A direct main olfactory bulb projection to the 'vomeronasal' amygdala in female mice selectively responds to volatile pheromones from males

Abstract

The main olfactory system, like the accessory olfactory system, responds to pheromones involved in social communication. Whereas pheromones detected by the accessory system are transmitted to the hypothalamus via the medial ('vomeronasal') amygdala, the pathway by which pheromones are detected and transmitted by the main system is not well understood. We examined in female mice whether a direct projection from mitral/tufted (M/T) cells in the main olfactory bulb (MOB) to the medial amygdala exists, and whether medial amygdala-projecting M/T cells are activated by volatile urinary odors from conspecifics or a predator (cat). Simultaneous anterograde tracing using Phaseolus vulgaris leucoagglutinin and Fluoro-Ruby placed in the MOB and accessory olfactory bulb (AOB), respectively, revealed that axons of MOB M/T cells projected to superficial laminae of layer Ia in anterior and posterodorsal subdivisions of the medial amygdala, whereas projection neurons from the AOB sent axons to non-overlapping, deeper layer Ia laminae of the same subdivisions. Placement of the retrograde tracer cholera toxin B into the medial amygdala labeled M/T cells that were concentrated in the ventral MOB. Urinary volatiles from male mice, but not from female conspecifics or cat, induced Fos in medial amygdala-projecting MOB M/T cells of female subjects, suggesting that information about male odors is transmitted directly from the MOB to the 'vomeronasal' amygdala. The presence of a direct MOB-to-medial amygdala pathway in mice and other mammals could enable volatile, opposite-sex pheromones to gain privileged access to diencephalic structures that control mate recognition and reproduction.

Fig. 1

Neurolucida drawings (2.25 mm² brain space) show labeled axons 7 days following a PHA-L injection into the ventral part of the MOB (A) in a female mouse. The photos shown in (A) and (F) were taken from the same animal. Medial amygdala-projecting fibers can be seen running along the ventral surface of the brain and innervating the MeAV (B and C). At the level of the MeA (D), labeled fibers were concentrated on the medial edge of the MeA (solid arrow), with a noticeable thinning of fibers (open arrow) present between the MeA and the ACo. MePD-projecting fibers (solid arrow in E) were separated from those projecting to the ACo by the unlabeled MePV. A photo taken from the boxed region in (E) shows labeled fibers with varicosities (two arrows in F). A thin layer of PHA-L-positive fibers was seen on the medial edge of the MePD (solid arrow in G). In the most caudal MePD, layer Ia was sparsely labeled (solid arrow in H). Numbers at the right bottom of each drawing indicate distances (in mm) behind bregma. Open and closed arrowheads in (A) point to the anterior olfactory nucleus and lateral olfactory tract, respectively. ACo, anterior cortical amygdala; AOB, accessory olfactory bulb; BAOT, bed nucleus of the accessory olfactory tract; MeA, anterior medial amygdala; MeAD, anterodorsal medial amygdala; MeAV, anteroventral medial amygdala; MePD, posterodorsal medial amygdala; MePV, posteroventral medial amygdala; MOB, main olfactory bulb; opt, optic tract; PLCo, posterolateral cortical amygdala.

Fig. 2

Fluorescently labeled MOB and AOB projection cell axons travel to adjacent laminae in subdivisions of the medial amygdala of a female mouse. (A) A PHA-L injection site in the MOB (solid arrow) and a Fluoro-Ruby injection site in the AOB (open arrow) are shown in a sagittal section of the olfactory bulb 7 days after injection of anterograde tracers into these respective sites (counterstained with TO-PRO-3 – see blue pseudo color in on-line graphic). Open and closed arrowheads indicate the anterior olfactory nucleus and lateral olfactory tract, respectively. (B and C) PHA-L-labeled MOB M/T cell axons (solid arrows) and Fluoro-Ruby-labeled AOB mitral cell axons (open arrows) can be seen abutting each other in the superficial and deeper regions of layer Ia in coronal sections taken through the MeA (B; 1.2 mm behind bregma) and the MePD (C; 1.46 mm behind bregma), respectively.

Fig. 3

Retrograde labeling of the AOB and MOB after injection of CTb into the Me or ACo. (A) 7 days after tracer injection, CTb (dark staining) was centered in layer I of the Me. (B) 7 days after injection of CTb into the Me, CTb-labeled cells were seen in the AOB and the ventral portion of the MOB. (C) A higher magnification image shows that the dorsal MOB contained no CTb-labeled cells. (D) A higher magnification image shows that CTb-labeled cells (arrows) were located in the mitral cell layer of the ventral MOB. Following an injection of CTb into the ACo (E), M/T cells were labeled throughout the MOB (F). (G and H) Higher magnification images show CTb-labeled M/T cells (arrows) in the dorsal and ventral segments of the MOB, respectively, after an ACo injection of CTb. Tissue was counterstained with neutral red.

Fig. 4

Collapsed stacks of sagittal Neurolucida drawings from a representative female mouse show the distribution of CTb-labeled cells (black dots) in the lateral, central and medial segments of the MOB (shown in the sagittal plane; rostral surface on the left side) following an injection of CTb into the medial amygdala (Me; center image). The central segment of the MOB is further divided into dorsal and ventral regions by an imaginary line drawn parallel to the ventral surface of the olfactory bulb and across the arch of the mitral cell layer at the most rostral position. The drawing of a coronal section of the olfactory bulb (top left panel) is used to illustrate the four segments. CTb-IR cells were concentrated in three regions, including the caudal region that surrounds the stem of the olfactory bulb (area number 1 in the lateral and medial segments), the central-ventral region (area number 2 in the central segment), and the anterior medial region (area number 3 in the medial segment). Numerous CTb-labeled AOB mitral cells were not plotted.

Fig. 5

Effect of volatile urinary odor exposure on the expression of Fos in MOB M/T cells of estrous female mice that were retrogradely labeled by a prior injection of cholera toxin B (CTb) into the Me. (A) Three possible types of labeling occurred in MOB M/T cells following Me CTb injections and urinary odor exposure: CTb/Fos double-labeled (CTb + Fos), CTb-single labeled (CTb) and Fos-single labeled (Fos) M/T cells. (B) The percentage of CTb-labeled (Me-projecting) M/T cells that co-expressed Fos is shown across three segments of the MOB following exposure to different volatile urinary odors or clean air. The number (mean ± SEM) of CTb-labeled M/T cells is shown above the error bar for each group. *P = 0.001; ^#P < 0.05, Student’s Newman–Keuls post hoc comparisons between groups following a significant overall ANOVA.