Chemical Senses Vol. 29 No. 8 © Oxford University Press
2004; all rights reserved
Effects of Vomeronasal Organ Removal on Olfactory Sex Discrimination and Odor Preferences of Female Ferrets
Department of Biology, Boston University, Boston, MA 02215, USA
Correspondence to be sent to: Dr Michael Baum, Department of Biology, 5 Cummington St, Boston, MA 02215, USA. e-mail: baum{at}bu.edu
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Previous research suggests that body odorants, including anal scents and urinary odors, contribute to sex discrimination and mate identification in European ferrets of both sexes. We assessed the possible role of the vomeronasal organ (VNO) in these functions by surgically removing the organ bilaterally in sexually experienced female ferrets. Lesioned (VNOx) and sham-operated control (VNOi) females reliably discriminated between male- and female-derived anal scent gland as well as fresh urinary odors in habituation/dishabituation tests. However, VNOi females spent significantly more time than VNOx subjects investigating male urinary odors in these tests. Also, VNOi females, but not VNOx subjects, preferred to investigate day-old male versus female urine spots as well as wooden blocks that had previously been soiled by male versus female ferrets. Both groups of female ferrets preferred to approach volatile odors from a breeding male instead of an estrous female in Y-maze tests and both groups showed similar levels of receptive sexual behavior in response to a male’s neck grip. The VNO is apparently not required for olfactory sex discrimination or mate recognition in this carnivore, but instead may play a role in promoting continued contact with nonvolatile body odors previously deposited by opposite-sex conspecifics during territorial scent marking.
Key words: carnivore, main olfactory epithelium, mate recognition, pheromone, scent marking
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Numerous studies (Halpern and Martinez-Marcos, 2003
) carried out primarily in rodent species have shown that receptor
neurons in the vomeronasal organ (VNO) are activated by non-volatile components of urine
or scent gland secretions which are dissolved in mucous and transported to the VNO lumen
from the nasal cavity by an active pumping mechanism (Meredith et al., 1980). VNO-mediated signaling has
been associated in male mice, for example, with the display of territorial marking
behavior (Labov and Wysocki, 1989),
luteinizing hormone secretion (Johnston and
Bronson, 1982) and the production of ultrasonic vocalizations (Wysocki et al., 1982) in response to
estrous female urinary odorants as well as the display of aggression in response to
urinary odors from a male intruder (Clancy et
al., 1984). An ion channel of the transient receptor potential channel
type 2 (TRPC2), which is selectively expressed in microvilli of VNO sensory neurons of
the mouse (Liman et al.,
1999), is essential for normal VNO function. Mice with a null mutation of
TRPC2 (TRPC2–/–) displayed high levels of mounting behavior and reduced
aggression towards an intruder male (Leypold
et al., 2002;
Stowers et al., 2002).
TRPC2–/– males also showed high levels of male-oriented mounting behavior
when tested simultaneously with a castrated (urine swabbed) male and a female, leading to
the suggestion that a functional VNO is required for sex discrimination (Stowers et al., 2002) and for
heterosexual partner preference (Leypold et
al., 2002).
The ferret (Mustela putorius furo) is a seasonally breeding carnivore in
which males and females usually live separately, coming together only during the breeding
season to mate (Moors and Lavers,
1981). Ferrets of both sexes, when primed with sex steroids, preferred to
seek out and mate with opposite-sex conspecifics in operant T-maze tests (Stockman et al., 1985;
Baum et al., 1990). A similar
preference was also displayed for volatile odorants emitted from anesthetized ferrets
(Kelliher and Baum, 2002) as well as
towards anal scent gland and urinary odorants (Cloe et al., 2004) from opposite-sex animals. The
chemical composition of anal scent gland secretions (Clapperton et al., 1988) as well as urine
(Soini et al., 2004) differs
between male and female ferrets. The ferret possesses a VNO and an associated accessory
olfactory bulb (AOB;
Weiler et al., 1999;
Kelliher et al., 2001),
although both structures are smaller in absolute volume and relative to body size than in
rodent species. This latter characteristic led to the suggestion (Weiler et al., 1999) that the ferret’s VNO
may be functionally less involved in pheromonal communication in ferrets than it is in
rodent species. Support for this view was provided by studies (Wersinger and Baum, 1997;
Kelliher et al., 1998)
showing that neither mating nor exposure to odors from soiled male or female bedding
augmented the number of Fos-immunoreactive (IR) cells in the mixed, mitral and granule
cell layer of the ferret’s AOB. This contrasts with studies carried out in rodent
species including the rat (Bressler and Baum,
1996), mouse (Halem et al.,
2001) and hamster (Swann et
al., 2001) in which exposure to soiled female bedding significantly
augmented the number of Fos-IR mitral and granule cells in the AOB.
