Odorant receptors (ORs) are thought to act in a combinatorial fashion, in which odor identity is encoded by the activation of a subset of ORs and the olfactory sensory neurons (OSNs) that express them. The extent to which a single OR contributes to chemotaxis behavior is not known. This question was investigated in Drosophila larvae, which represent a powerful genetic system to analyze the contribution of individual OSNs to odor coding. Twenty-five larval OR genes expressed in 21 OSNs were identified and genetic tools were generate that allow engineering of larvae missing a single OSN or having only a single or a pair of functional OSNs. Ablation of single OSNs disrupts chemotaxis behavior to a small subset of the odors tested. Larvae with only a single functional OSN are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the combinatorial code. Behavioral evidence is provided that an OSN not sufficient to support chemotaxis behavior alone can act in a combinatorial fashion to enhance chemotaxis along with a second OSN. It is concluded that there is extensive functional redundancy in the olfactory system, such that a given OSN is necessary and sufficient for the perception of only a subset of odors. This study is the first behavioral demonstration that formation of olfactory percepts involves the combinatorial integration of information transmitted by multiple ORs (Fishilevich, 2005).
The 'nose' of the Drosophila larva resides in a pair of dorsal organs at the anterior tip of the animal, each containing 21 OSNs. Previous studies showed that up to 23 of the 61 Drosophila ORs are expressed in larvae by PCR and transgenic analysis. RNA in situ hybridization was performed to provide direct evidence that OR genes are expressed in larval OSNs. Or83b, which is necessary for the proper localization and function of conventional ORs, is broadly expressed throughout the dorsal-organ ganglion. Twenty-four of the 30 ORs tested in this study are expressed in a single larval neuron in the dorsal organ. The expression of Or10a, Or43b, or Or49a mRNA or OR43b protein was not detected, although RT-PCR analysis detects these transcripts in larvae. Or92a and Or98b are also not detected by RNA in situ hybridization. Most larval OSNs express a single OR along with Or83b, but two OSNs coexpress a pair of ORs along with Or83b: Or33b/Or47a and Or94a/Or94b. Such OR coexpression has also been documented in the adult olfactory system (Fishilevich, 2005).
In parallel with the RNA in situ hybridization analysis, a collection of 42 different OR-Gal4 transgenes were examined that drive the expression of Gal4 under the control of OR promoter elements. To visualize gene expression in the dorsal organ, individual OR-Gal4 lines were crossed to UAS-GFP, encoding cytoplasmic green fluorescent protein (GFP) and the olfactory-neuron marker Or83b-Myc. Or83b-Gal4 labels all 21 larval OSNs. Per dorsal organ, 19 of the remaining 41 OR-Gal4 transgenes label a single larval OSN that is also positive for Or83b-Myc. Although Or49a mRNA was not detected in larvae, Or49a-Gal4 labels one dorsal-organ OSN along with a single terminal-organ gustatory neuron. Gustatory receptor (GR) genes are expressed in both olfactory and gustatory organs of the adult fly. GR-Gal4 transgenes are expressed only in the gustatory terminal organ or in nonolfactory dorsal-organ neurons that do not express Or83b-Myc. A total of 25 Drosophila ORs expressed in the larval dorsal organ were identified and direct evidence is provided that 24 of these OR mRNAs are expressed in situ. Of these, 14 are expressed only at the larval stage, whereas 11 are utilized by both larval and adult olfactory systems (Fishilevich, 2005).
Larval OSNs project axons to the larval antennal lobe of the brain. Patterns of axonal projections to the larval antennal lobe were examined in larvae carrying each of 20 larval OR-Gal4 transgenes along with UAS-GFP or UAS-CD8-GFP. Each OR-Gal4 line reveals a single labeled axonal arbor that terminates in an antennal-lobe glomerulus whose position is conserved between animals (Fishilevich, 2005).
The availability of genetic tools that uniquely label 19 of the 21 larval OSNs allows manipulation of the odor code by deconstructing the peripheral olfactory input and examining effects on behavioral output. Toward this end, a chemotaxis assay was establised of sufficient sensitivity to quantify differences in odor-evoked behavior. Chemotaxis of wild-type larvae was measured in response to 53 synthetic monomolecular odorants and three natural Drosophila attractants. The assay involves single-animal analysis in which the position of individual chemotaxing larvae is tracked over the course of a 5 min experiment (Fishilevich, 2005).
This assay was used to screen larval chemotaxis to a panel of 53 synthetic odors and quantified the median distance to odor for Or83b−/− and Or83b+/+ larvae. Forty of the 53 odors are naturally present in fruit, and of these 40, 13 are known to elicit behavioral and electrophysiological responses in Drosophila. Anosmic Or83b−/− larvae do not respond to any odors, but wild-type (yw) larvae respond to many odors with strong chemotaxis (Fishilevich, 2005).
It was next asked how sensitive larvae are to odors by performing chemotaxis experiments at various odor concentrations. The responses to 1-hexanol are weak and not statistically different from anosmic controls for low dilutions, whereas responses increase steeply between 0.02 μl and 0.2 μl doses and appear to reach a plateau for higher concentrations. No evidence was found that higher concentrations elicit repulsion. Response thresholds to heptanal and isoamyl acetate are one and two log orders, respectively, below that of 1-hexanol (Fishilevich, 2005).
To test whether the weak responses observed for some odors at 2 μl could be explained by high detection thresholds, seven of these odors were further tested with 20 μl. Under these conditions, 1-butanol and 2,3-butanediol elicit chemotaxis, whereas the remaining five odors do not. Thus the 2 μl stimulus dose elicits robust chemotaxis across a large group of different odors, in accord with previous behavioral studies (Fishilevich, 2005).
Upon loading of an odorant stimulus in the closed-dish assay, the spatial distribution and average airborne concentration of this odor in the dish will be partly determined by the odor's vapor pressure. Vapor pressure is thus likely to affect the behavioral response observed for a particular odorant stimulus. In addition to this factor, it is anticipated that the olfactory system of the larva may be differentially tuned to different stimuli. In the initial phases of this study, no clear correlation was found between the vapor pressure of a given odor and its corresponding behavioral efficacy. It was therefore decided to avoid any normalization of stimulus concentration and used the same quantity of odor (2 μl) for all 53 stimuli tested (Fishilevich, 2005).
