InteractiveFly: GeneBrief

Ionotropic receptor 64a: Biological Overview | References

BIOLOGICAL OVERVIEW

The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating. How acidity is sensed and translated into an appropriate behavioural response is poorly understood. This study describes a functionally segregated population of olfactory sensory neurons in Drosophila, that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors (Benton, 2009). In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery (Ai, 2010).

Many aversive odorants activate combinations of olfactory sensory neurons (OSNs) (Ng, 2002; Wang, 2003), complicating the dissection of the circuits that translate odour recognition into behaviour. By contrast, carbon dioxide (CO₂), an odorant that is salient for many insect behaviours, activates a single population of dedicated sensory neurons expressing the gustatory receptors GR21a and GR63a (see Gustatory receptor 21a and Gustatory receptor 63a). These neurons are essential for mediating avoidance behaviour of Drosophila to CO₂ at concentrations lower than about 2%. However, this study found that flies in which GR21a/GR63a+ neurons were inactivated still avoided CO₂ at concentrations higher than about 5%. Avoidance of high CO₂ concentrations required the antennae, indicating that another population of antennal neurons mediates avoidance to high CO₂ concentrations (Ai, 2010).

To identify these sensory neurons, a functional screen was performed for neurons required for responsiveness to CO₂ by crossing a collection of GAL4 enhancer traps to UAS-Shibire^ts. A line, GC16-GAL4, was isolated that failed to avoid 1% and 5% CO₂. GC16-GAL4 is expressed in OSNs that project to the V glomerulus among others, which is consistent with its defect in avoidance to 1% CO₂. To test whether other glomeruli labelled by GC16-GAL4 besides V are activated by CO₂, in vivo calcium imaging was conducted. Using this approach, an additional pair of dorsal glomeruli, termed DC4 were identified, that were activated by about 5% CO₂ (Ai, 2010).

Because CO₂, when dissolved in the lymph fluid inside the antennal sensilla that harbour OSNs, can generate metabolites, such as carbonic acid and bicarbonate ions, tests were performed to see whether DC4 could be activated by CO₂ metabolites. DC4 was stimulated by carbonic acid but not by bicarbonate, suggesting that these neurons detect acidosis produced by increased CO₂ concentrations, rather than CO₂ itself (Ai, 2010).

Axonal projections to DC4 originate from a population of OSNs that reside in coeloconic sensilla and express neither insect odorant receptors nor gustatory receptors. Instead, it was found that these neurons express a novel receptor, IR64a, a member of the chemosensory ionotropic glutamate receptor family (Benton, 2009). The IR64a promoter, IR64a-GAL4, driving UAS-CD8GFP, labelled the DC4 glomerulus and another glomerulus, DP1m. Anti-IR64a immunohistochemistry demonstrated that the IR64a-GAL4 driver recapitulated the endogenous IR64a expression. About 16±0.9 IR64a+ cells were detected surrounding the third chamber of the sacculus (Shanbhag, 1995), which is a three-chamber pit organ that opens to the posterior surface of the antenna. These IR64a+ cells send their dendrites to grooved sensilla that project to the interior of the sacculus (Ai, 2010).

Because IR64a+ neurons project to the DC4 and DP1m glomeruli, it was determined whether only DC4, or both DC4 and DP1m, were activated by acids by calcium imaging on flies carrying IR64a-GAL4 and UAS-GCaMP. All acids examined, but not non-acidic odorants, activated DC4. In contrast, DP1m was activated by acidic and non-acidic odorants. It was asked whether DP1m and DC4 might be activated by the functional side chains of some organic acids, rather than by the protons. Therefore tests were performed to see whether inorganic acids such as hydrochloric acid (HCl) and nitric acid (HNO₃), which dissociate completely in water and generate protons without an organic moiety, could activate DP1m and DC4. These inorganic acids, probably free protons in water vapour, activated DC4 in a dosage-dependent manner but did not activate DP1m. This is consistent with the observation that only DC4 is activated by CO₂, which contains no associated side chains. Furthermore, the strength of the DC4 activation was inversely correlated with the pH of one odorant, sodium acetate. These results demonstrate that the neurons projecting to the DC4 glomerulus are highly specific for the detection of acidity (Ai, 2010).

To determine whether the IR64a gene is required for acid detection, a mutation (IR64a^mi) was obtained with a transposable Minos element (Franz, 1994) inserted into the third intron of the IR64a locus. Flies homozygous for the IR64a^mi allele had significantly decreased IR64a messenger RNA transcript and IR64a protein in the antennae compared with wild-type flies. IR64a^mi is therefore a strong loss-of-function mutation. This mutation abrogated glomerular activation of DC4 by acids. IR64a^mi also attenuated the activation of DP1m to acidic and non-acidic odorants. Two different IR64a transgenes -- the genomic IR64a-HA (where HA stands for haemagglutinin) driven by its own regulatory elements and a UAS-IR64a complementary DNA driven by IR64a-GAL4 -- rescued the odour-sensing defects of IR64a^mi mutants. These results demonstrated that IR64a has a cell autonomous function as a component of the acid-sensing machinery required for DC4 activation and the machinery through which other odorants activate DP1m (Ai, 2010).

