Fig. 5

Low [K⁺] and gap junctions may facilitate presynaptic activity. a Presynaptic Ca²⁺ jRCaMP1a Ca²⁺ signals during a 2-s 5?Hz stimulus. Spatial patterns of jRCaMp1a Ca²⁺ signals during stimulation are colorized according to the ?F/F heat maps and superimposed onto a pre-stimulus baseline jRCaMP1a image (a, left panel). b Image of the same neuromast as a after labeling with the K⁺ indicator, APG-2. The active cells in a and b are marked with asterisks. c Quantification of APG-2 intensity shows that active cells (341.1 a.u.?±?33.17, n?=?14 cells) have lower resting [K⁺]_in levels than silent cells (520.1 a.u.?±?28.37, n?=?46 cells) p?=?0.001. d APG-2 dye labeling before (d, left panel) and after (d, right panel) FFA treatment to block gap junctions. e, f Quantification of APG-2 intensity shows 25?µm FFA significantly increases [K⁺]_in levels in hair cells (naïve: 950.2 a.u.?±?67.9; after FFA: 1105 a.u.?±?99.7, n?=?48 cells, p?=?0.01) and in supporting cells (naïve: 767.1 a.u.?±?34.4; after FFA: 845.1 a.u.?±?50.3, n?=?48 cells, p?=?0.015). g?i, k?m Mechanosensitive and presynaptic GCaMP6s Ca²⁺ signals within the same cells before and after application of 25??M FFA. Mechanosensative (h) and presynaptic (l) Ca²⁺ signals during a 2-s 5?Hz stimulus prior to drug treatment; 25??M FFA does not alter mechanotransduction (i) but decreases presynaptic Ca²⁺ responses (m). Spatial patterns of GCaMP6s Ca²⁺ signals during stimulation are colorized according to the ?F/F heat maps and superimposed onto baseline GCaMP6s images (g, k). j Quantifications of the mechanosensitive Ca²⁺ signals show no significant differences before (86.10%?±?7.55) and after 25??M FFA (85.18%?±?6.64), n?=?60 hair cells, p?=?0.76. n In the same cells as j, presynaptic Ca²⁺ signals (41.55%?±?4.85) are significantly decreased after FFA application (20.27%?±?3.07), n?=?60 cells, p?<?0.0001. A Mann?Whitney test was used in c, a Wilcoxon test was used in e, f, j, and n; *p?<?0.05, **p?<?0.01, ****p?<?0.0001. Scale bars?=?5??m