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. Author manuscript; available in PMC: 2008 Mar 6.
Published in final edited form as: Physiol Behav. 2006 Mar 3;87(4):781–788. doi: 10.1016/j.physbeh.2006.01.020

Accessory olfactory neural Fos responses to a conditioned environment are blocked in male mice by vomeronasal organ removal

Diana E Pankevich 1, James A Cherry 1, Michael J Baum 1,*
PMCID: PMC2263135  NIHMSID: NIHMS41043  PMID: 16516252

Abstract

The ability of an anesthetized estrous female to induce a conditioned place preference (CPP) response was assessed in male mice from which the vomeronasal organ (VNO) had either been removed (VNOx) or left intact (VNOi) in an initial effort to assess the possible contribution of VNO-accessory olfactory inputs to the intrinsically rewarding properties of opposite-sex body odorants. Both VNOi and VNOx male mice acquired a CPP after repeated pairing of an initially non-preferred test chamber with an anesthetized estrous female mouse, suggesting that odorants detected by the main olfactory system and/or visual and tactile cues from the anesthetized estrous female can compensate for absent VNO inputs to establish a CPP. Subsequent exposure to this conditioning chamber alone caused significant increases in the number of Fos-immunoreactive cells in the mitral and granule cell layers of the accessory olfactory bulb as well as in the medial amygdala and ventral tegmental area of VNOi but not of VNOx males. These results suggest that activity in distal segments of the VNO-accessory olfactory pathway, in addition to the mesolimbic dopamine reward system, can be conditioned to respond to non-odor cues.

Keywords: Conditioned place preference, Accessory olfactory bulb, Ventral tegmental area, c-fos, Pheromones, Anesthetized estrous female

1. Introduction

Environmental odors provide animals with information allowing the identification of food, prey, predators and mating partners. Odor cues from these sources are processed either via the main olfactory system after detection by receptor neurons located on the main olfactory epithelium or via the accessory olfactory system after detection by vomeronasal organ (VNO) receptor neurons [17,42]. Olfactory cues detected by the VNO cause pregnancy block [9], estrous induction [50], puberty acceleration [49] and delay [19,35] in female mice. We [36] found that whereas surgical removal of the VNO (VNOx) did not disrupt the ability of male mice to discriminate sex based on urinary odorants, it did eliminate males’ preference to approach and maintain physical contact with non-volatile urinary odorants from estrous females as opposed to males. More recently, we [37] found in Y-maze studies that male mice with an intact VNO (VNOi) approached more quickly and spent significantly more time in direct nasal contact with estrous female versus male urine whereas VNOx males showed identical responses to these two stimuli. Also, following direct nasal contact with estrous female urine VNOx male subjects had significantly fewer Fos immunoreactive cells than VNOi controls in the nucleus accumbens core region, an important segment of the mesolimbic dopamine reward system [43]. On the presumption that rewarding stimuli elicit and reinforce approach responses and consummatory behaviors [46], these results raise the possibility that VNO inputs communicate information about intrinsic rewarding properties of urinary odorants.

The conditioned place preference (CPP) paradigm [11] has been extensively used to assess the reward value of natural stimuli for male rodents. Such stimuli include sexual interaction with an estrous female [26–28,31,39], ejaculation with a female [1,4], victorious fighting with other males [24], water [2,13,41], sucrose [3,5,41] and food [12,22,38,41]. To our knowledge, the ability of odors from conspecifics to serve as the rewarding stimuli in acquiring a CPP and the role of VNO-accessory olfactory inputs in such learning have not previously been assessed. We hypothesized that direct physical contact with an anesthetized estrous female, a stimulus previously shown to activate AOB mitral cells in male mice [23], would be rewarding for males and that VNO removal would attenuate the development of a CPP to this stimulus.

