The Coupling between Ca²⁺Channels and the Exocytotic Ca²⁺Sensor at Hair Cell Ribbon Synapses Varies Tonotopically along the Mature Cochlea

Abstract

The cochlea processes auditory signals over a wide range of frequencies and intensities. However, the transfer characteristics at hair cell ribbon synapses are still poorly understood at different frequency locations along the cochlea. Using recordings from mature gerbils, we report here a surprisingly strong block of exocytosis by the slow Ca2+ buffer EGTA (10 mM) in basal hair cells tuned to high frequencies (∼30 kHz). In addition, using recordings from gerbil, mouse, and bullfrog auditory organs, we find that the spatial coupling between Ca2+ influx and exocytosis changes from nanodomain in low-frequency tuned hair cells (∼<2 kHz) to progressively more microdomain in high-frequency cells (∼>2 kHz). Hair cell synapses have thus developed remarkable frequency-dependent tuning of exocytosis: accurate low-latency encoding of onset and offset of sound intensity in the cochlea's base and submillisecond encoding of membrane receptor potential fluctuations in the apex for precise phase-locking to sound signals. We also found that synaptic vesicle pool recovery from depletion was sensitive to high concentrations of EGTA, suggesting that intracellular Ca2+ buffers play an important role in vesicle recruitment in both low- and high-frequency hair cells. In conclusion, our results indicate that microdomain coupling is important for exocytosis in high-frequency hair cells, suggesting a novel hypothesis for why these cells are more susceptible to sound-induced damage than low-frequency cells; high-frequency inner hair cells must have a low Ca2+ buffer capacity to sustain exocytosis, thus making them more prone to Ca2+-induced cytotoxicity.SIGNIFICANCE STATEMENT In the inner ear, sensory hair cells signal reception of sound. They do this by converting the sound-induced movement of their hair bundles present at the top of these cells, into an electrical current. This current depolarizes the hair cell and triggers the calcium-induced release of the neurotransmitter glutamate that activates the postsynaptic auditory fibers. The speed and precision of this process enables the brain to perceive the vital components of sound, such as frequency and intensity. We show that the coupling strength between calcium channels and the exocytosis calcium sensor at inner hair cell synapses changes along the mammalian cochlea such that the timing and/or intensity of sound is encoded with high precision.

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses

“…The amplitudes of I Ca TB, measured for a voltage step from −80 to −10 mV, were 28.0 ± 7.1 pA, equivalent to 13.7% ± 3.5% of the peak Ca 2+ current of 204.3 ± 5.6 pA (n = 9) for gerbil IHCs, and 29.3 ± 3.2 pA, equivalent to 15.4% ± 1.7% of the peak Ca 2+ current of 190.0 ± 5.6 pA (n = 17) for mice IHCs (p = 0.1, unpaired t test). The exocytotic responses were also found to have similar amplitudes in gerbil and mouse IHCs: 27.0 ± 2.7 and 31.9 ± 1.9 fF, respectively (Figures 2E and 2F; p = 0.1, unpaired t test; see also Johnson et al, 2017). …”

Hair Cell Afferent Synapses: Function and Dysfunction

“…Recent experiments in mature IHCs have demonstrated that 5-10 mM intracellular EGTA progressively reduced the release of the RRP from the apical to the base of the gerbil cochlea ( Fig. 6; Johnson et al 2017b). Therefore, the spatial coupling between Ca 2+ channels and the vesicle fusion machinery changes from a nanodomain configuration in the low-frequency apical regions (∼<2 kHz) to a microdomain configuration in the high-frequency basal regions (∼>2 kHz) (Johnson et al 2017b).…”

“…After the onset of hearing, Ca 2+ channels cluster beneath the ribbon within the IHC pre-synaptic active zones, resulting in a tighter spatial coupling between Ca 2+ influx and exocytosis at mature synapses (e.g., Wong et al 2014;Neef et al 2018). However, recent evidence suggests that very tight coupling is only present in relatively low frequency IHCs (Johnson et al 2017b).…”

“…6; Johnson et al 2017b). Therefore, the spatial coupling between Ca 2+ channels and the vesicle fusion machinery changes from a nanodomain configuration in the low-frequency apical regions (∼<2 kHz) to a microdomain configuration in the high-frequency basal regions (∼>2 kHz) (Johnson et al 2017b). These findings are corroborated by postsynaptic recordings from rat type I SGN terminals tuned to sounds in the several kHz range (Müller 1991), where the fastest component of vesicle release was greatly reduced by 5 mM EGTA (Goutman and Glowatzki 2007).…”

A sensory cell diversifies its output by varying Ca2+influx-release coupling among presynaptic active zones for wide range intensity coding

“…However, the heterogeneity was preserved in all recorded ages (P21-26, data not shown). Here, we studied synapses of apicocochlear IHCs and, given the previously described tonotopic differences in Ca 2+ channelrelease coupling on the whole-cell level 39 , single-synapse analysis in basocochlear IHCs remains an important task for the future. Taken together, we propose a model by which IHCs vary the topographies of Ca 2+ -channels and SV release sites at the AZs likely to diversify synaptic transmission beyond the heterogeneity of AZ size and Ca 2+ -signaling.…”