从产前小鼠海马神经元的分离和培养
Summary
我们没有从产前的老鼠大脑海马神经元的神经胶质细胞饲养层的使用高度纯化的文化提供了一个协议。
Abstract
被广泛用于揭示小学文化的大鼠和小鼠海马神经元细胞在神经生物学机制。通过隔离和不断增长的单个神经元，研究人员能够分析贩运细胞，细胞结构和个别蛋白质本地化，使用各种生化技术相关的属性。从这些实验的结果是测试解决学习和记忆功能的神经基础理论的关键。然而，从这些形式的实验结果明确的增长能力，神经细胞与其他脑细胞类型的最低污染的前提。在这个协议中，我们使用特定的神经细胞生长和胚胎海马组织小心剥离，旨在优化健康的神经细胞的生长，同时最大限度地降低污染类型的细胞（即星形胶质细胞）的媒体。胚胎小鼠海马组织能更加困难比DI大小的样品，由于类似的灭鼠组织隔离开来ssection。我们发现从胚胎19天（E19）小鼠幼仔海马的详细解剖技术。一旦海马组织是孤立的，温柔的神经细胞的分离，实现稀浓度的胰蛋白酶和机械设计单独从结缔组织细胞的破坏，同时提供单个细胞的损害最小。包括一个详细说明如何准备用于在中断移液器。免疫荧光协议，以最大限度地提高成功的细胞培养提供最佳的电镀密度。该协议提供了一个快速（约2小时）和有效的技术从小鼠海马组织神经细胞文化。
Protocol
1。设置收获前为了产生神经元的收获产前幼仔，安排成年鼠的繁殖19天前神经隔离的日子。 （只C57BL / 6小鼠年龄2-8个月内被用来发展这个协议的目的交配）。在怀孕的女性，心悸或视觉确认，确认成功的交配可以通过检测阴道塞。 前一天神经隔离： 对于免疫的应用，在24孔板以3:1的胶原蛋白，鼠尾光涂层的外衣盖玻片：聚-D-赖氨酸的解决方案。 用于…
Discussion
海马文化已被应用于分子生物学，为20年以上。虽然原则上，神经细胞可以从大脑的任何部分，海马文化已被证明是最流行 ，由于在7海马神经细胞的人口相对简单的架构。海马文化通常是由后期的胚胎组织。这个组织是容易分解和包含不少于成熟的脑组织的神经胶质细胞。从胚胎组织的海马神经元的分离，也减少了由于更少的黏附接触3的轴突和树突的剪切破坏。虽然海马的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢他在帮助准备手稿博士迈克尔·伍滕。这项工作是由NIH 2RO1NS033661（MWW）支持。
Materials
| Name of Reagent | Vendor | Catalog Number |
| Rat Tail Collagen 1 | BD Biosciences | 354236 |
| Poly-D-lysine Solution | Chemicon | A-003-E |
| Hanks Balanced Salt Solution | Invitrogen | 14175-095 |
| Trypsin Solution (1X) 0.25%, liquid | Invitrogen | 15050-065 |
| NeuroBasal Medium (1X) liquid | Invitrogen | 21103-049 |
| B27 Supplement (50X) liquid | Invitrogen | 17504-044 |
| L-Glutamine 200 mM (100X) liquid | Invitrogen | 25030-149 |
| Penicillin (10,000 units/ml) / Streptomycin (10,000 μg/ml) | Invitrogen | 15140-148 |
| HI-Donor Horse Serum | Atlanta Biologicals | S12150H |
Referências
- Banker, G. A., Cowan, W. M. Rat hippocampal neurons in dispersed cell culture. Brain Res. 126, 397-442 (1977).
- Brewer, G. J. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674-683 (1995).
- Brewer, G. J. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Methods. 71, 143-155 (1997).
- Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567-576 (1993).
- Burwell, R. D., Saddoris, M. P., Bucci, D. J., Wiig, K. A. Corticohippocampal contributions to spatial and contextual learning. J. Neurosci. 24, 3826-3836 (2004).
- Gluck, M. A., Myers, C., Meeter, M. Cortico-hippocampal interaction and adaptive stimulus representation: a neurocomputational theory of associative learning and memory. Neural Netw. 18, 1265-1279 (2005).
- Kaech, S., Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406-2415 (2006).
- Mao, L., Wang, J. Q. Gliogenesis in the striatum of the adult rat: alteration in neural progenitor population after psychostimulant exposure. Brain Res. Dev. Brain Res. 130, 41-51 (2001).
- Mao, L., Wang, J. Q. Upregulation of preprodynorphin and preproenkephalin mRNA expression by selective activation of group I metabotropic glutamate receptors in characterized primary cultures of rat striatal neurons. Brain Res. Mol. Brain Res. 86, 125-137 (2001).
- Oorschot, D. E. Effect of fluorodeoxyuridine on neurons and non-neuronal cells in cerebral explants. Exp. Brain Res. 78, 132-138 (1989).
- Price, P. J., Brewer, G. J., Federoff, S., Richardson, A. Serum -free media for neural cell cultures. Protocols for Neural Cell Culture. , (2001).
- Shen, J., Watanabe, S., Kaneko, A. Cell dissociation with papain reduces the density of cGMP-activated channels of the retinal rod. Jpn. J. Physiol. 45, 151-164 (1995).
- Stratmann, G., Sall, J. W., May, L. D., Loepke, A. W., Lee, M. T. Beyond anesthetic properties: The effects of isoflurane on brain cell death, neurogenesis and long-term neurocognitive function. Anesthesia and Analgesia. 110, 431-437 (2009).
- Wallace, T. L., Johnson, E. M. Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis. J. Neurosci. 9, 115-124 (1989).
Tags
IsolationCultureHippocampal NeuronsPrenatal MicePrimary CulturesRatMurineNeurobiologyCellular MechanismsCellular TraffickingCellular StructureProtein LocalizationBiochemical TechniquesMemoryLearningContaminationBrain Cell TypesSpecific MediaNeuron GrowthDissection TechniquesEmbryonic Day 19 (E19)Mouse PupsTrypsinMechanical DisruptionPipettes
Citar este artigo
Seibenhener, M. L., Wooten, M. W. Isolation and Culture of Hippocampal Neurons from Prenatal Mice. J. Vis. Exp. (65), e3634, doi:10.3791/3634 (2012).