed in at least 30 neurons. Although this number has not been confirmed by in situ hybridization, 30 is a larger number than is seen for the several members of the Gr family whose expression has been detected by in situ hybridization or reporter-gene expression. For these receptors, the number of cells exhibiting expression ranges from 4 to 22 in the labellum. Because response of all tested Gr5a mutant sugar cells was abnormal to trehalose but normal to sucrose, it seems likely that each of these cells expresses at least two receptor genes: Gr5a, which mediates response to trehalose, and another receptor that mediates response to sucrose. Colocalization of receptors within a single sugar cell might constrain the ability of the animal to discriminate among sugars through differential activation of distinct taste neurons. It remains possible, however, that discrimination might be achieved by insulation of different signaling pathways, as probably happens within individual chemosensory neurons of Caenorhabditis elegans (Dahanukar, 2001).
Although studies in mammals have implicated G protein-mediated signaling in the transduction of sweet taste, the mechanism in invertebrates is largely unknown. There is evidence that sweet taste in larger flies is mediated at least in part through a cGMP second messenger; however, there is also a report of a channel that is activated directly by sucrose without the mediation of second messengers or G proteins. The results support a role for G protein-mediated transduction of the disaccharide trehalose in Drosophila, as is found for sweet taste in mammals (Dahanukar, 2001).
In summary, the simplest interpretation of the data is that a member of the Gr gene family encodes a taste receptor required for response to the sugar trehalose, as indicated by both electrophysiological and behavioral analysis of mutant and transgenic flies. The association of a particular ligand with a particular Gr taste receptor now allows for a variety of studies, including detailed functional studies of the receptor and of the mechanism by which it transduces gustatory information. It will be interesting to determine whether the other genes in the Gr5a subfamily encode sweet taste receptors for other sugars (Dahanukar, 2001).
The molecular basis of sugar reception in Drosophila has been analyzed. The response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons has been defined. Using in situ hybridization and reporter gene expression, members of the Gr5a-related taste receptor subfamily were identified that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of these results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars (Dahanukar, 2007).
A major problem in neurobiology is how an animal decides what to eat. The fruit fly evaluates gustatory input to assess the nutritive value of a potential food source. In particular, the detection of sugars is a crucial factor in determining whether a food source is accepted. Despite its critical importance to the survival of the species, little is known about the molecular basis of sugar perception in the fly. A central goal in the field has been to define the receptors that mediate sugar detection (Dahanukar, 2007).
Sugars, salts, bitter compounds, and certain other molecules are detected by gustatory neurons, which are widely distributed in the body of the fly. Neurons that influence feeding behavior are present in the labellum as well as the tarsal segments of each of the legs. Activation of either labellar or tarsal gustatory neurons with a sugar solution results in proboscis extension, which is a component of feeding behavior (Dahanukar, 2007).
Gustatory neurons are housed in sensory hairs called sensilla. Each half of the labellum is covered with ~31 prominent taste hairs, arranged in a stereotypical pattern, and a number of smaller structures called taste pegs. Each of the 31 sensilla is typically innervated by four gustatory neurons and a single mechanosensory neuron. Physiological analysis has shown that one of the chemosensory neurons is activated by sucrose and other sugars, and has been referred to as the 'sugar' neuron. Another neuron is activated by salts and has been named the 'salt' neuron. A third neuron is activated by pure water but not by solutions of high osmolarity; it has been named the 'water' neuron. The fourth chemosensory neuron responds to aversive compounds such as caffeine, and has been named the (Dahanukar, 2007 and references therein).
In Drosophila, a large, highly diverse family of gustatory receptor (Gr) genes was identified by genomic analysis. The family consists of 60 genes encoding 68 predicted seven-transmembrane-domain receptors. In previous studies, Gr5a was identified as a receptor for trehalose, a disaccharide sugar. Gr5a is expressed in a large number of gustatory neurons in the labellum, and recent studies have shown that Gr5a serves as a marker for the sugar neuron in each sensillum. Bitter neurons express Gr66a, also a member of the Gr gene family, which is required for physiological and behavioral responses to caffeine. Promoter expression analysis of several other gustatory receptor genes in the labellum suggested that all of those tested were coexpressed with Gr66a in subsets of bitter neurons (Dahanukar, 2007 and references therein).