IR64a protein is localized in the cell bodies and dendrites but not in axonal processes in the antennal lobe. It is highly enriched in the tip of the dendritic terminals that innervate coeloconic sensilla protruding into the lumen of the third chamber of the sacculus. The subcellular localization of IR64a is consistent with its direct involvement in acid detection. To determine the role of IR64a as a putative acid receptor, IR64a was ectopically expressed in another population of sensory neurons that is normally insensitive to acids, and it was asked whether IR64a is capable of conferring acid sensitivity in these neurons. IR64a was expressed by using the IR76a-GAL4 driver (Benton, 2009), which is expressed in coeloconic sensory neurons that project to the VM4 glomerulus in the antennal lobe. Calcium imaging experiments showed that ectopic expression of IR64a induces odour sensitivity in VM4 to organic acids and octan-3-ol, which normally activate DP1m, substantiating the notion that IR64a is the direct determinant in odour detection. However, IR64a alone was not capable of conferring sensitivity to DC4-specific stimuli such as inorganic acids or CO₂. This result suggests that although IR64a alone can induce responsiveness to several odorants that activate DP1m, it probably requires a co-receptor in DC4 neurons to mediate the specificity to acidity (Ai, 2010).

Having shown that IR64a is part of the acid-sensing machinery, it was next determined whether IR64a+ neurons are necessary for the flies' behavioural response to acids. Flies were engineered in which IR64a+ cells were silenced by the targeted expression of tetanus toxin (TNT). In a T-maze, these flies showed significant decreases in avoidance to several acids, whereas responses to non-acidic odorants such as benzaldehyde and octan-3-ol were unaffected. However, this experiment could not determine whether it is DC4 or DP1m that is required for acid avoidance, because IR64a-GAL4 is expressed in both populations of sensory neurons. Nonetheless, DP1m is unlikely to be important because it is not activated by acidity. To confirm the importance of DC4 in acid avoidance, a GAL80 transgene was generated under the control of the IR64a promoter and was crossed to flies carrying GC16-GAL4 and UAS-Shibire^ts. Because GC16-GAL4 is expressed in DC4 neurons, but not in DP1m neurons, the IR64a-GAL80 transgene selectively relieves neuronal inhibition only in DC4 neurons. It was confirmed that IR64a-GAL80 suppresses GC16-GAL4 activity only in DC4 neurons by using a UAS-CD8GFP transgene. The IR64a-GAL80 transgene rescued the behavioural defects of flies carrying GC16-GAL4 and UAS-Shibire^ts, supporting the specific role of DC4 in mediating behavioural responses to acids (Ai, 2010).

Next, whether artificial activation of IR64a+ neurons is sufficient to trigger avoidance responses was examined. GR63a¹;IR64a^mi double mutants were generated expressing the CO₂ receptors UAS-GR21a and UAS-GR63a, and a calcium-sensitive GFP, UAS-GCaMP, by using the IR64a-GAL4 driver. Expression of the two CO₂ receptors in CO₂-insensitive sensory neurons was previously shown to be sufficient to confer ectopic sensitivity to CO₂. Indeed, the DC4 glomerulus in these flies was artificially stimulated by 5% CO₂. However, the activation of DP1m by CO₂ could not be detected in these flies, possibly because GR21a and GR63a receptors do not function properly in DP1m neurons. Behavioural experiments demonstrated that GR63a¹;IR64a^mi double mutant flies failed to distinguish ambient air from 5% CO₂. However, the CO₂-blind flies with CO₂ receptors expressed in IR64a+ neurons showed robust avoidance to CO₂. This suggests that avoidance behaviour is hardwired into the olfactory circuitry that detects acidity. Because DP1m in these flies does not seem to be activated by CO₂, it was reasoned that activation of DC4 neurons alone is sufficient for generating avoidance responses. These data, together with the observation that acidity evoked calcium responses only in DC4, firmly establish that IR64a+ neurons projecting to the DC4 glomerulus are necessary for acid sensation and sufficient for avoidance behaviour. These results provide strong evidence for the functional segregation of acid sensing at the periphery that drives innate avoidance behaviour (Ai, 2010).

Consistent with the physiological defects was the observation that IR64a^mi flies had impaired avoidance to acids but normal responses to an unrelated odorant. Conversely, flies in which the Minos element was precisely excised from the IR64a locus (IR64a revertants) and those carrying an IR64a transgene in the IR64a^mi mutant background showed robust avoidance to acids. Although the average avoidance indices of IR64a^mi and IR64a-GAL4 --> UAS-TNT flies were significantly different from those of the wild type, they still showed moderate avoidance responses to acids (avoidance index of 20%-25%). This residual response is unlikely to be mediated by the olfactory system, because flies lacking antennae had avoidance responses similar to those of IR64a^mi. Thus, additional acid sensors probably exist elsewhere in the fly (Ai, 2010).

