急性片的成年小鼠前脑的神经细胞迁移的延时成像
Summary
我们描述了一个协议，用于在小鼠前脑的神经细胞迁移的实时videoimaging。病毒标记的或接枝的神经元前体的迁移，在急性活片，使用具有较快的采集间隔的宽视场荧光成像研究细胞迁移的不同阶段，其中包括固定和迁移阶段的持续时间和速度的记录迁移。
Abstract
有相当多的证据表明新的功能性神经元组成产生的内源性神经干细胞库在禁区内的成年哺乳动物大脑。新生儿脑室下区（SVZ）的神经母细胞迁移沿喙迁徙的流（RMS）到其最终目的地，在嗅球（OB）1。在RMS中，神经母细胞迁移切向在链ensheathed的星形胶质细胞的过程2,3血管的结构支撑和源的分子因素需要迁移的4,5。在OB的枷锁，神经母细胞分离和放射状迁移到不同的延髓它们分化 成的interneurons和集成到现有的网络中，6层。
在这个手稿中，我们描述监测细胞在急性迁移的啮齿类动物的脑组织切片的过程。急性片的使用允许assessm耳鼻喉科中的细胞迁移的微环境，密切在体内条件和难以进入体内成像的大脑区域，是相似的。此外，它避免了长期的培养条件，如在器官和细胞培养物的情况下，最终可能会改变的迁移特性的细胞。神经前体在急性片可以应用DIC光学或荧光蛋白。病毒标签在SVZ，接枝记者小鼠神经母细胞从野生型小鼠的SVZ，以及使用表达荧光蛋白的转基因小鼠，在成神经细胞的神经元前体是用于可视化的成神经细胞，并按照它们的迁移的所有合适的方法。然而，后一种方法，不允许进行跟踪各个细胞的很长一段时间，因为标记的细胞的高密度。我们使用了一个宽视场荧光直立显微镜配备有CCD照相机，达到了较快的采集时间间隔（1伊马GE每15或30秒），以可靠地识别静止和迁徙阶段。精确的识别的固定和迁徙阶段的持续时间是至关重要的明确的解释结果。我们还进行了多个z步收购成神经细胞迁移的3D监视。宽视场荧光成像技术已广泛用于可视化神经细胞迁移的7-10。在这里，我们将详细介绍标签的神经母细胞，进行实时视频成像在急性片的成年小鼠前脑的神经细胞迁移，分析细胞迁移的协议。虽然所描述的协议例举在成人RMS的成神经细胞的迁移，它也可以被用于跟踪在胚胎和产后早期大脑细胞迁移。
Protocol
1。标记神经元前体细胞神经母细胞可以可视化使用转基因小鼠，选择性地表达荧光蛋白在神经母细胞（ 即 ，DCX-GFP，GAD67-GFP），立体定向注射的SVZ或RMS编码荧光蛋白的病毒颗粒，或通过嫁接记者小鼠的神经前体（ 即 DCX-GFP，GAD67-GFP）的野生型小鼠的SVZ。我们描述的程序的嫁接和病毒标记的神经元前体。 记者小鼠的SVZ的神经母细胞的?…
Discussion
正确的定位到相应的大脑区域神经元前体是一个基本的程序，合适的组装和功能的神经回路的基本。绝大多数胚胎发育过程中细胞迁移，并在出生后的大脑只在少数地区，如OB，齿状回和小脑，神经元的位移仍然需要地方。在出生后的大脑编排细胞迁移的机制依然存在，然而，知之甚少。知识的机制和成熟的神经组织细胞迁移导向的分子途径，有助于了解在出生后大脑的神经元的定位和发展的新?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由加拿大卫生研究院（CIHR）授予ASJK部分由拉瓦尔大学的奖学金支持。 AS是一个加拿大研究主席在出生后的神经发生的收件人。
Materials
| Name of the reagent | Company | Catalogue number |
| Sucrose | Sigma | S9378 |
| Glucose (ACSF) | EMD | DX0145-3 |
| NaCI | Sigma | S9625 |
| KCI | Sigma | P9541 |
| MgCI2x6H2O | Sigma-Aldrich | M2670 |
| NaHCO3 | Sigma | S5761 |
| NaH2PO4xH2O | EMD | SX0710-1 |
| CaCI2x2H2O | Sigma-Aldrich | C3881 |
| Dextran TexasRed | Invitrogen | D1864 |
| Dextran CascadeBlue | Invitrogen | D1976 |
| Glucose (40X solution) | Sigma | G8769 |
| Sodium pyruvat | Gibco | 11360-070 |
| HEPES | Sigma | H3375 |
| HBSS | Gibco | 14170-112 |
| DNase I | Sigma | D-5025 |
| Trypsin-EDTA | Gibco | 25300-054 |
| Neurobasal medium | Gibco | 21103-049 |
| BSA | EMD | 2930 |
| Pen/Strep | Life Technologies | 15140-122 |
| Ketamine/Xylazine | CDMV | 5230 |
| Pasteur pipette | VWR | 14672-380 |
| 15 ml conical tube | Sarstedt | 62.553.205 |
| 50 ml conical tube | Sarstedt | 62.547.205 |
| Glass capillaries (stereotaxic injection) | WPI | 4878 |
| Paraffin oil | EMD | PX0045-3 |
| Proviodine | Rougier | 65655-1370 |
| Suture | Stoelting | 50487 |
| Anafen | CDMV | 11508 |
| 20 cc Syringe | VWR | SS-20L2 |
| Petri dish | VWR | 25384-094 |
| Agar | Laboratoire Mat | AP-0108 |
| Glue | Permabond | 910 |
| 95% O2/5% CO2 | Linde | 24068835 |
| Blade | WPI | 501901 |
| Nylon mesh | Warner Instruments | 64-0198 |
| Centrifuge | Eppendorf | 5702 000.019 |
| Pipette puller | Sutter Instrument | P-97 |
| Nanoliter injector | WPI | B203MC4 |
| Stereotaxic injection apparatus | WPI | 502900 |
| Micro drill system | WPI | 501819 |
| Vibratome | Thermo Scientific | 920110 |
| Wide-field fluorescent microscope | Olympus | BX61WIF |
| CCD camera | Photometrics | CS-HQ2-D |
| Ultra-quiet imaging chamber | Harvard Apparatus | 64-1487 |
| PH-1 Series 20 heater platform | Harvard Apparatus | 64-0284 |
| Heating system | Warner Instruments | TC-344B |
| 40X water immersion objective | Olympus | 1-UM587 |
| 10X water immersion objective | Olympus | 1-UM583 |
| Lambda DG-4 | Sutter Instruments | DG-4/OF |
| MetaMorph software | Molecular Devices | 40000 |
| Imaris software | Bitplane | BPI-IM70-F1 |
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Cite This Article
Khlghatyan, J., Saghatelyan, A. Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain. J. Vis. Exp. (67), e4061, doi:10.3791/4061 (2012).