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From Pheromones to Behavior

ROBERTO TIRINDELLI, MICHELE DIBATTISTA, SIMONE PIFFERI, AND ANNA MENINI

Department of Neuroscience, University of Parma, Parma; and International School for Advanced Studies,

Scuola Internazionale Superiore di Studi Avanzati (SISSA) and Italian Institute of Technology, SISSA Unit,

Trieste, Italy

I. Introduction 921II. Mammalian Pheromones 922

A. Pheromones in various animal orders 922B. Major urinary proteins and major histocompatibility complex molecules 925C. Exocrine gland-secreting peptides 926

III. Chemosensory Systems Detecting Pheromones 927A. Vomeronasal epithelium 927B. Olfactory epithelium 929C. Other olfactory organs 929

IV. Putative Pheromone Receptors 930A. Vomeronasal receptors 930B. Odorant receptors 933C. Trace amine-associated receptors 934D. Olfactory-specific guanylyl cyclase type D receptor 934

V. Pheromone Transduction 935A. Vomeronasal sensory neurons 935B. Olfactory sensory neurons 939C. Grueneberg ganglion 940

VI. Signaling From Sensory Epithelia to the Brain 940A. Main and accessory olfactory bulbs 940B. Signaling beyond the olfactory bulbs 943C. Pheromone-Activated hormonal systems 944

VII. Pheromone-Mediated Behaviors 944A. Male behaviors 945B. Female behaviors 946

VIII. Conclusions and Future Challenges 947

Tirindelli R, Dibattista M, Pifferi S, Menini A. From Pheromones to Behavior. Physiol Rev 89: 921–956, 2009;doi:10.1152/physrev.00037.2008.—In recent years, considerable progress has been achieved in the comprehension ofthe profound effects of pheromones on reproductive physiology and behavior. Pheromones have been classified asmolecules released by individuals and responsible for the elicitation of specific behavioral expressions in membersof the same species. These signaling molecules, often chemically unrelated, are contained in body fluids like urine,sweat, specialized exocrine glands, and mucous secretions of genitals. The standard view of pheromone sensing wasbased on the assumption that most mammals have two separated olfactory systems with different functional roles:the main olfactory system for recognizing conventional odorant molecules and the vomeronasal system specificallydedicated to the detection of pheromones. However, recent studies have reexamined this traditional interpretationshowing that both the main olfactory and the vomeronasal systems are actively involved in pheromonal communi-cation. The current knowledge on the behavioral, physiological, and molecular aspects of pheromone detection inmammals is discussed in this review.

I. INTRODUCTION

Chemosensation is one of the sensory modalities thatanimals have evolved to analyze the chemical properties

of the external world, enabling the detection and thediscrimination of a very high number of molecules withdifferent structures. Substances carrying a chemical mes-sage among animals are termed “semiochemicals,” from

Physiol Rev 89: 921–956, 2009;doi:10.1152/physrev.00037.2008.

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the Greek “semeion” (sign). An important class of semio-chemicals is constituted by pheromones. The term pher-

omone, based on the Greek “pherein” (to carry or trans-fer) and “hormon” (to stimulate or excite), was intro-duced in 1959 by the entomologists Peter Karlson andMartin Luscher (151) to identify specific biologically ac-tive substances “which are secreted to the outside by anindividual and received by a second individual of the samespecies, in which they release a specific reaction, forexample, a definite behavior or a developmental process.”In the same year, the first insect pheromone bombykol,released by the female silkworm to attract mates, wascharacterized by Butenandt et al. (43). The use of phero-mones among insects has been extensively investigated,and interested readers may consult several reviews on thesubject (4, 111, 130, 379).

Another class of semiochemicals is constituted by al-lelomones: substances produced by members of one speciesthat influence the behavior of members of another species(for a review, see Ref. 333). Although in this review we willmainly discuss the effects elicited by pheromones in mam-mals, we will also briefly mention some examples of allel-omonal communication (5, 107, 108, 169).

In most mammals, the nasal cavity contains at least twomajor sensory systems that may be involved in the detectionof pheromones: the vomeronasal and the main olfactorysystems. Evidence indicates that some behaviors are medi-ated exclusively by one or by a complementary action ofboth systems. For example, Bruce (37) reported that, whenfemale mice that have been recently inseminated are ex-posed to urine of a male different from the one they matedwith, the pregnancy is interrupted and the female returns toestrus. This effect depends on a functional vomeronasalsystem (15). Differently, the nipple search in rabbit pups isone of the typical examples of behavior strictly dependenton an intact main olfactory system (119). In other instances,behavioral responses are mediated by both the vomeronasaland the main olfactory systems. This is the case of femalehamsters, whose ultrasonic calling during estrus is abol-ished by the inactivation of either the vomeronasal or themain olfactory system (145). In recent years, the discovery ofdistinct pheromone receptor families coupled to a complexnetwork of signal transduction pathways, together with ad-vances in molecular genetics and functional electrophysiologi-cal and imaging techniques, have greatly advanced our under-standing of the physiological role of pheromones.

II. MAMMALIAN PHEROMONES

A. Pheromones in Various Animal Orders

Pheromones have a very large chemical variability(Table 1), and therefore they are preferably grouped ac-cording to their functions rather than to their structural

determinants. For example, trace pheromones allow at-tracting a conspecific, while alarm pheromones alertother conspecifics about an external challenge and canevoke aggressive behaviors.

A first classification groups intraspecific signalingmolecules in releaser and primer pheromones (239). Re-leaser pheromones lead to individual recognition andevoke short-latency behavioral responses in the conspe-cific like in aggressive attacks, or mating, or territorymarking. Conversely, primer pheromones induce delayedresponses that are commonly mediated through the acti-vation of the neuroendocrine system. Thus, through theemission of chemical cues, an individual forces the hor-monal balance of a receiver, thus modulating its repro-ductive status.

In mammals, pheromones have been reported to beinvolved in a variety of behavioral effects also in unrelatedspecies and, surprisingly, some pheromonal molecules areshared, with different purposes, between species.

1. Proboscidea

Despite their size, which would obviously precludeextensive behavioral studies, Asian and African elephantshave been largely investigated for their pheromonal re-sponses by the late Bets Rasmussen. During musth, aperiodic condition in which mature males are character-ized by highly aggressive behavior, elephants producelarge quantities of a specific pheromone, frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane), that is releasedin temporal gland secretions, urine, and breath (307).Adult males are mostly indifferent to frontalin, whereassubadult males are highly reactive, often exhibiting repul-sion or avoidance. Female chemosensory responses tofrontalin vary with the hormonal status. Females in thefollicular phase are the most responsive and often dem-onstrate mating-related behaviors subsequent to frontalinstimulation (304). Furthermore, female Asian elephantsexcrete a urinary pheromone, (Z)-7-dodecen-L-yl acetate,to signal to males their readiness to mate (305). Interest-ingly, females of more than 100 species of insects use thesame compound as part of their pheromone blends toattract insect males. The finding that the same moleculemay be shared by unrelated species for a different phero-monal purpose is only apparently surprising, owing to thefact that structural constraints are often dictated by com-mon metabolic pathways.

2. Marsupialia

In marsupials, the gray short-tailed opossum (Mono-

delphis domestica) communicates by scent marking. Themale opossum possesses a prominent suprasternal scentgland, whose extracts strongly attract female opossums(131), stimulating the estrus in anestrous females via thevomeronasal system.

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3. Soricomorpha (Insectivores)

In musk shrew (Suncus murinus), virgin females ex-posed to cages recently vacated by an adult male receive

mounts from males significantly sooner than femalesexposed to cages soiled by a castrated male, by anotherfemale, or to a clean cage (310).

TABLE 1. Table of pheromones

Pheromone Origin Organ Target Cellular/Biochemical Response Behavior

2,5-Dimethyl pyrazine Mouse female urine VNO V1R neurons IP3 production in the VNO (377) Suppression of estrus cycle (Lee Boot-effect) (219, 281)

Electrical response and calciuminflux in VNO neurons (187)

2-Sec-butyl-4,5-dihydrothiazole (BT)

Mouse male urine VNO V1R neurons Electrical response and calciuminflux in VNO neurons (187)

Estrus induction and synchronization(Whitten effect) (138)

Protooncogene expression inthe AOB (32, 95)

Puberty acceleration (Vandenbergh effect)(266, 283)

Intermale aggressiveness (280)Attractiveness to females (139)

2,3-Dehydro-exo-brevicomin (DB)

Mouse male urine VNO V1R neurons Electrical response and calciuminflux in VNO neurons (187)

Estrus induction and synchronization(Whitten effect) (138)

Protooncogene expression inthe AOB (32, 95)


Intermale aggressiveness (280)Attractiveness to females (139)

�- and �-Farnesene Mouse preputial glands VNO V1R neurons Electrical response and calciuminflux in VNO neurons (187)


Territorial marking (279)Attractiveness to females (139)Suppression of estrus cycle (Lee Boot-

effect) (219)6-Hydroxyl-6-methyl-3-

heptanoneMouse male urine VNO V1ra and V1rb (V1Rs)

neuronsElectrical response and calcium

influx in VNO neurons (61,187)


2-Heptanone Mouse urine VNO V1R neurons Electrical response and calciuminflux in VNO neurons (187)

Estrus extension (137)

IP3 production in the VNO (377)n-Pentyl acetate Mouse urine VNO V1ra and V1rb (V1Rs)

neuronsElectrical response in VNO

neurons (61)Suppression of estrus cycle (Lee Boot-

effect) (281)Isobutylamine Mouse male urine VNO V1ra and V1rb (V1Rs)

neuronsElectrical response in VNO

neurons (61)Estrus acceleration and vaginal opening

(276, 301)3-Amino-s-triazole (similar

to BT)Mouse male urine ? ? ? Attractiveness to females (2)

4-Ethyl phenol Mouse male urine ? ? ? Attractiveness to females (2)3-Ethyl-2,7-dimethyl octane

(similar to DB)Mouse male urine ? ? ? Attractiveness to females (2)

Repulsion to mouse (territorial marking?)(2)

3-Cyclohexene-1-methanol Mouse male urine ? ? ? Attractiveness to females (2)Major urinary proteins

(MUPs)Mouse urine VNO V2R neurons Electrical response and calcium

influx in VNO neurons (47,163)


In vitro survival andproliferation of VNO neurons(408)

Individual recognition (49, 123, 375)

Intermale aggressivenessMHC class I peptides Mouse urine VNO V2R neurons Electrical response and calcium

influx in MOE and VNOneurons (364)

Attractiveness to individuals of a differentmouse strain (358)

(SYFPEITHE;AAPDNRETF)

MOE Single-cell recording in VNOneurons (156, 185)

Pregnancy block (Bruce effect)

Exocrine gland secretingpeptides

Extraorbital lacrimal glands VNO V2R neurons Electrical response in VNOneurons (162, 163)

?

�2-Urinary proteins (�2u) Rat urine VNO V2R neurons G protein activation and IP3

production in the VNO (176,321)

?

Dodecyl-propionate Rat preputial gland ? ? ? Ano-genital licking (36)Aphrodisin Hamster vaginal glands VNO ? IP3 production in the VNO and

c-fos expression in AOBneurons (136, 177)

Copulatory behavior in male (356)

2-Methylbut-2-enal Rabbit milk ? ? ? Attraction and oral grasping in pups (335)(Z)-7-Dodecen-1-yl acetate Elephant female urine ? ? ? Mating-associated behaviors in males (306)(�)(�)1,5-Dimethyl-6,8-

dioxabicyclo[3.2.1]octane(frontaline)

Male asian elephant temporalgland, urine, and breath

? ? ? Repulsion, attractiveness, and sexualbehavior depending on the racemic ratio(92, 304)

VNO, vomeronasal organ; MOE, main olfactory epithelium; AOB, accessory olfactory bulb. Reference numbers are given in paren-theses.

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In tree shrew (Tupaia belangeri), both males andfemales mark their surroundings with urine and skingland secretions. Males mark more frequently than fe-males in the absence of conspecific scent; moreover, malescent stimulates marking in females. Interestingly, in-creasing amounts of male scent result in a correspondingincrease in marking by females (114), suggesting that, inthe wild, pheromones may not exert an all-or-none effectbut rather act in a dose-specific fashion.

4. Carnivora and Ungulata

Both carnivores (dogs and cats) and ungulates com-monly mark their territory with urine and display a spe-cific behavior called “flehmen” (67, 88, 391). In the fleh-men reaction, animals, after the physical contact with aconspecific or with scents emanating from its excrements(urine or feces), lift the head, draw back their lips, andpush the tongue towards the anterior region of the palatein a manner that makes them appear to be “grimacing.” Itis commonly believed that this attitude would allow afaster transfer of pheromones into the olfactory organs.

The saliva of the adult male pig contains a mixture ofsteroids (boar odor). When a boar becomes aggressive orsexually aroused, it produces copious amount of salivawhose pungent odor is attractive to estrus females (287)and facilitates the display of the estrous female’s matingposture (352). The major component of the boar odor isandrostenone, a sexually dimorphic steroid that elicitsboth behavioral responses (65). Curiously, androstenonehas been hypothesized to be a human pheromone as well(18, 96).

5. Primata

Primates scarcely rely on chemical communication;however, the ring-tailed lemur (Lemur catta) exhibits themost highly developed olfactory system among all speciesbelonging to this order (75, 83, 338). Lemur catta pos-sesses a suite of specialized scent glands and a variety ofscent-marking displays (147, 258, 338). Both sexes haveapocrine, located on the wrists, and sebaceous glandfields in their genital regions and adopt distinctive hand-stand postures to deposit glandular secretions on sub-strates. Males mix the glandular secretions and then de-posit this mixture via “wrist marking.” They also impreg-nate their tail fur with their own secretions and then wafttheir tail at opponents during characteristic “stink fights”(147).

