InteractiveFly: GeneBrief

Ionotropic receptor 56d: Biological Overview | References

Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food prior to ingestion. While animals can discriminate between 1000's of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and innately attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, sweet taste neurons expressing the Ionotropic Receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Further, it was found that flies can discriminate between a fatty acid and a sugar in aversive memory assays, establishing a foundation to investigate the capacity of the Drosophila gustatory system to differentiate between various appetitive tastants. This study tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. The results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+GR64f+ neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. This study uncovers a capacity for the taste system to encode tastant identity within a taste category (Brown, 2021).

Previous studies identified IR56d as a receptor for hexanoic acid and carbonation (Ahn, 2017; Sanchez-Alcaniz, 2018). The current findings suggest that IR56d is selectively involved in responses to medium-chain fatty acids, including 6C, 7C, and 8C fatty acids, and dispensable for responses to shorter and longer-chain fatty acids. Such receptor specificity for different classes of fatty acids based on chain length has not been documented in other systems. In flies, both sugars and fatty acids evoke activity in neurons that co-express the receptors GR64f and IR56d. The finding that short- and long-chain fatty acids also evoke activity in IR56d-expressing neurons posits that additional fatty acid receptors are present in these neurons. Previously, it has been found that deletion of Phospholipase C (PLC) signaling selectively impairs fatty acid response while leaving sweet taste intact, raising the possibility that activation of distinct intracellular signaling pathways could serve as a mechanism for discrimination between sucrose and fatty acid, while another suggests TRPA1 and GR64e are targets of PLC and are generally required for fatty acid sensing. Determining whether or not short- and long-chain fatty acids also signal through PLC may provide insight into whether signaling mechanisms are shared between different fatty acid receptors expressed in IR56d-expressing neurons (Brown, 2021).

An aversive taste memory assay confirmed previous findings that flies can discriminate between sugars and fatty acids, and led to the surprising observation that flies can distinguish between different classes of fatty acids, even though the baseline responsiveness to short-, medium-, and long-chain fatty acids was similar in innate preference assays. Fatty acids are a natural by-product of yeast fermentation, and their abundance in peaches, for example, declines after ripening . Further, fatty acids have antifungal activity, which scales with chain length (i.e., the greater the chain length, the greater the antifungal efficiency). Thus, the ability to discriminate between different classes of fatty acids is likely to be important in determining the stage of fruit ripeness, degree of fermentation, and the general palatability of a potential food source/oviposition site (Brown, 2021).

The finding that flies can distinguish between different classes of fatty acids contrasts with the results of a previous study that applied a similar assay and found that flies were unable to discriminate between different sugars or bitter compounds. One possibility is that this is due to differences in fatty acid detection, which is dependent on IRs, and sweet and bitter tastant detection, which relies on GRs.The findings of this study highlight the complexity of taste discrimination, which extends beyond simple proboscis extension response (PER) as a readout for taste. For example, all types of fatty acids tested increase GR64f neural responsiveness; however, only GR64f neurons are required for PER to medium-chain fatty acids, thereby raising the possibility that short-, medium-, and long-chain fatty acid taste discrimination occurs through different neural channels. These findings stress the need to define the fatty acid receptors and neural circuits that govern responses to short- and long-chain fatty acid taste. Furthermore, the ability of the Drosophila taste system to discriminate suggests it may be more like the olfactory system than previously appreciated. Flies are able to distinguish between many different odorants, likely due to the complexity of olfactory coding at the level of the receptor as well as in the antennal lobe. However, flies can also discriminate between odorants sensed by a single olfactory receptor, suggesting that temporal coding also plays a role in discrimination. It is possible that similar mechanisms underlie discrimination between different classes of fatty acid tastants (Brown, 2021).

The Drosophila genome encodes 66 IRs, which comprise a recently identified family of receptors implicated in taste, olfaction, and temperature sensation. IRs are involved in the detection of many different tastants and function as heteromers that confer sensory specificity. While IR56d expression is restricted to a subset of sweet taste neurons, it likely functions in a complex with IR25a and IR76b, all three of which are required fatty acid taste. Other tastants whose responses are mediated by IRs are also likely to be detected by IR complexes. For example, roles for IR25a, IR62a, and IR76b have been described for Ca²⁺ taste. The broad degree of co-expression of IRs in the brain and periphery can provide candidates for those involved in detecting short- and long-chain fatty acids (Brown, 2021).

