Equipe Neurophysiologie de la Synapse Auditive, Unité Mixte de Recherche, Inserm U587 et Université Victor Segalen, Institut des Neurosciences de Bordeaux, Centre Hospitalier Universitaire Pellegrin, Bordeaux, France.
PLoS One. 2011;6(10):e25714. doi: 10.1371/journal.pone.0025714. Epub 2011 Oct 6.
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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.
听觉毛细胞（HCs）具有在持续声音刺激期间无限期维持高突触囊泡释放率的显著特性。允许在带状活跃区进行这种不知疲倦的胞吐作用的囊泡供应机制在很大程度上仍然未知。为了解决这个问题，我们在发育中的鸡听觉 HCs 中表征了囊泡募集和释放的动力学。通过在各种电压刺激期间监测膜电容和 Ca(2+)电流的变化，使用从 E10（胚胎第 10 天）到 P2（孵化后两天）的完整鸡基底乳头进行实验。与不成熟的前听力 HCs（E10-E12）相比，成熟的后听力 HCs（E18-P2）可以更稳定地动员具有更快动力学和更高 Ca(2+)效率的更大的易释放池（RRP）囊泡。通过改变 100 ms 配对脉冲去极化方案的脉冲间隔来评估，发现成熟 HCs 中 RRP 补充的动力学快得多。与成熟 HCs 不同，不成熟 HCs 中的胞吐作用在重复刺激期间显示出较大的抑制。值得注意的是，当细胞内 EGTA 浓度从 0.5 升高到 2 mM 时，不成熟 HCs 中的配对脉冲抑制水平保持不变，但在成熟 HCs 中却大大增加，表明囊泡补充过程的 Ca(2+)敏感性在成熟过程中增加。同时，钙传感器 otoferlin 的免疫反应性和 HC 质膜上的带状数量大大增加，在 E18-P2 时达到最大值。我们的结果表明，成熟 HCs 中有效的 Ca(2+)依赖性囊泡释放和供应本质上依赖于突触带和质膜上 otoferlin 的同时参与。
