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Department of Biology, Boston University, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Scott R. Wersinger, Department of Biology, University of Virginia, Gilmer Hall, Charlottesville, Virginia 22903. E-mail: srw4h{at}virginia.edu
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Introduction |
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It seems likely that genital-somatosensory as well as chemosensory cues associated with the receipt of an intromission contribute to the activation of LHRH neurons and the resultant generation of a preovulatory LH surge in estrous female ferrets. Using Fos-IR as a marker of neuronal activation, Wersinger and Baum (5) found that the receipt of intromissive stimulation augmented neuronal Fos-IR in the caudal thalamic central tegmental field (CTF) of estrous females in addition to the forebrain regions mentioned above. This site has not yet been investigated in the male ferret. Studies using several different rodent species have shown that chemosensory cues from an opposite sex conspecific augment neuronal Fos-IR in the accessory olfactory bulb (AOB) in addition to all of the forementioned forebrain regions (6, 7, 8). Other results suggest that chemosensory stimuli from a male activate LHRH neurons in female conspecifics. For instance, housing a naive female musk shrew across a wire mesh screen from a sexually active male increased the number of LHRH neurons visualized using immunocytochemistry (ICC) in the female, suggesting that the LHRH content in these neurons is increased above the threshold for detection (9). In female mink, olfactory cues from male conspecifics may augment the preovulatory secretion of LH (10). Finally, chemosensory cues from a male are required for the activation of LHRH neurons in female rats that are mated repetitively (11). Activation of the olfactory system by mating-associated stimuli or by chemosensory cues alone has not yet been examined in ferrets of either sex.
Studies in the rat (12, 13) have shown that genital-somatosensory stimuli activate neurons in the CTF. The CTF, in turn, projects to and activates neurons in the MA and MPOA. Chemosensory cues augment neuronal Fos-IR in the olfactory bulbs of hamsters and rats (6, 7, 8), which then project both directly, in the case of the main olfactory bulb (MOB), and indirectly, in the case of the AOB, to the MA and, via the BNST, to the MPOA (14). In the female ferret, mating-associated stimuli activate mediobasal hypothalamic LHRH neurons in addition to neurons in the above-mentioned brain regions. Based on results obtained from the rat and ferret, it seems likely that non-LHRH neurons in these forebrain regions comprise an excitatory input pathway to the mediobasal hypothalamic LHRH neurons.
Regions including the MOB, AOB, and CTF either directly receive or relay primary sensory stimuli associated with mating. These regions, in turn, project directly or indirectly to the MA, where sensory inputs are probably processed further. All of these brain regions are situated relatively early in the putative input pathway that excites mediobasal hypothalamic LHRH neurons in the female ferret; thus, we designate them more distal components of this pathway. Results of anatomical studies using several rodent species (14, 15, 16) and cats (17, 18, 19) suggest that sensory inputs from the CTF and MA activate more proximal components of the putative input pathway to LHRH neurons, which include the MPOA, BNST, and VLH. These more proximal components project, in turn, to mediobasal hypothalamic LHRH neurons or terminals via routes that are poorly understood.
We asked whether the chemosensory and genital-somatosensory stimuli associated with mating differentially augment Fos-IR in non-LHRH neurons located in distal vs. proximal components of the putative input pathway to mediobasal hypothalamic LHRH neurons in breeding female and male ferrets? In previous studies (2, 3, 4, 5), plasma LH and forebrain Fos responses were monitored in males that achieved and in females that received penile intromissions lasting 5 min. In the present study we examined Fos-IR in mediobasal hypothalamic LHRH neurons as well as in forebrain non-LHRH neurons in male-female pairs of breeding ferrets in which the male achieved an intromission lasting 16–90 min. We also quantified neuronal Fos-IR in the distal vs. proximal components of the putative input pathway to hypothalamic LHRH neurons as well as in LHRH neurons themselves in groups of males and females that were exposed only to chemosensory cues from an opposite sex ferret in breeding condition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sensory stimulation
All sensory stimulation was administered between 1000–1300 h.
Heterosexual pairs (n = 5) of breeding ferrets were placed in a
modified rabbit cage (30 x 67 x 51 cm) with clean bedding.
