A functional circuit underlying male sexual
behaviour in the female mouse brain
Tali Kimchi1, Jennings Xu1& Catherine Dulac1
In mice, pheromone detection is mediated by the vomeronasal organ and the main olfactory epithelium. Male mice that are
deficient for Trpc2, an ion channel specifically expressed in VNO neurons and essential for VNO sensory transduction, are
impaired in sex discrimination and male–male aggression. We report here that Trpc22/2female mice show a reduction
in female-specific behaviour, including maternal aggression and lactating behaviour. Strikingly, mutant females display
unique characteristics of male sexual and courtship behaviours such as mounting, pelvic thrust, solicitation, anogenital
olfactory investigation, and emission of complex ultrasonic vocalizations towards male and female conspecific mice. The
same behavioural phenotype is observed after VNO surgical removal in adult animals, and is not accompanied
by disruption of the oestrous cycle and sex hormone levels. These findings suggest that VNO-mediated pheromone
inputs act in wild-type females to repress male behaviour and activate female behaviours. Moreover, they imply that
functional neuronal circuits underlying male-specific behaviours exist in the normal female mouse brain.
Males and females within a given animal species display identifiable
differences in behaviours, mostly but not exclusively pertaining to
sexual and social responses. Although these represent the most obvi-
ous examples of behavioural variability within a species, the basic
principles underlying sexual dimorphism of brain function are lar-
gely unknown. Moreover, with few exceptions, the search for unique
structures and circuits in male and female brains that parallel the
dimorphism of peripheral sexual organs has so far met little
In many animals species- and sex-specific behaviours are orche-
strated by pheromonal cues. Recent studies in rodents have uncov-
ered the dual role of the main olfactory epithelium and the
vomeronasal organ (VNO) in pheromones controlling mating,
aggression and gender identification6. Genetic ablation of the
TRPC2 channel, a signalling component essential to VNO function,
leads to indiscriminate courtship and mounting behaviour of
Trpc22/2male mice towards both males and females, suggesting an
essential role of the vomeronasal system in sex identification7–10.
Furthermore, recent recording, genetic silencing and tracing experi-
ory epithelium and associated central pathways in pheromone-
To study the role of the VNO in female sexual receptivity, we
introduced a sexually experienced male to the home cage of either
Trpc21/1, Trpc21/2or Trpc22/2females. As expected, oestrous
Trpc21/1and Trpc21/2females were sexually receptive, allowing
leading to successful mating within minutes. However, in a striking
role reversal, Trpc22/2females were observed intensively investi-
gating the anogenital region of the intruder males and vigorously
attempting to mount them, eliciting aggressive responses from the
Male-like behaviours of Trpc22/2females
Female–female and female–male mounting in rodents has been
observed mainly in laboratory rats as part of dominance or sexual
solicitation, respectively15–17. The behaviour observed in Trpc22/2
females may thus represent either the exaggeration of normal female
responses, or abnormal male-like displays. We monitored unique
characteristics of male sexual and courtship behaviours in
type males while controlling for the presence of pheromones,
castrated or bulbectomized male swabbed with male urine were used
as male intruders.
Male-like sexual display was investigated by scoring the number of
animals mounting the intruder (Fig. 1a), the average duration of
mounting (Fig. 1b) and the latency (time taken) to mount (Sup-
plementary Fig. 1a) in a 15min assay. Because mounting can include
all four tests demonstrate that Trpc21/1and Trpc21/2females very
rarely displayed characteristics of male-like sexual behaviour towards
female intruders, while the majority of Trpc22/2females (Supple-
mentary Video 1), Trpc21/2and Trpc22/2males showed robust
mounting, pelvic thrusts and a short latency to mount. Remarkably,
the behaviour of Trpc22/2females towards other females was statist-
ically indistinguishable from that of heterozygous and mutant males.
males and females (Supplementary Video 2) displayed significant
levels of male-like sexual behaviour towards males, and that their
behaviour is both statistically indistinguishable from each other,
and from their response to female intruders
We further assessed male-specific courtship behaviours. Male
mice engage and solicit females by raising the female rear with their
snout. Also, when interacting with females, adult males emit ultra-
sonic vocalizations at high (30–110kHz) frequencies, while adult
females produce only a limited range of ultrasounds during
female–female social investigation18–22. In addition, males perform
intense olfactory investigation of the female rear, while females focus
on the head and body.
