Both olfactory epithelial and vomeronasal inputs are essential for activation of the medial amygdala and preoptic neurons of male rats

Neuroscience 199 (2011) 225–234

BOTH OLFACTORY EPITHELIAL AND VOMERONASAL INPUTS ARE ESSENTIAL FOR ACTIVATION OF THE MEDIAL AMYGDALA AND PREOPTIC NEURONS OF MALE RATS S. DHUNGEL,a M. MASAOKA,b D. RAI,a Y. KONDOa,c* AND Y. SAKUMAa

sexual maternal behaviors, and approaching and escaping responses. A considerable body of evidence has shown that the olfactory epithelium (OE) and the vomeronasal organ (VNO) are highly sensitive to such social information. In general, it has been believed that general olfaction involves the OE and the pheromones are detected in the VNO. However, that dichotomy is denied in recent studies, both the OE and VNO receive pheromonal signals in social context (Keller et al., 2009). It is also suggested that those two systems receive different pheromones, for example, in the female mouse, signal of dominant male urine are detected in the VNO, while that of subordinate urine are perceived in the OE (Veyrac et al., 2011). Surgical ablation of the OE and VNO suggests that they also have different behavioral functions. Removal of the VNO temporarily impairs social recognition in male rats (Bluthé and Dantzer, 1993). Some studies have reported that VNO dysfunction affects sex recognition and expression of sexual behavior in mice (Kimchi et al., 2007; Stowers et al., 2002), although other reports showed that vomeronasal-organ removal (VNOx) disrupted lordosis in female mice whereas no disruptive effect of VNOx has been reported in male mice that were studied under optimal conditions (Kelliher et al., 1999; Pankevich et al., 2004, 2006). OE destruction in male rats suppressed noncontact penile erection but removal of VNO had no effect on this response (Kondo et al., 1999). In female hamsters, removal of the VNO did not impair preference for male odors over female odors (Petrulis et al., 1999). Projections from the OE and VNO are distinct, the so-called main and accessory olfactory systems (MOB and AOB), respectively. Because the accessory olfactory system and a part of the main olfactory system converge on certain common areas (Pro-Sistiaga et al., 2007), it is hard to segregate the functions of one from those of the other. Indeed, receptors specifically sensing social cues were found in the rat olfactory epithelium (Liberles and Buck, 2006; Lin et al., 2004; Mandiyan et al., 2005; Wang et al., 2006). VNO and OE inputs may complement each other (Brennan and Keverne, 2004; Brennan and Zufall, 2006). Activation of both the MOB and AOB by volatile urinary odors (Martel and Baum, 2007; Muroi et al., 2006) and pheromone stimulation (Xu et al., 2005) has been reported. In this study, we investigated the effect of sensory deprivation from the OE or VNO on preference behavior for conspecific odors and neuronal activation in the AOB, the medial and cortical nuclei of the amygdala (MeA and CoA),

a Department of Physiology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8602, Japan b Department of Life Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan c Department of Animal Sciences, Teikyo University of Science, 2-2-1 Senju-Sakuragi, Adachi, Tokyo 120-0045, Japan

Abstract—Chemosensory inputs signaling volatile and nonvolatile molecules play a pivotal role in sexual and social behavior in rodents. We have demonstrated that olfactory preference in male rats, that is, attraction to receptive female odors, is regulated by the medial amygdala (MeA), the cortical amygdala (CoA), and the preoptic area (POA). In this paper, we investigated the involvement of two chemosensory organs, the olfactory epithelium (OE) and the vomeronasal organ (VNO), in olfactory preference and copulatory behavior in male rats. We found that olfactory preferences were impaired by zinc sulfate lesion of the OE but not surgical removal of the VNO. Copulatory behaviors, especially intromission frequency and ejaculation, were also suppressed by zinc sulfate treatment. Neuronal activation in the accessory olfactory bulb (AOB), the MeA, the CoA, and the POA was analyzed after stimulation by airborne odors or soiled bedding of estrous females using cFos immunohistochemistry. Although the OE and VNO belong to different neural systems, the main and accessory olfactory systems, respectively, both OE lesion and VNO removal almost equally suppressed the number of cFos-immunoreactive cells in those areas that regulate olfactory preference. These results suggest that signals received by the OE and VNO interact and converge in the early stage of olfactory processing, in the AOB and its targets, although they have distinct roles in the regulation of social behaviors. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: olfactory preference, olfactory epithelium, vomeronasal organ, amygdala, preoptic area.

