Understanding sensory systems that perceive environmental inputs and neural circuits that select appropriate motor outputs is essential for studying how organisms modulate behavior and make decisions necessary for survival. Drosophila melanogaster oviposition is one such important behavior, in which females evaluate their environment and choose to lay eggs on substrates they may find aversive in other contexts. This study employed neurogenetic techniques to characterize neurons that influence the choice between repulsive positional and attractive egg-laying responses toward the bitter-tasting compound lobeline. Surprisingly, it was found that neurons expressing Gr66a, a gustatory receptor normally involved in avoidance behaviors, receive input for both attractive and aversive preferences. It was hypothesized that these opposing responses may result from activation of distinct Gr66a-expressing neurons. Using tissue-specific rescue experiments, it was found that Gr66a-expressing neurons on the legs mediate positional aversion. In contrast, pharyngeal taste cells mediate the egg-laying attraction to lobeline, as determined by analysis of mosaic flies in which subsets of Gr66a neurons were silenced. Finally, inactivating mushroom body neurons disrupted both aversive and attractive responses, suggesting that this brain structure is a candidate integration center for decision-making during Drosophila oviposition. This study thus defines sensory and central neurons critical to the process by which flies decide where to lay an egg. Furthermore, the findings provide insights into the complex nature of gustatory perception in Drosophila. Tissue-specific activation of bitter-sensing Gr66a neurons provides one mechanism by which the gustatory system differentially encodes aversive and attractive responses, allowing the female fly to modulate her behavior in a context-dependent manner (Joseph, 2012).

Proper perception of the environment is essential for an organism to modulate its behavior and make choices necessary to both survival of individuals and propagation of the species. In Drosophila melanogaster, the selection of appropriate oviposition sites that will benefit survival of the progeny is one such behavior. Recent studies have demonstrated that during egg-laying site selection female fruit flies actively explore the different options available before choosing where to lay their eggs (Yang, 2008; Miller, 2011; Schwartz, 2012). Interestingly, females do not always remain on the substrate where they have deposited their eggs and will often choose to lay eggs on substrates they normally find aversive for foraging and feeding. Since a fly cannot be in two places at once, a choice must be made between these competing preference pathways. Thus, with regard to oviposition behavior, a decision is defined as the selection between one of two mutually exclusive responses: (1) avoid the substrate and hold eggs, or (2) choose the substrate in order to lay eggs. Taken together, these findings suggest that during oviposition, female Drosophila employ an evaluation process that meets the criteria of simple decision-making (Joseph, 2012).

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).

Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. The expression of eight Gr genes in the labial palps, the fly's main taste organ, has been characterized in detail. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities -- the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. It is concluded that iscrimination between two taste modalities -- sweet and bitter -- requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons (Thorne, 2004).

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

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

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

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

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

A crucial determinant for discerning chemical cues present in the environment is embedded in the peripheral expression pattern of cell surface receptors in sensory epithelia. In the olfactory systems of Drosophila and mice, each olfactory receptor neuron expresses only one of 60 or one of approximately 1000 Or genes, respectively, enabling these animals to discriminate between hundreds or thousands of different odors. In contrast, taste cells of the tongue allow mammals to distinguish only a few taste qualities: bitter, sweet, umami, salty, and acidic taste. Lack of discrimination between the hundreds of diverse chemical compounds -- all perceived as bitter -- is thought to be caused by coexpression of the approximately 40 T2R receptors in a single set of taste cells. 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. (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. 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. 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). 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).

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. 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). 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).