InteractiveFly: GeneBrief

Gustatory receptor 5a: Biological Overview | Developmental Biology | Effects of Mutation | References


Gene name - Gustatory receptor 5a

Synonyms - Trehalose-sensitivity

Cytological map position - 5A11-B1

Function - transmembrane receptor

Keywords - taste receptors, G-protein coupled receptor

Symbol - Gr5a

FlyBase ID: FBgn0003747

Genetic map position - 1-13.6

Classification - G-protein coupled receptor

Cellular location - transmembrane



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Miyazaki, T., Lin, T. Y., Ito, K., Lee, C. H. and Stopfer, M. (2015). A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain. J Neurogenet: 1-26. PubMed ID: 26004543
Summary:
This study identified and characterized a bilateral pair of gustatory second-order neurons in Drosophila. GRASP (GFP reconstitution across synaptic partners) was combined with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate gustatory second-order neurons (G2Ns) and the axonal terminals of Gr5a-expressing gustatory sensory neurons GSNs, which are known to respond to sucrose. Results of this analysis revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, fluorescence-tagged acetylcholine receptor subunit (Dalpha7) and active-zone marker (Brp) were expressed in the G2Ns. G2N input sites were found to overlay GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. The identified G2Ns were found to receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the gnathal ganglia (GNG), and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). These findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.
BIOLOGICAL OVERVIEW

Taste receptors are likely to belong to the superfamily of G protein-coupled receptors (GPCRs). 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 (Tanimura, 1982). The Tre gene has been cytologically mapped to the region between 5A10 and 5B1-3 on the X chromosome (Tanimura, 1988). 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. The Tre gene has been shown to be expressed in taste sensory cells. These results provide direct evidence that Tre encodes a putative taste receptor for trehalose in Drosophila (Ishimoto, 2000). An independent study has identified a family of taste receptors in Drosophila that is unrelated to the Trehalose-sensitivity gene described here (Clyne, 2000).

This review is divided into two halves. The first describes the identification of CG3171 as Tre by Ishimoto (2000). This identification has been called into question by Ueno (2001). Ueno's identification of Tre as the gene adjacent to CG3171, the gustatory receptor Gr5a, is presented in the second half of this essay (see below).

To identify the putative Tre gene, a differential screen was performed for genes cloned into P1 vectors that might be specifically expressed in chemosensory cells. Advantage was taken of the pox-neuro (poxn) gene, which is involved in the developmental decision pathway between mechanosensory and chemosensory cell fates. In an adult-viable allele of the poxn mutant, all external chemosensilla are either transformed into mechanosensilla or are deleted. In the legs of the wild-type fly, chemosensilla exist on the tarsus, but there are no chemosensilla on the femur. A differential screening was carried out with cDNA probes derived from labella, tarsi, and femurs of wild-type and poxn mutant flies. The purpose of this screen was to distinguish genes expressed in wild-type but not expressed in poxn mutants (Ishimoto, 2000).

Southern blot analysis of the subcloned P1 DNA fragments identified one clone that hybridized to the wild-type labella and tarsi probes, but not to the other probes. A portion of the 8.2-kb clone displayed conserved features of the superfamily of seven-transmembrane domain receptor proteins. The full-length putative Tre1 cDNA was obtained by reverse transcriptase-dependent polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (Ishimoto, 2000).

By searching the Drosophila DNA database with the 5'-flanking genomic sequences of the putative Tre gene, flanking genomic sequences of the P-element were found in one previously isolated transposon-inserted strain [EP(X)] that completely matched the genomic sequence of Tre. The EP element is inserted 113 bp upstream of the transcription initiation site in the EP(X)0496 strain. The taste sensitivity of this strain to trehalose was tested with the two-choice preference test and the strain was found to highly sensitive, suggesting that the P-element insertion leaves the gene function intact (Ishimoto, 2000).

One way to isolate a mutation is to screen for imprecise excision of a P-element inserted near the gene that one wants to disrupt. It was expected that imprecise excision of the P-element should disrupt the promoter region of the Tre gene, and this event might change sensitivity to trehalose. The EP element carries w+ as a genetic marker, and the element was jumped out by genetically supplying a transposase source. w male flies were tested by two-choice preference tests, using as choices 30 mM trehalose and 2 mM sucrose. At this concentration of trehalose, nearly 98% of the parental EP(X)0496 flies preferred trehalose. Most of the w flies preferred trehalose, indicating that the precise excision of the P-element does not impair trehalose sensitivity. Flies that consumed the sucrose side were selected and individually crossed to C(1)DX attached-X females. From about 3000 w flies, 90 lines were isolated that were confirmed as showing low sensitivity to trehalose. The extent of deletion was determined in all the 90 lines by PCR, using primers flanking the P-element insertion site. There were no amplification products in most of these lines, indicating that a deletion eliminated the primer site(s) (Ishimoto, 2000).

Next, several lines were selected, and the extent of deletion was determined by Southern blotting. The results indicate that the deletions removed the putative promoter region and the first exon. In fact, RT-PCR analyses indicate that the Tre mRNAs are undetectable in all these lines. The sequence surrounding the insertion site was determined and it was confirmed that the strain that showed high sensitivity to trehalose had undergone a precise excision event. Tre mRNA is normal in this line (Ishimoto, 2000).

The taste sensitivity to trehalose of two DeltaTre lines was tested by the two-choice preference test with different concentrations of trehalose. The sensitivity to trehalose can be defined as the PI50, the concentration of trehalose that gives a 50% preference index (PI). For Canton-S, a typical high-sensitive strain, PI50 is 10 mM. In the original EP(X)0496 flies, the PI50 value is 12 mM, whereas the value is 80 mM in the two DeltaTre lines. Taken together, the disruption of the Tre gene leads to a lowering of the taste sensitivity to trehalose. Results of the two-choice preference test cannot discern whether trehalose sensitivity alone is altered in the DeltaTre strains. The proboscis extension reflex was examined by using four different sugar solutions: glucose, fructose, sucrose, and trehalose. The results demonstrate that the response to trehalose is specifically reduced in the DeltaTre lines. Since sensitivity to other sugars is unaffected, the sensitivity difference to trehalose should be attributed to a defect in the trehalose receptor. This conclusion is supported by the observation that the nerve responses to trehalose in the labellar chemosensilla are reduced in the DeltaTre mutant, whereas the sucrose sensitivity is unaffected. This electrophysiological evidence indicates that Tre is directly involved in trehalose sensation (Ishimoto, 2000).

To further confirm that the Tre gene is directly involved in the taste response to trehalose, transgenic lines were established carrying the hs-Tre cDNA gene so that Tre gene expression could be induced by heat shock. The P[hs-Tre]#1 line shows the highest expression of Tre mRNA after heat shock. Heat shock was tested in the background of the DeltaTre deletion mutant and was found to restore the trehalose sensitivity of the DeltaTre deletion mutant (Ishimoto, 2000).

In summary, a putative taste receptor gene, Tre, has been identified in Drosophila and the product of the Tre gene likely functions as a taste receptor for trehalose. Three findings are noted: (1) disruption of the Tre gene lowers the trehalose sensitivity of sugar receptor cells while leaving sensitivity to other sugars intact; (2) overexpression of the Tre transgene restores the response to trehalose; (3) the Tre gene is specifically expressed in putative sugar receptor cells. Because the Tre gene identified in this study was isolated from the genomic clone where Tre was initially mapped mapped, it is thought that the mutation(s) of the Tre gene is involved in the natural variation. If it is assumed that Tre1 is the sole receptor for trehalose, the null mutant of Tre (DeltaTre) should show no response to trehalose. The DeltaTre flies still respond to higher concentrations of trehalose, and this response would be mediated by another unidentified receptor for trehalose, although the possibility cannot be excluded that deletion mutants are not null. In fact, two other genes in the Drosophila genome have been identified with similarity to Tre; it is thought that TRE1 belongs to a novel family of G protein-linked transmembrane receptors that may operate as taste receptors. The function of the cloned gene should be investigated using expression systems, as has been successfully applied in the studies of olfactory receptors (Ishimoto, 2000).

The identification of CG3171 as Tre by Ishimoto (2000) has been called into question by Ueno (2001). Ueno's identification of Tre as the gene adjacent to CG3171, the gustatory receptor Gr5a, is presented below. The argument is rather compelling, but the two sets of data are still in conflict because of Ishimoto's convincing overexpression data. Nevetheless, a confirmation of Ueno's result has been provided by Dahanukar (2001), who has also identified Gr5a as Tre. Dahanukar's results are given in the Effects of Mutation section.

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor, and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor. Molecular analyses of mutations in Tre were carried out in order to elucidate their involvement in the gustatory phenotype. Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression is not always associated with mutations in Tre. In flies with the spontaneous mutation Tre01, both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre01. Taken together, the mutation analyses support that Gr5a is allelic to Tre (Ueno, 2001).

Flies carrying a spontaneous mutation Tre01 show a decreased gustatory sensitivity to trehalose. Two seven-transmembrane receptor genes Gr5a (Scott, 2001 and Dunipace, 2001) and CG3171 (Ishimoto, 2000) have been identified in the region where the Tre01 is mapped. A single P element insert [EP(X)496] (GenBank accession number AQ025347) that maps directly between the two genes was used to generate Tre mutations by activating imprecise P element excisions. A total of 22 independent Tre mutations were recovered. The gustatory phenotype has been assessed by a feeding preference test. Males of a wild-type Tre+ strain, w cx Tre+, produced the mean proportion of flies choosing the trehalose solution, or preference index (PI), of 0.90, while w cv Tre01 males carrying a spontaneous mutation Tre01 gave a low PI value of 0.30 (p < 0.01). EP(X)496 was wild-type for Tre, since the strain gave a PI value of 0.95. Males carrying the P excised Tre mutations TreDeltaEP3, TreDeltaEP5, or TreDeltaEP19 gave significantly low PIs of 0.06 or less (p < 0.01). Complementation tests between the P mutations and Tre01 confirm that the P mutations are allelic to Tre, but these P mutations of Tre apparently show a more severe phenotype than the spontaneous mutation Tre01 (p < 0.01) (Ueno, 2001).

Induced Tre mutations are genomic deletions uncovering CG3171 and/or Gr5a. The two Tre candidates CG3171 and Gr5a are adjacent genes on the genome facing the 5' end (GenBank accession number AE003435). The insertion of EP(X)496 is located between the two genes and is less than 0.1 kbp upstream of the transcription start site (GenBank accession number AB042625) of CG3171 and 0.7 kbp upstream of the putative start codon of Gr5a. TreDeltaEP3 (GenBank accession number AB066610) has a 2.1 kbp genomic deletion uncovering 0.7 kbp toward Gr5a and 1.4 kbp toward CG3171. In TreDeltaEP5 (GenBank accession number AB066611), a 2.5 kbp deletion was identified that spans 0.9 and 1.6 kbp in the directions of Gr5a and CG3171, respectively. The gene structure of CG3171 in TreDeltaEP3 and TreDeltaEP5 is disrupted by the absence of the promoter, exon 1, and part of intron 1. Gr5a gene is also disrupted in TreDeltaEP5 since it uncovers the promoter, the 5' leader, and 133 bp downstream of the start codon. Deletion in TreDeltaEP3 leaves 44 bp intact in the 5' leader, but at least 58 bp are truncated in addition to the deletion of the promoter. Therefore, TreDeltaEP3 and TreDeltaEP5 are expected to be double mutations of both CG3171 and Gr5a (Ueno, 2001).