Whether chemotaxis elicited by single odors is comparable to that obtained with natural stimuli was examined. Chemotaxis was measured in the same assay to banana mush, balsamic vinegar, and yeast paste at different concentrations. It was found that attraction elicited by single synthetic odors is qualitatively similar to that obtained with natural odor blends and that the same steep threshold and stable plateau properties are seen for both stimulus types (Fishilevich, 2005).
The relative contribution of any given OSN to the formation of an odor percept was examined. Diphtheria toxin (DTI), an attenuated version of the cell-autonomous protein-translation inhibitor diphtheria toxin, was used to ablate identified OSNs selectively. Most but not all larval OSNs are ablated by the expression of DTI along with GFP under control of the Or83b-Gal4 driver in all 21 larval OSNs. In Or83b-ablated animals, GFP expression is not detected, and sensory dendrites are severely atrophied but not completely absent. In Or49a-ablated animals, the Or49a-GFP marker is not visible, and expression of other ORs is not perturbed (Fishilevich, 2005).
Chemotaxis of animals with single neurons ablated (Or1a, Or42a, or Or49a) was measured with a panel of 20 odors and compared to results obtained with the Or83b-ablation. Or83b-ablated larvae fail to respond to 17/20 odors. If a single false discovery (FD) is allowed for, Or83b-ablated animals fail to respond to 19/20 odors. Or1a-ablated and Or49a-ablated animals each show reduced chemotaxis to a single different odor, (E)-2-hexenal and 1-hexanol, respectively, but show normal chemotaxis to the other 19 odors. In contrast, ablation of the Or42a OSN causes decreases in chemotaxis to four of 20 odors. If FD = 1 is allowed, Or1a-ablated animals are impaired in responses to three of 20 and Or42-ablated animals to five of 20 odors (Fishilevich, 2005).
It was next asked which OSNs are sufficient to produce chemotaxis to a given odor by constructing animals with only one or combinatorials of two functional OSNs. This was achieved by exploiting the Or83b mutation, which prevents OR trafficking to the sensory dendrite. Or83b function was restored in individual OSNs by crossing animals with specific OrX-Gal4 drivers to UAS-Or83b animals, allowing assessment of the contribution of single neurons to odor-evoked behavior in the OrX-functional progeny (Fishilevich, 2005).
Only a single OR83b-expressing neuron is seen in Or42a-functional, Or49a-functional, and Or1a-functional animals, whereas two OR83b-positive neurons are visible in Or1a-/Or42a-functional and Or1a-/Or49a-functional animals. The remaining OSNs are present but unlabeled in these animals because the Or83b mutation eliminates OR83b protein expression. No evidence was found that the glomerular map is distorted by the activation of a single OSN in a background of nonfunctional neurons as evidenced by the normal position and volume of the Or1a glomerulus in Or1a-functional and Or83b mutant larvae (Fishilevich, 2005).
These animals along with genetically matched control animals were screened for chemotaxis to 53 odors by using the same behavioral assay and nonparametric statistical analysis employed for the ablation experiments. Consistent with the strong Or42a-ablated phenotypes, Or42a-functional animals respond to 22 odors compared to 36 odors in Or83b+/+ controls possessing 21 functional OSNs. Or42a-functional animals respond to three of four odors to which Or42a-ablated animals are anosmic. The broad behavioral response profile observed for Or42a-functional larvae is in agreement with the broad ligand specificity of this OR as defined by electrophysiological experiments (Fishilevich, 2005).
In contrast to the broad odor response profile of Or42a-functional larvae, Or1a- and Or49a-functional animals do not show significant chemotaxis to any of the 53 odors tested, consistent with the weak phenotype of ablating either the Or49a-expressing or Or1a-expressing neuron. These behavioral results are in accord with the ligand profiling of Or49a, which does not show strong electrophysiological responses to any of 27 odors tested (Fishilevich, 2005).
Although Or1a- and Or49-functional larvae do not chemotax to any odors tested, it was asked whether these neurons contribute to chemotaxis in concert with the Or42a neuron. Chemotaxis performance of larvae with two functional neurons was compared to data from animals with only a single functional neuron. Larvae with two functional neurons respond to a somewhat different subset of odors than animals having either single functional neuron alone (Fishilevich, 2005).
To examine the existence of interactions between these neurons and identify cases of combinatorial enhancement, a linear regression model was developed to compare chemotaxis data across genetically matched controls for larvae with one or two functional OSNs. The model was designed to identify potential cases where single-neuron chemotaxis behavior differs from two-neuron behavior. The linear model suggests six cases of potential positive cooperativity between Or1a and Or42a chemotaxis that merited further experimental investigation. Additional chemotaxis experiments were carried out with four odors (1-pentanol, 2-pentanol, 2-hexanol, and 3-octanone) at three concentrations. 1-pentanol shows significantly stronger chemotaxis in Or1a/Or42a-functional animals than Or42a-functional or Or1a-functional animals at all three concentrations. A qualitative view of this behavioral enhancement is seen in the sector-plot distributions comparing the anosmia of Or83b mutants to the progressive increase in chemotaxis to 1-pentanol of Or1a-functional or Or42a-functional compared to Or1a/Or42-functional. The Or1a/Or42-functional animals spend comparatively more time in the sector containing the odor than animals having either single functional neuron alone. For the other three odors, this cooperative effect is significant at a single odor concentration (Fishilevich, 2005).
This study has used behavioral analysis to measure the contribution of individual neurons to the odor code and provide a missing link between the understanding of the molecular biology of ORs, the neurophysiological properties of the olfactory network, and complex odor-evoked behaviors. The goal was to approach the question of how the combinatorial activation of ORs encodes odor stimuli and elicits olfactory behavior. The results suggest that there is a high level of redundancy in the larval olfactory system, such that ablating a single neuron has minimal effects on odor detection. Among these olfactory inputs, the Or42a neuron plays a more important role in odor detection than the Or1a or Or49a neuron. Animals engineered to have the Or42a neuron functional are able to chemotax to multiple odors. The addition of a second OSN to such animals results in enhanced chemotaxis for several odors. Whereas Or1a-functional animals show no significant responses to any odor tested, it was observed that responses of Or1a/Or42a-functional animals to four odors are enhanced relative to Or42a-functional animals. This suggests that although olfactory input contributed by the Or1a-expressing OSN is not sufficient alone to elicit robust chemotaxis, it enhances the perception of odors in conjunction with the information transmitted by the Or42a-expressing OSN. (Fishilevich, 2005).