Fruit flies are often called 'vinegar flies' because of their attraction to vinegar. Indeed, flies were attracted to certain concentrations of vinegar in a T-maze. However, a major ingredient of vinegar is acetic acid, which flies avoid. It is possible that flies are not repelled by vinegar because other constituents in vinegar inhibit DC4 activation by acetic acid. Alternatively, constituents other than acetic acid in vinegar might elicit an attraction response that overrides DC4-mediated avoidance. To distinguish between these possibilities, in vivo calcium imaging was performed to measure the activation of DC4 after exposure to vinegar. Apple cider vinegar (ACV), which contains about 5% acetic acid, activated DC4 as effectively as pure 5% acetic acid. These results suggest that vinegar contains attractants capable of overcoming DC4-mediated avoidance by activating other olfactory receptors. It is predicted that neutralized vinegar would not activate DC4 and should be more attractive to flies because it still contains attractants. Consistent with this prediction, calcium imaging showed that DC4 was not stimulated by neutralized vinegar. Moreover, wild-type flies avoided a high concentration of vinegar in a T-maze but became attracted to neutralized vinegar at the same concentration. A similar behavioural switch was observed in other D. melanogaster strains such as Berlin and OregonR, and with another type of vinegar. Furthermore, flies were attracted to 25% ACV, but not to acetic acid that had been diluted to the same concentration of acidity. This further supports the model that flies are attracted to components other than acid in vinegar. Avoidance of vinegar requires a functional IR64a+ circuit, because IR64a^mi mutants were equally attracted to vinegar and to neutralized vinegar (Ai, 2010).

Animals across various phyla show innate aversion to a plume of acid, often emanating from spoiled food or unripe fruit. This characterization of Drosophila IR64a provides a cellular and molecular mechanism that can explain the distinct olfactory sensation of acidity. In the mammalian taste system, acid detection is mediated by a unique cell type, independently of other taste modalities (Huang, 2006). This labelled-line organization is similar to those of acid and CO2 receptors in the fly olfactory system. Both acid and CO2 sensors are highly specific to their ligands and mediate similar avoidance behaviour. This raises a further question: where are these two similar aversive stimuli represented in the brain? The identification of neural substrates in the central nervous system mediating acid and CO2 sensation will facilitate future mapping of the avoidance circuitry (Ai, 2010).

Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

Ionotropic glutamate receptors mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. This paper characterizes a family of iGluR-related genes in Drosophila, which have been named ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead to the proposal that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues (Benton, 2009).

Species as diverse as bacteria, plants, and humans have the capacity to sense small molecules in the environment. Chemical cues can transmit the presence of food, alarm signals, and messages from conspecifics that signify mating compatibility. Peripheral chemical recognition largely relies on membrane receptor proteins that interact with external ligands and convert this binding into intracellular responses. The vast majority of identified chemosensory receptors in multicellular organisms belong to the seven transmembrane domain G protein-coupled receptor (GPCR) superfamily, including odorant, gustatory and pheromone receptors in mammals, birds, reptiles, amphibians, fish, and nematodes. Unicellular organisms also use GPCRs for chemoreception, such as the pheromone receptors in budding yeast (Benton, 2009).

Insects can detect a wide range of environmental chemicals: bitter, sweet, and salty tastants, odors, pheromones, humidity, carbon dioxide, and carbonated water. Most of these chemosensory stimuli are recognized by members of two evolutionarily related insect-specific chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs). These proteins contain seven predicted transmembrane domains but are evolutionarily unrelated to GPCRs and adopt a distinct membrane topology. Recent analysis has indicated that insect ORs function as odor-gated ion channels (Sato, 2008; Wicher, 2008), setting them mechanistically apart from metabotropic vertebrate ORs (Benton, 2009).

Comprehensive analysis of the expression of these receptors in Drosophila, has hinted at the existence of other types of insect chemosensory receptors (Couto, 2005; Yao, 2005). This is particularly apparent in the major olfactory organ, the third segment of the antenna, which bears three types of olfactory sensory hairs (sensilla): basiconic, trichoid, and coeloconic. All olfactory sensory neurons (OSNs) innervating basiconic and trichoid sensilla generally express one OR, along with the OR83b co-receptor. However, with the exception of OR35a/OR83b-expressing neurons (Yao, 2005), OSNs housed in coeloconic sensilla do not express OR83b or members of the OR or GR gene families. Nevertheless, electrophysiological analysis has revealed the existence of multiple types of coeloconic OSNs tuned to acids, ammonia and humidity (Yao, 2005), suggesting that other types of insect chemosensory receptors exist (Benton, 2009).

