Article Figures & Data
Figures
- Figure 1.
Effective concentrations of urine that activate the VNO. A, Schematic diagram showing the surgical procedure to remove the blood vessel (BV) to expose the underlying neuroepithelium (NE). The orientation of VNO is marked as follows: D, dorsal; V, ventral; R, rostral; C, caudal. The rectangular frame indicates the area in which confocal imaging is performed, shown in B. B, Fluorescent images of dissected VNO from animals exposed to dye-labeled urine painted on the urogenital areas of anesthetized mice. The images are maximal projections of multiple Z-stacks obtained at different depths. The differences in curvature of the images reflect geometric variations in dissected VNO. Scale bar, 200 μm. C, Quantification of fluorescent intensities in the VNO. Straight line is the standard curve derived from measurements from solutions containing dye at different concentrations (open circles). Measurements from six animals, three with one-bout investigation and three with three-bout investigations (filled diamonds) are plotted. Control (no dye) is also shown. For each animal, eight ROIs are measured, and the means ± SEM value are plotted. D, EVG traces in response to 10−6 to 10−2 dilution of B6 male urine. Arrowheads indicate 1 s applications of diluted urine. E, Bar graph showing averaged responses from multiple recordings. Numbers of recordings for each concentration are indicated above the bars. Data are represented as mean ± SEM.
- Figure 2.
Dose-dependent response to mouse urine. A, B, Images of VNO neurons responding to male (M.U.) and female (F.U.) urine at various concentrations (10−6 to 10−2 dilutions). Scale bar, 100 μm. C, Correspondence between calcium signal and electrical signal. Recordings from a G-CaMP2-expressing neuron in response to 1:100 female urine were shown. Urine triggered calcium signal (top) was simultaneously recorded with electrical responses (bottom). Black bar in the top indicates the duration of urine application. D, Line graph shows increases in the number of responsive cells with increasing concentrations of urine for all urine samples (black square), male urine alone (red diamond), and female urine alone (blue triangle). E, F, Responses of individual cells to different concentration of urine are plotted as heat maps. The heat maps are based on the amplitude of individual responses. In this example, a total of 84 cells responded to female urine (E), and 50 cells responded to male urine (F). Different types of responses (types I–IV; see Results) are boxed and marked. G, H, Representative dose–response curves of individual neurons for type I, II, and IV (G) and type III and IV (H) cells. I, Distribution of percentages of type I–IV cells for the overall responsive neuron population. Examples (A, B, E–H) are from animal 2 listed in supplemental Table 1 (available at www.jneurosci.org as supplemental material). Ensemble data (D, I) are from total 557 responsive cells of nine slices and six animals (supplemental Table 1, available at www.jneurosci.org as supplemental material). Color key: 0, ΔF/F < 10%; 1, 10% ≤ ΔF/F < 20%; 2, 20% ≤ ΔF/F < 30%; 3, 30% ≤ ΔF/F < 40%; 4, ΔF/F ≥ 40%.
- Figure 3.
Dose-dependent activation of MUSCs and FUSCs. A, Heat map of identified MUSCs and FUSCs in response to various concentrations of B6 male or female urine, as well as to 10−2 dilution of CBA and CD-1 male and female urine. B, Response traces of two MUSCs to different concentrations of B6 male urine. C, Response traces of two FUSCs to different concentrations of B6 female urine. Horizontal lines in B and C indicate background level activation. The results are obtained from a 6-month-old male and a 5-month-old female mouse.
- Figure 4.
Dose-dependent activation of VNO neurons by individual urine samples. A, Heat map of VNO neurons by 10−2 dilution of individual urines of different sex and strain. B, Cluster analyses show grouping of urine samples according to sex at 10−2. C, Heat map of VNO neurons by 10−4 dilution of individual urines of different sex and strain. D, Cluster analyses of urine samples indicate no apparent grouping at 10−4. The results are from a 6-month-old female mouse.
- Figure 5.
Dose-dependent responses single pheromones. A, Images of VNO neurons responding to DMP at various concentrations (10−11 to 10−5 m, 0.1% DMSO solution as negative control). B, Histogram shows an increase in the number of responsive neurons activated by increasing concentrations of DMP. C, Examples of dose–response curves of different individual neurons indicated in A. D, Images of the VNO neurons responding to ESP-1 at various concentrations (10−13 to 10−7 m, Ringer's solution as negative control). E, Histogram shows an increase in the number of responsive neurons activated by increasing concentrations of ESP-1. F, Examples of dose–response curves of different individual neurons as indicated in D. The results are from six animals (3 male and 3 female). Scale bars, 100 μm.
- Figure 6.
Masking of low-concentration urine by high-concentration urine in mixtures. A, Simultaneous electrophysiological recording and calcium imaging from a type I cell in response to three different concentrations of urine. Vertical lines indicate individual spikes recorded from this cell. The traces indicate calcium signals. Arrows indicate the starting points of 5 s urine applications. The recordings were obtained from a slice prepared from a 6-month-old male mouse. B, Heat map of activated VNO neurons by urine from two individual males at different concentrations. C, Response traces for three cells indicated in B. D, Response traces for two cells that are activated by low-concentration urine. They are activated again immediately after application of high concentration of urine and mixture, which did not elicit any responses. E, Response trace for a cell activated by low concentration of B6 urine but not high concentration of CD-1 urine. The responses to 10−4 B6 urine mixed with different concentrations of CD-1 urine are shown. The data are from a total of nine animals (5 male and 4 female; 2–5 months old), and examples shown here are from 3-month-old female mice.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Material
- supplemental material - Supplemental Movie 1
- supplemental material - Supplemental Movie 2
- supplemental material - Supplemental Movie 3
- supplemental material - Supplemental Movie 4
