From Pheromones to Behavior

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

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 921 II. Mammalian Pheromones 922

A. Pheromones in various animal orders 922 Downloaded from B. Major urinary proteins and major histocompatibility complex molecules 925 C. Exocrine gland-secreting peptides 926 III. Chemosensory Systems Detecting Pheromones 927 A. Vomeronasal epithelium 927 B. Olfactory epithelium 929 C. Other olfactory organs 929 IV. Putative Pheromone Receptors 930 physrev.physiology.org A. Vomeronasal receptors 930 B. Odorant receptors 933 C. Trace amine-associated receptors 934 D. Olfactory-specific guanylyl cyclase type D receptor 934 V. Pheromone Transduction 935 A. Vomeronasal sensory neurons 935 B. Olfactory sensory neurons 939 C. Grueneberg ganglion 940 VI. Signaling From Sensory Epithelia to the Brain 940 A. Main and accessory olfactory bulbs 940 on February 9, 2011 B. Signaling beyond the olfactory bulbs 943 C. Pheromone-Activated hormonal systems 944 VII. Pheromone-Mediated Behaviors 944 A. Male behaviors 945 B. 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 of the profound effects of pheromones on reproductive physiology and behavior. Pheromones have been classified as molecules released by individuals and responsible for the elicitation of specific behavioral expressions in members of 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 was based 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 specifically dedicated to the detection of pheromones. However, recent studies have reexamined this traditional interpretation showing that both the main olfactory and the vomeronasal systems are actively involved in pheromonal communication. The current knowledge on the behavioral, physiological, and molecular aspects of pheromone detection in mammals is discussed in this review.

I. INTRODUCTION of the external world, enabling the detection and the discrimination of a very high number of molecules with Chemosensation is one of the sensory modalities that different structures. Substances carrying a chemical mes- animals have evolved to analyze the chemical properties sage among animals are termed “semiochemicals,” from www.prv.org 0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society 921 922 TIRINDELLI ET AL. the Greek “semeion” (sign). An important class of semio- determinants. For example, trace pheromones allow at- chemicals is constituted by pheromones. The term pher- tracting a conspecific, while alarm pheromones alert omone, based on the Greek “pherein” (to carry or trans- other conspecifics about an external challenge and can fer) and “hormon” (to stimulate or excite), was intro- evoke aggressive behaviors. duced in 1959 by the entomologists Peter Karlson and A first classification groups intraspecific signaling Martin Lu¨scher (151) to identify specific biologically ac- molecules in releaser and primer pheromones (239). Re- tive substances “which are secreted to the outside by an leaser pheromones lead to individual recognition and individual and received by a second individual of the same evoke short-latency behavioral responses in the conspe- species, in which they release a specific reaction, for cific like in aggressive attacks, or mating, or territory example, a definite behavior or a developmental process.” marking. Conversely, primer pheromones induce delayed In the same year, the first insect pheromone bombykol, responses that are commonly mediated through the acti- released by the female silkworm to attract mates, was vation of the neuroendocrine system. Thus, through the characterized by Butenandt et al. (43). The use of phero- emission of chemical cues, an individual forces the hormones among insects has been extensively investigated, monal balance of a receiver, thus modulating its repro- and interested readers may consult several reviews on the ductive status. subject (4, 111, 130, 379). In mammals, pheromones have been reported to be

Another class of semiochemicals is constituted by al- involved in a variety of behavioral effects also in unrelated Downloaded from lelomones: substances produced by members of one species species and, surprisingly, some pheromonal molecules are that influence the behavior of members of another species shared, with different purposes, between species. (for a review, see Ref. 333). Although in this review we will mainly discuss the effects elicited by pheromones in mam- 1. Proboscidea mals, we will also briefly mention some examples of allel- Despite their size, which would obviously preclude omonal communication (5, 107, 108, 169). physrev.physiology.org extensive behavioral studies, Asian and African elephants In most mammals, the nasal cavity contains at least two have been largely investigated for their pheromonal re- major sensory systems that may be involved in the detection sponses by the late Bets Rasmussen. During musth, a of pheromones: the vomeronasal and the main olfactory periodic condition in which mature males are character- systems. Evidence indicates that some behaviors are medi- ized by highly aggressive behavior, elephants produce ated exclusively by one or by a complementary action of large quantities of a specific pheromone, frontalin (1,5- both systems. For example, Bruce (37) reported that, when dimethyl-6,8-dioxabicyclo[3.2.1]octane), that is released female mice that have been recently inseminated are ex-

in temporal gland secretions, urine, and breath (307). on February 9, 2011 posed to urine of a male different from the one they mated Adult males are mostly indifferent to frontalin, whereas with, the pregnancy is interrupted and the female returns to subadult males are highly reactive, often exhibiting repul- estrus. This effect depends on a functional vomeronasal sion or avoidance. Female chemosensory responses to system (15). Differently, the nipple search in rabbit pups is frontalin vary with the hormonal status. Females in the one of the typical examples of behavior strictly dependent follicular phase are the most responsive and often demon an intact main olfactory system (119). In other instances, onstrate mating-related behaviors subsequent to frontalin behavioral responses are mediated by both the vomeronasal stimulation (304). Furthermore, female Asian elephants and the main olfactory systems. This is the case of female excrete a urinary pheromone, (Z)-7-dodecen-L-yl acetate, hamsters, whose ultrasonic calling during estrus is abol- to signal to males their readiness to mate (305). Interest- ished by the inactivation of either the vomeronasal or the ingly, females of more than 100 species of insects use the main olfactory system (145). In recent years, the discovery of same compound as part of their pheromone blends to distinct pheromone receptor families coupled to a complex attract insect males. The ﬁnding that the same molecule network of signal transduction pathways, together with ad- may be shared by unrelated species for a different phero- vances in molecular genetics and functional electrophysiologi- monal purpose is only apparently surprising, owing to the cal and imaging techniques, have greatly advanced our under- fact that structural constraints are often dictated by com- standing of the physiological role of pheromones. mon metabolic pathways.

II. MAMMALIAN PHEROMONES 2. Marsupialia In marsupials, the gray short-tailed opossum (Mono- A. Pheromones in Various Animal Orders delphis domestica) communicates by scent marking. The male opossum possesses a prominent suprasternal scent Pheromones have a very large chemical variability gland, whose extracts strongly attract female opossums (Table 1), and therefore they are preferably grouped ac- (131), stimulating the estrus in anestrous females via the cording to their functions rather than to their structural vomeronasal system.

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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 calcium influx in VNO neurons (187) 2-Sec-butyl-4,5- Mouse male urine VNO V1R neurons Electrical response and calcium Estrus induction and synchronization dihydrothiazole (BT) influx in VNO neurons (187) (Whitten effect) (138) Protooncogene expression in Puberty acceleration (Vandenbergh effect) the AOB (32, 95) (266, 283) Intermale aggressiveness (280) Attractiveness to females (139) 2,3-Dehydro-exo- Mouse male urine VNO V1R neurons Electrical response and calcium Estrus induction and synchronization brevicomin (DB) influx in VNO neurons (187) (Whitten effect) (138) Protooncogene expression in Puberty acceleration (Vandenbergh effect) the AOB (32, 95) (266, 283) Intermale aggressiveness (280) Attractiveness to females (139) ␣- and ␤-Farnesene Mouse preputial glands VNO V1R neurons Electrical response and calcium Puberty acceleration (Vandenbergh effect) influx in VNO neurons (187) (266, 283) Territorial marking (279) Attractiveness to females (139) Downloaded from Suppression of estrus cycle (Lee Boot- effect) (219) 6-Hydroxyl-6-methyl-3- Mouse male urine VNO V1ra and V1rb (V1Rs) Electrical response and calcium Puberty acceleration (Vandenbergh effect) heptanone neurons influx in VNO neurons (61, (266, 282) 187) 2-Heptanone Mouse urine VNO V1R neurons Electrical response and calcium Estrus extension (137) influx in VNO neurons (187)

IP3 production in the VNO (377) physrev.physiology.org n-Pentyl acetate Mouse urine VNO V1ra and V1rb (V1Rs) Electrical response in VNO Suppression of estrus cycle (Lee Boot- neurons neurons (61) effect) (281) Isobutylamine Mouse male urine VNO V1ra and V1rb (V1Rs) Electrical response in VNO Estrus acceleration and vaginal opening neurons neurons (61) (276, 301) 3-Amino-s-triazole (similar Mouse male urine ? ? ? Attractiveness to females (2) to BT) 4-Ethyl phenol Mouse male urine ? ? ? Attractiveness to females (2) 3-Ethyl-2,7-dimethyl octane Mouse male urine ? ? ? Attractiveness to females (2) (similar to DB) Repulsion to mouse (territorial marking?) (2) 3-Cyclohexene-1-methanol Mouse male urine ? ? ? Attractiveness to females (2) on February 9, 2011 Major urinary proteins Mouse urine VNO V2R neurons Electrical response and calcium Puberty acceleration (Vandenbergh effect) (MUPs) inﬂux in VNO neurons (47, (266, 283) 163) In vitro survival and Individual recognition (49, 123, 375) proliferation of VNO neurons (408) Intermale aggressiveness MHC class I peptides Mouse urine VNO V2R neurons Electrical response and calcium Attractiveness to individuals of a different inﬂux in MOE and VNO mouse strain (358) neurons (364) (SYFPEITHE; MOE Single-cell recording in VNO Pregnancy block (Bruce effect) AAPDNRETF) neurons (156, 185) Exocrine gland secreting Extraorbital lacrimal glands VNO V2R neurons Electrical response in VNO ? peptides neurons (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 Copulatory behavior in male (356) c-fos expression in AOB neurons (136, 177) 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) (ϩ)(Ϫ)1,5-Dimethyl-6,8- Male asian elephant temporal ? ? ? Repulsion, attractiveness, and sexual dioxabicyclo[3.2.1]octane gland, urine, and breath behavior depending on the racemic ratio (frontaline) (92, 304)

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

3. Soricomorpha (Insectivores) mounts from males signiﬁcantly sooner than females In musk shrew (Suncus murinus), virgin females ex- exposed to cages soiled by a castrated male, by another posed to cages recently vacated by an adult male receive female, or to a clean cage (310).

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In tree shrew (Tupaia belangeri), both males and sure to odorless axillary’s secretions from females in the females mark their surroundings with urine and skin late follicular and ovulatory phase of the menstrual cycle, gland secretions. Males mark more frequently than fe- respectively, shortens and lengthens the menstrual cycles males in the absence of conspecific scent; moreover, male of recipient females, accelerating or delaying the lutein- scent stimulates marking in females. Interestingly, in- izing hormone (LH) surge (367). Women smelling an an- creasing amounts of male scent result in a corresponding drogen-like compound activate the preoptic and ventro- increase in marking by females (114), suggesting that, in medial nuclei of the hypothalamus in contrast to men who the wild, pheromones may not exert an all-or-none effect activate the paraventricular and dorsomedial nuclei hypo- but rather act in a dose-specific fashion. thalamus when smelling an estrogen-like substance (332). Although a robust hypothalamic response is commonly 4. Carnivora and Ungulata observed with ordinary odorants, such a dramatic dimorphic difference can be interpreted as a hormonal response Both carnivores (dogs and cats) and ungulates com- to pheromone stimulation. monly mark their territory with urine and display a spe- The apocrine gland secretion of the axilla in combi- cific behavior called “flehmen” (67, 88, 391). In the fleh- nation with the microbial flora is probably the most rele- men reaction, animals, after the physical contact with a vant source of candidate compounds for human phero- conspecific or with scents emanating from its excrements mones, which consists of a blend of molecules character- Downloaded from (urine or feces), lift the head, draw back their lips, and izing the individual body odor (14). However, salivary and push the tongue towards the anterior region of the palate seminal secretions and urine represent other reservoirs of in a manner that makes them appear to be “grimacing.” It putative human pheromones. The male sweat and salivary is commonly believed that this attitude would allow a progesterone derivative androstadienone (4,16-androsta- faster transfer of pheromones into the olfactory organs. dien-3-one) (AND) has been indicated as a putative hu- The saliva of the adult male pig contains a mixture of man pheromone influencing brain activity (20, 331, 332), physrev.physiology.org steroids (boar odor). When a boar becomes aggressive or endocrine levels (406), physiological arousal, context-de- sexually aroused, it produces copious amount of saliva pendent mood, and sexual orientation (132, 133, 212, 232, whose pungent odor is attractive to estrus females (287) 300). Woman urine contains the estrogen like steroid and facilitates the display of the estrous female’s mating estra-1,3,5(10),16-tetraen-3-ol (EST) that also appears to posture (352). The major component of the boar odor is be a good candidate compound for human pheromones androstenone, a sexually dimorphic steroid that elicits (20, 132, 331, 332). both behavioral responses (65). Curiously, androstenone In studies of brain activity by positron emission to- has been hypothesized to be a human pheromone as well on February 9, 2011 mography, as above reported, smelling AND and EST (18, 96). activates regions primarily incorporating the sexually dimorphic nuclei of the anterior hypothalamus (332), and 5. Primata this activation is differentiated with respect to sex and Primates scarcely rely on chemical communication; compound. Very interestingly, homosexual males process however, the ring-tailed lemur (Lemur catta) exhibits the AND similarly to heterosexual women rather than to het- most highly developed olfactory system among all species erosexual men (20). Conversely, lesbian women process belonging to this order (75, 83, 338). Lemur catta pos- AND stimuli as common odorants and, when smelling sesses a suite of specialized scent glands and a variety of EST, they partly share activation of the anterior hypothal- scent-marking displays (147, 258, 338). Both sexes have amus with heterosexual men (20). These observations apocrine, located on the wrists, and sebaceous gland indicate the involvement of the anterior hypothalamus in fields in their genital regions and adopt distinctive hand- physiological processes related to sexual orientation/pref- stand postures to deposit glandular secretions on sub- erence in humans. strates. Males mix the glandular secretions and then deposit this mixture via “wrist marking.” They also impreg- 6. Rodentia nate their tail fur with their own secretions and then waft their tail at opponents during characteristic “stink fights” The paradigmatic model for mammalian pheromone (147). investigations involves laboratory species, among which There is nowadays a general consensus for the exis- rodents and especially mice are the most employed, be- tence of a chemical communication in humans, although cause they are easy to handle, have extremely well-devel- there is still a great controversy about the existence, oped olfactory systems, and offer an almost unlimited extent, and nature of human pheromones. Among the best possibility to act on behavioral responses by genetic ma- and first known effects attributed to human pheromones nipulation. In these species, several effects have been is the synchronization of menstrual cycle (237, 238, 367) carefully described, and the mechanisms of their action and the dimorphic activation of brain areas (332). Expo- have even been dissected at a molecular level.

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The vaginal secretion and saliva of female golden signal alert to the presence of a potential danger, promot- hamsters (Mesocricetus auratus) contain an aphrodisiac ing dispersion and the adoption of defensive actions in the substance that facilitates the mating behavior of the male group (400). Usually, mice escape from places in which (223, 356). This pheromonal effect has been clearly dem- the odors from the urine of a physically stressed conspe- onstrated in assays that measure the mounting behavior cific are present (418). Among other ketones, 2-heptanone of a male in the presence of an anesthetized male hamster is detectable in the mouse and rat urine (97, 342). In rat, (defined as surrogate female) scented with the stimuli 2-heptanone was found to be increased in urine of (222). Evidence shows that biologically active compounds stressed subjects and to induce immobility in a recipient were present in the proteinaceous fraction, whereas aque- conspecific forced to swim (97). ous fractions, organic extracts, and distillates containing Several other urinary compounds have been indi- volatile compounds did not exert detectable effects. After cated as activators of distinct anatomical regions of the purification, pheromonal activity was attributed to a 17- pheromonal pathway in rats (176, 321, 372); however, kDa soluble glycosylated protein appropriately termed none of the effects exerted by these substances is report- aphrodisin (356). The complete primary structure of aph- edly linked to obvious behavioral responses. rodisin shows that this protein belongs to the lipocalin Mouse ecology is largely triggered by pheromonal superfamily. A prominent feature of almost all lipocalins signals, which have been investigated for a long time, and is the presence of a hydrophobic pocket that accommo- therefore, mice represent the world-wide animal model to Downloaded from dates endogenous molecules with a relatively high li- study intraspecies communication. In the last decade, an pophilic coefficient. The biochemically purified aphrodi- enormous burden of data has been collected and, how it sin carries natural molecules that are contained in the is often the case, there is yet an open and sometimes vaginal secretions (33, 34) and that may be responsible for contrasting debate on the specific properties of the dif- the pheromonal effect observed in males (136). Biochem- ferent signaling molecules. ical and immunohistochemical data suggest that aphrodi- Early studies have demonstrated a number of incon- physrev.physiology.org sin may act through G protein-coupled receptors that are trovertible effects of primer pheromones in mice. It is expressed in the vomeronasal system (136, 177). known that grouped female mice modify or suppress their Fossorial mammals, like the mole-rat, are noted for estrous cycle (Lee-Boot effect) (387), while male urine sensory specializations, such as exquisite tactile senses can restore and synchronize the estrus cycle of noncyc- (45, 60). Although suppression of mole-rat reproduction ling females (Whitten effect) (401) or accelerate puberty in females is thought to be meditated by behavioral, onset in females (Vandenbergh effect) (388). In addition, rather than pheromonal, cues from the breeding females the exposure of a recently mated female mouse to a male, (360), the chemosensory systems retain demonstrable different from the stud, prevents implantation of fertilized on February 9, 2011 functions of scent marking in common nesting and toilet eggs (Bruce effect) (38), implying that the stud or its loci (359) that have been associated with pheromonal individual odor must be memorized at the moment of communication. mating to be recognized later. These pheromonal effects In rats, several pheromones are used in different are commonly believed to be mediated by stimuli present contexts as territory marking (309), alarm (1), and mater- in urine that act via the vomeronasal system. nal behaviors (188, 254). The chemically characterized rat Signaling pheromones also account for many behav- pheromones include compounds that are produced by ioral responses in the mouse. For example, male as well pup’s preputial glands and urine. A particular pattern of as lactating female aggressiveness towards an intruder rat maternal behavior is the specific licking and cleaning male are both believed to be triggered by molecules that of pup’s anogenital area, an action crucial to pup survival, are present in male urine. as nonlicked pups cannot defecate and run into death. Thus dodecyl propionate, a compound isolated from pups’ preputial glands, can sustain pups’ anogenital lick- B. Major Urinary Proteins and Major ing by dams and therefore pups’ survival (36). Histocompatibility Complex Molecules The odors emanating from physically stressed rodents are recognized by conspecifics (220, 386). Once the Male mouse urine contains an unusually high quan- chemical signal is perceived, the receiver undergoes sig- tity of proteins, called major urinary proteins (MUPs). nificant changes. For example, lymphocyte T-killers are MUPs form a large family of highly homologous androgen- suppressed, whereas B-cells proliferate (57), rats develop dependent proteins that are synthesized in the liver and hyperthermia (160), c-Fos protein expression is increased excreted with urine (46, 208). MUPs and the rat hortologs ␣ in the mitral cell layer of the accessory olfactory bulb 2 urinary globulins belong to the lipocalin superfamily, (164), reproductive behavior and sexual maturation initi- and accordingly, they bind and release small (volatile) ate (389a), and social hierarchy may be modified (140). pheromones that are also produced in an androgen-de- These bodily compounds, defined as alarm pheromones, pendent fashion (9, 23). Although MUPs are also ex-

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 926 TIRINDELLI ET AL. pressed in exocrine glands as mammary, parotid, sublin- Thom et al. (375) indicated that female mice use gual, submaxillary, lachrymal, nasal, and in modified se- MUPs as a specific genetic marker to identify and prefer- baceous glands like preputial and perianal glands (344), entially associate with heterozygous males. The primary their biological effects have been exclusively demon- function of MUPs is in signaling social information strated when these proteins were purified from urine or through scent, including individual genetic identity. Mice added to urine that does not possess pheromonal activity are highly sensitive to differences in the fixed patterns of (i.e., of a prepubertal or castrated mouse) (123, 266). multiple MUP isoforms expressed by each individual and MUPs have been reported to act either as primer or also avoid inbreeding with close relatives that share the releaser pheromones; however, some of their effects ap- same MUP type as themselves. Heterozygosis assessment, pear contradictory. This is in part due to the tight binding in contrast, most likely involves recognition of the greater of MUPs with volatile pheromones that have been proven diversity of MUP isoforms expressed by heterozygous to be directly implicated in estrus synchronization (138), animals. attractiveness to females (139), intermale aggression Other biological effects of MUPs include attractive- (280), induction of estrus (␣- and ␤-farnesene) (219), pu- ness to females and repulsion to males (265), modification berty acceleration in females (283), and territory marking of light-avoidance behavior in both males and females (21, 122). The advantage of such a pheromone-protein (263, 264), the aggressive reactions of males towards complex may include concentration and slow release of adult and newborn mice (264, 267), and intermale aggres- Downloaded from chemosignals, stabilization of labile pheromones, or de- siveness (47). Mate choice and parent-offspring interac- livery to the reception sites. Common chromatographic tions have been demonstrated to be influenced by MHC procedures for purification of MUPs are ineffectual to genotype B (411). For example, house mice avoid mating remove these pheromones, and solvent extraction of vola- with individuals that are genetically similar at the MHC tiles appears the only way to obtain the native apopro- locus. Mice are able to recognize MHC-similar individuals through specific odorant cues. Moreover, females avoid physrev.physiology.org teins. Therefore, in literature, the term of MUPs com- mating with males carrying MHC genes of their foster monly defines the protein with the bound pheromones. family, supporting a familial imprinting hypothesis (288). While a general consensus exists about the source, Mate preference for MHC-dissimilar individuals can be male urine, that releases the pheromonal stimuli respon- adaptive as it would increase offspring MHC heterozygos- sible for the puberty acceleration (Vandenbergh effect), a ity, with beneficial influences on offspring viability through debate is currently open about the molecules that deter- increased resistance to infectious disease or avoidance of mine this behavioral response. Evidence suggests that inbreeding effects (180, 297).