After being paired, the male generally investigates the female’s
vulva. He then grips the female’s neck, mounts her, and displays
pelvic thrusting until he attains an intromission. A single
intromission (without pelvic thrusting) often lasts for more than
1 h, although ejaculation probably occurs within 1–2 min after
the onset of intromission. A previous study using female ferrets (5)
demonstrated that Fos-IR in both LHRH and non-LHRH neurons was maximal
90 min after the receipt of one 5-min intromission. Therefore, the male
as well as the female subjects used in the present experiment were
perfused 90 min after the onset of the male’s intromission. Different
males were allowed to intromit undisturbed with their female partners;
however, if the intromission did not terminate spontaneously, the
mating pair was separated by the experimenter just before the time of
perfusion. The actual intromission durations were 16 (n = 1 pair),
47 (n = 1 pair), and 90 (n = 3 pairs) min. Additional groups
of males (n = 5) and females (n = 5) were placed alone in a
cage in which an opposite sex ferret in breeding condition had been
housed for 48 h. Unstimulated male (n = 5) and female (n
= 5) control ferrets were placed alone in a clean cage. Subjects were
all perfused 90 min after the onset of each of these respective
treatments.
Perfusion
Subjects were injected ip with pentobarbital (100 mg/kg;
Somlethol, J. A. Webster, Leominster, MA). The heart was exposed, 0.1
cc sodium heparin (10,000 U/ml; Henry Schein, New York, NY) was
injected into the left ventricle, and a 5-cc blood sample was taken for
LH RIA. The blood sample was centrifuged, and the plasma was stored at
-10 C until RIA. The descending aorta was clamped, the tip of the
heart was cut off, and a perfusion cannula was inserted into the aorta
through the left ventricle. The subject was perfused using a forced air
apparatus (pressure, 100 mm Hg) with 50 ml 0.1 M PBS (pH
7.4) followed by 750 (female) or 1000 (male) ml 4% paraformaldehyde in
0.1 M phosphate buffer. The brains were quickly removed and
postfixed in the fixative for 4 h at room temperature. The brains
were transferred to 30% sucrose-PBS and, after they sank, were
sectioned coronally at 30 µm on a Reichert-Jung SM2000R (Leica,
Deerfield, IL) tabletop sliding microtome. Every sixth section was
saved for Fos/LHRH ICC.
ICC
As labeling can vary between runs of ICC, one animal from each
group was processed in each run of Fos/LHRH ICC. The sections were
incubated in 3% normal goat serum-1% H2O2-PBS
for 120 min at 4 C. The sections were immediately placed into the Fos
primary antiserum (DCH-1; 1:5000 in 0.4% Triton X-100–0.1 sodium
azide-PBS; gift from Dr. Gerard Evan) for 16 h at room
temperature. The sections were rinsed four times for 15 min each time
in PBS between each incubation, and all incubations were performed at
room temperature. The sections were incubated for 2 h with
biotinylated goat antirabbit IgG (1:200; Vector Laboratories,
Burlingame, CA) followed by avidin-biotin-peroxidase complex (1:100;
Vector Laboratories, Burlingame, CA) for 2 h. Sections were then
reacted for 9 min with nickel-diaminobenzidene (prepared according to
the manufacturer’s recommendation; Vector Laboratories). After being
incubated with the LHRH primary antiserum (1:100,000 in 0.4% Triton
X-100, 0.1 sodium azide, and PBS; LR-1, gift of Dr. Robert Benoit) for
16 h, the sections were incubated with biotinylated goat
antirabbit IgG (1:200; Vector Laboratories) for 2 h, followed by
avidin-biotin-peroxidase complex (1:100; Vector Laboratories) for
2 h. The sections were reacted for 32 min with AEC (prepared
according to the manufacturer’s recommendation; Vector Laboratories).
Finally, the sections were mounted onto gelatin-coated slides, dried,
rinsed in water, treated according to the manufacturer’s
recommendation with Crystal Mount (Biomeda, Foster City, CA), and
coverslipped using Permount.
The specificity of both the LHRH and Fos primary antibodies has been demonstrated previously in the ferret (4, 5). Preincubation of both antibodies with their respective antigens completely eliminated immunoreactive staining. Although the absolute percentage of Fos/LHRH double labeled neurons reported by Lambert et al. (4) and Wersinger et al. (5) and in the present study differ, the pattern of mating-induced augmentation of Fos-IR in both non-LHRH and LHRH neurons is consistent among the studies. The variability in the percentage of double labeled neurons among the studies is probably attributable to methodological differences, including different pretreatments, fixations, primary antibodies, chromogens, and reaction times in the chromogens.
