1716 S. L. Johnson and others J Physiol 587.8 2005) change with development in order to transmit the different physiological responses generated by pre- (spontaneous APs) and post-hearing (graded receptor potentials) cells. One example of functional maturation is the steeper exocytotic Ca2+ dependence of immature IHCs (Ca2+ cooperativity of about 4) compared to that observed in adult cells (mouse: Johnson et al. 2005; gerbils: Johnson et al. 2008). Although this Ca2+ dependence was similar in IHCs along the immature cochlea, tonotopic differences in the kinetics and Ca2+ sensitivity of exocytosis were present in adult gerbil IHCs (Johnson et al. 2008), which could enhance the signalling of receptor potentials in the low-frequency (phasic) and high-frequency (tonic) cells. Despite the investigations on gerbils and mice, there remains a shortage of information regarding the developmental time course for changes in the exocytotic Ca2+ sensitivity in IHCs as a function of their position along the mammalian cochlea. Moreover, very little is known about the kinetic properties and topographic organization of the synaptic machinery in immature hair cells. Therefore, the aims of the present study were to determine whether tonotopic differences in the kinetics of exocytosis and its Ca2+ coupling exist in immature IHCs and to study the maturation of the synaptic Ca2+ sensitivity. Finally, we investigated whether exocytosis in apical and basal immature OHCs differs from that of IHCs. The information presented provides the ﬁrst biophysical and tonotopic correlation of hair cell ribbon synapse functional development before and at around the onset of hearing in mammals. All recordings, apart from those designed to investigate OHCs, were performed in near physiological conditions (body temperature and using 1.3 mM extracellular Ca2+) to ensure a more realistic estimation of exocytosis at mammalian hair cell ribbon synapses. Methods The methods have been described fully before (Johnson et al. 2008) but important details are speciﬁed below. Electrophysiology Tissue preparation. Apical- and basal-coil IHCs (n = 78) from gerbils were studied in acutely dissected cochleae from postnatal day 5 (P5) to P16, where the day of birth is P0. This includes 13 IHCs (apical n = 6, P5–P6; basal: n = 7, P6–P7), used to determine the kinetics of exocytosis in Fig. 1C and D, that have been imported from Johnson et al. (2008) where they were only used to measure the Ca2+ dependence of vesicle release. The gerbil was preferred to the more commonly used mouse because of its extended low-frequency hearing range would emphasize any tonotopic differences (adult gerbil: ∼0.1–60 kHz; adult mouse: ∼2–100 kHz: Greenwood, 1990; M u¨ller, 1996). A few P3 OHCs from gerbils (n = 9), C57B mice (apical-coil: n = 6) and Wistar rats (apical-coil: n = 6) were also investigated. All animals used for electrophysiology and some of those used for immuno- labelling experiments were killed by cervical dislocation in accordance with UK Home Ofﬁce regulations. The cochleae were dissected using the following extracellular solution (in mM): 135 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 D-glucose, 10 Hepes-NaOH, 2 sodium pyruvate. Amino acids and vitamins were added from concentrates (Eagle’s minimal essential medium). The pH was adjusted to 7.5. The approximate position of apical and basal hair cells was measured as a fractional distance from the cochlear apex (for details see Johnson et al. 2008). Patch clamp recording. All patch clamp recordings were performed near body temperature (34–37◦C) using an Optopatch ampliﬁer (Cairn Research Ltd, Faversham, UK). Patch pipettes were made from soda glass capillaries (2–3 M ) and coated with surf wax. The patch pipette ﬁlling solution contained (in mM): 106 caesium glutamate, 20 CsCl, 3 MgCl2, 1 EGTA-CsOH, 5 Na2ATP, 0.3 Na2GTP, 5 Hepes-CsOH, 10 Na2-phosphocreatine (pH 7.3). For some experiments BAPTA (0.1 mM, 0.6 mM, 1 mM) was used instead of 1 mM EGTA. Command protocols were applied and data acquired using pCLAMP software and a Digidata 1322A (Molecular Devices, Sunnyvale, CA, USA) and analysed with Origin (OriginLab Corp., Northampton, MA, USA). Synaptic vesicle exocytosis was measured as an increase in cell membrane capacitance ( C m) that is generally interpreted as a sign of neuro- transmitter release from presynaptic cells (Parsons et al. 1994; Moser & Beutner, 2000). Real-time C m was measured as previously described (Johnson et al. 2005). Brieﬂy, a 4 kHz sine wave of 13 mV RMS was applied to cells from −81 mV and was interrupted for the duration of the voltage step. The capacitance signal from the Optopatch was ampliﬁed (×50), ﬁltered at 250 Hz, sampled at 5 kHz and measured by averaging the C m traces after the voltage step (around 200 ms) and subtracting from pre-pulse baseline. Experiments were performed during the superfusion of 30 mM TEA and 15 mM 4-AP to block most of the K+ currents. Apamin (300 nM) and linopirdine (80–100 μM) were also added to the solution in order to block speciﬁcally I SK and I K,n, respectively. Calcium currents were corrected off-line for the linear leak conductance (g leak) typically calculated between −81 mV and −71 mV (P5–P16 IHCs: 2.3 ± 0.1 nS, n = 77; P3 gerbil OHCs: 0.7 ± 0.1 nS, n = 9; P3 mouse OHCs: 1.0 ± 0.2 nS, n = 6; P3 rat OHCs: 1.0 ± 0.1 nS, n = 6). Membrane potentials were corrected for the voltage drop across the uncompensated series C 2009 The Authors. Journal compilation C 2009 The Physiological Society