Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 64, 1100-1105 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Mating Induces Gonadotropin-Releasing Hormone Neuronal Activation in Anosmic Female Ferrets1

Julie Bakker2,a, Kevin R. Kellihera, and Michael J. Bauma

a Department of Biology, Boston University, Boston, Massachusetts 02215

ABSTRACT

In females of both spontaneously and induced ovulating species, pheromones from male conspecifics can directly stimulate GnRH neuronal activity, thereby inducing pituitary LH secretion and stimulating the onset of estrus. However, whether pheromones contribute to the steroid- or mating-induced preovulatory activation of GnRH neurons is less clear. Previous studies in the ferret, an induced ovulator, raised the possibility that olfactory cues contribute to the ability of genital-somatosensory stimulation to activate GnRH neurons in the mediobasal hypothalamus (MBH). In the present study the percentage of GnRH neurons colabeled with Fos-immunoreactivity (IR), used as a marker for neuronal activation, was investigated in the MBH of mated gonadectomized, estradiol-treated female ferrets in which both nares were occluded. In addition, the percentage of GnRH neurons colabeled with Fos-IR was examined in the MBH of gonadectomized, estradiol-treated female ferrets exposed to male bedding. Bilateral nares occlusion successfully blocked mating or odor-induced increments in Fos-IR in central olfactory regions, including the cortical and medial amygdala. By contrast, the percentage of GnRH neurons expressing Fos-IR did not differ between mated nares- and sham-occluded females. Exposure to male bedding alone failed to induce Fos-IR in MBH GnRH neurons. Thus, the mating-induced preovulatory activation of GnRH neurons in the female ferret's MBH appears to rely solely on genital-somatosensory as opposed to olfactory inputs.

GnRH, hypothalamus, ovulation, pheromones, reproductive behavior

INTRODUCTION

Olfactory signals influence a variety of social and reproductive functions by carrying information regarding species, gender, and hormonal status. In many mammalian species, olfactory cues serve to attract opposite sex conspecifics by inducing sexual arousal, which results in the display of mating behaviors [1]. In addition, pheromones can directly induce hormonal changes in the recipient animal [2].

Females of spontaneously ovulating species (e.g., mice, hamsters, sheep, and humans) undergo ovarian cycles in which the preovulatory LH surge and subsequent ovulation is induced by ovarian steroid hormones at regular intervals. The periodicity of these ovarian cycles can be influenced by pheromonal cues. For example, exposure of anestrous female mice, sheep, or goats to pheromones derived from male conspecifics stimulates pituitary LH secretion and consequent steroidogenesis, which culminate in a steroid-induced preovulatory LH surge [3–8]. Females of induced ovulating species (e.g., shrews, voles, rabbits, and ferrets) undergo periods of behavioral estrus when ovulation is induced by sensory stimulation associated with mating. As in spontaneously ovulating species, the periodicity of these periods of behavioral estrus can be influenced by pheromonal cues. For example, in virgin female prairie voles, pheromonal and tactile stimuli from a male caused significant increases in uterine weights [9] and circulating estradiol levels [10], leading to the onset of estrus and the display of female sexual behavior. Furthermore, exposure to male pheromones activated GnRH neurons and subsequent behavioral receptivity in the musk shrew [11–13]. Thus, in both spontaneously and induced ovulating species, the periodicity of ovarian cycles or behavioral estrus is strongly influenced by pheromonal cues.

The contribution of pheromonal cues to the steroid- or mating-induced preovulatory LH surge is less clear. There is some evidence from rat and shrew studies that olfactory cues are necessary for the preovulatory activation of GnRH neurons [14, 15]. Removal of the vomeronasal organ (VNO) significantly suppressed the expression of Fos-immunoreactivity (IR), which is used as a marker for neuronal activity, in GnRH neurons and subsequent LH release in repetitively mated female rats [14]. Furthermore, Rissman and Li [15] found that olfactory bulbectomy blocked mating-induced ovulation in the musk shrew.

