Drosophila gene families: Taste receptors

The Interactive Fly

Zygotically transcribed genes

Taste receptors and taste processing: Antenna, The labial palps, labellum, legs and subesophageal ganglion and central brain

  • G-protein coupled receptors
  • A map of sensilla and neurons in the taste system of Drosophila larvae
  • Characterization of Drosophila taste receptors
  • Taste perception and coding in Drosophila
  • Behavioral analysis of bitter taste perception in Drosophila larvae
  • Gustatory receptor 21a and Gustatory receptor 63a confer CO2-chemosensation in Drosophila
  • Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants
  • Two Gr genes underlie sugar reception in Drosophila
  • Deciphering the genes for taste receptors for fructose in Drosophila
  • Enhancing perception of contaminated food through acid-mediated modulation of taste neuron responses
  • Presynaptic gain control drives sweet and bitter taste integration in Drosophila
  • Starvation-induced depotentiation of bitter taste in Drosophila
  • Taste representations in the Drosophila brain
  • Imaging taste responses in the fly brain reveals a functional map of taste category and behavior
  • Activity-dependent plasticity in an olfactory circuit
  • An inhibitory sex pheromone tastes bitter for Drosophila males
  • Molecular and cellular organization of the taste system in the Drosophila larva
  • Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity
  • The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors
  • The molecular and cellular basis of bitter taste in Drosophila
  • Functional dissociation in sweet taste receptor neurons between and within taste organs of Drosophila
  • A Drosophila gustatory receptor required for strychnine sensation
  • Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing
  • Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila
  • A fructose receptor functions as a nutrient sensor in the Drosophila brain
  • Drosophila learn opposing components of a compound food stimulus
  • Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila
  • Dopaminergic modulation of sucrose acceptance behavior in Drosophila
  • Food experience-induced taste desensitization modulated by the Drosophila TRPL channel
  • Gustatory receptor 28b controls rapid warmth avoidance
  • Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1
  • Histone methyltransferase G9a is a key regulator of the starvation-induced behaviors in Drosophila melanogaster
  • Drosophila life span and physiology are modulated by sexual perception and reward
  • Gr33a modulates Drosophila male courtship preference
  • High-NaCl perception in Drosophila melanogaster
  • Representations of taste modality in the Drosophila brain
  • Immediate perception of a reward is distinct from the reward's long-term salience
  • Bitter taste receptors confer diverse functions to neurons
  • Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain
  • Mechanosensory neurons control sweet sensing in Drosophila
  • Long-range projection neurons in the taste circuit of Drosophila
  • The taste response to ammonia in Drosophila
  • Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster
  • Neofunctionalization of "Juvenile Hormone Esterase Duplication" in Drosophila as an odorant-degrading enzyme towards food odorants
  • Physiological responses of the Drosophila labellum to amino acids
  • Molecular and cellular organization of taste neurons in adult Drosophila pharynx
  • Internal amino acid state modulates yeast taste neurons to support protein homeostasis in Drosophila
  • The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool
    Gustatory Receptors



    A map of sensilla and neurons in the taste system of Drosophila larvae

    In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. This study consisted of a systematic anatomical and molecular analysis of the TO. Morphological types of TO sensilla were precisely defined taking advantage of volume electron microscopy and 3D image analysis. The presence of five external types of sensilla were corroborated: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes: three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, the repertoire of receptor genes for each sensillum was determined by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. A map of the TO was constructed, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment (Rist, 2017).

    A major challenge in the field of neurobiology is to find out how animals detect, discriminate, and respond to the huge variety of sensory stimuli in the environment. Reception of those stimuli starts in the periphery, where specialized receptor proteins recognize certain stimuli and translate these into neuronal activity patterns that are then reported to the central nervous system. In insects, the first structures that come into contact with external stimuli, like tastants, are the sensilla on the body surface. These small organs are formed by cuticle structures associated with sensory neurons for the reception of chemical or mechanical stimuli. Sensilla might form long or short hairs, small pegs or shallow domes. Despite variations in form and shape, across insect species structural properties of sensilla were identified that are required for a particular sensory modality. The reception of chemical molecules requires contact between the molecule and the specific receptors on the dendritic membrane. Therefore, the cuticle wall of olfactory sensilla invariably is perforated by multiple pores that enable olfactory molecules to enter. Inside the sensillum, the dendrites of olfactory receptor neurons most often are highly branched but might also be unbranched. In contrast, reception of taste molecules seems to require only a single but larger pore opening that is located at the tip of taste sensilla (terminal pore). The taste-sensitive neurons do not branch and most often are accompanied by one mechanosensitive neuron (Rist, 2017).

    The major taste sensilla of cyclorrhaphan fly larvae are supposed to cluster in the terminal organ (TO) at the tip of the cephalic lobes. A detailed description of TO ultrastructure has been provided for Musca domestica larvae. Hence, the TO consists of 14 external sensilla grouped into five types which divide into a dorso-lateral group and a distal group. Gustatory function was inferred for most of the sensilla due to relevant structural properties. Investigations on the ultrastructure of the TO of Drosophila melanogaster larvae, however, have been rare. This paucity of information might derive from the technical difficulty to examine sensillar ultrastructure in three dimensions using transmission electron microscopy (TEM). Only recently a number of advanced techniques to effectively acquire volume electron microscopy data have been introduced to the field of biology (Rist, 2017).

    Nevertheless, in Drosophila larvae, a function in taste reception of the TO was confirmed by behavioral, physiological and anatomical studies. Importantly, a receptor-to-neuron map of gustatory receptors (Grs) and neurons of the TO was constructed using the GAL4/UAS system. In this map, eight neurons of the TO are defined by their combinatorial expression of 28 Gr-GAL4 drivers. Subsequent studies assigned a role to mapped TO neurons in the reception of bitter compounds and also of other tastants. In addition to Grs, neurons of the TO might express Ionotropic receptors (Irs) and receptors of the Pickpocket receptor (Ppk) gene family (Rist, 2017).

    Despite the increasing number of investigations on the neuronal and molecular basis of larval taste reception, the functional organization of the TO is still unclear. In particular, the specific stimuli for most neurons of the TO remain unknown. Previous work has not defined which receptor neurons are housed in individual sensilla nor in which sensilla individual receptor genes are expressed. Thus, it has been difficult to carry out well-designed experiments on the function of sensory neurons and sensilla (Rist, 2017).

    This study define morphological types of TO sensilla based on their ultrastructure taking advantage of focused-ion-beam scanning-electron microscopy (FIB-SEM) that allows for automated acquisition of serial sections with highest resolution. Further, receptor genes of the Ir, Ppk and Gr family were mapped to sensilla using the GAL4/UAS system. A neuron-to-sensillum map was created that lays a foundation for subsequent investigations on how Drosophila larvae use sensory information to perceive their environment (Rist, 2017).

    The results clarify and alter several of the previous findings by light and electron microscopy. The TO consists of two groups of sensilla, the distal and the dorso-lateral group. In addition to the external sensilla of both groups, mechanosensitive scolopidia sensilla lie internally below the cuticle surface of the TO. Neurons of the TO-distal group cluster in the TO-ganglion. Neurons of the dorso-lateral group lie in the DO-ganglion, in the TO-ganglion or independently of both ganglia. The number of neurons belonging to the TO, and DO has been estimated in a previous study according to anti-Elav-stainings of neuronal cell nuclei. A total of 32-33 anti-Elav-stained neurons was in the ganglion of the TO. Of these, 30 neurons were assumed to be associated with the TO-distal group including 26 neurons that belong to the external sensilla and four neurons that belong to internal scolopidia sensilla. The FIB-SEM analysis in the present study is restricted to the external sensilla of the TO. It was found that the external sensilla of the TO-distal group comprise in total 28 neurons by counting the dendrites in the sensilla. This number is slightly different from the previous study but consistent with another study that also examined the ultrastructure of external sensilla of the TO of Drosophila larvae. The external sensilla of the dorso-lateral group comprise five neurons in agreement with all previous studies (Rist, 2017).

    Twenty-six neurons of the TO-distal and dorso-lateral group were assumed to be gustatory according to interpretations on the fine structure of associated sensilla. This study considered 25 neurons of the TO to be gustatory based on their structural and molecular characteristics. Despite the similar number, in contrast to the previous studies this study include the neurons of the knob sensilla and exclude the neurons of Pmodand the spot sensilla. Pmod and spot sensilla most likely are not gustatory because they lack fine structural characteristics of gustatory sensilla and also lack chemosensory receptor gene expression. This study waw able to define 19 of the 25 putative chemosensory neurons by GAL4 driver expression thereby expanding the established receptor-to-neuron map by 11 neurons. Six neurons remain 'orphan'. These neurons might be labeled by one of the GAL4 drivers which could not be mapped to a single neuron but to the sensillum, or they might express receptor genes not covered by the screened GAL4 lines (Rist, 2017).

    Characterization of Drosophila taste receptors

    A computer algorithm that seeks proteins with particular structural properties, as opposed to proteins with particular sequences, has identified a large family of candidate odorant receptors from the Drosophila genomic database. Further analysis of genes identified by this algorithm has revealed one gene that defines a distinct large family of membrane proteins; 43 members of this family have been identified in the first 60% of the Drosophila genome that has been sequenced thus far. If the sequenced part of the genome is representative, then extrapolation suggests that the entire genome would encode on the order of 75 proteins, a figure comparable to the estimated number of odorant receptors. The previously unidentified family of proteins shows no sequence similarities to any known odorant receptors or to any other known proteins. This family has been tentatively named the gustatory receptor (GR) family, with each individual gene named according to its cytogenetic location in the genome. Thus, the GR59D.1 and GR59D.2 genes, which are abbreviated here as 59D.1 and 59D.2, refer to two family members located in cytogenetic region 59D on the second chromosome (Clyne, 2000).

    The amino acid sequences of 19 members of the GR family indicate the high degree of sequence divergence. Sequence alignment reveals only one residue conserved among a select group of the family and only 24 residues conserved among more than half of the genes in this group. Fifteen of these conserved residues lie in the vicinity of the COOH-terminus. Amino acid identity between individual genes ranges from a maximum of 34% to <10%. By contrast, other features of the gene family show substantial conservation. The positions of a number of introns are conserved, suggesting that the family originated from a common ancestral gene. Overall sequence length, ~380 amino acids, is another common feature. All of the genes encode approximately seven predicted transmembrane domains, a feature characteristic of G protein-coupled receptors (GPCRs). All 43 of the predicted GR gene products have been identified as GPCRs by an algorithm trained to distinguish between GPCRs and other multitransmembrane proteins (Clyne, 2000).

    The genes are widely dispersed in the genome, but at the same time, many are found in clusters. The two largest clusters each contain four genes; there are also several clusters of two or three genes. Genes within these clusters are closely spaced, with intergenic distances ranging from 150 to 450 base pairs (bp) in all cases for which the data are currently available. There is no rule specifying the orientation of genes within clusters, unlike the case of the Drosophila odorant receptors, in which genes within a cluster are in the same orientation in all clusters examined (Clyne, 2000).

    An unusual form of alternative splicing occurs in at least two chromosomal locations. Four large exons in cytogenetic region 39D each contain sequences specifying six predicted transmembrane domains, followed by three small exons that together specify a putative seventh transmembrane domain and the COOH-terminus. Reverse transcription-polymerase chain reaction (RT-PCR) analysis reveals that each of the four large exons is spliced to the smaller exons, thereby generating four predicted seven transmembrane domain proteins. These four proteins are thus distinct through the first six transmembrane domains and identical in the seventh and in the COOH-terminal sequences. Likewise, in cytogenetic region 23A, there are two large exons, each of which specifies six transmembrane domains and each is spliced to two small exons that together encode a seventh transmembrane domain and the COOH-terminus. Thus, the gene in region 23A encodes two related proteins. This pattern of splicing, in which alternative large 5' exons encoding most of the protein are joined to common short 3' exons encoding only a small portion of the protein, is unusual among genes encoding GPCRs and proteins in general. This pattern of splicing provides a mechanism at a single locus for generating products that exhibit a pattern observed for this family in general: extreme diversity among all sequences of the proteins except in a small region in the vicinity of the COOH-terminus (Clyne, 2000).

    To assess the tissue specificity of expression, RT-PCR was performed with primers that span introns in the coding regions. Of the 19 transcripts tested, 18 are expressed in the labellum, the major gustatory organ of the fly. Moreover, for most of these genes, expression is labellum-specific: only 1 of the 19 yielded amplification products from heads depleted of taste organs and only 2 showed expression in the thorax, which contains the thoracic nervous system but no characterized taste sensilla. Likewise, expression in several other tissues, including the abdomen, wings, and legs, is limited to a small fraction of genes (Clyne, 2000).

    To further analyze gene expression by in situ hybridization, 12 GR transcripts were used as probes. Each probe was used individually and in mixtures of multiple probes. Sequences encompassing all, or nearly all, of each transcript were used, and several diverse methods of signal amplification and detection were used, with a variety of experimental conditions. None of the genes shows detectable expression in any tissue, including the taste organs. As positive controls, the pheromone-binding protein-related protein-2 gene (pbprp-2), which may encode a carrier of hydrophobic molecules, shows hybridization in taste sensilla on the labellum, and the Drosophila olfactory receptor gene 22A.2 (DOR22A.2) hybridizes to olfactory sensilla on the antenna. The simplest interpretation of these results is that expression levels of the GR genes are exceedingly low. Consistent with this interpretation is the fact that no expressed sequence tags have been identified for any of the 43 GR transcripts (Clyne, 2000).

    To further analyze the tissue specificity of GR expression, a microdissection experiment was performed in which the labral sense organ (LSO), a small taste organ that lines the pharynx, was surgically excised from each of 50 animals. The LSO consists of a very limited number of cells and is highly enriched in taste neurons; it does not, for example, contain muscle cells. By RT-PCR amplification, the expression of seven GR transcripts in this taste organ was detected. These results indicate that expression of the GR family extends to include at least one additional taste organ besides the labellum. The data are also fully consistent with the notion that the GR genes are expressed in taste neurons (Clyne, 2000).

    To confirm the gene expression in taste receptor neurons, a Drosophila mutant, pox-neuro70 (poxn70), in which chemosensory bristles are transformed into mechanosensory bristles, was used. Specifically, in poxn70, which behaves as a null mutation with respect to adult chemosensory organs, chemosensory bristles are transformed into mechanosensory bristles with respect to various morphological and developmental criteria. In particular, most chemosensory bristles in wild-type Drosophila are innervated by five neurons: four chemosensory neurons and one mechanosensory neuron. In contrast, wild-type mechanosensory bristles contain a single mechanosensory neuron. In chemosensory bristles transformed to mechanosensory bristles by poxn70, the number of neurons is reduced from five to one. It was predicted that if the GR family is in fact expressed in the chemosensory neurons of taste sensilla, their expression would likely be eliminated in the poxn70 mutant. Consistent with this prediction, 18 of 19 GR transcripts examined were not expressed in the labellum of the poxn70 mutant. These results indicate that the GR gene family is expressed in labellar chemosensory neurons (Clyne, 2000).

    The large size of this protein family likely reflects the diversity of compounds that flies can detect. The labellar hairs of larger flies are not only sensitive to a variety of simple and compound sugars, but also to a wide variety of other molecules, such as amino acids. Behavioral studies have shown that Drosophila is sensitive to quinine, which is perceived by humans as bitter, and other insects have been shown to be sensitive to an array of structurally diverse bitter compounds. Moreover, an individual insect taste receptor cell can respond to a broad range of structurally heterogeneous alkaloids and other bitter molecules. The extreme diversity of these receptors may not only reflect diversity among the ligands that they bind, but also diversity in the signal transduction components with which they interact. For example, the lack of conserved intracellular regions suggests the possibility that, during the evolution of this sensory modality, multiple G proteins arose, each interacting with a different subset of receptors. Finally, it seems likely that the Drosophila genome encodes taste receptors in addition to those of the GR family. Although expression in the labellum and the LSO has been detected, few if any family members are expressed in the leg or wing chemosensory hairs, some of which are morphologically similar to labellar taste hairs. The Drosophila olfactory system also contains two sense organs: the antenna and the maxillary palp. Both olfactory organs respond to all, or nearly all, of the same odorants and both are derived from the same imaginal discs. However, most individual members of the DOR gene family are expressed in one or the other but not in both olfactory organs. Perhaps the distinction among taste receptor genes is even more extreme in the gustatory system, whose organs derive from different imaginal discs. For example, the legs may express a completely distinct family of genes or a subfamily whose similarities to the present family are sufficiently tenuous as to place it slightly beyond the boundaries that define the GR family (Clyne, 2000).

    Gustatory (taste) neurons sense soluble chemical cues that elicit feeding behaviors. In insects, taste neurons also initiate innate sexual and reproductive responses. In Drosophila, for example, sweet compounds are recognized by chemosensory hairs on the proboscis and legs that activate proboscis extension and feeding. Sexually dimorphic chemosensory bristles on the foreleg of males recognize cues from receptive females that are thought to elicit the embrace of mating. Females have yet a third set of specialized bristles on their genitalia that may cause oviposition in response to nutrients. In this manner, gravid females will preferentially deposit their eggs on a rich environment that enhances survival of their offspring. These robust and innate gustatory responses provide the opportunity to understand how chemosensory information is recognized in the periphery and ultimately translated into specific behaviors (Scott, 2001 and references therein).

    Taste in Drosophila is mediated by sensory bristles that reside on the proboscis, legs, wing, and genitalia. Most chemosensory bristles are innervated by four bipolar gustatory neurons and a single mechanoreceptor cell. The dendrites of gustatory neurons extend into the shaft of the bristle and are the site of taste recognition that translates the binding of tastants into alterations in membrane potential. The sensory axons from the proboscis project to the brain where they synapse on projection neurons within the subesophageal ganglion (SOG), the first relay station for gustatory information in the fly brain. Sensory axons from taste neurons at other sites along the body project locally to peripheral ganglia. Drosophila larvae, whose predominant activity is eating, sense their chemical environment with gustatory neurons that reside in chemosensory organs on the head and are also distributed along the body surface. What remains unknown is the pattern of projection of functionally distinct classes of taste cells and therefore the nature of the representation of gustatory information in the Drosophila brain (Scott, 2001 and references therein).

    The identification of the genes encoding taste receptors and the analysis of the patterns of receptor expression may provide insight into the logic of taste discrimination in the fly. In Drosophila, the recognition of odorants is thought to be accomplished by about 60 seven-transmembrane domain proteins encoded by the Drosophila odorant receptor (DOR) gene family. Recently, a large family of putative G protein-coupled receptors was identified by searching the Drosophila genome with an algorithm designed to detect seven-transmembrane domain proteins (Clyne, 2000). These genes were suggested to encode gustatory receptors (GRs) because members of this gene family were detected in the proboscis by RT-PCR experiments (Scott, 2001).

    This study has characterized and extended the family of putative G protein-coupled receptors originally identified by Clyne (2000) and provides evidence that they encode both olfactory and gustatory receptors. In situ hybridization, along with transgene experiments, reveals that some receptors are expressed in topographically restricted sets of neurons in the proboscis, whereas other members are expressed in spatially fixed olfactory neurons in the antenna. Members of this gene family are also expressed in chemosensory bristles on the leg and in larval chemosensory organs. The projections of different subsets of larval chemosensory neurons have been traced to the subesophageal ganglion and the antennal lobe. These data provide insight into the diversity of chemosensory recognition in the periphery and afford an initial view of the representation of gustatory information in the fly brain (Scott, 2001).

    The GR gene family identified by Clyne (2000), has been extended by analyzing the recently completed euchromatic genome sequence of Drosophila using reiterative BLAST searches, transmembrane domain prediction programs, and hidden Markov model (HMM) analyses . These searches have identified a total of 56 candidate GR genes in the Drosophila genome, including 23 GRs not previously described. Gene sequences are available at the Columbia University site titled Newly identified GRs. The family as a whole is extremely divergent and reveals an overall sequence identity ranging from 7%-50%. However, all genes share significant sequence similarity within a 33 amino signature motif in the putative seventh transmembrane domain in the C terminus. Analysis of the sequence of the 56 genes reveals the existence of four discrete subfamilies (containing ten, six, four, and three genes) whose members exhibit greater overall sequence identity ranging from 30%-50%. Twenty-two of the GR genes reside as individual sequences distributed throughout each of the Drosophila chromosomes, whereas the remaining genes are linked in the genome in small tandem arrays of two to five genes (Scott, 2001).

    The GR family shares little sequence similarity outside of the conserved C-terminal signature in the putative seventh transmembrane domain and therefore the searches of the genome database described here are unlikely to be exhaustive. Thus, this family of candidate gustatory receptors consists of a minimum of 56 genes. Moreover, this analysis would not detect alternatively spliced transcripts, a feature previously reported for some members of this gene family (Clyne, 2000). cDNAs or RT PCR products could be detected from only six genes and verification of the gene predictions therefore awaits the isolation and sequencing of additional cDNAs (Scott, 2001).

    Interestingly, the 33 amino acid signature motif characteristic of the GR genes is present but somewhat diverged in 33 of the 60 members of the family of Drosophila odorant receptor (DOR) genes. However, the DOR genes possess additional conserved motifs not present in the GR genes and these motifs define a distinct family. These observations suggest that the putative gustatory and olfactory receptor gene families may have evolved from a common ancestral gene (Scott, 2001).

    Insight into the specific problem of the function of these candidate receptor genes and the more general question as to how tastants are recognized and discriminated by the fly brain initially requires an analysis of the patterns of expression of the individual GR genes in chemosensory cells. In situ hybridization was performed on sagittal sections of the adult fly head with RNA probes obtained from all 56 family members. Six of the genes are expressed in discrete, topographically restricted subpopulations of neurons within the proboscis. Three of the genes reveal no hybridization to the proboscis but are expressed in spatially-defined sets of neurons within the third antennal segment, the major olfactory organ of the adult fly. The remaining genes show no hybridization to adult head tissues (Scott, 2001).

    This analysis of the pattern of GR gene expression by in situ hybridization demonstrates that a small number of GR genes is transcribed in either the proboscis or the antenna, suggesting that this family encodes chemosensory receptors involved in smell as well as taste. However, expression of over 80% of the family members could not be detected using the available in situ hybridization conditions. The sequence of these GR genes does not reveal nonsense or frameshift mutations that characterize pseudogenes. The inability to detect transcripts from the majority of the GR genes by in situ hybridization might result from low levels of expression of GR genes, expression in populations of chemosensory cells not amenable to analysis by in situ hybridization (e.g., leg, wing, or vulva), or expression at other developmental stages (Scott, 2001).

    Lines of flies expressing GR promoter transgenes were generated to visualize the expression in a wider range of cell types with higher sensitivity. Transgenes were constructed in which putative GR promoter sequences (0.5-9.5 kb of DNA immediately upstream of the translational start) were fused to the Gal4 coding sequence. Flies bearing GR transgenes were mated to transgenic flies that contain either ß-galactosidase (lacZ) or green fluorescent protein (GFP) under the control of the Gal4-responsive promoter, UAS. GR promoter-Gal4 lines were constructed with upstream sequences from 15 chemoreceptor genes and transgene expression was detected for 7 lines. Five of the genes that were expressed by transgene analyses were also detected by in situ hybridization (Scott, 2001).

    Expression of the GR transgenes in the proboscis was initially visualized using the UAS-lacZ reporter. The labellum of the proboscis is formed from the fusion of two labial palps, each containing 31-36 bilaterally symmetric chemosensory bristles arranged in four rows. The sensilla of the first three columns contains four chemosensory neurons and a single mechanoreceptor cell whereas the sensilla in the most peripheral row are composed of only two chemosensory neurons and one mechanoreceptor. Each labial palp therefore contains approximately 120 chemosensory neurons (Scott, 2001 and references therein).

    The GR promoter-Gal4 lines were crossed to UAS-lacZ flies and the progeny were examined for lacZ expression by staining of whole-mount preparations of the labial palp. Five transgenic lines exhibit lacZ expression in sensory neurons of the labial sensilla. The expression of each transgene is restricted to a single row of chemosensory bristles. Gr47A1, for example, is expressed in sensilla innervating the most peripheral row of bristles, whereas Gr66C1 is expressed in sensilla that occupy a medial column. Flies bearing a GR promoter-Gal4 gene were also crossed with UAS-GFP stocks. The expression of GFP allows greater cellular definition and reveals that each receptor is expressed in a single neuron within a sensillum. The pattern of GR gene expression determined by GR promoter transgenes resembles that seen by in situ hybridization. However, coexpression of the transgene reporter and the endogenous gene could not be directly demonstrated by dual label in situ hybridization due to low levels of GR gene expression. Nevertheless, this pattern of expression, in which a receptor is expressed in only one neuron in a sensillum and in one sensillar row, is maintained in over 50 individuals examined for each transgenic line and is also maintained in independent transformed lines for each GR transgene (Scott, 2001).

    Chemosensory bristles reside at multiple anatomic sites in the fly including the taste organs in the mouth, the legs and wings, as well as in the female genitalia. Three sensory organs reside deep in the mouth: the labral sense organ (comprised of 10 chemosensory neurons) and the ventral and dorsal cibarial organs of the mouth (each containing six chemosensory neurons). The function of these specialized sensory organs is unknown, but their anatomic position and CNS projection pattern suggests that they participate in taste recognition. Three of the five GR promoter-Gal4 lines that are expressed in the proboscis are also expressed in the cibarial organs. One gene, Gr2B1, is expressed solely in the labral sense organ and is not detected in the proboscis labellum or in the cibarial organs (Scott, 2001).

    Chemosensory bristles also decorate both the legs and wings of Drosophila with about 40 chemosensory hairs on each structure. One gene, Gr32D1, expressed both in the proboscis and cibarial organ, is also expressed in two to three neurons in the most distal tarsal segments of all legs. These results are consistent with the observation that exposure of the legs to tastants results in proboscis extension and feeding behavior. The observation that members of this gene family are expressed in the proboscis and in chemosensory cells of the internal mouth organs and leg suggests that this gene family encodes gustatory receptors (Scott, 2001).

    GR transgene expression has also been examined in larvae. The detection of food in larvae is mediated by chemosensors that reside largely in the antennal-maxillary complex, a bilaterally symmetric anterior structure composed of the dorsal and terminal organs. Each of the two larval chemosensory organs comprises about 40 neurons. Neurons of the dorsal organ primarily detect volatile odorants, whereas the terminal organ is thought to detect both soluble and volatile chemical cues (Scott, 2001 and references therein).

    It was asked whether members of the GR family are expressed in larval chemosensory cells by examining the larval progeny that result from crosses between GR promoter-Gal4 and UAS-GFP flies. Examination of live larvae by direct fluorescent microscopy reveals that five of the seven GRs expressed in the adult are expressed in single neurons within the terminal organ. GR-promoter fusions from each of the 5 genes show bilateral expression of GFP both in the neuronal cell body and in the dendrite. The dendrites extend anteriorly to terminate in the terminal organ, a dome-shaped structure that opens to the environment. In about 5% of the larvae, a second positive cell is observed in each of the lines (Scott, 2001).

    Gr2B1 is expressed in only a single neuron in the labral sense organ of the adult, but is expressed in an extensive population of chemosensory cells in larvae. This gene is expressed in two neurons innervating the dorsal organ, one neuron innervating the terminal organ, and a single bilaterally symmetric neuron innervating the ventral pit in each thoracic hemisegment. The ventral pit contains a single sensory neuron that may be involved in contact chemosensation. The GR genes are therefore likely to play a significant role in chemosensory recognition in larvae as well as adults (Scott, 2001).

