小鼠大脑皮层细胞组织的大尺度三维成像
Summary
在这里, 我们描述一个程序, 组织清除, 荧光标记, 和大规模的小鼠脑组织成像, 从而使可视化的三维组织的细胞类型在大脑皮层。
Abstract
哺乳动物大脑皮层由许多类型的兴奋和抑制神经元组成, 每一种都有特定的电生理和生化特性, 突触连接和体内功能, 但其基本功能和解剖学组织从细胞到网络规模是不太了解。在这里, 我们描述了一个方法, 三维图像的荧光标记神经元跨大区域的大脑, 以调查皮质细胞组织。在转基因小鼠中, 用荧光逆行神经元示踪剂或荧光蛋白的表达来标记特定类型的神经元。块脑标本,例如,半球, 是在固定后准备的, 用组织清除方法透明, 并受到特定细胞类型的荧光 immunolabeling。大面积扫描使用共焦或双光子显微镜, 配备了较大的工作距离目标和机动阶段。该方法可以解决小鼠大脑皮层细胞型特定柱功能模块的周期性组织问题。该方法可用于研究不同脑区和其他复杂组织中的三维细胞结构。
Introduction
哺乳动物大脑皮层由大量的细胞类型组成, 每一个有特定的基因表达模式, 电生理和生化特性, 突触连接, 和在体内功能1,2 ,3,4,5,6,7。这些细胞类型是否被组织成重复的结构是不清楚的。皮质柱, 包括视觉定向柱和体感桶, 有重复的结构, 但其细胞组织仍然不清楚8,9。这些是存在于特定的皮质区域, 不是大脑范围的系统。
在皮层5层, 大部分神经元被分为四大类。一种主要类型的兴奋神经元, 亚脑投射神经元, 项目轴突到皮层下靶, 包括桥脑, 脊髓, 和优越的丘, 因此, 代表了主要的皮质输出通路10。皮质投射神经元, 另一种主要类型的兴奋神经元, 支配皮层10。抑制神经元也包含两大类: parvalbumin 表达和生长抑素表达细胞11。
最近的分析表明, 四细胞类型被组织成重复结构12,13,14。次级脑投射神经元12、13、14和皮层投射神经元14组织成细胞型特异性 microcolumns, 有一直径的细胞。Parvalbumin 表达和生长抑素表达细胞与 microcolumns 的亚脑投射神经元, 而不是与 microcolumns 皮层投射神经元14。Microcolumns 自己周期性地排列形成一个六角格子阵列14并且在多皮层区域包括视觉, 感觉和马达区域在老鼠脑子12,14并且在语言人脑的区域13。神经元在个体柱陈列同步活动并且有相似的感觉反应14。这些观察表明, 5 层细胞类型组织成一个柱晶格结构, 代表第一个已知的大脑范围内重复功能模块的组织。
Microcolumns 有大约10µm 的半径并且有大约40µm 的空间周期性。此外, microcolumns 的方向与它们的顶端树突平行, 并根据它们在皮层14中的位置而变化。因此, 柱系统很难分析使用传统的皮质切片的典型厚度的几微米。此外, 周期性分析需要来自广泛脑区的三维数据, 因此, 共焦显微镜和活体2 光子成像的典型成像区域太窄。
最近, 技术已经发展到清除厚组织15,16。在这里, 我们描述了这些方法的应用, 以获得大型, 三维图像的主要细胞类型在小鼠皮层层5组成的柱系统。Subcerebral 投射神经元用逆行标记或增强绿色荧光蛋白在Crym-egfp转基因小鼠12中的表达进行标记, 皮质投射神经元用逆行标记标记或由 tdTomato 表达在Tlx3/Ai9小鼠17。Parvalbumin 表达和生长抑素表达细胞用免疫组化标记。(抗体标度 S) AbScale 方法18用于抗体染色实验, 而 (见深脑) SeeDB 方法19用于其他实验。这些方法克服了传统成像方法的难点, 揭示了5层14的精确细胞组织。
Protocol
所有实验程序均经山本理瓦科动物实验委员会和山本理基因重组实验安全委员会批准, 并根据山本理脑科学动物设施的机构指南进行。研究所。 1. 成像室的制备 成像室19 使用硅橡胶板, 准备一个房间的厚度约5毫米和地板板的各种厚度。此外, 准备有和没有玻璃底部的培养皿 (图 1A)。 ?…
Representative Results
我们标记皮层投射神经元的 tdTomato 在Tlx3/Ai9 转基因小鼠和可视化的亚脑投射神经元, 通过注射逆行示踪 CTB488 进入脑桥。大脑的左半球受 SeeDB 方法扫描, 使用双光子显微镜, 配备了水浸泡长工作距离目标 (25X, 1.1, 工作距离2毫米) 和机动阶段。一叠401张图像 (512 x 512 像素; 像素大小 = 0.99 µm) 在 z 步 = 2.8 µm 在每30个扫描位置被获得了。两种细胞的细胞体在广泛的?…
Discussion
我们已经提出的程序, 以获得大规模的三维图像的细胞类型特定组织的主要细胞类型的小鼠皮层层5。与传统的切片染色相比, 该方法在确定大脑皮层的三维组织方面更为有用。该方法使图像获得从更广泛和更深层次的大脑区域相比, 典型的活体2 光子显微镜或常规共焦显微术, 从而可以让皮层细胞的综合分析组织。
该方法的一个关键步骤是抗体穿透。抗体的子集显示, 渗?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢淳宫胁和哈马为他们的建议在 AbScale 实验, 查尔斯横山为编辑手稿, eriko 公司 Ohshima 和美雪 Kishino 为他们的技术协助。这项工作得到了从山本理到金斗勋的研究资金和日本教育、文化、体育、科学和技术部 (下个) 为金斗勋 (“介观 Neurocircuitry” 的创新领域) 的科学研究资助; 22115004) 和(25890023)。
Materials
| Crym-egfp transgenic mice | MMRRC | 012003-UCD | |
| Tlx3-cre transgenic mice | MMRRC | 36547-UCD | |
| ROSA-CAG-flox-tdTomato mice | Jackson Laboratory | JAX #7909 | |
| Silicone rubber sheet | AS ONE | 6-611-01 | 0.5 mm thickness |
| Silicone rubber sheet | AS ONE | 6-611-02 | 1.0 mm thickness |
| Silicone rubber sheet | AS ONE | 6-611-05 | 3.0 mm thickness |
| Petri dishes | Falcon | 351008 | |
| Cover glass | Matsunami | C022241 | |
| Cholera toxin subunit B (recombinant), Alexa Fluor 488 conjugate | Invitrogen | C22841 | |
| Cholera toxin subunit B (recombinant), Alexa Fluor 555 conjugate | Invitrogen | C22843 | |
| Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate | Invitrogen | C22842 | |
| Cholera toxin subunit B (recombinant), Alexa Fluor 647 conjugate | Invitrogen | C34778 | |
| 26G Hamilton syringe | Hamilton | 701N | |
| Injector pump | KD Scientific | KDS 310 | Pons injection |
