Experimental design and examples of single unit recordings.

A. Icons describing the different subject and stimulus donors. In these icons, mouse colors denote the strain, while the background circles denote the status (estrus, non-estrus for females, naïve, dominant, castrated for males). B. Schematic of the recording setup. A multisite electrode probe is advanced to the external cell layer of the AOB. Stimuli (yellow drop) are applied to the nostril and, after a delay, the sympathetic nerve trunk (SNT) is stimulated. Between stimulus applications, the nasal cavity and VNO are flushed with Ringer’s solution. VNO: Vomeronasal organ. C. Timeline of an individual trial. After a 20 second delay, the SNT is activated to induce VNO activation. Following another 40 seconds, the flushing procedure is initiated. An inter-trial interval (ITI) of 10 seconds is applied between consecutive stimulus applications. During the experiment, stimuli are delivered repeatedly in blocks. Within each block, each of the individual stimuli are presented in a random order. D. Examples of single unit responses to all stimuli. For each unit, responses to each of 11 stimuli are shown. Left panels show the mean firing rate (PSTH, peri-stimulus time histogram), while the right panels shown raster displays for each of the individual trials. The red vertical line indicates stimulus application. For unit 2, time 0 indicate stimulus application, as in this case, responses began after stimulus application, prior to stimulation.

Basic response characteristics.

A. Colormap of responses. Each neuron (n=546) is represented by one row, and each stimulus by one column, as indicated at the bottom. Responses are normalized between −1 and 1. Non-significant responses are assigned a value of 0. B. Classical multidimensional scaling of population level responses. The mean error due to the reduced 2-D representation is 0.29 (arbitrary units). C. Distribution of number of significant responses across all neurons in the dataset. D. Number of neurons responding to each of the stimuli, separately for rate increases (upper bars, red), and decreases (lower bars, green). E. Mean response magnitude, across all recorded neurons to each of the stimuli.

Analysis of response patterns.

A. Graphical representation of response pattern definitions. In these representations, each of the stimuli indicated in red should elicit a stronger response than each of the stimuli in blue. The left and center panels show the basic response patterns and their complementary patterns, respectively. The panels on the right provide two examples of adjusted response patterns. For example, a dominant/strain pattern requires fulfilment of three conditions, so that within each strain, responses will be strongest to the dominant stimulus. Within a given condition, gray squares indicate stimuli that are irrelevant. The complementary adjusted responses patterns are not shown here. B. All response patterns across neurons. A neuron may fulfill more than one pattern and thus may appear in more than one row. The right panel is an expanded view of predefined response patterns. The adjusted categories are not shown in this representation. C. Frequency analysis of each of the basic and complementary response patterns. The response pattern name is indicated next to each panel. Within each panel, the blue histogram shows the shuffled distribution of pattern frequency (n = 100,000). The vertical blue lines show the mean value of the shuffled distribution. Red vertical lines indicate the actual number of observed patterns. Green panels indicate significant cases (i.e., the estimated probability to obtain the observed number of cases by chance is less than 5%). In most cases, the probability is considerably less (as indicated by the numbers within each panel).

Comparison of population level representations in estrus and non-estrus females.

A. Responses of individual neurons. Same data as in Fig. 2A, divided according to reproductive state. B. Correlation of population level pairwise distances across the two reproductive states, using the correlation distance measure. C. Same as in B using the Euclidean distance. In both B and C, the correlation coefficient (CC) and the probability to obtain it by chance (p-value) are indicated within each panel.

Comparison of responses in estrus and non-estrus females.