In rodents, olfactory signatures play a key role in social recognition. Chemical signals derived from their body or genital secretions activate the neuroendocrine pathway involved in various social behaviors including aggression, *Corresponding author. Tel: ⫹81-3-6910-3804; fax: ⫹81-3-69103800. E-mail address: [email protected] (Y. Kondo). Abbreviations: AOB, accessory olfactory bulb; cFos-ir, cFos-immunoreactive; CoA, cortical amygdale; FITC-SBA, fluorescein isothiocyanate conjugate soybean agglutinin; GRL, granular cell layer; MeA, medial amygdale; MOB, main olfactory systems; MTL, mitral cell layer; OE, olfactory epithelium; OEx, olfactory epithelium lesions; PBS, phosphate-buffered saline; POA, preoptic area; VNO, vomeronasal organ; VNOx, vomeronasal-organ removal.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.09.051

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and the preoptic area (POA) in response to odors of estrous females in male rats.

EXPERIMENTAL PROCEDURES Animals Male and female Long-Evans rats aged 8 weeks were purchased from the Institute for Animal Reproduction, Ibaraki, Japan and maintained under controlled temperature (23⫾2 °C) and reversed light/dark photoperiod (lights off from 11:00 –23:00) conditions with free access to food and water. The experimental protocols were approved and carried out under the guidelines for the care and use of laboratory animals of Nippon Medical School.

Preparation of animals Prior to the experiment, all females were ovariectomized under ether anesthesia. Some were brought into estrus by a s.c. injection of 5 ␮g of estradiol benzoate (dissolved in 0.1 ml of sesame oil, Sigma-Aldrich, St. Louis, MO, USA) at 48 h, and 500 ␮g of progesterone (dissolved in 0.1 ml of sesame oil, Sigma-Aldrich, St. Louis, MO, USA) at 3–7 h prior to each behavioral test. Following a 1-week acclimation to our laboratory, all males were subjected to three mating sessions with estrous females. Those who failed to ejaculate in the third session were excluded. After the sexual experience, olfactory preference tests (described below) followed by copulation tests were carried out twice weekly as pre-surgery baseline tests.

Surgery After the baseline behavioral tests, 15 male rats were subjected to olfactory epithelium lesions (OEx) induced by intranasal infusion of 10% zinc sulfate (ZnSO4·7H2O in 0.5% NaCl solution) under ketamine HCl (25 mg/kg) and sodium pentobarbital (25 mg/kg) anesthesia (Alberts, 1974; Margolis et al., 1974). During infusion, animals were placed in the prone position on an inclined surface while one end of a polyethylene tube was inserted to deliver 100 ␮l per nostril (at a rate of 0.02 ml/min) and another end of the tube was fitted to a Hamilton syringe attached to a microinfusion pump. Sham-OEx rats were made in the same way but infused with saline. All animals were allowed 4 –5 days to recover before post-surgery experiments. To exclude the possible involvement of regenerated OE sensory neurons, all behavioral tests were completed within 6 days of the zinc sulfate treatment. Another subset of 15 males was subjected to VNOx following the method previously described (Saito and Moltz, 1986). Briefly, under ketamine and sodium pentobarbital anesthesia, each male was held in the supine position on a homemade operating board. Their jaws and tongues were restrained by wire hooks used to keep their mouths open. The vomer bone, which encapsulates the VNO, was exposed by a midline incision of the soft palate and drilled at two points (approximately 5– 6 mm apart) to excise the VNO. The incised palate was stitched using an absorbable suture. Corresponding Sham-VNOx males received the same operation without vomer bone removal. Because sham-OEx (n⫽7) and sham-VNOx (n⫽6) were statistically indiscernible in all behavioral tests, they were combined as a single sham group (n⫽13) for subsequent analyses to compare with OEx and VNOx rats.

