Sensing Odorants and Pheromones with Chemosensory Receptors

ANRV369-PH71-15 ARI 5 January 2009 15:25

Sensing Odorantsand Pheromones withChemosensory ReceptorsKazushige Touhara1 and Leslie B. Vosshall21Department of Integrated Biosciences, The University of Tokyo, Chiba, 277-8562 Japan;email: [email protected] Hughes Medical Institute, Laboratory of Neurogenetics and Behavior,The Rockefeller University, New York, NY 10065; email: [email protected]

Annu. Rev. Physiol. 2009. 71:307–32

The Annual Review of Physiology is online atphysiol.annualreviews.org

This article’s doi:10.1146/annurev.physiol.010908.163209

Copyright c© 2009 by Annual Reviews.All rights reserved

0066-4278/09/0315-0307$20.00

Key Words

vomeronasal organ, olfaction, behavior, G protein–coupled receptors,olfactory bulb, olfactory sensory neuron

AbstractOlfaction is a critical sensory modality that allows living things to ac-quire chemical information from the external world. The olfactory sys-tem processes two major classes of stimuli: (a) general odorants, smallmolecules derived from food or the environment that signal the pres-ence of food, fire, or predators, and (b) pheromones, molecules releasedfrom individuals of the same species that convey social or sexual cues.Chemosensory receptors are broadly classified, by the ligands that ac-tivate them, into odorant or pheromone receptors. Peripheral sensoryneurons expressing either odorant or pheromone receptors send sig-nals to separate odor- and pheromone-processing centers in the brainto elicit distinct behavioral and neuroendocrinological outputs. Gen-eral odorants activate receptors in a combinatorial fashion, whereaspheromones activate narrowly tuned receptors that activate sexuallydimorphic neural circuits in the brain. We review recent progress onchemosensory receptor structure, function, and circuitry in vertebratesand invertebrates from the point of view of the molecular biology andphysiology of these sensory systems.

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Odorant: a volatilechemical compound,usually of molecularweight 300 or smaller,that activates olfactoryneurons and inducesthe percept of an odor

Pheromone: asubstance released by amember of the samespecies that elicits astereotyped behaviorand/orendocrinologicalresponse in anothermember of the samespecies. Pheromonescan be proteins, smallmolecules, or acombination thereof

Conspecifics:members of the sameanimal species

INTRODUCTION

What Is an Odorant?What Is a Pheromone?

An odorant is a volatile chemical compoundwith a molecular weight of lower than ∼300that humans or other animals perceive as odor-ous via the olfactory system. The number ofpossible odorant molecules that exist on earthis unknown, but essentially all living thingssuch as plants, insects, animals, and microbesemit smells—purposefully or as by-products ofmetabolism or waste excretion. There are alsoinorganic sources of odorants: Sulfur dioxideand some metals have a distinct odor. All theseodorants can potentially be exploited by animalsto improve their chances for survival.

How many odorants are there, and howmany can humans detect and distinguish? Thescientific and popular literature is full of claimsthat the number of odorants that exist and thatwe can detect ranges from hundreds to thou-sands or even hundreds of thousands. Mean-while, most laboratory studies in the field ofolfaction use a few dozen odorants from astandard set of approximately 500 chemicals(1). The most oft-cited statistic is that hu-mans can distinguish approximately 10,000 dif-ferent odors. In his engaging book on smell(2), Avery Gilbert painstakingly traces the fa-miliar 10,000 odors claim back through thetwentieth-century scientific literature to a se-ries of questionable assumptions in a theoreticalmodel developed in 1927 by Ernest C. Crockerand Lloyd F. Henderson. Therefore, no one ap-pears to have catalogued the exact number ofknown smells or how many such smells we canperceive. Regardless of exactly how many dif-ferent odorants there may be, or how many agiven animal can detect and discriminate, thesesmall molecules are clearly important for an-imal survival, allowing animals to locate foodand to avoid predators and environmental dan-gers such as fire.

In contrast to general odorants, apheromone is defined as a specific substancethat is secreted by an individual and receivedby a second individual of the same species, or

conspecific, to induce a specific reaction suchas a stereotyped behavior or endocrinologicalchange (3) [see also the book by Wyatt (4) for anexcellent review on the topic of pheromones].The term was originally coined on the basisof the identification of a volatile sex attractant,bombykol, which is released by the female silkmoth Bombyx mori and elicits the full sequenceof sexual behaviors in male moths (5). Thedefinition avoids any use of the terms odor orodorant because a pheromone does not have tobe odorous or volatile as long as the signal is achemical substance that is transferred betweenconspecifics. Pheromones can be nonvolatilesubstances with a molecular weight of largerthan a few hundred, including relatively largeorganic compounds, peptides, and proteins.

In some cases, the distinctions between gen-eral odorants and pheromones are blurred. Forinstance, a pheromone can be an odorous com-pound, and an odorant can be a pheromone.As such, a pheromone released and utilized byone species can be produced as a general odor-ant for a second species but can be an infor-mative odor that a third species uses to predatethe second species. General odorants can alsoinduce behavioral or physiological changes ina fashion similar to a pheromone. Insects suchas moths and flies are attracted by the smell offlowers and plants, which induces stereotypedfeeding behavior. We similarly experience phys-iological effects of general odorants elicited bytrees, plants, and flowers, an effect on whichthe aromatherapy industry is based. These be-havioral and physiological effects, however, arenot categorized as pheromonal effects becausethe active chemical substances are not derivedfrom conspecifics. There is an ongoing trend inthe chemosensory field to extend the definitionof pheromones beyond the strict definition ofKarlson & Luscher (3) because there is increas-ing evidence that pheromones play much morediverse functional roles than are included in theabove definition (4). Thus, a pheromone couldbe more broadly defined as a substance thatis utilized for intraspecies communication eventhough it does not elicit obvious behavioral orendocrinological changes. We list below some

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possible criteria for a more inclusive definitionof a pheromone:

1. Pheromones are released by one individ-ual and received by conspecifics;

2. pheromones themselves can send infor-mation about sex, strain, and species tothe receiver; and

3. pheromonal effects must be meaningfulor informative for the species.

As molecular biologists, chemical ecologists,and neurobiologists continue to work togetherto understand the link between pheromonesand behavior, the field is likely to arrive at anoptimal definition of a pheromone in the future.

How Are Odorants andPheromones Detected?

If one assumes that there are thousands of im-portant odorants, how do animals recognizesuch a large diversity of chemical cues? TitusLucretius Carus, a Roman poet and philoso-pher, proposed in 50 bce that a large variety ofodors exists because each odorant possesses aunique structure (6). In the mid-twentieth cen-tury, Amoore (7) formalized this concept as thestereospecific receptor theory, which attemptedto explain the molecular mechanisms underly-ing the remarkable olfactory sensing system.The receptor theory postulates that there aremany receptor sites for odorants and that odorperception occurs only when the structure ofan odorant molecule and the structure of itsbinding site match. Buck & Axel (8) discov-ered in 1991 a large multigene family encodingreceptor proteins for odorants in rat; this find-ing was later extended to all vertebrates studied.Thus, the large number and diversity of olfac-tory (or odorant) receptors (ORs) confirm theessential features of Amoore’s receptor theory.Although we still cannot predict what odorantswill bind to a particular OR, or how a partic-ular odorant will smell, the stereochemical re-ceptor theory remains the dominant theory inthe field. Alternate theories proposed to explainodor perception, including vibrational, punc-turing, radiational, and absorption theories, arehotly debated (9, 10) but remain unproven.

OR: olfactoryreceptor or odorantreceptor

VNO: vomeronasalorgan

GPCR: G protein–coupled receptor

Odorants and pheromones are detected byolfactory sensory neurons in the olfactory sys-tem. Mammals usually detect general odorantsby the nasal olfactory epithelium via the mainolfactory system. Rodents and a number ofother nonprimate species possess a secondaryolfactory system called the vomeronasal path-way, which detects signals via the vomeronasalorgan (VNO), located at the bottom of the nasalcavity. The appearance of the VNO coincidedwith the acquisition of the lung respiratory sys-tem during the Cambrian explosion (11), andthe VNO became genetically vestigial duringthe evolution of the primate lineage (12). Sim-ilarly, most insects also have two olfactory or-gans, the antenna and the maxillary palp, al-though insects differ in the extent to whicheach organ is dedicated to general odorants,pheromones, or even nonolfactory cues such astaste and mechanical stimuli. Recent evidencesuggests that the labial palps, typically thoughtto be exclusively taste organs, can also senseodors (13).

