Differential activation of glomeruli in the ferret's main olfactory bulb by anal scent gland odours from males and females: an early step in mate identification

Abstract

Peripheral anosmia was previously found to disrupt sex discrimination and partner preference in male and female ferrets. Here we show directly that volatile anal scent gland odourants from male and female ferrets activated overlapping but distinguishable clusters of glomeruli located in the ventral-caudal portion of the main olfactory bulb (MOB) of breeding ferrets of both sexes. No glomerular activation was seen in the accessory olfactory bulb (AOB). The profile of MOB glomerular activation induced in oestrous females by male anal scents was very similar to that induced by direct contact with a male during mating, and oestrogen treatment failed to alter the profile of glomerular activation induced in ovo-hysterectomized females by male anal scents. In rodents, 'atypical' MOB glomeruli, which have dense acetylcholinesterase (AChE) activity in the neuropil, may be activated by body odours from conspecifics. No such AChE-staining 'atypical' glomeruli were found in the ferret's MOB, suggesting that in this carnivore they do not constitute a subset of MOB glomeruli that respond to body odourants. In ferrets of both sexes, volatile body odourants that are detected by the main as opposed to the vomeronasal-AOB accessory olfactory system may play a critical role in mate identification.

Fig. 1

Identifying and mapping activated MOB glomeruli based on the presence of Fos-IR juxtaglomerular cells following exposure to anal scents. (A) A representative coronal section (taken at level ‘c’ from the diagram in B) showing Fos-IR in juxtaglomerular cells in the MOB of an oestrous female ferret after exposure to volatile components of male anal scent. The upper inset shows the AOB at a higher magnification to illustrate the absence of Fos-IR cells in the glomerular layer. The lower inset shows the MOB at a higher magnification to illustrate the presence of an activated glomerulus with a ≥ 180° arc of Fos-IR juxtaglomerular cells. The upper boundary of a second, non-activated glomerulus in which the arc of Fos-IR juxtaglomerular cells did not exceed 180° is also shown at the bottom of the inset. The radial angle corresponding to each activated glomerulus was determined according to a vertical axis and point of origin, as shown. (B) The rostro-caudal location and radial angle of each activated glomerulus was used to map patterns of odour-induced glomerular activity. The location of the activated glomerulus shown in A is shown with an X on the map. Letters a–c indicate the rostro-caudal location of different coronal sections in the sagittal diagram of the olfactory bulb and the map. D, dorsal; M, medial; FB, forebrain; Gl, AOB glomerular layer. Scale bars, 100 μm.

Fig. 2

Patterns of glomerular activation in the MOB of gonadally intact, oestrous female ferrets after exposure to male vs. female anal scents. Data are plotted in bins defined by radial angle and rostro-caudal distance through the olfactory bulb. (A–C) The average number of activated glomeruli per bin after exposure to unscented air (A), female anal scent (B) or male anal scent (C); scale above the left column indicates the number of activated glomeruli per bin. No activated glomeruli were seen in white areas. (D–F) P-values from bin-wise Mann–Whitney U-tests comparing differences between female vs. male anal scent (D), control vs. female anal scent (E) and control vs. male anal scent (F). The scale above the right column indicates P-values; P ≤ 1 × 10⁻⁵ was significantly different after Bonferroni correction. The black outlines in B and C indicate the areas of maximal activation in response to female and male anal scent, respectively. These black outlines are superimposed on the Mann–Whitney U-plots (D–F) and indicate whether areas of maximal activation were significantly different between groups.

Fig. 3

Patterns of glomerular activation in the MOB of gonadally intact, breeding male ferrets after exposure to male vs. female anal scents. Data are plotted in bins defined by radial angle and rostro-caudal distance through the olfactory bulb. (A–C) The average number of activated glomeruli per bin after exposure to unscented air (A), female anal scent (B) or male anal scent (C); scale above the left column indicates the number of activated glomeruli per bin. No activated glomeruli were seen in white areas. (D–F) P-values from bin-wise Mann–Whitney U-tests comparing differences between female vs. male anal scent (D), control vs. female anal scent (E) and control vs. male anal scent (F). The scale above the right column indicates P-values; P ≤ 1 × 10⁻⁵ was significantly different after Bonferroni correction. The black outlines in B and C indicate the areas of maximal activation in response to female or male anal scent, respectively. These black outlines are superimposed on the Mann–Whitney U-plots (D–F) and indicate whether areas of maximal activation were significantly different between groups.

Fig. 4

Patterns of glomerular activation in the MOB of gonadally intact, oestrous female ferrets after mating. Data are plotted in bins defined by radial angle and rostro–caudal distance through the olfactory bulb. (A and B) The average number of activated glomeruli per bin after exposure to a clean cage (A) or after receiving a neck grip, mount and intromission from a male (B). The scale above A indicates the number of activated glomeruli per bin. No activated glomeruli were seen in white areas. (C) P-values from bin-wise Mann–Whitney U-tests comparing differences between the two conditions. The scale above C indicates P-values; P ≤1 × 10⁻⁵ was significantly different after Bonferroni correction.

Fig. 5

Effect of oestrogen treatment on patterns of MOB glomerular activation induced by exposure to male anal scents in ovo-hysterectomized (Ovex) female ferrets. Data are plotted in bins defined by radial angle and rostro-caudal distance through the olfactory bulb. (A and B) The average number of activated glomeruli per bin after exposure to male anal scents in ovo-hysterectomized female ferrets previously injected daily with either oil vehicle (A) or oestradiol benzoate (EB) (B). The scale above A indicates the number of activated glomeruli per bin. No activated glomeruli were seen in white areas. (C) P-values from bin-wise Mann–Whitney U-tests comparing differences between the two conditions. The scale above C indicates P-values; P ≤ 1 × 10⁻⁵ was significantly different after Bonferroni correction.

Fig. 6

Absence of ‘atypical’ glomeruli in the MOB of ferrets. Representative coronal sections show AChE histochemistry in the MOB and AOB of an oestrous female ferret (A) and a male mouse (B). Insets show boxed areas at higher magnification. Note the presence of AChE staining in the glomerular (Gl) and internal plexiform layers (IPL) in both ferret and mouse. Arrows in B indicate the location of densely staining ‘atypical’ glomeruli in the mouse, located adjacent to the AOB. D, dorsal; M, medial. Scale bars, 250 μm.