Behavioral testing Hidden food finding test. To examine the abilities of experimental males to smell, hidden food finding tests were carried out after 48 h of food deprivation. In each test, a pellet of laboratory chow was located in a transparent observation cage (50 cm long⫻30 cm wide⫻40 cm high) and covered with 5-cm-thick

wooden bedding. Each male was placed in the center of the cage and allowed to seek the buried food for 5 min. Time holding the pellet with the forepaws was recorded as latency. Tests were carried out twice, 1 week before and 3 days after the zinc sulfate treatment. Olfactory preference test. Olfactory preference was assessed using an alternate choice paradigm. The details of the preference chamber have been described previously (Xiao et al., 2004). The apparatus was a three-chambered acrylic observation box (110 cm long⫻12 cm wide⫻30 cm high). Each experimental male was placed in the middle compartment and two stimulus rats were placed in the side compartments. Three opaque plates with holes of 3-cm diameter at different levels were assembled into a partition to create divisions between the compartments. A blower connected to the ceiling of the middle compartment through a corrugated flexible tube and maintaining negative pressure in the middle compartment allowed airflow (approximately 0.2 m3/min) from the two side compartments into the middle compartment. A 2-cm-deep transparent tube at 2 cm from the floor was attached to the holes in both side partition plates facing the middle compartment. On the day of the olfactory preference test, each experimental male was subjected to three preference tests with different pairs of stimulus animals: (1) a receptive female and an intact male; (2) a receptive female and an ovariectomized female; and (3) an intact male and a castrated male. The test order and the positions of the stimulus pairs were counterbalanced, and the intervals between tests were more than 1 h. Before each test, the apparatus was cleaned with 70% ethanol (v/v) and bedded with fresh paper chips (Alpha-dri, Shepherd Speciality Papers; Kalamazoo, MI, USA). During a period of 5 min for acclimation to the apparatus, downstream airflow was made from the middle to the side compartments. Behavioral observations were made for 5 min and recorded by a video camera fixed in front of the preference chamber. Time spent nose-poking in each of the left and right inlets was calculated by an event recorder on a personal computer. Preference scores in each stimulus pair were calculated by determining percent time spent nose-poking toward receptive females (Pairs 1 and 2) or castrated males (Pair 3) relative to total time spent nose-poking toward both stimulus animals. All males were subjected to twice weekly repetitive olfactory preference tests before surgery. As a post-surgery test, olfactory preference was tested within 1 week after OEx surgery to exclude the influence of regenerated olfactory neurons, and within 2 weeks after VNOx surgery to allow for sufficient recovery (see timeline scheme of behavioral testing shown in Table 1). Table 1. Timeline schema of behavioral tests Week

First session

1 2 3 4

Sexual behavior test Sexual behavior test Olfactory preference test Olfactory preference test surgery of OEx and VNOxa Olfactory preference testb Olfactory preference testc Stimulation with estrous odor

5 6 7

Second session

Sexual behavior test

Sexual behavior testb Sexual behavior testc Sacrifice after 2 h

Tests appeared in each line were carried out within the same day in order with an interval more than 2 h. a Surgery was conducted within 1 or 2 d after olfactory preference test. b Recovery period was 1 wk in OEx males and 2 wk in VNOx males (see Method). c In OEx males, the second test of post-surgery was omitted in order to exclude an influence of possible neuronal regeneration.

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Sexual behavior test. Observation of sexual behavior with estrous females was performed three times weekly as mating experience preceding olfactory preference tests. Each male was put into a transparent observation cage (50 cm long⫻30 cm wide⫻40 cm high) bedded with wooden shavings, and acclimated for 5 min. The test was started by the introduction of estrous females, and terminated after the first ejaculation or time reaching 60 min. The numbers and latencies of mounts and intromissions, and latencies of ejaculation were recorded. Sexual behavior was also tested in the following olfactory preference tests. Observations were started 1 h after finishing all olfactory preference tests.