In this review, we summarize the cur-rent knowledge of chemosensory receptors forodorants and pheromones, point out what iscommon and what is different in chemosensingmechanisms between invertebrate and verte-brate animals, and discuss how sexually dimor-phic responses to chemosignals are encoded inthe brain.

CHEMOSENSORY RECEPTORGENES

Vertebrate Olfactory Receptors

The vertebrate OR genes encode a large familyof seven-transmembrane-domain G protein–coupled receptors (GPCRs) that play a role inrecognizing odorant molecules in the olfactoryepithelium (14). OR proteins were classified asmembers of the GPCR superfamily because ofthe presence of structural features common toGPCRs (8, 15) and because the ORs coupleto and activate heterotrimeric G proteins (16,17). The size of the OR family was estimatedto be at least several hundred genes when the

www.annualreviews.org • Comparative Chemosensory Perception 309

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VR: vomeronasalreceptor

family was originally identified in the rat (8). Inthe past several years, whole-genome sequenc-ing projects have allowed for a comprehen-sive analysis of the OR gene family in diversespecies and have made it possible to study thegenomic structure and distribution of OR genesfrom various organisms (18). In mammals, theOR repertoire comprises ∼800–1500 members,whereas fish have a relatively small OR familyof ∼100 genes (19). Thus, the OR gene expan-sion likely occurred when animals shifted fromaqueous to terrestrial environments.

In every vertebrate in which the expressionof OR genes has been examined, each olfactorysensory neuron expresses only a single memberof the OR gene family. This is hypothesized tobe important for olfactory coding, such that agiven population of olfactory neurons respondsto a restricted number of odorants and com-municates ligand binding to central olfactorycircuits. The mechanism by which an olfactoryneuron selects a single OR, and represses the ex-pression of all remaining ORs in the genome,remains a fascinating and controversial ques-tion (20–23).

A significant portion of the OR gene familyhas been pseudogenized in vertebrates, leadingto the loss of a large fraction of the potentiallyfunctional ORs in a given species. Hominoidspossess high pseudogene content in their ORs(∼50%), whereas ∼20% of mouse and dog ORgenes and 25–35% of primate OR genes arepseudogenes (19, 24, 25). The OR family in thelineage of many species appears to be undergo-ing a rapid molecular evolution by tandem geneduplication and pseudogenization. The fractionof pseudogenes has increased during the evolu-tion from rodents, monkeys, and humans, sug-gesting that the reduced function of the senseof smell correlates with the loss of functionalOR genes. Indeed, in whale and dolphin, an-imals for which the auditory system is domi-nant over the olfactory system, 70–80% of ORgenes appear to be pseudogenes (Y. Go, per-sonal communication). Furthermore, OR genesare highly polymorphic, as suggested by hu-man leukocyte antigen–linked OR genes (26,27) and dog OR genes (28). Recent studies have

revealed that single-nucleotide polymorphismsin human OR genes account in part for individ-ual differences in the ability to detect specificodors (29, 30).

Vertebrate Vomeronasal Receptors

The VNO expresses vomeronasal receptors(VRs), comprising two distinct subfamilies ofGPCRs, the V1Rs and V2Rs (31–36). Thevomeronasal sensory epithelium in the VNOcan be divided into apical and basal layers,which express V1R- and V2R-type receptors,respectively. V1Rs and V2Rs begin to be ex-pressed after birth (31), whereas ORs beginto be expressed during embryogenesis (37).The segregation of the vomeronasal epitheliuminto two layers occurs during the postnatal pe-riod (32), consistent with the development ofthe VNO as an organ that functions in adultcommunication.

The V1R family consists of a total of 308sequences in mice, 187 of which appear to en-code full-length open reading frames (38). Mostof the gene family is located on chromosomes6, 7, and 13, and like ORs, V1Rs are encodedby a single exon (39, 40). In humans, more than90% of the V1R genes have been converted topseudogenes, so only five V1R genes appear tobe intact (41, 42). V1Rs do not show signifi-cant homology to the ORs, but they are weaklyrelated to the T2R family of bitter receptors(31). The expression of V1Rs is restricted tothe apical neuroepithelium in mice (43); a givenvomeronasal neuron expresses just one memberof the V1R family, as previously documented forthe main olfactory system. In humans, who lacka functional VNO, V1Rs are expressed in theolfactory epithelium (41). The genomic struc-ture and expression patterns of V1Rs appear tohave undergone rapid changes during the pro-cess of the evolution (44).

In mice, the V2R family comprises 121 in-tact genes out of a total of 279 genes, includ-ing pseudogenes (38, 45). Several years wereneeded for the comprehensive genomic analy-sis of the V2R family because of the complexintron/exon structures of V2Rs (33–35). V2Rs


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possess a long extracellular N terminus that iscommon to calcium-sensing and metabotropicglutamate receptors and the T1R family ofsweet and umami receptors (18). Intact V2Rgenes have not been found in the humangenome. There appear to be only ∼20 V2Rpseudogenes in humans, suggesting that V2Rgenes have changed more dramatically thanV1R genes. The expression of V2Rs is restrictedto the basal neuroepithelium in mice (33–35),and as in the case of ORs and V1Rs, the onecell–one receptor rule seems conserved (46).One notable exception to the one-receptor-per-cell rule is a small subfamily of V2R genes, theV2R2 genes, that appear to be broadly coex-pressed with other V2Rs (47). The function ofthe V2R2s in the V2R2-expressing portion ofthe VNO remains a mystery.

Insect Olfactory Receptorsand Gustatory Receptors

The initial description of vertebrate ORs byBuck & Axel (8) rapidly led to the identifi-cation of ORs in essentially every vertebratespecies studied. However, all attempts to iso-late insect homologs of vertebrate GPCR-typeORs selectively expressed in olfactory tissuesfailed. Instead, three groups used the unbiasedapproach of plus-minus screening and genomemining to identify candidate ORs in the fruit flyDrosophila melanogaster (48–50). The insect ORgenes, comprising 62 members in the fruit fly,encode a novel family of seven-transmembrane-domain receptor proteins selectively expressedin subsets of olfactory neurons in the insectolfactory organs, the antennae and maxillarypalps.

Subsequent to the discovery of DrosophilaORs, a family of divergent seven-trans-membrane-domain receptor genes, distantlyrelated to the ORs, was isolated (51). This genefamily was named the gustatory receptor (GR)family because many of the GR genes were ex-pressed in taste organs such as the labial palps(51–53).

The number of chemosensory receptorgenes is smaller in insects than in most mam-

GR: gustatoryreceptor

mals and more closely approaches the numberfound in fish. Genomic analysis has identified62 ORs and 68 GRs in D. melanogaster (54), 79ORs and 72 GRs in the malaria vector mosquitoAnopheles gambiae (55), 170 ORs and 13 GRs inthe honeybee Apis mellifera (56), 131 ORs and88 GRs in the yellow fever and dengue virusvector Aedes aegyptii (57), 341 ORs (58) and 62GRs (59) in the beetle Tribolium castaneum, and,most recently, 66 ORs and 14 GRs in the silkmoth B. mori (International Silkworm GenomeConsortium, submitted).

The numbers of ORs and GRs and the rel-ative ratios of these related chemosensory re-ceptor gene families differ from insect to in-sect, suggesting that sociosexual behavior andlifestyle may have positively influenced thesegene families during the insect evolution. Forinstance, the honeybee has a severely reducedrepertoire of taste receptors, comprising only ahandful of putative sugar receptors, and an ex-panded OR gene family. Robertson & Wanner(56) suggested that the absence of bitter tastereceptors in the honeybee could be explainedby the cooperative relationship these pollina-tors have with host plants, which would notproduce the bitter alkaloids that would harmbees and thus would not need to be detected bybitter-sensing bee GRs. Fewer chemosensoryreceptor genes in insects than in vertebratesare pseudogenes, with the exception of the Tri-bolium beetle genome, in which many OR andGR pseudogenes exist. To examine the molec-ular evolution and dynamics of OR genes, ORsfrom more than 10 Drosophila species were iden-tified, and phylogenetic analysis was performed(60). The insect OR genes appear to be evo-lutionarily more stable than the vertebrate ORgenes, but there are some lineage-specific geneduplications and losses that are reflected by pos-itive selection possibly because of environmen-tal and behavioral differences between species.