| Injector pump | KD Scientific | KDS 100 | Superior colliculus injection |
| Manipulator | Narishige | SM-15 | |
| Sodium pentobarbital | Kyoritsu Seiyaku | Somnopentyl | |
| Isoflurane | Pfizer | ||
| Lidocaine | AstraZeneca | Xylocaine injection 1% with epinephrine | |
| Drill | Toyo Associates | HP-200 | |
| Avitene microfibrillar hemostat | Davol Inc | 1010090 | |
| Alonalfa | Daiichi-Sankyo | Alonalpha A | |
| Surgical silk | Ethicon | K881H | |
| Incubator | UVP | HB-1000 Hybridizer | |
| Glass pipette | Drummond Scientific Company | 2-000-075 | |
| Electrode puller | Sutter Instrument Company | P-97 | |
| Paraffin Liquid, light | Nacalai tesque | 26132-35 | |
| Saline | Otsuka | 1326 | |
| Paraformaldehyde | Nacalai tesque | 26126-54 | |
| Tungsten needle | Inter medical | Φ0.1 *L200 mm | |
| Vibratome | Leica | VT1000S | |
| 50 mL plastic tube | Falcon | 352070 | |
| α-thioglycerol | Nacalai tesque | 33709-62 | |
| D(-) Fructose | Nacalai tesque | 16315-55 | |
| BluTack | Bostik | CKBT-450000 | |
| Two-photon microscope | Nikon | A1RMP | |
| Water-immersion long working distance objectives | Nikon | CFI Apo LWD 25XW, NA 1.1, WD 2 mm | |
| Water-immersion long working distance objectives | Nikon | CFI LWD 16XW, NA 0.8, WD 3 mm | |
| Motorized stage | COMS | PT100C-50XY | |
| Filter | Semrock | FF01-492/SP-25 | |
| Filter | Semrock | FF03-525/50-25 | |
| Filter | Semrock | FF03-575/25-25 | |
| Filter | Semrock | FF01-629/56 | |
| Filter | Chroma | D605/55m | |
| 5 mL plastic tube | AS ONE | VIO-5B | |
| 2 mL plastic tube | Eppendorf | 0030120094 | |
| Urea | Nacalai tesque | 35905-35 | |
| Triton X-100 | Nacalai tesque | 35501-15 | |
| Glyserol | Sigma-aldrich | 191612 | |
| D(-)-sorbitol | Wako | 191-14735 | |
| Methyl-β-cyclodextrin | Tokyo chemical industry | M1356 | |
| γ-Cyclodextrin | Wako | 037-10643 | |
| N-acetyl-L-hydroxyproline | Skin Essential Actives | 33996-33-7 | |
| DMSO | Nacalai tesque | 13445-45 | |
| Bovine Serum Albumin | Sigma-aldrich | A7906 | |
| Tween-20 (1.1 g/mL) | Nacalai tesque | 35624-15 | |
| Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A21422 | |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A21428 | |
| Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A21235 | |
| Goat anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A11029 | |
| Donkey anti-Rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A21206 | |
| Confocal microscope | Olympus | FV1000 | |
| Water-immersion long working distance objectives | Olympus | XLUMPLFLN 20XW, NA 1.0, WD 2 mm | |
| Anti-NeuN | Millipore | MAB377 | |
| Anti-NeuN | Millipore | ABN78 | |
| Anti-CTIP2 | Abcam | ab18465 | |
| Anti-Statb2 | Abcam | ab51502 | |
| Anti-GAD67 | Millipore | MAB5406 | |
| Anti-GABA | Sigma | A2052 | |
| Anti-Parvalbumin | Swant | 235 | |
| Anti-Parvalbumin | Frontier Institute | PV-Go-Af460 | |
| Anti-Parvalbumin | Sigma | P3088 | |
| Anti-Parvalbumin | Abcam | ab11427 | |
| Anti-Somatostatin | Peninsula Laboratories | T-4103 | |
| Anti-c-Fos | CalbioChem | PC38 | |
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Citar este artigo
Yoneda, T., Sakai, S., Maruoka, H., Hosoya, T. Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex. J. Vis. Exp. (139), e58027, doi:10.3791/58027 (2018).