    Olfactory neurons of mammals as well as Drosophila express a single odorant receptor such that the brain can discriminate odor by determining which neurons have been activated. In contrast, nematode olfactory neurons and mammalian gustatory cells coexpress multiple receptor genes. Therefore the diversity of GR gene expression has been examined in individual larval taste neurons. In larvae, most receptors are expressed in only one neuron in the terminal organ. Crosses between five GR promoter-Gal4 lines and flies bearing UAS-GFP reveal a single intensely stained neuron within each terminal organ. Seven lines were generated bearing two different GR promoter-Gal4 transgenes along with the UAS-GFP reporter. In every line bearing two GR promoter-Gal4 fusions, two GFP positive cells per terminal organ were identified. These experiments demonstrate that individual gustatory neurons of larvae express different complements of receptors and are likely to respond to different chemosensory cues (Scott, 2001).

    In other sensory systems, a spatial map of receptor activation in the periphery is maintained in the brain such that the quality of a sensory stimulus may be encoded in spatially defined patterns of neural activity. GR promoter-Gal4 transgenes have been used to drive the expression of UAS-nSyb-GFP to visualize the projections of sensory neurons expressing different GR genes. nSyb-GFP is a C-terminal fusion of green fluorescent protein to neuronal synaptobrevin that selectively labels synaptic vesicles, allowing the visualization of terminal axonal projections. Whole-mount brain preparations from transgenic flies were examined by immunofluorescence with an antibody against GFP and a monoclonal antibody, nc82, which labels neuropil and identifies the individual glomeruli in the antennal lobe. These experiments were initially performed with larvae because of the relative simplicity of the larval brain and the observation that a given GR is expressed in only a small number of gustatory neurons (Scott, 2001).

    The Drosophila larval brain is composed of two dorsal brain hemispheres fused to the ventral hindbrain. The brain hemispheres and the hindbrain contain an outer shell of neuronal cell bodies and a central fibrous neuropil. Determination of the number of neuroblasts and the number of cell divisions suggest that there are ~10,000-15,000 neurons in the larval brain, a value 10- to 20-fold lower than in the adult. Chemosensory neurons send axonal projections to two distinct regions of the larval brain, the antennal lobe and the subesophageal ganglion (SOG). The antennal lobe is a small neuropil in the medial aspect of the deuterocerebrum within each brain hemisphere. The antennal lobe receives input from neurons of the dorsal and terminal organ and presumably participates in processing olfactory information. The SOG resides in the most anterior aspect of the hindbrain, at the juncture of the hindbrain with the brain hemispheres. The SOG receives input from the terminal organ and mouthparts and is thought to process gustatory information. Whereas the projections of populations of chemosensory cells have been traced to the antennal lobe and the SOG, the patterns of axonal projections for individual sensory cells have not been described. Moreover, the connections of chemosensory axons with second order brain neurons is unknown for the larval brain (Scott, 2001).

    Gr32D1-Gal4 is expressed in multiple neurons in the proboscis of the adult, but it is expressed in only a single neuron in the terminal organ of larvae. In larvae containing the Gr32D1-Gal4 and UAS-nSyb-GFP transgenes, it is possible to visualize the axons of Gr32D1-expressing cells as they course posteriorly to enter the subesophageal ganglion. The axons then turn dorsally and intensely stained fibers terminate in the medial aspect of the SOG. A similar pattern is observed for neurons expressing Gr66C1, a gene expressed in the adult proboscis and in a single neuron in the larval terminal organ and two neurons in the larval mouth. However, the terminal arbors of Gr66C1 neurons are consistently thicker than those observed for Gr32D1, perhaps reflecting the increased number of Gr66C1-bearing neurons. The reporter nSyb-GFP stains axons only weakly but shows intense staining of what are likely to be terminal projections of sensory neurons that synapse on second order neurons in the neuropil of the SOG. This terminal arbor extends for about 40 µm and reveals a looser, more distributed pattern that the tight neuropil of the olfactory glomerulus. The position and pattern of the terminal projections from individual chemosensory cells in the terminal organ show bilateral symmetry and are maintained in over 20 larvae examined (Scott, 2001).

    A more complex pattern of projections is observed for Gr2B1, a gene expressed in one neuron in the terminal organ, two in the dorsal organ, and a single bilaterally symmetric neuron in each thoracic hemisegment. One set of fibers appears to terminate in the antennal lobe. A second more posterior set of fibers can be traced from the thorax into the hindbrain, with fibers terminating posterior to the antennal lobe. This pattern of projections is of interest for it implies that neurons in different locations in larvae that express the same receptor project to discrete locations in the larval brain, suggesting the possibility that the same chemosensory stimulus can elicit distinct behavioral outputs (Scott, 2001).

    Attempts have been made to determine whether neurons in the terminal organ that express different GRs project to discrete loci within the SOG. Therefore larvae that express two promoter fusions, Gr66C1-Gal4 and Gr32D1-Gal4, along with a UAS-nSyb-GFP transgene were generated. The projections in these flies are broadened, suggesting that these sets of neurons terminate in overlapping but nonidentical regions of the SOG. More definitive data to support the existence of a topographic map of taste quality will require two-color labeling of the different fibers to discern whether the projections from neurons expressing different GRs are spatially segregated in the SOG (Scott, 2001).

    Are GRs also odorant receptors? A large family of presumed olfactory receptor genes in Drosophila (the DOR genes) has been identified that is distinct from the GR gene family. Expression of the DOR genes is only observed in olfactory sensory neurons within the antenna and maxillary palp, where a given DOR gene is expressed in a spatially invariant subpopulation of cells. In situ hybridization experiments demonstrate that three members of the GR gene family are also expressed in subpopulations of antennal neurons. These observations suggest either that the odorant receptors in Drosophila are encoded by at least two different gene families or that previously unidentified taste responsive neurons reside within the antenna (Scott, 2001).

    In Drosophila, olfactory information is transmitted to the antennal lobe, whereas gustatory neurons in the proboscis and mouth relay sensory information to the subesophageal ganglion. Therefore the spatial pattern of expression of GRs in the antenna was examined and the pattern of projections of their sensory axons in the brain. In situ hybridization with the three GR genes reveals that each gene is expressed in about 20-30 cells/gene in the antenna. Similar results are obtained in a cross between an antennal GR promoter-Gal4 line, Gr21D1-Gal4, and UAS-LacZ or UAS-GFP lines. This pattern of GR gene expression is maintained in over 50 antennae analyzed. The GR-positive cells occupy regions of the antenna that do not express identified members of the DOR gene family, suggesting that there is spatial segregation of these two receptor families (Scott, 2001).

    It was then asked whether antennal neurons expressing a GR gene project to the antennal lobe in a manner analogous to that observed for cells expressing the DOR genes. Transgenic flies expressing a Gr21D1 promoter-Gal4 fusion were crossed to animals bearing the UAS-nSyb-GFP transgene. These studies demonstrate that neurons expressing the Gr21D1 transgene project to a single, bilaterally symmetric glomerulus in the ventral-most region of the antennal lobe (the V glomerulus) and do not project to the SOG. Thus, as in the case of the family of DOR genes, neurons expressing the same receptor project to a single spatially invariant glomerulus (Scott, 2001).

    Gr21D1 is also expressed in one cell of the terminal organ of larvae. Therefore, the projections of Gr21D1-bearing neurons to the larval brain have been traced. Gr21D1 axons enter the larval brain and terminate in the antennal lobe rather than the SOG. The segregation of projections from presumed olfactory and gustatory neurons is apparent in larvae that contain Gr21D1-Gal4 and Gr66C1-Gal4 along with UAS-nSyb-GFP. In these transgenic flies, two distinct sets of termini are observed, one entering the SOG, and a second entering the antennal lobe (Scott, 2001).

    Thus, a member of the GR gene family is expressed in sensory neurons of the antenna and the terminal organ of larvae, and GR-bearing neurons project to the antennal lobe. These data suggest that at least two independent gene families, the DORs and the GRs, recognize olfactory information. The GR gene family is therefore likely to encode both olfactory and gustatory receptors, and neurons expressing distinct classes of GR receptors project to different regions of the fly brain (Scott, 2001).

    A common gene family encoding both olfactory and taste receptors is not present in vertebrates, where the main olfactory epithelium, the vomeronasal organ, and the tongue express receptors encoded by independent gene families. The observations presented here are more reminiscent of the chemosensory receptor families in C. elegans that encode odorant receptors expressed in the amphid neurons and taste receptors in sensory neurons responsive to soluble chemicals (Scott, 2001).

    The size of the family of candidate taste receptors and the pattern of expression in chemosensory cells provides insight into the problem of the recognition and discrimination of gustatory cues. On average, each GR is expressed in 5% of the cells in the proboscis labellum, suggesting that the proboscis alone will contain at least 20 distinct taste cells expressing about 20 different GR receptors. Moreover, a given receptor is expressed in one of the four rows of sensilla such that the sensilla in different rows are likely to be functionally distinct. Electrophysiologic studies have suggested that all sensilla are identical and contain four distinct cells, each responsive to a different category of taste. The data presented here are not consistent with these conclusions and argue that different rows of sensilla are likely to contain cells with different taste specificities (Scott, 2001).

    At present, the nature of the ligands recognized by these GR receptors are not known, nor is it known whether all taste modalities are recognized by this gene family. In mammals, gustatory cues have classically been grouped into five categories: sweet, bitter, salt, sour, and glutamate (umami). Sugar and bitter taste are likely to be mediated by G protein-coupled receptors since these modalities require the function of a taste cell-specific Ga subunit, gustducin. Recently, two novel families of seven transmembrane proteins (the T1Rs and T2Rs) were shown to be selectively expressed in taste cells in the tongue and palate epithelium. Genetic experiments have implicated members of the T2R family in the recognition of bitter tastants and functional studies have directly demonstrated that members of the T2R family serve as gustducin-linked bitter taste receptors. A large number of candidate genes have been suggested to encode receptors for other taste modalities, but in only a few instances have functional data and expression patterns supported these assumptions. In mammals, an amiloride-sensitive sodium channel has been suggested as the salt receptor, a degenerin homolog (MDEG-1) and a potassium channel as sour or pH sensors, and a rare splice form of the metabotropic glutamate receptor as the umami sensor. In Drosophila, genetic analysis of mutant flies defective in the recognition of the sugar trehalose has led to the identification of a transmembrane receptor distinct from GRs that reduces the sensitivity to one class of sugars. The interpretation of the role of these putative taste receptors in taste perception awaits a more definitive association between tastant and gene product (Scott, 2001).

    How does the fly discriminate among multiple tastants? One mechanism of chemosensory discrimination, thought to operate in the olfactory system of insects and vertebrates, requires that individual sensory neurons express only one of multiple receptor genes. Neurons expressing a given receptor project axons that converge on topographically invariant glomeruli such that different odors elicit different patterns of spatial activity in the brain. The nematode C. elegans uses a rather different logic, in which a given sensory neuron dictates a specific behavior but expresses multiple receptors. In the worm olfactory system, discrimination is necessarily more limited and exploits mechanisms to diversify the limited number of sensory cells. A similar logic has been suggested for mammalian taste. Several members of the T2R family of about 50 receptor genes, each thought to encode bitter sensors, are coexpressed in sensory cells within the tongue. This organization allows the organism to recognize a diverse repertoire of aversive tastants but limits the ability to discriminate among them (Scott, 2001).

    What can be discerned about the logic of taste discrimination from the pattern of GR gene expression in Drosophila? First, the number of GR genes, 56, approximates the number of DOR genes, suggesting that the fly recognizes diverse repertoires of both soluble and volatile chemical cues. Moreover, the data argue that individual sensory neurons differ with respect to receptor gene expression and are therefore functionally distinct. Experiments with Drosophila larvae demonstrate that a given GR gene is expressed in one neuron in the larval terminal organ. Strains bearing two different GR-promoter fusions reveal twice the number of expressing cells. Similar results are obtained in adult gustatory organs. More definitive experiments to examine the diversity of receptor expression in a single neuron, employed successfully in the olfactory system, have been difficult since the levels of GR RNA are 10- to 20-fold lower than odorant receptor RNA levels. Nevertheless, these experiments demonstrate that different gustatory neurons express different complements of GR genes and at the extreme are consistent with a model in which gustatory neurons express only a single receptor gene (Scott, 2001).

    How does the brain discern which of the different gustatory neurons is activated by a given tastant? As in other sensory systems, it is possible that axons from different taste neurons segregate to spatially distinct loci in the subesophageal ganglion. In such a model, taste quality would be represented by different spatial patterns of activity in the brain. Preliminary experiments suggest that neurons expressing different GRs project to spatially segregated loci within the brain. Clear segregation of axonal termini is observed for presumed taste neurons that project to the SOG and olfactory neurons that project to the antennal lobe. A second interesting pattern of projections is observed for the presumed gustatory receptor Gr2B1, a gene expressed in neurons in the terminal and dorsal organs and in a single neuron in the ventral pit present bilaterally in each thoracic segment. At least two spatially segregated targets are observed for these neurons in the larval brain: one set of fibers terminates in glomeruli of the antennal lobe and a second set of fibers (from the ventral pits) project to the SOG. Thus, neurons expressing the same receptor in different chemosensory organs project to distinct brain regions. In this manner, the same chemosensory cue could elicit distinct behaviors depending upon the cell it activates. Sucrose, for example, could elicit chemoattraction upon exposure to the thoracic neurons and eating behavior upon activation of neurons in the terminal and dorsal organ (Scott, 2001).

    These data establish that presumed olfactory neurons and gustatory neurons expressing GR genes project to different regions of the larval brain. Taste neurons expressing different GR genes, however, all project to the SOG. The current data do not permit a discernment of whether axons from neurons expressing different GR genes project to spatially distinct loci within the SOG. The axon termini of gustatory neurons terminate in more diffuse, elongated structures than the tightly compacted glomeruli formed by olfactory sensory axons, rendering it difficult at present to discern a topographic map of gustatory projections in the larval brain (Scott, 2001).

    An additional study (Dunipace, 2001) provides evidence for spatially restricted expression of candidate taste receptors in the Drosophila gustatory system. BLAST searches with the predicted amino acid sequences of 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. The sequences of all genes are deposited at the Amrein web-site at Duke University. Expression of eight of these genes was examined by using a Gal4 reporter gene assay; five of them are expressed in the gustatory system of the fly. Four genes are expressed in 1%-4% of taste sensilla, located in well-defined regions of either the proboscis, the legs, or both. The fifth gene is expressed in about 20% of taste sensilla in all major gustatory organs, including the taste bristles on the anterior wing margin. Axon-tracing experiments have demonstrated that neurons expressing a given Gr gene project their axons to a spatially restricted domain of the subesophageal ganglion in the fly brain. These 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 (Dunipace, 2001).

    The genomic organization of the Gr genes was investigated. Thirty-six Gr genes are arranged in clusters of two to six members. Genes within a cluster are more conserved (up to 50% identity; 70% similarity). The intergenic distance between clustered genes is very short, in many cases between 150-300 base pairs from the end of one open reading frame to the beginning of the next. Two genes appear to be pseudogenes (Gr22b and Gr22d) since their coding sequence is interrupted by a stop codon and a frame shift mutation, respectively. The number of introns varies widely because some genes have five or more introns and others have none. About half of the genes have a single conserved intron near the carboxy terminus. There are potentially alternative spliced genes at two loci, but in principle all genes that are arranged head to tail within a cluster and have conserved introns might be subjected to alternative splicing (Dunipace, 2001).

    Because Gr gene expression could not be detected by in situ hybridization, a transgenic approach was used. Putative promoter fragments of the Gr22a, Gr22c, Gr22e, and Gr22f genes were cloned from genomic DNA by PCR and inserted into the expression vector SM1 in front of the GAL4 gene. These 'drivers', in combination with a UAS-lacZ reporter, allow visualization of transgene expression in vivo. At least three independent transgenic lines of flies homozygous for each of these drivers were crossed to flies homozygous for a UAS-lacZ reporter, and the double-hemizygous progeny were analyzed by whole-mount staining to monitor the activity of the promoter fragments. No ß-gal activity was observed in the main body parts, such as the head, thorax, and abdomen. Analysis of the appendages of flies with the driver P [22e]-Gal4, however, demonstrates that this gene is expressed in many cells in the antenna, maxillary palps, proboscis, legs, and wings. Specifically, P [22e]-Gal4 is expressed in cells at the base of many chemosensory bristles of the labial palps and in cells located in the dorsal and ventral cibarial sense organs. It is also expressed along the tibiae and tarsi of all legs and the anterior wing margin in cells that are located at the base of chemosensory bristles. This highly specific, localized expression is particularly evident in the anterior wing margin, which contains two morphologically distinct types of bristles. Thinner and slightly bent chemosensory bristles occasionally interrupt thick and straight mechanosensory bristles. Only cells at the base of chemosensory bristles express ß-Gal. Finally, a number of cells in the third antennal segment and the maxillary palp also express Gr22e. Their expression pattern in olfactory organs is similar to the one observed for most Or genes, but it appears to cover a somewhat larger area (Dunipace, 2001).

    Expression of the driver P [22c]-Gal4 is restricted to the tarsi of the foreleg at the base of bristles, which are gustatory, based on morphological criteria. No transgene expression is observed in the labial palps, the labral and cibarial sense organs, or the anterior wing margin. Yet another expression pattern was observed with the driver P [22f]-Gal4, for which only four to eight positive cells are found on the labial palps in chemosensory sensilla. The cell bodies of these neurons are farther from the epithelial surface than are those of neurons in the wing margin, and it is therefore not always possible to correlate a LacZ-positive cell with a particular bristle. LacZ staining can be seen within the taste bristles themselves. Only chemosensory, not mechanosensory, neurons extend their dendrite into the bristle cavity, and this observation suggests that these LacZ-positive cells are gustatory neurons (Dunipace, 2001).

    These studies have shown that three Gr genes located at 22B are excellent candidates for encoding taste receptors. To generalize these findings for the entire Gr gene family, four additional transgenes were created by cloning the promoters of Gr10a, Gr59b, Gr63a, and Gr66a into the GAL4 expression vector. Using these constructs, three independent lines of transgenic flies were generated and analyzed. Two of these drivers were expressed exclusively in the gustatory neurons of the adult. The driver P [66a]-Gal4 is expressed in sensilla of the foreleg, the labial palps, and the labral and ventral cibarial sense organs but not in the mid leg, hind leg, or wing. In the labial palps, the expression of Gr66a is not restricted to a single row of bristles, as is Gr22f expression, but extends more laterally through two rows of sensory bristles. The driver P [59b]-Gal4 is expressed only in the labial palps and at weaker levels than either Gr22f or Gr66a (Dunipace, 2001).

    Taken together, these analyses show that five of eight analyzed genes (62.5%) are expressed in distinct gustatory sensilla in all major taste organs of Drosophila. Most of the genes are expressed in a very small fraction (1%-4%) of gustatory sensilla in a spatially restricted region of the fly, whereas one gene is expressed in about 20% of sensilla distributed all over the fly. Thus, they are excellent candidates for taste receptor genes (Dunipace, 2001).

    To establish the neuronal identity of the Gr-expressing cells, immunolocalization experiments were performed. Each sensillum contains not only gustatory neurons (and in most cases one mechanosensory neuron) but also three non-neuronal cells that form the hair, socket, and sheet surrounding the dendrites of the neurons. Confocal microscopy was performed and all neurons were visualized with an anti-Elav antibody. The Gr-expressing cell was visualized with an anti-ß-Gal antibody. Both drivers, P [22e]-Gal4 and P [22f]-Gal4, stain neurons that project a dendrite toward the epithelium and an axon toward the brain. Interestingly, none of the more than 50 sensilla analyzed contained more than one ß-Gal-positive cell (Dunipace, 2001).

    The projection patterns of neurons expressing the putative gustatory receptors were investigated. The gustatory neurons in the labial palps project their axons through the labial nerve to the subesophageal ganglion (SOG) in the brain. It was asked whether axons expressing a given receptor converge to a specific region within the SOG. Flies with the drivers P [22e]-Gal4 and P [66a]-Gal4 were crossed to a UAS-lacZ or UAS-Tau-lacZ line. Tau-LacZ is a Tau-ß-Gal fusion protein that is preferentially localized in axons and dendrites. Flies with the driver P [66a]-Gal4 show distinct staining of the labial and accessory pharyngeal nerves, and this staining reflects the expression of this gene in neurons of the labial palps and the labral/cibarial sense organs. As the nerves enter the brain, the axons terminate in two distinct regions, occupying only a fraction of the SOG. Axon projections of the driver P [22e]-Gal4 were visualized with confocal microscopy. These axons converge to a somewhat larger domain within the SOG, and this finding presumably reflects the wider expression domain of Gr22e when compared to Gr66a. Nevertheless, the ß-Gal-positive region occupies only a part of the SOG. Axonal convergence of gustatory receptor neurons, however, is not as defined as in the olfactory system, where axons expressing an individual Or gene project to one glomerulus in the antennal lobe (Dunipace, 2001).

    A genetic approach was used to show that the neurons expressing the Gr genes are indeed chemosensory neurons. Pox-n is a paired-box-containing transcription factor involved in several steps of neuron specification in all developmental stages. Some pox-n alleles do not interfere with the early steps in neurogenesis but affect the determination of chemosensory neurons in the adult. For example, flies carrying the poxn70-23 allele are viable but show complete transformation of gustatory neurons into mechanosensory neurons. pox-n mutant flies were generated carrying three Gal4 drivers and a single copy of a UAS-lacZ reporter and reported expression was analyzed. None of the drivers was expressed in the gustatory organs in these flies, whereas siblings that carried one wild-type pox-n allele expressed each driver in a normal pattern. Antenna and maxillary palp expression of Gr22e remained normal in pox-n flies (Dunipace, 2001).

    Thus, by several different criteria, the Gr genes are expressed in gustatory neurons of the adult. Furthermore, these experiments also demonstrate that neurons in the labial palps and the labral/cibarial sense organs expressing individual taste receptors project their axon to a distinct region in the SOG of the brain (Dunipace, 2001).

    The location of neurons expressing a given receptor is conserved between individuals. This was determined by comparing the location and number of LacZ-positive sensilla of flies from three independent lines containing either the P [22f]-Gal4 or the P [22c]-Gal4 driver. The expression patterns of both transgenes were highly reproducible, but small variations were observed. For example, most flies with the P [22c]-Gal4 driver had two positive cells at the tip of the foreleg; however, occasionally only one LacZ-positive cell was found at that location or one additional cell at a more proximal location in the foreleg or on the tarsi of the second and third leg. No positive cells were recorded on the labial palps, the labral and cibarial sense organs, or the wing. Flies with the P [22f]-Gal4 driver had between two and four ß-Gal-positive cells in a discrete row of taste bristles on the labial palps; again, this pattern is very restricted, since no ß-Gal-positive cells were encountered in the legs or wings (Dunipace, 2001).

    Variations in receptor gene expression might reflect a stochastic mechanism underlying the transcriptional control of Gr gene expression. For example, a neuron within a given sensillum might have a certain probability of expressing Gr X, a lower probability of expressing Gr Y, and virtually no probability of expressing any of the remaining Gr genes. The possibility that these differences are transgene-dependent effects and that the endogenous gene is precisely expressed in the same cells in each animal cannot be excluded. Whatever the reason for these modest variations, these experiments demonstrate that there exists a relatively precise topographic map for individual receptor gene expression. It should be noted that Gr22e is expressed in many more neurons than any other gene analyzed and that such a topographic map might not apply to this gene (Dunipace, 2001).

    What is the neuron-to-receptor ratio in the Drosophila gustatory system? Based on analysis of about 15% (8/54) of all Gr genes, a global expression profile of this gene family can be predicted. The ratio of expressed genes to total number of genes appears to be similar in the gustatory (62.5%) and olfactory systems (66.7%). Thus, about 30-35 Gr genes might be expressed in the gustatory system. These studies reveal two distinct expression profiles. One gene, Gr22e, is expressed in about 20% of the taste sensilla throughout the gustatory system, whereas the remaining genes are expressed in more defined regions that occupy only about 1%-4% of all taste sensilla. If the remaining Gr genes are expressed in a similar profile, about 6-7 genes would be found to be expressed in 20% of sensilla and 27-28 genes would be found to be expressed in 1%-4% of sensilla (about 20 Gr genes would not be expressed at all). Such a breakdown would ultimately require that a given sensilla express about two Gr genes. However, since each sensillum contains on average of three gustatory neurons, the one-neuron-to-one-Gr-gene rule would still apply. In fact, the remaining neuron in each sensillum might express another type of receptor, such as the trehalose receptor. Therefore, these data are consistent with a model found in the olfactory system of both Drosophila and mammals in which each sensory neuron expresses only one receptor gene. Convergence of axons expressing a specific Gr gene to a specific domain within the SOG is also found. This situation is similar to that found in the olfactory system in which neurons expressing a specific Or project their axons to a single glomerulus in the antennal lobe (Dunipace, 2001).

    An independent study (Ishimoto, 2000) has identified a Trehalose-sensitivity gene that is unrelated to the GR family discussed above. In Drosophila, taste sensilla are present on the labellum, tarsi, and wing margins. In a typical chemosensillum on the labellum, there are four taste sensory cells, each of which responds to either water, salt, or sugar. The Trehalose-sensitivity (Tre) gene was identified through studies on natural variants. The Tre gene has been cytologically mapped to the region between 5A10 and 5B1-3 on the X chromosome. Because the Tre gene controls taste sensitivity to trehalose without affecting the responses to other sugars, the gene product of Tre should function in sugar receptor cells. Disruption of the Tre gene lowers the taste sensitivity to trehalose, whereas sensitivities to other sugars are unaltered. Overexpression of the Tre gene restores the taste sensitivity to trehalose in the Tre deletion mutant (Ishimoto, 2000).

    Although several conserved regions are found between Tre1 and other GPCRs, the structures of the third and fourth cytoplasmic domains may be unique, because they are longer than the corresponding domains of typical GPCRs. The Tre1 gene most closely resembles two other orphan receptors of Drosophila: EG:22E5.11 and EG:22E5.10. It is suggested that the Tre gene may represent a new subclass of taste receptors (Ishimoto, 2000)

    Taste perception and coding in Drosophila

    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. The expression of eight Gr genes in the labial palps, the fly's main taste organ, has been characterized in detail. 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. It is concluded that iscrimination 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 (Thorne, 2004).

    The labellum, considered to be the main taste organ in Drosophila, has approximately 62 chemosensory bristles (sensilla) that are arranged in a stereotyped pattern. These sensilla are morphologically identified as short (S), intermediate (I), and long (L). S and L bristles house dendrites of four chemosensory neurons, whereas I bristles are associated with two chemosensory neurons. To determine expression of Gr genes in these chemosensory neurons, the Gal4/UAS system has been used. This indirect method of expression analysis has proven far superior to RNA in situ hybridization due to low levels of Gr transcripts per cell and the wide distribution of taste neurons in tissues not amenable to sectioning procedures. The Gal4/UAS analyses revealed that a given Gr gene is expressed in a small number of chemosensory neurons per labial palp and, in each case, in only one neuron per chemosensory bristle. Hiroi (2002) demonstrated an association of specific Gr genes with certain bristles of the labellum. The majority of receptors examined are expressed in one of the four neurons of S type sensilla. For example, several Gr genes are strongly expressed in a single neuron associated with three S type sensilla (S1, S3, and S6) (Thorne, 2004).