Like ferrets, dogs of both sexes are attracted to anal scent gland and urinary odors
from opposite-sex conspecifics (Doty and Dunbar,
1974). In another carnivore, the cat, males investigate and display flehmen
responses in response to females’ urinary odors (Verberne and de Boer, 1976) and occlusion of the VNO duct
disrupted this odor-induced flehmen behavior (Verberne, 1976). Otherwise, to our knowledge, nobody has
previously studied the effects of disrupting VNO function on behavioral responses to
same- versus opposite-sex body odorants in a carnivore. The present experiment was
conducted to determine whether surgical removal of the female ferret’s VNO would
disrupt aspects of between-sex olfactory communication. Ferrets, unlike cats, do not
display flehmen-like behaviors in response to social odors. However, using home-cage
habituation/dishabituation tests, we previously found that gonadectomized,
estrogen-primed ferrets of both sexes could discriminate between volatile anal scent
gland odorants (Woodley and Baum,
2003) and between volatile urinary odors (S. Woodley, M. Batterton and M.J.
Baum, unpublished results) of males and females when they were presented sequentially. In
the present study we hypothesized that VNO removal would not disrupt the ability of
sexually experienced, ovohysterectomized, estrogen-treated female ferrets to discriminate
these same types of volatile body odors which are presumably detected by receptors in the
main olfactory epithelium as opposed to the VNO. We (Kelliher and Baum, 2001) also previously found that
blocking odorant access to receptor neurons in the main olfactory epithelium of
estrogen-primed female ferrets by nares occlusion eliminated their preference to approach
volatile odors emitted by an anesthetized male in the goal box of a Y-maze. In the
present study we hypothesized, again, that VNO removal would not disrupt female
subject’s preference to approach volatile male body odorants in similar tests.
Obtaining these predicted outcomes would argue against an essential role (Leypold et al., 2002;
Stowers et al., 2002) of the
VNO in olfactory sex discrimination or heterosexual partner preference in ferrets and
would be in agreement with a recent observation (Pankevich et al., 2004) that the VNO was not
needed for male mice to discriminate urinary odors from the two sexes. In male mice VNO
removal reduced the preference to investigate volatile plus non-volatile urinary odors
from estrous females versus males when the stimuli were presented simultaneously inside
the home cage (Pankevich et al.,
2004). In the present study we hypothesized that in female ferrets, as in
male mice, VNO removal would disrupt subjects’ preference to investigate anal
scents and urinary odors from males versus females. We also hypothesized that VNO removal
would have its greatest disruptive effect on females’ motivation to investigate
non-volatile urinary odors that persist in urine samples from which volatile odorants had
been allowed to dissipate for 1 day prior to presentation. We (Chang et al., 2000) previously found that
estrogen-primed female ferrets preferred to investigate and show scent marking towards
wooden blocks previously soiled by a male as opposed to a female. Other workers
(Petrulis et al., 1999)
showed that VNO removal reduced flank marking behavior in female hamsters. We therefore
hypothesized that VNO removal would reduce female ferrets’ motivation to
investigate and to display scent marking behaviors towards wood blocks that were
previously soiled by a male. Finally, previous research showed that the display of
sexually receptive lordosis behavior was significantly reduced in female rats (Saito and Moltz, 1986) and hamsters (Mackay-Sim and Rose, 1986) after VNO removal. We
hypothesized that VNO removal would also disrupt receptive sexual behavior shown by
ovohysterectomized, estrogen-treated female ferrets in response to receipt of a
male’s neck grip.
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Animals
Retired breeder female ferrets (Mustela putorius furo) were purchased from
Marshall Farms (North Rose, NY) and housed in modified rabbit cages under a long-day (16
h light, 8 h dark) photoperiod. Ferrets were fed Ralston Purina (St Louis, MO)
ferret chow once a day and water was available ad libitum. The females had
previously been ovohysterectomized at Marshall Farms and upon arrival at Boston
University were injected daily with estradiol benzoate (EB; 12 µg/kg, s.c.) in
sesame oil. This treatment elicits the full range of sexual behaviors in gonadectomized
females and maintains plasma levels of sex steroids that resemble those of gonadally
intact estrous females. Steroid treatment began 4 weeks before the onset of behavioral
testing and continued for the entire study until just before tests of sexual behavior
were conducted. Beginning 1 week prior to sexual behavior tests, all females received
injections of estradiol in sesame oil (5 µg/kg, s.c.) daily in the early morning
and late afternoon. This dose of estradiol, which effectively activates sexual behavior
while allowing for the mating-induced stimulation of luteinizing hormone secretion in
ovariectomized ferrets (Carroll et al.,
1987), continued until the termination of the study. Stimulus animals were
either castrated males given daily injections of testosterone proprionate (TP; 5 mg/kg,
s.c.) or ovohysterectomized females given daily injections of EB (12 µg/kg, s.c.).