MUPs and not the bound pheromones are effective in on February 9, 2011 The polymorphism of MHC molecules is reflected accelerating the puberty onset in females. These results into structurally diverse binding sites, such that different are supported by the biological activity shown by MUPs MHC binds to distinct peptides. Like other membrane- when stripped of its pheromones and by a short synthetic bound proteins, these MHC molecules are anchored in the hexapeptide homologous to the NH2-terminal sequence of lipid bilayer. However, it has been demonstrated that the many MUPs variants (266). Conclusions from a different peptide-binding pocket releases its peptide in the extra- laboratory, performing similar but not identical experi- cellular fluid when the MHC is cleaved from the cell. Thus ments, seem, however, to argue against this hypothesis peptides or their fragments are constitutively excreted in (283). urine and other bodily secretions (358) that in turn can be It has been also demonstrated that the combinatorial used for interindividual communication (357). diversity of urinary MUPs among wild mice (a male can produce up to 12 variants from the polymorphic MUP family, that includes a total of 21 functional isoforms) is C. Exocrine Gland-Secreting Peptides virtually as great as for molecules of the major histocompatibility complex (MHC) (see below) (48, 123). However, Mice secrete a family of peptides of different length although MHC has been long regarded as the primary called exocrine gland-secreting peptides (ESPs) from ex- determinant for individual recognition via bodily odors, traorbital, Harderian and submaxillary glands into tear, this evidence has now been challenged by recent studies nasal mucus, or saliva. The ESP family consists of 38 in which it appears that recognition, at least in the wild, is members in mice and 10 in rats, while it is absent from the instead strictly and exclusively dependent on the MUPs human genome. variant profile contained in each individual urine (49). Very Moreover, at least two of these peptides, produced by intriguing is the observation that recognition requires the phys- the extraorbital lacrimar gland, are gender specific: ESP1 ical contact with the donor urine, thus discarding the is exclusively secreted by males (162), while ESP36 is hypothesis of this effect being exclusively attributed to female specific (163). Despite the fact that these peptides the volatile pheromones released from MUPs (49). are recognized by systems dedicated to pheromone per-

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 927 ception, until now there were no clues regarding their cased in a protective bony capsule located at the base of physiological function. the nasal septum (68, 134) (Fig. 2). Given its recessed nature, the vomeronasal epithelium cannot be reached by III. CHEMOSENSORY SYSTEMS the airstream that regularly flows through the nasal cav- DETECTING PHEROMONES ity. Thus, to target the vomeronasal epithelium, molecules dissolved in the nasal mucus must be sucked into the It has been believed for a long time that two chemo- lumen of the organ. Lateral to the lumen are blood vessels sensory systems, the main olfactory and the vomeronasal and sinuses, innervated by the autonomic nervous sys- system, were responsible for different functions. The tem, that induces vasodilation and vasoconstriction, main olfactory system was considered to be responsible thereby producing a pumplike action for stimulus access for recognizing the conventional volatile odorant mole- to the lumen (72, 109, 244, 248, 249, 328). In ungulates, cules, whereas the vomeronasal system was thought to be as described in section IIA4, the “flehmen” behavior is tuned for sensing pheromones. Recent studies have dem- thought to be associated with promoting stimulus access onstrated that both chemosensory systems, together with to the vomeronasal organ (268). additional olfactory organs, are involved in pheromone Norepinephrine accumulation has been measured in detection. In these systems, peripheral chemosensory the vomeronasal organ of mice after exposure to phero- Downloaded from neurons located in the nasal cavity express distinct fam- monal cues (419). It is possible that this hormonal release ilies of receptors that are believed to bind pheromones alters the vascular tone, or changes the glandular secre- and trigger a cascade of molecular and electrical events tions, thus increasing the receptivity of the sensory epi- that, ultimately, influence some aspects of the social be- thelium in particular behavioral contexts. The functioning havior of the individual. of the vomeronasal organ is thus not simply a passive Although the main olfactory and vomeronasal sys- event, but it can be actively modulated. tems share a similar histological organization, they also The medial, concave side of the vomeronasal lumen physrev.physiology.org display relevant differences with regard to the receptor is lined by a pseudostratified epithelium composed of repertoire that they express and to the connections to three types of cells: sensory neurons, supporting cells, specific central areas (Fig. 1). Each system possesses and basal cells. The basal stem cells are located both primary sensory neurons that send axons to second-order along the basal membrane of the sensory epithelium and neurons (mitral cells) in specific regions of the main at the boundary with the nonsensory epithelium (85). olfactory bulb (main olfactory system) or of the accessory Supporting cells are found in the uppermost superficial

olfactory bulb (vomeronasal system). The mitral cells of layer of the sensory epithelium, while vomeronasal neu- on February 9, 2011 the main olfactory bulb project to several higher centers rons form two overlapping populations, basal and apical, including the piriform cortex and the cortical amygdala. that are further described in detail in sections IV and V. Instead, projections from the accessory olfactory bulb In the mouse, the vomeronasal sensory epithelium only reach the medial amygdala and the posterior-medial increases from birth to puberty and becomes fully devel- part of the cortical amygdala. From here ﬁbers terminate oped and completely active at 2 mo after birth (117). The in the hypothalamus either directly or via the bed nucleus sensory neurons, on the mucosal side, bear dendrites and of the stria terminalis. apical microvilli, whereas, on the opposite side, their In the following sections we summarize how the axons cross the basal membrane and merge together, various sensory epithelia are functionally organized. For forming vomeronasal nerves that run between the paired more exhaustive anatomical description, readers may olfactory bulbs and enter the accessory olfactory bulb at consult reviews speciﬁcally dedicated to these subjects the posterior dorsal side of the main olfactory bulb (see (66, 229). Figs. 2 and 3). The vomeronasal sensory neurons take origin from A. Vomeronasal Epithelium the olfactory placode together with gonadotropin-releasing hormone (GnRH, also known as LH-RH or luteinizing The vomeronasal epithelium is part of the vomero- hormone-releasing hormone) neurons and ␥-aminobu- nasal organ (VNO) of Jacobson, a tubular structure en- tyric acid (GABA)-containing neurons (405). The GnRH

FIG. 1. A schematic diagram showing the anatomical pathways of the rodent vomeronasal and main olfactory systems.

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FIG. 2. Chemosensory systems in rodents. A simplified sagittal section of a rodent head showing the location of sensory epithelia: the main olfactory epithelium (MOE), the vomeronasal organ (VNO), the septal organ of Masera (SO), the Grueneberg ganglion (GG), and the location of the main olfactory bulb (MOB) with necklace glomeruli (NG) and of the accessory olfactory bulb (AOB). Insets show images obtained from the intrinsic green fluorescent protein (GFP) fluorescence from olfactory marker protein (OMP)-GFP gene-targeted mice. Top left: the Grueneberg ganglion has an 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 and Strotmann (191).] Bottom left: mature vomeronasal sensory neurons in a coronal section of the vomeronasal organ. Bottom right: mature olfactory sensory neurons in a coronal section of the main olfactory epithelium. neurons traverse the developing accessory olfactory bulb reports showed that in human adults it becomes a blind- and migrate to the mediobasal hypothalamus. The vome- ended diverticulum in the septal mucosa (246, 383). More- ronasal sensory neurons maintain a functional relation- over, the epithelium lining the vomeronasal organ in hu- ship with the hypothalamic areas in the adult mammal, man adults is different from that of other species and influencing neuroendocrine function and behavior. from the olfactory or respiratory epithelium (259, 366). Do humans have a vomeronasal organ? The presence Several studies showed that vomeronasal cells were not of a vomeronasal organ in human embryos, containing stained by an antibody against the olfactory marker pro- bipolar neurons similar to developing vomeronasal neu- tein (259, 366, 383, 403), a protein abundantly expressed rons of other species, was clearly demonstrated (24, 25, in mature neurons in the olfactory and vomeronasal epi- 166, 240). However, the vomeronasal structure becomes thelia of other species (41, 144). In addition, Trotier et al. more simplified later in development (24), and several (383) did not identify vomeronasal nerve bundles using a

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 929 specific antibody against the S-100 protein, a characteris- C. Other Olfactory Organs tic axonal marker (7). Therefore, even if vomeronasal cells were chemosensitive, they would not have any ob- 1. Septal organ of Masera vious way to communicate with the brain. Therefore, the The septal organ of Masera (SO) (35, 311) is a small development of human vomeronasal structures seems to island of olfactory epithelium within the nasal mucosa be limited to the embryonic stage, when they possibly and is located on each side of the septum, posterior to the play a role in the migration of GnRH-secreting neurons nasopalatine duct that connects the oral to the nasal toward the brain (383; for review, see Ref. 246). cavity (Fig. 2). The anatomical organization of the septal organ appears similar to that of the main olfactory epi- B. Olfactory Epithelium thelium, although its neuroepithelium appears thinner (218, 396). Furthermore, sensory neurons of the septal organ are provided with cilia that bear shorter dendrites New evidence suggests that some pheromones are and larger dendritic knobs than those of the main olfac- detected by the main olfactory epithelium, a structure tory epithelium (218); their axons project to a subset of specifically dedicated to sensing volatile molecules, usu- glomeruli in the posterior, ventromedial main olfactory ally named general odorants. The main olfactory epithe- bulb (Fig. 3). Interestingly, some of these glomeruli ex- lium lines cartilaginous protuberances in the posterior Downloaded from clusively receive input from the septal organ sensory nasal cavity (Fig. 2), called turbinates, and is a pseudostrati- neurons, while others are also innervated by fibers origi- fied epithelium composed, similarly to the vomeronasal nating from the main olfactory epithelium (191). The an- epithelium, of three main types of cells: olfactory sensory terior position of the septal organ, its vicinity to the neurons, supporting cells, and basal cells. The olfactory nasopalatine duct, and the wide distribution in many sensory neurons are bipolar neurons with a single ciliated mammalian species suggest that this organ may be func- dendrite extending to the mucosal surface. Cilia are the physrev.physiology.org tionally specialized for the early detection of biologically site of primary transduction for odorants and, possibly, relevant molecules as those, for example, resulting from for pheromones. Although most olfactory sensory neu- licking behavior. rons have the same type of transduction cascade, it has been recently discovered that subsets of olfactory neu- 2. Grueneberg ganglion rons express different types of transduction molecules (see sect. VB2) (reviewed in Refs. 31, 217, 269). Axons of The Grueneberg ganglion is located bilaterally in the sensory neurons are segregated in the epithelium and, anterodorsal area of the nasal cavity in the corners after crossing the basal membrane, bundle together to formed by the septum and the nasal roof of several mam- on February 9, 2011 form the fascicles of the olfactory nerves. Axons project malian species (93a) (Fig. 2). Differently from other che- to distinct domains of the main olfactory bulb as further mosensory organs, neurons of the Grueneberg ganglion described in section V. cluster in small groups (30, 79, 80, 317). Only two types of In the mouse, the olfactory system is functionally established during the prenatal phase as the first axons contact the telencephalic vesicle around embryonic day 13 (59, 320). Conversely, axonal targeting to specific domains within the main olfactory bulb occurs as early as embryonic day 15.5 (59). In the mouse, both vomeronasal and olfactory sensory neurons undergo a continuous renewal process throughout adult life, and their rate of neurogenesis is also regulated by environmental factors (11, 59, 90, 91, 402). Readers interested in the neurogenesis of olfactory and vomeronasal sensory neurons may consult some recent 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 boundary FIG. 3. Projections of sensory neurons to the olfactory bulbs. Each olfactory sensory neuron sends a single axon to the main olfactory bulb between olfactory and respiratory epithelium is not as (MOB). The apical and basal sensory neurons in the vomeronasal organ sharp as in other mammals; instead, there is an irregular (VNO) send axons, respectively, to the anterior and posterior accessory zone where olfactory and respiratory cells intermingle. olfactory bulb (AOB), a posterior dorsal region of the main olfactory bulb. Neurons of the septal organ of Masera (SO) project to the posterior Moreover, patches of respiratory epithelium have been ventromedial main olfactory bulb. Neurons of the Grueneberg ganglion identified in purely olfactory areas (262). (GG) project to necklace glomeruli (NG) in the main olfactory bulb.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 930 TIRINDELLI ET AL. cells have been identified in the Grueneberg ganglion: ily compared with other species (313, 346, 415, 422). The glial cells and ciliated neurons (30). Each neuron has a gene tree of functional V1Rs in placental mammals, based single axon that fasciculates with others into nerve bun- on the mouse repertoire, is split into 12 clades (a-l) (Fig. 4) dles that project to ϳ10 glomeruli in the main olfactory (313). All rat V1R receptor genes, except for one, are bulb (Fig. 3). These glomeruli are a subpopulation of the distributed within 10 of these 12 clades (a-g and j-l). Thus necklace glomeruli. Since necklace glomeruli are active the mouse h and i clades represent an instructive example during the suckling behavior of the pup, it was suggested of species specificity, exclusively including mouse se- that the Grueneberg ganglion may sense maternal phero- quences. Given that one rat pseudogene is placed in each mones (82, 173, 317). However, a very recent study has of the h and i subfamilies, it is likely that the absence of rejected this hypothesis, suggesting instead that alarm rat functional genes in these clades is the result of dele- pheromones, volatile molecules released by animals to tion in this species and of postspeciation expansion in the alert conspecifics about danger, are the likely substrates mouse lineage. of the Grueneberg ganglion neurons. These results are V1Rs in the mouse account for almost 200 potentially discussed in more detail in section VB. functional receptors and are highly expressed in vomeronasal neurons, at least at mRNA level. The mouse has a larger V1R repertoire than the rat (Fig. 5).

IV. PUTATIVE PHEROMONE RECEPTORS In the mouse V1R phylogenetic tree, clades share Downloaded from from 15 to 40% of their amino acid residues between each A. Vomeronasal Receptors other, with intrafamily identities varying between 40 and 99%. High sequence variability is found in transmembrane In the vomeronasal organ of rodents, detection of domain 2, in intracellular loop 1, and in extracellular pheromones is mediated by two anatomically indepen- loops 2 and 3, while transmembrane domain 3 is highly dent pathways that originate from chemosensory neurons conserved. A few amino acid residues mostly located physrev.physiology.org of distinct compartments, basal and apical, of the neuro- between transmembrane domains 5 and 6, and a potential epithelium. The organization of the vomeronasal epithe- glycosylation in extracellular loop 2, are common features lium of rodents in apical and basal neurons was initially to virtually all members of the 12 V1R clades, while spe- based on the specific expression pattern of marker mol- cific amino acid signatures are characteristic of each of ecules (261, 340, 341). More recently, exhaustive data the 12 families (313). established that also distinct transduction components characterize these two subsets of chemosensory neurons. ␣ on February 9, 2011 The G protein subunit G i2 was found to enrich the apical ␣ neurons, whereas G o was identified in the basal ones (19, 143). These important findings are credited to have addressed the efforts towards the search of receptors that could specifically couple to G proteins. Thus two large families of vomeronasal G protein-coupled receptors, named V1R and V2R, have been discovered, and their expression pattern was extensively analyzed. Moreover, other putative receptors as the major histocompatibility complex class Ib molecules (H2-Mv) have been identified in the vomeronasal organ, and their expression was demonstrated to be tightly linked to that of V2Rs.

1. V1Rs The first family of vomeronasal receptors, V1Rs, was discovered with elegant experiments of differential screening, based on the assumption that the genetic pro- ␣ file of two vomeronasal neurons expressing G i2 only differs for the receptor RNA that they express (71, 285). It was also evident that V1R expression is restricted to the apical neurons of the vomeronasal organ which express FIG. 4. The mouse vomeronasal receptor families. Top: phyloge- G␣ (71, 285). From the structural point of view, V1Rs are netic tree of V1Rs showing 12 phylogenetically isolated subfamilies. i2 Bottom: phylogenetic tree of the 4 V2R families showing their clades. related to group I of G protein-coupled receptors and, in Numbers of receptors for each clade and family are shown in red. the mouse, represent a strikingly expanded receptor fam- [Modified from Silvotti et al. (355).]

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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 teleost fishes (green spotted pufferfish, fugu, and zebrafish). The numbers of intact genes for V1R, V2R, OR, and TAAR receptors are given together with the numbers of nonintact genes shown in parentheses. Numbers of receptor genes in each species were taken from Grus et al. (94), except for chimpanzee which were obtained from Nei et al. 2008; V1Rs and V2Rs in green spotted pufferfish, fugu, and zebrafish from Shi and Zhang (346); ORs in fugu from Niimura and Nei (274); and TAARs in fugu and zebrafish from Hashiguchi and Nishida (105).

V1R genes are scattered in regions of many chromo- presence of V1Rs (Fig. 5). Interestingly, V1R genes were somes, where members of a given family are found almost found in all species except in chicken and chimpanzee, in invariably clustered. It has been recently reported that which the presence of a functional vomeronasal organ has V1R clusterization may not simply reﬂect gene expansion not been consistently demonstrated. The number of func- but may be actively linked to the mechanisms underlying tional V1R genes drops dramatically from rodents to other the tight regulation of receptors’ expression. V1R expres- species. The human genome contains several V1R pseu- sion is, indeed, monoallelic and monogenic, both of these dogenes, only ﬁve human V1R genes that have an intact being tools that achieve the expression of one receptor open reading frame, and at least one V1R that is tran- type per cell, thus contributing to the narrow tuning of the scribed in the olfactory epithelium. The chimpanzee ge- vomeronasal neurons (318). nome has no obvious functional V1R genes but a large At present, some vertebrate genomes of species of collection of pseudogenes. This is surprising if we con- different orders have been extensively scrutinized for the sider that the massive loss of V1R genes in chimpanzee

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 932 TIRINDELLI ET AL. appears even greater than for the human repertoire, in human pseudogenes), suggesting a relatively recent loss which V1R genes have been identified. However, the most of function. surprising observation is that the dog only possesses eight In rodents, this receptor superfamily is largely ex- functional V1R genes spread among four clades. This panded and, in contrast to V1Rs, it appears completely finding is unexpected because the dog is traditionally degenerated in primates, dogs, and cows (Fig. 5). In the renowned to have evolved an extraordinary well-devel- dog, the lack of a sizable V1R and V2R family does not oped olfactory system and a complex social behavior probably reflect a reduction in the pheromonal relevance (346). Although it is plausible that V1Rs emerged as dom- in triggering intraspecific behavior, but rather a general inant receptors for triggering specific rodent behaviors decline of the vomeronasal system, a hypothesis that is (multiple losses of ancestral V1R genes in carnivores and supported by anatomical data (63). In contrast, in the artiodactyls and gains of many new genes by gene dupli- opossum, V2Rs are abundantly represented (Fig. 5). Thus cation in rodents), it cannot be alternatively ruled out that it appears that the evolution of opossum and rodent V2Rs a loss of dog V1Rs is due to the breeding and domestica- genes parallel that of the V1R ones in these species, which tion processes of this species. Therefore, diversification expanded independently. Moreover, most opossum and into subfamilies probably occurred prior to the rodent- rodent V2R and V1R genes arose by duplication (rodent dog-primate split early in the mammalian evolution (415). V2R and V1R genes are found in genomic clusters, and The question as to whether V1Rs are exclusively closely related V2Rs tend to reside near one another in Downloaded from expressed in the vomeronasal epithelium or also in other the genome) after these lineages diverged. This receptor tissues is still controversial. Karunadasa et al. (152) re- diversification and the consequent detection of additional ported the expression of V1Rd RNA in some cells scat- pheromones may have resulted in a selective advantage. tered within the main olfactory epithelium of embryonic The expression pattern of V2Rs makes them different from V1Rs and odorant receptors in that each basal neu- and postnatal mouse and suggested that pheromone de- ron expresses two V2Rs, one of which is a member of physrev.physiology.org tection may also take place in this structure. V1R-ho- family C (231). Moreover, there is evidence for a combi- molog RNAs have also been identified in the main olfac- natorial scheme for the detection of chemosensory cues tory epithelium of goats and humans (316, 394). However, in the basal neurons of the vomeronasal organ. Recent caution must be taken before raising any hypothesis as, data indicate the existence of multimolecular complexes until now, there are no clues that a functional V1R is in individual basal neurons, constituted by MHC mole- expressed at a detectable level in the main olfactory cules, by family A, B, or D and family C V2Rs (355). For epithelium. example, neurons expressing family A receptors of the highly represented clades I, III, and IV almost exclusively on February 9, 2011 2. V2Rs express the family C receptor Vmn2r2, whereas neurons expressing clade VIII receptors specifically express the A second family of vomeronasal receptors, V2Rs, was family C receptor Vmn2r1. Interestingly, coexpression of discovered in 1997 (110, 234, 323) and, in mouse, it ac- Vmn2r1 and Vmn2r2 with family A V2Rs seems to involve ϳ counts for 120 potentially functional members. V2Rs whole clades rather than individual receptors. Thus a belong to group III G protein-coupled receptors and are peculiar modality of expression subsists in the basal layer characterized by a long NH2-terminal domain. This region of the vomeronasal organ, where two family receptors are has been demonstrated to represent the binding domain mutually exclusively expressed in the same basal neu- in most receptors of group III (for review, see Ref. 293). rons. V2Rs are strictly coexpressed with the G protein subunit It has been suggested that V2Rs bind MHC peptides ␣ G o in the basal neurons of the vomeronasal organ. (185) and MUPs (47), as further discussed in section VA. In the mouse, intact V2Rs can be grouped in four A V2R receptor, V2R83, is also expressed outside the distinct families (A-D) according to sequence homology vomeronasal organ, in a large subset of neurons of the (Fig. 4). Together, receptors of families A, B, and D rep- Grueneberg ganglion (79), and it is a good candidate resent 95% of all V2Rs. Their expression pattern strongly receptor for alarm pheromones (30). suggests that these receptors are expressed by mutual At present, however, direct evidence that V2Rs are exclusion in the vomeronasal organ. A phylogenetic anal- directly involved in pheromone signaling is still missing. ysis shows that family C V2Rs (also known as V2R2 At least 50% of the basal vomeronasal neurons ex- family) are very divergent from those of families A, B, and press another multigene family representing nonclassical D (3, 231, 323), and these differences are also functional class I major histocompatibility complex (H2-Mv) genes. (354). This family is also noteworthy for the high se- Each of the nine identified H2-Mv genes is present in a quence conservation between species. Moreover, com- subset of vomeronasal neurons, and multiple H2-Mv pared with other V2R pseudogenes, family C pseudogenes genes can be coexpressed in the same neuron (127, 207). have one or very few inactivating mutations (also in the H2-Mv molecules are also coexpressed in a combinatorial

