Figure 5The 2D plots of the first three principal components (PCs), labeled based on the clusters obtained by K-means clustering algorithm. 3D plot showing the first three PCs. Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters).Dynamic ranges of corresponding Ca2+-influx (gray) and glutamate release (color-coded) with V1/2 depicted. Note that the synapses from cluster 1 (purple), employing Ca2+-nanodomain-like control of release (m = 1.3), exhibit wider dynamic range than the other synapses. Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses). Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters). E' The relation between Ca2+-influx and glutamate release of given synapses in the voltage range of -57 to -17 mV plotted with 1 mV increments. A power function was fitted until the 25% of normalized iGluSnFR AUC(ΔF/F0). Synapses showed different Ca2+-dependencies (m = 2.0, 1.3, 7.4; green, purple, orange). '' Dynamic ranges of corresponding Ca2+-influx (gray) and glutamate release (color-coded) with V1/2 depicted. Note that the synapses from cluster 1 (purple), employing Ca2+-nanodomain-like control of release (m = 1.3), exhibit wider dynamic range than the other synapses. Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses). The Ca2+-cooperativity (m) (G) show differences for the three clusters. Cluster 1 is composed of linear synapses with wider dynamic range and lower threshold compared to the other clusters. Pillar synapses are depicted as white-filled circles, and modiolar ones are depicted as gray-filled circles. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. The clusters were compared The dynamic range (H), and glutamate release threshold (V10) (I) show differences for the three clusters. Cluster 1 is composed of linear synapses with wider dynamic range and lower threshold compared to the other clusters. Pillar synapses are depicted as white-filled circles, and modiolar ones are depicted as gray-filled circles. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. The clusters were compared by one-way ANOVA test, followed by a posthoc Tukey's test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 The proposed model for sound intensity encoding in an IHC. Differences in the presynaptic control of release, in terms of Ca2+-signaling and Ca2+ channel-exocytosis coupling, enable IHC to diversify the transfer functions of individual synapses for the same receptor potentials.