The scoring of solicitation (Fig. 1d), ultrasound duration and
complexity, latency to whistle, number of animals emitting ultra-
sounds (Fig. 1e, Fig. 2, Supplementary Fig. 1b–d), and olfactory
1Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Vol 448|30 August 2007|doi:10.1038/nature06089
investigation of anogenital region (Supplementary Fig. 1e) further
confirmed that the behaviour of Trpc22/2females towards females
cannot be distinguished from that of Trpc22/2and Trpc21/2males,
and is very different from that of Trpc21/1and Trpc21/2females.
Moreover, the behaviour of Trpc22/2males and females towards
male and female intruders was similar and, when intruders were
presented simultaneously, Trpc22/2females, as previously shown
with Trpc22/2males9,10, display no preference for either sex, enga-
ging indiscriminately in sexual behaviour with both male and female
intruders with equal frequency (n56, data not shown).
Thus, the behaviour of Trpc22/2females highly resembles that
typically exhibited by wild-type males interacting with females.
Remarkably, in clear contrast with the normal, though rare occur-
rences of female mounting described in rodents15–17, the mounting
behaviour of the Trpc22/2females was not influenced by their oes-
trous stage (not shown), and rather than disappearing, it was main-
tained after sexual experience (Supplementary Fig. 2), and was not
associated with dominance and aggression (see arena observation
below). Moreover, some male-like behaviours shown by Trpc22/2
females, such as pelvic thrusts and sexual solicitation with the snout
(Supplementary Videos 1 and 2), are very rarely displayed by normal
females. These observations strongly argue that the behaviour of
Trpc22/2females does not simply result from a female hypersexual
titatively and qualitatively different from normal female behaviour.
Surgical ablation of the VNO in adults
Because the Trpc2 mutation eliminates VNO function throughout
life, we reasoned that the male-like sexual behaviour of Trpc22/2
females may result from the abnormal development of female beha-
viour circuits. Alternatively, it may reveal the de-repression of an
brain by inhibitory vomeronasal inputs. To distinguish between
these hypotheses, we compared the behaviour of mice in which the
VNO has been surgically removed in the adult to the behaviour of
genetically deficient animals of the same age.
Adult olfactory marker protein (OMP)-TLZ1/2males and fe-
males, in which olfactory expression of tau-LacZ permits direct
visualization of main olfactory epithelium and VNO projections23,
week after surgery. Behavioural tests were performed three weeks
after surgery, and animals were killed to assess the extent of VNO
removal and accessibility of nasal airways (Fig. 3). These additional
steps were made necessary by our observation that VNO ablation
may cause bleeding and obstruction of the nasal cavity (Supple-
mentary Fig. 3), resulting in behaviour patterns similar to that of
mice, and showed that mutant animals with or without intact VNOs
of a given gender (n59 each gender) exhibited identical behaviours
(not shown), ensuring that no remnants of VNO function can be
identified in Trpc22/2animals.
Our data showed that the sexual and courtship behaviours of
VNOx males towards male and female intruders are indistinguish-
and Supplementary Fig. 1, right side of each histogram). Further,
Trpc21/1and Trpc21/2VNOx females exhibited most male-like
traits at levels similar to that of Trpc22/2females. These included
mounting, pelvic thrust, latency to mount, ultrasound vocalization
and olfactory investigation (Supplementary Video 3). We obtained
similar results when the VNO removal was performed on C57BL/6J
adults (Supplementary Fig. 4), in direct contrast with previously
published studies24,25. Because these earlier studies did not control
for the possible occlusion of the nasal cavity (a frequent occurrence
after standard VNO surgical removal), it is possible that additional
olfactory deficits have confounded the interpretation of the data.
Thus, the loss of VNO function in adulthood resulted in altered
sexual behaviourinmalesand insudden sex-reversal offemale beha-
viour, demonstrating the requirement for sustained control by VNO
behaviour of Trpc22/2and VNOx females suggest that VNO activity
plays a minor role during development.
Behaviour under semi-natural conditions
When studying complex and dynamic behaviours, such as social
interactions, confined experimental conditions could cause the
Mounting time (s)
** ** *
** ** **
Pelvic thrust (s)
Animals mounting (%)
n = 12
n = 10
n = 9
n = 4
n = 7
n = 12
n = 10
n = 9
n = 4
n = 7
** ** **
** ** **
Solicitation time (s)
Ultrasonic whistling (%)
Figure 1 | Male-like sexual and courtship behaviours are displayed by
Trpc22/2and VNOx females. Sexual (a–c) and courtship (d and e)
behaviours towards female (left) and male (right) intruders observed in
sexually naive adult Trpc21/1, Trpc21/2and Trpc22/2mice. VNOx mice
had VNO surgical ablation. Responses to female intruders by Trpc22/2and
VNOx females was indistinguishable from control and mutant males while
responses to intruder males was similar to that of mutant males (error bars
are s.e.m; *P,0.05; **P,0.01, one-way ANOVA followed by post-hoc
Tukey Honest tests), suggesting a role of the VNO in sex discrimination in
both males and females, and in repressing male-like behaviour in females.
