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Neurociência

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

Published: April 20, 2018
doi:
10.3791/57526
, , ,
1Centre de recherche du CHU Sainte-Justine, 2Department of Neuroscience,Université de Montréal, 3Department of pathology and cellular biology,Université de Montréal, 4Department of Pediatrics,Université de Montréal

Summary

在这里, 我们提供了一个低成本和可靠的方法来产生转脑脊髓切片培养的小鼠胚胎适合共焦显微镜和实时成像技术。

Abstract

GABAergic 中间神经元 (INs) 是神经网络的重要组成部分, 它驱动着认知和行为。用于填充皮层的 INs 在腹侧前脑 (包括从内侧和尾部节显赫人士 (梅兰日兰、CGE)) 向背皮质板块迁移, 以响应各种内在和外在线索。多年来, 已经制定了不同的方法来基因操作特定的途径, 并调查它们如何调节在迁移过程中适当的动态骨架变化。在子宫内电穿孔广泛用于研究基因抑制或过度表达在亚型中的作用, 同时评估对形态学和最终位置的影响。然而, 虽然这种方法很容易用于修改径向迁移的锥体细胞, 但它在技术上更具挑战性, 当靶向 INs.在子宫内电穿孔产生低产量, 因为幼崽的存活率降低时电穿孔是在 e14.5 之前进行的, 就像研究梅兰日兰的习惯一样。在另一种方法中, 梅兰日兰外植体为梅兰日兰提供了方便的途径, 并促进了基因修饰的 INs 的成像。然而, 在这些外植体中, INs 迁移成一个人工基质, 缺乏内源性指导线索和丘脑输入。这促使我们优化一种方法, 使 INs 可以在更自然的环境中迁移, 同时规避在子宫内方法中的技术挑战。本文描述了胚胎小鼠脑中的前宫内电穿孔与脊髓切片培养的结合, 以方便地跟踪、图像和重建基因改造的 INs 沿着自然路径迁徙, 以应对内源性线索。这种方法既可以量化的动态方面的迁移与时间推移共焦成像, 以及详细分析各种形态学参数使用神经元重建的固定 immunolabeled 组织。

Introduction

皮质 GABAergic 中间神经元 (INs) 不同于它们的生化特性、生理特性和连通性, 它们在成熟网络中调解不同的功能 1, 2, 3 ,4,5。通过对1、2进行了广泛研究的遗传级联, 对皮质 INs 的不同亚型进行了严格的规范。大多数 (70%) 皮质 GABAergic 的起源于祖细胞的内侧节隆起 (梅兰日兰), 腹部定位胚胎结构, 并必须跨越较长的距离迁移到皮质板1,2,6. 当皮质锥体细胞从心室区 (VZ) 向皮质板块沿径向胶质组织支架进行径向迁移时, 不依附于这种支架的 INs 的切向迁移需要多种内在和外部提示, 吸引迁移神经元朝向皮质板块, 同时引导他们远离非皮层结构2,7,8。在退出细胞周期后, 在梅兰日兰的 VZ 中表达的化疗排斥信号会使 INs 被排斥在梅兰日兰中, 这会触发向皮质板块9,10的切向迁移。迁移 INs 避免纹状体与不同的排斥线索11 , 并在到达皮质板块后, 他们从一个切线切换到径向迁移模式, 并达到他们的最终层流位置, 部分是响应从金字塔的线索单元格12和其他蜂窝群13。对于其他神经元种群, INs 的迁移涉及各种动态形态学变化, 以允许神经元的实际运动。这种所谓的神经元运动的特点是重复循环三个连续步骤: 一个领先的进程的伸长, 核的主动顺运动 (nucleokinesis), 并撤回的尾随进程14。在迁移过程中, 通过许多内在和外在的线索来调节引导进程的分支和主动重塑, 以正确的方向指导 INs, 同时确定迁移的方向和速度14,15 ,16。

