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
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
Representative Results
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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