Insect ORs and GRs do not show any signif-icant sequence similarity to vertebrate ORs andalso lack homology to chemosensory GPCRsin vertebrates such as the typical DRY andNPXXY amino acid motifs. Furthermore, bothcomputer prediction and experimental analysis


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suggest that the insect ORs and GRs possess atransmembrane topology distinct from that ofGPCRs, with the N terminus located intracel-lularly and the C terminus located extracellu-larly (61–63).

Another feature that distinguishes insectORs from their molecularly distinct mam-malian OR counterparts is that a highly con-served OR, called Or83b after its name in D.melanogaster, is coexpressed with other conven-tional ORs in single olfactory neurons (64–66). Or83b is an essential constant subunitof the heteromultimeric insect OR that formsa receptor complex with the variable ligand-binding ORs (62, 65, 67). We discuss the func-tional consequences of these differences in thestructure and expression profile between ver-tebrate and invertebrate ORs (see Insect Ol-factory Receptors subsection in ChemosensorySignal Transduction section, below). Later inthis review, we continue to develop the conceptthat chemosensory receptors appear to havearisen independently in the evolution of ver-tebrate and invertebrate species.

CHEMOSENSORY RECEPTORFUNCTION: LIGAND-RECEPTORPAIRS

Mouse Olfactory Receptors

When originally cloned by Buck & Axel (8),the OR genes were hypothesized to encode thereceptor proteins underlying the molecular ba-sis of smell. Functional proof that ORs recog-nize odorants has been assessed by both ho-mologous and heterologous expression systemsthat seek to link a given OR with its cognateodor ligand(s). Olfactory neurons themselvesprovide the homologous expression system: ho-mologous because they possess the appropriatecellular machinery for OR expression and thetransmission of odorant signals. Thus, olfactoryneurons identified to express a certain OR ofinterest respond to the cognate ligands for theOR (68–71).

ORs have also been functionally expressed inheterologous expression systems such as mam-

malian cell lines and Xenopus laevis frog oocytes,making it possible to assess OR responsivenessto odorants (16, 17, 72–76). Thus, in HEK293cells coexpressing tagged ORs and the promis-cuous G protein Gα15, Ca2+ responses were ob-served when the cells were stimulated with theirligands (16, 17, 73, 74). Without coexpressionof Gα15, ORs activate endogenous Gαs uponligand stimulation, resulting in an increase incAMP levels in various mammalian cell lines in-cluding HEK293, COS-7, and CHO-K1 cells(16, 72–74). A luciferase-reporter assay systemusing the zif268 promoter allows luminescentdetection of cAMP increases upon stimulationwith an odorant (72–74, 77).

Although convenient for rapid and high-throughput expression outside of native olfac-tory neurons, the expression of ORs in heterol-ogous cells is tricky because ORs appear to beinefficiently translocated to the plasma mem-brane. In some cases, adding the N-terminalleader sequence of rhodopsin, another GPCR,resulted in improved targeting of functionalORs to the plasma membrane and in successfulodorant-response recording in a heterologoussystem such as HEK293 cells (16, 17, 74, 75).

To improve functional heterologous expres-sion, several groups have searched for cofactorssuch as protein chaperones and other proteinsto facilitate and stabilize cell-surface expres-sion of ORs. The one-transmembrane-domainprotein RTP1 expressed in olfactory neuronsenhances cell-surface expression of ORs, andsome ORs have been deorphanized in cell linescoexpressing RTP1 (77). In addition, Ric8B, aputative guanine nucleotide exchange factor forGαs and Gαolf, promotes efficient signal trans-duction of ORs (78). Moreover, coexpression ofmyristylated Ric8A, a Ric8B homolog that actson Gαq-type G proteins, greatly enhances Gα15-mediated odorant signaling (79). These effortsto enhance cell-surface expression and signaltransduction of ORs have greatly facilitated theprocess of deorphanizing ORs in the past fewyears (74).

What can we learn from the comparativeanalysis of OR structure and function and re-gions that are variable versus those that are


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conserved? ORs possess seven hydrophobictransmembrane domains, a disulfide bond be-tween the conserved cysteines in the extracel-lular loops, a conserved glycosylation site inthe N-terminal region (80), and several aminoacid sequence patterns that are conserved in theOR family (81, 82). These conserved motifslikely contribute to proper folding and mem-brane trafficking of ORs to the plasma mem-brane so that ORs can function in binding odor-ants and coupling to the appropriate G proteins.In contrast, the transmembrane regions are rel-atively variable and may play a role in formingan odorant-binding pocket, such that variabilityin these domains allows ORs to cover a wide va-riety of odorant molecules in the ligand recog-nition spectra (83–88).

Touhara and coworkers (89) undertook asystematic experimental approach to decipherthe OR odorant-binding site. They employedfunctional analysis of site-directed mutants andligand docking simulation studies, revealingthat most of the critical residues involved inodorant recognition are hydrophobic and lo-cated within the binding pocket formed bytransmembrane (TM) domains TM3, TM5,and TM6 (89). Furthermore, the accuracy ofthe binding model was validated by the fact thatsingle-amino-acid changes caused predictablechanges in agonist and antagonist specificity(89). These results allowed us to conclude thatvertebrate ORs recognize the size, shape, andfunctional group of an odorant, using both hy-drogen bonds and hydrophobic interactions inthe odor-binding pocket formed by transmem-brane helices.

As more and more vertebrate ORs have beenpaired with ligands, the results have revealeda combinatorial coding strategy in which eachodorant is recognized by a subset of ORs that isunique for the odorant (90). ORs that recognizemany odorants with a wide range of structuresare defined as broadly tuned or generalist re-ceptors. This overlapping coding strategy mayrepresent a molecular basis for the discrimina-tive power of the olfactory system. In contrast,some ORs detect certain odorant moleculeswith high specificity and are referred to as nar-

TM: transmembrane

rowly tuned or specialist receptors. Such nar-rowly tuned receptors may mediate signals thatactivate specialized circuits in the brain, result-ing in discrete behaviors or neuroendocrino-logical changes reminiscent of a pheromoneeffect. As discussed above, the differences be-tween pheromones and odorants are in partsemantic and relate to the source and effectof the stimulus. With recent advances in an-notating OR genes in diverse species and inthe efficient functional expression of ORs inheterologous cells, it seems feasible that thecomplete odorant-OR matrix will eventually beconstructed for several species. Such informa-tion will reveal how broad the universe of odor-ant molecules recognized by each species is andwhich odorant-OR combinations elicit innatereactions that are essential for individuals tosurvive in various environments.

Mouse Vomeronasal Receptors

VRs are receptors that mark two neural com-partments of the VNO, but how good is theevidence that either V1Rs or V2Rs functionas pheromone receptors? In fact, recent evi-dence for V1Rs as pheromone receptors hasemerged (18, 91). First, a mouse V1R expressedectopically in vomeronasal sensory neurons re-sponds to urine (92), a bodily substance shownto contain a number of molecules that elicitpheromonal effects in rodents. Second, mutantmice lacking a cluster of V1Rs fail to respond tosome volatile pheromones and show decreasedaggressiveness and sexual behavior (93). Third,V1Rb2 expressed in vomeronasal neurons re-sponds to 2-heptanone, one of the componentsof urine that cause extension of the estrous cy-cle (94). Together with evidence from calciumimaging of intact VNO epithelial slices show-ing that vomeronasal neurons in the apical layerrespond to various volatile pheromones (95),these findings suggest that V1Rs serve as sen-sors for volatile pheromones.

Vomeronasal neurons, however, can alsodetect nonpheromonal odorants (96), imply-ing that V1R-expressing neurons may not berestricted to the detection of species-specific


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ESP: exocrinegland–secretingpeptide

AOB: accessoryolfactory bulb

volatile pheromones for mating but also mayperceive non-species-related signals such asthose from prey, predators, and the physical en-vironment. Five V1Rs expressed in the humanolfactory epithelium responded strongly to C9-C10 aliphatic alcohols or aldehydes in a com-binatorial fashion (97). Whether these humanreceptors function as pheromone receptors andwhat ligands activate them remain to be eluci-dated. Although the available data are relativelylimited, it seems that the strategy for V1Rs torecognize volatile pheromones or odorants issimilar to that of ORs. Thus, how a combina-torial code of V1R activation is processed inthe brain to elicit innate behavior or neuroen-docrine changes remains to be studied.