There is nowadays a general consensus for the exis-tence of a chemical communication in humans, althoughthere is still a great controversy about the existence,extent, and nature of human pheromones. Among the bestand first known effects attributed to human pheromonesis the synchronization of menstrual cycle (237, 238, 367)and the dimorphic activation of brain areas (332). Expo-

sure to odorless axillary’s secretions from females in thelate follicular and ovulatory phase of the menstrual cycle,respectively, shortens and lengthens the menstrual cyclesof recipient females, accelerating or delaying the lutein-izing hormone (LH) surge (367). Women smelling an an-drogen-like compound activate the preoptic and ventro-medial nuclei of the hypothalamus in contrast to men whoactivate the paraventricular and dorsomedial nuclei hypo-thalamus when smelling an estrogen-like substance (332).Although a robust hypothalamic response is commonlyobserved with ordinary odorants, such a dramatic dimor-phic difference can be interpreted as a hormonal responseto pheromone stimulation.

The apocrine gland secretion of the axilla in combi-nation with the microbial flora is probably the most rele-vant source of candidate compounds for human phero-mones, which consists of a blend of molecules character-izing the individual body odor (14). However, salivary andseminal secretions and urine represent other reservoirs ofputative human pheromones. The male sweat and salivaryprogesterone derivative androstadienone (4,16-androsta-dien-3-one) (AND) has been indicated as a putative hu-man pheromone influencing brain activity (20, 331, 332),endocrine levels (406), physiological arousal, context-de-pendent mood, and sexual orientation (132, 133, 212, 232,300). Woman urine contains the estrogen like steroidestra-1,3,5(10),16-tetraen-3-ol (EST) that also appears tobe a good candidate compound for human pheromones(20, 132, 331, 332).

In studies of brain activity by positron emission to-mography, as above reported, smelling AND and ESTactivates regions primarily incorporating the sexually di-morphic nuclei of the anterior hypothalamus (332), andthis activation is differentiated with respect to sex andcompound. Very interestingly, homosexual males processAND similarly to heterosexual women rather than to het-erosexual men (20). Conversely, lesbian women processAND stimuli as common odorants and, when smellingEST, they partly share activation of the anterior hypothal-amus with heterosexual men (20). These observationsindicate the involvement of the anterior hypothalamus inphysiological processes related to sexual orientation/pref-erence in humans.

6. Rodentia

The paradigmatic model for mammalian pheromoneinvestigations involves laboratory species, among whichrodents and especially mice are the most employed, be-cause they are easy to handle, have extremely well-devel-oped olfactory systems, and offer an almost unlimitedpossibility to act on behavioral responses by genetic ma-nipulation. In these species, several effects have beencarefully described, and the mechanisms of their actionhave even been dissected at a molecular level.



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The vaginal secretion and saliva of female goldenhamsters (Mesocricetus auratus) contain an aphrodisiacsubstance that facilitates the mating behavior of the male(223, 356). This pheromonal effect has been clearly dem-onstrated in assays that measure the mounting behaviorof a male in the presence of an anesthetized male hamster(defined as surrogate female) scented with the stimuli(222). Evidence shows that biologically active compoundswere present in the proteinaceous fraction, whereas aque-ous fractions, organic extracts, and distillates containingvolatile compounds did not exert detectable effects. Afterpurification, pheromonal activity was attributed to a 17-kDa soluble glycosylated protein appropriately termedaphrodisin (356). The complete primary structure of aph-rodisin shows that this protein belongs to the lipocalinsuperfamily. A prominent feature of almost all lipocalinsis the presence of a hydrophobic pocket that accommo-dates endogenous molecules with a relatively high li-pophilic coefficient. The biochemically purified aphrodi-sin carries natural molecules that are contained in thevaginal secretions (33, 34) and that may be responsible forthe pheromonal effect observed in males (136). Biochem-ical and immunohistochemical data suggest that aphrodi-sin may act through G protein-coupled receptors that areexpressed in the vomeronasal system (136, 177).

Fossorial mammals, like the mole-rat, are noted forsensory specializations, such as exquisite tactile senses(45, 60). Although suppression of mole-rat reproductionin females is thought to be meditated by behavioral,rather than pheromonal, cues from the breeding females(360), the chemosensory systems retain demonstrablefunctions of scent marking in common nesting and toiletloci (359) that have been associated with pheromonalcommunication.

In rats, several pheromones are used in differentcontexts as territory marking (309), alarm (1), and mater-nal behaviors (188, 254). The chemically characterized ratpheromones include compounds that are produced bypup’s preputial glands and urine. A particular pattern ofrat maternal behavior is the specific licking and cleaningof pup’s anogenital area, an action crucial to pup survival,as nonlicked pups cannot defecate and run into death.Thus dodecyl propionate, a compound isolated frompups’ preputial glands, can sustain pups’ anogenital lick-ing by dams and therefore pups’ survival (36).

The odors emanating from physically stressed ro-dents are recognized by conspecifics (220, 386). Once thechemical signal is perceived, the receiver undergoes sig-nificant changes. For example, lymphocyte T-killers aresuppressed, whereas B-cells proliferate (57), rats develophyperthermia (160), c-Fos protein expression is increasedin the mitral cell layer of the accessory olfactory bulb(164), reproductive behavior and sexual maturation initi-ate (389a), and social hierarchy may be modified (140).These bodily compounds, defined as alarm pheromones,

signal alert to the presence of a potential danger, promot-ing dispersion and the adoption of defensive actions in thegroup (400). Usually, mice escape from places in whichthe odors from the urine of a physically stressed conspe-cific are present (418). Among other ketones, 2-heptanoneis detectable in the mouse and rat urine (97, 342). In rat,2-heptanone was found to be increased in urine ofstressed subjects and to induce immobility in a recipientconspecific forced to swim (97).

Several other urinary compounds have been indi-cated as activators of distinct anatomical regions of thepheromonal pathway in rats (176, 321, 372); however,none of the effects exerted by these substances is report-edly linked to obvious behavioral responses.

Mouse ecology is largely triggered by pheromonalsignals, which have been investigated for a long time, andtherefore, mice represent the world-wide animal model tostudy intraspecies communication. In the last decade, anenormous burden of data has been collected and, how itis often the case, there is yet an open and sometimescontrasting debate on the specific properties of the dif-ferent signaling molecules.

Early studies have demonstrated a number of incon-trovertible effects of primer pheromones in mice. It isknown that grouped female mice modify or suppress theirestrous cycle (Lee-Boot effect) (387), while male urinecan restore and synchronize the estrus cycle of noncyc-ling females (Whitten effect) (401) or accelerate pubertyonset in females (Vandenbergh effect) (388). In addition,the exposure of a recently mated female mouse to a male,different from the stud, prevents implantation of fertilizedeggs (Bruce effect) (38), implying that the stud or itsindividual odor must be memorized at the moment ofmating to be recognized later. These pheromonal effectsare commonly believed to be mediated by stimuli presentin urine that act via the vomeronasal system.

Signaling pheromones also account for many behav-ioral responses in the mouse. For example, male as wellas lactating female aggressiveness towards an intrudermale are both believed to be triggered by molecules thatare present in male urine.

B. Major Urinary Proteins and Major

Histocompatibility Complex Molecules

Male mouse urine contains an unusually high quan-tity of proteins, called major urinary proteins (MUPs).MUPs form a large family of highly homologous androgen-dependent proteins that are synthesized in the liver andexcreted with urine (46, 208). MUPs and the rat hortologs�2 urinary globulins belong to the lipocalin superfamily,and accordingly, they bind and release small (volatile)pheromones that are also produced in an androgen-de-pendent fashion (9, 23). Although MUPs are also ex-



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pressed in exocrine glands as mammary, parotid, sublin-gual, submaxillary, lachrymal, nasal, and in modified se-baceous glands like preputial and perianal glands (344),their biological effects have been exclusively demon-strated when these proteins were purified from urine oradded to urine that does not possess pheromonal activity(i.e., of a prepubertal or castrated mouse) (123, 266).

MUPs have been reported to act either as primer orreleaser pheromones; however, some of their effects ap-pear contradictory. This is in part due to the tight bindingof MUPs with volatile pheromones that have been provento be directly implicated in estrus synchronization (138),attractiveness to females (139), intermale aggression(280), induction of estrus (�- and �-farnesene) (219), pu-berty acceleration in females (283), and territory marking(21, 122). The advantage of such a pheromone-proteincomplex may include concentration and slow release ofchemosignals, stabilization of labile pheromones, or de-livery to the reception sites. Common chromatographicprocedures for purification of MUPs are ineffectual toremove these pheromones, and solvent extraction of vola-tiles appears the only way to obtain the native apopro-teins. Therefore, in literature, the term of MUPs com-monly defines the protein with the bound pheromones.

While a general consensus exists about the source,male urine, that releases the pheromonal stimuli respon-sible for the puberty acceleration (Vandenbergh effect), adebate is currently open about the molecules that deter-mine this behavioral response. Evidence suggests thatMUPs and not the bound pheromones are effective inaccelerating the puberty onset in females. These resultsare supported by the biological activity shown by MUPswhen stripped of its pheromones and by a short synthetichexapeptide homologous to the NH2-terminal sequence ofmany MUPs variants (266). Conclusions from a differentlaboratory, performing similar but not identical experi-ments, seem, however, to argue against this hypothesis(283).

It has been also demonstrated that the combinatorialdiversity of urinary MUPs among wild mice (a male canproduce up to 12 variants from the polymorphic MUPfamily, that includes a total of 21 functional isoforms) isvirtually as great as for molecules of the major histocom-patibility complex (MHC) (see below) (48, 123). However,although MHC has been long regarded as the primarydeterminant for individual recognition via bodily odors,this evidence has now been challenged by recent studiesin which it appears that recognition, at least in the wild, isinstead strictly and exclusively dependent on the MUPsvariant profile contained in each individual urine (49). Veryintriguing is the observation that recognition requires the phys-ical contact with the donor urine, thus discarding thehypothesis of this effect being exclusively attributed tothe volatile pheromones released from MUPs (49).

Thom et al. (375) indicated that female mice useMUPs as a specific genetic marker to identify and prefer-entially associate with heterozygous males. The primaryfunction of MUPs is in signaling social informationthrough scent, including individual genetic identity. Miceare highly sensitive to differences in the fixed patterns ofmultiple MUP isoforms expressed by each individual andalso avoid inbreeding with close relatives that share thesame MUP type as themselves. Heterozygosis assessment,in contrast, most likely involves recognition of the greaterdiversity of MUP isoforms expressed by heterozygousanimals.

Other biological effects of MUPs include attractive-ness to females and repulsion to males (265), modificationof light-avoidance behavior in both males and females(263, 264), the aggressive reactions of males towardsadult and newborn mice (264, 267), and intermale aggres-siveness (47). Mate choice and parent-offspring interac-tions have been demonstrated to be influenced by MHCgenotype B (411). For example, house mice avoid matingwith individuals that are genetically similar at the MHClocus. Mice are able to recognize MHC-similar individualsthrough specific odorant cues. Moreover, females avoidmating with males carrying MHC genes of their fosterfamily, supporting a familial imprinting hypothesis (288).Mate preference for MHC-dissimilar individuals can beadaptive as it would increase offspring MHC heterozygos-ity, with beneficial influences on offspring viability throughincreased resistance to infectious disease or avoidance ofinbreeding effects (180, 297).

The polymorphism of MHC molecules is reflectedinto structurally diverse binding sites, such that differentMHC binds to distinct peptides. Like other membrane-bound proteins, these MHC molecules are anchored in thelipid bilayer. However, it has been demonstrated that thepeptide-binding pocket releases its peptide in the extra-cellular fluid when the MHC is cleaved from the cell. Thuspeptides or their fragments are constitutively excreted inurine and other bodily secretions (358) that in turn can beused for interindividual communication (357).

C. Exocrine Gland-Secreting Peptides

Mice secrete a family of peptides of different lengthcalled exocrine gland-secreting peptides (ESPs) from ex-traorbital, Harderian and submaxillary glands into tear,nasal mucus, or saliva. The ESP family consists of 38members in mice and 10 in rats, while it is absent from thehuman genome.

Moreover, at least two of these peptides, produced bythe extraorbital lacrimar gland, are gender specific: ESP1is exclusively secreted by males (162), while ESP36 isfemale specific (163). Despite the fact that these peptidesare recognized by systems dedicated to pheromone per-



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ception, until now there were no clues regarding theirphysiological function.

III. CHEMOSENSORY SYSTEMS

DETECTING PHEROMONES

It has been believed for a long time that two chemo-sensory systems, the main olfactory and the vomeronasalsystem, were responsible for different functions. Themain olfactory system was considered to be responsiblefor recognizing the conventional volatile odorant mole-cules, whereas the vomeronasal system was thought to betuned for sensing pheromones. Recent studies have dem-onstrated that both chemosensory systems, together withadditional olfactory organs, are involved in pheromonedetection. In these systems, peripheral chemosensoryneurons located in the nasal cavity express distinct fam-ilies of receptors that are believed to bind pheromonesand trigger a cascade of molecular and electrical eventsthat, ultimately, influence some aspects of the social be-havior of the individual.

