Summary

神经肌肉接头处:测量突触大小,碎片和改变突触蛋白密度利用共聚焦荧光显微镜

Published: December 26, 2014
doi:

Summary

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Abstract

神经肌肉接头(NMJ)为大,胆碱能突触继电器通过哺乳动物运动神经元控制随意肌收缩。结构变化在NMJ可导致神经传导故障,导致无力,萎缩,甚至死亡的肌纤维。许多研究已经调查了遗传修饰或疾病如何改变鼠标NMJ的结构。不幸的是,它可以是难以直接从这些研究中,因为他们经常使用不同的参数和分析方法比较结果。三个协议描述。第一种使用最大强度投影共焦图象以测量乙酰胆碱受体(AChR的)的区域富含突触后膜结构域在端板和突触小泡染色上覆突触前神经末端的区域。所述第二协议进行比较的免疫染色在突触后膜突触蛋白的相对强度。第三公关otocol使用荧光共振能量转移(FRET)来检测改变突触后乙酰胆碱受体的终板包装。该协议已经制定和完善了一系列的研究。影响的质量和结果的一致性的因素进行了讨论,并提供了用于在健康的年轻成年小鼠NMJs标准数据。

Introduction

神经肌肉接头(NMJ)是临界中继突触介导的神经系统和骨骼肌之间的通信。它是必需的所有随意运动。荧光显微镜长期以来被用于研究转基因的作用,对小鼠NMJ 1-3或之上啮齿类NMJs 4-11进行比较的年龄,饮食,运动和疾病的影响。这样的研究已经告诉我们很多关于NMJ的生理与病理生理学,但不同的参数报告( 如,乙酰胆碱受体区,终板面积,周长,碎片指数)往往让人很难比较这些研究结果。有越来越多的期望的临床前研究人员能够证明再现性,特别是与疾病12的啮齿动物模型的研究。这里描述的方案通过一系列的研究,研究的发展,生理和病理CH进行了细化安格斯的NMJ。这样的研究需要突触专业的鼠标运动终板的面积和突触蛋白的填料的突触后特13-15内的相对密度的测定。

这些方法的效用是由最近的研究中的抗麝香重症肌无力的小鼠模型中示出。 IgG抗体从抗麝香阳性重症每日注射肌无力患者到成年小鼠造成他们2周16内变弱。肌肉切片是被双重标示突触(神经 – 终端)和突触后乙酰胆碱受体共聚焦最大投影图像揭示了乙酰胆碱受体染色作为主要变化领域逐渐下降。下降重要率为足以解释可比下降突触电位,突触传递和肌肉无力17,18的故障的幅度。定性类似的发现报告了其他研究小组10,19。同样NMJ测量方法,至今已用于评估治疗抗麝香重症三种药物的影响肌无力这种小鼠模型20,21。

久坐老化可导致神经肌肉连接的损失。这里所描述的协议已经透露,目前运动终板的年龄相关下降神经末梢突触的面积小鼠进展到老年。同样的方法发现,志愿工作可以在很大程度上防止减少突触前神经末端区域22,与以前的工作由其他组4保持一致。神经肌肉连接的损失也发生在肌萎缩侧索硬化症9,23的SOD1G93A小鼠模型。

上述研究表明,各种健康状况可能导致减少前或突触后专业在NMJ的面积。这可能会导致受损突触乐趣ction或可能预示着神经肌肉连接的完全丧失。三种协议的描述,使该地区和突触专业的密度定量。第一协议的目的是提供前和突触后专业并且在哺乳动物NMJs其对准的区域的实际和可重复的措施,使用荧光显微镜。二维最大投影共焦图象和图象分析与美国国立卫生研究院的ImageJ用于检测变化的突触染色(突触小泡),突触后乙酰胆碱受体和突触的重叠区域的面积。共焦成像参数(增益和偏移电平)为每个NMJ优化以便最大化用于辨别突触专业化的区域中的可视信息。神经肌肉故障也可导致改变突触后乙酰胆碱受体和/或其他突触蛋白的密度。第二协议可应用到检测变化后突触蛋白例如相对密度麝香,rapsyn,Dystroglycan基因,磷酸化Src激酶和磷酸乙酰胆碱受体18,21。

在重症肌无力,乙酰胆碱受体的突触后膜中的密度降低是突触失效和肌肉无力的直接原因。第三协议描述了一种荧光共振能量转移(FRET)的方法来评估的变化在邻近乙酰胆碱受体的突触后膜14,15内的接近。此方法检测标记荧光-α银环蛇毒素(BGT)相邻乙酰胆碱受体之间的能量转移。当荧光供体和受体探针小于10nm相距FRET只发生。这可以揭示(亚显微)变化乙酰胆碱受体的包装的密封性,可能直接涉及突触电位的振幅。