Stimulation with estrous odors After the completion of all preference tests, each group was divided into three conditions for odor stimulation: (1) clean bedding (OEx, n⫽3; VNOx, n⫽5; Sham, n⫽4); (2) soiled bedding of estrous female cages (OEx, n⫽4; VNOx, n⫽5; Sham, n⫽5); and (3) airborne estrous odors (OEx, n⫽3; VNOx, n⫽5; Sham, n⫽4). Odor stimulation was made in the same apparatus used for the olfactory preference test. In the clean bedding condition, male subjects were placed in the middle compartment the floor of which was covered with clean wood shavings, and an air blower was turned on during a 1.5-h stimulation period without stimulus animals. In the soiled bedding condition, male subjects were subjected to the same procedure as the clean bedding males, but the wood shavings on the floor were collected from the floors of cages in which ovariectomized female rats primed with estrogen and progesterone (see animal preparation) had lived for 1 week. In the airborne odor condition, male subjects were subjected to the same procedure as the clean bedding males, but each side compartment contained an estrous female during stimulation. After stimulation, all males were immediately anesthetized with an overdose of sodium pentobarbital, and perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed and kept in the same fixative overnight. After replacing fixative with 30% sucrose (in 0.1 M PB), 40-␮m-thick frontal sections were cut using a freezing microtome. Every third sagittal section of the AOB was processed for fluorescein isothiocyanate conjugate soybean agglutinin (FITC-SBA) staining, cFos immunohistochemistry, and Nissl staining to identify the different layers of the AOB. Blocks including the amygdala and POA were sectioned coronally, and every second section of the same thickness (40 ␮m) was processed for cFos immunohistochemistry and Nissl staining.

FITC-SBA staining Each VNOx was confirmed by histological methods in which sections were stained with fluorescent soybean agglutinin (SBA) for glomerular layer visualization as described previously (Key and Giorgi, 1986). A lack of fluorescence in the glomerular layer indicates loss of innervation of VNO sensory neurons. Sections mounted on slides were washed with 0.01 M PBS (phosphatebuffered saline), and incubated in FITC-SBA (20 ␮g/ml in 0.01 M PBS, 1:100 dilutions, Vector Lab; Burlingame, CA, USA) for 40 min at room temperature. The sections were then rinsed with 0.01 M PBS and cover slipped with fluoro-guard antifade reagent (Biomeda Corporation; Foster City, CA, USA).

Immunohistochemistry In cFos immunohistochemistry, free-floating sagittal sections including the AOB, and coronal sections including the POA, the MeA, and the CoA, were incubated in 3% normal goat serum and 0.25% Triton X-100 in 0.01 M PBS, and subsequently incubated with cFos antibody (rabbit polyclonal IgG; 1:20,000, Calbiochem; Gibbstown, NJ, USA) at 4 °C for 4 days. Then, the

Fig. 1. Schematic representation of the cFos counted regions of brain. (A) The mitral and granular cell layers of the AOB (sagittal section, rostral to the left side), (B) the POA, and (C) the MeA and CoA. Squares in panels (B) and (C) indicate counting areas of 330⫻330 ␮m2 in the POA, MeA, and CoA regions. MOB, main olfactory bulb; GL, glomerular layer of AOB; Ctx, cerebral cortex; ac, anterior commissure; ox, optic chiasm; VMN, ventromedial nucleus of hypothalamus (redrawn from Paxinos and Watson, 2008).

sections were processed using an ABC kit (Vectastain ABC Elite standard; Vector Lab) and developed with 0.25 mg/ml diaminobenzidine in 0.01 M PBS containing 0.03% H2O2). Areas of the AOB used for counting cFos-positive cells were the complete mitral and granular layers seen on sagittal sections. Areas of the amygdala and POA used for cell counting were a 330⫻330 ␮m2 within the amygdala and a square of the same size located below the anterior commissure and just rostral to the disappearance of the anterior commissure, respectively (Fig. 1). The area of the MeA used for cell counting was located between the stria terminalis and the optic tract and just rostral to the appearance of the lateral ventricle, while that of the CoA was located directly below the stria terminalis on the same section used for cell counting in the MeA. The counting of cFos-positive cells was conducted in those areas of three consecutive sections.

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VNOx males maintained general olfaction to find hidden pellets and hence the latency was comparable with that of Sham males (ns Kruskal–Wallis test, X2⫽0.034). Histological examination of the VNOx Sham and OEx males had visible fluorescence in the glomerular layer of the AOB of FITC-SBA-stained sagittal sections, whereas fluorescence was absent in those of VNOx males, indicating loss of innervations of VNO sensory neurons (Fig. 3). Fig. 2. Latency to grasp buried laboratory chow in a 5-min food finding test. Each diamond (open, Sham; black, OEx and VNOx males) represents time required to grasp the chow. After OEx surgery, five males were excluded from data analyses because of normosmic behavior (gray diamonds). Asterisks indicate a significant difference from Sham and VNOx males (P⬍0.001, Kruskal–Wallis test). The horizontal bar in each panel indicates median values and the shaded zone indicates interquartile range (IQR) of the groups.

