BIOLOGICAL OVERVIEW

Animals are able to sense and discriminate among a remarkable number of odors. Olfactory information is received and encoded by olfactory receptor neurons (ORNs). These neurons encode the quality and intensity of odors, as well as aspects of their spatiotemporal distribution. The code is in the form of action potentials and is based on the differential responses of ORNs to different olfactory stimuli. The signals generated by ORNs are transmitted to the brain, where processing takes place (Dobritsa, 2003).

ORNs vary in their odor specificity, sensitivity, and response dynamics. The cellular basis of the odor code has been explored in detail in Drosophila, whose relatively simple olfactory system allows precise physiological measurements of individual ORNs in vivo. Flies contain two olfactory organs, the antenna and the maxillary palp, which contain ~1200 and ~120 ORNs, respectively (Stocker, 1994; Shanbhag, 1999, 2000). These ORNs are compartmentalized in olfactory sensilla, which divide into morphologically distinct classes, including large basiconic sensilla, small basiconic sensilla, trichoid sensilla, and coeloconic sensilla. Each sensillum contains up to four neurons, whose activities can be defined by extracellular electrophysiological recordings (Dobritsa, 2003 and references therein).

In Drosophila, extensive recordings have revealed that ORNs fall into distinct functional classes based on their odor response spectra. Sixteen functional classes of ORNs, each with a unique response spectrum to a panel of 47 odors, were identified from recordings of antennal basiconic sensilla (De Bruyne, 2001). These ORNs exhibit diverse response dynamics, including excitatory and inhibitory responses, and various modes of termination kinetics. The 16 ORN classes are found in stereotyped combinations in seven functional types of basiconic sensilla, each mapping to a defined subregion of the antennal surface (Dobritsa, 2003).

Functional differences among ORN classes are believed to arise from the expression of different odor receptors. A family of at least 60 seven-transmembrane-domain receptor genes, the Or genes, was discovered in Drosophila and proposed to encode odor receptors (Clyne, 1999a; Gao, 1999; Vosshall, 1999, 2000). Individual Or genes are expressed in different subsets of ORNs. A mutation that alters the expression of a subset of Or genes alters the odor specificity of a subset of ORNs (Clyne, 1999b), and direct evidence (Störtkuhl and Kettler, 2001; Wetzel, was recently found for the involvement of one Or gene in olfactory signaling (Dobritsa, 2003).

The isolation of Or genes and the functional identification of discrete ORN classes by physiological analysis now allows a critical problem to be explored in Drosophila: the integration of the molecular and cellular maps of the olfactory system. Three means of mapping the receptor repertoire to the neuronal repertoire are demonstrated in this study. Moreover, since the odor specificities of the neurons are defined, the results by extension map receptor space to odor space. This approach allows an integrated molecular and cellular definition of the basis of odor coding (Dobritsa, 2003).

Individual receptors are demonstrated to map to individual neuronal classes through a genetic and molecular analysis of two Or genes, Or22a and Or22b. There are three functional types of large basiconic sensilla, ab1, ab2, and ab3, defined on the basis of electrophysiological recordings from the ORNs they contain. The sensilla expressing Or22a and Or22b contain an A neuron whose strongest responses are to ethyl butyrate, pentyl acetate, and ethyl acetate and a B neuron whose strongest responses are to heptanone, hexanol, and octenol. The Or22a receptor is shown to map to the ab3A neuron, by using the Or22a promoter and the GAL4-UAS system to drive expression of GFP or the cell death gene reaper, followed by physiological recordings from individual sensilla. The Or22a receptor is thereby linked to the odor ethyl butyrate, to which ab3A is highly sensitive (Dobritsa, 2003).

Analysis of a mutant lacking Or22a, together with rescue experiments using an Or22a transgene, confirm the mapping of Or22a to the ab3A neuron. This genetic analysis provides direct evidence that an Or gene is required in vivo for normal odor detection (Dobritsa, 2003).

Evidence is provided that Or22b is coexpressed with Or22a in the same cell but that Or22b is neither necessary nor sufficient for ab3A function; rather, the broad response spectrum of the ab3A neuron is accounted for by a single receptor. Ectopic expression of another receptor, Or47a, in the mutant ab3A neuron is used to identify the ORN from which Or47a derives and to determine its odor specificity. These results show that the odor response spectrum of an ORN in Drosophila depends on the Or gene that it expresses. Expression of Or47a in a wild-type ORN shows that two receptors are able to function in a single cell (Dobritsa, 2003).

