可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征
Özet
该协议展示了一种使用具有不同生物标志物（即神经丝200，水泡乙酰胆碱转运蛋白，α-蹦极毒素和S100）的荧光免疫组织化学来检查大鼠内侧腓肠肌中突触前末端，突触后受体和突触周围施万细胞之间空间相关性的方法。
Abstract
神经肌肉接头（NMJ）是一种复杂的结构，用于从运动神经元到骨骼肌的信号通信，由三个基本的组织学成分组成：突触前运动轴突末端，突触后烟碱乙酰胆碱受体（AchRs）和突触周围施万细胞（PSC）。为了证明NMJ的形态学特征，选择大鼠内侧腓肠肌作为靶组织，并使用多种生物标志物进行荧光染色，包括神经丝200（NF200）和用于运动神经纤维及其突触前末端的水泡乙酰胆碱转运蛋白（VAChT），突触后烟碱AchR的α-蹦极毒素（α-BTX），进行检查， 和 S100 用于 PSC。在这项研究中，在两组中进行染色：在第一组中，用NF200，VAChT和α-BTX染色样品，在第二组中，样品用NF200，α-BTX和S100染色。结果表明，这两种协议都能有效地证明NMJ的详细结构。利用共聚焦显微镜观察突触前终末梢、突触后受体和PSC的形态特征，并以三维模式重建其Z-stacks图像，以进一步分析不同标记之间的空间相关性。从方法论上看，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，也适合于评价NMJ的病理改变，如周围神经损伤和再生。
Introduction
由于神经肌肉接头（NMJ）1，2，3，4的三个基本结构成分，突触前运动轴突末端的形态学方面，含有烟碱乙酰胆碱受体（AchRs）的突触后膜和突触周围施万细胞（PSC）已被广泛研究。骨骼肌的薄切片和整个安装标本已经用不同的组织学技术进行了检查，例如电子显微镜5，6，共聚焦显微镜7，8和光片显微镜9，10。虽然这些技术已经从不同方面证明了NMJ的形态特征，但作为比较，共聚焦显微镜仍然是对NMJ的详细形态进行成像的理想选择。
最近，已经开发了许多新技术来显示NMJ的结构部件。例如，thy1-YFP转基因荧光小鼠已被直接用于观察体内和体外的运动轴突和运动端板10，11。此外，静脉注射荧光α-BTX已应用于揭示野生型和转基因荧光小鼠全安装骨骼肌中运动端板的空间分布，方法是使用组织光学清除处理用光片显微镜检查9，12。然而，除了可以通过这些高级方法查看的突触前和突触后元素外，PSC不能同时被证明。
越来越多的证据表明，PSC作为外周神经胶质细胞，与突触前末梢密切相关，有助于NMJ的发展和稳定性，调节生理条件下NMJ的突触活性，以及神经损伤后NMJ的再生13，14，15.考虑到NMJ的细胞结构，该协议是同时标记PSC，突触前和突触后元素的适当候选者，并且可能用于评估NMJ在正常和病理条件下的完整性和可塑性。 例如，比较 NMJ 的强度、突触后运动端板的形态和体积、NMJ 的神经支配和去神经支配以及生理和病理状态肌肉中 PSC 的数量。
腓肠肌是形成小腿凸起的最大肌肉，通过从肢体上去除皮肤和股二头肌很容易解剖。肌肉通常被选择来评估肌肉萎缩、神经肌肉变性、肌肉表现和离体或体内运动单位力16、17、18。然而，该技术也适用于从各种骨骼肌中揭示NMJ的形态特征。同时，与薄切片7，8和挑逗的肌肉纤维19相比，厚厚的肌肉切片可以揭示更完整的NMJ形态和数量。
根据这些研究，本研究选择大鼠内侧腓肠肌作为靶组织，并根据NMJ的结构成分，以80μm的厚度切片，用各种生物标志物进行多次荧光染色。这里分别使用神经丝200（NF200）20，21，水泡乙酰胆碱转运蛋白（VAChT）22，α-蹦珠毒素（α-BTX）23，24和S10025，26分别用于标记神经纤维，突触前终末梢，突触后AchR和PSC。此外，肌肉组织和细胞核的背景进一步用鬼瘠瘩素和DAPI进行复染。
在这项研究中，我们期望开发一种改进的方案，用于在较厚的固定标本上同时染色NMJ的细胞结构及其相应的生物标志物，这更便于在共聚焦显微镜中使用，并有助于获得有关PSC，突触前和突触后元素的详细结构以及它们彼此之间的空间相关性的更多信息。从方法论的角度来看，该方案可能有助于评估NMJ在正常和病理条件下的形态特征。
Protocol
Representative Results
Discussion
我们已经描述了成功进行肌肉切片多次染色和使用荧光免疫组织化学在大鼠内侧腓肠肌厚切片上揭示NMJ的形态学特征所需的技术细节。通过使用这种方法，可以在共聚焦显微镜下分析和理解PSC与突触前后元素的精细细节和空间相关性，并以三维模式进一步重建。这里使用各种生物标志物来揭示NMJ的形态特征，其中，NF200在有髓鞘轴突20，21上高度表达，VA…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
本研究由CACMS创新基金资助（No.CI2021A03407），国家自然科学基金（第82004299号），中央公益事业单位基础研究基金（编号：ZZ13-YQ-068;ZZ14-YQ-032;ZZ14-YQ-034;ZZ201914001;ZZ202017006;ZZ202017015）。
Materials