V2Rs have a long N-terminal region relatedto the N-terminal region found in metabotropicglutamate receptors and calcium sensors, andtherefore potential ligands for V2Rs have beenthought to be nonvolatile pheromones such aspeptides and proteins. Candidate ligands forV2Rs include three polypeptide ligands thathave been implicated in VNO function: majorurinary proteins (MUPs), major histocompat-ibility complex (MHC) peptides, and the ex-ocrine gland–secreting peptide (ESP) family(98).

Biochemical studies in rats have demon-strated that some MUPs activate vomeronasalneurons in the basal layer, which express Gαo

and V2Rs (99). In a recent paper, Stowers andcoworkers (100) demonstrated that highly pu-rified MUPs in the absence of volatile urinecomponents activated VNO neurons derivedfrom the basal zone. Interestingly, the syntheticMUPs alone sufficed to elicit aggressive behav-ior in male mice, confirming that MUPs ful-fill the basic definition of a pheromone. How-ever, there have been some contradictions in thevarious MUP studies. One study showed thatMUPs activate isolated V2R-expessing basalneurons in calcium imaging (100), whereasother studies showed that urine, which containsMUPs, does not elicit electrical responses in theVNO or neural spikes in V2R-expressing neu-rons (101). The basis for these differences is notcurrently known.

For MHC peptides, calcium-imaging stud-ies in mice indicated that vomeronasal sen-sory neurons responding to the peptides are lo-cated in the basal neuroepithelium, where V2Rsare expressed (102, 103). Moreover, these pep-tides sufficed to induce the physiological effectof pregnancy block, a pheromonal behavior inwhich exposure to a foreign male blocks im-plantation of embryos sired by a familiar male(102). These peptides, however, also activatethe main olfactory epithelium, which does notexpress VRs (104).

ESP1 is a male-specific peptide that acti-vates V2R neurons in four different assay sys-tems. First, ESP1 induced c-Fos expression, atranscriptional readout of neuronal activation,both in V2R neurons (105) and in the accessoryolfactory bulb (AOB) (S. Haga & K. Touhara,unpublished observations). Second, ESP1 ac-tivated V2R-expressing neurons as assayed bycalcium imaging (K. Touhara & C.R. Yu, un-published observation). Third, ESP1 evokedpotentials in the vomeronasal epithelium asmeasured in electro-vomeronasogram (EVG)recordings (105). Fourth, ESP1 activated neu-ral spikes as measured with electrical record-ings of vomeronasal neurons (106). Althoughthese studies do not provide direct evidence forpheromone-V2R interactions, the results sup-port the idea that V2R-expressing neurons re-spond to nonvolatile pheromones.

Further studies were performed to identifywhich V2R(s) is expressed in vomeronasal neu-rons that recognize ESP1. Vomeronasal neu-rons were double-labeled with an anti-c-Fos an-tibody and an in situ RNA probe for V2Rs. Of12 different RNA probes, one of them, V2Rp,which was designed to hybridize with fivehighly homologous V2Rp genes, clearly rec-ognized c-Fos-positive neurons that respondedto ESP1 (105). In contrast, none of the otherprobes overlapped with c-Fos. Further studiesusing specific probes to discriminate the fiveV2Rp genes revealed that a single V2R, namedV2Rp5, was expressed in all the c-Fos neuronsinduced by exposure to ESP1 (107). V2Rp5-expressing neurons tagged with DsRed respondto ESP1, clearly demonstrating that V2Rp5 is


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a receptor for ESP1 (S. Haga, Y. Yoshihara &K. Touhara, unpublished observation). Theseresults provide evidence that each V2R pos-sesses a narrow ligand spectrum and thus is re-sponsible for the detection of a specific pep-tide pheromone. Multielectrode recordings ofthe spike firing rate of vomeronasal neuronsalso demonstrated that sensory neurons, eachof which expresses a single type of V2R, showedresponses to the specific ESP ligand, and there-fore the V2R neurons are narrowly tuned (106).This putative narrow tuning of the VNO con-trasts with the combinatorial strategy observedin the main olfactory system.

Insect Olfactory Receptors

Initial attempts to characterize insect OR lig-ands were carried out by a strategy similar tothat used for vertebrate ORs. Drosophila Or43awas expressed in a homologous system, in thiscase in the antenna (108), or in a heterologoussystem, Xenopus oocytes, which led to the iden-tification of ligands for Or43a, including ben-zaldehyde and cyclohexanone (109). Later, re-searchers carried out a large-scale analysis ofthe Drosophila antennal ORs by genetically in-troducing individual ORs into an antennal neu-ron lacking the endogenous ligand-binding ORbut retaining the Or83b coreceptor (110). Re-sponses of 24 ORs (out of a total of 62 ORs)expressed in the adult fly olfactory system to110 odorants revealed combinatorial receptorcodes for odorants, similar to those in the ver-tebrate olfactory system (111, 112). Twenty-fiveORs were detected in the larva, and 14 showedlarva-specific expression, whereas 11 ORs wereexpressed in both larvae and adults (113, 114).Most larval ORs are also broadly tuned but canbe divided into two general classes of sensi-tivity: aromatic or aliphatic compounds (114,115). This suggests that the vast majority of in-sect ORs are generalist-type sensors that de-tect a variety of odorous molecules from foodsources.

As we have seen, vertebrates use dis-tinct receptor gene families to detect putativepheromones, but what detection strategy have

cVA: 11-cis-vaccenylacetate

insects evolved to respond to pheromones? Twoindependent studies were carried out to identifyan insect pheromone receptor that specificallyrecognizes moth sex pheromones (116–119).Both studies were based on the assumptionthat a sex-pheromone receptor would be ex-pressed sex-specifically in the male antenna. In-deed, two male-specific members of the insectOR gene family, named BmOR1 and BmOR3,were discovered (116, 117). These were shownto function as sensors for bombykol and bom-bykal, two pheromone components in the silkmoth B. mori, respectively (117). The functionof these two proteins as pheromone sensors wasconfirmed by heterologous expression in Xeno-pus oocytes by coexpressing each receptor withBmOR2, the B. mori Or83b ortholog (117). Thebinding site for bombykol seems to reside inBmOR1, not the BmOR2 coreceptor, becauseOr83b orthologs from different species act as afunctional partner to BmOR1 (117). BmOR1ectopically expressed in Drosophila antennaealso responded to bombykol (120), furtherconfirming that BmOR1 is the pheromone-binding site. A second study showed that sex-pheromone receptors from a different mothspecies (Heliothis virescens), HR13/15/16, arealso expressed male-specifically and recog-nize female-producing pheromone compounds(118).

After these initial reports of mothpheromone receptors, other studies linkingidentified insect pheromones to receptorswere published. A male drone–biased OR,AmOr11, in honeybees was shown to be thereceptor for the queen retinue pheromone9-oxo-2-decenoic acid by coexpression ofAmOr11 together with AmOr2, the honeybeeOr83b ortholog, in Xenopus oocytes (121). TheDrosophila aggregation and sex pheromone11-cis-vaccenyl acetate (cVA) is also recognizedby an OR named Or67d, which functionstogether with Or83b (122, 123). These resultssuggest that insects have selected pheromonereceptors from a repertoire of ORs, rather thancreating a new family of specific pheromone re-ceptors. This is in contrast to vertebrate speciesin which structurally distinct chemosensory


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receptor families, the V1Rs and V2Rs, play arole in pheromone detection.

Although insects use members of the ORfamily to detect pheromones, there is recent ev-idence that cVA-sensing neurons in Drosophilaare specialized and that additional accessoryfactors are crucial to detecting pheromones.These include the CD36 homolog sensoryneuron membrane protein (SNMP) and thepheromone-binding protein LUSH. SNMP isa two-transmembrane-domain protein that isrequired to tune the spontaneous activity of theOr67/Or83b complex (124, 125), and LUSH isincreasingly viewed as a critical component ofthe ligand recognition of the cVA pheromone(126). It will be interesting to see if vertebratepheromone receptors similarly use specific co-factors to recognize pheromone ligands.