NA, not applicable.
NATURE|Vol 448|30 August 2007
animal to present a limited and even impaired behavioural rep-
ertoire. Therefore, we also decided to test the role of VNO-mediated
pheromone detection infemale mice under semi-natural conditions.
Groups of four Trpc21/2and Trpc22/2females were first kept
separated in each half of a large enclosure with enriched environ-
ment, and were scored for social behaviours. In contrast to the
interactions such as mounting, anogenital olfactory investigation, as
well as defensive behaviour resulting from mounting attempts by
other females (Fig. 4, stage 1). Importantly, neither the Trpc22/2
nor the Trpc21/2group established any recognizable dominant–
subordinate social hierarchy.
Sexually experienced wild-type males were then introduced into
each side of the enclosure. Remarkably, unlike Trpc21/2females
(Supplementary Video 4), Trpc22/2females (Supplementary Video
regionand tomount(Fig. 4, stage2).Again,wefound nodominant–
subordinate social hierarchy established in either group, and no cor-
relation between the level of female–male mounting in Trpc22/2
individuals and the level of aggression towards each other or towards
males. Thus, in contrast with normally occurring female–female
mounting, the mounting behaviour of Trpc22/2females is unrelated
250270290 310330 341342 343
5070 90 110 13072 73
100 120140 160180165 166167168
Time (s) Time (s)
20 4060 80100 2425 2627
Figure 2 | Ultrasonic vocalization by male and
female mice in resident-intruder assays.
a, Ultrasonic complexity index. Whistle clusters
with no pitch jump and temporal overlap in
frequency received a low complexity score (1–2),
while clusters of whistles with pitch jump and
extensive temporal overlap received a high
complexity score (3–4). b, Representative
examplesof ultrasonic vocalizationsemitted by a
Trpc21/2male and female, a Trpc22/2female
female intruder. Except for the Trpc21/2female,
complexity score (3–4). Power below 35kHz was
Figure 3 | Surgery leads to a complete removal of the vomeronasal organ
(VNOx) while the nasal airways stay clear. a, Coronal sections (50mm)
were stained with haematoxylin and eosin, showing full removal of the
bilateral VNO structure while the nasal airway is kept open. b, c, X-Gal
staining of the olfactory bulb of OMP-ires-tauLacZ (OMP-TLZ) control
sections (50mm) served as controls for the complete disappearance of all
VNO projections to the accessory olfactory bulb. A, anterior; P, posterior;
AOB, accessory olfactory bulb; MOB, main olfactory bulb.
NATURE|Vol 448|30 August 2007
of non-responsive males by oestrous females as described in rats15,16,
and instead has characteristics of genuine male sexual behaviour.
Most females within the arena became pregnant and15 to 22 pups
were delivered per group. The successful mating of males with
and mating attempts of the males with the Trpc22/2females. We
subsequently tested maternal aggression of lactating females towards
intruder males by adding a strange male from the CD1 strain to the
enclosure. All lactating Trpc21/2females attacked the intruder male
and showed low sexual receptivity. In contrast, the intruder male
evoked a low level of aggression from the Trpc22/2females, which
appeared highly sexually receptive (Fig. 4, stage 3). Our findings
confirm previous results showing low aggression from lactating
Trpc22/2females10while contradicting the described deficiency in
sexual receptivity of VNOx females24
Finally, we investigated female maternal and lactating behaviours.
Both Trpc21/2and Trpc22/2females kept their litters in one com-
mon breeding nest where the females nursed the pups in turn. In the
first and second days after birth, both groups spent a similar amount
of time with their pups. However, during the following days,
Trpc22/2females exhibited a significant decrease in time spent in
thebreeding nest, whileTrpc21/2femalesspent most oftheirtimein
the breeding nest. On the last day (day 14), the partition between the
Trpc22/2and Trpc21/2females was lifted to allow both groups to
interact. Strikingly, the Trpc22/2females immediately abandoned
the breeding nest to explore the Trpc21/2territory, while Trpc21/2
females remained in breeding nests and continued to nurse their
pups (Fig. 4, stage 4). These findings indicate that Trpc22/2females
display a deficiency in maternal behaviour.