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 933 manner with receptors of speciﬁc subfamily of V2Rs (127, To deorphanize V1Rs and study their binding prop- 207). It has been initially proposed that H2-Mv may func- erties, it is necessary to develop an efﬁcient bioassay. At tion as indispensable escort molecules in the transport of present, it is only plausible to assume that V1Rs are V2Rs to the plasma membrane (207). However, this chap- vomeronasal receptors narrowly tuned to bind small vol- erone-like function is not generalized to all V2Rs (127, atile pheromones, possibly through a hydrophobic pocket 354), and moreover, it is likely to be exclusively restricted located in their transmembrane region. Nevertheless, to the mouse or rat as the opossum genome contains a achievements in this direction have been very recently hundred functional V2R genes and no H2-Mv genes (346). accomplished by Shirokova and colleagues who managed

to deorphanize the human V1Rs employing NH2-termi- 3. Ligands of vomeronasal receptors nally tagged receptors expressed in the cell line HeLa/Olf (350). It appears that, in sharp contrast to V1Rs, each Although an initial report provided clues on the pos- human recombinant V1R receptor responds to several sibility that V1Rs could be expressed in a heterologous aliphatic alcohols or aldehydes via the olfactory G protein ␣ system (99), the problem of expression later became the G olf (350). Unfortunately, a pheromonal function of hu- major barrier to the identification of ligands for vomero- man V1R ligands has yet to be demonstrated. nasal receptors, as well as for odorant receptors (for reviews, see Refs. 224, 256, 312). Some information about 4. Formyl peptide receptors Downloaded from ligands for V1Rs was obtained from physiological ap- Recently, a group of receptors belonging to the fam- proaches to the problem. Results from calcium imaging ily of formyl peptide receptors (FPRs) has been exclu- experiments on slices of the mouse vomeronasal organ sively identified in the vomeronasal organ (310a). FPRs indicated that some molecules credited as pheromones are G protein-coupled receptors expressed in all mam- stimulated specific vomeronasal neurons that, for their mals, with three genes present in the human genome and topographic position in the vomeronasal organ, were physrev.physiology.org seven in the mouse genome (249a). At least two of the likely to express the V1R repertoire (187). Furthermore, mouse FPRs have been reportedly involved in immune these authors reasoned that, if neurons responding to and inflammation responses being expressed in granulo- these pheromones indeed reflected a V1R-dependent ac- cytes, monocytes, and macrophages (183a). An in situ tivity, V1Rs must be narrowly tuned to bind one or very analysis of the vomeronasal organ of the mouse indicates few pheromones. This contrasts with the broad selectivity that five FPRs are expressed in a small subset of chemo- of olfactory sensory neurons that can be stimulated by a sensory neurons of the apical layer. Antibody staining large array of odorants. Further and more direct investi-

reveals that FPRs are featured by a subcellular distribu- on February 9, 2011 gations on the V1R ligand specificity lead to the conclu- tion similar to that of V2R receptors including, besides the sion that V1Rs are indeed qualified as pheromone recep- microvillar surface, the dendrite and somata. Although tors: Del Punta et al. (61) succeeded to delete a large Fpr expression is restricted to the apical layer and, as genomic region containing ϳ16 V1R genes of subfamily a well as VIRs, adopts monogenic transcription, no coex- and b, including the receptor V1rb2. The behavior of these pression is evident with receptors of this large family, mutant animals was also thoroughly investigated showing suggesting that FPRs characterize a new and specific remarkable deficits (see sect. VII). Moreover, electrophys- class of chemosensory neurons. Experiments based on iological responses of vomeronasal neurons to some calcium imaging on the whole vomeronasal epithelium pheromones, including 6-hydroxyl-6-methyl-3-heptanone, and in dissociated chemosensory neurons establish that n-pentylacetate, isobutylamine, and 2-heptanone, were al- indeed FPR agonists, such as some microbial, antimicro- tered (Table 1). Thus the repertoire of V1R receptor can- bial, and viral peptides, stimulate neuronal subsets with a didates to respond to these pheromones was restricted to nonidentical affinity profile. These findings indirectly a relatively limited number. prove that these receptors play a role in offering a barrier Another approach, developed by Boschat et al. (28), against immunological diseases, or perhaps also in phero- led to the identification of the first pheromone-V1R pair in monal communication itself, given that formyl peptides mammals. These authors employed mouse mutant lines have been detected in urine (52a) and that, in the olfac- with a GFP-targeted V1rb2 allele, allowing the visualiza- tory and vomeronasal epithelium, two FPR isotypes ap- tion and the isolation of the V1Rb2-expressing neurons for pear to be expressed in the glandular but not chemosen- calcium imaging based experiments. Thus a direct corre- sory compartment (310a). lation was established between V1Rb2 expression and the response of vomeronasal neurons to the mouse pheromone 2-heptanone (Table 1). As a confirmation of the B. Odorant Receptors strong selectivity of V1Rs above reported, 2-heptanone- related compounds were found to be ineffectual to stim- Odorant receptor genes were discovered in 1991 by ulate V1Rb2-expressing sensory neurons. Buck and Axel (39). The vast majority of the olfactory

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 934 TIRINDELLI ET AL. neurons express only 1 of the 1,000 odorant receptor same neurons, suggesting that subsets of neurons exist in genes (51, 226, 273, 308, 370, 389, 389). Neurons express- the olfactory epithelium that are not exclusively dedi- ing different receptor types are usually found in one of cated to the detection of conventional odorants. By the four partially overlapping zones of the olfactory epithe- expression of olfactory TAARs in heterologous cell sys- lium, within each of which a given odorant receptor ap- tems it was demonstrated that at least four of them pears to be randomly distributed (128, 251, 308, 369, 389). (mTAAR3, mTAAR4, mTAAR5, mTAAR7f) bind small- An olfactory sensory neuron typically responds to molecule amines and that each of these receptors recog- more than one type of odorant molecules, and a given nizes a specific set of ligands, among which are ␤-phenyl- odorant can activate neurons with different specificity ethylamine, isoamylamine, and trimethylamine that are (70, 77, 226, 351). For the discrimination and perception natural components of the mouse urine. ␤-Phenylethyl- of odorants, the odorant receptor family seems to employ amine, a ligand for mTAAR4, is contained in human and a combinatorial scheme, with one single odorant mole- rodent urine, where its concentration rises during stress- cule activating several types of receptors and each recep- ful situations (93, 286, 361). Isoamylamine and trimethyl- tor sensing several different types of odorants, thereby amine bind to mTAAR3 and mTAAR5, respectively, and producing a unique combination of receptors activated by are present at higher concentrations in male compared each odorant molecule. This combinatorial scheme al- with female urine (84, 276). Noteworthy, isoamylamine lows the olfactory system to recognize an enormous num- has been reported to act as a pheromone accelerating Downloaded from ber of odorants (40, 256). puberty onset in female mice (276). Therefore, stimula- Odorant receptor genes have been identified in re- tion of mTAARs seems to affect the capacity of discrim- gions outside the main olfactory epithelium as the septal inating gender and social status of an individual. These organ and the vomeronasal epithelium. About 50 odorant findings strongly contribute to confirm that the main ol- receptor genes are expressed in the mouse septal organ factory epithelium is involved in the elicitation of physi-

(150, 378). Moreover, a subset of 44 odorant receptor ological responses and innate behaviors. physrev.physiology.org genes is also expressed in the vomeronasal epithelium In the mouse, TAARs are also expressed in subsets of (190), although their functional role has not been estab- neurons of the Grueneberg ganglion (80). As in the main lished. olfactory epithelium, also in the Grueneberg ganglion, At present, we do not possess evidence that odorant distinct TAAR subtypes are expressed in nonoverlapping receptors can also function as pheromone receptors. subsets of neurons that do not coexpress the vomeronasal However, intriguingly, recent experiments linked the ex- receptors V2Rs. Since the number of neurons expressing pression of a subclass of odorant receptors with stereo- TAARs and V2Rs in the Grueneberg ganglion is much typed behavioral expressions in the mouse, thus suggest- higher during the perinatal stage than in the adult, it was on February 9, 2011 ing that odorant receptors can convey information both suggested that these neurons may be important for inter- about the quality of an odorant and the individual that has actions between pups and their mother (80). emitted it (169). TAARs have also been identiﬁed in the genome of For additional information about odorant receptors, many species including humans and ﬁsh, with the number the reader is referred to several reviews that extensively of putatively functional TAAR genes greatly varying discuss this subject (86, 224, 225, 256). among species (105) (Fig. 5).

C. Trace Amine-Associated Receptors D. Olfactory-Specific Guanylyl Cyclase Type D Receptor In 2006, Liberles and Buck (194) reported the expression of a second class of chemosensory receptors in the A small subset of olfactory sensory neurons localized mouse olfactory epithelium. These receptors are the in the dorsal region of the main olfactory epithelium “trace amine-associated receptors” (TAARs) (27, 192, expresses a specific subtype of the transmembrane recep- 203), a family of G protein-coupled receptors discovered tor guanylyl cyclase, named guanylyl cyclase type D in 2001 (27, 42) (for review, see Ref. 425). TAARs are (GC-D) (81, 149). The GC-D receptor is encoded by one of unrelated to odorant receptors but have similarities with the seven guanylyl cyclase genes with whom it shares a receptors for biogenic amines, such as serotonin and similar topology: all receptor guanylyl cyclases retain an dopamine receptors. The TAAR gene family includes 15 NH2-terminal ligand binding region, a single transmem- members in the mouse (Fig. 5), and each of them, except brane loop, and a COOH terminus that includes the cata- for TAAR1, is expressed in the olfactory epithelium (194). lytic domain (81, 296). Specific ligands have been identi- Moreover, the level of expression of TAAR genes is sim- fied for some guanyly cyclases, but not for GC-D yet; ilar to that of odorant receptors, although TAARs and however, potential ligands for this receptor have been odorant receptors do not appear to be coexpressed in the recently assessed. In the mouse, for example, neurons

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 935 expressing GC-D respond to dilute urine and to the intes- mice, hamsters, opossum, and platypus that possess func- tinal peptide hormones uroguanylyn and guanylin, while tional V2R genes (Fig. 5). neurons in GC-D knockouts are unresponsive to these Krieger et al. (176) reported that whole male urine compounds (186). Given their localization in the enteric induces inositol 1,4,5-trisphosphate (IP3) production via ␣ ␣ tract, uroguanylyn and guanylin are supposed to contrib- the activation of G i2 as well as G o, suggesting a role for ute to the maintenance of salt and water homeostasis or both G protein subtypes in transducing pheromonal re- to the detection of cues related to hunger, satiety, or thirst sponses mediated by urinary components. Moreover, ac- ␣ ␣ (186). GC-D neurons also express carbonic anhydrase II cording to these authors, the activation of G i2 and G o and respond to CO2 stimuli (118). It is not clear if there is subtypes in the vomeronasal epithelium appears to be a possible interplay between these two chemosensory caused by two structurally different classes of molecules. ␣ responses. Whereas G i2 activation was only observed upon stimu- ␣ Despite its exquisite expression in the main olfactory lation with lipophilic volatile components, G o activation ␣ epithelium, GC-D appears to be a pseudogene in species was elicited by the rat major urinary protein, 2-globulin. lacking a functional vomeronasal organ such as humans, A conﬁrmatory role of these G proteins in pheromone apes, Old World and New World monkeys, and tarsier. transduction issues from behavioral (see sect. VII) and ␣ ␣ This suggests that a functional GC-D gene was lost early anatomical studies performed on G i2 and G o negative in primate evolution and that chemical detection in most mutant mice (278, 373): both mouse lines show a remark- Downloaded from primate species is unlikely to involve GC-D (416). able reduction in the size of the neuronal layers where ␣ ␣ G o and G i2 were originally expressed, indicating that G protein-mediated activity is required for survival of vome- V. PHEROMONE TRANSDUCTION ronasal sensory neurons.

Since PLC stimulation and IP3 production appear to

Distinct neuronal subsets of the olfactory organs ex- be predominantly mediated by the release of the ␤␥ com- physrev.physiology.org press specific types of receptors, second messenger sys- plex of the heterotrimeric G proteins, Go and Gi (56), it tems, and ion channels to transform the pheromone bind- has been proposed that G␤␥ may play an important role in ing into electrical signals. the transduction process of the vomeronasal epithelium. The electrical activity of chemosensory neurons in One ␤-subunit (G␤2) and two ␥-subunits (G␥2 and G␥8) response to stimuli can be measured with a number of have been described in the vomeronasal epithelium (321, approaches, including recording of the field potential, of 322, 380). G␥2 immunopositive neurons are exclusively the firing activity with an array of extracellular electrodes localized in the apical layer, whereas G␥8 neurons are and by patch-clamp technique at the single-cell level. preferentially restricted to the basal one. G␤2, on the on February 9, 2011 Nonetheless, calcium imaging either in dissociated cells other hand, seems to be active in both neuronal subsets or epithelium slices is often employed as well as the (Fig. 6). Biochemical studies reveal that IP3 formation metabolic activity of stimulated neurons is frequently induced by stimulation of membrane preparations with monitored upon pheromonal stimulation by evaluating volatile urinary compounds was selectively blocked by the expression of the early protooncogenes. an anti-G␤2 antibody, whereas second messenger production induced by stimulation with major urinary pro- A. Vomeronasal Sensory Neurons teins was inhibited by preincubation of the vomeronasal membranes with an antibody recognizing G␥8 (321). ␣ ␤ ␥ 1. Signal transduction molecules Thus it appears that the G o 2 8 complex may control PLC activation by proteinaceous pheromones, whereas ␣ ␤ ␥ The detailed signal transduction cascade in the G i2 2 2 neurons respond to urinary volatile com- vomeronasal organ is still largely unknown, although it pounds. has been clearly demonstrated that pheromone stimuli Other lines of evidence indicate that, at least in some cause membrane depolarization and increase the action vomeronasal sensory neurons, transduction is mediated potential firing rate in vomeronasal sensory neurons (28, by a phosphatidylinositide signaling pathway. Some stud-

53, 61, 68, 115, 124, 125, 185, 187, 211, 363, 374). As ies showed that electrical responses, IP3 production, and described in previous sections, the sensory epithelium of calcium entry upon pheromonal stimulation (53, 176, 177, the vomeronasal organ presents two subsets of neurons: 215, 330, 397) are speciﬁcally blocked by the phospho- apical and basal. Apical neurons express members of the lipase C inhibitor U73122 (115, 363). Recently, Thompson V1R family of vomeronasal receptor genes together with et al. (377) demonstrated that compounds known to in- ␣ G i2, whereas basal neurons express V2R receptors and duce pregnancy block (Bruce effect), such as MHC pep- ␣ G o (19, 102, 142, 143, 321, 348, 371, 398). The anatomical tides and male urine, also increased the IP3 formation, and perhaps functional segregation in the vomeronasal conﬁrming a major role of the phosphatidylinositide path- epithelium is likely to be a prerogative of species as rats, way in the vomeronasal signal transduction.

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The ion channel TRPC2, a member of the superfamily among TRP channels in that a functional gene has been of transient receptor potential channels (390, 412), is lost from the Old World monkey and human genomes expressed in the microvilli of both apical and basal neu- (198, 420). Proposed mechanisms for the activation of the rons (197, 241, 271) and plays an important role in the TRPC2 channel in the vomeronasal neurons are similar to transduction process of some, but not all, vomeronasal those involved in the signaling cascade of Drosophila sensory neurons. Interesting to note, TRPC2 is unique photoreceptors (103, 250): pheromones trigger the activa- Downloaded from physrev.physiology.org on February 9, 2011

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 937 tion of G protein-coupled receptors that in turn stimulate level in the gartner snake (215) and in rodents (215, 319, phospholipase C and the production of the lipid messen- 424). However, the decrease in cAMP occurs with a longer ger diacylglycerol (DAG) (211) and possibly of polyunsat- delay with respect to pheromone stimulation (319). Pos- urated fatty acids such as arachidonic acid (363, 421) and sible targets for modulatory actions by cAMP include the linolenic acid (Fig. 6) (363). Ca2ϩ-activated cation channel (195) and the recently Genetic ablation of the TRPC2 channel results in a characterized hyperpolarization-activated cyclic nucleoti- strong, but not complete, reduction of electrophysiologi- de-gated (HCN) cation channels (64). cal responses of the vomeronasal neurons to urine or its It is intriguing that a subpopulation of apical sensory volatile components (156, 193, 368). Moreover, male neurons expresses members of the odorant receptor gene ␣ TRPC2-knockout mice showed severe behavioral abnor- family, together with G i2 and TRPC2 (190). Since some malities (see sect. VII) (157, 193, 236, 368), indicating that conventional odorants are detected by the vomeronasal the TRPC2 channel may be an important component of epithelium (329, 382, 409), it was suggested that vomero- the transduction of pheromone signals. nasal neurons expressing odorant receptors might be Nevertheless, the genetic ablation of TRPC2 does not those that respond to odorants (190). completely abolish the response of the vomeronasal or- Following the initial transduction events, when the gan to pheromones, since electrophysiological recordings membrane potential of vomeronasal sensory neurons have demonstrated that basal neurons of mutant mice still reaches threshold, action potentials are generated and Downloaded from respond to MHC peptide ligands as controls (156). In propagate along the axons to the accessory olfactory addition, TRPC2-knockout mice exhibit the Bruce effect bulb. Interestingly, most vomeronasal neurons ﬁre toni- (156), a neuroendocrine response that requires a func- cally for several seconds, without sign of adaptation, tional vomeronasal organ. All together, these studies sug- when small current injections (64, 196, 347, 384) or dilute gest that at least a subset of basal neurons uses a different urine are applied for up to 2–3 s (211, 421). However, in channel from TRPC2 for the transduction of some phero- one study, longer stimulations of 10–60 s caused spike- physrev.physiology.org monal stimuli. frequency adaptation (384), and in addition, a recent work Interestingly, Liman (195) showed that a cation chan- suggested that sensory adaptation in vomeronasal sen- nel activated by a high concentration of Ca2ϩ is expressed sory neurons requires the inﬂux of Ca2ϩ and is mediated in hamster vomeronasal sensory neurons. This channel is by calmodulin (362). permeant to both Naϩ and Kϩ and is blocked by ATP and cAMP, sharing many features with the TRPM4 ion channel 2. Responses to urinary pheromones (275). These properties make this channel a good candidate to be directly involved in sensory transduction or in Using an array of extracellular electrodes, Holy et al. on February 9, 2011 amplifying a primary sensory response. At present, the (115) simultaneously recorded the activity of a large num- molecular nature of this channel and its relation with ber of mouse vomeronasal neurons in response to dilute

TRPC2 or with IP3 production are not yet resolved. urine. Neurons responded to urine with an increase in the Some components of the cAMP signaling cascade firing frequency that remained approximately unchanged have been identified in vomeronasal sensory neurons. if the stimulus was applied for as long as 100 s, indicating These include adenylyl cyclase types 2 (19), 4, 5 (184), and that the response of these neurons do not adapt to pro- 6 (184, 319); the subunit CNGA4 of the olfactory cyclic longed stimulus exposure. Furthermore, they identified nucleotide-gated channel (19, 184); and phosphodiester- distinct and nonoverlapping subsets of neurons that were ase types 1 and 4 (50, 183), suggesting that cAMP may play selectively activated by substances contained in female or a physiological role in vomeronasal sensory neurons. In- male urine; in contrast, about half of the vomeronasal deed, some pheromones induce a reduction of the cAMP neurons 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 rat vomeronasal 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 2 ␣ ␤ ␥ ␤␥ in 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. The gating of the TRPC2 channel allows an influx of Naϩ and Ca2ϩ, causing a membrane depolarization. An additional cation-selective channel activated by Ca2ϩ may be involved in the transduction cascade. The TRPC2 channel appears to be the principal transduction channel in V1R-expressing neurons, whereas its role in V2R-expressing neurons in unclear. C: a scanning electron micrograph of the knob of a human olfactory sensory neuron showing the protrusion of several cilia. Scale bar, 1 ␮m. [From Morrison and Costanzo (262).] D: olfactory transduction takes place in the cilia, where ␣ ligands 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 carried by Naϩ and Ca2ϩ.Ca2ϩ entry amplifies the signal by causing the efflux of ClϪ through Ca2ϩ-activated ClϪ channels (Cl-Ca). These ion fluxes cause a depolarization. Inhibitory actions are played by the complex Ca2ϩ-calmodulin (CaM) by increasing the activity of the phosphodiesterase (PDE) 2ϩ ϩ 2ϩ that hydrolyzes cAMP and by reducing the affinity for cAMP of the CNG channels. Ca is extruded by the Na /Ca exchanger (Na/Caex).

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Thus these findings establish the capacity of the vomero- an exceptionally low-noise imaging of large neuronal pop- nasal organ to identify differences between sexes. ulations, unexpectedly found that responses to dilute A recent study by He et al. (106) further stressed the urine are predominantly, and perhaps exclusively, gener- concept of the vomeronasal organ being subtly dedicated ated by neurons in the apical layer, where V1Rs are ex- to the identification of conspecifics. These authors quan- pressed. tified the number of neurons responding to individual Leinders-Zufall et al. (187) showed that some volatile urine of either sex and of different strains in slices of the hydrophobic pheromones, known to be tightly bound to vomeronasal epithelium of mice genetically modified to the hydrophobic pocket of MUPs, produced a calcium express a calcium indicator. It was observed that only a increase in neurons of slice preparations of the vomero- very small subpopulation of neurons (1–3%) exclusively nasal organ. The distribution pattern of the activated responded to urine samples from all individuals of the neurons by any of these molecules reflects a localization same sex. The gender-specific neurons were evenly dis- that can be associated with the expression of the vome- tributed both in the apical and basal layers of the epithe- ronasal receptors V1Rs (187), and the percentage of neu- lium and did not differ in males or females, consistent rons responding to an individual pheromone (0.2–3%) with the little dimorphism generally observed at the level matches the proportion of neurons expressing one type of of receptor expression in the vomeronasal epithelium. V1R. Therefore, when volatile molecules are released by

Interestingly, neurons specific to female urine showed MUPs, in the external environment or in the nasal cavity, Downloaded from different activation patterns depending on the phase of they are likely to bind and activate V1Rs. the estrus cycle; in contrast, urine from castrated mice Urine contains compounds whose physiological role failed to elicit a response in male-specific neurons. Fur- is largely unknown. Nodari et al. (277) applied chromato- thermore, information about individuals and strains is not graphic fractions of female mouse urine to isolated vome- encoded by specialized neurons but, rather, by the com- ronasal epithelia mounted on a multielectrode array and binatorial activation of vomeronasal neurons, as no urine measured the spiking activity of neurons (277). Increase physrev.physiology.org samples from different mice of the same sex elicited the in the firing rate was associated with compounds with same pattern of neuronal activity. Thus the information homogeneous chemical properties that, after further pu- contained in the mouse urine about gender is encoded by rifications, appeared to correspond to sulfated steroids. dedicated neurons; strains and individuals are recognized Sulfatase-treated urine extracts lost Ͼ80% of their activ- by a combinatorial code of neuronal activation (106), with ity, indicating that sulfated compounds are the predomi- the obvious advantage to discriminate a large population nant ligands for vomeronasal neurons in female urine. of individuals with a restricted repertoire of receptors. Interestingly, a collection of 31 synthetic sulfated steroids He et al. (106) also compared responses elicited by triggered responses 30-fold more frequently than did a on February 9, 2011 synthetic strain-specific MHC class I peptides with those similarly sized stimulus set containing the majority of all induced by urine of the same strain and found that neu- previously reported ligands. Vomeronasal neurons seemed rons activated by urine and by the peptides did not over- to have a high tuning specificity for sulfated steroids, since lap. Furthermore, the same authors (106) pointed out that each neuron responded only to one or to a few structur- only fragments, but not the peptides, of the MHC com- ally similar compounds, but collectively, neurons de- plexes are detectable in urine (358). This result strongly tected all major classes of sulfated steroids. Some of contrasts with what was observed by Leinders-Zufall et al. these compounds may be indicators of a status of stress. (185), who identified a specific subset of basal neurons Indeed, the concentration of two sulfated glucocorticoids highly sensitive to MHC class I peptides and to urine from in urine increased about threefold in animals subjected to mice of the relevant haplotype. stress compared with unstressed controls, indicating that Other studies investigated the response of the vome- information about the status of stress of these mice is ronasal epithelium to specific urinary components, such encoded in urine and could be detected by vomeronasal as MUPs, that represent another class of candidates for sensory neurons (277). strain recognition, as well as for heterozygosity signals (375). Kimoto et al. (163) found that a recombinant iso- 3. Responses to secretory peptides form of MUPs elicits electrical responses in the vomeronasal epithelium, and Chamero et al. (47) showed that Kimoto et al. (163) showed that mouse recombinant native and recombinant MUPs evoke calcium influx in exocrine gland-secreting peptides (see sect. IIC) evoked ␣ sensory neurons expressing the G protein subunit G o. electrical activity in the vomeronasal but not in the olfac- ␣ Since G o coexpresses with V2Rs, it is likely that MUPs tory epithelium. Interestingly, when the electrical re- directly activate a response in vomeronasal sensory neu- sponses of individual neurons to ESP1 were recorded rons by binding to a subset of these receptors (47). In with a multielectrode array, only a small percentage of sharp contrast to these observation, Holekamp et al. neurons, 1.6%, responded to ESP1 (163). This result is con- (113), by using a new microscopical technique that allows sistent with the possibility that ESP1 is recognized by one

