Center of Anatomy and Cell Biology, MIC, Medical University Vienna, Vienna, Austria.
Department of Anatomy and Biomechanics, Division of Anatomy and Developmental Biology, Karl Landsteiner University of Health Science, Krems an der Donau, Austria.
Invest Ophthalmol Vis Sci. 2020 Dec 1;61(14):31. doi: 10.1167/iovs.61.14.31.
The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown.
Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope.
All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings.
This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.
本研究旨在检测边缘终末是否表达胞吐机制的结构和分子特征，以及是否与乙酰胆碱受体和神经递质分解的酶相关。
本研究使用了 6 只猫的外直肌。使用突触相关蛋白 25kDa（SNAP25）、突触融合蛋白、突触小泡相关蛋白、突触结合蛋白和复合蛋白等胞吐蛋白的标记物对眼外直肌的整体标本进行免疫标记。乙酰胆碱受体（AChRs）用α-银环蛇毒素和针对 AChRs 的抗体进行可视化，乙酰胆碱酯酶（AChE）用抗 AChE 标记。在共聚焦扫描显微镜下分析多色标记的边缘终末的分子特征，并在透射电子显微镜下揭示其超微结构特征。
所有的边缘终末均表达胞吐蛋白 SNAP25、突触融合蛋白、突触小泡相关蛋白、突触结合蛋白和复合蛋白。在超微结构水平上，囊泡在边缘终末的末端膨体的质膜上停靠。如α-银环蛇毒素和抗 AChR 结合不存在所示，没有 AChRs 与边缘终末相关。乙酰胆碱酯酶是乙酰胆碱的降解酶，在边缘终末中活性低或不存在。轴突追踪显示，具有多个 en grappe 运动终末的轴突与边缘终末连续。
本研究表明，边缘终末表现出胞吐机制的结构和分子特征，提示神经递质释放。然而，AChRs 与边缘终末无关，因此没有乙酰胆碱的结合位点，而且由于 AChE 活性低/不存在，神经递质去除不足。因此，目前的研究结果表明，边缘终末属于与其他骨骼肌中发现的效应器系统非常不同的系统。
