Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles.
Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles.
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DOI：
10.1523/jneurosci.3426-12.2013
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发表时间：
2013-01-30
期刊：
影响因子：
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通讯作者：
Thoreson WB
中科院分区：
文献类型：
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作者：
Chen M;Van Hook MJ;Zenisek D;Thoreson WB
视杆光感受器的囊泡释放受到 Ca2+ 通过突触带附近的 L 型通道进入的调节。我们通过使用全内反射荧光显微镜来观察单个突触囊泡的融合，从而表征了蝾螈杆中囊泡释放的位点和动力学。通过与 FM1-43 或葡聚糖偶联、pH 敏感形式的罗丹明 pHrodo 短暂孵育，加载少量囊泡。标记的细胞器与荧光微球的衍射极限尺寸相匹配，并在刺激过程中迅速消失。与融合一致，去极化引起的囊泡消失与电生理释放动力学平行，并通过抑制 Ca2+ 流入而被阻断。杆体在接近黑暗的静息膜电位下维持强直释放，导致膜相关囊泡的耗竭，除非 Ca2+ 进入被抑制。释放位点的耗尽意味着持续释放可能受到囊泡递送的速率限制。在去极化刺激过程中，新出现的囊泡以~800 nm/s的速度接近膜，它们在融合前暂停~60 ms。随着融合，囊泡前进约 18 nm，更接近膜。释放事件集中在带状区域附近，但长时间的去极化也引发了更远的非带状区域的释放。与较长去极化过程中非色带位点的更大贡献一致，Ribeye 的荧光团辅助激光灭活 (FALI) 对色带的破坏仅对 200 毫秒去极化测试步骤引起的胞吐电容增加产生较弱的抑制，而 FALI 更强烈地抑制由 25 毫秒步骤引起的胞吐电容增加。当杆在黑暗中去极化时，通过使用非带位点来放大释放可以改善对光超极化时释放减少的检测。
Vesicle release from rod photoreceptors is regulated by Ca2+ entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1– 43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca2+ influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca2+ entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.