亚细胞钙荧光和生物发光成像<sup> 2+</sup>老年海马神经元
Summary
Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.
Abstract
易感性相关的神经退行性疾病和局部缺血神经元细胞死亡的中老年大脑是极其增加，但负责的机制都是很知名。兴奋性毒性，认为有助于诱导既损伤神经元损害的方法，通过激活谷氨酸受体，促进钙离子内流和线粒体的 Ca 2+超载的介导。在细胞内钙离子稳态细胞内钙离子稳态或改造的显着变化可能有利于老神经细胞神经元损伤。在老化研究的 Ca 2+重塑我们已经使用活细胞成像在大鼠海马神经元的，类似于在老化体内神经元的某些方面的长期培养物。为这个目的，海马细胞，在第一个地方，新鲜从新生大鼠海马分散并涂布于POLI-D-赖氨酸包被的玻璃盖玻片。然后培养物保持在控制的媒体的数天或数瓦特eeks调查年轻人和老年人的神经元，分别。第二，培养的神经元中装入fura2并进行使用数字荧光比率成像细胞内的Ca 2+浓度的测量。第三，培养的神经元被转染质粒表达低亲和力水母发光蛋白和绿色荧光蛋白靶向线粒体串联。 24小时后，水母发光蛋白在细胞内重构与腔肠素和神经元受到生物发光成像监测线粒体Ca 2+浓度的。这三个步骤的过程允许细胞质和线粒体的 Ca 2+响应的监测，以相关的刺激如例如谷氨酸受体激动剂NMDA和比较是否这些和其它响应由老化的影响。此过程可产生新的见解，如何老化影响细胞溶质和线粒体的 Ca 2+响应所选的刺激以及所选药物旨在防止神经元细胞的测试死亡年龄相关的疾病。
Introduction
兴奋性毒性是有助于神经元损伤和细胞死亡的神经系统损伤，如局部缺血的最重要机制之一，并且在某些神经变性疾病如阿尔茨海默氏病1。这种类型的神经毒性的主要是对Ca介导的谷氨酸演技2+ -permeable，离子型NMDA受体（NMDA受体）2。曝光培养神经元对谷氨酸的可导致兴奋性中毒3，这会导致神经细胞凋亡4。我们和其他人先前认为神经易受NMDA诱导的凋亡可能与发育的体外和老化5-8变化。
它已被广泛接受的增加胞质无Ca 2+浓度（[Ca2 +浓度cyt的 ）导致细胞的活化。但是，如果此温升过高和/或持续的话，它可以触发细胞死亡9。此外，已经提出该兴奋性中毒，需要线粒体的Ca 2+摄取10中，由于治疗神经元对谷氨酸诱导的细胞死亡11线粒体解偶联剂保护神经元。如果线粒体占用过多的钙离子，线粒体通透性转换孔的开放，可能会出现，导致释放细胞色素C等促凋亡因子，并诱导细胞凋亡。我们最近已表明，这种线粒体的Ca 2+摄取直接相关的年龄依赖性易感性兴奋性毒性，通过直接测量NMDA诱导的线粒体的Ca 2+摄取在单个海马神经元5，其被报告在本文中的方法。海马，涉及生理过程，如学习，记忆等认知过程12，极易受到衰老和神经退行性疾病13。已经提出的是，数周体外后，培养海马神经元表现出了一些老化神经元14的典型特征。因此，长期培养海马神经元可以提供一个全面的模型来研究衰老增强兴奋性毒性的钙离子介导的机制。
提出的方法的总的目标是，因此，调查在细胞内Ca 2+体内稳态或Ca 2+重塑大脑老化实质性的变化包括由NMDA受体激动剂在一个长期培养海马神经元激发的差的 Ca 2+响应。该方法包括大鼠海马神经元的培养物的详细描述和胞浆和线粒体的 Ca 2+浓度的单个神经元的监测荧光和生物发光成像，分别。细胞内钙离子在培养的神经元荧光成像是一个标准的程序。然而，该方法是用于子不太可靠细胞钙测量，包括线粒体钙 。原因包括缺乏合成探针的适当的定位和不恰当的亲和性钙离子浓度可在线粒体从低μM级改连到毫水平的。以蛋白质作为例如水母发光蛋白的使用钙离子探针，已经允许靶向亚细胞器和使用使用不同的coelenterazines或突变的探针缺乏具体的钙离子结合位点15衍生物不同的钙亲和力。以这种方式，表达线粒体靶向水母发光蛋白的生物发光成像可允许的线粒体的 Ca 2+浓度在单个神经元的监测。然而，这个过程可能需要使用光子计数摄像机或超灵敏CCD相机对生物发光成像16-18。这种方法可能会产生新的结果，应该在更establ确认ished大脑衰老模型作为，例如，从旧的动物的大脑切片。
Protocol
Representative Results
Discussion
细胞内钙的大脑老化的重塑2+稳态已涉及到认知的损失，易感性增加缺血性损伤，兴奋性毒性和神经退行性疾病。为了验证这种假设在体外 ， 钙成像程序可用。不幸的是，旧的海马神经元的活培养是不可靠的。最近，人们已经观察到大鼠海马神经元老化体内包括ROS的积聚，形成脂褐素颗粒，异色灶，活化的pJNK和p53 / p21的通路，胆固醇损失的典型标志的存在许多，和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Ministerio de Economìa y competitividad (BFU2012-37146) and Junta de Castilla y Leòn (BIO103/VA45/11, VA145U13 and BIO/VA33/13), Spain. MCR was supported by Junta de Castilla y Leòn (Spain) and the European Social Fund. We thank the late Dr. Philippe Brûlet (1947-2013) for the mitochondrial GFP Aequorin plasmid.
