1. A Molecular Code for Identity in the Vomeronasal System
Xiaoyan Fu, Yuetian Yan, Pei S. Xu, Ilan Geerlof-Vidavsky, Wongi Chong, Michael L. Gross, Timothy E. Holy 
Cell 
Volume 163, Issue 2, Pages (October 2015)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 CAM for Ligand Identification
A diverse collection of samples (here, urine extracts from male and female mice of different strains) are analyzed to extract the relative concentrations of each component by LC-ESI mass spectrometry and subjected to an assay of activity (left, by recordings of spiking responses). For a selected neuron, physiological responses (neuronal firing rate, orange bars) to the different stimuli are compared to the abundances of individual components (blue and green bars). Component 1 (blue bars) is not distributed in a manner that could explain the firing rate responses and is therefore an implausible candidate. In contrast, component 2 (green bars) has an across-sample distribution consistent with the measured neuronal firing rates and is thus a plausible candidate to explain the response.
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4. Figure 2 Categorization of VSN Responses
(A) Electrophysiological recording with a multi-electrode array. The voltage signal recorded from a representative electrode (indicated by red color) is illustrated.
(B) Experimental design: firing rate versus time of one neuron, across five cycles of stimulus presentation. A collection of extracts from males (M) and females (F) of four strains, encoded by color, are presented at three different concentrations (encoded by stimulus bar height). Stimulus order is randomized across cycles, and each stimulus is presented once per cycle. Note that collecting this number of sensory responses required more than 2.5 hr of continuous recording.
(C) Overlays of all neurons assigned to one of eight repeatedly identified response patterns. Single exemplar neurons are shown in bold colors, and other neurons of similar functional type are shown in faint colors. Red indicates responses to female urine; blue indicates responses to male; and responses to all three dilutions are shown. The primary differentiator of the first two classes is their concentration dependence. Error bars represent firing rate SEM.
See also Figure S1.
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5. Figure 3
Matching Component Concentrations to Single-Neuron Activity Patterns
(A) Firing rate of a neuron from the second class in Figure 2C, marked with a yellow arrow in Figure S1.
(B) Relative abundance across samples of a component with m/z eluting between 34%−37% acetonitrile.
(C) Incorporation of the abundance with the dilution factor for each stimulus leads to a putative dose-response curve as a function of the concentration of this particular component. Fitting the points to a Hill model leads to a goodness-of-fit measured by χ2. This example represents a very poor fit (p < 10−50).
(D) Another component with m/z eluting between 52%−56% acetonitrile is most abundant in BALB/c female urine.
(E) The dose-response curve from this second component fits well (p = 0.18).
(F) χ2 for a class 1 neuron (Figure 2C, marked with a green arrow in Figure S1) for each of 1,634 components. Only a single component (marked with a green circle), the one in (D) and (E), represents a good fit (χ2 < 75).
See also Figure S2.
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6. Figure 4 Candidate Ligands for Each Class of VSN Response
Goodness-of-fit plotted for the 400 most abundant components for each neuron shown at right. In each case, at most a handful of components represent plausible ligands. For each candidate with χ2 < 75, m/z and elution data are listed in Table S1. Circles highlight a previously identified CAM ligand m/z (4-pregnene-11,20,21-triol-3-one 21-sulfate, green) and the two novel CAM candidates to explain a pan-female response (class 5), m/z (red) and m/z (purple).
See also Figure S3 and Table S1.
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7. Figure 5 M377 Excites Female-Selective Neurons
(A) Experimental paradigm for calcium-imaging experiments. Each stimulus presentation is marked by a color-coded bar, each repeated four times. GCaMP2 fluorescence intensity for a particular neuron is shown.
(B) Single optical sections of the whole-mount VNO, with the dendritic knob layer near the top of the image. ΔF/F is encoded voxel-wise using a red color scale. Female extracts excite more neuronal activity than male extracts; M377 potently excites a subset of neurons. Scale bar, 50 μm.
(C) Overlay of ΔF/F responses across stimuli. Note responses to M377 overlap responses to those cells activated by female cues.
(D) Comparison of response amplitudes to C57BL/6J female extract and purified M377. Each neuron sensitive to C57BL/6J female extract is shown as a separate circle. Diagonal line represents equal response to the two stimuli.
(E) Heatmap of all urine- or M377-responsive neurons imaged in two male VNOs. Each cell is represented as a single column, and cells with similar response patterns were grouped. The first three groups correspond to three different types of female-selective responses. Note that all neurons showing roughly equal responsiveness to both BALB/c and C57BL/6J female urine are also responsive to M377.
See also Figure S4.
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8. Figure 6 Structural Validation of M377 and M361
(A) Proposed chemical structure of purified M377 and its MS2 product-ion spectrum.
(B) Structure of synthetic cortigynic acid and its MS2 spectrum.
(C) Calcium imaging to purified and synthetic M377 over a range of matched concentrations.
(D) Response amplitude of each neuron to purified and synthetic M377, pooled across all concentrations. The average response across four trials by each neuron to each matched-concentration stimulus combination is shown as a circle.
(E and F) Chemical structure of purified and synthetic M361 and the MS2 product-ion spectrum.