TreDeltaEP19 (GenBank accession number AB066612) is distinct from TreDeltaEP3 or TreDeltaEP5 since the 3' end of the P element has been precisely excised out, leaving the CG3171 gene structure intact. TreDeltaEP19 uncovers a 1.0 kbp sequence in the direction of Gr5a that includes the promoter, the 5' leader, and a 253 bp sequence of the entire exon 1, intron 1, and part of exon 2. Therefore, only the Gr5a gene structure is specifically and most severely disrupted in TreDeltaEP19. Taking the genomic analyses together, disruption of Gr5a but not CG3171 is associated with Tre mutations (Ueno, 2001).

The transcripts of CG3171 and Gr5a were then investigated in the P excised mutants. RT-PCR with total RNAs isolated from the head amplified a CG3171 mRNA sequence in Tre+ and Tre01 flies. No amplification was observed with TreDeltaEP3 or TreDeltaEP5 templates. With TreDeltaEP1, TreDeltaEP13, and TreDeltaEP18 templates, the RT-PCR also failed to amplify the fragment. However, there was another class of mutations in which the expression of CG3171 mRNA was observed. TreDeltaEP7, TreDeltaEP11, TreDeltaEP12, TreDeltaEP14, and TreDeltaEP19 belong to this class. Among these, TreDeltaEP19 was predicted to express CG3171 from the intact genomic structure. In the Northern blot analysis, the antisense RNA probe of CG3171 labeled a 1.6 kbp mRNA from wild-type heads. The 1.6 kbp band was not detectable in TreDeltaEP3 or TreDeltaEP5, but was present in TreDeltaEP19, as in the RT-PCR analysis. CG3171 mRNA was further analyzed in various wild-type tissues (Ueno, 2001).

CG3171 mRNA is normally expressed in the labella, the head, and the appendages of a mutant poxn in which chemosensory neurons are transformed to mechanosensory neurons. The low tissue specificity of CG3171 is inconsistent with the claim of Ishimoto (2000) that CG3171 mRNA was identified from the differential screen of cDNA libraries between wild-type and poxn mutant tissues. A CG3171 cDNA clone LD12308 (GenBank accession number AA438512) was isolated from an embryonic library. CG3171 mRNA is expressed throughout embryonic and adult stages. A different line of evidence supporting CG3171 as a developmental gene was provided by Toba (1999) who showed that developmental phenotypes are induced in various tissues of the transformants carrying a misexpression construct inserted adjacent to CG3171 locus when the expression is activated by Gal4 drivers (Ueno, 2001).

Gr5a mRNA was examined in wild-type and mutant flies. RT-PCR was successful with poly(A)+ RNA prepared from 300 heads and 100 labella. An 856 bp Gr5a mRNA fragment was not identified in the deletions TreDeltaEP3, TreDeltaEP5, and TreDeltaEP19, while Gr5a mRNA is transcribed in wild-type, EP(X)496, and Tre01 heads. It is therefore suggested that intact mRNAs are not transcribed in those mutants. Gr5a mRNAs from wild-type and poxn labella were compared by the RT-PCR. Gr5a mRNA was identified in the wild-type labella, as observed in the heads. In the poxn labella, however, it was either absent or present in severely decreased amounts, suggesting that Gr5a is predominantly expressed in the gustatory sensory neurons in the labella. The expression analyses support that Gr5a is expressed in the gustatory neurons and that the mutation of Gr5a is associated with the Tre phenotype (Ueno, 2001).

Both CG3171 and Gr5a mRNAs were identified in the flies carrying the spontaneous mutation Tre01. The nucleotide polymorphisms leading to amino acid changes were investigated. A 1.5 kbp genomic sequence downstream of the start codon of CG3171 was analyzed in w cx Tre+, w cv Tre01, and four isofemale wild strains. The gustatory phenotype was also investigated for the wild strains. One strain (HG84) was Tre+, and the other three strains were Tre01. Sequence analysis has revealed a total of 12 single-nucleotide polymorphisms (SNPs) and an 18 bp insertion/deletion in exons. In addition, a total of 18 SNPs and two oligonucleotide insertions/deletions were also identified in the introns (GenBank accession numbers AB066613). The insertion of the 18 bp oligonucleotide ATGGATATGGATATGGGA, leading to an insertion of six amino acids in the N-terminal region, was identified in w cv Tre01 and two wild strains, HG84(Tre+) and Singapore (Tre01). In these three strains, a SNP in exon 2 leading to a Phe12Ile substitution was also identified. A second nonsynonymous SNP in exon 7 leading to a Leu348Met substitution was identified in all strains, except in w cx Tre+. None of the three sites, however, was linked to the gustatory dimorphism (Ueno, 2001).

Similarly, Gr5a polymorphisms were also analyzed for the 1.7 kbp genomic region in the following six strains: w cx Tre+, Canton-S (Tre+), HG84 (Tre+), w cv Tre01, Oregon-R (Tre01), and Singapore (Tre01). There were a total of 25 SNPs within the exons of the Gr5a gene. Nineteen SNPs and two single-nucleotide insertions/deletions were also found in the introns (GenBank accessions numbers AB066619-24). Among the exon polymorphisms, four nonsynonymous SNPs were identifed. The SNPs Met23Ile and Leu216His were identified only in Oregon-R (Tre01) and w cv Tre01, respectively. Val19Ile was identified in all three Tre01 strains but also in HG84 (Tre+). The only SNP that cosegregated with the Tre phenotype was Ala218Thr. Additional experiments have shown that Ala218Thr is significantly correlated with the gustatory Tre phenotype. The Ala218 is located in the predicted second intracellular loop domain of GR5a. Since the second or the third loop is known to be critically important in the activation of G protein both by rhodopsin and by mGluR1, GR5a may activate G protein through a similar mechanism, and the Ala218 may be involved in the activation process. Although functional characterization of Gr5a has yet to be performed, it is proposed that Gr5a encodes a gustatory sugar receptor controlled by the locus Tre (Ueno, 2001).

By Clustal W analysis, Gr5a has been shown to be most closely related phylogenetically to Drosophila candidate gustatory receptor gene Gr61a and a gene cluster of Gr64a-f on the third chromosome. Since Tre affects the taste response to a limited subset of sugars, some of these receptors may also be involved in the sweet taste response to different subsets of sugars. Physiological studies show that the input from sugar-sensitive neurons in the labellar, tarsal, and other gustatory organs controls the proboscis extension reflex and the feeding behavior. GRs have been shown to be expressed in gustatory neurons in distinct subsets of gustatory organs. Future studies on the localization and the projection of gustatory neurons expressing GR5a and the related GR members may provide clues to understanding the neuronal mechanism underlying feeding behavior in flies (Ueno, 2001).

Taste perception and coding in Drosophila

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


REGULATION

Drosophila Gr5a encodes a taste receptor tuned to trehalose

Recent studies have suggested that Drosophila taste receptors are encoded by a family of G protein-coupled receptor genes comprising at least 56 members. One of these genes, Gr5a, has been shown by genetic analysis to be required by the fly for behavioral and sensory responses to a sugar, trehalose. Gr5a is expressed in neurons of taste sensilla located on the labellum and legs. Expression is observed in most if not all labellar sensilla and suggests that many taste neurons express more than one receptor. It was demonstrate by heterologous expression in a Drosophila S2 cell line that Gr5a encodes a receptor tuned to trehalose. This is the first functional expression of an invertebrate taste receptor (Chyb, 2003).

To test the hypothesis that Gr5a plays a direct role in trehalose reception, it was asked whether it is expressed in taste neurons. Because previous attempts at in situ hybridization have proved unsuccessful with the great majority of Gr genes Gr5a promoter-GAL4 lines were generated. An 8.5-kb genomic region upstream of Gr5a was used to supply a promoter, and GAL4 was used to drive expression of both UAS-lacZ and UAS-GFP reporters. Wide expression was observed in taste neurons of the labellum as well as in four to six neurons in the tarsi of adult flies. No sexual dimorphism was observed in the expression pattern. Six independently derived lines were examined and all gave equivalent results (Chyb, 2003).

To examine Gr5a function at the cellular level, Gr5a cDNA was expressed in Drosophila S2 cells. This cell line was chosen for two reasons: (1) chemosensory receptors have been notoriously difficult to express in heterologous systems and it was predicted that use of a Drosophila cell line might improve the possibility of functional expression of a Drosophila receptor; (2) previous studies have documented Ca2+ release after activation of G protein-coupled receptors that couple to the endogenous Gq protein of S2 cells. In this system, ligand binding to the receptor results in the activation of the phosphoinositide (PI) pathway: hydrolysis of PIP2 by phospholipase C into InsP3 and 4,5-diacylglycerol, and release of Ca2+ from intracellular stores. The stimulus-activated change in [Ca2+]i can be monitored with Ca2+-sensitive fluorescent ratiometric indicators, such as fura 2 (Chyb, 2003).

Gr5a was transiently expressed in S2 cells: they were loaded with 100 µM fura 2, and 100 mM trehalose was applied via puffer pipette. Stimulation evoked Ca2+ release: cell response developed within ~5 s of ligand application and reached a peak intensity within ~15 s of application. Upon removal of the ligand, the level of intracellular calcium gradually returned to the baseline. These data provided initial evidence that Gr5a encodes a functional trehalose receptor when expressed in S2 cells. The results also suggested the possibility that Gr5a-encoded receptor protein couples to the endogenous phosphoinositide pathway. S2 cells were then cotransfected with Gr5a and promiscuous G proteins: Galpha15,Galpha16, or Galpha15 and Galpha16 together. These G proteins are known to couple a wide variety of G protein-coupled receptors to intracellular Ca2+ release. There was no significant increase in the response intensity to trehalose stimulation compared with the S2-Gr5a cells, consistent with the possibility that Gr5a couples efficiently to Gq (Chyb, 2003).

Therefore, Gr5a is expressed in all, or almost all, of the ~33 sensilla present on the labellum. Because the labellum responds to a variety of sugars, and because the sensilla each contain a single sugar-sensitive neuron, the broad expression observed is consistent with a model in which many, if not all, of the sugar-sensitive taste neurons express more than one receptor. This model is supported by the finding that mutation of Gr5a affects the physiological response of the sugar cell to trehalose, but not to sucrose, as if many of the sugar-sensitive cells contain both a trehalose receptor, Gr5a, and a sucrose receptor (Chyb, 2003).

