J Physiol 587.8 Exocytosis at hair cell ribbon synapses 1721 corresponding I Ca between −71 mV and −11 mV (see Fig. 1B). Data were approximated using a power function: Cm = c I CNa where c is a scaling coefﬁcient and the power is N . For this comparison the peak I Ca was used, instead of its time integral, to avoid possible errors due to the pre- sence of the residual unblocked K+ current as previously described (Johnson et al. 2005, 2008). Figure 4A and B shows averaged transfer functions from apical and basal IHCs that have been grouped into pre- and post-hearing stages (see Fig. 4C for a better overview of the age range selected). For basal IHCs, an additional group showing the transfer function at the onset of hearing is included. A detailed representation of the developmental change in the exocytotic Ca2+ dependence as a function of days after birth is shown in Fig. 4C. Fits to the data in Fig. 4C are according to a sigmoidal logistic growth curve: (Nmax − Nmin) N = Nmin + 1 + exp (−k(t − thalf )) where k is a slope factor, t half is the age where N is halfway between the maximal (N max) and minimal (N min) power. The ﬁts gave values for t half and k of P13.7 and 2.6 day−1 in apical and P10.6 and 2.5 day−1 in basal IHCs. The linearization in Ca2+ dependence occurred very rapidly from around the beginning of the second postnatal week in basal IHCs and was complete by the onset of hearing at P12. Contrary to basal IHCs, the high Ca2+ dependence of exocytosis in apical cells was only slightly reduced from just after the onset of hearing. (1) (2) Functional coupling between Ca2+ channels and the RRP in pre-hearing IHCs The developmental changes in the exocytotic Ca2+ dependence in apical and basal IHCs (Fig. 4) could arise from variations in the coupling between Ca2+ inﬂux and the release-ready pool (RRP) of vesicles. In adult gerbil IHCs Ca2+ channels are localized within a nanodomain of the synaptic machinery (Johnson et al. 2008). Therefore, we investigated whether the higher exocytotic Ca2+ dependence in pre-hearing IHCs resulted from a weaker (microdomain) coupling to Ca2+ channels. The distance between Ca2+ channels and presynaptic release sites was estimated by measuring exocytosis in both apical and basal IHCs whilst using either intracellular EGTA (1 mM) or different concentrations of the faster Ca2+ chelator BAPTA (Fig. 5A–D), which is capable of buffering Ca2+ elevations closer to their source (Neher, 1998). For these experiments we used a similar voltage protocol to that described in Fig. 1D (voltage steps between 5 ms and 100 ms) in order to recruit mainly the RRP. The release of the RRP was unaffected when pre-hearing C 2009 The Authors. Journal compilation C 2009 The Physiological Society IHCs were buffered with 0.1 and 0.6 mM BAPTA (Fig. 5E). However, 1 mM BAPTA almost completely blocked RRP exocytosis in both cochlear regions (apical: P < 0.01; basal: P < 0.001; relative to recordings in 1 mM EGTA). This indicates a similar distance between Ca2+ channels and release sites in immature IHCs positioned at each end of the cochlea (in the range of 40 nm: Naraghi & Neher, 1997). Neurotransmitter release in immature OHCs A recent investigation has shown that pre-hearing OHCs from Otofoferlin (Otof ) control mice exhibit robust neuro- transmitter release (Beurg et al. 2008). This led us to look for possible position-dependent differences in OHC exocytosis using immature P3 gerbils. Initially, we performed experiments using physiological 1.3 mM Figure 4. Changes in Ca2+ dependence of exocytosis in gerbil IHCs A and B, examples of synaptic transfer functions obtained by plotting average C m against the corresponding ICa from I–V and C m–V curves (as in Fig. 1B: voltage range –71 mV to −11 mV) of apical and basal IHCs, respectively. Data were grouped into age ranges at around the onset of hearing at P12. Fits are according to eqn (1). Values of N were: apical 4.3 (P8–P12, n = 12) and 3.0 (P14–P16, n = 10); basal 4.2 (P6–P8, n = 4), 1.7 (P10–P12, n = 12) and 1.1 (P14–P16, n = 10). C, developmental changes in N from both apical and basal IHCs as a function of days. Data points were obtained by averaging N values from ﬁts to synaptic transfer functions of single cells using eqn (1). Note that basal IHCs start to mature a few days earlier than apical IHCs. The ﬁts to the averaged data points are according to eqn (2). Numbers of cells: apical (P5–P34): 2, 7, 1, 1, 4, 4, 5, 5, 2, 1, 2, 3, 5, 2, 1; basal (P6–P30): 3, 6, 3, 4, 4, 4, 5, 5, 2, 2, 3, 2, 2, 2, 1.