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. 2018 Jan 12;7:e29275. doi: 10.7554/eLife.29275

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© 2018, Jean et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — (A, B) IHCs were patch-clamped at the modiolar basolateral face, loaded with TAMRA-CtBP2-binding peptide and the low affinity Ca²⁺-indicator Fluo-4FF, and scanned in the red channel after loading for 4 min to image TAMRA-labeled ribbons, nuclei, and cytosol. 3D projection of TAMRA fluorescence shows the absence of ribbons in RBE^KO/KO IHCs (B: 3D projection and red channel). Voltage-ramps from −87 to +63 mV during 150 ms (A: left top) were used to trigger synaptic hotspots of Fluo-4FF fluorescence (A: left middle, 10 AZs in one exemplary RBE^WT/WT IHC, B: green channel, marked by arrowheads; ΔF: average of the nine brightest pixels (red square)) and IHC Ca²⁺-influx (A, left bottom). Ca²⁺-imaging proceeded from the IHC bottom to the most apical ribbon in RBE^WT/WT, and from IHC bottom to +12 µm (typically reaching the bottom of nucleus) in RBE^KO/KO. Scale bar = 5 µm. (C) FV-relationship (ΔF/F₀ vs. depolarization level in ramp, protocol as in A): approximating the voltage-dependence of synaptic Ca²⁺-influx.Mean (line) ± S.E.M. (shaded areas) are displayed as for (D). (Ci) ΔF_max/F₀ was calculated by averaging 5 values at the FV-peak (between the dotted lines) and was comparable between RBE^WT/WT (n = 78 AZs for 15 cells, N = 8) and RBE^KO/KO IHCs (n = 61 AZs for 15 cells, N = 7) (p=0.20, Mann-Whitney-Wilcoxon test). Box plots show 10, 25, 50, 75 and 90^th percentiles with individual data points overlaid, means are shown as crosses, as for (D, F). (D) Fractional activation curves derived from fits to the FV-relationships (C) were fitted to a Boltzmann function. Mean (line) ± S.E.M. (shaded areas) are displayed. (Di) The voltage for half-maximal activation V_h was significantly different between RBE^WT/WT (n = 68 AZs for 15 IHCs, N = 8) and RBE^KO/KO (n = 55 AZs for 15 IHCs, N = 7) AZs (p=0.0029, t-test), while the voltage-sensitivity or slope factor k (Dii) not (p=0.42, t-test). (E) Exemplary ΔF pictures of Fluo-4FF hotspots at RBE^WT/WT (left) and RBE ^KO/KO (right) synapses fitted and overlaid by 2D-Gaussian functions to estimate spatial extent as full width at half maximum (FWHM) for the short axis (S.A.) and the long axis (L.A.). Scale bar = 1 µm. (F) Ribbonless synapses of RBE^KO/KO IHCs showed a greater spatial spread of the Fluo-4FF fluorescence change. FWHM calculated from the Gaussian fitting to the Fluo-4FF fluorescence hotspot was larger for both axes in RBE^KO/KO (n = 61 AZs for 15 IHCs, N = 8) compared to RBE^WT/WT (n = 74 AZs for 15 IHCs, N = 7) (L.A.: p=0.00016; S.A.: p=0.0029, t-test).

Figure 6—figure supplement 1. — (A, B) IHCs were patch-clamped at the modiolar basolateral face, loaded with TAMRA-CtBP2-binding peptide and the low affinity Ca²⁺-indicator Fluo-4FF, and scanned in the red channel after loading for 4 min to image TAMRA-labeled ribbons, nuclei, and cytosol. 3D projection of TAMRA fluorescence shows the absence of ribbons in RBE^KO/KO IHCs (B: 3D projection and red channel). Voltage-ramps from −87 to +63 mV during 150 ms (A: left top) were used to trigger synaptic hotspots of Fluo-4FF fluorescence (A: left middle, 10 AZs in one exemplary RBE^WT/WT IHC, B: green channel, marked by arrowheads; ΔF: average of the nine brightest pixels (red square)) and IHC Ca²⁺-influx (A, left bottom). Ca²⁺-imaging proceeded from the IHC bottom to the most apical ribbon in RBE^WT/WT, and from IHC bottom to +12 µm (typically reaching the bottom of nucleus) in RBE^KO/KO. Scale bar = 5 µm. (C) FV-relationship (ΔF/F₀ vs. depolarization level in ramp, protocol as in A): approximating the voltage-dependence of synaptic Ca²⁺-influx.Mean (line) ± S.E.M. (shaded areas) are displayed as for (D). (Ci) ΔF_max/F₀ was calculated by averaging 5 values at the FV-peak (between the dotted lines) and was comparable between RBE^WT/WT (n = 78 AZs for 15 cells, N = 8) and RBE^KO/KO IHCs (n = 61 AZs for 15 cells, N = 7) (p=0.20, Mann-Whitney-Wilcoxon test). Box plots show 10, 25, 50, 75 and 90^th percentiles with individual data points overlaid, means are shown as crosses, as for (D, F). (D) Fractional activation curves derived from fits to the FV-relationships (C) were fitted to a Boltzmann function. Mean (line) ± S.E.M. (shaded areas) are displayed. (Di) The voltage for half-maximal activation V_h was significantly different between RBE^WT/WT (n = 68 AZs for 15 IHCs, N = 8) and RBE^KO/KO (n = 55 AZs for 15 IHCs, N = 7) AZs (p=0.0029, t-test), while the voltage-sensitivity or slope factor k (Dii) not (p=0.42, t-test). (E) Exemplary ΔF pictures of Fluo-4FF hotspots at RBE^WT/WT (left) and RBE ^KO/KO (right) synapses fitted and overlaid by 2D-Gaussian functions to estimate spatial extent as full width at half maximum (FWHM) for the short axis (S.A.) and the long axis (L.A.). Scale bar = 1 µm. (F) Ribbonless synapses of RBE^KO/KO IHCs showed a greater spatial spread of the Fluo-4FF fluorescence change. FWHM calculated from the Gaussian fitting to the Fluo-4FF fluorescence hotspot was larger for both axes in RBE^KO/KO (n = 61 AZs for 15 IHCs, N = 8) compared to RBE^WT/WT (n = 74 AZs for 15 IHCs, N = 7) (L.A.: p=0.00016; S.A.: p=0.0029, t-test).