这三个协议,细化在过去十年中,提供了完整的NMJ以一致的和可重复的方式补充措施。采用标准化协议的第二参数应促进在哺乳动物NMJ基因和环境干预的影响比较。

Protocol

注:动物实验的设计,实施和报告应采取的现行准则24账户。这样的工作必须由当地动物福利机构(在我们的例子悉尼大学的动物伦理委员会)事先批准。 1.安乐死的动物和肌肉的解剖传送鼠标从保持室以一个单独的房间,其中它被安乐死与腹膜内注射戊巴比妥溶液 ​​(30毫克/千克)使用由清水25详述的小鼠的处理方式。将鼠标放回笼子。 一旦鼠标的呼吸停…

Representative Results

突触面积的测量在NMJ 区域的任何估计依赖的边界的绘制时以限定突触专业化的程度。在健康的年轻成年肌肉NMJ图像应显示良好定义的边界为乙酰胆碱受体和突触素染色( 图2A和B)。荧光强度为乙酰胆碱受体和突触素在电机板( 图5A'和B')的周围突触和突触部之间的边界处急剧上升。对于这样的图像的最小阈值(正上?…

Discussion

这里所述的协议,使我们能够可靠地测量和在一系列条件,包括正常的老化和疾病状态量化变化NMJ的属性。用于连接所描述的方法面临NMJ图像将允许研究来比较前和突触后专业领域和突触重叠和/对齐的区域。比较前和突触后的蛋白质的第二协议,它使用横向光学切片的相对强度,是优选的。第三个协议专门用于测试的变化乙酰胆碱受体的包装在突触后膜的接近。

特异性控制?…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Health and Medical Research Council [570930]. Imaging was carried out at the Bosch Institute Advanced Microscopy Facility. Former members of the lab, whose work is cited, are thanked for their contributions to developing these methods.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Scanning confocal microscope Leica DM IRE2 with  TCS SP2 system Most scanning confocal microscopes should be suitable. 
Zeiss LSM 510 Meta 
Leica SPE-II
Alexa555-a-bungarotoxin (red-BGT) Life technologies B35451 Used for labelling AChRs
Alexa647-α-bungarotoxin (far-red-BGT) Life technologies B35450 Far red fluorescence: barely visible through the eyepiece 
rabbit anti-synaptophysin Life technologies 18-0130 Different batches of primary antibody differ in effective working dilution
FITC-anti-rapsyn mab1234 Milipore FCMAB134F Monoclonal antibody conjugated to FITC
FITC-donkey anti-rabbit IgG Jackson 711-095-152 Polyclonal secondary antibodies can vary in quality according to source and batch
Optimal Cutting Temperature compound (O.T.C.) ProSciTech IA018 Cryostat embedding matrix for freezing  muscles
DABCO Sigma 10981 Mounting medium that slows photobleaching of fluorophors

