Kimchi and co-workers (Kimchi et al.,
2007) reported that female mice with a null mutation of the Trp2C gene (initially reported to cause a total elimination of VNO function) failed to discriminate male from female conspecifics (Trp2C mutant females reportedly directed mounting behavior indiscriminately toward a castrated male and an estrous female). However, several other studies showed that surgical VNO destruction failed to disrupt the ability of female mice to discriminate body and/or urinary odors of male vs female or of testes-intact vs castrate male specifics (Lloyd-Thomas and Keverne,
1982; Keller et al.,
2006b; Martel and Baum,
2009a). The Kimchi et al. study can be criticized on two grounds. First, it seems likely that some VNO neurons remain functional even after the Trp2C channel is knocked out: thus pregnancy block in response to pheromones from a strange (non-mating) male was retained in Trp2C female mice (Kelliher et al.,
2006) even though surgical VNO removal has been shown to eliminate the pregnancy block otherwise induced in recently mated female mice that are exposed to urinary odors from a strange (non-mating) male (Lloyd-Thomas and Keverne,
1982). A recent study (Kim et al.,
2011) showed that calcium activated chloride currents may also generate action potentials in murine VNO neurons. Second, Kimchi and co-workers (Kimchi et al.,
2007) never directly assessed the ability of their Trp2C null mutant females to discriminate between pheromonal cues derived from male vs female conspecifics; instead females' motivation to direct mounts toward a castrated male vs an estrous female was assessed. By itself, this does not constitute a rigorous assessment of females' female-typical sexual motivation or of its signaling by sex-specific pheromonal cues. Systematic analysis of olfactory preferences in female mice (Keller et al.,
2006b; Martel and Baum,
2009a) showed that surgical VNO destruction eliminated females' preference to investigate male vs female non-volatile urinary odors without disrupting their preference to approach volatile odor cues from a male. VNO destruction also dramatically reduced the capacity of female mice, when tested while in estrus, to display lordosis in response to the receipt of mounts from a male. A similar disruption of lordosis was seen in female mice given bilateral lesions of the AOB (Martel and Baum,
2009a). Likewise, in estrous female rats VNO lesions (Rajendren et al.,
1990) as well as AOB lesions (Dudley and Moss,
1994) significantly reduced the expression of lordosis behavior. Further evidence of VNO involvement in the control of lordosis in mice comes from the work of Haga et al. (
2010) who identified a pheromone secreted in male tears (exocrine gland-secreted pepide; ESP1). These workers showed that ESP1 stimulated immediate early gene expression in the female's VNO, after being detected by Vmn2r116 receptor. Further behavioral studies showed that application of ESP1 to WT females in estrus enhanced their lordosis behavior; however, no such facilitation was seen in females in which the Vmn24116 receptor was knocked out.
Several studies (Edwards and Burge,
1973; Keller et al.,
2006a) showed that zinc sulfate lesions of the MOE eliminated the capacity of female mice to show a preference for volatile body odors emitted from male vs female or from testes-intact male vs castrated male conspecifics. These investigators also reported significant reductions in females' lordosis capacity after MOE lesions. Thus both the accessory and main olfactory inputs to the MeA may mediate the pheromonal facilitation of lordosis capacity in female mice. Results of a recent study (DiBenedictis et al.,
2012) showed that bilateral lesions of the MeA (like lesions of the VNO, MOE, or AOB) also significantly diminished lordosis in ovariectomized females following priming with ovarian hormones. Thus central disruption of pheromonal inputs (e.g., ESP-1 males' tears; other yet to be determined male urinary volatiles) that are initially detected by either the VNO or the MOE are potentially as disruptive to the display of females' lordosis behavior as eliminating the ovarian sex hormones (e.g., after ovariectomy).