Midbrain dopamine neurons are activated after ingestion of water or food [45]. Dopamine neurons are also activated by conditioned cues that predict a reward [47] [14], and show graded responses to rewards of different value [48]. Based on this previous research, we hypothesized that the conditioned visual and tactile features of the CPP testing chambers would acquire the ability to predict a reward (i.e., olfactory cues as well as other stimuli derived from contact with an anesthetized estrous female) such that exposure to these conditioned cues might themselves activate segments of the mesolimbic dopamine system, in VNOi but not VNOx males. We therefore assessed the ability of visual and tactile cues from the CPP chamber to augment neuronal Fos-IR in segments of the mesolimbic dopamine reward pathway as well as in the AOB and its projection to the forebrain in VNOi and VNOx males that either had or had not previously acquired a CPP for access to an anesthetized estrous female.

2. Methods

2.1. Subjects

All procedures were approved by the Boston University Animal Care and Use Committee. 39 male and 32 female Swiss Webster (Taconic, Germantown, PA) were purchased at 5–6 weeks of age and were group housed under a reversed 12h light/dark (L/D) photoperiod with food and water provided ad libitum. Gonadally intact male subjects were paired overnight 1–2 times with an ovariectomized female made sexually receptive by s.c. injections of estradiol benzoate (20μg, 48 and 24h before pairing) and progesterone (500μg, 4h before pairing). Subjects in this study were given mating experience in order to maximize the appetitive value assigned to the unconditioned stimulus, an anesthetized estrous female mouse, which was later used to establish a CPP. After sexual experience, subjects underwent either bilateral removal of the VNO or sham surgery, as described in Ref. [36] under ketamine (120mg/kg) and xylazine (12mg/kg) anesthesia. A midline incision was made in the soft palette, and the underlying bone was exposed. In VNOi males, the incision was closed at this point with absorbable sutures. For VNOx males, the VNO was removed, the cavity was packed with gel foam and the incision was closed with absorbable sutures. Animals were closely monitored after surgery for bleeding and/or breathing difficulties and were allowed 1 week to recover before the onset of behavioral testing.

2.2. Experiment 1 — acquisition of a conditioned place preference

2.2.1. Conditioned place preference apparatus

Conditioned place preference tests were carried out using three-chambered wooden boxes. The conditioning chambers (8.5” long×4.5” wide) at either end of the apparatus were distinguished from each other by color and floor texture: one conditioning chamber was painted black and had a smooth rubber floor while the other was painted white and had a roughened Plexiglas floor. The middle neutral chamber (20.5” long×4.5” wide) was painted gray and had a smooth floor. Guillotine doors could be lowered to separate the gray compartment from the two conditioning chambers on either side. Each chamber had a clear Plexiglas top with breathing holes to enclose the animal within the box during tests. The apparatus was cleaned with 70% ethanol between tests for each animal.

2.2.2. Biased conditioned place preference paradigm

Subjects were tested at the same time each day, starting 4h into the dark portion of the 12L:12D cycle. Subjects were transferred to the testing room in their home cages approximately 15min prior to testing. At the end of testing, subjects were placed back into their home cages and transported back to the colony room. Male mice were habituated to the entire testing apparatus (guillotine doors raised) for 15min daily for three days. On the subsequent day subjects’ preference for either the black or white chamber was assessed in a 5min test during which subjects were able to freely explore the entire apparatus while the guillotine doors were raised. The end chamber (black or white) in which each subject spent the greater number of seconds was designated as that subject’s preferred chamber and the other as the non-preferred chamber. Eighty-two percent of all subjects preferred the black chamber during this initial assessment. Conditioning occurred over the next ten days during which the guillotine doors were lowered and subjects were placed on alternate days in either the initially preferred or non-preferred chamber. On days 1, 3, 5, 7, and 9 groups of VNOi and VNOx male subjects were confined for 15min in the initially non-preferred chamber together with an anesthetized estrous female mouse. The stimulus female mice had previously received estradiol benzoate (20μg, 48 and 24h before conditioning trials) and progesterone (500 μg, 4 h before conditioning trials). On days 2, 4, 6, 8, and 10 these same male subjects were confined for 15min in the initially preferred chamber without any stimulus present. An additional group of control VNOi subjects was confined on alternate days in these respective chambers (total of 10days) with no social stimulus ever present. One day after the final conditioning session, subjects were allowed to explore the entire testing apparatus for 5min with the guillotine doors raised to assess any postconditioning shift in their preference to spend time in either the black or white chambers.