Behavior is the ultimate output of a sensory system that integrates all aspects of external-information processing. These experiments demonstrate the feasibility and value of integrating behavioral analysis into the study of odor coding. It is proposed that the simple olfactory system of Drosophila larvae will be an invaluable model in any attempt to correlate the cellular basis of the odor code with its behaviorally relevant output (Fishilevich, 2005).
Drosophila is a holometabolous insect that undergoes dramatic changes in lifestyle from the larval to adult stage. In a sense, these animals can be considered to occupy completely separate ecological niches. Larvae maintain constant contact with food until pupation, whereas adults are flying insects that use their sense of smell to identify suitable food sources and appropriate sites for egg-laying. In essence, larvae are specialized for feeding and growth, whereas adults are devoted to breeding and dispersal. To what extent have these two life stages of the same species evolved a different chemosensory system? This study shows 14 of 25 larval OR genes are stage specific and not used again by the adult animal. All larval OSNs are histolyzed in metamorphosis and replaced in the adult by newly differentiated antennal and maxillary palp OSNs. Perhaps this developmental changeover has led to largely separate OR genes with transcriptional regulatory regions specific for either larval or adult olfactory organs. Alternatively, the segregation of larval- and adult-expressed ORs could be functional and relate to the different ecological niches that these life stages occupy: larvae may cope with much higher odor concentrations because of their direct contact with food (Fishilevich, 2005).
Odor processing occurs at various levels in the nervous system, from peripheral sensory neurons to primary processing centers, such as the olfactory bulb in vertebrates and the antennal lobe in insects, and further to higher brain centers of the olfactory cortex in vertebrates and mushroom body and lateral horn in insects. How the combinatorial code established by the ORs at the periphery is transmitted through this olfactory circuitry to produce the perception of an odor in any species is unknown. The data support the notion that peripheral sensory neurons constitute information channels that are not independent but subject to interactions in the olfactory circuit. Otherwise, one would expect that the behavioral response profile observed for the Or1a/Or42a-functional genotype be given by the union of the best performances of the single Or1a- and Or42a-functional genotypes. Where and how the information is processed remains unclear, but part of this transformation may occur in the antennal lobe (Fishilevich, 2005).
A number of conclusions about odor coding in the Drosophila larva can be drawn from this work. There appears to be no clear structural relationship between the odors that elicit chemotaxis mediated by a given OSN, as has been previously shown in an analysis of the ligand response properties of ORs in the adult fly. The Or42a-expressing neuron differs from other neurons studied here in the large number of odors that attract animals having only this neuron active. Interestingly, the behavioral response profile of the Or42a-functional genotype indicates that an OR may not need to be strongly activated by a given odor to allow for chemotaxis toward the odor source. This point is best illustrated by 3-octanol and anisole, which both elicit strong chemotaxis in Or42a-functional animals whereas they seem to induce relatively weak electrophysiological activity (Fishilevich, 2005).
Finally, the behavioral receptive field of animals having combinatorials of functional neurons cannot be predicted from a simple model where the responses of animals having either single OSN functional are added. The chemotaxis results reported in this study highlight the existence of strong nonlinearities in the processing of olfactory information in such a way that in the arithmetic of sensory coding, the whole is greater than what the parts can produce independently. Such a scheme would be consistent with the extraordinary needs of the olfactory system to detect numbers of odors that greatly exceed the number of OR genes in any given animal. The functional redundancy observed here could buffer the olfactory system against mutations and allow animals to adapt to changing or new odor environments (Fishilevich, 2005).
The genetic tools presented in this study should permit a systematic analysis of the peripheral and central components that generate an odor response in the Drosophila larva. A number of key unanswered questions remain for future studies. Electrophysiological or optical imaging tools must be used to analyze the neuronal correlates of the observed behavior. Greater understanding of the second- and third-order neurons that communicate information from the antennal lobe to eventual motor output is needed. This study has been restricted to simple chemotaxis assays, and no attempt has been made to query larvae for their powers of odor discrimination. Animals missing a single OSN may chemotax normally but experience olfactory-perception not uncovered in these chemotaxis assays. By coupling associative learning of odors in intact animals followed by generalization tests in the same animals that conditionally lack a single OSN, it should be possible to determine whether odor salience is altered in larvae missing a single OSN. Finally, it will be important to determine whether the phenomena reported in this study can be considered general olfactory-coding principles that also apply to more complex animals (Fishilevich, 2005).
Despite increasing knowledge about dimerization of G-protein-coupled receptors, nothing is known about dimerization in the largest subfamily, odorant receptors. Using a combination of biochemical and electrophysiological approaches, this study demonstrates that odorant receptors can dimerize. DOR83b, an odorant receptor that is ubiquitously expressed in olfactory neurons from Drosophila and highly conserved among insect species, forms heterodimeric complexes with other odorant-receptor proteins, which strongly increases their functionality (Neuhaus, 2005).
Generally, a given odorant-receptor gene is expressed in only a small fraction of olfactory receptor neurons (ORNs), and each ORN expresses a very small number of odorant-receptor genes. In insects, however, one highly conserved member of the odorant-receptor gene family is expressed in nearly all ORNs in addition to the conventional odorant receptor or receptors. The encoded proteins DOR83b in Drosophila, AgOr7 in Anopheles gambiae, HvirR2 in Heliothis virescens and AmelR2 in Apis mellifera have between 64% and 88% amino acid-sequence identity, a level not observed for any other insect odorant-receptor gene. The function of this ubiquitously expressed protein is still unknown. Antennal neurons in Drosophila that express only DOR83b do not respond to any of a large panel of odors, so it is unlikely that the receptor itself has ligand-binding properties. Alternatively, DOR83b could act as a subunit in heterodimeric receptor complexes, either modulating the binding specificity of the coexpressed receptor or regulating the assembly of functional receptor transduction complexes, both of which are known functions of G-protein-coupled receptors. Heterodimerization might alternatively be necessary for the folding or membrane targeting of conventional odorant-receptor proteins. Using a combination of electrophysiological and biochemical approaches, this study investigated the role of DOR83b in the olfactory system of Drosophila (Neuhaus, 2005).
