The crayfish claw opener neuromuscular junction is a biological model for studying presynaptic neuromodulation by serotonin (5HT) and synaptic vesicle recycling. It has been hypothesized that 5HT enhances release by recruiting a population of either previously nonrecycling or "reluctant" vesicles to increase the readily releasable pool. To determine if 5HT activates a distinct population of synaptic vesicles, recycling membranes were labeled with the membrane dye, FM1-43. Unloading (destaining) protocols could not resolve a population of vesicles that were only releasable in the presence of 5HT. Instead, we conclude synaptic vesicles change behavior in axon terminals independent of 5HT, becoming less likely to exocytose and unload dye over periods of >1 hr after recycling. We hypothesized this to be due to the slow conversion of a portion of recycled vesicles to a difficult to release state. The possibility that vesicles in these pools were spatially separated within the terminal was tested using photoconversion of FM1-43 and transmission electron microscopy. The location of FM1-43-labeled vesicles fixed 2 min following 3 min of 20-Hz stimulation did not reveal preferential localization of recycling vesicles specifically near release sites and the distribution of labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 s following stimulation contained a significant proportion of vesicular structures equivalent in diameter to 2-5 regular vesicles, with multivesicular bodies and calveoli rarely seen, suggesting that endocytosis during sustained release at crayfish terminals occurs via multiple routes, most commonly through large "vesicle" intermediates.

中文翻译：

---

时间和血清素暴露影响小龙虾开爪器肌肉突触处回收囊泡的可释放性。

小龙虾开爪神经肌肉接头是一种生物模型，用于研究血清素 (5HT) 和突触小泡回收的突触前神经调节。已经假设 5HT 通过招募以前不可回收或“不情愿”囊泡的群体来增加容易释放的池来增强释放。为了确定 5HT 是否激活不同的突触囊泡群，回收膜用膜染料 FM1-43 标记。卸载（脱色）协议无法解决一群仅在 5HT 存在时才能释放的囊泡。相反，我们得出结论，突触囊泡在独立于 5HT 的轴突末端改变行为，在回收后超过 1 小时的时间内胞吐和卸载染料的可能性降低。我们假设这是由于一部分回收的囊泡缓慢转化为难以释放的状态。使用 FM1-43 的光转换和透射电子显微镜测试了这些池中的囊泡在终端内空间分离的可能性。在 20 Hz 刺激 3 分钟后固定 2 分钟的 FM1-43 标记囊泡的位置没有显示回收囊泡的优先定位，特别是在释放位点附近，标记囊泡的分布在早期（2 分钟）和晚期之间没有显着差异（180 分钟）时间点。刺激后 30 s 固定的末端包含相当大比例的泡状结构，其直径相当于 2-5 个规则泡，多泡体和小泡很少见，