From Pheromones to Behavior - Physiological Reviews

Transcription

Physiol Rev 89: 921–956, 2009;
doi:10.1152/physrev.00037.2008.
From Pheromones to Behavior
ROBERTO TIRINDELLI, MICHELE DIBATTISTA, SIMONE PIFFERI, AND ANNA MENINI
Department of Neuroscience, University of Parma, Parma; and International School for Advanced Studies,
Scuola Internazionale Superiore di Studi Avanzati (SISSA) and Italian Institute of Technology, SISSA Unit,
Trieste, Italy
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 13, 2016
I. Introduction
II. Mammalian Pheromones
A. Pheromones in various animal orders
B. Major urinary proteins and major histocompatibility complex molecules
C. Exocrine gland-secreting peptides
III. Chemosensory Systems Detecting Pheromones
A. Vomeronasal epithelium
B. Olfactory epithelium
C. Other olfactory organs
IV. Putative Pheromone Receptors
A. Vomeronasal receptors
B. Odorant receptors
C. Trace amine-associated receptors
D. Olfactory-specific guanylyl cyclase type D receptor
V. Pheromone Transduction
A. Vomeronasal sensory neurons
B. Olfactory sensory neurons
C. Grueneberg ganglion
VI. Signaling From Sensory Epithelia to the Brain
A. Main and accessory olfactory bulbs
B. Signaling beyond the olfactory bulbs
C. Pheromone-Activated hormonal systems
VII. Pheromone-Mediated Behaviors
A. Male behaviors
B. Female behaviors
VIII. Conclusions and Future Challenges
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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
Chemosensation is one of the sensory modalities that
animals have evolved to analyze the chemical properties
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of the external world, enabling the detection and the
discrimination of a very high number of molecules with
different structures. Substances carrying a chemical message among animals are termed “semiochemicals,” from
0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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determinants. For example, trace pheromones allow attracting a conspecific, while alarm pheromones alert
other conspecifics about an external challenge and can
evoke aggressive behaviors.
A first classification groups intraspecific signaling
molecules in releaser and primer pheromones (239). Releaser pheromones lead to individual recognition and
evoke short-latency behavioral responses in the conspecific like in aggressive attacks, or mating, or territory
marking. Conversely, primer pheromones induce delayed
responses that are commonly mediated through the activation of the neuroendocrine system. Thus, through the
emission of chemical cues, an individual forces the hormonal balance of a receiver, thus modulating its reproductive status.
In mammals, pheromones have been reported to be
involved in a variety of behavioral effects also in unrelated
species and, surprisingly, some pheromonal molecules are
shared, with different purposes, between species.
II. MAMMALIAN PHEROMONES
2. Marsupialia
A. Pheromones in Various Animal Orders
Pheromones have a very large chemical variability
(Table 1), and therefore they are preferably grouped according to their functions rather than to their structural
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1. Proboscidea
Despite their size, which would obviously preclude
extensive behavioral studies, Asian and African elephants
have been largely investigated for their pheromonal responses by the late Bets Rasmussen. During musth, a
periodic condition in which mature males are characterized by highly aggressive behavior, elephants produce
large quantities of a specific pheromone, frontalin (1,5dimethyl-6,8-dioxabicyclo[3.2.1]octane), that is released
in temporal gland secretions, urine, and breath (307).
Adult males are mostly indifferent to frontalin, whereas
subadult males are highly reactive, often exhibiting repulsion or avoidance. Female chemosensory responses to
frontalin vary with the hormonal status. Females in the
follicular phase are the most responsive and often demonstrate mating-related behaviors subsequent to frontalin
stimulation (304). Furthermore, female Asian elephants
excrete a urinary pheromone, (Z)-7-dodecen-L-yl acetate,
to signal to males their readiness to mate (305). Interestingly, females of more than 100 species of insects use the
same compound as part of their pheromone blends to
attract insect males. The finding that the same molecule
may be shared by unrelated species for a different pheromonal purpose is only apparently surprising, owing to the
fact that structural constraints are often dictated by common metabolic pathways.
In marsupials, the gray short-tailed opossum (Monodelphis domestica) communicates by scent marking. The
male opossum possesses a prominent suprasternal scent
gland, whose extracts strongly attract female opossums
(131), stimulating the estrus in anestrous females via the
vomeronasal system.
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the Greek “semeion” (sign). An important class of semiochemicals is constituted by pheromones. The term pheromone, based on the Greek “pherein” (to carry or transfer) and “hormon” (to stimulate or excite), was introduced in 1959 by the entomologists Peter Karlson and
Martin Lüscher (151) to identify specific biologically active substances “which are secreted to the outside by an
individual and received by a second individual of the same
species, in which they release a specific reaction, for
example, a definite behavior or a developmental process.”
In the same year, the first insect pheromone bombykol,
released by the female silkworm to attract mates, was
characterized by Butenandt et al. (43). The use of pheromones among insects has been extensively investigated,
and interested readers may consult several reviews on the
subject (4, 111, 130, 379).
Another class of semiochemicals is constituted by allelomones: substances produced by members of one species
that influence the behavior of members of another species
(for a review, see Ref. 333). Although in this review we will
mainly discuss the effects elicited by pheromones in mammals, we will also briefly mention some examples of allelomonal communication (5, 107, 108, 169).
In most mammals, the nasal cavity contains at least two
major sensory systems that may be involved in the detection
of pheromones: the vomeronasal and the main olfactory
systems. Evidence indicates that some behaviors are mediated exclusively by one or by a complementary action of
both systems. For example, Bruce (37) reported that, when
female mice that have been recently inseminated are exposed to urine of a male different from the one they mated
with, the pregnancy is interrupted and the female returns to
estrus. This effect depends on a functional vomeronasal
system (15). Differently, the nipple search in rabbit pups is
one of the typical examples of behavior strictly dependent
on an intact main olfactory system (119). In other instances,
behavioral responses are mediated by both the vomeronasal
and the main olfactory systems. This is the case of female
hamsters, whose ultrasonic calling during estrus is abolished by the inactivation of either the vomeronasal or the
main olfactory system (145). In recent years, the discovery of
distinct pheromone receptor families coupled to a complex
network of signal transduction pathways, together with advances in molecular genetics and functional electrophysiological and imaging techniques, have greatly advanced our understanding of the physiological role of pheromones.
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FROM PHEROMONES TO BEHAVIOR
TABLE
1.
Table of pheromones
Pheromone
Origin
Organ
Mouse female urine
VNO
2-Sec-butyl-4,5dihydrothiazole (BT)
Mouse male urine
VNO
2,3-Dehydro-exobrevicomin (DB)
Mouse male urine
VNO
␣- and ␤-Farnesene
Mouse preputial glands
VNO
6-Hydroxyl-6-methyl-3heptanone
Mouse male urine
VNO
2-Heptanone
Mouse urine
VNO
n-Pentyl acetate
Mouse urine
VNO
Isobutylamine
Mouse male urine
VNO
3-Amino-s-triazole (similar
to BT)
4-Ethyl phenol
3-Ethyl-2,7-dimethyl octane
(similar to DB)
Mouse male urine
?
Mouse male urine
Mouse male urine
?
?
3-Cyclohexene-1-methanol
Major urinary proteins
(MUPs)
MHC class I peptides
Mouse male urine
Mouse urine
?
VNO
Mouse urine
VNO
Behavior
V1R neurons
IP3 production in the VNO (377) Suppression of estrus cycle (Lee Booteffect) (219, 281)
Electrical response and calcium
influx in VNO neurons (187)
V1R neurons
Electrical response and calcium Estrus induction and synchronization
(Whitten effect) (138)
Protooncogene expression in
Puberty acceleration (Vandenbergh effect)
the AOB (32, 95)
(266, 283)
Intermale aggressiveness (280)
Attractiveness to females (139)
V1R neurons
Electrical response and calcium Estrus induction and synchronization
(Whitten effect) (138)
Protooncogene expression in
the AOB (32, 95)
(266, 283)
Intermale aggressiveness (280)
V1R neurons
Electrical response and calcium Puberty acceleration (Vandenbergh effect)
(266, 283)
Territorial marking (279)
Suppression of estrus cycle (Lee Booteffect) (219)
V1ra and V1rb (V1Rs) Electrical response and calcium Puberty acceleration (Vandenbergh effect)
(266, 282)
neurons
influx in VNO neurons (61,
187)
V1R neurons
Electrical response and calcium Estrus extension (137)
IP3 production in the VNO (377)
V1ra and V1rb (V1Rs) Electrical response in VNO
Suppression of estrus cycle (Lee Bootneurons
neurons (61)
effect) (281)
V1ra and V1rb (V1Rs) Electrical response in VNO
Estrus acceleration and vaginal opening
neurons
neurons (61)
(276, 301)
?
?
?
?
?
V2R neurons
V2R neurons
(SYFPEITHE;
AAPDNRETF)
Exocrine gland secreting
peptides
␣2-Urinary proteins (␣2u)
MOE
Extraorbital lacrimal glands
VNO
V2R neurons
Rat urine
VNO
V2R neurons
Dodecyl-propionate
Aphrodisin
Rat preputial gland
Hamster vaginal glands
?
VNO
?
?
?
?
?
?
?
?
2-Methylbut-2-enal
Rabbit milk
(Z)-7-Dodecen-1-yl acetate Elephant female urine
Male asian elephant temporal
(⫹)(⫺)1,5-Dimethyl-6,8gland, urine, and breath
dioxabicyclo[3.2.1]octane
(frontaline)
Cellular/Biochemical Response
?
?
?
influx in VNO neurons (47,
163)
In vitro survival and
proliferation of VNO neurons
(408)
influx in MOE and VNO
neurons (364)
Single-cell recording in VNO
neurons (156, 185)
Electrical response in VNO
neurons (162, 163)
G protein activation and IP3
production in the VNO (176,
321)
?
IP3 production in the VNO and
c-fos expression in AOB
neurons (136, 177)
?
?
?
Repulsion to mouse (territorial marking?)
(2)
(266, 283)
Individual recognition (49, 123, 375)
Intermale aggressiveness
Attractiveness to individuals of a different
mouse strain (358)
Pregnancy block (Bruce effect)
?
?
Ano-genital licking (36)
Copulatory behavior in male (356)
Attraction and oral grasping in pups (335)
Mating-associated behaviors in males (306)
Repulsion, attractiveness, and sexual
behavior depending on the racemic ratio
(92, 304)
VNO, vomeronasal organ; MOE, main olfactory epithelium; AOB, accessory olfactory bulb. Reference numbers are given in parentheses.
3. Soricomorpha (Insectivores)
In musk shrew (Suncus murinus), virgin females exposed to cages recently vacated by an adult male receive
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mounts from males significantly sooner than females
exposed to cages soiled by a castrated male, by another
female, or to a clean cage (310).
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2,5-Dimethyl pyrazine
Target
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In tree shrew (Tupaia belangeri), both males and
females mark their surroundings with urine and skin
gland secretions. Males mark more frequently than females in the absence of conspecific scent; moreover, male
scent stimulates marking in females. Interestingly, increasing amounts of male scent result in a corresponding
increase in marking by females (114), suggesting that, in
the wild, pheromones may not exert an all-or-none effect
but rather act in a dose-specific fashion.
4. Carnivora and Ungulata
5. Primata
Primates scarcely rely on chemical communication;
however, the ring-tailed lemur (Lemur catta) exhibits the
most highly developed olfactory system among all species
belonging to this order (75, 83, 338). Lemur catta possesses a suite of specialized scent glands and a variety of
scent-marking displays (147, 258, 338). Both sexes have
apocrine, located on the wrists, and sebaceous gland
fields in their genital regions and adopt distinctive handstand postures to deposit glandular secretions on substrates. Males mix the glandular secretions and then deposit this mixture via “wrist marking.” They also impregnate their tail fur with their own secretions and then waft
their tail at opponents during characteristic “stink fights”
(147).
There is nowadays a general consensus for the existence of a chemical communication in humans, although
there is still a great controversy about the existence,
extent, and nature of human pheromones. Among the best
and first known effects attributed to human pheromones
is the synchronization of menstrual cycle (237, 238, 367)
and the dimorphic activation of brain areas (332). ExpoPhysiol Rev • VOL
6. Rodentia
The paradigmatic model for mammalian pheromone
investigations involves laboratory species, among which
rodents and especially mice are the most employed, because they are easy to handle, have extremely well-developed olfactory systems, and offer an almost unlimited
possibility to act on behavioral responses by genetic manipulation. In these species, several effects have been
carefully described, and the mechanisms of their action
have even been dissected at a molecular level.