在迁移中调节皮质的决定因素近年来得到了广泛的研究1,2,7,17,18,19,20,一些分子行为者的中断被假定导致神经发育紊乱, 如小儿难治性癫痫或自闭症频谱紊乱1,2,21,22,23,24. 因此, 开发了各种体外和体内方法, 以极大地提高我们研究这个动态过程的能力, 如前所述25。体外方法, 包括博伊登室法和条纹选择法, 提供了评估神经元迁移过程中特定基因或蛋白质的需求和细胞自主影响的最快和最可重现的手段,没有其他因素的影响25。当与实时成像8、26、27结合使用时, 这些检测尤其有用。通过这些技术, INs 可以很容易地从 e13.5 梅兰日兰中提取出来, 并通过酶和机械分离被分离, 然后通过不同的信号通路和指导线索进行研究, 如前所示8,28.然而, 这些化验发生在一个人工细胞外基质中缺乏三维组织结构, 这可能会改变神经细胞的行为和细胞的性质, 可能影响细胞迁移和/或生存25。为了规避这些限制,前体梅兰日兰外植体已被开发为一种替代工具, 用于量化迁移过程中发生的动态形态学变化以及诸如速度和方向14等参数, 29。生成梅兰日兰外植体相对简单, 在其他地方也被广泛描述为30。它需要在有吸引力或令人反感的提示25的情况下, 将梅兰日兰的小提取物涂在混合皮质细胞的单层上, 或者在胶和胶原蛋白的混合物中, 尽管后者是可选的31。梅兰日兰外植体允许高分辨率成像的疏生标记细胞, 简化了细胞内过程的研究, 如骨架重塑在领先的过程分支, 如前所示32,33 、34和本研究中。梅兰日兰外植体已成功地用于评估在2D 环境中迁移过程中的动态骨架变化, 例如在特定的药理或趋化操作之后 (参见, 例如, Tielens et 201633).然而, 通过这种方法, INs 在一个人工矩阵中迁移, 这可能改变行为和实验结果的再现性和意义。

相比之下,在子宫内电穿孔能够在其本土环境中对 INs 进行遗传操作, 是一种广泛使用的方法, 可以快速有效地评估基因功能的增益和丢失的影响, 同时规避成本高昂且耗时的挖空和敲入策略25,35。在子宫内电穿孔可能会偏向于祖细胞, 通过使用单元类型特定的促进器, 并将电极定位到丘脑结构, 包括梅兰日兰36。此外,在子宫内电穿孔允许在 1-2 天内及时表达实验性构造, 与使用病毒载体25构建表达式所需的 7-10 天相比。然而, 在祖细胞中的在子宫内电穿孔往往是低产量。的确, 虽然位于背心室区的锥体细胞祖体可以用在子宫内电穿孔中有效地转染, 但针对更多的腹部定位结构, 如梅兰日兰, 在技术上更具挑战性,特别是在小 e13.5 胚胎中, 胚胎致死率的高速度进一步降低了实验产量25。

为了规避与体外梅兰日兰植块实验和体内在子宫内的的一些技术局限性电穿孔,前体脊髓切片培养已经开发8,37, 38,39。大脑脊髓切片区域性提供了模拟体内条件的优势, 同时它比生成动物模型25更便宜, 而且耗时。事实上, 这些准备工作可以方便地访问梅兰日兰, 同时还可以通过特定的 INs 可视化, 并可与焦电穿孔结合, 调查在更具生理环境中迁移的 ins 中的特定分子通路8,39,40,41. 因此, 我们优化了脊髓文化的方法38, 我们结合前子宫电穿孔和时间推移共焦成像, 进一步评估的形态学和动态过程中发生在梅兰日兰的切向迁移过程中。本议定书是根据使用了前子宫或子宫内脑电穿孔和脊髓切片培养的其他人进行调整和优化的, 以研究锥体细胞的迁移42,43和皮质 INs36,39,44。具体来说, 小鼠胚胎被斩首, 梅兰日兰是转的前体在脑室注射实验质粒后, 允许更有效和精确的靶向梅兰日兰祖细胞比什么可以达到在子宫内电穿孔。然后将大脑提取并切片成全脑冠状切片, 可以培养几天, 从而允许连续跟踪和成像转染的 INs。这种方法通常标签为每脑切片 5-20 个切向迁移的 INs, 尽量减少达到统计学意义所需的实验迭代次数, 同时标记足够稀疏的神经元种群, 以确保容易分离单个神经元进行重建和精细形态学评估。此外, 与梅兰日兰外植体相比, 脊髓文化确保迁徙的 INs 接触到更自然的环境, 包括局部分泌的趋化因子和丘脑传入的投入。因此, 这种方法非常适合于量化转染的 INs 所采用的方向性和迁徙路径, 同时提供足够的解剖细节, 以便能够刻画出更精细的动态过程, 如领先的过程分支,nucleokinesis 和尾随过程撤回。

