This value is similar to that obtained when photolysis of caged calcium was used to stimulate release and to that observed in mouse IHCs (Beurg et al., 2010 and Beutner et al., 2001). Interestingly, this value is predominated by the superlinear component of release that is at least in part a reflection of vesicle trafficking and not only of release. The nonlinearity in

release differs from previous measurements (Schnee et al., 2005). However, a limitation to those experiments was the use of the single-sine method, which provided no direct kinetic information; rather, kinetics were inferred from responses measured after the pulse by combining responses from multiple cells and/or multiple pulses to individual cells. A comparison of data collected by using the two-sine wave technique to that previously Selisistat clinical trial obtained by using the single-sine technique confirmed that variability between and within cells may have masked the superlinear behavior of individual cells (Figure 2F). These data point out the limitations of using a technique that requires

multiple sampling to intuit kinetic information as compared to direct measurements of kinetics. To determine whether the superlinear release component was an artifact of whole-cell recording, we performed perforated-patch experiments to maintain endogenous buffering. We observed two release components in both perforated-patch recordings and whole-cell recordings by using 1 mM EGTA (Figure 3), selleck chemical indicating that the observed release properties are not due to the whole-cell recording technique. To ensure that the superlinear release component is not next unique to turtle, we recorded from rat and mouse inner hair cells (ages postnatal day P7–P15) and observed two components of release in these preparations (Figure 3C). Previous work in chick auditory hair cells also documented two release components (Eisen et al., 2004), suggesting multiple release components may be a ubiquitous feature of vesicle release in hair cells. Quantitative comparison of release properties between frequency positions requires knowing the number

of synapses present. Whole-mount papillae were double labeled with Ctbp2+PSD-95 or Ribeye+PSD-95 antibodies to count the number of functional ribbon synapses at the same tonotopic positions used in the electrophysiological analysis (n = 6). Examples from a high-frequency position are shown in Figures 4A–4F. Ribbon synapses were localized in hair cell basolateral regions (Figures 4D and 4E), were scarce above the nucleus and were typically present in series, probably corresponding to the fingerlike projections of the afferent fiber (Figures 4D and 4F, inset). No synapses were included that did not positively label adjacent pre- and postsynaptic markers (Figure 4F, inset). PSD-95 puncta that did not have a corresponding Ribeye component accounted for less than 5% of the observed puncta.