Although the main olfactory and vomeronasal sys-tems share a similar histological organization, they alsodisplay relevant differences with regard to the receptorrepertoire that they express and to the connections tospecific central areas (Fig. 1). Each system possessesprimary sensory neurons that send axons to second-orderneurons (mitral cells) in specific regions of the mainolfactory bulb (main olfactory system) or of the accessoryolfactory bulb (vomeronasal system). The mitral cells ofthe main olfactory bulb project to several higher centersincluding the piriform cortex and the cortical amygdala.Instead, projections from the accessory olfactory bulbonly reach the medial amygdala and the posterior-medialpart of the cortical amygdala. From here fibers terminatein the hypothalamus either directly or via the bed nucleusof the stria terminalis.

In the following sections we summarize how thevarious sensory epithelia are functionally organized. Formore exhaustive anatomical description, readers mayconsult reviews specifically dedicated to these subjects(66, 229).

A. Vomeronasal Epithelium

The vomeronasal epithelium is part of the vomero-nasal organ (VNO) of Jacobson, a tubular structure en-

cased in a protective bony capsule located at the base ofthe nasal septum (68, 134) (Fig. 2). Given its recessednature, the vomeronasal epithelium cannot be reached bythe airstream that regularly flows through the nasal cav-ity. Thus, to target the vomeronasal epithelium, moleculesdissolved in the nasal mucus must be sucked into thelumen of the organ. Lateral to the lumen are blood vesselsand sinuses, innervated by the autonomic nervous sys-tem, that induces vasodilation and vasoconstriction,thereby producing a pumplike action for stimulus accessto the lumen (72, 109, 244, 248, 249, 328). In ungulates,as described in section IIA4, the “flehmen” behavior isthought to be associated with promoting stimulus accessto the vomeronasal organ (268).

Norepinephrine accumulation has been measured inthe vomeronasal organ of mice after exposure to phero-monal cues (419). It is possible that this hormonal releasealters the vascular tone, or changes the glandular secre-tions, thus increasing the receptivity of the sensory epi-thelium in particular behavioral contexts. The functioningof the vomeronasal organ is thus not simply a passiveevent, but it can be actively modulated.

The medial, concave side of the vomeronasal lumenis lined by a pseudostratified epithelium composed ofthree types of cells: sensory neurons, supporting cells,and basal cells. The basal stem cells are located bothalong the basal membrane of the sensory epithelium andat the boundary with the nonsensory epithelium (85).Supporting cells are found in the uppermost superficiallayer of the sensory epithelium, while vomeronasal neu-rons form two overlapping populations, basal and apical,that are further described in detail in sections IV and V.

In the mouse, the vomeronasal sensory epitheliumincreases from birth to puberty and becomes fully devel-oped and completely active at 2 mo after birth (117). Thesensory neurons, on the mucosal side, bear dendrites andapical microvilli, whereas, on the opposite side, theiraxons cross the basal membrane and merge together,forming vomeronasal nerves that run between the pairedolfactory bulbs and enter the accessory olfactory bulb atthe posterior dorsal side of the main olfactory bulb (seeFigs. 2 and 3).

The vomeronasal sensory neurons take origin fromthe olfactory placode together with gonadotropin-releas-ing hormone (GnRH, also known as LH-RH or luteinizinghormone-releasing hormone) neurons and �-aminobu-tyric acid (GABA)-containing neurons (405). The GnRH

FIG. 1. A schematic diagram showingthe anatomical pathways of the rodentvomeronasal and main olfactory systems.



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neurons traverse the developing accessory olfactory bulband migrate to the mediobasal hypothalamus. The vome-ronasal sensory neurons maintain a functional relation-ship with the hypothalamic areas in the adult mammal,influencing neuroendocrine function and behavior.

Do humans have a vomeronasal organ? The presenceof a vomeronasal organ in human embryos, containingbipolar neurons similar to developing vomeronasal neu-rons of other species, was clearly demonstrated (24, 25,166, 240). However, the vomeronasal structure becomesmore simplified later in development (24), and several

reports showed that in human adults it becomes a blind-ended diverticulum in the septal mucosa (246, 383). More-over, the epithelium lining the vomeronasal organ in hu-man adults is different from that of other species andfrom the olfactory or respiratory epithelium (259, 366).Several studies showed that vomeronasal cells were notstained by an antibody against the olfactory marker pro-tein (259, 366, 383, 403), a protein abundantly expressedin mature neurons in the olfactory and vomeronasal epi-thelia of other species (41, 144). In addition, Trotier et al.(383) did not identify vomeronasal nerve bundles using a

FIG. 2. Chemosensory systems in rodents. A simplified sagittal section of a rodent head showing the location of sensory epithelia: the mainolfactory epithelium (MOE), the vomeronasal organ (VNO), the septal organ of Masera (SO), the Grueneberg ganglion (GG), and the location of themain olfactory bulb (MOB) with necklace glomeruli (NG) and of the accessory olfactory bulb (AOB). Insets show images obtained from the intrinsicgreen fluorescent protein (GFP) fluorescence from olfactory marker protein (OMP)-GFP gene-targeted mice. Top left: the Grueneberg ganglion hasan arrowlike shape with neurons clustered in small groups. Axons fasciculate and form a single nerve bundle. [Modified from Fuss et al. (82).] Top

right: the septal organ of Masera is an island of sensory epithelium. Axons from sensory neurons form two bundles. [Modified from Levai andStrotmann (191).] Bottom left: mature vomeronasal sensory neurons in a coronal section of the vomeronasal organ. Bottom right: mature olfactorysensory neurons in a coronal section of the main olfactory epithelium.



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specific antibody against the S-100 protein, a characteris-tic axonal marker (7). Therefore, even if vomeronasalcells were chemosensitive, they would not have any ob-vious way to communicate with the brain. Therefore, thedevelopment of human vomeronasal structures seems tobe limited to the embryonic stage, when they possiblyplay a role in the migration of GnRH-secreting neuronstoward the brain (383; for review, see Ref. 246).

B. Olfactory Epithelium

New evidence suggests that some pheromones aredetected by the main olfactory epithelium, a structurespecifically dedicated to sensing volatile molecules, usu-ally named general odorants. The main olfactory epithe-lium lines cartilaginous protuberances in the posteriornasal cavity (Fig. 2), called turbinates, and is a pseudostrati-fied epithelium composed, similarly to the vomeronasalepithelium, of three main types of cells: olfactory sensoryneurons, supporting cells, and basal cells. The olfactorysensory neurons are bipolar neurons with a single ciliateddendrite extending to the mucosal surface. Cilia are thesite of primary transduction for odorants and, possibly,for pheromones. Although most olfactory sensory neu-rons have the same type of transduction cascade, it hasbeen recently discovered that subsets of olfactory neu-rons express different types of transduction molecules(see sect. VB2) (reviewed in Refs. 31, 217, 269). Axons ofsensory neurons are segregated in the epithelium and,after crossing the basal membrane, bundle together toform the fascicles of the olfactory nerves. Axons projectto distinct domains of the main olfactory bulb as furtherdescribed in section V.

In the mouse, the olfactory system is functionallyestablished during the prenatal phase as the first axonscontact the telencephalic vesicle around embryonic day13 (59, 320). Conversely, axonal targeting to specific do-mains within the main olfactory bulb occurs as early asembryonic day 15.5 (59).

In the mouse, both vomeronasal and olfactory sen-sory neurons undergo a continuous renewal processthroughout adult life, and their rate of neurogenesis isalso regulated by environmental factors (11, 59, 90, 91,402). Readers interested in the neurogenesis of olfactoryand vomeronasal sensory neurons may consult some re-cent reviews on the subject (101, 205).

The human olfactory epithelial region is a small area(1–5 cm2) covering part of the superior turbinate, septum,and roof of the nasal cavity. The transition boundarybetween olfactory and respiratory epithelium is not assharp as in other mammals; instead, there is an irregularzone where olfactory and respiratory cells intermingle.Moreover, patches of respiratory epithelium have beenidentified in purely olfactory areas (262).

C. Other Olfactory Organs

1. Septal organ of Masera

The septal organ of Masera (SO) (35, 311) is a smallisland of olfactory epithelium within the nasal mucosaand is located on each side of the septum, posterior to thenasopalatine duct that connects the oral to the nasalcavity (Fig. 2). The anatomical organization of the septalorgan appears similar to that of the main olfactory epi-thelium, although its neuroepithelium appears thinner(218, 396). Furthermore, sensory neurons of the septalorgan are provided with cilia that bear shorter dendritesand larger dendritic knobs than those of the main olfac-tory epithelium (218); their axons project to a subset ofglomeruli in the posterior, ventromedial main olfactorybulb (Fig. 3). Interestingly, some of these glomeruli ex-clusively receive input from the septal organ sensoryneurons, while others are also innervated by fibers origi-nating from the main olfactory epithelium (191). The an-terior position of the septal organ, its vicinity to thenasopalatine duct, and the wide distribution in manymammalian species suggest that this organ may be func-tionally specialized for the early detection of biologicallyrelevant molecules as those, for example, resulting fromlicking behavior.

2. Grueneberg ganglion

The Grueneberg ganglion is located bilaterally in theanterodorsal area of the nasal cavity in the cornersformed by the septum and the nasal roof of several mam-malian species (93a) (Fig. 2). Differently from other che-mosensory organs, neurons of the Grueneberg ganglioncluster in small groups (30, 79, 80, 317). Only two types of

FIG. 3. Projections of sensory neurons to the olfactory bulbs. Eacholfactory sensory neuron sends a single axon to the main olfactory bulb(MOB). The apical and basal sensory neurons in the vomeronasal organ(VNO) send axons, respectively, to the anterior and posterior accessoryolfactory bulb (AOB), a posterior dorsal region of the main olfactorybulb. Neurons of the septal organ of Masera (SO) project to the posteriorventromedial main olfactory bulb. Neurons of the Grueneberg ganglion(GG) project to necklace glomeruli (NG) in the main olfactory bulb.



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cells have been identified in the Grueneberg ganglion:glial cells and ciliated neurons (30). Each neuron has asingle axon that fasciculates with others into nerve bun-dles that project to �10 glomeruli in the main olfactorybulb (Fig. 3). These glomeruli are a subpopulation of thenecklace glomeruli. Since necklace glomeruli are activeduring the suckling behavior of the pup, it was suggestedthat the Grueneberg ganglion may sense maternal phero-mones (82, 173, 317). However, a very recent study hasrejected this hypothesis, suggesting instead that alarmpheromones, volatile molecules released by animals toalert conspecifics about danger, are the likely substratesof the Grueneberg ganglion neurons. These results arediscussed in more detail in section VB.

IV. PUTATIVE PHEROMONE RECEPTORS

A. Vomeronasal Receptors

In the vomeronasal organ of rodents, detection ofpheromones is mediated by two anatomically indepen-dent pathways that originate from chemosensory neuronsof distinct compartments, basal and apical, of the neuro-epithelium. The organization of the vomeronasal epithe-lium of rodents in apical and basal neurons was initiallybased on the specific expression pattern of marker mol-ecules (261, 340, 341). More recently, exhaustive dataestablished that also distinct transduction componentscharacterize these two subsets of chemosensory neurons.The G protein subunit G�i2 was found to enrich the apicalneurons, whereas G�o was identified in the basal ones(19, 143). These important findings are credited to haveaddressed the efforts towards the search of receptors thatcould specifically couple to G proteins. Thus two largefamilies of vomeronasal G protein-coupled receptors,named V1R and V2R, have been discovered, and theirexpression pattern was extensively analyzed. Moreover,other putative receptors as the major histocompatibilitycomplex class Ib molecules (H2-Mv) have been identifiedin the vomeronasal organ, and their expression was dem-onstrated to be tightly linked to that of V2Rs.

1. V1Rs

The first family of vomeronasal receptors, V1Rs, wasdiscovered with elegant experiments of differentialscreening, based on the assumption that the genetic pro-file of two vomeronasal neurons expressing G�i2 onlydiffers for the receptor RNA that they express (71, 285). Itwas also evident that V1R expression is restricted to theapical neurons of the vomeronasal organ which expressG�i2 (71, 285). From the structural point of view, V1Rs arerelated to group I of G protein-coupled receptors and, inthe mouse, represent a strikingly expanded receptor fam-

ily compared with other species (313, 346, 415, 422). Thegene tree of functional V1Rs in placental mammals, basedon the mouse repertoire, is split into 12 clades (a-l) (Fig. 4)(313). All rat V1R receptor genes, except for one, aredistributed within 10 of these 12 clades (a-g and j-l). Thusthe mouse h and i clades represent an instructive exampleof species specificity, exclusively including mouse se-quences. Given that one rat pseudogene is placed in eachof the h and i subfamilies, it is likely that the absence ofrat functional genes in these clades is the result of dele-tion in this species and of postspeciation expansion in themouse lineage.

V1Rs in the mouse account for almost 200 potentiallyfunctional receptors and are highly expressed in vomer-onasal neurons, at least at mRNA level. The mouse has alarger V1R repertoire than the rat (Fig. 5).

In the mouse V1R phylogenetic tree, clades sharefrom 15 to 40% of their amino acid residues between eachother, with intrafamily identities varying between 40 and99%. High sequence variability is found in transmembranedomain 2, in intracellular loop 1, and in extracellularloops 2 and 3, while transmembrane domain 3 is highlyconserved. A few amino acid residues mostly locatedbetween transmembrane domains 5 and 6, and a potentialglycosylation in extracellular loop 2, are common featuresto virtually all members of the 12 V1R clades, while spe-cific amino acid signatures are characteristic of each ofthe 12 families (313).

FIG. 4. The mouse vomeronasal receptor families. Top: phyloge-netic tree of V1Rs showing 12 phylogenetically isolated subfamilies.Bottom: phylogenetic tree of the 4 V2R families showing their clades.Numbers of receptors for each clade and family are shown in red.[Modified from Silvotti et al. (355).]