2.2.3. Statistical analysis

For each subject, a preference ratio (time spent in the initially non-preferred chamber over the total test time) was calculated for tests given both before and after the conditioning sessions. A two-way repeated measures ANOVA followed by post hoc Student–Newman–Keuls comparisons of pairs of group means were used to assess the effect of VNO removal on the acquisition of a CPP.

2.3. Experiment 2 — forebrain neuronal fos responses to conditioned cues

One day after the post-conditioning assessment of CPP the entire testing apparatus was cleaned with 70% alcohol followed by a hot water wash. The apparatus was allowed to dry and then cleaned again with 70% alcohol with the aim of removing any traces of mouse body odors. Male subjects were exposed for 15min to the initially non-preferred chamber where they had previously been exposed to no stimulus in the case of the VNOi male control subjects or to the anesthetized estrous female in the case of other VNOi as well as VNOx males. Subjects were then returned to their home cages for an additional 75min prior to sacrifice. Mice were sacrificed using carbon dioxide followed by transcardiac perfusion with 0.1M PBS, pH 7.4, followed by 4% paraformaldehyde in 0.1M PBS. Brains were postfixed for 4h in 4% paraformaldehyde, followed by 3days in 30% sucrose in 0.1M PBS at 4°C. Olfactory bulbs were separated from the forebrains, individually frozen in OCT (Tissue–Tek; Miles, Elkhart, IN), and stored at −80°C until sectioning. Both olfactory bulbs were cut sagittally at 30μm using a freezing sledge microtome. Every section from one bulb and every other section from the second bulb were processed for soybean–agglutinin conjugated with horseradish peroxidase (SBA–HRP) staining to verify successful VNO removal [52]. Male subjects that showed no SBA–HRP staining throughout both olfactory bulbs were included in the VNOx group [36]. There were traces of SBA–HRP staining in a majority of olfactory bulb sections from 4 of 15 mice that had received VNO removal surgery; Data from these mice were excluded from the experiment.

The right hemisphere of each forebrain was sectioned coronally at 30μm, and every other section along with the remaining olfactory bulb sections were processed immunohistochemically [16] to visualize Fos protein in neuronal nuclei, which was taken as an index of neuronal activation. Briefly, tissue was pretreated with 7.5% normal goat serum (NGS) in 0.1% Triton X-100/PBS solution for 3h. Tissue was then incubated for 18h at 4°C in primary Fos antibody (sc-52, Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:3000 in 2% NGS in 0.1% Triton X-100/PBS. Tissue was rinsed three times in 1.5% NGS in 0.1% Triton X-100/PBS followed by a 1h incubation in biotinylated goat anti-rabbit immunoglobulin G (Vector Laboratories, Burlington, CA) diluted 1:200 in 2% NGS in 0.1% Triton X-100/PBS. Tissue was rinsed in PBS prior to incubation in avidin–biotin–peroxidase solution (ABC Kit; Vector Laboratories) for 45min. Tissue was rinsed again in PBS followed by reaction with 3,3′-diaminobenzidine with nickel intensification (DAB Kit; Vector Laboratories) for 5min. After a final rinse tissue was mounted on gelatin-coated slides and cover slipped. Slides were coded to conceal the subjects’ treatments prior to further analysis.