| 4',6-diamidino-2-phenylindole dihydrochloride | ThermoFisher | D3571 | |
| Confocal laser scanning microscope | Olympus | FV1200 | |
| Donkey anti-chicken AF488 | Jackson | 149973 (703-545-155) | |
| Donkey anti-goat AF546 | ThermoFisher | A11056 | |
| Donkey anti-rabbit AF488 | ThermoFisher | A21206 | |
| Donkey anti-rabbit AF546 | ThermoFisher | A10040 | |
| Frozen Section Medium | ThermoFisher | Neg-50 | Colorless |
| Microscope cover glass | Citotest | 10212450C | |
| Microtome | Yamato | REM-710 | |
| Neurofilament 200 | Sigma-Aldrich | N4142 | Rabbit |
| Neurofilament 200 | Abcam | ab4680 | Chicken |
| Normal donkey serum | Jackson ImmunoResearch Laboratories | 017-000-12 | 10 ml |
| Normal saline | Shandong Hualu Pharmaceutical Co.Ltd | H37022750 | 250 ml |
| Paraformaldehyde | Macklin | P804536 | 500g |
| Phalloidin AF350 | ThermoFisher | A22281 | |
| Precision peristaltic pump | Longer | BT100-2J | |
| S100-β | Abcam | ab52642 | Rabbit |
| Sodium phosphate dbasic dodecahydrate | Macklin | S818118 | 500g |
| Sodium phosphate monobasic dihydrate | Macklin | S817463 | 500g |
| Sucrose | Macklin | S818046 | 500g |
| Superfrost plus microscope slides | ThermoFisher | 4951PLUS-001E | |
| Triton X-100 | Solarbio Life Sciences | 9002-93-1 | 100 ml |
| Vesicular Acetylcholine Transporter | Milipore | ANB100 | Goat |
| α-bungarotoxin AF647 conjugate | ThermoFisher | B35450 |
Referanslar
- Kawabuchi, M., et al. The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats. TheAnatomical Record. 264 (2), 183-202 (2001).
- Nishimune, H., Shigemoto, K. Practical anatomy of the neuromuscular junction in health and disease. Neurologic Clinics. 36 (2), 231-240 (2018).
- Guarino, S. R., Canciani, A., Forneris, F. Dissecting the extracellular complexity of neuromuscular junction organizers. Frontiers in Molecular Biosciences. 6, 156 (2020).
- Cruz, P. M. R., Cossins, J., Beeson, D., Vincent, A. The neuromuscular junction in health and disease: molecular mechanisms governing synaptic formation and homeostasis. Frontiers in Molecular Neuroscience. 13, 610964 (2020).
- Matthews-Bellinger, J., Salpeter, M. Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. The Journal of Neuroscienc : the Official Journal of the Society for Neuroscience. 3 (3), 644-657 (1983).