Axonal projections of Gr5a-positive and Gr66a-positive neurons have been mapped to the subesophageal ganglion (SOG) of the brain. The two classes of neurons project to nonoverlapping regions in the SOG, suggesting that at the first level of processing, attractive and aversive inputs may be segregated. Evidence that Gr5a neurons mediate attractive signals and Gr66a neurons mediate aversive signals was provided by expression of a capsaicin receptor in each of these classes of neurons. In the first instance, flies showed behavioral attraction to capsaicin, and in the second instance they were repelled by it (Dahanukar, 2007).
Gr5a-labeled neurons are responsive not only to trehalose, but to sucrose and other sugars. Physiological and behavioral analysis showed that sucrose response is not affected in flies lacking Gr5a, suggesting that these neurons express at least one other receptor; however, other receptors in sugar neurons were not identified (Dahanukar, 2007).
This study examined the responses of sugar neurons in the largest sensilla of the labellum, the 'L' sensilla. Of 50 compounds tested, including 34 diverse sugars, a small number were identified, primarily disaccharides and oligosaccharides, which elicit robust electrophysiological responses in sugar neurons. In situ hybridization and reporter gene expression determined that two other Gr genes, both phylogenetically related to Gr5a, are coexpressed with Gr5a in sugar neurons. Neurons expressing reporters of each receptor gene show distinct projection patterns, providing a mechanism by which information from different subpopulations of sugar cells in the periphery could be spatially represented in the brain (Dahanukar, 2007).
Having found coexpression of Gr5a-related genes in sugar neurons, mutants of Gr5a and two related genes were examined by electrophysiology and behavioral analysis. Gr5a was found to be required for detection of a small subset of sugars including trehalose. Deletion mutants lacking Gr64a shows that it is required for response to a complementary subset of sugars. Strikingly, flies lacking both Gr5a and Gr64a do not show electrophysiological or behavioral responses to any tested sugar. These results demonstrate that the sugars divide into two classes that are dependent either on Gr5a or on Gr64a for their responses. The simplest interpretation of these results is that these two receptors are capable of operating independently of each other in an individual sugar neuron, and that they constitute the primary basis of sugar reception in the fly (Dahanukar, 2007).
Sucrose generated the strongest responses among a panel of 50 compounds tested at 100 mM. Sucrose is present at comparable concentrations in many fruits, including citrus, peaches, and pineapples. Turanose, palatinose, and leucrose are all isomers of sucrose and also elicit responses of various strengths. Many of the sugars that evoke responses, including glucose and trehalose, are found in fruits and vegetables or in yeasts and may thus be encountered by the fly in its natural environment (Dahanukar, 2007).
The responses depend on sugar concentration as well as identity. The neurons are sensitive to a number of sugars over concentrations that span three orders of magnitude. The dose-response curves of different sugars, however, are distinct: they differ in threshold, slope, and maximal firing rate observed. Many of these sugars are present in fruits at concentrations of 100-300 mM, and at these concentrations the responses lie well within the dynamic range of the neurons. Surprisingly, responses to fructose and glucose, which are particularly abundant in fruits, are much weaker than those of sucrose, even when compared at concentrations that have equal caloric values. However, the concentrations of both fructose and glucose are typically higher than that of sucrose in fruits such as apples, bananas, and grapes, suggesting that sugar neurons may be most sensitive to changes in sugar concentrations over a range that is ecologically relevant (Dahanukar, 2007).
Molecular analysis has revealed coexpression of Gr61a and Gr64f with Gr5a, and genetic analysis of a double mutant has provided evidence for coexpression of Gr64a with Gr5a in sugar neurons. These results suggest that at least some labellar sugar neurons, including those of L-type sensilla, coexpress four receptors of the Gr5a subfamily (Dahanukar, 2007).
Molecular and genetic evidence indicates that Gr5a is expressed in essentially all labellar sensilla. Molecular analysis has provided evidence that Gr64f is also broadly expressed, and functional evidence suggests that Gr64a is as well. Specifically, an electrophysiological survey showed that all labellar sensilla in wild-type flies respond to sucrose, a sugar that acts via Gr64a. In a Gr5a;Gr64a mutant all morphological types of sensillum (L, M, S, I, P) showed no activity in response to sucrose; moreover, nearly all of the L, M, I, and P sensilla were tested, suggesting that Gr64a acts in all, or almost all, of the 31 sensilla on the labellum. Furthermore, the double mutants are also behaviorally unresponsive to sugars. Thus Gr5a and Gr64a seem likely to be expressed in all or almost all sugar neurons in the labellum, and perhaps Gr64f is as well (Dahanukar, 2007).
