1720 S. L. Johnson and others J Physiol 587.8 each step. The overall individual C m (Fig. 2E) was found to be similar between apical (7.2 ± 0.4 fF, n = 6) and basal (7.9 ± 0.3 fF, n = 11) immature IHCs. Following repetitive long-lasting (1 s) voltage steps (200 ms interstep intervals), the cumulative C m showed saturation of SRP release in both apical and basal IHCs (Fig. 2D), which was also evident from the individual C m (Fig. 2F). The depletion time constant was 8.6 s and 4.8 s in apical and basal IHCs, respectively. The absence of the initial potentiation of SRP exocytosis in pre-hearing IHCs, which was found in adult cells (Johnson et al. 2008), is likely to be due to the recruitment of the entire pool in response to the ﬁrst step. Potentiation was evident by P16 in IHCs from both cochlear regions (data not shown). Although long-lasting (1 s) protocols are of less physiological relevance for immature spiking IHCs, it allowed us to compare the properties of SRP reﬁlling to adult IHCs (Table 1). Developmental changes in the Ca2+ sensitivity of exocytosis The Ca2+ sensitivity of neurotransmitter release in gerbil IHCs changes between immature and adult stages (Johnson et al. 2008). However, the time course of this transition is still unknown. In order to present a more comprehensive picture of the development in exocytotic Ca2+ sensitivity, values from immature (apical: P5–P6; basal: P6–P7) and adult (>P20) IHCs used in Figs 3 and 4C (see below) are derived from cells included in Johnson et al. (2008). Figure 3A shows the average size of I Ca, measured near the peak of the I –V curves (see Fig. 1B), as a function of postnatal age and IHC position in the cochlea. In order to obtain a better representation of the change in I Ca during the main stages of IHC functional development, values were pooled into three age groups (Fig. 3A and B: immature, intermediate and adult). Apical and basal IHCs showed a signiﬁcant decrease in the size of I Ca with development (Fig. 3B, overall P < 0.0001). However, during the same period, no signiﬁcant difference in C m was found between IHCs from both cochlear regions (Fig. 3C and D). The difference between the development of I Ca and C m was indicative of an increased Ca2+ efﬁciency of exocytosis (deﬁned as C m/I Ca) with maturation as shown in Fig. 3E and F (overall P < 0.0001 for both apical and basal IHCs). The Ca2+ dependence of synaptic vesicle release in immature IHCs (Fig. 4), deﬁned as the change in C m as a function of I Ca, was measured using the synaptic transfer curve (Augustine et al. 1985; Johnson et al. 2008). This was obtained by plotting C m values against the Figure 3. Changes in Ca2+ efﬁciency of exocytosis in developing gerbil IHCs A, size of ICa from near the peak of individual I–V curves (see Fig. 1B) as function of postnatal day. B, average size of ICa grouped into the three main stages of cochlear development shown in panel A: Immature, Intermediate and Adult. One-way ANOVA with Tukey’s post hoc test indicates P < 0.001 for all combinations apart from immature vs. intermediate basal cells, which was not signiﬁcant. C and D, development of C m. E and F, development of the Ca2+ efﬁciency of exocytosis. In panel F the post hoc test indicates P < 0.01 or higher apart from immature vs. intermediate cells, which was not signiﬁcant. Numbers of cells in panels A–F: apical (P5–P34): 2, 7, 1, 1, 4, 4, 5, 5, 6, 4, 3, 2, 3, 5, 6, 1; basal (P6–P30): 3, 6, 3, 4, 4, 4, 5, 5, 2, 4, 5, 3, 4, 3, 2, 1. C 2009 The Authors. Journal compilation C 2009 The Physiological Society