All procedures used in this study were approved by the Boston University Institutional
Animal Care and Use Committee.
VNO surgery
Ferrets were anesthetized using i.p. injections of ketamine (35 mg/kg) and xylazine (4
mg/kg) and were given periodic subcutaneous booster injections of these drugs to maintain
anesthesia. Ferrets were secured in the supine orientation with the head fixed in place
using ear bars in a stereotaxic apparatus. The lower jaw was gently retracted with
elastic bands. VNO removal was carried out under a dissecting stereomicroscope. A midline
incision was made in the hard palate starting from the base of the incisors, moving
caudally 
1 cm. The skin was retracted to expose the nasopalatine ducts. A triangular
piece of bone, to which both VNOs were attached, was removed by drilling the hard palate
rostral-medially and caudal-medially from the nasopalatine ducts on each side of the
midline. To ensure that the VNO was entirely removed bilaterally, the area behind the
incisors was cauterized. Gelfoam was packed into the wound and the incison was closed
using 4.0 Vicryl sutures. Nexaband, a medial adhesive, was applied to the wound to
facilitate healing. Sham VNO removal involved making the initial incision of the skin
overlying the hard palate, whereupon the wound was immediately sutured. All surgical
techniques were performed under sterile conditions and animals were injected with
penicillin for 3 days thereafter to prevent infection.
Sources of odor stimuli
Anal scent glands were removed from gonadally intact, breeding male ferrets and from
estrous females by veterinarians at Marshall Farms. Immediately after removal, anal scent
glands were frozen on dry ice and shipped to Boston University where they were stored at
–20°C. To obtain anal scent secretions, anal scent glands were briefly thawed,
the contents removed and sonicated. Scent gland secretions from two males were combined
and diluted 1:100 in mineral oil, aliquoted and frozen at –20°C until the day
of use. Likewise, 1:100 dilutions of female anal scent were prepared by combining anal
scent gland secretions from two females. These particular concentrations of anal scents
from male and female ferrets were readily discriminated by ferrets of both sexes
(Woodley and Baum, 2003).
Urine was collected from two castrated stimulus males injected with TP and from two ovohysterectomized stimulus females injected with EB. These donor animals were placed alone in a clean cage over a stainless steel collecting tray. Undiluted urine that had not been contaminated with fecal matter from the two stimulus males was combined, divided into 30 µl aliquots and frozen at –20°C until use. Urine derived from the two stimulus females was similarly prepared.
Presentation of olfactory stimuli in the home cage
A wire mesh rectangle (13 x 8 cm) was attached to the outside of the ferret’s home cage and folded so that it would hold a glass slide (7.5 x 2.5 cm) that was used to present different olfactory stimuli. Two (2 x 2 cm) circular holes were cut out of the wire mesh holder adjacent to the subject’s cage. Anal scent gland secretions or urine (17 µl aliquots of each compound) were applied to circular pieces of filter paper (1.5 cm diameter) affixed to a glass slide using double-sided tape. For habituation-dishabituation tests of odor detection and discrimination, particular odor stimuli were presented sequentially using one piece of filter paper per slide. In other tests, subjects’ preference to investigate particular odors was assessed by presenting pairs of odors simultaneously using two pieces of filter paper placed at opposite ends of a glass slide (5.5 cm apart). In all cases, slides were positioned in the wire mesh holder so that subjects could potentially touch the stimuli presented on pieces of filter paper with their noses.
Habituation/dishabituation tests of odorant detection and discrimination
Habituation/dishabituation tests are commonly used to assess subjects’ ability
to detect particular odorants or to discriminate between two different odorants
(Woodley and Baum, 2003). Animals
were first given three presentations (90 s/stimulus presentation with 30 s intervals
between each stimulus) of mineral oil vehicle followed by a single presentation of
menthone diluted 1:10 in mineral oil to familiarize subjects with the testing procedure.