The identification of taste discrimination between different classes of fatty acids provides the opportunity to identify how different tastants are encoded in the brain and how these circuits are modified with experience. Although projections of primary taste neurons to the SEZ have been mapped in some detail, little is known about connectivity with downstream neurons and whether sensory neurons responsive to different appetitive tastants can activate different downstream circuits. Recent studies have identified a number of interneurons that modify feeding, including IN1, a cholinergic interneuron responsive to sucrose, E564 neurons that inhibit feeding, and Fdg neurons that are required for sucrose-induced feeding. Future work can investigate whether these and other downstream neurons are shared for fatty acid taste. Previous studies have found that incoming sensory information is selectively modulated within the SEZ in accordance with feeding state. It will be interesting to determine if similar modulation promotes differentiation of sugars and fatty acids, which are sensed by shared GRNs. Large-scale brain imaging has now been applied in flies to measure responsiveness to different tastants, and a comparison of brain activity patterns elicited by different classes of fatty acids may provide insight into differences in their sensory input and processing (Brown, 2021).

All experiments in this study tested flies under starved conditions, which is necessary to elicit the PER that is used as a behavioral readout of taste acceptance. However, responses to many tastants and odorants are altered in accordance with feeding state. For example, the taste of acetic acid is aversive to fed flies but attractive to starved flies, revealing a hunger-dependent switch. Similarly, hexanoic acid evokes activity in both sweet and bitter-sensing taste neurons, and the activity of bitter taste neurons is dependent on different receptors from those involved in the appetitive response. Further, hunger enhances activity in sweet taste circuits and suppresses that of bitter taste circuits, providing a mechanism for complex state-dependent modulation of taste response that increase activity of both appetitive and deterrent neurons (Brown, 2021).

The neural circuits that are required for aversive taste memory have been well defined for sugar, yet little is known about how fatty acid taste is conditioned. The pairing of sugar with bitter quinine results in aversive memory to sugar. Optogenetic activation of bitter taste neurons that are activated by quinine, in combination with the presentation of sugar, is sufficient to induce sugar avoidance. Further studies have elucidated that aversive taste memories are dependent on mushroom body neurons that form the gamma and alpha lobes, the PPL1 cluster of dopamine neurons, and alpha lobe output neurons, revealing a circuit regulating taste memory that differs from that controlling appetitive olfactory memory. It will be interesting to determine whether shared components regulate conditioning to fatty acids or whether distinct mushroom body circuits regulate sweet and fatty acid taste conditioning. Further, examination of the central brain circuits that regulate aversive taste conditioning to different classes of fatty acids will provide insight into how taste discrimination is processed within the brain (Brown, 2021).

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, it was revealed that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. This study characterize IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. It was further demonstrated that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. This work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour (Sanchez-Alcaniz, 2018).

IRs are best-characterised in Drosophila melanogaster, which possesses 60 intact Ir genes. Of these, the most thoroughly understood are the 17 receptors expressed in the adult antenna. Thirteen of these are expressed in discrete populations of sensory neurons, and function as olfactory receptors for volatile acids, aldehydes and amines or in humidity detection. The remaining four are expressed in multiple, distinct neuron populations and function, in various combinations, as co-receptors with the selectively-expressed tuning IRs (Sanchez-Alcaniz, 2018).

By contrast, little is known about the sensory functions of the remaining, large majority of non-antennal IRs. Previous analyses described the expression of transgenic reporters for subsets of these receptors in small groups of gustatory sensory neurons (GSNs) in several different contact chemosensory structures. While these observations strongly implicate these genes as having gustatory functions, the evidence linking specific taste ligands to particular receptors, neurons and behaviours remains sparse. For example, IR52c and IR52d are expressed in sexually-dimorphic populations of leg neurons and implicated in male courtship behaviours, although their ligands are unknown. Reporters for IR60b, IR94f and IR94h are co-expressed in pharyngeal GSNs that respond to sucrose, which may limit overfeeding or monitor the state of externally digested food. IR62a is essential for behavioural avoidance of high Ca2+ concentrations, but the precise neuronal expression of this receptor is unclear. As in the olfactory system, these selectively-expressed IRs are likely to function with the IR25a and/or IR76b co-receptors, which are broadly-expressed in contact chemosensory organs, and required for detection of multiple types of tastants, including polyamines, inorganic, carboxylic and amino acid and Ca2+ (Sanchez-Alcaniz, 2018).

