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Functional organization of glomerular maps in the mouse accessory olfactory bulb

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Abstract

The mammalian accessory olfactory system extracts information about species, sex and individual identity from social odors, but its functional organization remains unclear. We imaged presynaptic Ca2+ signals in vomeronasal inputs to the accessory olfactory bulb (AOB) during peripheral stimulation using light sheet microscopy. Urine- and steroid-responsive glomeruli densely innervated the anterior AOB. Glomerular activity maps for sexually mature female mouse urine overlapped maps for juvenile and/or gonadectomized urine of both sexes, whereas maps for sexually mature male urine were highly distinct. Further spatial analysis revealed a complicated organization involving selective juxtaposition and dispersal of functionally grouped glomerular classes. Glomeruli that were similarly tuned to urines were often closely associated, whereas more disparately tuned glomeruli were selectively dispersed. Maps to a panel of sulfated steroid odorants identified tightly juxtaposed groups that were disparately tuned and dispersed groups that were similarly tuned. These results reveal a modular, nonchemotopic spatial organization in the AOB.

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Figure 1: Functional presynaptic Ca2+ imaging in the AOB.
Figure 2: Overlap and distribution of adult BALB/c male and female urine activity maps.
Figure 3: Glomerular maps activated by mouse urine across sex and maturity.
Figure 4: Absolute positions and relative distances between populations of urine-responsive glomeruli.
Figure 5: Sulfated steroid VNO stimulation generates robust activity in the aAOB.
Figure 6: Glomerular activity patterns identify functional VSN classes.
Figure 7: Glomeruli of similarly tuned VSN classes are distributed across the AOB.
Figure 8: Glomerular juxtaposition does not imply sensitivity to similar odorants.

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Acknowledgements

We thank X. Fu for mass spectrometry advice and assistance. Work was funded by US National Institutes of Health, Awards F32DC009352 (J.P.M.), K99/R00DC011780 (J.P.M.), R01DC010381 (T.E.H.), R01NS068409 (T.E.H.) and F30DC011673 (G.F.H.), and National Science Foundation IGERT: Cognitive, Computational, and System Neuroscience Award DGE-0548890 (G.F.H.).

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Author notes

  1. Timothy E Holy and Julian P Meeks: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri, USA

    Gary F Hammen, Diwakar Turaga & Timothy E Holy

  2. MD-PhD Program, Washington University School of Medicine, St. Louis, Missouri, USA

    Gary F Hammen & Diwakar Turaga

  3. Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Julian P Meeks

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  1. Gary F Hammen
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  2. Diwakar Turaga
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Contributions

J.P.M. and D.T. developed the AOB imaging infrastructure, J.P.M. and G.F.H. conducted the experiments, and J.P.M., G.F.H. and T.E.H. analyzed results and wrote the manuscript.

Corresponding authors

Correspondence to Timothy E Holy or Julian P Meeks.

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Integrated supplementary information

Supplementary Figure 1 Response overlap and spatial distribution of urine-responsive glomeruli

(a) Pairwise comparison matrix for the overlapping ROI volume between all tested urine sources (ΔF/F 2%). Columns represent the reference groups and rows represent the test groups. Note: sulfatase-treated adult female urine (final row/column) activated extremely few glomeruli at this threshold. (b) Urine-selective glomerular maps for 5 experimental animals. Each glomerulus is colored using the strategy indicated in Figure 3f. The first column shows maps for clusters 1 and 2, the second 3 and 4, and the third 5 and 6. The smallest clusters, Clusters 7 and 8 are omitted from this figure. The right-most column shows clusters 1-6. Figure 3a displays experiment “2013_02_14,” Figure 3g displays experiments “2013_02_13a” and “2013_02_14,” and Figure 4 displays experiment “2013_02_13b.” Dotted lines indicate the position of the linea alba. Scale bars: 100 μm.

Supplementary Figure 2 Sulfated steroid glomerular activity across concentrations and at fast time scales.