Investigations of the function of DOR83b were done in a heterologous HEK293 expression system. Bioluminescence resonance energy transfer (BRET) experiments showed that DOR83b interacts with other odorant receptors. Energy transfer occurred when a DOR83b-luciferase (Luc) fusion construct was expressed together with DOR43a-green fluorescent protein (GFP) or with DOR22a-GFP in HEK293 cells, indicating that dimers or oligomers were forming between DOR83b and the conventional Drosophila odorant receptors DOR43a or DOR22a. The energy transfer was similar to the one measured with GFP and Luc-tagged β2-adrenergic receptors, G-protein-coupled receptors for which homodimerization is already known to occur. Similar results were obtained for coexpression of DOR83b-GFP and DOR83b-Luc and for coexpression of DOR43a-GFP and DOR43a-Luc, indicating that the receptors can also homodimerize. Receptor oligomerization was specific and not the result of receptor overexpression; no BRET was detected when DOR83b-Luc was coexpressed with the GFP-tagged cyclic nucleotide-gated channel from Drosophila or with the GFP-tagged rat β2-adrenergic receptor. Dimeric complexes persisted following denaturing gel electrophoresis and western blotting (Neuhaus, 2005).
To test the functional consequences of coexpression of DOR83b with DOR43a, calcium imaging measurements were performed in the heterologous HEK293 system, which has frequently been used to assay odorant receptor function. DOR43a can be activated by cyclohexanone, but in HEK293 cells responses were obtained only when ligand concentrations were in the millimolar range, and with poor efficiency (below 1% of cells responding). In contrast, when DOR43a was cotransfected with DOR83b, the threshold dropped to the micromolar range and the number of cells responding significantly increased to 10%-15%, even though expression of the receptors remained unchanged (30%-40% of cells after transfection). The DOR43a-DOR83b heterodimer had a spectrum of active ligands similar to that reported for DOR43a-expressing neurons and for recombinantly expressed DOR43a alone. Active substances were cyclohexanone, benzaldehyde, isoamyl acetate, cineole and cyclohexanol; inactive substances were ethylbutyrate, octanal and 2-octanal. To generalize these findings, the responses of DOR22a transfected alone and with DOR83b (DOR22a/DOR83b) was tested in HEK293 cells, and similar effects were found. DOR22a/DOR83b cotransfected cells responded to lower (micromolar compared to millimolar) concentrations of ethylbutyrate, one of the best ligands for DOR22a-expressing neurons, and the number of responding cells was significantly higher despite similar transfection rates. To check the specificity of the effect of the DOR83b cotransfection, DOR43a together with DOR22a was transfected into HEK293 cells and, again, obtained responses only at ligand concentrations in the millimolar range and with poor efficiency. DOR83b did not respond to any of the odorants tested, even in the millimolar range (Neuhaus, 2005).
If, as the BRET and calcium imaging data suggest, DOR83b functions as a co-receptor, a reduction in DOR83b expression in vivo should lead to a reduction in odor responsiveness of the ORNs. To investigate the influence of DOR83b expression in the fly, RNA interference was used to reduce the level of DOR83b, and the odor-evoked response of the antennae was monitored using electroantennogram (EAG) recordings. In flies hatched from embryos injected with DOR83b double-stranded RNA (RNAi flies), the EAG amplitude was reduced compared to control flies . Six different, commonly used odors evoked significantly smaller EAG responses in RNAi flies compared to control flies, with average reductions ranging from 56% for 1-butanol to 30% for ethanol. The residual response is likely to reflect incomplete knockdown of DOR83b mRNA, as suggested by the absence of detectable responses in DOR83b mutant flies. Moreover, in situ hybridization on the antennae of the flies (RNAi versus non-RNAi) on which EAG recordings had been done showed that DOR83b mRNA levels were much lower, but not completely abolished, in flies with lowered EAG responses (Neuhaus, 2005).
This study shows that insect odorant receptors form dimers and that heterodimerization improves the functionality of the receptors. The results suggest that DOR83b functions as a co-receptor of conventional Drosophila odorant receptors. Further investigation will be needed to determine whether dimerization is a general property of odorant receptors in vertebrates as well (Neuhaus, 2005).
Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. This study demonstrates that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. These results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors (Benton, 2006).
These results define Drosophila OR83b and ORs as a novel family of TM proteins with sequence and membrane topology that is distinct from mammalian GPCR-family ORs. OR83b associates with ORs through conserved cytoplasmic loops previously believed to be extracellular; ORs and OR83b form heteromeric complexes within OSNs. These complexes form early in the membrane-trafficking pathways but persist and concentrate in the sensory cilia. An essential role is shown for OR83b in targeting and maintaining these complexes within the ciliary membranes at the site of odorant signal transduction. These results define OR83b as an integral part of the functional odorant receptor in insects. Furthermore, despite the striking similarities in the anatomy and physiology of mammalian and insect olfactory systems, they reveal important distinctions in the molecular nature of the odorant receptor in these organisms (Benton, 2006).
The role of ORs in translating the odorous environment into neuronal activity depends critically on their localization to the surface of the ciliated sensory endings of OSN dendrites. How ORs navigate from their site of synthesis in the ER to these specialized sensory compartments is poorly characterized but has long been suspected to depend upon olfactory-specific cofactors because of the difficulty in functionally expressing these proteins in heterologous cells. The data show that in insects an OR protein has been adapted to subserve a new cellular function: to traffic structurally similar ligand-binding ORs to olfactory cilia (Benton, 2006).
The observation that OR83b can localize to chemosensory cilia in the absence of associated ORs rules out the possibility that only the heteromeric OR/OR83b complex is transport-competent. Instead, the data indicate that OR83b itself can associate with the transport pathway in OSNs and functions to link ORs to this transport machinery. Because OR83b can promote OR localization to cilia in mechanosensory neurons, it likely couples to a general ciliary transport pathway, without the requirement for additional OSN-specific cofactors (Benton, 2006).