    Several issues with broad implications for taste coding remain to be elucidated. For example, it is still not known whether some Gr genes are coexpressed in the same neurons and, if so, to what extent. Similarly, it is not known what kind of taste properties are mediated by GRNs expressing these receptors. Finally, experiments to visualize axonal targets in the CNS of neurons expressing individual Gr genes have not been performed in any detail. To further advance understanding of Drosophila taste perception, these questions were addressed: the number of neurons expressing novel and previously characterized Gr genes was addressed, their extent of coexpression was investigated, the projection patterns of GRNs expressing these genes was visualized, and taste perception of flies lacking specific sets of GRNs was determined (Thorne, 2004).

    Gal4 drivers (p[Gr]-Gal4) for eight Gr genes, Gr5a, Gr22b, Gr22e, Gr22f, Gr28be, Gr32a, Gr59b, and Gr66a, were combined with a UAS-nucGFP reporter gene encoding a green fluorescent protein tagged with a nuclear localization signal and images of optical sections through the entire labellum were collected by using confocal microscopy after anti-GFP antibody staining. By using the map generated by Hiroi (2002) as a guide, detailed analysis of confocal stacks allowed the organization and number of neurons expressing each of these genes to be more accurately determined. The expression patterns fell into two broad groups: Gr5a (representing the first group) was expressed in a large number of neurons throughout the entire labial palp, whereas the other Gr genes had restricted expression to relatively few neurons (Thorne, 2004).

    Of the second group, Gr66a was expressed in the largest number (n = 22 ± 1)of cells per palp. Significantly, only a single neuron per S and I type sensillum stained positive for this driver. The neurons associated with S type bristles, which are located more medially, appeared larger in size compared to more laterally located neurons of I type sensilla. Gr22b, Gr22e, Gr22f, Gr28be, Gr32a, and Gr59b were expressed in fewer neurons than Gr66a. Expression of these receptors appears more restricted to larger neurons associated mostly with S type bristles. These expression studies provided the groundwork necessary to determine whether two or more Gr genes are actually coexpressed in the same neuron associated with an S type bristle (Thorne, 2004).

    Ideally, coexpression of Gr genes may be addressed by labeling individual Gr gene-specific probes with different markers. However, expression levels of these genes are too low for reliable detection of transcripts by RNA in situ hybridization. Attempts were made to use the Gal4/UAS system along with a second reporter system, the tetracycline transactivator/tet-O reporter system. The sensitivity of this system, however, was too low to obtain reliable cell staining in taste neurons. Therefore, the issue of coexpression was addressed by quantification of labeled cells using the Gal4/UAS system, an approach that seemed feasible given the relatively low number of cells in which each receptor is expressed. Transgenic fly lines were made expressing UAS-nucGFP under the control of two different Gal4 drivers and then the number of labeled neurons was counted and compared to that of flies containing each driver alone. Surprisingly, in all cases where such double-driver experiments were carried out, the number of labeled cells expressing two drivers was close or equal to the number of labeled cells of flies containing the single driver with the higher cell count. For example, in flies that express either the p[Gr66a]-Gal4 or p[Gr22e]-Gal4 driver, an average of 22 and 14 neurons/labial palp are labeled, respectively. In flies that express both drivers, again approximately 22 neurons are detected per palp, which indicates that most if not all cells that express Gr22e also express Gr66a (Thorne, 2004).

    A crucial determinant for discerning chemical cues present in the environment is embedded in the peripheral expression pattern of cell surface receptors in sensory epithelia. In the olfactory systems of Drosophila and mice, each olfactory receptor neuron expresses only one of 60 or one of approximately 1000 Or genes, respectively, enabling these animals to discriminate between hundreds or thousands of different odors. In contrast, taste cells of the tongue allow mammals to distinguish only a few taste qualities: bitter, sweet, umami, salty, and acidic taste. Lack of discrimination between the hundreds of diverse chemical compounds -- all perceived as bitter -- is thought to be caused by coexpression of the approximately 40 T2R receptors in a single set of taste cells (Chandrashekar, 2000; Zhang, 2003). Therefore, activation of the bitter taste cells by any one of the T2Rs is likely to generate a single activation pattern in taste centers of the brain, leading to a similar, repulsive behavioral output. Associating primary taste centers in the mammalian brain with specific taste modalities has, as of yet, proved challenging (Thorne, 2004).

    Insect taste is still rather poorly understood, especially at the molecular level. Drosophila, which exhibits remarkably similar taste preferences with humans, is the only insect for which candidate receptors have been characterized experimentally. The investigations presented here provide significant new insights into insect taste perception (Thorne, 2004).

    Initial expression studies suggested that the fly gustatory receptors are not simply coexpressed in three sets of cells dedicated to bitter, sweet, and umami taste like the T2Rs, T1R2/T1R3, and T1R1/T1R3 receptors of mammals. Instead,these experiments suggested that they either are expressed according to the one receptor (the 'one neuron' hypothesis well established for insect and mammalian olfactory systems) or they are expressed in partially overlapping sets of neurons. The current analysis supports the latter of these possibilities. Most labellar Gr genes (seven out of eight) are expressed in a single neuron of mostly S and some I type bristles. Most interestingly, coexpression studies provide evidence that individual neurons express anywhere from one to six receptors. In this way, S bristle-associated neurons are defined by unique receptor gene codes, thereby outfitting the labellum with an array of sensory assemblies that may exhibit distinct, albeit overlapping, ligand specificities (Thorne, 2004).

    The functional implications of distinct neuronal receptor codes on taste perception are currently unclear and will require analysis of mutations of individual Gr genes. However, a general role for these neurons in feeding inhibition ('avoidance neurons') can be inferred from experiments presented in this study and supported through analogy with the mammalian taste system/receptors. (1) Avoidance neurons express the majority of analyzed Gr genes -- and by extension -- the majority of the genes in the entire Gr gene family. In mammals, bitter taste receptors far outnumber the sweet taste receptors (40:3). (2) Avoidance neurons associated with S type bristles do not express the receptors for the sugar trehalose encoded by the Gr5a gene. In fact, avoidance neurons associated with S type bristles have a distinct appearance compared to neurons expressing the Gr5a gene. In mammals, the sweet/umami taste receptors and the bitter taste receptors are expressed in distinct group of cells (Chandrashekar, 2000; Zhang, 2003). (3) Inactivation of avoidance neurons has no effect on sucrose or trehalose sensitivity in flies but significantly reduces their sensitivity to caffeine. (4) Avoidance neurons and Gr5a-expressing neurons have distinct targets in the subesophageal ganglion (SOG), a feature consistent with the detection of different taste qualities by these neurons (Thorne, 2004).

    If the avoidance neurons have a general function in the detection of toxic or otherwise undesirable chemicals, what is the rationale for a complex and distinct Gr gene code among different groups of such neurons? It is proposed that the receptor code allows a fly to discriminate among different chemicals, which are in general avoided but might have distinct consequences on their health if ingested. According to such a proposal, a fly encountering a food source rich in nutrients (sugars) but contaminated with toxic chemicals may choose between feeding and avoidance, depending on the impact the particular toxic compound may have on its health. There is indirect evidence from feeding studies in Maduca sexta larvae that discrimination between the bitter substrates caffeine and aristolochic acid does occur in insects, even though actual taste preference, adaptation, or both may contribute to this phenomenon (Glendinning, 2001). Thus, discrimination among toxic/bitter-tasting compounds might be possible in insects including Drosophila (Thorne, 2004).

    It was somewhat surprising that the sensitivity to other compounds known to be avoided by insects -- denatonium benzoate, quinine hydrochloride, and berberine -- was not affected in animals lacking Gr66a-expressing neurons. This may simply be explained by the presence of additional neurons expressing receptors that recognize these particular substrates. Alternatively, one or a few neurons coexpressing Gr66a along with a receptor for one (of these) ligand(s) might not have been completely inactivated by TNT. Finally, studies in rodents indicate that caffeine may directly affect neurons in the brain, circumventing activation of taste cells altogether. This is not likely to be the case in the current experiments, because none of the Gr genes examined is expressed in the CNS (Thorne, 2004).

    Relatively few studies have investigated bitter taste sensitivity in insects, particularly Drosophila. Electrophysiological studies have identified bristles in the legs, but not the labellum of Drosophila, that respond to bitter-tasting chemicals (Meunier, 2003). However, S type sensilla are notoriously difficult to record from, because their bristles are extremely difficult to access for this type of experiment (Thorne, 2004).

    Gr5a-expressing neurons represent more than half of chemosensory cells in the labellum and appear to be associated with all sensilla types, including the taste pegs. In fact, association of Gr5a with taste pegs provides the best evidence yet that these sensilla have a specific chemosensory function in the detection of trehalose. Significantly, Gr5a-expressing neurons define a largely distinct set of neurons from the avoidance neurons. This observation is consistent with the results from behavioral investigations of flies lacking the function of specific sets of neurons. Specifically, inactivation of Gr5a-expressing neurons leads to a reduction in trehalose sensitivity, but the sensitivity to any bitter substrate tested was unaffected. These flies did not exhibit reduced sucrose sensitivity, another nutrient-relevant sugar for Drosophila. This result is somewhat unexpected, since electrophysiological investigations have led to the proposal that a single neuron in L, I, and S bristles is responsive to several sugars including trehalose and sucrose (the 'sugar' neuron) (Dahanukar, 2001; Hiroi, 2002; Rodrigues, 1981; Dethier, 1976). According to these studies, sugar neurons may express a single, broadly tuned sugar receptor, or more likely, they may coexpress several distinct sugar receptors, each of which recognizes a specific sugar (i.e., sucrose, trehalose, fructose, etc.). This latter possibility is favored from genetic studies, which have shown that mutation in the Gr5a gene reduces the sensitivity of flies to trehalose, but not to sucrose. However, the proposition of a single sugar neuron per bristle is also not consistent with expression studies, which show that two to three neurons within a bristle can express Gr5a. The possibility that the p[Gr5a]-Gal4 drivers do not represent endogenous Gr5a expression cannot be excluded, but this is unlikely to be the case for two reasons: (1) several lines with p[Gr5a]-Gal4 show the same expression, and (2) the p[Gr5a_C]-Gal4 driver containing a much larger promoter fragment produces a similar expression profile, with many clusters of Gr5a-expressing neurons associated with the same bristle (Thorne, 2004).

    In order to realign the electrophysiological data with expression analysis, another explanation is proposed: the 'sugar neuron' identified in electrophysiological studies expresses many (possibly all) distinct sugar receptors, including GR5a. However, one or two additional neurons per bristle express only a fraction, or possibly just one, of the sugar receptors present in the sugar neuron. Worth noting in this context is the fact that electrophysiological recordings are carried out at significantly higher substrate concentrations (up to 100 mM for sucrose and trehalose) than behavioral experiments (2 mM for sucrose and 25 mM for trehalose). The model is also more consistent with recent experiments (Hiroi, 2002) that noted different electrophysiological sugar responses among labellar sensilla (Thorne, 2004).

    Approximately 45 labellar neurons have not yet been associated with any Gr gene, and some of these neurons might express putative candidate receptors for sucrose or additional sugars. These genes are likely to be encoded by members of the Gr64 gene cluster, which share much higher sequence similarity with Gr5a than any other Gr genes. Gal4 drivers for two of these genes (Gr64a and Gr64e) were analyzed and found to be expressed in the pharyngeal taste organs, but not in the labellum. Whether these two receptors are indeed involved in sugar detection remains to be seen, but it is predicted that other Gr genes for sugars like sucrose and fructose are be expressed more broadly and in taste neurons of labellar bristles and pegs (Thorne, 2004).

    In summary, expression and behavioral studies suggest two fundamentally different roles for neurons expressing nonoverlapping groups of Gr genes in the detection of substrates that lead to feeding or avoidance behavior. According to this new model, S and I bristles on the labial palps contain one avoidance neuron and one or more feeding neurons (depending on the number of neurons associated with the particular bristle). The avoidance neuron expresses multiple Gr genes, and avoidance neurons of different bristles express these Gr genes in different combinations (the Gr gene code). The feeding neurons, which appear morphologically smaller than the avoidance neurons, express an entirely different set of receptors that includes Gr5a and possibly Gr genes encoding receptors for other sugars, amino acids, and peptides (Thorne, 2004).

    The different functions for GRNs expressing Gr66a and Gr5a are also supported by their different projection patterns in the brain. Neurons expressing Gr66a or any of the partially coexpressed receptors target similar regions in the SOG/tritocerebrum, though the number of termini differs significantly depending on the number of peripheral sensory neurons the Gr is expressed in. For example, Gr66a-expressing neurons show a robust array of termini in the SOG/tritocerebrum, whereas the termini of Gr59b- and Gr22f-expressing neurons are significantly less numerous. In all cases, dense, contralaterally projecting fibers provide extensive innervation of both halves of the SOG by labellar neurons, as demonstrated by labial palp ablation experiments (Thorne, 2004).

    An entirely different projection pattern is observed for feeding neurons that express Gr5a. Most strikingly, the axon termini of these neurons are distributed over a very large area of the SOG and extend into regions not innervated by avoidance neurons. A second striking difference is the poorly established contralateral connective between the two halves of the SOG, suggesting that neurons located in the right labial palp preferentially terminate in the right half of the SOG. This idea was tested and confirmed through ablation studies and has interesting implications, namely that spatially restricted activation of neurons in one palp will preferentially stimulate the same side of the SOG; this could potentially allow for spatial discrimination of taste input in the brain. This feature might allow the fly to orient its labellum in the direction of a food source, identifying regions with high concentrations of trehalose or other sugars (Thorne, 2004).

    The distinct pattern of axon termini in the SOG of neurons required for feeding and avoidance suggests that these behaviors are mediated through different neuronal pathways. Anatomical studies in honeybees have identified second-order neurons that mediate synaptic activity of primary taste neurons to higher brain centers. It will be interesting to see whether second-order neurons contacting synapses of avoidance and feeding neurons define different target regions in these higher brain centers (Thorne, 2004).

    Taste is an ancient sense, which exists in bacteria in the form of chemotaxis. Neuroanatomical and molecular comparison of taste systems between mammals and insects imply that this sense has evolved independently in these phyla. In mammals, for example, taste ligands are perceived through sensory epithelial cells in the lingual epithelium of the tongue. These cells then activate secondary neurons that innervate taste centers in the brain. In insects, tastants are detected by primary sensory neurons that directly innervate the CNS. Moreover, insects have multiple taste organs (legs, wings, and in some cases, the female genitalia) for which no counterparts exist in mammals. Finally, sequence comparison of the Gr and T1R/T2R genes has failed to reveal any direct kinship between mammalian and insect taste receptors (Thorne, 2004).

    However, a remarkable convergence of anatomical as well as molecular features of gustatory systems between mammals and insects (Drosophila) appears to emerge from these studies. The functional taste units, the taste buds in the tongue and the taste bristles of the labellum, are composed of 30 to 100 taste cells and two to four chemosensory neurons, respectively. Individual taste cells in each taste bud are dedicated to the perception of sweet, umami, or bitter taste sensation based on the T1R or T2R receptors they express. Similarly, the data indicate that taste bristles of the labellum contain neurons that either respond to repulsive or attractive stimuli, properties that are likely determined by the specific (set of) taste receptors they express (Thorne, 2004).

    Despite the sequence divergence of mammalian and insect taste receptors, it is believed there are intriguing similarities at the molecular level as well. The number of taste receptors in mammals and Drosophila is very similar. The eight genes described in this study probably encode a significant number of the functional labellar taste receptors. Some of the 60 Gr genes are likely to encode taste receptors only expressed in the pharynx, legs, and wings or might only be expressed in the larva. Other Gr genes are likely to function as pheromone receptors, or might recognize internal ligands based on their restricted expression in the CNS. Considering these alternative functions for some Gr genes, it is estimated that the fly has about 30 to 45 labellar taste receptors, a number close to the total number of T1Rs and T2Rs (30 in humans and 45 in mice) (Thorne, 2004).

    In addition to the similar size of the Gr and T1R/T2R gene families, taste receptors of mammals and Drosophila fall into similar functional groups. Only three mammalian T1R receptors are thought to be dedicated to the detection of attractive stimuli (sugars and amino acids/proteins), whereas the large majority -- the T2Rs -- are thought to be exclusively involved in the detection of repulsive (bitter) ligands. If the current expression analysis is more or less representative of the entire Gr gene family, it might be expected that 25 to 40 Gr genes will be expressed in the avoidance neurons, whereas just three to six are expected to be expressed in feeding neurons. Identification and analysis of Gr genes encoding receptors for known ligands, combined with biochemical analyses, should reveal whether additional molecular features are shared between the GRs and T1Rs and T2Rs, such as whether Drosophila also possess a specific receptor for amino acids and whether some receptors also function as multimers, as is proposed for mammalian T1Rs (Thorne, 2004).

    Behavioral analysis of bitter taste perception in Drosophila larvae

    Insect larvae, which recognize food sources through chemosensory cues, are a major source of global agricultural loss. Gustation is an important factor that determines feeding behavior, and the gustatory receptors (Grs) act as molecular receptors that recognize diverse chemicals in gustatory receptor neurons (GRNs). The behavior of Drosophila larvae is relatively simpler than the adult fly, and a gustatory receptor-to-neuron map was established in a previous study of the major external larval head sensory organs. This study extensively examined The bitter taste responses of larvae using 2-choice behavioral assays. First, a panel of 23 candidate bitter compounds was tested to compare the behavioral responses of larvae and adults. Nine bitter compounds were tested, which elicit aversive behavior in a dose-dependent manner. A functional map of the larval GRNs was constructed with the use of Gr-GAL4 lines that drive expression of UAS-tetanus toxin and UAS-VR1 in specific gustatory neurons to identify bitter tastants-GRN combinations by suppressing and activating discrete subsets of taste neurons, respectively. The results suggest that many gustatory neurons act cooperatively in larval bitter sensing, and that these neurons have different degrees of responsiveness to different bitter compounds (Kim, 2015).

    Gustatory receptor 21a and Gustatory receptor 63a confer CO2-chemosensation in Drosophila

    Blood-feeding insects, including the malaria mosquito Anopheles gambiae, use highly specialized and sensitive olfactory systems to locate their hosts. This is accomplished by detecting and following plumes of volatile host emissions, which include carbon dioxide (CO2). CO2 is sensed by a population of olfactory sensory neurons in the maxillary palps of mosquitoes and in the antennae of Drosophila. The molecular identity of the chemosensory CO2 receptor, however, remains unknown. This study reports that CO2-responsive neurons in Drosophila co-express a pair of chemosensory receptors, Gr21a and Gr63a, at both larval and adult life stages. Mosquito homologues of Gr21a and Gr63a, GPRGR22 and GPRGR24 have been identified; these are co-expressed in A. gambiae maxillary palps. Gr21a and Gr63a together are sufficient for olfactory CO2-chemosensation in Drosophila. Ectopic expression of Gr21a and Gr63a together confers CO2 sensitivity on CO2- insensitive olfactory neurons, but neither gustatory receptor alone has this function. Mutant flies lacking Gr63a lose both electrophysiological and behavioural responses to CO2. Knowledge of the molecular identity of the insect olfactory CO2 receptors may spur the development of novel mosquito control strategies designed to take advantage of this unique and critical olfactory pathway. This in turn could bolster the worldwide fight against malaria and other insect-borne diseases (Jones, 2007).

    CO2 elicits a response from many insects, including mosquito vectors of diseases such as malaria and yellow fever, but the molecular basis of CO2 detection is unknown in insects or other higher eukaryotes. Gr21a and Gr63a, members of a large family of Drosophila seven-transmembrane-domain chemoreceptor genes, are coexpressed in chemosensory neurons of both the larva and the adult. The two genes confer CO2 response when coexpressed in an in vivo expression system, the 'empty neuron system.' The response is highly specific for CO2 and dependent on CO2 concentration. The response shows an equivalent dependence on the dose of Gr21a and Gr63a. None of 39 other chemosensory receptors confers a comparable response to CO2. The identification of these receptors may now allow the identification of agents that block or activate them. Such agents could affect the responses of insect pests to the humans they seek (Kwon, 2007; full text of article).

    Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants

    The fruitfly Drosophila melanogaster exhibits a robust and innate olfactory-based avoidance behaviour to CO2, a component of odour emitted from stressed flies. Specialized neurons in the antenna and a dedicated neuronal circuit in the higher olfactory system mediate CO2 detection and avoidance. However, fruitflies need to overcome this avoidance response in some environments that contain CO2 such as ripening fruits and fermenting yeast, which are essential food sources. Very little is known about the molecular and neuronal basis of this unique, context-dependent modification of innate olfactory avoidance behaviour. This study identified a new class of odorants present in food that directly inhibit CO2-sensitive neurons in the antenna. Using an in vivo expression system it was established that the odorants act on the Gr21a/Gr63a CO2 receptor. The presence of these odorants significantly and specifically reduces CO2-mediated avoidance behaviour, as well as avoidance mediated by 'Drosophila stress odour'. A model is proposed in which behavioural avoidance to CO2 is directly influenced by inhibitory interactions of the novel odours with CO2 receptors. Furthermore, differences were observed in the temporal dynamics of inhibition: the effect of one of these odorants lasts several minutes beyond the initial exposure. Notably, animals that have been briefly pre-exposed to this odorant do not respond to the CO2 avoidance cue even after the odorant is no longer present. Related odorants were shown to be effective inhibitors of the CO2 response in Culex mosquitoes that transmit West Nile fever and filariasis. These findings have broader implications in highlighting the important role of inhibitory odorants in olfactory coding, and in their potential to disrupt CO2-mediated host-seeking behaviour in disease-carrying insects like mosquitoes (Turner, 2009).

    CO2 is an important sensory cue for many animals, including insects, in a variety of behavioural contexts. In Drosophila, CO2 is exclusively detected by a unique heteromeric receptor encoded by Gr21a and Gr63a that is expressed in a single population of antennal olfactory receptor neurons (ORNs), called ab1C, which innervate the ab1 class of large basiconic sensilla. These neurons send stereotypical axonal projections to the V glomerulus, and activation of this dedicated uni-glomerular circuit leads to an innate avoidance of CO2 (Turner, 2009).

    In fact, CO2 is a major component of Drosophila stress odour (dSO), which is emitted by flies subjected to vigorous shaking or electric shock, and which elicits an immediate escape response in naive flies (Suh, 2004). However, CO2 is also present in significant quantities in several important food sources that elicit behavioural attraction of Drosophila. Fruits and plants emit CO2 as a by-product of respiration, as do fruits undergoing fermentation by microorganisms and yeasts. Flies are attracted to headspace odours containing CO2 collected from over-ripe fruits, fermenting yeast and beer when presented with a choice between two tubes in a T-maze assay, one containing air and the other containing headspace odours. However, flies avoid headspace odours collected from green fruits, which also emit CO2. A subset of specialized gustatory neurons mediate a small degree of attraction to carbonated water upon contact (Fischler, 2007); however, they do not respond to CO2 in the gas phase and are not likely to contribute to long-range or short-range behavioural attraction towards a food source. Therefore, olfactory avoidance to CO2 may be modified by context for some CO2-rich sources such as over-ripe fruit, yeast and beer (Turner, 2009).

    Little is known about the molecular and neuronal mechanisms that lead to such a dramatic modification of innate avoidance behaviour. Two alternative models, although not mutually exclusive, may be evoked to explain this phenomenon. In the first model, avoidance to CO2 is overcome simply by detection of attractive odorants emitted by the same food sources. In the second model, some components of food volatiles may also directly inhibit the CO2-responsive circuit, and thereby suppress avoidance behaviour to CO2 (Turner, 2009).

    To test whether odorants present in fruits and other natural environments of Drosophila can directly inhibit CO2-sensitive ab1C neurons, a simple electrophysiology screen was performed. Several individual odorants were tested for their ability to inhibit the baseline activity of the ab1C neuron (to about 0.03% CO2 present in room air) using single-sensillum electrophysiology. These experiments were performed using Or83b2 mutant flies in which the ab1C neuron remains the sole functional neuron in the ab1 sensillum. In a screen with 46 odorants, two, 1-hexanol and 2,3-butanedione, were identified that strongly inhibit the baseline activity of ab1C neurons. Both of these compounds are present in Drosophila food sources including various types of fruit. More interestingly, the abundance of both these compounds is greatly increased during the ripening process of fruits: for example, in banana, 1-hexanol increases by 777% and 2,3-butanedione by 14,900%. 1-Hexanol is formed during ripening by lipid oxidation of unsaturated fatty acids, whereas 2,3-butanedione is a natural by-product of fermentation of carbohydrates through pyruvate by yeasts and bacteria and is thus also present in fermenting fruit, wine (Turner, 2009).

    Both 1-hexanol and 2,3-butanedione inhibit CO2 response in a dose-dependent manner, irrespective of whether their application is initiated before, or after, the presentation of the CO2 stimulus at relatively low, physiologically relevant concentrations (Turner, 2009).

    A fly approaching an odour source from a distance likely contacts plumes of CO2, which will vary widely in concentration over baseline atmospheric levels. When several concentrations of CO2 were tested, it was found that the presence of 2,3-butanedione (10-1 dilution) completely inhibits responses up to 3.2% CO2; 1-hexanol (10-1 dilution) also causes a significant reduction of CO2 response across most tested concentrations, but complete inhibition occurs only at 0.1% CO2 (Turner, 2009).

    To understand odorant structural features that might have a role in inhibition, a rationally designed panel of odorants was tested that varied in the number of carbon atoms and in the nature of the functional group. On the basis of this analysis, additional odorants were identified that also inhibit CO2 response. The inhibitory effects of each of the compounds identified so far are specific to the CO2-sensitive neuron; previous studies have shown that all of them can excite other classes of Drosophila ORNs, which suggests that they are not general inhibitors of ORN function. Surprisingly, these compounds are structurally quite different from CO2, thus raising the possibility that they may act through allosteric binding sites within the Gr21a/Gr63a receptor, or on other components of the CO2 detection pathway such as factors present in the sensillar lymph or in ab1C neurons (Turner, 2009).

    To investigate whether the inhibitors act directly on the CO2 receptor, Gr21a and Gr63a were expressed in an in vivo decoder system called the 'empty neuron'. It was found that expression of Gr21a and Gr63a in the empty ab3A neuron is sufficient to impart a robust and reproducible dose-dependent CO2 response, comparable to the levels reported previously. Upon application of each of the four inhibitory odorants along with CO2, dose-dependent inhibition was observed of CO2 response of the ab3A neuron in a Gr21a/Gr63a-dependent manner. The simplest interpretation of these results is that the odorants that were identified inhibit CO2 response by direct interaction with the CO2 receptor, Gr21a/Gr63a. However, the inhibitory effect appears shorter in duration than observed in the endogenous ab1C neurons, suggesting that additional neuron- or sensillum-specific factors may also influence the temporal aspects of the inhibition (Turner, 2009).

    Next it was asked whether the inhibitory odorants identified using electrophysiology could disrupt avoidance behaviour of Drosophila to CO2. Using a T-maze choice assay, it was found that wild-type Drosophila show a robust avoidance behaviour to 0.67% CO2. Inclusion of 2,3-butanedione with CO2 completely abolishes avoidance to CO2. Importantly, 2,3-butanedione by itself does not elicit any significant attraction or avoidance behaviour. In wild-type Drosophila, however, several other ORN classes are activated by 2,3-butanedione, raising the possibility that behavioural avoidance to CO2 may be overcome by activation of these other classes of ORN, and not solely by inhibition of CO2-responsive neurons (Turner, 2009).

