Encoding gender and individual information in the mouse vomeronasal organ

Abstract

The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.

Fig. 1

Detection of urine-elicited responses in the VNO of G-CaMP2-expressing mice. (A) Expression of G-CaMP2 in the neurons of the mainol-factory epithelium (MOE), the vomeronasal organ (VNO), and their axonal projections to the olfactory bulb (OB). (B) Twophoton image of a VNO slice used in an imaging experiment. (C) VNO responses to pooled C57BL/6 female urine (green) or male urine (red). The VNO slice (from a 4-month-old male) was stimulated with female urine (F.U.) under control (c1), treatment with 50 μM 2-APB (c2), and recovery (c3) conditions and male urine (c4, M.U.). Merge shows cells that respond to both male and female urine (c5). C6 shows the response traces of the three cells indicated in c1 to c5. (D) Fluorescence changes for a neuron responding to female urine applied for 10 (black), 20 (red), and 30 (blue) seconds, 10 s application following 2-APB treatment (black dot), and recovery (black dash), respectively. (E) The patterns of activation of a VNO slice by six different urine samples from different sex and strain animals are color-coded and shown in a merged picture.

Fig. 2

VNO responses to individual male and female mouse urine. (A) Heat map of 134 VNO neurons from a single slice (from a 3-month-old male) that responded to male and female urine from C57BL/6 (B6), CBA, and CD-1 strains. (B and C) Principal components analysis of the data shown in two-dimensional plots for PC1 and PC2 in (B), and PC1 and PC3 in (C). Urine from males and females is labeled with black and red, respectively. (D) Hierarchical cluster analysis of responses shown in (A) is plotted as a dendrogram based on distance obtained from Pearson correlations between responses to different urine applications.

Fig. 3

Hormone regulation of sex pheromones. (A) Responses to urine from two C57BL/6 males and castrated males in a VNO slice from a 2-month-old female. Two cells with differential responses are indicated. (B) Response traces of the cells indicated in (A). (C) A heat map showing all identified MUSCs, none of which responded to castrate urine. (D) Responses to female urine collected from a C57BL/6 mouse after injection of pregnant mare serum gonadotrophin in a VNO slice (from a 3-month-old male). Responses to urine collected on day 1 and day 4 are shown. Three cells with differential responses are indicated. (E) Response traces of the cells marked in (A). (F) A heat map showing the identified FUSCs (19 cells from two slices from a 3-month-old male and a 2-month-old female). Group A cells were activated by estrous urine from all three strains. Group B was activated by both estrous and diestrus urine, but not castrate urine. Group C responded to estrus, diestrus, and castrate urine. ♂, ♀, and ○ represent male, female, and castrate animals, respectively; E, day 1 (estrus); D, day 4 (diestrus).

Fig. 4

Response of VNO neurons to different individuals and to MHC peptide. (A and B) Heat maps and pie charts of responses to urine from non-littermates (A) and littermates (B). The pie charts show percentages of cells activated by different numbers of urine samples. (C) Response patterns of a VNO slice to urine from a C57BL/6 male, a C57BL/6 female, and 10^-9 M AAPDNRETF peptide, identified in the C57BL/6 strain. (D) A heat map for the responses summarizing the responses. The slice was from a 2-month-old female.