1724 S. L. Johnson and others J Physiol 587.8 the faster depletion of the SRP compared to the more sustained activity of adult cells (Table 1). The above ﬁndings indicate that the synaptic machinery of pre-hearing IHCs lacks any position-dependent biophysical specialization that has been described in the adult gerbil cochlea (Johnson et al. 2008). This could indicate that in pre-hearing IHCs, possible differences in signalling characteristics such as the frequency and/or pattern of spontaneous action potentials along the gerbil cochlea (Johnson & Marcotti, 2008) do not require specialized synaptic machinery. This is in contrast to more mature IHCs where the receptor characteristics of low- and high-frequency cells are different, with basal cells responding to sound with graded receptor potentials (Russell & Sellick, 1978) while apical cells show an additional phasic component representing the sound frequency (Dallos, 1985). Although the role of exocytosis in immature OHCs is less clear, they express CaV1.3 Ca2+ channels (Michna et al. 2003), appear to show robust exocytosis (Beurg et al. 2008) and have presynaptic specializations opposite to type I afferent ﬁbres (Shnerson et al. 1982), which are only transiently present during early postnatal stages (Pujol et al. 1998). This suggests that OHCs could potentially contribute to the reﬁnement of synaptic connections within the immature cochlea, a role only suggested for electrically active IHCs (Kros et al. 1998). In the present study we have shown that early postnatal OHCs from wild-type rodents (gerbils, C57B mice and Wistar rats) exhibited relatively small exocytotic responses, which could be about an order of magnitude smaller than that of IHCs if OHCs were to be recorded under similar conditions (physiological 1.3 mM Ca2+). This came as a surprise considering the large RRP size recently described in OHCs from Otoferlin control mice (Beurg et al. 2008). Moreover, Otof control OHCs ﬁred spontaneous action potentials that are absent in wild-type rodents when working in near-physiological Ca2+ concentrations (CD-1 mice: Marcotti & Kros 1999; rats: Oliver et al. 1997). Together these observations suggest that the biophysical properties of OHCs in the mixed Otof background mouse could be somehow different to those of other wild-type animals. Moreover, the linear exocytotic Ca2+ dependence reported in OHCs of both mice (Beurg et al. 2008) and gerbils (Fig. 6C) is unlikely to be suitable for encoding the spiking activity (on–off events) since the release of neurotransmitter may also occur in the intervals between spikes. Despite the relatively small C m responses of OHCs, the estimated size of the RRP at each active zone was comparable to that of immature IHCs, consisting of 15 and 13 vesicles in apical and basal cells, respectively. These numbers correlate with those obtained at IHC ribbon synapses using morphological observations (≤30 vesicles per active zone in mouse: Khimich et al. 2005), electro- physiological recordings (∼12 vesicles in rat: Goutman & Glowatzki, 2007) and also at ribbon synapses in the retina (22 vesicles: von Gersdorff et al. 1996). Although the release of single quanta is likely to trigger action potential activity in the afferent ﬁbres (IHCs: Glowatzki & Fuchs, 2002), it remains unclear how immature OHCs drive afferent activity since in wild-type rodents they are not spontaneously active. The exocytotic Ca2+ sensitivity at gerbil IHC synapses changes with development The onset of hearing in most rodents occurs at around P10–P12. While immature IHCs exhibit intrinsic spontaneous Ca2+-dependent action potential activity (Glowatzki & Fuchs, 2002; Marcotti et al. 2003), adult cells respond to sound vibration with rapid and graded receptor potentials (Russell & Sellick, 1978). It has been suggested that developmental and tonotopic differences in the exocytotic Ca2+ dependence are important for IHC function, allowing them to encode these different receptor potentials (mouse: Johnson et al. 2005; gerbil: Johnson et al. 2008). Our ﬁndings indicate that the transition between a high-order (immature) and a near linear (adult) relation is achieved in 2–3 days just before the onset of hearing in high-frequency cells. Although low-frequency IHCs maintain an overall higher order exocytotic Ca2+ dependence, its moderate decline occurred a few days later (Fig. 5: between P12 and P16), reﬂecting the delayed development of the apical coil (Pujol et al. 1998). In contrast to the Ca2+ dependence, the exocytotic Ca2+ efﬁciency was not fully mature until ≥P20, due to the size of I Ca reaching a steady level at around this time. The high Ca2+ efﬁciency of exocytosis in adult IHCs is likely to drive spontaneous activity in auditory afferents, the frequency of which gradually increases only from around the onset of hearing (Woolf & Ryan, 1985). These results also suggest that some caution should be taken when investigating and comparing the biophysical properties of exocytosis at intermediate stages (P13–P18) with either immature or adult stages. Functional Ca2+ coupling of gerbil IHC ribbon synapses Although the basic biophysical development of IHC synapses is now better understood, very little information is available on the factors responsible for the developmental changes in the exocytotic Ca2+ dependence. It has been suggested that a close coupling between Ca2+ channels and release sites (creating a Ca2+ nanodomain) could be responsible for the linear Ca2+ dependence of exocytosis in adult IHCs (Brandt et al. 2005). Under this scenario, high Ca2+ cooperativity C 2009 The Authors. Journal compilation C 2009 The Physiological Society