Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster

Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials.     

Sugar receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.     

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.     

A Drosophila Gustatory Receptor Essential for Aversive Taste and Inhibiting Male-to-Male Courtship

Contact chemosensation is required for several behaviors that promote insect survival. These include evasive behaviors such as suppression of feeding on repellent compounds, known as antifeedants, and inhibition of male-to-male courtship. However, the gustatory receptors (GRs) required for responding to nonvolatile avoidance chemicals are largely unknown. Exceptions include Drosophila GR66a and GR93a, which are required to prevent ingestion of caffeine [1] and [2], and GR32a, which is necessary for inhibiting male-to-male courtship [3]. However, GR32a is dispensable for normal taste. Thus, distinct GRs may function in sensing avoidance pheromones and antifeedants. Here, we describe the requirements for GR33a, which is expressed widely in gustatory receptor neurons (GRNs) that respond to aversive chemicals. Gr33a mutant flies were impaired in avoiding all nonvolatile repellents tested, ranging from quinine to denatonium, lobeline, and caffeine. Gr33a mutant males also displayed increased male-to-male courtship, implying that it functioned in the detection of a repulsive male pheromone. In contrast to the broadly required olfactory receptor (OR) OR83b, which is essential for trafficking other ORs [4], GR66a and GR93a are localized normally in Gr33a mutant GRNs. Thus, rather than regulating GR trafficking, GR33a may be a coreceptor required for sensing all nonvolatile repulsive chemicals, including tastants and pheromones.     

Rat gustatory neurons in the geniculate ganglion express glutamate receptor subunits

Taste receptor cells are innervated by primary gustatory neurons that relay sensory information to the central nervous system. The transmitter(s) at synapses between taste receptor cells and primary afferent fibers is (are) not yet known. By analogy with other sensory organs, glutamate might a transmitter in taste buds. We examined the presence of AMPA and NMDA receptor subunits in rat gustatory primary neurons in the ganglion that innervates the anterior tongue (geniculate ganglion). AMPA and NMDA type subunits were immunohistochemically detected with antibodies against GluR1, GluR2, GluR2/3, GluR4 and NR1 subunits. Gustatory neurons were specifically identified by retrograde tracing with fluorogold from injections made into the anterior portion of the tongue. Most gustatory neurons in the geniculate ganglion were strongly immunoreactive for GluR2/3 (68%), GluR4 (78%) or NR1 (71%). GluR1 was seen in few cells (16%). We further examined if glutamate receptors were present in the peripheral terminals of primary gustatory neurons in taste buds. Many axonal varicosities in fungiform and vallate taste buds were immunoreactive for GluR2/3 but not for NR1. We conclude that gustatory neurons express glutamate receptors and that glutamate receptors of the AMPA type are likely targeted to synapses within taste buds.     

The molecular and cellular basis of bitter taste in Drosophila

The extent of diversity among bitter-sensing neurons is a fundamental issue in the field of taste. Data are limited and conflicting as to whether bitter neurons are broadly tuned and uniform, resulting in indiscriminate avoidance of bitter stimuli, or diverse, allowing a more discerning evaluation of food sources. We provide a systematic analysis of how bitter taste is encoded by the major taste organ of the Drosophila head, the labellum. Each of 16 bitter compounds is tested physiologically against all 31 taste hairs, revealing responses that are diverse in magnitude and dynamics. Four functional classes of bitter neurons are defined. Four corresponding classes are defined through expression analysis of all 68 gustatory taste receptors. A receptor-to-neuron-to-tastant map is constructed. Misexpression of one receptor confers bitter responses as predicted by the map. These results reveal a degree of complexity that greatly expands the capacity of the system to encode bitter taste.     

Hedonic taste in Drosophila revealed by olfactory receptors expressed in taste neurons

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, we tested the assumption 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, we show 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.     

