For this study we used 66 female Wistar rats, aged more than 12 weeks of age. To obtain chemically naïve female rats, females were reared in the absence of mature males or their derived chemicals signals (Moncho-Bogani et al., 2002). Briefly, we housed pregnant females in a room without male rats, sexed the litters and separated the male siblings 19 days after delivery, early before puberty. Experimental females were housed in the same room without males, so they were both sexually and chemically inexperienced. A previous study in mice showed that ovariectomized females treated with either oil, estradiol or estradiol + progesterone displayed similar levels of innate attraction toward male chemosignals, while showing the expected differences in receptivity to a stud male (Moncho-Bogani et al., 2004). Thus, we deemed unnecessary to track the phase of the estral cycle of the rats.

Rats were housed in plastic cages (48 cm × 38 cm × 21 cm) in groups of four to six, with controlled humidity and temperature (22°C), a 12:12-h light/dark cycle, and water and food available ad libitum. All the procedures were carried out in strict accordance with the EEC Council Directive 86/609, Spanish laws (RD 53/2013) and animal protection policies. The protocols were approved by the Animal Care Committee of the Faculty of Pharmacy at the University of Valencia, Spain.

The irreversible antagonist of the μ-opioid receptor, β-funaltrexamine, and the broad-spectrum antagonist of the opioid receptors, naltrexone, were obtained from Tocris (Bristol, UK). Kynurenic acid, an antagonist of NMDA, AMPA and kainate glutamate receptors, was obtained from Sigma–Aldrich Co. Stock solutions of the drugs were prepared by dissolving the compound in the proper volume of distilled water. These solutions were aliquoted and kept frozen at -40°C until use. Prior to use, aliquots of the stock solutions were conveniently diluted in artificial cerebrospinal fluid solution (aCSF) (Sánchez-Catalán et al., 2009).

Rats were anesthetized with 95 mg/kg of ketamine plus 10 mg/kg of xylazine intraperitoneally (i.p.) and placed in a stereotaxic apparatus (Stoelting, USA). An incision (8–10 mm) was made in the skin above the skull and the wound margin was infiltrated with lidocaine (3%).

Animals for Experiment 1b were implanted unilaterally with a 28-gage guide cannula (Plastics One, USA) aimed at 1.0 mm above the pVTA. A stainless steel stylet (33-gauge) extending 1.0 mm beyond the tip of the guide cannula was introduced at the time of surgery and removed at the time of testing. After surgery, rats were left to recover for 3 days before the experiment. For Experiments 2 and 3, animals were implanted with one concentric microdialysis probe with 2 mm of permeable membrane (Hospal, AN69) in the AcbC (Experiment 2) or the AcbSh (Experiment 3). The brain coordinates related to bregma and skull surface were: pVTA, A/P: -6.0 mm, L: -2.1 mm, V: 7.9 mm (10° from the vertical midline); AcbC, A/P: +1.3 mm, L: -1.4 mm, V: -8.1 mm; AcbSh, A/P: +1.3 mm, L: -0.8 mm, V: -8.3 mm, according to Paxinos and Watson (2007).

Dialysis experiments were performed 24 h after surgery and rats were used for only one experiment. The use of this recovery period was shown to be sufficient in several previous published studies (Santiago and Westerink, 1990; Santiago et al., 2000; Hipólito et al., 2008, 2009a,b). PE10 inlet tubing was attached to a 2.5 mL syringe (Hamilton), mounted on a syringe pump (Harvard Instruments, South Natick, MA, USA) and connected to the dialysis probes that were perfused at 3.5 μL/min with aCSF solution. Fractions of dialysate were on-line analyzed for DA content every 20 min using an HPLC system with electrochemical detection, as previously described (Hipólito et al., 2008). The HPLC system consisted of a Waters 510 series pump in conjunction with an electrochemical detector (Mod. Intro, Antec, Leyden, The Netherlands). The applied potential was +0.55V (vs. Ag/AgCl). Dialysates were injected onto a 5 mm RP-18 column (LiChroCART 125-4, Merck, Darmstadt, Germany) via a VALCO valve fitted with a 65 μL sample loop. The mobile phase consisted of a sodium acetate/acetic acid buffer (Hipólito et al., 2008), which was pumped through the column at a flow rate of 0.2 mL/min. Chromatograms were integrated and compared with separately run standards on each experimental day, using the AZUR 4.2 software (Datalys, France). Detection limit was defined by a signal to noise ratio of 2:1, being approximately 6 fmol/sample.

