J Physiol 587.8 Exocytosis at hair cell ribbon synapses 1717 resistance (P5–P16 IHCs: 5.8 ± 0.1 M , n = 78; gerbil OHCs: 6.5 ± 0.6 M , n = 9; mouse OHCs: 6.7 ± 0.5 M , n = 6; rat OHCs: 7.3 ± 0.2 M , n = 6) and for a liquid junction potential (−11 mV). IHC membrane capacitance (C m) was 8.8 ± 0.2 pF (P5–P16: n = 78). OHC C m was 6.2 ± 0.3 pF in gerbils (n = 9), 6.0 ± 0.2 pF in mice (n = 6) and 6.9 ± 0.8 pF in rats (n = 6). Statistical comparisons of means were made by Student’s two-tailed t -test or, for multiple comparisons, analysis of variance, one-way ANOVA followed by Tukey’s test. Two-way ANOVA, followed by Bonferroni’s test, was used to compare data sets from apical and basal IHCs. Means are quoted ± S.E.M. and P < 0.05 indicates statistical signiﬁcance. Immunocytochemistry Immunostaining of gerbil IHCs was performed using otoferlin and CtBP2 antibodies as previously described (Schug et al. 2006; Johnson et al. 2008). Animals not processed in the UK were killed by CO2 asphyxiation in accordance with the ethical guidelines approved by the University of T u¨bingen and the Tierschutzgesetz (Germany). Sections were viewed using an Olympus AX70 microscope equipped with epiﬂuorescence illumination and motorization in the z-axis. Images were acquired using a CCD camera and the imaging software Cell∧F (OSIS GmbH, M u¨nster, Germany). For ribbon counts, cryo-sectioned cochleae were imaged over a distance of 8 μm with the complete coverage of the IHC nucleus and beyond in an image-stack along the z-axis (z-stack). Typically the z-stack consisted of 30 layers with a z-increment of 0.276 μm, for each layer one image per ﬂuorochrome was acquired. To perform ribbon counts, z-stacks were three-dimensionally deconvoluted using Cell∧F’s RIDE module with the Nearest Neighbour algorithm and VoxelViewer. Figures 1E and 6D are representative single-layer images at the hair-cell nuclear plane. CtBP2 immunopositive ribbons and otoferlin staining were from apical and mid-basal immature IHCs from three gerbils (P4, P7 and P8). For each age tested between two and nine IHCs and one and ﬁve OHCs were analysed. Results Neurotransmitter release from pre-hearing gerbil IHCs Tonotopic differences in the kinetic properties of synaptic vesicle release have recently been shown in adult gerbil IHCs (Johnson et al. 2008). Therefore, in this study we ﬁrst investigated whether such kinetic differences were already present during pre-hearing stages (≤P12). A typical example of Ca2+ currents (I Ca) and the corresponding C 2009 The Authors. Journal compilation C 2009 The Physiological Society C m recorded from immature apical- and basal-coil gerbil IHCs is shown in Fig. 1A and B. Responses were obtained using a 100 ms depolarizing voltage step (10 mV nominal increments) from −81 mV. The kinetics of neurotransmitter release in pre-hearing IHCs (P5–P7) was investigated by measuring C m near the peak I Ca by varying the stimulus duration (from 2 ms to 1.5 s), which allowed the investigation of synaptic vesicle pool emptying (Fig. 1C). While relatively short stimuli revealed the number of vesicles in the readily releasable pool (RRP), longer steps induced the release of vesicles from a secondarily releasable pool (SRP) that is located further away from the Ca2+ channels (Moser & Beutner, 2000). Voltage steps of up to about 100 ms in apical and 50 ms in basal IHCs seemed to recruit mainly the RRP since C m responses could be approximated with a single exponential (Fig. 1D). The higher C m measured at 100 ms from some basal IHCs is likely to be caused by the earlier recruitment of the SRP. This was responsible for the apparently higher Ca2+ efﬁciency of exocytosis in early postnatal basal IHCs compared to age-matched apical cells (Johnson et al. 2008). The available RRP consisted of 400 and 510 synaptic vesicles in apical and basal IHCs, respectively, and had a similar initial release rate (Table 1). In order to determine whether the single ribbon synapse vesicle release rate varied as a function of IHC position along the cochlea, we performed ribbon counts on immature gerbil IHCs (Fig. 1E) using an antibody against the presynaptic ribbon component RIBEYE (CtBP2: Schmitz et al. 2000). The number of synaptic ribbons per IHC was similar in both regions of the cochlea (apical IHCs: 36.4 ± 1.2, n = 15; basal: 37.3 ± 0.8, n = 16) giving a release rate per ribbon of 355 and 360 vesicles s−1 in apical and basal IHCs, respectively, signiﬁcantly different from that measured in basal adult cells only (Table 1: P < 0.005). The estimated number of readily releasable vesicles per ribbon was 10 in apical and 14 in basal immature IHCs, assuming that each active zone contributes equally to transmitter release. The size of the SRP (Fig. 1C) was also similar among immature IHCs and showed signs of saturation within the 1.5 s tested, not observed in adult IHCs (Johnson et al. 2008). An estimation of the SRP release rate was obtained by ﬁtting the linear range of the data points (Fig. 1C, apical: 200–800 ms; basal: 100–600 ms). Linear regression indicated slopes of 216 vesicles s−1 (apical IHCs) and 214 vesicles s−1 (basal) per ribbon. Figure 1E also shows the distribution of otoferlin, the proposed Ca2+ sensor of exocytosis in cochlear hair cells (Roux et al. 2006; Beurg et al. 2008). Otoferlin was detected throughout the cytoplasm of immature IHCs with a higher expression level at the active zones (in the same region as RIBEYE) and usually below the cell’s cuticular plate, where synaptic vesicle internalization is likely to occur (Griesinger et al. 2005). Although some variability in staining intensity was observed among preparations, we