The expression pattern of Gr5a is broader than that observed for previously described GAL4 lines established by using promoters of other Gr genes. The broad pattern is consistent with physiological data, which indicates that Gr5a is required for trehalose response in all L- and M-type sensilla. Most sensilla on the labellum respond to trehalose (Chyb, 2003).

The simplest interpretation of the results is that Gr5a functions as a homodimer, unlike the mammalian sweet receptors, which function as heterodimers. Furthermore, in contrast to the T1R2/T1R3 mammalian receptor that is rather broadly tuned to diverse sweet-tasting molecules such as sucrose, saccharin, dulcin, and acesulfame-K, the Gr5a receptor is tuned to trehalose and shows much less, if any, response to other sugars, such as sucrose, fructose, and glucose, which the fly encounters in its natural habitat (Chyb, 2003).

The relatively narrow tuning of Gr5a has implications for the mechanism of taste coding. If other Drosophila taste receptors are as specific as Gr5a, then an individual tastant is likely to be encoded largely by the activity of one or a small number of receptors, as opposed to the integrated activity of many receptors, each exhibiting a varying degree of response to a ligand. In the olfactory system of Drosophila, many individual odorants activate several distinct classes of receptor neurons, each expressing distinct odor receptors. This model of taste coding is also supported by the severe loss of trehalose response after mutation of a single receptor gene, Gr5a. Analysis of further Gr proteins will be required to determine whether the narrow tuning of Gr5a is representative of Gr receptors at large or of those that recognize tastants of particular metabolic significance to the fly, such as trehalose (Chyb, 2003).

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

The sense of taste allows animals to distinguish nutritious and toxic substances and elicits food acceptance or avoidance behaviors. In Drosophila, taste cells that contain the Gr5a receptor are necessary for acceptance behavior, and cells with the Gr66a receptor are necessary for avoidance. To determine the cellular substrates of taste behaviors, taste cell activity in vivo was monitored with the genetically encoded calcium indicator G-CaMP. These studies reveal that Gr5a cells selectively respond to sugars and Gr66a cells to bitter compounds. Flies are attracted to sugars and avoid bitter substances, suggesting that Gr5a cell activity is sufficient to mediate acceptance behavior and that Gr66a cell activation mediates avoidance. As a direct test of this hypothesis, different taste neurons were inducibly activated by expression of an exogenous ligand-gated ion channel, and it was found that cellular activity is sufficient to drive taste behaviors. These studies demonstrate that taste cells are tuned by taste category and are hardwired to taste behaviors (Marella, 2006).

In Drosophila, cells with the Gr5a taste receptor are necessary for sugar acceptance behaviors, and those with Gr66a are necessary for avoidance. These taste cells selectively recognize different taste modalities, such that there is functional segregation of taste qualities in the periphery and at the first relay in the brain. Moreover, activation of these different taste neurons is sufficient to elicit different taste behaviors. Thus, activity of the sensory neuron, rather than the receptor, is the arbiter of taste behavior. These studies argue that animals distinguish different tastes by activation of dedicated neural circuits that dictate behavioral outputs (Marella, 2006).

The patterns of sensory projections provide internal representations of the external world. For example, there is an odotopic map of olfactory projections in flies and mammals. Drosophila gustatory projections are segregated by taste organ such that there is an anterior-posterior map in the subesophageal ganglion of mouthpart, proboscis, and leg projections. Within the proboscis, two different populations of taste neurons can be defined by their expression of either the Gr5a receptor or the Gr66a receptor. Neurons with these different receptors show segregated projections in the brain, with Gr5a projections lateral and anterior to Gr66a projections. Genetic cell ablation experiments revealed that Gr5a cells are required for sugar acceptance behavior and Gr66a for avoidance of bitter compounds. These experiments suggest that in addition to the organotopic map of taste projections, there is also an anatomical map of different taste modalities (Marella, 2006).

This study directly demonstrates that there is functional segregation of different taste modalities in the fly brain. Taste responses were monitored in the living fly by expressing the calcium-sensitive indicator G-CaMP in different classes of taste neurons; Gr5a projections respond to a large number of sugars and Gr66a termini respond to several bitter compounds. Monitoring the responses of subsets of Gr66a cells to a panel of bitter compounds did not reveal striking differences in ligand recognition profiles. Although the possibility that different subsets of Gr5a or Gr66a cells show more selective responses cannot be rule out, clear spatial segregation of sugar and bitter responses was found in the SOG. This argues that there is a spatial activity map of different taste modalities in the fly brain that corresponds to the anatomical projections of Gr5a and Gr66a cells (Marella, 2006).

The character of the taste map in the fly brain is very different from the olfactory map. In the olfactory system, 70 receptors in flies and ~1000 in mammals are used to detect odors. Neurons generally express one receptor, and neurons with the same odorant receptor in the periphery form functional synapses at the same glomerulus in the first relay of the brain. Functional imaging experiments demonstrate that a given odor will activate multiple glomeruli, and one glomerulus will respond to multiple odors. This has led to a spatial model for odor coding in the brain in which the unique combination of activated glomeruli specifies a smell. An animal is thus able to distinguish thousands of different smells by the activation of thousands of different combinations of glomeruli. By contrast, in the fly taste system, sugars activate Gr5a taste projections and bitter compounds activate Gr66a projections. This suggests that there is not a combinatorial code for different tastes in the fly. Instead, the activation of segregated neural populations encodes different taste modalities. This simple map may allow the fly to distinguish sugars from bitter compounds, but may limit the ability to distinguish compounds within the same modality (Marella, 2006).

Sugars elicit food acceptance behavior, and bitter compounds elicit avoidance. The segregation of sugar and bitter responses in the fly brain suggests that activation of different classes of sensory neurons may be sufficient to generate different taste behaviors. This hypothesis was directly tested. Gr5a or Gr66a cells were inducibly activated by expression of a cationic ion channel, VR1E600K, in taste cells and application of its ligand, capsaicin, at the proboscis. G-CaMP imaging experiments demonstrated that taste cells show calcium increases in response to capsaicin. Behavioral studies showed altered taste preferences in flies containing the VR1E600K channel: flies with VR1E600K in Gr5a cells are attracted to capsaicin, and those with VR1E600K in Gr66a cells avoid it. This demonstrates that activation of different taste neurons is sufficient to generate different taste behaviors. Recent studies in C. elegans chemosensory neurons and mammalian gustatory cells demonstrate that exogenous activation of these cells is sufficient to generate acceptance and avoidance behaviors as well. The picture that is emerging from these studies is that the activity of selective sensory cells in the periphery generates behavioral programs through the activation of dedicated neural circuits (Marella, 2006).

G-CaMP imaging and behavioral studies have important implications for understanding how taste information is encoded in the periphery. Three different models have been suggested for how taste information is encoded in the brain: the labeled-line model, the population-coding model (or mixed-lines model), and the temporal-coding model. In the labeled-line model of taste coding, cells are dedicated to detecting different taste ligands, and this information remains segregated as it is relayed to the brain, such that different tastes are distinguished by the selective activation of nonoverlapping cells. In population-coding models, the comparative activity of many cell types rather than activation of one type conveys taste information. This model proposes that the ensemble activity encodes taste quality. In temporal-coding models, it is the precise pattern of action potentials that communicates taste quality (Marella, 2006).

The labeled-line model can be distinguished from the other models by the requirement for a neuron to have a unique identity in terms of recognition properties and behavior. The observation that neurons express subsets of receptors and selectively recognize different taste categories argues that taste neurons have different identities. Moreover, the finding that activation of an exogenous ion channel in discrete taste cell populations elicits specific behaviors argues that selective cell activation is sufficient to mediate behavior, under conditions that do not activate the entire taste cell population and are unlikely to mimic endogenous firing patterns. Taken together, these studies strongly favor the labeled-line model of taste coding in the periphery, although they cannot rule out a role for spike timing or ensemble encoding in fine-tuning the responses (Marella, 2006).

Seminal studies in the gustatory system of mammals strongly argue in favor of the labeled-line model of taste coding in the mammalian gustatory system in the periphery as well. Taste cells on the tongue selectively express either sugar, bitter, or amino acid receptors, such that different taste qualities are detected by different cells in the periphery. Activation of these different taste cells is sufficient to generate specific taste behaviors, with artificial activation of sugar cells eliciting acceptance behavior and artificial activation of bitter cells eliciting avoidance. The observation that cells are dedicated to detecting a specific taste modality and mediate a specific behavior suggests that there are labeled lines of taste information from peripheral detection to behavior. Thus, taste behaviors are hardwired to selective cell activation on the tongue in mammals and the proboscis in flies (Marella, 2006).

The advantage of having taste cell activation innately coupled to behavioral outputs via labeled lines is that the valence of a taste compound is dictated by the neural circuit and requires no previous association. The stereotypy of taste behaviors affords the opportunity to examine how neural connectivity elicits distinct behaviors. It is anticipated that live imaging of neural responses will be a powerful approach to dissect higher-order taste processing in the fly brain (Marella, 2006).

Gsalpha is involved in sugar perception in Drosophila melanogaster

In Drosophila, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. This study proposes that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: (1) immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. (2) trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescues those impairments. (3) Expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depresses both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. It is suggested that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila (Ueno, 2006: full text of article).

It was found that DGsalpha is localized not only in Gr5a-GRNs but also in non-Gr5a GRNs (~40 GRNs in a labelum). In labela, there are at least four types of GRNs sensitive to sugar, low concentrations of salt, bitter-substances/high concentrations of salt, water, and mechanosensory neurons. Then, two questions arise: (1) which GRN, other than Gr5a-GRNs, contains DGsalpha? (2) Is DGsalpha in unknown GRNs involved in the taste signaling of GRNs? The behavioral responses to bitter solutions were not different between heterozygous DGsalpha-null mutant and control flies, and the behavioral and electrophysiological responses to water were not different among all DGsalpha strains examined in this study. It is known that salt responses in larvae require amiloride-sensitive channels encoded by ppk11 and ppk19, and the low and high concentrations of salt responses do not require Ggamma1 in adult flies. These findings together with the current results suggest that DGsalpha in non-Gr5a GRNs serves for other signaling than taste or that the non-Gr5a GRNs containing DGsalpha are mechanosensory neurons. However, because the bitter and water responses in the homozygous DGsalphaR19 mutant were not studied, the possibility that DGsalpha is involved in bitter and/or water tastes cannot be rigorously excluded (Ueno, 2006).

It is suggested that, in Drosophila, the Gs-mediated cAMP transduction pathway is the main signaling route in sugar-sensitive GRNs. In contrast, the PLC/IP3 mediating pathway is involved in sugar-taste signaling in the fleshfly (Boettcherisca peregrina) and the guanosine-3',5'-cyclic monophosphate/nitric oxide pathway in the blowfly (Phormia regina). The cAMP pathway may be involved in sugar-taste perception in the frog, rat, and pig, whereas a recent study on T1R2/T1R3 gustatory sugar receptors of the mouse supports involvement of the PLC pathway. Additional comparative studies are necessary to elucidate the diversity of molecular mechanisms of sugar-taste signaling in various animals (Ueno, 2006).