Experiments were recorded using a video camera. For Experiment 1, we automatically measured the time that females spent in a defined area (6 cm × 5.5 cm) by means of the video tracking software Raddot (University of Valencia, Spain), during the 5 min of the test. Furthermore, an experimenter who was blind to the treatment of the animal and type of bedding measured the time that the animal spent digging on the bedding contained on the dish using a stopwatch. Previous data showed that female mice spent a significantly longer proportion of time digging on male-soiled bedding than in female-soiled bedding (Agustín-Pavón et al., 2007). Thus, digging was taken as an approximate measure of the attractive value of the stimulus for the animal. For Experiment 3, a person who was unaware of the treatment recorded by means of a stopwatch the time in seconds that each rat spent investigating the dish, i.e., the time that females spent sniffing and digging on the male-soiled bedding, as a measure of exposure to the olfactory and vomeronasal cues contained in the it.

At the end of the experiments, animals were deeply anesthetized and killed by decapitation. Brains were quickly removed, frozen in isopentane and cut in a cryostat into 40 μm thick coronal sections. The slices were mounted, stained with cresyl violet and evaluated histologically to confirm the position of the cannula tips and the microdialysis probes. Only rats with the cannula tip or probe correctly placed were included in the statistical analysis. The position of the tips of the cannulae and microdialysis probes is depicted in Figures 1E, 2A, 3A, and 4A. Representative samples of Nissl-stained coronal sections are provided in Supplementary Figure 1.

Data are represented as mean ± SEM. In Experiment 1, we analyzed the time that females spent in the defined area around the dishes and digging on the bedding in seconds by means of Student’s t-tests. In Experiments 2 and 3, the level of DA was expressed as percentage of baseline, defined as 100% DA concentration in the Acb. The effects of treatments and bedding exposure on DA levels were analyzed through a mixed two-way analysis of variance (ANOVA) of repeated measures, with time as within-subject factor and treatment as between-subject factor. This analysis was followed by Dunnet’s post hoc test to identify the time points that differed significantly from the respective baseline (third baseline time point). Significant time × treatment interactions were analyzed by post hoc analyses with the Bonferroni correction when appropriate. Areas under the curve (AUC) for DA change (%) were calculated from 60 to 220 min and analyzed by means of a Student’s t-test. The level of significance was set at p < 0.05. All the analyses were performed using SPSS, v. 15.0 (SPSS, Inc., Chicago, IL, USA).

Our data from Experiment 1a shows that chemically naïve female rats prefer to investigate male- to female-soiled bedding in a two-choice test, suggesting that females display an innate attraction for male chemosignals. Thus, the time that females spent around both dishes containing female-soiled bedding was identical in the control (p = 0.92), whereas females spent significantly more time around male-soiled bedding than around female-soiled bedding in the male preference test (p = 0.026) (Figure 1A). In addition, rats spent more time digging on male-soiled bedding as compared to female-soiled bedding (p = 0.0009) (Figure 1B). Moreover, the proportion of time digging on the bedding with respect to the time spent in each zone was significantly higher for male-soiled bedding (female-soiled bedding = 4.16 ± 1.27%, male-soiled bedding = 15 ± 2.8%, p = 0.003).

For Experiment 1B, we first evaluated the cannulae placements and animals with the tip of the cannula in the pVTA were included in the statistical analysis (Figure 1E). Female rats treated with β-funaltrexamine spent more time in the male zone (p = 0.044, Figure 1C) and digging on male-soiled bedding (p = 0.02, Figure 1D) than in the female zone, whereas no differences were observed in the control test, neither in time spent in the zone nor in digging (p > 0.1 in both cases) (Figures 1C,D). Moreover, rats treated with β-funaltrexamine displayed similar percentages of digging on the bedding than non-treated rats of Experiment 1A, and the percentage of digging on male-soiled bedding was significantly higher than on female-soiled bedding (female = 4.90 ± 2.15%, male = 14.14 ± 2.88%, p = 0.02). Thus, intra-pVTA microinjection β-funaltrexamine did not affect the attraction of female rats for male chemosignals, suggesting that μ-opioid receptors in the pVTA are not involved in the expression of this innate behavior.