A bioinformatic screen has been carried out for insect-specific genes enriched in OSNs (Benton, 2007). Among these, a large expansion was found of the ionotropic glutamate receptor (iGluR) gene family of unknown biological function (Littleton, 2000). This study provides evidence that these variant iGluRs represent a novel class of chemosensory receptor (Benton, 2009).

The screen identified 6 antennal-expressed genes encoding proteins annotated as ionotropic glutamate receptors (iGluRs) (Littleton, 2000). Using these novel receptor sequences as queries, exhaustive BLAST searches of the Drosophila genome identified a family of 61 predicted genes and 1 pseudogene. These genes are distributed throughout the genome, both as individual sequences and in tandem arrays of up to four genes. This family was named the Ionotropic Receptors (IRs), and individual gene names were assigned to the IRs using nomenclature conventions of Drosophila ORs (Benton, 2009).

Phylogenetic analysis of predicted IR protein sequences revealed that they are not closely related to members of the canonical families of iGluRs (AMPA, kainate, NMDA, or delta). However, they appear to have a similar modular organization to iGluRs, comprising an extracellular N-terminus, a bipartite ligand-binding domain, whose two lobes (S1 and S2) are separated by an ion channel domain, and a short cytoplasmic C-terminus. It is noted that the gene structure and protein sequence of most receptors are presently only computational predictions. Nevertheless, the family is extremely divergent, exhibiting overall amino acid sequence identity of 10-70%. The most conserved region between IRs and iGluRs spans the ion channel pore, suggesting that IRs retain ion-conducting properties (Benton, 2009).

The ligand-binding domains are considerably more variable, although alignment of small regions of the S1 and S2 lobes of IRs and iGluRs allowed examination of conservation in amino acid positions that make direct contact with glutamate or artificial agonists in iGluRs. While all iGluRs have an arginine (R) residue in S1 that binds the glutamate α-carboxyl group, only 19/61 (31%) IRs retain this residue. In the first half of the S2 domain, 9/61 (15%) of IRs retain a threonine (T), which contacts the glutamate γ-carboxyl group in all AMPA and kainate receptors. Interestingly, the iGluRs that lack this T residue (NR1, NR3A, delta) have glycine or serine and not glutamate as a preferred ligand (Mayer, 2006; Naur, 2007). Finally, in the second half of the S2 domain, 100% of the iGluRs have a conserved aspartate (D) or glutamate (E) that interacts with the α-amino group of the glutamate ligand, compared with 10/61 (16%) IRs. Of 61 IRs, only three (IR8a, IR75a, IR75c) retain the R, D/E, and T residues characteristic of iGluRs, although these residues lie within a divergent structural backbone. Other IRs have a diversity of different amino acids at one or more of these positions. Thus, the ligand-binding specificity of most or all IRs is likely to be both distinct from that of iGluRs and varied within the IR family (Benton, 2009).

The expression of the IR family was determined by both tissue-specific RT-PCR and RNA in situ hybridization. Fifteen IR genes are expressed in the antenna. Transcripts of these genes were not detected elsewhere in the adult head, body or appendages, except for IR25a and IR76b, which are also expressed in the proboscis. Expression of the remaining 46 IR genes was not reproducibly detected in any adult tissue. It is unclear whether these genes are not expressed, expressed at different life stages, or expressed in at levels below the detection threshold of these assays (Benton, 2009).

Analysis of where in the antenna IR genes are expressed compared to ORs was performed by double RNA in situ hybridization with probes for the OR co-receptor OR83b and one of several IR genes, including IR64a, IR76b, IR31a, and IR40a. IRs are not expressed in basiconic and trichoid sensilla, as they are not co-expressed with OR83b, and IR expression persists in mutants for the proneural gene absent md neurons and olfactory sensilla (amos), which completely lack these sensilla types. However, expression of these IRs is dependent upon the proneural gene atonal, which specifies the coeloconic sensilla as well as a feather-like projection called the arista, and a three-chambered pocket called the sacculus. Thus, ORs and IRs are expressed in developmentally distinct sensory lineages in the antenna. One exception is the subpopulation of coeloconic OSNs that expresses both IR76b and OR35a and OR83b. It was confirmed that IR-expressing cells in the antenna are neurons by demonstrating that they co-express the neuronal marker elav (Benton, 2009).

A comprehensive map was generated of IR expression. Each IR was observed to have a topologically-defined expression pattern that is conserved across individuals of both sexes. IR8a and IR25a, which encode closely related receptors, are broadly expressed, detected in overlapping populations of neurons around the sacculus and in the main portion of the antenna. IR25a but not IR8a is also detected in the arista. IR21a is expressed in approximately 6 neurons in the arista, as well as 5-10 neurons near the third chamber of the sacculus. Three IRs display specific expression in neurons surrounding the sacculus: IR40a and IR93a are co-expressed in 10-15 neurons adjacent to the first and second sacculus chambers, while IR64a is found in 10-15 neurons surrounding the third chamber (Benton, 2009).