Two anatomically matched sections were selected for each subject, and the location of each Fos-immunoreactive (IR) cell in the following brain areas was recorded on paper sheets using a camera lucida microscope attachment: regions receiving either direct or indirect inputs from the VNO including the mitral and granule cell layers of the AOB, the anterior portion (MeA) and posterior dorsal portion (MePD) of the medial amygdala, two portions of the bed nucleus of the stria terminalis (BNST, vBNST), the ventral lateral ventromedial hypothalamus (VMHVL), and the medial preoptic area (MPA) [17]; corticoamygdaloid sites that receive input from the main olfactory bulb including the anterior (ACo) and posterolateral (PLCo) cortical amygdaloid nucleus; areas of the corticolimbic amygdala including the posterior part of the basomedial amygdaloid nucleus (BMP), the anterior (BLA) and posterior (BLP) parts of the basolateral amygdaloid nucleus that have been shown to be involved in vomeronasal–olfactory reward [34]; and areas of the mesolimbic dopamine reward pathway including the nucleus accumbens core (AcbC) and shell (AcbSh), and the ventral tegmental area (VTA). See Fig. 1 for drawings of the area where Fos-IR cells were counted in each of these brain regions. Fos results for all areas were analyzed using one-way ANOVA, and post hoc comparisons of pairs of means were made using Student–Neuman–Keuls tests.

Fig. 1.

Fig. 1

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Drawings modified from the mouse brain atlas of Paxinos and Franklin [40] showing the location of forebrain regions in which Fos-IR cells were counted in Experiment 2. The distance rostral to the interaural line is given in each panel. The standard counting area of 0.1mm2 is shown as a gray circle in each panel. (A1) Accumbens nucleus, core (AcbC); (A2) Accumbens nucleus, shell (AcbSh); (B1) ventral portion, Bed nucleus of the stria terminalis (vBNST); (B2) Medial preoptic area (MPA); (C) Bed nucleus of the stria terminalis (BNST); (D) Medial amygdaloid nucleus, anterior part (MeA); (E) Anterior cortical amygdaloid nucleus (ACo); (F) Posterolateral cortical amygdaloid nucleus (PLCo); (G) Basolateral amygdaloid nucleus, anterior part (BLA); (H) Medial amygdaloid nucleus, posterodorsal part (MePD); (I) Ventromedial hypothalamic nucleus, ventrolateral part (VMHVL); (J) Basomedial amygdaloid nucleus, posterior part (BMP); (K) Basolateral amygdaloid nucleus, posterior part (BLP); (L) Ventral tegmental area (VTA).

3. Results

3.1. Experiment 1 — conditioned place preference

Both VNOi and VNOx male subjects spent significantly more time in the initially non-preferred chamber after repeated pairing of characteristics of that chamber with an anesthetized female, thus showing a significant CPP (Fig. 2). By contrast, other VNOi control males that had been placed alone on alternate days in the black and white chambers of the apparatus showed no CPP. A two-way repeated measures ANOVA revealed a significant effect of group (F(2, 32) =4.380, p < 0.05) and conditioning (F(1, 32) = 14.457, p<0.05), as well as a significant group×conditioning interaction (F(2, 32) =7.245, p<0.05). Post hoc tests revealed significant increases in the preference ratios for both VNOi and VNOx male mice exposed to an anesthetized estrous female, indicating acquisition of a CPP in both instances.

Fig. 2.

Fig. 2

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Effect of bilateral removal of the VNO (VNOx) or sham operation (VNOi) on the acquisition by male mice of a conditioned place preference for a chamber in which they previously had physical access to an anesthetized estrous female or an empty chamber (Nothing). Preference ratios (the number of seconds spent in the initially non-preferred chamber over the total time spent in the entire apparatus) are shown before (pre; open symbols) and after (post; black symbols) conditioning. Values for individual subjects (circles) as well as the group means (rectangles;±SEM) are shown before and after conditioning. *p<0.05, Student–Newman–Keuls post hoc within group comparisons.