Following histological evaluation, animals with correct microdialysis probe placement were included for analysis (Figures 2A and 3A). The statistical analysis of the data revealed that exposure to a new dish with clean bedding elicited a mild increase in DA efflux in the AcbC with respect to baseline (Figure 2B, filled symbols). The administration of naltrexone did not affect DA levels after the introduction of a new dish with clean bedding, since the analysis revealed no differences for treatment [F_(1,12) = 0.146, p = 0.709], or the interaction time × treatment [F_(10,120) = 1.434, p = 0.178]. The effect of the new dish was reflected in a significant effect of main factor time [F_(10,120) = 8.258, p < 0.001]. Thus, DA efflux peaked with a 20% increase over baseline 180 min after the onset of the experiment, i.e., 100 min after the first manipulation of the dish (Figure 2B). Finally, the comparison of AUCs of DA change revealed no significant differences between vehicle and naltrexone-treated animals after exposure to clean bedding (Figure 2C).

Male-soiled bedding evoked a significant increase of DA in the AcbC (Figure 3B). The mixed two-way ANOVA revealed statistically significant main effects of time [F_(10,110) = 46.955, p < 0.001] and treatment [F_(1,11) = 8.349, p = 0.015], as well as a significant interaction [time × treatment, F_(10,110) = 5.759, p < 0.001]. In the vehicle-treated animals, DA efflux was significantly increased with respect to baseline 20 min after the introduction of the dish containing male-soiled bedding, and peaked with a 33% increase over baseline 160 min after the onset of the experiment, i.e., 80 min after the introduction of the male-soiled bedding.

We also compared the increase in DA efflux after clean bedding and male soiled bedding exposure in the vehicle-treated groups. The ANOVA revealed a significant effect of the type of bedding, showing that DA levels were significantly higher after the introduction of male-soiled bedding than after the introduction of a new dish with clean bedding [F_(1,12) = 6.421, p = 0.026, compare Figures 2B and 3B].

Furthermore, we explored the differences between vehicle and naltrexone-treated groups exposed to male-soiled bedding. A post hoc comparison revealed that DA level in the naltrexone-treated group was higher than in vehicle-treated animals from minute 160 after the onset of the experiment, and this higher level was maintained until the end of the experiment, when DA level peaked in the naltrexone-treated animals with an increase of 66% with respect to baseline (at minute 220, Figure 3B). Moreover, the comparison of AUC’s of dopamine change showed that the percentage DA change in the AcbC following male-soiled bedding exposure was higher in the naltrexone-treated animals than in the vehicle group (p = 0.014) (Figure 3C).

In this experiment, we investigated the possible changes in DA efflux in the AcbSh and its regulation by glutamate receptors. Animals with correct probe placement were included in the analysis (Figure 4A). The ANOVA revealed a significant effect of the factor treatment [F_(1,10) = 16.345, p = 0.002] and the interaction between time × treatment [F_(10,100) = 7.34, p < 0.001], but no significant effect of time [F_(10,100) = 0.597, p = 0.813] (Figure 4B). Post hoc analysis revealed that the exposure to male-soiled bedding increased DA levels over baseline 20 min after the onset of the exposure to bedding in vehicle-treated animals, but returned to baseline before the end of the experiment, 100 min after the onset of exposure to male bedding. By contrast, the treatment with kynurenic acid blocked the DA response and reduced DA levels in the AcbSh (Figure 4B). This difference between treatments was additionally confirmed following the comparison of AUC’s of DA change (p < 0.001) (Figure 4C).

Since kynurenic acid decreased the dopaminergic response in the AcbSh to male chemosignals, we wondered whether this drug had some behavioral effect. To check this, we measured the time that females spent investigating the dish containing male-soiled bedding. A Student’s t-test revealed that females treated with kynurenic acid spent significantly less time investigating male bedding than females treated with vehicle (Figure 4D). To investigate the dynamics of this reduction, we divided the 40 min of exposure in eight slots of 5 min, and compared these slots between groups. An ANOVA for repeated measures using time slot as within-subject factor revealed a significant main effect of this factor (F_7,4 = 60.860, p = 0.001) and also of the between-subject factor group (F_1,10 = 0.025) as well as a significant effect of the interaction time × group (F_7,4 = 12.704, p = 0.014). Pairwise comparisons revealed that time spent investigating the male bedding was not significantly different between groups during the first slot (vehicle-treated group, 94.2 ± 12.7 s; kynurenic acid-treated group, 81.6 ± 21.2 s, p = 0.623), but became significantly lower in the kynurenic acid-treated animals during the second slot (vehicle-treated group, 99.3 ± 15.9 s; kynurenic acid-treated group, 45.6 ± 17.4 s, p = 0.046). Further, time spent investigating by the animals in the vehicle group was similar across the first 35 min, and it declined to 0 only during the last 5 min of exposure. By contrast, the time spent investigating the bedding in the kynurenic acid group declined rapidly, so it was 0 during the last 15 min.