Early studies using female mice showed that females placed on soiled male bedding showed an increase in Fos and/or EGR-1 expression in VNO sensory neurons (Halem et al.,
1999,
2001; Kimoto et al.,
2005) as well as in central target sites of these neurons including the AOB, the vomeronasal amygdala and hypothalamic regions including the BNST and VMH. These results have recently (Isogai et al.,
2011) been confirmed and extended. In so far as subjects used in these studies had direct nasal contact with pheromones deposited in soiled bedding, it seems likely that the VNO played a central role in the pheromone detection Other early studies (Schaefer et al.,
2001,
2002) showed, however, that urinary volatiles from male mice of different major histocompatibility complex (MHC) genotypes elicited significantly different profiles of MOB glomerular activation in females, as indexed by odor-induced expression of Fos in periglomerular cells surrounding activated glomeruli. The pheromone-activated glomeruli were concentrated in the ventral portion of the MOB. Numerous studies (Boehm and Zufall,
2006; Spehr et al.,
2006) conducted over the past 30 years have shown that volatile MHC molecules as well as small peptide ligands for these molecules, contribute to individual mate recognition and mate choice in female mice. The detection of these individual MHC odortypes likely occurs after their detection by the MOE, since female mice from which the VNO was removed continued to successfully discriminate urinary volatiles from males of two MHC haplotypes (Wysocki et al.,
2004). It is not known which of the ~1000 different classical olfactory receptor genes (Buck and Axel,
1991) expressed in the MOE detect MHC molecules. A second family of receptors (trace amine-associated receptors; TAARs) was recently found to be expressed in the MOE of mice (Liberles and Buck,
2006). These investigators also showed that trimethylamine is elevated in the urine of male vs female mice and is bound by mTAAR5-expressing cells. Liberles and Buck (
2006) raised the possibility that trimethylamine, which like methylthio methanethiol (MTMT; see below) is a volatile component of male mouse urine, may serve as a signaling pheromone to attract males to female conspecifics. Other recent work (Lin et al.,
2007) showed that many of the pheromone-responsive glomeruli in the MOB of both sexes are innervated by axons whose cell bodies in the MOE express the transient receptor potential channel M5 (TRPM5). It is not known whether TRPM5 expression occurs in subsets of MOE receptor neurons that express classical olfactory receptor proteins, TAARs, or both types of receptors. Electrophysiological recording from mitral cells in the ventral MOB of female mice revealed that a subset of these neurons were activated by male urinary volatiles (Lin et al.,
2005). These same neurons in the female MOB were also reliably activated by MTMT which was also found to attract sexually experienced female mice in behavioral tests. In another study (Xu et al.,
2005) functional magnetic resonance imaging (fMRI) was used to reveal glomerular activation in both the MOB and AOB of female mice in response to male urinary volatiles, with the MOB activation occurring slightly earlier than that in AOB. Slotnick and colleagues (Slotnick et al.,
2010) suggested that the VNO responses to volatile pheromones typically are preceded by MOE detection which then leads the animal to make the nasal contact with a non-volatile pheromone, leading to VNO activation. Early work (Luo et al.,
2003) showed used
in vivo recordings from the mouse AOB in which neuronal activation was only seen after direct nasal contact with pheromonal stimuli.
The observation (Xu et al.,
2005) that AOB activation (indexed by fMRI) occurred in response to urinary volatiles, which are thought to be detected solely by the MOE as opposed to the VNO, was surprising. However, this outcome was confirmed and extended by a study (Martel and Baum,
2007) which compared the ability of urinary volatiles from male vs female mice to activate MOB glomeruli (indexed by Fos expression in the periglomerular cells) and augment Fos expression in AOB mitral and granule cells as well as in targets of VNO olfactory input in the vomeronasal amygdala and hypothalamus of gonadectomized (non-hormone treated) male and female mice. As predicted from the outcome of several above-mentioned studies, both male and female urinary volatiles activated multiple glomeruli located in the ventral MOB of both male and female subjects; the distribution of these activated glomeruli, while overlapping, was distinct in mice of both sexes exposed to male vs female urinary volatiles. In contrast to the MOB, only opposite-sex urinary volatiles stimulated Fos expression in mitral and granule cells of the AOB of each sex (i.e., female subjects showed AOB Fos responses to male urinary volatiles whereas male subjects showed AOB Fos responses to urinary volatiles from estrous females). In female subjects the selective Fos responsiveness of the AOB to male urinary volatiles extended to other forebrain targets of pheromone processing, including the vomeronasal amygdala and hypothalamus. By contrast, in male subjects exposure to either female or male urinary volatiles stimulated Fos expression in forebrain regions including the vomeronasal amygdala and hypothalamus. It is noteworthy that all of the MOB, AOB, and other forebrain Fos responses to urinary volatiles were absent in groups of gonadectomized female and male mice that 4 days earlier had received intranasal infusions of the toxic compound, zinc sulfate, which killed MOE sensory neurons. Zinc sulfate lesions of the MOE did not attenuate the ability of direct nasal contact with male urine to augment Fos expression in AOB mitral and/or granule cells. These results suggest that all observed Fos responses to urinary volatiles resulted from their detection by MOE, as opposed to VNO sensory neurons, and that there exists an input pathway whereby main olfactory signals reach the AOB.