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Both carnivores (dogs and cats) and ungulates commonly mark their territory with urine and display a specific behavior called “flehmen” (67, 88, 391). In the flehmen reaction, animals, after the physical contact with a
conspecific or with scents emanating from its excrements
(urine or feces), lift the head, draw back their lips, and
push the tongue towards the anterior region of the palate
in a manner that makes them appear to be “grimacing.” It
is commonly believed that this attitude would allow a
faster transfer of pheromones into the olfactory organs.
The saliva of the adult male pig contains a mixture of
steroids (boar odor). When a boar becomes aggressive or
sexually aroused, it produces copious amount of saliva
whose pungent odor is attractive to estrus females (287)
and facilitates the display of the estrous female’s mating
posture (352). The major component of the boar odor is
androstenone, a sexually dimorphic steroid that elicits
both behavioral responses (65). Curiously, androstenone
has been hypothesized to be a human pheromone as well
(18, 96).
sure to odorless axillary’s secretions from females in the
late follicular and ovulatory phase of the menstrual cycle,
respectively, shortens and lengthens the menstrual cycles
of recipient females, accelerating or delaying the luteinizing hormone (LH) surge (367). Women smelling an androgen-like compound activate the preoptic and ventromedial nuclei of the hypothalamus in contrast to men who
activate the paraventricular and dorsomedial nuclei hypothalamus when smelling an estrogen-like substance (332).
Although a robust hypothalamic response is commonly
observed with ordinary odorants, such a dramatic dimorphic difference can be interpreted as a hormonal response
to pheromone stimulation.
The apocrine gland secretion of the axilla in combination with the microbial flora is probably the most relevant source of candidate compounds for human pheromones, which consists of a blend of molecules characterizing the individual body odor (14). However, salivary and
seminal secretions and urine represent other reservoirs of
putative human pheromones. The male sweat and salivary
progesterone derivative androstadienone (4,16-androstadien-3-one) (AND) has been indicated as a putative human pheromone influencing brain activity (20, 331, 332),
endocrine levels (406), physiological arousal, context-dependent mood, and sexual orientation (132, 133, 212, 232,
300). Woman urine contains the estrogen like steroid
estra-1,3,5(10),16-tetraen-3-ol (EST) that also appears to
be a good candidate compound for human pheromones
(20, 132, 331, 332).
In studies of brain activity by positron emission tomography, as above reported, smelling AND and EST
activates regions primarily incorporating the sexually dimorphic nuclei of the anterior hypothalamus (332), and
this activation is differentiated with respect to sex and
compound. Very interestingly, homosexual males process
AND similarly to heterosexual women rather than to heterosexual men (20). Conversely, lesbian women process
AND stimuli as common odorants and, when smelling
EST, they partly share activation of the anterior hypothalamus with heterosexual men (20). These observations
indicate the involvement of the anterior hypothalamus in
physiological processes related to sexual orientation/preference in humans.
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signal alert to the presence of a potential danger, promoting dispersion and the adoption of defensive actions in the
group (400). Usually, mice escape from places in which
the odors from the urine of a physically stressed conspecific are present (418). Among other ketones, 2-heptanone
is detectable in the mouse and rat urine (97, 342). In rat,
2-heptanone was found to be increased in urine of
stressed subjects and to induce immobility in a recipient
conspecific forced to swim (97).
Several other urinary compounds have been indicated as activators of distinct anatomical regions of the
pheromonal pathway in rats (176, 321, 372); however,
none of the effects exerted by these substances is reportedly linked to obvious behavioral responses.
Mouse ecology is largely triggered by pheromonal
signals, which have been investigated for a long time, and
therefore, mice represent the world-wide animal model to
study intraspecies communication. In the last decade, an
enormous burden of data has been collected and, how it
is often the case, there is yet an open and sometimes
contrasting debate on the specific properties of the different signaling molecules.
Early studies have demonstrated a number of incontrovertible effects of primer pheromones in mice. It is
known that grouped female mice modify or suppress their
estrous cycle (Lee-Boot effect) (387), while male urine
can restore and synchronize the estrus cycle of noncycling females (Whitten effect) (401) or accelerate puberty
onset in females (Vandenbergh effect) (388). In addition,
the exposure of a recently mated female mouse to a male,
different from the stud, prevents implantation of fertilized
eggs (Bruce effect) (38), implying that the stud or its
individual odor must be memorized at the moment of
mating to be recognized later. These pheromonal effects
are commonly believed to be mediated by stimuli present
in urine that act via the vomeronasal system.
Signaling pheromones also account for many behavioral responses in the mouse. For example, male as well
as lactating female aggressiveness towards an intruder
male are both believed to be triggered by molecules that
are present in male urine.
B. Major Urinary Proteins and Major
Histocompatibility Complex Molecules
Male mouse urine contains an unusually high quantity of proteins, called major urinary proteins (MUPs).
MUPs form a large family of highly homologous androgendependent proteins that are synthesized in the liver and
excreted with urine (46, 208). MUPs and the rat hortologs
␣2 urinary globulins belong to the lipocalin superfamily,
and accordingly, they bind and release small (volatile)
pheromones that are also produced in an androgen-dependent fashion (9, 23). Although MUPs are also ex-
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The vaginal secretion and saliva of female golden
hamsters (Mesocricetus auratus) contain an aphrodisiac
substance that facilitates the mating behavior of the male
(223, 356). This pheromonal effect has been clearly demonstrated in assays that measure the mounting behavior
of a male in the presence of an anesthetized male hamster
(defined as surrogate female) scented with the stimuli
(222). Evidence shows that biologically active compounds
were present in the proteinaceous fraction, whereas aqueous fractions, organic extracts, and distillates containing
volatile compounds did not exert detectable effects. After
purification, pheromonal activity was attributed to a 17kDa soluble glycosylated protein appropriately termed
aphrodisin (356). The complete primary structure of aphrodisin shows that this protein belongs to the lipocalin
superfamily. A prominent feature of almost all lipocalins
is the presence of a hydrophobic pocket that accommodates endogenous molecules with a relatively high lipophilic coefficient. The biochemically purified aphrodisin carries natural molecules that are contained in the
vaginal secretions (33, 34) and that may be responsible for
the pheromonal effect observed in males (136). Biochemical and immunohistochemical data suggest that aphrodisin may act through G protein-coupled receptors that are
expressed in the vomeronasal system (136, 177).
Fossorial mammals, like the mole-rat, are noted for
sensory specializations, such as exquisite tactile senses
(45, 60). Although suppression of mole-rat reproduction
in females is thought to be meditated by behavioral,
rather than pheromonal, cues from the breeding females
(360), the chemosensory systems retain demonstrable
functions of scent marking in common nesting and toilet
loci (359) that have been associated with pheromonal
communication.
In rats, several pheromones are used in different
contexts as territory marking (309), alarm (1), and maternal behaviors (188, 254). The chemically characterized rat
pheromones include compounds that are produced by
pup’s preputial glands and urine. A particular pattern of
rat maternal behavior is the specific licking and cleaning
of pup’s anogenital area, an action crucial to pup survival,
as nonlicked pups cannot defecate and run into death.
Thus dodecyl propionate, a compound isolated from
pups’ preputial glands, can sustain pups’ anogenital licking by dams and therefore pups’ survival (36).
The odors emanating from physically stressed rodents are recognized by conspecifics (220, 386). Once the
chemical signal is perceived, the receiver undergoes significant changes. For example, lymphocyte T-killers are
suppressed, whereas B-cells proliferate (57), rats develop
hyperthermia (160), c-Fos protein expression is increased
in the mitral cell layer of the accessory olfactory bulb
(164), reproductive behavior and sexual maturation initiate (389a), and social hierarchy may be modified (140).
These bodily compounds, defined as alarm pheromones,
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Thom et al. (375) indicated that female mice use
MUPs as a specific genetic marker to identify and preferentially associate with heterozygous males. The primary
function of MUPs is in signaling social information
through scent, including individual genetic identity. Mice
are highly sensitive to differences in the fixed patterns of
multiple MUP isoforms expressed by each individual and
also avoid inbreeding with close relatives that share the
same MUP type as themselves. Heterozygosis assessment,
in contrast, most likely involves recognition of the greater
diversity of MUP isoforms expressed by heterozygous
animals.
Other biological effects of MUPs include attractiveness to females and repulsion to males (265), modification
of light-avoidance behavior in both males and females
(263, 264), the aggressive reactions of males towards
adult and newborn mice (264, 267), and intermale aggressiveness (47). Mate choice and parent-offspring interactions have been demonstrated to be influenced by MHC
genotype B (411). For example, house mice avoid mating
with individuals that are genetically similar at the MHC
locus. Mice are able to recognize MHC-similar individuals
through specific odorant cues. Moreover, females avoid
mating with males carrying MHC genes of their foster
family, supporting a familial imprinting hypothesis (288).
Mate preference for MHC-dissimilar individuals can be
adaptive as it would increase offspring MHC heterozygosity, with beneficial influences on offspring viability through
increased resistance to infectious disease or avoidance of
inbreeding effects (180, 297).
The polymorphism of MHC molecules is reflected
into structurally diverse binding sites, such that different
MHC binds to distinct peptides. Like other membranebound proteins, these MHC molecules are anchored in the
lipid bilayer. However, it has been demonstrated that the
peptide-binding pocket releases its peptide in the extracellular fluid when the MHC is cleaved from the cell. Thus
peptides or their fragments are constitutively excreted in
urine and other bodily secretions (358) that in turn can be
used for interindividual communication (357).
C. Exocrine Gland-Secreting Peptides
Mice secrete a family of peptides of different length
called exocrine gland-secreting peptides (ESPs) from extraorbital, Harderian and submaxillary glands into tear,
nasal mucus, or saliva. The ESP family consists of 38
members in mice and 10 in rats, while it is absent from the
human genome.
Moreover, at least two of these peptides, produced by
the extraorbital lacrimar gland, are gender specific: ESP1
is exclusively secreted by males (162), while ESP36 is
female specific (163). Despite the fact that these peptides
are recognized by systems dedicated to pheromone per-
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pressed in exocrine glands as mammary, parotid, sublingual, submaxillary, lachrymal, nasal, and in modified sebaceous glands like preputial and perianal glands (344),
their biological effects have been exclusively demonstrated when these proteins were purified from urine or
added to urine that does not possess pheromonal activity
(i.e., of a prepubertal or castrated mouse) (123, 266).
MUPs have been reported to act either as primer or
releaser pheromones; however, some of their effects appear contradictory. This is in part due to the tight binding
of MUPs with volatile pheromones that have been proven
to be directly implicated in estrus synchronization (138),
attractiveness to females (139), intermale aggression
(280), induction of estrus (␣- and ␤-farnesene) (219), puberty acceleration in females (283), and territory marking
(21, 122). The advantage of such a pheromone-protein
complex may include concentration and slow release of
chemosignals, stabilization of labile pheromones, or delivery to the reception sites. Common chromatographic
procedures for purification of MUPs are ineffectual to
remove these pheromones, and solvent extraction of volatiles appears the only way to obtain the native apoproteins. Therefore, in literature, the term of MUPs commonly defines the protein with the bound pheromones.
While a general consensus exists about the source,
male urine, that releases the pheromonal stimuli responsible for the puberty acceleration (Vandenbergh effect), a
debate is currently open about the molecules that determine this behavioral response. Evidence suggests that
MUPs and not the bound pheromones are effective in
accelerating the puberty onset in females. These results
are supported by the biological activity shown by MUPs
when stripped of its pheromones and by a short synthetic
hexapeptide homologous to the NH2-terminal sequence of
many MUPs variants (266). Conclusions from a different
laboratory, performing similar but not identical experiments, seem, however, to argue against this hypothesis
(283).
It has been also demonstrated that the combinatorial
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
virtually as great as for molecules of the major histocompatibility complex (MHC) (see below) (48, 123). However,
although MHC has been long regarded as the primary
determinant for individual recognition via bodily odors,
this evidence has now been challenged by recent studies
in which it appears that recognition, at least in the wild, is
instead strictly and exclusively dependent on the MUPs
variant profile contained in each individual urine (49). Very
intriguing is the observation that recognition requires the physical contact with the donor urine, thus discarding the
hypothesis of this effect being exclusively attributed to
the volatile pheromones released from MUPs (49).
ception, until now there were no clues regarding their
physiological function.