This study describes a set of transgenic reporters for the entire Ir repertoire. These were used to survey the expression of this receptor family in both larval and adult stages. Using this molecular map, IR56d was identified as a selectively-expressed receptor that acts with IR25a and IR76b to mediate physiological and attractive behavioural responses to carbonation, a previously orphan taste class. Furthermore, this study extends recent studies to show that IR56d is also required in sugar-sensing GR neurons to mediate distinct behavioural responses to fatty acids (Sanchez-Alcaniz, 2018).

This work describes that non-antennal IRs function to detect a myriad of chemical stimuli to evoke a variety of behavioural responses. Such properties presumably apply to the vast, divergent IR repertoires of other insect species, for example, the 455 family members in the German cockroach Blatella germanica, or the 135 IRs in the mosquito Aedes aegypti. Within Drosophila no obvious relationships were detected between IR phylogeny and stage- or organ-specific expression patterns. Phylogenetic proximity may therefore be the most indicative of functional relationships between IRs, as is the case for those expressed in the antenna. If this hypothesis is correct, the expression data presented in this study suggest that functionally-related clades of receptors act in several types of chemosensory organ (Sanchez-Alcaniz, 2018).

An important caveat to the transgenic approach used to reveal expression is the faithfulness of these reporters to the endogenous expression pattern of Ir genes. Although this strategy has been widely (and successfully) used for antennal IRs and other chemosensory receptor families, it is impossible to determine reporter fidelity without a complementary tool (e.g. receptor-specific antibodies or tagging of the endogenous genomic locus). Discrepancies were noted between the expression of some of the Ir-Gal4 lines and those described previously; many of these probably reflect differences in the length of regulatory regions used to create these distinct transgenes. Precise comparison of independently-constructed transgenic constructs may in fact be useful in informing the location of enhancer elements directing particular temporal or spatial expression patterns. Moreover, transgenic reporters provide powerful genetic tools for visualisation and manipulation of specific neuronal populations. The reagents generated in this study should therefore provide a valuable resource for further exploration of the IRs in insect gustation (Sanchez-Alcaniz, 2018).

Using the atlas, IR56d—together with the broadly-expressed co-receptors IR25a and IR76b- were identified as essential for responses of labellar taste peg (Pit-like sensilla) neurons to carbonation. Such observations implicate IR56d as the previously unknown tuning receptor for this stimulus. However, these IRs do not appear to be sufficient for carbonation detection, as their misexpression in other neurons failed to confer sensitivity to carbonated stimuli. This observation suggests that additional molecules or cellular specialisations are required. Such a factor may be rather specific to taste pegs, given the minimal/absent responses of Ir56d-expressing taste bristle/leg neurons to carbonation, but does not appear to be another IR, as no other IR reporters were detected that expressed in this population of cells (Sanchez-Alcaniz, 2018).

While precise mechanistic insights into carbonation sensing will require the ability to reconstitute IR56d-dependent carbonation responses in heterologous systems, it is interesting to compare how insects and mammals detect this stimulus. The main mammalian gustatory carbonation sensor, the carbonic anhydrase Car4 is an enzyme tethered to the extracellular surface of sour (acid) taste receptor cells in lingual taste buds, where it is thought to catalyse the conversion of aqueous CO2 into hydrogencarbonate (bicarbonate) ions (HCO3-) and protons (H+). The resulting free protons, but not hydrogencarbonate ions, provide a relevant signal for the sour-sensing cells. By contrast, IR56d neurons are not responsive to low pH, suggesting a different chemical mechanism of carbonation detection. The observation that IR56d is also essential for sensitivity to hexanoic acid suggests that IR56d could recognise the common carboxyl group of hydrogencarbonate and fatty acid ligands. However, IR56d neurons are not responsive to all organic acids, indicating that this cannot be the only determinant of ligand recognition (Sanchez-Alcaniz, 2018).