Data analysis
The number of Fos-IR non-LHRH neurons was counted in the
granular layer of both the MOB and AOB, four subdivisions of the
preoptic area [POA; the medial (M), the lateral (L), the dorsal (D),
and the ventral (V) subregions], the BNST, the MA, the VLH, and the
CTF. The ferret has a vomeronasal organ (Dr. Michael Meredith, personal
communication) that presumably projects to an AOB. We based our
distinction between the MOB and AOB in the ferret on a previous report
in another carnivore, the dog (20), in conjunction with our own
analysis of serial cresyl violet-stained sections of the ferret
olfactory bulb cut in the coronal, horizontal, and sagittal planes. We
also used tyrosine hydroxylase immunoreactivity, which is not present
in the AOB of most species (21), as well as the distribution of soybean
agglutinin binding, which is heavy in the AOB but light in the MOB
(22), to confirm our morphological analysis (data not shown here).
Regions corresponding to the MPOA and LPOA were both quantified because
there is heterogeneity among the various subdivisions of the POA in
their inputs and projections (23), their neurotransmitter and
neuropeptide contents (24), and their steroid receptor content (25).
The DPOA was quantified because this is the location of the sexually
dimorphic male nucleus of the POA/anterior hypothalamus (AH), and the
VPOA was quantified because it is a nonsexually dimorphic region at the
same AP level as the DPOA (26). The other regions were selected because
previous studies in the ferret (4, 5), rat (6, 7, 8, 11, 12, 13), and hamster
(8) had shown that mating or exposure to chemosensory cues alone
induced neuronal Fos-IR in them.
All slides were coded so that the investigator was unaware of the sex
or treatment of the animals. Sections were selected for analysis by
being matched as closely as possible to a set of standard template
drawings. These templates were camera lucida drawings of cresyl
violet-stained sections at the level of each brain region we quantified
(see Fig. 1
). The Fos-IR nuclei of non-LHRH neurons in
one field of view under the x40 objective (0.10 mm2) were
traced onto a blank sheet of paper using a camera lucida microscope
attachment. In addition, each forebrain LHRH neuron was examined under
the x100 objective using oil. A LHRH neuron was identified as double
labeled only if a black round Fos-IR nucleus was seen within the red
LHRH-IR cytoplasm. LHRH neurons without a clearly identifiable black
nucleus were scored as single labeled. Figure 2 shows
representative photomicrographs of single and double labeled LHRH
neurons. Each LHRH neuron was mapped according to its location using a
set of standard template camera lucida drawings of cresyl
violet-stained coronal brain sections taken at 400-µm intervals. The
brain was divided into six partitions based upon anterior-posterior
position as in the report by Wersinger and Baum (5), and the number and
percentage of double labeled LHRH neurons were counted within each
partition. In addition, the number of and percentage of double labeled
LHRH neurons were determined for all six regions combined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). There was a significant
effect of treatment, but not of sex, on the mean number of Fos-IR
nuclei in the granular layer of the MOB [F(2,29) = 12.6;
P < 0.01] and in the MA [F(2,29) = 57.4;
P < 0.01]. In both sexes, mating augmented Fos-IR in
the MA more strongly than chemosensory cues alone. Mating, but not
exposure to chemosensory cues, significantly augmented Fos-IR in the
CTF in both sexes, with the response being significantly greater in the
female [F(2,29) = 3.8; P < 0.01].
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). The
number of Fos-IR nuclei was significantly augmented by mating in the
medial [F(2,29) = 33.5; P < 0.01], dorsal [F(2,29)
= 4.7; P < 0.05], and ventral [F(2,29) = 6.6;
P < 0.01] POA, the BNST [F(2,29) = 15.2;
P < 0.01], and the VLH [F(2,29) = 21.9;
P < 0.01] of female, but not male, ferrets.
Chemosensory cues alone also augmented Fos-IR in a sexually dimorphic
pattern in proximal components of this putative input pathway (Table 2). In females, Fos-IR was augmented in the MPOA, DPOA, VPOA, and VLH
by chemosensory cues, but in each of these regions significantly fewer
Fos-IR nuclei were counted after exposure to chemosensory cues alone
than after mating. Figure 3 shows representative
photomicrographs from the MPOA (top row), MA (middle
row), and VLH (bottom row) of a female (left
column) and a male (center column) ferret that,
respectively, received or achieved a 90-min intromission as well as of
an unpaired control female (right column).