III. CHEMOSENSORY SYSTEMS
DETECTING PHEROMONES
A. Vomeronasal Epithelium
The vomeronasal epithelium is part of the vomeronasal organ (VNO) of Jacobson, a tubular structure en-
cased in a protective bony capsule located at the base of
the nasal septum (68, 134) (Fig. 2). Given its recessed
nature, the vomeronasal epithelium cannot be reached by
the airstream that regularly flows through the nasal cavity. Thus, to target the vomeronasal epithelium, molecules
dissolved in the nasal mucus must be sucked into the
lumen of the organ. Lateral to the lumen are blood vessels
and sinuses, innervated by the autonomic nervous system, that induces vasodilation and vasoconstriction,
thereby producing a pumplike action for stimulus access
to the lumen (72, 109, 244, 248, 249, 328). In ungulates,
as described in section IIA4, the “flehmen” behavior is
thought to be associated with promoting stimulus access
to the vomeronasal organ (268).
Norepinephrine accumulation has been measured in
the vomeronasal organ of mice after exposure to pheromonal cues (419). It is possible that this hormonal release
alters the vascular tone, or changes the glandular secretions, thus increasing the receptivity of the sensory epithelium in particular behavioral contexts. The functioning
of the vomeronasal organ is thus not simply a passive
event, but it can be actively modulated.
The medial, concave side of the vomeronasal lumen
is lined by a pseudostratified epithelium composed of
three types of cells: sensory neurons, supporting cells,
and basal cells. The basal stem cells are located both
along the basal membrane of the sensory epithelium and
at the boundary with the nonsensory epithelium (85).
Supporting cells are found in the uppermost superficial
layer of the sensory epithelium, while vomeronasal neurons form two overlapping populations, basal and apical,
that are further described in detail in sections IV and V.
In the mouse, the vomeronasal sensory epithelium
increases from birth to puberty and becomes fully developed and completely active at 2 mo after birth (117). The
sensory neurons, on the mucosal side, bear dendrites and
apical microvilli, whereas, on the opposite side, their
axons cross the basal membrane and merge together,
forming vomeronasal nerves that run between the paired
olfactory bulbs and enter the accessory olfactory bulb at
the posterior dorsal side of the main olfactory bulb (see
Figs. 2 and 3).
The vomeronasal sensory neurons take origin from
the olfactory placode together with gonadotropin-releasing hormone (GnRH, also known as LH-RH or luteinizing
hormone-releasing hormone) neurons and ␥-aminobutyric acid (GABA)-containing neurons (405). The GnRH
FIG. 1. A schematic diagram showing
the anatomical pathways of the rodent
vomeronasal and main olfactory systems.
Physiol Rev • VOL
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It has been believed for a long time that two chemosensory systems, the main olfactory and the vomeronasal
system, were responsible for different functions. The
main olfactory system was considered to be responsible
for recognizing the conventional volatile odorant molecules, whereas the vomeronasal system was thought to be
tuned for sensing pheromones. Recent studies have demonstrated that both chemosensory systems, together with
additional olfactory organs, are involved in pheromone
detection. In these systems, peripheral chemosensory
neurons located in the nasal cavity express distinct families of receptors that are believed to bind pheromones
and trigger a cascade of molecular and electrical events
that, ultimately, influence some aspects of the social behavior of the individual.
Although the main olfactory and vomeronasal systems share a similar histological organization, they also
display relevant differences with regard to the receptor
repertoire that they express and to the connections to
specific central areas (Fig. 1). Each system possesses
primary sensory neurons that send axons to second-order
neurons (mitral cells) in specific regions of the main
olfactory bulb (main olfactory system) or of the accessory
olfactory bulb (vomeronasal system). The mitral cells of
the main olfactory bulb project to several higher centers
including the piriform cortex and the cortical amygdala.
Instead, projections from the accessory olfactory bulb
only reach the medial amygdala and the posterior-medial
part of the cortical amygdala. From here fibers terminate
in the hypothalamus either directly or via the bed nucleus
of the stria terminalis.
In the following sections we summarize how the
various sensory epithelia are functionally organized. For
more exhaustive anatomical description, readers may
consult reviews specifically dedicated to these subjects
(66, 229).
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neurons traverse the developing accessory olfactory bulb
and migrate to the mediobasal hypothalamus. The vomeronasal sensory neurons maintain a functional relationship with the hypothalamic areas in the adult mammal,
influencing neuroendocrine function and behavior.
Do humans have a vomeronasal organ? The presence
of a vomeronasal organ in human embryos, containing
bipolar neurons similar to developing vomeronasal neurons of other species, was clearly demonstrated (24, 25,
166, 240). However, the vomeronasal structure becomes
more simplified later in development (24), and several
Physiol Rev • VOL
reports showed that in human adults it becomes a blindended diverticulum in the septal mucosa (246, 383). Moreover, the epithelium lining the vomeronasal organ in human adults is different from that of other species and
from the olfactory or respiratory epithelium (259, 366).
Several studies showed that vomeronasal cells were not
stained by an antibody against the olfactory marker protein (259, 366, 383, 403), a protein abundantly expressed
in mature neurons in the olfactory and vomeronasal epithelia of other species (41, 144). In addition, Trotier et al.
(383) did not identify vomeronasal nerve bundles using a
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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.
specific antibody against the S-100 protein, a characteristic axonal marker (7). Therefore, even if vomeronasal
cells were chemosensitive, they would not have any obvious way to communicate with the brain. Therefore, the
development of human vomeronasal structures seems to
be limited to the embryonic stage, when they possibly
play a role in the migration of GnRH-secreting neurons
toward the brain (383; for review, see Ref. 246).
B. Olfactory Epithelium
Physiol Rev • VOL
C. Other Olfactory Organs
1. Septal organ of Masera
The septal organ of Masera (SO) (35, 311) is a small
island of olfactory epithelium within the nasal mucosa
and is located on each side of the septum, posterior to the
nasopalatine duct that connects the oral to the nasal
cavity (Fig. 2). The anatomical organization of the septal
organ appears similar to that of the main olfactory epithelium, although its neuroepithelium appears thinner
(218, 396). Furthermore, sensory neurons of the septal
organ are provided with cilia that bear shorter dendrites
and larger dendritic knobs than those of the main olfactory epithelium (218); their axons project to a subset of
glomeruli in the posterior, ventromedial main olfactory
bulb (Fig. 3). Interestingly, some of these glomeruli exclusively receive input from the septal organ sensory
neurons, while others are also innervated by fibers originating from the main olfactory epithelium (191). The anterior position of the septal organ, its vicinity to the
nasopalatine duct, and the wide distribution in many
mammalian species suggest that this organ may be functionally specialized for the early detection of biologically
relevant molecules as those, for example, resulting from
licking behavior.
2. Grueneberg ganglion
The Grueneberg ganglion is located bilaterally in the
anterodorsal area of the nasal cavity in the corners
formed by the septum and the nasal roof of several mammalian species (93a) (Fig. 2). Differently from other chemosensory organs, neurons of the Grueneberg ganglion
cluster in small groups (30, 79, 80, 317). Only two types of
FIG. 3. Projections of sensory neurons to the olfactory bulbs. Each
olfactory sensory neuron sends a single axon to the main olfactory bulb
(MOB). The apical and basal sensory neurons in the vomeronasal organ
(VNO) send axons, respectively, to the anterior and posterior accessory
olfactory bulb (AOB), a posterior dorsal region of the main olfactory
bulb. Neurons of the septal organ of Masera (SO) project to the posterior
ventromedial main olfactory bulb. Neurons of the Grueneberg ganglion
(GG) project to necklace glomeruli (NG) in the main olfactory bulb.
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New evidence suggests that some pheromones are
detected by the main olfactory epithelium, a structure
specifically dedicated to sensing volatile molecules, usually named general odorants. The main olfactory epithelium lines cartilaginous protuberances in the posterior
nasal cavity (Fig. 2), called turbinates, and is a pseudostratified epithelium composed, similarly to the vomeronasal
epithelium, of three main types of cells: olfactory sensory
neurons, supporting cells, and basal cells. The olfactory
sensory neurons are bipolar neurons with a single ciliated
dendrite extending to the mucosal surface. Cilia are the
site of primary transduction for odorants and, possibly,
for pheromones. Although most olfactory sensory neurons have the same type of transduction cascade, it has
been recently discovered that subsets of olfactory neurons express different types of transduction molecules
(see sect. VB2) (reviewed in Refs. 31, 217, 269). Axons of
sensory neurons are segregated in the epithelium and,
after crossing the basal membrane, bundle together to
form the fascicles of the olfactory nerves. Axons project
to distinct domains of the main olfactory bulb as further
described in section V.
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
between olfactory and respiratory epithelium is not as
sharp as in other mammals; instead, there is an irregular
zone where olfactory and respiratory cells intermingle.
Moreover, patches of respiratory epithelium have been
identified in purely olfactory areas (262).
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IV. PUTATIVE PHEROMONE RECEPTORS
A. Vomeronasal Receptors
In the vomeronasal organ of rodents, detection of
pheromones is mediated by two anatomically independent pathways that originate from chemosensory neurons
of distinct compartments, basal and apical, of the neuroepithelium. The organization of the vomeronasal epithelium of rodents in apical and basal neurons was initially
based on the specific expression pattern of marker molecules (261, 340, 341). More recently, exhaustive data
established that also distinct transduction components
characterize these two subsets of chemosensory neurons.
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.
ily compared with other species (313, 346, 415, 422). The
gene tree of functional V1Rs in placental mammals, based
on the mouse repertoire, is split into 12 clades (a-l) (Fig. 4)
(313). All rat V1R receptor genes, except for one, are
distributed within 10 of these 12 clades (a-g and j-l). Thus
the mouse h and i clades represent an instructive example
of species specificity, exclusively including mouse sequences. Given that one rat pseudogene is placed in each
of the h and i subfamilies, it is likely that the absence of
rat functional genes in these clades is the result of deletion in this species and of postspeciation expansion in the
mouse lineage.
V1Rs in the mouse account for almost 200 potentially
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).
In the mouse V1R phylogenetic tree, clades share
from 15 to 40% of their amino acid residues between each
other, with intrafamily identities varying between 40 and
99%. High sequence variability is found in transmembrane
domain 2, in intracellular loop 1, and in extracellular
loops 2 and 3, while transmembrane domain 3 is highly
conserved. A few amino acid residues mostly located
between transmembrane domains 5 and 6, and a potential
glycosylation in extracellular loop 2, are common features
to virtually all members of the 12 V1R clades, while specific amino acid signatures are characteristic of each of
the 12 families (313).
1. V1Rs
The first family of vomeronasal receptors, V1Rs, was
discovered with elegant experiments of differential
screening, based on the assumption that the genetic profile 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
G␣i2 (71, 285). From the structural point of view, V1Rs are
related to group I of G protein-coupled receptors and, in
the mouse, represent a strikingly expanded receptor famPhysiol Rev • VOL
FIG. 4. The mouse vomeronasal receptor families. Top: phylogenetic tree of V1Rs showing 12 phylogenetically isolated subfamilies.
Bottom: phylogenetic tree of the 4 V2R families showing their clades.
Numbers of receptors for each clade and family are shown in red.
[Modified from Silvotti et al. (355).]
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cells have been identified in the Grueneberg ganglion:
glial cells and ciliated neurons (30). Each neuron has a
single axon that fasciculates with others into nerve bundles that project to ⬃10 glomeruli in the main olfactory
bulb (Fig. 3). These glomeruli are a subpopulation of the
necklace glomeruli. Since necklace glomeruli are active
during the suckling behavior of the pup, it was suggested
that the Grueneberg ganglion may sense maternal pheromones (82, 173, 317). However, a very recent study has
rejected this hypothesis, suggesting instead that alarm
pheromones, volatile molecules released by animals to
alert conspecifics about danger, are the likely substrates
of the Grueneberg ganglion neurons. These results are
discussed in more detail in section VB.
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V1R genes are scattered in regions of many chromosomes, where members of a given family are found almost
invariably clustered. It has been recently reported that
V1R clusterization may not simply reflect gene expansion
but may be actively linked to the mechanisms underlying
the tight regulation of receptors’ expression. V1R expression is, indeed, monoallelic and monogenic, both of these
being tools that achieve the expression of one receptor
type per cell, thus contributing to the narrow tuning of the
vomeronasal neurons (318).