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 939 type of receptor only, most likely by a specific V2R, as ESP1 Since TRPM5 channels are directly activated by intracel- induced c-fos production in vomeronasal neurons express- lular Ca2ϩ (112, 204, 299, 423), it is tempting to speculate ing V2Rp5, a V2R receptor of the family A, clade III (98). that Ca2ϩ entry through CNG channels could gate TRPM5 channels. Neurons expressing TRPM5 are located in the ven- B. Olfactory Sensory Neurons trolateral zones of the olfactory epithelium and project to domains of the ventral region of the main olfactory bulb, 1. The cAMP transduction cascade an area responsive to urine and other putative phero- In contrast to a common belief, some sensory neu- mones (199–201, 336, 409). Unfortunately, electrical re- rons of the main olfactory epithelium respond to phero- sponses from individual olfactory sensory neurons ex- mones (364, 395). A large majority of olfactory sensory pressing TRPM5 have not yet been investigated. neurons express a member of the odorant receptor family As previously mentioned (see sect. IVD), another sub- that is coupled to a signal transduction pathway mediated set of neurons in the main olfactory epithelium expresses by cAMP (Fig. 6). The binding of ligands to the odorant components typical of a cGMP instead than a cAMP sec- ␣ ond messenger system, namely, the receptor guanylyl receptor activates the G protein subunit G olf, which stimulates adenylyl cyclase-3 activity producing an in- cyclase type D (GC-D), the cGMP phosphodiesterase crease in cAMP concentration. cAMP causes the opening PDE2 (149), and the cyclic nucleotide-gated channel Downloaded from of the ciliary cyclic nucleotide-gated channels composed CNGA3, that was originally discovered in cone photore- of three subunits: CNGA2, CNGA4, and CNGB1b (for ceptors (417). These neurons are located in the central reviews, see Refs. 29, 153, 292). These channels allow an area of the olfactory epithelium and project to an anatom- influx of Naϩ and Ca2ϩ inside the cilia. Ca2ϩ-activated ClϪ ically distinct group of interconnected glomeruli, the channels are then activated in turn and, due to the unusu- necklace glomeruli, that have been implicated in the suck- ally high intracellular ClϪ concentration, allow an outflow ling response of mammals (149). Leinders-Zufall et al. physrev.physiology.org of ClϪ, contributing to the inward current (167, 179). As a (186) showed that neurons expressing GC-D can be acti- result of the odorant binding to receptors, the olfactory vated by the peptide hormones uroguanylin and guanylin sensory neuron depolarizes. The CNG channel allows and by natural urine stimuli, while Hu et al. (118) demon- Ca2ϩ entry not only for excitatory but also for inhibitory strated that these neurons may be involved in the detec- purposes. The complex Ca2ϩ-calmodulin activates a phos- tion of carbon dioxide. In both studies, GC-D neurons phodiesterase (PDE1C2) that hydrolyzes cAMP and, im- were shown to use a cGMP signaling cascade (118, 186). portantly, produces a negative-feedback effect on the A subset of neurons in the main olfactory epithelium on February 9, 2011 CNG channel itself, that has been shown to mediate ol- of humans and mice expresses some vomeronasal recep- factory adaptation (178, 242). tors of the V1R family (152, 313, 315). Ligands correspond- When the depolarization following the activation of ing to aliphatic alcohols or aldehydes have been identified the transduction cascade reaches the threshold for acti- for human V1Rs (350); however, it is still unresolved vation of action potentials, they will travel along the axon whether neurons expressing these receptors project their to the main olfactory bulb. Differently from vomeronasal axons to the main or the accessory olfactory bulb. sensory neurons, a maintained current injection will only A small population of neurons in the main olfactory elicit a short burst of action potentials in olfactory sen- epithelium expresses the TRPC2 channel. At present, sory neurons, instead of a tonic firing (196). there are no clues whether these neurons have chemo- For more details about the cAMP transduction cascade sensory properties, but this finding is certainly relevant in olfactory sensory neurons, the reader may consult previ- and must be seriously taken into account when compar- ous comprehensive reviews (40, 168, 235, 242, 292, 337). ing the behavioral effects produced by the genetic dele- tions of the TRPC2 channel with those of the surgical 2. Neuronal subsets removal of the vomeronasal organ (P. Mombaert, per- sonal communication). The subset of olfactory sensory neurons expressing Early microscopical and immunological studies have ␣ TAARs also express G olf, and therefore, it is possible revealed the existence of a novel cell type in the olfactory that they use a signal transduction pathway coupled to epithelium that is morphologically different from chemo- adenylyl cyclase and cAMP (194), although additional sensory neurons or supporting cells (44, 148, 260). The components of the transduction pathway in these neu- main feature of these cells is represented by the microvilli rons are still unknown. that they bear on the mucosal side. It now appears that The ion channel TRPM5 is expressed in another sub- olfactory microvillar cells do not represent a homoge- set of olfactory sensory neurons that coexpress the neous population of cells but rather they include different CNGA2 channel, and some components of the PLC path- cellular subsets expressing distinct signal transduction way, such as PLC-␤2 and the G protein ␥13 subunit (202). molecules (6, 74, 201). It is very unlikely that these cells

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 940 TIRINDELLI ET AL. are true neurons as they do not seem to project their long Brechbuhl et al. (30) recently uncovered at least one foot process to the olfactory bulb (74, 201). However, of the functional roles of the Grueneberg ganglion. In- although their role is enigmatic, a possible chemosensory deed, a subpopulation of neurons was shown to respond function for microvillar cells has been reported (74). to the yet unidentified “alarm pheromones,” a group of volatile compounds that are released by animals sub- 3. Responses of the main olfactory epithelium jected to stress. Lesions of this organ completely abolish to pheromones the so-called freezing reaction, a typical fear response elicited by alarm pheromones. Curiously, in this study, Wang et al. (395) showed that milk and urinary phero- lesions of the Grueneberg ganglion did not affect mother- mones that are responsible for puberty delay aggressiveness pup recognition (30), a behavior that was believed to be in the mouse (279, 281) and evoke an electrical response mediated by this organ (118, 173, 317). from the main olfactory epithelium, as measured by field potential recordings. Moreover, when the same experimen- VI. SIGNALING FROM SENSORY EPITHELIA TO tal approach was adapted to mice in which the adenylyl THE BRAIN cyclase type 3 gene had been knocked out, no electrical response was recorded, indicating that receptors responding A. Main and Accessory Olfactory Bulbs to these compounds are likely to be coupled to the cAMP Downloaded from signaling pathway. Since behavioral deficits are also evident 1. Anatomical organization in adenylyl cyclase type 3 mutant mice, it can be envisaged The main olfactory bulb is the primary target region of that a cAMP-mediated pheromone signaling may play an the olfactory neurons, whereas vomeronasal sensory neu- important role in the olfactory epithelium. rons project to a well-defined dorsal and posterior region of MHC peptides can also be detected by sensory neu- the olfactory bulb, namely, the accessory olfactory bulb. The rons of the main olfactory epithelium via the cAMP sig- main olfactory bulb exists in all vertebrates, except for physrev.physiology.org naling cascade, as shown by Spehr et al. (364). Two MHC aquatic mammals. In contrast, the accessory olfactory bulb peptide ligands, SYFPEITHI and AAPDNRETF, elicited an is absent in adult Old World monkeys, apes, and humans (for electrical response, measured by field potential record- more information, see Table 1 in Ref. 240). In rodents, both ings in the main olfactory epithelium at a concentration the main and the accessory olfactory bulbs have a largely near 10Ϫ11 M (364). Mutant mice lacking the CNGA2 gene similar laminar organization consisting of a superficial nerve failed to display electrical responses when stimulated layer formed by the axonal projections of the chemosensory with both peptides, suggesting that a subset of olfactory neurons, the glomerular layer that represents the first-order neurons expressing CNGA2 are actively involved in the on February 9, 2011 synaptic region between sensory neurons and mitral cell detection of pheromones besides conventional odorants. dendrites, and the external and internal plexiform layers that In contrast to that, Lin et al. (200) have demonstrated are regions where soma of, respectively, mitral/tufted and that at least two pheromones, 2-heptanone and dimeth- granule cells reside (for review, see Ref. 240; see also Ref. ylpyrazine, stimulate the main olfactory epithelium with- 182 for suggested changes to the nomenclature of the acces- out the intervention of the CNGA2 channel, suggesting sory olfactory bulb). In the main olfactory bulb, glomeruli that pheromone detection in the olfactory epithelium re- are quite well anatomically separated, encapsulated by peri- lies on multiple transduction pathways. glomerular cells, and rather uniform in size (ϳ50 ␮m diameter in mice). Conversely, in the accessory olfactory bulb, C. Grueneberg Ganglion these synaptic structures are very diffusely organized, sur- rounded by a small number of periglomerular cells, and The transduction pathways in the Grueneberg ganglion highly variable in size (10–30 ␮m diameter in mice). In the are still largely uncharacterized. Fleischer and colleagues main olfactory bulb, mitral/tufted cells form a distinct mono- showed that the majority of neurons express the vomerona- layer, whereas in the accessory olfactory bulb they are much sal receptor V2R83 (V2R2 family), together with the G pro- less organized. An important difference between mitral/ ␣ ␣ tein G i (79, 80), while a distinct subpopulation of G i- tufted cells of the two olfactory systems is their arborization: expressing neurons also expresses TAAR receptors (80). in the main olfactory bulb these neurons bear only one Another study investigated whether neuronal subsets of the apical dendrite, whereas in the accessory olfactory bulb Grueneberg ganglion express transduction components sim- mitral/tufted cells are equipped with up to six apical den- ilar to those of GC-D neurons, based on the observation that drites, each entering a different glomerulus (Fig. 7; for re- both types of neurons project to the necklace domain of the view, see Ref. 240). main olfactory bulb (Fig. 3). Fleischer et al. (78) reported that a subset of Grueneberg ganglion neurons expressing the 2. Topographic projections V2R83 receptor expresses also the GC-D receptor and the Axons of all the olfactory sensory neurons expressing a cGMP-stimulated phosphodiesterase PDE2A. particular odorant receptor converge to only two glomeruli

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FIG. 7. Main and accessory olfactory bulbs. A and B show the general organization of the main olfactory bulb (MOB) and of the accessory olfactory bulb (AOB) in the adult rat from Nissl-stained specimens. A: each mitral cell (MC) of the main olfactory bulb has a single apical dendrite projecting to a single glomerulus and several lateral dendrites that contact granule cell dendrites. B: each mitral cell (here indicated as large principal cell, LPC) of the accessory olfactory bulb has several apical dendrites projecting to multiple glomeruli. EPL, external plexiform layer; MC, mitral cells; EGC, external granule cell; asterisk, tufted cell axon; IGC, internal granule cell dendrite; S, superficial centrifugal fiber; LOT, lateral olfactory tract. [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 (or glomerular pair). Three populations of olfactory sensory neurons, each expressing one different type of odorant receptor, are represented in different 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. in the main olfactory bulb (257, 308, 389). This has been work (255, 256, 381). In mice there are ϳ2,000 glomeruli, and experimentally demonstrated at a single-neuron resolution their localization is roughly conserved among individuals. by using transgenic mice in which the expression of a given Therefore, the olfactory bulb is topographically organized, odorant receptor was linked to that of the green fluorescent with each glomerulus representing a single type of odorant protein, thus allowing the visualization of the axonal net- receptor (Fig. 7C).

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In rodents, V1Rs and V2Rs are individually expressed through glutamatergic synapses, but the chemical infor- in vomeronasal sensory neurons, and each neuron ex- mation is further processed by the activity of inhibitory pressing a given vomeronasal receptor (or vomeronasal interneurons as well as periglomerular and granule cells receptor pair) sends its axonal projection toward the (141, 210, 339) that contact mitral cells via glutamatergic anterior (V1Rs) or posterior (V2Rs) accessory olfactory dendrodendritic synapses. These consist of an excitatory bulb (100, 143, 414). Gene targeting approaches were used glutamatergic input from mitral cells to granule cells, to visualize the topographical map of each targeted recep- inducing the release of GABA from granule cells, that in tor in the mouse accessory olfactory bulb (16, 62, 314). turn provides inhibition to the mitral cells (141, 182, 303). When a fluorescent marker protein was inserted down- For more information, the reader may consult previous stream of a vomeronasal receptor gene, the resulting comprehensive reviews (339, 345, 385). visualized axonal projections of neurons expressing that specific receptor showed that several glomeruli in subre- 3. Encoding pheromonal information in the main and gions of the anterior or posterior accessory olfactory bulb accessory olfactory bulbs received inputs. A more accurate analysis indicated that axons from neurons expressing the same receptor coa- To simultaneously examine the responses to phero- lesce into 10–30 glomeruli and that a single glomerulus mones and odorants in the mouse main and accessory receives input from neurons expressing different receptor olfactory bulbs, Xu et al. (409) used high-resolution func- Downloaded from types (Fig. 7D). Moreover, the capacity to converge and to tional magnetic resonance imaging (fMRI), a very power- form these glomeruli depends on the possibility to ex- ful technique that can show dynamic responses in both press a specific vomeronasal receptor gene, suggesting entire olfactory bulbs to various stimuli in the same ani- that the receptor proteins themselves play a role in axonal mals. Mouse urine activated restricted areas in both olfac- wiring to the bulb (16, 314). In fact, vomeronasal neurons tory bulbs, eliciting signals mainly in the ventral region of the expressing a nonfunctional (truncated) V1R gene no main olfactory bulb and in the anterior part of the main physrev.physiology.org longer converge in the accessory olfactory bulb. This accessory bulb. Since the anterior region of the mouse ac- phenotype, also observed for odorant receptors (343), cessory olfactory bulb receives input from V1R-expressing could be possibly due to the inability of the truncated vomeronasal sensory neurons, this study is in agreement gene to prevent the expression of other V1Rs, resulting in with recent results from Holekamp et al. (113) who sug- a population of fluorescently tagged neurons randomly gested that urine mainly activates V1Rs (see sect. VA2). expressing different V1Rs . Indeed, Roppolo et al. (318) The encoding of pheromonal information in the mitral showed that the expression of a nonfunctional V1R allele

cells of the accessory olfactory bulb has been investigated on February 9, 2011 allows the coexpression of other V1R genes. Although no by recording responses in awake mice using a miniature V1R expression has been reported in other subcellular motorized drive to move microelectrodes and record the compartments besides the projecting dendrite, it is possible to envisage that V1Rs may direct axon guidance, activity of single neurons (213, 214). Male mice were allowed perhaps through a mechanism that involves their expres- to investigate conspecifics differing by sex, genetic makeup, sion in the growth cones. and hormonal status. Interestingly, an increase in mitral cell Wagner et al. (393) generated transgenic mouse lines activity occurred after test animals directly contacted and in which projections from various populations of neurons actively investigated the head and the anogenital regions of expressing distinct V1Rs were differentially labeled with the stimulus animal. The recorded mitral cell responses fluorescent proteins. They found a glomerular map di- were found to be specific for sex and strain of the stimulus vided in multiple nonoverlapping domains, with each do- mouse. In addition, mitral cells exhibited both highly spe- main clustering glomeruli formed by neurons expressing cific excitatory and inhibitory responses, these latter likely V1Rs of the same subfamily, randomly intermingled (393). originating from the inhibitory interneuron activity. There- Moreover, these authors (393) found that a mitral cell is fore, the activity of mitral cells appears to be tuned by the not only connected to glomeruli innervated by neurons combination of sex and genetic makeup (213, 214), and expressing the same V1R (62) but also to those associated therefore, information about sex and strain is already inte- with receptors of the same subfamily. Therefore, in the grated in the accessory olfactory bulb. anterior accessory olfactory bulb, the spatial map origi- Several studies have revealed that the exposure to nated from vomeronasal organ inputs seems to rely on urinary components from opposite-sex conspecifics in- receptor subfamilies, rather than individual receptors as creased the number of c-fos-immunoreactive mitral and in the main olfactory bulb. This wiring scheme may help granule and periglomerular cells in the accessory olfac- to discriminate molecules with highly similar chemical tory bulb (116, 126, 228, 295, 410). Volatile urinary odors structure at different ratios in a pheromonal blend. from male as well as female mice also stimulate c-fos In both the main and accessory olfactory systems, expression in distinct clusters of glomeruli in the main mitral cells are activated by primary sensory neurons olfactory bulb in both sexes (228, 270).

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Lin et al. (199) recorded the electrical activity in the olfactory cortex, without a thalamic relay. The primary ol- accessory olfactory bulb in response to urine of different factory cortex, defined as the ensemble of brain regions that mouse strains. A subset of mitral cells was found to be receive direct input from the olfactory bulb, is composed of very selective, responding only to male urine. Moreover, several anatomically distinct areas: the piriform cortex, the by combining electrophysiology and gas chromatographic olfactory tubercle, the anterior olfactory nucleus, the corti- separation of male mouse urine components, these au- cal amygdala, and the entorhinal cortex. In turn, most of thors identified a male specific compound, methyl-thio- these cortical regions project back to the main olfactory methanethiol (MTMT), that was indeed responsible for bulb and to other areas of the brain, including the thalamus, this effect. Interestingly, mitral cells responding to MTMT the hypothalamus, the hippocampus, the frontal cortex, and were not stimulated by any other urinary compound, the orbitofrontal cortex (Fig. 8; reviewed in Ref. 349). An- allowing the appellation of “specialist” cells. Further- atomical studies and genetic approaches indicate that the more, behavioral experiments also established the biolog- topographical organization of odorant information in the ical activity of this molecule, as female mice developed olfactory bulb is not reflected in the olfactory cortex. interest and attraction towards castrated mouse urine Instead, odorant receptors seem to be mapped to multi- after the addition of synthetic MTMT. ple, discrete clusters of cortical neurons. Mitral cells of the accessory olfactory bulb project to Downloaded from B. Signaling Beyond the Olfactory Bulbs areas of the limbic system: the medial amygdala and the posteromedial cortical amygdala, which are also called In the main olfactory bulb, mitral/tufted cells directly “vomeronasal amygdala,” the accessory olfactory tract, and transmit signals from glomeruli to pyramidal neurons in the the bed nucleus of the stria terminalis (Fig. 8) (272, 334, 392). physrev.physiology.org on February 9, 2011

FIG. 8. Central pathways involved in processing chemical information by the vomeronasal and main olfactory systems. [Based on data from Martinez-Marcos (229) and Yoon et al. (413).]

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Although vomeronasal sensory neurons expressing viral tracing allowed the identification of a major projec- V1Rs or V2Rs, respectively, project to distinct areas of the tion pathway from the main olfactory system. accessory olfactory bulb, this segregation, at least in some Both studies (26, 413) came to the conclusion that species, does not seem to be completely maintained in the the small pool of GnRH cells (800 neurons) was surpris- amygdalic regions, where clusters of neurons may com- ingly synaptically connected to some tens of thousand bine signal information originating from both families of neurons in ϳ50 diverse brain areas, including the main vomeronasal receptors (230, 252, 253, 327, 392). olfactory ones, suggesting that GnRH neurons may influ- In essence, the majority of these studies provide ence a large variety of brain functions. evidence that, excluding areas of convergence, the two Interestingly, Yoon et al. (413) reported the ab- vomeronasal pathways project to specific areas of the sence of synaptic connections between the vomerona- amygdala and hypothalamus, thus suggesting a partial sal pathway and GnRH neurons. This could be due to functional separation (252, 253). different experimental approaches used in the two In the vomeronasal system, projections to the hypothal- studies or, perhaps, presynaptic inputs from the vome- amus from the limbic regions are directed to the ventrome- ronasal pathway identified in one study (26) might de- dial and the medial preoptic areas, which are involved in rive from a subset of GnRH neurons distinct from those reproductive and social behaviors (158, 159, 289). targeted in the other study (413). However, the obser-

The medial amygdala receives direct input from the vations of Yoon et al. (413) do not refute previous Downloaded from accessory olfactory bulb, but also from the main olfactory neuroanatomical tracing data indicating that the vome- bulb. However, other rostral basal telencephalic regions ronasal pathway projects to the hypothalamic area seem to play a relevant role in the integration of signals where GnRH neurons are located. Indeed, Yoon et al. originating from both the vomeronasal and the main olfac- (413) conﬁrmed that neurons in this area receive inputs tory epithelia. from the vomeronasal pathway. It could be speculated

that the connections with GnRH are, at least in part, physrev.physiology.org mediated by nonsynaptic (e.g., hormonal or neuro- C. Pheromone-Activated Hormonal Systems modulatory) mechanisms. As a confirmation of these observations, stimulation of Recent experiments have revolutionized the common male mice with urinary pheromones induced the activation beliefs about the control of hormonal responses by pher- of neurons in the anterior cortical amygdala as well as in the omones (26, 413). The effects of pheromones on the neu- dorsal and ventral endopiriform nucleus. Therefore, signals roendocrine status are mediated by a group of neurons in in response to pheromonal cues also occur via the main the hypothalamus, which constitutes the endocrine con- olfactory epithelium and are transmitted to GnRH neurons on February 9, 2011 trol center (for reviews, see Refs. 89, 245, 353). These through the olfactory cortex. Moreover, feedback loops neurons secrete the GnRH, also known as LH-RH, which have been identified so that GnRH neurons could influence in turn stimulates the anterior pituitary gland to release both odorant and pheromone signaling. two gonadotrophins: the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH). Both hormones control the development and the function of the gonads in VII. PHEROMONE-MEDIATED BEHAVIORS males and females, although with different effects. A genetic approach has been used to visualize the From results presented in the previous sections, it GnRH neuronal network and, surprisingly, GnRH neurons now appears evident that both the vomeronasal and the were found to be connected with areas relaying informa- main olfactory systems may be activated by chemicals tion from the main olfactory system, in contrast to the that are known to produce pheromonal effects. The traditional view that only ascribes connections with involvement of the vomeronasal and main olfactory GnRH neurons from the vomeronasal system (26, 413). systems on specific behaviors is usually investigated by Boehm et al. (26) identified neurons that synapse inactivating one or both systems. In the past, lesions with GnRH by using a genetic transneuronal tracer, barley were made only by surgical removal of the vomeronasal lectin, a glycoprotein that is transferred across synapses. organ (VNOx) or by chemical ablation of the main The barley lectin gene was positioned under the control of olfactory epithelium (MOEx) (Table 2). The introduc- the GnRH promoter so that only GnRH neurons would tion of a molecular genetic approach allowed the dele- express this peptide. In another study, Yoon et al. (413) tion of specific transduction molecules, although it used a different experimental approach to anatomically must be taken into account that the knowledge of genes trace the afferent pathways to GnRH neurons in the hy- involved in transduction in each olfactory organ is still pothalamus. Only neurons expressing GnRH were in- incomplete, and therefore, results of behavioral exper- fected with a modified pseudo-rabies virus and green iments from knockout animals must be interpreted very fluorescent protein reporter. The genetically controlled cautiously.

