Spatially restricted expression of candidate taste receptors in the Drosophila gustatory system

BACKGROUND: Taste is an important sensory modality in most animals. In Drosophila, taste is perceived by gustatory neurons located in sensilla distributed on several different appendages throughout the body of the animal. Here we show that the gustatory receptors are encoded by a family of at least 54 genes (Gr genes), most of which are expressed exclusively in a small subset of taste sensilla located in narrowly defined regions of the fly's body. RESULTS: BLAST searches with the predicted amino acid sequences of 6 7-transmembrane-receptor genes of unknown function and 20 previously identified, putative gustatory receptor genes led to the identification of a large gene family comprising at least 54 genes. We investigated the expression of eight genes by using a Gal4 reporter gene assay and found that five of them were expressed in the gustatory system of the fly. Four genes were expressed in 1%-4% of taste sensilla, located in well-defined regions of the proboscis, the legs, or both. The fifth gene was expressed in about 20% of taste sensilla in all major gustatory organs, including the taste bristles on the anterior wing margin. Axon-tracing experiments demonstrated that neurons expressing a given Gr gene project their axons to a spatially restricted domain of the subesophageal ganglion in the fly brain. CONCLUSIONS: Our findings suggest that each taste sensillum represents a discrete, functional unit expressing at least one Gr receptor and that most Gr genes are expressed in spatially restricted domains of the gustatory system. These observations imply the potential for high taste discrimination of the Drosophila brain.     

Taste perception and coding in Drosophila

BACKGROUND: Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.     

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Putative Drosophila odor receptor OR43b localizes to dendrites of olfactory neurons

To gain insight into the role of the recently identified Drosophila seven transmembrane receptor family, we analyzed the cellular and subcellular localization of a member of this family, OR43b. The OR43b receptor is expressed exclusively in a subset of olfactory neurons in the third antennal segment. Consistent with a direct role in odorant transduction, receptor protein is concentrated within the dendrites, but is also present in the axons of the olfactory neurons in which it is expressed. OR43b protein is only detectable relatively late in development suggesting it may not be required for synaptic target choice of the olfactory neurons in which it is expressed. Flies carrying deletions removing one copy of OR43b have the same number of OR43b positive cells in the antenna as flies with two copies, suggesting that simple allelic exclusion of odor receptors may not occur in Drosophila. We show the OR43b gene on the balancer chromosome SM5 is expressed at reduced levels and contains nucleotide polymorphisms predicted to alter two amino acids in the receptor, including an arginine(128) to proline substitution in the first extracellular loop. The subcellular localization of OR43b in olfactory neurons supports the idea that some of the recently identified family of seven transmembrane receptors are odor receptors, and that Drosophila and vertebrates may differ in the developmental processes used to establish the neuronal architecture of the olfactory system.     

阻断大鼠杏仁中央核AMPA受体对臂旁核味觉反应的影响

以往的研究表明,电刺激或损毁杏仁中央核明显改变臂旁核味觉神经元的活动。为了研究杏仁中央核内的兴奋性受体是否参与此调节,本实验应用细胞外记录方法,在乌拉坦麻醉的大鼠观察了杏仁中央核内微量注射6-氰基-7-硝基喹喔啉-2,3- 二酮(CNQX)前后臂旁核味觉神经元对四种基本味觉刺激反应的变化。结果表明,杏仁中央核内注射 CNQX 对 30% 的臂旁核神经元产生时间依赖性的抑制作用,此抑制作用以对盐酸和盐酸奎宁刺激引起的反应尤为明显(P<0.05)。根据对味觉刺激的优势反应,40% 的NaCl优势、30% 的HCl优势和20% 的奎宁优势反应神经元在注射CNQX 后对至少一种味觉刺激的反应降低;盐酸优势和奎宁优势反应神经元对各自的优势反应在杏仁中央核内注药后均明显降低(P<0.01)。相关性分析表明,在注射 CNQX 后,臂旁核味觉神经元对 NaCl 和其它三种味觉刺激物之间的分辨能力降低。以上结果表明,杏仁中央核内的AMPA 受体可能参与杏仁核对臂旁核味觉神经元的下行调控。     

Imaging taste responses in the fly brain reveals a functional map of taste category and behavior

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, we monitored taste cell activity in vivo 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, we inducibly activated different taste neurons by expression of an exogenous ligand-gated ion channel and 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.     