Characterisation of IR56d neurons extends previous reports to reveal an unexpected complexity in the molecular and neuronal basis by which attractive taste stimuli are encoded. The taste bristle population of IR56d neurons represents a subset of sugar-sensing cells that are also responsive to fatty acids, glycerol and, minimally, to carbonation. Although activation of these neurons promotes PER, it was found that carbonation-evoked stimulation is insufficient to trigger this behaviour, which suggests that taste bristles are not a relevant sensory channel for this stimulus. While members of a specific clade of GRs are well-established to mediate responses to sugars and glycerol, the detection mechanisms of fatty acids appear to be more complex. Earlier work demonstrated an important role of a phospholipase C homologue (encoded by norpA) in labellar fatty acid responses. More recently, GR64e was implicated as a key transducer of fatty acid-dependent signals, but suggested to act downstream of NorpA, rather than as a direct fatty acid receptor. By contrast, an independent study of the legs showed that all sugar-sensing Gr genes (including Gr64e) were dispensable for fatty acid detection and provided evidence instead for an important role of IR25a and IR76b in these responses. Analysis of Ir56d mutants indicates an IR-dependent fatty acid-detection mechanism also exists in the labellum; future work will be needed to relate this to the roles of GR64e and NorpA (Sanchez-Alcaniz, 2018).

The IR56d taste peg population is, by contrast, sensitive to carbonation and fatty acids (but not sugars or glycerol), and these responses can be ascribed to IR56d (a Gr64eLexA reporter is not expressed in taste peg neurons). Although these neurons mediate taste-acceptance behaviour, they do not appear to promote proboscis extension or food ingestion. Recent work using optogenetic neuronal silencing experiments provided evidence that taste peg neuron activity is important for sustaining, rather than initiating, feeding on yeast, by controlling the number of sips an animal makes after proboscis extension. These observations are concordant with the internal location of taste pegs on the labellum, as they will not come into contact with food until the proboscis has been extended, and could explain the positional preference for carbonated substrates that were observed. This study has attempted to determine whether carbonation can influence sipping behaviour using flyPAD. Although these experiments did not reveal a statistically-significant effect, interpretation is complicated by the difficulty of providing and maintaining carbonation stimuli in the solid medium used in flyPAD assays. Future development of other approaches to provide this stimulus in feeding assays will be necessary. Nevertheless, the data strengthen the view that carbonation, a non-nutritious microbial fermentation product, regulates—via activation of IR56d taste peg neurons—a distinct motor programme to PER as part of a multicomponent behavioural response (Sanchez-Alcaniz, 2018).

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry. A broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. This study reports a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. Neurons identified by expression of Ionotropic Receptor 56d (IR56d) were found to be necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants (Tauber, 2017).

Sweet-sensing neurons in Drosophila have been broadly classified as those responding to sugars and other attractive tastants such as glycerol and amino acids. The findings presented in this study further localize the reflexive feeding response to hexanoic and octanoic acids, both medium-chain FAs, to a small population of FA-responsive taste neurons that partially overlap with sweet-sensing neurons. Previous work has shown that genetic silencing of most sweet-sensing neurons using Gr64f-GAL4 abolished FA response, suggesting that these neurons detect sugars and FAs. In flies, some subpopulations of Gr64f neurons are selectively sensitive to certain tastants including a Gr64e population that is sensitive to glycerol and a Gr5a subset that is sensitive to trehalose. To localize the Gr64f neurons responsible for FA taste, a targeted screen was conducted and neurons were silenced that are believed to overlap with Gr64f neurons, which led to a study of the IR56d population of neurons. Silencing IR56d neurons appears to selectively disrupt HxA response without affecting response to sucrose, supporting the notion that independent mechanisms within the Gr64f population mediate responses to sugars and FAs (Tauber, 2017).

