高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用
Summary
在这里, 我们提出了一个完善的协议, 以有效地揭示生物素化葡聚糖胺 (BDA) 标签的荧光染色方法通过一个相互的神经通路。它适用于分析 BDA 标签的精细结构, 并在共焦激光扫描显微镜下将其与其他神经元进行区分。
Abstract
高分子量生物素化葡聚糖胺 (BDA) 已被用作高度敏感的神经解剖学示踪剂数十年。由于其标签的质量受到各种因素的影响, 在这里, 我们提供了一个完善的协议, 以应用高分子量的 BDA 研究最佳神经标记在中枢神经系统。经立体定向注入内侧核 (VPM) 的大鼠丘脑通过一个微妙的玻璃吸管, bda 染色的荧光链亲和素 Alexa (AF) 594 和复染荧光尼斯尔染色 AF500/525。在绿色尼斯尔染色的背景下, 在体感皮层显示了红色的 BDA 标记, 包括神经元细胞体和轴突末端。此外, 对 bda 和钙结合蛋白 parvalbumin (pv) 进行了双荧光染色, 观察了 bda 标记与中间神经元在皮质靶上的相关性, 为研究局部神经电路及其化学特性。因此, 这种精制方法不仅适用于通过丘脑和大脑皮层之间的相互神经通路对高品质的神经标记进行可视化, 而且还可以同时证明其他具有荧光组织化学或免疫化学的神经标记。
Introduction
高分子量 BDA (1万分子量), 一种高度灵敏的示踪剂, 已用于在中枢神经系统中追踪神经通路20年以上的1。虽然 bda 的使用是一种常见的神经道追踪技术, 但在动物中, bda 标签的质量可能受到各种因素的影响, 如1,2,3。最近的研究表明, BDA 标签的优化结构不仅与适当的注射后生存时间有关, 而且与染色方法4相关。到目前为止, 传统的亲和-生物素-过氧化物酶复合物 (ABC), 链亲和素异硫氰酸酯, 和 streptavidin-AF594 染色方法, 以揭示 BDA 标签在以前的研究2, 3, 4,5。相比之下, BDA 的荧光染色可以很容易地进行。
为了推广高分子量 BDA 的应用, 本研究引入了一种精细化的协议。在将 bda 注射液注入大鼠脑丘脑的 VPM 后, 采用常规 ABC 染色法和双荧光染色技术对 bda 标签进行了分析, 观察了 bda 标签的相关性和基本streptavidin-AF594 和荧光尼斯尔组织化学或 PV 免疫化学的皮质靶上的神经元素或中间神经元。通过 VPM 与原发性体感皮层 (S1)6、7、8之间的相互神经通路, 我们集中观察了在丘脑-皮质预测轴突和 corticothalamic 中的 BDA 标记。投射的细胞 somas 在 S1。通过这一过程, 我们希望不仅提供一个详细的协议, 以获得高品质的神经标记的高分子量 bda, 而且还有一个完善的协议, 结合荧光 BDA 标签和其他荧光神经标记与组织化学或免疫化学。这种方法在共聚焦激光扫描显微镜下研究局部神经回路及其化学特性是比较可取的。
Protocol
Representative Results
Discussion
选择合适的示踪剂是成功的神经追踪实验的关键步骤。在 bda 家族中, 建议通过顺神经通路优先输送高分子量的 bda (1万分子量), 与低分子量 bda (3000 分子量)2,3,11,12,13. 然而, 许多研究还表明, 高分子量的 BDA 也可能会与顺追踪1,2,</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由中国国家自然科学基金 (项目编号: 81373557; 81403327) 资助。
Materials
| Biotinylated dextran amine (BDA) | Molecular Probes | D1956 | 10,000 molecular weight |
| Streptavidin-Alexa Fluor 594 | Molecular Probes | S32356 | Protect from light |
| 500/525 green fluorescent Nissl stain | Molecular Probes | N21480 | Protect from light |
| Brain stereotaxis instrument | Narishige | SR-50 | |
| Freezing microtome | Thermo | Microm International GmbH | |
| Confocal imaging | Olympus | FV1200 | |
| system | |||
| Micro Drill | Saeyang Microtech | Marathon-N7 | |
| Sprague Dawley | Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences | SCKX (JUN) 2012-004 | |
| Vectastain ABC Kit | Vector Laboratories | PK-4000 | |
| superfrost plus microscope slides | Thermo | #4951PLUS-001 | 25x75x1mm |
| Photoshop and Illustration | Adobe | CS5 |
References
- Veenman, C. L., Reiner, A., Honig, M. G. Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. J Neurosci Methods. 41, 239-254 (1992).