Sexual behavior is under strict hormonal control in female rodents (Giuliano et al., 2010). However, previous studies showed that both freely cycling and ovariectomized, steroid- or oil-treated female mice, reared in complete absence of male mice, prefer investigating male-soiled bedding (Moncho-Bogani et al., 2002, 2004). These results suggest that intersexual attraction mediated by male chemosignals is innate and might be independent on the hormonal status in female mice. Furthermore, male-soiled bedding was effective as a reinforcer to induce conditioned place preference in freely cycling female mice (Agustín-Pavón et al., 2007; Martínez-Ricós et al., 2007). In the present study, we extend this observation to female rats, showing that chemically naïve, freely cycling adult females innately prefer investigating male chemosignals. This preferential investigation is unlikely to be due to a novelty effect, since a novel neutral odor did not induce preferential investigation in female mice using this protocol (Martínez-Ricós et al., 2007). Moreover, preference for male bedding was persistent for 4 consecutive days, whereas preference for castrated male or female chemosignals disappeared with repeated testing (Martínez-Ricós et al., 2007). Further experiments, however, should be carried out to investigate whether, in female rats, preference for male chemosignals is persistent in consecutive tests.

Sexual receptivity and intersexual attraction mediated by chemosignals seem to be under different regulatory mechanisms. In fact, ovariectomized female mice treated with vehicle or progesterone displayed similar levels of preference for male chemosignals than ovariectomized females primed with estradiol or estradiol + progesterone, although only the latter were receptive in direct encounters with a male, whereas vehicle and progesterone-treated females displayed high levels of refusal behavior (Moncho-Bogani et al., 2004). In this sense, male sexual pheromones can promote effective tracking of a sexual partner enhancing the probability of copulation in the short period of ovulation. In fact, sexual maturation and ovulation are induced by male pheromones in mice (Vandenbergh, 1969), giving adaptive value to the fact that females are attracted by male pheromones independently on their endocrine status. However, other studies do not fully support these results, since progesterone might be inhibitory for pheromone attraction (Dey et al., 2015). It is likely that hormonal regulation is less important in inexperienced females, since both in the present study and in the studies by Moncho-Bogani et al. (2002, 2004) and Martínez-Ricós et al. (2007), females were confronted for the first time with male odors, whereas in the study by Dey et al. (2015) it was not disclosed whether females were raised in the absence of males. Thus, the phase of estral cycle might have an impact in sexually experienced females, but not in our inexperienced females. Further experiments are necessary to test this possibility.

Another key difference relies on the stimuli that were used: the studies showing independency on the hormonal status, including the present one, used male-soiled bedding, containing a wide variety of chemosignals, whereas the study showing the inhibitory effect of progesterone used male urinary proteins (MUPs). Although MUPs alone are sufficient to elicit strong attraction in mice (Roberts et al., 2010), and female rats find those males which excrete a higher proportion of MUPs more attractive (Kumar et al., 2014), other chemosignals may contribute to modulate behavioral preference.

Anyhow, MUPs are detected by the vomeronasal system (Papes et al., 2010; Chamero et al., 2011; Kaur et al., 2014), which is necessary for the display of innate attraction toward male chemosignals, at least in female mice (Martínez-Ricós et al., 2008). The detection of vomeronasal stimuli requires specific behaviors, such as tongue-flick in snakes (Martínez-Marcos et al., 2001) or nuzzling in opossums (Poran et al., 1993), elements that have not been described in mice or rats. In this respect, we found that females dig on male-soiled bedding a significantly higher proportion of the time they spent in the vicinity of male bedding than in female bedding –in particular, the percentage of time females devoted to digging on male-soiled bedding was almost four times higher than the percentage of time digging on female bedding. This result is in agreement with a previous study in mice, showing that the proportion of time that females devoted to digging on male-soiled with respect to total investigation was twofold of the proportion they spent digging on female-soiled bedding (Agustín-Pavón et al., 2007). We hypothesize that digging might be related to the searching and detection of non-volatile, vomeronasal-detected pheromones, since the snout is in the closest contact with the substrate when the animal is digging on it. Further, sniffing opposite-sex chemosignals might be viewed as a reward-seeking behavior (Agustín-Pavón et al., 2007; Malkesman et al., 2010), to which the contribution of the mesolimbic dopaminergic system has been largely known (Salamone and Correa, 2012).