3.2. Experiment 2 — forebrain neuronal Fos responses to conditioned visual and tactile stimuli

Exposure to the chamber previously paired with an anesthetized female caused significant increases in the number of Fos-IR AOB mitral and granule cells present in VNOi males when compared to VNOi males previously exposed only to an empty chamber. Following exposure to the chamber previously paired with an anesthetized female, VNOx males had significantly fewer Fos-IR cells than either the conditioned VNOi group or the unconditioned control group in the mitral (F(2, 38) = 13.335, p < 0.001) and granule (F(2, 32) = 11.997, p<0.001) cell layers of the AOB (Fig. 3A–C; Table 1).

Fig. 3.

Fig. 3

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Representative photomicrographs show Fos-immunoreactive cells in sagittal sections of the accessory olfactory bulb (AOB; panels A–C) and coronal sections of the anterior medial amygdala (MeA; panels D–F) and the ventral tegmental area (VTA; panels G–I) of male mice that received a sham removal of the vomeronasal organ (VNOi) or bilateral removal of these organs (VNOx). Panels A, D, and G show minimal Fos responses in the AOB (A), MeA (D), and VTA (G) of VNOi males following exposure to the conditioned place preference (CPP) chamber in which no previous pairing with an estrous female had ever occurred. Panels B, E, and H show maximal Fos responses in the AOB (B), MeA (E), and VTA (H) of VNOi males following exposure to the CPP chamber which had previously been paired with an anesthetized estrous female. Panels C, F, and I show minimal Fos responses in the AOB (C), MeA (F), and VTA (I) of VNOx males following exposure to the CPP chamber which had previously been paired with an anesthetized estrous female. Fos counting areas for the MeA and VTA are shown in all panels. Additional abbreviations: Gl, Glomerular layer of the AOB; Mi, Mitral cell layer of the AOB; Gr, Granule cell layer of the AOB. Scale bars=100μm.

Table 1.

Effect of bilateral vomeronasal organ removal on Fos protein levels in forebrain regions of male mice following exposure to conditioned visual and tactile cues

Conditioning stimulus:
Empty chamber
Anesthetized estrous female
Brain region VNOi
VNOi
VNOx
(n=12) (n=12) (n=11)
Accessory olfactory bulb
 Mitral layer 6±2a 22±3b 3±1c
 Granule layer 13±4a 38±8b 2±1c
Mesolimbic dopamine system
 AcbC 2±1 2±0.4 1±1
 AcbSh 5±1 9±1 5 ±1
 VTA 6±1a 13±2b 6±2a
Amygdaloid reward system
 BMP 2±1 3±1 3±1
 BLA 3±1 7±2 7±2
 BLP 2±1 3±1 3±1
Vomeronasal projection pathway
 MeA 3±1a 6±1b 3±1a
 MePD 2±0.4a 5±1b 2±0.3a
 BNST 2±0.4 3±1 2±01
 vBNST 4±1 5±1 4±1
 VMHVL 3±1 4±1 6±2
 MPA 5±1 5±1 5±1
Amygdaloid targets of main olfactory input
 Aco 4±1a 10±2b 8±2ab
 PLCo 5±1a 12±2b 7±2a
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Data are expressed as the mean (±SEM) number of Fos-IR cells/standard area. VNOi=sham-operated mice with an intact vomeronasal organ; VNOx=mice from which the vomeronasal organ was bilaterally removed. Means with different superscript letters (a, b, c) differed significantly from each other (p<.05) by Fisher’s least significant difference post hoc tests following a significant overall ANOVA.