Finally, the possibility of a developmental role for Or genes is addressed, a possibility that has not been systematically investigated by expression analysis and that can be rigorously determined only by functional analysis. The ab3A ORN is able to navigate toward its target in the CNS if Or22a and Or22b are deleted or substituted by other receptor genes (Dobritsa, 2003).

The demonstration that deletion mutants lacking Or22a and Or22b are defective in odor response and that the response is restored upon introduction of an Or22a transgene provide direct evidence that Drosophila Or genes are in fact critical components of olfactory signal transduction. The effect of the deletion mutation is specific: the mutation has a profound effect on the ab3A neuron but no other ORN among the large basiconic sensilla. The response of the ab3A cell is eliminated for all odors tested. Most ab3A neurons in mutant flies exhibited spontaneous electrical activity, albeit at a low rate, and thus the absence of Or22a/b does not lead to the immediate death of these neurons. The loss of Or22a/b does not appear to lead to the de novo expression of another functional receptor, suggesting that the process of receptor gene choice does not include a receptor-mediated negative feedback mechanism (Dobritsa, 2003).

A central problem in odor coding concerns the distribution of odor receptors among ORNs. A priori, there are several ways of distributing n receptor types among m functional classes of ORNs. Each receptor type could be expressed in a single ORN class, or, by contrast, in multiple, distinct ORN classes. If expressed in a single ORN class, then a receptor, R_i, could in principle be the only receptor expressed in its class, or it could be one of multiple receptors, e.g., (R_i, R_j), that are invariably coexpressed in that class. If a receptor is expressed in multiple, distinct ORN classes, then the ORN classes may be functionally distinct either because they express different combinations of receptors, e.g., (R_i, R_j) versus (R_i, R_k), or, conceivably, because each expresses the same receptor in different molecular contexts, (R_i; X) versus (R_i; Y), containing ORN class-specific differences in local OBPs, RAMPs, or heterodimerization partners, for example (Dobritsa, 2003).

The expression of the Or22a receptor was found to be limited to a single morphological subtype of olfactory sensillum (LB-I), a single functional type of sensillum (ab3), and a single class of ORN (ab3A). The mapping of Or22a to a single functional type of neuron argues against a model in which different neuronal classes acquire their diverse identities through the combinatorial expression of different receptors, or through the expression of a single receptor in different molecular contexts; according to these models, an individual receptor would be expressed in multiple, distinct neuronal classes (Dobritsa, 2003).

ORNs vary in the breadth of their odor response spectrum. Physiological recordings from individual ORNs have shown that some are narrowly tuned, whereas others are broadly tuned with respect to a panel of test odors (De Bruyne, 1999, 2001). For example, the ab5A cell responds to only one of 47 odors tested at relatively high doses, whereas the ab3A neuron responds to a variety of esters, alcohols, ketones, and other odors of varying chain lengths. The broad tuning specificity of ORNs such as ab3A could in principle be due either to the expression of multiple receptors or to the expression of a single receptor that is broadly tuned. The finding that deletion of Or22a and Or22b eliminates response to all tested odors, and that the full response spectrum can be rescued by Or22a alone, suggests that the broad response spectrum documented for ab3A can be attributed to one receptor, Or22a (Dobritsa, 2003).

The results with Or47a are also consistent with a model in which a single receptor accounts for the odor response profile of a particular ORN class. When expression of a single Or gene, Or47a, is driven in an ab3A cell lacking expression of Or22a and Or22b, Or47a confers an odor response profile like that of ab5B. These results support an interpretation in which the odor response profile of ab5B derives from the expression of a single Or gene, Or47a. Functional analysis of other Or genes will be required to determine the generality of these results, but they are consistent with observations made with mammalian ORNs, which are able to respond to diverse odors, apparently by virtue of expression of a single odor receptor in many (Malnic, 1999; Araneda, 2000; Bozza, 2002), if not all (Rawson, 2000; Spehr, 2002), cases (Dobritsa, 2003).

While these results are consistent with a model in which Or22a is the only receptor that functions in the ab3A neuron, the data force consideration of the possibility that Or22a is not the only receptor that is expressed in the ab3A neuron. Both Or22a-GAL4 and Or22b-GAL4 drive expression of GFP in ab3 sensilla and of rpr in ab3A neurons. The driver constructs were designed to mimic the expression of the endogenous Or22a or Or22b genes, respectively. In each construct, GAL4 coding sequences replace the coding sequences of the respective Or genes, and they include a substantial amount of DNA upstream of either Or22a or Or22b (8.2 and 10.3 kb, respectively). While it is formally possible that the expression of Or22b-GAL4 does not mimic the expression of Or22b in vivo, there is no evidence for expression of Or22b in ORNs of the fly other than ab3A: (1) in situ labeling with Or22b probes revealed no labeling outside the region of the antenna that contains ab3 sensilla; (2) immunolabeling showed no staining of sensilla other than LB-I in the wild-type antenna, nor the maxillary palp nor the larval olfactory organ. At the same time, it is clear that the antibody can recognize an Or22b product, since immunolabeling is observed in antennae lacking Or22a but expressing Or22b. Moreover, Or22b transcripts have been amplified from the antenna by RT-PCR or found in antennal/maxillary palp cDNA libraries in multiple laboratories (Clyne, 1999a; Vosshall, 1999). The simplest interpretation of all these data, taken together, is that Or22b is coexpressed with Or22a in ab3A neurons, but that Or22b is neither necessary nor sufficient for response to the odors used in this study (Dobritsa, 2003).

