体内新生小鼠皮层神经元的双光子成像
Summary
我们提出了一种用于成像新生小鼠大脑皮层的活体双光子成像协议。该方法适用于分析皮层神经元的发育动态、控制神经元动力学的分子机制以及疾病模型神经元动力学的变化。
Abstract
双光子成像是一种强大的工具, 用于体内分析哺乳动物大脑中的神经元电路。然而, 存在着有限数量的活体成像方法来检查活的新生哺乳动物的脑组织。本文综述了一种用于活体新生小鼠单个皮层神经元成像的协议。本议定书包括以下两种方法: (1) 在发育中脑皮层神经元稀疏和明亮标记的超新星系统, 以及 (2) 易碎新生儿颅骨的外科手术。该协议允许在高信噪比的新生儿阶段观察单个皮质突起的时间变化。通过将超新星与 RNA 干扰和 CRISPR/Cas9 基因编辑系统相结合, 也可以实现标记细胞特异基因的沉默和挖空。因此, 该协议可用于分析皮层神经元的发展动态、控制神经元动力学的分子机制, 以及疾病模型中神经元动力学的变化。
Introduction
脑皮层神经元电路的精确布线对于更高的大脑功能 (包括知觉、认知、学习和记忆) 至关重要。皮质电路在产后发育过程中动态地被提炼。通过组织学和体外培养分析, 研究了皮层电路的形成过程。然而, 活哺乳动物中的电路形成的动力学仍然主要是未知的。
双光子显微术已广泛应用于成人小鼠脑1、2神经元回路的体内分析。然而, 由于技术上的挑战, 只有有限数量的研究解决了新生小鼠神经元回路的形成。例如, Carrillo et 等执行了在第二个产后周3的小脑中攀爬纤维的延时成像。Portera-他等报道了第一个产后周4皮层层1的轴突成像。在本研究中, 我们总结了一个协议, 观察4层皮层神经元及其在新生小鼠的树突。在我们最近发表的5号出版物中报告了应用本议定书所获得的结果, 其中包括两种方法。首先, 我们使用超新星矢量系统5,6用于标记新生儿大脑中的单个神经元。在超新星系统中, 用于神经元标记的荧光蛋白是可交换的, 标记细胞特异基因敲除和编辑/挖空分析也是可能的。其次, 我们描述了在脆弱的新生小鼠颅窗准备的外科手术。这些方法结合在一起, 允许在体内观察新生儿大脑中的单个神经元。
Protocol
Representative Results
Discussion
协议和故障排除中的关键步骤:
该协议最关键的步骤是删除头骨 (协议步骤 3.2)。插入时, 刀片通常附着硬脑膜, 导致硬脑膜出血和大脑损伤。这可以避免通过添加一滴皮质缓冲液在头骨和删除头骨在皮质缓冲区。
颅窗准备后, 硬脑膜和皮肤出血导致窗口闭塞。为了避免这种, 在继续下一步之前, 应允许使用组织粘合剂和牙科水泥完全干燥。由于很难完全避免出…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢佐藤、m. 上林和 s. Kouyama 的技术援助。这项工作得到了 jsp KAKENHI 赠款号 JP15K14322 和 JP16H06143、武田科学基金会、上原纪念基金会以及新泻大学脑科学研究所 2017-2923 (陛下) 的合作研究项目的支持, 并由KAKENHI JP16K14559、JP15H01454、JP15H04263 和赠款–在创新领域的科学研究 “从 JP16H06459 (MEXT) 的废料 & 构建系统的动态调节大脑功能” (T.I.)。
Materials
| pK031. TRE-Cre | 저자 | – | Available from RIKEN BRC and Addgene |
| pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE | 저자 | – | Available from RIKEN BRC and Addgene |
| pK273. CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE | 저자 | – | Available from authors |
| Isoflurane | Wako | 099-06571 | |
| 410 Anaesthesia Unit (isoflurane gas machine) | Univentor | 8323101 | |
| Vetbond (tissue adhesive) | 3M | 084-1469SB | |
| MµltiFlex Round (loading tip) | Sorenson | 13810 | |
| Gelfoam (gelatin sponge) | Pfizer | 09-0353-01 | |
| Agarose | Sigma | A9793 | Low melting point |
| Round-shaped coverslip | Matsunami | – | Custom made |
| Unifast 2 (dental cement) | GC | – | |
| Titanium bar | 저자 | – | Custom made (see Figure 1G) |
| Rimadyl (carprofen) | Zoetis | – | Injectable |
| 2-photon microscope | Zeiss | LSM7MP | |
| Titanium-sapphire laser | Spertra-Physics | Mai-Tai eHPDS | |
| Titanium plate | 저자 | – | Custom made (see Figure 2A) |
| IMARIS, FilamentTracer, MeasurementPro | BITPLANE | ||
| Goniometer stage | Thorlabs | GN2/M |
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