延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em>在Utero</em>电穿孔
Summary
该方案提供了直接观察径向迁移皮质神经元的指导。 在子宫电穿孔中，组织切片培养和延时共聚焦成像结合直接动态地研究过度表达或下调基因在迁移神经元中的作用并分析其在发育过程中的分化。
Abstract
在子宫电穿孔是研究小鼠胚胎大脑皮层径向迁移过程的快速有力的方法。它有助于描述径向迁移的不同步骤，并表征控制该过程的分子机制。为了直接和动态地分析迁移神经元，他们必须随时间追踪。该协议描述了将子宫电穿孔与器官切片培养和延时共焦成像相结合的工作流程，其允许直接检查和动态分析径向迁移的皮质神经元。此外，迁移神经元的详细表征，例如迁移速度，速度分布以及径向取向变化是可能的。该方法可以容易地适应于通过损失和功能增益以及救援实验来进行径向迁移的皮层神经元感兴趣的基因的功能分析。时间推移迁移神经元的成像是一种最先进的技术，一旦建立，这是一种有效的工具，用于研究小鼠神经元迁移障碍模型中大脑皮质的发育。
Introduction
新皮质是认知，情感和感觉运动功能的主要部位。它由六个水平层组成，平行于大脑的表面。在后期端脑侧壁发育的祖细胞期间，引起径向迁移到侧面的投影神经元并获得层型特异性神经元的身份。在心室/室下区（VZ / SVZ）产生后，这些神经元变得瞬时多极化，并减慢其迁移。在中间区（IZ）短暂停留后，它们切换到双极形态，附着于径向胶质支架，并继续径向定向迁移到皮质板（CP）中。在达到其最终目标投影神经元离开径向胶质细胞过程并获得层特异性身份时。影响不同步骤的神经元迁移的基因突变会引起严重的皮层畸形，如lissencephaly或白质异位1,2。
在子宫内的电是一个快速和强大的技术转染神经祖细胞在啮齿动物胚胎3,4的脑发育。使用这种技术，可以敲除和/或过度表达感兴趣的基因，以研究其在发育神经元中的功能。该方法已具体地有助于描述形态细节和表征径向迁移5，6，7，8，9的处理的分子机制。径向迁移的神经元经历细胞形态，迁移速度以及迁移方向的动态变化，这需要随时间直接和连续观察。器官切片培养电穿孔脑的再次和延时共焦成像允许随着时间的推移直接观察迁移神经元。使用这种组合方法，可以分析在电穿孔脑的固定组织切片中不能研究的迁移神经元的不同特征。
我们最近迁移应用在电穿孔的大脑切片培养的神经元皮质发育期间10来研究转录因子B细胞白血病/淋巴瘤11A（BCL11A）的作用时间推移共焦成像。 Bcl11a在年轻的迁移皮质神经元中表达，我们使用条件突变体Bcl11a等位基因（ Bcl11a flox ） 11来研究其功能。将Cre重组酶与绿色荧光蛋白（GFP）电穿孔到Bcl11a flox / flox脑的皮质祖细胞中，使得我们能够产生镶嵌突变体情况，其中只有少数细胞在否则野生型背景。以这种方式，可以在单细胞水平研究Bcl11a的细胞自主功能。我们发现Bcl11a突变体神经元显示出降低的速度，速度变化的变化，以及在其迁移期间的随机取向变化10 。在概述的协议中，我们描述了成功的电穿孔和切片培养制备12小鼠脑的工作流程，以及皮质切片培养物的延时共焦成像。
Protocol
所有实验程序均经动物福利委员会（RegierungspräsidiumTübingen）批准，并按照“德国动物福利法”和欧盟指令2010/63 / EU进行。 在Utero电穿孔 显微注射针 使用具有盒状细丝（2.5mm×2.5mm）的微量移液管拉拔器将硼硅酸盐玻璃毛细管（外径：1.0mm，内径：0.58mm，长度：100mm）拉入显微注射针，并且以下程序：HEAT：540，PULL ：125，VELOCITY：20和DELAY：140.通…
Representative Results
以前，我们已经表明， 在子宫电穿孔中Bcl11a的遗传缺失会损害晚期出生的上层投影神经元的径向迁移10 。含有Cre-IRES-GFP的DNA质粒载体的电穿孔有效地在条件Bcl11a flox / flox脑中删除Bcl11a 11 。当我们在电穿孔后三天分析E14.5电穿孔大脑时，大多数控制神经元已经迁移到CP，而许多Bcl11a突变体?…
Discussion
径向迁移是新皮质发育的关键过程。在影响该过程的不同步骤基因中的突变可以引起严重的皮质畸形，包括无脑回畸形和白质异位1,2。我们最近表明，在年轻的迁移性皮层投影神经元中表达的Bcl11a在径向迁移中起作用。我们使用电穿孔大脑急性皮质切片中迁移神经元的延时共聚焦成像，直接证明迁移神经元中Bcl11a的遗传缺失会导致极化和迁移缺?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
感谢Jacqueline Andratschke，Elena Werle，Sachi Takenaka和Matthias Toberer的出色技术帮助，以及Victor Tarabykin的有益讨论。这项工作得到了德意志银行授予SB（BR-2215）的支持。
Materials
| isoflurane | Abbott Laboratories | 506949 | Forene |
| 6-well plate | Corning | 351146 | |
| 12-well plate | Corning | 351143 | |
| non-absorbable surgical suture | Ethicon | K890H | 3/8 circle, 13 mm, taper point |
| Micro Adson Forceps | Fine Science Tools | 11018-12 | serrated, length: 12 cm |
| fine scissors | Fine Science Tools | 14063-09 | angled to side, length: 9 cm |