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V1R genes are scattered in regions of many chromo-somes, where members of a given family are found almostinvariably clustered. It has been recently reported thatV1R clusterization may not simply reflect gene expansionbut may be actively linked to the mechanisms underlyingthe tight regulation of receptors’ expression. V1R expres-sion is, indeed, monoallelic and monogenic, both of thesebeing tools that achieve the expression of one receptortype per cell, thus contributing to the narrow tuning of thevomeronasal neurons (318).

At present, some vertebrate genomes of species ofdifferent orders have been extensively scrutinized for the

presence of V1Rs (Fig. 5). Interestingly, V1R genes werefound in all species except in chicken and chimpanzee, inwhich the presence of a functional vomeronasal organ hasnot been consistently demonstrated. The number of func-tional V1R genes drops dramatically from rodents to otherspecies. The human genome contains several V1R pseu-dogenes, only five human V1R genes that have an intactopen reading frame, and at least one V1R that is tran-scribed in the olfactory epithelium. The chimpanzee ge-nome has no obvious functional V1R genes but a largecollection of pseudogenes. This is surprising if we con-sider that the massive loss of V1R genes in chimpanzee

FIG. 5. Evolution of nasal chemosensory receptor gene repertoires in vertebrates. The tree shows the phylogeny of 13 species of vertebrates:8 mammals (platypus, chimpanzee, human, mouse, rat, dog, cow, and opossum), 1 bird (chicken), 1 amphibian (western clawed frog), and 3 teleostfishes (green spotted pufferfish, fugu, and zebrafish). The numbers of intact genes for V1R, V2R, OR, and TAAR receptors are given together withthe numbers of nonintact genes shown in parentheses. Numbers of receptor genes in each species were taken from Grus et al. (94), except forchimpanzee which were obtained from Nei et al. 2008; V1Rs and V2Rs in green spotted pufferfish, fugu, and zebrafish from Shi and Zhang (346); ORsin fugu from Niimura and Nei (274); and TAARs in fugu and zebrafish from Hashiguchi and Nishida (105).



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appears even greater than for the human repertoire, inwhich V1R genes have been identified. However, the mostsurprising observation is that the dog only possesses eightfunctional V1R genes spread among four clades. Thisfinding is unexpected because the dog is traditionallyrenowned to have evolved an extraordinary well-devel-oped olfactory system and a complex social behavior(346). Although it is plausible that V1Rs emerged as dom-inant receptors for triggering specific rodent behaviors(multiple losses of ancestral V1R genes in carnivores andartiodactyls and gains of many new genes by gene dupli-cation in rodents), it cannot be alternatively ruled out thata loss of dog V1Rs is due to the breeding and domestica-tion processes of this species. Therefore, diversificationinto subfamilies probably occurred prior to the rodent-dog-primate split early in the mammalian evolution (415).

The question as to whether V1Rs are exclusivelyexpressed in the vomeronasal epithelium or also in othertissues is still controversial. Karunadasa et al. (152) re-ported the expression of V1Rd RNA in some cells scat-tered within the main olfactory epithelium of embryonicand postnatal mouse and suggested that pheromone de-tection may also take place in this structure. V1R-ho-molog RNAs have also been identified in the main olfac-tory epithelium of goats and humans (316, 394). However,caution must be taken before raising any hypothesis as,until now, there are no clues that a functional V1R isexpressed at a detectable level in the main olfactoryepithelium.

2. V2Rs

A second family of vomeronasal receptors, V2Rs, wasdiscovered in 1997 (110, 234, 323) and, in mouse, it ac-counts for �120 potentially functional members. V2Rsbelong to group III G protein-coupled receptors and arecharacterized by a long NH2-terminal domain. This regionhas been demonstrated to represent the binding domainin most receptors of group III (for review, see Ref. 293).V2Rs are strictly coexpressed with the G protein subunitG�o in the basal neurons of the vomeronasal organ.

In the mouse, intact V2Rs can be grouped in fourdistinct families (A-D) according to sequence homology(Fig. 4). Together, receptors of families A, B, and D rep-resent 95% of all V2Rs. Their expression pattern stronglysuggests that these receptors are expressed by mutualexclusion in the vomeronasal organ. A phylogenetic anal-ysis shows that family C V2Rs (also known as V2R2family) are very divergent from those of families A, B, andD (3, 231, 323), and these differences are also functional(354). This family is also noteworthy for the high se-quence conservation between species. Moreover, com-pared with other V2R pseudogenes, family C pseudogeneshave one or very few inactivating mutations (also in the

human pseudogenes), suggesting a relatively recent lossof function.

In rodents, this receptor superfamily is largely ex-panded and, in contrast to V1Rs, it appears completelydegenerated in primates, dogs, and cows (Fig. 5). In thedog, the lack of a sizable V1R and V2R family does notprobably reflect a reduction in the pheromonal relevancein triggering intraspecific behavior, but rather a generaldecline of the vomeronasal system, a hypothesis that issupported by anatomical data (63). In contrast, in theopossum, V2Rs are abundantly represented (Fig. 5). Thusit appears that the evolution of opossum and rodent V2Rsgenes parallel that of the V1R ones in these species, whichexpanded independently. Moreover, most opossum androdent V2R and V1R genes arose by duplication (rodentV2R and V1R genes are found in genomic clusters, andclosely related V2Rs tend to reside near one another inthe genome) after these lineages diverged. This receptordiversification and the consequent detection of additionalpheromones may have resulted in a selective advantage.

The expression pattern of V2Rs makes them differentfrom V1Rs and odorant receptors in that each basal neu-ron expresses two V2Rs, one of which is a member offamily C (231). Moreover, there is evidence for a combi-natorial scheme for the detection of chemosensory cuesin the basal neurons of the vomeronasal organ. Recentdata indicate the existence of multimolecular complexesin individual basal neurons, constituted by MHC mole-cules, by family A, B, or D and family C V2Rs (355). Forexample, neurons expressing family A receptors of thehighly represented clades I, III, and IV almost exclusivelyexpress the family C receptor Vmn2r2, whereas neuronsexpressing clade VIII receptors specifically express thefamily C receptor Vmn2r1. Interestingly, coexpression ofVmn2r1 and Vmn2r2 with family A V2Rs seems to involvewhole clades rather than individual receptors. Thus apeculiar modality of expression subsists in the basal layerof the vomeronasal organ, where two family receptors aremutually exclusively expressed in the same basal neu-rons.

It has been suggested that V2Rs bind MHC peptides(185) and MUPs (47), as further discussed in section VA.

A V2R receptor, V2R83, is also expressed outside thevomeronasal organ, in a large subset of neurons of theGrueneberg ganglion (79), and it is a good candidatereceptor for alarm pheromones (30).

At present, however, direct evidence that V2Rs aredirectly involved in pheromone signaling is still missing.

At least 50% of the basal vomeronasal neurons ex-press another multigene family representing nonclassicalclass I major histocompatibility complex (H2-Mv) genes.Each of the nine identified H2-Mv genes is present in asubset of vomeronasal neurons, and multiple H2-Mv

genes can be coexpressed in the same neuron (127, 207).H2-Mv molecules are also coexpressed in a combinatorial



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manner with receptors of specific subfamily of V2Rs (127,207). It has been initially proposed that H2-Mv may func-tion as indispensable escort molecules in the transport ofV2Rs to the plasma membrane (207). However, this chap-erone-like function is not generalized to all V2Rs (127,354), and moreover, it is likely to be exclusively restrictedto the mouse or rat as the opossum genome contains ahundred functional V2R genes and no H2-Mv genes (346).

3. Ligands of vomeronasal receptors

Although an initial report provided clues on the pos-sibility that V1Rs could be expressed in a heterologoussystem (99), the problem of expression later became themajor barrier to the identification of ligands for vomero-nasal receptors, as well as for odorant receptors (forreviews, see Refs. 224, 256, 312). Some information aboutligands for V1Rs was obtained from physiological ap-proaches to the problem. Results from calcium imagingexperiments on slices of the mouse vomeronasal organindicated that some molecules credited as pheromonesstimulated specific vomeronasal neurons that, for theirtopographic position in the vomeronasal organ, werelikely to express the V1R repertoire (187). Furthermore,these authors reasoned that, if neurons responding tothese pheromones indeed reflected a V1R-dependent ac-tivity, V1Rs must be narrowly tuned to bind one or veryfew pheromones. This contrasts with the broad selectivityof olfactory sensory neurons that can be stimulated by alarge array of odorants. Further and more direct investi-gations on the V1R ligand specificity lead to the conclu-sion that V1Rs are indeed qualified as pheromone recep-tors: Del Punta et al. (61) succeeded to delete a largegenomic region containing �16 V1R genes of subfamily aand b, including the receptor V1rb2. The behavior of thesemutant animals was also thoroughly investigated showingremarkable deficits (see sect. VII). Moreover, electrophys-iological responses of vomeronasal neurons to somepheromones, including 6-hydroxyl-6-methyl-3-heptanone,n-pentylacetate, isobutylamine, and 2-heptanone, were al-tered (Table 1). Thus the repertoire of V1R receptor can-didates to respond to these pheromones was restricted toa relatively limited number.

Another approach, developed by Boschat et al. (28),led to the identification of the first pheromone-V1R pair inmammals. These authors employed mouse mutant lineswith a GFP-targeted V1rb2 allele, allowing the visualiza-tion and the isolation of the V1Rb2-expressing neurons forcalcium imaging based experiments. Thus a direct corre-lation was established between V1Rb2 expression and theresponse of vomeronasal neurons to the mouse phero-mone 2-heptanone (Table 1). As a confirmation of thestrong selectivity of V1Rs above reported, 2-heptanone-related compounds were found to be ineffectual to stim-ulate V1Rb2-expressing sensory neurons.

To deorphanize V1Rs and study their binding prop-erties, it is necessary to develop an efficient bioassay. Atpresent, it is only plausible to assume that V1Rs arevomeronasal receptors narrowly tuned to bind small vol-atile pheromones, possibly through a hydrophobic pocketlocated in their transmembrane region. Nevertheless,achievements in this direction have been very recentlyaccomplished by Shirokova and colleagues who managedto deorphanize the human V1Rs employing NH2-termi-nally tagged receptors expressed in the cell line HeLa/Olf(350). It appears that, in sharp contrast to V1Rs, eachhuman recombinant V1R receptor responds to severalaliphatic alcohols or aldehydes via the olfactory G proteinG�olf (350). Unfortunately, a pheromonal function of hu-man V1R ligands has yet to be demonstrated.

4. Formyl peptide receptors

Recently, a group of receptors belonging to the fam-ily of formyl peptide receptors (FPRs) has been exclu-sively identified in the vomeronasal organ (310a). FPRsare G protein-coupled receptors expressed in all mam-mals, with three genes present in the human genome andseven in the mouse genome (249a). At least two of themouse FPRs have been reportedly involved in immuneand inflammation responses being expressed in granulo-cytes, monocytes, and macrophages (183a). An in situanalysis of the vomeronasal organ of the mouse indicatesthat five FPRs are expressed in a small subset of chemo-sensory neurons of the apical layer. Antibody stainingreveals that FPRs are featured by a subcellular distribu-tion similar to that of V2R receptors including, besides themicrovillar surface, the dendrite and somata. AlthoughFpr expression is restricted to the apical layer and, aswell as VIRs, adopts monogenic transcription, no coex-pression is evident with receptors of this large family,suggesting that FPRs characterize a new and specificclass of chemosensory neurons. Experiments based oncalcium imaging on the whole vomeronasal epitheliumand in dissociated chemosensory neurons establish thatindeed FPR agonists, such as some microbial, antimicro-bial, and viral peptides, stimulate neuronal subsets with anonidentical affinity profile. These findings indirectlyprove that these receptors play a role in offering a barrieragainst immunological diseases, or perhaps also in phero-monal communication itself, given that formyl peptideshave been detected in urine (52a) and that, in the olfac-tory and vomeronasal epithelium, two FPR isotypes ap-pear to be expressed in the glandular but not chemosen-sory compartment (310a).

B. Odorant Receptors

Odorant receptor genes were discovered in 1991 byBuck and Axel (39). The vast majority of the olfactory



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neurons express only 1 of the 1,000 odorant receptorgenes (51, 226, 273, 308, 370, 389, 389). Neurons express-ing different receptor types are usually found in one offour partially overlapping zones of the olfactory epithe-lium, within each of which a given odorant receptor ap-pears to be randomly distributed (128, 251, 308, 369, 389).

An olfactory sensory neuron typically responds tomore than one type of odorant molecules, and a givenodorant can activate neurons with different specificity(70, 77, 226, 351). For the discrimination and perceptionof odorants, the odorant receptor family seems to employa combinatorial scheme, with one single odorant mole-cule activating several types of receptors and each recep-tor sensing several different types of odorants, therebyproducing a unique combination of receptors activated byeach odorant molecule. This combinatorial scheme al-lows the olfactory system to recognize an enormous num-ber of odorants (40, 256).

Odorant receptor genes have been identified in re-gions outside the main olfactory epithelium as the septalorgan and the vomeronasal epithelium. About 50 odorantreceptor genes are expressed in the mouse septal organ(150, 378). Moreover, a subset of 44 odorant receptorgenes is also expressed in the vomeronasal epithelium(190), although their functional role has not been estab-lished.

At present, we do not possess evidence that odorantreceptors can also function as pheromone receptors.However, intriguingly, recent experiments linked the ex-pression of a subclass of odorant receptors with stereo-typed behavioral expressions in the mouse, thus suggest-ing that odorant receptors can convey information bothabout the quality of an odorant and the individual that hasemitted it (169).