The remaining 9 IRs are expressed in coeloconic OSNs distributed across the antenna. Double and triple RNA in situ hybridization revealed that individual neurons express between 1 and 3 different IR genes and are organized into specific clusters of two or three neurons. Four distinct clusters (cluster A-cluster D), containing two (cluster C) or three (cluster A, B, and D) neurons, could be defined by their expression of stereotyped combinations of IR genes. Cluster C includes a coeloconic neuron that expresses OR35a and OR83b in addition to IR76b. Although each cluster is distinct, there is overlap between the IRs they express. IR76b is expressed in one neuron in all four clusters, IR75d in three clusters and IR75a in two clusters. In additional to these selectively-expressed receptors, individual neurons are likely to express one or both of the broadly-expressed IR8a and IR25a. The combinatorial expression patterns of the IRs raise the possibility that these genes define specific functional properties of these neurons (Benton, 2009).

Definition of four distinct clusters of IR-expressing neurons in the antenna is consistent with the identification of four types of coeloconic sensilla, named ac1-ac4, which have distinct yet partially overlapping sensory specificities (Yao, 2005). To examine whether IR expression correlates with the chemosensory properties of these OSNs, the spatial organization of IR-expressing neurons was compared using probes for unique IR markers for each cluster type to these functionally distinct sensilla types. As a unique molecular marker for Cluster B is lacking, this cluster was defined as those containing IR75a-expressing OSNs (present in Cluster B and Cluster C) that are not paired with OR35a-expressing cluster C neurons. It was found that each cluster has a different, though overlapping, spatial distribution in the antenna. For example, Cluster A neurons (marked by IR31a) are restricted to a zone at the anterior of the antenna, just below the arista, while cluster C neurons (marked by IR75b) are found exclusively in the posterior of the antenna. These stereotyped IR neuron distributions were observed in antennae from over 20 animals (Benton, 2009).

The initial description of the coeloconic sensilla classes did not describe their spatial distribution (Yao, 2005). This study therefore recorded odor-evoked responses in >100 coeloconic sensilla in several dozen animals across most of the accessible antennal surface, using a panel of odorants that allowed identification unambiguously of each sensilla type (ammonia for ac1, 1,4-diaminobutane for ac2, propanal and hexanol for ac3, and phenylacetaldehyde for ac4) (Yao, 2005). After electrophysiological identification, the location of the sensilla on the antennal surface was noted (Benton, 2009).

This mapping process allowed a correlation of the electrophysiological and molecular properties of the coeloconic sensilla. For example, ac1 sensilla were detected only in a region on the anterior antennal surface just ventral to the arista, and therefore are most likely correspond to cluster A, containing IR31a-IR75d-IR76b/IR92a-expressing neurons. The data fit well with the previous assignment of the OR35a-expressing neuron to the ac3 sensillum (Yao, 2005), which is found on the posterior of the antenna and is the only coeloconic sensillum class that unambiguously houses two neurons (Yao, 2005). While these results allow initial assignment of IRs to different coeloconic sensilla classes, it is noted that assignment of specific odor responses to individual IR-expressing OSNs is not possible from these data alone (Benton, 2009).

All neurons expressing a given OR extend axons that converge upon a single antennal lobe glomerulus, resulting in the representation of a cognate odor ligand as a spatially-defined pattern of neural activity within the brain. To ask whether IR-expressing neurons have the same wiring logic, the targeting of OSNs expressing IR76a was investigated by constructing an IR76a-promoter GAL4 driver that recapitulates the endogenous expression pattern. Labeling of these neurons with mCD8:GFP revealed convergence of their axons on to a single glomerulus, ventral medial 4 (VM4), in the antennal lobe. This glomerulus is one of approximately eight that was previously unaccounted for by maps of axonal projections of OR-expressing OSNs (Benton, 2009).

To determine where IRs localize in sensory neurons, antibodies were generated against IR25a. Broad expression of IR25a protein was detected in sensory neurons of the arista, sacculus, and coeloconic sensilla. All anti-IR25a immunoreactivity was abolished in an IR25a null mutant. Low levels of IR25a could be detected in the axon segment adjacent to the cell body in some neurons but no staining was observed along the axons as they entered the brain, or at synapses within antennal lobe glomeruli. In coeloconic neurons, prominent anti-IR25a staining was detected both in the cell body and in the distal tip of the dendrite, which corresponds to the ciliated outer dendritic segment innervating the sensory hair. Relatively low levels were detected in the inner dendrites, suggesting the existence of a transport mechanism to concentrate receptor protein in cilia. A similar subcellular localization was observed in sacculus and aristal sensory neurons. The specific targeting of an IR to sensory cilia suggests a role for these proteins in sensory detection (Benton, 2009).

