1718 S. L. Johnson and others J Physiol 587.8 could not detect any consistent difference in the expression of otoferlin between apical and basal IHCs (judged from the qualitative observations of more than 50 sections). This labelling pattern was also similar to that previously described for IHCs of adult gerbils (Johnson et al. 2008), rats and mice (Schug et al. 2006). Vesicle pool depletion in apical and basal immature IHCs Immature IHCs are known to produce trains of spontaneous Ca2+ action potentials (Marcotti et al. 2003), the frequency of which could increase along the length of the cochlea up to a maximum of just below 10 Hz in basal cells (Johnson & Marcotti, 2008). The ability of these IHCs to maintain bouts of vesicle release was investigated by using repetitive stimuli of different durations (designed to release predominantly RRP or SRP: Fig. 2A and B, respectively). These experiments were carried out in order to determine whether vesicle pool reﬁlling could become rate limiting for vesicle release in these cells. Following repeated 50 ms steps (stimulus frequency about 7 Hz), which induce RRP exocytosis in IHCs and correspond to about the width of an action potential (Marcotti et al. 2003), the cumulative C m showed a near linear increase (Fig. 2C). This indicates that both apical and basal IHCs were able to replenish their RRP following Figure 1. Neurotransmitter release in immature IHCs A, ICa (middle panel) and C m (bottom panel) responses recorded from apical (grey traces) and basal (black traces) IHCs of the gerbil cochlea at around the onset of hearing. Recordings were obtained in response to 100 ms voltage steps from the holding potential of −81 mV to near −10 mV. The voltage protocol is shown in the top panel. B, current–voltage (I–V, bottom panel) and capacitance–voltage ( C m–V, top panel) curves from the IHCs shown in A. Recordings were obtained in response to 10 mV depolarizing voltage steps (100 ms) from −81 mV. C, average C m responses from six apical (P5–P6) and ten basal (P6–P7) IHCs. Recordings were obtained by applying voltage steps from 2 ms to 1.5 s (to around −11 mV) in order to elicit both the RRP and SRP. D, expanded version of the ﬁrst 100 ms shown in C where data up to 50 ms have been approximated with single exponential functions (apical: maximum C m = 14.1 ± 2.7 fF, τ = 30.7 ± 12.5 ms; basal: maximum C m = 16.3 ± 4.2 fF, τ = 30.9 ± 17.2 ms). The RRP consisted of 400 ± 45 (apical: n = 6, P5–P6) and 510 ± 96 (basal: n = 10, P6–P7) synaptic vesicles using a conversion factor of 37 aF per vesicle (Lenzi et al. 1999). Unless otherwise stated, in this and the following ﬁgures recordings were obtained at body temperature and using physiological 1.3 mM extracellular Ca2+. E, immunolocalization of synaptic ribbons (CtBP2: green dot-like pattern indicated by open arrowheads) and otoferlin (red: ﬁlled arrowheads) in apical and basal P4 IHCs. Nuclei were stained with DAPI (blue). Scale bars represent: 10 μm. C 2009 The Authors. Journal compilation C 2009 The Physiological Society