We have shown here that Trpc22/2females exhibit robust male-like
sexual and courtship behaviours and display a reduction in female-
specific behaviours such as maternal aggression and nesting. These
findings suggest that VNO-mediated inputs repress male-like sexual
and courtship behaviours in females, and activate and sustain female
Previous studies have suggested a central role for sex hormones in
the regulation of male- and female-specific behaviours26. We found
that body weight and oestrous cycles of Trpc22/2females (Fig. 5a)
appeared normal. Moreover, radioimmunoassay for testosterone
and 17b-estradiol in the serum of Trpc22/2and Trpc21/2males
and females reveal that sex steroid levels remain within the normal
range for each gender (Fig. 5a). A minor increase in free testosterone
level was observed in Trpc22/2females that still vastly differs from
typical male levels and from the amounts of exogenous testosterone
6810 12 14
Relative time in nest (%)
Total interaction time (s)
Total interaction time (s)100
Total interaction time (s)
Total interaction time (s)
Total interaction time (s)
Figure 4 | Social behaviour of Trpc21/2and Trpc22/2females in semi-
natural conditions. Stages 1 and 2 show duration of defensive (white) and
sexual (grey) behaviours of resident females with each other (stage 1) and
towards a C57BL/6J adult male (stage 2). Stage 3 shows duration of sexual
behaviour (grey), olfactory investigation (black) and aggression (white) of
the resident lactating females towards an intruder CD1 adult male (left two
two columns). Stage 4 shows relative time spent by lactating Trpc21/2and
Trpc22/2females in the breeding nest before and after (day 14) the divider
of male-like sexual behaviour (stages 1 to 2). They exhibit no apparent
lactating behaviour (stage 4). Stages 1–4, error bars are s.e.m.
NATURE|Vol 448|30 August 2007
required to affect normal female behaviour27,28Thus, hormonal
changes do not seem to underlie the expression of male-like beha-
viours in Trpc22/2females.
The prevailing model for the sexual dimorphism of behaviours is
that thesexhormone testosterone initiates thedevelopment ofmale-
specific circuitry in the central nervous system and the activation of
male-specific neuronal networks in adulthood26. However, our
results clearly reveal that a functional neuronal network mediating
male sexual behaviour develops and persists in females. These find-
ings suggest a new model of sexual dimorphism in which the effector
circuits of both male and female behaviours exist in the brain of each
been shown in Drosophila to mediate the specificity of male and
female sexual responses in a similar way29,30. Further support for
our model in the mouse will involve the identification of specific
receptor inputs that inhibit the expression of male responses in the
female brain. Moreover, a similar inhibitory control of female beha-
viour may yet be found in the male brain.
Behavioural assays. We used sexually naive, 2–3-month-old Trpc21/1,
Trpc21/2, Trpc22/2(ref. 9), and C57BL/6J3129/Sv mice of mixed genetic
background as well as C57BL/6J mice in the behaviour experiments. Mice were
tested in 15min intruder–resident assaysin which individually housed residents
mice were exposed to sexually naive female, and either castrated or olfactory
bulboctemized male C57BL/6J intruders. Ultrasound vocalizations by the res-
ident mice were recorded during intruder–resident assays. Stored recordings
were processed using a custom MATLAB-based program as described18. Social
behaviour within a colony of Trpc21/2and Trpc22/2mice was recorded under
tally enriched enclosure. The behaviour of the mice was recorded by low-light-
sensitive video cameras that were connected to a custom-designed PC-based
recording unit (Protech PC). The social behaviour (courtship and sexual beha-
viours) in both the intruder–resident assays and in the semi-natural conditions
old, sexually naive mice (Trpc21/23OMP-tauLacZ and Trpc22/23OMP-
tauLacZ). For the first week after surgery, VNOx mice were anaesthetized daily,
their nostrils were rinsed with 0.9% saline solution, and any blood clots were
gently aspirated. VNOx mice were allowed at least three weeks to recover before
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 15 May; accepted 17 July 2007.
