J Physiol 587.8 (2009) pp 1715–1726 1715 Functional maturation of the exocytotic machinery at gerbil hair cell ribbon synapses Stuart L. Johnson1, Christoph Franz2, Marlies Knipper2 and Walter Marcotti1 1Department of Biomedical Science, University of Shefﬁeld, Shefﬁeld S10 2TN, UK 2Department of Otolaryngology, Tu¨bingen Hearing Research Centre, Laboratory of Molecular Neurobiology and Cell Biology of the Inner Ear, University of Tu¨bingen, D-72076 Tu¨bingen, Germany Auditory afferent ﬁbre activity in mammals relies on neurotransmission at hair cell ribbon synapses. Developmental changes in the Ca2+ sensitivity of the synaptic machinery allow inner hair cells (IHCs), the primary auditory receptors, to encode Ca2+ action potentials (APs) during pre-hearing stages and graded receptor potentials in adult animals. However, little is known about the time course of these changes or whether the kinetic properties of exocytosis differ as a function of IHC position along the immature cochlea. Furthermore, the role of afferent transmission in outer hair cells (OHCs) is not understood. Calcium currents and exocytosis (measured as membrane capacitance changes: C m) were measured with whole-cell recordings from immature gerbil hair cells using near-physiological conditions. The kinetics, vesicle pool depletion and Ca2+ coupling of exocytosis were similar in apical and basal immature IHCs. This could indicate that possible differences in AP activity along the immature cochlea do not require synaptic specialization. Neurotransmission in IHCs became mature from postnatal day 20 (P20), although changes in its Ca2+ dependence occurred at P9–P12 in basal and P12–P15 in apical cells. OHCs showed a smaller C m than IHCs that was reﬂected by fewer active zones in OHCs. Otoferlin, the proposed Ca2+ sensor in cochlear hair cells, was similarly distributed in both cell types despite the high-order exocytotic Ca2+ dependence in IHCs and the near-linear relation in OHCs. The results presented here provide a comprehensive study of the function and development of hair cell ribbon synapses. (Received 5 January 2009; accepted after revision 17 February 2009; ﬁrst published online 23 February 2009) Corresponding author W. Marcotti: Department of Biomedical Science, University of Shefﬁeld, Shefﬁeld S10 2TN, UK. Email: w.marcotti@shefﬁeld.ac.uk Abbreviations APs, action potentials; C m, membrane capacitance changes; I Ca, calcium current; IHCs, inner hair cells; OHCs, outer hair cells; P, postnatal day; RRP, readily releasable pool; SRP, secondarily releasable pool. Sensory cells of hearing organs transduce sound into electrical responses. Two different sensory cells are present in the mammalian cochlea: inner hair cells (IHCs) and outer hair cells (OHCs). IHCs, the primary sensory receptors, respond to sound stimuli with sustained and graded receptor potentials. These responses are relayed to the brain with temporal precision (Fuchs, 2005) via the coordinated release of vesicles from IHC ribbon synapses onto type I spiral ganglion neurons (Glowatzki et al. 2008). Pre-hearing IHCs (≤P12 in most rodents) do not respond to sound but instead generate spontaneous Ca2+-dependent action potentials (APs: Marcotti et al. 2003). Intracellular Ca2+ signalling associated with APs could regulate a variety of cellular responses involved in the cell’s functional differentiation and/or maturation (Berridge et al. 2000; Spitzer et al. 2000), as proposed for the exocytotic machinery (Johnson et al. 2007). Neuro- transmitter release from pre- and post-hearing IHCs is controlled by Ca2+ inﬂux through L-type CaV1.3 Ca2+ channels (Platzer et al. 2000; Brandt et al. 2003), known to cluster at the cell’s presynaptic site (Roberts et al. 1990; Tucker & Fettiplace, 1995). In contrast to IHCs, mature OHCs provide electromechanical ampliﬁcation of the cochlear partition (Dallos, 1992) that is modulated by efferent ﬁbres representing the majority of OHC synaptic connections (Guinan, 1996). However, during early post- natal development OHCs are transiently innervated by the same afferent ﬁbres as IHCs (Pujol et al. 1998), express CaV1.3 Ca2+ channels (Michna et al. 2003) and show exocytotic responses (Beurg et al. 2008), indicating the possible presence of functional afferent synapses. The synaptic machinery at mammalian hair cell ribbon synapses undergoes morphological (Sobkowicz et al. 1982) and biophysical (Beutner & Moser, 2001; Johnson et al. C 2009 The Authors. Journal compilation C 2009 The Physiological Society DOI: 10.1113/jphysiol.2009.168542