- Desaki, J., Uehara, Y. Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: a scanning and transmission electron microscopical study. Gelişim Biyolojisi. 119 (2), 390-401 (1987).
- Marques, M., Santo Neto, H. Imaging neuromuscular junctions by confocal fluorescence microscopy: individual endplates seen in whole muscles with vital intracellular staining of the nerve terminals. Journal of Anatomy. 192, 425-430 (1998).
- Magill, C. K., et al. Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice. Experimental Neurology. 207 (1), 64-74 (2007).
- Yin, X., et al. Spatial distribution of motor endplates and its adaptive change in skeletal muscle. Theranostics. 9 (3), 734-746 (2019).
- Cai, R., et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nature Neuroscience. 22 (2), 317-327 (2019).
- Feng, G., et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 28 (1), 41-51 (2000).
- Chen, W. T., et al. In vivo injection of -bungarotoxin to improve the efficiency of motor endplate labeling. Brain and Behavior. 6 (6), 00468 (2016).
- Sugiura, Y., Lin, W. Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function. Bioscience Reports. 31 (5), 295-302 (2011).
- Alvarez-Suarez, P., Gawor, M., Proszynski, T. J. Perisynaptic schwann cells – The multitasking cells at the developing neuromuscular junctions. Seminars in Cell & Developmental Biology. 104, 31-38 (2020).
- Walker, C. L. Progress in perisynaptic Schwann cell and neuromuscular junction research. Neural Regeneration Research. 17 (6), 1273-1274 (2022).
- Michaud, M., et al. Neuromuscular defects and breathing disorders in a new mouse model of spinal muscular atrophy. Neurobiology of Disease. 38 (1), 125-135 (2010).
- Sharma, S., et al. Heat-induced endoplasmic reticulum stress in soleus and gastrocnemius muscles and differential response to UPR pathway in rats. Cell Stress Chaperones. 26 (2), 323-339 (2021).
- Raikova, R., Celichowski, J., Angelova, S., Krutki, P. A model of the rat medial gastrocnemius muscle based on inputs to motoneurons and on an algorithm for prediction of the motor unit force. Journal of Neurophysiology. 120 (4), 1973-1987 (2018).
- Marinello, M., et al. Characterization of neuromuscular junctions in mice by combined confocal and super-resolution microscopy. Journal of Visualized Experiments: JoVE. (178), e63032 (2021).
- Perrot, R., Berges, R., Bocquet, A., Eyer, J. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Molecular Neurobiology. 38 (1), 27-65 (2008).
- Yuan, A., Rao, M. V., Nixon, R. A. Neurofilaments and neurofilament proteins in health and disease. Cold Spring Harbor Perspectives in Biology. 9 (4), 018309 (2017).
- Petrov, K. A., Proskurina, S. E., Krejci, E. Cholinesterases in tripartite neuromuscular synapse. Frontiers in Molecular Neuroscience. 14, 811220 (2021).
- Karlin, A. Emerging structure of the nicotinic acetylcholine receptors. Nature Reviews. Neuroscience. 3 (2), 102-114 (2002).
- Rudolf, R., Straka, T. Nicotinic acetylcholine receptor at vertebrate motor endplates: Endocytosis, recycling, and degradation. Neuroscience Letters. 711, 134434 (2019).
- Barik, A., Li, L., Sathyamurthy, A., Xiong, W. C., Mei, L. Schwann cells in neuromuscular junction formation and maintenance. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 36 (38), 9770-9781 (2016).
- Kang, H., Tian, L., Thompson, W. J. Schwann cell guidance of nerve growth between synaptic sites explains changes in the pattern of muscle innervation and remodeling of synaptic sites following peripheral nerve injuries. Journal of Comparative Neurology. 527 (8), 1388-1400 (2019).
- Wang, J., et al. A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater. Journal of Molecular Histology. 51 (5), 541-548 (2020).
- Hughes, B. W., Kusner, L. L., Kaminski, H. J. Molecular architecture of the neuromuscular junction. Muscle Nerve. 33 (4), 445-461 (2006).
- Boehm, I., et al. Comparative anatomy of the mammalian neuromuscular junction. Journal of Anatomy. 237 (5), 827-836 (2020).