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

Taste and olfaction are each tuned to a unique set of chemicals in the outside world, and their corresponding sensory spaces are mapped in different areas in the brain. This dichotomy matches categories of receptors detecting molecules either in the gaseous or in the liquid phase in terrestrial animals. However, in Drosophila olfactory and gustatory neurons express receptors which belong to the same family of 7-transmembrane domain proteins. Striking overlaps exist in their sequence structure and in their expression pattern, suggesting that there might be some functional commonalities between them. In this work, the assumption was tested that Drosophila olfactory receptor proteins are compatible with taste neurons by ectopically expressing an olfactory receptor (OR22a and OR83b) for which ligands are known. Using electrophysiological recordings, this study shows that the transformed taste neurons are excited by odor ligands as by their cognate tastants. The wiring of these neurons to the brain seems unchanged and no additional connections to the antennal lobe were detected. The odor ligands detected by the olfactory receptor acquire a new hedonic value, inducing appetitive or aversive behaviors depending on the categories of taste neurons in which they are expressed i.e. sugar- or bitter-sensing cells expressing either Gr5a or Gr66a receptors. Taste neurons expressing ectopic olfactory receptors can sense odors at close range either in the aerial phase or by contact, in a lipophilic phase. The responses of the transformed taste neurons to the odorant are similar to those obtained with tastants. The hedonic value attributed to tastants is directly linked to the taste neurons in which their receptors are expressed (Hiroi, 2008).

These experiments provide the first direct evidence that olfactory receptors are functional in true taste neurons of Drosophila. These neurons respond to odorants dissolved in paraffin oil upon contact, as if odorants were sapid molecules, and they can even respond to these molecules in air at close range. These observations indicate that the hedonic value that was associated with the detection of the odor is changed according to the identity of the GRNs expressing this receptor (Hiroi, 2008)

The results are consistent with and extend previous results published by Benton (2006) who expressed olfactory receptors in several classes of antennal neurons, including mechanosensory neurons of the Johnston organ and CO2-sensing neurons. Olfactory receptors like Or22a or Or43a need to be co-expressed with Or83b to be correctly addressed to the dendritic membranes and to induce functional responses to the proper odorant ligands. Benton expressed the olfactory receptor Or43a (with Or83b) in antennal neurons expressing Gr21a; these neurons respond to CO2 in the air and acquire the property of responding to cyclohexanol which is a ligand for Or43a. Although Gr21a and its partner Gr63a are classified as a taste receptors, these neurons should be considered as olfactory: (1) they are housed into sensilla ab1C which are lacking a terminal pore considered as characteristic to taste sensilla and (2) they project into the antennal lobe to the DM2-glomerulus while antennal taste sensilla in other insects project into the subesophageal ganglion. Nonetheless, these CO2-sensing sensilla express 'gustatory' receptors which are functional in the absence of Or83b. While Benton demonstrated that ectopic olfactory receptors are functional by population measurement using calcium imaging on the antennal lobe, this study used single-sensillum recordings that gives a greater temporal resolution. Lastly, this work extends Benton's work, by analyzing how the hedonic value of the odorants is changed after miss-expressing ORs into GRNs (Hiroi, 2008).

One important aspect of these experiments is that altered GRNs transduce odorants despite the obvious structural differences between olfactory and taste sensilla e.g. a single terminal pore for taste sensilla vs. a host of minute pores on the hair shaft for olfactory sensilla. The fact that volatile molecules can enter the terminal pore and stimulate taste neurons has received scant attention, except for reports showing that plant odors stimulate taste receptor neurons of tobacco hornworm larvae, Manduca sexta, the Colorado potato beetle, Leptinotarsa decemlineata (Say) and the blowfly. Further indications that taste sensilla may sense lipophilic molecules and odorants come from molecular studies that repeatedly report the presence of odorant-binding proteins in various taste sensilla of insects, which contribute to the transfer of chemicals from air to the sensillum lymph. While the tip-recording technique requires the use of lipophilic solvents that may damage the distal membrane of the taste cells, the technique used in this study should be suitable to record the responses of GRNs to other lipophilic compounds like cuticular pheromones or water-insoluble compounds from plants (Hiroi, 2008).

OR83b is an essential partner to OR22a and other odorant receptor proteins. These molecules form a dimer and adopt in vivo, a topology where their N-termini and most conserved loops are in the cytoplasm; this observation has been confirmed by another approach. This conformation suggested that signaling downstream of the ORs is non-canonical, a prediction that has been recently confirmed by two independent studies using in vitro heterologous expression systems. That OR receptors can induce spiking activities in taste neurons is therefore not surprising: these dimers form channels that when gated by an odorant, may generate current sufficient to induce a receptor potential and trigger the firing of action potentials. However, evidence is still missing about how these ORs are activated in vivo, especially considering that in addition to the odorant-gated channel activation, ORs may interact with more classical transduction pathways like cAMP or cGMP. From this perspective, Drosophila taste neurons represent a useful expression system to evaluate the specificity of olfactory receptors, since it provides cells fully equipped with compatible transduction pathways whose activities can be monitored by extracellular recording techniques or possibly by patch-clamp as done in fleshfly sugar-sensing GRNs (Hiroi, 2008).

Flies expressing olfactory receptors within subsets of taste neurons sharing the expression of the same GR should be particularly useful for understanding how the taste modalities are encoded at the periphery. Although the functional separation between sugar-sensing and bitter-sensing seems quite natural, it rests on chemical characteristics that may overlap. For example, NaCl was found to stimulate sugar-sensing cells at low concentration and bitter-sensing cells at high concentrations. Likewise, a number of artificial sweeteners are stimulating both sugar-sensing cells and bitter sensing-cells in humans and in flies. Because several Gr are co-expressed in Gr66a-GRNs and in Gr5a-GRNs, it is likely that more than one neuron detects the same molecule within a sensillum. The use of a heterologous receptor as a reporter gene for a given Gr has the advantage of activating only one cell without the confounding activity of the other cells (Hiroi, 2008).

While previous observations showed that impairing the expression of Gr5a or Gr66a in taste neurons changed the behavioral responses to sugars or to bitter substances and as well as the activities of the neurons projecting in the brain after 'ensemble' stimulations, the current experiments directly demonstrate that individual GRNs which express Gr5a and Gr66a are different and respond to sugar and to bitter compounds. This study indicates that taste sensory cells of insects encode broad qualities similar to those found in vertebrates (Hiroi, 2008).

How food controls aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. This study used the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. Food was shown to promote aggression in flies, and it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, it was shown that detection of sugar via Gr5a+ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs (Lim, 2014).


DEVELOPMENTAL BIOLOGY

Because the Tre gene was isolated on the basis of its specific expression in taste sensory cells, the gene is likely to be expressed in taste cells. RT-PCR analyses on isolated labella and tarsi preparations have shown that the mRNA is expressed in the labella and tarsi of original EP(X) lines but is absent in DeltaTre and poxn flies with no taste sensory cells. In situ hybridization experiments have shown that the Tre mRNA is present in one of the taste sensory cells beneath a taste bristle. There were no signals in the labellum preparation of poxn and in the central brain. Thus, Tre seems to be specifically expressed in taste sensory cells (Ishimoto, 2000).

Taste representations in the Drosophila brain

Fruit flies taste compounds with gustatory neurons on many parts of the body, suggesting that a fly detects both the location and quality of a food source. For example, activation of taste neurons on the legs causes proboscis extension or retraction, whereas activation of proboscis taste neurons causes food ingestion or rejection. Whether the features of taste location and taste quality are mapped in the fly brain was studied using molecular, genetic, and behavioral approaches. Projections were found to be segregated by the category of tastes that they recognize: neurons that recognize sugars project to a region different from those recognizing noxious substances. Transgenic axon labeling experiments also demonstrate that gustatory projections are segregated based on their location in the periphery. These studies reveal the gustatory map in the first relay of the fly brain and demonstrate that taste quality and position are represented in anatomical projection patterns (Wang, 2004).

Sixty-eight gustatory receptor (GR) genes have been identified in the sequenced Drosophila genome. These receptors are likely to recognize subsets of taste cues and therefore serve as molecular markers to distinguish neurons recognizing different taste stimuli. To determine whether there is a map of taste quality in the fly brain, the distribution of GRs in sensory neurons was examined. The potential number of tastes that a neuron may recognize was investigated and then the projections of different taste neurons in the brain were examined (Wang, 2004).

One of the difficulties in determining receptor expression patterns in the Drosophila taste system is that GR genes are expressed at very low levels. Most GR genes are not detectable by in situ hybridization experiments, and it has been necessary to generate transgenic flies in which GR promoters drive expression of reporters using the Gal4/UAS system to determine receptor expression. Two transgenic reporter systems were used to simultaneously detect the expression of different receptors. The Gal4/UAS system was used to label one set of neurons, using Gr-Gal4 to drive expression of UAS-CD2. Nine different GR promoters were used that have been reported to drive robust reporter expression in subsets of taste neurons (Dunipace, 2001; Scott, 2001; Chyb, 2003). To label the second set of GR-bearing neurons, transgenic flies were generated in which a GR promoter drives expression of multiple copies of GFP (e.g., Gr66a-GFP-IRES-GFP-IRES-GFP; for simplification, subsequently referred to as Gr-GFP) (Halfon, 2002). Although it is not known how much amplification multiple copies provide, this approach successfully allowed visualization of taste projections whereas direct promoter fusions to a single GFP did not. Transgenic flies for three different GR promoters (Gr32a, Gr47a, Gr66a) were generated and crossed to seven different Gr-Gal4, UAS-CD2 lines to generate a matrix of 21 double receptor combinations (Wang, 2004).

GRs are expressed in subsets of taste neurons, suggesting that one or a few receptors are expressed per cell. This hypothesis was tested by direct comparison of reporter expression for the matrix of three Gr-GFP by seven Gr-Gal4, UAS-CD2 receptor combinations. Focus was placed on the proboscis to compare reporter expression driven by different GR promoters. These studies revealed several surprising findings. (1) Many GR promoters drive reporter expression in partially overlapping cell populations. Gr66a-Gal4 drives expression in approximately 25 neurons per labial palp, in a single neuron in most or all sensilla. Of the six other GRs tested, five show expression in subsets of Gr66a-positive neurons. Of these five, four show largely overlapping expression with each other and one shows mostly non-overlapping expression. Therefore, Gr66a defines a population of gustatory neurons that express overlapping patterns of multiple receptors. (2) Some receptors are segregated into different cells. Gr5a-Gal4 drives reporter expression in approximately 30 neurons per labial palp, in one neuron in most sensilla. Gr5a is not expressed in Gr66a-positive cells. Thus, two non-overlapping neural populations can be identified by Gr66a and Gr5a. Together, these cells account for two of the four gustatory neurons in each taste sensillum (Wang, 2004).