For additional information about odorant receptors,the reader is referred to several reviews that extensivelydiscuss this subject (86, 224, 225, 256).

C. Trace Amine-Associated Receptors

In 2006, Liberles and Buck (194) reported the expres-sion of a second class of chemosensory receptors in themouse olfactory epithelium. These receptors are the“trace amine-associated receptors” (TAARs) (27, 192,203), a family of G protein-coupled receptors discoveredin 2001 (27, 42) (for review, see Ref. 425). TAARs areunrelated to odorant receptors but have similarities withreceptors for biogenic amines, such as serotonin anddopamine receptors. The TAAR gene family includes 15members in the mouse (Fig. 5), and each of them, exceptfor TAAR1, is expressed in the olfactory epithelium (194).Moreover, the level of expression of TAAR genes is sim-ilar to that of odorant receptors, although TAARs andodorant receptors do not appear to be coexpressed in the

same neurons, suggesting that subsets of neurons exist inthe olfactory epithelium that are not exclusively dedi-cated to the detection of conventional odorants. By theexpression of olfactory TAARs in heterologous cell sys-tems it was demonstrated that at least four of them(mTAAR3, mTAAR4, mTAAR5, mTAAR7f) bind small-molecule amines and that each of these receptors recog-nizes a specific set of ligands, among which are �-phenyl-ethylamine, isoamylamine, and trimethylamine that arenatural components of the mouse urine. �-Phenylethyl-amine, a ligand for mTAAR4, is contained in human androdent urine, where its concentration rises during stress-ful situations (93, 286, 361). Isoamylamine and trimethyl-amine bind to mTAAR3 and mTAAR5, respectively, andare present at higher concentrations in male comparedwith female urine (84, 276). Noteworthy, isoamylaminehas been reported to act as a pheromone acceleratingpuberty onset in female mice (276). Therefore, stimula-tion of mTAARs seems to affect the capacity of discrim-inating gender and social status of an individual. Thesefindings strongly contribute to confirm that the main ol-factory epithelium is involved in the elicitation of physi-ological responses and innate behaviors.

In the mouse, TAARs are also expressed in subsets ofneurons of the Grueneberg ganglion (80). As in the mainolfactory epithelium, also in the Grueneberg ganglion,distinct TAAR subtypes are expressed in nonoverlappingsubsets of neurons that do not coexpress the vomeronasalreceptors V2Rs. Since the number of neurons expressingTAARs and V2Rs in the Grueneberg ganglion is muchhigher during the perinatal stage than in the adult, it wassuggested that these neurons may be important for inter-actions between pups and their mother (80).

TAARs have also been identified in the genome ofmany species including humans and fish, with the numberof putatively functional TAAR genes greatly varyingamong species (105) (Fig. 5).

D. Olfactory-Specific Guanylyl Cyclase

Type D Receptor

A small subset of olfactory sensory neurons localizedin the dorsal region of the main olfactory epitheliumexpresses a specific subtype of the transmembrane recep-tor guanylyl cyclase, named guanylyl cyclase type D(GC-D) (81, 149). The GC-D receptor is encoded by one ofthe seven guanylyl cyclase genes with whom it shares asimilar topology: all receptor guanylyl cyclases retain anNH2-terminal ligand binding region, a single transmem-brane loop, and a COOH terminus that includes the cata-lytic domain (81, 296). Specific ligands have been identi-fied for some guanyly cyclases, but not for GC-D yet;however, potential ligands for this receptor have beenrecently assessed. In the mouse, for example, neurons



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expressing GC-D respond to dilute urine and to the intes-tinal peptide hormones uroguanylyn and guanylin, whileneurons in GC-D knockouts are unresponsive to thesecompounds (186). Given their localization in the enterictract, uroguanylyn and guanylin are supposed to contrib-ute to the maintenance of salt and water homeostasis orto the detection of cues related to hunger, satiety, or thirst(186). GC-D neurons also express carbonic anhydrase IIand respond to CO2 stimuli (118). It is not clear if there isa possible interplay between these two chemosensoryresponses.

Despite its exquisite expression in the main olfactoryepithelium, GC-D appears to be a pseudogene in specieslacking a functional vomeronasal organ such as humans,apes, Old World and New World monkeys, and tarsier.This suggests that a functional GC-D gene was lost earlyin primate evolution and that chemical detection in mostprimate species is unlikely to involve GC-D (416).

V. PHEROMONE TRANSDUCTION

Distinct neuronal subsets of the olfactory organs ex-press specific types of receptors, second messenger sys-tems, and ion channels to transform the pheromone bind-ing into electrical signals.

The electrical activity of chemosensory neurons inresponse to stimuli can be measured with a number ofapproaches, including recording of the field potential, ofthe firing activity with an array of extracellular electrodesand by patch-clamp technique at the single-cell level.Nonetheless, calcium imaging either in dissociated cellsor epithelium slices is often employed as well as themetabolic activity of stimulated neurons is frequentlymonitored upon pheromonal stimulation by evaluatingthe expression of the early protooncogenes.

A. Vomeronasal Sensory Neurons

1. Signal transduction molecules

The detailed signal transduction cascade in thevomeronasal organ is still largely unknown, although ithas been clearly demonstrated that pheromone stimulicause membrane depolarization and increase the actionpotential firing rate in vomeronasal sensory neurons (28,53, 61, 68, 115, 124, 125, 185, 187, 211, 363, 374). Asdescribed in previous sections, the sensory epithelium ofthe vomeronasal organ presents two subsets of neurons:apical and basal. Apical neurons express members of theV1R family of vomeronasal receptor genes together withG�i2, whereas basal neurons express V2R receptors andG�o (19, 102, 142, 143, 321, 348, 371, 398). The anatomicaland perhaps functional segregation in the vomeronasalepithelium is likely to be a prerogative of species as rats,

mice, hamsters, opossum, and platypus that possess func-tional V2R genes (Fig. 5).

Krieger et al. (176) reported that whole male urineinduces inositol 1,4,5-trisphosphate (IP3) production viathe activation of G�i2 as well as G�o, suggesting a role forboth G protein subtypes in transducing pheromonal re-sponses mediated by urinary components. Moreover, ac-cording to these authors, the activation of G�i2 and G�o

subtypes in the vomeronasal epithelium appears to becaused by two structurally different classes of molecules.Whereas G�i2 activation was only observed upon stimu-lation with lipophilic volatile components, G�o activationwas elicited by the rat major urinary protein, �2-globulin.A confirmatory role of these G proteins in pheromonetransduction issues from behavioral (see sect. VII) andanatomical studies performed on G�i2 and G�o negativemutant mice (278, 373): both mouse lines show a remark-able reduction in the size of the neuronal layers whereG�o and G�i2 were originally expressed, indicating that Gprotein-mediated activity is required for survival of vome-ronasal sensory neurons.

Since PLC stimulation and IP3 production appear tobe predominantly mediated by the release of the �� com-plex of the heterotrimeric G proteins, Go and Gi (56), ithas been proposed that G�� may play an important role inthe transduction process of the vomeronasal epithelium.One �-subunit (G�2) and two �-subunits (G�2 and G�8)have been described in the vomeronasal epithelium (321,322, 380). G�2 immunopositive neurons are exclusivelylocalized in the apical layer, whereas G�8 neurons arepreferentially restricted to the basal one. G�2, on theother hand, seems to be active in both neuronal subsets(Fig. 6). Biochemical studies reveal that IP3 formationinduced by stimulation of membrane preparations withvolatile urinary compounds was selectively blocked byan anti-G�2 antibody, whereas second messenger pro-duction induced by stimulation with major urinary pro-teins was inhibited by preincubation of the vomerona-sal membranes with an antibody recognizing G�8 (321).Thus it appears that the G�o�2�8 complex may controlPLC activation by proteinaceous pheromones, whereasG�i2�2�2 neurons respond to urinary volatile com-pounds.

Other lines of evidence indicate that, at least in somevomeronasal sensory neurons, transduction is mediatedby a phosphatidylinositide signaling pathway. Some stud-ies showed that electrical responses, IP3 production, andcalcium entry upon pheromonal stimulation (53, 176, 177,215, 330, 397) are specifically blocked by the phospho-lipase C inhibitor U73122 (115, 363). Recently, Thompsonet al. (377) demonstrated that compounds known to in-duce pregnancy block (Bruce effect), such as MHC pep-tides and male urine, also increased the IP3 formation,confirming a major role of the phosphatidylinositide path-way in the vomeronasal signal transduction.



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The ion channel TRPC2, a member of the superfamilyof transient receptor potential channels (390, 412), isexpressed in the microvilli of both apical and basal neu-rons (197, 241, 271) and plays an important role in thetransduction process of some, but not all, vomeronasalsensory neurons. Interesting to note, TRPC2 is unique

among TRP channels in that a functional gene has beenlost from the Old World monkey and human genomes(198, 420). Proposed mechanisms for the activation of theTRPC2 channel in the vomeronasal neurons are similar tothose involved in the signaling cascade of Drosophila

photoreceptors (103, 250): pheromones trigger the activa-



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tion of G protein-coupled receptors that in turn stimulatephospholipase C and the production of the lipid messen-ger diacylglycerol (DAG) (211) and possibly of polyunsat-urated fatty acids such as arachidonic acid (363, 421) andlinolenic acid (Fig. 6) (363).

Genetic ablation of the TRPC2 channel results in astrong, but not complete, reduction of electrophysiologi-cal responses of the vomeronasal neurons to urine or itsvolatile components (156, 193, 368). Moreover, maleTRPC2-knockout mice showed severe behavioral abnor-malities (see sect. VII) (157, 193, 236, 368), indicating thatthe TRPC2 channel may be an important component ofthe transduction of pheromone signals.

Nevertheless, the genetic ablation of TRPC2 does notcompletely abolish the response of the vomeronasal or-gan to pheromones, since electrophysiological recordingshave demonstrated that basal neurons of mutant mice stillrespond to MHC peptide ligands as controls (156). Inaddition, TRPC2-knockout mice exhibit the Bruce effect(156), a neuroendocrine response that requires a func-tional vomeronasal organ. All together, these studies sug-gest that at least a subset of basal neurons uses a differentchannel from TRPC2 for the transduction of some phero-monal stimuli.

Interestingly, Liman (195) showed that a cation chan-nel activated by a high concentration of Ca2� is expressedin hamster vomeronasal sensory neurons. This channel ispermeant to both Na� and K� and is blocked by ATP andcAMP, sharing many features with the TRPM4 ion channel(275). These properties make this channel a good candi-date to be directly involved in sensory transduction or inamplifying a primary sensory response. At present, themolecular nature of this channel and its relation withTRPC2 or with IP3 production are not yet resolved.

Some components of the cAMP signaling cascadehave been identified in vomeronasal sensory neurons.These include adenylyl cyclase types 2 (19), 4, 5 (184), and6 (184, 319); the subunit CNGA4 of the olfactory cyclicnucleotide-gated channel (19, 184); and phosphodiester-ase types 1 and 4 (50, 183), suggesting that cAMP may playa physiological role in vomeronasal sensory neurons. In-deed, some pheromones induce a reduction of the cAMP

level in the gartner snake (215) and in rodents (215, 319,424). However, the decrease in cAMP occurs with a longerdelay with respect to pheromone stimulation (319). Pos-sible targets for modulatory actions by cAMP include theCa2�-activated cation channel (195) and the recentlycharacterized hyperpolarization-activated cyclic nucleoti-de-gated (HCN) cation channels (64).

It is intriguing that a subpopulation of apical sensoryneurons expresses members of the odorant receptor genefamily, together with G�i2 and TRPC2 (190). Since someconventional odorants are detected by the vomeronasalepithelium (329, 382, 409), it was suggested that vomero-nasal neurons expressing odorant receptors might bethose that respond to odorants (190).

Following the initial transduction events, when themembrane potential of vomeronasal sensory neuronsreaches threshold, action potentials are generated andpropagate along the axons to the accessory olfactorybulb. Interestingly, most vomeronasal neurons fire toni-cally for several seconds, without sign of adaptation,when small current injections (64, 196, 347, 384) or diluteurine are applied for up to 2–3 s (211, 421). However, inone study, longer stimulations of 10–60 s caused spike-frequency adaptation (384), and in addition, a recent worksuggested that sensory adaptation in vomeronasal sen-sory neurons requires the influx of Ca2� and is mediatedby calmodulin (362).

2. Responses to urinary pheromones

Using an array of extracellular electrodes, Holy et al.(115) simultaneously recorded the activity of a large num-ber of mouse vomeronasal neurons in response to diluteurine. Neurons responded to urine with an increase in thefiring frequency that remained approximately unchangedif the stimulus was applied for as long as 100 s, indicatingthat the response of these neurons do not adapt to pro-longed stimulus exposure. Furthermore, they identifieddistinct and nonoverlapping subsets of neurons that wereselectively activated by substances contained in female ormale urine; in contrast, about half of the vomeronasalneurons detected stimuli that were independent of sex.

