Summary

低密度原海马神经元文化

Published: April 18, 2017
doi:

Summary

本文介绍用于培养低密度初级海马神经元上倒置在神经胶质单层玻璃盖玻片生长的协议。神经元和神经胶质层通过石蜡珠分离。通过该方法生长的神经元适合于高分辨率光学成像和功能测定法。

Abstract

探测单个神经细胞的培养物中的结构和生理的能力对于神经生物学的研究是至关重要的,并且允许在单个细胞或定义的网络的遗传和化学操作灵活性。相比于完整的脑组织时操纵这样便于在减少培养系统简单。尽管存在这些初级神经元的分离和生长方法很多,每个人都有其自身的局限性。这个协议描述了一种用于在玻璃盖玻片上,然后将其悬浮在神经胶质细胞的单层培养低密度和高纯度的啮齿动物胚胎海马神经元的方法。这种“三明治文化”允许同时允许从底层的神经胶质单层营养支持的神经元群的独家长期增长。当神经元是足够的年龄或到期水平的,神经元盖玻片可翻转时的胶质菜和在成像或功能测定法中使用。神经元摹rown通过该方法通常存活数周和发展广泛的乔木,突触连接,以及网络属性。

Introduction

大脑分为神经元的复杂网络。单个神经元到网络活动和脑功能的贡献可以通过它们的分子组成和它们的生理特性的扰动的选择性改变进行研究。单个神经元的遗传和化学处理是在培养的神经元比完整的脑组织受到后者的细胞的异质性和复杂性可以说是容易,无牵无挂。在培养神经细胞发展良好定义的轴突和树突乔木和彼此形成广泛的突触连接。

而来自成年动物或来自神经系统的其他区域神经元培养物是可能的,胚胎海马培养物经常由于优选为所定义的锥体细胞群体和相对低的密度胶质1,2。在培养低密度生长的海马神经元是特别AMEnable到亚细胞定位,蛋白质运输,神经元极性和突触发育的研究。在培养神经细胞也在突触可塑性3,4,5,6研究分子过程被广泛使用。从与不出生后存活的全球基因缺失的小鼠神经元文化的准备工作已经在研究某些基因7的细胞和突触的角色特别有用。

由于在脑,海马神经元依赖于从神经胶质细胞营养支持。这他们的文化复杂化,并导致由这种支持提供几种不同的方法的发展。一种常用的方法包括直接电镀到神经元的神经胶质细胞8,或允许从所获取的HIPP污染物神经胶质细胞的单层ocampal组织增殖并形成神经元9下方的单层。虽然这种方法已经发现了一些成功,得到的神经元培养物中的杂质是不利的成像实验。神经元培养物的另一种常用的方法是将离开时的胶质饲养层完全,而是提供在限定的生长培养基10的形式营养支持。

在这里,我们描述了“三明治”或神经元培养2,11的“银行家”的方法。此方法涉及镀覆在玻璃盖玻片上,然后将其悬浮在由石蜡珠分离的神经胶质细胞的单层的海马神经元。这有利于神经细胞的同质群体的长期培养,而不会污染的神经胶质细胞,同时允许从底层的神经胶质单层营养支持。当神经元是足够的年龄和成熟程度的,神经元盖玻片可翻转时的胶质菜和在成像或功能测定法中使用。

Protocol

所有实验和使用实验动物方案由曼尼托巴大学动物伦理委员会批准并且是符合加拿大动物保护协会的指导方针。 1.仪器,缓冲液和溶液的制备通过高压灭菌所有解剖设备,玻璃巴斯德移液管,移液管尖端,过滤装置,和去离子水进行消毒。 制备20%(w / v的1.1 M)在去离子水和过滤灭菌库存葡萄糖溶液。储存于4℃。 制备在去离子水和过滤灭菌100mM的丙酮?…

Representative Results

在初级神经细胞培养物的这种“三明治”方法,海马神经元( 图3)上生长由石蜡珠( 图2)中分离的神经胶质细胞( 图1)的床层。这确保了神经元在玻璃盖玻片上选择性地生长以最小的神经胶质细胞污染,但接收来自胶质细胞上的组织培养皿生长足够营养支持。通常情况下,神经元可在培养基中维持> 3周,开发具有发达的轴突和?…

Discussion

虽然培养神经元的“夹心层”的方法已在别处良好描述2, 如图11所示 ,在整个协议的几个步骤是相当困难的独自描述的文字,这可能会导致沮丧谁希望采纳调查。

该方法可分为三大工作流程:神经胶质培养,盖玻片制备和神经元培养物和维护。这三个准备的是高品质的神经元培养和几个重要的关键因素,必须牢记。电镀神经元前?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由CIHR MOP-142209到TJS支持。