Extracellular recordings of taste responses from proboscis chemosensory bristles have suggested that all taste sensilla are equivalent and that each of the four taste neurons within a sensillum recognizes a different taste modality, with one neuron responding to sugars, two to salts, and one to water. However, more recent experiments suggest a greater diversity of responsiveness. Because Gr5a and Gr66a are expressed in different cells in a sensillum, it was wondered whether they might mark neurons recognizing different classes of tastes. Interestingly, Drosophila defective in Gr5a show reduced responses to the sugar trehalose both in behavioral and electrophysiological studies, and heterologous expression of Gr5a in tissue culture cells confers trehalose responsiveness, strongly arguing that the ligand for Gr5a is trehalose. Given that Gr5a marks a cell that responds to a sugar, it was hypothesized that Gr66a might mark a cell responding to a different taste category. This type of segregation has been demonstrated in the mammalian taste system, where taste cells that respond to sweet are different from those responding to bitter or umami tastants. The taste ligands that Gr5a and Gr66a cells recognize were examined using genetic cell ablation and behavioral studies. It was discovered that Gr66a cells participate in the recognition of bitter compounds (Wang, 2004).

How is taste quality represented in the brain? Because Drosophila taste receptor neurons need not only recognize different tastes but most likely also the gustatory source (e.g., proboscis, internal mouthparts, legs, and wings), gustatory projections were examined to determine whether taste quality or location is represented in sensory projection patterns (Wang, 2004).

The adult Drosophila brain contains approximately 100,000 neurons, with cell bodies in an outer shell surrounding the dense fibrous core. The primary gustatory relay is the subesophageal ganglion/tritocerebrum (SOG) located in the ventral region of the fly brain. It receives input from three peripheral nerves. Neurons from the proboscis labellum project through the labial nerve; mouthpart neurons project through the pharyngeal/accessory pharyngeal nerve, and neurons from thoracic ganglia project via the cervical connective. Early studies employing cobalt backfills provided evidence that mouthpart neurons project more anteriorly in the SOG than proboscis neurons, suggesting that there might be a map of different taste organs in the fly brain (Wang, 2004).

To examine whether taste neurons in different locations project to different brain regions, GR promoters that drive reporter expression in different peripheral tissues were exploited to follow gustatory projections from the proboscis, mouthparts, or leg. Brains of Gr-Gal4, UAS-GFP were stained by anti-GFP immunohistochemistry, and a series of 1 μm optical sections through the SOG was collapsed to produce a two-dimensional representation of projections. These studies reveal differences in projections for neurons in different peripheral tissues. For instance, Gr2a is expressed only in the mouthparts and these neurons exit the pharyngeal nerve and arborize anteriorly. Gr59b, however, is expressed only in proboscis neurons that arborize in a ringed web. Notably, some receptors are expressed both in the proboscis and mouthparts. Interestingly, their neural projections seem to be the composite of Gr2a and Gr59b projections (Wang, 2004).

Two color labeling approaches were used to examine whether projections are segregated by peripheral tissue. For example, differential labeling of Gr2a neural projections, expressed in the mouthparts, and Gr66a neurons expressed in the proboscis, mouthparts, and legs illustrate overlap of the mouthpart projections but not of proboscis projections. Similarly, when the projections of Gr59b, expressed only in the proboscis, and Gr66a are differentially labeled, there is overlap of projections in the ventral proboscis region but not the dorsal mouthparts region (Wang, 2004).

The different axonal patterns from mouth, proboscis, and leg are also seen in different optical sections through the SOG of Gr32a-Gal4, UAS-GFP flies, arguing that projections are segregated by peripheral tissue even if they contain the same receptor. To better resolve the projections of individual neurons with the same receptor, taste neurons were labeled using a genetic mosaic strategy that relies on postmitotic recombination to induce expression of reporters in single cells. The Gr32a receptor is expressed in proboscis, mouthpart, and leg neurons. Single Gr32a-positive neurons from each tissue were labeled and their arborizations were examined in the SOG. A single mouthpart neuron sends an axon that arborizes in a discrete arbor in the most anterior aspect of the SOG. However, a proboscis neuron with the same receptor sends an axon that shows diffuse branching in the medial SOG, a region different from mouthpart projections. Gr32a-positive leg neurons project through the thoracic ganglia and directly terminate in the most posterior part of the SOG (Wang, 2004).

Overall, these studies demonstrate that taste neurons in different tissues project to different locations in the SOG, with mouthpart projections more anterior than proboscis projections, which are more anterior than leg projections. The demonstration that neurons that express the same receptor in different parts of the body project to distinct locations argues that they elicit different spatial patterns of brain activity and provide a means for encoding different behaviors in response to the same tastant (Wang, 2004).

It was next asked whether neurons from the same peripheral tissue that recognize different tastes project to the same or a different brain region to evaluate if taste quality is encoded in sensory projection patterns. Two-color labeling strategy was used to differentially label projections from Gr5a neurons that recognize sugars and Gr66a neurons that recognize bitter compounds. Remarkably, the projections of proboscis neurons with these receptors are clearly segregated in the SOG: Gr5a projections are more lateral and anterior to Gr66a projections. The Gr5a projections are ipsilateral and resemble two hands holding onto the medial, ringed web of Gr66a projections. Interestingly, leg taste projections for Gr5a and Gr66a neurons are segregated: Gr66a neurons project to the SOG whereas Gr5a neurons project to thoracic ganglia (Wang, 2004).

By contrast, when receptors are contained in partially overlapping populations, there is no obvious segregation of projections. For example, Gr32a is contained in a small fraction of Gr66a-positive cells in the proboscis, yet Gr32a-positive fibers colocalize with Gr66a-positive fibers in all optical sections. Moreover, Gr32a and Gr47a are expressed in mostly non-overlapping subsets of Gr66a-positive proboscis neurons, and their projections overlap, showing that smaller populations of Gr66a-positive cells are not spatially segregated. The lack of segregation suggests that these cell types are not functionally distinct (Wang, 2004).

These studies demonstrate that receptors that are expressed in subsets of cells that recognize bitter substances do not show segregated projections. However, different projection patterns are clearly discernible for proboscis neurons that recognize bitter compounds versus those that recognize sugars. The segregated projections from Gr5a and Gr66a cells reveal that there is a spatial map of taste quality in the brain (Wang, 2004).

The patterns of Drosophila taste receptor expression resemble those of the mammalian taste system and the C. elegans chemosensory system, where multiple receptors are also expressed per cell. In the mammalian taste system, multiple bitter receptors are coexpressed in one population of cells on the tongue whereas receptors for sugars are expressed in a different population of taste cells, arguing that different sensory cells recognize different taste modalities. Remarkably, the concept of distinct sweet and bitter cells also applies to the fly (Wang, 2004).

This study identified two populations of proboscis neurons that show spatially segregated projection patterns in the SOG. These different patterns correspond to different taste categories: neurons that recognize bitter substances are mapped differently in the fly brain from those that recognize sugars, suggesting that there is a map of taste modalities or behaviors in the fly brain. In addition, several subpopulations of Gr66a-positive cells show convergent projections. Two different models could account for this convergence. (1) In the simplest model, convergence could imply similar function. For example, all neurons mediating avoidance behaviors might project to the same region and synapse on a second order neuron that conveys avoidance. (2) The apparent convergence could still yield segregated gustatory information if there is a molecular identity code such that second order neurons synapse exclusively with gustatory neurons containing the same receptors. This second model is akin to what is seen in the mammalian pheromone system and sensory-motor connectivity in the spinal cord. Future experiments examining synaptic connectivity will be essential to determine how gustatory information is transmitted to higher brain centers. Nevertheless, the observation that there is spatial segregation of Gr5a sugar cells and Gr66a bitter cells, but not of smaller populations of Gr66a-positive cells, suggests that the diversity of recognition afforded by 68 or so receptor genes may be simplified into only a few different taste categories in the fly brain (Wang, 2004).

Gustatory projections are also segregated according to the peripheral position of the neuron. Early studies employing cobalt backfills argue that mouthpart neurons project more anteriorly in the SOG than proboscis neurons. The results are consistent with, and extend, these observations. Using genetic mosaic approaches, single taste neurons were labeled, and it was found that projections from different organs are segregated even from neurons containing the same receptor. These studies argue that the same taste stimulus will produce different patterns of brain activity depending on the stimulus' location in the periphery and may mediate different behaviors, consistent with the observation that sugar on the leg causes proboscis extension whereas sugar on the ovipositor causes egg laying. An organotopic map of gustatory projections may provide a means for the fly to distinguish different taste locations (Wang, 2004).

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

This study examined the molecular and cellular basis of taste perception in the Drosophila larva through a comprehensive analysis of the expression patterns of all 68 Gustatory receptors (Grs). Gr-GAL4 lines representing each Gr are examined, and 39 show expression in taste organs of the larval head, including the terminal organ (TO), the dorsal organ (DO), and the pharyngeal organs. A receptor-to-neuron map is constructed. The map defines 10 neurons of the TO and DO, and it identifies 28 receptors that map to them. Each of these neurons expresses a unique subset of Gr-GAL4 drivers, except for two neurons that express the same complement. All of these neurons express at least two drivers, and one neuron expresses 17. Many of the receptors map to only one of these cells, but some map to as many as six. Conspicuously absent from the roster of Gr-GAL4 drivers expressed in larvae are those of the sugar receptor subfamily. Coexpression analysis suggests that most larval Grs act in bitter response and that there are distinct bitter-sensing neurons. A comprehensive analysis of central projections confirms that sensory information collected from different regions (e.g., the tip of the head vs the pharynx) is processed in different regions of the s ganglion, the primary taste center of the CNS. Together, the results provide an extensive view of the molecular and cellular organization of the larval taste system (Kwon, 2011).

Of the 67 Gr-GAL4 transgenes, 43 showed expression in the larva, of which 39 were expressed in the major taste organs of the head. The 39 Gr-GAL4 drivers are expressed in combinatorial fashion. Individual Gr-GAL4 drivers are expressed in up to 12 cells, in the case of Gr33a- and Gr66a-GAL4; approximately one-half, however, are expressed in only one cell (Kwon, 2011).

For some Gr-GAL4 drivers the observed pattern of expression may not be identical with that of the endogenous Gr gene. It was precisely with this concern in mind that a mean of 7.6 independent lines were analyzed for each of the 67 Gr drivers, and a rigorous, quantitative protocol was establised for identifying a representative line for each gene. In the absence of an effective in situ hybridization protocol, the approach used here seemed likely to be the most informative in providing a comprehensive systems-level view of larval taste reception (Kwon, 2011).

The Gr receptor-to-neuron map of the dorsal and terminal organs identified 10 neurons. Two neurons have cell bodies in the DOG and innervate the DO, two have cell bodies in the DOG and innervate the TO, and six have cell bodies in the TOG and innervate the TO (Kwon, 2011).

