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. 2020 Jun 25;11:3208. doi: 10.1038/s41467-020-17003-z

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Coupled IHCs (delineated in color) were patch-clamped and loaded with the calcium indicator Fluo-4FF (F4FF) and TAMRA-fluorescent CtBP2-binding peptide. C1 (red) is the patched IHC, C2 (green) are coupled and adjacent to C1, C3 (blue) is coupled to C2 and C4 (violet). The red box shows the imaging plane. b Ca²⁺-signals co-localizing with the presynaptic ribbons are observable. Example ΔF/F₀ pictures from a single plane of the Ca²⁺-increments (black arrows) of 2 (left), 3 (middle), and 4 (right) coupled IHCs. Non-coupled IHCs are delineated in dashed lines. c The graph displays the fluorescence versus voltage-relationship (ΔF/F₀ versus depolarization level), showing presynaptic activity in the coupled IHCs. Left inset show individual values and percentiles (10th, 25th, 50th, 75th and 90th) of ΔF_max/F₀ of synapses in C1 (37 spots in 8 IHCs, N = 8) and C2 (43 spots in 9 IHCs, N = 8). Means are indicated as crosses. Right inset displays averaged fractional activation curves of a subset of the spots for which the ΔF/F₀ could be fitted faithfully (C1: 17 spots in 7 IHCs, N = 7; C2: 30 spots in 8 IHCs, N = 8). Mean (line) ± S.E.M. (shaded areas) are displayed. IHC inner hair cell. Scale bar: b = 5 µm.