FIG. 6. Transduction mechanisms in vomeronasal and olfactory sensory neurons. A: a scanning electron micrograph of the knob of a ratvomeronasal sensory neuron showing several microvilli. Scale bar, 1 �m. [From K. Doving and D. Trotier, unpublished data; see also Trotier et al.(382a).] B: binding of pheromone molecules to vomeronasal receptors in the microvilli activates a transduction cascade involving V1R, G�i2�2�2in apical neurons, or V2R, G�o�2�8 in basal neurons. The �� complex activates the PLC that in turn may cause an elevation of IP3 and/or DAG. Thegating of the TRPC2 channel allows an influx of Na� and Ca2�, causing a membrane depolarization. An additional cation-selective channel activatedby Ca2� may be involved in the transduction cascade. The TRPC2 channel appears to be the principal transduction channel in V1R-expressingneurons, whereas its role in V2R-expressing neurons in unclear. C: a scanning electron micrograph of the knob of a human olfactory sensory neuronshowing the protrusion of several cilia. Scale bar, 1 �m. [From Morrison and Costanzo (262).] D: olfactory transduction takes place in the cilia, whereligands bind to odorant receptors (OR) located in the ciliary membrane, thus activating a G protein (G�olf) that stimulates adenylyl cyclase type 3(ACIII), producing an increase in the generation of cAMP from ATP. cAMP directly gates ion channels (CNG), causing an inward current carriedby Na� and Ca2�. Ca2� entry amplifies the signal by causing the efflux of Cl� through Ca2�-activated Cl� channels (Cl-Ca). These ion fluxes causea depolarization. Inhibitory actions are played by the complex Ca2�-calmodulin (CaM) by increasing the activity of the phosphodiesterase (PDE)that hydrolyzes cAMP and by reducing the affinity for cAMP of the CNG channels. Ca2� is extruded by the Na�/Ca2� exchanger (Na/Caex).



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Thus these findings establish the capacity of the vomero-nasal organ to identify differences between sexes.

A recent study by He et al. (106) further stressed theconcept of the vomeronasal organ being subtly dedicatedto the identification of conspecifics. These authors quan-tified the number of neurons responding to individualurine of either sex and of different strains in slices of thevomeronasal epithelium of mice genetically modified toexpress a calcium indicator. It was observed that only avery small subpopulation of neurons (1–3%) exclusivelyresponded to urine samples from all individuals of thesame sex. The gender-specific neurons were evenly dis-tributed both in the apical and basal layers of the epithe-lium and did not differ in males or females, consistentwith the little dimorphism generally observed at the levelof receptor expression in the vomeronasal epithelium.Interestingly, neurons specific to female urine showeddifferent activation patterns depending on the phase ofthe estrus cycle; in contrast, urine from castrated micefailed to elicit a response in male-specific neurons. Fur-thermore, information about individuals and strains is notencoded by specialized neurons but, rather, by the com-binatorial activation of vomeronasal neurons, as no urinesamples from different mice of the same sex elicited thesame pattern of neuronal activity. Thus the informationcontained in the mouse urine about gender is encoded bydedicated neurons; strains and individuals are recognizedby a combinatorial code of neuronal activation (106), withthe obvious advantage to discriminate a large populationof individuals with a restricted repertoire of receptors.

He et al. (106) also compared responses elicited bysynthetic strain-specific MHC class I peptides with thoseinduced by urine of the same strain and found that neu-rons activated by urine and by the peptides did not over-lap. Furthermore, the same authors (106) pointed out thatonly fragments, but not the peptides, of the MHC com-plexes are detectable in urine (358). This result stronglycontrasts with what was observed by Leinders-Zufall et al.(185), who identified a specific subset of basal neuronshighly sensitive to MHC class I peptides and to urine frommice of the relevant haplotype.

Other studies investigated the response of the vome-ronasal epithelium to specific urinary components, suchas MUPs, that represent another class of candidates forstrain recognition, as well as for heterozygosity signals(375). Kimoto et al. (163) found that a recombinant iso-form of MUPs elicits electrical responses in the vomero-nasal epithelium, and Chamero et al. (47) showed thatnative and recombinant MUPs evoke calcium influx insensory neurons expressing the G protein subunit G�o.Since G�o coexpresses with V2Rs, it is likely that MUPsdirectly activate a response in vomeronasal sensory neu-rons by binding to a subset of these receptors (47). Insharp contrast to these observation, Holekamp et al.(113), by using a new microscopical technique that allows

an exceptionally low-noise imaging of large neuronal pop-ulations, unexpectedly found that responses to diluteurine are predominantly, and perhaps exclusively, gener-ated by neurons in the apical layer, where V1Rs are ex-pressed.

Leinders-Zufall et al. (187) showed that some volatilehydrophobic pheromones, known to be tightly bound tothe hydrophobic pocket of MUPs, produced a calciumincrease in neurons of slice preparations of the vomero-nasal organ. The distribution pattern of the activatedneurons by any of these molecules reflects a localizationthat can be associated with the expression of the vome-ronasal receptors V1Rs (187), and the percentage of neu-rons responding to an individual pheromone (0.2–3%)matches the proportion of neurons expressing one type ofV1R. Therefore, when volatile molecules are released byMUPs, in the external environment or in the nasal cavity,they are likely to bind and activate V1Rs.

Urine contains compounds whose physiological roleis largely unknown. Nodari et al. (277) applied chromato-graphic fractions of female mouse urine to isolated vome-ronasal epithelia mounted on a multielectrode array andmeasured the spiking activity of neurons (277). Increasein the firing rate was associated with compounds withhomogeneous chemical properties that, after further pu-rifications, appeared to correspond to sulfated steroids.Sulfatase-treated urine extracts lost �80% of their activ-ity, indicating that sulfated compounds are the predomi-nant ligands for vomeronasal neurons in female urine.Interestingly, a collection of 31 synthetic sulfated steroidstriggered responses 30-fold more frequently than did asimilarly sized stimulus set containing the majority of allpreviously reported ligands. Vomeronasal neurons seemedto have a high tuning specificity for sulfated steroids, sinceeach neuron responded only to one or to a few structur-ally similar compounds, but collectively, neurons de-tected all major classes of sulfated steroids. Some ofthese compounds may be indicators of a status of stress.Indeed, the concentration of two sulfated glucocorticoidsin urine increased about threefold in animals subjected tostress compared with unstressed controls, indicating thatinformation about the status of stress of these mice isencoded in urine and could be detected by vomeronasalsensory neurons (277).

3. Responses to secretory peptides

Kimoto et al. (163) showed that mouse recombinantexocrine gland-secreting peptides (see sect. IIC) evokedelectrical activity in the vomeronasal but not in the olfac-tory epithelium. Interestingly, when the electrical re-sponses of individual neurons to ESP1 were recordedwith a multielectrode array, only a small percentage ofneurons, 1.6%, responded to ESP1 (163). This result is con-sistent with the possibility that ESP1 is recognized by one



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type of receptor only, most likely by a specific V2R, as ESP1induced c-fos production in vomeronasal neurons express-ing V2Rp5, a V2R receptor of the family A, clade III (98).

B. Olfactory Sensory Neurons

1. The cAMP transduction cascade

In contrast to a common belief, some sensory neu-rons of the main olfactory epithelium respond to phero-mones (364, 395). A large majority of olfactory sensoryneurons express a member of the odorant receptor familythat is coupled to a signal transduction pathway mediatedby cAMP (Fig. 6). The binding of ligands to the odorantreceptor activates the G protein subunit G�olf, whichstimulates adenylyl cyclase-3 activity producing an in-crease in cAMP concentration. cAMP causes the openingof the ciliary cyclic nucleotide-gated channels composedof three subunits: CNGA2, CNGA4, and CNGB1b (forreviews, see Refs. 29, 153, 292). These channels allow aninflux of Na� and Ca2� inside the cilia. Ca2�-activated Cl�

channels are then activated in turn and, due to the unusu-ally high intracellular Cl� concentration, allow an outflowof Cl�, contributing to the inward current (167, 179). As aresult of the odorant binding to receptors, the olfactorysensory neuron depolarizes. The CNG channel allowsCa2� entry not only for excitatory but also for inhibitorypurposes. The complex Ca2�-calmodulin activates a phos-phodiesterase (PDE1C2) that hydrolyzes cAMP and, im-portantly, produces a negative-feedback effect on theCNG channel itself, that has been shown to mediate ol-factory adaptation (178, 242).

When the depolarization following the activation ofthe transduction cascade reaches the threshold for acti-vation of action potentials, they will travel along the axonto the main olfactory bulb. Differently from vomeronasalsensory neurons, a maintained current injection will onlyelicit a short burst of action potentials in olfactory sen-sory neurons, instead of a tonic firing (196).

For more details about the cAMP transduction cascadein olfactory sensory neurons, the reader may consult previ-ous comprehensive reviews (40, 168, 235, 242, 292, 337).

2. Neuronal subsets

The subset of olfactory sensory neurons expressingTAARs also express G�olf, and therefore, it is possiblethat they use a signal transduction pathway coupled toadenylyl cyclase and cAMP (194), although additionalcomponents of the transduction pathway in these neu-rons are still unknown.

The ion channel TRPM5 is expressed in another sub-set of olfactory sensory neurons that coexpress theCNGA2 channel, and some components of the PLC path-way, such as PLC-�2 and the G protein �13 subunit (202).

Since TRPM5 channels are directly activated by intracel-lular Ca2� (112, 204, 299, 423), it is tempting to speculatethat Ca2� entry through CNG channels could gate TRPM5channels.

Neurons expressing TRPM5 are located in the ven-trolateral zones of the olfactory epithelium and project todomains of the ventral region of the main olfactory bulb,an area responsive to urine and other putative phero-mones (199–201, 336, 409). Unfortunately, electrical re-sponses from individual olfactory sensory neurons ex-pressing TRPM5 have not yet been investigated.

As previously mentioned (see sect. IVD), another sub-set of neurons in the main olfactory epithelium expressescomponents typical of a cGMP instead than a cAMP sec-ond messenger system, namely, the receptor guanylylcyclase type D (GC-D), the cGMP phosphodiesterasePDE2 (149), and the cyclic nucleotide-gated channelCNGA3, that was originally discovered in cone photore-ceptors (417). These neurons are located in the centralarea of the olfactory epithelium and project to an anatom-ically distinct group of interconnected glomeruli, thenecklace glomeruli, that have been implicated in the suck-ling response of mammals (149). Leinders-Zufall et al.(186) showed that neurons expressing GC-D can be acti-vated by the peptide hormones uroguanylin and guanylinand by natural urine stimuli, while Hu et al. (118) demon-strated that these neurons may be involved in the detec-tion of carbon dioxide. In both studies, GC-D neuronswere shown to use a cGMP signaling cascade (118, 186).

A subset of neurons in the main olfactory epitheliumof humans and mice expresses some vomeronasal recep-tors of the V1R family (152, 313, 315). Ligands correspond-ing to aliphatic alcohols or aldehydes have been identifiedfor human V1Rs (350); however, it is still unresolvedwhether neurons expressing these receptors project theiraxons to the main or the accessory olfactory bulb.

A small population of neurons in the main olfactoryepithelium expresses the TRPC2 channel. At present,there are no clues whether these neurons have chemo-sensory properties, but this finding is certainly relevantand must be seriously taken into account when compar-ing the behavioral effects produced by the genetic dele-tions of the TRPC2 channel with those of the surgicalremoval of the vomeronasal organ (P. Mombaert, per-sonal communication).

Early microscopical and immunological studies haverevealed the existence of a novel cell type in the olfactoryepithelium that is morphologically different from chemo-sensory neurons or supporting cells (44, 148, 260). Themain feature of these cells is represented by the microvillithat they bear on the mucosal side. It now appears thatolfactory microvillar cells do not represent a homoge-neous population of cells but rather they include differentcellular subsets expressing distinct signal transductionmolecules (6, 74, 201). It is very unlikely that these cells



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are true neurons as they do not seem to project their longfoot process to the olfactory bulb (74, 201). However,although their role is enigmatic, a possible chemosensoryfunction for microvillar cells has been reported (74).

3. Responses of the main olfactory epithelium

to pheromones

Wang et al. (395) showed that milk and urinary phero-mones that are responsible for puberty delay aggressivenessin the mouse (279, 281) and evoke an electrical responsefrom the main olfactory epithelium, as measured by fieldpotential recordings. Moreover, when the same experimen-tal approach was adapted to mice in which the adenylylcyclase type 3 gene had been knocked out, no electricalresponse was recorded, indicating that receptors respondingto these compounds are likely to be coupled to the cAMPsignaling pathway. Since behavioral deficits are also evidentin adenylyl cyclase type 3 mutant mice, it can be envisagedthat a cAMP-mediated pheromone signaling may play animportant role in the olfactory epithelium.

MHC peptides can also be detected by sensory neu-rons of the main olfactory epithelium via the cAMP sig-naling cascade, as shown by Spehr et al. (364). Two MHCpeptide ligands, SYFPEITHI and AAPDNRETF, elicited anelectrical response, measured by field potential record-ings in the main olfactory epithelium at a concentrationnear 10�11 M (364). Mutant mice lacking the CNGA2 genefailed to display electrical responses when stimulatedwith both peptides, suggesting that a subset of olfactoryneurons expressing CNGA2 are actively involved in thedetection of pheromones besides conventional odorants.

In contrast to that, Lin et al. (200) have demonstratedthat at least two pheromones, 2-heptanone and dimeth-ylpyrazine, stimulate the main olfactory epithelium with-out the intervention of the CNGA2 channel, suggestingthat pheromone detection in the olfactory epithelium re-lies on multiple transduction pathways.

C. Grueneberg Ganglion

The transduction pathways in the Grueneberg ganglionare still largely uncharacterized. Fleischer and colleaguesshowed that the majority of neurons express the vomerona-sal receptor V2R83 (V2R2 family), together with the G pro-tein G�i (79, 80), while a distinct subpopulation of G�i-expressing neurons also expresses TAAR receptors (80).Another study investigated whether neuronal subsets of theGrueneberg ganglion express transduction components sim-ilar to those of GC-D neurons, based on the observation thatboth types of neurons project to the necklace domain of themain olfactory bulb (Fig. 3). Fleischer et al. (78) reportedthat a subset of Grueneberg ganglion neurons expressing theV2R83 receptor expresses also the GC-D receptor and thecGMP-stimulated phosphodiesterase PDE2A.