CHEMOSENSORY SIGNALTRANSDUCTION

Vertebrate Olfactory Receptors

The odorant signal received by ORs is con-verted to an electrical signal in olfactory sen-sory neurons (127). In vertebrates, the first stepin olfactory signal transduction is the activationof the G protein Gαolf by odorant-bound acti-vated ORs. Some amino acids in the C-terminaldomain and third intracellular loop of ORs ap-pear to be involved in coupling and activationof Gαolf (80, 128). Unlike the case of rhodopsin,signal amplification does not occur in the OR–G protein activation cycle (129). Instead, Ric8B,a guanine nucleotide exchange factor, enhancesthe accumulation of Gαolf at the cell membrane,thus improving the efficiency of OR couplingto Gαolf (78). The activated Gαolf in turn stim-ulates adenylyl cyclase III, resulting in a cAMPincrease (130). The elevated levels of cAMP in-teract with and gate a cyclic nucleotide–gated(CNG) channel, leading to cation influx andto depolarization of the receptor neuron. Fur-thermore, calcium activates a chloride chan-nel that leads to an efflux of Cl−, contributingto the amplification of the sensory depolariza-

tion. Bestrophin-2 is a candidate for a molecularcomponent of the olfactory calcium-activatedchloride channel (131, 132).

Gene knockout mice lacking any of thethree molecular components (i.e., Gαolf, adeny-lyl cyclase III, or CNG channel) failed to re-spond to odorants, suggesting that the cAMPcascade is dominant in transmitting the odor-ant signal in olfactory neurons (133–135).However, investigators later demonstrated thatCNGA2 knockout mice could detect a sub-set of odorants including 2-heptanone and 2,5-dimethylpyrazine, which are constituents ofurine (136). Two additional olfactory pathwayshave been proposed: One involves membrane-bound guanylyl cyclase and phosphodiesterasetype 2 (137–139), and another involves phos-pholipase C and TRPM5 channel (140). Mul-tiple pathways in olfactory neurons may allowmice to respond readily to complex biologicalsignals for social and sexual communication.

The activated olfactory neuron must returnto the steady state in a process known as desen-sitization. As is the case for other GPCRs, Gprotein–coupled ORs may be phosphorylatedupon odorant binding by protein kinases such asprotein kinase A (PKA) and G protein–coupledreceptor kinase (GRK), resulting in desensiti-zation (141–143). Indeed, knockout mice lack-ing GRK showed slower recovery of cAMP in-creases, implicating the involvement of GRKin OR desensitization (144). In addition, PKA-mediated phosphorylation and subsequent in-ternalization of ORs along with β-arrestin ledto a reduction of the number of ORs in themembrane (145). Although direct evidence forOR phosphorylation has not been obtained, de-sensitization mechanisms likely exist at the levelof ORs. In addition, elevated intracellular Ca2+

that enters the cell via the influx through CNGchannels causes these channels to downregu-late themselves and thus close (146–148). Ca2+

also negatively regulates adenylyl cyclase activ-ity (149). These Ca2+-mediated negative feed-back mechanisms allow activated olfactory neu-rons to return to the steady state and to preparefor the next odor stimulus.


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Vertebrate Vomeronasal Receptors

The mouse vomeronasal epithelium is dividedinto two functionally distinct layers: The neu-rons in the apical layer coexpress V1Rs and theG protein Gαi2, and the neurons in the basallayer coexpress V2Rs and Gαo (32). V1Rs arelikely to couple Gαi2 and to stimulate Gβγ-mediated calcium signaling, whereas V2Rs arelikely to couple to Gαo and also to stimu-late a calcium signaling cascade. Indeed, mouseV1rb2, one member of the V1R subfamily,elicits an inhibitory signal for the cAMP cas-cade and Gβγ-mediated calcium increases uponstimulation with 2-heptanone via coupling withGαi2 in HeLa cells coexpressing CNG channels(97). Gβγ-mediated activation of phospholipaseC resulted in the production of inositol-1,4,5-triphosphate (IP3), diacylglycerol (DAG), andpolyunsaturated fatty acids, all of which havebeen implicated in vomeronasal sensory sig-naling (150–152). In contrast, human V1Rscouple to stimulatory G proteins such as Gαs

and Gαolf and stimulate the cAMP pathway inHeLa/CNG cells (97). This observation is con-sistent with the fact that human V1Rs are coex-pressed with Gαolf in the human olfactory ep-ithelium. The VRs in various species appearto have evolved so that each VR can coupleto the appropriate G proteins in the expressedneurons.

The primary transduction channel down-stream of these products is thought to be thetransient receptor potential channel TRPC2,which is strongly expressed in both apical andbasal compartments of the VNO (153). Indeed,involvement of TRPC2 in VNO-mediatedpheromone signaling was obtained by analysisof knockout mice whose sociosexual behaviorwas impaired (154–156). Caution is warrantedin interpreting such results because expressionof VRs in the VNO of TRPC2 knockout miceappears to be downregulated. Thus, a primaryeffect of TRPC2 function in transduction ver-sus a secondary effect of TRPC function on thecellular viability of the VNO needs to be disen-tangled (155). The mechanisms underlying ac-tivation of TRPC2 via a VR/PLC pathway are

currently unknown, and additional ion channelsmay be involved in producing the pheromone-evoked conductance of vomeronasal sensoryneurons. Recently, we have shown that V2Rp5-mediated ESP1 signal is completely impaired inTRPC2 knockout mice, indicating that TRPC2exists downstream of VRs (S. Haga & K.Touhara, manuscript in preparation).

Insect Olfactory Receptors

Vertebrate chemosensory signal transduction,as we discuss above, utilizes familiar signalingelements downstream of canonical GPCRs. Towhat extent do insects share canonical GPCRsignal transduction mechanisms with verte-brates? Because insect OR genes encode pro-teins that span the membrane seven times, mostscientists assume that insect ORs, like ORsin all other species, are seven-transmembrane-domain GPCRs. This assumption seems rea-sonable because the vast majority of proteinswith seven transmembrane domains are in factGPCRs [the light-activated ion channel chan-nelrhodopsin is a notable exception (157)]. Ac-cordingly, it is generally thought that the insectolfactory system utilizes a signal transductionmechanism similar to that in the vertebrate ol-factory system: G protein activation and sub-sequent second-messenger production lead toan increase in dendritic membrane conductanceand the generation of action potentials followedby membrane depolarization via ion channelopening. How strong is the evidence that Gprotein signaling is part of the primary signaltransduction cascade in insect olfaction?

Here we review the existing evidence forand against metabotropic coupling of insectORs to G protein signaling pathways. Electro-physiological experiments in insect antennaesuggested that olfactory neurons have multipletypes of ion channels sensitive to various sec-ond messengers such as IP3, DAG, cGMP, andcalcium (158–160). Biochemical analysis alsoindicated that IP3 is produced upon odorant orpheromone simulation of insect moth antennae(161). However, none of these observationaldata really proved that such second messengers


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were necessary for insect olfaction. Geneticapproaches using Drosophila made it possible toknock down, knock out, or overexpress variouselements of canonical G protein signalingcascades. Overexpression of the dunce adenylylcyclase produced dominant effects on olfactorybehavior, leading Alcorta and colleagues (162)to conclude that cAMP signaling is crucial forfly smell. Carlson and colleagues (163, 164)used mutants defective in phospholipase sig-naling to reach the opposite conclusion: InsectORs use phospholipase C signaling pathways.Genetic knockdown of Gq protein subunitsresulted in reduced olfactory responses inelectrophysiological recordings from antennae(165) and in odor-evoked behavior (166). Noneof the phenotypes obtained to date appear tobe as strong as any of the mouse knockouts ofprimary olfactory signal transduction elementsreviewed above (see Vertebrate OlfactoryReceptors subsection in this section, above).Thus, the mechanisms mediating insect olfac-tory transduction are controversial, and theevidence for the involvement of G protein–mediated second-messenger molecules ininsect olfaction remains equivocal andconfusing.

Several groups have tried to clarify mat-ters by expressing insect ORs in heterologouscells in a reductionist approach to understandwhat signaling elements are necessary and suf-ficient for insect ORs to respond to their cog-nate odor ligands. Initial reports using het-erologous expression suggested that insect ORsfunctioned in heterologous cells without thecoreceptor encoded by Or83b in Drosophilaand its orthologs in other insects. Thus, theodor responsiveness of Drosophila Or43a wasreconstituted in oocytes without Or83b (109).A Bombyx pheromone receptor, BmOR1, re-sponded to bombykol in the presence of BmGαq

without BmOR2, the Bombyx Or83b ortholog(116). BmOR1 and HR13 were functionallyexpressed when Gα15 was cotransfected inHEK293 cells (167). These results suggestedthat the ligand-binding subunit of the insect ORcould function as a GPCR in vitro, but these invitro results were at odds with functional mech-

anisms in vivo in which the complex formationof conventional ORs and Or83b was requiredfor insect OR function (65). Indeed, the re-sponse amplitude obtained by BmOR1/BmGαq

signaling in the absence of the BmOR2 core-ceptor (116) was 100 times lower than that ob-served for BmOR1/BmOR2 (117). This led ourgroups and others to speculate that G protein–mediated pathways via the ligand-binding ORsubunit contribute little to insect smell and thatinstead an unknown signaling cascade medi-ated by the OR/OR83b complex predominatesin vivo.