A functional role for Or22b is nonetheless suggested by the observation that an Or22b ortholog is present in D. simulans, which diverged from D. melanogaster ~2.5 million years ago. Most important, in neither species has the gene accumulated stop codons, frameshift mutations, or deletions. One possibility is that Or22b recognizes odors not tested in this study, such as pheromones. Another possibility is that it functions only under a specific set of epigenetic, e.g., environmental, conditions (Dobritsa, 2003).

Does the odor response spectrum of a cell depend exclusively on its receptor expression or on a more complex molecular context? In the case of Or47a, the substitution of Or47a for Or22a and Or22b engenders a transformation of the response spectrum from that of ab3A to that of ab5B. These results are consistent with evidence from other organisms that the odor response spectrum of an ORN depends on the odor receptor gene that it expresses (Dobritsa, 2003).

ORNs in insects are intimately associated with each other in sensillar compartments. In this study the activity of an ORN was measured following genetic manipulation of its neighbor. When the function of ab3A was severely compromised, either by mutation of Or22a and Or22b or by expression of the cell death gene rpr, the neighboring ab3B cell showed strong responses to odors. Thus, the ability of ab3B to respond to odors does not depend absolutely on the presence of a functional neighboring cell (Dobritsa, 2003).

At the same time, however, in both cases a large increase was noted in the response of ab3B to pentyl acetate. It is formally possible that this effect may arise to some extent from difficulties in counting the small ab3B spikes during intense activity of the ab3A cell. However, another interpretation is that in wild-type the activity of ab3A inhibits the response of ab3B; when this inhibition is relieved, ab3B exhibits an increased response. In some insects, neighboring ORNs in certain sensilla have been shown to respond to odors whose behavioral significance is related (Wojtasek, 1998; Grant, 1998). It seems plausible that information transmission between adjacent ORNs represents an early step in the processing of information carried by neighboring ORNs (Dobritsa, 2003 and references therein).

An emerging body of evidence in mammals indicates that odor receptor expression is essential to normal axonal pathfinding in the vertebrate olfactory system. The results of this study indicate, however, that the ab3A neuron finds its normal glomerular target in a mutant that lacks Or22a and Or22b expression, demonstrating that these receptors are not required for targeting. Moreover, ectopic expression of either of two other receptors did not cause alterations in the targeting. The possibility that Or22a and Or22b play a subtle role cannot be excluded, or that ab3A expresses an additional Or gene that plays a role in pathfinding, such as Or83b, which is expressed widely among ORNs (Vosshall, 1999, 2000) and whose function is unknown. However, the simplest interpretation of these results is that ab3A finds its glomerular targets through mechanisms independent of Or expression (Dobritsa, 2003).

One striking difference between the insect and mammalian olfactory systems is that in mammals, but not insects, ORNs are regenerated throughout adult life. Thus, pathfinding of mammalian ORNs toward their target glomeruli occurs during adult life, whereas in insects axonal pathfinding occurs only during development. Insect ORN pathfinding likely depends on a system of navigational cues that are expressed in an orchestrated temporal and spatial program. Perhaps the mammalian dependence on Ors, which are expressed both during development and during adult life, reflects the evolution of a mechanism designed to operate independently of signals that occur only transiently in development (Dobritsa, 2003).

A second difference between insect and mammalian olfactory systems is the greater numerical complexity of mammals. The number of ORNs and glomeruli in mammals exceeds that of Drosophila by more than an order of hmagnitude, and it is likely that the mammalian olfactory system accordingly requires more information to specify the larger number of connections. Perhaps the use of extant Ors to provide developmental cues may have been the most economical means of expanding the informational content of the navigational system during evolution (Dobritsa, 2003).