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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 2Sexual arousal (146) (365) 2Individual recognition (146) 2Male copulatory behavior (146) Rat 2Stress-induced 2Penile erection (172) hyperthermia (165) Maternal aggression (170) 2Male induced sexual receptivity (302) 2Nursing behavior (36) 2Infanticide (243) 2Sexual arousal (325) 2Lordosis behavior (326) Mouse 2Lordosis behavior to 2Sex discrimination (trpc2) (193, 2Lordosis behavior (154) 2Copulatory behavior (ac- Downloaded from male mount (155) 284, 368) 3) (395) 1Sperm motility (175) 2Intermale aggressiveness (trpc2; 2Sex discrimination (154) 2Intermale aggressiveness g␣i2) (278, 368) (ac-3) (395) 2Urine marking (181, 233) 2Maternal aggression (trpc2; v1ra- (73, 376) 2Individual recognition v1rb; g␣i2) (61, 161, 193, 278) (cnga2) (364) 2Intermale aggressiveness 2Lactating behavior (trpc2) (161) (54, 233)

2Marking Male-like sexual behavior in females physrev.physiology.org (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 on February 9, 2011 in females (161) Ferret 2Sexual investigation (404) Lemur 2Copulatory behavior (8) 2Intermale aggressiveness (8) Opossum 2Male-mediated induction of estrus (131) Prairie vole 2Intermale aggressiveness (399) 2Pairing (189) Rabbit 1Maternal behavior (87) 2Suckling behavior 1Maternal behavior (52) Cat 2Catnip reaction (104)

Reference numbers are given in parentheses.

Here, we will discuss experimental results mainly A. Male Behaviors related to sexual and aggressive behaviors and will try to interpret to what extent the vomeronasal and/or the 1. Male sexual behavior main olfactory systems appear to be involved. It is very Sexual behavior has been intensively approached by important to note that behavioral expressions largely many studies in the last 20 years, particularly in rodents. In vary among species; moreover, responses to phero- male mice, the typical mating sequence consists of olfactory mones cannot be simply described as innate or stereo- exploration of estrus females followed by multiple episodes typed, but may also depend on contextual situations of ultrasound vocalization, mounting and intromission, and and can be modiﬁed by some forms of learning and eventually ejaculation. All these parameters can be evalu- memory. ated in a typical test experiment (120, 121).

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The genetic ablation of the TRPC2 channel greatly cation as scent or flank marking are likely to be primarily impairs some features of the sexual behavior. Indeed, mediated by the main olfactory system (145). although male mice mate normally with females, they also show an indiscriminate courtship and mounting behavior 2. Male aggressiveness toward females (161, 193, 368). A similar situation was In mice, male to male territorial aggression is a re- observed in VNOx male mice (161). Furthermore, individ- markable innate social behavior that is triggered by com- ual recognition, through the discrimination of nonvolatile pounds present in urine (see sect. II, Table 1). In a com- odorants, is also affected in VNOx mice (155, 284). On the mon assay employed to test male aggressiveness, a male other hand, the simultaneous deletion of a group of V1R intruder is introduced in a cage containing a singly housed receptors results in a significant decrease of male copu- male resident. After a period of olfactory investigation, to latory behavior (61), although this latter appears normal allow individual recognition, the resident mouse initiates ␣ in G i2 mutants, suggesting that apical neurons are likely a series of attacks against the intruder. The number and not to be of relevant importance for mating motivation. All duration of the attacks and the latency to the first attack these findings reveal that the mouse vomeronasal organ is represent standard parameters to evaluate the aggressive indeed involved in individual recognition and reproductive behavior (58, 233). activity, but that signaling through the vomeronasal organ is The surgical ablation of the vomeronasal organ has not required for initiating sexual behavior. been long known to nearly abolish aggressiveness in male Downloaded from The chemical ablation of the main olfactory system mice and prairie voles (12, 54, 55, 233, 399, 407), an effect (via nasal irrigation with zinc sulfate) produces a strik- that is recapitulated in mutant mice bearing the deletion ing reduction in male reproductive behavior (227). Fur- of the gene encoding TRPC2 (161, 193, 368). Indeed, male thermore, the genetic deletion of the CNGA2 channel mice bearing the genetic ablation of the TRPC2 channel (227), or of adenylyl cyclase type 3 (395), entirely abol- fail to display aggression toward male intruders (193, ishes male mating performances producing severe de- 368), a behavioral effect that, in an attenuated form, is physrev.physiology.org ␣ ficiencies in each step involved in mating: olfactory also evident in mice lacking G i2 (278). exploration, mounting, and intromission, compared The main olfactory system also plays a very impor- with normal mice. Thus the main olfactory system tant role in male aggressiveness. Indeed, mice that either seems to play an essential role for male mouse mating have undergone the chemical ablation of the olfactory behavior. epithelium (via nasal irrigation with zinc sulfate) (154) or In rats, individual recognition and sexual arousal is have been genetically mutated in the genes encoding the

only temporary or moderately depressed by lesions of the odorant transduction molecules adenylyl cyclase type 3 on February 9, 2011 vomeronasal organ (22, 325) as well as the overall copu- and CNGA2 (227, 395) show abnormal sexual and aggres- latory behaviors appear to be slightly or partially affected sive behaviors (227). (171, 324, 325). Thus both the olfactory and the vomeronasal systems Sexual behavior in male hamsters is totally abolished appear to be necessary for eliciting male aggressiveness. by bilateral removal of the olfactory bulbs, an operation that eliminates sensory input from both the olfactory and B. Female Behaviors the vomeronasal systems (298). The destruction of the main olfactory epithelium did not impair male hamster 1. Female sexual behavior mating behavior, whereas the peripheral deafferentation In females of different species, sexual behavior is of the vomeronasal system produces severe sexual behav- characterized by ultrasonic vocalization and lordosis elic- ior deﬁcits in approximately one-third of the treated ani- ited by lumbosacral tactile stimulation (174, 294). mals. Combined deafferentation of both the vomeronasal In mice, the genetic ablation of the TRPC2 channel and the olfactory systems completely abolishes copula- did not alter female sexual receptivity to males but, sur- tion in all experimental animals (298). Mating behavior prisingly, caused a female sexual behavior similar to that alterations appear after removal of the vomeronasal or- of males, i.e., females attempted to copulate as males gan in sexually inexperienced males, and this effect is (161). Kimchi et al. (161) also showed that similar sexual reversed by intracerebroventricular injections of GnRH behaviors were displayed by females in which the vome- (76, 247), indicating that GnRH acts as an intermediate in ronasal organ was surgically removed, in contrast to a the facilitation of mating behavior by vomeronasal sen- previous study that described a reduced sexual receptiv- sory input. However, sexual experience prior to the re- ity in VNOx females (155). The chemical ablation of the moval of the vomeronasal organ mitigates the effect of the main olfactory epithelium (with zinc sulfate) reduces sex- lesion on sexual behavior, indicating that behavioral re- ual behavior in female mice (154). sponses are inﬂuenced both by VNOx and by sexual ex- In female rats, both chemical ablation of the main perience (291). In this species, other ways of communi- olfactory epithelium and vomeronasal organ removal af-

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 947 fect sexual receptivity and the male-induced release of have developed independently for the detection of pher- GnRH (17, 302). omones, and only the main olfactory epithelium has fur- In female hamsters, the removal of the vomeronasal ther specialized also for the perception of conventional organ disrupts the elicitation of lordosis by lumbosacral odorants. Proof of this hypothesis will require additional tactile stimulation, an effect reversed by injection of studies especially in species in which olfaction is not a GnRH (221). primary sensory process as humans, for example; however, it is interesting to note that part of the main olfac- 2. Maternal aggressive behavior tory central pathways seem to converge in regions that control the sexual status of the individual. Female mice are normally not aggressive towards intruders, but during lactation, they violently attack non- ACKNOWLEDGMENTS resident males. This type of maternal aggressive behavior We thank Didier Trotier for kindly providing the original has been shown to be dependent on chemosensory cues micrograph for figure 6A. (13). The surgical removal of the vomeronasal organ af- Address for reprint requests and other correspondence: A. fects aggression of lactating females (13, 161), whereas it Menini, Scuola Internazionale Superiore di Studi Avanzati, Via has little or no effect on other maternal behavior param- Beirut 2, 34014 Trieste, Italy (e-mail: [email protected]). eters as nest building or pup retrieval (13). The genetic Downloaded from ␣ GRANTS ablation of the TRPC2 channel (161, 193), the G i2 subunit (278), or a subfamily of V1R receptors (61), also results in This work was supported by grants from the Italian Minis- a low expression of maternal aggression, suggesting that try of Research and by the Italian Institute of Technology. this type of behavior is possibly linked to the apical subset of chemosensory neurons. REFERENCES physrev.physiology.org 1. Abel EL. Alarm substance emitted by rats in the forced-swim test VIII. CONCLUSIONS AND is a low volatile pheromone. Physiol Behav 50: 723–727, 1991. FUTURE CHALLENGES 2. Achiraman S, Archunan G. Characterization of urinary volatiles in Swiss male mice (Mus musculus): bioassay of identified compounds. J Biosci 27: 679–686, 2002. Although we are only beginning to understand the 3. Alioto TS, Ngai J. The odorant receptor repertoire of teleost fish. overlapping role of the main olfactory and vomeronasal BMC Genomics 6: 173, 2005. systems for pheromone sensing, there is now overwhelm- 4. Amrein H. Pheromone perception and behavior in Drosophila. Curr Opin Neurobiol 14: 435–442, 2004. ing evidence that the main olfactory system is involved in 5. Apfelbach R, Blanchard CD, Blanchard RJ, Hayes RA, McGregor a variety of important physiological mechanisms underly- IS. The effects of predator odors in mammalian prey species: a review on February 9, 2011 ing pheromonal communication. of field and laboratory studies. Neurosci Biobehav Rev 29: 1123–1144, 2005. In the early 1970s, different studies developed a 6. Asan E, Drenckhahn D. Immunocytochemical characterization of model that led to the so-called dual olfactory hypothesis, two types of microvillar cells in rodent olfactory epithelium. His- sustained by the observation that efferent and afferent tochem Cell Biol 123: 157–168, 2005. 7. Astic L, Pellier-Monnin V, Godinot F. Spatio-temporal patterns connections of the main and accessory olfactory bulb of ensheathing cell differentiation in the rat olfactory system during project in nonoverlapping areas of the brain, according to development. Neuroscience 84: 295–307, 1998. which the olfactory and vomeronasal systems were orga- 8. Aujard F. Effect of vomeronasal organ removal on male socio- sexual responses to female in a prosimian primate (Microcebus nized as parallel anatomical axes subserving different murinus). Physiol Behav 62: 1003–1008, 1997. functions. Recent anatomical and functional studies have 9. Bacchini A, Gaetani E, Cavaggioni A. Pheromone binding pro- indicated that this distinction is not as strict as commonly teins of the mouse, Mus musculus. Experientia 48: 419–421, 1992. 10. Ballard CL, Wood RI. Partner preference in male hamsters: ste- thought (229). There seems to be, in fact, areas of large roids, sexual experience and chemosensory cues. Physiol Behav convergence in the anterior medial amygdala. Moreover, 91: 1–8, 2007. in the rostral basal telencephalon, both inputs converged 11. Barber PC, Raisman G. Replacement of receptor neurones after section of the vomeronasal nerves in the adult mouse. Brain Res in areas that were classically categorized as exclusively 147: 297–313, 1978. vomeronasal or olfactory. As for the anatomical observa- 12. Bean NJ. Modulation of agonistic behavior by the dual olfactory tion, behavioral and biochemical studies seem to point to system in male mice. Physiol Behav 29: 433–437, 1982. 13. Bean NJ, Wysocki CJ. Vomeronasal organ removal and female a complementary role of the two systems. For example, mouse aggression: the role of experience. Physiol Behav 45: 875– the functional ablation of the olfactory and vomeronasal 882, 1989. organs leads to similar behavioral changes. Chemosen- 14. Beier K, Ginez I, Schaller H. Localization of steroid hormone receptors in the apocrine sweat glands of the human axilla. Histo- sory neurons sensitive to pheromone have been identified chem Cell Biol 123: 61–65, 2005. in both subsystems. Not last, humans as well as other 15. Bellringer JF, Pratt HP, Keverne EB. Involvement of the vome- species in which the vomeronasal system is absent can ronasal organ and prolactin in pheromonal induction of delayed implantation in mice. J Reprod Fertil 59: 223–228, 1980. probably detect pheromones via the main olfactory epi- 16. Belluscio L, Koentges G, Axel R, Dulac C. A map of pheromone thelium. It is therefore possible that the two systems may receptor activation in the mammalian brain. Cell 97: 209–220, 1999.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 948 TIRINDELLI ET AL.

17. Beltramino C, Taleisnik S. Release of LH in the female rat by 42. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, olfactory stimuli. Effect of the removal of the vomeronasal organs Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, or lesioning of the accessory olfactory bulbs. Neuroendocrinology Kennedy JL, Olson SB, Magenis RE, Amara SG, Grandy DK. 36: 53–58, 1983. Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid 18. Benton D, Wastell V. Effects of androstenol on human sexual diethylamide, and metabolites of the catecholamine neurotransmit- arousal. Biol Psychol 22: 141–147, 1986. ters are agonists of a rat trace amine receptor. Mol Pharmacol 60: 19. Berghard A, Buck LB. Sensory transduction in vomeronasal neu- 1181–1188, 2001. rons: evidence for G alpha o, G alpha i2, and adenylyl cyclase II as 43. Butenandt A, Beckmann R, Stamm D, Hecker E. u¨ber den major components of a pheromone signaling cascade. J Neurosci Sexuallockstoff des Seidenspinners Bombyx mori. Reindarstellung 16: 909–918, 1996. und Konstitution. Naturforsch 14: 283–284, 1959. 20. Berglund H, Lindstrom P, Savic I. Brain response to putative 44. Carr VM, Farbman AI, Colletti LM, Morgan JI. Identiﬁcation of pheromones in lesbian women. Proc Natl Acad Sci USA 103: 8269– a new non-neuronal cell type in rat olfactory epithelium. Neuro- 8274, 2006. science 45: 433–449, 1991. 21. Beynon RJ, Hurst JL. Urinary proteins and the modulation of 45. Catania KC. Evolution of sensory specializations in insectivores. chemical scents in mice and rats. Peptides 25: 1553–1563, 2004. Anat Rec A Discov Mol Cell Evol Biol 287: 1038–1050, 2005. 22. Bluthe RM, Dantzer R. Role of the vomeronasal system in vaso- 46. Cavaggioni A, Mucignat-Caretta C. Major urinary proteins, pressinergic modulation of social recognition in rats. Brain Res alpha(2U)-globulins and aphrodisin. Biochim Biophys Acta 1482: 604: 205–210, 1993. 218–228, 2000. 23. Bocskei Z, Groom CR, Flower DR, Wright CE, Phillips SE, 47. Chamero P, Marton TF, Logan DW, Flanagan K, Cruz JR, Cavaggioni A, Findlay JB, North AC. Pheromone binding to two Saghatelian A, Cravatt BF, Stowers L. Identiﬁcation of protein rodent urinary proteins revealed by X-ray crystallography. Nature pheromones that promote aggressive behaviour. Nature 450: 899–

360: 186–188, 1992. 902, 2007. Downloaded from 24. Boehm N, Gasser B. Sensory receptor-like cells in the human 48. Cheetham SA, Smith AL, Armstrong SD, Beynon RJ, Hurst foetal vomeronasal organ. Neuroreport 4: 867–870, 1993. JL. Limited variation in the major urinary proteins of laboratory 25. Boehm N, Roos J, Gasser B. Luteinizing hormone-releasing hor- mice. Physiol Behav 96: 253–261, 2009. mone (LHRH)-expressing cells in the nasal septum of human fe- 49. Cheetham SA, Thom MD, Jury F, Ollier WE, Beynon RJ, Hurst tuses. Brain Res 82: 175–180, 1994. JL. The genetic basis of individual-recognition signals in the 26. Boehm U, Zou Z, Buck LB. Feedback loops link odor and pher- mouse. Curr Biol 17: 1771–1777, 2007. omone signaling with reproduction. Cell 123: 683–695, 2005. 50. Cherry JA, Pho V. Characterization of cAMP degradation by 27. Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, phosphodiesterases in the accessory olfactory system. Chem physrev.physiology.org Ogozalek KL, Durkin MM, Lakhlani PP, Bonini JA, Pathirana Senses 27: 643–652, 2002. S, Boyle N, Pu X, Kouranova E, Lichtblau H, Ochoa FY, 51. Chess A, Simon I, Cedar H, Axel R. Allelic inactivation regulates Branchek TA, Gerald C. Trace amines: identification of a family olfactory receptor gene expression. Cell 78: 823–834, 1994. of mammalian G protein-coupled receptors. Proc Natl Acad Sci 52. Chirino R, Beyer C, Gonzalez-Mariscal G. Lesion of the main USA 98: 8966–8971, 2001. olfactory epithelium facilitates maternal behavior in virgin rabbits. 28. Boschat C, Pelofi C, Randin O, Roppolo D, Luscher C, Broillet Behav Brain Res 180: 127–132, 2007. MC, Rodriguez I. Pheromone detection mediated by a V1r vome- 52a.Chromek M, Slamova´ Z, Bergman P, Kovaćs L, Podracka´L, ronasal receptor. Nat Neurosci 5: 1261–1262, 2002. Ehreń I, Ho¨kfelt T, Gudmundsson GH, Gallo RL, Agerberth B, 29. Bradley J, Reisert J, Frings S. Regulation of cyclic nucleotide- Brauner A. The antimicrobial peptide cathelicidin protects the gated channels. Curr Opin Neurobiol 15: 343–349, 2005. urinary tract against invasive bacterial infection. Nat Med 12: 636– 30. Brechbuhl J, Klaey M, Broillet MC. Grueneberg ganglion cells 641, 2006. on February 9, 2011 mediate alarm pheromone detection in mice. Science 321: 1092– 53. Cinelli AR, Wang D, Chen P, Liu W, Halpern M. Calcium tran- 1095, 2008. sients in the garter snake vomeronasal organ. J Neurophysiol 87: 31. Breer H, Fleischer J, Strotmann J. The sense of smell: multiple 1449–1472, 2002. olfactory subsystems. Cell Mol Life Sci 63: 1465–1475, 2006. 54. Clancy AN, Coquelin A, Macrides F, Gorski RA, Noble EP. 32. Brennan PA, Schellinck HM, Keverne EB. Patterns of expres- Sexual behavior and aggression in male mice: involvement of the sion of the immediate-early gene egr-1 in the accessory olfactory vomeronasal system. J Neurosci 4: 2222–2229, 1984. bulb of female mice exposed to pheromonal constituents of male 55. Clancy AN, Macrides F, Singer AG, Agosta WC. Male hamster urine. Neuroscience 90: 1463–1470, 1999. copulatory responses to a high molecular weight fraction of vaginal 33. Briand L, Blon F, Trotier D, Pernollet JC. Natural ligands of discharge: effects of vomeronasal organ removal. Physiol Behav hamster aphrodisin. Chem Senses 29: 425–430, 2004. 33: 653–660, 1984. 34. Briand L, Trotier D, Pernollet JC. Aphrodisin, an aphrodisiac 56. Clapham DE, Neer EJ. G protein beta gamma subunits. Annu Rev lipocalin secreted in hamster vaginal secretions. Peptides 25: 1545– Pharmacol Toxicol 37: 167–203, 1997. 1552, 2004. 57. Cocke R, Moynihan JA, Cohen N, Grota LJ, Ader R. Exposure 35. Broman I. Uber die Entwickelung der konstanten grosseren Nas- to conspecific alarm chemosignals alters immune responses in enhohlendr usen der Nagetiere. Zeitschriftf ur Anatomie und En- BALB/c mice. Brain Behav Immun 7: 36–46, 1993. twicklungs-Geschichte 60: 439–586, 1921. 58. Connor JL, Lynds PG. Mouse aggression and the intruder-famil- 36. Brouette-Lahlou I, Godinot F, Vernet-Maury E. The mother iarity effect: evidence for multiple-factor determination c57bl. rat’s vomeronasal organ is involved in detection of dodecyl propi- J Comp Physiol Psychol 91: 270–280, 1977. onate, the pup’s preputial gland pheromone. Physiol Behav 66: 59. Conzelmann S, Malun D, Breer H, Strotmann J. Brain targeting 427–436, 1999. and glomerulus formation of two olfactory neuron populations 37. Bruce HM. An exteroceptive block to pregnancy in the mouse. expressing related receptor types. Eur J Neurosci 14: 1623–1632, Nature 184: 105, 1959. 2001. 38. Bruce HM. Pheromones and behavior in mice. Acta Neurol Psy- 60. Crish SD, Rice FL, Park TJ, Comer CM. Somatosensory orga- chiatr Belg 69: 529–538, 1969. nization and behavior in naked mole-rats I: vibrissa-like body hairs 39. Buck L, Axel R. A novel multigene family may encode odorant comprise a sensory array that mediates orientation to tactile stim- receptors: a molecular basis for odor recognition. Cell 65: 175–187, uli. Brain Behav Evol 62: 141–151, 2003. 1991. 61. Del Punta K, Leinders-Zufall T, Rodriguez I, Jukam D, 40. Buck LB. The molecular architecture of odor and pheromone Wysocki CJ, Ogawa S, Zufall F, Mombaerts P. Deficient pher- sensing in mammals. Cell 100: 611–618, 2000. omone responses in mice lacking a cluster of vomeronasal receptor 41. Buiakova OI, Krishna NS, Getchell TV, Margolis FL. Human genes. Nature 419: 70–74, 2002. and rodent OMP genes: conservation of structural and regulatory 62. Del Punta K, Puche A, Adams NC, Rodriguez I, Mombaerts P. motifs and cellular localization. Genomics 20: 452–462, 1994. A divergent pattern of sensory axonal projections is rendered

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 949

convergent by second-order neurons in the accessory olfactory 89. Gore AC. GnRH: The Master Molecule of Reproduction. Norwell, bulb. Neuron 35: 1057–1066, 2002. MA: Kluwer Academic, 2002. 63. Dennis JC, Allgier JG, Desouza LS, Eward WC, Morrison EE. 90. Graziadei PP, Graziadei GA. Neurogenesis and neuron regener- Immunohistochemistry of the canine vomeronasal organ. J Anat ation in the olfactory system of mammals. I. Morphological aspects 203: 329–338, 2003. of differentiation and structural organization of the olfactory sen- 64. Dibattista M, Mazzatenta A, Grassi F, Tirindelli R, Menini A. sory neurons. J Neurocytol 8: 1–18, 1979. Hyperpolarization-activated cyclic nucleotide-gated channels in 91. Graziadei PP, Monti A, Graziadei G. Neurogenesis and neuron mouse vomeronasal sensory neurons. J Neurophysiol 100: 576– regeneration in the olfactory system of mammals. III. Deafferenta- 586, 2008. tion and reinnervation of the olfactory bulb following section of the 65. Dorries KM, Adkins-Regan E, Halpern BP. Olfactory sensitivity fila olfactoria in rat. J Neurocytol 9: 145–162, 1980. to the pheromone, androstenone, is sexually dimorphic in the pig. 92. Greenwood DR, Comeskey D, Hunt MB, Rasmussen LE. Chem- Physiol Behav 57: 255–259, 1995. ical communication: chirality in elephant pheromones. Nature 438: 66. Doty R. Handbook of Olfaction and Gustation. New York: Dekker, 1097–1098, 2005. 2003. 93. Grimsby J, Toth M, Chen K, Kumazawa T, Klaidman L, Adams 67. Doty RL, Dunbar I. Attraction of beagles to conspecific urine, JD, Karoum F, Gal J, Shih JC. Increased stress response and vaginal and anal sac secretion odors. Physiol Behav 12: 825–833, beta-phenylethylamine in MAOB-deficient mice. Nat Genet 17: 206– 1974. 210, 1997. 68. Doving KB, Trotier D. Structure and function of the vomeronasal 93a.Gru¨neberg H. A ganglion probably belonging to the N. terminals organ. J Exp Biol 201: 2913–2925, 1998. system in the nasal mucosa of the mouse. Z Anat Entwicklungs- 70. Duchamp-Viret P, Chaput MA, Duchamp A. Odor response gesch 140: 39–52, 1973. properties of rat olfactory receptor neurons. Science 284: 2171– 94. Grus WE, Shi P, Zhang J. Largest vertebrate vomeronasal type 1

