Summary

原发性脑多巴胺能神经元的分离,培养和长期维持胚胎脑鼠类

Published: February 19, 2015
doi:

Summary

The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Abstract

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Introduction

多巴胺能神经元从黑质损耗致密部导致帕金森氏病(PD),所述第二最常见的神经变性疾病的基本运动症状。这种脑的神经元群的灭亡的根本原因尚不清楚。研究负责开发和调制神经生理特性和mesDA神经元,几个细胞培养和动物模型系统的生存已使用的生化途径。永生化细胞系,包括大鼠多巴胺能细胞系1RB 3 AN 27(N27),所述的SH-SY5Y人的多巴胺能神经母细胞瘤细 ​​胞系,小鼠的多巴胺能杂交细胞系MN9D和人脑LUHMES细胞已被用于生化和有限的机理研究1 -5。对于mesDA神经元的具体损失的研究,几种神经毒素为基础和遗传模型已开发6-8。原发性脑腹侧文化,提供一个不可缺少的工具用于研究多巴胺能神经元的神经元和突触的属性和参与这一常见的疾病的发病机制中的通路。

这里我们提出了一个详细的协议,用于中脑多巴胺能神经元的分离,它含有导致更高的生存能力和每个胚胎盖玻片的增加的产量的修改。使用预成熟E12.5鼠脑(E14.5大鼠)提高了生存能力。在这个年龄段的神经元还没有开发轴突的是,它在清扫树叶细胞的完整,减少应力,从而提高显著生存能力。此外,小心解​​剖腹侧中脑,如在该协议的第2节描述的,进一步提高的生存能力。增加每个胚胎盖玻片的数量,另一种电镀方法,提出在该协议的第4条。这导致每个胚胎高达10盖玻片的收率相比,下4盖玻片标准电镀条件从而减少每个实验动物的数量。

在轴突和树突的文化展品生长的神经元,形成突触联系,揭示了神经元和突触的标记使这些文化适合活细胞成像,免疫细胞化学和电生理研究的存在。此外,使用的神经元培养物的促进基因和药理学操纵。 体外 2天的轴突生长允许发育研究。此外,培养物的长期存活(最多6周),使它们适合于这些神经元的速度慢,进行性变性的研究。

Protocol

注:该动物保持并遵守处理与制度的指导方针,所有动物的程序批准了帝国学院的动物福利和伦理审查机构(AWERB)以及内政部和哈佛大学的机构动物护理和使用委员会(IACUC)符合联邦和州法规。 1,试剂和设备安装解冻,分装和存储1日-80ºC毫克/毫升层粘连蛋白的解决方案。溶解2微升在1ml DMEM / F12,导致在1-2微克/厘米2的涂布的浓度。 注:解冻层粘连慢…

Representative Results

免疫组织化学对酪氨酸羟化酶(TH)表明,该细胞在培养中的0.5-1%之间的多巴胺。电镀后的2小时的神经元突起出现并通过第一天,轴突和树突是可区分( 图2),使用酪氨酸羟化酶(TH)和微管相关蛋白2(MAP2)抗体( 图3)。神经元存活超过六个星期,并显示大量的产物。在培养的神经元 – 神经胶质细胞比直接相关的血清的培养基中的浓度,如前所示。同时消除FBS从培?…

Discussion

在中脑多巴胺能神经元是多巴胺在中枢神经系统中的主要来源。它们被分成三组,黑质致密部(黑质致密部),腹侧被盖区(VTA)和retrorubral字段(RRF)10,11。在黑质致密部和VTA的神经元产生重大多巴胺能通路,mesocortical,脑边缘和黑质纹状体,涉及的功能,如情感,动机和运动行为的控制。在黑质致密部和黑质纹状体系统的功能性破坏的神经元死亡是第二个最显着的神经变性疾病,帕?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Dulbecco's modified Eagle medium nutrient mixture F-12 Invitrogen 11330
Hanks' Balanced Salt Solution (HBSS) (1X), liquid Invitrogen 24020-117)
Fetal bovine serum, heat-inactivated (FBS) Invitrogen 16140
N2 Supplement (100X), liquid Invitrogen 17502-048)
D-(+)-Glucose solution (45% (wt/vol) in water Sigma G8769
Bovine serum albumin BSA Sigma A9430
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane Sigma L2020
Penicillin/streptomycin Invitrogen 15070
Trypsin (0.05% (wt/vol) Invitrogen 25300
Bovine serum albumin (BSA) cell culture tested Sigma  A9418
Phosphate-buffered saline (PBS) Sigma P3813
Anti-Tyrosine hydroxylase (Th) antibody Pel-Freez Biologicals P60101-0 
Poly-L-ornithine, 0.01% solution Sigma P4957
Anti-Map2 (Microtubule associated protein-2A and -2B) antibody Millipore MAB3418
Anti-Synapsin-1 antibody Millipore AB1543P
Alexa Fluor 488 donkey anti-rabbit IgG   antibody Molecular Probes A-21206
Alexa Fluor 594 donkey anti-rabbit IgG   antibody Molecular Probes A-21207
Alexa Fluor 488 donkey anti-sheep IgG   antibody Molecular Probes A-11015
Alexa Fluor 594 donkey anti-sheep IgG   antibody Molecular Probes A-11016
Alexa Fluor 594 donkey anti-mouse IgG   antibody Molecular Probes A-21203
Trypan blue solution (0.4% (wt/vol) Biowhittaker 17-942E
Stereo Microscope Carl Zeiss Stemi 2000-C
Inverted phase contrast microscope Carl Zeiss Axiovert 40 C
Dumont Forceps Fine Scientific Tools May-45
Cover Slip Forceps – Dumoxel Fine Scientific Tools 11251-33
Two Dumont #45 Forceps – Dumoxel Fine Scientific Tools 11245-30
Blade Holder/Breaker Flat Grip – 11cm Fine Scientific Tools 10052-11
Student Iris Scissors – Straight 11.5cm Fine Scientific Tools 91460-11
Fiber optic halogen illuminator Nikon MKII
Disposable Borosilicate Glass Pasteur Pipettes Fisherbrand 13-678-20C
Hemocytometer Proscitech SVZ2NIOU
0.2 µm sterile filter units Nalgene NL-CE-156-4020
100x20mm Petri dishes BD Biosciences 351005
Round Cover Slip #1 Thickness German Glass 12mm Bellco Glass 1943-10012 

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Cite This Article
Weinert, M., Selvakumar, T., Tierney, T. S., Alavian, K. N. Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains. J. Vis. Exp. (96), e52475, doi:10.3791/52475 (2015).

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