Then subjects’ ability to detect and discriminate male versus female anal scents
was assessed. Initially, all subjects were presented consecutively with mineral oil
vehicle three times, then with three presentations of freshly thawed male anal scent
gland secretion and finally with a single presentation of freshly-thawed female anal
scent. On a subsequent day all subjects received a second test comprised of three
presentations of oil vehicle followed by three presentations of freshly-thawed female
anal scent gland secretion followed by a single presentation of freshly-thawed male anal
scent. Next all subjects were given two additional tests, similar to those using anal
scents, in which deionized water served as the vehicle and freshly thawed, undiluted male
and female urine served as the odrant stimuli. During each vehicle or stimulus
presentation, an investigator used a Psion Observer Workabout hand-held computer to
record the total time spent subjects spent investigating the filter paper containing each
stimulus. Investigation was scored whenever a subject made bobbing movements of the snout
within 1 cm of the stimulus. One-tailed, dependant Wilcoxon signed-rank tests were used
to check for significant increases in investigation times upon the first presentation of
a social odorant after the third vehicle presentation or upon the presentation of a
second social odorant after the third presentation of odorant No. 1. These non-parametric
tests were used because of the large number of zero scores obtained for the third
presentation of either vehicle or the first social odorant. One-tailed tests were used
because we had a strong hypothesis, based on our previous study (Woodley and Baum, 2003) that VNO-intact ferrets would be
able to detect and discriminate the anal scents and urinary odorants presented in this
study. One-tailed, independent t-tests were used to compare the times that VNOi
and VNOx animals spent investigating the first presentation of male urine odors. Again,
one-tailed tests were used because we had a strong hypothesis, based on a previous study
using male mice (Pankevich et al.,
2004), that VNOx ferrets would spend less time than VNOi control females
investigating social stimuli from opposite-sex conspecifics.
Odorant preference tests
Female-derived and male-derived anal scent gland secretions or urine (17 µl
aliquots) were presented simultaneously on pieces of filter paper taped to opposite ends
of a glass slide. The slide was placed in the wire-mesh slide holder for 5 min and the
time that subjects spent investigating each stimulus was recorded using a Psion Observer
Workabout. Again, investigation was scored whenever a subject made bobbing movements of
the snout within 1 cm of the stimulus. On separate days subjects were presented with
freshly thawed anal scent gland, freshly thawed urine and day-old urine. The latter
stimuli were prepared by thawing urine from each sex, placing 17 µl at the two ends
of a glass slide and leaving the slide for 24 h in a fume hood so that volatile
components of the urine would evaporate (Sipos
et al., 1993). We reasoned that the non-volatile components of urine
which remained on the slides might be detected by VNOi, but not by VNOx, subjects. For
each of the three pairs of different stimuli presented, the location of the male and
female odors on the glass slide was alternated in successive presentations. There was a
strong hypothesis based on previous findings (Chang et al., 2000) that females would prefer to
investigate scents from males. Therefore the amount of time that subjects within each
group spent investigating male versus female odors was compared using one-tailed paired
t-tests.
Y-maze Tests of Partner Preference
Subjects were tested for their preference to approach Y-maze goal boxes containing an
anesthetized stimulus male versus a female. The maze consisted of a stainless steel box
(120 x 90 x 30cm) plus two goal boxes and one start box (45 x 30
x 30cm) (Kelliher and Baum,
2002). A stainless steel triangle centered at one end of the box divided it
into a Y-shaped maze. The top edges of the Y-maze were covered with waterproof weather
stripping and the entire maze was covered with Plexiglas panels that created an airtight
seal. Stimulus animals were placed in goal boxes separated from the rest of the maze by
perforated, opaque doors. Air was drawn through the maze from the goal boxes to the start
box with an exhaust fan and vented from the room. Stimulus animals were anesthetized to
eliminate auditory cues. In this way, visual and auditory cues were eliminated and
subjects’ choice to approach one or the other subject was based only on volatile
odor cues emitted from the stimulus ferrets. The maze and start box were washed with a
dilute bleach solution and 70% ethanol between subjects. Prior to the experiment,
subjects were placed in the empty Y-maze for five min to familiarize them with the
apparatus.