After exposure to the chamber previously paired with the anesthetized female, VNOi males had significantly more Fos-IR cells in the VTA (the segment of the mesolimbic dopamine system that contains dopamine neurons and sends projections to the nucleus accumbens) than unconditioned VNOi controls; however, this conditioning effect was absent in VNOx males (F(2, 32) =6.191, p<0.05; Fig. 3 G–I; Table 1). There was a statistically non-significant trend in VNOi males, but not in VNOx males, for exposure to the chamber previously paired with a female to stimulate Fos-IR in the AcbSH (F(2, 32) =2.943, p=0.067; Table 1). Areas that are part of the vomeronasal projection pathway, including the MeA (F(2, 32) = 7.020, p<0.05; Fig. 3D–F; Table 1) and the MePD (F(2, 32) =7.475, p<0.05; Table 1) also showed significant increases in Fos-IR cells in conditioned VNOi males when compared with conditioned VNOx males and non-conditioned VNOi controls. Conditioned VNOi males showed a significant increase in the number of Fos-IR cells as compared to unconditioned control VNOi males in the ACo (F(2, 32) =4.846, p < 0.05) and PLCo (F(2, 32) =5.545, p <0.05), both targets of input from the main olfactory bulb. Post hoc tests showed that conditioned VNOx males had Fos values that fell between those of conditioned VNOi males and unconditioned VNOi controls in the ACo while in the PLCo conditioned VNOx males had significantly fewer Fos-IR cells than conditioned VNOi males.

4. Discussion

Repeated pairing of odor, visual and tactile cues from an anesthetized estrous female with the visual and tactile characteristics of an initially non-preferred chamber led to the acquisition of a significant CPP in both VNOi and VNOx male mice. Previous studies using social stimuli in CPP tests have found that male rats allowed to intromit [18,26,31] and/or ejaculate [1,4] with a female acquired a CPP. No CPP was established in male rats that were either prevented from intromitting with an estrous female [18] or from controlling the rate of intromission with the female [10]. In the present study using male mice the use of an anesthetized female as the rewarding stimulus eliminated any sensory information that male mice might have received from mating—leaving only odor, visual and tactile stimuli from the female as potential rewarding cues. When contrasted with the empty test chamber presented on alternate days, these cues were sufficiently rewarding to both VNOi and VNOx male mice to establish a significant CPP. The specific role that olfactory cues (detected and processed via either the main or accessory olfactory systems) played in this learning is unclear because visual and tactile cues from the stimulus female may by themselves have motivated male subjects to acquire the CPP. A recent study from our laboratories (C. Bann and J.A. Cherry, unpublished data) showed that male mice acquired a CPP to estrous female urinary odors, provided they had direct physical access to the stimulus. Additional studies are needed to determine whether VNO inputs are required in order for non-volatile female urinary odors to motivate successful CPP learning in male mice.

Unexpectedly, in the absence of conspecific body odors, conditioned visual and tactile stimuli previously associated with access to an anesthetized estrous female caused significant increases in the number of Fos-IR mitral and granule cells in the AOB, provided the VNO was intact. No such Fos responses occurred in non-conditioned VNOi control males that were exposed to visual and tactile cues not previously associated with an estrous female. One possible explanation for the lack of Fos responses in the AOB of conditioned VNOx male mice is that VNO removal caused cell death in AOB mitral and granule cells due to a chronic lack of synaptic input from VNO sensory neurons. This seems unlikely for two reasons. First, mating with an estrous female induced Fos-IR in the AOB mitral cell layer of male rats 7–10 days after VNO removal [21]. Thus a precedent exists for stimulus-induced Fos expression in the AOB mitral cell layer in the absence of VNO inputs. Second, after cresyl violet staining we (D.E. Pankevich and M.J. Baum, unpublished findings) observed that the number and location of AOB mitral and granule cells appeared to be very similar in VNOi and VNOx male mice killed 2 weeks postoperatively. Two other possible explanations for the observed activation of AOB mitral and granule cells in VNOi males by conditioned, non-olfactory stimuli follow: First, the conditioned stimuli may have acquired the ability to activate the VNO ‘pumping’ mechanism [29,30] that normally delivers mucous-bound odorants to the VNO neuroepithelium. It is possible, though not yet proven, that activation of the pump augments the activity of VNO sensory neurons which in turn activates mitral and granule cells in the AOB. A second alternative that we find more compelling is that conditioned activation of centrifugal inputs to the AOB activated these cells. The posteromedial cortical amygdala (PMCo) extends a projection that terminates in the internal granule cell layer of the mouse AOB while the bed nucleus of the accessory olfactory tract (bnAOT) and the medial amygdala (MeA) send projections to the internal plexiform layer of the AOB [6]. An additional centrifugal input to the AOB originates in the locus coeruleus, where noradrenergic neurons send dense projections to the external plexiform layer, granule cell layer and the internal part of the mitral cell layer of the AOB [25]. Any of these centrifugal pathways may have mediated the conditioned activation of the AOB observed in the present study. It is not obvious, however, why removal of the VNO would have blocked the ability of centrifugal inputs to activate AOB mitral and granule cells.