2174, 1999. receptor gene repertoire in the semiaquatic platypus. Mol Biol Evol Downloaded from 71. Dulac C, Axel R. A novel family of genes encoding putative 24: 2153–2157, 2007. pheromone receptors in mammals. Cell 83: 195–206, 1995. 95. Guo J, Zhou A, Moss RL. Urine and urine-derived compounds 72. Eccles R. Autonomic innervation of the vomeronasal organ of the induce c-fos mRNA expression in accessory olfactory bulb. Neuro- cat. Physiol Behav 28: 1011–1015, 1982. report 8: 1679–1683, 1997. 73. Edwards DA, Burge KG. Olfactory control of the sexual behavior 96. Gustavson AR, Dawson ME, Bonett DG. Androstenol, a putative of male and female mice. Physiol Behav 11: 867–872, 1973. human pheromone, affects human (Homo sapiens) male choice 74. Elsaesser R, Montani G, Tirindelli R, Paysan J. Phosphatidyl- performance. J Comp Psychol 101: 210–212, 1987. Gutierrez-Garcia AG, Contreras CM, Mendoza-Lopez MR, inositide signalling proteins in a novel class of sensory cells in the 97. physrev.physiology.org mammalian olfactory epithelium. Eur J Neurosci 21: 2692–2700, Garcia-Barradas O, Cruz-Sanchez JS. Urine from stressed rats 2005. increases immobility in receptor rats forced to swim: role of 2-heptanone. Physiol Behav 91: 166–172, 2007. 75. Epple G. Communication by chemical signals. In: Comparative 98. Haga S, Yoshihara Y, Touhara K. Male-specific exocrine peptide Primate Biology: Behavior, Conservation and Ecology, edited by ESP1 activates a selective neural pathway via V2Rp5 receptor in Mitchell G and Erwin J. New York: Liss, 1986, vol. 2A, p. 531–580. the mouse vomeronasal system. Chem Senses 33: S90, 2008. 76. Fernandez-Fewell GD, Meredith M. Facilitation of mating be- 99. Hagino-Yamagishi K, Matsuoka M, Ichikawa M, Wakabayashi havior in male hamsters by LHRH and AcLHRH5–10: interaction Y, Mori Y, Yazaki K. The mouse putative pheromone receptor was with the vomeronasal system. Physiol Behav 57: 213–221, 1995. specifically activated by stimulation with male mouse urine. J Bio- 77. Firestein S, Picco C, Menini A. The relation between stimulus chem 129: 509–512, 2001. and response in olfactory receptor cells of the tiger salamander. on February 9, 2011 100. Halem HA, Baum MJ, Cherry JA. Sex difference and steroid J Physiol 468: 1–10, 1993. modulation of pheromone-induced immediate early genes in the 78. Fleischer J, Mamasuew K, Breer H. Expression of cGMP signal- two zones of the mouse accessory olfactory system. J Neurosci ing elements in the Grueneberg ganglion. Histochem Cell Biol 131: 21: 2474–2480, 2001. 75–88, 2009. 101. Halpern M, Martinez-Marcos A. Structure and function of the 79. Fleischer J, Schwarzenbacher K, Besser S, Hass N, Breer H. vomeronasal system: an update. Prog Neurobiol 70: 245–318, 2003. Olfactory receptors and signalling elements in the Grueneberg 102. Halpern M, Shapiro LS, Jia C. Differential localization of G ganglion. J Neurochem 98: 543–554, 2006. proteins in the opossum vomeronasal system. Brain Res 677: 157– 80. Fleischer J, Schwarzenbacher K, Breer H. Expression of trace 161, 1995. amine-associated receptors in the Grueneberg ganglion. Chem 103. Hardie RC. Vision: dynamic platforms. Nature 450: 37–39, 2007. Senses 32: 623–631, 2007. 104. Hart BL, Leedy MG. Analysis of the catnip reaction: mediation by 81. Fulle HJ, Vassar R, Foster DC, Yang RB, Axel R, Garbers DL. olfactory system, not vomeronasal organ. Behav Neural Biol 44: A receptor guanylyl cyclase expressed specifically in olfactory 38–46, 1985. sensory neurons. Proc Natl Acad Sci USA 92: 3571–3575, 1995. 105. Hashiguchi Y, Nishida M. Evolution of trace amine associated 82. Fuss SH, Omura M, Mombaerts P. The Grueneberg ganglion of receptor (TAAR) gene family in vertebrates: lineage-specific expan- the mouse projects axons to glomeruli in the olfactory bulb. Eur sions and degradations of a second class of vertebrate chemosen- J Neurosci 22: 2649–2654, 2005. sory receptors expressed in the olfactory epithelium. Mol Biol Evol 83. Ganzhorn JU, Kappeler PM. [Lemurs of Madagascar. Tests on 24: 2099–2107, 2007. evolution of primate communities.] Naturwissenschaften 80: 195– 106. He J, Ma L, Kim S, Nakai J, Yu CR. Encoding gender and 208, 1993. individual information in the mouse vomeronasal organ. Science 84. Gavaghan McKee CL, Wilson ID, Nicholson JK. Metabolic phe- 320: 535–538, 2008. notyping of nude and normal (Alpk:ApfCD, C57BL10J) mice. J 107. Hebb AL, Zacharko RM, Dominguez H, Trudel F, Laforest S, Proteome Res 5: 378–384, 2006. Drolet G. Odor-induced variation in anxiety-like behavior in mice 85. Giacobini P, Benedetto A, Tirindelli R, Fasolo A. Proliferation is associated with discrete and differential effects on mesocorti- and migration of receptor neurons in the vomeronasal organ of the colimbic cholecystokinin mRNA expression. Neuropsychopharma- adult mouse. Brain Res 123: 33–40, 2000. cology 27: 744–755, 2002. 86. Godfrey PA, Malnic B, Buck LB. The mouse olfactory receptor 108. Hebb AL, Zacharko RM, Gauthier M, Trudel F, Laforest S, gene family. Proc Natl Acad Sci USA 101: 2156–2161, 2004. Drolet G. Brief exposure to predator odor and resultant anxiety 87. Gonzalez-Mariscal G, Chirino R, Beyer C, Rosenblatt JS. Re- enhances mesocorticolimbic activity and enkephalin expression in moval of the accessory olfactory bulbs promotes maternal behav- CD-1 mice. Eur J Neurosci 20: 2415–2429, 2004. ior in virgin rabbits. Behav Brain Res 152: 89–95, 2004. 109. Hedgewig R. [Comparative anatomical studies on the Jacobson 88. Goodwin M, Gooding KM, Regnier F. Sex pheromone in the dog. organs of Nycticebus coucang Boddaert, 1785 (Prosimiae, Lorisi- Science 203: 559–561, 1979. dae) and Galago crassicaudatus E. Geoffroy, 1812 (Prosimiae,

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 950 TIRINDELLI ET AL.

Lorisidae). I. Nycticebus coucang.] Gegenbaurs Morphol Jahrb 126: 134. Jacobson L, Trotier D, Doving KB. Anatomical description of a 543–593, 1980. new organ in the nose of domesticated animals by Ludvig Jacobson 110. Herrada G, Dulac C. A novel family of putative pheromone re- (1813). Chem Senses 23: 743–754, 1998. ceptors in mammals with a topographically organized and sexually 135. Jakupovic J, Kang N, Baum MJ. Effect of bilateral accessory dimorphic distribution. Cell 90: 763–773, 1997. olfactory bulb lesions on volatile urinary odor discrimination and 111. Hoffmann KH, Dettner K, Tomaschko KH. Chemical signals in investigation as well as mating behavior in male mice. Physiol insects and other arthropods: from molecular structure to physio- Behav 93: 467–473, 2008. logical functions. Physiol Biochem Zool 79: 344–356, 2006. 136. Jang T, Singer AG, O’Connell RJ. Induction of c-fos in hamster 112. Hofmann T, Chubanov V, Gudermann T, Montell C. TRPM5 is accessory olfactory bulbs by natural and cloned aphrodisin. Neu- a voltage-modulated and Ca2ϩ-activated monovalent selective cat- roreport 12: 449–452, 2001. ion channel. Curr Biol 13: 1153–1158, 2003. 137. Jemiolo B, Andreolini F, Xie TM, Wiesler D, Novotny M. 113. Holekamp TF, Turaga D, Holy TE. Fast three-dimensional fluo- Puberty-affecting synthetic analogs of urinary chemosignals in the rescence imaging of activity in neural populations by objective- house mouse, Mus domesticus. Physiol Behav 46: 293–298, 1989. coupled planar illumination microscopy. Neuron 57: 661–672, 2008. 138. Jemiolo B, Harvey S, Novotny M. Promotion of the Whitten 114. Holst DV, Eichmann F. Sex-specific regulation of marking behav- effect in female mice by synthetic analogs of male urinary constit- ior by sex hormones and conspecifics scent in tree shrews (Tupaia uents. Proc Natl Acad Sci USA 83: 4576–4579, 1986. belangeri). Physiol Behav 63: 157–164, 1998. 139. Jemiolo B, Xie TM, Novotny M. Socio-sexual olfactory prefer- 115. Holy TE, Dulac C, Meister M. Responses of vomeronasal neu- ence in female mice: attractiveness of synthetic chemosignals. rons to natural stimuli. Science 289: 1569–1572, 2000. Physiol Behav 50: 1119–1122, 1991. 116. Honda N, Sakamoto H, Inamura K, Kashiwayanagi M. Changes 140. Jemiolo B, Xie TM, Novotny M. Urine marking in male mice: in Fos expression in the accessory olfactory bulb of sexually responses to natural and synthetic chemosignals. Physiol Behav experienced male rats after exposure to female urinary phero- 52: 521–526, 1992. Downloaded from mones. Eur J Neurosci 27: 1980–1988, 2008. 141. Jia C, Chen WR, Shepherd GM. Synaptic organization and neu- 117. Horowitz LF, Montmayeur JP, Echelard Y, Buck LB. A genetic rotransmitters in the rat accessory olfactory bulb. J Neurophysiol approach to trace neural circuits. Proc Natl Acad Sci USA 96: 81: 345–355, 1999. 3194–3199, 1999. 142. Jia C, Goldman G, Halpern M. Development of vomeronasal 118. Hu J, Zhong C, Ding C, Chi Q, Walz A, Mombaerts P, Matsu- receptor neuron subclasses and establishment of topographic pro- nami H, Luo M. Detection of near-atmospheric concentrations of jections to the accessory olfactory bulb. Brain Res 102: 209–216, 1997. CO2 by an olfactory subsystem in the mouse. Science 317: 953–957, 2007. 143. Jia C, Halpern M. Subclasses of vomeronasal receptor neurons: physrev.physiology.org differential expression of G proteins [Gi alpha 2 and G(o alpha)] 119. Hudson R, Distel H. Pheromonal release of suckling in rabbits and segregated projections to the accessory olfactory bulb. Brain does not depend on the vomeronasal organ. Physiol Behav 37: Res 719: 117–128, 1996. 123–128, 1986. 144. Johnson EW, Eller PM, Jafek BW. An immuno-electron micro- 120. Hull EM, Dominguez JM. Getting his act together: roles of glu- scopic comparison of olfactory marker protein localization in the tamate, nitric oxide, and dopamine in the medial preoptic area. supranuclear regions of the rat olfactory epithelium and vomero- Brain Res 1126: 66–75, 2006. nasal organ neuroepithelium. Acta Otolaryngol 113: 766–771, 1993. 121. Hull EM, Dominguez JM. Sexual behavior in male rodents. Horm 145. Johnston RE. Vomeronasal and/or olfactory mediation of ultra- Behav 52: 45–55, 2007. sonic calling and scent marking by female golden hamsters. 122. Hurst JL, Beynon RJ. Scent wars: the chemobiology of compet- Physiol Behav 51: 437–448, 1992. on February 9, 2011 itive signalling in mice. Bioessays 26: 1288–1298, 2004. 146. Johnston RE, Rasmussen K. Individual recognition of female 123. Hurst JL, Payne CE, Nevison CM, Marie AD, Humphries RE, hamsters by males: role of chemical cues and of the olfactory and Robertson DH, Cavaggioni A, Beynon RJ. Individual recogni- vomeronasal systems. Physiol Behav 33: 95–104, 1984. tion in mice mediated by major urinary proteins. Nature 414: 147. Jolly A. Lemur social behavior and primate intelligence. Science 631–634, 2001. 153: 501–506, 1966. 124. Inamura K, Kashiwayanagi M. Inward current responses to uri- 148. Jourdan F. [Ultrastructure of the olfactory epithelium of the rat: nary substances in rat vomeronasal sensory neurons. Eur J Neu- polymorphism of the receptors]. C R Acad Sci Hebd Seances Acad rosci 12: 3529–3536, 2000. Sci D 280: 443–446, 1975. 125. Inamura K, Kashiwayanagi M, Kurihara K. Inositol-1,4,5- 149. Juilfs DM, Fulle HJ, Zhao AZ, Houslay MD, Garbers DL, trisphosphate induces responses in receptor neurons in rat vome- Beavo JA. A subset of olfactory neurons that selectively express ronasal sensory slices. Chem Senses 22: 93–103, 1997. cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cy- 126. Inamura K, Kashiwayanagi M, Kurihara K. Regionalization of clase-D define a unique olfactory signal transduction pathway. Proc Fos immunostaining in rat accessory olfactory bulb when the Natl Acad Sci USA 94: 3388–3395, 1997. vomeronasal organ was exposed to urine. Eur J Neurosci 11: 150. Kaluza JF, Gussing F, Bohm S, Breer H, Strotmann J. Olfac- 2254–2260, 1999. tory receptors in the mouse septal organ. J Neurosci Res 76: 127. Ishii T, Mombaerts P. Expression of nonclassical class I major 442–452, 2004. histocompatibility genes defines a tripartite organization of the 151. Karlson P, Luscher M. Pheromones’: a new term for a class of mouse vomeronasal system. J Neurosci 28: 2332–2341, 2008. biologically active substances. Nature 183: 55–56, 1959. 128. Iwema CL, Fang H, Kurtz DB, Youngentob SL, Schwob JE. 152. Karunadasa DK, Chapman C, Bicknell RJ. Expression of pher- Odorant receptor expression patterns are restored in lesion-recov- omone receptor gene families during olfactory development in the ered rat olfactory epithelium. J Neurosci 24: 356–369, 2004. mouse: expression of a V1 receptor in the main olfactory epithe- 130. Jackson DE, Ratnieks FL. Communication in ants. Curr Biol 16: lium. Eur J Neurosci 23: 2563–2572, 2006. R570–R574, 2006. 153. Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. 131. Jackson LM, Harder JD. Vomeronasal organ removal blocks Physiol Rev 82: 769–824, 2002. pheromonal induction of estrus in gray short-tailed opossums (Mo- 154. Keller M, Douhard Q, Baum MJ, Bakker J. Destruction of the nodelphis domestica). Biol Reprod 54: 506–512, 1996. main olfactory epithelium reduces female sexual behavior and 132. Jacob S, Kinnunen LH, Metz J, Cooper M, McClintock MK. olfactory investigation in female mice. Chem Senses 31: 315–323, Sustained human chemosignal unconsciously alters brain function. 2006. Neuroreport 12: 2391–2394, 2001. 155. Keller M, Pierman S, Douhard Q, Baum MJ, Bakker J. The 133. Jacob S, Zelano B, Gungor A, Abbott D, Naclerio R, McClintock vomeronasal organ is required for the expression of lordosis be- MK. Location and gross morphology of the nasopalatine duct in haviour, but not sex discrimination in female mice. Eur J Neurosci human adults. Arch Otolaryngol Head Neck Surg 126: 741–748, 2000. 23: 521–530, 2006.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 951

156. Kelliher KR, Spehr M, Li XH, Zufall F, Leinders-Zufall T. lebacks: an immuno-ecological approach. Proc Biol Sci 273: 1407– Pheromonal recognition memory induced by TRPC2-independent 1414, 2006. vomeronasal sensing. Eur J Neurosci 23: 3385–3390, 2006. 181. Labov JB, Wysocki CJ. Vomeronasal organ and social factors 157. Keverne EB. Pheromones, vomeronasal function, and gender- affect urine marking by male mice. Physiol Behav 45: 443–447, speciﬁc behavior. Cell 108: 735–738, 2002. 1989. 158. Kevetter GA, Winans SS. Connections of the corticomedial 182. Larriva-Sahd J. The accessory olfactory bulb in the adult rat: a amygdala in the golden hamster. I. Efferents of the “vomeronasal cytological study of its cell types, neuropil, neuronal modules, and amygdala.” J Comp Neurol 197: 81–98, 1981. interactions with the main olfactory system. J Comp Neurol 510: 159. Kevetter GA, Winans SS. Connections of the corticomedial 309–350, 2008. amygdala in the golden hamster. II. Efferents of the “olfactory 183. Lau YE, Cherry JA. Distribution of PDE4A and G(o) alpha immu- amygdala.” J Comp Neurol 197: 99–111, 1981. noreactivity in the accessory olfactory system of the mouse. Neu- 160. Kikusui T, Takigami S, Takeuchi Y, Mori Y. Alarm pheromone roreport 11: 27–32, 2000. enhances stress-induced hyperthermia in rats. Physiol Behav 72: 183a.Le Y, Murphy PM, Wang JM. Formyl-peptide receptors revisited. 45–50, 2001. Trends Immunol 23: 541–548, 2002. 161. Kimchi T, Xu J, Dulac C. A functional circuit underlying male 184. Lee SJ, Mammen A, Kim EJ, Kim SY, Park YJ, Park M, Han sexual behaviour in the female mouse brain. Nature 448: 1009– HS, Bae YC, Ronnett GV, Moon C. The vomeronasal organ and 1014, 2007. adjacent glands express components of signaling cascades found in 162. Kimoto H, Haga S, Sato K, Touhara K. Sex-speciﬁc peptides sensory neurons in the main olfactory system. Mol Cells 26: 2008. from exocrine glands stimulate mouse vomeronasal sensory neu- 185. Leinders-Zufall T, Brennan P, Widmayer P, PCS, Maul-Pavicic rons. Nature 437: 898–901, 2005. A, Jager M, Li XH, Breer H, Zufall F, Boehm T. MHC class I 163. Kimoto H, Sato K, Nodari F, Haga S, Holy TE, Touhara K. Sex- peptides as chemosensory signals in the vomeronasal organ. Sci-

and strain-speciﬁc expression and vomeronasal activity of mouse ence 306: 1033–1037, 2004. Downloaded from ESP family peptides. Curr Biol 17: 1879–1884, 2007. 186. Leinders-Zufall T, Cockerham RE, Michalakis S, Biel M, Gar- 164. Kiyokawa Y, Kikusui T, Takeuchi Y, Mori Y. Partner’s stress bers DL, Reed RR, Zufall F, Munger SD. Contribution of the status inﬂuences social buffering effects in rats. Behav Neurosci receptor guanylyl cyclase GC-D to chemosensory function in the 118: 798–804, 2004. olfactory epithelium. Proc Natl Acad Sci USA 104: 14507–14512, 165. Kiyokawa Y, Takeuchi Y, Mori Y. Two types of social buffering 2007. differentially mitigate conditioned fear responses. Eur J Neurosci 187. Leinders-Zufall T, Lane AP, Puche AC, Ma W, Novotny MV, 26: 3606–3613, 2007. Shipley MT, Zufall F. Ultrasensitive pheromone detection by

166. Kjaer I, Fischer HB. The human vomeronasal organ: prenatal mammalian vomeronasal neurons. Nature 405: 792–796, 2000. physrev.physiology.org developmental stages and distribution of luteinizing hormone-re- 188. Leon M, Moltz H. Endocrine control of the maternal pheromone leasing hormone. Eur J Oral Sci 104: 34–40, 1996. in the postpartum female rat. Physiol Behav 10: 65–67, 1973. 167. Kleene SJ. Origin of the chloride current in olfactory transduc- 189. Lepri JJ, Wysocki CJ. Removal of the vomeronasal organ dis- tion. Neuron 11: 123–132, 1993. rupts the activation of reproduction in female voles. Physiol Behav 168. Kleene SJ. The electrochemical basis of odor transduction in 40: 349–355, 1987. vertebrate olfactory cilia. Chem Senses 33: 839–859, 2008. 190. Levai O, Feistel T, Breer H, Strotmann J. Cells in the vomero- 169. Kobayakawa K, Kobayakawa R, Matsumoto H, Oka Y, Imai T, nasal organ express odorant receptors but project to the accessory Ikawa M, Okabe M, Ikeda T, Itohara S, Kikusui T, Mori K, olfactory bulb. J Comp Neurol 498: 476–490, 2006. Sakano H. Innate versus learned odour processing in the mouse 191. Levai O, Strotmann J. Projection pattern of nerve ﬁbers from the

olfactory bulb. Nature 450: 503–508, 2007. septal organ: DiI-tracing studies with transgenic OMP mice. Histo- on February 9, 2011 170. Kolunie JM, Stern JM. Maternal aggression in rats: effects of chem Cell Biol 120: 483–492, 2003.

olfactory bulbectomy, ZnSO4-induced anosmia, and vomeronasal 192. Lewin AH. Receptors of mammalian trace amines. AAPS J 8: organ removal. Horm Behav 29: 492–518, 1995. E138–E145, 2006. 171. Kondo Y, Sudo T, Tomihara K, Sakuma Y. Activation of acces- 193. Leypold BG, Yu CR, Leinders-Zufall T, Kim MM, Zufall F, Axel sory olfactory bulb neurons during copulatory behavior after de- R. Altered sexual and social behaviors in trp2 mutant mice. Proc privation of vomeronasal inputs in male rats. Brain Res 962: 232– Natl Acad Sci USA 99: 6376–6381, 2002. 236, 2003. 194. Liberles SD, Buck LB. A second class of chemosensory receptors 172. Kondo Y, Tomihara K, Sakuma Y. Sensory requirements for in the olfactory epithelium. Nature 442: 645–650, 2006. noncontact penile erection in the rat. Behav Neurosci 113: 1062– 195. Liman ER. Regulation by voltage and adenine nucleotides of a 1070, 1999. Ca2ϩ-activated cation channel from hamster vomeronasal sensory 173. Koos DS, Fraser SE. The Grueneberg ganglion projects to the neurons. J Physiol 548: 777–787, 2003. olfactory bulb. Neuroreport 16: 1929–1932, 2005. 196. Liman ER, Corey DP. Electrophysiological characterization of 174. Kow LM, Florea C, Schwanzel-Fukuda M, Devidze N, Kami chemosensory neurons from the mouse vomeronasal organ. J Neu- KH, Lee A, Zhou J, Maclaughlin D, Donahoe P, Pfaff D. Devel- rosci 16: 4625–4637, 1996. opment of a sexually differentiated behavior and its underlying 197. Liman ER, Corey DP, Dulac C. TRP2: a candidate transduction CNS arousal functions. Curr Top Dev Biol 79: 37–59, 2007. channel for mammalian pheromone sensory signaling. Proc Natl 175. Koyama S, Kamimura S. Effects of vomeronasal organ removal Acad Sci USA 96: 5791–5796, 1999. on the sperm motility in male mice. Zool Sci 20: 1355–1358, 2003. 198. Liman ER, Innan H. Relaxed selective pressure on an essential 176. Krieger J, Schmitt A, Lobel D, Gudermann T, Schultz G, component of pheromone transduction in primate evolution. Proc Breer H, Boekhoff I. Selective activation of G protein subtypes in Natl Acad Sci USA 100: 3328–3332, 2003. the vomeronasal organ upon stimulation with urine-derived com- 199. Lin DY, Zhang SZ, Block E, Katz LC. Encoding social signals in pounds. J Biol Chem 274: 4655–4662, 1999. the mouse main olfactory bulb. Nature 434: 470–477, 2005. 177. Kroner C, Breer H, Singer AG, O’Connell RJ. Pheromone- 200. Lin W, Arellano J, Slotnick B, Restrepo D. Odors detected by

induced second messenger signaling in the hamster vomeronasal mice deﬁcient in cyclic nucleotide-gated channel subunit A2 stim- organ. Neuroreport 7: 2989–2992, 1996. ulate the main olfactory system. J Neurosci 24: 3703–3710, 2004. 178. Kurahashi T, Menini A. Mechanism of odorant adaptation in the 201. Lin W, Margolskee R, Donnert G, Hell SW, Restrepo D. Olfac- olfactory receptor cell. Nature 385: 725–729, 1997. tory neurons expressing transient receptor potential channel M5 179. Kurahashi T, Yau KW. Co-existence of cationic and chloride (TRPM5) are involved in sensing semiochemicals. Proc Natl Acad components in odorant-induced current of vertebrate olfactory Sci USA 104: 2471–2476, 2007. receptor cells. Nature 363: 71–74, 1993. 202. Lin W, Ogura T, Margolskee RF, Finger TE, Restrepo D. 180. Kurtz J, Wegner KM, Kalbe M, Reusch TB, Schaschl H, Has- TRPM5-expressing solitary chemosensory cells respond to odorous selquist D, Milinski M. MHC genes and oxidative stress in stick- irritants. J Neurophysiol 99: 1451–1460, 2008.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 952 TIRINDELLI ET AL.