Statistical analyses ANOVA followed by the Student–Newman–Keuls test, the Mann– Whitney test, the Kruskal–Wallis test and the Student t-test were used where appropriate. The threshold for significance was P⬍.05.

RESULTS Hidden food finding testing In the pre-surgery food finding test, latencies to grasp a hidden pellet were not significantly different among the groups. Following zinc sulfate treatment, OEx rats were anosmic and could not find pellets within a 5-min observation period, with latencies significantly longer than that of Sham males (P⬍0.001 Kruskal–Wallis test, X2⫽17.69) and VNOx males (P⬍.001, ⌾2⫽18.48) (Fig. 2). In contrast,

GL

Sham OEx

Olfactory preference All sexually experienced experimental male rats displayed a masculine type of olfactory preference during baseline pre-surgery tests. They spent a significantly longer time nose-poking toward receptive females than toward intact males or anestrous females and nosepoking toward castrated males than toward intact males (paired t-test). After surgical operations, OEx rats failed to show preference for airborne odors in any of the three stimulus pairs (Fig. 4). Overall, post hoc analysis using the Student– Newman–Keuls test showed that OEx rats spent less time nose-poking toward receptive females than Sham and VNOx rats in the male–female stimulus pair experiments (P⬍0.05, ANOVA F(2,35)⫽21.18, P⬍0.001) and in the female stimulus pair experiments (P⬍0.05, ANOVA F(2,35)⫽20.50, P⬍0.001). Similarly, OEx rats spent significantly less time nose-poking toward castrated males than Sham and VNOx males in the male stimulus pair experiments(P⬍0.05, ANOVA F(2,35)⫽12.82, P⬍0.001). On the other hand, VNOx males still showed significant olfactory preference, similar to Sham males (Fig. 4).

Sham VNOx

MTL GRL OEx

VNOx

Fig. 3. Photomicrographs of sagittal sections of the AOB stained with FITC-labeled soybean agglutinin. Lack of innervations results in diminished fluorescence in the glomerular cell layer indicating complete loss of the vomeronasal organ (VNOx). GL, glomerular cell layer; MTL, mitral cell layer; GRL, granule cell layer. Bar 200 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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Fig. 4. Olfactory preference behavior of all groups in the post-surgery test. Gender symbols on both sides of each panel indicate the pairs of stimulus animals tested. Cross-over male or female symbols indicate gonadectomy. Asterisks and plus signs indicate a significant difference from Sham and VNOx animals (ANOVA followed by the Student–Newman–Keuls test); * and ** indicate significant decreases relative to Sham at P⬍.05 and .001, respectively; ⫹ indicates a significant decrease relative to VNOx at P⬍.05. The horizontal bar in each column indicates SEM.

Sexual behavior Sexually experienced Sham, OEx, and VNOx rats showed a high level of sexual behavior, and no difference was found in mount, intromission, and ejaculation among the groups before surgery. Destruction of the OE resulted in a mild suppressive effect on copulatory behavior. In the post-surgery copulatory test, OEx and VNOx males showed no difference in numbers of mounts and intromissions compared with Sham males. Although all OEx males could ejaculate, their latencies for mount and ejaculation were significantly longer than those of Sham and VNOx males (P⬍0.05, ANOVA F(2,35)⫽3.43, P⬍0.05; P⬍0.05, ANOVA F(2,35)⫽4.18, P⬍0.05, respectively) (Fig. 5). Intromission and mount frequencies per 5 min were also significantly decreased in OEx males compared with Sham and VNOx males (P⬍0.05, ANOVA F(2,35)⫽6.63, P⬍0.01; P⬍0.05, ANOVA F(2,35)⫽3.63, P⬍0.05, respectively). However, their intromission ratio (the number of