To test the hypothesis that IR genes encode chemosensory receptors, whether ectopic IR expression could induce novel olfactory specificities was investigated. Three IRs expressed in ac4 sensilla (IR84a, IR76a and IR75d) were individually mis-expressed in ac3 sensilla using the OR35a-GAL4 driver. Single sensillum recordings were used to examine which, if any, of these three IRs, could confer sensitivity to phenylacetaldehyde, the only known robust ligand for ac4 but not ac3 sensilla (Yao, 2005). Mis-expression of IR84a conferred a strong response to phenylacetaldehyde that was not observed in control strains or in animals mis-expressing either IR76a or IR75d. Ectopically-expressed IR84a did not confer sensitivity to the structurally related odor, phenylacetonitrile, which does not activate either ac3 or ac4 neurons (Yao, 2005). This indicates that mis-expressed IR84a does not simply generate non-specific ligand sensitivity in these neurons (Benton, 2009).

Next the novel odor responses conferred by IR84a mis-expression were compared to the endogenous phenylacetaldehyde responses of ac4 sensilla by generating dose-response curves. Stimulus evoked spike frequencies of ac3 sensilla ectopically expressing IR84a are quantitatively very similar to those in ac4 sensilla, even exceeding the endogenous ac4 responses at higher odor concentrations. These elevated responses are likely to be due to the contribution of weak endogenous phenylacetaldehyde responses that were observed in ac3 sensilla at high stimulus concentrations, as subtraction of these values produces an IR84a-dependent phenylacetaldehyde dose-response curve that is statistically the same as that of ac4 sensilla. Thus, ectopic expression of a single IR in ac3 is sufficient to confer a novel ligand- and receptor-specific odor sensitivity that is physiologically indistinguishable from endogenous responses (Benton, 2009).

To extend this analysis to a second IR, whether mis-expression of one of the IR genes uniquely expressed in ammonia-sensitive ac1 neurons (IR31a and IR92a) was sufficient to confer ectopic responsiveness to this odor was examined. Because ac3 sensilla neurons display endogenous ammonia-evoked responses at modest stimulus concentrations, these experiments used the IR76a-promoter GAL4 transgene to mis-express these receptors in ammonia-insensitive ac4 sensilla (Yao, 2005). ac4 sensilla mis-expressing IR92a, but not IR31a, displayed responses to ammonia. 1,4-diaminobutane, a control stimulus that does not activate either ac1 or ac4 neurons (Yao, 2005), did not stimulate ac4 sensilla mis-expressing IR92a. It was noted that the magnitude of the ectopic IR92a ammonia response is lower than native ammonia-evoked responses of ac1 sensilla (Yao, 2005). This may be due to the lack of co-factors present in ac1 sensilla but not in ac4 sensilla. Nevertheless, these results suggest that IR92a comprises at least part of an ammonia-specific chemosensory receptor (Benton, 2009).

The specific combinatorial expression patterns of IRs in sensory neurons and the diversity in their ligand-binding domains is difficult to rationalize with a general role in signal transduction, independent of ligand recognition. More importantly, the novel olfactory sensitivity induced by ectopic expression of IR84a and IR92a provides evidence that IR proteins function directly as ligand-specific, chemosensory receptors. While these experiments demonstrate a sufficiency of IRs for conferring odor-responsiveness, definitive proof of their necessity will require analysis of loss-of-function mutations (Benton, 2009).

In animal nervous systems, iGluRs mediate neuronal communication by forming glutamate-gated ion channels, and it is speculated that IRs also form ion channels, gated by odors and other chemosensory stimuli. A growing number of ionotropic mechanisms in chemoreception are known. For example, members of the transient receptor potential (TRP) family of ion channels are the primary receptors for nociceptive compounds including capsaicin and menthol and have also been implicated in gustatory detection of acids. Insect ORs also display functional properties of ion channels. Proof that IRs function as ion channels will necessitate electrophysiological characterization of these receptors in heterologous expression systems, and evidence for direct binding of chemosensory ligands to IRs will require biochemical assays in vitro (Benton, 2009).

iGluRs normally function as heterotetrameric assemblies of variable subunit composition that exhibit differing functional properties such as ligand sensitivity and ion permeability. The current analysis indicates that up to five different IRs may be co-expressed in a single sensory neuron, raising the possibility that these receptors also form multimeric protein assemblies with subunit-dependent characteristics. Of particular interest are the two broadly-expressed members of the family, IR8a and IR25a, which may represent common subunits in many different types of IR complexes. Their function is unclear, but it is possible that they have a co-receptor function with other IRs, analogous to that of OR83b. Preliminary analysis of IR25a mutants revealed no obvious defects in odor-evoked responses in coeloconic sensilla, but this may be due to redundancy of IR25a with IR8a or the existence of homomeric IR receptors without IR8a or IR25a. Other IRs, such as IR75a and IR76b, are expressed in two or more types of coeloconic sensory neurons. In these cases, the response properties may be defined by the combination of IRs expressed in these distinct neuronal populations. However, the present lack of knowledge of relevant ligands for several coeloconic OSNs makes it difficult to match specific ligands to individual IR neurons based on the expression map alone (Benton, 2009).