Studies (Pro-Sistiaga et al.,
2007; Kang et al.,
2009) from rat and mouse point to the existence of a population of MOB mitral cells that extend axons directly to portions of the “vomeronasal (MeA) amygdala.” These mitral cells are separate from the population of MOB mitral cells, described in the classic paper of Kevetter and Winans (
1981b), which target subdivisions of the “olfactory amygdala,” including the anterior cortical amygdala. In studies using female mice (Kang et al.,
2009) dual labeling of the MOB (PHA-L) and AOB (Fluoro-Ruby) with anterograde tracers led to labeling of abutting superficial laminae of the ipsilateral MeA and MePD. In additional females, injection of the retrograde tracer, Cholera Toxin B (CTb) into the MeA led to retrograde labeling of a large number of AOB mitral cells as well as a restricted population of mitral cells located in the ventral and medial subdivisions of the MOB. In a functional study (Kang et al.,
2009) exposure of ovariectomized, hormone-primed female mice to urinary volatiles from male, but not from female mice, significantly augmented the population of MeA projecting MOB mitral cells that co-expressed Fos protein. More recently, Thompson et al. (
2012) found using both male and female mice that MOB glomeruli which receive synaptic inputs from MOE olfactory sensory neurons that express the cation channel TRPM5 (and which are thought to respond selectively to several different pheromones) are more likely to be innervated by an apical dendrite from MOB mitral cells that extend axons to the vomeronasal amgydala (MeA), although this overlap was not complete. Finally, evidence of a direct projection pathway of MOB mitral cells to the granule cells of the adjacent (ipsilateral) AOB has been provided in male rats (Larriva-Sahd,
2008).
We asked whether the MOB-MeA projection pathway passes information about male urinary volatiles on to the AOB, thereby accounting for our previous observation (Martel and Baum,
2007) that opposite-sex urinary volatiles (detected by the MOE, and not the VNO) augmented Fos expression in AOB mitral and granule cells? An anatomical study (Fan and Luo,
2009) used an anterograde tracer to show that axons extend from MeA neurons to innervate granule cells in the ipsilateral AOB of mice. We found (Martel and Baum,
2009b) that exposure to male, but not female, urinary volatiles stimulated the expression of Fos in the cell bodies of MeA neurons that were co-labeled with CTb which had been injected 1 week earlier into the ipsilateral AOB. As in our previous study (Martel and Baum,
2007) exposure to male, but not to female, urinary volatiles stimulated Fos expression in several brain regions, including the vomeronasal amygdala and several hypothalamic regions including the BNST, MPA, and VMH. Taken together, these data raised the possibility that opposite-sex (male) urinary volatiles are detected in the MOE by sensory neurons that express TRPM5. These neurons convey their inputs to a subset of MOB glomeruli where information about “maleness” is transferred directly to a subset of MOB mitral cells that target the MeA. The intregration of these main olfactory inputs and signaling from ovarian hormones (estradiol and progesterone) likely occurs in the posterior dorsal portion of the MeA, as indicated by our recent finding (DiBenedictis et al.,
2012) that bilateral lesions of the caudal (PD), but not the rostral, subdivision of the MeA disrupted the preference of estrous females to approach urinary volatiles from testes-intact vs castrated male mice.
It is widely agreed that even in the absence of previous nasal contact with male pheromonal cues, adult female mice are inherently motivated to investigate/make nasal contact with non-volatile urinary pheromones deposited by testes-intact adult males (Ramm et al.,
2008). The attraction has been attributed to the presence of a particular major urinary protein (MUP), named darcin (Roberts et al.,
2010), which is excreted in male mouse urine. This female-typical preference for male urine/darcin is hard wired, and likely depends on the absence of sex steroid signaling around the time of females' birth (which contrasts with an organizational action of perinatal testosterone and/or estrogenic metabolites of testosterone in the developing male hypothalamus) coupled with the later presence in females of estradiol signaling over a prepubertal period (P15-P25); (Brock et al.,
2010,
2011). The strongest preference for male odors is expressed in adult cycling females on the night of proestrus, thanks to the central “activational” actions of estradiol and progesterone in the projection circuit that processes pheromonal cues. Additional studies (Ramm et al.,
2008; Martinez-Garcia et al.,
2009) suggested that female mice which had never had nasal contact or mating experience with a male showed no preference to seek out airborn urinary volatiles from male vs female conspecifics. By contrast, a robust preference for airborn (volatile) scents from males (vs castrated males or females) was seen in female mice that previously had experienced nasal contact with male urine or had mated with a male. This preference was strongest for volatile body odors from socially dominant males, provided females had previously had direct nasal access with the suite of odors deposited on cage bedding by dominant males (Mak et al.,
2007; Veyrac et al.,
2011). As already stated, female mice were strongly attracted to the volatile component of male urine, MTMT (Lin et al.,
2005), a behavioral effect that was correlated with the ability of MTMT to augment electrical activity of MOB mitral cells in female mice. All of the females used in that study had received prior mating experience with males, thus it is not known whether naïve females which had not previously received either mating experience or nasal contact with male body parts/male urine would have been attracted to the putative volatile male urinary pheromone. Martinez-Ricos et al. (