28 receptors were mapped to these 10 neurons. All of these neurons express at least two Gr-GAL4 drivers. Two receptors, Gr21a and Gr63a, are coreceptors for CO2; neither is sufficient to confer chemosensory function alone. It is conceivable that many other Grs may also require a coreceptor, which may explain the lack of neurons expressing a single Gr-GAL4. The number of receptors per neuron ranges up to 17, in the case of C1. This number is comparable with the maximum number of Gr-GAL4s observed in a labellar neuron (29), and much greater than the number of Ors observed in individual neurons of either the larval or adult olfactory system (Kwon, 2011).

Among the 10 identified cells, individual Gr-GAL4 drivers are expressed in as few as one cell and as many as six cells. Most of the drivers are expressed in only one of these 10 cells. The drivers expressed in six cells, Gr33a-GAL4 and Gr66a-GAL4, are expressed in all bitter neurons of the adult labellum. It is noted that Gr33a-GAL4 and Gr66a-GAL4 are the only drivers expressed in B1, arguing against the possibility that both of these receptors function exclusively as chaperones or as coreceptors that require another Gr for ligand specificity (Kwon, 2011).

There is little cellular redundancy. Only two neurons, A1 and A2, express the same complement of receptors. All other neurons contain a unique subset of the Gr repertoire. In this respect, the larval taste system differs from the adult taste system but is similar to the larval olfactory system, which also contains little if any cellular redundancy (Kwon, 2011).

Analysis of the central projections of all 39 Gr-GAL4 drivers provided evidence for a systematic difference among projection patterns between TO/DO neurons and pharyngeal neurons. These results support the conclusion that sensory information collected from the tip of the head is processed in different regions of the SOG than information collected in the pharynx, i.e., that evaluation of a potential food source before ingestion and the testing of food quality during ingestion are functionally partitioned in the brain. Similar inferences were drawn in an elegant study of a limited number of Gr-GAL4 transgenes (Colomb, 2007; Kwon, 2011 and references therein).

Conspicuously absent from the list of Gr-GAL4 drivers expressed in the larval taste system are those representing the eight members of the sugar receptor subfamily (Gr5a, Gr61a, Gr64a-f). The founding member of this family, Gr5a, mediates response to the sugar trehalose, and two other members of the subfamily have been shown to encode sugar receptors as well. No GFP expression for these genes was observed in cells of the taste organs or in neural fibers in the brain or ventral ganglion. Most of these Gr-GAL4 transgenes drive expression in the adult, but it is acknowledged that these transgenes may not faithfully reflect expression in the larva (Kwon, 2011).

Given that Drosophila larvae respond to sugars, as do larvae of other insect species, how do they detect them without members of the sugar receptor subfamily? Other Grs, including the recently identified fructose receptor Gr43a, may underlie sugar detection in the larva. It is noted that Gr59e-GAL4 and Gr59f-GAL4 are coexpressed in a cell that does not express the bitter cell markers Gr33a-GAL4 or Gr66a-GAL4. Sugar reception may also be mediated by other kinds of receptors, such as those of the TRPA family (Kwon, 2011).

In adult Drosophila, Gr33a-GAL4 and Gr66a-GAL4 are coexpressed with other Gr-GAL4s in bitter neurons; the simplest interpretation of expression and functional analysis is that multiple bitter receptors are coexpressed (Kwon, 2011).

In the larva, it ws found that most larval Gr-GAL4s are coexpressed with Gr33a- and Gr66a-GAL4, suggesting the possibility that most larval Grs act in bitter response. It is noted that, of the 17 Gr-GAL4s coexpressed in the C1 neuron, 15 are coexpressed in a bitter neuron of the labellum. It was also establish that there are distinct molecular classes of Gr33a-GAL4, Gr66a-GAL4-expressing neurons. The simplest interpretation of these results is that there are distinct bitter-sensing neurons in larvae (Kwon, 2011).

Larvae must determine whether to accept or reject a food source, and in principle this determination could be made by a simple binary decision-making circuit. However, the existence of six Gr33a-GAL4, Gr66a-GAL4-expressing neurons expressing distinct subsets of Gr-GAL4s suggests a greater level of complexity in the processing of gustatory information. One possibility is that C1, which expresses the largest subset of drivers among the TO/DO neurons, may activate an aversive behavior in response to many of the bitter compounds that the larva encounters, while C2, C3, C4, or B2 either potentiates the response or activates a different motor program in response to chemical cues of particular biological significance or exceptional toxicity. The existence of heterogenous bitter-sensing cells, some more specialized than others, is a common theme in insect larvae. In particular, many species contain a taste cell that responds physiologically to many aversive compounds and whose activity deters feeding. C1 could be such a cell, and its coexpression of many receptors may provide the molecular basis of a broad response spectrum (Kwon, 2011).

It is striking that the number of TO/DO neurons that express Gr-GAL4s is small compared with the total number of TO/DO neurons. Gr-GAL4 expression was mapped to only 10 cells in the TO/DO (although Gr2a-GAL4 and Gr28a-GAL4 were each expressed in two TO neurons that were not mapped). The DOG and TOG contain 36-37 and 32 sensory neurons, respectively, among which 21 in the DOG are olfactory. Thus, of the nonolfactory cells, on the order of 20%-30% express Gr-GAL4 drivers. It will be interesting to determine how many of the other DOG/TOG cells express other chemoreceptor genes, such as Ppk, Trp, or IR genes, and how many of the other neurons have mechanosensory, thermoreceptive, hygrosensory, or other sensory functions (Kwon, 2011).

The role of Gr genes in the larval pharyngeal organs is unknown. In adult pharyngeal sensilla, the TRPA1 channel, which detects irritating compounds, regulates proboscis extension. It is possible that Grs expressed in larval pharyngeal organs may also play a role in modulating feeding behavior. Of the 24 Gr-GAL4 drivers expressed in the larval pharyngeal organs, 9 are coexpressed with Gr33a-GAL4 and Gr66a-GAL4 in the TO/DO; it seems plausible that they may monitor ingested food for the presence of aversive compounds (Kwon, 2011).

In summary, this study has analyzed essential features of the molecular and cellular organization of a numerically simple taste system in a genetic model organism. Ten gustatory receptor neurons were described and evidence was provided that they express Grs in combinatorial fashion, with most of these neurons and receptors acting in the perception of bitter compounds. The results lay a foundation for a molecular and genetic analysis of how these receptors and neurons, and the downstream circuitry, underlie a critical decision: whether to accept or reject a food source (Kwon, 2011).


EFFECTS OF MUTATION

Functional evidence is provided that one of the gustatory receptor genes, Gr5a, encodes a taste receptor required for response to the sugar trehalose. In two different mutants that carry deletions in Gr5a, electrophysiological and behavioral responses to trehalose are diminished but the response to sucrose is unaffected. Transgenic rescue experiments show that Gr5a confers response to trehalose. The results correlate a particular taste ligand with a Gr receptor and indicate a role for G protein-mediated signaling in the transduction of sweet taste in Drosophila (Dahanukar, 2001).

To test the hypothesis that Gr genes encode taste receptors, it was asked whether any Gr genes reside at loci implicated in taste perception. The trehalose response locus, whose alleles confer different levels of response to the disaccharide trehalose, has been mapped to cytogenetic region 5A on the X chromosome. One Gr gene, Gr5a, is also located in region 5A and is thus tightly linked to this locus. Gr5a is a member of a small subfamily of eight Gr genes. The intron-exon structure of Gr5a was determined by 5' and 3' RACE and RT-PCR experiments. The experiments also show expression of Gr5a in the proboscis and the legs, both of which contain sensilla that respond to trehalose. The 5' end of Gr5a lies less than 900 base pairs from CG3171, which has previously been reported to encode the trehalose receptor and has been named Tre1 (Ishimoto, 2000; Dahanukar, 2001).

To investigate the possible role of Gr5a in trehalose reception, two strains of flies, each with a deletion in the Gr5a-Tre1 genomic region, were used. The two deletion lines, DeltaEP(X)-19 and DeltaEP(X)-5, henceforth referred to as Delta19 and Delta5, were generated by imprecise excision of a P element that lies in the region between the two genes in strain EP(X)0496, henceforth referred to as 496. Sequence analysis determined that in Delta19, the proximal endpoint of the deletion lies within the second exon of Gr5a (3' to the codon specifying Ala 62), and in Delta5, the proximal endpoint lies in the first intron (which lies 3' to the codon specifying Arg 36) (CG15779 in Gadfly). Thus, in each deletion strain, the translation initiation codon of Gr5a is removed. Flies that are either homozygous or hemizygous for these deletions are viable (Dahanukar, 2001).

It was next asked whether the deletion mutants lacking Gr5a were defective in trehalose response, using two independent assays. First, electrophysiological response was measured to sugars in individual labellar taste hairs of the large (L) or medium (M) class, using the tip-recording method. L and M sensilla each have four chemosensory neurons, classified according to the stimuli that elicit a response from them: a sugar cell, two salt cells and a water cell. The sugar cell responds to a number of different sugars, including disaccharides such as sucrose and trehalose, which is present in yeast (an important food source of Drosophila). Individual sensilla were stimulated by placing an electrode containing the sugar over the tip of the sensillum. Action potentials are elicited in both the sugar and the water cell, but the activities of the two cells can be distinguished by their different amplitudes. Dose-response curves for sucrose and trehalose in the various strains reveal that sucrose response is the same for all strains across a broad range of concentrations. In the same cells, however, the trehalose responses of Delta5 and Delta19 were drastically reduced compared to that of the parental strain 496 (Dahanukar, 2001).

Next the behavioral response to trehalose was tested using the two-choice preference test. This protocol compares the consumption of two sugars offered simultaneously to populations of flies. In the first control experiment, the preference for different concentrations of sucrose versus a standard concentration of 2 mM sucrose was tested. Flies of all strains tested preferred 3 mM (10-2.5 M) sucrose to 2 mM sucrose, with a preference index (PI) of 0.9, and they preferred 2 mM sucrose to 1 mM sucrose. Consistent with the physiological data, there was no difference in the response to sucrose between strain 496 and the strains that are mutant for Gr5a. To investigate trehalose response, several concentrations of trehalose were tested against 2 mM sucrose. The PI50 value, the concentration of trehalose at which flies consume as much trehalose as sucrose, was 11 mM for 496, but 77 mM for Delta5 and 76 mM for Delta19. Thus, in this behavioral protocol both mutants were severely defective in their response to trehalose (Dahanukar, 2001).

To determine whether loss of Gr5a expression is responsible for the reduced response to trehalose in the deletion mutants, a 10-kb genomic DNA rescue construct was engineered that includes both the Gr5a and the Tre1 coding regions. To assess the contributions of each gene separately, derivatives of this 10-kb construct were generated that include a stop codon near the N-terminus of either Gr5a or Tre1. Transgenic flies were generated containing these constructs and crossed into Delta5 and Delta19 genetic backgrounds. The resulting flies were tested for rescue of the trehalose response defect using the physiological and behavioral assays described above (Dahanukar, 2001).