Materials
| Neurobasal Culture Medium | Gibco | 21103-049 | |
| HBSS medium | Gibco | 14170-088 | |
| Ham's F-12 medium | Gibco | 31330-038 | |
| DNase I (from bovine pancreas) | Sigma | D5025-15KU | |
| Fetal Bobine Serum | Lonza | DE14-801E | |
| B27 | Gibco | 17504-044 | |
| Gentamicin | Gibco | 15750 | |
| L-glutamine | Gibco | 25030-032 | |
| 12 mm glass coverslips | Labolan | 0111520/20012 | |
| Papain | Worthington | LS003127 | |
| 4-well multidish plaques | Nunc | 176740 | |
| Petry dishes | JD Catalan s.l. | 2120044T | |
| Sterile pipettes | Fisher Scientific | 431030 | |
| Fura2-AM | Life Technologies | F1201 | |
| Lipofectamine2000 | Invitrogen | 11668-027 | |
| Coelenterazine n | Biotium | BT-10115-2250 uG | |
| Digitonin | Sigma | D5628 | |
| NMDA | Sigma | M3262 | |
| Glycine | Sigma | 50046 | |
| Zeiss Axiovert S100 TV microscope | Carl Zeiss Inc. | ||
| Xcite ilumination system | EXFO | ||
| ORCA ER fluorescence camera | Hamamatsu | ||
| VIM photon counting CCD camera | Hamamatsu | ||
| VC-8 valve controller | Warner Instruments | ||
| SH-27B heating system | Warner Instruments | ||
| Aquacosmos Software | Hamamatsu Photonics |
References
- Sattler, R., Tymianski, M. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Molecular Neurobiology. 24 (1-3), 107-129 (2001).
- MacDermott, A. B., Mayer, M. L., Westbrook, G. L., Smith, S. J., Barker, J. L. NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature. 321 (6069), 519-522 (1986).
- Choi, D. W., Maulucci-Gedde, M., Kriegstein, A. R. Glutamate neurotoxicity in cortical cell culture. The Journal of Neuroscience. 7 (2), 357-368 (1987).
- Kure, S., Tominaga, T., Yoshimoto, T., Tada, K., Narisawa, K. Glutamate triggers internucleosomal DNA cleavage in neuronal cells. Biochemical and Biophysical Research Communications. 179 (1), 39-45 (1991).
- Calvo, M., Sanz-Blasco, S., Caballero, E., Villalobos, C., Nunez, L. Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: role of mitochondrial calcium. Journal of Neurochemistry. 132 (4), 403-417 (2015).
- Liu, Y., et al. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. The Journal of Neuroscience. 27 (11), 2846-2857 (2007).
- Zhou, M., Baudry, M. Developmental changes in NMDA neurotoxicity reflect developmental changes in subunit composition of NMDA receptors. The Journal of Neuroscience. 26 (11), 2956-2963 (2006).
- Brewer, L. D. Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels. Brain Research. 1151, 20-31 (2007).
- Berridge, M. J. Calcium signalling remodelling and disease. Biochemical Society Transactions. 40 (2), 297-309 (2012).
- Stout, A. K., Raphael, H. M., Kanterewicz, B. I., Klann, E., Reynolds, I. J. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nature Neuroscience. 1 (5), 366-373 (1998).
- Pivovarova, N. B., et al. Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons. The Journal of Neuroscience. 24 (24), 5611-5622 (2004).
- Morris, R. G., Garrud, P., Rawlins, J. N., O’Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature. 297 (5868), 681-683 (1982).
- Geinisman, Y., Detoledo-Morrell, L., Morrell, F., Heller, R. E. Hippocampal markers of age-related memory dysfunction: behavioral, electrophysiological and morphological perspectives. Progress in Neurobiology. 45 (3), 223-252 (1995).
- Sodero, A. O., Weissmann, C., Ledesma, M. D., Dotti, C. G. Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro. Neurobiology of Aging. 32 (6), 1043-1053 (2011).
- Brini, M., et al. Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. The Journal of Biological Chemistry. 270 (17), 9896-9903 (1995).
- Villalobos, C., Alonso, M. T., Garcìa-Sancho, J. Bioluminescence imaging of calcium oscillations inside intracellular organelles. Methods in Molecular Biology. 574, 203-214 (2009).
- Villalobos, C., et al. Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells. FASEB Journal. 16, 343-353 (2002).
- Rogers, K. L., et al. Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters. The European Journal of Neuroscience. 21 (3), 597-610 (2005).
- Barreto-Chang, O. L., Dolmetsch, R. E. Calcium imaging of cortical neurons using Fura-2 AM. Journal of Visualized Experiments. (23), 1067 (2009).