| Mathieu Needle Holder | Fine Science Tools | 12510-14 | tungsten carbide, length: 14 cm |
| fine tipped forceps | Fine Science Tools | 11370-40 | straight, 11 cm |
| Vannas Tübingen Spring Scissors | Fine Science Tools | 15005-08 | angled up, 9.5 cm |
| ring forceps | Fine Science Tools | 11103-09 | OD: 3mm, ID, 2.2 mm, length: 9 cm |
| HBSS (10X) | Gibco | 14180046 | |
| L-Glutamine | Gibco | 25030081 | |
| Penicillin/Streptomycin | Gibco | 15140122 | |
| horse serum | Gibco | 26050088 | |
| BME | Gibco | 41010026 | |
| borosilicate glass capillaries | Harvard Apparatus | 30-0016 | 1.0 OD x 0.58 ID x 100 L mm |
| anesthsesia system | Harvard Apparaus | 72-6471 | |
| anesthetizing chamber | Harvard Apparaus | 34-0460 | |
| fluosorber filter canister | Harvard Apparaus | 34-0415 | |
| low melting point agarose | Invitrogen | 16520100 | |
| vibrating blade microtome | Leica | VT1200 S | |
| fluorescence stereo microscope | Leica | M205 FA | |
| stereo microscope | Leica | M125 | |
| inverted fluorescence tissue culture microscope | Leica | DM IL LED | |
| confocal laser scanning microscope | Leica | TCS SP5II | |
| hybrid detector | Leica | HyD | |
| objective, 40x/0.60 NA | Leica | 11506201 | |
| microscope temperature control system | Life Imaging Services | Cube, Brick & Box | |
| cell culture insert | Millipore | PICM0RG50 | |
| microgrinder | Narishige | EG-45 | use 38° angle for beveling |
| microinjector | Parker Hannifin | 052-0500-900 | Picospritzer III |
| carprofen | Pfizer Animal Health | NDC 61106-8507 | Rimadyl |
| emdedding mold | Polysciences | 18986-1 | |
| endotoxin-free plasmid maxi kit | Qiagen | 12362 | |
| fast green | Sigma | F7252 | |
| laminin | Sigma | L2020 | |
| poly-L-lysine | Sigma | P5899 | |
| HEPES | Sigma | H4034 | |
| D-glucose | Sigma | G6152 | |
| calcium chloride | Sigma | C7902 | |
| magensium sulfate | Sigma | M2643 | |
| sodium bicarbonate | Sigma | S6297 | |
| square wave electroporator | Sonidel | CUY21EDIT | |
| tweezers with 5 mm platinum disk electrodes | Sonidel | CUY650P5 | |
| micropipette puller | Sutter Instrument | P-97 | |
| box filament | Sutter Instrument | FB255B | 2.5 mm x 2.5 mm |
| micro-spoon spatula | VWR | 231-0191 | 185 mm x 5 mm |
| glass bottom dish, 50 mm | World Precision Instruments | FD5040-100 |
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Citazione di questo articolo
Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation. J. Vis. Exp. (125), e55886, doi:10.3791/55886 (2017).