    To distinguish between these possibilities, the behaviour of Or83b2 mutant flies was tested under conditions in which most ORNs are non-functional, but electrophysiological responses to CO2 are not affected. Consistent with the electrophysiological analysis, flies lacking Or83b have a robust avoidance response to CO2, which is absent when 2,3-butanedione is included with CO2 or is presented alone. Similar results, albeit with weaker effects, are obtained using 1-hexanol. Taken together, these results show that inhibitory odorants can effectively block CO2-mediated innate avoidance behaviour (Turner, 2009).

    CO2 is one of the main components of dSO, which is emitted by stressed flies, and which triggers a robust avoidance behaviour in naive flies. Therefore, whether 2,3-butanedione can disrupt avoidance to dSO was tested. It was found that naive flies avoid odour collected from a tube of vortexed flies (dSO), but not that collected from a tube of untreated flies (mock), in a T-maze assay. Remarkably, addition of 2,3-butanedione to dSO effectively abolishes avoidance behaviour (Turner, 2009).

    Interestingly, it was observed that with increasing concentrations of 2,3-butanedione, the CO2 neuron is silenced well beyond the period of application. This effect is specific to 2,3-butanedione and is not observed for 1-hexanol. To investigate this further, the fly was exposed to a 3-s stimulus of 2,3-butanedione (10-1 dilution) and subsequently tests were performed for the recovery of ab1C neuron responsiveness by applying a 0.5-s stimulus of 0.3% CO2 every 30 s, over a period of 10 min. Surprisingly, the inhibitory effect of the initial exposure to 2,3-butanedione persisted for an extended period (Turner, 2009).

    It was of interest to test whether behaviour was also affected in a similar manner. Flies were exposed for 1 min to 2,3-butanedione and then transferred them to clean air for 2 min before testing for CO2-mediated avoidance behaviour. Remarkably, CO2 avoidance is almost abolished in pre-treated flies. Prior exposure to another odorant 2-methyl phenol, which does not inhibit the CO2 response, does not have any effect on behaviour. Moreover, pre-exposure to 2,3-butandione does not have a significant effect on behavioural attraction towards a different odorant, ethyl acetate. Taken together, these observations show that exposure to a long-term CO2 response inhibitor can exert a profound and specific effect on the behaviour of the animal, even after it is no longer present in the environment. Similar observations were made with Or83b mutant flies (Turner, 2009).

    To demonstrate unambiguously that 2,3-butanedione causes behaviour modification primarily by inhibiting CO2 responsiveness of ab1C neurons and not by other peripheral or central mechanisms, the following experiment was performed. The ab1C neuron was activated in a manner that is not inhibited by 2,3-butanedione, and it was asked whether 2,3-butanedione inhibits avoidance behaviour in this context. The odorant, butanone, which activates ab1C neurons strongly at 10-1 dilution in a Gr63a-dependent manner, was identified. It was found that Or83b mutant flies strongly avoid butanone (10-1 dilution) whereas flies lacking both Or83b and Gr63a do not, as predicted from the electrophysiology data. However, electrophysiological response to butanone is not affected by pre-exposure to, or the presence of, 2,3-butanedione, unlike what was observed for CO2. In a T-maze behaviour assay, 2,3-butanedione has no effect on behavioural avoidance of Or83b mutant flies to butanone, regardless of whether it is used to pre-treat the flies as described above or is included in a mixture with butanone. These results demonstrate that 2,3-butanedione disrupts CO2 avoidance behaviour by directly inhibiting the CO2 responsiveness of ab1C neurons, rather than by other indirect mechanisms (Turner, 2009).

    CO2 emitted in human breath is a critical component of odour blends used as host-seeking cues by many vector insect species that carry deadly diseases, including Culex quinquefasciatus mosquitoes that transmit filarial parasites in tropical countries, and West Nile virus in the USA and various parts of the world. Culex mosquitoes have three conserved proteins that are closely related to the Drosophila CO2 receptors, Gr21a and Gr63a. To test whether odorants that inhibit Drosophila CO2 receptors can also inhibit CO2 response in Culex, CO2-sensitive A neurons in peg sensilla on the surface of the maxillary palps of Culex mosquitoes were tested using a panel of structurally related odours. It was found found that electrophysiological response to CO2 is not inhibited by 2,3-butanedione, but is strongly inhibited by 1-butanal and 1-hexanol. These odours are the first reported inhibitors of CO2-sensitive neurons in mosquitoes and may provide a valuable resource for the identification of economical, environmentally safe, volatile compounds that may reduce mosquito-human contact by blocking responsiveness to CO2 (Turner, 2009).

    Two Gr genes underlie sugar reception in Drosophila

    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 (Chyb, 2003). Gr5a is expressed in a large number of gustatory neurons in the labellum (Chyb, 2003), 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 (Moon, 2006). 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 (Thorne, 2004; Wang, 2004; 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 (Thorne, 2004; Wang, 2004). 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 (Marella, 2006). 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 (Wang, 2004; Marella, 2006). 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 (Slone, 2007), but not by Gr64a alone (Jiao, 2007). 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 (Jiao, 2007; Slone, 2007), particularly that of Gr64a in response to sugars including sucrose, maltose, and glucose (Jiao, 2007). 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 (Jiao, 2007) 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).

    Deciphering the genes for taste receptors for fructose in Drosophila

    Taste sensitivity to sugars plays an essential role in the initiation of feeding behavior. In Drosophila melanogaster, recent studies have identified several gustatory receptor (Gr) genes required for sensing sweet compounds. However, it is as yet undetermined how these GRs function as taste receptors tuned to a wide range of sugars. Among sugars, fructose has been suggested to be detected by a distinct receptor from other sugars. While GR43A has been reported to sense fructose in the brain, it is not expressed in labellar gustatory receptor neurons that show taste response to fructose. In contrast, the Gr64a-Gr64f gene cluster was recently shown to be associated with fructose sensitivity. This study sought to decipher the genes required for fructose response among Gr64a-Gr64f genes. Unexpectedly, the qPCR analyses for these genes show that labellar expression levels of Gr64d and Gr64e are higher in fructose low-sensitivity flies than in high-sensitivity flies. Moreover, gustatory nerve responses to fructose in labellar sensilla are higher in Gr64d and Gr64f mutant lines than in mutant flies of the other Gr64a-Gr64f genes. These data suggest the possibility that deletion of GR64D or GR64F may indirectly induce enhanced fructose sensitivity in the labellum. Finally, it is concluded that response to fructose cannot be explained by a single one of the Gr64a-Gr64f genes (Uchizano, 2017).

    Enhancing perception of contaminated food through acid-mediated modulation of taste neuron responses

    Natural foods contain not only nutrients, but also nonnutritious and potentially harmful chemicals. Thus, animals need to evaluate food content in order to make adequate feeding decisions. This study investigated the effects of acids on the taste neuron responses and on taste behavior of desirable, nutritious sugars and sugar/bitter compound mixtures in Drosophila melanogaster. Using Ca2+ imaging, acids were shown to activate neither sweet nor bitter taste neurons in tarsal taste sensilla. However, they suppress responses to bitter compounds in bitter-sensing neurons. Moreover, acids reverse suppression of bitter compounds exerted on sweet-sensing neurons. Consistent with these observations, behavioral analyses show that bitter-compound-mediated inhibition on feeding behavior is alleviated by acids. To investigate the cellular mechanism by which acids modulate these effects, bitter-sensing gustatory neurons were silenced. Surprisingly, this intervention had little effect on acid-mediated derepression of sweet neuron or feeding responses to either sugar/bitter compound mixtures or sugar/bitter compound/acid mixtures, suggesting that there are two independent pathways by which bitter compounds are sensed. These investigations reveal that acids, when presented in dietary relevant concentrations, enhance the perception of sugar/bitter compound mixtures. Drosophila's natural food sources - fruits and cohabitating yeast - are rich in sugars and acids but are rapidly colonized by microorganisms, such as fungi, protozoan parasites, and bacteria, many of which produce bitter compounds. It is proposed that the acids present in most fruits counteract the inhibitory effects of these bitter compounds during feeding (Chen, 2014).

    Presynaptic gain control drives sweet and bitter taste integration in Drosophila

    The sense of taste is critical in determining the nutritional suitability of foods. Sweet and bitter are primary taste modalities in mammals, and their behavioral relevance is similar in flies. Sweet taste drives the appetitive response to energy sources, whereas bitter taste drives avoidance of potential toxins and also suppresses the sweet response. Despite their importance to survival, little is known about the neural circuit mechanisms underlying integration of sweet and bitter taste. This study describes a presynaptic gain control mechanism in Drosophila that differentially affects sweet and bitter taste channels and mediates integration of these opposing stimuli. Gain control is known to play an important role in fly olfaction, where GABAB receptor (GABABR) mediates intra- and interglomerular presynaptic inhibition of sensory neuron output. In the taste system, gustatory receptor neurons (GRNs) responding to sweet compounds were found to express GABABR, whereas those that respond to bitter do not. GABABR mediates presynaptic inhibition of calcium responses in sweet GRNs, and both sweet and bitter stimuli evoke GABAergic neuron activity in the vicinity of GRN axon terminals. Pharmacological blockade and genetic reduction of GABABR both lead to increased sugar responses and decreased suppression of the sweet response by bitter compounds. A model is proposed in which GABA acts via GABABR to expand the dynamic range of sweet GRNs through presynaptic gain control and suppress the output of sweet GRNs in the presence of opposing bitter stimuli (Chu, 2014).

    Starvation-induced depotentiation of bitter taste in Drosophila

    Nutrient deprivation can lead to dramatic changes in feeding behavior, including acceptance of foods that are normally rejected. In flies, this behavioral shift depends in part on reciprocal sensitization and desensitization of sweet and bitter taste, respectively. However, the mechanisms for bitter taste modulation remain unclear. This study identified a set of brain ventrolateral octopaminergic/tyraminergic neurons, named OA-VLs, that directly modulate bitter sensory neuron output in response to starvation. OA-VLs are in close proximity to bitter sensory neuron axon terminals and show reduced tonic firing following starvation. It was found that octopamine and tyramine potentiate bitter sensory neuron responses, suggesting that starvation-induced reduction in OA-VL activity depotentiates bitter taste. Consistent with this model, artificial silencing of OA-VL activity induces a starvation-like reduction in bitter sensory neuron output. These results demonstrate that OA-VLs mediate a critical step in starvation-dependent bitter taste modulation, allowing flies to dynamically balance the risks associated with bitter food consumption against the threat of severe starvation (LeDue, 2016).

    Taste representations in the Drosophila brain

    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).

    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, 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).

    An inhibitory sex pheromone tastes bitter for Drosophila males

    Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, this study shows that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, this study shows that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. It is concluded that the inhibitory pheromone tastes bitter to the fly (Lacaille, 2007).

    Sexual behavior requires animals to distinguish between the sexes and respond appropriately to each of them. The genetic potential of Drosophila has made it a focus of study to investigate the role of genes that affect male courtship behavior and sexual orientation. Most studies have centred on the stereotyped male courtship, rather than on the range of sensory cues that are integrated to produce changes in the male's behavior. In Drosophila melanogaster, as in many insect species, chemical signals likely play a key role in sex recognition, and in the initiation and progress of courtship: males are thought to be excited by a range of chemical substances produced by the females, including long-chain hydrocarbons, and to be inhibited by hydrocarbons on the male cuticle (Ferveur, 1996), especially by Z-7-tricosene (7-T) (Lacaille, 2007).

    These substances, which have low or very low volatility are thought to be detected by gustatory receptors (Gr) on the male's legs and proboscis, but there is no direct neuronal evidence of their pheromonal role. The strongest evidence thus far obtained comes from the manipulation of male-specific neurons expressing the GR68A gustatory receptor protein, found in gustatory sensilla on fore-tarsi, which apparently altered the duration of male courtship of females (Bray, 2003). In support of this finding, a putative pheromone-binding protein expressed specifically in these gustatory sensilla has been shown to be required for male discrimination of sex objects (Xu, 2002; Svetec, 2005; Park, 2006). Strikingly, the pheromone(s) causing these effects has not been identified, nor has the nature of the responses shown by these sensilla been determined (Lacaille, 2007).

    Cuticular hydrocarbons are lipophilic and difficult to manipulate using the classical electrophysiological 'tip-recording' method, which uses a single electrode filled with hydrophilic solution eventually mixed with a detergent. A device consisting of two electrodes was designed, with which taste sensilla could be separately stimulated and their physiological activity in response to synthetic 7-T recorded. The pheromonal effect of pure 7-T on male courtship intensity was investigated. It was found that (1) the same taste neurons respond to both 7-T and to bitter substances and (2) these two types of molecules similarly inhibit male behavior in a dose-dependent manner (Lacaille, 2007).

    To assess the effect of sex pheromones on male courtship behavior, the courtship index (CI) that wild-type Canton-S (WT) males directed towards immobilized target flies with various cuticular hydrocarbon profiles was measured. All tests were carried out under dim red light and with decapitated targets so as to enhance the behavioral effect of pheromones. Under these experimental conditions, WT males produced relatively high levels of homosexual courtship to sibling target WT males which produce high levels of 7-T. However, as expected, these tester males showed significantly higher CIs toward WT females, and also to Tai and desat1 (encoding a stearoyl-CoA 9-desaturase) males. These three types of fly all have low levels of 7-T. However, in other respects they have different cuticular hydrocarbon profiles: Tai males (from West Africa) are rich in Z-7-pentacosene (7-P, a stimulatory pheromone for D. melanogaster males), whereas males from the desat1 mutant line have very low levels of 7-P. WT females have also very low levels of 7-P but produce high levels of 7,11 heptacosadiene (7,11-HD) and 7,11 nonacosadiene (7,11-ND) which tend to enhance male courtship stimulation (Lacaille, 2007).

    These data confirm previous suggestions that 7-T tends to inhibit homosexual courtship by WT males. To demonstrate this, 7-T was synthesized, and various doses of this substance were placed on the cuticle of a desat1 mutant male which show almost no 7-T. The results further supported the hypothesis: tester WT males showed a negative correlation between their courtship of 7-T covered desat1 males, and the dose of synthetic 7-T placed on the target male. 0.5 micrograms 7-T did not affect the CI whereas 1 microgram 7-T—which roughly corresponds to one WT male-equivalent—induced a CI that was similar to that induced by WT target males, and 2 micrograms 7-T induced a strong inhibition of WT male courtship. The solvent (pentane) used to dissolve 7-T had no significant effect on male courtship. These data demonstrate that 7-T induces a dose-dependent inhibition of the homosexual courtship shown by WT tester males (Lacaille, 2007).

    To investigate the role of gustatory neurons in detecting male inhibitory pheromones, the electrophysiological activity of these neurons in response to pure synthetic 7-T was measured. The tungsten electrode method allowed separate stimulation and recording of the electrophysiological activity of a sensillum on the labial palp. The stimulating electrode was filled with a lipophilic buffer (paraffin oil) containing 7-T, and the tip of the taste sensillum was tapped, while the tungsten recording electrode was inserted at the base of the sensillum (Lacaille, 2007).

    Recordings were taken from the three types of sensilla present on the labial palps: i-type (which have two gustatory receptor neurons), s- and l-type taste sensilla (which have four gustatory receptor neurons), and the sensilla were mapped according to their physiological responses. Six out of six s- and four out of eight i-type sensilla so far examined consistently responded to 7-T at the 10-8M concentration. This provides the first direct evidence of a neuronal response to a cuticular hydrocarbon sex pheromone in an insect. In i-type sensilla, only one of the two gustatory neurons responded to 7-T. The active cell was the L2 cell, which produced small spikes. In contrast, the second gustatory neuron (the L1-S or L1 cell), housed in the same i-type sensilla, did not respond to 7-T but responded normally to salt and sucrose. The firing frequency of the L2 cell, measured in a s-type sensilla, increased with 7-T concentration (between 10-12 and 10-8 M). Conversely, l-type sensilla on the labial palp responded to NaCl, KCl and sucrose, but not to 10-8M 7-T (Lacaille, 2007).

    The L2 cell in i-type sensilla is known to respond to bitter molecules. To investigate whether the same neuron processes both 7-T and bitter substances, a series of experiments was performed. When stimulated with a mixture of 7-T and caffeine, the L2 cell elicited an increased number of spikes with the same amplitude than when stimulated with either substance alone. 7-T and bitter substances also show cross-adaptation: pre-stimulation with 7-T significantly reduced the response of L2 cells to caffeine but not to sucrose. Taken together, these additive and cross-adaptative effects strongly indicate that 7-T and bitter stimuli are processed by the same taste neuron, the L2 cell, in the responsive i-type sensilla of the labial palps (Lacaille, 2007). thumbnail

    To confirm this apparent dual sensory processing at the neuronal level, the reciprocal cross-effects were observed of (1) bitter stimuli on sexual behavior and (2) 7-T on feeding-related behavior. Painting desat1 males with any of three bitter substances-caffeine, quinine or berberine-strongly inhibited male courtship of these painted flies by WT control males. Tester males were sensitive to these bitter stimuli just as they were to synthetic 7-T: the three bitter substances induced dose-dependent effects very similar to those induced by 7-T. Berberine induced a more potent inhibition than the two other bitter substances: 1 µg and 2 µg of the former molecule respectively inhibited 50% and 100% males; similar doses of the latter molecules respectively inhibited 35% and 80% males. These results clearly show that bitter substances induce dose-dependent inhibition of male homosexual courtship (Lacaille, 2007).

    To assess whether the sensory processing of 7-T affected feeding behavior the Proboscis Extension Reflex (PER) was performed. When sensilla on the tarsi were unilaterally stimulated with 0.1M sucrose, a positive PER was shown by a majority of male and female flies. When flies were bilaterally stimulated, on one side with 0.1M sucrose and on the contralateral side with 10-8M 7-T, PER was highly reduced in both sexes. PER was also significantly reduced in both sexes by a contralateral application of 0.1M caffeine. These data are supported by a previous PER experiment carried out with berberine. Together, they indicate that both 7-T and bitter compounds similarly affect the appetitive behavior of male flies (Lacaille, 2007).

    Finally, courtship behavior was measured in males with genetically altered taste neurons. To target neurons potentially involved in the perception of bitter substances and of 7-T, the Gr66a-Gal4 transgene, which contains the promoter of the gustatory receptor Gr66a gene fused with the yeast Gal4 sequence, was used. This choice was based on two observations: firstly, both the GR66A-receptor protein and Gr66a-Gal4-expressing neurons are involved in the detection of caffeine. Second, using UAS-GFP, the expression of the Gr66a-Gal4 transgene was visualized and found in approximately 22 i- and s-type taste sensilla symmetrically arranged on each labial plate (these were the same sensilla in which a dual response to 7-T and caffeine was observed) and in 7 to 8 taste sensilla on each front leg. In the labial palps, a single neuron in each sensillum expressed GFP under the control of Gr66a-Gal4 (Lacaille, 2007).

    To assess the effect of Gr66a-Gal4 expressing neurons on male courtship and taste perception, the courtship index (CI) that males with targeted Gr66a-Gal4 expressing neurons directed towards immobilized target WT males was measured. When Gr66a-Gal4 expressing cells were deleted using the pro-apoptotic transgene reaper (UAS-rpr), Gr66a-Gal4; UAS-rpr males showed a significantly enhanced homosexual CI to WT target males. The amplitude of this effect was similar to that shown by Gr66a-Gal4/WT males carrying the Gr66a-Gal4 transgene alone, indicating that the GAL4 protein directly or indirectly affects the targeted taste cells and changes pheromonal perception. Strikingly, the effect of the transgene alone was specific to WT target males: the CIs of Gr66a-Gal4/WT tester males toward WT females and toward Tai and desat1 males were not significantly different to those shown by WT tester males, reinforcing the suggestion that Gr66a-Gal4-expressing neurons are specifically involved in detecting inhibitory chemical stimuli (Lacaille, 2007).

    The inhibitory effect induced by pure 7-T was measured on Gr66a-Gal4/WT males using desat1 target males painted with various amounts of 7-T. The results further supported the hypothesis: contrary to the strong dose-dependent effect induced in WT males, 7-T had a much weaker (if any) inhibitory effect on Gr66a-Gal4/WT flies. As with the effects of 7-T, the three bitter molecules had less (if any) effect on Gr66a-Gal4/WT males compared to WT males. Taken together, these data suggest that Gr66a-Gal4 expressing neurons are required to detect the aversive effect induced by 7-T and by bitter stimuli on male courtship behavior (Lacaille, 2007).

    This study has provided the first direct neuronal evidence of cuticular hydrocarbon sex pheromone processing in an insect. This study has also shown that an inhibitory sex pheromone and repulsive gustatory stimuli are processed by the same neurons (The L2 cells corresponding to some Gr66a-Gal4 expressing neurons) and induce similar behavioral responses. For the male fly, there is apparently no difference between the sensation induced by bitter stimuli and that induced by the inhibitory pheromone 7-T (Lacaille, 2007).

    Given that the two types of substances have little structural similarity (7-T is a straight chain hydrocarbon whereas berberine, caffeine, and quinine are oxygenated, oligocyclic alkaloids) it is unlikely that they are detected by the same receptor molecule. Since multiple types of GR molecules are probably co-expressed in Gr66a-Gal4 expressing labellar taste neurons, and the GR66A receptor molecule is involved in the detection of caffeine, this suggests that 7-T and perhaps other bitter substances are detected by other, unknown receptor molecule(s). In this case, cross-adaptation would presumably take place through common activation of second-messenger systems or of calcium trafficking (Lacaille, 2007).

    These findings provide experimental support for Darwin's hypothesis that sexual selection operates on pre-existing structures and behaviors, co-opting them into new functions. In the present case, it is proposed that pre-existing neuronal networks responsible for detecting and responding to bitter substances became able to detect stimuli produced by other males, with the result that these stimuli inhibited male courtship by activating aversive behaviors that were previously solely induced by bitter stimuli. A similar—likely convergent—process may have taken place in vertebrates, when some semiochemicals produced by male mice are detected by specialized receptor molecules in the main olfactory bulb, not in the accessory olfactory system, which had previously been considered to be the sole site of pheromone processing. This study has shown that identical peripheral neurons are involved in detecting inhibitory pheromones and aversive gustatory stimuli. The next challenge will be to understand how these signals are represented in the fly brain (Lacaille, 2007).

    Molecular and cellular organization of the taste system in the Drosophila larva

    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).

    Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity

    Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, different Drosophila OSNs were challenged with both varying stimulus durations (10-2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1-10 Hz). The results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment (Getahun, 2012).

    The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors

    Insects use taste to evaluate food, hosts, and mates. Drosophila has many "orphan" taste neurons that express no known taste receptors. The Ionotropic Receptor (IR) superfamily is best known for its role in olfaction, but virtually nothing is known about a clade of approximately 35 members, the IR20a clade. Here, a comprehensive analysis of this clade reveals expression in all taste organs of the fly. Some members are expressed in orphan taste neurons, whereas others are coexpressed with bitter- or sugar-sensing Gustatory receptor (Gr) genes. Analysis of the closely related IR52c and IR52d genes reveals signatures of adaptive evolution, roles in male mating behavior, and sexually dimorphic expression in neurons of the male foreleg, which contacts females during courtship. These neurons are activated by conspecific females and contact a neural circuit for sexual behavior. Together, these results greatly expand the repertoire of candidate taste and pheromone receptors in the fly (Koh, 2014).

    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. This study provides 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 (see The Drosophila labellum and its physiological responses). 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 (Weiss, 2011).

    This study has defined five distinct classes of sensilla in the Drosophila labellum on the basis of their responses to bitter compounds (see Labellar sensilla fall into five expression classes that are similar to the functional classes). Four of these sensillar classes contain bitter-sensing neurons; other sensilla did not respond physiologically to any of our bitter tastants. This analysis, then, has defined four classes of bitter-sensing neurons that are diverse in their response profiles. Some are broadly tuned with respect to a panel of bitter compounds and some are more narrowly tuned. The neurons also vary in the temporal dynamics of their responses. Different neurons respond to the same tastant with different onset kinetics, and an individual neuron responds to distinct tastants with diverse dynamics. The functional diversity of bitter-sensing neurons expands the coding capacity of the system: different tastants elicit responses from different subsets of neurons, and distinct tastants elicit diverse temporal patterns of activity from these neurons (Weiss, 2011).

    This systematic analysis does not support previous models that suggest functional uniformity among bitter neurons. A previous physiological study of the labellum did not reveal functionally distinct neuronal classes, but was limited in the number of sensilla and tastants that were examined. There are major technical challenges in recording from I and S sensilla; the S sensilla in particular are small, curved, and difficult to access because of their position on the labellar surface. The finding of functional heterogeneity in labellar sensilla is consistent with the finding that two taste sensilla on the prothoracic leg responded to berberine but not quinine, whereas another sensillum responded to quinine but not berberine. A recent study found that DEET elicited different responses from several labellar sensilla tested. Functionally distinct bitter neurons have also been described in taste organs of caterpillars, and in the case of the Manduca larva, aristolochic acid and salicin activate spike trains that differ in dynamics (Weiss, 2011).

    The functional differences among neurons in the Drosophila labellum suggested underlying molecular differences. In particular, it was asked whether the four classes of bitter taste neurons defined by physiological analysis could be distinguished by molecular analysis. A receptor-to-neuron map of the entire Gr repertoire was constructed, and it was found that four classes of bitter taste neurons emerged on the basis of receptor expression, classes that coincided closely with the four functional classes. Moreover, the neuronal classes that were more broadly tuned expressed more receptors (Weiss, 2011).

    While the physiological and molecular analyses support each other well, there are limitations to each analysis that raise interesting considerations. The functional analysis is based on a limited number of taste stimuli. Bitter tastants were selected that were structurally diverse, but bitter compounds vary enormously in structure and only a small fraction of them can be sampled. It is possible that by testing more tastants, by testing them over a greater concentration range, or by analyzing temporal dynamics in greater detail, that even more diversity would become apparent among the bitter-sensing neurons (Weiss, 2011).

    There are also limitations to the receptor-to-neuron map. First, the map considers exclusively the 68 Grs. There are at least two additional receptors that can mediate bitter taste. DmXR, a G-protein coupled receptor, is expressed in bitter neurons of the labellum and is required for behavioral avoidance of L-canavanine, a naturally occurring insecticide (Mitri, 2009); the TRPA1 cation channel, also expressed in a subset of bitter neurons in the labellum, is required for behavioral and electrophysiological responses to aristolochic acid (Kim, 2010). Second, Gr-GAL4 drivers may not provide a fully accurate representation of Gr gene expression in every case. Genetic analysis has shown that Gr64a is required for the physiological responses of labellar sensilla to some sugars and is therefore expected to be expressed in labellar sugar neurons. The Gr64a-GAL4 driver, however, is not expressed in these neurons, suggesting the lack of a regulatory element. In light of the limitations to the use of the GAL4 system to assess receptor expression, it was encouraging that drivers representing all 68 Grs were expressed in chemosensory neurons, with very few exceptions, and that the expression patterns in the labellum agreed well with the patterns of physiological responses. In addition, it was possible to integrate the functional and expression data and predict a function for one Gr (Weiss, 2011).