A. Percent responding neurons to each of the stimuli in non-estrus (gray) and estrus (red) neurons. B. Mean response magnitude to each stimulus under the two states. In A and B, p-values correspond to the binominal exact test. Bonferroni adjusted p-value given 11 comparisons for the 0.05 level is 0.00454. The one significant difference is indicated by red text. In A and B, n = 305 non-estrus, and n = 241 estrus. C. Correlation between mean pairwise preference indices under the two reproductive states. The correlation coefficient and the probability to obtain it by chance are indicated. D. Comparison of pairwise preference indices under the two states. Each pair of bars corresponds to one comparison. The stimuli are indicated by the icons and the text. For example, the first pair of bars on the left corresponds to pairwise comparison of dominant and naive ICR male urine. In both estrus states, there is a stronger response to the dominant stimulus as indicated by the downward pointing bars. However, the difference is not significant. E. Comparison of pairwise preference indices between stimulus pairs comprising male and female stimuli. In D and E, significance is determined using a shuffling test (n = 100,000). Bonferroni adjusted p-values at the 0.05 level are 0.0056 and 0.01 for D, and E, respectively. Significant differences among the states are indicated in red. Note that in most cases, significant differences correspond to cases where responses during estrus are stronger to the less virile male stimulus.

Response selectivity under the two reproductive states.

A. Lifetime sparseness distributions for non-estrus (left, n = 305, mean: 0.29 median: 0.27), and estrus (right, n = 241, mean: 0.33, median: 0.31), neurons. Non-parametric ANOVA p-value: 0.0078, reflecting higher selectivity during estrus. B. Triangle plots showing relative response magnitude to stimuli from each of the three male states, separated according to strains. Neurons recorded in non-estrus and estrus females are indicated in gray and red, respectively. C. Distributions of the male state selectivity index (calculated from the data shown in B) under the two reproductive states, calculated for each strain separately. p- values corresponding to non-parametric analysis of variance (Kruskal Wallis test): (ICR: KW p: 0.0017, BC: KW p: 0.24, C57: KW p: 0.08). Higher selectivity during estrus is observed for the stimuli from ICR male mice (p = 0.0017). Mean selectivity scores: ICR: estrus: 0.52, n = 161, non-estrus: 0.42, n = 181. BC: estrus: 0.47, n = 153, non-estrus: 0.43, n = 181. C57: estrus: 0.37, n = 139, non-estrus: 0.41, n = 206.

Comparison of response frequency under the two reproductive states.

A. Frequencies of basic patterns (including complementary patterns). B. Frequencies of adjusted patterns (including complementary patterns). In both panels, frequencies in estrus and non-estrus females are shown above and below the horizontal axis, respectively. Significantly represented categories (using a shuffling test as described in Fig. 3) are indicated in green. P-values for differences for each pattern under the two states, using the binomial exact test, are listed. The Bonferroni adjusted p-values at the 0.05 level are 0.0031 (0.05/16) and 0.0042 (0.05/12), for A and B, respectively, and thus none of the differences are significant. Relaxing the correction, three categories fulfil the 0.05 criterion. None of them correspond to increased representation of dominant male stimuli during estrus. Note that in some cases, a category is overrepresented only during one of the states, but this is not necessarily reflected by a significant difference among the two states (e.g., ∼female).

Schematic illustration of sampling of trait selective molecules.

A. Molecular profiles associated with 3 different individuals sharing a common trait. Trait selective molecules are shown in red and all others (unique molecules) are shown in green, within a 30 x 30 grid representing all (900) molecules in the world. The three individuals are indicated on the three columns on the left. The rightmost column shows their union. Rows correspond to different proportions of individual specific and trait selective molecules. B. Different sampling scenarios by AOB neurons. The three rows correspond to the scenarios shown in A. The left column is the union of all molecules under each scenario, as shown in A. The three other columns represent sampling schemes that differ in the proportion of trait selective and other molecules sampled, moving from trait avoiding, to balanced, to trait selective sampling. The two values above each column indicate the probability of sampling unique (left), or common (trait-selective) molecules (right). We speculate that sampling properties are matched to component frequencies, thereby balancing robustness of trait detection with redundancy. While only AOB-MCs are depicted here, sampling actually involves VNSs and the manner by which they are sampled by AOB-MCs.