In the ferret, an induced ovulator, several observations ([16] and unpublished results) suggest that olfactory cues may be necessary for the mating-induced preovulatory activation of GnRH neurons in the mediobasal hypothalamus (MBH). Vaginal-cervical stimulation using a glass rod was only successful in augmenting LH release when females were simultaneously neck-gripped by a stud male. On the other hand, Carroll et al. [17, 18] found that receipt of an intromission was required in order for a preovulatory LH surge to occur in estrous females. No increase in plasma LH was observed in estrous females that received only neck-grips and mounts from a male. Accordingly, Wersinger and Baum [19] found that mating with intromission significantly augmented the percentage of MBH GnRH neurons expressing Fos-IR in the female ferret, whereas male odors, by themselves, did not induce Fos-IR in MBH GnRH neurons. Moreover, exposure to male bedding induced higher levels of neuronal Fos-IR in the medial preoptic area (POA) and ventrolateral hypothalamus (VLH) in female than in male ferrets [19, 20]. This enhanced olfactory responsiveness of the female hypothalamus may contribute to the ability of genital-somatosensory stimulation, derived from receipt of intromission, to increase the activity of neurons that project to MBH GnRH neurons. As a result, male odors may facilitate the intromission-induced activation of GnRH neurons, thereby augmenting LH release. Therefore, in the present study we asked whether olfactory cues contribute to the mating-induced activation of MBH GnRH neurons in the estrous ferret. Levels of mating-induced Fos-IR in MBH GnRH neurons as well as in non-GnRH neurons were compared between estrous female ferrets in which both nares were either occluded or were left open. In addition, levels of neuronal Fos-IR in MBH GnRH as well as in non-GnRH neurons were compared between mated estrous female ferrets and estrous female ferrets exposed to either male-soiled bedding or clean bedding.

MATERIALS AND METHODS

Animals

Gonadally intact European male and female ferrets were purchased from Marshall Farms (North Rose, NY). Subjects were housed individually in modified rabbit cages under a long-day photoperiod (16L:8D lights-on at 0700 h). All ferrets were fed moistened Purina ferret chow (Ralston Purina Co., St. Louis, MO) once a day. Water was available ad libitum. Ferrets were ovo-hysterectomized under ketamine (35 mg/kg) and xylazine (4 mg/kg) anesthesia and subsequently injected subcutaneously twice daily with estradiol (E2; 5 µg/kg). Previous studies in our laboratory [17, 18] demonstrated that this pulsed regimen of E2 enabled an intromission-induced preovulatory LH surge to occur in ovo-hysterectomized females. Stimulus males were gonadally intact and in breeding condition. All experiments were conducted in accordance with the guidelines set forth by the National Institutes of Health Guiding Principles for the Care and Use of Research Animals and were approved by Boston University (protocol 99-017).

Experimental Protocol

Nares of ovo-hysterectomized female ferrets (nares-occluded; n = 4) were occluded using a method that was slightly modified from Buchman et al. [21]. Following general anesthesia with ketamine and xylazine, a topical anesthetic (Cetacaine; Cetylite Industries Inc., Pennsauken, NJ) was sprayed into both nares (1-sec spray = 200 mg) to suppress the sneezing reflex. VP mix dental impression material (Henry Schein, Melville, NY) was injected using silastic tubing into each nasal cavity. The injection tubing was placed at a depth of 5 mm, and the impression material was continuously injected while the tubing was slowly withdrawn from the nasal cavity to ensure complete occlusion of the naris. Sham-occluded females (n = 5) were treated in the same manner as the nares-occluded females except that the impression material was not injected. Subjects were observed closely both during and after recovery from the general anesthesia for signs of respiratory distress. Nares-occluded ferrets had no trouble breathing via their mouths. Anosmia in nares-occluded females was confirmed using an olfactory discrimination task [22]. Briefly, prior to nares-occlusion, all subjects were trained to approach a peppermint odor in order to obtain a food reward in a Y-maze. Subjects were food-deprived for 24 h before each trial. After nares-occlusion, the percentage of correct choices made by the nares-occluded females dropped to less than 50%, whereas sham-occluded females continued to identify the location of the food reward using the peppermint odor. After allowing 2 wk for ferrets to recover from surgery, nares- and sham-occluded females were used in a behavioral study [22]. Following the last behavioral test, both nares- and sham-occluded females were paired with a male in breeding condition, which was allowed to intromit undisturbed (a single intromission can last longer than 1 h in this species). Previous studies in our laboratory [19, 23] demonstrated that Fos-IR in forebrain GnRH and non-GnRH neurons was maximal 90 min after onset of intromission. Therefore, in the present study all females were perfused 90 min after onset of intromission. If intromissions did not end spontaneously, the mating pair was separated by the investigator just prior to perfusion. The occurrence of intromission was always confirmed by feeling the genital region of the two ferrets. Additional ovo-hysterectomized, E2-treated females were placed in a cage that either contained wood chip bedding that had been soiled overnight by a male ferret in breeding condition (n = 4) or clean wood chip bedding (n = 4), and were perfused 90 min later.