Vomeronasal information reaches the Acb through sparse projections from the posteromedial cortical amygdala (Ubeda-Bañon et al., 2008; Gutiérrez-Castellanos et al., 2014). In addition, olfactory and vomeronasal information are conveyed to the Acb through conspicuous afferents from the BLA (Novejarque et al., 2011). Finally, sparse projections from the medial amygdala reach both the Acb and VTA (Pardo-Bellver et al., 2012).

Our results show that blocking μ receptors in the pVTA with the irreversible antagonist β-funaltrexamine does not affect preference for male-soiled bedding. Systemic treatment with the opioid antagonist naloxone did not affect the behavioral preference for male chemosignals in female mice (Agustín-Pavón et al., 2008), a result that is in agreement with our data showing that local application of an opioidergic antagonist does not affect the behavioral preference of female rats. The use of unilateral injections of β-funaltrexamine in the pVTA was effective in previous studies in blocking the locomotor-stimulant effects of ethanol and its metabolites/derivatives, while leaving basal locomotion unaffected (Sánchez-Catalán et al., 2009; Hipólito et al., 2010). Moreover, preliminary data in our laboratory have shown that the microinjection of β-funaltrexamine attenuates the stimulating effects of DAMGO, a μ-opioid agonist, into the pVTA throughout 1 week (unpublished results). Likewise, the pretreatment with this antagonist has also been shown to attenuate the effects of several drugs of abuse, remaining this effect for up 3–6 days, depending on the administered site (Ward et al., 2003; Martin et al., 2008). Thus, it could be assumed that the lack of effect shown in the present results reflects a lack of involvement of the opioidergic modulation in the pVTA in the preference for male chemosignals. In fact, innate attraction toward male sexual pheromones is independent from the integrity of the dopaminergic neurons of the VTA in female mice (Martínez-Hernández et al., 2006) and VTA is not activated by the first exposure to male chemosignals as measured by Fos in a study (Moncho-Bogani et al., 2005). We thus hypothesize that pheromonal information might be able to bypass the VTA and be conveyed directly to the Acb via amygdaloid projections (Figure 5). However, DiBenedictis et al. (2015) recently showed that male chemosignals, but not female chemosignals, induce c-Fos in the VTA of female mice. Thus, further experiments are needed to reassess the role of the VTA in the preference for male chemosignals.

Our results extend to the female rat an older observation made in males showing an increased DA signal in the AcbC of male rats exposed for 20 min to estrous female odors, but not odors from ovariectomized females or males (Mitchell and Gratton, 1991). A close analysis of the results of Mitchell and Gratton (1991) reveals that the increase in DA efflux quickly returned to baseline 5 min after the termination of the exposure, whereas in our study the DA levels did not return to baseline before termination of the experiment in the AcbC. This difference might be related to the technique, since in the study by Mitchell and Gratton they used chronoamperometry, detecting fast, phasic DA release, whereas in our study we employed microdialysis, which allows measuring more sustained changes in the neurotransmitter content.

Male chemosignals were able to induce an increase in DA efflux in both divisions of the Acb of female rats, although we found a different time course of the dopaminergic response. Thus, DA efflux in the AcbC was significantly higher than baseline already 20 min after the exposure to male chemosignals and remained higher than baseline for the whole experiment, 100 min after the male chemosignals were removed. By contrast, the increase in DA levels in the AcbSh returned to baseline 90 min after the removal of the male chemosignals. Although the maximum increase in DA efflux was similar in the AcbC and AcbSh, i.e., around 30% with respect to baseline (see Supplementary Figure 2), the more sustained effect in the AcbC might be related to conditioning processes, whereas the increase in AcbSh could be related to novelty and consummatory responses, as suggested by previous evidence (Bassareo et al., 2002; Cacciapaglia et al., 2012). It should be noted that presenting a new dish with clean bedding also produced an increase in DA release in the AcbC, but this increase was significantly lower than the increase induced by male chemosignals. In addition, it is well established that not only reinforcing, but also aversive and novel stimuli, induce DA efflux in the Acb (Rebec et al., 1997; Horvitz, 2000; McCutcheon et al., 2012).