    While the data support the hypothesis that Gr59c encodes a bitter receptor for berberine, denatonium and lobeline, Gr59c is not sufficient for responses to these compounds in sugar neurons. It is also apparently not necessary, in the sense that physiological responses to these tastants were observed in S-a sensilla that do not express the Gr59c driver. These observations suggest that there is another receptor for berberine, denatonium and lobeline that may recognize a different moiety of these tastants, providing multiple means of detecting some of the most behaviorally aversive bitter tastants in the panel (Weiss, 2011).

    It is noted that 38 of the Gr-GAL4 drivers, slightly more than half, showed expression in the labellum. The other Grs are likely expressed in other chemosensory neurons of the adult and larva. Of the 38 labellar Gr-GAL4 drivers, 33 are expressed in bitter neurons, and only a few in sugar neurons. It seems likely that a high fraction of Grs are devoted to bitter perception because of the number and structural complexity of bitter compounds. Sugars are simpler and more similar in structure. In order to detect the wide diversity of noxious bitter substances that an animal may encounter, a larger and more versatile repertoire of receptors is likely needed. It is noted that in mice and rats, 36 bitter receptors have been identified (Weiss, 2011).

    Among the Grs mapped to bitter neurons, five map to all bitter neurons: Gr32a, Gr33a, Gr39a.a, Gr66a, and Gr89a. Some or all of these 'core bitter Grs' may function as coreceptors, perhaps forming multimers with other Grs. These core Grs might play a role analogous to Or83b, an Or that is broadly expressed in olfactory receptor neurons and that functions in the transport of other Ors and as a channel, rather than conferring odor-specificity per se. If so, the core Grs may be useful in deorphanizing other Grs in heterologous expression systems. It is noted that in mammals, T1R3 functions as a common coreceptor with either T1R1 or T1R2 to mediate gustatory responses to amino acids or sugars, respectively (Weiss, 2011).

    Finally it is noted that the receptor-to-neuron map defines intriguing developmental problems. How do the five classes of sensilla acquire their diverse functional identities? How does an individual taste neuron select, from among a large Gr repertoire, which receptor genes to express? In the olfactory system of the fly, the expression of each receptor gene is dictated by a combinatorial code of cis-regulatory elements and by a combinatorial code of transcription factors. Mechanisms of receptor gene choice were elucidated in part by identifying upstream regulatory elements that were common to coexpressed Or genes. The receptor-to-neuron map that this study has established for the taste system lays a foundation for identifying regulatory elements shared by coexpressed Gr genes, which in turn may elucidate mechanisms of receptor gene choice in the taste system. It will be interesting to determine whether the mechanisms used in the olfactory and taste systems are similar (Weiss, 2011).

    In principle the design of the Drosophila taste system could have been extremely simple. Every sensillum could be identical, and all sensilla could report uniformly the valence of each tastant, e.g. positive for most sugars and negative for bitter compounds. Such a design would be economical to encode in the genome and to execute during development (Weiss, 2011).

    The design of the Drosophila olfactory system is not so simple. Physiological analysis of the fly has identified ~17 functionally distinct types of olfactory sensilla. This design allows for the combinatorial coding of odors. A recent study of the Drosophila larva defined an odor space in which each dimension represents the response of each component of olfactory input . The distance between two odors in this space was proportional to the perceptual relationship between them. In principle, a coding space of high dimension may enhance sensory discrimination and allow for a more adaptive behavioral response to a sensory stimulus (Weiss, 2011).

    This study has found that the fly's taste system is similar to its olfactory system in that its sensilla fall into at least five functionally distinct types, four of which respond to bitter stimuli. This heterogeneity provides the basis for a combinatorial code for tastes and for a multidimensional taste space. A recent report has suggested that flies can not discriminate between pairs of bitter stimuli when applied to leg sensilla (Masek, 2010); it will be interesting to extend such analysis to the labellum, and especially to examine pairs of stimuli that have been shown to activate distinct populations of neurons. Physiological analysis thus invites an extensive behavioral analysis, beyond the scope of the current study, which explores the extent to which such a taste space supports taste discrimination in the fly (Weiss, 2011).

    Why might there be selective pressure to enhance the coding of bitter taste? Why not simply coexpress all bitter receptors in one type of neuron that activates a single circuit, thereby triggering equivalent avoidance of all bitter compounds? Not all bitter compounds are equally toxic, and it is not clear that there is a direct correlation between bitterness and toxicity. It is even possible that in certain contexts, such as the selection of egg-laying sites or self-medication, some bitter tastants may have a positive valence. It is noted that in the behavioral analysis carried out in this study, flies tended to be more sensitive to bitter compounds that activate I-a than I-b neurons, suggesting that I-a ligands are perceived to be more bitter than those of I-b ligand, as if I-a ligands were more toxic. A more nuanced behavioral decision based on the intensities of bitter compounds may exist within the complex milieu of rotting fruit (Weiss, 2011).

    The olfactory and taste systems of the fly differ in the anatomy of their projections to the brain. Olfactory receptor neurons (ORNs) project to the antennal lobe, which consists of spherical modules called glomeruli. ORNs of a particular functional specificity converge upon a common glomerulus, and there is a distinct glomerulus for each type of ORN. Taste neurons project from the labellum to a region of the ventral brain called the subesophageal ganglion (SOG) that does not have such an obviously modular structure. A study using Gr66a-GAL4, which marks all or almost all bitter cells in the labellum, and Gr5a-GAL4, which marks all or almost all sugar cells, revealed that the two classes of cells project to spatially segregated regions of the SOG (Thorne, 2004; Wang, 2004). However, subsets of bitter cells labeled by Gr-GAL4 drivers did not show obvious spatial segregation within the region of the SOG labeled by Gr66a-GAL4. Markers of different subsets of sugar cells also showed overlapping projections in the SOG. These studies did not, then, reveal at a gross level the kind of spatially discrete projections that are characteristic of the olfactory system (Weiss, 2011).

    However, analysis of the SOG at higher resolution has recently revealed more detailed substructure (Miyazaki, 2010). Different sets of Gr66a-expressing neurons, such as those expressing Gr47a, an I-b-specific receptor, showed distinguishable projection patterns, leading to the suggestion that different subregions process different subsets of bitter compounds. Moreover, similarity in projection patterns does not imply identity of function. For example, in the antennal lobe, ORNs that express the odor receptor Or67d converge on the DA1 glomerulus in both males and females, but the projections from DA1 to the protocerebrum are sexually dimorphic. Activation of these ORNs elicits different behaviors in males and females. Taste neurons that project to similar locations in the SOG could also activate different circuits, with distinguishable behavioral consequences. Like the fly taste system, the C. elegans olfactory system does not contain glomeruli and its sensory neurons coexpress many receptors, yet the worm is able to discriminate odors. Finally, it is noted that different sensory neurons that project to similar positions may carry distinguishable information by virtue of differences in the temporal dynamics of their firing. Differences have been identified in the temporal dynamics elicited by different tastants. In summary, it is difficult to draw definitive conclusions about the functional roles of taste neurons from the currently available anatomical analysis (Weiss, 2011).

    A final consideration raised by this analysis is how the responses of the different functional classes of taste sensilla are temporally integrated to control feeding behavior. The different functional classes of sensilla differ in length and are located in different regions of the labellar surface. Moreover, during the course of feeding the labellum expands, changing the positions of the various sensilla with respect to the food source. It seems likely that there is a temporal order in which labellar taste sensilla send information to the CNS (Weiss, 2011).

    In summary, this study has provided a systematic behavioral, physiological, and molecular analysis of the primary representation of bitter compounds in a major taste organ. This study has defined the molecular and cellular organization of the bitter-sensitive neurons, and extensive functional diversity was found in their responses. The results provide a foundation for investigating how this primary tastant representation is transformed into successive representations in the CNS and ultimately into behavior (Weiss, 2011).

    Functional dissociation in sweet taste receptor neurons between and within taste organs of Drosophila

    Finding food sources is essential for survival. Insects detect nutrients with external taste receptor neurons. Drosophila possesses multiple taste organs that are distributed throughout its body. However, the role of different taste organs in feeding remains poorly understood. By blocking subsets of sweet taste receptor neurons, receptor neurons in the legs were shown to be required for immediate sugar choice. Furthermore, two anatomically distinct classes of sweet taste receptor neurons were identified in the leg. The axonal projections of one class terminate in the thoracic ganglia, whereas the other projects directly to the brain. These two classes are functionally distinct: the brain-projecting neurons are involved in feeding initiation, whereas the thoracic ganglia-projecting neurons play a role in sugar-dependent suppression of locomotion. Distinct receptor neurons for the same taste quality may coordinate early appetitive responses, taking advantage of the legs as the first appendages to contact food (Thoma, 2016).

    A Drosophila gustatory receptor required for strychnine sensation
    Summary:
    Strychnine is a potent, naturally occurring neurotoxin that effectively protects plants from animal pests by deterring feeding behavior. In insects, such as the fruit fly, Drosophila melanogaster, bitter-tasting aversive compounds are detected primarily through a family of
    gustatory receptors (GRs), which are expressed in gustatory receptor neurons. Multiple GRs that eliminate the behavioral avoidance to several bitter compounds, with the exception of strychnine, have been previously described. This study reports the identity of a strychnine receptor, referred to as GR47a. A mutation in Gr47a, eliminates strychnine repulsion and strychnine-induced action potentials. GR47a is narrowly tuned, as the responses to other avoidance compounds are unaffected in the mutant animals. This analysis supports an emerging model that Drosophila GRs fall broadly into two specificity classes-one class is comprised of core receptors that are broadly required, whereas the other class, which includes GR47a, consists of narrowly tuned receptors that define chemical specificity (Lee, 2015).

    Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing

    Behavior cannot be predicted from a 'connectome' because the brain contains a chemical 'map' of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. This study describes a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms (Inagaki, 2012).

    The Tango system transforms a transient ligand/receptor interaction into a stable, anatomical readout of reporter gene expression. The reporter gene is activated by a 'private,' synthetic signal transduction pathway, using a bacterial transcription factor (lexA) that is covalently coupled (via a specific tobacco etch virus [TEV] protease-sensitive cleavage site) to the exogenous DA receptor expressed in the cells of interest. The transcription factor is cleaved from the DA receptor following ligand binding, by recruitment of an arrestin-TEV protease fusion protein, and translocates to the nucleus where it activates a lexAop-driven reporter. This system was originally developed to detect receptor activation in cultured mammalian cell lines, but it used to detect receptor activation in this system (Inagaki, 2012).

    The data presented here provide proof-of-principle for the utility of a new method, called TANGO-map, to identify, in a brain-wide and relatively unbiased manner, circuit-level substrates of neuromodulation relevant to a particular state-dependent influence on behavior (Inagaki, 2012).

    Sweet taste sensitivity in the labellum is enhanced with increasing duration of food deprivation in Drosophila. This observation confirms and extends previous reports in Drosophila and is consistent with observations in many other animal species. This phenomenon was used as a prototypic case of a state-dependent change in behavior to investigate the ability of TANGO-map to identify underlying neuromodulatory mechanisms (Inagaki, 2012).

    The results indicate that starvation enhances endogenous DA release onto primary gustatory receptor neurons (GRNs) in the sub-esophageal ganglion, as detected by increased expression of the DopR-Tango reporter in vivo. In contrast, starvation did not increase the DopR-Tango reporter in the MB or AL, although L-dopa feeding did so. These data indicate that DopR-Tango is capable of revealing selective sites of endogenous DA release in a brain-wide manner, under specific behavioral conditions (Inagaki, 2012).

    The results indicate that a mutation in the DA receptor DopEcR, as well as specific knockdown of this receptor in sugar-sensing GRNs, eliminates the effect of starvation to enhance the sucrose sensitivity of the proboscis extension reflex (PER). However, this phenotype was only observed at 6 hr of starvation; after 24 hr of food deprivation, these genetic manipulations no longer had an effect. This is not because these manipulations themselves became ineffective at later times, as the same manipulations did attenuate the increased PER sensitivity caused by L-dopa feeding for 24 hr. This suggests that at an early stage of starvation, DA is necessary to enhance the sugar sensitivity of the PER, whereas at later stages additional factors come into play. The slow kinetics of Tango reporter accumulation preclude the detection of statistically significant increases in signal as early as 6 hr following an experimental manipulation. However, the level of reporter expression detected in animals examined after 48 hr of treatment likely reflects the integration of increases in dopaminergic signaling occurring throughout the first 12-24 hr of the treatment period. Thus, although an increase was detected in DopR-Tango signal at a starvation time point when genetic reduction of DopEcR levels no longer impaired the behavioral effect of starvation and observed a behavioral phenotype at a time point too early to be evaluated directly by the TANGO-map method, this should not be taken to imply that no DA release occurred after 6 hr of starvation. Importantly, given the kinetics of the system, the DopR-Tango signals detected in vivo are likely to reflect primarily changes in tonic levels of DA signaling, rather than brief episodes of phasic DA release. Further improvements of the TANGO-map method are required to increase its temporal resolution. Nevertheless, the present methodology provides a powerful method to identify sites where dopaminergic modulation of a given behavior may occur, even if it cannot reveal precisely how quickly such regulation is exerted (Inagaki, 2012).

    Several lines of evidence suggest that the dopaminergic modulation of sugar-sensing GRNs revealed in this study may involve an enhancement of Ca2+ influx at the nerve terminal. Both starvation and L-dopa feeding increased sucrose-evoked Ca2+ influx, without changing the frequency of action potentials measured extracellularly at GRN somata. Furthermore, it was found that direct exposure of the brain to DA increased Ca2+ influx at the presynaptic terminals of sugar-sensing GRNs in a DopEcR-dependent manner. A model consistent with these data is that starvation leads to increased DA release, which increases calcium influx into sugar-sensing GRNs via DopEcR, leading to increased neurotransmitter release. The fact that DopEcR signals via the cAMP/PKA pathway, and that this pathway has been reported to increase Ca2+ channel currents in Drosophila, is also consistent with this scenario. Nevertheless, the genetic data suggest that there are additional pathways through which starvation modulates feeding behavior in this system. The finding that DA modulates primary GRNs to control starvation-dependent changes in behavioral sensitivity to sugar echoes the observation of a similar influence of food deprivation on odorant sensitivity in Drosophila. Such neuromodulatory gain control at the level of primary sensory neurons has also been reported in a variety of other invertebrate as well as vertebrate species. Although the possibility that hunger also influences PER behavior at higher-order synapses in the circuit cannot be excluded, the data add to a growing body of information indicating that modulation of primary sensory neurons is a general mechanism for implementing state-dependent changes in behavioral responses to the stimuli detected by these neurons (Inagaki, 2012).

    TANGO-map affords a number of unique advantages to study neuronal modulation in the brain. First, and most importantly, it permits the detection of increases in endogenous neuromodulator release in vivo, in an organism in which the application of conventional methods is not feasible. Second, it provides an anatomical readout of neuromodulation at the neural circuit level. The use of a pan-neuronal GAL4 driver to express the sensor permits, in principle, an unbiased survey of potential sites of neuromodulatory activity throughout the brain. Third, the sensor has ligand specificity. The modular design of the Tango system affords the ability to develop in vivo Tango reporters for other biogenic amines and neuropeptides that work via G protein-coupled receptors (GPCRs). Importantly, because the method employs a synthetic, 'private' signal transduction pathway, the readout of the reporter should be relatively insensitive to interference from conventional signal transduction pathways activated by other endogenous receptors. Systematic and comprehensive application of this approach could, in principle, provide an overview of anatomic patterns of neuromodulation in the brain in a given behavioral setting. Finally, because the Tango system is transcriptionally based, in principle it permits the expression not only of neutral reporters but also of effectors such as RNAi’s or ion channels in the neurons receiving neuromodulatory input. Although the TANGO-map system can certainly benefit from improvements in its kinetics and SNR, it affords a means of identifying points-of-entry for studying circuit-level mechanisms of behaviorally relevant neuromodulation that are currently difficult to access in any otherway. The extension of this methodology to other neuromodulators and model organisms should further understanding of state-dependent control of neural activity and behavior (Inagaki, 2012).

    Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila

    An organism's behavioral decisions often depend upon the relative strength of appetitive and aversive sensory stimuli, the relative sensitivity to which can be modified by internal states like hunger. However, whether sensitivity to such opposing influences is modulated in a unidirectional or bidirectional manner is not clear. Starved flies exhibit increased sugar and decreased bitter sensitivity. It is widely believed that only sugar sensitivity changes, and that this masks bitter sensitivity. This study used gene- and circuit-level manipulations to show that sweet and bitter sensitivity are independently and reciprocally regulated by starvation in Drosophila. Orthogonal neuromodulatory cascades were detected that oppositely control peripheral taste sensitivity for each modality. Moreover, these pathways are recruited at increasing hunger levels, such that low-risk changes (higher sugar sensitivity) precede high-risk changes (lower sensitivity to potentially toxic resources). In this way, state-intensity-dependent, reciprocal regulation of appetitive and aversive peripheral gustatory sensitivity permits flexible, adaptive feeding decisions (Inagaki, 2014).

    A fructose receptor functions as a nutrient sensor in the Drosophila brain

    Internal nutrient sensors play important roles in feeding behavior, yet their molecular structure and mechanism of action are poorly understood. Using Ca2+ imaging and behavioral assays, this study shows that the Gustatory receptor 43a (Gr43a) functions as a narrowly tuned fructose receptor in taste neurons. Remarkably, Gr43a also functions as a fructose receptor in the brain. Interestingly, hemolymph fructose levels are tightly linked to feeding status: after nutritious carbohydrate consumption, fructose levels rise several fold and reach a concentration sufficient to activate Gr43a in the brain. By using different feeding paradigms and artificial activation of Gr43a-expressing brain neurons, this study shows that Gr43a is both necessary and sufficient to sense hemolymph fructose and promote feeding in hungry flies but suppress feeding in satiated flies. Thus, these studies indicate that the Gr43a-expressing brain neurons function as a nutrient sensor for hemolymph fructose and assign opposing valence to feeding experiences in a satiation-dependent manner (Miyamoto, 2012).

    The taste sensory system plays a central role in identifying and evaluating potential foods by discriminating between nutritious chemicals that promote feeding, and structurally diverse, harmful or even toxic compounds, that inhibit feeding. Despite the distinct evolutionary origin of taste receptors of mammals and invertebrates, the cellular organization underlying taste discrimination is largely conserved. In Drosophila, gustatory receptor neurons (GRNs) express distinct sets of gustatory receptors (GRs), providing the basis for discrimination between sweet and bitter taste, respectively. Specifically, the sweet taste of sugars is thought to be exclusively mediated by members of a small conserved subfamily of eight putative sugar Gr (psGr) genes (Gr5a, Gr61a and Gr64a-f), which are partially co-expressed in a single GRN of each taste sensillum, the 'sweet' neuron. Conversely, the bitter taste of alkaloids, terpenoids and phenols is mediated by receptors encoded by most other Gr genes, including Gr66a, Gr33a, and Gr93a, which are partially co-expressed in a second GRN of each sensillum, the 'bitter/high salt' neuron. Most sensillae have two additional GRNs not associated with any characterized Gr gene and are thought to detect low salt solutions and water, respectively (Miyamoto, 2012).

    In addition to evaluating external chemicals by the taste sensory system, cells located in internal organs, including the gut, liver/fat body and the brain, express receptors that detect nutrients or their metabolically processed derivates to regulate energy homeostasis and feeding behaviors. Interestingly, internal nutrient sensing in the gut of rodents is in part mediated by taste receptors. Moreover, some bitter taste receptors were shown to be expressed in the mammalian brain and glucose sensing neurons were identified in the hypothalamus. In Drosophila, evidence of an internal nutrient sensor was recently suggested by work of several laboratories. Two groups showed that flies are able to evaluate tasteless carbohydrates based solely on their nutritional content. Suh and co-workers reported that hungry flies with severely impaired sugar-sensing ability can still discriminate sweet tasting sugars based on their nutritious content. However, the molecular identity of the proposed nutrient sensor, the anatomical structure in which it resides, and its ligand, are not known (Miyamoto, 2012).

    This paper reports that Gr43a, one of the most conserved insect gustatory receptor genes, is expressed in the brain. Using a Ca2+ imaging assay, GR43a was found to be a narrowly tuned fructose receptor. Although circulating fructose is approximately 100 times less abundant than the main hemolymph sugars glucose/trehalose, it rises to levels high enough after a sugar meal to activate the Gr43a-expressing brain neurons. Feeding experiments reveal that these neurons promote feeding in hungry flies, but suppress feeding in satiated flies. It was shown that artificial activation of Gr43a expressing brain neurons assign positive valence in hungry flies, but negative valence in satiated flies. Thus, this work establishes a precedent of a new nutrient sensing system that regulates food consumption in a satiation-dependent manner (Miyamoto, 2012).

    Fructose is the most abundant dietary carbohydrate in many fruits, including apples, grapes and blueberries, major food sources of many Drosophila species. This study has shown that flies have two receptors for this sugar, GR43a, and one encoded by the putative sugar Gr genes (Gr5a, Gr61a and Gr64a-f). While Ca2+ imaging experiments indicate that GR43a is more potent in sensing fructose, it has a minor role as an external sugar receptor in adult flies. However, Gr43a plays a prominent role in sugar sensing during the larval stage. Recent patch-clamp recordings in heterologous expression systems showed that the bombyx mori gustatory receptor BmGr-9 and its Drosophila ortholog GR43a function as a ligand-gated ion channel that is selectively activated by fructose. Curiously, BmGr-9 was not activated by sucrose (GR43a was not tested), and it will be interesting to see whether this difference is due to sequence variation between the two orthologs, or a reflection of suboptimal conditions in a heterologous expression system (Miyamoto, 2012).

    Like blood glucose in mammals, levels of glucose and trehalose, the main hemolymph sugars in Drosophila, are kept relatively stable, regardless of feeding state. Analogous to the insulin and glucagon pathways in mammals, flies counteract rising or decreasing glucose/trehalose in the hemolymph by secreting insulin-like peptides or adipokinetic hormone to maintain energy homeostasis. Interestingly, glucose responsive neurons have been identified in the hypothalamus, and it has been proposed that an internal sugar sensor in mice modulates feeding behavior via the dopamine reward system). However, the neural circuits and the molecular nature of internal nutrient sensors are unknown, both in mammals and insects (Miyamoto, 2012).

    The internal fructose sensing system described in this study establishes a precedent. About six Gr43aGAL4 neurons in the posterior superior lateral protocerebrum are specifically activated by fructose. Although GR43a functions independently of any of the known psGRs, ectopic expression of Gr43a in other brain neurons does not render them fructose sensitive, implying that this receptor acts in concert with another GR protein, or requires a cell-specific transducer. Regardless, these observations indicate that Gr43aGAL4 brain neurons detect fructose in the hemolymph that derive directly from dietary fructose, or indirectly from other nutritious carbohydrates (Miyamoto, 2012).

    Why do flies use fructose, rather glucose (or its disaccharide, trehalose), which is 100 fold more abundant than fructose in the hemolymph, as a signal for an internal nutrient sensor? The data indicate that a glucose sensor would be difficult to activate in a robust fashion because a single sugar meal, even if completely absorbed and converted into glucose would increase overall amount of hemolymph glucose by less than 1/3. Specifically, the amount of hemolymph glucose in a fly (1 mg weight) is ~9.4 μg, compared to ~3 μg of glucose or ~2.4 μg of fructose present in a single meal. In contrast, the amount of hemolymph fructose is very low and therefore increases several fold after a single sugar meal. For example, the ~2.4 μg of fructose in a meal is more than 30 times the amount of fructose present in the hemolymph (0.07 μg). The actual increase observed in the hemolymph sugar assay is ~10 fold, which nevertheless is sufficient for strong activation of the fructose sensor GR43a. Therefore, the internal fructose sensor described here provides a robust system that takes advantage of a steep increase in hemolymph fructose after a sugar meal to evaluate the nutritious content of food (Miyamoto, 2012).

    A remarkable property of the Gr43aGAL4 brain neurons is their ability to both promote feeding in hungry flies and suppress feeding in satiated flies. This was directly demonstrated in the odor-conditioning paradigm, which revealed that these neurons assign diametrically opposite valence that is dependent on the satiation status. These observations lead to proposal of the following model: Ingestion and conversion of nutritious carbohydrates leads to an increase of hemolymph fructose, resulting in activation of Gr43aGAL4 brain neurons. In hungry flies, this activation is perceived positively, thereby reinforcing feeding behavior. In contrast, the activation in satiated flies is perceived negatively and leads to feeding termination. How can the satiation status so drastically change the behavioral output through a single group of neurons? At least two distinct mechanisms could account for these opposite effects. The first mechanism invokes direct modulation of some, but not all of the Gr43aGAL4 brain neurons. For example a subset of these neurons may respond to a factor present in hemolymph of satiated, but not hungry flies. Such a factor may act on a second receptor co-expressed in these neurons and modulate GR43a-mediated activity. Alternatively, Gr43aGAL4 brain neurons may target distinct regions of the brain. Consistent with this idea is the observation that axons of Gr43aGAL4 brain neurons project along two separate paths, connecting to distinct, spatially segregated neural ensembles. Distinct output of Gr43aGAL4 brain neurons could be achieved if one of these neural ensembles is regulated by a satiation-dependent signal such as dopamine or octopamine, which are required for aversive and appetitive learning, respectively. Even though Gr43aGAL4 neurons are neither dopaminergic nor octopaminergic, it is possible that their downstream targets use these neurotransmitters to convert output from Gr43aGAL4 neurons into positive or negative valence (Miyamoto, 2012).

    In both scenarios, the satiety signal may derive from the fat cells or neurosecretory cells, such as the Drosophila insulin like peptide (DILP) expressing cells in the brain. Interestingly, TOR signaling of larvae fed with a protein rich diet was shown to induce secretion of a humoral signal from fat cells that acts on the DILP expressing cells to control growth and glucose homeostasis. The same signaling pathway might be used to regulate the intake of carbohydrates in adult flies by imposing positive and negative valence via the Gr43aGAL4 brain neurons. Alternatively, satiation-dependent factors, such as neuropeptide F (NPF), may modulate the Gr43aGAL4 brain circuitry. NPF and the mammalian ortholog, neuropeptide Y (NPY) are known to increase food consumption. Moreover, the NPF/NPF receptor system also provides the neural framework that integrates the state of satiety and appetitive memory in adult flies (Miyamoto, 2012).

    Gr43a is likely to have additional roles, due to its highly specific expression in proventriculus- and uterus-associated neurons. For example, detection of dietary fructose in the foregut may induce digestive processes, such as peristaltic movements of the gut musculature, or activation of metabolic enzymes in secretory cells. Gr43a expression in the uterus suggests a role for this receptor in female physiology and/or behavior that are linked to mating or reproduction. Fructose, like sex-peptide and other male-specific proteins, might be present in seminal fluid and serve as a ligand to modulate female behaviors associated with reproduction. Whatever the roles of Gr43a may be, the biological functions of this receptor are predicted to be conserved across insect species (Miyamoto, 2012).