Analysis of the temporal requirement of OR83b also reveals an essential role for OR83b in maintaining ORs within this sensory compartment, since OR22a/b is never detected in cilia in the absence of OR83b. Together with observation of the persistence of OR/OR83b heteromeric complexes in the sensory compartment, these results strongly suggest that OR83b is an integral and stable component of the insect OR complex necessary for both proper localization and stability of the conventional ORs in dendrites, rather than a transient chaperone that shuttles and deposits monomeric ORs in the ciliary membrane (Benton, 2006).
While Drosophila ORs do not contain any known protein motifs, the strongest homology within members of this family spans predicted TM6 and TM7, suggesting that this region might mediate a function common to all ORs. Consistent with this conservation, it was found that the loop linking these predicted TM domains (IC3) forms at least part of the interaction interface between ORs and OR83b. In OR83b, this region is almost fully conserved between insect orthologues, and this may explain why OR83b orthologues from diverse insects can functionally substitute for OR83b in Drosophila. Comparison of OR83b with other ORs reveals the presence of a 70-amino-acid insertion in IC2 that is unique to OR83b. The data suggest this loop is located in the cytoplasm, and it is speculated that the insertion links OR83b to the intracellular transport machinery (Benton, 2006).
Most mammalian ORs fail to reach the cell surface when expressed in heterologous cells but are largely trapped in aggregates in the ER and are eventually degraded. This is remarkably similar to the fate of Drosophila ORs in Or83b mutants. OR trafficking in mammals appears to have been solved differently from insects. Screens for olfactory-specific genes that facilitate OR localization have identified a number of single TM domain accessory factors that associate with but are structurally unrelated to the ORs: REEP, RTP1, and RTP2 for mouse ORs and MHC class 1b proteins for mouse V2R pheromone receptors (Loconto, 2003; Saito, 2004). A single TM domain protein, ODR-4, has also been shown to be required for OR trafficking in C. elegans but, unlike OR83b, ODR-4 is not present in sensory cilia (Benton, 2006 and references therein).
Heterodimerization of structurally related chemosensory receptors is important for mammalian taste perception, but this modulates the ligand-binding properties of these receptors rather than their subcellular localization. Thus, the requirement for a universal co-receptor for chemoreception appears to be unique to the Drosophila OR family, and this relies on the remarkable property of OR83b to couple both to the conserved ciliary trafficking machinery and probably to all 61 members of the highly divergent OR family (Benton, 2006).
The presence of OR/OR83b complexes in the sensory compartment raises the possibility that OR83b has additional functions in olfactory perception. This is difficult to address in vivo because of the essential requirement for OR83b in OR localization. However, the diversity in odor response profiles of different classes of OR83b-expressing neurons makes it unlikely that OR83b itself recognizes ligands. This has been confirmed experimentally in Or22a/b mutant neurons, which do not respond to odors even though OR83b is present in these sensory cilia. In vitro studies have revealed that some ectopically expressed ORs in heterologous cells are capable of recognizing odors in the absence of OR83b, albeit with low efficiency. The odor-response profiles of these ORs is similar to that observed in vivo, which suggests that OR83b is unlikely to influence OR ligand specificity but could facilitate efficient ligand-receptor interactions by, for example, maintaining the conformation of ORs within the ciliary membrane (Benton, 2006).
Although the site of ligand interaction in insect ORs and GRs is unknown, a naturally occurring single amino acid polymorphism (A218T) in GR5a influences the sensitivity of this receptor to trehalose. Previously, this residue was thought to lie in the second intracellular loop, where it was proposed to affect coupling of GR5a to G proteins. In the topology model for insect chemosensory receptors proposed here, this residue is predicted to be extracellular and may instead influence ligand binding (Benton, 2006).
The molecular components of primary olfactory signal transduction pathways in insects are unknown. However, in vivo misexpression experiments indicate that ORs can function in other OR-expressing OSNs. This suggests that ORs converge on a common signaling cascade in Or83b neurons, and it is attractive to suggest that OR83b might form part of this common pathway. The capacity of OR83b to couple to downstream signal transduction components is supported by the observation that OSNs expressing OR83b but lacking conventional ORs show spontaneous activity while Or83b mutant OSNs do not. The demonstration that OR/OR83b complexes can function in Gr21a neurons suggests that OR83b may not define a signal transduction pathway that is unique to OR-expressing sensory neurons, but rather that the divergent OR and GR chemosensory receptor families can couple to a common cascade (Benton, 2006).
The structural distinction between insect and mammalian ORs begs the question of whether G proteins are involved in insect chemosensory signaling. In both mammals and C. elegans, loss-of-function genetics provides strong support for a role of G proteins in olfactory signal transduction. In contrast, no direct experimental evidence exists for G protein signaling downstream of insect ORs. Several G alpha subunits, in particular Gαq, are expressed in insect antennae, but they are not specifically enriched in the ciliated dendrite of OSNs. Reduction of Gαq levels in Drosophila OSNs produces defective behavioral responses to some odor stimuli, although it is not known whether this is due to a primary defect in olfactory signal transduction. Surprisingly, OR/OR83b odor-evoked signaling is observed in heterologous cells with or without co-expression of exogenous insect Gαq proteins, suggesting that these proteins have the capacity to couple to endogenous, but unknown, signaling molecules. Thus, despite the widespread assumption that insect chemoreception employs a canonical G protein-signaling cascade, the evidence in support of this is inconclusive (Benton, 2006).
While defining the molecular nature of insect olfactory signaling remains an important goal, the observation that insect ORs are structurally unrelated to the GPCR superfamily raises two equally intriguing possibilities. Insect chemosensory receptors may represent a second family of polytopic TM proteins that has evolved independently to couple to G proteins. Alternatively, these receptors may not couple to G proteins but activate a distinct signaling cascade in response to odor stimulation (Benton, 2006).