The first evidence for atypical signal trans-duction characteristics of insect ORs camefrom studies of the silk moth bombykol recep-tor BmOR1. The BmOR1/BmOR2 complexexpressed in Xenopus oocytes elicited a non-selective cation channel activity previouslyunobserved in oocyte membranes (117). Thisfinding led to the intriguing hypothesis that theinsect OR/OR83b complex forms ionotropic-type ligand-gated channels. Indeed, not onlyin oocytes, but also in mammalian cells such asHeLa and HEK293 cells, ligand-binding ORsfrom silk moth, fruit fly, and mosquito (i.e.,BmOR1, Or47a, and GPROR2) combined withthe orthologous Or83b coreceptor from theseinsect species exhibited similar ion channelproperties in evoking nonselective ion conduc-tance upon odorant or pheromone stimulation(168). Outside-out patch-clamp recordingsof membranes expressing insect OR/Or83bcomplexes showed transient unitary currents,which were independent of intracellular factorssuch as cAMP, IP3, DAG, ATP, and GTP.Whole-cell currents and influx of extracellularcalcium persisted in the presence of generalinhibitors of G protein signaling (168), a resultalso confirmed by Newcomb and colleagues(169). The latency of current responses wasmuch faster than that of G protein–coupled ol-factory responses, and moreover, ion selectivityof the insect OR complex was dependent onsubunit combination (168). Taken together, weconcluded that insect ORs form heteromericcomplexes and that the complex itself evokescurrents directly upon ligand stimulation.


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This is by no means the end of this intrigu-ing story because Hansson’s group (170) useda similar approach to examine signaling down-stream of the fruit fly Or22a/Or83b complex inheterologous cells and reached somewhat dif-ferent conclusions. Wicher et al. (170), like Satoet al. (168), noticed the same rapid, ionotropiccurrent that was independent of G proteins, butcharacterized a later metabotropic current in-duced by cyclic nucleotide–dependent signal-ing via Gαs. On the basis of their data, Wicheret al. (170) concluded that Or22a couples to Gproteins and that the Or83b coreceptor func-tions as a CNG channel. Sato et al. (168)observed that some insect OR complexes ex-hibit a small ligand-independent sensitivity tocyclic nucleotides such as cAMP and cGMP;these investigators observed no clear increasein cAMP levels upon ligand stimulation (168).Thus, there is continued controversy as towhether cyclic nucleotides modulate activity ofthe OR/OR83b ion channel complex indepen-dently of ligand signal transduction or whetherthey are involved in primary olfactory signaltransduction.

Despite the differences between the two re-cent papers discussed above (168, 170), bothgroups propose that the insect ORs have the ca-pacity to act as ligand-gated ion channels. Likechannelrhodopsin, these seven-transmembraneproteins thus represent an exception to therule that seven-transmembrane-domain pro-teins are always GPCRs, and instead areproposed to be seven-transmembrane-domainproteins that form a ligand-gated ion channelcomplex. No known ion channel pore or se-lectivity filter motifs have been found in insectORs, and therefore a molecular basis for thechannel activity is unknown. Small deletionsin the sixth transmembrane domain of Or83bthat produced a GYG motif found in potas-sium channel selectivity filters altered the ionselectivity of the Or22a/Or83b complex (170),but the details of how these proteins form acation-selective pore remain to be elucidated.Although an untested hypothesis, it seems rea-sonable to propose that insect GRs, proteinsthat are in the same receptor superfamily as

the ORs and that sense bitter and sweet tas-tants as well as carbon dioxide may also sharean ionotropic coupling mechanism with the in-sect ORs. Support for this idea can be foundin an earlier paper that documented ionotropicsugar-gated currents on the blowfly antenna(171). If future data confirm this proposal, theinsect chemosensory gene family may representthe largest single family of ion channels in anygenome.

How and why did insects acquire such an ol-factory signaling mechanism completely differ-ent from that in the vertebrate olfactory system?The speed of G protein–mediated transductionutilized in the vertebrate olfactory system isrelatively slow [50–100 ms for vertebrate ORs(172) versus 18–25 ms for insect ORs (168)].The fast responses to the olfactory environmentvia odor-gated ion channels may be an advan-tage in the evolution of flying insects, such asflies and moths, that need to find mating part-ners and food sources while flying. Anotherpossible advantage for using ionotropic recep-tors is to avoid energy consumption in second-messenger cascades using ATP and GTP insmall cellular compartments like the olfactorydendrites. However, ionotropic coupling doescome with a cost. Direct receptor activationwithout G protein amplification is not as effi-cient in generating depolarizing membrane po-tential changes unless the receptor moleculesare highly concentrated in dendritic mem-branes. Indeed, immunohistochemical studieshave shown that the insect ORs are highly en-riched in membranes (65, 110).

The OR/OR83b complex is spontaneouslyactive in outside-out patch membranes (168),which appears to account for previous ob-servations that olfactory sensory neurons ex-hibit both ligand-activated and ligand-inhibitedelectrical activity in vivo (110, 111). Insectspecies have fewer ORs in comparison with ver-tebrate species. We speculate that these bipo-lar characteristics derived from the ionotropicproperties of insect ORs may provide a greatervariety of temporal response profiles andpatterns, compensating for the small num-bers of ORs. The distinct signal transduction


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Glomerulus:describes theorganization of thefirst olfactory neuropilin both vertebrates andinvertebrates. Thisspherical structurerepresents theconvergence ofincoming olfactorysensory neurons aswell as the dendrites ofsecond-orderprojection neuronsand processes of localinterneurons

OB: olfactory bulb

mechanisms between invertebrates and verte-brates may reflect different evolutionary andenvironmental pressures on the olfactory sys-tems in these different animals.

INFORMATION TRANSMISSIONTO THE BRAIN

Olfactory Receptor Circuitsin Mammals and Insects

In mammalian species, the olfactory bulb (OB)is the first brain region that relays neural sig-nals of olfactory sensory neurons to secondaryneurons, called mitral/tufted cells, that in turnsend their axons to the central olfactory system(173, 174). The OB corresponds to the antennallobe in insects, and the two olfactory process-ing centers are organized in a similar manner(175). Individual odorants activate distinct sub-sets of ORs, resulting in the construction of aglomerular activation pattern that is unique foreach odorant in a stereotyped region of the OBcalled an odor map (176–178). Different odor-ants elicit different glomerular activity patterns,but structurally related odorants activate simi-lar sets of glomeruli because similar odorantsare recognized by similar sets of ORs in theolfactory epithelium. Even for the same odor-ant, different concentrations result in differentpatterns in a way such that more glomeruli arerecruited by higher concentrations of odorants(179). In mice, spatial and functional mappingof OR-defined glomeruli has revealed that thepositional relationship of glomeruli varies con-siderably between individual mice and even be-tween the left and right OBs in the same animal(179). Unlike the stereotyped odor map, precisebulbar OR maps appear to differ between indi-viduals, and the odor map can be truly describedonly by repeated examinations using many ani-mals. Odor maps that change according to odorconcentration have also been documented in in-sect species (180, 181).

Mitral and tufted cells that synapse with ol-factory sensory neurons in glomeruli projecttheir axons to the olfactory cortex throughthe lateral olfactory tract (182). Classical neu-

roanatomical tracing studies have revealed thatthe regions in the primary olfactory cortex re-ceiving input from the OB include the anteriorolfactory nucleus (AON), taenia tecta (TT), ol-factory tubercle (OT), piriform cortex (PYR),anterior cortical amygdaloid nucleus (ACN),posterolateral cortical amygdaloid nucleus(PLCN), and entorhinal cortex (EC) (183, 184).In insects, the secondary neurons are projectionneurons that project the axons to higher brainregions of the mushroom body and the lateralhorn of the protocerebrum. It is not yet entirelyclear whether the orderly spatial representa-tions created in olfactory bulb or antennal lobeglomeruli are preserved in higher brain regionsor whether the representations are distributed.Cortical representations of odor informationwill be an important field for future study.

