1722 S. L. Johnson and others J Physiol 587.8 extracellular Ca2+. Under these experimental conditions C m was very small and could not be accurately measured. Therefore, I Ca and C m were recorded from OHCs in 5 mM extracellular Ca2+ (Fig. 6A). Despite this, C m was still relatively small and in order to obtain reliable measurements, especially for small I Ca, traces recorded from all OHCs within each cochlear region were averaged. Higher Ca2+ concentrations could not be tested because they rapidly cause the preparation to deteriorate when working at body temperature (Johnson et al. 2005). The size of both the peak I Ca (apical OHCs: −113 ± 18 pA, n = 4; basal OHCs: −83 ± 15 pA, n = 4) and maximal C m (apical: 5.0 fF; basal: 5.3 fF), elicited using 100 ms steps, did not vary signiﬁcantly along the cochlea (Fig. 6B). This indicates that the Ca2+ efﬁciency of exocytosis was similar between apical and basal OHCs. Although the size of I Ca was comparable to that recently reported in Otof control OHCs (Beurg et al. 2008), C m was surprisingly about 9 times smaller. At ﬁrst we thought that this could be down to the different animal species used for the two studies (control Otof mice and gerbils), so we performed additional experiments in P3 OHCs from normal C57B mice and Wistar rats. Despite their larger peak I Ca (mouse: −190 ± 19 pA, n = 6; rat: −151 ± 26 pA, n = 6), the maximal size of C m remained relatively small (mouse: 7.0 ± 0.7 fF; rat: 7.8 ± 1.0 fF) and similar to that of gerbil OHCs (Fig. 6C). For the results obtained from gerbil OHCs, synaptic transfer functions (see above) were used to measure the Ca2+ dependence of exocytosis (Fig. 6C). The power values were 0.7 ± 0.1 (n = 4) in apical and 1.1 ± 0.1 (n = 4) in basal OHCs (Fig. 6C), indicating a near linear relation between Ca2+ entry and exocytosis as previously described (mouse Otof control OHCs: Beurg et al. 2008). Although we did not measure directly the release kinetics of the RRP (the very small C m in OHCs limited the accuracy of recording responses to very short stimuli) we estimated its overall size using a 100 ms voltage step. This is likely to be a reliable measure since the same stimulus almost completely isolated the RRP in mouse OHCs (Beurg et al. 2008). Using this method, the RRP in gerbil OHCs consisted of about 135 vesicles and 143 vesicles in apical and basal cells, respectively. In agreement with the ﬁndings in IHCs (Fig. 1E), the number of CtBP2 positive dot-like spots per OHC (i.e. active zones) was similar along the cochlea (Fig. 6D: 3rd row OHCs: apical 9; basal 11). The size of these spots were usually larger than that seen in IHCs (see also Beurg et al. 2008) most likely reﬂecting multiple synaptic ribbons at OHC active zones (Shnerson et al. 1982). Considering that on average two synaptic ribbons could be present at each active zone, the number of vesicles per ribbon in the RRP in immature OHCs is likely to be about eight. The intensity and distribution of otoferlin labelling was similar among OHCs (Fig. 6D). Figure 5. Coupling between Ca2+ channels and the RRP in immature gerbil IHCs A and B, C m recordings from apical (A) and basal (B) immature IHCs in response to a 100 ms voltage step (from −81 mV to around −11 mV) using 0.1 mM, 0.6 mM and 1 mM intracellular BAPTA. C and D, average C m from apical and basal IHCs, respectively, in response to voltage steps between 5 ms and 100 ms using the above BAPTA concentrations. E, average C m (top panel) and peak ICa (bottom panel) at 50 ms, in order to measure the isolated RRP, from data shown in C and D. C m values obtained in EGTA from Fig. 1C were also included. Number of cells in EGTA (1 mM) and BAPTA (0.1, 0.6, 1 mM) are: apical: 6, 3, 3, 3 (P5–P8); basal: 10, 1, 4, 4 (P6–P8). C 2009 The Authors. Journal compilation C 2009 The Physiological Society