While ORs in mammals and insects share a common function in translating odor stimuli into neuronal activity, these findings reveal fundamental differences in the molecular basis of olfactory perception in these organisms. That their OR families should have unrelated evolutionary origins highlights the remarkable convergence in the anatomical and physiological mechanisms that mammals and insects display in the representation of odors in their peripheral circuits. This work raises important questions about the mechanism of odor recognition and olfactory signal transduction in insects. Furthermore, the demonstration that the OR/OR83b complex is the essential molecular unit of olfactory perception in insects makes this complex an attractive target for the development of highly selective insect repellents to interrupt chemosensory-driven, host-seeking behaviors of insect vectors of human disease (Benton, 2006).
The expression of Or83bhas been described only anecdotally (Vosshall, 1999; Vosshall, 2000): a detailed characterization of the distribution and subcellular localization of this protein is presented in this study. The fly has olfactory sensory neurons (OSNs) in three specialized structures: the dorsal organ of the larva and the maxillary palps and third antennal segments on the adult head. Or83b expression is restricted to OSNs and is not detected in other tissues, including gustatory neurons. The expression of the Or83b mRNA was examined throughout the four main developmental stages of Drosophila. Or83b is first detected late in embryonic development at stage 15, where its expression is limited to the antennal-maxillary complex, an anterior structure that contains the OSNs. Later in development, it is expressed in all 21 OSNs of the larval dorsal organ located at the anterior tip of the larva. During metamorphosis, all of these larval OSNs are destroyed and replaced by new OSNs that populate the antenna and maxillary palp. In the pupa, Or83b is first detected in antennal OSNs at 80 hr after puparium formation, which is late in pupal development and approximately coincident with the onset of expression of conventional OR genes. Or83b expression in all adult maxillary palp neurons and ~70%-80% of antennal OSNs. The exact proportion of neurons that express Or83b in the antenna is not known and is difficult to estimate because Or83b is expressed at different levels in different subpopulations of OSNs. Levels are highest at the dorsal-medial region (Larsson, 2004).
Both in the antenna and maxillary palp, each Or83b positive OSN expresses Or83b along with at least one conventional OR gene. Or22a/band Or83b mRNA overlap in a group of dorsal-medial neurons. An anti-Or83b antibody was raised against loop residues between transmembrane domains three and four and this antibody was used to determine the subcellular localization of Or83b. Or83b protein is found in ciliated OSN dendrites, the chemosensory specialization in which OR proteins are localized and where olfactory signal transduction occurs. Double immunostaining with a rabbit anti-Or22a/b antibody and a mouse anti-Or83b antibody demonstrates that these two OR proteins colocalize in the distal portion of the dendrite inserted into the sensory hair. Or83b is also detected in OSN cell bodies but is not seen in proximal axons in the antenna or at axonal termini in the antennal lobe of the brain, suggesting that its function lies in the ligand-detecting region of these polarized sensory neurons (Larsson, 2004).
To examine the connectivity of Or83b-expressing neurons, an Or83b promoter-enhancer trans-gene (Wang, 2003) in combination with the synaptic marker, n-synaptobrevin-Green Fluorescent Protein was used to visualize projections of these neurons in the antennal lobe. To do this, advantage was taken of the two-component gene regulation system that uses the yeast Gal 4 transcription factor fused to the Or83b regulatory regions and flies carrying this driver transgene were crossed to a UAS-nsyb-GFP responder transgene. Consistent with the apparent medial to lateral gradient of Or83b expression in the antenna, there is a similar gradient of fibers innervating the antennal lobe. Dorsal and medial glomeruli, including those that receive input from Or22a/b-expressing OSNs were brightly labeled. Lateral glomeruli receive considerably less innervation. Confocal sections through central and posterior aspects of the antennal lobe clearly show the sparsely innervated lateral and ventral glomeruli. Notably, the V glomerulus receives no apparent input from Or83b-expressing neurons (Larsson, 2004).
To study the loss of function of Or83b, null mutants were generated in which the putative transcription start site and the first five transmembrane domains of this seven transmembrane domain receptor protein were deleted by gene-targeting techniques designed to replace these portions of the Or83b coding region with the white gene, a selectable eye color marker. 14 homozygous mutants were obtained and confirmed to be null by Southern blotting with probes that lie both 5' and 3' of the targeted locus and by PCR directed against the Or83b coding region. The homozygous mutants produce viable and fertile adult progeny with no gross developmental or physical defects. Or83b mutant larvae show normal locomotor and gustatory behavior. Three mutant lines (Or83b1, Or83b2, Or83b3) were selected for further characterization, in addition to a genetically matched control line in which the targeting construct integrated but left the Or83b region intact (Larsson, 2004).
No Or83b mRNA or Or83b protein is detectable in the homozygous Or83b2 mutant strain in Or83b-expressing neurons. Antibodies directed against the terminal cytoplasmic loop of Or83b, encoded by DNA sequences left intact by the targeted deletion, also show no detectable residual Or83b protein. This rules out the possibility that partial expression of a truncated protein could interfere with mutant analysis by causing dominant-negative effects. Similar expression results were obtained with the Or83b2 and Or83b3 strains (Larsson, 2004).
In animals carrying an Or83b rescuing transgene under the control of the Or83b-Gal4 driver, Or83b expression is partially restored. The medial to lateral gradient of Or83b expression is exaggerated in these animals, producing lower than normal levels of Or83b expression in the lateral-distal domains of the antenna. Rescue of Or83b expression in mutants under control of this transgenic driver in larvae also show lower than wild-type levels in certain dorsal organ neurons (Larsson, 2004).
Because Or83b is broadly coexpressed with other ORs, it was asked whether the loss of Or83b function has an effect on the distribution or levels of expression of other coexpressed ORs. In adult olfactory neurons, both Or22a/b and Or43b proteins localize to OSN dendrites in wild-type antenna. Or22a/b is particularly efficiently transported into the dendrite with little protein detected in the cell body, while Or43b is distributed in a manner similar to Or83b in both sensory dendrite and cell body. In all three Or83b mutants both Or22a/b and Or43b proteins are weakly detected in the cell body but no dendritic staining is seen. The available reagents do not permit determination of whether residual Or22a/b and Or43b proteins are intracellular or on the surface of the cell body in Or83b mutants. In control experiments, no signal is detected with these antibodies in Or22a/b and Or43b null mutants. Restoring Or83b gene function by transgenic rescue restores the subcellular localization of both Or22a/b and Or43b to the chemosensory dendrite (Larsson, 2004).
