Article Figures & Data
Figures
- Figure 1.
TMEM16A and TMEM16B are expressed at the apical surface of the vomeronasal epithelium. A, Confocal micrographs of coronal sections of the VNO from WT, Tmem16a cKO, or Tmem16b KO mice, as indicated, immunostained with antibody against TMEM16A (green) and TMEM16B (red). No immunoreactivity to TMEM16A or TMEM16B was detectable in Tmem16a cKO or Tmem16b KO mice, respectively. B, Confocal micrographs of VNO sections from Tmem16b KO mice expressing mCherry in the membrane of cells that normally express TMEM16B, immunostained with antibody against mCherry (red) and OMP (green). At the bottom, a magnification showing the co-expression of mCherry and OMP. Note that mCherry is expressed, as expected, in the membrane of entire VSNs and not only at the apical side. Cell nuclei were stained by DAPI (blue).
- Figure 2.
Ca2+-activated chloride currents in VSNs. A, Representative whole-cell recordings obtained with an intracellular solution containing 0 or 1.5 μm Ca2+ from VSNs from WT, Tmem16a cKO, or Tmem16b KO mice, as indicated. The voltage protocol is in the center of the figure. B, Scatter dot plot with average ± SEM of steady-state current amplitudes measured at −100 or +100 mV with intracellular pipette solution containing nominally 0 Ca2+ from Tmem16a cKO (red, n = 6), or 1.5 μm free Ca2+ from Tmem16a cKO (green, n = 14), WT (black, n = 10), and Tmem16b KO (brown, n = 8) mice (**p < 0.01; Dunn–Hollander–Wolfe test after Kruskal–Wallis analysis at –100 and +100 mV), ns: not significant.
- Figure 3.
Tmem16a or Tmem16b deletion does not affect voltage-gated currents and membrane properties of VSNs. A, Representative whole-cell recordings of VSNs obtained from the indicated mouse lines. The holding potential was −90 mV and voltage steps from −110 to +30 mV with 20-mV increment were applied. B, Average ± SEM of the IV relationships of inward currents (circles) and steady-state outward currents (triangles) from WT (black, n = 31), Tmem16a cKO (green, n = 23), or Tmem16b KO (brown, n = 23) VSNs. Values were taken at the negative peak and at the end of voltage step as indicated by the symbols in A. C, Scatter dot plot with average ± SEM of the membrane resistance (Rm) recorded in voltage-clamp mode (WT, n = 31; Tmem16a cKO, n = 23; Tmem16b KO, n = 23. Kruskal–Wallis analysis p = 0.21). D, Scatter dot plot with average ± SEM of resting membrane potential (RMP) recorded in current-clamp configuration (WT, n = 28; Tmem16a cKO, n = 22; Tmem16b KO, n = 20. Kruskal–Wallis analysis p = 0.68).
- Figure 4.
Tmem16a deletion modifies VSNs spontaneous firing activity. A, Raster plots of recordings of the spontaneous activity of VSNs from WT (black, n = 50), Tmem16a cKO (green, n = 52), or Tmem16b KO (brown, n = 34) mice. Each row shows spike activity from a different VSN. B, Mean frequency of spontaneous activity from the cells shown in A. C, ISI distributions of spontaneous firing from a subset of cells with mean frequency >0.1 Hz (bin = 10 ms). Values were normalized to the area under each curve to show the spike percentages for WT (black, n = 25), Tmem16a cKO (green, n = 18), or Tmem16b KO (brown, n = 20) mice. D, Cumulative fraction of ISI distributions (***p < 0.001, Kolmogorov–Smirnov test).
- Figure 5.
Tmem16a cKO effect on VSN density and glomerular volume. A–C, top, Representative 3D projection image of a VNE from a V2R1b-WT mouse. Zoom-in indicated by white rectangles. Arrows indicate example fluorescently labeled V2R1b neurons. D, Scatter dot plot (average ± SEM) of VSN density in V2R1b-WT and V2R1b-Tmem16a cKO mice (Student’s t test, p = 0.1876). E–G, top, Representative 3D projection image of AOB from V2R1b-WT mouse. Zoom-in indicated by white rectangles. Arrows indicate exemplary fluorescently labeled V2R1b glomerular structures. H, Scatter dot plot with average ± SEM of glomerular volume in V2R1b-WT and V2R1b-Tmem16a cKO mice (Student’s t test, p = 0.2573).
- Figure 6.
Evoked firing activity. A–C, Representative loose-patch recordings of a VSN from each indicated mouse line stimulated with high K+ (A), diluted artificial urine as control (B), or diluted urine (C). Black bars indicate the time of stimulus presentation. D, Average firing activity (1-s bin width) for the responses to diluted artificial urine (empty circles) or urine (filled circles) from B, C. Error bars represent SEM.
- Figure 7.
Patterns of evoked firing activity are modulated by TMEM16A and TMEM16B. A, Raster plots showing the responses of VSNs from each indicated mouse line to diluted urine. Black bars indicate the time of stimulus presentation (10 s). Every neuron was tested at least three times. Horizontal dotted lines delimit multiple recordings from each VSNs. Numbers on the left indicate the neuron number. B, Histograms of the number of spikes from neurons in A (100-ms bin width). Each neuron was averaged to equally contribute to spikes count. C, D, Scatter dot plots with average ± SEM of the mean frequency (C, Kruskal–Wallis analysis p = 0.37) and duration (D, Kruskal–Wallis analysis p = 0.79) of the responses to urine of VSNs from WT (black, n = 17), Tmem16a cKO (green, n = 22), or Tmem16b KO (brown, n = 15) mice. E, Normalized ISI distributions of firing activity (20-ms bin width). F, Cumulative fraction of the ISI distributions shown in D (***p < 0.001, Kolmogorov–Smirnov test).
Movies
- Movie 1.
Light-sheet fluorescence microscopy of cleared VNOs. Examples show intact transparent organs from wild type control (V2R1b-GFP - TMEM16Afl/fl - OMP/OMP; left) and TMEM16A knockout (V2R1bGFP - TMEM16Afl/fl - OMP/Cre; right) animals. Three-dimensional rendering displays the VNOs rotating around their longitudinal axes.
- Movie 2.
Light-sheet fluorescence imaging of cleared AOBs. Examples show intact transparent olfactory bulb samples from wild type control (V2R1b-GFP - TMEM16Afl/fl - OMP/OMP; left) and TMEM16A knockout (V2R1bGFP - TMEM16Afl/fl - OMP/Cre; right) mice. Nuclei are stained with DRAQ5 (grey scale). Green fluorescence of V2R1b-positive vomeronasal sensory neuron axons becomes apparent upon fiber coalescence and convergence in AOB glomeruli.
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