Perfusion

Subjects were injected intraperitoneally with pentobarbital (100 mg/kg; JA Webster, Leominster, MA). The heart was exposed and 1000 units of heparin (10 000 U/ml; Elkins-Sinn, Cherry Hill, NJ) were injected into the left ventricle. Subjects were perfused via the ascending aorta with approximately 50 ml of 0.1 M PBS pH 7.4, followed by approximately 800 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) pH 7.2. After perfusion, the brains were immediately removed and postfixed in 4% paraformaldehyde for 2 h. Brains were then placed for cryoprotection into a 30% sucrose/PBS solution at 4°C until they sank. Then brains were frozen with dry ice and sectioned coronally at 30 µm using a Reichert-Jung SM2000R tabletop sliding microtome (Nossloch, Germany). Each brain section was saved in antifreeze solution [20] and maintained at -20°C for later dual Fos-GnRH-immunocytochemistry.

Immunocytochemistry

Every fourth brain section was run for dual-label Fos-GnRH-immunocytochemistry. Free-floating sections were incubated in 3% normal goat serum/1% H2O2/PBS for 90 min at room temperature and then in rabbit-anti-Fos primary antiserum (SC52, 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 16 h at room temperature. Sections were rinsed three times for 15 min with 0.1 M PBS between each incubation. All incubations were carried out at room temperature. Sections were incubated with biotinylated goat-anti-rabbit antibody (1:200; Vector Laboratories, Burlingame, CA) for 2 h followed by avidin-biotin-complex (ABC; 1:100; Vector Laboratories) for 1.5 h, and reacted with nickel diaminobenzidine (DAB; Vector Laboratories, prepared according to the manufacturer's recommendation) for 7 min. Then sections were incubated with monoclonal mouse-anti-GnRH primary antiserum (1:10 000; QED Bioscience Inc., San Diego, CA) for 16 h, incubated with biotinylated goat-anti-mouse (1:200; Vector Laboratories) for 2 h followed by ABC (1:100; Vector Laboratories) for 1.5 h. The sections were then reacted with NovaRed (Vector Laboratories; prepared according to the manufacturer's recommendation) for 1–2 min. Sections were mounted onto gelatin-coated slides, rinsed in distilled water, and coverslipped with Permount (Fisher Scientific Co., Pittsburgh, PA).

Data Analysis

All slides were coded to conceal the treatment received by each subject. The number and percentage of Fos-IR GnRH neurons were analyzed by dividing the brain into four partitions from anterior to posterior: the diagonal band of broca (DBB)/POA, the anterior hypothalamus (AH), the arcuate region (Arc) including median eminence (Me), and the posterior hypothalamus (PH) [24]. Each GnRH neuron was examined under a 40x objective (standard area, 0.1 mm2). A GnRH neuron was identified as Fos-IR only if a black, round Fos-IR nucleus was seen within the red GnRH-IR (Fig. 1) cytoplasm. For each subject, the total number of GnRH-IR neurons (including Fos-IR GnRH and non-Fos-IR GnRH neurons) were counted for each brain region (DBB-POA, AH, Arc including Me, and PH). The number of sections analyzed per brain region ranged from 20 to 24 for the DBB/POA, 10–12 for the AH, 13–16 for the Arc including the Me, and 9–12 for the PH, respectively. For each subject, a percentage of Fos/GnRH double-labeling was calculated for each brain region by dividing the number of Fos-IR GnRH neurons by the total number of GnRH-IR neurons x 100%. Then GnRH-IR cell counts and percentages of Fos/GnRH double-labeling were combined for each experimental group for each brain region to calculate group means and standard errors of the mean.