In this complex panorama, dopaminergic activity in the mesolimbic pathway has been linked to learning through prediction of the rewarding outcome (Schultz, 2002), to the signaling of the incentive motivational properties of reinforcing stimuli (Berridge and Robinson, 1998) and to behavioral activation (Salamone and Correa, 2012). Although it is out of the scope of this study to contribute to the debate on the role of DA to these processes, it could be speculated that the release of DA in the Acb elicited by male chemosignals could be involved in the motivational process enabling pheromone-seeking behavior. If this were the case, blocking DA transmission might blunt the preference for male chemosignals. Previous studies seem contradictory on that point. On the one hand, systemic DA antagonists did not affect the preference of female mice for male-soiled bedding (Agustín-Pavón et al., 2007), whereas DA agonists induced a decrease in preference for unreachable opposite-sex subjects in female rats and male mice (Landauer and Balster, 1982; Ellingsen and Ågmo, 2004). On the other hand, selective 6-OHDA lesions of the anteromedial Acb plus OT disrupted the preference of female mice for male chemosignals (DiBenedictis et al., 2014), although similar lesions failed to affect this preference (Martínez-Hernández et al., 2012). This latter discrepancy might be related to differences in the protocol used, since in the study by Martínez-Hernández et al. (2012) the control stimulus was clean bedding, a stimulus with low incentive value, whereas in the study by DiBenedictis et al. (2014), they used female chemosignals as control. If, on the other hand, DA is related to the effort that an animal has to put to obtain the reinforcing stimulus, the effect of manipulation of the dopaminergic system would only be discovered in tests requiring an effort to obtain the reinforcing chemosignals, which is not the case in our tests, where male chemosignals are readily available. To investigate these possibilities, it would be interesting to carry out future experiments exploring whether the release of DA is correlated with preference or instrumental responses directed to obtain the reinforcing chemosignals.

A final question that we sought to address in our study was related to the pharmacological modulation of the dopaminergic response to male chemosignals. Our results show that locally blocking opioid receptors in the AcbC by naltrexone resulted in a higher increase of DA in AcbC 40 min after termination of the exposure to male chemosignals until the end of the experiment, i.e., the levels of DA were not affected by naltrexone during and immediately after exposure to male-soiled bedding. This delayed effect of naltrexone might be related to the pharmacological profile of this drug, which is both a μ- and κ-opioid receptor antagonist. In this sense, it has been shown that κ-opioid inhibition increases DA efflux in the Acb (Spanagel et al., 1992); therefore, the delayed increase in DA levels might be caused by the pharmacological action of naltrexone over κ-opioid receptors, which would act further disinhibiting the significant rise in DA levels provoked by male chemosignals. By contrast, DA efflux during exposure to clean bedding was unaffected by naltrexone. It is likely that we did not observe this disinhibition due to a more moderate increase in DA efflux elicited by clean bedding.

This result fits into a scenario in which inhibitory opioidergic receptors might be located presynaptically in the excitatory afferents promoting DA efflux and/or in the dopaminergic terminals, so the administration of an opioidergic antagonist would prevent an opioid-mediated termination of the DA release induced by male chemosignals. Since naltrexone failed to affect the DA efflux during the exposure to male-soiled bedding, it is likely that the opioidergic modulation takes place way after the initial increase in DA efflux elicited by the sensory stimulus, to help returning the DA levels to baseline.

To the best of our knowledge, there are only scarce detailed descriptions of the distribution of opioid receptors in AcbC to support this hypothesis. Regarding this, μ-opioid receptors have been described in axon terminals in the AcbSh, providing a possible site of action for naltrexone in disinhibiting the DA efflux. However, previous results showed that activation of opioidergic receptors in AcbC enhances, rather than decreases, extracellular levels of DA, presumably by activation of presynaptic receptors on local inhibitory neurons in the Acb, or by inhibition of GABA projection neurons to the VTA (Hipólito et al., 2008). It should be noted that in that latter study, DA levels were measured in response to an application of agonists of μ- and δ-opioid receptors, whereas in the present study we measured DA efflux after olfactory stimulation. Further studies characterizing in detail the location of opioid receptors in the AcbC are needed to provide anatomical grounds to our result. Finally, our result seems contradictory with the study by Mitchell and Gratton (1991) in male rats showing that systemic treatment with the opioidergic antagonist naloxone attenuated the dopaminergic release in the AcbC induced by female odors. This discrepancy might be related to the site of action of opiates, which is ubiquitous and cannot be determined by using a systemic approach.