The IR repertoire is remarkably similar in size, overall genomic organization and sequence divergence to Drosophila ORs. Like the ORs, individual IRs are specifically expressed in small subpopulations of chemosensory neurons, and this expression is regulated by relatively short (< 1-2 kb) upstream regulatory regions. Furthermore, at least one population of IR-expressing neurons converges on to a single glomerulus in the antennal lobe, similar to the wiring logic established for OR-expressing neurons both in invertebrate and vertebrate olfactory systems. Some differences are observed, however, in the organizational logic of IR and OR expression. Most OR-expressing neurons express a single OR gene, along with OR83b, in distinct clusters that innervate specific olfactory hairs. In contrast, many IR-expressing neurons identified in the antenna express 2 or 3 IR genes, in addition to one or both of the broadly-expressed IR8a and IR25a genes. Moreover, overlap is observed both between the molecular composition of different IR neurons and the combination of neurons that innervate a given sensillum. For example, IR76b is co-expressed with at least two other different IR genes in at least two different sensilla - with IR92a in ac1 and with IR76a in ac4 - as well as being co-expressed with OR35a and OR83b in ac3. While the precise biological logic of IR co-expression awaits the matching of specific chemosensory ligands to IR-expressing neurons, combinatorial expression of IRs may contribute more significantly to their role in sensory detection than for ORs (Benton, 2009).

Why does Drosophila possess two types of antennal chemosensory receptors? Although both may be ionotropic, IRs and ORs are not simply slight evolutionary variants. The receptor families are molecularly unrelated, are under the control of distinct developmental programs, and housed within sensory structures of radically different morphology. Thus, it seems likely that these chemosensory receptors fulfill distinct functions in chemosensation. Analysis of the chemosensory behaviors mediated by IR sensory circuits (now possible with the identification of specific molecular markers for these pathways) may provide insights into the contributions of these different olfactory subsystems. IRs may also have functions in other chemosensory modalities, as two antennal IRs are also detected in the proboscis, and the expression of 46 members of the repertoire remains unknown (Benton, 2009).

Chemosensation is an ancient sensory modality that predates the evolution of the eukaryotes. Are there traces of conservation in the molecular mechanism by which prokaryotes and eukaryotes sense external chemicals? iGluRs have long been recognized to have prokaryotic origins. Their ion channel domain is homologous to bacterial potassium channels, and the ligand binding domain is structurally related to bacterial periplasmic binding proteins (PBPs), extracellular proteins that scavenge or sense amino acids, carbohydrates and metal ions by coupling to transporters or chemotaxis receptors. Evolutionary connections between iGluRs and PBP function have not often been considered, perhaps in part due to their very weak primary sequence similarity, the widespread occurrence of the PBP fold -- also present, for example, in bacterial transcription regulators -- and the dedicated role for iGluRs in mediating or regulating synaptic transmission, a process seemingly distant from bacterial solute uptake and chemotaxis (Benton, 2009).

This discovery of a family of divergent iGluR-like proteins that may act as peripheral chemosensors provides a link between the disparate functions of these protein modules. While a role for IRs in detecting diverse external ligands is analogous to the function of bacterial PBPs, the primary sequence and neuronal expression of IRs is clearly closer to the properties of iGluRs. Intriguingly, a large family of iGluR-related proteins, the GLRs has also been identified in the plant Arabidopsis thaliana (Lam, 1998; Chiu, 1999). Almost nothing is known about their physiological functions, but bioinformatic analysis of GLRs suggests that glutamate is unlikely to be their natural ligand (Dubos, 2003; Qi, 2006). It is possible that GLRs may have roles as chemosensors, for example in detection of soil nutrients or airborne volatiles. Thus, while iGluRs have been intensely studied for their roles in synaptic communication, this characterization of the IRs leads to the suggestion that the ancestral function of this protein family may have been in detecting diverse chemical ligands to mediate both intercellular communication and environmental chemical sensing (Benton, 2009).

Drosophila tracks carbon dioxide in flight

Carbon dioxide (CO₂) elicits an attractive host-seeking response from mosquitos yet is innately aversive to Drosophila melanogaster despite being a plentiful byproduct of attractive fermenting food sources. Prior studies used walking flies exclusively, yet adults track distant food sources on the wing. This study shows that a fly tethered within a magnetic field allowing free rotation about the yaw axis actively seeks a narrow CO₂ plume during flight. Genetic disruption of the canonical CO₂-sensing olfactory neurons does not alter in-flight attraction to CO₂; however, antennal ablation and genetic disruption of the Ir64a acid sensor do. Surprisingly, mutation of the obligate olfactory coreceptor (Orco; Or83b) does not abolish CO₂ aversion during walking yet eliminates CO₂ tracking in flight. The biogenic amine octopamine regulates critical physiological processes during flight, and blocking synaptic output from octopamine neurons inverts the valence assigned to CO₂ and elicits an aversive response in flight. Combined, these results suggest that a novel Orco-mediated olfactory pathway that gains sensitivity to CO₂ in flight via changes in octopamine levels, along with Ir64a, quickly switches the valence of a key environmental stimulus in a behavioral-state-dependent manner (Wasserman, 2013).