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  		FIG. 1. Representative photomicrographs of a single-labeled GnRH (A) and a double-labeled Fos/GnRH neuron (B). Fos-IR was visualized using nickel-DAB as the chromogen; GnRH-IR was visualized in the same sections using NovaRed as the chromogen. The arrowhead indicates the Fos-IR nucleus of the GnRH neuron, whereas the arrow points to GnRH-IR cytoplasm

The number of Fos-IR non-GnRH neurons was counted in the medial preoptic area (mPOA), the bed nucleus of the stria terminalis (BNST), and the ventrolateral hypothalamus (VLH) using a 40x objective. We selected these brain areas because previous studies [20, 23, 25] have shown that mating or exposure to olfactory cues induced Fos-IR in these sites. For each brain region, one section on the left hemisphere was analyzed. All Fos-IR nuclei visible in one field of view were traced onto a blank sheet of paper with the aid of a camera lucida microscope attachment. In addition, the presence of Fos-IR non-GnRH neurons in the anterior cortical (ACo) and anterior and posterior medial amygdala (MA) was recorded using either a 20x objective (ACo and anterior MA; standard area, 0.38 mm2) or a 10x objective (posterior MA, standard area 0.60 mm2).

Data Analysis

All group means were compared using a one-way ANOVA; post hoc comparisons of group means were made using Student-Newman-Keuls tests.

RESULTS

Fos-IR in GnRH Neurons

Receipt of an intromission induced Fos-IR in GnRH neurons throughout the hypothalamus of both nares-occluded and sham-occluded females (Fig. 2); the percentage of GnRH neurons colabeled with Fos-IR did not differ between mated nares- and sham-occluded females. Exposure to male odors alone did not induce Fos-IR in GnRH neurons. There was a significant effect of treatment at the level of the DBB/POA (F[3/16] = 5.3, P = 0.013), the AH (F[3/16] = 5.3, P = 0.013), the Arc (F[3/16] = 6.3, P = 0.007), and the PH (F[3/16] = 4.5, P = 0.022). No differences were observed in number of GnRH-IR neurons among mated, male odor-exposed, or clean bedding females.



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  		FIG. 2. Top: Mean number (± SEM) of GnRH-IR neurons counted in the DBB/POA, AH, Arc, and the PH. Bottom: The mean percentage (± SEM) of GnRH-IR neurons colabeled with nuclear Fos-IR for each brain area. Groups included gonadectomized, estradiol-treated female ferrets that were either placed in a clean testing cage (clean bedding) or exposed for 90 min to soiled bedding from a breeder male (male bedding); and gonadectomized, estradiol-treated female ferrets in which both nares were either occluded (nares-occluded) or left open (sham-occluded) and allowed to receive an intromission from a breeder male. *P < 0.05 compared with all other groups by post hoc Newman-Keuls tests after a significant one-way ANOVA

Fos-IR in Non-GnRH Neurons

Bilateral occlusion of the nares decreased mating-induced Fos-IR in the central brain regions that receive olfactory inputs, including the ACo and the anterior and posterior MA (Fig. 3). Exposure to male bedding alone induced Fos-IR in the MA and VLH, but not the BNST and mPOA. There was a significant effect of treatment on number of Fos-IR neurons in the mPOA (F[3/16] = 6.3, P = 0.007), BNST (F[3/16] = 8.6, P = 0.002), ACo (F[3/16] = 6.0, P = 0.009), anterior MA (F[3/16] = 12.5, P < 0.001), posterior MA (F[3/16] = 12.6, P < 0.001) and VLH (F[3/16] = 13.9, P < 0.001). Mating with intromission induced neuronal Fos-IR in the mPOA, BNST, and VLH of nares- and sham-occluded females. Furthermore, levels of neuronal Fos-IR were augmented in the anterior and posterior MA of females that either received intromission or were exposed to male odors (Fig. 3).



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  		FIG. 3. Mean (± SEM) number of Fos-IR neurons in the ACo, MA, mPOA, BNST, and the VLH. See caption to Figure 2 for an explanation of different group treatments. *P < 0.05 from clean bedding females; {dagger}P < 0.05 from nares-occluded females, and #P < 0.05 from clean bedding and mated females using post hoc Newman-Keuls tests after a significant one-way ANOVA

DISCUSSION

Role of Olfactory Cues in Activation of the GnRH System

The present data clearly show that depriving estrous female ferrets of olfactory cues failed to diminish the mating-induced preovulatory activation of MBH GnRH neurons. Bilateral occlusion of the nares, a procedure that successfully diminished Fos-IR in brain sites receiving olfactory input, including the cortical and medial amygdala, did not reduce the percentage of Fos-IR GnRH colabeled neurons in the MBH. Thus, the preovulatory activation of the GnRH neuronal system in the ferret appears to rely solely on genital-somatosensory cues derived from the receipt of intromission.