Brechbuhl et al. (30) recently uncovered at least oneof the functional roles of the Grueneberg ganglion. In-deed, a subpopulation of neurons was shown to respondto the yet unidentified “alarm pheromones,” a group ofvolatile compounds that are released by animals sub-jected to stress. Lesions of this organ completely abolishthe so-called freezing reaction, a typical fear responseelicited by alarm pheromones. Curiously, in this study,lesions of the Grueneberg ganglion did not affect mother-pup recognition (30), a behavior that was believed to bemediated by this organ (118, 173, 317).

VI. SIGNALING FROM SENSORY EPITHELIA TO

THE BRAIN

A. Main and Accessory Olfactory Bulbs

1. Anatomical organization

The main olfactory bulb is the primary target region ofthe olfactory neurons, whereas vomeronasal sensory neu-rons project to a well-defined dorsal and posterior region ofthe olfactory bulb, namely, the accessory olfactory bulb. Themain olfactory bulb exists in all vertebrates, except foraquatic mammals. In contrast, the accessory olfactory bulbis absent in adult Old World monkeys, apes, and humans (formore information, see Table 1 in Ref. 240). In rodents, boththe main and the accessory olfactory bulbs have a largelysimilar laminar organization consisting of a superficial nervelayer formed by the axonal projections of the chemosensoryneurons, the glomerular layer that represents the first-ordersynaptic region between sensory neurons and mitral celldendrites, and the external and internal plexiform layers thatare regions where soma of, respectively, mitral/tufted andgranule cells reside (for review, see Ref. 240; see also Ref.182 for suggested changes to the nomenclature of the acces-sory olfactory bulb). In the main olfactory bulb, glomeruliare quite well anatomically separated, encapsulated by peri-glomerular cells, and rather uniform in size (�50 �m diam-eter in mice). Conversely, in the accessory olfactory bulb,these synaptic structures are very diffusely organized, sur-rounded by a small number of periglomerular cells, andhighly variable in size (10–30 �m diameter in mice). In themain olfactory bulb, mitral/tufted cells form a distinct mono-layer, whereas in the accessory olfactory bulb they are muchless organized. An important difference between mitral/tufted cells of the two olfactory systems is their arborization:in the main olfactory bulb these neurons bear only oneapical dendrite, whereas in the accessory olfactory bulbmitral/tufted cells are equipped with up to six apical den-drites, each entering a different glomerulus (Fig. 7; for re-view, see Ref. 240).

2. Topographic projections

Axons of all the olfactory sensory neurons expressing aparticular odorant receptor converge to only two glomeruli



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in the main olfactory bulb (257, 308, 389). This has beenexperimentally demonstrated at a single-neuron resolutionby using transgenic mice in which the expression of a givenodorant receptor was linked to that of the green fluorescentprotein, thus allowing the visualization of the axonal net-

work (255, 256, 381). In mice there are �2,000 glomeruli, andtheir localization is roughly conserved among individuals.Therefore, the olfactory bulb is topographically organized,with each glomerulus representing a single type of odorantreceptor (Fig. 7C).

FIG. 7. Main and accessory olfactory bulbs. A and B show the general organization of the main olfactory bulb (MOB) and of the accessoryolfactory bulb (AOB) in the adult rat from Nissl-stained specimens. A: each mitral cell (MC) of the main olfactory bulb has a single apical dendriteprojecting to a single glomerulus and several lateral dendrites that contact granule cell dendrites. B: each mitral cell (here indicated as large principalcell, LPC) of the accessory olfactory bulb has several apical dendrites projecting to multiple glomeruli. EPL, external plexiform layer; MC, mitralcells; EGC, external granule cell; asterisk, tufted cell axon; IGC, internal granule cell dendrite; S, superficial centrifugal fiber; LOT, lateral olfactorytract. [From Larriva-Sahd (182).] C and D: schematic topographical representations of sensory projections in the main olfactory (C) and vomeronasal(D) systems. C: in the main olfactory system, olfactory sensory neurons expressing a given odorant receptor project to the same glomerulus (orglomerular pair). Three populations of olfactory sensory neurons, each expressing one different type of odorant receptor, are represented indifferent colors. D: in the vomeronasal system, sensory neurons that express the same vomeronasal receptor project to multiple small glomeruli.Six populations of vomeronasal sensory neurons, each expressing one different type of vomeronasal receptor, are represented in different colors.



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In rodents, V1Rs and V2Rs are individually expressedin vomeronasal sensory neurons, and each neuron ex-pressing a given vomeronasal receptor (or vomeronasalreceptor pair) sends its axonal projection toward theanterior (V1Rs) or posterior (V2Rs) accessory olfactorybulb (100, 143, 414). Gene targeting approaches were usedto visualize the topographical map of each targeted recep-tor in the mouse accessory olfactory bulb (16, 62, 314).When a fluorescent marker protein was inserted down-stream of a vomeronasal receptor gene, the resultingvisualized axonal projections of neurons expressing thatspecific receptor showed that several glomeruli in subre-gions of the anterior or posterior accessory olfactory bulbreceived inputs. A more accurate analysis indicated thataxons from neurons expressing the same receptor coa-lesce into 10–30 glomeruli and that a single glomerulusreceives input from neurons expressing different receptortypes (Fig. 7D). Moreover, the capacity to converge and toform these glomeruli depends on the possibility to ex-press a specific vomeronasal receptor gene, suggestingthat the receptor proteins themselves play a role in axonalwiring to the bulb (16, 314). In fact, vomeronasal neuronsexpressing a nonfunctional (truncated) V1R gene nolonger converge in the accessory olfactory bulb. Thisphenotype, also observed for odorant receptors (343),could be possibly due to the inability of the truncatedgene to prevent the expression of other V1Rs, resulting ina population of fluorescently tagged neurons randomlyexpressing different V1Rs . Indeed, Roppolo et al. (318)showed that the expression of a nonfunctional V1R alleleallows the coexpression of other V1R genes. Although noV1R expression has been reported in other subcellularcompartments besides the projecting dendrite, it is pos-sible to envisage that V1Rs may direct axon guidance,perhaps through a mechanism that involves their expres-sion in the growth cones.

Wagner et al. (393) generated transgenic mouse linesin which projections from various populations of neuronsexpressing distinct V1Rs were differentially labeled withfluorescent proteins. They found a glomerular map di-vided in multiple nonoverlapping domains, with each do-main clustering glomeruli formed by neurons expressingV1Rs of the same subfamily, randomly intermingled (393).Moreover, these authors (393) found that a mitral cell isnot only connected to glomeruli innervated by neuronsexpressing the same V1R (62) but also to those associatedwith receptors of the same subfamily. Therefore, in theanterior accessory olfactory bulb, the spatial map origi-nated from vomeronasal organ inputs seems to rely onreceptor subfamilies, rather than individual receptors asin the main olfactory bulb. This wiring scheme may helpto discriminate molecules with highly similar chemicalstructure at different ratios in a pheromonal blend.

In both the main and accessory olfactory systems,mitral cells are activated by primary sensory neurons

through glutamatergic synapses, but the chemical infor-mation is further processed by the activity of inhibitoryinterneurons as well as periglomerular and granule cells(141, 210, 339) that contact mitral cells via glutamatergicdendrodendritic synapses. These consist of an excitatoryglutamatergic input from mitral cells to granule cells,inducing the release of GABA from granule cells, that inturn provides inhibition to the mitral cells (141, 182, 303).For more information, the reader may consult previouscomprehensive reviews (339, 345, 385).

3. Encoding pheromonal information in the main and

accessory olfactory bulbs

To simultaneously examine the responses to phero-mones and odorants in the mouse main and accessoryolfactory bulbs, Xu et al. (409) used high-resolution func-tional magnetic resonance imaging (fMRI), a very power-ful technique that can show dynamic responses in bothentire olfactory bulbs to various stimuli in the same ani-mals. Mouse urine activated restricted areas in both olfac-tory bulbs, eliciting signals mainly in the ventral region of themain olfactory bulb and in the anterior part of the mainaccessory bulb. Since the anterior region of the mouse ac-cessory olfactory bulb receives input from V1R-expressingvomeronasal sensory neurons, this study is in agreementwith recent results from Holekamp et al. (113) who sug-gested that urine mainly activates V1Rs (see sect. VA2).

The encoding of pheromonal information in the mitralcells of the accessory olfactory bulb has been investigatedby recording responses in awake mice using a miniaturemotorized drive to move microelectrodes and record theactivity of single neurons (213, 214). Male mice were allowedto investigate conspecifics differing by sex, genetic makeup,and hormonal status. Interestingly, an increase in mitral cellactivity occurred after test animals directly contacted andactively investigated the head and the anogenital regions ofthe stimulus animal. The recorded mitral cell responseswere found to be specific for sex and strain of the stimulusmouse. In addition, mitral cells exhibited both highly spe-cific excitatory and inhibitory responses, these latter likelyoriginating from the inhibitory interneuron activity. There-fore, the activity of mitral cells appears to be tuned by thecombination of sex and genetic makeup (213, 214), andtherefore, information about sex and strain is already inte-grated in the accessory olfactory bulb.

Several studies have revealed that the exposure tourinary components from opposite-sex conspecifics in-creased the number of c-fos-immunoreactive mitral andgranule and periglomerular cells in the accessory olfac-tory bulb (116, 126, 228, 295, 410). Volatile urinary odorsfrom male as well as female mice also stimulate c-fos

expression in distinct clusters of glomeruli in the mainolfactory bulb in both sexes (228, 270).



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Lin et al. (199) recorded the electrical activity in theaccessory olfactory bulb in response to urine of differentmouse strains. A subset of mitral cells was found to bevery selective, responding only to male urine. Moreover,by combining electrophysiology and gas chromatographicseparation of male mouse urine components, these au-thors identified a male specific compound, methyl-thio-methanethiol (MTMT), that was indeed responsible forthis effect. Interestingly, mitral cells responding to MTMTwere not stimulated by any other urinary compound,allowing the appellation of “specialist” cells. Further-more, behavioral experiments also established the biolog-ical activity of this molecule, as female mice developedinterest and attraction towards castrated mouse urineafter the addition of synthetic MTMT.

B. Signaling Beyond the Olfactory Bulbs

In the main olfactory bulb, mitral/tufted cells directlytransmit signals from glomeruli to pyramidal neurons in the

olfactory cortex, without a thalamic relay. The primary ol-factory cortex, defined as the ensemble of brain regions thatreceive direct input from the olfactory bulb, is composed ofseveral anatomically distinct areas: the piriform cortex, theolfactory tubercle, the anterior olfactory nucleus, the corti-cal amygdala, and the entorhinal cortex. In turn, most ofthese cortical regions project back to the main olfactorybulb and to other areas of the brain, including the thalamus,the hypothalamus, the hippocampus, the frontal cortex, andthe orbitofrontal cortex (Fig. 8; reviewed in Ref. 349). An-atomical studies and genetic approaches indicate that thetopographical organization of odorant information in theolfactory bulb is not reflected in the olfactory cortex.Instead, odorant receptors seem to be mapped to multi-ple, discrete clusters of cortical neurons.

Mitral cells of the accessory olfactory bulb project toareas of the limbic system: the medial amygdala and theposteromedial cortical amygdala, which are also called“vomeronasal amygdala,” the accessory olfactory tract, andthe bed nucleus of the stria terminalis (Fig. 8) (272, 334, 392).

FIG. 8. Central pathways involved in pro-cessing chemical information by the vomerona-sal and main olfactory systems. [Based on datafrom Martinez-Marcos (229) and Yoon et al.(413).]



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Although vomeronasal sensory neurons expressingV1Rs or V2Rs, respectively, project to distinct areas of theaccessory olfactory bulb, this segregation, at least in somespecies, does not seem to be completely maintained in theamygdalic regions, where clusters of neurons may com-bine signal information originating from both families ofvomeronasal receptors (230, 252, 253, 327, 392).

In essence, the majority of these studies provideevidence that, excluding areas of convergence, the twovomeronasal pathways project to specific areas of theamygdala and hypothalamus, thus suggesting a partialfunctional separation (252, 253).

In the vomeronasal system, projections to the hypothal-amus from the limbic regions are directed to the ventrome-dial and the medial preoptic areas, which are involved inreproductive and social behaviors (158, 159, 289).

The medial amygdala receives direct input from theaccessory olfactory bulb, but also from the main olfactorybulb. However, other rostral basal telencephalic regionsseem to play a relevant role in the integration of signalsoriginating from both the vomeronasal and the main olfac-tory epithelia.

C. Pheromone-Activated Hormonal Systems

Recent experiments have revolutionized the commonbeliefs about the control of hormonal responses by pher-omones (26, 413). The effects of pheromones on the neu-roendocrine status are mediated by a group of neurons inthe hypothalamus, which constitutes the endocrine con-trol center (for reviews, see Refs. 89, 245, 353). Theseneurons secrete the GnRH, also known as LH-RH, whichin turn stimulates the anterior pituitary gland to releasetwo gonadotrophins: the luteinizing hormone (LH) andthe follicle-stimulating hormone (FSH). Both hormonescontrol the development and the function of the gonads inmales and females, although with different effects.

A genetic approach has been used to visualize theGnRH neuronal network and, surprisingly, GnRH neuronswere found to be connected with areas relaying informa-tion from the main olfactory system, in contrast to thetraditional view that only ascribes connections withGnRH neurons from the vomeronasal system (26, 413).