(G) Combinatorial coding of female-specific cues. Neuronal responses to 100-fold dilute urine stimuli, M377, M361, and their mixture at three different concentrations.
See also Figures S5 and S6 and Table S2.
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9. Figure 7 Male Investigatory and Mounting Behavior
(A and B) Schematic of the experiment. Male “sniff time” to stimuli presented on a cotton swab is measured by an optical beam-break detector; male mounting behavior was scored manually.
(C) Mean and SEM of the fraction of time spent investigating the swab after initial contact. Males spent longer investigating the sample doped with M377, M377 + M361, female extract, or female urine (∗p < 0.001, ∗∗p < , n = 11, t test).
(D) Removing M377 from female urine extract significantly reduced wild-type males’ investigation time (∗p < 0.01, n = 12, t test).
(E) Adding M377 back to the “deficient” female urine extract robustly increased male’s investigation time (∗p < 0.05, n = 12, t test).
(F) Duration of mounting toward females painted with vehicle (light blue) and M377 (salmon) (∗p < 0.05, n = 12, Mann-Whitney test).
(G) Duration of mounting toward females painted with vehicle (light blue) and M361 (lime).
(H) Duration of mounting toward females painted with vehicle (light blue) and M361 + M377 (pink) (∗p < 0.02, n = 17, Mann-Whitney test).
(I) Frequency of mounting toward females painted with vehicle (light blue) and M361 + M377 (pink) (∗p < 0.02, n = 17, Mann-Whitney test).
(J) Mounting duration of individual males toward females painted with vehicle (light blue) and M377 + M361 (pink).
(K) Mounting frequency of individual males toward females painted with vehicle (light blue) and M377 + M361 (pink).
See also Figure S7.
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10. Figure S1
All Responsive VNO Neurons to Urine Samples, Related to Figure 2
These 121 neurons responded (Δr > 5Hz and distinguishable from the response to the standard at a level of p < 0.05, t test) to at least one stimulus. No neuron passing this test responded significantly to the independent negative control.
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11. Figure S2
Distribution of Components across Samples, Related to Figure 3
(A) Ion count as a function of elution time (high-resolution nanoLC-MS/MS) from the column (horizontal axis, expressed as % acetonitrile) and m/z (vertical axis). Results from two urine extracts, female mice of the BALB/c and DBA strains, are overlaid in two colors; their overlap renders as black, indicating peaks that are present in both samples at similar abundance. Intensity of color is proportional to the square root of the ion count, to allow visualization of a wider range of component abundances.
(B) Zoomed view of one small region from (A), illustrating the consequences of high mass resolving power. A component of m/z , plus its two co-eluting isotopologues at +1 and +2 mass units, is present in just BALB/c female mouse urine. While not visible at this resolution, the two major +2 isotopologues (34S and 13C2) are distinguishable from each other and another component of m/z Several other components can also be seen, including the 425 standard (corticosterone-21-sulfate).
(C) Distribution of ion counts for each component (blue). In green is the distribution of counts for each distinct m/z, summed across components eluting at different times.
(D) Distribution of sex-specificities across components; the sex-specificity index is defined in supplementary methods.
(E) Entropy versus abundance for each component. The majority of components have high entropy, indicating relatively little specificity; specificity is not strongly coupled to abundance.
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12. Figure S3 M377 Is Present in Only Female Urine, Related to Figure 4
High-resolution m/z data for fraction eluting at 44%–46% acetonitrile.
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13. Figure S4
Validation of M377 Structure and Synthesis by HPLC-UV and Periodic Acid Reaction, Related to Figure 5 and Experimental Procedures
(A) HPLC-UV spectra of synthetic cortigynic acid (green) and purified M377 (blue). Approximately 10% of the synthetic sample corresponds to the endogenous epimer.
(B) HPLC-UV spectra of synthetic corticosteronic acid (blue) and purified M361 (red).
(C) Mass spectra for products of periodic reaction with 1, 4-pregnadien-11β, 17, 20-triol-3-one 21-carboxylic acid.
(D) Mass spectra for products of periodic reaction with M377.
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14. Figure S5
Validation of M377 Structure by HRMS and MS3, Related to Figure 6
(A) HRMS for M377in the negative ion mode.
(B) Negative ion mode after H/DX.
(C) Positive ion mode after H/DX.
(D) MS3 analysis of the product ion at m/z 189 from M377 MS2.
(E) Product ion of m/z 331 from M377 MS2.
(F) Product ion of m/z 301 from M377 MS2.
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15. Figure S6
Scheme of Fragmentation Processes Proposed for the [M-H]− Ion of M377, Related to Figure 6 and Experimental Procedures
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16. Figure S7
Specificity of Male Investigatory Behavior, Related to Figure 7
(A) Episodes of beam-breaking (black bars), with each mouse displayed on a separate row. The detector and scented swab were inserted at the beginning of the trial.
(B) LC-MS/MS mass spectrum of an HPLC fraction (water/methanol gradient) of C57BL/6 female urine extract that contains M377.
(C) P0750 did not excite additional investigation (p > 0.7, n = 10, t test).
(D) TrpC2−/−male mice did not investigate M377 and M361 more than the vehicle control.
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