(a) Stimulation of the VNO with 1, 10, or 100 μM P8200 or Q1570 resulted in increased fluorescent intensity and number of active glomeruli. At 100 μM, the activity increased dramatically, making discrimination of individual glomeruli more difficult. Scale bars 100 μm. (b) Percentage of signal strength (sum of suprathreshold ΔF/F intensity, normalized per experiment) for each steroid across 5 log orders of concentration. Shaded areas represent mean ± standard error of the mean across multiple experiments (P8200, N = 4; Q1570, N = 2). (c) At higher steroid concentrations, the number of steroid-responsive glomeruli in the pAOB increased for all steroids. Posterior AOB glomeruli responsive to sulfated pregnanolone P3817 were evident at lower concentrations. (d) Average change in fluorescence during stimulation during continuous 10 Hz imaging of a single frame. Colored arrowheads indicate the location of two glomeruli analyzed in e. Scale bars 100 μm. (e) Intensity changes in glomeruli indicated by the colored arrowheads in d. Stimuli were delivered for 10 s (gray regions).

Supplementary Figure 3 Absolute positions of sulfated steroid-responsive ROIs and pAOB responses to sulfated pregnanolones.

(a) Orthogonal plot of views of the AOB as visualized from the surface (top-left), posterior (bottom) and lateral (right) viewpoints. The color of the glomeruli reflects their responsivities (threshold ΔF/F > 1%). Yellow, magenta, and cyan regions indicate glomeruli responding to more than one steroid at this threshold. (b) Aggregated position information for all 1078 steroid-responsive ROIs along each of the 3 orthogonal coordinates at ΔF/F > 1% (n = 10). The dotted line along the medial/lateral (M/L) and posterior/anterior (P/A) axes refers to their position relative to the landmarks indicated by dotted lines in a. Depth was measured relative to the actual tissue surface. Asterisks denote populations which were statistically different than the majority of the other populations (p < 0.05, one-way ANOVA).

Supplementary Figure 4 VSN co-activation by sulfated steroids and intact BALB/c female urine.

(a-b) Several aAOB glomeruli were co-activated by 1:100 intact adult BALB/c female urine (bottom, red arrowheads) and 10 μM Q1570 (purple arrowhead, a) or 10 μM epitestosterone sulfate (A6940, yellow arrowhead, b). Right insets in a and b show single responses of the indicated glomeruli across 3 consecutive frames of the stack at higher zoom (image size 35 μm square). (c-d) Rendered maps of glomerular ROIs responding to 10 μM Q1570 (c, purple) or A6940 (d, orange). Steroid-responsive maps are overlaid on glomerular ROIs responsive to 1:100 dilutions of the indicated urines (ΔF/F > 2%) with overlapping voxels labeled yellow. All scale bars 100 μm. A: anterior, L: lateral. (e) The number of VSNs responding to sulfated steroids and 1:100 BALB/c female urine as measured on multielectrode arrays (p < 0.05 Wilcoxon rank-sum test, firing rate change > 1 Hz compared to controls; Meeks et al, 2010). These estimates are normalized to the total number of neurons responsive to the steroid at each concentration or 1:100 urine (left-most plots). Note the concentration-dependent decrease in the percentage of co-responsive neurons for Q1570. Because nearly all 100 nM Q1570-responsive VSNs also respond to BALB/c female-urine, this would be consistent with an endogenous concentration of Q1570 in BALB/c female urine of 1 to 10 μM (100 x [10 to 100 nM]), consistent with estimates from Nodari et al, 2008. (f) The percentage of steroid-responsive voxels that overlapped urine-responsive voxels (N = 7 for intact adult urines, N = 4 for all others). (f) Normalized voxel overlap between steroid-responsive glomeruli and mouse urine across sex and sexual maturity. Error bars reflect s.e.m.

Supplementary Figure 5 ROI functional clusters across threshold.