Stimulus animals were anesthetized using ketamine (35 mg/kg) and xylazine (4
mg/kg). Each testing session began by placing the subject in the start box for 30 s and
then raising the door, allowing the animal to approach a goal box (free trial). A choice
was recorded when the subject touched one of the goal box doors with her nose or front
paw and the latency to approach the goal box was timed. After a choice was made, the door
to the goal box was raised and the subject was allowed to approach and investigate the
anesthetized stimulus animal for 5 s prior to being returned to the start box. If the
subject did not make a choice within 120 s, she was returned to the start box and the
trial was repeated; this rarely happened. After completion of a free trial, the subject
was placed back into the start box, the arm of the Y-maze leading to the previously
chosen goal box was blocked off using a wooden block and the start box door was raised,
forcing the animal to approach the goal box not chosen on the previous trial (guided
trial). In this way subjects were reminded of which stimulus lay behind each goal box
door. Again, when the animal approached this goal box, the door was lifted and the animal
again investigated the stimulus animal. Each day’s testing session consisted of
eight free trials alternating with seven guided trials. Three such test sessions were
given to each female subject for a total of 24 free and 21 guided trials. The locations
of the male and female stimuli were alternated each day. The mean percentage of free
trials (out of 24) on which the subject approached each goal box was computed for each
animal. Binomial tests were used to determine whether females in each group preferred to
approach anesthetized males on >50% of free trials. One-tailed tests were used
because there was a strong hypothesis based on previous studies (Kelliher and Baum, 2001, 2002) that estrogen-primed females
would prefer to approach the goal box that emitted odors from an anesthetized male as
opposed to a female.
Investigation and scent marking of previously soiled wood blocks
Nasal investigation (sniffing) of and scent making behavior directed towards wooden
blocks that had recently been soiled by a stimulus male and female was assessed in all
subjects (Chang et al.,
2000). Clean wood blocks (38 x 20 cm) with a 5 cm raised bar down
the middle were placed in the home cage of a stimulus animal for 30 min, during which
time the stimulus animal scent marked, urinated and defecated on them. Beginning 1 h
later, soiled blocks from a male and a female were placed side by side in a clean cage.
The subject was then put into the cage for 30 min and the total investigation time and
the frequency of flank marks and urogenital wipes directed towards each wood block were
recorded using a Psion Observer Workabout. Again, investigation was scored whenever a
subject made bobbing movements of the snout within 1 cm of the surface of a wood block.
The location of the male- and female-soiled wood blocks was alternated between subjects
and the blocks were washed after each test session using soap and hot water and a
70% ethanol–bleach mixture. Blocks were then allowed to dry for 1 h before
they were used again. Scent marking and investigative behaviors directed to the male- and
female-soiled blocks were compared using one-tailed t-tests. One-tailed tests
were used because there was a strong hypothesis based on our previous study (Chang et al., 2000) that females would
respond more to blocks soiled by males.
Mating Behavior
A stimulus male was placed in a clean test cage with a Plexiglas front for 10 min. A
female subject was then added to the cage and the social interaction was videotaped for
45 min. In the event that mating behavior (neck gripping by the male) ceased for >3
min , the videotaping was paused and the male was replaced with a different male, in an
effort to provide 45 min of persistent male-initiated mating behavior. Videotaped
behavior was later scored using the Psion Pocket Observer Program. The total time that
the stimulus male spent sniffing the subject’s anogenital region, attempting a neck
grip, successfully neck gripping and mounting the subject were recorded. The total time
that the female subject spent sniffing the stimulus male’s anogenital region as
well as the time spent showing a limp, unresisting receptive posture or a resisting,
unreceptive response to a neck grip from the stimulus male were also recorded. In
addition, aggressive behavior (bites and swats) initiated by the female subject was
recorded. A female ferret’s ‘acceptance quotient’, which is analogous
to the lordosis quotient as an index of receptivity in female rodents, was computed by
dividing the time that a female was receptive by the amount of time that the stimulus
male either successfully or unsuccessfully attempted to grip the female’s neck
(Baum, 1976). The investigative and
aggressive behaviors, as well as the receptivity quotients of VNOi and VNOx females were
compared using two-tailed independent t-tests.
Histological assessment of the success of VNO removal
Animals were killed with an overdose (100 mg/kg) of sodium pentobarbital and perfused transcardially with 0.1 M phosphate buffered saline (PBS; pH = 7.4) followed immediately by 4% paraformaldehyde. The brains were removed and immersed in 4% paraformaldehyde for 2 h. Afterwards, the brains were cryoprotected in 30% sucrose/PBS solution until they sank. The olfactory bulbs were removed from the brain, frozen and sectioned coronally at 30 µm on a Reichert-Jung sm200R table-top sliding microtome.
Every other section was stained for soybean agglutinin conjugated with horseradish
peroxidase (SBA–HRP) and the remaining sections were stained with cresyl violet.