At present, some vertebrate genomes of species of
different orders have been extensively scrutinized for the
Physiol Rev • VOL
presence of V1Rs (Fig. 5). Interestingly, V1R genes were
found in all species except in chicken and chimpanzee, in
which the presence of a functional vomeronasal organ has
not been consistently demonstrated. The number of functional V1R genes drops dramatically from rodents to other
species. The human genome contains several V1R pseudogenes, only five human V1R genes that have an intact
open reading frame, and at least one V1R that is transcribed in the olfactory epithelium. The chimpanzee genome has no obvious functional V1R genes but a large
collection of pseudogenes. This is surprising if we consider that the massive loss of V1R genes in chimpanzee
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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).
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2. V2Rs
A second family of vomeronasal receptors, V2Rs, was
discovered in 1997 (110, 234, 323) and, in mouse, it accounts for ⬃120 potentially functional members. V2Rs
belong to group III G protein-coupled receptors and are
characterized by a long NH2-terminal domain. This region
has been demonstrated to represent the binding domain
in most receptors of group III (for review, see Ref. 293).
V2Rs are strictly coexpressed with the G protein subunit
G␣o in the basal neurons of the vomeronasal organ.
In the mouse, intact V2Rs can be grouped in four
distinct families (A-D) according to sequence homology
(Fig. 4). Together, receptors of families A, B, and D represent 95% of all V2Rs. Their expression pattern strongly
suggests that these receptors are expressed by mutual
exclusion in the vomeronasal organ. A phylogenetic analysis shows that family C V2Rs (also known as V2R2
family) are very divergent from those of families A, B, and
D (3, 231, 323), and these differences are also functional
(354). This family is also noteworthy for the high sequence conservation between species. Moreover, compared with other V2R pseudogenes, family C pseudogenes
have one or very few inactivating mutations (also in the
Physiol Rev • VOL
human pseudogenes), suggesting a relatively recent loss
of function.
In rodents, this receptor superfamily is largely expanded and, in contrast to V1Rs, it appears completely
degenerated in primates, dogs, and cows (Fig. 5). In the
dog, the lack of a sizable V1R and V2R family does not
probably reflect a reduction in the pheromonal relevance
in triggering intraspecific behavior, but rather a general
decline of the vomeronasal system, a hypothesis that is
supported by anatomical data (63). In contrast, in the
opossum, V2Rs are abundantly represented (Fig. 5). Thus
it appears that the evolution of opossum and rodent V2Rs
genes parallel that of the V1R ones in these species, which
expanded independently. Moreover, most opossum and
rodent V2R and V1R genes arose by duplication (rodent
V2R and V1R genes are found in genomic clusters, and
closely related V2Rs tend to reside near one another in
the genome) after these lineages diverged. This receptor
diversification and the consequent detection of additional
pheromones may have resulted in a selective advantage.
The expression pattern of V2Rs makes them different
from V1Rs and odorant receptors in that each basal neuron expresses two V2Rs, one of which is a member of
family C (231). Moreover, there is evidence for a combinatorial scheme for the detection of chemosensory cues
in the basal neurons of the vomeronasal organ. Recent
data indicate the existence of multimolecular complexes
in individual basal neurons, constituted by MHC molecules, by family A, B, or D and family C V2Rs (355). For
example, neurons expressing family A receptors of the
highly represented clades I, III, and IV almost exclusively
express the family C receptor Vmn2r2, whereas neurons
expressing clade VIII receptors specifically express the
family C receptor Vmn2r1. Interestingly, coexpression of
Vmn2r1 and Vmn2r2 with family A V2Rs seems to involve
whole clades rather than individual receptors. Thus a
peculiar modality of expression subsists in the basal layer
of the vomeronasal organ, where two family receptors are
mutually exclusively expressed in the same basal neurons.
It has been suggested that V2Rs bind MHC peptides
(185) and MUPs (47), as further discussed in section VA.
A V2R receptor, V2R83, is also expressed outside the
vomeronasal organ, in a large subset of neurons of the
Grueneberg ganglion (79), and it is a good candidate
receptor for alarm pheromones (30).
At present, however, direct evidence that V2Rs are
directly involved in pheromone signaling is still missing.
At least 50% of the basal vomeronasal neurons express another multigene family representing nonclassical
class I major histocompatibility complex (H2-Mv) genes.
Each of the nine identified H2-Mv genes is present in a
subset of vomeronasal neurons, and multiple H2-Mv
genes can be coexpressed in the same neuron (127, 207).
H2-Mv molecules are also coexpressed in a combinatorial
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appears even greater than for the human repertoire, in
which V1R genes have been identified. However, the most
surprising observation is that the dog only possesses eight
functional V1R genes spread among four clades. This
finding is unexpected because the dog is traditionally
renowned to have evolved an extraordinary well-developed olfactory system and a complex social behavior
(346). Although it is plausible that V1Rs emerged as dominant receptors for triggering specific rodent behaviors
(multiple losses of ancestral V1R genes in carnivores and
artiodactyls and gains of many new genes by gene duplication in rodents), it cannot be alternatively ruled out that
a loss of dog V1Rs is due to the breeding and domestication processes of this species. Therefore, diversification
into subfamilies probably occurred prior to the rodentdog-primate split early in the mammalian evolution (415).
The question as to whether V1Rs are exclusively
expressed in the vomeronasal epithelium or also in other
tissues is still controversial. Karunadasa et al. (152) reported the expression of V1Rd RNA in some cells scattered within the main olfactory epithelium of embryonic
and postnatal mouse and suggested that pheromone detection may also take place in this structure. V1R-homolog RNAs have also been identified in the main olfactory epithelium of goats and humans (316, 394). However,
caution must be taken before raising any hypothesis as,
until now, there are no clues that a functional V1R is
expressed at a detectable level in the main olfactory
epithelium.
manner with receptors of specific subfamily of V2Rs (127,
207). It has been initially proposed that H2-Mv may function as indispensable escort molecules in the transport of
V2Rs to the plasma membrane (207). However, this chaperone-like function is not generalized to all V2Rs (127,
354), and moreover, it is likely to be exclusively restricted
to the mouse or rat as the opossum genome contains a
hundred functional V2R genes and no H2-Mv genes (346).
3. Ligands of vomeronasal receptors
Physiol Rev • VOL
To deorphanize V1Rs and study their binding properties, it is necessary to develop an efficient bioassay. At
present, it is only plausible to assume that V1Rs are
vomeronasal receptors narrowly tuned to bind small volatile pheromones, possibly through a hydrophobic pocket
located in their transmembrane region. Nevertheless,
achievements in this direction have been very recently
accomplished by Shirokova and colleagues who managed
to deorphanize the human V1Rs employing NH2-terminally tagged receptors expressed in the cell line HeLa/Olf
(350). It appears that, in sharp contrast to V1Rs, each
human recombinant V1R receptor responds to several
aliphatic alcohols or aldehydes via the olfactory G protein
G␣olf (350). Unfortunately, a pheromonal function of human V1R ligands has yet to be demonstrated.
4. Formyl peptide receptors
Recently, a group of receptors belonging to the family of formyl peptide receptors (FPRs) has been exclusively identified in the vomeronasal organ (310a). FPRs
are G protein-coupled receptors expressed in all mammals, with three genes present in the human genome and
seven in the mouse genome (249a). At least two of the
mouse FPRs have been reportedly involved in immune
and inflammation responses being expressed in granulocytes, monocytes, and macrophages (183a). An in situ
analysis of the vomeronasal organ of the mouse indicates
that five FPRs are expressed in a small subset of chemosensory neurons of the apical layer. Antibody staining
reveals that FPRs are featured by a subcellular distribution similar to that of V2R receptors including, besides the
microvillar surface, the dendrite and somata. Although
Fpr expression is restricted to the apical layer and, as
well as VIRs, adopts monogenic transcription, no coexpression is evident with receptors of this large family,
suggesting that FPRs characterize a new and specific
class of chemosensory neurons. Experiments based on
calcium imaging on the whole vomeronasal epithelium
and in dissociated chemosensory neurons establish that
indeed FPR agonists, such as some microbial, antimicrobial, and viral peptides, stimulate neuronal subsets with a
nonidentical affinity profile. These findings indirectly
prove that these receptors play a role in offering a barrier
against immunological diseases, or perhaps also in pheromonal communication itself, given that formyl peptides
have been detected in urine (52a) and that, in the olfactory and vomeronasal epithelium, two FPR isotypes appear to be expressed in the glandular but not chemosensory compartment (310a).
B. Odorant Receptors
Odorant receptor genes were discovered in 1991 by
Buck and Axel (39). The vast majority of the olfactory
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Although an initial report provided clues on the possibility that V1Rs could be expressed in a heterologous
system (99), the problem of expression later became the
major barrier to the identification of ligands for vomeronasal receptors, as well as for odorant receptors (for
reviews, see Refs. 224, 256, 312). Some information about
ligands for V1Rs was obtained from physiological approaches to the problem. Results from calcium imaging
experiments on slices of the mouse vomeronasal organ
indicated that some molecules credited as pheromones
stimulated specific vomeronasal neurons that, for their
topographic position in the vomeronasal organ, were
likely to express the V1R repertoire (187). Furthermore,
these authors reasoned that, if neurons responding to
these pheromones indeed reflected a V1R-dependent activity, V1Rs must be narrowly tuned to bind one or very
few pheromones. This contrasts with the broad selectivity
of olfactory sensory neurons that can be stimulated by a
large array of odorants. Further and more direct investigations on the V1R ligand specificity lead to the conclusion that V1Rs are indeed qualified as pheromone receptors: Del Punta et al. (61) succeeded to delete a large
genomic region containing ⬃16 V1R genes of subfamily a
and b, including the receptor V1rb2. The behavior of these
mutant animals was also thoroughly investigated showing
remarkable deficits (see sect. VII). Moreover, electrophysiological responses of vomeronasal neurons to some
pheromones, including 6-hydroxyl-6-methyl-3-heptanone,
n-pentylacetate, isobutylamine, and 2-heptanone, were altered (Table 1). Thus the repertoire of V1R receptor candidates to respond to these pheromones was restricted to
a relatively limited number.
Another approach, developed by Boschat et al. (28),
led to the identification of the first pheromone-V1R pair in
mammals. These authors employed mouse mutant lines
with a GFP-targeted V1rb2 allele, allowing the visualization and the isolation of the V1Rb2-expressing neurons for
calcium imaging based experiments. Thus a direct correlation was established between V1Rb2 expression and the
response of vomeronasal neurons to the mouse pheromone 2-heptanone (Table 1). As a confirmation of the
strong selectivity of V1Rs above reported, 2-heptanonerelated compounds were found to be ineffectual to stimulate V1Rb2-expressing sensory neurons.
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same neurons, suggesting that subsets of neurons exist in
the olfactory epithelium that are not exclusively dedicated to the detection of conventional odorants. By the
expression of olfactory TAARs in heterologous cell systems it was demonstrated that at least four of them
(mTAAR3, mTAAR4, mTAAR5, mTAAR7f) bind smallmolecule amines and that each of these receptors recognizes a specific set of ligands, among which are ␤-phenylethylamine, isoamylamine, and trimethylamine that are
natural components of the mouse urine. ␤-Phenylethylamine, a ligand for mTAAR4, is contained in human and
rodent urine, where its concentration rises during stressful situations (93, 286, 361). Isoamylamine and trimethylamine bind to mTAAR3 and mTAAR5, respectively, and
are present at higher concentrations in male compared
with female urine (84, 276). Noteworthy, isoamylamine
has been reported to act as a pheromone accelerating
puberty onset in female mice (276). Therefore, stimulation of mTAARs seems to affect the capacity of discriminating gender and social status of an individual. These
findings strongly contribute to confirm that the main olfactory epithelium is involved in the elicitation of physiological responses and innate behaviors.
In the mouse, TAARs are also expressed in subsets of
neurons of the Grueneberg ganglion (80). As in the main
olfactory epithelium, also in the Grueneberg ganglion,
distinct TAAR subtypes are expressed in nonoverlapping
subsets of neurons that do not coexpress the vomeronasal
receptors V2Rs. Since the number of neurons expressing
TAARs and V2Rs in the Grueneberg ganglion is much
higher during the perinatal stage than in the adult, it was
suggested that these neurons may be important for interactions between pups and their mother (80).
TAARs have also been identified in the genome of
many species including humans and fish, with the number
of putatively functional TAAR genes greatly varying
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
mouse olfactory epithelium. These receptors are the
“trace amine-associated receptors” (TAARs) (27, 192,
203), a family of G protein-coupled receptors discovered
in 2001 (27, 42) (for review, see Ref. 425). TAARs are
unrelated to odorant receptors but have similarities with
receptors for biogenic amines, such as serotonin and
dopamine receptors. The TAAR gene family includes 15
members in the mouse (Fig. 5), and each of them, except
for TAAR1, is expressed in the olfactory epithelium (194).