The role of contact chemoreception in the host location process of an egg parasitoid

Taste allows insects to detect palatable or toxic foods, identify a mate, and select appropriate oviposition sites. The gustatory system strongly contributes to the survival and reproductive success of many species, yet it is rarely studied in insect parasitoids. In order to locate and assess a host in which they will lay their eggs, female wasps actively search for chemical cues using their sensory organs present mainly on the antennae. In this paper, we studied the role of antennal taste sensilla chaetica in the perception of contact semiochemicals in Trissolcus brochymenae (Hymenoptera: Platygastridae), an egg parasitoid of the brassicaceae pest Murgantia histrionica (Heteroptera: Pentatomidae). Methanolic extracts obtained from male and female hosts elicited action potentials in taste neurons housed in antennal sensilla chaetica, indicating that these sensilla are involved in the perception of non volatile host kairomones. In behavioural assays, wasp females displayed an intense searching behaviour in open arenas treated with host extracts, thus confirming that these kairomones are soluble in polar solvents. We further investigated the extracts by Gas Chromatography-Mass Spectrometry (GC-MS) and found that they contain several compounds which are good candidates for these contact kairomones. This study contributes to better understanding contact chemoreception in egg parasitoids and identifying gustatory receptor neurons involved in the host location process.     

Neural coding of gustatory information.

The nervous system encodes information relating chemical stimuli to taste perception, beginning with transduction mechanisms at the receptor and ending in the representation of stimulus attributes by the activity of neurons in the brain. Recent studies have rekindled the long-standing debate about whether taste information is coded by the pattern of activity across afferent neurons or by specifically tuned 'labeled lines'. Taste neurons are broadly tuned to stimuli representing different qualities and are also responsive to stimulus intensity and often to touch and temperature. Their responsiveness is also modulated by a number of physiological factors. In addition to representing stimulus quality and intensity, activity in taste neurons must code information about the hedonic value of gustatory stimuli. These considerations suggest that individual gustatory neurons contribute to the coding of more than one stimulus parameter, making the response of any one cell meaningful only in the context of the activity of its neighbors.     

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.     

Adaptive variability of the gustatory system receptor part of the carp Cyprinus carpio (Cyprinidae, Teleostei) after chronic anosmia

To confirm the existence in fish of the olfactogustatory interactions of adaptive significance, the study was performed of reactions of the receptor part of the carp gustatory system to the complete olfactory deafferentation with use of the method of scanning electron microscopy. Peculiarities of morphological changes of the taste receptor apparatus of the external and oral localization were revealed, and the dynamic of development of the taste receptor adaptive changes was traced for 1–9 months after production of anosmia. Receptors of the intraoral gustatory system had reactions of low statistical significance which were delayed in time by 5–6 months. The receptor apparatus of the external gustatory system receptors of the intraoral taste system showed pronounced hypertrophic changes that appeared since 2–3 months after the beginning of anosmia. The changes consisted in an increase of the area of the taste bud (TB) sensory field and of their number in the sensory zones of the maxillary and mandibular parts. The taste receptors located in the maxillary barbels and in the mandible gular zone had the most expressed reactions to the olfactory deafferentation. In the fish with anosmia, formation of the additional structures of the “external taste” was detected in the form of epidermal processes under the lower lip, which were covered with taste papillae. Thus, it has been shown that the fish olfactory and gustatory systems are functionally interrelated; under conditions of olfactory deprivation the external gustatory system is able to undergo compensatory morphofunctional changes aimed at vicariation of the lost distant chemical reception.     

G protein-coupled receptors in human fat taste perception

In contrast to carbohydrates and proteins, which are detected by specialized taste receptors in the forms of their respective building blocks, sugars, and L-amino acids, the third macronutrient, lipids, has until now not been associated with gustatory receptors. Instead, the recognition of fat stimuli was believed to rely mostly on textural, olfactory, and postingestive cues. During the recent years, however, research done mainly in rodent models revealed an additional gustatory component for the detection of long-chain fatty acids (LCFAs), the main taste-activating component of lipids. Concomitantly, a number of candidate fat taste receptors were proposed to be involved in rodent's gustatory fatty acid perception. Compared with rodent models, much less is known about human fat taste. In order to investigate the ability of the human gustatory system to respond to fat components, we performed sensory experiments with fatty acids of different chain lengths and derivatives thereof. We found that our panelists discriminated a "fatty" and an irritant "scratchy" taste component, with the "fatty" percept restricted to LCFAs. Using functional calcium-imaging experiments with the human orthologs of mouse candidate fat receptors belonging to the G protein-coupled receptor family, we correlated human sensory data with receptor properties characterized in vitro. We demonstrated that the pharmacological activation profile of human GPR40 and GPR120, 2 LCFA-specific receptors associated with gustatory fat perception in rodents, is inconsistent with the "scratchy" sensation of human subjects and more consistent with the percept described as "fatty." Expression analysis of GPR40 and GPR120 in human gustatory tissues revealed that, while the GPR40 gene is not expressed, GPR120 is detected in gustatory and nongustatory epithelia. On a cellular level, we found GPR120 mRNA and protein in taste buds as well as in the surrounding epithelial cells. We conclude that GPR120 may indeed participate in human gustatory fatty acid perception.     

