培养胚胎高级宫颈神经群中的大鼠交感神经元进行形态学和蛋白质学分析
Summary
本文描述了胚胎大鼠交感神经元从优越的宫颈神经的分离和培养。它还为免疫细胞化学染色和制备用于质谱分析的神经元提取物提供了详细的方案。
Abstract
来自胚胎大鼠高级宫颈节（SCG）的交感神经元已被用作周围神经元的体外模型系统，用于研究共培养系统中的突触生长、斧质贩运、突触、树突生长、树突增合和神经靶相互作用。该协议描述了神经元与E21大鼠胚胎的优越宫颈神经分离，随后在无血清培养中制备和维持纯神经元培养。由于神经元不粘附于未涂覆的塑料，神经元将在 12 mm 玻璃盖玻片或涂有聚 D-lysine 的 6 孔板上培养。在用抗米理剂（Ara-C，细胞素β-D-阿拉伯富拉诺赛德）治疗后，该协议产生健康神经元培养，非神经元细胞少于5%，可以在体外维持一个多月。虽然胚胎大鼠SCG神经元是多极的，体内有5-8个树突;在无血清条件下，这些神经元在培养中只延伸单个轴突，并在培养期间继续是单极的。然而，这些神经元可以诱导扩大树突在地下膜提取物，骨形态遗传蛋白（BPS），或10%胎儿小牛血清的存在。这些同质神经元培养物可用于免疫细胞化学染色和生化研究。本文还介绍了这些神经元中微管相关蛋白-2（MAP-2）的免疫细胞化学染色的优化方案，以及用于质谱学的神经元提取物的制备。
Introduction
来自胚胎优越的宫颈神经群（SCG）的交感神经元已被广泛用作研究神经元发育的初级神经元培养系统，包括生长因子依赖、神经元靶向相互作用、神经递质信号、突克生长、树突发育和可塑性、突触发生和信号机制，其基础神经靶点/神经胶质相互作用,,,1,1、2、3、4、5、6、7、8、9。7,8,95,6,234尽管它们体积小（大约10000个神经元/神经），但有三个主要原因，发展和广泛使用这种文化系统是一）作为第一个神经群在同情链，他们更大，因此更容易隔离，比其余的同情神经10;ii） 与中央神经元不同，SCG 中的神经元相当均匀，所有神经元都来自神经峰，大小相似，依赖于神经生长因子，并且无肾上腺素。这使得它们成为形态学和基因组研究的宝贵模型10，11,11和iii）这些神经元可以保持在一个定义的无血清介质包含神经生长因子超过一个月10，12。10,12围产期SCG神经元已被广泛使用，用于研究树突2的启动和维持机制。这主要是因为，虽然SCG神经元在体内有广泛的树突状，但在缺乏血清的情况下，它们不在体外延伸树突，但在某些生长因子（如骨形态遗传蛋白,2、12、13）,12的存在下，可以诱导树突生长树突。13
本文介绍了胚胎大鼠SCG神经元的隔离和培养方案。在过去50年中，SCG的原发神经元培养主要用于形态学研究，研究大规模基因组或蛋白质组变化的研究数量有限。这主要是由于组织体积小，导致低量的DNA或蛋白质的分离，这使得很难对这些神经元进行基因组和蛋白质组分析。然而，近年来，检测敏感性的提高使得在树突生长发育过程中，在SCG神经元中检查基因组、miRNome和蛋白质组的方法的发展已经能够发展14、15、16、17。,15,16,17本文还将描述利用免疫细胞化学和获取神经蛋白提取物进行质谱分析的神经元形态分析方法。
Protocol
加州圣玛丽学院的机构动物护理和使用委员会（IACUC）批准了有关动物研究的所有程序。圣玛丽学院的动物护理和使用指南是根据国家卫生研究所实验室动物福利办公室提供的准则（https://olaw.nih.gov/sites/default/files/PHSPolicyLabAnimals.pdf和https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf） https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf。 …
Representative Results
隔离和维护胚胎SCG神经元的神经元培养大鼠胚胎SCG分离的细胞被镀在多D-lysine涂层板或盖玻片中，并保留在含有b神经生长因子的血清自由培养基中。含有神经元和胶质细胞混合物的分离细胞在电镀时看起来是圆形的（图1A）。在电镀的24小时内，神经元扩展小轴突过程与胶质细胞扁平化，并在相位对比度显微镜下出现相暗（图1B）。在使?…
Discussion
本文介绍了从胚胎大鼠幼崽的上颈神经群中培养交感神经元的规程。使用这个模型系统的优点是，有可能获得一个均匀的神经元群，提供类似的反应生长因子，并且由于这些神经元的生长因子要求已经很好地特征化，有可能在无血清条件下在定义的介质中体外生长这些神经元。虽然该协议描述了从E21大鼠幼崽隔离SCG的过程，但该协议可用于从E17到E21的鼠幼幼解剖SCG，以及解剖胚?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加州圣玛丽学院的教师发展基金和暑期研究计划赠款的支持。作者还要感谢加州大学戴维斯分校的帕梅拉·莱因博士和加州大学伯克利分校质谱设施的安东尼·伊瓦龙博士在开发这些协议期间提供的建议。作者还要感谢加州圣玛丽学院传播办公室的海莉·纳尔逊在视频制作和编辑方面的帮助。
Materials
| 2D nanoACQUITY | Waters Corporation | ||
| Ammonium bicarbonate | Sigma-Aldrich | 9830 | |
| BMP-7 | R&D Systems | 354-BP | |
| Bovine Serum Alumin | Sigma-Aldrich | 5470 | |
| Cell scraper | Corning | CLS-3010 | |
| Collagenase | Worthington Biochemical | 4176 | |
| Corning Costar or Nunc Flat bottomed Cell culture plates | Fisher Scientific | 07-200, 140675, 142475 | |
| Cytosine- β- D-arabinofuranoside | Sigma-Aldrich | C1768 | |
| D-phosphate buffered saline (Calcium and magnesium free) | ATCC | 30-2200 | |
| Dispase II | Roche | 4942078001 | |
| Distilled Water | Thermo Fisher Scientific | 15230 | |