intromissions divided by the number of total mounts) was not significantly different from those of Sham and VNOx males (Fig. 5). In contrast to OEx males, sexual behavior in VNOx males did not differ in any parameter of copulatory behaviors from that of Sham males (Fig. 5). cFos expression cFos-immunoreactive (cFos-ir) cells were counted in the mitral cell layer (MTL) and the granular cell layer (GRL) of the AOB, the MeA, the CoA, and the POA (Fig. 1). In the MTL and GRL, sham males exposed to estrous-soiled bedding and airborne estrous odor had a significantly increased number of cFos-ir cells compared with those exposed to clean bedding (P⬍0.05, ANOVA F(2,10)⫽6.107, P⬍0.05; P⬍0.05 ANOVA F(2,10)⫽19.02, P⬍0.001, respectively). In contrast, neither estrous-soiled bedding nor estrous airborne odor increased the number of cFos-ir cells in either layer of the AOB in OEx and VNOx

Fig. 5. Mean frequency of intromissions (per 5 min), ejaculation latency, and intromission ratio (number of intromissions/total number of mounts and intromissions before the first ejaculation). Letters above the columns indicate a significant difference from columns with a different letter (ANOVA followed by the Student–Neuman–Keuls test). The vertical bar in each column indicates SEM.

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Fig. 6. (A) Mean number of cFos-ir neurons in the AOB after exposure to clean or estrous-soiled bedding or estrous odor. Different letters above the vertical bars indicate a significant difference from columns with a different letter (ANOVA followed by Student–Neuman–Keuls test). The vertical bar in each column indicates SEM. (B) Photomicrographs showing representative cFos activities in animals exposed to clean bedding (first row), estrous-soiled bedding (middle row) and estrous odor (third row) for each group. MTL, mitral cell layer; GRL, granule cell layer. Bar 200 ␮m.

males, when compared with those exposed to clean bedding (Fig. 6). In the MeA, estrous-soiled bedding increased the number of cFos-ir cells in sham males, but not in OEx and VNOx males, although both of the latter groups showed a similar tendency toward an increment. They had significantly fewer cFos-ir cells in the MeA after exposure to estrous-soiled bedding (P⬍0.05, ANOVA F(2,11)⫽5.717; P⬍0.05). Exposure to estrous airborne odor failed to produce a significant increment in the number of cFos-ir cells in the MeA in any of the groups. Sham males also showed a significantly in-

creased number of cFos-ir cells in the CoA after exposure to estrous-soiled bedding and estrous airborne odor, but OEx and VNOx males did not. Both OEx and VNOx males had significantly fewer cFos-ir cells in the CoA than sham males after exposure to either estrous-soiled bedding or estrus airborne odor (P⬍.05, ANOVA F(2,11)⫽19.709, P⬍0.001; P⬍0.05, ANOVA F(2,9)⫽10.107, P⬍0.01, respectively). Additionally, post hoc analysis revealed that there were significantly fewer cFos-ir cells in OEx males than in sham males but that the number was higher than that in VNOx males after exposure to estrous-soiled bedding (Fig. 7).

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The number of cFos-ir cells in the POA was significantly increased in sham males after exposure to both estrous-soiled bedding and estrous airborne odor, but this increase was not seen in OEx and VNOx males. The number of cFos-ir cells in the POA of OEx males after exposure to estrous airborne odor was significantly lower than that in sham males (P⬍0.05, ANOVA F(2,9)⫽12.461, P⬍0.01), as were the numbers in the POA of VNOx males after exposure to both estrous-soiled bedding and airborne odor (P⬍0.05, ANOVA F(2,11)⫽4.389, P⬍0.05).