Materials

Dissection Instruments
Micro Dissecting Scissors Roboz RS-5910
Micro Dissecting Spring Scissors Roboz RS-5650
Micro Dissecting Spring Scissors Roboz RS-5605
Dumont Forceps (#5) Roboz RS-5045
Dumont Forceps (#PP) Roboz RS-4950
Name Company Catalog Number Comments
Tissue Preparation
Trypsin (2.5%) Gibco 15-090-046
Trypsin-EDTA (0.25%) Gibco 25-200-072
Swinnex Filter Holder, 25 mm EMD Millipore SX0002500 Used as cell strainer. Assemble first with filter and autoclave
Isoflorane Pharmaceutical Partners of Canada Inc. CP0406V2
Hemocytometer Hausser Scientific 1492
Grade 105 Lens Cleaning Tissue GE Healthcare 2105-841 Used as cell strainer. Assemble first in filter holder and autoclave
Glass Pasteur pipettes with cotton filter VWR 14672-412
HEPES (1 M) Gibco 15-630-080
Hank's Balanced Salt Solution without Calcium, Magnesium, Phenol Red (HBSS, 10x) Gibco 14-185-052
Glass Pasteur pipettes VWR 14672-380
Deoxyribonuclease I from bovine pancreas (Dnase) Sigma-Aldrich DN25-100mg
Butane bunsen burner Wall-Lenk Mfg. Co. Model 65
Centrifuge Eppendorf 5810R
Name Company Catalog Number Comments
Tissue Culture
Penicillin-Streptomycin (10,000 U/mL) Gibco 15-140-122
Petri Dish (100 mm) Fisher FB0875712
Petri Dish (60 mm) Fisher FB0875713A
Horse Serum Gibco 16050-122 Can be used in place of BGS, but each lot must be tested due to inter-lot variation. Heat-inactivation of serum is recommended.  
Sodium Hydroxide Fisher Scientific S318-1
Sodium Pyruvate Sigma-Aldrich P2256
Minimum Essential Medium (MEM) Gibco 11-095-080
Neurobasal Medium Gibco 21-103-049
L-Glutamine (200 mM) Gibco 25-030-081
GlutaMAX Supplement Gibco 35050061 Can be used in place of L-Glutamine in the NBG medium
Culture Dish (60 mm) Corning, Inc 353002
Culture Flasks (75 cm^2) Greiner Bio-One 658170
Cytarabine (Ara-C) Sigma-Aldrich C3350000
D-(+)-Glucose Sigma-Aldrich G8270
Bovine Growth Serum (BGS) HyClone SH3054103 Heat-inactivation is recommended before use.
B27 Supplement (50x) Gibco 17-504-044
Dimethyl Sulfoxide (DMSO) Sigma-Aldrich D8418-100ml
Cryogenic Vials VWR 89094-806
DL-2-Amino-5-phosphonopentanoic acid (APV) Sigma-Aldrich A5282
Name Company Catalog Number Comments
Coverslip Preparation
Sodium tetraborate decahydrate (borax) Sigma-Aldrich B9876-1KG
Poly-L-Lysine Hydrobromide Sigma-Aldrich P2636
Histoplast Paraffin Wax Fisher 22-900-700
Gravity Convection Oven VWR 89511-404 Used for alternative coverslip cleaning method discussed in protocol
Ultrasonic Bath (Sonicator) Fisher Scientific 15337400
Nitric Acid Anachemia 62786-460
Ceramic Staining Racks Thomas Scientific 8542E40 Used for alternative coverslip cleaning method discussed in protocol
Coverslips Glaswarenfabrik Karl Hecht GmbH 1001/18 Manufacturer is very important, as neurons do not adhere well to lower quality glass 
Boric Acid Sigma-Aldrich B0252
Name Company Catalog Number Comments
Miscellaneous
Sterile Syringe Filters VWR 28145-477 Used with BD syringe for filter-sterilization
Syringe  BD 302832 Used with VWR sterile syringe filters for fliter-sterilization
Water Bath Fisher Scientific 15-460-16Q
Inverted Microscope Olympus CKX41
15 mL Conical Sterile Centrifuge Tubes ThermoScientific 339650
50 mL Conical Sterile Centrifuge Tubes ThermoScientific 339652

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Cite This Article
Roppongi, R. T., Champagne-Jorgensen, K. P., Siddiqui, T. J. Low-Density Primary Hippocampal Neuron Culture. J. Vis. Exp. (122), e55000, doi:10.3791/55000 (2017).

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