Gr61a, however, appeared to be restricted in its expression among labellar sensilla, both by in situ hybridization and by analysis of a Gr61a-GAL4 driver. These results suggest a subdivision of labellar sugar neurons into two classes based on the presence or absence of Gr61a. No function was defined for Gr61a; however, mutational analysis suggests that it does not play a role in responses to any of the sugars in the panel. It is possible that Gr61a is required for response to other sugars or sugar derivatives that have not yet been tested or for responses to another class of behaviorally attractive compounds. Further electrophysiological analysis with an expanded panel of tastants may provide insight into whether there are functional differences among sugar-sensing neurons and whether these differences correlate with the expression of Gr61a (Dahanukar, 2007).
Gr5a and Gr64a are both required for normal responses of sugar neurons, but for different subsets of sugars. Flies lacking Gr5a are severely defective in physiological and behavioral responses to one subset of sugars, including trehalose; flies lacking Gr64a are severely defective in responses to a complementary subset of sugars, including sucrose. All tested sugars fall into one of these two subsets. These results suggest that Gr5a and Gr64a function as distinct receptors in the same neurons, rather than as obligate heterodimeric coreceptors, as in the mammalian sugar receptor T1R2+T1R3 (Dahanukar, 2007).
It is possible that Gr5a and Gr64a function as heterodimeric receptors with other members of the Gr family, such as Gr64f. Two recent studies report deletions of part or all of the Gr64 cluster that result in reduced behavioral responses to trehalose; the phenotype is rescued by supplying a transgene containing five of the six receptors encoded by this cluster, but not by Gr64a alone. These data support the idea that one of the receptors in this cluster other than Gr64a may function in concert with Gr5a to mediate trehalose response. There is precedent for such interactions from Or proteins, which dimerize with the noncanonical receptor Or83b (Dahanukar, 2007).
The neat subdivision of sugars into those dependent on Gr5a and those dependent on Gr64a was surprising. A simple structural criterion to distinguish the two classes of sugars is not immediately evident upon inspection. The Gr64a-dependent sugars are remarkably diverse in structure, with some containing glucose units and some containing fructose subunits; they ranged in size from one to four subunits. Gr5a-dependent sugars also vary in size, subunit composition, and linkage types (Dahanukar, 2007).
In Gr5a mutants, there are some weak residual responses to the affected subset of sugars; likewise, in Gr64a mutants, some of the affected sugars continue to elicit some response. Since there is no residual response in the Gr5a;Gr64a double mutant, the simplest interpretation of these results is that each receptor provides the residual function observed when the other is eliminated, i.e., the two receptors exhibit some limited redundancy (Dahanukar, 2007).
Gr5a and Gr64a share 28% amino acid identity and 47% amino acid similarity. Both receptors are evolutionary conserved and are found in all of the 12 Drosophila species for which genome sequences are available, with the exception that D. pseudoobscura appears to have lost Gr5a. The receptor most closely related to Gr5a is Gr64f (40% amino acid identity), and the receptor most closely related to Gr64a is Gr61a (36% amino acid identity). Although evidence was found that Gr64f and Gr61a are both expressed in sugar neurons, no functions have been identifed for them. The possibility cannot be excluded of a role for Gr61a or Gr64f in response to compounds not tested, such as glycoproteins or glycolipids, or in neurons whose responses have not been measured, such as those of internal chemosensory cells. It is noted that in mammals, an amino acid receptor (T1R1+T1R3) comprises a subunit, T1R3, of the heterodimeric sugar receptor (T1R2+T1R3). However, L-type sensilla did not respond to any of 18 amino acids tested, making it unlikely that either Gr61a or Gr64f mediates responses to this class of compounds (Dahanukar, 2007).
Classic physiological and biochemical studies led to the proposal of a 'fructose' site in sugar-sensing neurons. The current studies provide a molecular and genetic identity to this site: fructose response is completely abolished by loss of Gr64a and is completely restored by the addition of a Gr64a transgene. These results also provide a molecular explanation for the earlier finding that sucrose responses were not affected in a Gr5a mutant. These results suggested the presence of another receptor within the sugar neuron, a receptor that has now been identified as Gr64a (Dahanukar, 2007).
It is noted that two recent studies have identified a role for members of the Gr64 cluster in mediating sugar responses, particularly that of Gr64a in response to sugars including sucrose, maltose, and glucose. Consistent with the observations, physiological and behavioral responses to sucrose were restored to wild-type levels in transgenic rescue experiments; no role was observed for Gr64a in glucose response. One of these studies also provided biochemical evidence that Gr5a-related receptors are expressed in sugar-sensitive neurons (Dahanukar, 2007).