Genetic and functional subdivision of the Drosophila antennal lobe

Olfactory systems confer the recognition and discrimination of a large number of structurally distinct odor molecules. Recent molecular analysis of odorant receptor (OR) genes and circuits has led to a model of odor coding in which a population of olfactory sensory neurons (OSNs) expressing a single OR converges upon a unique olfactory glomerulus. Activation of the OR can thus be read out by the activation of its cognate glomerulus. Drosophila is a powerful system in which to test this model because the entire repertoire of 62 ORs can be manipulated genetically. However, a complete understanding of how fly olfactory circuits are organized is lacking. Here, we present a nearly complete map of OR projections from OSNs to the antennal lobe (AL) in the fly brain. Four populations of OSNs coexpress two ORs along with Or83b, and a fifth expresses one OR and one gustatory receptor (GR) along with Or83b. One glomerulus receives coconvergent input from two separate populations of OSNs. Three ORs label sexually dimorphic glomeruli implicated in sexual courtship and are thus candidate Drosophila pheromone receptors. This olfactory sensory map provides an experimental framework for relating ORs to glomeruli and ultimately behavior.     

Taste receptor cell-based biosensor for taste specific recognition based on temporal firing

Taste receptor cells are the taste sensation elements expressing sour, salty, sweet, bitter and umami receptors, respectively. There are cell-to-cell communications between different types of cells. Nevertheless, the mechanism of taste sensation and taste information coded by taste receptor cell is not well understood at present and it is a long-standing issue. In order to explore taste sensation and analyze taste-firing responses from another point of view, we present a promising biomimetic taste receptor cell-based biosensor. The temporal firing responses to different tastants are recorded. Meanwhile, we investigate the firing rate and temporal firing of taste receptor cells. The experimental results are consistent with that from patch clamp and molecular biology experiment. Firing rate is dependent on the concentration of stimulus. PCA analysis (principal component analysis) of the temporal firing responses shows that the responses from different types of taste receptor cells can be distinguished. Furthermore, exogenous ATP is applied to mimic the effects of transmitter ATP (adenosine triphosphate) released from type II cells onto type III cells. Both enhanced and inhibitory effects on spontaneous firing are observed. This novel biomimetic hybrid biosensor provides a potential solution to investigate the taste sensation and coding mechanisms in a non-invasive way.     

The molecular logic of olfaction in Drosophila

Drosophila fruit flies display robust olfactory-driven behaviors with an olfactory system far simpler than that of vertebrates. Endowed with 1300 olfactory receptor neurons, these insects are able to recognize and discriminate between a large number of distinct odorants. Candidate odorant receptor molecules were identified by complimentary approaches of differential cloning and genome analysis. The Drosophila odorant receptor (DOR) genes encode a novel family of proteins with seven predicted membrane-spanning domains, unrelated to vertebrate or nematode chemosensory receptors. There are on the order of 60 or more members of this gene family in the Drosophila genome, far fewer than the hundreds to thousands of receptors found in vertebrates or nematodes. DOR genes are selectively expressed in small subsets of olfactory neurons, in expression domains that are spatially conserved between individuals, bilaterally symmetric and not sexually dimorphic. Double in situ RNA hybridization with a number of pairwise combinations of DOR genes fails to reveal any overlap in gene expression, suggesting that each olfactory neuron expresses one or a small number of receptor genes and is therefore functionally distinct. How is activation of such a subpopulation of olfactory receptor neurons in the periphery sensed by the brain? In the mouse, all neurons expressing a given receptor project with precision to two of 1800 olfactory bulb glomeruli, creating a spatial map of odor quality in the brain. We have employed DOR promoter transgenes that recapitulate expression of endogenous receptor to visualize the projections of individual populations of receptor neurons to subsets of the 43 glomeruli in the Drosophila antennal lobe. The results suggest functional conservation in the logic of olfactory discrimination from insects to mammals.     

Sweet and bitter tastants specific detection by the taste cell-based sensor

Sweet and bitter tastants specific detection by cell-based sensor is investigated in this paper. Human enteroendocrine NCI-H716 cells, expressing G protein-coupled receptors and sweet receptors (type 1, member 2/type 1, member 3), and human enteroendocrine STC-1 cells, expressing G protein-coupled receptors and bitter receptors (type 2 members) are used as sensing devices. The HEK-293 cells, without taste receptor expression, are used as negative control. The electrochemical impedance spectrum data is recorded and processed by bistable stochastic resonance for signal-to-noise ratio calculation. NCI-H716 cell-based sensor selectively responds to sweeteners and sweet tastant mixtures. STC-1 cell-based sensor selectively responds to bitter tastants and bitter tastant mixtures. The tastants species and concentrations can be decided by signal-to-noise ratio parameters. HEK-293 cell-based sensor lacks the tastants discriminating ability. The taste cell-based sensor is easy to prepare and operate. This work offers a useful way in gustatory mechanism research.     