    Drosophila learn opposing components of a compound food stimulus

    Dopaminergic neurons provide value signals in mammals and insects. During Drosophila olfactory learning, distinct subsets of dopaminergic neurons appear to assign either positive or negative value to odor representations in mushroom body neurons. However, it is not known how flies evaluate substances that have mixed valence. This study shows that flies form short-lived aversive olfactory memories when trained with odors and sugars that are contaminated with the common insect repellent DEET. This DEET-aversive learning required the MB-MP1 dopaminergic neurons that are also required for shock learning. Moreover, differential conditioning with DEET versus shock suggests that formation of these distinct aversive olfactory memories relies on a common negatively reinforcing dopaminergic mechanism. Surprisingly, as time passed after training, the behavior of DEET-sugar-trained flies reversed from conditioned odor avoidance into odor approach. In addition, flies that were compromised for reward learning exhibited a more robust and longer-lived aversive-DEET memory. These data demonstrate that flies independently process the DEET and sugar components to form parallel aversive and appetitive olfactory memories, with distinct kinetics, that compete to guide learned behavior (Das, 2014).

    DEET has been reported to drive aversive behavior in flies through olfactory and gustatory pathways. Therefore a low concentration presented in solid medium (1% agar) was used to decrease the effects of volatile DEET and increase the chance that flies would taste and perhaps ingest it. To further encourage flies to sample DEET, its palatability was increased by adding it to a mixture of sweet sugars-3 M xylose and 100 mM sucrose (from here on referred to as 'carrier'). Xylose is detected by sweet-sensitive gustatory neurons and is palatable to flies, but it contributes no measurable nutrient value. The low concentration of sweet and nutritious sucrose was added to further increase palatability. The optimum DEET concentration was determined by adding increasing amounts to sugar carrier and conditioning hungry flies by pairing the exposure of the second of two odors with DEET presentation (Das, 2014).

    Flies trained with only the sugar carrier showed a significant appetitive memory. In contrast, those trained with increasing amounts of DEET formed aversive memory, with the score rising in line with the increase in DEET concentration, up to 0.4%. Surprisingly, flies trained with 0.8% DEET did not exhibit significantly negative aversive memory scores, suggesting a change in the flies' perception of DEET at this concentration. Therefore the effect of 0.4% and 0.8% DEET on fly feeding was tested by measuring ingestion marked with blue food dye. Whereas flies ate significant amounts of food containing sugar carrier, both 0.4% and 0.8% DEET strongly suppressed feeding behavior. However, whereas flies ate a measurable amount of dye with 0.4% DEET, ingestion was abolished with 0.8% DEET. These data suggest that the failure to train flies with 0.8% DEET reflects an inhibition of sampling by the proboscis and perhaps ingestion of DEET and sugar. To further test a requirement for feeding in learning, attempts were made to train flies that were not hungry or with 0.4% DEET without sugar carrier. Both of these conditions significantly impaired aversive learning when compared to hungry flies trained with 0.4% DEET in sugar carrier. A similar concentration-dependent aversive memory formation was also observed when flies were trained with bitter-tasting quinine that was mixed with sugar carrier. Furthermore, flies that were defective in the IR40a olfactory route of DEET detection displayed normal DEET learning. It was therefore concluded that robust learning with 0.4% DEET-laced sugar requires the flies to attempt to eat DEET and that low DEET concentrations convert the conditioned approach that is formed when flies are trained with the sugar carrier into a conditioned aversion (Das, 2014).

    The persistence of DEET memory was tested by conditioning flies and testing their odor preference at extended times after training. Whereas aversive memory performance was robust immediately after training, no statistically significant performance was evident 15 min later. Aversive memory formed with 0.4% DEET is therefore surprisingly labile. DEET and quinine can be sensed by bitter-taste neurons, and ablation of bitter-sensing neurons with Gr66a-GAL4-directed expression of cell-death genes partially impaired DEET, but not sugar, learning. Therefore whether flies could be aversively conditioned by pairing odor presentation with artificial bitter-taste neuron activation, achieved by expression of UAS-dTrpA1, was tested. The dTrpA1 gene encodes a transient receptor potential (TRP) channel that conducts Ca2+ and depolarizes neurons when flies are exposed to temperature >25°C. Gr66a-GAL4, UAS-dTrpA1, and Gr66a-GAL4; UAS-dTrpA1 flies were conditioned by presentation of the first odor with activating 32°C and were immediately tested for memory. Gr66a-GAL4; UAS-dTrpA1 flies exhibited aversive memory that was statistically different from that of all other groups. However, unlike flies conditioned with DEET, significant memory remained 3 hr after training. The differing persistence could result from artificial stimulation of bitter neurons being stronger than DEET activation, in addition to lacking plausible competition from a copresented sugar stimulus (Das, 2014).

    Octopamine is required to convey the reinforcing effects of sweet taste. Therefore DEET learning was tested in TbhM18 mutant flies that cannot synthesize octopamine. Whereas appetitive conditioning with 1 M sucrose was significantly impaired in TbhM18 flies, aversive learning with 0.4% DEET was indistinguishable from that of wild-type flies. Therefore, octopamine is not required for DEET learning (Das, 2014).

    Electric-shock-reinforced aversive memory formation also requires specific dopaminergic neurons and the DopR1 dopamine receptor. It was therefore first determined whether DEET learning required the DopR1 receptor. Mutant dumb1 flies that are defective for the DopR1 dopamine receptor did not display aversive learning with DEET. Similarly, aversive learning with artificial activation of bitter-taste neurons was abolished in dumb1 flies (Das, 2014).

    The MB-MP1, MB-MV1, and MB-M3 classes of dopamine neuron have been previously implicated in shock learning. To test whether either of these neurons were required for DEET learning, the dominant temperature-sensitive UAS-shibirets1 transgene was tested in MP1, MV1, and M3 neurons using the c061; MBGAL80, R73F07, and NP5272 and NP1528 GAL4 drivers, respectively. The shibirets1 transgene permitted blockade of the respective neurons by performing DEET conditioning experiments at the restrictive temperature of 31°C. This analysis revealed significantly impaired DEET learning performance when MP1 neurons were blocked but nonsignificant effects when either MV1 or M3 neurons were compromised. Blockade of MP1 neurons, however, did not significantly affect DEET avoidance in naive flies. To further support a role for the dopaminergic MP1 neurons in c061; MBGAL80, they were removed from the expression pattern by including a TH-GAL80 transgene. When the remaining cells were blocked during conditioning, flies exhibited levels of DEET learning that were indistinguishable from those of wild-type flies. It is therefore concluded that MP1 neurons are critical for DEET learning, whereas MV1 and M3 neurons contribute a lesser role. It was noted that prior work implicated the MV1 and M3 neurons in the formation of more persistent forms of shock-reinforced aversive memory (Das, 2014).

    Next, live imaging was used to determine whether DEET ingestion activated the MP1 dopamine neurons. UAS-GCaMP3 was expressed in dopaminergic neurons with TH-GAL4 and imaged DEET-evoked changes in fluorescence in the dopaminergic neuron processes on the mushroom body. These analyses revealed strong activation of the MP1 innervated heel and MV1 innervated junction regions of the mushroom body while presenting flies with both 0.4% DEET in sugar carrier, sugar carrier alone, and DEET alone. In comparison, water presentation did not activate the MP1 and MV1 neurons. Therefore, functional imaging does not reveal obvious valence specificity of MP1 and MV1 signals, being activated by both sugar and DEET. It should be noted that the MP1 neurons have been previously implicated in shock- and sugar-reinforced learning and memory expression. Since a strong requirement was observed for MP1 neurons in behavioral DEET learning, it is concluded that MP1 activity is likely to represent aversive reinforcement signals to mushroom body neurons. As expected, transmission from mushroom body neurons is required for the expression of DEET memory (Das, 2014).

    Finding a role in DEET learning for dopamine neurons that are also required for shock learning suggests a common reinforcement process, despite the different nature of the external unconditioned stimulus. Therefore a differential conditioning paradigm was designed to further test this model. Flies were trained by pairing of one odor with DEET and the other odor with a varying intensity of electric shock. These experiments revealed an avoidance of the previously DEET-associated odor when countered with 30 or 60 V but an avoidance of the shock-paired odor when countered with 80 or 90 V. Extrapolation of a curve fit between the tested points predicted 70 V as being equivalent to 0.4% DEET-which was subsequently confirmed in direct experiments. Having established the point of reinforcer equivalence, it was reasoned that if the shock and DEET reinforcement processes were common, blocking some of the responsible dopamine neurons would equally impair shock and DEET learning and therefore not alter equivalence. If, on the other hand, MP1 neurons contribute differently to DEET and shock reinforcement, blocking them would unevenly affect learned behavior and would skew performance toward one or the other, reflecting the imbalance. Strikingly, differential learning remained balanced in c061; MBGAL80; UAS-shits1 flies in which MP1 neurons were blocked. Importantly, this balanced valuation does not reflect a 'zero versus zero' learning because the same c061; MBGAL80; UAS-shits1 flies only display a partial defect if they were trained with 70 V shock alone. Therefore, these experiments support a model in which the reinforcing systems for 0.4% DEET and 70 V shock are similar, with MP1 being part of the system for both. In addition, it is notable that despite the relative magnitude of immediate memory scores (∼0.6 for 70 V shock and <0.3 for DEET) and the difference in respective memory persistence (hours for shock and minutes for DEET), the immediate learned value of these two aversive stimuli is comparable (Das, 2014).

    Next whether the apparent fragility of aversive DEET memory could be explained by the coformation of a more persistent sugar memory was tested. Reasoning that these analyses would benefit from the induction of a more robust sugar memory, optimal conditions for aversive memory formation was first established with DEET-laced 1 M sucrose. Flies trained with DEET in 1 M sucrose showed a similar dose-dependent aversive learning to those trained in prior experiments with DEET in xylose and sucrose carrier, although the optimal DEET concentration for learning shifted from 0.4% to 0.6%. The DEET memory performance was next thested of TbhM18 mutant flies that are impaired in appetitive learning. Strikingly, whereas the behavior of wild-type flies became conditioned approach within 30 min, TbhM18 flies showed a more persistent aversive memory performance, with scores remaining significantly negative 30 and 60 min after training. However, the performance still converted from odor avoidance to approach by 24 hr. Since octopamine only provides short-term sweet-taste reinforcement, it was hypothesized that persistent nutrient-dependent memory must be independently formed in TbhM18 flies. Indeed, TbhM18 flies trained with 1 M sucrose did not display immediate memory, but significant performance emerged 1 hr after training and remained for at least 24 hr. These data support the prior model of octopamine specifically conveying short-term appetitive reinforcement and not the nutrient-dependent long-term signal. In addition, they suggest that the DEET learning protocols form parallel aversive and appetitive memories. To further test a parallel memory trace model, flies were trained with 0.3% DEET and 1 M sucrose, a combination with which no immediate odor avoidance or approach performance is evident, and either the rewarding or aversive dopaminergic neurons were blocked during training. Strikingly, blockade of the rewarding dopaminergic neurons with 0104; UAS-shits1 revealed significant conditioned avoidance. In contrast, blockade of the negatively reinforcing MB-MP1 dopaminergic neurons with c061; MBGAL80; UAS-shits1 uncovered significant conditioned odor approach performance. It was therefore concluded that training with the compound DEET and sugar stimulus leads to the independent formation of aversive and appetitive memories. The differing stability of these competing memories subsequently determines which one of them guides learned behavior after training (Das, 2014).

    The extent to which rewarding and aversive stimuli are coded in mammalian dopaminergic neurons is hotly debated. Recordings in the monkey have shown that some dopaminergic neurons respond to either bitter taste or an aversive air puff, suggesting that the quality of an aversive reinforcer may be represented. Work in flies has functionally split dopaminergic neurons into groups that are critical for reward learning and others for aversive learning. However, recent studies suggested a requirement for modulation of the aversive system in appetitive learning and demonstrated a role for rewarding dopaminergic neurons in relative aversive learning. In addition, imaging activity in negatively reinforcing MB-MP1 neurons revealed responses to both sweet sugar and bitter DEET. Nevertheless, the DEET reinforcement data presented in this study, when taken with published knowledge of shock reinforcement, imply that flies utilize the same, or at least an overlapping, evaluation system to convey the reinforcing effects of discrete aversive stimuli. It will be interesting to determine the respective input pathways to the negatively reinforcing dopaminergic neurons. These experiments also highlight the importance of being able to both record from and control recognizable subpopulations of dopaminergic neurons. Without intervention, it is difficult to understand whether a given dopaminergic neuron provides a reinforcement or motivational salience signal (Das, 2014).

    Perhaps most surprisingly, the data demonstrate that during learning flies independently assign the value of individual components of a compound food stimulus to an odor. Rather than forming a single memory of the relative quality of the tainted sugar, they learn the bitter and sugar components in parallel. This multiplexing is further illustrated by sugars in which octopamine distinguishes between memories of sweet taste and nutrient components. These results suggest that despite the integration of tastant information that occurs within the first layers of the gustatory system and provides control over food ingestion, each component also gains unprocessed access to the negative and positive arms of the reinforcement system. The fly therefore appears to retain as much information of foraging history as possible, while allowing the relative persistence of the resultant constituent memories to inform later behavior. Such a mechanism might help the fly to direct short-term foraging away from food sources that happen to be unpalatable but remember that they are usually nutritious (Das, 2014).

    Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila

    The easy accessibility of energy-rich palatable food makes it difficult to resist food temptation. Drosophila larvae are surrounded by sugar-rich food most of their lives, raising the question of how these animals modulate food-seeking behaviors in tune with physiological needs. This study describes a circuit mechanism defined by neurons expressing tdc2-Gal4 (a tyrosine decarboxylase 2 promoter-directed driver) that selectively drives a distinct foraging strategy in food-deprived larvae. Stimulation of this otherwise functionally latent circuit in tdc2-Gal4 neurons was sufficient to induce exuberant feeding of liquid food in fed animals, whereas targeted lesions in a small subset of tdc2-Gal4 neurons in the subesophageal ganglion blocked hunger-driven increases in the feeding response. Furthermore, regulation of feeding rate enhancement by tdc2-Gal4 neurons requires a novel signaling mechanism involving the VEGF2-like receptor, octopamine, and its receptor. These findings provide fresh insight for the neurobiology and evolution of appetitive motivation (T. Zhang, 2013).

    Modulation of feeding responses to food sources is heavily influenced by nutritional quality, taste, and the energy costs of foraging. The current findings suggest that Drosophila larvae have evolved a complex neural network to regulate appetitive motivations. In hungry fly larvae, OA neurons seem to mediate a specialized circuit that selectively promotes persistent feeding of readily ingestible sugar food. This OA circuit functions in parallel to the previously characterized mechanism coregulated by the fly insulin and NPY-like systems that drives feeding response to non-preferred solid food. Because food deprivation triggers simultaneous activation of both circuits, hungry larvae become capable of adaptively responding to diverse energy sources of high or low quality. It remains to be determined how OA signaling promotes persistent feeding response to liquid sugar food in hungry larvae. One possible scenario is that OA neurons in the SOG may be conditionally activated by gustatory cues associated with rich palatable food to promote appetitive motivation (T. Zhang, 2013).

    This study has has provided evidence, at both molecular and neuronal levels, that the OA-mediated feeding circuit has two opposing effects on food motivation. When surrounded by liquid sugar media, the OA circuit is essential to prevent fed animals from excessive feeding. Because targeted lesions in VUM1 neurons caused excessive feeding response, these neurons may define an inhibitory subprogram within the OA feeding circuit. However, targeted lesions in VUM2 neurons attenuated hunger-induced increases of feeding response, suggesting that VUM2 neurons, along with the OA receptor Octβ3R, may define a subprogram that enhances feeding in fasted larvae. Several lines of evidence suggest that the VUM2 neuron-mediated subprogram may be suppressed by the VUM1 neuron-mediated subprogram. First, fed larvae with double lesions in both VUM1 and VUM2 neurons failed to display excessive feeding, suggesting that increased feeding response of fed larvae deficient for VUM1 neuronal signaling requires VUM2 neurons. Second, targeted lesions in VUM2 neurons of fed tdc2-Gal4/UAS- dTrpA1 larvae completely blocked the increased feeding response induced by genetic activation of tdc2-Gal4 neurons. Finally, the anatomical data also show that VUM1 and VUM2 neurons project to many common regions of the larval brain implicated in the control of feeding. Future work will be needed to determine whether VUM1 neurons inhibit directly or indirectly the activity of VUM2 neurons (T. Zhang, 2013).

    Genetic and pharmacological evidence has been obtained for the critical role of OA in the regulation of acquiring readily accessible sugar media. OA has been reported to mediate diverse neurobiological functions including appetitive memory formation and modulation of the dance of honey bee foragers to communicate floral or sucrose rewards. It is postulated that the different OA receptors may mediate diverse OA-dependent behavioral responses to high-quality foods (T. Zhang, 2013).

    Norepinephrine (NE), the vertebrate counterpart of OA, has been shown to promote ingestion of carbohydrate-rich food at the beginning of a natural feeding cycle. This feeding activity of NE resides in the paraventricular nucleus (PVN) of the feeding control center. In the PVN, α1 and α2 adrenergic receptors are organized in an antagonistic pattern. Activation of α1 receptor inhibits food intake, whereas activation of the α2 receptor stimulates food intake. The current results suggest that the insect OA system, like the NE system in mammals, exerts both positive and negative effects on the intake of preferred food. The activity of NE in PVN has been shown to antagonize that of 5-HT, which suppresses intake of carbohydrate- rich food. In Drosophila, 5-HT is also known to suppress feeding response. These findings suggest that the homeostatic control of intake of preferred food is likely mediated by a conserved neural network in flies and mammals (T. Zhang, 2013).

    This study has identified a unique role of Pvr in physiological regulation of hunger-motivated feeding of preferred food (liquid sugar media). The feeding-related activity of the Pvr pathway involves two regulatory proteins, Drk and Ras, and oral introduction of OA restores the hunger-driven feeding response in tdc2-Gal4/ UAS-drkdsRNA larvae. Together, these results suggest that the Pvr pathway positively regulates OA release by tdc2-Gal4 neurons. Among the three identified ligands of Pvr, Pvf2 is enriched in the larval CNS. The current finding suggests that Pvf2 regulates the feeding-related activity of the Pvr pathway. It is possible that Pvf2 may transduce a metabolic stimulus to Pvr/tdc2-Gal4 neurons that signals the energy state of larvae. In the honey bee brain, OA neurons from the SOG have been reported to respond to sugar stimulation. Therefore, it would be interesting to test whether the Pvf2/ Pvr pathway is responsive to sugar stimuli (T. Zhang, 2013).

    Previous studies have shown that the fly insulin and NPY-like systems coregulate hunger-elicited motivation to acquire solid sugar media. This study has now provided evidence that the fly VEGFR2- and NE-like systems control larval motivation to acquire liquid sugar media. These findings strongly suggest that the neural activities of different RTK systems play critical roles in different aspects of adaptive feeding decisions under various food and metabolic conditions. Therefore, further investigation of the mechanistic details of the food-related functions of RTK systems in the Drosophila model may provide novel insights into the neurobiology and evolution of appetitive control as well as pathophysiology of eating-related disorders (T. Zhang, 2013).

    Dopaminergic modulation of sucrose acceptance behavior in Drosophila

    For an animal to survive in a constantly changing environment, its behavior must be shaped by the complex milieu of sensory stimuli it detects, its previous experience, and its internal state. Although taste behaviors in the fly are relatively simple, with sugars eliciting acceptance behavior and bitter compounds avoidance, these behaviors are also plastic and are modified by intrinsic and extrinsic cues, such as hunger and sensory stimuli. This study shows that dopamine modulates a simple taste behavior, proboscis extension to sucrose. Conditional silencing of dopaminergic neurons reduces proboscis extension probability, and increased activation of dopaminergic neurons increases extension to sucrose, but not to bitter compounds or water. One dopaminergic neuron with extensive branching in the primary taste relay, the subesophageal ganglion, triggers proboscis extension, and its activity is altered by satiety state. These studies demonstrate the marked specificity of dopamine signaling and provide a foundation to examine neural mechanisms of feeding modulation in the fly (Marella, 2012).

    Invertebrate models with less complex nervous systems and robust sensory-motor behaviors may illuminate simple neural modules that regulate behavior. This study examined flexibility in a gustatory-driven behavior and found that a dopaminergic neuron is a critical modulator. Loss-of-function studies involving dopamine receptor mutants and gain-of-function studies argue that increased dopaminergic activity promotes proboscis extension to sucrose, and decreased dopaminergic activity inhibits it. These studies show that a single dopaminergic neuron in the SOG, TH-VUM, can drive proboscis extension. TH-VUM does not respond to sugars, arguing that it is not directly in the pathway from taste detection to behavior, but instead acts over a longer timescale or in response to other cues to modulate proboscis extension to sucrose. Consistent with this idea, satiety state influences TH-VUM activity, promoting activity when the animal is food deprived and the probability of proboscis extension is increased. These studies suggest that dopaminergic activity regulates the probability of extension according to an animal's nutritional needs (Marella, 2012).

    The finding that dopamine neural activity affects proboscis extension to sucrose, but not water, argues that dopamine regulation occurs upstream of shared motor neurons involved in proboscis extension. The pathway selectivity also argues that different molecular mechanisms modulate food and water intake independently in the fly, with parallels to hunger and thirst drives in mammals. Where dopamine acts in the sugar pathway is not known. Experiments to test for proximity between sugar sensory neurons and TH-VUM using the GRASP approach suggested that a few fibers are in close proximity, but the significance is unclear. The broad arborizations of TH-VUM suggest it may have many targets (Marella, 2012).

    Dopamine is a potent modulator of a variety of behaviors in mammals and flies. In mammals, functions of dopamine include motor control, reward, arousal, motivation, and saliency. Dopamine also critically regulates feeding behavior. Mice mutant for tyrosine hydroxylase fail to initiate feeding, although they distinguish sucrose concentrations and have the motor ability to consume (Szczypka, 1999). Dopamine pathways that regulate feeding are complex, with the tuberoinfundibular, nigrostriatal, and mesolimbic and mesocortical pathways implicated in different aspects of feeding regulation (Vucetic and Reyes, 2010). Although several studies show that dopamine promotes positive aspects of feeding, there is debate over whether dopamine is involved in pleasure ('liking'), motivation or salience ('wanting'), associative learning, or sensory-motor activation (Berridge, 2007). With 20,000–30,000 TH-positive neurons in mice and 400,000-600,000 in humans (Björklund and Dunnett, 2007), the complexity of dopaminergic regulation makes it difficult to parse the function of different neurons (Marella, 2012).

    In Drosophila, as in mammals, dopamine participates in conditioning and arousal, and this study highlights a shared role in feeding regulation. There are only a few hundred TH-positive neurons in Drosophila, and recent studies have begun to elucidate the function of different dopaminergic neural subsets. This work demonstrates that a single dopaminergic neuron in the SOG potently modulates proboscis extension behavior. Other dopaminergic neurons have cell bodies near TH-VUM and extensive projections in the SOG, yet activation of these neurons is not associated with proboscis extension. It is possible that additional dopaminergic neurons regulate other aspects of taste behavior, but they are insufficient to drive proboscis extension (Marella, 2012).

    In mammals, dopamine levels in the nucleus accumbens, the target of the mesolimbic pathway, increase upon sugar detection in the absence of consumption or upon nutrient consumption in the absence of detection, suggesting that dopamine encodes multiple rewarding aspects of sugar: intensity on the tongue and nutritional value. Recent studies in Drosophila also show that they sense nutritional content independent of taste detection, and this influences ingestion. It will be interesting to determine whether dopamine plays a role in sensing internal nutritional state and regulates other aspects of ingestion in addition to its role in proboscis extension (Marella, 2012).

    The anatomical location of the dopaminergic interneuron highlights the central role of the SOG in taste processing and suggests that local SOG circuits may control proboscis extension behavior. Future studies identifying the downstream targets of TH-VUM will ultimately enable a deeper understanding of how dopamine achieves spatial and temporal modulation of extension probability. Our current study identifies an essential role for dopamine in gain control of proboscis extension to sucrose and underscores the exquisite specificity of single neurons as thin threads to behavior (Marella, 2012).

    Food experience-induced taste desensitization modulated by the Drosophila TRPL channel

    Animals tend to reject bitter foods. However, long-term exposure to some unpalatable tastants increases acceptance of these foods. This study shows that dietary exposure to an unappealing but safe additive, camphor, caused the fruit fly to decrease camphor rejection. The transient receptor potential-like (TRPL) cation channel is a direct target for camphor in gustatory receptor neurons, and long-term feeding on a camphor diet leads to reversible downregulation of TRPL protein concentrations. The turnover of TRPL is controlled by an E3 ubiquitin ligase, Ube3a. The decline in TRPL levels and increased acceptance of camphor reversed after returning the flies to a camphor-free diet long term. It is proposed that dynamic regulation of taste receptors by ubiquitin-mediated protein degradation comprises an important molecular mechanism that allows an animal to alter its taste behavior in response to a changing food environment (Y. V. Zhang, 2013).

    Depending on the properties of a food, an animal decides to accept or reject it. In most terrestrial animals, sweet substances are assumed to provide nutrients, whereas many bitter compounds are correlated with poisons. However, this latter assumption is flawed, as many bitter foods are safe and nutritious7. Consequently, many animals learn to accept formerly unpalatable, bitter tasting foods, but only if they are safe and if more appealing options are unavailable. Although changes in the mammalian gustatory cortex have been found to be associated with diet-induced changes in taste preference, the nature of the molecular modifications in taste-receptor cells that contribute to environmentally induced modifications of food selection was previously unclear (Y. V. Zhang, 2013).

    This study found that fruit flies, similarly to many other animals, including insects such as locusts and moths, decrease food avoidance to certain bitter foods after prolonged exposure. The flies decreased their aversion to the unappealing tastant, camphor, in response to dietary experience. However, the flies did not form adaptation to all bitter tastants, including quinine, strychnine and lobeline (Y. V. Zhang, 2013).

    The decline in rejection of camphor was controlled in the peripheral sensory neurons through a reversible decline in the concentration of the camphor-activated TRPL channel in dendrites. Because TRPL was activated by camphor but not other unpalatable tastants such as quinine, downregulation of this channel selectively affected aversion to camphor. Two observations support the conclusion that the downregulation of TRPL contributes to taste adaptation. First, removal of camphor from the diet resulted in a return to the original TRPL levels and a restoration of the formerly held aversion to camphor. Second, during camphor exposure, an E3 ubiquitin ligase, Ube3a, targeted TRPL for degradation, thereby decreasing TRPL expression levels in the GRNs. Loss of Ube3a eliminated the camphor diet–induced downregulation of TRPL and prevented taste desensitization. This finding also highlights that the diet-induced reduction in TRPL expression is mediated by protein turnover. Consistent with this mechanism underlying the decline in TRPL levels rather than a reduction in trpl transcription, the activity of the trpl reporter was indistinguishable between flies maintained on normal as compared to camphor diets. Thus, this study identified a molecular mechanism in peripheral sensory neurons that underlies plastic, diet-induced alterations in food preference. A question that remains open concerns the link between TRPL activation and downregulation of the channel. Given that TRPL is a Ca2+-permeable channel, Ube3a activity might be directly or indirectly activated by a rise in Ca2+ levels (Y. V. Zhang, 2013).

    During the formation of taste adaptation at the behavioral level, there was a second change that occurred. After downregulation of TRPL, the number of boutons at the GRN axonal terminals in the SOG declined. Thus, synapse loss appeared to be a secondary consequence of the decline in TRPL levels. In further support of this conclusion, the number of synapses was unchanged in the ube3a mutant, which did not show a reduction in TRPL protein levels. Because the morphological change was reversible by withdrawal of a sustained camphor diet, it is concluded that GRNs in adult Drosophila undergo cellular modification. An important question still to be answered concerns the identity of the molecular pathway bridging the long-distance communication between the decline of TRPL levels in the dendrites and bouton pruning in the axons. It is suggested that synapse elimination may be insufficient to cause camphor desensitization, but it might synergize with TRPL downregulation to decrease distaste for camphor. In summary, this study revealed that food experience can modify behavior by altering signaling at both the dendrites and axons of the GRNs (Y. V. Zhang, 2013).