It is possible that FA-sensitive neurons are broadly tuned to FAs or selectively respond to distinct classes of FAs. Ca2+ imaging experiments indicate that IR56d neurons are responsive to medium-chain HxA (C6, saturated) and octanoic acid (C8, saturated) in both anterior and posterior regions if the SEZ, and to short-chain pentanoic acid (C5, saturated), but only in the anterior projections. No IR56d neurons were found that were responsive to long-chain oleic acid (C18, mono-unsaturated). These findings are supported by behavioral data revealing that flies exhibit PER in response to pentanoic acid, HxA, and octanoic acid, but not oleic acid. Therefore, it seems likely that flies are strongly responsive to short/medium-chain FAs, but are less responsive to long-chain and/or unsaturated FAs. The finding that PER elicited by pentanoic acid occurs even when Ir56d neurons are genetically silenced suggests independent populations of taste neurons drive PER in response to short-chain and medium-chain FAs. Further, IR56d neurons may be activated by long-chain FAs that were not tested, and these could modulate feeding response and induce PER. Nevertheless, the findings presented in this study reveal specificity for medium-chain FAs within a defined population of taste neurons (Tauber, 2017).

Many of the neurons identified by IR56d expression express multiple taste receptors including IR56d, Gr64f and Gr5a. These neurons likely express many additional candidate taste receptors, and future studies are needed to identify the receptor(s) that are responsive to FAs. IRs are related to ionotropic Glutamate receptors and respond to diverse tastants and odorants, making them excellent candidates for detecting FAs. While IR56d remains an excellent candidate, it will be necessary to examine potential IR co-receptors that may be critical for IR-dependent sensation. For example, IR25a is relatively broadly expressed and likely functions as a co-receptor for numerous IR-dependent sensory processes including temperature sensing and hygrosensation. It is possible that multiple IRs are required for FA taste, with some acting as co-receptors and others detecting specific FAs. While future work is required to identify the molecular bases for FA taste, the identification of FA sensitivity in IR56d neurons provides a system to interrogate the cellular mechanisms of FA taste (Tauber, 2017).

The PER response induced by two different medium-chain FAs, hexanoic and octanoic acids, suggests they may be part of Drosophila melanogaster diet. Typical dietary fats, including many plant based oils, such as coconut oil, are rich in longer-chain FAs including palmitic acid, oleic acid and linoleic acid. However, medium-chain FAs are present in fermenting fruits such as guava and also in pollen. Moreover, the medium-chain FAs (mostly C6-C10) are excreted by yeast during fermentation, possibly helping with finding yeast-rich feeding substrates, raising the possibility that flies have developed a response to FAs in order to locate suitable fermented food sources. Further, previous work has shown that a diet of HxA alone is sufficient for survival. Therefore, it is possible that FA attraction evolved to promote consumption of calorically rich fermenting fruits consumed by Drosophila (Tauber, 2017).

The use of sucrose and hexanoic acid (HxA) in an aversive taste memory paradigm reveals flies can discriminate between these attractive tastants. Sugars induce broad activation of Gr64f neurons, while the activation induced by HxA appears more restricted, and therefore it is possible that differences in activation allow for differentiation. Alternatively, it was found that HxA also activates anterior-projecting IR56d neurons that emanate from the taste pegs and do not co-express Gr64f, raising the possibility that differential response of these neurons to sucrose and FAs allows discrimination. Considering the different biochemical pathways involved in sugar and FA sensing, their identity may also be coded by unique temporal and spatial dynamics of sensory neuron activation. Differences in activation are suggested to provide a mechanism for olfactory discrimination within defined neural populations, and it is possible that similar mechanisms are utilized for attractive tastants. In Drosophila, attractive tastants have been found to induce a wide range of excitatory responses ranging from acute to prolonged firing, providing a potential mechanism for discrimination. While the sensillar response to FAs has not been reported, the differences in Ca2+ response to sugar or HxA presentation within the SEZ suggest differences in temporal activation (Tauber, 2017).