Exposure to a conditioned, reward-predicting environment stimulated Fos-IR in the ventral tegmental area (VTA) of VNOi but not VNOx male mice. The VTA Fos response of these latter subjects resembled the low levels of Fos seen in unconditioned VNOi control subjects. Activation of dopamine neurons in the VTA and the resultant activation of post-synaptic nucleus accumbens neurons has been linked to both natural reward [7,51] and its prediction by conditioned stimuli [45]. Likewise, exposure to an environment that had previously been paired with ingestion of either cocaine [15] or morphine [44] caused significant increases in Fos cell number in the nucleus accumbens, among other brain structures. In another study [32] application of K+ to the vomeronasal nerve layer and accessory olfactory bulb stimulated dopamine release in the nucleus accumbens of male rats, and this same group [33] reported that repeated exposure to estrous bedding progressively augmented the level of odor-induced dopamine release in the nucleus accumbens of male rats. These previous studies along with the present profile of conditioned Fos responses in the VTA suggest that during acquisition of a CPP olfactory stimuli detected by the VNO and processed by the accessory olfactory system provided sufficient information to encode the incentive value of the stimulus, an anesthetized estrous female. Obviously, however, such inputs were not absolutely required in order for mice to acquire such a CPP in that such learning persisted in VNOx subjects.

It is noteworthy that we observed only a non-significant trend for the conditioned stimuli to augment Fos in the nucleus accumbens (i.e., AcbSh) of VNOi male mice. In previous studies using male rats [8,20] direct contact with estrous female bedding stimulated Fos expression in the nucleus accumbens core and/or shell. Likewise, in a recent study using male mice [37] nasal contact with estrous urinary odors stimulated Fos in the nucleus accumbens core, provided the VNO was intact. Apparently non-olfactory conditioned stimuli, by themselves, cannot duplicate the activational effects of naturally rewarding odor stimuli (estrous female urinary odors) on the nucleus accumbens. A similar discrepancy exists in the ability of conditioned vs. naturally rewarding stimuli to stimulate Fos responses in the BNST. Thus in previous studies exposure to male soiled bedding [16] or to nasally applied estrous female urine [37] augmented Fos in the BNST whereas in the present study conditioned stimuli associated with an anesthetized female failed to do so. Note that in our previous study [37] and in the present experiment urinary odors and conditioned stimuli, respectively, activated Fos expression in AOB mitral cells as well as in the MeA and MePD, and these responses were blocked by VNO removal. The BNST receives direct inputs from AOB mitral cells as well downstream inputs from projection neurons in the medial amygdala [17]. More research is needed to understand these discrepancies in the ability of intrinsically rewarding versus conditioned stimuli to activate Fos expression in more proximal segments of the VNO projection pathway as well as in the target regions of the mesolimbic dopamine pathway.

Acknowledgments

This research was supported by NIH grant MH59200 awarded to James Cherry. We thank Esther Kim for technical assistance.

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