The physiological response of the sugar cell to trehalose was rescued by the construct with wild-type copies of both Tre1 and Gr5a. Although rescue occurs in both the Tre1+Gr5a+ and the Tre1-Gr5a+ transgenic flies, it does not occur in the Tre1+Gr5a- transgenic flies, indicating that rescue required a wild-type Gr5a but not a wild-type Tre1. None of the transgenes affected response to sucrose (Dahanukar, 2001).

Next, the behavioral phenotype of the transgenic flies was tested. The preference for trehalose was measured at a 10-1.5 M (31.6 mM) concentration, which gave the maximal difference in PI between the parental and deletion strains. Rescue again occurred, and the pattern of rescue was consistent with the physiological data: it was dependent on Gr5a and not on Tre1 in both the Delta19 and Delta5 flies (Dahanukar, 2001).

Thus, Gr5a, a member of a large family of candidate taste receptors, corresponds to the Tre locus, a genetic locus that affects response to the sugar trehalose. The most compelling evidence that Gr5a is responsible for the trehalose sensitivity phenotype is that deletion mutations affecting Tre function can be rescued by a transgenic construct containing a wild-type copy of Gr5a, but not by a construct containing a mutant copy of Gr5a. Rescue has been shown both by measurements of single-cell electrophysiology and by behavioral assays. The equivalence of Gr5a and the Tre locus is also consistent with mapping data indicating that all reported Tre deletions remove portions of Gr5a (Dahanukar, 2001).

The simplest interpretation of these results is that Gr5a encodes a taste receptor for trehalose. The absence of a functional Gr5a affects the physiological response of a taste neuron to trehalose, but not the response of the same neuron to another disaccharide sugar, sucrose. Thus, the specificity of Gr5a function is consistent with that expected of a taste receptor, and inconsistent with that expected of a GPCR playing a general role in taste neuron development or function. Trehalose reception could also be mediated in part by additional receptors; however, the severely reduced trehalose response in flies lacking Gr5a and the rescue data suggest that the response to trehalose is mediated primarily by Gr5a (Dahanukar, 2001).

A previous report identified the product of Tre1 as the trehalose receptor (Ishimoto, 2000). That report used a heat shock-inducible Tre1 cDNA construct to rescue the phenotype of a deletion mutant similar to Delta5. Although the results do not formally exclude a role for Tre1 in trehalose response, neither do they support such a role. In considering the differing conclusions of these two studies, it is noted that although Tre1 mutants were shown to be abnormal in both a two-choice preference test and a proboscis extension test, rescue was described only in the two-choice preference test and only for a single pair of concentrations (2 mM sucrose and 80 mM trehalose). Moreover, although some limited data are reported to indicate a physiological phenotype for a Tre1 mutant, rescue of the peripheral physiological defect by the hs-Tre1 transgene has not been shown. It is noted that Tre1 is expressed in embryonic EST collections, suggesting that it is expressed in early Drosophila development. Tre1 is also expressed ubiquitously in adult tissue, according to a paper published while the current manuscript was under review (Ueno, 2001). Polymorphisms in the sequence of Gr5a, but not Tre1, correlate with the trehalose phenotype (Dahanukar, 2001).

The sugar cells in all wild-type L- or M-class sensilla from which recording were taken responded to both sucrose and trehalose. In recordings from Gr5a deletion mutants, no sensilla of these types were found whose sugar cells respond at wild-type levels to trehalose. Though not exhaustive, these data suggest that Gr5a is expressed in the sugar cells of all the L and M sensilla on the labellum. These two sensillar classes together constitute ~30 of the ~66 sensilla on the labellum; thus, Gr5a seems to be expressed in at least 30 neurons. Although this number has not been confirmed by in situ hybridization, 30 is a larger number than is seen for the several members of the Gr family whose expression has been detected by in situ hybridization or reporter-gene expression. For these receptors, the number of cells exhibiting expression ranges from 4 to 22 in the labellum. Because response of all tested Gr5a mutant sugar cells was abnormal to trehalose but normal to sucrose, it seems likely that each of these cells expresses at least two receptor genes: Gr5a, which mediates response to trehalose, and another receptor that mediates response to sucrose. Colocalization of receptors within a single sugar cell might constrain the ability of the animal to discriminate among sugars through differential activation of distinct taste neurons. It remains possible, however, that discrimination might be achieved by insulation of different signaling pathways, as probably happens within individual chemosensory neurons of Caenorhabditis elegans (Dahanukar, 2001).

Although studies in mammals have implicated G protein-mediated signaling in the transduction of sweet taste, the mechanism in invertebrates is largely unknown. There is evidence that sweet taste in larger flies is mediated at least in part through a cGMP second messenger; however, there is also a report of a channel that is activated directly by sucrose without the mediation of second messengers or G proteins. The results support a role for G protein-mediated transduction of the disaccharide trehalose in Drosophila, as is found for sweet taste in mammals (Dahanukar, 2001).

In summary, the simplest interpretation of the data is that a member of the Gr gene family encodes a taste receptor required for response to the sugar trehalose, as indicated by both electrophysiological and behavioral analysis of mutant and transgenic flies. The association of a particular ligand with a particular Gr taste receptor now allows for a variety of studies, including detailed functional studies of the receptor and of the mechanism by which it transduces gustatory information. It will be interesting to determine whether the other genes in the Gr5a subfamily encode sweet taste receptors for other sugars (Dahanukar, 2001).

Two Gr genes underlie sugar reception in Drosophila

The molecular basis of sugar reception in Drosophila has been analyzed. The response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons has been defined. Using in situ hybridization and reporter gene expression, members of the Gr5a-related taste receptor subfamily were identified that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of these results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars (Dahanukar, 2007).

A major problem in neurobiology is how an animal decides what to eat. The fruit fly evaluates gustatory input to assess the nutritive value of a potential food source. In particular, the detection of sugars is a crucial factor in determining whether a food source is accepted. Despite its critical importance to the survival of the species, little is known about the molecular basis of sugar perception in the fly. A central goal in the field has been to define the receptors that mediate sugar detection (Dahanukar, 2007).

Sugars, salts, bitter compounds, and certain other molecules are detected by gustatory neurons, which are widely distributed in the body of the fly. Neurons that influence feeding behavior are present in the labellum as well as the tarsal segments of each of the legs. Activation of either labellar or tarsal gustatory neurons with a sugar solution results in proboscis extension, which is a component of feeding behavior (Dahanukar, 2007).

Gustatory neurons are housed in sensory hairs called sensilla. Each half of the labellum is covered with ~31 prominent taste hairs, arranged in a stereotypical pattern, and a number of smaller structures called taste pegs. Each of the 31 sensilla is typically innervated by four gustatory neurons and a single mechanosensory neuron. Physiological analysis has shown that one of the chemosensory neurons is activated by sucrose and other sugars, and has been referred to as the 'sugar' neuron. Another neuron is activated by salts and has been named the 'salt' neuron. A third neuron is activated by pure water but not by solutions of high osmolarity; it has been named the 'water' neuron. The fourth chemosensory neuron responds to aversive compounds such as caffeine, and has been named the (Dahanukar, 2007 and references therein).

In Drosophila, a large, highly diverse family of gustatory receptor (Gr) genes was identified by genomic analysis. The family consists of 60 genes encoding 68 predicted seven-transmembrane-domain receptors. In previous studies, Gr5a was identified as a receptor for trehalose, a disaccharide sugar. Gr5a is expressed in a large number of gustatory neurons in the labellum, and recent studies have shown that Gr5a serves as a marker for the sugar neuron in each sensillum. Bitter neurons express Gr66a, also a member of the Gr gene family, which is required for physiological and behavioral responses to caffeine. Promoter expression analysis of several other gustatory receptor genes in the labellum suggested that all of those tested were coexpressed with Gr66a in subsets of bitter neurons (Dahanukar, 2007 and references therein).

Axonal projections of Gr5a-positive and Gr66a-positive neurons have been mapped to the subesophageal ganglion (SOG) of the brain. The two classes of neurons project to nonoverlapping regions in the SOG, suggesting that at the first level of processing, attractive and aversive inputs may be segregated. Evidence that Gr5a neurons mediate attractive signals and Gr66a neurons mediate aversive signals was provided by expression of a capsaicin receptor in each of these classes of neurons. In the first instance, flies showed behavioral attraction to capsaicin, and in the second instance they were repelled by it (Dahanukar, 2007).

Gr5a-labeled neurons are responsive not only to trehalose, but to sucrose and other sugars. Physiological and behavioral analysis showed that sucrose response is not affected in flies lacking Gr5a, suggesting that these neurons express at least one other receptor; however, other receptors in sugar neurons were not identified (Dahanukar, 2007).

This study examined the responses of sugar neurons in the largest sensilla of the labellum, the 'L' sensilla. Of 50 compounds tested, including 34 diverse sugars, a small number were identified, primarily disaccharides and oligosaccharides, which elicit robust electrophysiological responses in sugar neurons. In situ hybridization and reporter gene expression determined that two other Gr genes, both phylogenetically related to Gr5a, are coexpressed with Gr5a in sugar neurons. Neurons expressing reporters of each receptor gene show distinct projection patterns, providing a mechanism by which information from different subpopulations of sugar cells in the periphery could be spatially represented in the brain (Dahanukar, 2007).

Having found coexpression of Gr5a-related genes in sugar neurons, mutants of Gr5a and two related genes were examined by electrophysiology and behavioral analysis. Gr5a was found to be required for detection of a small subset of sugars including trehalose. Deletion mutants lacking Gr64a shows that it is required for response to a complementary subset of sugars. Strikingly, flies lacking both Gr5a and Gr64a do not show electrophysiological or behavioral responses to any tested sugar. These results demonstrate that the sugars divide into two classes that are dependent either on Gr5a or on Gr64a for their responses. The simplest interpretation of these results is that these two receptors are capable of operating independently of each other in an individual sugar neuron, and that they constitute the primary basis of sugar reception in the fly (Dahanukar, 2007).

Sucrose generated the strongest responses among a panel of 50 compounds tested at 100 mM. Sucrose is present at comparable concentrations in many fruits, including citrus, peaches, and pineapples. Turanose, palatinose, and leucrose are all isomers of sucrose and also elicit responses of various strengths. Many of the sugars that evoke responses, including glucose and trehalose, are found in fruits and vegetables or in yeasts and may thus be encountered by the fly in its natural environment (Dahanukar, 2007).