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). Exposure to chemosensory cues alone did not affect
the colocalization of Fos-IR and LHRH in either sex. There was a
significant effect of sex x treatment at the level of the POA
[F(2,29) = 30.2; P < 0.01], the anterior
hypothalamus [F(2,29) = 45.1; P < 0.01], the arcuate
region [F(2,29) = 30.2; P < 0.01], and the posterior
hypothalamus [F(2,29) = 45.1; P < 0.01] as well as
in all of these brain regions combined [F(2,29) = 101.4;
P < 0.01]. Consistent with previous reports in the
ferret (5, 27, 28, 29), LHRH neurons extended caudally at the base of
the brain from the level of the diagonal band of Broca to the
mammillary bodies, and as Fig. 4 illustrates, more LHRH neurons were
concentrated caudally in the mediobasal hypothalamus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). By contrast, the capacity of mating to induce Fos-IR
in neurons situated in more proximal components of this input pathway
(i.e. POA, BNST, and VLH) as well as in LHRH neurons
themselves is sexually differentiated; significant increments in Fos-IR
in the latter neurons occurred only in females that received an
intromission. A similar profile of augmented Fos-IR in non-LHRH neurons
was observed in male and female ferrets exposed only to chemosensory
cues derived from an opposite sex ferret in breeding condition.
Chemosensory cues alone failed, however, to augment Fos-IR in
mediobasal hypothalamic LHRH neurons even in estrous females.
Considered together, our results suggest that both chemosensory and
genital-somatosensory stimuli associated with mating are processed
differently in male and female ferrets beginning with neurons located
in the MA and/or in regions that receive projections from the MA.
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The inputs to the MA place it in a unique position to integrate sensory information and direct the activation of other forebrain regions as well as, via an unknown pathway, LHRH neurons located in the mediobasal hypothalamus. In a number of species, including the rat, rabbit, and opossum, Scalia and Winans (14) showed that the vomeronasal organ projects to the AOB, which, in turn, projects to the MA. Neurons in the primary olfactory epithelium project to the MOB, which, via the cortical nucleus of the amygdala, then projects to the MA (14). In the rat the MA also receives projections from the subparafascicular and the lateral subparafascicular nuclei of the thalamus, (15), regions that include the CTF (23). Studies using the rat also suggest that the CTF receives genital-somatosensory inputs (12, 13). Assuming that similar projections exist in the ferret, the MA receives both chemosensory and genital-somatosensory stimuli. Indeed, in ferrets of both sexes, neural Fos-IR was augmented in the MA by mating (which provides both of these types of stimuli) as well as by exposure to chemosensory cues alone. The MA, in turn, has multiple projections to brain regions involved in the regulation of reproductive behavior and physiology, including the POA, BNST, and VLH (16).
In estrous females, neuronal Fos-IR was augmented in the MA, VLH, and MPOA by exposure to chemosensory cues alone, but not as strongly as by the receipt of an intromission. Chemosensory cues alone failed to augment Fos-IR in LHRH neurons. It is possible that the olfactory stimulation received during mating is stronger than or different from that received from the soiled cages used in this study and activates more neurons throughout the putative input pathway to the mediobasal hypothalamic LHRH neurons. There was no difference in the number of neuronal Fos-IR nuclei in the granular layer of the MOB between subjects allowed to achieve or receive intromissive stimulation and those exposed to chemosensory cues alone. This suggests that both stimuli were sufficient for full activation of brain regions involved in the initial processing of chemosensory cues. Thus, the observed differences between neuronal Fos-IR induced by chemosensory vs. mating stimulation are probably due to differential processing or integration of sensory information. Another possibility is that the MA contains two separate sets of neurons: one responsive to chemosensory cues, the other responsive to genital/somatosensory stimuli. Mating would activate both sets of neurons and result in a greater number of Fos-IR nuclei than would exposure to chemosensory cues alone. Our results are consistent with this hypothesis, as mating induced significantly more Fos-IR nuclei in the MA than exposure to chemosensory cues alone. Alternatively, neurons may exist that receive inputs from both the MOB and CTF and that require excitatory stimuli from both to express Fos. If there are two sets of amygdaloid neurons, they must be diffusely distributed throughout the MA and overlap extensively, as a close inspection of our slides revealed no regional differences in the distribution of Fos-IR neurons between mated females and females exposed only to chemosensory cues.