203. Lindemann L, Hoener MC. A renaissance in trace amines in- 226. Malnic B, Hirono J, Sato T, Buck LB. Combinatorial receptor spired by a novel GPCR family. Trends Pharmacol Sci 26: 274–281, codes for odors. Cell 96: 713–723, 1999. 2005. 227. Mandiyan VS, Coats JK, Shah NM. Deficits in sexual and aggres- 2ϩ 204. Liu D, Liman ER. Intracellular Ca and the phospholipid PIP2 sive behaviors in Cnga2 mutant mice. Nat Neurosci 8: 1660–1662, regulate the taste transduction ion channel TRPM5. Proc Natl Acad 2005. Sci USA 100: 15160–15165, 2003. 228. Martel KL, Baum MJ. Sexually dimorphic activation of the acces- 205. Lledo PM, Saghatelyan A. Integrating new neurons into the adult sory, but not the main, olfactory bulb in mice by urinary volatiles. olfactory bulb: joining the network, life-death decisions, and the Eur J Neurosci 26: 463–475, 2007. effects of sensory experience. Trends Neurosci 28: 248–254, 2005. 229. Martinez-Marcos A. On the organization of olfactory and vome- 206. Lloyd-Thomas A, Keverne EB. Role of the brain and accessory ronasal cortices. Prog Neurobiol 87: 21–30, 2009. olfactory system in the block to pregnancy in mice. Neuroscience 7: 230. Martinez-Marcos A, Halpern M. Differential projections from 907–913, 1982. the anterior and posterior divisions of the accessory olfactory bulb 207. Loconto J, Papes F, Chang E, Stowers L, Jones EP, Takada T, to the medial amygdala in the opossum, Monodelphis domestica. Kumanovics A, Fischer LK, Dulac C. Functional expression of Eur J Neurosci 11: 3789–3799, 1999. murine V2R pheromone receptors involves selective association 231. Martini S, Silvotti L, Shirazi A, Ryba NJ, Tirindelli R. Co- with the M10 and M1 families of MHC class Ib molecules. Cell 112: expression of putative pheromone receptors in the sensory neu- 607–618, 2003. rons of the vomeronasal organ. J Neurosci 21: 843–848, 2001. 208. Logan DW, Marton TF, Stowers L. Species specificity in major 232. Martins Y, Preti G, Crabtree CR, Runyan T, Vainius AA, urinary proteins by parallel evolution. PLoS ONE 3: e3280, 2008. Wysocki CJ. Preference for human body odors is influenced by 209. Lomas DE, Keverne EB. Role of the vomeronasal organ and gender and sexual orientation. Psychol Sci 16: 694–701, 2005. prolactin in the acceleration of puberty in female mice. J Reprod 233. Maruniak JA, Wysocki CJ, Taylor JA. Mediation of male mouse

Fertil 66: 101–107, 1982. urine marking and aggression by the vomeronasal organ. Physiol Downloaded from 210. Lowe G. Flash photolysis reveals a diversity of ionotropic gluta- Behav 37: 655–657, 1986. mate receptors on the mitral cell somatodendritic membrane. 234. Matsunami H, Buck LB. A multigene family encoding a diverse J Neurophysiol 90: 1737–1746, 2003. array of putative pheromone receptors in mammals. Cell 90: 775– 211. Lucas P, Ukhanov K, Leinders-Zufall T, Zufall F. A diacylglyc- 784, 1997. erol-gated cation channel in vomeronasal neuron dendrites is im- 235. Matthews HR, Reisert J. Calcium, the two-faced messenger of paired in TRPC2 mutant mice: mechanism of pheromone transduc- olfactory transduction and adaptation. Curr Opin Neurobiol 13: tion. Neuron 40: 551–561, 2003. 469–475, 2003. McCarthy MM, Auger AP. 212. Lundstrom JN, Hummel T, Olsson MJ. Individual differences in 236. He’s a lover, not a fighter–smell, sex physrev.physiology.org sensitivity to the odor of 4,16-androstadien-3-one. Chem Senses 28: and civility. Trends Endocrinol Metab 13: 183–184, 2002. 643–650, 2003. 237. McClintock MK. Menstrual synchorony and suppression. Nature 229: 244–245, 1971. 213. Luo M, Fee MS, Katz LC. Encoding pheromonal signals in the 238. McClintock MK. Estrous synchrony: modulation of ovarian cycle accessory olfactory bulb of behaving mice. Science 299: 1196–1201, length by female pheromones. Physiol Behav 32: 701–705, 1984. 2003. 239. McClintock MK. On the nature of mammalian and human phero- 214. Luo M, Katz LC. Encoding pheromonal signals in the mammalian mones. Ann NY Acad Sci 855: 390–392, 1998. vomeronasal system. Curr Opin Neurobiol 14: 428–434, 2004. 240. Meisami E, Bhatnagar KP. Structure and diversity in mammalian 215. Luo Y, Lu S, Chen P, Wang D, Halpern M. Identification of accessory olfactory bulb. Microsc Res Tech 43: 476–499, 1998. chemoattractant receptors and G-proteins in the vomeronasal sys- 241. Menco BP, Carr VM, Ezeh PI, Liman ER, Yankova MP. Ultra- tem of garter snakes. J Biol Chem 269: 16867–16877, 1994. on February 9, 2011 structural localization of G-proteins and the channel protein TRP2 216. Ma D, Allen ND, Van Bergen YC, Jones CM, Baum MJ, to microvilli of rat vomeronasal receptor cells. J Comp Neurol 438: Keverne EB, Brennan PA. Selective ablation of olfactory recep- 468–489, 2001. tor neurons without functional impairment of vomeronasal recep- 242. Menini A. Calcium signalling and regulation in olfactory neurons. tor neurons in OMP-ntr transgenic mice. Eur J Neurosci 16: 2317– Curr Opin Neurobiol 9: 419–426, 1999. 2323, 2002. 243. Mennella JA, Moltz H. Infanticide in the male rat: the role of the 217. Ma M. Encoding olfactory signals via multiple chemosensory sys- vomeronasal organ. Physiol Behav 42: 303–306, 1988. tems. Crit Rev Biochem Mol Biol 42: 463–480, 2007. 244. Meredith M. Chronic recording of vomeronasal pump activation 218. Ma M, Grosmaitre X, Iwema CL, Baker H, Greer CA, Shep- in awake behaving hamsters. Physiol Behav 56: 345–354, 1994. herd GM. Olfactory signal transduction in the mouse septal organ. 245. Meredith M. Vomeronasal function. Chem Senses 23: 463–466, J Neurosci 23: 317–324, 2003. 1998. 219. Ma W, Miao Z, Novotny MV. Role of the adrenal gland and 246. Meredith M. Human vomeronasal organ function: a critical review adrenal-mediated chemosignals in suppression of estrus in the of best and worst cases. Chem Senses 26: 433–445, 2001. house mouse: the lee-boot effect revisited. Biol Reprod 59: 1317– 247. Meredith M, Howard G. Intracerebroventricular LHRH relieves 1320, 1998. behavioral deficits due to vomeronasal organ removal. Brain Res 220. Mackay-Sim A, Laing DG. Discrimination of odors from stressed Bull 29: 75–79, 1992. rats by non-stressed rats. Physiol Behav 24: 699–704, 1980. 248. Meredith M, Marques DM, O’Connell RO, Stern FL. Vomero- 221. Mackay-Sim A, Rose JD. Removal of the vomeronasal organ nasal pump: significance for male hamster sexual behavior. Science impairs lordosis in female hamsters: effect is reversed by luteinis- 207: 1224–1226, 1980. ing hormone-releasing hormone. Neuroendocrinology 42: 489–493, 249. Meredith M, O’Connell RJ. Efferent control of stimulus access to 1986. the hamster vomeronasal organ. J Physiol 286: 301–316, 1979. 222. Macrides F, Singer AG, Clancy AN, Goldman BD, Agosta WC. 249a.Migeotte I, Communi D, Parmentier M. Formyl peptide recep- Male hamster investigatory and copulatory responses to vaginal tors: a promiscuous subfamily of G protein-coupled receptors con- discharge: relationship to the endocrine status of females. Physiol trolling immune responses. Cytokine Growth Factor Rev 17: 501– Behav 33: 633–637, 1984. 519, 2006. 223. Magert HJ, Cieslak A, Alkan O, Luscher B, Kauffels W, Forss- 250. Minke B, Parnas M. Insights on TRP channels from in vivo studies mann WG. The golden hamster aphrodisin gene. Structure, expres- in Drosophila. Annu Rev Physiol 68: 649–684, 2006. sion in parotid glands of female animals, and comparison with a 251. Miyamichi K, Serizawa S, Kimura HM, Sakano H. Continuous similar murine gene. J Biol Chem 274: 444–450, 1999. and overlapping expression domains of odorant receptor genes in 224. Malnic B. Searching for the ligands of odorant receptors. Mol the olfactory epithelium determine the dorsal/ventral positioning of Neurobiol 35: 175–181, 2007. glomeruli in the olfactory bulb. J Neurosci 25: 3586–3592, 2005. 225. Malnic B, Godfrey PA, Buck LB. The human olfactory receptor 252. Mohedano-Moriano A, Pro-Sistiaga P, Ubeda-Banon I, Crespo gene family. Proc Natl Acad Sci USA 101: 2584–2589, 2004. C, Insausti R, Martinez-Marcos A. Segregated pathways to the

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 953

vomeronasal amygdala: differential projections from the anterior 277. Nodari F, Hsu FF, Fu X, Holekamp TF, Kao LF, Turk J, Holy and posterior divisions of the accessory olfactory bulb. Eur J Neu- TE. Sulfated steroids as natural ligands of mouse pheromone- rosci 25: 2065–2080, 2007. sensing neurons. J Neurosci 28: 6407–6418, 2008. 253. Mohedano-Moriano A, Pro-Sistiaga P, Ubeda-Banon I, Rosa- 278. Norlin EM, Gussing F, Berghard A. Vomeronasal phenotype and Prieto C, Saiz-Sanchez D, Martinez-Marcos A. V1R and V2R behavioral alterations in G alpha i2 mutant mice. Curr Biol 13: segregated vomeronasal pathways to the hypothalamus. Neurore- 1214–1219, 2003. port 19: 1623–1626, 2008. 279. Novotny M, Harvey S, Jemiolo B. Chemistry of male dominance 254. Moltz H, Lee TM. The maternal pheromone of the rat: identity and in the house mouse, Mus domesticus. Experientia 46: 109–113, functional signiﬁcance. Physiol Behav 26: 301–306, 1981. 1990. 255. Mombaerts P. Seven-transmembrane proteins as odorant and che- 280. Novotny M, Harvey S, Jemiolo B, Alberts J. Synthetic phero- mosensory receptors. Science 286: 707–711, 1999. mones that promote inter-male aggression in mice. Proc Natl Acad 256. Mombaerts P. Genes and ligands for odorant, vomeronasal and Sci USA 82: 2059–2061, 1985. taste receptors. Nat Rev Neurosci 5: 263–278, 2004. 281. Novotny M, Jemiolo B, Harvey S, Wiesler D, Marchlewska-Koj 257. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendel- A. Adrenal-mediated endogenous metabolites inhibit puberty in sohn M, Edmondson J, Axel R. Visualizing an olfactory sensory female mice. Science 231: 722–725, 1986. map. Cell 87: 675–686, 1996. 282. Novotny MV, Jemiolo B, Wiesler D, Ma W, Harvey S, Xu F, Xie TM, Carmack M. A unique urinary constituent, 6-hydroxy-6-methyl- 258. Montagna W, Yun JS. The skin of primates. X. The skin of the 3-heptanone, is a pheromone that accelerates puberty in female mice. ring-tailed lemur (Lemur catta). Am J Phys Anthropol 20: 95–117, Chem Biol 6: 377–383, 1999. 1962. 283. Novotny MV, Ma W, Wiesler D, Zidek L. Positive identiﬁcation of 259. Moran DT, Jafek BW, Rowley JC III. The vomeronasal (Jacob- the puberty-accelerating pheromone of the house mouse: the vol- son’s) organ in man: ultrastructure and frequency of occurrence. J

atile ligands associating with the major urinary protein. Proc Biol Downloaded from Steroid Biochem Mol Biol 39: 545–552, 1991. Sci 266: 2017–2022, 1999. 260. Moran DT, Rowley JC III, Jafek BW. Electron microscopy of 284. Pankevich DE, Baum MJ, Cherry JA. Olfactory sex discrimina- human olfactory epithelium reveals a new cell type: the microvillar tion persists, whereas the preference for urinary odorants from cell. Brain Res 253: 39–46, 1982. estrous females disappears in male mice after vomeronasal organ 261. Mori K. Monoclonal antibodies (2C5 and 4C9) against lactoseries removal. J Neurosci 24: 9451–9457, 2004. carbohydrates identify subsets of olfactory and vomeronasal recep- 285. Pantages E, Dulac C. A novel family of candidate pheromone tor cells and their axons in the rabbit. Brain Res 408: 215–221, 1987. receptors in mammals. Neuron 28: 835–845, 2000. 262. Morrison EE, Costanzo RM. Morphology of the human olfactory 286. Paulos MA, Tessel RE. Excretion of beta-phenethylamine is ele- physrev.physiology.org epithelium. J Comp Neurol 297: 1–13, 1990. vated in humans after profound stress. Science 215: 1127–1129, 263. Mucignat-Caretta C. Modulation of exploratory behavior in fe- 1982. male mice by protein-borne male urinary molecules. J Chem Ecol 287. Pearce GP, Hughes PE. The influence of male contact on plasma 28: 1853–1863, 2002. cortisol concentrations in the prepubertal gilt. J Reprod Fertil 80: 264. Mucignat-Caretta C, Caretta A. Urinary chemical cues affect 417–424, 1987. light avoidance behaviour in male laboratory mice, Mus musculus. 288. Penn D, Potts W. MHC-disassortative mating preferences re- Anim Behav 57: 765–769, 1999. versed by cross-fostering. Proc Biol Sci 265: 1299–1306, 1998. 265. Mucignat-Caretta C, Caretta A, Baldini E. Protein-bound male 289. Petrovich GD, Canteras NS, Swanson LW. Combinatorial amyg- urinary pheromones: differential responses according to age and dalar inputs to hippocampal domains and hypothalamic behavior gender. Chem Senses 23: 67–70, 1998. systems. Brain Res 38: 247–289, 2001. on February 9, 2011 266. Mucignat-Caretta C, Caretta A, Cavaggioni A. Acceleration of 290. Petrulis A, Peng M, Johnston RE. Effects of vomeronasal organ puberty onset in female mice by male urinary proteins. J Physiol removal on individual odor discrimination, sex-odor preference, 486: 517–522, 1995. and scent marking by female hamsters. Physiol Behav 66: 73–83, 267. Mucignat-Caretta C, Cavaggioni A, Caretta A. Male urinary 1999. chemosignals differentially affect aggressive behavior in male 291. Pfeiffer CA, Johnston RE. Hormonal and behavioral responses mice. J Chem Ecol 30: 777–791, 2004. of male hamsters to females and female odors: roles of olfaction, 268. Muller-Schwarze D. Chemical Ecology: Odor Communication in the vomeronasal system, and sexual experience. Physiol Behav 55: Animals. New York: Elsevier, 1979. 129–138, 1994. 269. Munger SD, Leinders-Zufall T, Zufall F. Subsystem organization 292. Pifferi S, Boccaccio A, Menini A. Cyclic nucleotide-gated ion of the mammalian sense of smell. Annu Rev Physiol 71: 115–140, channels in sensory transduction. FEBS Lett 580: 2853–2859, 2006. 2008. 293. Pin JP, Kniazeff J, Goudet C, Bessis AS, Liu J, Galvez T, 270. Muroi Y, Ishii T, Komori S, Kitamura N, Nishimura M. Volatile Acher F, Rondard P, Prezeau L. The activation mechanism of class-C G-protein coupled receptors. Biol Cell 96: 335–342, 2004. female odors activate the accessory olfactory system of male mice 294. Portfors CV. Types and functions of ultrasonic vocalizations in without physical contact. Neuroscience 141: 551–558, 2006. laboratory rats and mice. J Am Assoc Lab Anim Sci 46: 28–34, 271. Murphy FA, Tucker K, Fadool DA. Sexual dimorphism and 2007. developmental expression of signal-transduction machinery in the 295. Portillo W, Diaz NF, Retana-Marquez S, Paredes RG. Olfac- vomeronasal organ. J Comp Neurol 432: 61–74, 2001. tory, partner preference and Fos expression in the vomeronasal 272. Newman R, Winans SS. An experimental study of the ventral projection pathway of sexually sluggish male rats. Physiol Behav striatum of the golden hamster. II. Neuronal connections of the 88: 389–397, 2006. olfactory tubercle. J Comp Neurol 191: 193–212, 1980. 296. Potthast R, Potter LR. Phosphorylation-dependent regulation of 273. Ngai J, Dowling MM, Buck L, Axel R, Chess A. The family of the guanylyl cyclase-linked natriuretic peptide receptors. Peptides genes encoding odorant receptors in the channel catfish. Cell 72: 26: 1001–1008, 2005. 657–666, 1993. 297. Potts WK, Wakeland EK. Evolution of MHC genetic diversity: a 274. Niimura Y, Nei M. Evolutionary dynamics of olfactory receptor tale of incest, pestilence and sexual preference. Trends Genet 9: genes in fishes and tetrapods. Proc Natl Acad Sci USA 102: 6039– 408–412, 1993. 6044, 2005. 298. Powers JB, Winans SS. Vomeronasal organ: critical role in me- 275. Nilius B, Vennekens R. From cardiac cation channels to the diating sexual behavior of the male hamster. Science 187: 961–963, molecular dissection of the transient receptor potential channel 1975. TRPM4. Pflu¨gers Arch 453: 313–321, 2006. 299. Prawitt D, Monteilh-Zoller MK, Brixel L, Spangenberg C, 276. Nishimura K, Utsumi K, Yuhara M, Fujitani Y, Iritani A. Iden- Zabel B, Fleig A, Penner R. TRPM5 is a transient Ca2ϩ-activated 2ϩ tification of puberty-accelerating pheromones in male mouse urine. cation channel responding to rapid changes in [Ca ]i. Proc Natl J Exp Zool 251: 300–305, 1989. Acad Sci USA 100: 15166–15171, 2003.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 954 TIRINDELLI ET AL.

300. Preti G, Wysocki CJ, Barnhart KT, Sondheimer SJ, Leyden 323. Ryba NJ, Tirindelli R. A new multigene family of putative pher- JJ. Male axillary extracts contain pheromones that affect pulsatile omone receptors. Neuron 19: 371–379, 1997. secretion of luteinizing hormone and mood in women recipients. 324. Saito TR, Hokao R, Imamichi T. Incidence of lordosis behavior Biol Reprod 68: 2107–2113, 2003. in the female rat following removal of the vomeronasal organ. 301. Price MA, Vandenbergh JG. Analysis of puberty-accelerating Jikken Dobutsu 37: 93–95, 1988. pheromones. J Exp Zool 264: 42–45, 1992. 325. Saito TR, Moltz H. Copulatory behavior of sexually naive and 302. Rajendren G, Dudley CA, Moss RL. Role of the vomeronasal sexually experienced male rats following removal of the vomero- organ in the male-induced enhancement of sexual receptivity in nasal organ. Physiol Behav 37: 507–510, 1986. female rats. Neuroendocrinology 52: 368–372, 1990. 326. Saito TR, Moltz H. Sexual behavior in the female rat following 303. Rall W, Shepherd GM, Reese TS, Brightman MW. Dendroden- removal of the vomeronasal organ. Physiol Behav 38: 81–87, 1986. dritic synaptic pathway for inhibition in the olfactory bulb. Exp 327. Salazar I, Brennan PA. Retrograde labelling of mitral/tufted cells Neurol 14: 44–56, 1966. in the mouse accessory olfactory bulb following local injections of 304. Rasmussen LE, Greenwood DR. Frontalin: a chemical message the lipophilic tracer DiI into the vomeronasal amygdala. Brain Res of musth in Asian elephants (Elephas maximus). Chem Senses 28: 896: 198–203, 2001. 433–446, 2003. 328. Salazar I, Sanchez-Quinteiro P, Aleman N, Prieto D. Anatom- 305. Rasmussen LE, Lee TD, Roelofs WL, Zhang A, Daves GD Jr. ical, immnunohistochemical and physiological characteristics of Insect pheromone in elephants. Nature 379: 684, 1996. the vomeronasal vessels in cows and their possible role in vome- 306. Rasmussen LE, Lee TD, Zhang A, Roelofs WL, Daves GD Jr. ronasal reception. J Anat 212: 686–696, 2008. Puriﬁcation, identiﬁcation, concentration and bioactivity of (Z)-7- 329. Sam M, Vora S, Malnic B, Ma W, Novotny MV, Buck Neuro- dodecen-1-yl acetate: sex pheromone of the female Asian elephant, pharmacology LB. Odorants may arouse instinctive behaviours. Elephas maximus. Chem Senses 22: 417–437, 1997. Nature 412: 142, 2001.