Protocol

所有涉及动物的实验均由 Institutionnel 女佣 Pratiques avec Animaux de 研究所 (CIBPAR) 在楚圣贾斯汀研究中心批准, 并按照加拿大动物保育委员会指南进行实验动物的护理和使用 (Ed 2)。 此处描述的协议是在胚胎日 (e) 13.5 中对胚胎的电穿孔进行优化的, 此时在梅兰日兰派生的 ins 被主动生成之前, 在 CGE 派生的 ins 生产45,46的峰值之前。此外, 为?…

Representative Results

在这一节中, 我们提供了具有代表性的结果后, 在前子宫内电穿孔的控制质粒, 或一个实验质粒靶向感兴趣的基因, 在 e13.5 小鼠胚胎梅兰日兰后脊髓切片培养孵化37°c 为48小时 (用于时间推移成像) 或 72 h (用于固定和免疫组化标记) (请参见图 1B用于示意图协议)。还说明了从梅兰日兰外植体迁移的 INs 的代表性示例 (参见图 1C?…

Discussion

在本文中, 我们提供了一个可靠的方法来执行前子宫内电穿孔的小鼠梅兰日兰在 e13.5 和脊髓培养胚胎脑切片。虽然体外方法 (如博伊登室化验) 相对容易执行, 可用于评估不同基因和蛋白质在不受其他因素干扰的情况下的具体作用, 但它们排除了在关于方向性和迁移路径的迁移动态25。梅兰日兰外植体提供了一种有用的方法来研究在迁移过程中发生的动态骨架变化, 正?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了萨伏地基金会和治疗癫痫基金会的业务赠款和加拿大创新基金会 (共聚焦显微镜) 和 G H (旋转圆盘共焦显微镜) 的设备赠款的支持。急诊室从全宗 de 研究所Santé (FRQ S) 获得职业奖;临床医生-科学家奖) 并且从加拿大学院为健康研究 (卫生研究院;年轻研究员奖)。伦理是 FRQ 的资深学者。L. E 是 Steriade-萨伏基博士后培训奖的接受者, 从萨伏依基金会, 楚圣-贾斯汀基金会博士后培训奖和 FRQ 博士后培训奖, 与明星基金会合作。加拿大脑研究基金在加拿大卫生部的财政支持下, 通过加拿大大脑研究所, 使这个项目成为可能, 授予 L.E。