In summary, the simplest interpretation of the results is that Gr5a and Gr64a are the primary sugar receptors in the labellum of the adult fly. Each is capable of mediating response to a subset of sugars independently of the other, and together they are able to identify the food sources that are sufficiently rich in caloric value as to sustain the life of the fly (Dahanukar, 2007).
Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. This study shows that the fruit fly selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway (Dus, 2011).
This taste-independent sugar-sensing pathway has several distinctive characteristics. First, this pathway is specifically associated with a starved state; taste-blind flies execute food-choice behavior after prolonged food deprivation of between 10 and 15 h of starvation. This time frame coincides with the onset of starvation-induced sleep suppression, indicating that these two behaviors might share a common metabolic trigger. Second, the taste-independent pathway operates on a different timescale from the gustatory pathway. Whereas WT flies made a food choice almost instantly, taste-blind flies chose sugars only after the ingestion of food. Third, this pathway responds to the nutritional content of sugars, but not to their orosensory value. Taste-blind flies chose metabolizable sugars over nonmetabolizable sugars and never consumed nonmetabolizable sugars. Furthermore, the fact that WT flies failed to distinguish a metabolizable sugar from a nonmetabolizable sugar, but shifted their preference to the metabolizable sugar after starvation, indicates that the taste-independent pathway is not an artifact associated with taste-blind flies, but functions in WT flies. Finally, the ability to detect the caloric content of sugars correlated under multiple experimental conditions with drops in hemolymph glycemia (Dus, 2011).
These results demonstrate that starvation directs the selection of nutrient-rich foods in the fly in the absence of the gustatory cues. Thus, as previously suggested in mice, postingestive cues can drive feeding behavior independently of gustatory information. The physiological factors that triggered the taste-independent food choices in mice are, however, unknown. In Drosophila, the internal energy state and carbohydrate metabolism play crucial roles in the metabolic sensing of food according to the results. A possible evolutionary purpose of taste-independent metabolic sensing is to ensure that animals select calorie-rich foods to quickly replenish energy, especially in times of food shortage (Dus, 2011).
How do starved sugar-blind flies preferentially ingest metabolizable sugar over nonmetabolizable sugar? It is plausible that sugar-blind flies are equally attracted to and feed on both sugars, but those on nonmetabolizable sugar resume foraging because of the lack of nutritional value in this sugar. These foraging flies are again equally attracted to both sugars, but those on nonmetabolizable sugar continue to forage until they find the correct food substrate. Food choice in this model is mediated by random selection and 'trapping' of the flies on the metabolizable sugar. Alternatively, sugar-blind flies might readily detect the metabolizable sugar without ingesting a large amount of food because nutrient information is rapidly conveyed to the brain within minutes of ingesting food. In this model, the flies select for metabolizable sugar over nonmetabolizable sugar by a metabolic sensor that operates on a fast timescale to mediate discrimination between the two sugar substrates. Tracking and monitoring the locomotor activity and feeding behavior that generates a preference for metabolizable sugar will address this question (Dus, 2011).
It is intriguing to speculate on the molecular nature of the metabolic sensor. This sensor could be expressed in a subset of neural, digestive, or other tissues. Among the organs and cells that have been proposed for their involvement in feeding regulation in the fly are the fat body, the insulin-producing cells (IPC), and the corpora cardiaca/allata complex. These cells may respond to the metabolic value of sugars in circulation, as seen with the glucose-excited and glucose-inhibited neuropeptide neurons in the arcuate nucleus of the mammalian hypothalamus. A model that explains how changes in circulating glucose levels alter the electrical and secretory properties of the hypothalamic glucose-responsive neurons could also describe how metabolizable sugars trigger the metabolic sensor. In mammals, glucose-sensitive cells detect glucose availability by responding to metabolites of glycolytic enzymes such as hexokinase or the energy-sensing AMP-activated protein kinase (Dus, 2011).
Almost all crucial metabolic functions in mammals are also conserved in Drosophila. During the past decade, researchers using the fruit fly as a model system for studying feeding behaviors and feeding-related disorders, including obesity, have shed much light on the molecular mechanisms of metabolism. By revealing the possibility of a metabolic sensing pathway in Drosophila, this study has introduced the possibility of understanding the molecular mechanism underlying this pathway. Identification of the cellular and genetic nature of this sensor might reveal the identity of the master switch that regulates many hunger-driven behaviors (Dus, 2011).
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date revised: 20 April 2012
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