The responses depend on sugar concentration as well as identity. The neurons are sensitive to a number of sugars over concentrations that span three orders of magnitude. The dose-response curves of different sugars, however, are distinct: they differ in threshold, slope, and maximal firing rate observed. Many of these sugars are present in fruits at concentrations of 100-300 mM, and at these concentrations the responses lie well within the dynamic range of the neurons. Surprisingly, responses to fructose and glucose, which are particularly abundant in fruits, are much weaker than those of sucrose, even when compared at concentrations that have equal caloric values. However, the concentrations of both fructose and glucose are typically higher than that of sucrose in fruits such as apples, bananas, and grapes, suggesting that sugar neurons may be most sensitive to changes in sugar concentrations over a range that is ecologically relevant (Dahanukar, 2007).

Molecular analysis has revealed coexpression of Gr61a and Gr64f with Gr5a, and genetic analysis of a double mutant has provided evidence for coexpression of Gr64a with Gr5a in sugar neurons. These results suggest that at least some labellar sugar neurons, including those of L-type sensilla, coexpress four receptors of the Gr5a subfamily (Dahanukar, 2007).

Molecular and genetic evidence indicates that Gr5a is expressed in essentially all labellar sensilla. Molecular analysis has provided evidence that Gr64f is also broadly expressed, and functional evidence suggests that Gr64a is as well. Specifically, an electrophysiological survey showed that all labellar sensilla in wild-type flies respond to sucrose, a sugar that acts via Gr64a. In a Gr5a;Gr64a mutant all morphological types of sensillum (L, M, S, I, P) showed no activity in response to sucrose; moreover, nearly all of the L, M, I, and P sensilla were tested, suggesting that Gr64a acts in all, or almost all, of the 31 sensilla on the labellum. Furthermore, the double mutants are also behaviorally unresponsive to sugars. Thus Gr5a and Gr64a seem likely to be expressed in all or almost all sugar neurons in the labellum, and perhaps Gr64f is as well (Dahanukar, 2007).

Gr61a, however, appeared to be restricted in its expression among labellar sensilla, both by in situ hybridization and by analysis of a Gr61a-GAL4 driver. These results suggest a subdivision of labellar sugar neurons into two classes based on the presence or absence of Gr61a. No function was defined for Gr61a; however, mutational analysis suggests that it does not play a role in responses to any of the sugars in the panel. It is possible that Gr61a is required for response to other sugars or sugar derivatives that have not yet been tested or for responses to another class of behaviorally attractive compounds. Further electrophysiological analysis with an expanded panel of tastants may provide insight into whether there are functional differences among sugar-sensing neurons and whether these differences correlate with the expression of Gr61a (Dahanukar, 2007).

Gr5a and Gr64a are both required for normal responses of sugar neurons, but for different subsets of sugars. Flies lacking Gr5a are severely defective in physiological and behavioral responses to one subset of sugars, including trehalose; flies lacking Gr64a are severely defective in responses to a complementary subset of sugars, including sucrose. All tested sugars fall into one of these two subsets. These results suggest that Gr5a and Gr64a function as distinct receptors in the same neurons, rather than as obligate heterodimeric coreceptors, as in the mammalian sugar receptor T1R2+T1R3 (Dahanukar, 2007).

It is possible that Gr5a and Gr64a function as heterodimeric receptors with other members of the Gr family, such as Gr64f. Two recent studies report deletions of part or all of the Gr64 cluster that result in reduced behavioral responses to trehalose; the phenotype is rescued by supplying a transgene containing five of the six receptors encoded by this cluster, but not by Gr64a alone. These data support the idea that one of the receptors in this cluster other than Gr64a may function in concert with Gr5a to mediate trehalose response. There is precedent for such interactions from Or proteins, which dimerize with the noncanonical receptor Or83b (Dahanukar, 2007).

The neat subdivision of sugars into those dependent on Gr5a and those dependent on Gr64a was surprising. A simple structural criterion to distinguish the two classes of sugars is not immediately evident upon inspection. The Gr64a-dependent sugars are remarkably diverse in structure, with some containing glucose units and some containing fructose subunits; they ranged in size from one to four subunits. Gr5a-dependent sugars also vary in size, subunit composition, and linkage types (Dahanukar, 2007).

In Gr5a mutants, there are some weak residual responses to the affected subset of sugars; likewise, in Gr64a mutants, some of the affected sugars continue to elicit some response. Since there is no residual response in the Gr5a;Gr64a double mutant, the simplest interpretation of these results is that each receptor provides the residual function observed when the other is eliminated, i.e., the two receptors exhibit some limited redundancy (Dahanukar, 2007).

Gr5a and Gr64a share 28% amino acid identity and 47% amino acid similarity. Both receptors are evolutionary conserved and are found in all of the 12 Drosophila species for which genome sequences are available, with the exception that D. pseudoobscura appears to have lost Gr5a. The receptor most closely related to Gr5a is Gr64f (40% amino acid identity), and the receptor most closely related to Gr64a is Gr61a (36% amino acid identity). Although evidence was found that Gr64f and Gr61a are both expressed in sugar neurons, no functions have been identifed for them. The possibility cannot be excluded of a role for Gr61a or Gr64f in response to compounds not tested, such as glycoproteins or glycolipids, or in neurons whose responses have not been measured, such as those of internal chemosensory cells. It is noted that in mammals, an amino acid receptor (T1R1+T1R3) comprises a subunit, T1R3, of the heterodimeric sugar receptor (T1R2+T1R3). However, L-type sensilla did not respond to any of 18 amino acids tested, making it unlikely that either Gr61a or Gr64f mediates responses to this class of compounds (Dahanukar, 2007).

Classic physiological and biochemical studies led to the proposal of a 'fructose' site in sugar-sensing neurons. The current studies provide a molecular and genetic identity to this site: fructose response is completely abolished by loss of Gr64a and is completely restored by the addition of a Gr64a transgene. These results also provide a molecular explanation for the earlier finding that sucrose responses were not affected in a Gr5a mutant. These results suggested the presence of another receptor within the sugar neuron, a receptor that has now been identified as Gr64a (Dahanukar, 2007).

It is noted that two recent studies have identified a role for members of the Gr64 cluster in mediating sugar responses, particularly that of Gr64a in response to sugars including sucrose, maltose, and glucose. Consistent with the observations, physiological and behavioral responses to sucrose were restored to wild-type levels in transgenic rescue experiments; no role was observed for Gr64a in glucose response. One of these studies also provided biochemical evidence that Gr5a-related receptors are expressed in sugar-sensitive neurons (Dahanukar, 2007).

In summary, the simplest interpretation of the results is that Gr5a and Gr64a are the primary sugar receptors in the labellum of the adult fly. Each is capable of mediating response to a subset of sugars independently of the other, and together they are able to identify the food sources that are sufficiently rich in caloric value as to sustain the life of the fly (Dahanukar, 2007).

Taste-independent detection of the caloric content of sugar in Drosophila

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. This study shows that the fruit fly selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway (Dus, 2011).

This taste-independent sugar-sensing pathway has several distinctive characteristics. First, this pathway is specifically associated with a starved state; taste-blind flies execute food-choice behavior after prolonged food deprivation of between 10 and 15 h of starvation. This time frame coincides with the onset of starvation-induced sleep suppression, indicating that these two behaviors might share a common metabolic trigger. Second, the taste-independent pathway operates on a different timescale from the gustatory pathway. Whereas WT flies made a food choice almost instantly, taste-blind flies chose sugars only after the ingestion of food. Third, this pathway responds to the nutritional content of sugars, but not to their orosensory value. Taste-blind flies chose metabolizable sugars over nonmetabolizable sugars and never consumed nonmetabolizable sugars. Furthermore, the fact that WT flies failed to distinguish a metabolizable sugar from a nonmetabolizable sugar, but shifted their preference to the metabolizable sugar after starvation, indicates that the taste-independent pathway is not an artifact associated with taste-blind flies, but functions in WT flies. Finally, the ability to detect the caloric content of sugars correlated under multiple experimental conditions with drops in hemolymph glycemia (Dus, 2011).

These results demonstrate that starvation directs the selection of nutrient-rich foods in the fly in the absence of the gustatory cues. Thus, as previously suggested in mice, postingestive cues can drive feeding behavior independently of gustatory information. The physiological factors that triggered the taste-independent food choices in mice are, however, unknown. In Drosophila, the internal energy state and carbohydrate metabolism play crucial roles in the metabolic sensing of food according to the results. A possible evolutionary purpose of taste-independent metabolic sensing is to ensure that animals select calorie-rich foods to quickly replenish energy, especially in times of food shortage (Dus, 2011).

How do starved sugar-blind flies preferentially ingest metabolizable sugar over nonmetabolizable sugar? It is plausible that sugar-blind flies are equally attracted to and feed on both sugars, but those on nonmetabolizable sugar resume foraging because of the lack of nutritional value in this sugar. These foraging flies are again equally attracted to both sugars, but those on nonmetabolizable sugar continue to forage until they find the correct food substrate. Food choice in this model is mediated by random selection and 'trapping' of the flies on the metabolizable sugar. Alternatively, sugar-blind flies might readily detect the metabolizable sugar without ingesting a large amount of food because nutrient information is rapidly conveyed to the brain within minutes of ingesting food. In this model, the flies select for metabolizable sugar over nonmetabolizable sugar by a metabolic sensor that operates on a fast timescale to mediate discrimination between the two sugar substrates. Tracking and monitoring the locomotor activity and feeding behavior that generates a preference for metabolizable sugar will address this question (Dus, 2011).

It is intriguing to speculate on the molecular nature of the metabolic sensor. This sensor could be expressed in a subset of neural, digestive, or other tissues. Among the organs and cells that have been proposed for their involvement in feeding regulation in the fly are the fat body, the insulin-producing cells (IPC), and the corpora cardiaca/allata complex. These cells may respond to the metabolic value of sugars in circulation, as seen with the glucose-excited and glucose-inhibited neuropeptide neurons in the arcuate nucleus of the mammalian hypothalamus. A model that explains how changes in circulating glucose levels alter the electrical and secretory properties of the hypothalamic glucose-responsive neurons could also describe how metabolizable sugars trigger the metabolic sensor. In mammals, glucose-sensitive cells detect glucose availability by responding to metabolites of glycolytic enzymes such as hexokinase or the energy-sensing AMP-activated protein kinase (Dus, 2011).

Almost all crucial metabolic functions in mammals are also conserved in Drosophila. During the past decade, researchers using the fruit fly as a model system for studying feeding behaviors and feeding-related disorders, including obesity, have shed much light on the molecular mechanisms of metabolism. By revealing the possibility of a metabolic sensing pathway in Drosophila, this study has introduced the possibility of understanding the molecular mechanism underlying this pathway. Identification of the cellular and genetic nature of this sensor might reveal the identity of the master switch that regulates many hunger-driven behaviors (Dus, 2011).


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Dahanukar, A., Lei, Y. T., Kwon, J. Y. and Carlson, J. R. (2007). Two Gr genes underlie sugar reception in Drosophila. Neuron 56(3): 503-16. PubMed citation: 17988633

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date revised: 20 April 2012

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