The sex dimorphism in the pattern of mating-induced Fos-IR in non-LHRH forebrain neurons could reflect a tonic inhibition of portions of the input pathway to the LHRH neurons in the ferret. This inhibition could be lifted by mating in the female, but not the male. Alternatively, the inhibitory input pathway could simply be absent in the female, but present and active in the male. One of the functions of the male nucleus of the POA/AH appears to be to inhibit female-typical proceptive behavior in the male. Lesions within this region significantly reduced the approach latency of estradiol benzoate (EB)-treated males to a stud male (31) and reversed the partner preference of EB-treated males from an estrous female to a stud male (32). Perhaps this region also inhibits female-typical neuronal responses to mating-associated cues. There are a number of neurotransmitter and neuropeptide systems that could inhibit the LHRH system, although it seems unlikely that endogenous opioids play a role because naloxone failed to increase plasma LH in gonadectomized, estradiol-treated male or female ferrets (33). It is also possible that the neural pathways underlying the mating-induced preovulatory surge in the female are simply absent in the male ferret. This seems unlikely, however, because the vast majority of sex dimorphisms reported in the brain do not reflect the absence of a structure or connection in one sex, but, rather, reflect differences in the number of neurons or in the morphology of neurons in a given region (34).
The sexual dimorphism in the augmentation of Fos-IR in non-LHRH forebrain neurons by mating observed in the ferret is unique among the various mammalian species studied to date. Wersinger et al. (12) showed that although the absolute number of mating-induced Fos-IR neurons differed between the sexes, the forebrain regions activated by mating were equivalent in gonadectomized, EB-treated male and female rats. Results of other studies suggest that mating augments neuronal Fos-IR in the forebrain of male rats (12, 13), hamsters (8), mice (35), and gerbils (36). In male rodents chemosensory cues alone also increase neuronal Fos-IR in the forebrain. Neuronal Fos-IR is augmented in the male rat forebrain after exposure to soiled bedding from an estrous female (6, 7). Similar results have been obtained for the male hamster (8). Thus, the ferret is unique in that no sexual stimulus used to date has successfully induced Fos-IR in neurons located in the male’s POA or hypothalamus.
Although there was no relationship in the present study between the duration of intromissions received by females and the induction of Fos-IR in either non-LHRH or LHRH neurons, there is some indication that longer intromissions activate the LHRH system more strongly than shorter ones. In the present experiment, in which intromissions lasted 16–90 min, there was a group of preoptic LHRH neurons activated by mating that was not present in previous studies (4, 5) in which the mating stimulus was always a single 5-min intromission. Lambert et al. (4) reported that LHRH neurons were activated equivalently throughout their anterior-posterior distribution; however, they analyzed fewer anterior-posterior segments of the mediobasal hypothalamus than in the present study. Also, they did not examine the most rostral group of LHRH neurons examined in the study by Wersinger and Baum (5) and in the present study. The increased Fos-IR in a small population of mediobasal LHRH neurons at the level of the organum vasculosum of the lamina terminalis/POA in addition to the more caudal sites suggests that the longer intromissions used in the present study activated more LHRH neurons than the 5-min intromissions used previously. It is unclear whether there is any physiological significance to the activation of these more rostral LHRH neurons. Carroll et al. (1) demonstrated that in estrous ferrets the receipt of intromissions lasting as little as 1.5 min elicited a preovulatory LH surge and ovulation as effectively as much longer intromissions. However, in the mink, a closely related mustelid species that also ovulates reflexively, females receiving a 6-min intromission shed significantly fewer ova than those allowed to intromit for 12 min (37). Although ovulation results from short intromissions (6 min), they are clearly not optimal for fertility in the mink (37). There may be differences in plasma LH levels and the number of ova shed between female ferrets receiving long vs. short intromissions. However, another experiment (38) suggests that the increased litter size in ferrets receiving long intromissions results from increased sperm transport rather than from increased LH secretion.
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Acknowledgments |
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Footnotes |
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Received July 2, 1996.
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