Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences.
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences;
J Vis Exp. 2022 May 17(183). doi: 10.3791/63954.
The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (α-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and α-BTX, and in the second group, samples were stained with NF200, α-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.
神经肌肉接头（NMJ）是一种复杂的结构，用于实现运动神经元到骨骼肌的信号通讯，它由三个基本的组织学成分组成：突触前运动轴突末梢、突触后烟碱型乙酰胆碱受体（AchR）和突触周许旺细胞（PSC）。为了展示 NMJ 的形态特征，选择大鼠内侧腓肠肌作为目标组织，并使用多种荧光标记物进行多重染色，包括神经丝 200（NF200）和囊泡乙酰胆碱转运体（VAChT）用于运动神经纤维及其突触前末梢，α-银环蛇毒素（α-BTX）用于突触后烟碱型 AchR，S100 用于 PSCs。在本研究中，对两组样本进行了染色：第一组样本用 NF200、VAChT 和 α-BTX 进行染色，第二组样本用 NF200、α-BTX 和 S100 进行染色。结果表明，这两种方案都能有效地显示 NMJ 的详细结构。使用共聚焦显微镜观察到突触前末梢、突触后受体和 PSC 的形态特征，并对其 Z 堆叠图像进行了三维重建，以进一步分析不同标记之间的空间相关性。从方法学的角度来看，这些方案为研究 NMJ 在生理条件下的形态特征提供了有价值的参考，也可能适用于评估 NMJ 的病理改变，如周围神经损伤和再生。