These results show that a single molecule can carry both negative and positive hedonic valence depending on the behavioral state of the animal. It is posited that flight behavior is accompanied by neuromodulatory activation of the olfactory system by octopamine that rapidly shifts the function of olfactory sensory pathways in a manner similar to the operational gain and frequency response shifts triggered by locomotor activity in fly visual interneurons. Recent work in other organisms has identified similar roles for neuromodulators that serve to alter the state of neuronal circuits in a behaviorally contextual manner, thereby enabling computational flexibility and behavioral robustness to ever-changing internal and external environmental conditions. These findings unravel the paradox of why D. melanogaster would find an environmental signal indicating a potential food source repellent instead of attractive; for Drosophila gathered on the ground, under crowded social conditions, CO2 secreted as part of a stress pheromone releases an innate avoidance response. Taking flight appears to fully and rapidly switch the valence of this stimulus, triggering CO2 attraction consistent with the search for sugar-rich food resources undergoing fermentation that robustly attract D. melanogaster vinegar flies. These findings lay the groundwork for further exploring the neural substrate for a rapid and robust switch in hedonic valence (Wasserman, 2013).

Search PubMed for articles about Drosophila Ionotropic receptors

Ai, M., Min, S., Grosjean, Y., Leblanc, C., Bell, R., Benton, R. and Suh, G. S. (2010). Acid sensing by the Drosophila olfactory system. Nature 468(7324): 691-5. PubMed ID: 21085119

Benton, R., Vannice, K. S. and Vosshall, L. B. (2007). An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature 450: 289-293. PubMed ID: 17943085

Benton, R., Vannice, K. S., Gomez-Diaz, C. and Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136(1): 149-62. PubMed ID: 19135896

Chiu, J., et al. (1999). DeSalle R, Lam H, Meisel L, Coruzzi G. Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged. Mol. Biol. Evol. 16: 826-838. PubMed ID: 10368960

Couto, A., Alenius, M. and Dickson, B. J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15: 1535-1547. PubMed ID: 16139208

Dubos, C., et al. (2003). Huggins D, Grant GH, Knight MR, Campbell MM. A role for glycine in the gating of plant NMDA-like receptors. Plant J. 35: 800-810. PubMed ID: 12969432

Franz, G., Loukeris, T. G., Dialektaki, G., Thompson, C. R. and Savakis, C. (1994). Mobile Minos elements from Drosophila hydei encode a two-exon transposase with similarity to the paired DNA-binding domain. Proc. Natl Acad. Sci. 91: 4746-4750. PubMed ID: 8197129

Huang, A. L. et al. (2006). The cells and logic for mammalian sour taste detection. Nature 442: 934-938. PubMed ID: 16929298

Lam, H. M., et al. (1998). Glutamate-receptor genes in plants. Nature 396: 125-126. PubMed ID: 9823891

Littleton, J. T. and Ganetzky, B. (2000). Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26: 35-43. PubMed ID: 10798390

Mayer, M. L., et al. (2006). Crystal structures of the kainate receptor GluR5 ligand binding core dimer with novel GluR5-selective antagonists. J. Neurosci. 26: 2852-2861 . PubMed ID: 16540562

Naur, P., et al. (2007). Ionotropic glutamate-like receptor delta2 binds D-serine and glycine. Proc. Natl. Acad. Sci. 104: 14116-14121. PubMed ID: 17715062

Ng, M., et al. (2002). Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36: 463-474. PubMed ID: 12408848

Qi, Z., Stephens, N. R. and Spalding, E. P. (2006). Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiol. 142: 963-971. PubMed ID: 17012403

Shanbhag, S. R., Singh, K. and Singh, R. N. (1995). Fine structure and primary sensory projections of sensilla located in the sacculus of the antenna of Drosophila melanogaster . Cell Tissue Res. 282: 237-249. PubMed ID: 8565054

Sato, K., Pellegrino, M., Nakagawa, T., Nakagawa, T., Vosshall, L. B. and Touhara, K. (2008). Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452(7190): 1002-6. PubMed ID: 18408712

Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B. and Axel, R. (2003). Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112: 271-282. PubMed ID: 12553914

Wasserman, S., Salomon, A. and Frye, M. A. (2013). Drosophila tracks carbon dioxide in flight. Curr Biol 23: 301-306. PubMed ID: 23352695

Wicher, D. et al. (2008). Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452(7190): 1007-11. PubMed ID: 18408711

Yao, C. A., Ignell, R. and Carlson, J. R. (2005). Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J. Neurosci. 25: 8359-8367. PubMed ID: 16162917

date revised: 15 July 2013

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