Although it has previously been recognized that amygdaloid subnuclei receive input from the MOB, the Me has traditionally been thought to receive only indirect MOB signals via afferents from the ACo (Scalia & Winans, 1975
; Licht & Meredith, 1987
). In principle, signals carried by a monosynaptic pathway may be less subject to modification and would enable MOB M/T cells to gain unconditioned access to the Me. Such a pathway may be more suitable for the regulation of innate, stereotyped behaviors evoked by pheromones or other biologically relevant chemosensory cues such as predator odors. We provide evidence using anatomical tracing that MOB M/T cells project directly to the Me in mice. Moreover, we corroborate previous reports (Schaefer et al., 2001
; Ma et al., 2003
; Xu et al., 2005
; Martel et al., 2007
) that the mouse MOB responds to volatile, biologically significant pheromones. Finally, by combining Fos immunohisto-chemistry with retrograde labeling of Me-projecting M/T cells, we provide evidence in estrous female mice that volatiles from male conspecifics, but not from other female mice or from a predator (cat), can stimulate these cells.
In male and female mice, urinary volatiles from the opposite sex induce Fos in the AOB via a pathway that requires a functional main olfactory system (Martel & Baum, 2007
). The Me-projecting MOB M/T cells identified in the present study may convey information about opposite-sex odors to the MeA and then back to the AOB via a centrifugal pathway (Barber, 1982
; Martel & Baum, 2009
). The present observation of a selective ability of male urinary volatiles to stimulate Fos expression in both the classic vomeronasal and olfactory subnuclei of the amygdala as well as in other segments of the vomeronasal projection pathway (e.g. BNST, ventrolateral portion of the ventromedial hypothalamus and medial preoptic area) and the mesolimbic dopamine system (e.g. nucleus accumbens shell) of estrous female subjects replicates and extends the results of a similar study (Martel & Baum, 2007
) carried out in ovariectomized female mice given no hormone priming. In that previous study, odor-induced stimulation of forebrain Fos expression was eliminated after chemical lesions of the main olfactory epithelium, further emphasizing the critical role of main olfactory epithelium receptors in mediating these effects of opposite-sex urinary volatiles. A recent study shows that female-typical central processing of sexually relevant olfactory cues was shaped by early estrogens (Pierman et al., 2008
). Presumably, the forebrain targets of this main olfactory input control essential aspects of courtship behavior and reproduction.
Double-labeling with anterograde tracers revealed that AOB and MOB M/T cell axonal projections converged in the Me in adjacent laminae of layer 1, a relatively cell-free zone rich in synapses (Ichikawa, 1987
). Although ventral MOB injections labeled the outermost layer, axonal branches also penetrated into deeper layers (e.g. and Fig. S3), suggesting that synaptic interactions between MOB and AOB afferents to the Me, as well as with intrinsic Me neurons, are likely. This interaction may be the means by which the accessory olfactory system, through a process of associative conditioning, transfers the saliency of social odor cues to the main olfactory system. For example, removal of the VNO in male mice prior to receiving heterosexual mating experience prevented subjects from emitting ultrasonic vocalizations (a component of mating behavior) in response to anesthetized females or female urine (Wysocki et al., 1982
). In chemically naïve female mice, lesions of the AOB disrupted the development of a preference for volatile male odors (Martinez-Ricos et al., 2008
). In male hamsters, VNO removal before, but not after, acquisition of sexual experience impaired mating behavior (Meredith, 1986
) and, in male guinea pigs, initial interest in female urinary odors declined in the weeks following VNO removal (but not in control subjects), suggesting an extinction in the salience of cues processed by the main olfactory system in the absence of reinforcement provided by the accessory system (Beauchamp et al., 1982
). Our evidence that MOB and AOB projections converge on the MeA and MePD now implicate these sites in such odor conditioning.
A recent report in the rat (Pro-Sistiaga et al., 2007
) has also demonstrated convergence of main and accessory olfactory inputs to medial amygdaloid nuclei. Unfortunately, due to differences between the location of these structures described in the mouse (Paxinos & Franklin, 2001
) and rat (Paxinos & Watson, 2005
) atlases, direct comparisons with the present study are difficult. Whereas we found in the mouse that the AOB and MOB both send axons to the MeA and MePD, convergent AOB/MOB projections in the rat were seen in the anterior, rather than the posterior, medial amygdala (Pro-Sistiaga et al., 2007
). In addition, we saw no AOB afferents to the ACo, which Pro-Sistiaga et al.
reported as a site of AOB/MOB convergence in the rat. Thus, despite general agreement that the Me receives projections from both the AOB and MOB, there may be species differences in the details of the specific locations involved.
Electrophysiological and anatomical evidence show that the main olfactory system and hypothalamus are linked (Pfaff & Pfaffmann, 1969
; Price et al., 1991
; Boehm et al., 2005
; Yoon et al., 2005
). These data, together with our results and a growing number of reports (Petrulis et al., 1999
; Pankevich et al., 2004
; Baxi et al., 2006
; Shepherd, 2006
), indicate that the accessory olfactory system is unlikely to be the sole access point for social odorants that are involved in intraspecific communication. The renewed focus on the main olfactory system as an important transducer of social odors is particularly relevant for humans. Neither an AOB nor a projection from the VNO to the brain has been identified in humans (Knecht et al., 2001
; Meredith, 2001
; Witt & Hummel, 2006
). Nevertheless, exposure to extracts from human sweat or to volatile steroids such as androstadienone (a putative pheromone excreted in male sweat) can produce in human subjects: (1) sex- and sexual orientation-specific activation of the hypothalamus (Savic et al., 2001
; Berglund et al., 2006
); and (2) an increased incidence of luteinizing hormone pulses (Shinohara et al., 2001
; Preti et al., 2003
; Witt & Hummel, 2006
). The lack of a functional accessory system in humans has raised the possibility that circuitry within the MOB is responsible for the initial processing and transmission of pheromones that result in these neurobehavioral responses. Our current findings in the mouse point to a MOB–Me pathway that may mediate such actions.
There is compelling evidence that the innate avoidance of aversive odors is regulated in mice by dorsal MOB glomeruli (Kobayakawa et al., 2007
). Our results suggest that different categories of behaviors, such as those involved in mating, are regulated by odors processed via a different set of MOB glomeruli. Thus, we observed that Me-projecting M/T cells that co-expressed Fos in response to male urinary odors were disproportionately located in the ventral MOB, which is consistent with other evidence (Schaefer et al., 2001
; Martel et al., 2007
) in mice, suggesting that volatiles in urine activate glomeruli located primarily in the ventral MOB. In another recent study in mice, it was reported that mature olfactory sensory neurons that express the transient receptor potential channel M5 (TRPM5) send axons to a small number of glomeruli in the ventral MOB (Lin et al., 2007
). These TRPM5-expressing olfactory sensory neurons were stimulated by a variety of semiochemicals, including the putative male urinary pheromone, 2,5-dimethylpyrazine, (methylthio) methanethiol, as measured by increased Fos expression in periglomerular cells of the MOB glomeruli that receive input from these neurons. This small population of TRPM5-expressing sensory neurons, which is concentrated in the ventrolateral olfactory epithelium, may represent the sensory segment of a pathway that signals information regarding volatile pheromones from the MOB to the Me and subsequently to hypothalamic targets that control heterosexual mate recognition and sexual arousal.