In mammals, each of the cortical subregionsprojects information to various areas in thebrain (185). One such region is the orbitofrontalcortex, which is a prominent site of olfactoryprocessing. Functional imaging studies usingfMRI (functional magnetic resonance imaging)or PET (positron emission tomography) madeit possible to identify specific areas in whicholfactory processing takes place upon odorantstimulation (186, 187).

By targeting of transneuronal tracersto gonadotropin-releasing hormone (GnRH)neurons in transgenic mice, the medial pre-optic area–anterior hypothalamus (MPOA-AH), in which many GnRH neurons arelocated, was shown to receive olfactory in-put from various cortical areas including theTT, the AON, and the PYR (188). In addi-tion, a virus-mediated approach successfullylabeled the olfactory-hypothalamus pathway(189). Trimethyl-thiazoline, an odorant thatinduces the freezing response in mice, acti-vates the hypothalamic-pituitary-adrenal path-way via the bed nucleus of the stria termi-nalis (BST) (190). Neuroanatomical, genetic,and imaging approaches are beginning to sug-gest that hard-wired circuits for inducing innatebehavior or reproductive changes exist in themain olfactory system. It will be of interest tostudy this circuitry at finer scales of resolution.


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Vomeronasal Receptor Circuits

VNO-mediated chemosensory signals are sentto the AOB, which then projects to the basalforebrain, which contains regions importantfor reproductive and mating behavior (11, 43,184, 191). To examine the projection patternof vomeronasal sensory neurons to the AOB,researchers generated various transgenic andknock-in mice in which specific VR-expressingneurons could be visualized. V1R- and V2R-expressing neurons project to the rostral andthe caudal regions of the AOB, respectively,and form several converged glomeruli whereinthe sensory neuronal axon terminals synapsewith the secondary neurons (46, 192). Thesecondary neurons then project to the BST,the BAOT, the nucleus of the accessory ol-factory tract, and the medial and posterome-dial cortical nuclei of the amygdala (MEAand PMCO, respectively), from which the in-formation is relayed to the MPOA-AH; be-havioral and endocrine responses ensue (184,193, 194). Because the MPOA contains manyGnRH or luteinizing hormone–releasing hor-mone (LHRH) neurons, the VNO inputs to theamygdala-hypothalamus pathway may regulatereproductive function. Recently, expression ofa targeted transneuronal tracer in transgenicmice was used to visualize the VNO-AOB neu-ronal circuit, which connects to GnRH/LHRHneurons (188). Although one study suggestedthat this input is negligible (189), VNO signal-ing appears to be processed at the basal fore-brain area, which may eventually lead to gen-der recognition and stimulation of mating andaggressive behaviors.

The emerging view of the higher-orderneural circuitry in the vomeronasal systemand main olfactory system in mice suggeststhat the underlying mechanisms for process-ing general odorant-induced behavioral or en-docrinological effects are shared with those forpheromone-induced innate behavior or repro-ductive changes via the vomeronasal system.As discussed above (see subsection: What Isan Odorant? What Is a Pheromone?), whetherthe observed effect is defined as pheromonal

or not is solely dependent on the informationpossessed by the chemical and whether it repre-sents meaningful communication between con-specific individuals.

Sexual Dimorphismin Olfactory Circuits

Many pheromones elicit sexually dimorphicbehaviors. A simple means to afford suchsex-specific effects is to build sensory systemsthat are sexually dimorphic at various lev-els of the circuit from input to output: thepheromone, the receptor, and/or higher braincircuitry. In some insects, sexual dimorphismis extreme at all levels. Female moths producefemale-specific pheromones that are detectedby male-specific pheromone receptors, and thisinformation is transmitted to a male-specificregion of the antennal lobe, resulting inspecific sex behavior. This mechanism is clearlydocumented in the B. mori silk moth: Themale-specific OR BmOR1 recognizes bom-bykol, the sex pheromone that only female silkmoths produce (116, 117), and male neuronsexpressing BmOR1 project to the male-specificmacroglomerular complex in the antennal lobe(195). In this case, the sexual dimorphism isensured at the three distinct levels.

In contrast, cVA is a male-specific phero-mone that elicits aggregation behavior in bothmale and female Drosophila but induces differ-ent courtship behavior between male and fe-male. cVA is detected by Or67d (122, 123),which is expressed in both male and female flies.Careful analysis of the higher-brain circuits up-stream of Or67d input indicated that sexuallydimorphic behavior elicited by cVA may be me-diated by a sexually dimorphic neural circuit inthe lateral horn (196).

Sexual dimorphism affording sex-specificpheromone perception is also inferred to ex-ist in the mouse brain, but neuroanatomicaldemonstration of this idea is not yet avail-able. For example, 2,5-dimethylpyrazine, acompound in female urine, delays the onset ofpuberty and induces longer estrous cycles in


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female mice (197, 198) but decreases testos-terone levels in male mice (see discussion inReference 199). 3,4-Dehydro-exo-brevicomin,2-sec-butyl-4,5-dihydrothiazole, 6-hydroxy-6-methyl-3-heptanone, and α- and β-farnesenesfrom male urine or the preputial gland acceler-ate the onset of puberty and induce estrous in

HUMAN PHEROMONES

The importance of pheromones to animals is unquestionable,but what is the evidence for and against the existence of humanpheromones? In 1971, Martha McClintock described the bio-logical phenomenon of menstrual synchrony and suppression inhuman females (207). Axial secretions from one woman were ob-served to change menstrual cycle timing when they were paintedon the upper lip of another woman. More than 35 years later, thecompound(s) responsible for this effect remains unknown, andeven the existence of the effect is controversial among psycholo-gists (despite the fact that the concept of synchronized estrus iswell-accepted in rodents).

Other studies have shown that odorous steroids such as an-drostenone and androstadienone, derived from male axial secre-tions, can selectively influence mood, physiological state, andneural activity in humans and that sexual orientation affects re-sponses to these male-derived odors (205, 208). The ability todetect androstenone and androstadienone differs enormously be-tween people and is in part genetically determined (209). Poly-morphisms in a single human odorant receptor were recentlylinked to perceptual variation in androstenone and androsta-dienone perception (29).

The strict definition of a pheromone may have to be relaxedif this field is to progress (see discussion in subsection: WhatIs an Odorant? What Is a Pheromone?). Humans are complexcreatures who use multiple sensory inputs in evaluating a po-tential mate. It is therefore unlikely that the kind of innate re-sponses one sees in a male silk moth smelling bombykol can beexpected in a human male. Nevertheless, the identification of ahuman pheromone that reliably modulates human sexual and so-cial behavior would be of obvious clinical and commercial impor-tance. Better chemistry to identify compounds produced by hu-mans in different reproductive states, as well as more quantitativereadouts of the effects of human-derived compounds on humanphysiology and behavior, are needed. If these two experimentalimprovements can be achieved, this area will certainly be one towatch in the years to come.

female mice (200–202). These male odors alsoinduce aggressive behavior in male mice (203).Although the production of these pheromonesis sex biased, there are no validated cases ofany V1Rs or V2Rs that are sex-specifically ex-pressed (but see Reference 33). Therefore, wecan assume that both male and female mice havethe same set of receptors for pheromones andthat different behavioral output is establishedby sex-specific neural circuits in the brain. Sup-port for this idea comes from an experiment inwhich researchers observed a sexually dimor-phic distribution of c-Fos-labeled neurons inthe brain of mice stimulated with soiled bed-ding or body odors of the opposite sex (204).This partial sexual dimorphism is also seen forthe mouse ESP1 peptide pheromone. ESP1 issecreted in male mouse tears but is receivedby both male and female vomeronasal sensoryneurons that express the ESP1 receptor, V2Rp5(107). Expression of V2Rp5 is not sex specific,but ESP1 and the higher-brain neural circuitappear to be sex specific (S. Haga & K. Touhara,unpublished observation).

The existence of pheromonal communica-tion in humans is hotly debated (see side bar:Human Pheromones). One clue indicating thathumans are sensitive to social signals came fromfunctional imaging studies that demonstratedthat the male-derived odor androstadienoneelicits a sex-specific pattern of hypothalamic ac-tivity that also depends on the sexual orien-tation of the subject (205, 206). Thus, sexu-ally dimorphic mechanisms for the detectionof physiologically important pheromones andodorant substances appear to be conserved be-tween invertebrates and vertebrates, and this di-morphism can be established at different levelsfrom chemical substances to receptors and fi-nally to the brain.