Published online 5 August 2007.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank R. Hellmiss for artistic work and illustrations,
S. Sullivan for assistance with behavioural analysis, A. E. Launjuin and S. Yao for
help with histochemistry, the Dulac laboratory for discussions and comments on
the manuscript, T. E. Holy, B. O¨lveczky and A. Kampf for help with the recording of
ultrasonic vocalizations. The work was supported by the Howard Hughes Medical
Institute (to C.D.), the NIH (to C.D.) and the Human Frontier Science Program (to
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to C.D.
(email@example.com) or T.K. (firstname.lastname@example.org).
NATURE|Vol 448|30 August 2007
METHODS Download full-text
Intruder–resident assay. Trpc21/2and Trpc22/2(ref. 9) sexually naive, 2–3-
background were housed individually in a cage for one to two weeks before the
beginning of the experiment. Experiments started at the beginning of the dark
phase and were performed under dim red light.
The following two kinds of intruders (C57BL/6J) were introduced to the res-
ident mouse cage: sexually naive, receptive female (as determined by vaginal
smear), 7–8 weeks old; and sexually naive, castrated or olfactory bulboctemized
assay was videotapedand scored for thefollowing three behaviours: sexual beha-
viour (mounting, and mounting with pelvic thrust); solicitation behaviour (res-
ident animal lifts intruder’s rear with its snout); and olfactory investigation.
Recording and processing of ultrasonic vocalization. Sounds over the fre-
(Bruel & Kjaer) and digitized at 250kHz, 16 bits (National Instruments) and
saved to disk within a custom-designed MATLAB-based program.
In brief, the MATLAB-based program converted the stored waveforms to
sonograms (512 sample/blockform, time resolution of 1.02ms and a frequency
of 0.98kHz), removing white noise outside the range 25–110kHz; it identified
used to estimate the whistling activity level by summing the overall whistling
were classified by the criteria that there existed at least five whistles where the
period of time between each whistle was less than 500ms, and additionally, that
the beginning and end whistle were separated from the previous and next whis-
tles, respectively, by at least 500ms.
To confirm that the resident mouse was the source of the ultrasonic vocaliza-
was anaesthetized. We were only able to record robust ultrasonic vocalizations
mouse was anaesthetized and not the resident.
Semi-natural experimental set-up and procedure. Sexually naive, 3–4-month-
old females (n54 each genotype) Trpc22/2and Trpc21/2mice, derived from
four different litters, were used. The female mice were housed individually in a
cage two weeks before the beginning of the test. To enable us to individually
recognize the animals, before the beginning of the experiment the animals were
anaesthetized (with 120mg of ketamine per kg of mouse body weight and
10mgkg21xylazine) and marked with commercial hair dye. The mice were
was conducted twice with different animals.
Semi-natural enclosure set-up. The enclosure consisted of a large central arena
constructed from transparent polycarbonate boards that were connected to six
peripheral standard mouse cages by short transparent tubes. A removable
oblique polycarbonate board (90380cm) served as a divider. The floor of the
pieces, and shelter boxes. In addition, each half of the arena contained a central
platform with rodent pellets and water freely supplied.
The enclosure was placed in a temperature- and light-controlled (12h:12h
light/dark cycle) room furnished with infrared lights and equipped with low-
light-sensitive cameras mounted above and around the enclosure. All cameras
were connected to a digital video recording unit (ProtechPC).
The behaviour was recorded daily for 10–20min every hour, from the begin-
the Observer Video Pro software (Noldus).
overdose of 2.5% avertin. Blood was removed from the heart, and serum or
plasma was separated and stored at 220uC until analysed. Concentrations of
testosterone and 17b-estradiol were measured using a radioimmunoassay RIA
Kit (MP Biomedicals).
Surgical VNO removal. Mice were anaesthetized (120 mgkg21ketamine and
10 mgkg21xylazine), placed supine in a head holder, and the lower jaw was
from behind the first palatal ridge to the incisors, and the underlying bone was
exposed. The caudal end of the vomer bone was cut and the VNO was removed
bilaterally. Low-pressure vacuum was used to clear blood from the mouse and
(Pharmacia) and the incision was closed with veterinary sterile tissue adhesive
After testing, VNOx mice were killed to confirm complete VNO removal. To
confirm complete degeneration of axonal projections to the accessory olfactory
bulb,we performed whole-mountX-Gal staining ofthe olfactorybulbs,as prev-
iously described23, followed by 50mm parasagittal sections of the stained tissue,
ing the olfactory airways, skulls of VNOx mice were decalcified in 10% EDTA
(pH57.4), sectioned coronally (50mm)on a cryostat, counterstained with hae-
matoxylin and eosin, and dehydrated.