Moreover, the level of expression of TAAR genes is similar to that of odorant receptors, although TAARs and
odorant receptors do not appear to be coexpressed in the
Physiol Rev • VOL
A small subset of olfactory sensory neurons localized
in the dorsal region of the main olfactory epithelium
expresses a specific subtype of the transmembrane receptor guanylyl cyclase, named guanylyl cyclase type D
(GC-D) (81, 149). The GC-D receptor is encoded by one of
the seven guanylyl cyclase genes with whom it shares a
similar topology: all receptor guanylyl cyclases retain an
NH2-terminal ligand binding region, a single transmembrane loop, and a COOH terminus that includes the catalytic domain (81, 296). Specific ligands have been identified for some guanyly cyclases, but not for GC-D yet;
however, potential ligands for this receptor have been
recently assessed. In the mouse, for example, neurons
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neurons express only 1 of the 1,000 odorant receptor
genes (51, 226, 273, 308, 370, 389, 389). Neurons expressing different receptor types are usually found in one of
four partially overlapping zones of the olfactory epithelium, within each of which a given odorant receptor appears to be randomly distributed (128, 251, 308, 369, 389).
An olfactory sensory neuron typically responds to
more than one type of odorant molecules, and a given
odorant can activate neurons with different specificity
(70, 77, 226, 351). For the discrimination and perception
of odorants, the odorant receptor family seems to employ
a combinatorial scheme, with one single odorant molecule activating several types of receptors and each receptor sensing several different types of odorants, thereby
producing a unique combination of receptors activated by
each odorant molecule. This combinatorial scheme allows the olfactory system to recognize an enormous number of odorants (40, 256).
Odorant receptor genes have been identified in regions outside the main olfactory epithelium as the septal
organ and the vomeronasal epithelium. About 50 odorant
receptor genes are expressed in the mouse septal organ
(150, 378). Moreover, a subset of 44 odorant receptor
genes is also expressed in the vomeronasal epithelium
(190), although their functional role has not been established.
At present, we do not possess evidence that odorant
receptors can also function as pheromone receptors.
However, intriguingly, recent experiments linked the expression of a subclass of odorant receptors with stereotyped behavioral expressions in the mouse, thus suggesting that odorant receptors can convey information both
about the quality of an odorant and the individual that has
emitted it (169).
For additional information about odorant receptors,
the reader is referred to several reviews that extensively
discuss this subject (86, 224, 225, 256).
V. PHEROMONE TRANSDUCTION
Distinct neuronal subsets of the olfactory organs express specific types of receptors, second messenger systems, and ion channels to transform the pheromone binding into electrical signals.
The electrical activity of chemosensory neurons in
response to stimuli can be measured with a number of
approaches, including recording of the field potential, of
the firing activity with an array of extracellular electrodes
and by patch-clamp technique at the single-cell level.
Nonetheless, calcium imaging either in dissociated cells
or epithelium slices is often employed as well as the
metabolic activity of stimulated neurons is frequently
monitored upon pheromonal stimulation by evaluating
the expression of the early protooncogenes.
A. Vomeronasal Sensory Neurons
1. Signal transduction molecules
The detailed signal transduction cascade in the
vomeronasal organ is still largely unknown, although it
has been clearly demonstrated that pheromone stimuli
cause membrane depolarization and increase the action
potential firing rate in vomeronasal sensory neurons (28,
53, 61, 68, 115, 124, 125, 185, 187, 211, 363, 374). As
described in previous sections, the sensory epithelium of
the vomeronasal organ presents two subsets of neurons:
apical and basal. Apical neurons express members of the
V1R family of vomeronasal receptor genes together with
G␣i2, whereas basal neurons express V2R receptors and
G␣o (19, 102, 142, 143, 321, 348, 371, 398). The anatomical
and perhaps functional segregation in the vomeronasal
epithelium is likely to be a prerogative of species as rats,
Physiol Rev • VOL
mice, hamsters, opossum, and platypus that possess functional V2R genes (Fig. 5).
Krieger et al. (176) reported that whole male urine
induces inositol 1,4,5-trisphosphate (IP3) production via
the activation of G␣i2 as well as G␣o, suggesting a role for
both G protein subtypes in transducing pheromonal responses mediated by urinary components. Moreover, according to these authors, the activation of G␣i2 and G␣o
subtypes in the vomeronasal epithelium appears to be
caused by two structurally different classes of molecules.
Whereas G␣i2 activation was only observed upon stimulation with lipophilic volatile components, G␣o activation
was elicited by the rat major urinary protein, ␣2-globulin.
A confirmatory role of these G proteins in pheromone
transduction issues from behavioral (see sect. VII) and
anatomical studies performed on G␣i2 and G␣o negative
mutant mice (278, 373): both mouse lines show a remarkable 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 vomeronasal sensory neurons.
Since PLC stimulation and IP3 production appear to
be predominantly mediated by the release of the ␤␥ complex of the heterotrimeric G proteins, Go and Gi (56), it
has been proposed that G␤␥ may play an important role in
the transduction process of the vomeronasal epithelium.
One ␤-subunit (G␤2) and two ␥-subunits (G␥2 and G␥8)
have been described in the vomeronasal epithelium (321,
322, 380). G␥2 immunopositive neurons are exclusively
localized in the apical layer, whereas G␥8 neurons are
preferentially restricted to the basal one. G␤2, on the
other hand, seems to be active in both neuronal subsets
(Fig. 6). Biochemical studies reveal that IP3 formation
induced by stimulation of membrane preparations with
volatile urinary compounds was selectively blocked by
an anti-G␤2 antibody, whereas second messenger production induced by stimulation with major urinary proteins was inhibited by preincubation of the vomeronasal membranes with an antibody recognizing G␥8 (321).
Thus it appears that the G␣o␤2␥8 complex may control
PLC activation by proteinaceous pheromones, whereas
G␣i2␤2␥2 neurons respond to urinary volatile compounds.
Other lines of evidence indicate that, at least in some
vomeronasal sensory neurons, transduction is mediated
by a phosphatidylinositide signaling pathway. Some studies showed that electrical responses, IP3 production, and
calcium entry upon pheromonal stimulation (53, 176, 177,
215, 330, 397) are specifically blocked by the phospholipase C inhibitor U73122 (115, 363). Recently, Thompson
et al. (377) demonstrated that compounds known to induce pregnancy block (Bruce effect), such as MHC peptides and male urine, also increased the IP3 formation,
confirming a major role of the phosphatidylinositide pathway in the vomeronasal signal transduction.
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expressing GC-D respond to dilute urine and to the intestinal peptide hormones uroguanylyn and guanylin, while
neurons in GC-D knockouts are unresponsive to these
compounds (186). Given their localization in the enteric
tract, uroguanylyn and guanylin are supposed to contribute to the maintenance of salt and water homeostasis or
to the detection of cues related to hunger, satiety, or thirst
(186). GC-D neurons also express carbonic anhydrase II
and respond to CO2 stimuli (118). It is not clear if there is
a possible interplay between these two chemosensory
responses.
Despite its exquisite expression in the main olfactory
epithelium, GC-D appears to be a pseudogene in species
lacking a functional vomeronasal organ such as humans,
apes, Old World and New World monkeys, and tarsier.
This suggests that a functional GC-D gene was lost early
in primate evolution and that chemical detection in most
primate species is unlikely to involve GC-D (416).
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The ion channel TRPC2, a member of the superfamily
of transient receptor potential channels (390, 412), is
expressed in the microvilli of both apical and basal neurons (197, 241, 271) and plays an important role in the
transduction process of some, but not all, vomeronasal
sensory neurons. Interesting to note, TRPC2 is unique
among TRP channels in that a functional gene has been
lost from the Old World monkey and human genomes
(198, 420). Proposed mechanisms for the activation of the
TRPC2 channel in the vomeronasal neurons are similar to
those involved in the signaling cascade of Drosophila
photoreceptors (103, 250): pheromones trigger the activa-
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level in the gartner snake (215) and in rodents (215, 319,
424). However, the decrease in cAMP occurs with a longer
delay with respect to pheromone stimulation (319). Possible targets for modulatory actions by cAMP include the
Ca2⫹-activated cation channel (195) and the recently
characterized hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels (64).
It is intriguing that a subpopulation of apical sensory
neurons expresses members of the odorant receptor gene
family, together with G␣i2 and TRPC2 (190). Since some
conventional odorants are detected by the vomeronasal
epithelium (329, 382, 409), it was suggested that vomeronasal neurons expressing odorant receptors might be
those that respond to odorants (190).
Following the initial transduction events, when the
membrane potential of vomeronasal sensory neurons
reaches threshold, action potentials are generated and
propagate along the axons to the accessory olfactory
bulb. Interestingly, most vomeronasal neurons fire tonically for several seconds, without sign of adaptation,
when small current injections (64, 196, 347, 384) or dilute
urine are applied for up to 2–3 s (211, 421). However, in
one study, longer stimulations of 10 – 60 s caused spikefrequency adaptation (384), and in addition, a recent work
suggested that sensory adaptation in vomeronasal sensory neurons requires the influx of Ca2⫹ and is mediated
by calmodulin (362).
2. Responses to urinary pheromones
Using an array of extracellular electrodes, Holy et al.
(115) simultaneously recorded the activity of a large number of mouse vomeronasal neurons in response to dilute
urine. Neurons responded to urine with an increase in the
firing frequency that remained approximately unchanged
if the stimulus was applied for as long as 100 s, indicating
that the response of these neurons do not adapt to prolonged stimulus exposure. Furthermore, they identified
distinct and nonoverlapping subsets of neurons that were
selectively activated by substances contained in female or
male urine; in contrast, about half of the vomeronasal
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 Na⫹ and Ca2⫹, causing a membrane depolarization. An additional cation-selective channel activated
by Ca2⫹ 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 Na⫹ and Ca2⫹. Ca2⫹ entry amplifies the signal by causing the efflux of Cl⫺ through Ca2⫹-activated Cl⫺ channels (Cl-Ca). These ion fluxes cause
a depolarization. Inhibitory actions are played by the complex Ca2⫹-calmodulin (CaM) by increasing the activity of the phosphodiesterase (PDE)
that hydrolyzes cAMP and by reducing the affinity for cAMP of the CNG channels. Ca2⫹ is extruded by the Na⫹/Ca2⫹ exchanger (Na/Caex).
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tion of G protein-coupled receptors that in turn stimulate
phospholipase C and the production of the lipid messenger diacylglycerol (DAG) (211) and possibly of polyunsaturated fatty acids such as arachidonic acid (363, 421) and
linolenic acid (Fig. 6) (363).
Genetic ablation of the TRPC2 channel results in a
strong, but not complete, reduction of electrophysiological responses of the vomeronasal neurons to urine or its
volatile components (156, 193, 368). Moreover, male
TRPC2-knockout mice showed severe behavioral abnormalities (see sect. VII) (157, 193, 236, 368), indicating that
the TRPC2 channel may be an important component of
the transduction of pheromone signals.
Nevertheless, the genetic ablation of TRPC2 does not
completely abolish the response of the vomeronasal organ to pheromones, since electrophysiological recordings
have demonstrated that basal neurons of mutant mice still
respond to MHC peptide ligands as controls (156). In
addition, TRPC2-knockout mice exhibit the Bruce effect
(156), a neuroendocrine response that requires a functional vomeronasal organ. All together, these studies suggest that at least a subset of basal neurons uses a different
channel from TRPC2 for the transduction of some pheromonal stimuli.
Interestingly, Liman (195) showed that a cation channel activated by a high concentration of Ca2⫹ is expressed
in hamster vomeronasal sensory neurons. This channel is
permeant to both Na⫹ and K⫹ and is blocked by ATP and
cAMP, sharing many features with the TRPM4 ion channel
(275). These properties make this channel a good candidate to be directly involved in sensory transduction or in
amplifying a primary sensory response. At present, the
molecular nature of this channel and its relation with
TRPC2 or with IP3 production are not yet resolved.
Some components of the cAMP signaling cascade
have been identified in vomeronasal sensory neurons.