The findings of this study reveal the population of IR56d neurons that innervate the anterior SEZ, which emanate from the taste pegs, are dispensable for PER in response to FAs. However, it is possible these neurons are still involved in discrimination between FAs and sugars. These neurons are not responsive to sucrose, therefore distinct anatomical activation may account for the gustatory discrimination between attractive substances. The taste pegs have previously been implicated in sensing non-sugar attractive tastants including polyamines and carbonation, raising the possibility that these neurons are responsive to multiple taste modalities (Hassain 2016; Fischler, 2007). Selectively silencing the IR56d taste peg neurons and measuring discrimination between FAs and sugars may determine whether distinct classes of IR56d neurons mediate taste feeding response and taste discrimination (Tauber, 2017).

This study found that flies can discriminate between sugars and FAs, but it is not known whether they can discriminate qualitatively between different classes of FAs. A previous study examining discrimination between different sugars found that flies are unable to discriminate based on quality, but could discriminate based on perceived palatability. This study found that pentanoic acid elicits a PER response that is independent of IR56d neurons. The findings, coupled with evidence that distinct populations of neurons respond to FAs from different classes, raise the possibility that flies may discriminate between FAs based on the identity of neurons activated by each FA, or classes of FAs (Tauber, 2017).

We previously reported that PLC signaling in sweet-sensing Gr64f neurons is required for FA taste (Masek, 201). Flies with mutation or knockdown of the PLC-β ortholog norpA do not respond to HxA or octanoic acid but respond normally to sugars, revealing independent intercellular signaling mechanisms likely underlie response to FAs and sugars. This study found that knockdown of norpA in IR56d neurons abolishes FA taste without disrupting the taste of sucrose. These findings phenocopy norpA mutants and broad knockdown of norpA in Gr64f neurons, fortifying the notion that PLC signaling is selectively required for FA taste. Testing the response of norpA deficient flies to FAs that are sensed independently of IR56d will inform whether PLC is more generally required for FA taste, or is only specific to medium-chain FAs detected by IR56d neurons (Tauber, 2017).

While taste coding within the SEZ has been extensively investigated, less is known about the higher order circuits governing taste. Sweet-sensing neurons connect to the antennal mechanosensory and motor center (AMMC) and downstream PAM dopamine neurons that are activated by sugar. In addition, a separate population of dopamine neurons, the PPL1 cluster, is required for olfactory appetitive memory and taste aversive conditioning. To date, higher order neurons responsive to FA taste have not been identified. It is possible that sugar and FA taste signal through shared higher order dopamine neurons or, alternatively, each taste modality may activate distinct populations of higher order neurons that convey valence to the mushroom bodies, the memory and sensory integration center in insects (Tauber, 2017).

While both sugars and FAs activate shared neurons, the ability to discriminate between these tastants provides a model for investigating sensory discrimination. There is growing evidence of multimodal coding within Drosophila sensory neurons, and in downstream targets. Flies harboring only a single functional type of olfactory receptor neurons are able to discriminate between odorants, presumably due to differences in temporal activation between the odorants. Further, in the larval taste system, a single gustatory receptor neuron is responsive to both attractive and aversive compounds, and mediates the integration of these competing stimuli. In addition to integration of distinct cues by the sensory system, the Drosophila mushroom bodies, and courtship circuitry integrate complex sensory cues within the brain. Future studies on how the central brain processes sugar and FA taste will help elucidate mechanisms of discrimination between sugars and FAs (Tauber, 2017).

Despite the role of FAs in promoting feeding, surprisingly little is known about how FAs promote taste in any model system. Fats contain many sensory cues and separating the taste of fat per se, from other cues such as texture, viscosity and smell is a particular challenge in mammals. A number of studies have implicated the lipid binding protein CD36 as contributing to FA taste. CD36 is expressed in gustatory oral tissue, and appears to be selectively involved in FA taste. CD36 knockout animals show no preference for FAs but retain preference for sugars. The Drosophila homolog of CD36, Sensory neuron membrane protein 1, is expressed in the olfactory system and required for sensation of the pheromone cis-vaccenyl acetate, and therefore is unlikely to mediate FA taste. Additionally, a number of FA-binding GPCRs are expressed in taste cells, but their role in FA taste has not been identified. The ability to selectively manipulate and ablate defined classes of sensory neurons in Drosophila allows for the disambiguation of taste from other sensory processes. Identifying FA receptors and neural circuitry mediating FA taste and discrimination will provide a framework for investigating similar processes in mammalian systems (Tauber, 2017).

