Abstract
Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.
Highlights
The ribbon synapse of cochlear hair cells (HCs) encodes sound information by tightly controlling the number and precise timing of postsynaptic spikes
For a 100 ms-depolarization, which is considered to entirely release the readily releasable pool (RRP) [22], the amplitude of DCm responses increased with HC maturation (Fig 1A–B)
We examined the developmental changes associated with the rate of vesicle supply to the RRP in apical and basal HCs using a 100 ms paired-pulse depolarization protocol (Fig. 3)
Summary
The ribbon synapse of cochlear hair cells (HCs) encodes sound information by tightly controlling the number (discharge rate) and precise timing (temporal coding) of postsynaptic spikes This synapse can drive postsynaptic auditory nerve fibers at extremely high instantaneous discharge rates (over several thousand spikes/s at stimulus onset) and, after rapid adaptation, can support sustained discharge rates over several hundred spikes/ s during ongoing sound stimulation [1], [2]. It was hypothesized that otoferlin, a multiC2 Ca2+ sensor that directly regulates SNARE-membrane fusion in vitro [4] and is required for HC vesicle exocytosis [5], controls the supply of synaptic vesicles at the active zones [6] During their early developmental period, immature auditory HCs transiently express several Ca2+-dependent synaptotagmins and do not require otoferlin to control phasic transmitter release driven by spontaneous action potentials [7]. The precise mechanisms of how otoferlin is engaged to produce fast vesicle supply and release in developing HCs remains to be elucidated
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