- Reiner, A., Veenman, C. L., Medina, L., Jiao, Y., Del Mar, N., Honig, M. G. Pathway tracing using biotinylated dextran amines. J Neurosci Methods. 103, 23-37 (2000).
- Ling, C., Hendrickson, M. L., Kalil, R. E. Resolving the detailed structure of cortical and thalamic neurons in the adult rat brain with refined biotinylated dextran amine labeling. PLoS One. 7, e45886 (2012).
- Zhang, W. J., et al. Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways. Microsc Res Tech. 80, 260-266 (2017).
- Han, X., et al. Biotinylated dextran amine anterograde tracing of the canine corticospinal tract. Neural Regen Res. 7, 805-809 (2012).
- Armstrong-James, M., Callahan, C. A. Thalamo-cortical processing of vibrissal information in the rat. II. spatiotemporal convergence in the thalamic ventroposterior medial nucleus (VPm) and its relevance to generation of receptive fields of S1 cortical "barrel" neurones. J Comp Neurol. 303, 211-224 (1991).
- Armstrong-James, M., Callahan, C. A., Friedman, M. A. Thalamo-cortical processing of vibrissal information in the rat. I. Intracortical origins of surround but not centre-receptive fields of layer IV neurones in the rat S1 barrel field cortex. J Comp Neurol. 303, 193-210 (1991).
- Agmon, A., Yang, L. T., Jones, E. G., O’Dowd, D. K. Topological precision in the thalamic projection to neonatal mouse barrel cortex. J Neurosci. 15, 549-561 (1995).
- Paxinos, G., Watson, C. . The rat brain in stereotaxic coordinates. , (1998).
- Davidoff, M., Schulze, W. Standard avidin-biotin-peroxidase complex (ABC) staining combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining. Histochemistry. 93, 531-536 (1990).
- Fritzsch, B. Fast axonal diffusion of 3000 molecular weight dextran amines. J Neurosci Methods. 50, 95-103 (1993).
- Kaneko, T., Saeki, K., Lee, T., Mizuno, N. Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR. J Neurosci Methods. 65, 157-165 (1996).
- Medina, L., Reiner, A. The efferent projections of the dorsal and ventral pallidal parts of the pigeon basal ganglia, studied with biotinylated dextran amine. 신경과학. 81, 773-802 (1997).
- DE Venecia, R. K., Smelser, C. B., McMullen, N. T. Parvalbumin is expressed in a reciprocal circuit linking the medial geniculate body and auditory neocortex in the rabbit. J Comp Neurol. 400, 349-362 (1998).
- Ojima, H., Takayanagi, M. Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity. J Neurosci Methods. 104, 177-182 (2001).
- Kobbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. Current concepts in neuroanatomical tracing. Prog Neurobiol. 62, 327-351 (2000).
- Vercelli, A., Repici, M., Garbossa, D., Grimaldi, A. Recent techniques for tracing pathways in the central nervous system of developing and adult mammals. Brain Res Bull. 51, 11-28 (2000).
- Liao, C. C., Reed, J. L., Kaas, J. H., Qi, H. X. Intracortical connections are altered after long-standing deprivation of dorsal column inputs in the hand region of area 3b in squirrel monkeys. J Comp Neurol. 524, 1494-1526 (2016).