As we pointed out above, the increase of DA efflux in the Acb of female rats elicited by male chemosignals might be due to the activity of glutamatergic projections from the amygdala conveying olfactory and vomeronasal information to the Acb rather than to the activity of VTA cells. In fact, afferent activity from the BLA increases DA efflux in the Acb (Floresco et al., 2001a), even in the absence of activation of the VTA neurons, e.g., after inactivation of VTA with lidocaine (Howland et al., 2002). If this were the mechanism by which DA is released by exposure to sexual chemosignals, then pre-synaptic opioid receptors in the glutamatergic afferents could blunt the dopaminergic response and, conversely, antagonists could enhance the DA efflux. In addition, the regulation of DA efflux in the Acb by glutamate is dependent on NMDA receptors (Floresco et al., 2001a; Howland et al., 2002), which are located in presynaptic TH-positive axons (Gracy and Pickel, 1996). Thus, we expected that the administration of kynurenic acid, an antagonist of this type of receptors, would blunt the DA increase. In agreement with our hypothesis, kynurenic acid completely blocked the release of DA elicited by male chemosignals, and even lowered the DA levels with respect to baseline. Further experiments are necessary to test the validity of our hypothesis and to investigate whether activation of the glutamatergic projections from the vomeronasal amygdala are able of to elicit the same DA response in the absence of VTA stimulation.

Finally, kynurenic acid administration in AcbSh via retrodyalisis decreased the overall time spent by female rats exploring male-soiled bedding as compared to vehicle-treated females. Strikingly, the investigation of male bedding was unaffected by the treatment for the first 5 min of the test. Although the two-choice tests and the microdialysis experiments are not comparable, it should be noted that those first 5 min represent the same time window for both type of experiments, when the rats are faced for the first time with male chemosignals. Thus, we wonder whether the initial preference response to male chemosignals is independent on dopaminergic signaling, a possibility which would fit the lack of effect of dopaminergic antagonists on preference for sexual chemosignals reported by Agustín-Pavón et al. (2007) and of VTA and Acb lesions reported by Martínez-Hernández et al. (2006, 2012), but would contradict the results by DiBenedictis et al. (2014). In this scenario, DA efflux to the Acb might be more related to persistent pheromone-seeking behavior than to innate preference for male chemosignals. In fact, kynurenic acid decreased investigation of male chemosignals after the initial 5 min of exposure.

However, an alternative explanation might be that kynurenic acid would reduce the investigation of chemosignals acting over glutamatergic receptors in striatal neurons. Given that DA efflux seems to be caused by chemosignal exposure, then blocking the exposure would prevent the increase in DA levels. To test these possibilities, further experiments should check the effect of blocking DA efflux to the Acb in preference tests and/or instrumental paradigms requiring an effort to gain access to sexual chemosignals, as suggested above. For example, it might be interesting to check whether optogenetic signaling of the amygdalar inputs into the Acb would disrupt the DA efflux and preference for male chemosignals.

Accumulated evidence suggest that the Acb participates in gating the impact of the sensory input on behavior, acting as a limbic-motor interface, and participates in controlling the motivation of an animal to get a reward or the effort that an animal is ready to allocate to obtaining or avoiding a particular stimulus (Salamone and Correa, 2012). Our finding that blocking DA efflux in the AcbSh reduces the investigation of male chemosignals in long exposures (40 min), but not during the first minutes of exposure is consistent with that interpretation.

In summary, our results show that olfactory/vomeronasal information, and in particular sexual chemosignals, are able to elicit a dopaminergic response in the Acb of female rats, and that blocking the release of DA in the AcbSh with a glutamate antagonist reduces the investigation of male chemosignals after the initial exposure. Further experiments should aim to investigate whether the dopaminergic response can be elicited by direct activation of the projections of the vomeronasal amygdala to the Acb, and further explore the role of other divisions of the ventral striatum, in particular the OT, which might be key to control behavior elicited by emotionally salient odors (Agustín-Pavón et al., 2014; DiBenedictis et al., 2014, 2015; Fitzgerald et al., 2014). The use of these olfactory/vomeronasal stimuli can provide an ethologically relevant approach to explore how the brain codes motivation and reward-directed behavior.