Referências

  1. Schmidt, N., et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J Cell Biol. 195, 1171-1184 (2011).
  2. Amenta, A. R., et al. Biglycan is an extracellular MuSK binding protein important for synapse stability. J Neurosci. 32, 2324-2334 (2012).
  3. Samuel, M. A., Valdez, G., Tapia, J. C., Lichtman, J. W., Sanes, J. R. Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions. PLOS ONE. 7, e46663 (2012).
  4. Valdez, G., et al. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci (USA). 107, 14863-14868 (2010).
  5. Yampolsky, P., Pacifici, P. G., Witzemann, V. Differential muscle-driven synaptic remodeling in the neuromuscular junction after denervation). Eur J Neurosci. 31, 646-658 (2010).
  6. Li, Y., Lee, Y., Thompson, W. J. Changes in Aging Mouse Neuromuscular Junctions Are Explained by Degeneration and Regeneration of Muscle Fiber Segments at the Synapse. J Neurosci. 31, 14910-14919 (2011).
  7. Zhu, H., Bhattacharyya, B. J., Lin, H., Gomez, C. M. Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals. J Neurosci. 31, 15269-15283 (2011).
  8. Valdez, G., Tapia, J. C., Lichtman, J. W., Fox, M. A., Sanes, J. R. Shared resistance to aging and ALS in neuromuscular junctions of specific muscles. PLoS ONE. 7, e34640 (2012).
  9. Perez-Garcia, M. J., Burden, S. J. Increasing MuSK Activity Delays Denervation and Improves Motor Function in ALS Mice. Cell reports. 2, 1-6 (2012).
  10. Klooster, R., et al. Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice. Brain. 135, 1081-1101 (2012).
  11. Pratt, S. J., Shah, S. B., Ward, C. W., Inacio, M. P., Stains, J. P., Lovering, R. M. Effects of in vivo injury on the neuromuscular junction in healthy and dystrophic muscles. J Physiol. 591, 559-570 (2013).
  12. Landis, S. C., et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature. 490, 187-191 (2012).
  13. Gervásio, O. L., Phillips, W. D. Increased ratio of rapsyn to ACh receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse. J Physiol. 562, 673-685 (2005).
  14. Gervásio, O. L., Armson, P. F., Phillips, W. D. Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability. Dev Biol. 305, 262-275 (2007).
  15. Brockhausen, J., Cole, R. N., Gervásio, O. L., Ngo, S. T., Noakes, P. G., Phillips, W. D. Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse. Dev Neurobiol. 68, 1153-1169 (2008).
  16. Cole, R. N., Reddel, S. W., Gervásio, O. L., Phillips, W. D. Anti-MuSK patient antibodies disrupt the mouse neuromuscular junction. Ann Neurol. 63, 782-789 (2008).
  17. Morsch, M., Reddel, S. W., Ghazanfari, N., Toyka, K. V., Phillips, W. D. Muscle Specific Kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors. Exp Neurol. 237, 237-286 (2012).
  18. Cole, R. N., Ghazanfari, N., Ngo, S. T., Gervasio, O. L., Reddel, S. W., Phillips, W. D. Patient autoantibodies deplete postsynaptic Muscle Specific Kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice. J Physiol. 588, 3217-3229 (2010).
  19. Viegas, S., et al. Passive and active immunization models of MuSK-Ab positive myasthenia: Electrophysiological evidence for pre and postsynaptic defects. Exp Neurol. 234, 506-512 (2012).
  20. Morsch, M., Reddel, S. W., Ghazanfari, N., Toyka, K. V., Phillips, W. D. Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by Muscle Specific Kinase autoantibody. J Physiol. 591, 2747-2762 (2013).
  21. Ghazanfari, N., Morsch, M., Reddel, S. W., Liang, S. X., Phillips, W. D. Muscle Specific Kinase autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction. J Physiol. 592, 2881-2897 (2014).
  22. Cheng, A., Morsch, M., Murata, Y., Ghazanfari, N., Reddel, S. W., Phillips, W. D. Sequence of age-associated changes to the mouse neuromuscular junction and the protective effects of voluntary exercise. PLoS One. 8, e67970 (2013).
  23. Schaefer, A. M., Sanes, J. R., Lichtman, J. W. A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis. J Comp Neurol. 490, 209-219 (2005).
  24. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLos Biol. 8, e1000412 (2010).
  25. Shimizu, S., Hedrich, H. J., Bullock, G. Routes of Administration. The Laboratory Mouse. , (2004).
  26. Chiasson, R. B. . Laboratory anatomy of the white rat. , (1988).
  27. Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J. Vis. Exp. (65), e3564 (2012).
  28. Mitra, A. K., Stroud McCarthy, M. P., M, R. Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22A by low dose electron microscopy and x-ray diffraction to 12.5A. J Cell Biol. 109, 755-774 (1989).
  29. Paas, Y., et al. Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid-detergent matrices. Proc. Natl Acad. Sci. (USA). 100, 11309-11314 (2003).
  30. Samson, A. O., Scherf, T., Eisenstein, M., Chill, J. H., Anglister, J. The mechanism for acetylhcoline receptor inhibition by α-neurotoxins and species-specific resistance to α-bungarotoxin revealed by NMR). Neuron. 35, 319-332 (2002).
  31. Ghazanfari, N., et al. Muscle Specific Kinase: Organiser of synaptic membrane domains. Int J Biochem Cell Biol. 43, 295-298 (2011).
  32. Ghazanfari, N., Morsch, M., Tse, N., Reddel, S. W., Phillips, W. D. Effects of the β2-adrenoceptor agonist, albuterol, in a mouse model of anti-MuSK myasthenia gravis. PLoS ONE. 9, e87840 (2014).
  33. Prakash, Y. S., Miller, S. M., Huang, M., Sieck, G. C. Morphology of diaphragm neuromuscular junctions on different fibre types. J Neurocytol. 25, 88-100 (1996).
  34. Salpeter, M. M., Harris, R. Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction. J Cell Biol. 96, 1781-1785 (1983).
  35. Soper, S. A., Nutter, H. L., Keller, R. A., Davis, L. M., Shera, E. B. The photophysical constants of several fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy. Photochem Photobiol. 57, 972-977 (1993).
  36. Panchuk-Voloshina, N., et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 47, 1179-1188 (1999).
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Tse, N., Morsch, M., Ghazanfari, N., Cole, L., Visvanathan, A., Leamey, C., Phillips, W. D. The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy. J. Vis. Exp. (94), e52220, doi:10.3791/52220 (2014).

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