Taken together, this study provides insight into the coding of FAs within the fly gustatory system. The results reveal a population of sweet-sensing neurons that are tuned for medium-chain FAs, but not short- or long-chain FAs. Further, the finding that flies are capable of discriminating between FAs and sugars suggests coding differences, either spatial or temporal neuronal activation, and provides a mechanism to distinguish between tastants of the same valence. Understanding how FAs are coded within the fly brain provides a model for understanding taste in more complex systems and will offer insight into generalizable mechanisms for taste discrimination (Tauber, 2017).

Molecular basis of fatty acid taste in Drosophila

Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). This study shows that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With previous report on sour taste, these studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits (Ahn, 2017).

IR genes have emerged as a second large gene family encoding chemoreceptors in insects. In the Drosophila olfactory system, IRs function as multimeric receptors in coeloconic olfactory sensory neurons (OSN) and are thought to sense volatile carboxylic acids, amines and aldehydes. Expression analyses have shown that each coeloconic OSN expresses up to four IR genes, including high levels of either IR8a or IR25a. IR25a and IR8a are distinct from other IRs in that they are more conserved to each other and iGluRs, and they share a long amino terminal domain absent in all other IRs. These observations, along with functional analyses of basiconic olfactory neurons that express combinations of IR genes, led to a model in which IR based olfactory receptors are tetrameric complexes thought to consist of up to three different subunits that contain at least one core unit and two additional IRs that determine ligand binding specificity. The findings presented in this paper expand this concept to taste receptors that sense fatty acids through the sweet GRNs found in tarsal taste sensilla (Ahn, 2017).

This analysis extends the multimodal role of IR25a and IR76b to the taste systems. Consistent with gene expression arrays, this study showed that up to three GRNs, including many sweet and bitter GRNs, co-express IR25a and IR76b. Functional studies have established a novel role for these two IR proteins in fatty acid taste, which revealed that these two subunits are not only critically important to elicit PER responses in flies when challenged with fatty acids, but are also necessary for fatty acid induced Ca2+ increases in tarsal sweet GRNs. Based on these findings and with consideration of their established role in other sensory systems, it is proposed that IR25a and IR76b play central roles in sweet GRNs in a multimeric receptor complex for initiating appetitive taste behavior to these chemicals. Intriguingly, both genes are also co-expressed in  two other GRNs of most tarsal taste sensilla, strongly arguing for additional taste functions. While the subset of tarsal bitter GRNs activated by hexanoic acid does not require either gene, this study has discovered that the third GRN (the sour GRN) is narrowly tuned to acids in an IR25a/IR76b dependent manner. These observations suggest that modality specific IRs are likely expressed in a cell-type specific fashion whereby they complement IR25a/IR76b to function as either a fatty acid or a sour taste receptor. Indeed, the screen identified IR56d, a gene that is expressed in sweet GRNs of tarsal taste sensilla, as a likely candidate encoding an IR subunit specific for a fatty acid taste receptor. It remains to be seen whether IR25a, IR76b and IR56d comprise all subunits that constitute this receptor or whether yet additional IRs are necessary to mediate responses to these chemicals (Ahn, 2017).

The fact that different food chemicals can activate a single class of neurons raises the question how flies discriminate between sugars and fatty acids. First, it is noted the difference in sensitivity of appetitive GRNs to sugars and fatty acids, respectively: The most responsive GRN for sugars is the one associated with the 5v sensilla, followed by that with the 5s and finally the 5b sensilla, while the responsiveness for fatty acids is the reverse (5b > 5s > 5v). Second, fatty acids induces weaker PER responses from stimulation of the labial palps as opposed to tarsi, while sugars induce equally strong PER responses from stimulation of either taste organ. Third, at least some fatty acids activate bitter GRNs, and hence, generate more complex activation patterns in the brain than sugars, which are not known to activate neurons other than sweet GRNs. These properties may provide a rationale for differential coding of these two classes of chemicals in the brain. Finally, sugars but not fatty acids are soluble in water, and hence, the specific solvents in which these chemicals are presented provides different textural quality, which was recently shown to play a role in taste perception (Ahn, 2017).