DISCUSSION The data in the current study demonstrated that OE lesions induced by intranasal irrigation of zinc sulfate completely abolished olfactory preference, but that the effect on copulatory behavior was confined to partial aspects. In our olfactory preference test, the presented stimuli were limited to airborne volatile odors of unanesthetized animals in adjacent compartments. During the test, although the experimental males also could sense noise and vocalization derived from neighboring stimulus animals, there was no evident response during the acclimation period prior to each test, in which airflow was reversed (neither experimental nor stimulus rats located in the olfactory preference test apparatus vocalized ultrasonics, Kondo (unpublished observation)). Thus, the nose-poking preference shown by the experimental males must be based on volatile odor cues conveyed with the airflow. OEx impaired olfactory preference, whereas VNOx did not. These findings support our common belief that the OE receives volatile chemicals, while the VNO detects involatile chemicals. Because of the regeneration ability of OE sensory neurons, the effectiveness of anosmia induced by zinc sulfate lesion is limited in period (Alberts, 1974; Alberts and Galef, 1971; Thor and Flannelly, 1977). After the application of zinc sulfate, we first confirmed the anosmic effect within 2 days and tested for olfactory preference within a week. This is why the experiment was carried out only once after OEx. A paper reported that destruction of olfactory mucosa by zinc sulfate application does not produce a complete loss of olfactory sensory neurons (Slotnick et al., 2000), anosmia in our males with OEx may also be incomplete similar to rats of that study. However, the reduced sensory inputs of the OEx males impaired hidden food finding and were insufficient to maintain olfactory preference in the present study. The VNO has been believed to detect sexual singals, namely pheromones. Previous studies reported that VNOx female rats and mice failed to show preference for odors of sexual active males’ urinary volatiles (Romero et al., 1990; Martel and Baum, 2009). In contrast, male VNOx mice resembled sham-operated controls in their ability to discriminate between volatile urinary odors of estrous females and intact males (Pankevich et al., 2004). The performance of our VNOx males may be due to a property of the stimuli and the restriction of stimulus to volatile odors. A similar paradigm is used in a test for noncontact erection (NCE), which was expressed in male rats located in the

Fig. 7. Mean number of cFos-ir neurons in the POA, MeA, and CoA regions after exposure to clean bedding, soiled bedding of estrous female cages and airborne estrus odor. Letters above the columns indicate a significant difference from columns with a different letter (ANOVA followed by the Student–Neuman–Keuls test). The vertical bar in each column indicates SEM.

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vicinity of receptive females. Induction of NCE requires sensory inputs from the OE rather than the VNO (Kelliher et al., 1999; Kondo et al., 1999). This indicates the importance of the OE in signal detection during sexual interaction. In mice, OEx induced by zinc sulfate (Keller et al., 2006a) or dysfunction of the OE induced by Cnga2 gene depletion (Mandiyan et al., 2005) also prevented olfactory preference. One report showed that VNO dysfunction induced by transient receptor potential cation 2 channel (Trpc2) knockout resulted in a lack of discrimination between male and female conspecifics in male mice (Stowers et al., 2002). However, contradictory studies also reported the failure of suppression of olfactory preference after surgical removal of the VNO (Keller et al., 2006b; Pankevich et al., 2004). Although the rat and mouse are different species, our current data support the latter findings. Partial effects of OEx on sexual behavior were observed; these include prolonged latencies of mount and ejaculation, and decreased intromission frequency per 5 min. The prolongation of ejaculatory latency reflected the decrease in intromission frequency because the numbers of intromissions achieving ejaculation were not different between Sham and OEx males. The decline in sexual behavior may be due to difficulty in detection of sexual signals in OEx males, rather than lowered sexual motivation. Although OEx in mice severely impairs sexual behavior (Keller et al., 2006a; Mandiyan et al., 2005), male rats with OEx can accomplish sexual behavior in spite of a loss of olfactory preference. In contrast, VNOx had no effect on sexual behavior. Some studies have reported partial impairment of sexual behavior in male rats (Kondo et al., 2003), although the extent of influence of VNOx might depend on the degree of sexual experience (Saito and Moltz, 1986). Following the behavioral tests, we also explored neuronal activation in the AOB, MeA, CoA, and POA by cFos immunohistochemistry after stimulation by either airborne odor from unanesthetized estrous females or soiled bedding collected from the home cages of estrous females. The former consisted of only volatile substances, while the latter included both relatively long-life volatile and involatile chemicals. Because the OE receives volatile substances and the VNO receives involatile chemicals, it was assumed that the airborne odor would stimulate the OE pathway and that the soiled bedding would stimulate both the OE and VNO pathways. In Sham males, however, increased numbers of cFos-ir neurons were observed in the AOB after both. If it is true that information regarding airborne chemicals is transmitted via the OE, crosstalk of those signal processing systems might occur in the AOB, a primitive sensory region of the vomeronasal system. Indeed, the number of cFos-ir cells following stimulation was suppressed after not only VNOx, but also OEx. A similar observation was reported in the mouse (Martel and Baum, 2007), and the interaction of these systems may exist across rodent genera. It has been reported that such biologically meaningful odorants as the odors of predators or spoiled foods are detected in zone I, a dorsal part of the

