从外围目标跨突触跟踪与伪狂犬病病毒其次是霍乱毒素和生物素葡聚糖胺双标
Summary
Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.
Abstract
跨突触跟踪已成为用于分析调节通过多突触电路周边靶中央传出神经的有力工具。这种方法已通过利用猪病原体伪狂犬病毒（PRV）1最广泛使用的大脑中的。 PRV不会感染大猿，包括人类，所以它是最常用的研究，小型哺乳动物，尤其是啮齿动物。的伪狂犬病应变PRV152表达增强的绿色荧光蛋白（EGFP）报告基因和只有逆行通过突触连接远离感染部位2,3的分层顺序跨过功能性突触。其他PRV毒株具有不同的微生物学性质，并且可以在两个方向（PRV-Becker和PRV-卡普兰）-4,5-运输。该协议将专门处理PRV152。通过提供该病毒在外围部位，如肌肉，也能够限制病毒的进入吨通过一组特定的神经元的他大脑。的eGFP信号的整个大脑所得图案然后解决了连接到最初感染的细胞的神经元。作为跨突触跟踪与伪狂犬病毒的分布特性使得解释中所标识的网络专用连接困难，我们目前采用生物素葡聚糖胺（BDA）和霍乱毒素B亚基（CTB）进行确认，使用鉴定细胞间的连接灵敏，可靠的方法PRV152。免疫化学检测BDA和CTB的具有过氧化物和DAB（3，3'-二氨基联苯胺）的选择是因为它们是在揭示细胞过程，包括远侧树突6-11有效。
Introduction
跨突触跟踪已成为用于分析调节通过多突触电路周边靶中央传出神经的有力工具。这种方法已通过利用猪病原体伪狂犬病毒（PRV），尤其是减毒株PRV-巴萨在1961年首先描述最广泛使用的啮齿动物的大脑12。在这里，我们提出了一个协议，确定具体肌肉的运动皮层表现或使用重组伪狂犬病病毒株（PRV152）表达绿色荧光蛋白（EGFP）报告基因2的肌肉群。所描述的方法利用的嗜神经病毒，产生感染性子代跨突触感染功能电路3,4,13中的其他神经细胞的行为。 PRV152，这是同基因与PRV-巴萨，只有穿越突触逆行通过突触连接的分层顺序远离感染部位3,5- </ SUP>。通过精确控制感染的周边部位也能够限制病毒的进入大脑通过运动神经元的特定子集。作为依次病毒感染连接神经元的链，eGFP的信号的整个大脑所得图案然后将解决在连接到最初感染的细胞的神经元网络。
使用病毒神经跟踪的一个附加的优点是报告蛋白（eGFP的在这种情况下）感染的细胞内的扩增。这个信号放大提供灵敏度的电平，它允许检测甚至稀疏突起。例如，一个稀疏突起从鼻毛运动皮层的面部运动神经元控制所述晶须被发现在使用病毒表达的绿色荧光蛋白14只大鼠 ;先前的研究没有使用经典的示踪剂没有报告基因扩增11,15找到这个投影。不幸的是，许多病毒追踪载体，如在引用的研究中使用的，不交叉的突触，从而限制了其用于跟踪多突触电路。
而呈现明显的优点，用于识别参与的电动机电路单元的网络，跨突触的分布式特性跟踪与PRV-152使得该电路难以解释内特定的连接。因此，我们提出了验证识别电路中的特定连接使用PRV-152双标使用生物素葡聚糖胺（BDA）和霍乱毒素B亚基（CTB）的简单方法。北京经济技术开发区和CTB的结合使用是一种行之有效的追踪神经元6-8,11的特定集合之间的连接方式。一起使用时，这两种示踪剂可以在相同的部分用双色DAB（3，3'-二氨基联苯胺）过程16可视化。高分子量BDA（BDA10kDa）被选定为这个协议，因为它ÿields神经元突起6,7,9进行详细标注。 BDA10kDa的其它优点包括以下：它是在顺行方向6-8优先运输;它可以通过离子电渗或压力注射6-8交付;它可以通过简单的抗生物素蛋白-生物素化辣根过氧化物酶（ABC）的程序17被可视化;它可以通过光或电子显微镜6,7,18进行成像。免疫化学检测CTB的具有过氧化物和DAB被选择为运动神经元逆行标记，因为它是在揭示细胞过程，包括远侧树突10,19有效。我们最近使用这种方法来识别发声运动通路在小鼠和以显示从主运动皮层稀疏连接到喉运动神经元，这是以前假定为缺席20。
Protocol
Representative Results
Discussion