Materials

Neurobasal Medium ThermoFisher Scientific 21103049 Commercially available neuron-specific culture medium. Complete formulation available on this website: https://www.thermofisher.com/ca/en/home/technical-resources/media-formulation.251.html
B-27 serum-free supplement ThermoFisher Scientific 17504044 50X Serum-free neuron specific supplement
15 mL sterile centrifuge tubes Sarstedt 62.554.002
Leibovitz's (1X) L-15 Medium (+ L-Glutamine) ThermoFisher Scientific 11415064 Commercially available neural-based culture medium supplemented with amino acids, vitamins and inorganic salts. Complete formulation available on the distributor's website 
L-Glutamine Invitrogen 25030-081
Horse serum, heat inactivated Millipore-Sigma H1138-500ML
Neurocell supplement N-2 100X Wisent 305-016 Botteinstein's N-2 Formulation
VWR Square PETG Media Bottles 125 mL VWR 89132-062
Class II Type A Biosafety Cabinet Nuaire NU-540
Sucrose BioShop SUC700.1
Sodium Chloride BioShop SOD001.1
Sodium bicarbonate ThermoFisher Scientific S233-500
D+ glucose Millipore-Sigma G7528-250G
Potassium Chloride ThermoFisher Scientific P217-500
Sodium phosphate monobasic anhydrous BioShop SPM400.500
Calcium chloride dihydrate  ThermoFisher Scientific C79-500
Magnesium sulfate heptahydrate BiosShop MAG522
Agarose BioShop AGA002.500
50 mL sterile centrifuge tubes Sarstedt 62.547.004
1.5 mL centrifuge tubes Sarstedt 72.690.001
P-97 Flaming/Brown Micropipette puller Sutter Instruments Co. Model P-97
0.4 mm I.D. x 75 mm Capillary Tube Drummond scientific 1-000-800/12
Ethanol VWR E193
5 mL syringe Becton Dickinson & Co 309646
Mineral Oil (heavy) Rougier Pharma
WPI Swiss Tweezers #5 World Precision Instruments 504511 11 cm, straight, 0.06×0.07mm tips, antimagnetic. You will need 2 of these.
WPI Swiss Tweezers #7 World Precision Instruments 504504 11.5 cm, 0.18×0.2mm, curved tips
HTC Tweezers World Precision Instruments 504617 11 cm, Straight, flat
Operating scissors World Precision Instruments 501225 16 cm, Sharp/sharp, straight. You will need 3 of these.
Dressing Forceps World Precision Instruments 501217 12.5 cm, straight, serrated
Iris Forceps World Precision Instruments 504478 10.2 cm, full curve, serrated
DeBakey Tissue Forceps World Precision Instruments 501996 15 cm, 45° angle, Delicate Jaw, 1.5mm wide
Fisherbran Microspatula with rounded ends FisherScientific 21-401-5 You will need 2 of these.
Nanoject II Auto-Nanoliter Injector Drummond scientific 3-000-204
TC Dish 60, Standard Sarstedt 83.3901 60-mm dish
Tissue culture dish Sarstedt 83.1800 35-mm dish
Black Wax FisherScientific S17432
Transfer pipettes  Ultident 170-CTB700-212 3 mL, small bulb
Stereo Microscope Leica Biosystems Leica M80 In replacement to our stereomicroscope which has been discontinued by the manufacturer (StereoMaster from FisherScientific)
Electro Square Porator BTX Harvard Apparatus ECM 830
Tweezertrodes, Plattinum Plated, 3mm BTX Harvard Apparatus 45-0487
25G 1 1/2 Becton Dickinson & Co 305127
Leica VT1000S Vibrating blade microtome Leica Biosystems VT1000S
GEM, Single edge razor blade Electron Microscopy Sciences 71952-10 Remove the blunt end before inserting in the blade designated space of the vibratome
µ-Slide 8 well Ibidi 80827 Pack of 15
Millicell cell culture insert Millipore-Sigma PICM0RG50 30 mm, hydrophilic PTFE, 0.4 µm pore, pack of 50. 
Leica DMi6000 microscope Leica Microsystems N/A
Spinning disk confocal head Ultraview Vox Perkin Elmer N/A
Volocity 6.0 acquisition software Improvision/Perkin Elmer N/A
LiveCell Stage top incubation system Pathology devices LC30030 Provides Temperature, CO2 and humidity control. 
SP8 confocal microscope Leica
mCherry-Lifeact-7 Addgene 54491 Gift from Michael Davidson
Fast Green FCF Millipore-Sigma F7258-25G 25G bottle, certified by the Biological Stain Commission
DOWNLOAD MATERIALS LIST

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Ex Utero ElectroporationOrganotypic Slice CulturesEmbryonic Mouse BrainsLive-imagingMigrating GABAergic InterneuronsDevelopmental NeuroscienceGenetic Neurodevelopmental DisordersAutismEpilepsyBiosafety CabinetDissectionCultureNeural Based MediumTransfer PipetteBlack Wax
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Eid, L., Lachance, M., Hickson, G., Rossignol, E. Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons. J. Vis. Exp. (134), e57526, doi:10.3791/57526 (2018).
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