多色揭示神经回路地形
Summary
我们提供了一个实用指南提供示踪剂<em>在体内</em>，并使用作为一个模型系统的脊髓小脑通路证明成功在小鼠神经元电路分析的基本步骤。我们详细描述了我们全方位的跟踪协议，它利用麦胚凝集素（WGA）的共轭Alexa的荧光团。
Abstract
被组织成神经回路的功能地形图。为了形象直观地查看复杂的电路架构，我们开发了一个方法多种地形预测全球的图案，以可靠的标签。小脑是一个理想的的模型来研究神经回路的有序安排。例如，房室脊髓小脑长满青苔的纤维组织已经被证明是一个不可缺少的系统研究苔藓纤维图案的。我们最近表明，麦胚凝集素（WGA）的共轭Alexa的555和488可以用来跟踪，在发展中国家和成年小鼠的脊髓小脑苔藓纤维预测（Reeber等。，2011年）。我们发现了三个主要物业，使标签神经预测的WGA Alexa的示踪剂的理想工具。首先，Alexa的荧光团激烈，其亮度wholemount成像允许后直接跟踪。二，WGA – Alexa的示踪剂标签的整个发展中国家和成人神经projectio轨迹NS。第三，WGA – Alexa的示踪剂正在迅速运逆行及顺行方向。在这里，我们详细描述了如何准备示踪剂和其他所需的工具，如何进行手术治疗脊髓小脑跟踪和如何以最佳形象追溯到三个方面的预测。综上所述，我们提供了一个一步一步跟踪协议，将破译的组织和功能的地图不仅在小脑，而且在皮质，脑干和脊髓的连通。
Protocol
1。示踪剂在体内提供使用无菌手术（第1.1-1.16） 整个过程中，我们建议使用标准的无菌手术技术。这包括使用无菌手套，隔离衣和口罩。工具应进行高压灭菌或使用前彻底清洁的水，然后乙醇。在手术过程中，特别是与动物之间，干净的纸巾碎片珠用热灭菌（无菌250＃18000-45，精细科学的工具）的工具和干燥消毒仪器关闭。此外，至关重要的是，您组织您的工作空间，?…
Discussion
我们已经描述了手术和技术的成功使用一种新型的荧光为基础的方法，迅速在发展中国家和成年小鼠的神经预测标签的轴突和树突跟踪所需的详细信息。使用WGA – Alexa的我们展示了如何示踪剂和标记可用于图案电路地形分析在高分辨率和在三个方面。
许多示踪剂追踪神经回路，包括霍乱毒素B亚基，生物胞素，Neurobiotin，菜豆leucoagglutinin（PHAL），fluororuby，fluoremerald和荧光金。?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了新的启动资金来自叶史瓦大学阿尔伯特爱因斯坦医学院的研究者RVS。
Materials
| Equipment/Reagents | Model/Catalogue number | Company |
| Bead sterilizer | Model Steri 250 Cat. # 18000-45 | Fine Science Tools |
| Cauterizer | Cat. # 18000-00 | Fine Science Tools |
| Borosilicate glass capillaries | Cat. # 300056 | Harvard Apparatus |
| Dual stage Glass Micropipette Puller | Model 001-PC-10 | Narishige |
| Micrometer syringe | Cat. # GS-1100 | Gilmont Instruments |
| Small Animal Stereotaxic Instrument | Model 940-A | Kopf Instrumentation |
| Electrode Manipulator | Model 960 | Kopf Instrumentation |
| Vetcare chamber | Cat. # 340508 | Harvard Apparatus |
| Heating Pad | Cat. # 341241 | Harvard Apparatus |
| Leica DFC360 FX camera | DFC360 FX | Leica |
| Leica DFC490 camera | DFC490 | Leica |
| Leica DM5500 microscope | DM5500 | Leica |
| Leica DFC3000 FX camera | DFC3000 FX | Leica |
| Leica MZ16 FA microscope | MZ16 FA | Leica |
| CY3 Filter | Model # 11600231 | Leica |
| FITC Filter | Model # 11513880 | Leica |
| A4 DAPI/UV filter | Model # 11504162 | Leica |
| Wheat germ agglutinin, Alexa Fluor 488 conjugate | Cat. #W11261 | Invitrogen |
| Wheat germ agglutinin, Alexa Fluor 555 conjugate | Cat. #W32464 | Invitrogen |
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Tags
Neural Circuit TopographyMulti-color Neural CircuitsFunctional Topographic MapsVisualizing Circuit ArchitectureLabeling Topographic ProjectionsCerebellumSpinocerebellar Mossy FibersWheat Germ Agglutinin (WGA)Alexa 555Alexa 488Tracing Neural ProjectionsRetrograde TransportAnterograde TransportTracers PreparationSpinocerebellar Tracing SurgeryThree-dimensional ImagingFunctional Maps OrganizationConnectivity
Diesen Artikel zitieren
Reeber, S. L., Gebre, S. A., Filatova, N., Sillitoe, R. V. Revealing Neural Circuit Topography in Multi-Color. J. Vis. Exp. (57), e3371, doi:10.3791/3371 (2011).