For the SBA–HRP staining, sections were first incubated in 3% normal goat
serum/1% H2O2/PBS for 120 min at room temperature, followed
by four washes in 0.1 M PBS, each for 10 min. The sections were then incubated in
SBA–HRP (15 µg/ml; Sigma) for 40 min at room temperature, followed by
four washes in 0.1 M PBS. After washing, sections were reacted with nickel–DAB
(Vector Labs) for 7 min. Sections were then mounted onto gelatin-coated slides and
coverslipped using Permount. SBA–HRP stains the axons of VNO neurons that project
to the glomerular layer of the accessory olfactory bulb and serves as a useful marker for
intact VNO neurons in mice and rats (Key and
Giorgi, 1986;
Ichikawa et al., 1992) and
ferrets (Kelliher et al.,
2001). The lack of SBA staining in the AOB glomerular layer of each
hemisphere provides evidence that the VNO was successfully removed ipsilaterally.
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Histological assessment of the success of VNO removal
Inspection of the olfactory bulb sections showed that 4 of the 12 females given VNO removal surgery had small remnants of SBA–HRP staining in the AOB of one or both hemispheres. Therefore, the behavioral data from these four subjects were dropped from the experiment, leaving eight females in which VNO removals were deemed to be complete bilaterally. Comparisons of the SBA–HRP staining present in the glomerular layer of the AOB from both a VNOi (middle panel) and a VNOx (bottom panel) female are shown in Figure 1. Intense SBA–HRP staining can be seen in the VNO nerve fibers and in the glomerular layer of the AOB of the VNOi, but not the VNOx, subject. The morphology of the ferret’s AOB is illustrated in a cresyl-violet stained section of a VNOi female (top panel of Figure 1).
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Habituation/dishabituation tests of odorant detection and discrimination
In habituation/dishabituation tests using anal scent gland odorants, both VNOi and VNOx animals showed significant dishabituation responses when either male or female anal scent was presented following the third mineral oil vehicle (Figure 2, both panels). Following habituation to male anal scents (Figure 2, top panel) VNOx females showed significant dishabituation responses to the presentation of a female anal scent; control VNOi females showed a non-significant trend to increase their investigation of this stimulus as well. When the reverse sequence of anal scent gland odorants was presented (Figure 2, bottom panel), both groups showed significant dishabituation responses to the presentation of male anal scents.
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A similar profile of results was obtained in habituation/dishabituation tests using urinary odorants. Thus both VNOi and VNOx females showed significant dishabituation responses when either male or female urine was first presented following the third deionized water stimulus (Figure 3, both panels). Following habituation to male urine (Figure 3, top panel), VNOx, but not VNOi, females showed a significant dishabituation response to the presentation of female urine. Following habituation to female urine (Figure 3, bottom panel), both VNOx and VNOi females showed a significant dishabituation response to male urine. Interestingly, VNOx female spent significantly less time than VNOi controls investigating the male urinary odors, regardless of whether they were presented first (Figure 3, top panel) or second (Figure 3, bottom panel) in sequence.
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P < 0.05 one-tailed
t-test comparisons between the two groups of subjects. The number of ferrets in
each group is shown in parentheses.Odorant preference tests
VNOx females investigated male anal scents significantly longer than female anal scents when they were presented simultaneously in the home cage (Figure 4, top panel). There was a non-significant trend for a similar preference in VNOi control females. Both groups investigated fresh male urine significantly longer than fresh female urine (Figure 4, middle panel). Finally, VNOi control females investigated day-old male urine significantly longer than day-old female urine, whereas the VNOx animals investigated these two urinary odors for equal times (Figure 4, bottom panel).
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Y-maze Tests of Partner Preference
The VNOi (76 ± 4; mean ± SEM) and VNOx (69 ± 4) females approached the goal box emitting the male odors on an equivalent percentage of free Y-maze trials. When data from all subjects were combined, they showed a significant preference to approach the male goal box over the female goal box (binomial test, P < 0.02).
Investigation and scent marking of previously soiled wood blocks
VNOi animals investigated wood blocks previously soiled by males for significantly
more time than they investigated blocks that had been soiled by females whereas VNOx
animals investigated these blocks equally (Table
1). Both groups showed low, but
equal, numbers of flank rubs directed towards the two types of wood block (Table
1). Very few anal wipes were
displayed by either female group towards either stimulus block (data not shown). In a
previous study (Chang et al.,
2000) estrogen-primed male ferrets displayed twice as many scent marking
behaviors as females. A more rigorous assessment of the possible contribution of
VNO-accessory olfactory inputs to ferret scent marking behaviors should be carried out in
a future study using male instead of female subjects.
