Hearing in mammals relies on the highly synchronous synaptic transfer between cochlear inner hair cells (IHCs) and the auditory nerve. We studied the presynaptic function of single mouse IHCs by monitoring membrane capacitance changes and voltage-gated Ca 2؉ currents. Exocytosis initially occurred at a high rate but then slowed down within a few milliseconds, despite nearly constant Ca 2؉ influx. We interpret the observed secretory depression as depletion of a readily releasable pool (RRP) of about 280 vesicles. These vesicles are probably docked close to Ca 2؉ channels at the ribbon-type active zones of the IHCs. Continued depolarization evoked slower exocytosis occurring at a nearly constant rate for at least 1 s and depending on ''long-distance'' Ca 2؉ signaling. Refilling of the RRP after depletion followed a biphasic time course and was faster than endocytosis. RRP depletion is discussed as a mechanism for fast auditory adaptation. influx at the ribbon-type active zones triggers exocytosis of synaptic vesicles, which probably release glutamate (2) onto glutamate receptors (3, 4) of the postsynaptic auditory nerve fibers. There is little information about the presynaptic function of IHCs, because the small diameter of auditory nerve fibers in mammals hinders postsynaptic recordings. Assumptions about transmitter release have, therefore, mainly been based on auditory nerve fiber spiking rate data (5) or on Furukawa's classical recordings of postsynaptic potentials from goldfish (6).To study the presynaptic function of mouse IHCs independently of postsynaptic recordings, we detected the exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (C m ; ref. 7). The specificity of C m measurements for reporting exocytosis of neurotransmitters has recently been confirmed in another ribbon-type presynapse, that of the goldfish retinal bipolar nerve terminal, by simultaneously recording transmitter release (8). In these neurons, as well as in neuroendocrine cells, several kinetic components of exocytosis have been observed and attributed to release of functionally different pools of vesicles (9-12). We compare the presynaptic properties of IHCs to the findings in other neurosecretory preparations and discuss them in the context of auditory adaptation and recovery from adaptation. ] e ; 10 mM CaCl 2 ) was used for all experiments except for those of Fig. 1b (2 mM CaCl 2 ). The extracellular solution further contained (in mM) 105 NaCl, 35 tetraethylammonium chloride (Pfaltz & Bauer), 2.8 KCl, 1 MgCl 2 , 10 NaOH-Hepes, and 10 D-glucose (pH 7.2). Solution changes were achieved by bath exchange. IHCs were stimulated electrically rather than mechanically, because C m measurements require voltage clamp. Unless stated otherwise, a resting period of Ϸ30 s was kept between depolarizations to allow recovery of exocytosis. EPC-9 amplifiers (HEKA Electronics, Lambrecht͞Pfalz, Germany) controlled by PULSE software (HEKA Electronics) were used for measurements. All voltages were co...

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.

Before mice start to hear at approximately postnatal day 10, their cochlear inner hair cells (IHCs) spontaneously generate Ca 2ϩ action potentials. Therefore, immature IHCs could stimulate the auditory pathway, provided that they were already competent for transmitter release. Here, we combined patchclamp capacitance measurements and fluorimetric [Ca 2ϩ ] i recordings to study the presynaptic function of IHCs during cochlear maturation. Ca 2ϩ -dependent exocytosis and subsequent endocytic membrane retrieval were already observed near the date of birth. Ca 2ϩ action potentials triggered exocytosis in immature IHCs, which probably activates the auditory pathway before it becomes responsive to sound. IHCs underwent profound changes in Ca 2ϩ -channel expression and secretion during their postnatal development. Ca 2ϩ -channel expression increased toward the end of the first week, providing for large secretory responses during this period and thereafter declined to reach mature levels. The efficacy whereby Ca 2ϩ influx triggers exocytosis increased toward maturation, such that vesicle fusion caused by a given Ca 2ϩ current occurred faster in mature IHCs. The observed changes in Ca 2ϩ -channel expression and synaptic efficacy probably reflected the ongoing synaptogenesis in IHCs that had been described previously in morphological studies.

Abstract. Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse -the electron-dense synaptic ribbon or synaptic body -has been recognized for decades, dissection of the synapseÕs molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years.