Searching for the Ligands of Odorant Receptors

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor–ligand interactions.     

Origin of basal activity in mammalian olfactory receptor neurons

Mammalian odorant receptors form a large, diverse group of G protein-coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain.     

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors^1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.     

Rapid encoding and perception of novel odors in the rat

To gain insight into which parameters of neural activity are important in shaping the perception of odors, we combined a behavioral measure of odor perception with optical imaging of odor representations at the level of receptor neuron input to the rat olfactory bulb. Instead of the typical test of an animal's ability to discriminate two familiar odorants by exhibiting an operant response, we used a spontaneously expressed response to a novel odorant—exploratory sniffing—as a measure of odor perception. This assay allowed us to measure the speed with which rats perform spontaneous odor discriminations. With this paradigm, rats discriminated and began responding to a novel odorant in as little as 140 ms. This time is comparable to that measured in earlier studies using operant behavioral readouts after extensive training. In a subset of these trials, we simultaneously imaged receptor neuron input to the dorsal olfactory bulb with near-millisecond temporal resolution as the animal sampled and then responded to the novel odorant. The imaging data revealed that the bulk of the discrimination time can be attributed to the peripheral events underlying odorant detection: receptor input arrives at the olfactory bulb 100–150 ms after inhalation begins, leaving only 50–100 ms for central processing and response initiation. In most trials, odor discrimination had occurred even before the initial barrage of receptor neuron firing had ceased and before spatial maps of activity across glomeruli had fully developed. These results suggest a coding strategy in which the earliest-activated glomeruli play a major role in the initial perception of odor quality, and place constraints on coding and processing schemes based on simple changes in spike rate.     

Coding of odors by a receptor repertoire

We provide a systematic analysis of how odor quality, quantity, and duration are encoded by the odorant receptor repertoire of the Drosophila antenna. We test the receptors with a panel of over 100 odors and find that strong responses are sparse, with response density dependent on chemical class. Individual receptors range along a continuum from narrowly tuned to broadly tuned. Broadly tuned receptors are most sensitive to structurally similar odorants. Strikingly, inhibitory responses are widespread among receptors. The temporal dynamics of the receptor repertoire provide a rich representation of odor quality, quantity, and duration. Receptors with similar odor sensitivity often map to widely dispersed glomeruli in the antennal lobe. We construct a multidimensional "odor space" based on the responses of each individual receptor and find that the positions of odors depend on their chemical class, concentration, and molecular complexity. The space provides a basis for predicting behavioral responses to odors.     

Molecular and cellular basis of human olfaction

The human olfactory systems recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein in the ciliary membrane of olfactory neurons. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed, and characterized some of the human olfactory receptors from chromosome 17. Our results show that a receptor protein is capable of recognizing the particular chemical substructure of an odor molecule and, therefore, is able to respond only to odorants that have a defined molecular structure. These findings represent the beginning of the molecular understanding of odorant recognition in humans. In the future, this knowledge could be used for the design of synthetic ideal receptors for specific odors (biosensors), or the perfect odor molecule for a given receptor.     

Combinatorial receptor codes for odors

The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT-PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its "code," potentially explaining how such changes can alter perceived odor quality.     

Odor recognition and second messenger signaling in olfactory receptor neurons

The detection of volatile odorants is supposed to begin with their interaction with soluble binding proteins which shuttle the hydrophobic ligands through the aqueous mucus layer towards specific odorant receptors in the ciliary membrane of olfactory neurons. A large family of receptors for odorants has been identified recently; individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, ultimately leading to an electrical response of the receptor neuron. Olfactory signaling is terminated by phosphorylation of receptors via a negative feedback reaction catalyzed by two types of kinases.     

Translation of sensory input into behavioral output via an olfactory system

We investigate the logic by which sensory input is translated into behavioral output. First we provide a functional analysis of the entire odor receptor repertoire of an olfactory system. We construct tuning curves for the 21 functional odor receptors of the Drosophila larva and show that they sharpen at lower odor doses. We construct a 21-dimensional odor space from the responses of the receptors and find that the distance between two odors correlates with the extent to which one odor masks the other. Mutational analysis shows that different receptors mediate the responses to different concentrations of an odorant. The summed response of the entire receptor repertoire correlates with the strength of the behavioral response. The activity of a small number of receptors is a surprisingly powerful predictor of behavior. Odors that inhibit more receptors are more likely to be repellents. Odor space is largely conserved between two dissimilar olfactory systems.     

Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors (hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1 and OR5P3. As it remains largely unknown how human perception of odorants influence or prevent diseases, we also developed an odorant-protein matrix to explore global relationships between chemicals, biological targets and disease susceptibilities. We successfully experimentally demonstrated interactions between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma (PPARγ). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.     

Signal recognition and chemoelectrical transduction in olfaction

The mimicking of olfaction is considered to be a promising approach for the construction of artificial odour-sensing systems. In the nose, the detection of volatile odorants begins when the odorant ligands interact with specific odorant receptors in the ciliary membrane of the olfactory neurons. A large family of genes encoding putative odorant receptors has been identified recently. Individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, resulting in a “pulse” of second messengers that initiates an electrical response from the receptor neuron. Olfactory signalling is terminated by phosphorylation of receptors via a negative feedback reaction, catalyzed by specific kinases.     

Biochemical Studies of Olfaction: Role of Cilia in Odorant Recognition

Chemoreception in vertebrates is beginning to be understood. Numerous anatomical, behavioral, and physiological studies are now available. Current research efforts are examining the molecular basis of chemoreception. Rainbow trout (Salmo gairdneri) have a functional olfactory system and are a suitable vertebrate model for studying odorant interactions with receptors. Using a biochemical approach, initial events of olfactory recognition were examined; the aim was to determine the location and specificity of odor receptors. Cilia occupy the distal region of the receptor neuron on the trout olfactory epithelium, and their membranes are the postulated locus of odorant receptor sites. A cilia preparation was isolated from the olfactory rosette. The preparation was characterized by quantifying biochemical markers for cilia, along with electron microscopy, all of which substantiated enrichment of cilia. Functional activity was assessed by quantifying binding of several radioactively labeled odorant amino acids. The odorants bound to the cilia in a manner similar to the sedimentable preparation previously isolated from t h e olfactory rosette of the same animal, thus verifying the presence of odor receptors in the cilia preparation. Evidence also confirmed a site TSA which binds L-threonine, L-serine, and L-alanine and a site L which binds L-lysine (and L-arginine). Binding of L-serine and D-alanine showed evidence for a single affinity site while the others showed two affinity sites. Separation of membrane fractions from the cilia preparation revealed that binding activity is associated with a very low density membrane fraction B.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Representation of odorants by receptor neuron input to the mouse olfactory bulb.

To visualize odorant representations by receptor neuron input to the mouse olfactory bulb, we loaded receptor neurons with calcium-sensitive dye and imaged odorant-evoked responses from their axon terminals. Fluorescence increases reflected activation of receptor neuron populations converging onto individual glomeruli. We report several findings. First, five glomeruli were identifiable across animals based on their location and odorant responsiveness; all five showed complex response specificities. Second, maps of input were chemotopically organized at near-threshold concentrations but, at moderate concentrations, involved many widely distributed glomeruli. Third, the dynamic range of input to a glomerulus was greater than that reported for individual receptor neurons. Finally, odorant activation slopes could differ across glomeruli, and for different odorants activating the same glomerulus. These results imply a high degree of complexity in odorant representations at the level of olfactory bulb input.     

Learning to smell the roses: experience-dependent neural plasticity in human piriform and orbitofrontal cortices

It is widely presumed that odor quality is a direct outcome of odorant structure, but human studies indicate that molecular knowledge of an odorant is not always sufficient to predict odor quality. Indeed, the same olfactory input may generate different odor percepts depending on prior learning and experience. Combining functional magnetic resonance imaging with an olfactory paradigm of perceptual learning, we examined how sensory experience modifies odor perception and odor quality coding in the human brain. Prolonged exposure to a target odorant enhanced perceptual differentiation for odorants related in odor quality or functional group, an effect that was paralleled by learning-induced response increases in piriform cortex and orbitofrontal cortex (OFC). Critically, the magnitude of OFC activation predicted subsequent improvement in behavioral differentiation. Our findings suggest that neural representations of odor quality can be rapidly updated through mere perceptual experience, a mechanism that may underlie the development of odor perception.     

The role of the coreceptor Orco in insect olfactory transduction

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. Our results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants. Or83b therefore encodes an atypical odorant receptor that plays an essential general role in olfaction.