OE, and that the signals are conveyed to the glomeruli in the dorsal part of the MOB (Kobayakawa et al., 2007). A study reported that neurons in the MOB expressed cFos in response to male urinary volatiles in female mice (Kang et al., 2009). Thus, social signals of opposite-sex odor may be received in the OE and sent to the MOB. Such crosstalk may occur directly and/or indirectly between the MOB and AOB. Interestingly, VNOx failed to suppress the increase in the number of cFos-ir cells after sexual behavior (Kondo et al., 2003). AOB activity is influenced by the POA, which critically regulates sexual behavior of male rats (Hurtazo and Paredes, 2005). Since there is no evidence of direct projection from the POA to the AOB, it may be achieved via the MeA afferent. Thus, the POA and MeA are possible candidates underlying such an interaction. Signals processed in the AOB travel to the MeA through the axons of mitral cells, while signals containing biological meaning in the dorsal MOB are conveyed to the CoA (Miyamichi et al., 2011). These signals converge on the POA via the stria terminalis. The results were very similar in these three areas, the MeA, CoA, and POA. Irrespective of what the stimuli were, the greatest increase in the number of cFos-ir cells was seen in Sham males, and this increase was suppressed by both OEx and VNOx. In the present study, although airborne estrous odor failed to yield statistical significance in terms of the increment of cFos-ir cells in the MeA, the pattern of differences among groups was similar to that in the CoA or POA. This might be due to insufficient duration or strength of the stimulation (Dhungel et al., 2011; Kelliher et al., 1999). Activation of the MeA by soiled bedding was higher than that due to airborne odor, probably because stimuli of soiled bedding were processed via dual pathways of the OE and VNO, and integrated in the MeA. Although we did not analyze MOB activation in this study, cFos expression in the CoA as well as the MeA was significantly suppressed by not only OEx but also VNOx, suggesting that MOB activation is also affected by inputs from both the OE and VNO. Our recent study demonstrated that small lesions confined to a single nucleus, the MeA or CoA, failed to suppress olfactory preference, while extended lesions including both the MeA and CoA did suppress it (Dhungel et al., 2011). In male hamsters, small lesions in either anterior or posterodorsal part in the medial amygdala impair volatilebased preference (Maras and Petrulis, 2010a), suggesting species difference exist in olfactory preference in rodents. In the rat, olfactory preference is carried by OE inputs, as shown in our study, indicating that signals that input to the OE diverge at the olfactory bulb to the MeA and CoA. Because the posterodorsal part of the MeA contains a dense distribution of steroid receptors, the MeA may play a role in the integration of olfactory and endocrine information (Maras and Petrulis, 2010b). The olfactory signals processed in the MeA and CoA may converge on the POA. The POA regulates not only olfactory preference (Dhungel et al., 2011), but also other social behaviors such as sexual behavior (Dhungel et al., 2011; Paredes et al., 1998), aggressive behavior (Albert et al., 1986), and maternal behavior (Jacobson et al., 1980).

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Although the increase in the number of cells showing cFos expression in the POA was induced by stimulation with estrous-soiled bedding (Hosokawa and Chiba, 2005; Hurtazo and Paredes, 2005; Swann and Fiber, 1997; Veening and Coolen, 1998), it also depends on sexual experience (Hosokawa and Chiba, 2005). The present study demonstrates that this activation of the POA requires chemosensory inputs from both the OE and the VNO. Furthermore, the POA receives somatosensory information from the lower brain stem, and integrates it with chemosensory signals (Baum and Everitt, 1992). Thus, the POA may regulate the final phenotype of social behavior. Acknowledgments—This study was partly supported by Grantsin-Aid for Scientific Research to Y.K. (No. 22590229) and Y.S. (No. 16086210) from the Japan Society for the Promotion of Science.

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(Accepted 26 September 2011) (Available online 1 October 2011)