Our results partially agree with findings in other induced ovulating species. In the rabbit, olfactory bulbectomy did not block mating-induced ovulation [26]. Artificial or mechanical stimulation of the female's genital tract induced preovulatory LH surges in several induced ovulating species, including rabbit, cat, and mink (reviewed in [27]). In contrast with rabbits, olfactory bulbectomy disrupted mating-induced ovulation in musk shrews [15], suggesting that olfactory cues are necessary for the preovulatory LH surge in this species. Differences in the amount and type of sensory stimuli needed to activate the reproductive axis may explain these species differences in olfactory responsiveness of the GnRH system. Female musk shrews typically show no ovarian cycles; onset of behavioral estrus appears to depend entirely on the presence of a male [11, 12]. By contrast, ferrets, minks, cats, and rabbits (hares and wild rabbits, but not the domestic rabbit) are all seasonal breeders in which reproductive activity is controlled by seasonal changes in photoperiod (reviewed in [28]). Thus the activation of the GnRH neuronal system of the ferret, mink, cat, and rabbit does not rely as much on male-associated stimuli as that of the musk shrew. Likewise, in the prairie vole, estrus and consequent sexual behavior and ovulation are induced by exposure to an unfamiliar male (reviewed in [29]). Mechanical stimulation of the female's genital tract failed to induce ovulation (reviewed in [30]), suggesting that sensory cues other than genital-somatosensory cues (e.g., olfactory cues) contribute to the activation of the GnRH neuronal system in this species.

Pheromones Are Processed by the Main Olfactory System in the Ferret

In most mammalian species, a main olfactory system (MOS) and an accessory olfactory system (AOS) can be distinguished neuroanatomically. In rodent species, primer pheromones are believed to be processed primarily through the AOS (i.e., the VNO and accessory olfactory bulb [AOB]), whereas nonreproductively relevant odors are believed to be processed through the MOS (i.e., the main olfactory epithelium [MOE]) and main olfactory bulb (MOB). In rats, exposure to male pheromones enhanced LH secretion in persistently estrous or ovariectomized, estradiol-treated females, but not after surgical occlusion or removal of the VNO [31, 32]. Furthermore, exposure to male pheromones rapidly induced neuronal Fos-IR in the AOB granule and mitral cell layers of female mice [33, 34] and rats [35, 36]. In contrast with rodent species, available evidence in the ferret [20], a carnivore, and the sheep [37], an ungulate, suggests that primer pheromones are primarily processed through the MOS instead of the AOS. A VNO [38, 39] as well as an AOB [20] have been identified in the ferret; however, neither exposure to olfactory cues nor mating with intromission induced neuronal Fos-IR in the AOB of female ferrets [19, 20]. These results suggest that the AOS is not important for processing of primer pheromones in the ferret. Likewise, in the sheep, electrocauterization of the VNO or vomeronasal nerve transection failed to block the male odor-induced LH response in the female [37], suggesting that the MOS is involved in the LH response to male odors in ewes.

Procedures for Eliminating Olfactory Inputs

Olfactory bulbectomy has most commonly been used to eliminate olfactory inputs to the forebrain. However, this procedure also damages the terminal nerve. For example, in the study by Rissman and Li [15], olfactory bulbectomy completely disrupted input from the terminal nerve, which is highly immunoreactive for GnRH in several species, including musk shrews [40]. Therefore, it is possible that elimination of GnRH-IR cells associated with the terminal nerve, as opposed to olfactory inputs to GnRH neurons, disrupted mating-induced ovulation in this species. In the present study, we used a relatively noninvasive technique to eliminate olfactory inputs to the MBH by permanently occluding the nares with dental impression material. Our procedure successfully produced anosmia in our ferrets. Nares-occluded females were no longer able to use peppermint odor as a discriminative stimulus to detect a food reward [22]. In addition, bilateral nares-occlusion completely eliminated the females' preference to approach volatile odors from a breeder male over those from an estrous female [22]. Finally, nares-occluded females showed decreased levels of mating-induced neuronal Fos-IR throughout the main olfactory pathway (i.e., MOB, piriform cortex [22], as well as the ACo and the MA [present study]). The attenuation of Fos-IR in these brain regions strongly suggests that olfactory cues received during mating were not detected by nares-occluded females. We acknowledge that our procedure of nares occlusion did not eliminate possible inputs from the AOS to MBH GnRH neurons. However, as stated earlier, we believe that the AOS is not functional in the ferret. Thus, mating with intromission did not augment neuronal Fos-IR in the AOB of nares- or sham-occluded females [22], suggesting that the VNO/AOB does not play an essential role in detecting or processing olfactory cues in the ferret. It is also interesting to note that nares-occluded females did not display any anogenital investigation [22], which would be necessary in order for odors to gain access to the VNO receptors.