307. Rasmussen LE, Schulte BA. Chemical signals in the reproduction 330. Sasaki K, Okamoto K, Inamura K, Tokumitsu Y, Kashiway- Downloaded from of Asian (Elephas maximus) and African (Loxodonta africana) anagi M. Inositol-1,4,5-trisphosphate accumulation induced by uri- elephants. Anim Reprod Sci 53: 19–34, 1998. nary pheromones in female rat vomeronasal epithelium. Brain Res 308. Ressler KJ, Sullivan SL, Buck LB. A zonal organization of odor- 823: 161–168, 1999. ant receptor gene expression in the olfactory epithelium. Cell 73: 331. Savic I. Brain imaging studies of the functional organization of 597–609, 1993. human olfaction. Chem Senses 30 Suppl 1: i222–i223, 2005. 309. Richards DB, Stevens DA. Evidence for marking with urine by 332. Savic I, Berglund H, Gulyas B, Roland P. Smelling of odorous rats. Behav Biol 12: 517–523, 1974. sex hormone-like compounds causes sex-differentiated hypotha- 310. Rissman EF, Crews D. Effect of castration on epididymal sperm lamic activations in humans. Neuron 31: 661–668, 2001. physrev.physiology.org storage in male musk shrews (Suncus murinus) and mice (Mus 333. Sbarbati A, Osculati F. Allelochemical communication in verte- musculus). J Reprod Fertil 86: 219–222, 1989. brates: kairomones, allomones and synomones. Cells Tissues Or- 310a.Rivie`re S, Challet L, Fluegge D, Spehr M, Rodriguez I. Formyl gans 183: 206–219, 2006. peptide receptor-like proteins are a novel family of vomeronasal 334. Scalia F, Winans SS. The differential projections of the olfactory chemosensors. Nature 2009 Apr 22. [Epub ahead of print] doi: bulb and accessory olfactory bulb in mammals. J Comp Neurol 161: 10.1038/nature08029. 31–55, 1975. 311. Rodolfo-Masera T. Sur l’esistenza di un particulare organo olfat- 335. Schaal B, Coureaud G, Langlois D, Ginies C, Semon E, Perrier tivo nel setto nasale della cavia e di altri roditori. Arch Ital Anat G. Chemical and behavioural characterization of the rabbit mam- Embryol 48: 157–212, 1943. mary pheromone. Nature 424: 68–72, 2003. 312. Rodriguez I. Pheromone receptors in mammals. Horm Behav 46: 336. Schaefer ML, Yamazaki K, Osada K, Restrepo D, Beauchamp on February 9, 2011 219–230, 2004. GK. Olfactory fingerprints for major histocompatibility complex- 313. Rodriguez I, Del Punta K, Rothman A, Ishii T, Mombaerts P. Multiple new and isolated families within the mouse superfamily of determined body odors II: relationship among odor maps, genetics, V1r vomeronasal receptors. Nat Neurosci 5: 134–140, 2002. odor composition, and behavior. J Neurosci 22: 9513–9521, 2002. 314. Rodriguez I, Feinstein P, Mombaerts P. Variable patterns of 337. Schild D, Restrepo D. Transduction mechanisms in vertebrate axonal projections of sensory neurons in the mouse vomeronasal olfactory receptor cells. Physiol Rev 78: 429–466, 1998. system. Cell 97: 199–208, 1999. 338. Schilling A. Olfactory communication in prosimians. In: The Study 315. Rodriguez I, Greer CA, Mok MY, Mombaerts P. A putative of Prosimian Behavior, edited by Doyle GA and Martin RD. New pheromone receptor gene expressed in human olfactory mucosa. York: Academic, 1979. Nat Genet 26: 18–19, 2000. 339. Schoppa NE, Urban NN. Dendritic processing within olfactory 316. Rodriguez I, Mombaerts P. Novel human vomeronasal receptor- bulb circuits. Trends Neurosci 26: 501–506, 2003. like genes reveal species-specific families. Curr Biol 12: R409– 340. Schwarting GA, Crandall JE. Subsets of olfactory and vomero- R411, 2002. nasal sensory epithelial cells and axons revealed by monoclonal 317. Roppolo D, Ribaud V, Jungo VP, Luscher C, Rodriguez I. antibodies to carbohydrate antigens. Brain Res 547: 239–248, 1991. Projection of the Gruneberg ganglion to the mouse olfactory bulb. 341. Schwarting GA, Deutsch G, Gattey DM, Crandall JE. Glyco- Eur J Neurosci 23: 2887–2894, 2006. conjugates are stage- and position-specific cell surface molecules 318. Roppolo D, Vollery S, Kan CD, Luscher C, Broillet MC, Ro- in the developing olfactory system. 1: The CC1 immunoreactive driguez I. Gene cluster lock after pheromone receptor gene glycolipid defines a rostrocaudal gradient in the rat vomeronasal choice. EMBO J 26: 3423–3430, 2007. system. J Neurobiol 23: 120–129, 1992. 319. Rossler P, Kroner C, Krieger J, Lobel D, Breer H, Boekhoff I. 342. Schwende FJ, Wiesler D, Novotny M. Volatile compounds asso- Cyclic adenosine monophosphate signaling in the rat vomeronasal ciated with estrus in mouse urine: potential pheromones. Experi- organ: role of an adenylyl cyclase type VI. Chem Senses 25: 313– entia 40: 213–215, 1984. 322, 2000. 343. Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, 320. Royal SJ, Key B. Development of P2 olfactory glomeruli in P2- Yoshihara Y, Sakano H. Negative feedback regulation ensures the internal ribosome entry site-tau-LacZ transgenic mice. J Neurosci one receptor-one olfactory neuron rule in mouse. Science 302: 19: 9856–9864, 1999. 2088–2094, 2003. 321. Runnenburger K, Breer H, Boekhoff I. Selective G protein beta 344. Shahan K, Denaro M, Gilmartin M, Shi Y, Derman E. Expres- gamma-subunit compositions mediate phospholipase C activation sion of six mouse major urinary protein genes in the mammary, in the vomeronasal organ. Eur J Cell Biol 81: 539–547, 2002. parotid, sublingual, submaxillary, and lachrymal glands and in the 322. Ryba NJ, Tirindelli R. A novel GTP-binding protein gamma- liver. Mol Cell Biol 7: 1947–1954, 1987. subunit, G gamma 8, is expressed during neurogenesis in the 345. Shepherd GM, Chen WR, Willhite D, Migliore M, Greer CA. olfactory and vomeronasal neuroepithelia. J Biol Chem 270: 6757– The olfactory granule cell: from classical enigma to central role in 6767, 1995. olfactory processing. Brain Res Rev 55: 373–382, 2007.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org FROM PHEROMONES TO BEHAVIOR 955

346. Shi P, Zhang J. Comparative genomic analysis identiﬁes an evo- receptor subtypes are spatially segregated in the nasal neuroepi- lutionary shift of vomeronasal receptor gene repertoires in the thelium. Cell Tissue Res 276: 429–438, 1994. vertebrate transition from water to land. Genome Res 17: 166–174, 370. Strotmann J, Wanner I, Krieger J, Raming K, Breer H. Expression 2007. of odorant receptors in spatially restricted subsets of chemosen- 347. Shimazaki R, Boccaccio A, Mazzatenta A, Pinato G, Migliore sory neurones. Neuroreport 3: 1053–1056, 1992. M, Menini A. Electrophysiological properties and modeling of 371. Sugai T, Sugitani M, Onoda N. Subdivisions of the guinea-pig murine vomeronasal sensory neurons in acute slice preparations. accessory olfactory bulb revealed by the combined method with Chem Senses 31: 425–435, 2006. immunohistochemistry, electrophysiological, and optical record- 348. Shinohara H, Kato K, Asano T. Differential localization of G ings. Neuroscience 79: 871–885, 1997. proteins, Gi and Go, in the olfactory epithelium and the main 372. Sugai T, Yoshimura H, Kato N, Onoda N. Component-dependent olfactory bulb of the rat. Acta Anat 144: 167–171, 1992. urine responses in the rat accessory olfactory bulb. Neuroreport 17: 349. Shipley MT, Ennis M. Functional organization of olfactory sys- 1663–1667, 2006. tem. J Neurobiol 30: 123–176, 1996. 373. Tanaka M, Treloar H, Kalb RG, Greer CA, Strittmatter SM. 350. Shirokova E, Raguse JD, Meyerhof W, Krautwurst D. The G(o) protein-dependent survival of primary accessory olfactory human vomeronasal type-1 receptor family: detection of volatiles neurons. Proc Natl Acad Sci USA 96: 14106–14111, 1999. and cAMP signaling in HeLa/Olf cells. FASEB J 22: 1416–1425, 374. Taniguchi M, Wang D, Halpern M. Chemosensitive conductance 2008. and inositol 1,4,5-trisphosphate-induced conductance in snake 351. Sicard G, Holley A. Receptor cell responses to odorants: similar- vomeronasal receptor neurons. Chem Senses 25: 67–76, 2000. ities and differences among odorants. Brain Res 292: 283–296, 375. Thom MD, Stockley P, Jury F, Ollier WE, Beynon RJ, Hurst 1984. JL. The direct assessment of genetic heterozygosity through scent 352. Signoret JP. Reproductive behaviour of pigs. J Reprod Fertil in the mouse. Curr Biol 18: 619–623, 2008.

Suppl 11: Suppl, 1970. 376. Thompson ML, Edwards DA. Olfactory bulb ablation and hor- Downloaded from 353. Silverman AJ, Livne I, Witkin JW. The gonadotropin-releasing monally induced mating in spayed female mice. Physiol Behav 8: hormone (GnRH), neuronal systems: immunocytochemistry and in 1141–1146, 1972. situ hybridization. In: The Physiology of Reproduction, edited by 377. Thompson RN, Robertson BK, Napier A, Wekesa KS. Sex- Neill JD. New York: Raven, 1994, p. 1683–1709. specific responses to urinary chemicals by the mouse vomeronasal 354. Silvotti L, Giannini G, Tirindelli R. The vomeronasal receptor organ. Chem Senses 29: 749–754, 2004. V2R2 does not require escort molecules for expression in heterol- 378. Tian H, Ma M. Molecular organization of the olfactory septal ogous systems. Chem Senses 30: 1–8, 2005. organ. J Neurosci 24: 8383–8390, 2004. Tillman JA, Seybold SJ, Jurenka RA, Blomquist GJ. 355. Silvotti L, Moiani A, Gatti R, Tirindelli R. Combinatorial co- 379. Insect physrev.physiology.org expression of pheromone receptors, V2Rs. J Neurochem 103: 1753– pheromones–an overview of biosynthesis and endocrine regula- 1763, 2007. tion. Insect Biochem Mol Biol 29: 481–514, 1999. 380. Tirindelli R, Ryba NJ. The G-protein gamma-subunit G gamma 8 356. Singer AG, Macrides F, Clancy AN, Agosta WC. Purification is expressed in the developing axons of olfactory and vomeronasal and analysis of a proteinaceous aphrodisiac pheromone from ham- neurons. Eur J Neurosci 8: 2388–2398, 1996. ster vaginal discharge. J Biol Chem 261: 13323–13326, 1986. 381. Treloar HB, Feinstein P, Mombaerts P, Greer CA. Specificity 357. Singh PB. Chemosensation and genetic individuality. Reproduc- of glomerular targeting by olfactory sensory axons. J Neurosci 22: tion 121: 529–539, 2001. 2469–2477, 2002. 358. Singh PB, Brown RE, Roser B. MHC antigens in urine as olfac- 382. Trinh K, Storm DR. Vomeronasal organ detects odorants in ab- tory recognition cues. Nature 327: 161–164, 1987. sence of signaling through main olfactory epithelium. Nat Neurosci 359. Smith TD, Bhatnagar KP, Dennis JC, Morrison EE, Park TJ. on February 9, 2011 6: 519–525, 2003. Het- Growth-deficient vomeronasal organs in the naked mole-rat ( 382a.Trotier D, Doving KB, Ore K, Shalchian-Tabrizi C. Scanning erocephalus glaber). Brain Res 1132: 78–83, 2007. electron microscopy and gramicidin patch calmp recordings of 360. Smith TE, Faulkes CG, Abbott DH. Combined olfactory contact microvillous receptor neurons dissociated from the rat vomerona- with the parent colony and direct contact with nonbreeding ani- sal organ. Chem Senses 23: 49–57, 1998. mals does not maintain suppression of ovulation in female naked 383. Trotier D, Eloit C, Wassef M, Talmain G, Bensimon JL, Dov- mole-rats (Heterocephalus glaber). Horm Behav 31: 277–288, 1997. ing KB, Ferrand J. The vomeronasal cavity in adult humans. 361. Snoddy AM, Heckathorn D, Tessel RE. Cold-restraint stress and Chem Senses 25: 369–380, 2000. urinary endogenous beta-phenylethylamine excretion in rats. Phar- 384. Ukhanov K, Leinders-Zufall T, Zufall F. Patch-clamp analysis of macol Biochem Behav 22: 497–500, 1985. gene-targeted vomeronasal neurons expressing a defined V1r or 362. Spehr J, Hagendorf S, Weiss J, Spehr M, Leinders-Zufall T, J Neu- 2ϩ V2r receptor: ionic mechanisms underlying persistent firing. Zufall F. Ca -calmodulin feedback mediates sensory adaptation rophysiol 98: 2357–2369, 2007. and inhibits pheromone-sensitive ion channels in the vomeronasal 385. Urban NN, Sakmann B. Reciprocal intraglomerular excitation organ. J Neurosci 29: 2125–2135, 2009. and intra- and interglomerular lateral inhibition between mouse 363. Spehr M, Hatt H, Wetzel CH. Arachidonic acid plays a role in rat olfactory bulb mitral cells. J Physiol 542: 355–367, 2002. vomeronasal signal transduction. J Neurosci 22: 8429–8437, 2002. 386. Valenta JG, Rigby MK. Discrimination of the odor of stressed 364. Spehr M, Kelliher KR, Li XH, Boehm T, Leinders-Zufall T, rats. Science 161: 599–601, 1968. Zufall F. Essential role of the main olfactory system in social 387. Van Der LEE, Boot LM. Spontaneous pseudopregnancy in mice. recognition of major histocompatibility complex peptide ligands. Acta Physiol Pharmacol Neerl 4: 442–444, 1955. J Neurosci 26: 1961–1970, 2006. 388. Vandenbergh JG. Male odor accelerates female sexual maturation 365. Steel E, Keverne EB. Effect of female odour on male hamsters in mice. Endocrinology 84: 658–660, 1969. mediated by the vomeronasal organ. Physiol Behav 35: 195–200, 389. Vassar R, Ngai J, Axel R. Spatial segregation of odorant receptor 1985. expression in the mammalian olfactory epithelium. Cell 74: 309– 366. Stensaas LJ, Lavker RM, Monti-Bloch L, Grosser BI, Berliner 318, 1993. DL. Ultrastructure of the human vomeronasal organ. J Steroid 389a.Venderbergh JG. Pheromones and mammalian reproduction. In: Biochem Mol Biol 39: 553–560, 1991. The Physiology and Reproduction, edited by E. Knobil and J. D. 367. Stern K, McClintock MK. Regulation of ovulation by human Neil. New York: Raven, 1994, p. 343–356. pheromones. Nature 392: 177–179, 1998. 390. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 368. Stowers L, Holy TE, Meister M, Dulac C, Koentges G. Loss of 76: 387–417, 2007. sex discrimination and male-male aggression in mice deficient for 391. Verberne G. Chemocommunication among domestic cats, medi- TRP2. Science 295: 1493–1500, 2002. ated by the olfactory and vomeronasal senses. II. The relation 369. Strotmann J, Wanner I, Helfrich T, Beck A, Meinken C, Ku- between the function of Jacobson’s organ (vomeronasal organ) and bick S, Breer H. Olfactory neurones expressing distinct odorant Flehmen behaviour. Z Tierpsychol 42: 113–128, 1976.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org 956 TIRINDELLI ET AL.

392. Von Campenhausen H, Mori K. Convergence of segregated 409. Xu F, Schaefer M, Kida I, Schafer J, Liu N, Rothman DL, pheromonal pathways from the accessory olfactory bulb to the Hyder F, Restrepo D, Shepherd GM. Simultaneous activation of cortex in the mouse. Eur J Neurosci 12: 33–46, 2000. mouse main and accessory olfactory bulbs by odors or phero- 393. Wagner S, Gresser AL, Torello AT, Dulac C. A multireceptor mones. J Comp Neurol 489: 491–500, 2005. genetic approach uncovers an ordered integration of VNO sensory 410. Yamaguchi T, Inamura K, Kashiwayanagi M. Increases in Fos- inputs in the accessory olfactory bulb. Neuron 50: 697–709, 2006. immunoreactivity after exposure to a combination of two male 394. Wakabayashi Y, Mori Y, Ichikawa M, Yazaki K, Hagino-Yam- urinary components in the accessory olfactory bulb of the female agishi K. A putative pheromone receptor gene is expressed in two rat. Brain Res 876: 211–214, 2000. distinct olfactory organs in goats. Chem Senses 27: 207–213, 2002. 411. Yamazaki K, Beauchamp GK. Genetic basis for MHC-dependent 395. Wang Z, Balet SC, Li V, Nudelman A, Chan GC, Storm DR. mate choice. Adv Genet 59: 129–145, 2007. Pheromone detection in male mice depends on signaling through 412. Yildirim E, Birnbaumer L. TRPC2: molecular biology and func- the type 3 adenylyl cyclase in the main olfactory epithelium. J Neu- tional importance. Handb Exp Pharmacol 53–75, 2007. rosci 26: 7375–7379, 2006. 413. Yoon H, Enquist LW, Dulac C. Olfactory inputs to hypothalamic 396. Weiler E, Farbman AI. The septal organ of the rat during post- neurons controlling reproduction and fertility. Cell 123: 669–682, 2005. natal development. Chem Senses 28: 581–593, 2003. 414. Yoshihara Y, Kawasaki M, Tamada A, Fujita H, Hayashi H, 397. Wekesa KS, Anholt RR. Pheromone regulated production of ino- Kagamiyama H, Mori K. OCAM: a new member of the neural cell sitol-(1,4,5)-trisphosphate in the mammalian vomeronasal organ. adhesion molecule family related to zone-to-zone projection of Endocrinology 138: 3497–3504, 1997. olfactory and vomeronasal axons. J Neurosci 17: 5830–5842, 1997. 398. Wekesa KS, Anholt RR. Differential expression of G proteins in 415. Young JM, Kambere M, Trask BJ, Lane RP. Divergent V1R the mouse olfactory system. Brain Res 837: 117–126, 1999. repertoires in ﬁve species: ampliﬁcation in rodents, decimation in 399. Wekesa KS, Lepri JJ. Removal of the vomeronasal organ reduces primates, and a surprisingly small repertoire in dogs. Genome Res

reproductive performance and aggression in male prairie voles. 15: 231–240, 2005. Downloaded from 416. Young JM, Waters H, Dong C, Fulle HJ, Liman ER. Degenera- Chem Senses 19: 35–45, 1994. tion of the olfactory guanylyl cyclase D gene during primate evo- 400. Weldon PJ. The evolution of alarm pheromones. In: Chemical lution. PLoS ONE 2: e884, 2007. Signals in Vertebrates, edited by Muller-Schwarze D and Silver- 417. Yu WP, Grunwald ME, Yau KW. Molecular cloning, functional stein RM. New York: Plenum, 1983, p. 309–312. expression and chromosomal localization of a human homolog of 401. Whitten WK. Modiﬁcation of the oestrous cycle of the mouse by the cyclic nucleotide-gated ion channel of retinal cone photorecep- external stimuli associated with the male. J Endocrinol 13: 399– tors. FEBS Lett 393: 211–215, 1996. 404, 1956. 418. Zalaquett C, Thiessen D. The effects of odors from stressed mice physrev.physiology.org 402. Wilson KC, Raisman G. Age-related changes in the neurosensory on conspeciﬁc behavior. Physiol Behav 50: 221–227, 1991. epithelium of the mouse vomeronasal organ: extended period of 419. Zancanaro C, Caretta CM, Bolner A, Sbarbati A, Nordera GP, postnatal growth in size and evidence for rapid cell turnover in the Osculati F. Biogenic amines in the vomeronasal organ. Chem adult. Brain Res 185: 103–113, 1980. Senses 22: 439–445, 1997. 403. Witt M, Georgiewa B, Knecht M, Hummel T. On the chemosen- 420. Zhang J, Webb DM. Evolutionary deterioration of the vomerona- sory nature of the vomeronasal epithelium in adult humans. His- sal pheromone transduction pathway in catarrhine primates. Proc tochem Cell Biol 117: 493–509, 2002. Natl Acad Sci USA 100: 8337–8341, 2003. 404. Woodley SK, Cloe AL, Waters P, Baum MJ. Effects of vomero- 421. Zhang P, Yang C, Delay RJ. Urine stimulation activates BK chan- nasal organ removal on olfactory sex discrimination and odor nels in mouse vomeronasal neurons. J Neurophysiol 100: 1824– preferences of female ferrets. Chem Senses 29: 659–669, 2004. 1834, 2008. 405. Wray S, Fueshko SM, Kusano K, Gainer H. GABAergic neurons 422. Zhang X, Rodriguez I, Mombaerts P, Firestein S. Odorant and on February 9, 2011 in the embryonic olfactory pit/vomeronasal organ: maintenance of vomeronasal receptor genes in two mouse genome assemblies. functional GABAergic synapses in olfactory explants. Dev Biol 180: Genomics 83: 802–811, 2004. 631–645, 1996. 423. Zhang Y, Hoon MA, Chandrashekar J, Mueller KL, Cook B, 406. Wyart C, Webster WW, Chen JH, Wilson SR, McClary A, Khan Wu D, Zuker CS, Ryba NJ. Coding of sweet, bitter, and umami RM, Sobel N. Smelling a single component of male sweat alters tastes: different receptor cells sharing similar signaling pathways. levels of cortisol in women. J Neurosci 27: 1261–1265, 2007. Cell 112: 293–301, 2003. 407. Wysocki CJ, Lepri JJ. Consequences of removing the vomerona- 424. Zhou A, Moss RL. Effect of urine-derived compounds on cAMP sal organ. J Steroid Biochem Mol Biol 39: 661–669, 1991. accumulation in mouse vomeronasal cells. Neuroreport 8: 2173– 408. Xia J, Sellers LA, Oxley D, Smith T, Emson P, Keverne EB. 2177, 1997. Urinary pheromones promote ERK/Akt phosphorylation, regener- 425. Zucchi R, Chiellini G, Scanlan TS, Grandy DK. Trace amine- ation and survival of vomeronasal (V2R) neurons. Eur J Neurosci associated receptors and their ligands. Br J Pharmacol 149: 967– 24: 3333–3342, 2006. 978, 2006.

Physiol Rev • VOL 89 • JULY 2009 • www.prv.org From Pheromones to Behavior Roberto Tirindelli, Michele Dibattista, Simone Pifferi and Anna Menini Physiol Rev 89:921-956, 2009. doi:10.1152/physrev.00037.2008

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