These include adenylyl cyclase types 2 (19), 4, 5 (184), and
6 (184, 319); the subunit CNGA4 of the olfactory cyclic
nucleotide-gated channel (19, 184); and phosphodiesterase types 1 and 4 (50, 183), suggesting that cAMP may play
a physiological role in vomeronasal sensory neurons. Indeed, some pheromones induce a reduction of the cAMP
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an exceptionally low-noise imaging of large neuronal populations, unexpectedly found that responses to dilute
urine are predominantly, and perhaps exclusively, generated by neurons in the apical layer, where V1Rs are expressed.
Leinders-Zufall et al. (187) showed that some volatile
hydrophobic pheromones, known to be tightly bound to
the hydrophobic pocket of MUPs, produced a calcium
increase in neurons of slice preparations of the vomeronasal organ. The distribution pattern of the activated
neurons by any of these molecules reflects a localization
that can be associated with the expression of the vomeronasal receptors V1Rs (187), and the percentage of neurons responding to an individual pheromone (0.2–3%)
matches the proportion of neurons expressing one type of
V1R. Therefore, when volatile molecules are released by
MUPs, in the external environment or in the nasal cavity,
they are likely to bind and activate V1Rs.
Urine contains compounds whose physiological role
is largely unknown. Nodari et al. (277) applied chromatographic fractions of female mouse urine to isolated vomeronasal epithelia mounted on a multielectrode array and
measured the spiking activity of neurons (277). Increase
in the firing rate was associated with compounds with
homogeneous chemical properties that, after further purifications, appeared to correspond to sulfated steroids.
Sulfatase-treated urine extracts lost ⬎80% of their activity, indicating that sulfated compounds are the predominant ligands for vomeronasal neurons in female urine.
Interestingly, a collection of 31 synthetic sulfated steroids
triggered responses 30-fold more frequently than did a
similarly sized stimulus set containing the majority of all
previously reported ligands. Vomeronasal neurons seemed
to have a high tuning specificity for sulfated steroids, since
each neuron responded only to one or to a few structurally similar compounds, but collectively, neurons detected all major classes of sulfated steroids. Some of
these compounds may be indicators of a status of stress.
Indeed, the concentration of two sulfated glucocorticoids
in urine increased about threefold in animals subjected to
stress compared with unstressed controls, indicating that
information about the status of stress of these mice is
encoded in urine and could be detected by vomeronasal
sensory neurons (277).
3. Responses to secretory peptides
Kimoto et al. (163) showed that mouse recombinant
exocrine gland-secreting peptides (see sect. IIC) evoked
electrical activity in the vomeronasal but not in the olfactory epithelium. Interestingly, when the electrical responses of individual neurons to ESP1 were recorded
with a multielectrode array, only a small percentage of
neurons, 1.6%, responded to ESP1 (163). This result is consistent with the possibility that ESP1 is recognized by one
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Thus these findings establish the capacity of the vomeronasal organ to identify differences between sexes.
A recent study by He et al. (106) further stressed the
concept of the vomeronasal organ being subtly dedicated
to the identification of conspecifics. These authors quantified the number of neurons responding to individual
urine of either sex and of different strains in slices of the
vomeronasal epithelium of mice genetically modified to
express a calcium indicator. It was observed that only a
very small subpopulation of neurons (1–3%) exclusively
responded to urine samples from all individuals of the
same sex. The gender-specific neurons were evenly distributed both in the apical and basal layers of the epithelium and did not differ in males or females, consistent
with the little dimorphism generally observed at the level
of receptor expression in the vomeronasal epithelium.
Interestingly, neurons specific to female urine showed
different activation patterns depending on the phase of
the estrus cycle; in contrast, urine from castrated mice
failed to elicit a response in male-specific neurons. Furthermore, information about individuals and strains is not
encoded by specialized neurons but, rather, by the combinatorial activation of vomeronasal neurons, as no urine
samples from different mice of the same sex elicited the
same pattern of neuronal activity. Thus the information
contained in the mouse urine about gender is encoded by
dedicated neurons; strains and individuals are recognized
by a combinatorial code of neuronal activation (106), with
the obvious advantage to discriminate a large population
of individuals with a restricted repertoire of receptors.
He et al. (106) also compared responses elicited by
synthetic strain-specific MHC class I peptides with those
induced by urine of the same strain and found that neurons activated by urine and by the peptides did not overlap. Furthermore, the same authors (106) pointed out that
only fragments, but not the peptides, of the MHC complexes are detectable in urine (358). This result strongly
contrasts with what was observed by Leinders-Zufall et al.
(185), who identified a specific subset of basal neurons
highly sensitive to MHC class I peptides and to urine from
mice of the relevant haplotype.
Other studies investigated the response of the vomeronasal epithelium to specific urinary components, such
as MUPs, that represent another class of candidates for
strain recognition, as well as for heterozygosity signals
(375). Kimoto et al. (163) found that a recombinant isoform of MUPs elicits electrical responses in the vomeronasal epithelium, and Chamero et al. (47) showed that
native and recombinant MUPs evoke calcium influx in
sensory neurons expressing the G protein subunit G␣o.
Since G␣o coexpresses with V2Rs, it is likely that MUPs
directly activate a response in vomeronasal sensory neurons by binding to a subset of these receptors (47). In
sharp contrast to these observation, Holekamp et al.
(113), by using a new microscopical technique that allows
type of receptor only, most likely by a specific V2R, as ESP1
induced c-fos production in vomeronasal neurons expressing V2Rp5, a V2R receptor of the family A, clade III (98).
B. Olfactory Sensory Neurons
1. The cAMP transduction cascade
2. Neuronal subsets
The subset of olfactory sensory neurons expressing
TAARs also express G␣olf, and therefore, it is possible
that they use a signal transduction pathway coupled to
adenylyl cyclase and cAMP (194), although additional
components of the transduction pathway in these neurons are still unknown.
The ion channel TRPM5 is expressed in another subset of olfactory sensory neurons that coexpress the
CNGA2 channel, and some components of the PLC pathway, such as PLC-␤2 and the G protein ␥13 subunit (202).
Physiol Rev • VOL
Since TRPM5 channels are directly activated by intracellular Ca2⫹ (112, 204, 299, 423), it is tempting to speculate
that Ca2⫹ entry through CNG channels could gate TRPM5
channels.
Neurons expressing TRPM5 are located in the ventrolateral zones of the olfactory epithelium and project to
domains of the ventral region of the main olfactory bulb,
an area responsive to urine and other putative pheromones (199 –201, 336, 409). Unfortunately, electrical responses from individual olfactory sensory neurons expressing TRPM5 have not yet been investigated.
As previously mentioned (see sect. IVD), another subset of neurons in the main olfactory epithelium expresses
components typical of a cGMP instead than a cAMP second messenger system, namely, the receptor guanylyl
cyclase type D (GC-D), the cGMP phosphodiesterase
PDE2 (149), and the cyclic nucleotide-gated channel
CNGA3, that was originally discovered in cone photoreceptors (417). These neurons are located in the central
area of the olfactory epithelium and project to an anatomically distinct group of interconnected glomeruli, the
necklace glomeruli, that have been implicated in the suckling response of mammals (149). Leinders-Zufall et al.
(186) showed that neurons expressing GC-D can be activated by the peptide hormones uroguanylin and guanylin
and by natural urine stimuli, while Hu et al. (118) demonstrated that these neurons may be involved in the detection of carbon dioxide. In both studies, GC-D neurons
were shown to use a cGMP signaling cascade (118, 186).
A subset of neurons in the main olfactory epithelium
of humans and mice expresses some vomeronasal receptors of the V1R family (152, 313, 315). Ligands corresponding to aliphatic alcohols or aldehydes have been identified
for human V1Rs (350); however, it is still unresolved
whether neurons expressing these receptors project their
axons to the main or the accessory olfactory bulb.
A small population of neurons in the main olfactory
epithelium expresses the TRPC2 channel. At present,
there are no clues whether these neurons have chemosensory properties, but this finding is certainly relevant
and must be seriously taken into account when comparing the behavioral effects produced by the genetic deletions of the TRPC2 channel with those of the surgical
removal of the vomeronasal organ (P. Mombaert, personal communication).
Early microscopical and immunological studies have
revealed the existence of a novel cell type in the olfactory
epithelium that is morphologically different from chemosensory neurons or supporting cells (44, 148, 260). The
main feature of these cells is represented by the microvilli
that they bear on the mucosal side. It now appears that
olfactory microvillar cells do not represent a homogeneous population of cells but rather they include different
cellular subsets expressing distinct signal transduction
molecules (6, 74, 201). It is very unlikely that these cells
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In contrast to a common belief, some sensory neurons of the main olfactory epithelium respond to pheromones (364, 395). A large majority of olfactory sensory
neurons express a member of the odorant receptor family
that is coupled to a signal transduction pathway mediated
by cAMP (Fig. 6). The binding of ligands to the odorant
receptor activates the G protein subunit G␣olf, which
stimulates adenylyl cyclase-3 activity producing an increase in cAMP concentration. cAMP causes the opening
of the ciliary cyclic nucleotide-gated channels composed
of three subunits: CNGA2, CNGA4, and CNGB1b (for
reviews, see Refs. 29, 153, 292). These channels allow an
influx of Na⫹ and Ca2⫹ inside the cilia. Ca2⫹-activated Cl⫺
channels are then activated in turn and, due to the unusually high intracellular Cl⫺ concentration, allow an outflow
of Cl⫺, contributing to the inward current (167, 179). As a
result of the odorant binding to receptors, the olfactory
sensory neuron depolarizes. The CNG channel allows
Ca2⫹ entry not only for excitatory but also for inhibitory
purposes. The complex Ca2⫹-calmodulin activates a phosphodiesterase (PDE1C2) that hydrolyzes cAMP and, importantly, produces a negative-feedback effect on the
CNG channel itself, that has been shown to mediate olfactory adaptation (178, 242).
When the depolarization following the activation of
the transduction cascade reaches the threshold for activation of action potentials, they will travel along the axon
to the main olfactory bulb. Differently from vomeronasal
sensory neurons, a maintained current injection will only
elicit a short burst of action potentials in olfactory sensory neurons, instead of a tonic firing (196).
For more details about the cAMP transduction cascade
in olfactory sensory neurons, the reader may consult previous comprehensive reviews (40, 168, 235, 242, 292, 337).
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are true neurons as they do not seem to project their long
foot process to the olfactory bulb (74, 201). However,
although their role is enigmatic, a possible chemosensory
function for microvillar cells has been reported (74).
3. Responses of the main olfactory epithelium
to pheromones
C. Grueneberg Ganglion
The transduction pathways in the Grueneberg ganglion
are still largely uncharacterized. Fleischer and colleagues
showed that the majority of neurons express the vomeronasal receptor V2R83 (V2R2 family), together with the G protein G␣i (79, 80), while a distinct subpopulation of G␣iexpressing neurons also expresses TAAR receptors (80).
Another study investigated whether neuronal subsets of the
Grueneberg ganglion express transduction components similar to those of GC-D neurons, based on the observation that
both types of neurons project to the necklace domain of the
main olfactory bulb (Fig. 3). Fleischer et al. (78) reported
that a subset of Grueneberg ganglion neurons expressing the
V2R83 receptor expresses also the GC-D receptor and the
cGMP-stimulated phosphodiesterase PDE2A.
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VI. SIGNALING FROM SENSORY EPITHELIA TO
THE BRAIN
A. Main and Accessory Olfactory Bulbs
1. Anatomical organization
The main olfactory bulb is the primary target region of
the olfactory neurons, whereas vomeronasal sensory neurons project to a well-defined dorsal and posterior region of
the olfactory bulb, namely, the accessory olfactory bulb. The
main olfactory bulb exists in all vertebrates, except for
aquatic mammals. In contrast, the accessory olfactory bulb
is absent in adult Old World monkeys, apes, and humans (for
more information, see Table 1 in Ref. 240). In rodents, both
the main and the accessory olfactory bulbs have a largely
similar laminar organization consisting of a superficial nerve
layer formed by the axonal projections of the chemosensory
neurons, the glomerular layer that represents the first-order
synaptic region between sensory neurons and mitral cell
dendrites, and the external and internal plexiform layers that
are regions where soma of, respectively, mitral/tufted and
granule cells reside (for review, see Ref. 240; see also Ref.