Neural Pathways Mediating Mating-Induced Activation of GnRH Release

The absence of any significant Fos-induction in the MA of mated, nares-occluded female ferrets suggests that amygdaloid inputs may not be essential for the mating-induced activation of MBH GnRH neurons. However, the absence of Fos in the MA does not exclude a possible role for non-Fos expressing amygdaloid neurons in conveying genital-somatosensory stimuli to MBH GnRH neurons. In the rat, bilateral lesions of the MA decreased the percentage of mating-induced Fos-IR in GnRH neurons in ovariectomized, estradiol-primed females [41], suggesting that the MA plays an important role in conveying both olfactory and genital-somatosensory inputs to forebrain GnRH neurons in this species. Future studies using amygdaloid lesions should help to elucidate the role of the MA in the mating-induced activation of the GnRH neuronal system in the ferret.

Several lines of evidence suggest that the mPOA, BNST, and VLH play a key role in conveying genital-somatosensory inputs to the MBH GnRH neurons in the ferret. Equivalent levels of neuronal Fos-IR were observed in the mPOA, BNST, and VLH among mated nares- and sham-occluded females, whereas neuronal Fos-IR levels were not augmented in these brain sites, with the exception of a slight increase in the VLH, in females exposed to male odors alone. The latter result differs from previous studies from our laboratory [19, 20] in which exposure to male odors rapidly induced Fos-IR in the mPOA and BNST. This discrepancy may reflect differences in baseline Fos-IR levels between the clean bedding controls used in different studies. In the present study, clean bedding controls had higher levels of Fos-IR compared with those in the reports by Wersinger and Baum [19] or Kelliher et al. [20]. This elevation in Fos expression might have been caused by the estradiol regimens administered in the present study. Indeed, studies in the female rat have shown that estradiol administration rapidly induces Fos expression in the mPOA [42–45].

The activation of the mPOA, BNST, and VLH in estrous ferrets receiving an intromission points to the existence of a genital-somatosensory neural circuit that promotes the activation of MBH GnRH neurons and the consequent preovulatory release of LH from the pituitary. Veening and Coolen [46] reported that intromissive stimulation induced dense clusters of neuronal Fos-IR in the parvicellular part of the subparafascicular nucleus (SPFp), and the posterodorsal MA, posteromedial BNST, and mPOA of female rats. Tract-tracing studies using both anterograde and retrograde tracers showed that the mPOA had extensive projections to each of the brain sites that expressed dense clusters of neuronal Fos-IR following intromissive stimulation [46]. All these projections were found to be bidirectional [47]. Assuming that similar projections exist in the female ferret, the mPOA probably receives direct genital-somatosensory inputs from midbrain sites, including the SPFp and the central tegmental field. The latter brain region is highly activated by intromissive stimulation in estrous ferrets [19]. The mPOA, in turn, projects to the BNST and VLH; increased activity in these brain regions perhaps promotes the activation of GnRH neurons in the female's MBH. More research using antero- and retrograde tracers is needed to elucidate the neural pathways that lead to the activation of GnRH neurons in the ferret MBH.

ACKNOWLEDGMENTS

We thank the animal care staff at Boston University for their care of our ferrets.

FOOTNOTES

First decision: 28 September 2000.

1 Supported by grant HD21094 from the National Institutes of Health. Back

2 Correspondence and current address: Julie Bakker, Department of Biochemistry, Université de Liège, 17 Place Delcour Bat L1, Liege B4020, Belgium. FAX: 32 4 366 5971; jbakker{at}ulg.ac.be Back

Accepted: November 10, 2000.

Received: August 25, 2000.

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K. R. Kelliher and M. J. Baum
Nares Occlusion Eliminates Heterosexual Partner Selection without Disrupting Coitus in Ferrets of Both Sexes
J. Neurosci., August 1, 2001; 21(15): 5832 - 5840.
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