Because odorant binding and signal transduction occurs in the dendrite, the OR mislocalization defect found in Or83b mutants would be expected to disrupt odor-evoked physiology. It is therefore of great importance to determine whether this failure of conventional OR proteins to be localized in the dendrite reflects a specific requirement of Or83b or is a nonspecific effect of a sick neuron. Therefore a number of control experiments were carried out to determine whether other aspects of the biology of OSNs is normal in Or83b mutants. The stereotyped targeting of Or22a-expressing axons to the DM2 glomerulus in the antennal lobe is not affected in the mutant. Or22a-nsyb-GFP-positive fibers are found restricted to only the DM2 glomerulus, and no ectopic innervation is seen. Levels of this synaptic marker are considerably lower in the mutant than in wild-type, which likely is a direct consequence of the disruption of neuronal activity in these neurons. Similar results were obtained on reduced trafficking of both membrane-targeted and synaptically targeted marker proteins in neurons that have been silenced genetically, perhaps because of a general effect on axonal and synaptic membrane protein transport in inactive neurons. This suggests that Or83b is not required for the establishment or maintenance of synaptic connections in glomeruli and that these connections persist in the absence of neuronal activity. The gross morphology of OSNs is normal in Or83b mutants, as judged by the levels and distribution of the neuron-specific microtubule associated protein, Futsch, recognized by the 22c10 monoclonal antibody. In an additional control experiment, a mix of antisense DOR gene probes was hybridized to wild-type and mutant antennae and it was found that the numbers, distribution, and levels of gene expression of these OR genes are comparable in both genotypes (Larsson, 2004).
Finally, the subpopulation of Or22a/b neurons was electrically silenced in wild-type animals to determine if loss of neuronal activity per se affects the accumulation or maintenance of Or22a/b and Or83b protein in dendrites. Expression of the inward rectifying potassium channel Kir2.1 in Or83b-expressing neurons eliminates EAG responses to all odors tested. However, normal dendritic localization of Or22a/b and Or83b is maintained under conditions of prolonged electrical silencing that mimic other aspects of the Or83b phenotype, including the reduced levels of synaptic markers in OSN axonal termini This suggests that the dendritic localization defect seen in Or83b animals reflects a specific role of this gene in protein localization and is not a secondary consequence of a loss of neuronal activity (Larsson, 2004).
The effect of the Or83b mutation on OR localization in the 21 neurons of the larval dorsal organ was investigated. These neurons are clustered into a ganglion containing the cell bodies, each of which extends a dendrite that inserts into the dorsal organ dome. Cytoplasmic lacZ marker protein labels the cell bodies and basal portions of the dendrites but is excluded from the distal tip of the dendrite inserted in the dome. The Or83b-Gal4 driver was used to misexpress an adult OR, Or22a, in all larval OSNs. Or22a protein is localized to the distal dendritic tips as seen in an exterior view of the dorsal organ dome. Lower levels of Or22a are detected in the cell bodies (Larsson, 2004).
In Or83b mutant larvae, no Or22a protein is detected in dendrites and low levels are seen in the cell bodies. In wild-type animals, Or83b protein is detected in distal dendritic tips inserted into the dorsal organ dome as well as the OSN cell bodies but is not detected in Or83b mutants. The accumulation of the membrane bound marker CD8-GFP in the distal dorsal organ dendrites is maintained in Or83b mutant OSNs, ruling out a general defect in dendritic morphology or protein sorting in the mutants. Therefore, larval OSNs have the same requirement for Or83b gene function in localizing ORs to the site of odor interaction in dendrites as adult OSNs (Larsson, 2004).
The dramatic and selective effect of the Or83b mutation on OR localization in olfactory dendrites would be expected to eliminate odor-evoked potentials in these neurons. To test this, electroantennograms (EAGs), which are thought to reflect broad domains of receptor potentials summed around the site of the recording electrode in the fly antenna, were measured. Control preparations show strong negative potentials in response to all odorants tested. EAGs recorded in Or83b mutant antennae produce no responses, and normal EAG responses are restored by transgenic rescue of Or83b gene function. These results are consistent with a general requirement for Or83b in adult olfactory function to a broad range of odor stimuli (Larsson, 2004).
EAGs provide a convenient measure of olfactory function across the entire antenna but suffer from a lack of single cell resolution and have low sensitivity. Therefore, to be certain that this mutation blocks odor-evoked activity at the single neuron level, extracellular single sensillum recordings were carried out on the identified ab1 sensillum, which contains three general odor-responsive neurons and a single neuron (ab1C) highly tuned to carbon dioxide. In wild-type antennae, all four neurons in the ab1 sensillum show characteristic odor responsivity. Or83b mutant antennae show no odor-evoked action potentials to the general odorants ethyl acetate (ab1A), acetoin (ab1B), or methyl salicylate (ab1D), but responses to carbon dioxide (ab1C) are completely normal. Unlike the case of neurons with deficiencies in conventional odorant receptors, Or83b mutant OSNs generally show very little spontaneous activity. Evidence of spontaneous or damage-induced activity of the general odor neurons were occasionally found in these animals, which may reflect low levels of spontaneous activity independent of receptor activation. Restoring Or83b gene function to antennal neurons by transgenic rescue also restores wild-type odor responses to the three affected neurons of the ab1 sensillum (Larsson, 2004).
The dramatic loss of odor-evoked potentials to a broad range of general odors at the level of the whole antenna and single neurons suggests a general and essential role for Or83b in odor detection. To determine the behavioral effects of this mutation on larval behavior, chemotaxis was monitored with a single-animal video-tracking assay, modified from a previously described population assay. Tracks of animals responding to ethyl acetate were examined. While the control yw animal chemotaxes toward the stimulus, the Or83b1 mutant larva does not respond to the odor and wanders at random around the plate. Expression of a rescuing UAS-Or83b transgene under control of the Or83b-Gal4 driver in the Or83b1 mutant restores chemotaxis behavior. Olfactory responses of animals with these three genotypes were tested to 36 odors. Control yw larvae chemotax to all 36 odors, while mutant larvae showed no response to 34 of these odors and weak responses to 2. The Or83b rescuing transgene partially restores chemotaxis in Or83b1 mutants, consistent with the nonuniform expression of the Or83b-Gal4 driver in the larval dorsal organ. Similar results were obtained with two additional mutant and control strains to a subset of these odors (Larsson, 2004).