182 for suggested changes to the nomenclature of the accessory olfactory bulb). In the main olfactory bulb, glomeruli
are quite well anatomically separated, encapsulated by periglomerular cells, and rather uniform in size (⬃50 ␮m diameter in mice). Conversely, in the accessory olfactory bulb,
these synaptic structures are very diffusely organized, surrounded by a small number of periglomerular cells, and
highly variable in size (10 –30 ␮m diameter in mice). In the
main olfactory bulb, mitral/tufted cells form a distinct monolayer, whereas in the accessory olfactory bulb they are much
less organized. An important difference between mitral/
tufted cells of the two olfactory systems is their arborization:
in the main olfactory bulb these neurons bear only one
apical dendrite, whereas in the accessory olfactory bulb
mitral/tufted cells are equipped with up to six apical dendrites, each entering a different glomerulus (Fig. 7; for review, see Ref. 240).
2. Topographic projections
Axons of all the olfactory sensory neurons expressing a
particular odorant receptor converge to only two glomeruli
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Wang et al. (395) showed that milk and urinary pheromones that are responsible for puberty delay aggressiveness
in the mouse (279, 281) and evoke an electrical response
from the main olfactory epithelium, as measured by field
potential recordings. Moreover, when the same experimental approach was adapted to mice in which the adenylyl
cyclase type 3 gene had been knocked out, no electrical
response was recorded, indicating that receptors responding
to these compounds are likely to be coupled to the cAMP
signaling pathway. Since behavioral deficits are also evident
in adenylyl cyclase type 3 mutant mice, it can be envisaged
that a cAMP-mediated pheromone signaling may play an
important role in the olfactory epithelium.
MHC peptides can also be detected by sensory neurons of the main olfactory epithelium via the cAMP signaling cascade, as shown by Spehr et al. (364). Two MHC
peptide ligands, SYFPEITHI and AAPDNRETF, elicited an
electrical response, measured by field potential recordings in the main olfactory epithelium at a concentration
near 10⫺11 M (364). Mutant mice lacking the CNGA2 gene
failed to display electrical responses when stimulated
with both peptides, suggesting that a subset of olfactory
neurons expressing CNGA2 are actively involved in the
detection of pheromones besides conventional odorants.
In contrast to that, Lin et al. (200) have demonstrated
that at least two pheromones, 2-heptanone and dimethylpyrazine, stimulate the main olfactory epithelium without the intervention of the CNGA2 channel, suggesting
that pheromone detection in the olfactory epithelium relies on multiple transduction pathways.
Brechbuhl et al. (30) recently uncovered at least one
of the functional roles of the Grueneberg ganglion. Indeed, a subpopulation of neurons was shown to respond
to the yet unidentified “alarm pheromones,” a group of
volatile compounds that are released by animals subjected to stress. Lesions of this organ completely abolish
the so-called freezing reaction, a typical fear response
elicited by alarm pheromones. Curiously, in this study,
lesions of the Grueneberg ganglion did not affect motherpup recognition (30), a behavior that was believed to be
mediated by this organ (118, 173, 317).
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in the main olfactory bulb (257, 308, 389). This has been
experimentally demonstrated at a single-neuron resolution
by using transgenic mice in which the expression of a given
odorant receptor was linked to that of the green fluorescent
protein, thus allowing the visualization of the axonal netPhysiol Rev • VOL
work (255, 256, 381). In mice there are ⬃2,000 glomeruli, and
their localization is roughly conserved among individuals.
Therefore, the olfactory bulb is topographically organized,
with each glomerulus representing a single type of odorant
receptor (Fig. 7C).
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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.
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through glutamatergic synapses, but the chemical information is further processed by the activity of inhibitory
interneurons as well as periglomerular and granule cells
(141, 210, 339) that contact mitral cells via glutamatergic
dendrodendritic synapses. These consist of an excitatory
glutamatergic input from mitral cells to granule cells,
inducing the release of GABA from granule cells, that in
turn provides inhibition to the mitral cells (141, 182, 303).
For more information, the reader may consult previous
comprehensive reviews (339, 345, 385).
3. Encoding pheromonal information in the main and
accessory olfactory bulbs
To simultaneously examine the responses to pheromones and odorants in the mouse main and accessory
olfactory bulbs, Xu et al. (409) used high-resolution functional magnetic resonance imaging (fMRI), a very powerful technique that can show dynamic responses in both
entire olfactory bulbs to various stimuli in the same animals. Mouse urine activated restricted areas in both olfactory bulbs, eliciting signals mainly in the ventral region of the
main olfactory bulb and in the anterior part of the main
accessory bulb. Since the anterior region of the mouse accessory olfactory bulb receives input from V1R-expressing
vomeronasal sensory neurons, this study is in agreement
with recent results from Holekamp et al. (113) who suggested that urine mainly activates V1Rs (see sect. VA2).
The encoding of pheromonal information in the mitral
cells of the accessory olfactory bulb has been investigated
by recording responses in awake mice using a miniature
motorized drive to move microelectrodes and record the
activity of single neurons (213, 214). Male mice were allowed
to investigate conspecifics differing by sex, genetic makeup,
and hormonal status. Interestingly, an increase in mitral cell
activity occurred after test animals directly contacted and
actively investigated the head and the anogenital regions of
the stimulus animal. The recorded mitral cell responses
were found to be specific for sex and strain of the stimulus
mouse. In addition, mitral cells exhibited both highly specific excitatory and inhibitory responses, these latter likely
originating from the inhibitory interneuron activity. Therefore, the activity of mitral cells appears to be tuned by the
combination of sex and genetic makeup (213, 214), and
therefore, information about sex and strain is already integrated in the accessory olfactory bulb.
Several studies have revealed that the exposure to
urinary components from opposite-sex conspecifics increased the number of c-fos-immunoreactive mitral and
granule and periglomerular cells in the accessory olfactory bulb (116, 126, 228, 295, 410). Volatile urinary odors
from male as well as female mice also stimulate c-fos
expression in distinct clusters of glomeruli in the main
olfactory bulb in both sexes (228, 270).
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In rodents, V1Rs and V2Rs are individually expressed
in vomeronasal sensory neurons, and each neuron expressing a given vomeronasal receptor (or vomeronasal
receptor pair) sends its axonal projection toward the
anterior (V1Rs) or posterior (V2Rs) accessory olfactory
bulb (100, 143, 414). Gene targeting approaches were used
to visualize the topographical map of each targeted receptor in the mouse accessory olfactory bulb (16, 62, 314).
When a fluorescent marker protein was inserted downstream of a vomeronasal receptor gene, the resulting
visualized axonal projections of neurons expressing that
specific receptor showed that several glomeruli in subregions of the anterior or posterior accessory olfactory bulb
received inputs. A more accurate analysis indicated that
axons from neurons expressing the same receptor coalesce into 10 –30 glomeruli and that a single glomerulus
receives input from neurons expressing different receptor
types (Fig. 7D). Moreover, the capacity to converge and to
form these glomeruli depends on the possibility to express a specific vomeronasal receptor gene, suggesting
that the receptor proteins themselves play a role in axonal
wiring to the bulb (16, 314). In fact, vomeronasal neurons
expressing a nonfunctional (truncated) V1R gene no
longer converge in the accessory olfactory bulb. This
phenotype, also observed for odorant receptors (343),
could be possibly due to the inability of the truncated
gene to prevent the expression of other V1Rs, resulting in
a population of fluorescently tagged neurons randomly
expressing different V1Rs . Indeed, Roppolo et al. (318)
showed that the expression of a nonfunctional V1R allele
allows the coexpression of other V1R genes. Although no
V1R expression has been reported in other subcellular
compartments besides the projecting dendrite, it is possible to envisage that V1Rs may direct axon guidance,
perhaps through a mechanism that involves their expression in the growth cones.
Wagner et al. (393) generated transgenic mouse lines
in which projections from various populations of neurons
expressing distinct V1Rs were differentially labeled with
fluorescent proteins. They found a glomerular map divided in multiple nonoverlapping domains, with each domain clustering glomeruli formed by neurons expressing
V1Rs of the same subfamily, randomly intermingled (393).
Moreover, these authors (393) found that a mitral cell is
not only connected to glomeruli innervated by neurons
expressing the same V1R (62) but also to those associated
with receptors of the same subfamily. Therefore, in the
anterior accessory olfactory bulb, the spatial map originated from vomeronasal organ inputs seems to rely on
receptor subfamilies, rather than individual receptors as
in the main olfactory bulb. This wiring scheme may help
to discriminate molecules with highly similar chemical
structure at different ratios in a pheromonal blend.
In both the main and accessory olfactory systems,
mitral cells are activated by primary sensory neurons
B. Signaling Beyond the Olfactory Bulbs
In the main olfactory bulb, mitral/tufted cells directly
transmit signals from glomeruli to pyramidal neurons in the
olfactory cortex, without a thalamic relay. The primary olfactory cortex, defined as the ensemble of brain regions that
receive direct input from the olfactory bulb, is composed of
several anatomically distinct areas: the piriform cortex, the
olfactory tubercle, the anterior olfactory nucleus, the cortical amygdala, and the entorhinal cortex. In turn, most of
these cortical regions project back to the main olfactory
bulb and to other areas of the brain, including the thalamus,
the hypothalamus, the hippocampus, the frontal cortex, and
the orbitofrontal cortex (Fig. 8; reviewed in Ref. 349). Anatomical studies and genetic approaches indicate that the
topographical organization of odorant information in the
olfactory bulb is not reflected in the olfactory cortex.
Instead, odorant receptors seem to be mapped to multiple, discrete clusters of cortical neurons.
Mitral cells of the accessory olfactory bulb project to
areas of the limbic system: the medial amygdala and the
posteromedial cortical amygdala, which are also called
“vomeronasal amygdala,” the accessory olfactory tract, and
the bed nucleus of the stria terminalis (Fig. 8) (272, 334, 392).
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).]
Physiol Rev • VOL
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Lin et al. (199) recorded the electrical activity in the
accessory olfactory bulb in response to urine of different
mouse strains. A subset of mitral cells was found to be
very selective, responding only to male urine. Moreover,
by combining electrophysiology and gas chromatographic
separation of male mouse urine components, these authors identified a male specific compound, methyl-thiomethanethiol (MTMT), that was indeed responsible for
this effect. Interestingly, mitral cells responding to MTMT
were not stimulated by any other urinary compound,
allowing the appellation of “specialist” cells. Furthermore, behavioral experiments also established the biological activity of this molecule, as female mice developed
interest and attraction towards castrated mouse urine
after the addition of synthetic MTMT.
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C. Pheromone-Activated Hormonal Systems
Recent experiments have revolutionized the common
beliefs about the control of hormonal responses by pheromones (26, 413). The effects of pheromones on the neuroendocrine status are mediated by a group of neurons in
the hypothalamus, which constitutes the endocrine control center (for reviews, see Refs. 89, 245, 353). These
neurons secrete the GnRH, also known as LH-RH, which
in turn stimulates the anterior pituitary gland to release
two gonadotrophins: the luteinizing hormone (LH) and
the follicle-stimulating hormone (FSH). Both hormones
control the development and the function of the gonads in
males and females, although with different effects.
A genetic approach has been used to visualize the
GnRH neuronal network and, surprisingly, GnRH neurons
were found to be connected with areas relaying information from the main olfactory system, in contrast to the
traditional view that only ascribes connections with
GnRH neurons from the vomeronasal system (26, 413).
Boehm et al. (26) identified neurons that synapse
with GnRH by using a genetic transneuronal tracer, barley
lectin, a glycoprotein that is transferred across synapses.
The barley lectin gene was positioned under the control of
the GnRH promoter so that only GnRH neurons would
express this peptide. In another study, Yoon et al. (413)
used a different experimental approach to anatomically
trace the afferent pathways to GnRH neurons in the hypothalamus. Only neurons expressing GnRH were infected with a modified pseudo-rabies virus and green
fluorescent protein reporter. The genetically controlled
Physiol Rev • VOL
viral tracing allowed the identification of a major projection pathway from the main olfactory system.
Both studies (26, 413) came to the conclusion that
the small pool of GnRH cells (800 neurons) was surprisingly synaptically connected to some tens of thousand
neurons in ⬃50 diverse brain areas, including the main
olfactory ones, suggesting that GnRH neurons may influence a large variety of brain functions.
Interestingly, Yoon et al. (413) reported the absence of synaptic connections between the vomeronasal pathway and GnRH neurons. This could be due to
different experimental approaches used in the two
studies or, perhaps, presynaptic inputs from the vomeronasal pathway identified in one study (26) might derive from a subset of GnRH neurons distinct from those
targeted in the other study (413). However, the observations of Yoon et al. (413) do not refute previous
neuroanatomical tracing data indicating that the vomeronasal pathway projects to the hypothalamic area
where GnRH neurons are located. Indeed, Yoon et al.
