The Interactive Fly
Zygotically transcribed genes
Taste perception and coding in Drosophila
Gustatory receptor 21a and Gustatory receptor 63a confer CO2-chemosensation in Drosophila
Two Gr genes underlie sugar reception 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
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)
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).
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).
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 we have not yet 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 our 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).
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).
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).
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).
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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).
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date revised: 25 February 2009
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