| Dithiothreitol | Sigma-Aldrich | D0632 | |
| DMEM – Low glucose + Glutamine, + sodium pyruvate | Thermo Fisher Scientific | 11885 | |
| Fatty Acid Free BSA | Calbiochem | 126609 | 20 mg/mL stock in low glucose DMEM |
| Fine forceps Dumont no.4 and no.5 | Ted Pella Inc | 5621, 5622 | |
| Forceps and Scissors for Dissection | Ted Pella Inc | 1328, 1329, 5002 | |
| Glass coverlips – 12mm | Neuvitro Corporation | GG-12 | |
| Goat-Anti Mouse IgG Alexa 488 conjugated | Thermo Fisher Scientific | A32723 | |
| Ham's F-12 Nutrient Mix | Thermo Fisher Scientific | 11765 | |
| Hank's balanced salt soltion (Calcium and Magnesium free) | Thermo Fisher Scientific | 14185 | |
| Insulin-Selenium-Transferrin (100X) | Thermo Fisher Scientific | 41400-045 | |
| Iodoacetamide | Sigma-Aldrich | A3221 | |
| L-Glutamine | Thermo Fisher Scientific | 25030 | |
| Leibovitz L-15 medium | Thermo Fisher Scientific | 11415064 | |
| Mounting media for glass coverslips | Thermo Fisher Scientific | P36931, P36934 | |
| Mouse-anti- MAP2 antibody (SMI-52) | BioLegend | SMI 52 | |
| Nerve growth factor | Envigo Bioproducts (formerly Harlan Bioproducts) | BT5017 | Stock 125 μg/mL in 0.2% Prionex in DMEM |
| Paraformaldehye | Spectrum Chemicals | P1010 | |
| Penicillin-Streptomycin (100X) | Thermo Fisher Scientific | 15140 | |
| Poly-D-Lysine | Sigma-Aldrich | P0899 | |
| Prionex | Millipore | 529600 | 10% solution, 100 mL |
| RapiGest SF | Waters Corporation | 186001861 | 5 X 1 mg |
| Synapt G2 High Definition Mass Spectrometry | Waters Corporation | ||
| Trifluoro acetic acid – Sequencing grade | Thermo Fisher Scientific | 28904 | 10 X 1 mL |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Trypsin | Promega or NEB | V511A, P8101S | 100 μg or 5 X 20 mg |
| Waters Total recovery vials | Waters Corporation | 186000385c |
References
- Rees, R. P., Bunge, M. B., Bunge, R. P. Morphological Changes in the neuritic growth cone and target neuron during synaptic junction development in culture. Journal of Cell Biology. 9, (1976).
- Chandrasekaran, V., Lein, P. J. Regulation of Dendritogenesis in Sympathetic Neurons. Autonomic Nervous System. , (2018).
- Higgins, D., Burack, M., Lein, P., Banker, G. Mechanisms of neuronal polarity. Current Opinion in Neurobiology. 7 (5), 599-604 (1997).
- Lein, P., Guo, X., Hedges, A. M., Rueger, D., Johnson, M., Higgins, D. The effects of Extracellular Matrix And Osteogenic Protein-1 on the morphological differentiation of rat sympathetic neurons. International Journal of Developmental Neuroscience. 14 (3), 203-215 (1996).
- Kobayashi, M., Fujii, M., Kurihara, K., Matsuoka, I. Bone morphogenetic protein-2 and retinoic acid induce neurotrophin-3 responsiveness in developing rat sympathetic neurons. Molecular Brain Research. 53 (1-2), 206-217 (1998).