Two Gr genes underlie sugar reception in Drosophila

We have analyzed the molecular basis of sugar reception in Drosophila. We define the response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons. Using in situ hybridization and reporter gene expression, we identify members of the Gr5a-related taste receptor subfamily 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 our 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.     

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.     

A taste receptor required for the caffeine response in vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.     

Insect chemoreception

Insect chemoreception is mediated by a large and diverse superfamily of seven-transmembrane domain receptors. These receptors were first identified in Drosophila, but have since been found in other insects, including mosquitoes and moths. Expression and functional analysis of these receptors have been used to identify receptor ligands and to map receptors to functional classes of neurons. Many receptors detect general odorants or tastants, whereas some detect pheromones. The non-canonical receptor Or83b, which is highly conserved across insect orders, dimerizes with odorant and pheromone receptors and is required for efficient localization of these proteins to dendrites of sensory neurons. These studies provide a foundation for understanding the molecular and cellular basis of olfactory and gustatory coding.     

Olfactory neurons expressing identified receptor genes project to subsets of glomeruli within the antennal lobe of Drosophila melanogaster

We have used green fluorescent protein to trace the projection patterns of olfactory neurons expressing identified candidate odorant receptors to the brain of Drosophila. At the periphery, receptor expression correlates with specific sense-organ subtype, independent of location on the antennal surface. The majority of neurons expressing a given receptor converge onto one or two major glomeruli as described previously. However, we detected a few additional glomeruli, which are less intensely innervated and also tend to be somewhat variable. This means that functionally similar olfactory neurons connect to small subsets of glomeruli rather than to a single glomerulus as believed previously. This finding has important implications for our understanding of odor coding and the generation of olfactory behavior.     

The independence and integration of olfaction and taste

To study the influences of smell on the scaling of taste, andvice versa, an unobtrusive device was developed which permitsthe independent varying of the olfactory and gustatory componentsof a flavor compound. With instant coffee and the method ofmagnitude estimation, the slopes of the olfactory, taste, andflavor curves produced by this device were similar to thoseproduced by more traditional delivery systems. To study theinteraction of taste on smell and vice versa, subjects scaledolfactory stimuli against different taste backgrounds and tastestimuli against different odorant backgrounds. Taste backgroundsdid not significantly alter the scaling of olfaction. Similarly,olfactory backgrounds did not significantly alter the scalingof taste. Finally, subjects scaled overall intensity (flavor)when presented with all combinations of four concentrationsof odorants and tastants. Smell and taste were shown to contributeindependently to the estimation of overall intensity.     

Using biosensors to detect the release of serotonin from taste buds during taste stimulation

CHO cells transfected with high-affinity 5HT receptors were used to detect and identify the release of serotonin from taste buds. Taste cells release 5HT when depolarized or when stimulated with bitter, sweet, or sour tastants. Sour- and depolarization-evoked release of 5HT from taste buds is triggered by Ca2+ influx from the extracellular fluid. In contrast, bitter- and sweet-evoked release of 5HT is triggered by Ca2+ derived from intracellular stores.     

Sex-specific non-pheromonal taste receptors in Drosophila

Taste receptors have recently been reported in Drosophila [1,2], but little is known of the relation between receptor and response. Morphological studies of the distribution of chemosensory sensilla indicate that the fruit fly has two major sites of gustation: the proboscis and the legs [3]. The taste sensilla on both these sites are similar in structure and each sensillum generally houses four gustatory neurons [4]. Early anatomical observations have demonstrated a sexual dimorphism in the number of tarsal sensilla [5] and in their central projections [6]. We measured the electrophysiological responses of the prothoracic taste sensilla to non-pheromonal substances--salts, sugars and water--and found a clear sexual dimorphism. From the response profile of individual sensilla, we were able to distinguish three types of tarsal sensilla in females as against only two types in males. The female-specific type, which responded specifically to sugar, was absent in males except when male gustatory neurons were genetically feminised. The fact that tarsal gustatory hairs exhibit a sexual dimorphism that affects the perception of non-pheromonal compounds suggests that sexual identity is more complex than has previously been thought [7,8].