诱导培养的交感神经元的树突状增长
Summary
我们描述了使用骨形态发生蛋白-7（BMP-7）或基底膜的协议，主要从围产期大鼠颈上神经节（SCG）分离交感神经元选择性诱导的树突状增长。
Abstract
树突状乔木的形状决定的总突触输入一个神经细胞可以接收1-3，并影响这些输入4-6的类型和分布。树突生长和可塑性的改变模式与7个实验模型中的神经行为功能受损，被认为有助于神经紊乱8-10和11-13的神经退行性疾病中观察到的临床症状。这种意见强调精确调节树突形态功能的重要性，并建议确定枝晶生长的机制，控制，不仅促进神经元的连接是如何在正常发展的规管的认识，但还可以提供各种神经系统疾病的新型治疗策略的洞察力。
枝晶生长的机理研究，将极大地促进了可用性Ø发模型系统，使神经元实验从一个国家，他们在不延长树突之一，在他们阐述树突状乔木与体内对口切换。从颈上神经节（SCG）围产期啮齿类动物中分离交感神经元的原代培养提供了这样一种模式。定义培养基中的血清和神经节神经胶质细胞的情况下培养时，交感神经元延长一个单一的过程，这是轴突，这个单极状态持续几周文化14,15个月。然而，无论是骨形态发生蛋白-7（BMP-7），16,17或18基底膜培养基除了引发这些神经元的延长，树突的形态，生化和功能的标准，满足多个进程。交感神经元脱离围产期啮齿动物的上海建工，并在特定条件下生长的同质人口的神经元19一致响应基底膜，BMP-7和其他骨形态发生蛋白decapentaplegic（民进党）和60A亚科17,18,20,21枝促进活动。重要的是，基底膜和BMP诱导的树突的形成发生在细胞的存活或轴突生长17,18变化的情况下。
在这里，我们描述了如何设置从上海建工围产期大鼠的交感神经元的分离培养，使他们顺应选择性枝促进基底膜或BMP的活动。
Protocol
1。培养基的制备（C2的介质） 中列出的顺序，添加以下，无菌的，一次性的500毫升三角瓶： 量 元件 终浓度 第190笔资料毫升 DMEM培养基（低糖） N / A 10毫升脂肪酸免费BSA（DMEM培养液20mg/mL时） 50微克/毫升 2.8毫升 L-谷氨酰胺 1.4?…
Discussion
这种模式的优点包括：（一）文化是一个没有其他细胞类型17,23交感神经元的同质人口组成;（二）交感神经神经生长因子的要求建立的，它允许使用工作定义和界定媒体的各种媒介，与基板增长和维持交感神经元23（三）在这些文化中的神经元统一回应基底膜，BMP-7和民进党的其他骨形态发生蛋白的树突促进活动60A亚科17,18,20,21;及（d）基底膜或BMP诱导的树突形成细胞的存?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从美国国立卫生研究院（赠款R21的NS45037和以R01 ES014901）的资金支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Glass coverslips | Bellco | 1943-10012 | 12 mm Round Coverslip #1 Thickness, German glass |
| Bent tip pipets | Bellco | 1273-50003 | |
| Poly-D-lysine | Sigma | P0899-100MG | Stock solution 1 mg/ml |
| Distilled water | Gibco | 15230 | |
| Fine forceps Dumont no. 4 and 5 | Fine Science Tools (FST) | 11252-30 11241-30 | |
| 20-gauge needles | Becton Dickinson | 305176 | |
| Leibovitz’s L-15 medium | Invitrogen | 11415 | |
| Collagenase | Worthington | 4176 | 1 mg/ml |
| Dispase | Roche | 04942078001 | 5 mg/ml |
| Hank’s balanced salt solution( -Ca,-Mg) | Invitrogen | 14185-052 | |
| DMEM Low glucose | Invitrogen | 11885 | |
| L-Glutamine | Invitrogen | 25030 | 20 mM |
| Insulin/Transferrin/ Selenium | Invitrogen | 51500-056 | 100X |
| Fatty-acid free BSA | Calbiochem | 126609 | |
| NGF | Harlan Bioproducts | 5017 | |
| Ham F-12 nutrient medium (F-12) | Invitrogen | 11765 | |
| Cytosine arabinoside | Sigma | C1768 | |
| Matrigel | BD Biosciences | 356234 | Avoid repeated freeze-thaw cycles |
| BMP-7 | R&D Systems Curis, Inc. | 345-BP BMP7-07H | BMPs 4, 5 and 6 work equally as well as BMP-7 and are sometimes easier to obtain commercially |
| Paraformaldehyde | Sigma | P6148 | |
| Triton X-100 | Fisher | BP151-500 | |
| Mouse anti-rat MAP2 antibody | Sternberg Monoclonals Inc. | SMI52 | 1/5000 |
| Alexa Fluor 546 goat-anti-mouse | Invitrogen | A11003 | 1/10000 |
| Mounting media | Prolong Antifade kit Molecular Probes | P7481 |
Table 1. Materials.
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Tags
Dendritic GrowthCultured Sympathetic NeuronsDendritic ArborSynaptic InputAltered PatternsDendritic PlasticityNeurobehavioral FunctionNeurodevelopmental DisordersNeurodegenerative DiseasesDendritic MorphologyNeuronal ConnectivityMechanisms Of Dendritic GrowthModel SystemSuperior Cervical Ganglia (SCG)Perinatal Rodents
Cite This Article
Ghogha, A., Bruun, D. A., Lein, P. J. Inducing Dendritic Growth in Cultured Sympathetic Neurons. J. Vis. Exp. (61), e3546, doi:10.3791/3546 (2012).