A gustatory receptor tuned to d-fructose in antennal sensilla chaetica of Helicoverpa armigera

Insect gustatory systems play important roles in food selection and feeding behaviors. In spite of the enormous progress in understanding gustation in Drosophila, for other insects one of the key elements in gustatory signaling, the gustatory receptor (GR), is still elusive. In this study, we report that fructose elicits behavioral and physiological responses in Helicoverpa armigera (Harm) to fructose and identify the gustatory receptor for this sugar. Using the proboscis extension reflex (PER) assays we found that females respond to fructose following stimulation of the distal part of the antenna, where we have identified contact chemosensilla tuned to fructose in tip recording experiments. We isolated three full-length cDNAs encoding candidate HarmGRs based on comparison with orthologous GR sequences in Heliothis virescens and functionally characterized the responses of HarmGR4 to 15 chemicals when this receptor was expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Among the tastants tested, the oocytes dose-dependently responded only to d-fructose (EC₅₀ = 0.045 M). By combining behavioral, electrophysiological and molecular approaches, these results provide basic knowledge for further research on the molecular mechanisms of gustatory reception.     

Topological and functional characterization of an insect gustatory receptor

Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

Synthesis and structure–activity relationships of novel indazolyl glucocorticoid receptor partial agonists

SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors.     

Immunohistochemical detection of TAS2R38 protein in human taste cells

The sense of taste plays an important role in the evaluation of the nutrient composition of consumed food. Bitter taste in particular is believed to serve a warning function against the ingestion of poisonous substances. In the past years enormous progress was made in the characterization of bitter taste receptors, including their gene expression patterns, pharmacological features and presumed physiological roles in gustatory as well as in non-gustatory tissues. However, due to a lack in TAS2R-specifc antibodies the localization of receptor proteins within gustatory tissues has never been analyzed. In the present study we have screened a panel of commercially available antisera raised against human bitter taste receptors by immunocytochemical experiments. One of these antisera was found to be highly specific for the human bitter taste receptor TAS2R38. We further demonstrate that this antibody is able to detect heterologously expressed TAS2R38 protein on Western blots. The antiserum is, however, not able to interfere significantly with TAS2R38 function in cell based calcium imaging analyses. Most importantly, we were able to demonstrate the presence of TAS2R38 protein in human gustatory papillae. Using double immunofluorescence we show that TAS2R38-positive cells form a subpopulation of PLCbeta2 expressing cells. On a subcellular level the localization of this bitter taste receptor is neither restricted to the cell surface nor particularly enriched at the level of the microvilli protruding into the pore region of the taste buds, but rather evenly distributed over the entire cell body.     

The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

GR64 genes are a cluster of neuronally expressed gustatory receptors normally involved in taste sensation in Drosophila melanogaster. This study reveals a surprising role for these receptors in regulating proteostasis and cell survival in epithelial cells exposed to proteotoxic stress.     

Gustatory responses to feeding‐ and non‐feeding‐stimulant chemicals, with an emphasis on amino acids, in rainbow trout

Specific receptor and fibre types of rainbow trout Oncorhynchus mykiss involved in the detection and discrimination of amino acids and a heterogeneous collection of compounds were investigated by recording the electrical activity of the maxillary branch of the facial nerve innervating taste buds inside the upper jaw. Proline (Pro), alanine (Ala), leucine (Leu), betaine (Bet) and 2‐amino‐3‐guanidinopropionic acid (Agp) were the major amino acids detected by the gustatory system. The two experimental approaches, concentration‐response curves and cross‐adaptations, showed that all amino acids were detected by three independent receptor types: Pro ‐, Agp/Bet ‐ and Leu ‐receptors. Bile acids, the most potent stimulants recorded, were detected by a single receptor type independent of those for amino acids, with threshold concentrations of 10⁻¹² M. Strychnine, quinine and tetrodotoxin may have partially shared a single receptor mechanism. The gustatory sensibility narrowly tuned towards the amino acid spectrum compared to those for a diverse array of non‐feeding stimulant chemicals, combined with feeding behaviour triggered primarily by vision and olfaction, suggest that in rainbow trout, and possibly other salmonid species, gustatory chemical cues, in addition to food finding and intake, play an important role in detecting poisonous prey and substances.