To determine whether the electrophysiological defects seen in the antenna translate to behavioral phenotypes, a modified trap assay was performed. Adult flies show strong behavioral responses to natural odors and synthetic odor blends but respond to only a subset of the single odors composing these blends. The chemosensory behavior of adult Or83b mutant flies was tested with two of these behaviorally active single odors, acetoin and 2-phenylethanol. As expected from the absence of odor-evoked physiological responses to these odors, Or83b mutants show severely impaired responses to acetoin and are anosmic to 2-phenylethanol. Normal responses to acetoin and 2-phenylethanol are restored in Or83b2 mutants carrying a UAS-Or83b rescuing transgene under control of the Or83b-Gal4 driver (Larsson, 2004).
A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants. OSNs also exhibit odor-evoked responses with complex temporal dynamics, but the contribution of this activity to behavioral odor discrimination has received little attention. This study investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. Flies can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies distinguish one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination (DasGupta, 2008).
In conclusion, multiple classes of OSNs are not required for flies to discriminate odorants. Although flies without functional Or83b-expressing neurons cannot learn to discriminate between a number of chemically distinct odorants, providing a single class of Or83b-expressing OSNs restores learned discrimination between two odorants that activate that particular OSN class. These flies cross-adapt to odorants that activate the restored OSNs, demonstrating that the relevant OSNs are the same, thus challenging a requirement for discrete spatial codes for odorants in the antennal lobe. As expected, flies with one class of Or83b-expressing OSNs have limitations and can apparently only encode one odorant receptor that activates the appropriate receptor at a time. These data suggest that a benefit of having multiple classes of OSNs is the ability to identify certain odorants present within a more complex milieu. Importantly, Or83b2 mutant flies with one functional class of Or83b-expressing OSNs choose appropriately between two odorants even though the absolute and relative concentration was changed between training and testing, implying that they encode odorant identity and do not only rely on encoding odorant intensity (DasGupta, 2008).
Finding that distinct combinatorial spatial patterns of OSN activation in the antennal lobe are not essential to represent odorant information implies an important role for odorant-evoked temporal dynamics. Previous studies in insects and vertebrates have documented considerable temporal complexity in odor-evoked activity at successive layers of the olfactory system, but few have investigated the behavioral relevance. Recent work has shown that excitatory and inhibitory lateral connectivity in the Drosophila antennal lobe can shape projection neuron responses; therefore, it can be expected that different temporal signals in the same OSNs generate distinct temporal, and perhaps spatial, patterns of projection neuron activity. However, because flies with a single functional class of Or83b-expressing OSNs lack the lateral input driven by additional classes of OSN, it will be important to determine how lateral connectivity within the antennal lobe contributes to odorant discrimination in Drosophila (DasGupta, 2008).
Reference names in red indicate recommended papers.
Benton, R., Sachse, S., Michnick, S. W. and Vosshall, L. B. (2006). Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 4(2): e20. 16402857
DasGupta, S. and Waddell, S. (2008). Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe. Curr. Biol. 18(21): 1668-74. PubMed Citation: 18951022
Dobritsa, A. A., van der Goes van Naters, W., Warr, C. G., Steinbrecht, R. A. and Carlson, J. R. (2003). Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37: 827-841. 12628173
Elmore, T., Ignell, R., Carlson, J. R. and Smith, D. P. (2003). Targeted mutation of a Drosophila odor receptor defines receptor requirement in a novel class of sensillum. J. Neurosci. 23: 9906-9912. 14586020
Fishilevich, E., Domingos, A. I., Asahina, K., Naef, F., Vosshall, L. B. and Louis, M. (2005). Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila. Curr. Biol. 15(23): 2086-96. 16332533
Hallem, E. A., Ho, M. G. and Carlson, J. R. (2004a). The molecular basis of odor coding in the Drosophila antenna. Cell 117: 965-979. 15210116
Hallem, E. A., Fox, A. N., Zwiebel, L. J. and Carlson, J. R. (2004b). Olfaction: mosquito receptor for human-sweat odorant. Nature 427: 212-213. 14724626
Hill, C. A., et al. (2002). G protein-coupled receptors in Anopheles gambiae. Science 298(5591): 176-8. 12364795
Ishii, T., Hirota, J. and Mombaerts, P. (2003). Combinatorial coexpression of neural and immune multigene families in mouse vomeronasal sensory neurons. Curr. Biol. 13: 394-400. 12620187
Krieger, J., Klink, O., Mohl, C., Raming, K. and Breer, H. (2003). A candidate olfactory receptor subtype highly conserved across different insect orders. J. Comp. Physiol. [A] 189: 519-526. 12827420
Larsson, M. C., Domingos, A. I., Jones, W. D., Chiappe, M. E., Amrein, H. and Vosshall, L. B. (2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43(5): 703-14. 15339651
Loconto, J., Papes, F., Chang, E., Stowers, L., Jones, E.P., Takada, T., Kumanovics, A., Fischer Lindahl, K. and Dulac, C. (2003). Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class molecules. Cell 112: 607-618. 12628182
Neuhaus, E. M., et al. (2005). Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster. Nat. Neurosci. 8(1): 15-7. Medline abstract: 15592462
Pitts, R. J., Fox, A. N. and Zwiebel, L. J. (2004). A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae. Proc. Natl. Acad. Sci. USA 101: 5058-5063. 15037749
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 citation: 18408712
Saito, H., Kubota, M., Roberts, R. W., Chi, Q. and Matsunami, H. (2004). RTP family members induce functional expression of mammalian odorant receptors. Cell 119: 679-691. 15550249
Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725-736. 10089887
Vosshall, L. B., Wong, A. M. and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102: 147-159. 10943836
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. 12553914
Wicher, D. et al. (2008). Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452(7190): 1007-11. PubMed citation: 18408711
date revised: 10 August 2009
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