(a-b) Plots of the normalized ΔF/F for all ROIs across all experiments (N = 10). Each colorized column represents 1 ROI. The order of the ROIs within each cluster grouping (delineated by vertical black bars) was randomized. Insets to the right of each plot show the first 3 multidimensional scaling dimensions from independent clustering runs. The color of each point matches the VSN class color values in c. (c) Proportion of total ROIs in each cluster across all thresholds tested. Colorized list at right indicates the color assigned to each VSN class.

Supplementary Figure 6 Absolute positions of steroid-responsive glomerular classes.

(a-c) Plots of ROI relative occupancy by steroid-responsive glomerular class (n = 10, cluster definitions shown in Fig. 6b). The observed relative occupancy of glomeruli of each class (solid colored lines) was compared to the 95th percentile (gray shaded region) from shuffled maps (100,000 shuffle tests per experiment, n = 10 experiments). Lines exceeding the shaded regions indicate preferential occupancy of particular positions by glomeruli of that class.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1853 kb)

Supplementary Methods Checklist

(PDF 425 kb)

Supplementary Table 1

Sulfated steroids used in this study. (XLSX 10 kb)

Animation of single-stack image acquisition using OCPI.

The left panel shows a side view cross-section through the tissue, with the tissue surface oriented towards the top. On the right, each full-frame image acquired at the light sheet position noted by the cyan line is shown. Note that although the movie plays in two scan directions, image stacks were acquired in only one, followed by a delay period during which images were written to disk and the light sheet was move back to the start position. (MOV 422 kb)

Raw ΔF/F GCaMP2 fluorescence visualized in synaptic terminals ramifying in the accessory olfactory bulb (AOB) in response to delivery of 10 μM sulfated steroids to the vomeronasal organ.

The image is viewed from above the AOB surface, with the medial side (including part of the vomeronasal nerve) on the left, and the anterior portion at the top. Movie is rendered at 10× actual speed. Changes in fluorescence are color-coded on a red–blue scale, with red indicating activation and blue indicating inhibition. Maximum redness indicates ΔF/F ≥ 6%, and maximum blueness indicates ΔF/F ≤ −6%. For clarity, only voxels with ΔF/F > 2% or ΔF/F < −2% are shown. During stimulus presentation, the scale bars (100 μm) change from colored to solid white. Note that ΔF/F signals in a small portion of the medial AOB was suppressed for display purposes. This region underwent spurious fluctuations in intensity due to movement of small untethered blood vessels (not seen) in the imaging path. (MOV 2145 kb)

Continuous 10-Hz imaging of the single image frame shown in Supplementary Figure 2d,e.

P8200 and Q1570 (10 μM concentration) were delivered at the times indicated by the presence of the text of the stimulus identity. Red and blue hues indicate increases and decreases in stimulus intensity compared to the pre-stimulus period. Maximum hue values represent changes of ±5% (ΔF/F). Striped bright red/blue regions at the tissue surface reflect changes in surface shadows (probably due to loose vasculature). These effects are not reproducible across trials, and are excluded from analysis of volumetric movies by virtue of having a low RRI. (MOV 1606 kb)

Functional glomerular maps produced by stimulating vomeronasal neurons with sulfated steroids.

Regions responding with RRI >0.6 across five randomized interleaved trials are highlighted in blue (E0893, 17α–estradiol-3-sulfate), green (P3817, allopregnanolone-3-sulfate) and red (P8200: epipregnanolone-3-sulfate). This static three-dimensional map is sequentially rotated about the medial–lateral axis (giving anterior–posterior tilt) and anterior–posterior axis (giving medial–lateral tilt) to visualize depth. Scale bars, 100 μm. (MOV 1947 kb)

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Hammen, G., Turaga, D., Holy, T. et al. Functional organization of glomerular maps in the mouse accessory olfactory bulb. Nat Neurosci 17, 953–961 (2014). https://doi.org/10.1038/nn.3738

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