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Mating behavior
There were no significant differences in females’ display of anogenital investigation, in their sexual receptivity (indexed by acceptance quotients), or in their display of aggressive behaviors towards the stimulus male (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The present results provide the first behavioral evidence that the ferret’s VNO is functional. Although surgical removal of the VNO did not disrupt the ability of female ferrets to detect and discriminate male versus female anal scent and urinary odorants, it did significantly reduce the time that female ferrets spent investigating male urinary odors when they were first presented in habituation/dishabituation tests. VNO removal also eliminated the significant preference, otherwise seen in VNOi control females, to investigate 1-day-old male urine spots in home cage tests and to investigate wood blocks that had previously been soiled by a male as opposed to a female conspecific. It is noteworthy that these significant effects of VNO removal were most evident when urinary as opposed to anal scent gland odorants were presented. Also, the effects of VNO removal were often best revealed in tests that involved the presentation of stimuli (e.g. 1-day-old urine; wood blocks that had been soiled 1.5–2 h previously) from which volatile components had presumably had some opportunity to dissipate. Any remaining non-volatile components of these odorants would have most likely activated VNO receptors in VNOi female ferrets that came into direct contact with them, resulting in a significant preference to investigate male-derived stimuli. Such non-volatile odorants may be important components of scents that serve to mark males’ territories. Females’ preference to remain in contact or close proximity to these odorants may be facilitated by the activation of VNO neurons.
The female ferrets used in the present study were retired breeders which had
previously produced several litters. In a previous study (Meredith, 1986) VNO removal disrupted mating behavior in
male hamsters more strongly when the surgery was carried out prior to as opposed to after
subjects received mating experience. In another experiment (Kelliher and Baum, 2002) the time that estrogen-primed
female ferrets spent investigating a Y-maze goal box previously soiled by a male was
significantly increased after mating experience. These observations imply that we might
have observed even more profound effects of VNO removal on olfactory responses to social
odors and on scent marking and mating behaviors had we carried out VNO removal surgery in
female ferrets prior to their receipt of mating experience.
Further evidence that a functional VNO is not required for sex discrimination derives
from our observation that VNOx female ferrets resembled VNOi controls in their preference
in Y-maze tests to approach volatile odors emitted from an anesthetized male, as opposed
to a female. This result resembles the observation in female pigs (Dorries et al., 1997) that occlusion of the VNO
duct failed to disrupt subjects’ preference to approach the male pheromone
androstenone, instead of oil vehicle in T-maze tests. Our result also resembles the
outcome of a study using female hamsters (Petrulis et al., 1999) in which VNOx failed to
disrupt the preference to approach and investigate soiled male as opposed to female
bedding. The results from these studies differ from our previous observation (Kelliher and Baum, 2001) that nares occlusion of
female ferrets eliminated subjects’ preference to approach a stimulus male in
Y-maze tests, even when visual and auditory cues were available to compensate for the
lack of olfactory cues in anosmic subjects. In that study dental impression material was
infused into the nasal sinuses and post-mortem dissection showed that this procedure did
not occlude the nasopalatine duct, thereby preserving potential access of odorants to VNO
receptors via the mouth. By contrast, the dental impression material effectively blocked
access to the main olfactory epithelium with the consequence that nares-occluded subjects
were unable to use peppermint odor as a discriminative stimulus for food reward in Y-maze
tests. Also, a dramatic reduction in Fos-IR granule and mitral cells was seen in the main
olfactory bulbs of nares-occluded subjects. Further evidence that the main as opposed to
accessory olfactory system is responsible for mate identification in ferrets stems from
our observation that volatile anal scent gland odorants from male and female ferrets
activated overlapping, but distinguishable, clusters of glomeruli in the ventral-caudal
portion of the MOB (Woodley and Baum,
2004). It has been claimed (Trinh and
Storm, 2003) that volatile components of urine can directly activate VNO
neurons in mice; however, a more definitive study (Luo et al., 2003) showed that electrical activity
in AOB mitral cells of male mice was only augmented when subjects came into direct
physical contact with the either the head or anogenital region of conspecifics. In the
present study we allowed subjects to have a brief physical contact with anesthetized male
and female stimulus ferrets after every Y-maze trial. In this way we provided the
non-volatile odorant stimuli for potential activation of VNO receptors in our VNOi
control ferrets. The similarity in the male-oriented preference of VNOx and VNOi subjects
further suggests, however, that an activation of VNO inputs to the forebrain is not
required for female ferrets to prefer approaching volatile odors from males.