2007) also reported that nasal contact with soiled male bedding reliably served as a stimulus that established a learned conditioned place preference (CCP) response in female mice whereas access to volatiles emitted from soiled male bedding failed to establish a CCP. In this latter study, as in the other studies (Martinez-Ricos et al.,
2008) that reported an absence of female preference for male vs female or male vs castrated male urinary or body volatile odorants the female subjects had been kept in a separate colony room from males beginning at the age of weaning. In these particular studies odor “naïve” female subjects were given behavioral tests in adulthood while ovary intact and at an unspecified stage of the estrous cycle. In another study (Ramm et al.,
2008) female mice derived from wild caught parents were carefully prevented from experiencing contact with male body odorants until adulthood. In the absence of direct nasal experience with urine from a particular male, these females showed no preference to approach male vs female urinary volatiles. After nasal experience with a specific male, females later preferred to approach male vs female urinary odors, provided the male odor presented was from a specific male. Finally, Ramm et al. (
2008) reported that they exposed all of their subjects to soiled bedding from an unfamiliar male for three consecutive days in order to bring their ovary-intact females into proestrous/estrous at the time their preference for male vs female urinary volatiles was assessed. No direct confirmation of the successful induction of proestrus/estrus (using vaginal smears) was carried out in this study, thus it remains a matter of speculation as to whether odor preferences were actually made while females were in an optimal hormonal condition to show a male-directed preference. Also, it is hard to argue that the subjects in this study were odor “naïve” given that direct nasal contact with soiled male bedding was reportedly used to bring all females in to proestrus/estrus at the time of testing.
In two recent studies (Martel and Baum,
2009a; DiBenedictis et al.,
2012) my colleagues and I examined the preference of young adult female Swiss-Webster mice to approach volatile urinary odors from testes intact males vs either estrous females or castrated males. Although our females may have previously been exposed to volatile male body odors in the colony room, they had never had direct nasal access to such male odorants, nor had they had any mating experience prior to these studies. In both studies such naïve female subjects showed a significant preference to investigate volatile urinary odors from testes intact males vs estrous females (Martel and Baum,
2009a) or from testes intact males vs castrated males (DiBenedictis et al.,
2012). In the former study the female subjects had been ovariectomized and treated chronically with a s.c. Silastic capsule releasing estradiol at the time odor preference was assessed. In the latter study, the female subjects had been ovariectomized, treated chronically with a s.c. Silastic capsule releasing estradiol, and given a s.c. injection progesterone 3–6 h prior to the assessment of odor preference. Thus in both of our studies male odor “naïve” females (no prior nasal contact with male odors) showed a preference to investigate male urinary volatiles—findings that conflict with previous reports (Ramm et al.,
2008; Martinez-Garcia et al.,
2009) that female mice require prior nasal contact with male body or urinary odors in order for male urinary volatiles associated with non-volatiles (presumably detected by the female's VNO-AOB-accessory olfactory system). The absence of a preference for male urinary volatiles in the absence of previous nasal contact with male urine may reflect the non-estrous status at the time of behavioral testing in a large proportion of the ovary-intact females used in those previous experiments. It will be important in future studies to systematically assess the role of ovarian hormones in the expression of females' preference for male urinary (or general body) volatile odorants. The suggestion (Martinez-Garcia et al.,
2009) that female mice attend to male urinary volatiles and find them rewarding only after a conditioning process in which these odors are paired with VNO detection of non-volatile male odorants that are processed via a hard-wired straight line pathway to the hypothalamus via the medial amygdala (Choi et al.,
2005) is an attractive one. Indeed, there are several lines of neuroanatomical data that indirectly support this proposed mechanism. Thus inputs from the VNO are passed directly on to the AOB which, in turn, directly targets the MeA, followed by the hypothalamus with little or no input to higher olfactory cortical structures (Kevetter and Winans,
1981a; Kang et al.,
2009). By contrast, MOB mitral cells that target the MeA invariably extend axon collaterals into the far reaches of anterior and posterior piriform cortex (Kang et al.,
2011). The intermingling of AOB and MOB inputs to the MeA, combined with parallel cortical processing of MOB inputs to the MeA may provide a substrate for the type of olfactory learning proposed by others (Ramm et al.,
2008; Martinez-Garcia et al.,
2009). It may also be, however, that ovarian hormones somehow obviate the need for this learning, leading females to express a preference for volatile male urinary odors in the absence of prior direct nasal contact with male urine. More research is needed to answer this question.
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sex mating partners