CONCLUSIONS

In this review, we examine how odorant andpheromone signals are distinguished and dis-criminated, focusing our discussion on threemolecular and cellular levels in chemosensorypathways: a chemosignaling molecule (odorant


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or pheromone), a receptor (OR, VR), and aneural circuit to the brain. The production ofchemosensory signals and the expression of thereceptor molecules are often carefully regu-lated to produce species- or sex-specific sig-nals. Pheromonal effects are sometimes ob-served in both male and female individualsbut in a sexually dimorphic fashion. In thiscase, sex specificity is likely encoded at thelevel of neural circuits in the brain. By com-paring the olfactory systems of invertebratessuch as flies and moths, and vertebrates suchas mice and humans, we note both commonstrategies for integrating olfactory signals aswell as completely different signal transductionmechanisms.

Although olfaction is a primitive sen-sory modality, the ligand-binding receptormolecules and associated signal transductionmechanisms are rapidly changing in each evo-

lutionary lineage. We suggest that such rapidevolution originates in positive pressure fromthe unique environment and the lifestyle of agiven species. Extreme differences in the under-lying mechanisms of OR transduction betweeninsect and mammal suggest that other, evenmore novel ORs and signal transduction mech-anisms may remain to be discovered. Such di-versity of chemosensory receptors and signalingsystems may account for species-specific differ-ences in olfaction. Deciphering the molecularand physiological mechanisms of olfactory sys-tems in various animal species, including aque-ous and terrestrial animals, would expand theview of various chemosensory systems. Theeventual goal of the field would be to under-stand how animals developed such sophisticatedand individually tailored chemical communica-tion strategies that afford optimal survival ofeach organism.

SUMMARY POINTS

1. General odors and pheromones derive from different sources and elicit distinct behaviorsand physiological effects when detected by an animal.

2. General odors and pheromones bind to structurally different receptors in different genefamilies in mammals and to different receptors in the same gene family in insects.

3. Vertebrate and insect olfactory receptors are structurally unrelated and signal throughdifferent pathways.

4. General odors in general are encoded in a combinatorial fashion, whereas pheromonesin general appear to activate a labeled-line pathway in which pheromone input leads toa direct behavioral or physiological output.

FUTURE ISSUES

1. At the time of this writing, the details of insect chemosensory signal transduction are stillhighly controversial. Do G proteins figure in insect chemosensory signal transduction,or do the involved receptors signal independently of G proteins?

2. Are there pheromones for individual and species recognition in mammals? If so, whatare they, and how are they processed by the brain?

3. In humans and nonhuman primates, which lack a functional VNO, does the main olfac-tory system perform the dual roles of general odor and pheromone detection?

4. Do humans use chemical signals to communicate? If so, what is the nature of thesepheromones, and how are they received?


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DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

Work in our laboratories is supported by grants from the Program for Promotion of Basic Re-search Activities for Innovative Biosciences, Japan (PROBRAIN) and a Grant-in-Aid for ScientificResearch on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Tech-nology (MEXT) of Japan (K.T.); by the National Institutes of Health (NIH) and the Foundationfor the National Institutes of Health through the Grand Challenges in Global Health Initiative(L.B.V.); and by joint grants to K.T. and L.B.V. from the NIH U.S.-Japan BRCP and the JSPSJapan-U.S. Cooperative Science Program.

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RELATED RESOURCES

1. Two databases provide molecular and genomic information on chemosensory receptors:The Human Olfactory Receptor Data Explore (HORDE) (http://bioportal.weizmann.ac.il/HORDE/) and Olfactory Receptor DataBase (ORDB) (http://senselab.med.yale.edu/ordb/default.asp)

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3. Brennan PA, Zufall F. 2006. Pheromonal communication in vertebrates. Nature 444:308–154. Vosshall LB, Stocker RF. 2007. Molecular architecture of smell and taste in Drosophila. Annu.

Rev. Neurosci. 30:505–335. Wilson RI, Mainen ZF. 2006. Early events in olfactory processing. Annu. Rev. Neurosci.

29:163–201


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AR369-FM ARI 9 January 2009 0:57

Annual Review ofPhysiology

Volume 71, 2009Contents

CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbbins, Section Editor

Sex-Based Cardiac PhysiologyElizabeth D. Luczak and Leslie A. Leinwand � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

CELL PHYSIOLOGY, David E. Clapham, Associate and Section Editor

Convergent Evolution of Alternative Splices at Domain Boundariesof the BK ChannelAnthony A. Fodor and Richard W. Aldrich � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �19

Mechanisms of Muscle Degeneration, Regeneration, and Repair in theMuscular DystrophiesGregory Q. Wallace and Elizabeth M. McNally � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �37

Plant Ion Channels: Gene Families, Physiology, and FunctionalGenomics AnalysesJohn M. Ward, Pascal Mäser, and Julian I. Schroeder � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Polycystins and Primary Cilia: Primers for Cell Cycle ProgressionJing Zhou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �83

Subsystem Organization of the Mammalian Sense of SmellSteven D. Munger, Trese Leinders-Zufall, and Frank Zufall � � � � � � � � � � � � � � � � � � � � � � � � � � � � 115

ECOLOGICAL, EVOLUTIONARY, AND COMPARATIVEPHYSIOLOGY, Martin E. Feder, Section Editor

Complementary Roles of the Main and Accessory Olfactory Systemsin Mammalian Mate RecognitionMichael J. Baum and Kevin R. Kelliher � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 141

Pheromone Communication in Amphibians and ReptilesLynne D. Houck � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

ENDOCRINOLOGY, Holly A. Ingraham, Section Editor

Transcriptional Control of Mitochondrial Biogenesis and FunctionM. Benjamin Hock and Anastasia Kralli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 177

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GASTROINTESTINAL PHYSIOLOGY, James M. Anderson, Section Editor

The Functions and Roles of the Extracellular Ca2+–Sensing Receptoralong the Gastrointestinal TractJohn P. Geibel and Steven C. Hebert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 205

Neuroendocrine Control of the Gut During Stress:Corticotropin-Releasing Factor Signaling Pathways in the SpotlightAndreas Stengel and Yvette Taché � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 219

Stem Cells, Self-Renewal, and Differentiation in theIntestinal EpitheliumLaurens G. van der Flier and Hans Clevers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241

NEUROPHYSIOLOGY, Roger Nicoll, Section Editor

Dendritic Spine DynamicsD. Harshad Bhatt, Shengxiang Zhang, and Wen-Biao Gan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 261

Endocannabinoid Signaling and Long-Term Synaptic PlasticityBoris D. Heifets and Pablo E. Castillo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 283

Sensing Odorants and Pheromones with Chemosensory ReceptorsKazushige Touhara and Leslie B. Vosshall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 307

Signaling at Purinergic P2X ReceptorsAnnmarie Surprenant and R. Alan North � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

RENAL AND ELECTROLYTE PHYSIOLOGY, Gerhard H. Giebisch, Section Editor

Activation of the Epithelial Sodium Channel (ENaC)by Serine ProteasesBernard C. Rossier and M. Jackson Stutts � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

Recent Advances in Understanding Integrative Control of PotassiumHomeostasisJang H. Youn and Alicia A. McDonough � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 381

RESPIRATORY PHYSIOLOGY, Richard C. Boucher, Jr., Section Editor

The Contribution of Epithelial Sodium Channels to Alveolar Functionin Health and DiseaseDouglas C. Eaton, My N. Helms, Michael Koval, Hui Fang Bao, and Lucky Jain � � � � � 403

The Role of CLCA Proteins in Inflammatory Airway DiseaseAnand C. Patel, Tom J. Brett, and Michael J. Holtzman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 425

Role of HDAC2 in the Pathophysiology of COPDPeter J. Barnes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 451

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SPECIAL TOPIC, ASTHMA, Jeffrey M. Drazen, Special Topic Editor

Aspirin-Sensitive Respiratory DiseaseSophie P. Farooque and Tak H. Lee � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 465

Immunobiology of AsthmaQutayba Hamid and Meri Tulic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 489

Noncontractile Functions of Airway Smooth Muscle Cells in AsthmaOmar Tliba and Reynold A. Panettieri, Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

Indexes

Cumulative Index of Contributing Authors, Volumes 67–71 � � � � � � � � � � � � � � � � � � � � � � � � � � � 537

Cumulative Index of Chapter Titles, Volumes 67–71 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 540

Errata

An online log of corrections to Annual Review of Physiology articles may be found athttp://physiol.annualreviews.org/errata.shtml

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Documents

Sensing Odorants and Pheromones with Chemosensory Receptors