There was a similarity between the profile of behavioral responses to VNO removal
seen in female ferrets and those previously reported in male guinea pigs (Beauchamp et al., 1982) and in male mice
(Pankevich et al., 2004). In
these latter studies VNO removal eliminated males’ preference to investigate urine
spots from female as opposed to male conspecifics, provided subjects had direct physical
access to the urine stimuli. In the present study, as in these two previous studies using
other species, VNO removal failed to disrupt subjects’ ability to discriminate male
versus female odorants (anal scents and urinary odors in the case of ferrets; urinary
odors in the case of guinea pigs and mice). Likewise, in female pigs (Dorries et al., 1997) occlusion of the
VNO duct failed to disrupt subjects’ ability to detect decreasing concentrations of
the male pheromone androstenone, in operant tests motivated with sucrose reward. To our
knowledge, only one study manipulating VNO function has been carried out in a primate
species. In that study (Aujard, 1997)
VNO removal from male lesser mouse lemurs reduced their activity level and motivation to
compete with a socially dominant male for access to a sexually receptive female. However,
there was no evidence that VNO removal disrupted the ability of male lemurs either to
identify or to successfully mate and ejaculate with a female once access was gained.
Taken together, data obtained from a wide variety of mammalian species support the view
(Powers et al., 1979;
O'Connell and Meredith, 1984)
that the main olfactory system is used to identify odorants from the opposite sex.
However, once physical contact is made with these odorants, non-volatile components reach
the VNO lumen via a pumping mechanism (Meredith
et al., 1980) where they bind to G-protein coupled VNO receptors
(Halpern and Martinez-Marcos, 2003)
leading to the generation of action potentials. Inputs are conveyed to the AOB which
leads, in turn, to an activation of limbic and hypothalamic circuits, including reward
mechanisms (Beauchamp et al.,
1985) that prolong contact with opposite-sex conspecifics.
In several previous studies using rats (Saito
and Moltz, 1986;
Rajendren et al., 1990,
1993;
Kelliher and Baum, 2002) and hamsters
(Mackay-Sim and Rose, 1986), VNOx
disrupted the ability of females to show sexual behavior. These reports contrast with the
present observation that VNOx failed to affect the display of receptive feminine sexual
behavior (indexed by acceptance quotients to male neck grips) in ovohysterectomized,
estradiol-primed female ferrets. Our negative results in the ferret correspond with
results obtained in the female pig (Dorries
et al., 1997) in which occlusion of the VNO ducts failed to disrupt
the ability of ‘back pressure’, combined with spraying the snout with
aerosolized androstenone, to induce receptive standing behavior. The inability of VNOx to
disrupt receptivity in the pig and ferret may reflect the species differences in the
neuroendocrine control of reflexive, arched back lordosis behaviors seen in rodent
species such as rat and hamster versus the standing (pig) or passive, limp acceptance
posture (ferret) shown by other mammals.
The original hypothesis that the VNO in ferrets may be less essential for social
communication than in rodents stemmed from the observation (Weiler et al., 1999;
Kelliher et al., 2001) that
the volume of the ferret’s VNO neuroepithelium is only 50% of that of the
rat and from studies (Wersinger and Baum,
1997;
Kelliher et al., 1998)
showing that neither mating nor exposure to odors from soiled male or female bedding
augmented the number of Fos-IR cells in the mixed, mitral and granule cell layer of the
ferret’s AOB. Despite the lack of any AOB Fos response in ferrets exposed to social
odors, the present behavioral experiments demonstrate quite clearly that VNOx disrupted
specific behavioral responses to such odors. These observations provide further evidence,
if it was needed, that the absence of increased expression of immediate-early genes such
as c-fos should not be taken as conclusive evidence that neurons in a particular
brain region remain inactive after exposure to a particular sensory stimulus. Likewise,
our results suggest that species differences in the relative size of the VNO and AOB are
not necessarily predictive of species differences in function. Indeed, as summarized
above, the present results suggest that in the ferret, a carnivore, as in mice, hamsters,
guinea pigs and other rodents, the VNO detects olfactory signals that activate neural
reward mechanisms leading to a prolongation of contact with opposite-sex conspecifics or
the odorants they deposit during scent marking.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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This study was supported by NIH grants NRSA DC00426 and HD21094. We thank Diana Pankevich for technical advice.
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Accepted July 22, 2004
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