Spontaneously Recycling Synaptic Vesicles Constitute Readily Releasable Vesicles in Intact Neuromuscular Synapses.
Spontaneously Recycling Synaptic Vesicles Constitute Readily Releasable Vesicles in Intact Neuromuscular Synapses.
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自发回收的突触小泡构成完整神经肌肉突触中易于释放的小泡。
DOI：
10.1523/jneurosci.2005-21.2022
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发表时间：
2022-04-27
影响因子：
5.3
通讯作者：
Ono, Fumihito
中科院分区：
文献类型：
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作者：
Egashira, Yoshihiro;Kumade, Ayane;Ojida, Akio;Ono, Fumihito
越来越多的证据表明，自发突触传递通过不同于动作电位诱发传递的突触前分子机制对神经元功能起着至关重要的作用。然而，突触囊泡（SV）群体是否经历两种形式的传递仍然存在争议，部分原因是在培养的神经元中观察到的结果相互矛盾。在这里，我们使用转基因斑马鱼幼虫在完整的神经肌肉突触中解决了这个问题，这些斑马鱼幼虫表达了SVs中两种不同的指标：ph敏感的荧光蛋白pHluorin和标记蛋白HaloTag。通过建立循环SV组分的定量测量，我们发现~ 85%的SV被高频AP放电调动。相比之下，在25°C条件下，自发循环的sv仅从<8%的sv中被动员，时间常数为45 min，尽管长时间的AP抑制动员了额外的延迟发病的人群。早发种群的动员对温度敏感性较低，对破伤风毒素具有抗性，而晚发种群的动员对温度更敏感，并受到破伤风毒素的抑制，这表明长时间的AP抑制激活了一种独特的分子机制，用于自发的SV融合。因此，限制在<8%的早发人群可能是生理上自发释放的唯一来源。我们进一步表明，这种有限的细胞群独立于那些在AP放电过程中不愿融合的细胞群，并且在高渗刺激和AP诱发释放的直接阶段都被使用，从而与易于释放的细胞群的特征相匹配。突触囊泡（SVs）根据它们对动作电位（AP）放电的反应方式被划分为功能不同的池。尽管在培养制剂中进行了大量的研究，用于自发融合的sv的起源仍然是一个谜。我们在完整的神经肌肉突触中解决了这个问题，并提供了两个发现。首先，长时间的AP抑制激活了不同的融合群体，这需要与由高度有限的部分引起的真正的自发融合区分开来。第二，在AP抑制期早期观察到的有限部分在随后的刺激中表现出易释放池的特征。我们的研究表明自发性SV融合的起源仅限于参与ap诱发融合的SV池中的易释放池。
Emerging evidence shows that spontaneous synaptic transmission plays crucial roles on neuronal functions through presynaptic molecular mechanisms distinct from that of action potential (AP)-evoked transmission. However, whether the synaptic vesicle (SV) population undergoing the two forms of transmission is segregated remains controversial due in part to the conflicting results observed in cultured neurons. Here we address this issue in intact neuromuscular synapses using transgenic zebrafish larvae expressing two different indicators targeted in the SVs: a pH-sensitive fluorescent protein, pHluorin, and a tag protein, HaloTag. By establishing a quantitative measure of recycled SV fractions, we found that ∼85% of SVs were mobilized by high-frequency AP firings. In contrast, spontaneously recycling SVs were mobilized only from <8% of SVs with a time constant of 45 min at 25°C, although prolonged AP inhibition mobilized an additional population with a delayed onset. The mobilization of the early-onset population was less temperature-sensitive and resistant to tetanus toxin, whereas that of the late-onset population was more sensitive to temperature and was inhibited by tetanus toxin, indicating that prolonged AP inhibition activated a distinct molecular machinery for spontaneous SV fusion. Therefore, the early-onset population limited to <8% was likely the only source of spontaneous release that occurred physiologically. We further showed that this limited population was independent from those reluctant to fuse during AP firing and was used in both the hypertonic stimulation and the immediate phase of AP-evoked releases, thereby matching the characteristics of the readily releasable pool. SIGNIFICANCE STATEMENT Synaptic vesicles (SVs) are divided into functionally distinct pools depending on how they respond to action potential (AP) firing. The origin of SVs used for spontaneous fusion remains enigmatic despite intensive studies in cultured preparations. We addressed this question in intact neuromuscular synapses and provided two findings. First, prolonged AP inhibition activated a distinct population of fusion, which needs to be distinguished from genuine spontaneous fusion arising from a highly limited fraction. Second, the limited fraction observed early in the AP inhibition period exhibited the characteristics of readily releasable pool in the subsequent round of stimulation. Our study revealed that the origin of spontaneous SV fusion is restricted to the readily releasable pool among the SV pools involved in AP-evoked fusion.