NorpA, which encodes a phospholipase C (PLC), plays a critical role in sweet GRNs for appetitive feeding responses to fatty acids, but it is dispensable for behavioral responses to sugars. This study found that its absence also selectively abolishes Ca2+ responses to fatty acids, but not sugars, in sweet cells. NorpA is known for its role as downstream effector of G-protein coupled receptors in the fly's visual system, but interestingly it is also required for olfactory responses in neurons of the maxillary palps, which express ORs that are thought to function as ligand-gated ion channels. It is noted that fatty acid taste in mammals is in part mediated by two G – protein coupled receptors, GPR40 and GPR120, and that one of these (GPR120) was found to signal through a phospholipase C. Thus, future studies will be necessary to gain insights for how PLC mediates chemosensory responses through ORs and the phylogenetically unrelated IRs (Ahn, 2017).

Multimeric IR based receptors were recently shown to be required in non-chemosensory processes. Specifically, Dorsal Organ Cool Cells (DOCCs) located in the larval brain, express and require the function of three IRs (IR21a IR25a and IR93a), thereby allowing larvae to avoid temperatures below ~20°C. Similarly, two sets of cells in the antennal sacculus of adult flies, requiring the functions of IR25a and IR93a and either IR40a or IR68a, were shown to mediate a fly's preferred humidity environment, which is generally in the dry range, but is also dependent on the fly's hydration state. Intriguingly, these non-chemosensory IR complexes share a common theme with the fatty acid and carboxylic acid taste receptors in that they all require a core unit (IR25a) and two additional IRs that mediate specificity for a particular stimulus type (i.e. temperature, humidity, fat, acid) (Ahn, 2017).

An IR76b based sodium channel and an IR76b based amino acid receptor appear to lack an obligate core unit (IR25a or IR8a) found in olfactory receptors or fatty acid and carboxylic acid taste receptors. The IR76b sodium channel mediates salt responses in a heterologous systems independently of any other IRs, while a proposed multimeric IR76b containing receptor mediates amino acids taste in wild type and IR25a mutant flies (IR8a is not expressed in taste neurons). It will be interesting to elucidate the compositions of complete IR based amino acid and sour taste receptors, and – with regard of amino acid receptors – to identify the neurons that mediate this taste modality (Ahn, 2017).

Search PubMed for articles about Drosophila Ir56d

Ahn, J. E., Chen, Y. and Amrein, H. (2017). Molecular basis of fatty acid taste in Drosophila. Elife 6. PubMed ID: 29231818

Brown, E. B., Shah, K. D., Palermo, J., Dey, M., Dahanukar, A. and Keene, A. C. (2021). Ir56d-dependent fatty acid responses in Drosophila uncovers taste discrimination between different classes of fatty acids. Elife 10. PubMed ID: 33949306

Fischler, W., Kong, P., Marella, S. and Scott, K. (2007). The detection of carbonation by the Drosophila gustatory system. Nature 448(7157): 1054-1057. PubMed ID: 17728758

Hussain, A., Zhang, M., Ucpunar, H. K., Svensson, T., Quillery, E., Gompel, N., Ignell, R. and Grunwald Kadow, I. C. (2016). Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines. PLoS Biol 14(5): e1002454. PubMed ID: 27145030

Masek, P. and Keene, A. C. (2013). Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons. PLoS Genet 9(9): e1003710. PubMed ID: 24068941

Sanchez-Alcaniz, J. A., Silbering, A. F., Croset, V., Zappia, G., Sivasubramaniam, A. K., Abuin, L., Sahai, S. Y., Munch, D., Steck, K., Auer, T. O., Cruchet, S., Neagu-Maier, G. L., Sprecher, S. G., Ribeiro, C., Yapici, N. and Benton, R. (2018). An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing. Nat Commun 9(1): 4252. PubMed ID: 30315166

Tauber, J. M., Brown, E. B., Li, Y., Yurgel, M. E., Masek, P. and Keene, A. C. (2017). A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste. PLoS Genet 13(11): e1007059. PubMed ID: 29121639

date revised: 14 May 2022

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