J Physiol 587.8 Discussion In the present study we have investigated the biophysical properties of neurotransmitter release at ribbon synapses of immature mammalian hair cells using capacitance measurements. Recordings were performed using body temperature and physiological extracellular Ca2+. We found that, in contrast to adult animals (Johnson et al. 2008), the kinetic properties of exocytosis in immature IHCs were similar between apical and basal cells. However, the developmental time course of the exocytotic Ca2+ dependence differed in IHCs from the two cochlear regions. We have also provided an indication of the coupling between Ca2+ channels and release sites in immature IHCs. Finally, C m in immature OHCs was much smaller compared to that of IHCs, consistent with there being fewer synaptic active zones in OHCs. Neurotransmitter release at ribbon synapses of immature hair cells Immature apical and basal gerbil IHCs showed a similar size of C m and at least two kinetic components of synaptic vesicle release could be isolated. The initial fast component (RRP: Moser & Beutner, 2000) exhibited a release time constant of about 31 ms that was comparable to adult gerbil IHCs (42–52 ms: Johnson et al. 2008). Figure 6. Neurotransmitter release in immature gerbil OHCs A, ICa and C m responses recorded from an apical P3 gerbil OHC. Voltage protocol as described in Fig. 1A but using 5 mM extracellular Ca2+. Traces are the average of three protocol repetitions. B, average peak I–V and C m–V curves from apical and basal OHCs (P3, n = 4 for each) using depolarizing voltage steps in 10 mV increments from −81 mV. The C m values do not have S.E.M. since they were directly measured from the averaged traces of pooled cells. C, synaptic transfer functions for apical and basal OHCs. Average C m was plotted against the corresponding ICa, between −71 mV and near −10 mV, from the I–V and C m–V curves shown in B. Fits to the data points are according to eqn (1). The open triangle and square indicate the peak responses obtained from C57B mouse (n = 6) and rat (n = 6) apical OHCs, respectively. D, immunolocalization of active zones (green: open arrowheads) and otoferlin (red: closed arrowheads) in apical and basal gerbil OHCs (P4). The number of active zones in OHCs was: 1st row 8.9 ± 1.1 (n = 8), 2nd row 8.6 ± 1.4 (n = 8), 3rd row 9.0 ± 1.3 (n = 5); in basal OHCs was: 1st row 9.3 ± 1.0 (n = 8), 2nd row 10.9 ± 0.7 (n = 9), 3rd row 11.0 ± 0.7 (n = 8). C 2009 The Authors. Journal compilation C 2009 The Physiological Society Exocytosis at hair cell ribbon synapses 1723 However, the larger number of synaptic ribbons in pre-hearing IHCs (Table 1) means that their release rate per ribbon is actually about half that of adult cells. The reduction in ribbon number with maturation is likely to be a consequence of the pruning of immature branched afferent endings to reach their characteristic unbranched conﬁguration with adult IHCs (Pujol et al. 1998). The larger pool size and greater release rate of the RRP per ribbon in adult IHCs (Table 1) could promote the coincidence of release events, which seems to be required for accurate temporal coding of sound (Moser et al. 2006, Wittig & Parsons, 2008). The reﬁlling of the RRP in pre-hearing IHCs is unlikely to become rate limiting for vesicle release for frequencies up to at least 7 Hz, which is in the range of the predicted frequency of spontaneous Ca2+ action potentials intrinsic to these cells (<10 Hz: Johnson & Marcotti, 2008). In contrast to the RRP, the SRP release rate per ribbon in immature IHCs was faster than that of adult cells (Table 1). This is unlikely to be a consequence of the larger I Ca in immature IHCs because it cannot explain their comparatively lower RRP release rate. The inverse relation of the RRP and SRP release rates between immature and adult IHCs appears to be correlated with the cell’s different exocytotic Ca2+ dependence (Fig. 4C), especially considering that the rates for adult apical cells lie in between (Table 1). The lower demand for maintained exocytosis in immature spiking IHCs could also explain