    Mechanisms similar to those described here in this study represent an evolutionarily conserved strategy that contributes to chemosensory desensitization. It is noteworthy that a worm TRP vanilloid (TRPV) channel, OSM-9, functions in both olfactory adaptation and adaptation to sodium chloride. Thus, a similar mechanism of ubiquitination-mediated downregulation of OSM-9 might contribute to chemosensory desensitization in Caenorhabditis elegans. In addition, diet-induced morphological changes in mammalian taste buds have been reported. The current work raises the possibility that changes in taste preference in other animals, including vertebrates, may be mediated by alterations in the concentration of receptors and channels in taste-receptor cells and subsequent modifications in synaptic connections (Y. V. Zhang, 2013).

    A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila

    Behavioural responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures. Preferred temperature selection promotes avoidance of adverse thermal environments in the short term and maintenance of optimal body temperatures over the long term, but its molecular and cellular basis is largely unknown. Recent studies have generated conflicting views of thermal preference in Drosophila, attributing importance to either internal or peripheral warmth sensors. This study reconciles these views by showing that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. Previously it was found that the transient receptor potential channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient. This study now finds that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the gustatory receptor GR28B(D), one of five splice variants of Gustatory receptor 28b), drives this behaviour through peripheral thermosensors. Gustatory receptors are a large gene family, widely studied in insect gustation and olfaction, and are implicated in host-seeking by insect disease vectors, but have not previously been implicated in thermosensation. At the molecular level, GR28B(D) misexpression confers thermosensitivity upon diverse cell types, suggesting that it is a warmth sensor. These data reveal a new type of thermosensory molecule and uncover a functional distinction between peripheral and internal warmth sensors in this tiny ectotherm reminiscent of thermoregulatory systems in larger, endothermic animals. The use of multiple, distinct molecules to respond to a given temperature, as observed in this study, may facilitate independent tuning of an animal's distinct thermosensory responses (Ni, 2013).

    In Drosophila two sets of warmth-sensing neurons (activated above ~25°C) have been proposed to control thermal preference: the anterior cell (AC) neurons, located inside the head, and the hot cell (HC) neuron, located peripherally in the arista. However, different studies suggest conflicting cellular and molecular mechanisms for thermal preference control. At the cellular level, primary importance has been attributed to either internal warmth sensors. At the molecular level, the internal AC neurons sense warmth via TrpA1, which encodes a warmth-activated transient receptor potential (TRP) channel, whereas the peripheral HC neurons seem to be TrpA1-independent. To clarify the mechanisms of thermal preference, this study sought to discover the molecular basis of HC neuron function (Ni, 2013).

    The arista contains six neurons: three warmth-responsive HC neurons (which can be labelled using cell-specific Gal4 expression in the HC-GAL4 strain) and three cool-responsive (cold cell; CC) neurons (labelled in the CC-GAL4 strain). Three unidentified cells in the arista have been reported to express Gr28b.d-GAL4, a transgene in which promoter sequences upstream of the gustatory receptor GR28B(D) control Gal4 expression. This study found that these Gr28b.d-GAL4-expressing cells resembled thermoreceptors, with cell bodies near the arista base and thin processes in the shaft. To determine the thermoreceptor subset labelled, Gr28b.d-GAL4 was combined with each thermoreceptor-specific Gal4. Gr28b.d-GAL4 plus HC-GAL4 labelled three neurons, whereas Gr28b.d-GAL4 plus CC-GAL4 labelled six neurons, indicating that Gr28b.d-GAL4 is expressed in the HC neurons. Although in situ hybridization was unsuccessful (common for gustatory receptors, GR28B(D) transcripts were robustly detected in dissected antennae/aristae from wild-type, but not Gr28b mutant, animals by reverse transcriptase PCR (RT–PCR), demonstrating expression in this tissue (Ni, 2013).

    Gustatory receptors are a large family of seven transmembrane proteins present in invertebrates, with 68 members in Drosophila melanogaster. Insects also contain multiple gustatory receptor-related odorant receptors (62 in D. melanogaster. Gustatory receptors and odorant receptors form a gene family distinct from, and apparently unrelated to, the G-protein-coupled receptor superfamily. Gustatory receptors and odorant receptors have been studied extensively as chemoreceptors for sweet and bitter tastants, food odours, carbon dioxide and other chemicals, but have not previously been implicated in thermosensation. This study examined gustatory receptor involvement in thermosensation using a two-temperature choice assay, exposing flies for 1 min to a steep thermal gradient (initially >5°C per cm) created using tubes of ~25.5 and ~31.0°C air (a preferred and an increased-but-innocuous temperature, respectively) separated by 1 cm. Flies normally prefer the cooler tube, a behaviour termed 'rapid negative thermotaxis'. This and a previous study showed that inhibiting HC neurons by cell-specific expression of tetanus toxin light chain (TNT), a vesicle release inhibitor, using HC-GAL4 strongly reduced such behaviour). In agreement with the importance of HC neurons, and in addition to previous studies, third antennal segment/arista removal strongly reduced this behaviour, whereas ablating other tissues expressing HC-GAL4 and Gr28b.d-GAL4 did not. By contrast, inhibiting AC neurons by TNT expression using TrpA1GAL4, a Gal4 knock-in at the TrpA1 locus, had no effect. (This manipulation disrupted a previously reported AC-dependent thermosensory behaviour) These data indicate that rapid negative thermotaxis depends on the peripheral HC warmth sensors (Ni, 2013).

    To probe the molecular basis of rapid negative thermotaxis, its dependence on TrpA1, which is required for AC neuron warmth-sensing, was examined. Consistent with the TrpA1-independence of HC neuron thermosensitivity, a strong loss-of-function TrpA1 mutation did not affect this behaviour. By contrast, strong loss-of-function mutations in the gene encoding GR28B(D) eliminated the response; Gr28b mutants distributed nearly equally between ~25.5°C and ~31.0°C. The defect was specific: excising the transposon in the Gr28bMi allele restored thermotaxis, and both a Gr28b-containing genomic transgene and Gr28b(D) complementary DNA expression rescued the mutant. Attempts were made rescue by expressing cDNAs for the other Drosophila GR28 family members (four other Gr28b isoforms and Gr28a) under Gr28b.d-GAL4 control. Although a negative result could reflect a failure to be properly expressed, only Gr28b(E) yielded significant rescue. However, endogenous Gr28b(E) transcripts were not detected in the antenna/arista, consistent with a previous analysis indicating that GR28B(E) is not expressed there. Together, these data demonstrate that rapid negative thermotaxis depends not on TrpA1, but on Gr28b, consonant with the specific dependence of this behaviour on HC neuron function. Notably, cell-specific GR28B(D) expression using HC-GAL4 strongly rescued the Gr28b mutant, indicating that GR28B(D) function in the HC thermosensors is sufficient to restore rapid negative thermotaxis (Ni, 2013).

    To test whether GR28B(D) might act as a thermosensor, whether it conferred warmth-sensitivity when ectopically expressed was examined. Unlike controls, flies broadly expressing GR28B(D) under Actin5C-GAL4 control were incapacitated when heated to 37°C for 3 min, recovering when returned to 23°C. This dramatic effect suggested that GR28B(D) might promote warmth-responsive neuronal activation. It has been shown previously that ectopic expression of the warmth-activated cation channel TRPA1(B), a product of Drosophila TrpA1, renders fly chemosensors warmth-responsive. Like TRPA1(B), chemosensor expression of GR28B(D) (using Gr5a-GAL4) conferred robust warmth-responsiveness. The behavioural consequences of such GR28B(D) expression were examined. When chemically activated, sweet-responsive chemosensors promote proboscis extension. When GR28B(D) was expressed in these cells, strong proboscis extension was elicited by warming to ~32°C. This ability to confer warmth-responsiveness is consistent with GR28B(D) acting as a warmth sensor (Ni, 2013).

    Whether GR28B(D) requires sensory neuron-specific cofactors was examined in the neuromuscular system. Unlike controls, motor neurons expressing GR28B(D) (using OK371-GAL4) triggered warmth-responsive excitatory junction potentials at the neuromuscular junction. Thus, GR28B(D)-mediated warmth-responsiveness does not require sensory neuron-specific cofactors. The threshold for GR28B(D)-dependent muscle stimulation was 26.0± 0.3°C, just above TRPA1(B)’s ~25°C threshold in this system, indicating that both molecules mediate responses to innocuous warming (Ni, 2013).

    To quantify the thermosensitivity of GR28B(D)-dependent responses, currents were monitored using whole-cell patch-clamp electrophysiology. Unlike controls, voltage-clamped motor neurons expressing GR28B(D) exhibited warmth-responsive inward currents. The response's temperature coefficient (Q10, fold change in current per 10°C change) was calculated by Arrhenius analysis. GR28B(D)-dependent currents were highly thermosensitive (Q10 of 25 ± 5, similar to mammalian neurons expressing thermosensitive TRP channels. Substituting N-methyl-d-glucamine (NMDG)+ for Na+ in the extracellular solution eliminated heat-responsiveness, consistent with cation channel activation (Ni, 2013).

    The potential dependence of GR28B(D) on neuron-specific cofactors was tested in muscle. Although control muscles voltage-clamped at −60 mV exhibited modest warmth-responsive outward currents, muscles expressing GR28B(D) (using Mhc-GAL4) exhibited robust warmth-responsive inward currents. The ability of GR28B(D) to confer warmth sensitivity across diverse cell types supports the hypothesis that GR28B(D) acts as a molecular thermoreceptor. It further suggests GR28B(D) as a new class of tool for thermogenetic neuronal activation, adding to the TRP-based toolbox currently used in Drosophila (Ni, 2013).

    Although GR28B(D) resembles TRPA1(B) in conferring warmth-sensitivity, these two proteins have distinct functions in the fly, with only Gr28b controlling rapid negative thermotaxis. TrpA1 was found previously to control the slowly developing thermal preference response of flies on a shallow, broad thermal gradient (~0.5°C per cm, 18-32°C). The contribution of Gr28b to this long-term body temperature selection behaviour was tested. As reported previously, TrpA1 mutants selected unusually warm temperatures after 30 min on the gradient, with many accumulating at ≥28°C. By contrast, strong loss-of-function Gr28b mutants behaved indistinguishably from wild type. This neatly distinguishes Gr28b and TrpA1, with the former controlling rapid negative thermotaxis and the latter long-term body temperature selection (Ni, 2013).

    These findings reconcile previously disparate views of Drosophila thermosensation by demonstrating that thermal preference is not a singular behaviour, but involves multiple systems relevant in different contexts. It suggests a model in which Gr28b, acting peripherally, controls rapid responses to ambient temperature jumps, whereas TrpA1, acting internally, controls responses to sustained temperature increases reaching the core. In the arista, Gr28b could experience ambient temperature fluctuations in advance of core changes, eliciting rapid avoidance. Such behaviour could be critical for a tiny animal in which ambient and core temperatures equalize rapidly. The dispensability of Gr28b for responses on the shallow gradient could relate to observations in other insects where peripheral thermoreceptors respond more to temperature fluctuations than absolute values. The fly's reliance on distinct sensors for distinct aspects of thermal preference is reminiscent of complex thermosensory systems of larger, endothermic animals. In the fly, these warmth-responsive pathways potentially converge in the brain, where both sets of sensors innervate overlapping regions (Ni, 2013).

    Finally, whether Gr28b and TrpA1 were uniquely suited to their roles in the fly was tested. Although TrpA1 was normally not required for rapid negative thermotaxis, when expressed in the arista using Gr28b.d-GAL4, TRPA1(B) significantly rescued the Gr28b mutant defect. (As expected, a less thermosensitive TrpA1 isoform, TRPA1(A), did not rescue the defect). Conversely, although Gr28b was not normally required for slowly developing thermal preference on the shallow gradient, GR28B(D) expression under TrpA1GAL4 control significantly rescued the TrpA1 mutant defect. Thus, when their expression is manipulated appropriately, GR28B(D) and TRPA1(B) can act in the same cells and support the same behaviours, indicating fundamental functional similarities (Ni, 2013).

    Although studied extensively, the mechanisms of gustatory receptor action are not fully resolved. Gustatory receptors have been reported to act as cation channels and via G-proteins. Whether GR28B(D) acts by either mechanism remains unknown. Although attempts to study GR28B(D) in heterologous cells (including Xenopus laevis oocytes and HEK cells) were unsuccessful, the ability of GR28B(D) to confer warmth-responsiveness upon diverse cell types argues against a requirement for cell-type-specific cofactors in the fly. Gr28b has been implicated in responses to strong illumination (Xiang, 2010). This seems to be unrelated to GR28B(D)-dependent thermosensation, as Gr28b-dependent photosensors are unresponsive to innocuous warming (Xiang, 2010) and appear to express other Gr28b isoforms. GR28B(D)-expressing muscles were not light-responsive (Ni, 2013).

    Previous studies have demonstrated the importance of TRP channels in Drosophila thermosensation, stimulating interest in their potential involvement in warmth-dependent host-seeking by insect disease vectors. This work raises the possibility that gustatory receptors, including GR28 receptors in disease vectors such as tsetse flies and mosquitoes, regulate thermosensation more broadly. GR28B(D) adds to a growing list of highly thermosensitive membrane proteins including not only TRPs, but the mammalian ANO1 chloride channel. The presence of exceptional thermosensitivity in diverse proteins may facilitate temperature-responsive modulation of diverse physiological responses. Furthermore, using multiple molecules to mediate behavioural responses to similar temperatures may facilitate independent tuning of distinct thermosensory responses (Ni, 2013).

    Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1

    Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ('antisymmetries') or found stereotypically on one particular side of an animal ('directional asymmetries'). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. This study describes an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs (Cochella, 2014).

    Histone methyltransferase G9a is a key regulator of the starvation-induced behaviors in Drosophila melanogaster

    Organisms have developed behavioral strategies to defend themselves from starvation stress. Despite of their importance in nature, the underlying mechanisms have been poorly understood. This study shows that Drosophila G9a (dG9a), one of the histone H3 Lys 9-specific histone methyltransferases, functions as a key regulator for the starvation-induced behaviors. RNA-sequencing analyses utilizing dG9a null mutant flies revealed that the expression of some genes relating to gustatory perception are regulated by dG9a under starvation conditions. Reverse transcription quantitative-PCR analyses showed that the expression of gustatory receptor genes for sensing sugar are up-regulated in starved dG9a null mutant. Consistent with this, proboscis extension reflex tests indicated that dG9a depletion increased the sensitivity to sucrose under starvation conditions. Furthermore, the locomotion activity was promoted in starved dG9a null mutant. It was also found that dG9a depletion downregulates the expression of insulin-like peptide genes that are required for the suppression of starvation-induced hyperactivity. Furthermore, refeeding of wild type flies after starvation conditions restores the hyperactivity and increased sensitivity to sucrose as well as dG9a expression level. These data suggest that dG9a functions as a key regulator for the decision of behavioral strategies under starvation conditions (Shimaji, 2017).

    Drosophila Life Span and Physiology Are Modulated by Sexual Perception and Reward

    Sensory perception modulates aging and physiology across taxa. This study found that perception of female sexual pheromones through a specific gustatory receptor expressed in a subset of foreleg neurons in male fruit flies rapidly and reversibly decreases fat stores, reduces resistance to starvation, and limits life span together with neurons that express the reward-mediating neuropeptide F. High-throughput RNA-seq experiments revealed a set of molecular processes that were impacted by the activity of the longevity circuit, thereby identifying new candidate cell non-autonomous aging mechanisms. Mating reversed the effects of pheromone perception, suggesting a model where life span is modulated through integration of sensory and reward circuits and where healthy aging may be compromised when the expectations defined by sensory perception are discordant with ensuing experience (Gendron, 2013).

    Sensory perception can modulate aging and physiology in multiple species. In Drosophila, exposure to food-based odorants partially reverses the anti-aging effect of dietary restriction, whereas broad reduction in olfactory function promotes longevity and alters fat metabolism. Even the well-known relation between body temperature and life span may have a sensory component (Gendron, 2013).

    To identify sensory cues and neuronal circuitry that underlie the effects of sensory perception on aging, this study focused on the perception of potential mates. Social interactions are prevalent throughout nature, and the influence of social context on health and longevity is well-known in several species, including humans. Such influences include behavioral interactions with mates and broader physiological 'costs of reproduction,' which often form the basis for evolutionary models of aging (Gendron, 2013).

    In Drosophila, the presence of potential mates is perceived largely through non-volatile cuticular hydrocarbons, which are produced by cells called oenocytes and are secreted to the cuticular surface where they function as pheromones. To test whether differential pheromone exposure influenced life span or physiology, 'experimental' flies of the same genotype were housed with 'donor' animals of the same sex that either expressed normal pheromone profiles or were genetically engineered to express pheromone profiles characteristic of the opposite sex. Donor males with feminized pheromone profiles were generated by targeting expression of the sex determination gene, tra, to the oenocytes (via OK72-GAL4 or Prom-E800-Gal4), whereas masculinization of female flies was accomplished by expressing tra-RNAi in a similar way. This design allowed manipulation of the experimental animals' perceived sexual environment without introducing complications associated with mating itself (Gendron, 2013).

    In Drosophila, sensory manipulations can affect life span, fat storage (as determined by baseline measures of triacylglyceride-TAG), and certain aspects of stress resistance. This study has found that flies exposed to pheromones of the opposite sex showed differences in these phenotypes. Experimental male flies exposed to male donor pheromone had higher amounts of TAG, were substantially more resistant to starvation, and exhibited a significantly longer life span than genetically identical male siblings exposed to female donor pheromone. Females exhibited similar phenotypes in response to male donor pheromone, but the magnitude of the effects was smaller. Subsequent experiments were therefore focused on males (Gendron, 2013).

    The characteristics of pheromone exposure were indicative of a mechanism involving sensory perception. Effects were similar in several genetic backgrounds, including a strain recently collected in the wild, and were largely unaffected by cohort composition. Pheromone-induced phenotypes were detected after as little as two days exposure to donor animals, persisted with longer manipulations, and were progressively reversed when female donor pheromone was removed. Pheromone effects appeared not to be mediated by aberrant or aggressive interactions with donor flies because no significant differences were observed in such behaviors and because continuous, vigorous agitation of the vials throughout the exposure period, which effectively disrupted observed behaviors, had no effect on the impact of donor pheromone. Furthermore, exposure of experimental males to the purified female pheromone 7-11-heptacosadiene (7-11 HD) produced physiological changes in the absence of donor animals (Gendron, 2013).

    To explore the sensory modality through which donor pheromone exerts its effects, this study tested whether a broadly-expressed olfactory co-receptor, Or83b, whose loss of function renders flies largely unable to smell, was required for pheromone effects. Or83b mutant flies exhibited similar changes in starvation resistance in response to donor pheromone as did control animals, indicating that olfaction was not required. To test whether taste perception was involved, flies were tested that were mutant for the gene Pox neuro (Poxn), a null mutation that putatively transforms all chemosensory neurons into mechanosensory neurons. Drosophila taste neurons are present in the mouthparts and distributed on different body parts including the wings, legs, and genitals, which allow sensation by contact. When the Poxn null mutation is coupled with a partially rescuing transgene, Poxn ΔM22-B5-ΔXB, flies are generally healthy, but gustatory perception is eliminated in the labelum, the legs, and the wing margins. Poxn ΔM22-B5XB flies showed no pheromone-induced changes in starvation resistance, TAG amounts, or life span. However, Poxn mutant flies that carried a transgene that restores taste function to the legs and wing margins (but not labelum; PoxnΔM22-B5-Full1 responses were similar to those of control flies. Thus, the effects of pheromone exposure appear to be mediated by taste perception through gustatory neurons outside of the mouthparts (Gendron, 2013).

    To identify specific gustatory receptors and neurons that might mediate the pheromone effects, candidate pheromone receptors were tested. Of the mutants that were examined, only flies that carried a loss of function mutation in the gene pickpocket 23 (ppk23) were resistant to the effects of pheromone exposure. Further analysis verified that ppk23 was required for the effects of pheromone exposure on starvation resistance, TAG amounts, and life span. Silencing ppk23-expressing neurons only during exposure to donor males by expressing a temperature-sensitive dominant negative allele of the dynamin gene shibire (via ppk23-GAL4; UAS-shits) also eliminated the differential response to pheromones. In male Drosophila, the transcription factor fruitless (fru) is expressed with ppk23 in pheromone-sensing neurons located in the animals' forelegs, and silencing fru-expressing neurons during exposure (via fru-GAL4;UAS-shits) abrogated pheromone effects. Consistent with a requirement for these neurons, it was found that surgical amputation of the forelegs, but not injury alone, was sufficient to reproducibly eliminate the effects of pheromone exposure. Moreover, acute, targeted activation of ppk23-expressing neurons using a temperature-sensitive TRPA1 channel (ppk23-GAL4;UAS-TRPA1) was sufficient to mimic the effects of female pheromone without exposure. Together, these data indicate that pheromone-sensing neurons in the foreleg of the male fly that express the gustatory receptor, ppk23, and the transcription factor, fruitless, influence stress resistance, physiology, and life span in response to perception of female pheromones (Gendron, 2013).

    To examine brain circuits that may function in transducing pheromone perception, UAS-shits was selectively expressed to block synaptic transmission in various neuro-anatomical regions with the goal of disrupting the physiological effects of donor pheromone exposure. The effects were abrogated when UAS-shits was driven in neurons characterized by expression of neuropeptide F (NPF, as represented by npf-GAL4). Further analysis verified that pheromone-induced changes in starvation resistance and TAG abundance were lost following silencing of npf-expressing neurons. Consistent with a possible role in transducing pheromone information, npf expression was significantly increased by 30% in experimental males after exposure to feminized donor males, and activation of npf-expressing neurons was sufficient to decrease life span in the absence of pheromone exposure (Gendron, 2013).

    NPF may function as a mediator of sexual reward in Drosophila, and its mammalian counterpart, neuropeptide Y (NPY), has been associated with sexual motivation and psychological reward. Tests were performed to see whether the effects of pheromone perception might be rescued by allowing males to successfully mate with females. Neither a small number of conjugal visits with virgin females nor housing with wild-type females in a 1:1 ratio was sufficient to ameliorate the effects of pheromone exposure. In this context, decreased longevity may be a consequence of pheromone perception and not of mating itself. Male Drosophila are willing and able to copulate up to five times in rapid succession before requiring a refractory period. It was found that supplementing donor cohorts with an excess of mating females (in a 5:1 ratio) was sufficient to significantly reduce the effects on mortality and TAG caused by female donor pheromone early in life. The benefits of mating on age-specific mortality decreased with age, suggesting that aging may reduce mating efficiency or may diminish effective mating reward (Gendron, 2013).

    To identify how sexual perception and reward may alter physiological responses in peripheral tissues, changes in gene expression were examined using whole-genome RNA-seq technology. 195 genes were found with significantly different expression (using an experiment-wise error rate of 0.05) in control male flies that were exposed to feminized or control donor males for 48 hours. Nearly all (188/195 = 96%) of the changes appeared to be due to pheromone perception because they were not observed in identical experiments using ppk23 mutant flies. Males exposed to female pheromones decreased transcription of genes encoding odorant-binding proteins and increased transcription of several genes with lipase activity. A significant enrichment was observed in secreted molecules, which includes genes encoding proteins mediating immune- and stress-responses. Many of these genes and pathways were highlighted in a recent meta-analysis of gene expression changes in response to stress and aging (Gendron, 2013).

    Activities of insulin and target of rapamycin (TOR) signaling, which modulate aging across taxa, increase sexual attractiveness in flies. The current demonstration that perception of sexual characteristics is sufficient to modulate life span and physiology suggests aging pathways in one individual may modulate health and life span in another. These types of indirect genetic effects have the potential to be influential agents of natural selection, suggesting that expectation/reward imbalance may have broad effects on health and physiology in humans and may present a potent evolutionary force in nature (Gendron, 2013).

    Gr33a modulates Drosophila male courtship preference

    In any gamogenetic species, attraction between individuals of the opposite sex promotes reproductive success that guarantees their thriving. Consequently, mate determination between two sexes is effortless for an animal. However, choosing a spouse from numerous attractive partners of the opposite sex needs deliberation. In Drosophila melanogaster, both younger virgin females and older ones are equally liked options to males; nevertheless, when given options, males prefer younger females to older ones. Non-volatile cuticular hydrocarbons, considered as major pheromones in Drosophila, constitute females' sexual attraction that act through males' gustatory receptors (Grs) to elicit male courtship. To date, only a few putative Grs are known to play roles in male courtship. This study reports that loss of Gr33a function or abrogating the activity of Gr33a neurons does not disrupt male-female courtship, but eliminates males' preference for younger mates. Furthermore, ectopic expression of human amyloid precursor protein (APP; see Drosophila Appl) in Gr33a neurons abolishes males' preference behavior. Such function of APP is mediated by the transcription factor Forkhead box O (dFoxO). These results not only provide mechanistic insights into Drosophila male courtship preference, but also establish a novel Drosophila model for Alzheimer's disease (AD) (Xue, 2015).

    To avoid futile reproductive efforts, an animal must distinguish conspecifics from other species and differentiate the sex of conspecific partners. It must also determine the most suitable mates from large amounts of available partners in order to maximize reproductive efficiency. Evolution endows Drosophila melanogaster males with the instinct to discriminate conspecifics from other Drosophila species1 and to discern females from males. It also bestows on them the ability to select the most favorable mates among masses of desirable virgin females (Xue, 2015).

    In Drosophila, non-volatile cuticular hydrocarbons (CHCs) have been recognized as a type of major sex pheromone, which convey information of an individual such as species and sex. Female-specific CHCs are detected by male gustatory receptors (Grs), a chemosensory receptor family mainly responsible for detecting non-volatile chemicals, during tapping and licking steps in stereotypical male courtship behavior. So far, only a few Grs, including Gr32a, Gr33a and Gr39a are reported to be engaged in Drosophila male courtship behavior. While Gr32a acts to assist males to discriminate conspecifics from other species, Gr39a is required for males to distinguish females from males. On the other hand, Gr33a functions to inhibit male-male courtship. Despite these findings, roles of the Grs in males' choices for the most favorable mates have remained largely unknown (Xue, 2015).

    A previous study (Hu, 2014) set a paradigm of choice model in which both options (younger virgin females and older ones) are proved to be attractive to Drosophila males, but males still intensely prefer younger mates to older ones. Using this model, this study explored the mechanisms by which males bias their potential mates. Gene loss-of-function, gain-of-function, and cell-inactivation experiments demonstrated that Gr33a and Gr33a neurons are essential for males' preference for younger mates. Since the previous data indicated that pan-neuronal expression of human amyloid precursor protein (APP) ablates males' preference for younger mates, this study sought to investigate whether APP expression in Gr33a neurons would affect this behavior. Indeed, it was found that Gr33a neurons-specific expression of APP abolished males' preference for younger mates, and this function of APP is mediated by the transcription factor forkhead box O (dFoxO) (Xue, 2015).