Boehm et al. (26) identified neurons that synapsewith GnRH by using a genetic transneuronal tracer, barleylectin, a glycoprotein that is transferred across synapses.The barley lectin gene was positioned under the control ofthe GnRH promoter so that only GnRH neurons wouldexpress this peptide. In another study, Yoon et al. (413)used a different experimental approach to anatomicallytrace the afferent pathways to GnRH neurons in the hy-pothalamus. Only neurons expressing GnRH were in-fected with a modified pseudo-rabies virus and greenfluorescent protein reporter. The genetically controlled

viral tracing allowed the identification of a major projec-tion pathway from the main olfactory system.

Both studies (26, 413) came to the conclusion thatthe small pool of GnRH cells (800 neurons) was surpris-ingly synaptically connected to some tens of thousandneurons in �50 diverse brain areas, including the mainolfactory ones, suggesting that GnRH neurons may influ-ence a large variety of brain functions.

Interestingly, Yoon et al. (413) reported the ab-sence of synaptic connections between the vomerona-sal pathway and GnRH neurons. This could be due todifferent experimental approaches used in the twostudies or, perhaps, presynaptic inputs from the vome-ronasal pathway identified in one study (26) might de-rive from a subset of GnRH neurons distinct from thosetargeted in the other study (413). However, the obser-vations of Yoon et al. (413) do not refute previousneuroanatomical tracing data indicating that the vome-ronasal pathway projects to the hypothalamic areawhere GnRH neurons are located. Indeed, Yoon et al.(413) confirmed that neurons in this area receive inputsfrom the vomeronasal pathway. It could be speculatedthat the connections with GnRH are, at least in part,mediated by nonsynaptic (e.g., hormonal or neuro-modulatory) mechanisms.

As a confirmation of these observations, stimulation ofmale mice with urinary pheromones induced the activationof neurons in the anterior cortical amygdala as well as in thedorsal and ventral endopiriform nucleus. Therefore, signalsin response to pheromonal cues also occur via the mainolfactory epithelium and are transmitted to GnRH neuronsthrough the olfactory cortex. Moreover, feedback loopshave been identified so that GnRH neurons could influenceboth odorant and pheromone signaling.

VII. PHEROMONE-MEDIATED BEHAVIORS

From results presented in the previous sections, itnow appears evident that both the vomeronasal and themain olfactory systems may be activated by chemicalsthat are known to produce pheromonal effects. Theinvolvement of the vomeronasal and main olfactorysystems on specific behaviors is usually investigated byinactivating one or both systems. In the past, lesionswere made only by surgical removal of the vomeronasalorgan (VNOx) or by chemical ablation of the mainolfactory epithelium (MOEx) (Table 2). The introduc-tion of a molecular genetic approach allowed the dele-tion of specific transduction molecules, although itmust be taken into account that the knowledge of genesinvolved in transduction in each olfactory organ is stillincomplete, and therefore, results of behavioral exper-iments from knockout animals must be interpreted verycautiously.



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Here, we will discuss experimental results mainlyrelated to sexual and aggressive behaviors and will tryto interpret to what extent the vomeronasal and/or themain olfactory systems appear to be involved. It is veryimportant to note that behavioral expressions largelyvary among species; moreover, responses to phero-mones cannot be simply described as innate or stereo-typed, but may also depend on contextual situationsand can be modified by some forms of learning andmemory.

A. Male Behaviors

1. Male sexual behavior

Sexual behavior has been intensively approached bymany studies in the last 20 years, particularly in rodents. Inmale mice, the typical mating sequence consists of olfactoryexploration of estrus females followed by multiple episodesof ultrasound vocalization, mounting and intromission, andeventually ejaculation. All these parameters can be evalu-ated in a typical test experiment (120, 121).

TABLE 2. Effects of surgical and genetic ablations of olfactory and vomeronasal systems

VNX VGENX OEX OGENX

Hamster 2Scent marking (290) 2Partner preference (10)2Male copulatory

behavior (55, 76, 298)2Lordosis behavior (221) 2Scent marking (145)2Individual recognition

(365)2Sexual arousal (146)

2Individual recognition (146)2Male copulatory behavior (146)

Rat 2Stress-inducedhyperthermia (165)

2Penile erection (172)

Maternal aggression (170)2Male induced sexual

receptivity (302)2Nursing behavior (36)2Infanticide (243)2Sexual arousal (325)2Lordosis behavior (326)

Mouse 2Lordosis behavior tomale mount (155)

2Sex discrimination (trpc2) (193,284, 368)

2Lordosis behavior (154) 2Copulatory behavior (ac-

3) (395)1Sperm motility (175) 2Intermale aggressiveness (trpc2;

g�i2) (278, 368)2Sex discrimination (154) 2Intermale aggressiveness

(ac-3) (395)2Urine marking (181, 233) 2Maternal aggression (trpc2; v1ra-

v1rb; g�i2) (61, 161, 193, 278)(73, 376) 2Individual recognition

(cnga2) (364)2Intermale aggressiveness

(54, 233)2Lactating behavior (trpc2) (161)

2Marking Male-like sexual behavior in females(trpc2) (161)

2Copulatory behavior (54) 2Copulatory behavior (v1ra-v1rb)(61)

1Puberty onset (209) 2�arking (trpc2)(193)2Strain recognition (206)2Pregnancy block (156,

206, 216)2Sexual investigation

(135)Male-like sexual behavior

in females (161)Ferret 2Sexual investigation

(404)Lemur 2Copulatory behavior (8)

2Intermale aggressiveness(8)

Opossum 2Male-mediated inductionof estrus (131)

Prairie vole 2Intermale aggressiveness(399)2Pairing (189)

Rabbit 1Maternal behavior (87) 2Suckling behavior1Maternal behavior (52)

Cat 2Catnip reaction (104)

Reference numbers are given in parentheses.



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The genetic ablation of the TRPC2 channel greatlyimpairs some features of the sexual behavior. Indeed,although male mice mate normally with females, they alsoshow an indiscriminate courtship and mounting behaviortoward females (161, 193, 368). A similar situation wasobserved in VNOx male mice (161). Furthermore, individ-ual recognition, through the discrimination of nonvolatileodorants, is also affected in VNOx mice (155, 284). On theother hand, the simultaneous deletion of a group of V1Rreceptors results in a significant decrease of male copu-latory behavior (61), although this latter appears normalin G�i2 mutants, suggesting that apical neurons are likelynot to be of relevant importance for mating motivation. Allthese findings reveal that the mouse vomeronasal organ isindeed involved in individual recognition and reproductiveactivity, but that signaling through the vomeronasal organ isnot required for initiating sexual behavior.

The chemical ablation of the main olfactory system(via nasal irrigation with zinc sulfate) produces a strik-ing reduction in male reproductive behavior (227). Fur-thermore, the genetic deletion of the CNGA2 channel(227), or of adenylyl cyclase type 3 (395), entirely abol-ishes male mating performances producing severe de-ficiencies in each step involved in mating: olfactoryexploration, mounting, and intromission, comparedwith normal mice. Thus the main olfactory systemseems to play an essential role for male mouse matingbehavior.

In rats, individual recognition and sexual arousal isonly temporary or moderately depressed by lesions of thevomeronasal organ (22, 325) as well as the overall copu-latory behaviors appear to be slightly or partially affected(171, 324, 325).

Sexual behavior in male hamsters is totally abolishedby bilateral removal of the olfactory bulbs, an operationthat eliminates sensory input from both the olfactory andthe vomeronasal systems (298). The destruction of themain olfactory epithelium did not impair male hamstermating behavior, whereas the peripheral deafferentationof the vomeronasal system produces severe sexual behav-ior deficits in approximately one-third of the treated ani-mals. Combined deafferentation of both the vomeronasaland the olfactory systems completely abolishes copula-tion in all experimental animals (298). Mating behavioralterations appear after removal of the vomeronasal or-gan in sexually inexperienced males, and this effect isreversed by intracerebroventricular injections of GnRH(76, 247), indicating that GnRH acts as an intermediate inthe facilitation of mating behavior by vomeronasal sen-sory input. However, sexual experience prior to the re-moval of the vomeronasal organ mitigates the effect of thelesion on sexual behavior, indicating that behavioral re-sponses are influenced both by VNOx and by sexual ex-perience (291). In this species, other ways of communi-

cation as scent or flank marking are likely to be primarilymediated by the main olfactory system (145).

2. Male aggressiveness

In mice, male to male territorial aggression is a re-markable innate social behavior that is triggered by com-pounds present in urine (see sect. II, Table 1). In a com-mon assay employed to test male aggressiveness, a maleintruder is introduced in a cage containing a singly housedmale resident. After a period of olfactory investigation, toallow individual recognition, the resident mouse initiatesa series of attacks against the intruder. The number andduration of the attacks and the latency to the first attackrepresent standard parameters to evaluate the aggressivebehavior (58, 233).

The surgical ablation of the vomeronasal organ hasbeen long known to nearly abolish aggressiveness in malemice and prairie voles (12, 54, 55, 233, 399, 407), an effectthat is recapitulated in mutant mice bearing the deletionof the gene encoding TRPC2 (161, 193, 368). Indeed, malemice bearing the genetic ablation of the TRPC2 channelfail to display aggression toward male intruders (193,368), a behavioral effect that, in an attenuated form, isalso evident in mice lacking G�i2 (278).

The main olfactory system also plays a very impor-tant role in male aggressiveness. Indeed, mice that eitherhave undergone the chemical ablation of the olfactoryepithelium (via nasal irrigation with zinc sulfate) (154) orhave been genetically mutated in the genes encoding theodorant transduction molecules adenylyl cyclase type 3and CNGA2 (227, 395) show abnormal sexual and aggres-sive behaviors (227).

Thus both the olfactory and the vomeronasal systemsappear to be necessary for eliciting male aggressiveness.

B. Female Behaviors

1. Female sexual behavior

In females of different species, sexual behavior ischaracterized by ultrasonic vocalization and lordosis elic-ited by lumbosacral tactile stimulation (174, 294).

In mice, the genetic ablation of the TRPC2 channeldid not alter female sexual receptivity to males but, sur-prisingly, caused a female sexual behavior similar to thatof males, i.e., females attempted to copulate as males(161). Kimchi et al. (161) also showed that similar sexualbehaviors were displayed by females in which the vome-ronasal organ was surgically removed, in contrast to aprevious study that described a reduced sexual receptiv-ity in VNOx females (155). The chemical ablation of themain olfactory epithelium (with zinc sulfate) reduces sex-ual behavior in female mice (154).

In female rats, both chemical ablation of the mainolfactory epithelium and vomeronasal organ removal af-



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fect sexual receptivity and the male-induced release ofGnRH (17, 302).

In female hamsters, the removal of the vomeronasalorgan disrupts the elicitation of lordosis by lumbosacraltactile stimulation, an effect reversed by injection ofGnRH (221).

2. Maternal aggressive behavior

Female mice are normally not aggressive towardsintruders, but during lactation, they violently attack non-resident males. This type of maternal aggressive behaviorhas been shown to be dependent on chemosensory cues(13). The surgical removal of the vomeronasal organ af-fects aggression of lactating females (13, 161), whereas ithas little or no effect on other maternal behavior param-eters as nest building or pup retrieval (13). The geneticablation of the TRPC2 channel (161, 193), the G�i2 subunit(278), or a subfamily of V1R receptors (61), also results ina low expression of maternal aggression, suggesting thatthis type of behavior is possibly linked to the apical subsetof chemosensory neurons.

VIII. CONCLUSIONS AND

FUTURE CHALLENGES

Although we are only beginning to understand theoverlapping role of the main olfactory and vomeronasalsystems for pheromone sensing, there is now overwhelm-ing evidence that the main olfactory system is involved ina variety of important physiological mechanisms underly-ing pheromonal communication.

In the early 1970s, different studies developed amodel that led to the so-called dual olfactory hypothesis,sustained by the observation that efferent and afferentconnections of the main and accessory olfactory bulbproject in nonoverlapping areas of the brain, according towhich the olfactory and vomeronasal systems were orga-nized as parallel anatomical axes subserving differentfunctions. Recent anatomical and functional studies haveindicated that this distinction is not as strict as commonlythought (229). There seems to be, in fact, areas of largeconvergence in the anterior medial amygdala. Moreover,in the rostral basal telencephalon, both inputs convergedin areas that were classically categorized as exclusivelyvomeronasal or olfactory. As for the anatomical observa-tion, behavioral and biochemical studies seem to point toa complementary role of the two systems. For example,the functional ablation of the olfactory and vomeronasalorgans leads to similar behavioral changes. Chemosen-sory neurons sensitive to pheromone have been identifiedin both subsystems. Not last, humans as well as otherspecies in which the vomeronasal system is absent canprobably detect pheromones via the main olfactory epi-thelium. It is therefore possible that the two systems may

have developed independently for the detection of pher-omones, and only the main olfactory epithelium has fur-ther specialized also for the perception of conventionalodorants. Proof of this hypothesis will require additionalstudies especially in species in which olfaction is not aprimary sensory process as humans, for example; how-ever, it is interesting to note that part of the main olfac-tory central pathways seem to converge in regions thatcontrol the sexual status of the individual.

ACKNOWLEDGMENTS

We thank Didier Trotier for kindly providing the originalmicrograph for figure 6A.

Address for reprint requests and other correspondence: A.Menini, Scuola Internazionale Superiore di Studi Avanzati, ViaBeirut 2, 34014 Trieste, Italy (e-mail: [email protected]).

GRANTS

This work was supported by grants from the Italian Minis-try of Research and by the Italian Institute of Technology.

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doi:10.1152/physrev.00037.2008 89:921-956, 2009.Physiol RevRoberto Tirindelli, Michele Dibattista, Simone Pifferi and Anna MeniniFrom Pheromones to Behavior

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