(413) confirmed that neurons in this area receive inputs
from the vomeronasal pathway. It could be speculated
that the connections with GnRH are, at least in part,
mediated by nonsynaptic (e.g., hormonal or neuromodulatory) mechanisms.
As a confirmation of these observations, stimulation of
male mice with urinary pheromones induced the activation
of neurons in the anterior cortical amygdala as well as in the
dorsal and ventral endopiriform nucleus. Therefore, signals
in response to pheromonal cues also occur via the main
olfactory epithelium and are transmitted to GnRH neurons
through the olfactory cortex. Moreover, feedback loops
have been identified so that GnRH neurons could influence
both odorant and pheromone signaling.
VII. PHEROMONE-MEDIATED BEHAVIORS
From results presented in the previous sections, it
now appears evident that both the vomeronasal and the
main olfactory systems may be activated by chemicals
that are known to produce pheromonal effects. The
involvement of the vomeronasal and main olfactory
systems on specific behaviors is usually investigated by
inactivating one or both systems. In the past, lesions
were made only by surgical removal of the vomeronasal
organ (VNOx) or by chemical ablation of the main
olfactory epithelium (MOEx) (Table 2). The introduction of a molecular genetic approach allowed the deletion of specific transduction molecules, although it
must be taken into account that the knowledge of genes
involved in transduction in each olfactory organ is still
incomplete, and therefore, results of behavioral experiments from knockout animals must be interpreted very
cautiously.
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Although vomeronasal sensory neurons expressing
V1Rs or V2Rs, respectively, project to distinct areas of the
accessory olfactory bulb, this segregation, at least in some
species, does not seem to be completely maintained in the
amygdalic regions, where clusters of neurons may combine signal information originating from both families of
vomeronasal receptors (230, 252, 253, 327, 392).
In essence, the majority of these studies provide
evidence that, excluding areas of convergence, the two
vomeronasal pathways project to specific areas of the
amygdala and hypothalamus, thus suggesting a partial
functional separation (252, 253).
In the vomeronasal system, projections to the hypothalamus from the limbic regions are directed to the ventromedial and the medial preoptic areas, which are involved in
reproductive and social behaviors (158, 159, 289).
The medial amygdala receives direct input from the
accessory olfactory bulb, but also from the main olfactory
bulb. However, other rostral basal telencephalic regions
seem to play a relevant role in the integration of signals
originating from both the vomeronasal and the main olfactory epithelia.
945
TABLE
2.
Effects of surgical and genetic ablations of olfactory and vomeronasal systems
VNX
Hamster
Rat
VGENX
OEX
2Scent marking (290)
2Male copulatory
behavior (55, 76, 298)
2Lordosis behavior (221)
2Individual recognition
(365)
2Partner preference (10)
2Scent marking (145)
2Sexual arousal (146)
2Individual recognition (146)
2Male copulatory behavior (146)
2Penile erection (172)
Cat
2Sex discrimination (154)
(73, 376)
2Copulatory behavior (ac3) (395)
2Intermale aggressiveness
(ac-3) (395)
2Individual recognition
(cnga2) (364)
2Suckling behavior
1Maternal behavior (52)
2Catnip reaction (104)
Reference numbers are given in parentheses.
Here, we will discuss experimental results mainly
related to sexual and aggressive behaviors and will try
to interpret to what extent the vomeronasal and/or the
main olfactory systems appear to be involved. It is very
important to note that behavioral expressions largely
vary among species; moreover, responses to pheromones cannot be simply described as innate or stereotyped, but may also depend on contextual situations
and can be modified by some forms of learning and
memory.
Physiol Rev • VOL
A. Male Behaviors
1. Male sexual behavior
Sexual behavior has been intensively approached by
many studies in the last 20 years, particularly in rodents. In
male mice, the typical mating sequence consists of olfactory
exploration of estrus females followed by multiple episodes
of ultrasound vocalization, mounting and intromission, and
eventually ejaculation. All these parameters can be evaluated in a typical test experiment (120, 121).
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2Stress-induced
hyperthermia (165)
Maternal aggression (170)
2Male induced sexual
receptivity (302)
2Nursing behavior (36)
2Infanticide (243)
2Sexual arousal (325)
2Lordosis behavior to
male mount (155)
1Sperm motility (175)
2Sex discrimination (trpc2) (193,
284, 368)
2Intermale aggressiveness (trpc2;
g␣i2) (278, 368)
2Urine marking (181, 233) 2Maternal aggression (trpc2; v1rav1rb; g␣i2) (61, 161, 193, 278)
2Intermale aggressiveness 2Lactating behavior (trpc2) (161)
(54, 233)
2Marking
Male-like sexual behavior in females
(trpc2) (161)
2Copulatory behavior (54) 2Copulatory behavior (v1ra-v1rb)
(61)
1Puberty onset (209)
2⌴arking (trpc2)(193)
2Strain recognition (206)
2Pregnancy block (156,
206, 216)
2Sexual investigation
(135)
Male-like sexual behavior
in females (161)
Ferret
2Sexual investigation
(404)
Lemur
2Copulatory behavior (8)
2Intermale aggressiveness
(8)
Opossum
2Male-mediated induction
of estrus (131)
Prairie vole 2Intermale aggressiveness
(399)
2Pairing (189)
Rabbit
1Maternal behavior (87)
Mouse
OGENX
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cation as scent or flank marking are likely to be primarily
mediated by the main olfactory system (145).
2. Male aggressiveness
In mice, male to male territorial aggression is a remarkable innate social behavior that is triggered by compounds present in urine (see sect. II, Table 1). In a common assay employed to test male aggressiveness, a male
intruder is introduced in a cage containing a singly housed
male resident. After a period of olfactory investigation, to
allow individual recognition, the resident mouse initiates
a series of attacks against the intruder. The number and
duration of the attacks and the latency to the first attack
represent standard parameters to evaluate the aggressive
behavior (58, 233).
The surgical ablation of the vomeronasal organ has
been long known to nearly abolish aggressiveness in male
mice and prairie voles (12, 54, 55, 233, 399, 407), an effect
that is recapitulated in mutant mice bearing the deletion
of the gene encoding TRPC2 (161, 193, 368). Indeed, male
mice bearing the genetic ablation of the TRPC2 channel
fail to display aggression toward male intruders (193,
368), a behavioral effect that, in an attenuated form, is
also evident in mice lacking G␣i2 (278).
The main olfactory system also plays a very important role in male aggressiveness. Indeed, mice that either
have undergone the chemical ablation of the olfactory
epithelium (via nasal irrigation with zinc sulfate) (154) or
have been genetically mutated in the genes encoding the
odorant transduction molecules adenylyl cyclase type 3
and CNGA2 (227, 395) show abnormal sexual and aggressive behaviors (227).
Thus both the olfactory and the vomeronasal systems
appear to be necessary for eliciting male aggressiveness.
B. Female Behaviors
1. Female sexual behavior
In females of different species, sexual behavior is
characterized by ultrasonic vocalization and lordosis elicited by lumbosacral tactile stimulation (174, 294).
In mice, the genetic ablation of the TRPC2 channel
did not alter female sexual receptivity to males but, surprisingly, caused a female sexual behavior similar to that
of males, i.e., females attempted to copulate as males
(161). Kimchi et al. (161) also showed that similar sexual
behaviors were displayed by females in which the vomeronasal organ was surgically removed, in contrast to a
previous study that described a reduced sexual receptivity in VNOx females (155). The chemical ablation of the
main olfactory epithelium (with zinc sulfate) reduces sexual behavior in female mice (154).
In female rats, both chemical ablation of the main
olfactory epithelium and vomeronasal organ removal af-
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The genetic ablation of the TRPC2 channel greatly
impairs some features of the sexual behavior. Indeed,
although male mice mate normally with females, they also
show an indiscriminate courtship and mounting behavior
toward females (161, 193, 368). A similar situation was
observed in VNOx male mice (161). Furthermore, individual recognition, through the discrimination of nonvolatile
odorants, is also affected in VNOx mice (155, 284). On the
other hand, the simultaneous deletion of a group of V1R
receptors results in a significant decrease of male copulatory behavior (61), although this latter appears normal
in G␣i2 mutants, suggesting that apical neurons are likely
not to be of relevant importance for mating motivation. All
these findings reveal that the mouse vomeronasal organ is
indeed involved in individual recognition and reproductive
activity, but that signaling through the vomeronasal organ is
not required for initiating sexual behavior.
The chemical ablation of the main olfactory system
(via nasal irrigation with zinc sulfate) produces a striking reduction in male reproductive behavior (227). Furthermore, the genetic deletion of the CNGA2 channel
(227), or of adenylyl cyclase type 3 (395), entirely abolishes male mating performances producing severe deficiencies in each step involved in mating: olfactory
exploration, mounting, and intromission, compared
with normal mice. Thus the main olfactory system
seems to play an essential role for male mouse mating
behavior.
In rats, individual recognition and sexual arousal is
only temporary or moderately depressed by lesions of the
vomeronasal organ (22, 325) as well as the overall copulatory behaviors appear to be slightly or partially affected
(171, 324, 325).
Sexual behavior in male hamsters is totally abolished
by bilateral removal of the olfactory bulbs, an operation
that eliminates sensory input from both the olfactory and
the vomeronasal systems (298). The destruction of the
main olfactory epithelium did not impair male hamster
mating behavior, whereas the peripheral deafferentation
of the vomeronasal system produces severe sexual behavior deficits in approximately one-third of the treated animals. Combined deafferentation of both the vomeronasal
and the olfactory systems completely abolishes copulation in all experimental animals (298). Mating behavior
alterations appear after removal of the vomeronasal organ in sexually inexperienced males, and this effect is
reversed by intracerebroventricular injections of GnRH
(76, 247), indicating that GnRH acts as an intermediate in
the facilitation of mating behavior by vomeronasal sensory input. However, sexual experience prior to the removal of the vomeronasal organ mitigates the effect of the
lesion on sexual behavior, indicating that behavioral responses are influenced both by VNOx and by sexual experience (291). In this species, other ways of communi-
fect sexual receptivity and the male-induced release of
GnRH (17, 302).
In female hamsters, the removal of the vomeronasal
organ disrupts the elicitation of lordosis by lumbosacral
tactile stimulation, an effect reversed by injection of
GnRH (221).
2. Maternal aggressive behavior
VIII. CONCLUSIONS AND
FUTURE CHALLENGES
Although we are only beginning to understand the
overlapping role of the main olfactory and vomeronasal
systems for pheromone sensing, there is now overwhelming evidence that the main olfactory system is involved in
a variety of important physiological mechanisms underlying pheromonal communication.
In the early 1970s, different studies developed a
model that led to the so-called dual olfactory hypothesis,
sustained by the observation that efferent and afferent
connections of the main and accessory olfactory bulb
project in nonoverlapping areas of the brain, according to
which the olfactory and vomeronasal systems were organized as parallel anatomical axes subserving different
functions. Recent anatomical and functional studies have
indicated that this distinction is not as strict as commonly
thought (229). There seems to be, in fact, areas of large
convergence in the anterior medial amygdala. Moreover,
in the rostral basal telencephalon, both inputs converged
in areas that were classically categorized as exclusively
vomeronasal or olfactory. As for the anatomical observation, behavioral and biochemical studies seem to point to
a complementary role of the two systems. For example,
the functional ablation of the olfactory and vomeronasal
organs leads to similar behavioral changes. Chemosensory neurons sensitive to pheromone have been identified
in both subsystems. Not last, humans as well as other
species in which the vomeronasal system is absent can
probably detect pheromones via the main olfactory epithelium. It is therefore possible that the two systems may
Physiol Rev • VOL
have developed independently for the detection of pheromones, and only the main olfactory epithelium has further specialized also for the perception of conventional
odorants. Proof of this hypothesis will require additional
studies especially in species in which olfaction is not a
primary sensory process as humans, for example; however, it is interesting to note that part of the main olfactory central pathways seem to converge in regions that
control the sexual status of the individual.
ACKNOWLEDGMENTS
We thank Didier Trotier for kindly providing the original
micrograph for figure 6A.
Address for reprint requests and other correspondence: A.
Menini, Scuola Internazionale Superiore di Studi Avanzati, Via
Beirut 2, 34014 Trieste, Italy (e-mail: [email protected]).
GRANTS
This work was supported by grants from the Italian Ministry of Research and by the Italian Institute of Technology.
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From Pheromones to Behavior - Physiological Reviews

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