- Burnham, P., Louis, J. C., Magal, E., Varon, S. Effects of ciliary neurotrophic factor on the survival and response to nerve growth factor of cultured rat sympathetic neurons. 발생학. 161 (1), 96-106 (1994).
- Hou, X. E., Li, J. Y., Dahlström, A. Clathrin light chain and synaptotagmin I in rat sympathetic neurons. Journal of the Autonomic Nervous System. 62 (1-2), 13-26 (1997).
- Harris, G. M., et al. Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix. Matrix Biology. , 176-189 (2017).
- Wingerd, K. L., et al. α4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. Journal of Neuroscience. 22 (24), 10772-10780 (2002).
- Higgins, D., Lein, P., Osterhout, D. J., Johnson, M., Banker, G., Goslin, K. Tissue culture of autonomic neurons. Culturing Nerve Cells. , 177-205 (1991).
- Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron. 15 (3), 597-605 (1995).
- Bruckenstein, D. A., Higgins, D. Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. 발생학. 128 (2), 337-348 (1988).
- Voyvodic, J. T. Development and regulation of dendrites in the rat superior cervical ganglion. The Journal of Neuroscience. 7 (3), 904-912 (1987).
- Garred, M. M., Wang, M. M., Guo, X., Harrington, C. A., Lein, P. J. Transcriptional Responses of Cultured Rat Sympathetic Neurons during BMP-7-Induced Dendritic Growth. PLoS ONE. 6 (7), 21754 (2011).
- Pravoverov, K., et al. MicroRNAs are Necessary for BMP-7-induced Dendritic Growth in Cultured Rat Sympathetic Neurons. Cellular and Molecular Neurobiology. 39 (7), 917-934 (2019).
- Natera-Naranjo, O., Aschrafi, A., Gioio, A. E., Kaplan, B. B. Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons. RNA (New York, N.Y.). 16 (8), 1516-1529 (2010).
- Aschrafi, A., et al. Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons. Journal of Neurochemistry. 150 (6), 666-677 (2019).
- Ghogha, A., Bruun, D. a., Lein, P. J. Inducing dendritic growth in cultured sympathetic neurons. Journal of Visualized Experiments. (61), 4-8 (2012).
- Caceres, A., Banker, G., Steward, O., Binder, L., Payne, M. MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Research. 315 (2), 314-318 (1984).
- Guo, X., Rueger, D., Higgins, D. Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons. Neuroscience Letters. 245 (3), 131-134 (1998).
- Mi, H., et al. PANTHER version 11: Expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Research. 45, 183-189 (2017).
- Chandrasekaran, V., et al. Retinoic acid regulates the morphological development of sympathetic neurons. Journal of Neurobiology. 42 (4), (2000).
- Courter, L. A., et al. BMP7-induced dendritic growth in sympathetic neurons requires p75 NTR signaling. Developmental Neurobiology. 76 (9), 1003-1013 (2016).
- Lein, P. J., Fryer, A. D., Higgins, D. Cell Culture: Autonomic and Enteric Neurons. Encyclopedia of Neuroscience. , 625-632 (2009).
- Neto, E., et al. Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In vitro Tools for Neurobiological Research. Journal of Neuroscience. 36 (46), 11573-11584 (2016).
- Sleigh, J. N., Weir, G. A., Schiavo, G. A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia. BMC Research Notes. 9 (1), 82 (2016).
- Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. Lectin-based isolation and culture of mouse embryonic motoneurons. Journal of Visualized Experiments. (55), e3200 (2011).
- Takeuchi, A., et al. Microfabricated device for co-culture of sympathetic neuron and iPS-derived cardiomyocytes. Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2013, 3817-3820 (2013).
- Takeuchi, A., et al. Development of semi-separated co-culture system of sympathetic neuron and cardiomyocyte. Proceedings of the 31st Annual International Conference of the IEEE Engineering in Medicine and Biology Society: Engineering the Future of Biomedicine, EMBC 2009. , 1832-1835 (2009).
- Chandrasekaran, V., Lea, C., Sosa, J. C., Higgins, D., Lein, P. J. Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons. Molecular and Cellular Neuroscience. 67, (2015).
- Kim, W. Y., et al. Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation. Journal of Neurochemistry. 108 (4), 1057-1071 (2009).
- Dalby, B., et al. Advanced transfection with Lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods. 33 (2), 95-103 (2004).
- Szpara, M. L., et al. Analysis of gene expression during neurite outgrowth and regeneration. BMC Neuroscience. 8 (1), 100 (2007).
- Pop, C., Mogosan, C., Loghin, F. Evaluation of rapigest efficacy for the digestion of proteins from cell cultures and heart tissue. Clujul Medical. 87 (4), 5 (2014).
- Vit, O., Petrak, J. Integral membrane proteins in proteomics. How to break open the black box. Journal of Proteomics. 153, 8-20 (2017).
Cite This Article
Holt, M., Adams, B., Chandrasekaran, V. Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis. J. Vis. Exp. (163), e61283, doi:10.3791/61283 (2020).