GENE STRUCTURE

Or22a and Or22b, the first Or genes identified in a computational screen for Drosophila odor receptors (Clyne, 1999a), are tightly clustered, lying within 650 bp of each other in the genome. Clustering is common among Or genes, with more than one-third of the family members located in clusters of up to three genes (Dobritsa, 2003).

cDNA clone length - 1305 (Or22a) and 1321 (Or22b)

Exons - 4 (both Or22a and Or22b)

PROTEIN STRUCTURE

Amino Acids - 397 (both Or22a and Or22b)

Structural Domains

A two-part strategy was adopted to identify odorant receptor genes from the genomic database. Initially, a computer algorithm was designed to search the Drosophila genomic sequence for open reading frames (ORFs) from candidate odorant receptor genes. Reverse transcription polymerase chain reaction (RT-PCR) was then used to see if transcripts from any of these ORFs were expressed in olfactory organs. For the computational screens, the genomic sequence data obtained by FTP from the Berkeley Drosophila Genome Project was used. ORFs of 300 bases or longer in all six frames were identified. Next, a program written to identify GPCRs statistically by their physicochemical profile was used to screen for candidate ORFs. The number of possible candidates was reduced by comparing them to Drosophila codon usage tables. Further analysis revealed that 8 of the 34 candidate ORFs correspond to genes of known function, as for example, a cyclic nucleotide-gated channel. RT-PCR with primers designed from two of the final candidates yielded amplification products from antennal cDNA. These two genes are located within 500 bp of each other at cytological position 22A, and their predicted proteins are 75% identical at the amino acid level (Clyne, 1999a).

To determine if these two candidates were part of a larger family of genes encoding seven transmembrane domain proteins, their sequences were used in BLAST searches of the Drosophila genome database to identify related genes. Homologs of the two candidates were found, and in turn, their sequences were used for further database searches. In total, 16 genes have been identified from the 16% genomic sequence currently available. This family of genes has been named DOR (for Drosophila olfactory receptor), and each individual gene is named based on its cytogenetic location in the genome. Thus, the two genes identified initially are DOR22A.1 and DOR22A.2, which are abbreviated here as 22A.1 and 22A.2 (The final digit in this nomenclature is used to distinguish the genes at a site and does not refer to the cytogenetic band number). Of the 16 family members, 13 have been found to be expressed in either the antenna or the maxillary palp, or in both, based upon RT-PCR analysis and in situ hybridizations to RNA in tissue sections (Clyne, 1999a).

The Drosophila OR genes have no significant similarities to any known genes, and do not appear in any of the Drosophila EST databases. However, hydropathy plots of the predicted proteins show that each has approximately seven peaks that could represent transmembrane domains. The lengths of the 16 proteins are between 369 and 403 amino acids, similar to the lengths of most previously described families of GPCRs. In addition, the spacing of the putative transmembrane domains gives rise to predicted intracellular and extracellular loops similar in size to those in many families of GPCRs. Amino acid sequence identity among the DOR genes ranges from 10%-75%, with many genes showing a relatively low level of identity to each other (20%). Two pairs of clustered genes, 22A.1/22A.2 and 33B.1/33B.2 show the highest identity, with 75% and 57% identities, respectively. However, not all clustered genes show high degrees of similarity. 33B.3, for example, is only 28% identical to both 33B.1 and 33B.2, and 46F.1 and 46F.2 are only 29% identical. In addition to exhibiting sequence identity, many of the genes contain introns in corresponding locations, consistent with their constituting a family derived from a common ancestral gene. There are 67 residues that are conserved among at least 50% of the genes, and most of these are in the C-terminal halves of the proteins. Among the conserved residues are a serine and a threonine in the intracellular C-terminal tail, residues frequently conserved in this region of GPCRs. The most divergent region in the sequences is a stretch of 30 amino acids representing part of the first extracellular loop and nearly all of transmembrane domain 3. The divergence in this region also occurs in the most conserved pairs of genes: 22A.1 and 22A.2 are 75% identical overall but only 50% identical in this region, and 33B.1 and 33B.2 are 57% identical overall but only 33% identical in this region. Transmembrane domains 3, 4, and 5 are exceptionally divergent in rat odorant receptors and have been proposed to play a role in odorant binding. Some of the genes are clustered in the genome, while others are apparently isolated. Within a cluster, the average intergenic distance is on the order of 500 bp. Clustered DOR genes do not necessarily have introns in corresponding locations (e.g., 46F.1 and 46F.2), but all clustered genes have their transcriptional orientations in the same direction (Clyne, 1999a).

Or22a and Or22b are among the most closely related members of the family, showing 78% amino acid identity. The average identity among Or genes in a cluster is ~45% (Dobritsa, 2003).

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors1 that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila6. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. This study shows that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca²⁺ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling (Wicher, 2008).

date revised: 30 May 2003

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