    Drosophila male courtship choice has been frequently applied for studying decision making in animals, yet most of the past studies have focused on male courtship choices between likes and dislikes, such as court towards females vs. males, or virgin vs. non-virgin females. Previous study has characterized a choice behavior between two equally-liked options: mature virgin females, whether younger or older, were similarly attractive to naive males; nevertheless, when given the option, males turn out to be picky and prefer younger virgin females to older ones. This study found that a gustatory receptor, Gr33a, is necessary for males' preference for younger mates. Gr33a is thought to be necessary to inhibit homosexual behavior; its role in heterosexual behavior, however, is rarely pondered. This study has revealed the critical role of Gr33a in males' preference for younger mates. Furthermore, ectopic expression of APP in Gr33a neurons eliminates males' preference behavior, and such function is mediated by dFoxO, a recently reported downstream factor of APP27. Therefore, this work demonstrates the genetic interaction of APP and dFoxO in Gr33a neurons, which modulates males' preference for younger mates. APP is identified as a potential causative protein of AD, a common progressive neurodegenerative disorder, in which cognitive decline is the prime symptom. Although Drosophila has long been utilized for building AD models to investigate the pathogenesis and possible cure for AD, accepted Drosophila AD models are limited to locomotion model and life span model, which have little correlation with cognitive ability. The current findings, however, have offered the possibility for establishing a novel Drosophila AD model that is related to cognitive ability (Xue, 2015).

    In all CHCs produced by files, 7, 11-HD and 7, 11-ND have been identified as female specific aphrodisiac pheromones to Drosophila melanogaster males. GC and MS studies suggest that both 7, 11-HD and 7, 11-ND are expressed at lower concentration in younger virgin females than the older ones. Hence, it appears unlikely that 7, 11-HD or 7, 11-ND is the cause that leads males to court younger virgin females more vigorously than the older ones. On the contrary, since Gr33a has been reported as a receptor of aversive odors, it is more likely that older females produce certain aversive odors that can be recognized by males and repel them. Consistent with this explanation, this study found that the concentrations of most detected CHCs are significantly higher on the older virgin females than the younger ones. Nevertheless, at this stage it was not possible to identify the CHC(s) that serves as the aversive pheromone to males. Besides, it cannot be excluded that younger virgin females produce unknown attractive pheromones other than 7, 11-HD or 7, 11-ND. However, all CHCs detected on younger virgin females also present on older ones at a similar or higher lever. Thus, the tentative conclusion is drawn that younger virgin females do not produce more attractive pheromones than the older ones. The results, taken together, unravel the role of bitter sensory Gr33a neurons in males' preference for younger mates and infer that older females might produce certain aversive odors that cause males to turn to younger mates (Xue, 2015).

    High-NaCl perception in Drosophila melanogaster

    Salt is a fundamental nutrient that is required for many physiological processes, including electrolyte homeostasis and neuronal activity. In mammals and Drosophila, the detection of NaCl induces two different behaviors: low-salt concentrations provide an attractive stimulus, whereas high-salt concentrations are avoided. A gene called serrano (sano) was identified as being expressed in the sensory organs of Drosophila larvae. A transgenic reporter line showed that sano was coexpressed with Gr66a in a subset of gustatory neurons in the terminal organ of third-instar larvae. The disruption of sano gene expression in gustatory neurons led to the specific loss of high-salt concentration avoidance in larvae, whereas the detection of other attractive or aversive substances was unaffected. Moreover, using a cellular marker sensitive to calcium levels, Sano function was shown to be required for neuronal activity in response to high-salt concentrations. In these neurons, the loss of the DEG/ENaC channel PPK19 function also eliminated the cellular response to high-salt concentrations. This study revealed that PPK19 and Sano are required in the neurons of the larval gustatory organs for the detection of high-salt concentrations (Alves, 2014).

    Representations of taste modality in the Drosophila brain

    Gustatory receptors and peripheral taste cells have been identified in flies and mammals, revealing that sensory cells are tuned to taste modality across species. How taste modalities are processed in higher brain centers to guide feeding decisions is unresolved. This study developed a large-scale calcium-imaging approach coupled with cell labeling to examine how different taste modalities are processed in the fly brain. These studies reveal that sweet, bitter, and water sensory cells activate different cell populations throughout the subesophageal zone, with most cells responding to a single taste modality. Pathways for sweet and bitter tastes are segregated from sensory input to motor output, and this segregation is maintained in higher brain areas, including regions implicated in learning and neuromodulation. This work reveals independent processing of appetitive and aversive tastes, suggesting that flies and mammals use a similar coding strategy to ensure innate responses to salient compounds (Harris, 2015).

    A central question in taste processing is how different taste modalities are encoded in the brain. In the mammalian gustatory system, labeled lines, mixed lines, and temporal dynamics have all been proposed as fundamental stategies used by the nervous system to process tastes, with recent evidence favoring labeled line encoding. In Drosophila, the limited understanding of neural circuits beyond sensory neurons and MNs has precluded examination of modality processing. This study takes advantage of recent improvements in genetically encoded calcium indicators and high-speed, multi-plane imaging to examine taste-induced activity throughout the SEZ and anterior brain, similar to whole-brain calcium-imaging approaches taken previously in zebrafish and C. elegans. This approach enabled examination of the neurons activated by different taste modalities and probe models of taste coding (Harris, 2015).

    Monitoring brain activity in response to stimulation of different gustatory classes revealed that the majority of central neurons responded selectively to bitter, sweet, or water sensory cell activation. In addition, some neurons responded to both water and sucrose and may represent positive valence or taste acceptance behavior, while a few responded to other taste pairs. This demonstrates that most central taste-processing neurons in the fly do not respond to multiple taste modalities and argues against models of taste coding based on broadly tuned cells. Instead, it was found that taste representations are largely modality specific, indicating separate processing streams for different taste qualities. Moreover, bitter and sweet stimulation activate different sets of proboscis MNs and activate different neurons in the higher brain. This argues that sweet and bitter tastes are processed by segregated pathways, suggesting a strategy that ensures innate responses to essential compounds (Harris, 2015).

    Examining responses to taste mixtures in the SEZ revealed that no additional cells are activated by mixtures that are not activated by single components, again arguing against non-selective cells. Instead, mixtures activated only a fraction of the cells that responded to the individual components. Mixture suppression occurred for sugar/bitter mixes and sugar/water mixes, demonstrating inhibition between appetitive and aversive stimuli as well between two appetitive stimuli. Recent studies have identified two mechanisms by which bitter compounds inhibit sweet sensory activation: (1) bitter compounds bind a chemosensory binding protein that acts on sweet sensory cells to inhibit activity; and (2) bitter compounds activate GABAergic neurons, causing GABAB-receptor-mediated pre-synaptic inhibition of sweet sensory axons. The current study shows that taste mixtures reduce sensory activation and reduce the number of taste-responsive cells in the SEZ, consistent with the notion that decreased sensory activity contributes to mixture suppression. Cross-inhibition of different gustatory pathways is an effective strategy to weight the activation of acceptance versus avoidance pathways based on the ratio of sugars versus bitter compounds present in food (Harris, 2015).

    This study represents the first large-scale analysis of pan-neural activity in the fly brain. The advantages of this approach are that it is possible to monitor the activity of large populations at single-cell resolution, which is not feasible by other approaches; it enables unbiased sampling that does not require specific Gal4 lines expressed in already known cells of interest; and it provides rapid evaluation of brain-wide activity. Whereas previous studies had identified four classes of taste-responsive cells, this study uncovered more than 100 taste-responsive cells, the vast majority of which are modality specific. As this approach relies on calcium imaging in the soma, the sensitivity of GCaMP6, and the expression levels driven byn Synaptobrevin-Gal4, detection limitations may exist. Nevertheless, this study provides a population overview of gustatory processing in the fly brain and a framework for future studies to determine the anatomy and connectivity of taste-responsive neurons (Harris, 2015).

    This study shows that different taste modalities in the periphery activate different pathways in the brain, consistent with labeled line taste processing. Information is processed in separate streams for appetitive and aversive tastes, which are maintained in the higher brain and are mutually inhibitory. Recent studies in the mammalian gustatory system argue for modality-specific representations in the gustatory cortex and are consistent with the labeled line model, suggesting that dedicated pathways may be a general strategy to process tastes used throughout evolution (Harris, 2015).

    Immediate perception of a reward is distinct from the reward's long-term salience

    Reward perception guides all aspects of animal behavior. However, the relationship between the perceived value of a reward, the latent value of a reward, and the behavioral response remains unclear. This study report that, given a choice between two sweet and chemically similar sugars-L- and D-arabinose-Drosophila melanogaster prefers D- over L-arabinose, but forms long-term memories of L-arabinose (the isomer present in ripening fruits) more reliably. Behavioral assays indicate that L-arabinose-generated memories require sugar receptor Gr43a, and calcium imaging and electrophysiological recording indicate that L- and D-arabinose differentially activate Gr43a-expressing neurons. It is posited that the immediate valence of a reward is not always predictive of the long-term reinforcement value of that reward, and that a subset of sugar-sensing neurons may generate distinct representations of similar sugars, allowing for rapid assessment of the salient features of various sugar rewards and generation of reward-specific behaviors. However, how sensory neurons communicate information about L-arabinose quality and concentration-features relevant for long-term memory-remains unknown (McGinnis, 2016).

    The observation that two similar sugars generate strikingly different behavioral responses can perhaps be best understood using the framework of 'incentive salience' in rewards, formulated by Berridge and Robinson (2003), who divided reward percepts into 'liking' (conscious pleasure, hedonic) and 'wanting' (incentive salience). According to Berridge and Robinson, 'wanting' (incentive salience) is a component of rewards that transforms mere sensory information about rewards and their cues into 'attractive, desired, riveting incentives' and 'emerged early in evolution as an elementary form of stimulus-guided goal direction, to mediate pursuit of a few innate food or sex unconditioned stimuli' (Berridge and Robinson, 2003). In most cases, rewards that are 'liked' are usually also 'wanted', and in conventional formulations, they are considered effectively identical. But work on addiction and monetary reward on human suggest that 'wanting' and 'liking' are in fact dissociable, and while, in many cases, a behavioral response to an experience can predict the likelihood of memory formation, people can be motivated by cues remaining outside conscious awareness. This study reports that a similar distinction in reward perception may also exist in Drosophila: D-arabinose appears to preferentially involve the 'liking' component of the reward percept and L-arabinose the 'wanting'. For Drosophila, the incentive to remember L-arabinose is perhaps owing to the fact that it can inform a specific attribute of food, such as the ripening status of a fruit. Moreover, work in humans suggests that although 'liking' and 'wanting' both represent a positive reward, they utilize distinct neural processing. The observations with D- and L-arabinose now provide an opportunity to explore the neural basis of 'liking' and 'wanting', and how these reward percepts strengthen memory in the accessible nervous system of Drosophila (McGinnis, 2016).

    The caloric value of a sugar has been found to be an important determinant of long-term appetitive memory, implying that flies quickly metabolize the sugar and that caloric evaluation somehow provides cues necessary to elicit long-term memory. This study found that sugar with no caloric value can also produce long-term appetitive memories. One obvious possibility is that memories of sweet nutritious sugars are distinct from memories of sweet non-nutritious sugars. However, this seems so far not to be the case: a subset of higher order dopaminergic neurons (R58E02GAL4) necessary for long-term memory of nutritious sucrose is also required for non-nutritious L-arabinose. Similarly, addition of sorbitol, a tasteless but nutritious sugar, enhances the memory of non-nutritious sugars like xylose and D-arabinose, but does not enhance the memory of nutritious sugars. Adding sorbitol to L-arabinose had no additive effect on long-term memory. It therefore appears that L-arabinose memory uses at least some of the same downstream neural circuitry as memory of nutritious sugars (McGinnis, 2016).

    Whether memory of L-arabinose, a non-nutritious sugar, is an exception or represents a more general phenomenon is unclear since this study tested only a limited number of sugars in a particular behavioral paradigm. However, in addition to L-arabinose, L-fucose can also produce memory; both are components of the pectin in many fruits' cell walls. It is therefore possible that these sugars may signal some specific attributes of ripening fruit ripening is accompanied by breakdown of the fruit's cell walls although neither of these sugars are present in fruits near the concentrations (1 M) used in memory assays. Nonetheless, these observations suggest that flies can quickly assess salient features of sugars a sort of leading indicator of nutritional value without the sugar's metabolic breakdown. This approach to memory formation may allow flies to quickly recognize and remember potential foods using specific cues, a time advantage that could be vital in natural contexts (McGinnis, 2016).

    Do insects distinguish structurally similar sugars? The taste modality of insects, particularly Drosophila, is reported to have limited discriminatory power and be primarily based on the intensity of the stimuli as opposed to the chemical nature of the sugar. Indeed it was found that, apart from flies' differential preference for various sugars at equal concentrations, for immediate and short-term behavior this is largely true. However, no obvious correlation was observed between immediate behavior and long-term memories: flies immediate preference is L-fucose > D-arabinose > L-arabinose > L-sorbose; for short-term memory, L-sorbose = D-arabinose ≥ L-arabinose = L-fucose; but in order of long-term memory score, L-arabinose ≥ L-fucose ≥ D-arabinose = L-sorbose. These results indicate that while short-term responses are guided by palatability, long-term behavioral responses are guided by additional attributes of the sugars. It is not yet clear why D-arabinose is a less effective stimulus. Since D- and L-arabinose are both sweet, they may generate positive sensations in a different manner, or perhaps D-arabinose carries a negative value that over time reduces the positive association formed initially (or dampens the behavioral output) (McGinnis, 2016).

    The gustatory receptors Gr5a, Gr43a, Gr61a, and Gr64a-f have been implicated in sugar detection. Although exactly which Gr receptors are responsible for detecting which sugar remains somewhat controversial, two features of sweet-sensing gustatory receptors are generally agreed upon: first, different gustatory neurons express a number of Gr receptors in unique combinations; second, more than one receptor is typically involved in detecting a sugar. However, the physiological consequences of this combinatorial expression of semi-redundant gustatory receptors remain uncertain. This study raises the possibility that gustatory neurons in different locations, expressing unique combinations of receptors, are responsible for discriminating chemically similar sugars and eliciting different behavioral responses. Consistent with this idea, previous studies suggested that Gr43a neurons in the central brain monitor hemolymph fructose levels and modulate feeding behavior, while this study found that these neurons are dispensable for L-arabinose memory, and that peripheral Gr43a-neurons are likely sufficient to signal the presence of a rewarding sugar and generate associative memories. These differences likely arise from the locations of these neurons, differentially expressed receptors, the presence or absence of various co-receptors, and the second-order neurons to which these neurons project. Exactly which or how many Gr43a-, Gr61a-, and Gr5a-expressing neurons in the periphery are sufficient for L-arabinose memory is currently unclear (McGinnis, 2016).

    This study also found that activation of Gr43a-expressing neurons by ReaChR but not dTrpA1 is able to generate appetitive memory, while artificially activating a subset of dopaminergic neurons (R58E02GAL4) by heat (dTrpA1) or light (ReaChR) both led to long-term memory. How a difference in activity at the sensory level is conveyed to higher-order neurons, and how that difference is interpreted by the higher-order neurons, remains unclear. More concretely, why is dTrpA1 activation of a subset of dopamine neurons sufficient to generate memory, but dTrpA1 activation of Gr43a-expressing neurons is not? One possibility is that the activity requirements of neuromodulatory systems are less stringent than those for sensory coding, and that temporal selectivity occurs before the signal reaches these dopamine neurons. Alternatively, recent studies have indicated that dopaminergic neurons are functionally diverse, and that distinct population of dopaminergic neurons are involved in appetitive associative memory. These reports raise the possibility that differing sensory inputs could activate different subsets of dopaminergic neurons (McGinnis, 2016).

    How can structurally similar sugars generate differential activation? It is likely that although these sugars bind to some of the same receptors, the relative affinity of the receptors vary. In this regard, the fly sweet taste system may be similar to that of the mammalian system, where a single heteromeric receptor (T1R2 and T1R3) is responsible for detecting a large number of sweet substances, with multiple discrete ligand-binding sites in each receptor responsible for generating diverse responses. It is suspected that the differential engagement of multiple gustatory receptors leads similar chemicals to generate differential activation of the same neurons, and that differential activation and different ensembles of activated neurons allows higher-order neurons to decode the relevant features of sugars. It is speculated that, at least in Drosophila, evaluation of a sugar's long-term salience may be encoded in the activation pattern of subsets of gustatory neurons, which allows rapid evaluation and remembering of nutritious food in complex environments (McGinnis, 2016).

    Bitter taste receptors confer diverse functions to neurons

    Bitter compounds elicit an aversive response. In Drosophila, bitter-sensitive taste neurons coexpress many members of the Gr family of taste receptors. However, the molecular logic of bitter signaling is unknown. This study used an in vivo expression approach to analyze the logic of bitter taste signaling. Ectopic or overexpression of bitter Grs increased endogenous responses or conferred novel responses. Surprisingly, expression of Grs also suppressed many endogenous bitter responses. Conversely, deletion of an endogenous Gr led to novel responses. Expression of individual Grs conferred strikingly different effects in different neurons. The results support a model in which bitter Grs interact, exhibiting competition, inhibition, or activation. The results have broad implications for the problem of how taste systems evolve to detect new environmental dangers (Delventhal, 2016).

    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, this study examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. The antifeedant effect of chloroquine was identified by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. Two molecular sensors, GR22e and GR33a, were found to act as chloroquine receptors, and chloroquine-mediated activation of GRNs was found to occur through S-type sensilla. At the same time, the chloroquine receptor was successfully recapitulated by expressing GR22e in ectopic gustatory receptor neurons. GR22e was found to form a part of the strychnine receptor. It is suggested that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials (Poudel, 2017).

    Neofunctionalization of "Juvenile Hormone Esterase Duplication" in Drosophila as an odorant-degrading enzyme towards food odorants

    Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. This study functionally characterized JHEdup in vivo. The jhedup gene is highly expressed in large basiconic sensilla, housed in the the maxillary palps, that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. These results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids (Steiner, 2017).

    Physiological responses of the Drosophila labellum to amino acids

    This study has systematically studied the physiological responses elicited by amino acids from the principal taste organ of the Drosophila head. Although the detection and coding of sugars and bitter compounds have been examined extensively in this organism, little attention has been paid to the physiology of amino acid taste. One class of sensilla, the labellar basiconic S sensilla, were found to yield the strongest responses to amino acids, although these responses were much weaker than the most robust responses to sugar or bitter compounds. S sensilla are heterogeneous in their amino acid responses and amino acids differ in the responses they elicit from individual sensilla. Tryptophan elicited relatively strong responses from S sensilla and these responses were eliminated when bitter-sensing neurons were ablated. Although tryptophan yielded little if any response in a behavioral paradigm, phenylalanine elicited a relatively strong response in the same paradigm and had a different physiological profile, supporting the notion that different amino acids are differentially encoded by the repertoire of taste neurons (Park, 2017).

    Molecular and cellular organization of taste neurons in adult Drosophila pharynx

    The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. This study generated receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. The mapping results were validated by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. These results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors (Chen, 2017).

    Internal amino acid state modulates yeast taste neurons to support protein homeostasis in Drosophila

    To optimize fitness, animals must dynamically match food choices to their current needs. For drosophilids, yeast fulfils most dietary protein and micronutrient requirements. While several yeast metabolites activate known gustatory receptor neurons (GRNs) in Drosophila melanogaster, the chemosensory channels mediating yeast feeding remain unknown. This study identified a class of proboscis GRNs required for yeast intake. Within this class, taste peg GRNs are specifically required to sustain yeast feeding. Sensillar GRNs, however, mediate feeding initiation. Furthermore, the response of yeast GRNs, but not sweet GRNs, is enhanced following deprivation from amino acids, providing a potential basis for protein-specific appetite. Although nutritional and reproductive states synergistically increase yeast appetite, reproductive state acts independently of nutritional state, modulating processing downstream of GRNs. Together, these results suggest that different internal states act at distinct levels of a dedicated gustatory circuit to elicit nutrient-specific appetites towards a complex, ecologically relevant protein source (Steck, 2018).

    The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool

    A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior. Five members of the alternatively spliced Gr28b gene cluster were examined for temperature-dependent properties via three approaches: biophysical characterization in Xenopus oocytes, functional calcium imaging in Drosophila motor neurons, and behavioral assays in adult Drosophila. Gr28bD expression in Xenopus oocytes produced a non-specific cationic current that is activated by elevated temperatures. This current is non-inactivating and non-voltage dependent. When expressed in Drosophila motor neurons, Gr28bD can be used to change the firing pattern of individual cells in a temperature-dependent fashion. Pan-neuronal or motor neuron expression of Gr28bD can be used to alter fruit fly behavior with elevated temperatures. Together, these results validate the potential of the Gr28bD gene as a founding member of a new class of thermogenetic tools (Mishra, 2018).

    Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain

    Bitter is a taste modality associated with toxic substances evoking aversive behaviour in most animals, and the valence of different taste modalities is conserved between mammals and Drosophila. Despite knowledge gathered in the past on the peripheral perception of taste, little is known about the identity of taste interneurons in the brain. This study shows that hugin neuropeptide-containing neurons in the Drosophila larval subesophageal zone are necessary for avoidance behaviour to caffeine, and when activated, result in cessation of feeding and mediates a bitter taste signal within the brain. Hugin neuropeptide-containing neurons project to the neurosecretory region of the protocerebrum and functional imaging demonstrates that these neurons are activated by bitter stimuli and by activation of bitter sensory receptor neurons. The study proposes that hugin neurons projecting to the protocerebrum act as gustatory interneurons relaying bitter taste information to higher brain centres in Drosophila larvae (Hückesfeld, 2016).

    The bitter taste rejection response is important for all animals that encounter toxic or harmful food in their environment. This study showed that the hugin neurons in the Drosophila larval brain function as a relay between bitter sensory neurons and higher brain centres. Strikingly, activation of the hugin neurons, located in the subesophageal zone, made the animals significantly more insensitive to substrates with negative valence like bitter (caffeine) and salt (high NaCl), as well as positive valence like sweet (fructose). In other words, when the hugin neurons are active these animals 'think' they are tasting bitter and therefore become insensitive to other gustatory cues. This is in line with observations made in mice, in which optogenetically activating bitter cortex neurons caused animals to avoid an empty chamber illuminated with blue light. In this situation, although mice do not actually taste something bitter, they avoid the empty chamber since the bitter perception has been optogenetically induced in the central nervous system (CNS) and the mice 'think' they are tasting a bitter substance (Hückesfeld, 2016).

    In previous work, activation of all hugin neurons led to behavioural and physiological phenotypes such as decreased feeding, decrease in neural activity of the antennal nerve (AN), and induction of a wandering-like behaviour (Schoofs, 2014). The neurons responsible specifically to those that project have now been pinpointed to the protocerebrum. These neurons not only respond to bitter stimuli, but also show a concentration dependent increase in calcium activity in response to caffeine. Dose dependent coding of bitter taste stimuli was previously shown to occur in peripheral bitter sensory neurons, where bitter sensilla exhibit dose dependent responses to various bitter compounds. Larvae in which the huginPC neurons have been ablated still showed some avoidance to caffeine. Whether this implies the existence of other interneurons being involved in caffeine taste processing remains to be determined. Interestingly, the huginPC neurons are inhibited when larvae taste other modalities like salt (NaCl), sugar (fructose) or protein (yeast). This may indicate that taste pathways in the brain are segregated, but influence each other, as previously suggested (Hückesfeld, 2016).

    Bitter compounds may be able to inhibit the sweet-sensing response to ensure that bitter taste cannot be masked by sweet tasting food. This provides an efficient strategy for the detection of potentially harmful or toxic substances in food. For appetitive tastes like fructose and yeast, bitter interneurons neurons like the huginPC neurons in the CNS may become inhibited to ensure appropriate behaviour to pleasant food. Salt is a bivalent taste modality, that is, low doses of salt drive appetitive behaviour, whereas high doses of salt are aversive to larval and adult Drosophila. Inhibition of huginPC neurons when larvae are tasting salt might be due to a different processing circuit for different concentrations of salt and the decision to either take up low doses or reject high doses (Hückesfeld, 2016).

    Taken together, it is proposed that hugin neuropeptide neurons projecting to the protocerebrum represent a hub between bitter gustatory receptor neurons and higher brain centres that integrate bitter sensory information in the brain, and through its activity, influences the decision of the animal to avoid a bitter food source. The identification of second order gustatory neurons for bitter taste will not only provide valuable insights into bitter taste pathways in Drosophila, but may also help in assigning a potentially novel role of its mammalian homologue, Neuromedin U, in taste processing (Hückesfeld, 2016).

    Mechanosensory neurons control sweet sensing in Drosophila

    Animals discriminate nutritious food from toxic substances using their sense of taste. Since taste perception requires taste receptor cells to come into contact with water-soluble chemicals, it is a form of contact chemosensation. Concurrent with that contact, mechanosensitive cells detect the texture of food and also contribute to the regulation of feeding. Little is known, however, about the extent to which chemosensitive and mechanosensitive circuits interact. This study shows Drosophila prefers soft food at the expense of sweetness and that this preference requires labellar mechanosensory neurons (MNs) and the mechanosensory channel Nanchung. Activation of these labellar MNs causes GABAergic inhibition of sweet-sensing gustatory receptor neurons, reducing the perceived intensity of a sweet stimulus. These findings expand understanding of the ways different sensory modalities cooperate to shape animal behaviour (Jeong, 2016).

    Long-range projection neurons in the taste circuit of Drosophila

    Taste compounds elicit innate feeding behaviors and act as rewards or punishments to entrain other cues. The neural pathways by which taste compounds influence innate and learned behaviors have not been resolved. This study identified three classes of taste projection neurons (TPNs) in Drosophila melanogaster distinguished by their morphology and taste selectivity. TPNs receive input from gustatory receptor neurons and respond selectively to sweet or bitter stimuli, demonstrating segregated processing of different taste modalities. Activation of TPNs influences innate feeding behavior, whereas inhibition has little effect, suggesting parallel pathways. Moreover, two TPN classes are absolutely required for conditioned taste aversion, a learned behavior. The TPNs essential for conditioned aversion project to the superior lateral protocerebrum (SLP) and convey taste information to mushroom body learning centers. These data identify taste pathways from sensory detection to higher brain that influence innate behavior and are essential for learned responses to taste compounds (Kim, 2017).

    The taste response to ammonia in Drosophila

    Ammonia is both a building block and a breakdown product of amino acids and is found widely in the environment. The odor of ammonia is attractive to many insects, including insect vectors of disease. The olfactory response of Drosophila to ammonia has been studied in some detail, but the taste response has received remarkably little attention. This study shows that ammonia is a taste cue for Drosophila. Nearly all sensilla of the major taste organ of the Drosophila head house a neuron that responds to neutral solutions of ammonia. Ammonia is toxic at high levels to many organisms, and it was found to have a negative valence in two paradigms of taste behavior, one operating over hours and the other over seconds. Physiological and behavioral responses to ammonia depend at least in part on Gr66a+ bitter-sensing taste neurons, which activate a circuit that deters feeding. The Amt transporter, a critical component of olfactory responses to ammonia, is widely expressed in taste neurons but is not required for taste responses. This work establishes ammonia as an ecologically important taste cue in Drosophila, and shows that it can activate circuits that promote opposite behavioral outcomes via different sensory systems (Delventhal, 2017).


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    date revised: 9 March 2018
     

    Zygotically transcribed genes

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