有一些必须规划使用PRV152 4,21的实验时，应考虑到的问题。最重要的是，伪狂犬病毒是致命的。正如前面提到的，大猿，包括人类不容易受到感染，但相应的必须小心保护其他动物。成年小鼠通常接种减毒PRV152应变后五至七天生存。因此，PRV152是不适合用于需要存活时间超过一周长的实验。此外，受感染的动物在生存期通常显示疾病的迹象，从而限制PRV152为需要观察的正常行为的实验工具…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢神户大学，日本的寺岛俊雄博士，教喉癌手术技术，以及普林斯顿大学的林恩·恩奎斯特供应PRV-巴萨博士。研究由美国国立卫生研究院先锋奖DP1 OD000448埃里希D.贾维斯和美国国家科学基金会研究生研究奖学金奖励给古斯塔沃·阿里亚加支持。从适当地记入以前的工作图是在公共科学图书馆ONE开放获取知识共享许可协议（CC-BY），根据该杂志的编辑方针使用。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| NanoFil Microinjection System | World Precision Instruments | IO-Kit | 34 gauge option |
| Stereotaxic frame | David Kopf Instruments | Model 900 | |
| Nanoject II Auto-Nanoliter Injector | Drummond Scientific Company | 3-000-204 | |
| Sliding microtome | Leica | SM2010 R | |
| [header] | |||
| VetBond | 3M | 1469SB | |
| Isofluorane (Forane) | Baxter | 1001936060 | |
| Betadine Swab Stick | Cardinal Health | 2130-01 | 200 count |
| Permount Mounting Medium | Fisher Scientific | SP15-500 | |
| SuperFrost Plus slides | Fisher Scientific | 12-550-15 | |
| Biotinylated dextran amines | Invitrogen | D-1956 | 10,000 MW |
| Pseudorabies virus | Laboratory of Dr. Lynn Enquist (Princeton University) | PRV152 | Titer > 1 x 107 |
| Anti-Cholera Toxin B Subunit (Goat) | List Biological Laboratories | 703 | |
| Cholera Toxin B Subunit | List Biological Laboratories | 103B | |
| Anti-eGFP | Open Biosystems | ABS4528 | |
| 3, 3'-diaminobenzidine | Sigma-Aldrich | D5905 | 10 mg tablets |
| Ethanol | Sigma-Aldrich | E7023 | 200 proof |
| Formaldehyde | Sigma-Aldrich | F8775 | Dilute to 4% |
| Hydrogen peroxide | Sigma-Aldrich | H3410 | 30% |
| Ketamine HCl & Xylazine HCl | Sigma-Aldrich | K4138 | 80 mg/mL & 6 mg/mL |
| Nickel chloride | Sigma-Aldrich | 339350 | |
| Phosphate buffer | Sigma-Aldrich | P3619 | 1.0 M; pH 7.4 |
| Phosphate buffered saline | Sigma-Aldrich | P5493 | 10X; pH 7.4 |
| Sodium Pentobarbital | Sigma-Aldrich | P3761 | 50 mg/mL dose |
| Sucrose | Sigma-Aldrich | S9378 | |
| Tween 20 | Sigma-Aldrich | P1379 | |
| Xylenes | Sigma-Aldrich | 534056 | Histological grade |
| VECTASTAIN Elite ABC Kit | Vector Laboratories | PK-6101 (rabbit); PK-6105 (goat) | |
| Optixcare opthalmic ointment | Vet Depot | 1017992 |
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