病毒遗传背景明确的神经回路跟踪
Summary
跟踪突触连接的神经元的方法。我们使用TVA特异性的上游电池，探测是否感兴趣的接收的细胞群体突触输入从基因定义的细胞类型。
Abstract
古典方法研究神经回路是相当低的吞吐量。病毒跨突触，特别是伪狂犬（PRV）和狂犬病毒（RABV），，最近水泡性口炎病毒（VSV），学习电路，正变得越来越流行。这些高通量的方法使用顺行或逆行方向的神经元之间传递的病毒。
近日，RABV突触逆行追踪。 （ 图1A）。在这种方法中，糖蛋白（G）的基因被删除从病毒基因组中，仅在目标神经元和补给。感染特异性达到代的嵌合G，ASLV-阿糖蛋白和胞质域的RABV-G（A / RG）的胞外结构域组成，正常RABV-G 1。特别是感染这种嵌合G TVA受体1的表达。该基因编码TVA已经Delivered通过各种方法2-8。 RABV-G感染的TVA表达神经元，RABV可以传输到其他突触连接的神经元在逆行方向的性质，这是交付的TVA受体的G。这种技术标签相对大量的输入（5-10％）2到一个定义的细胞类型，提供了一个采样到一个定义的起动器的细胞类型中的所有的输入。
我们最近修改了这个技术的使用VSV作为一个跨突触示踪9。 VSV有几个好处，包括快速的基因表达。在这里，我们详细介绍了新的病毒跟踪系统，使用VSV有用的探测微型电路与更高的分辨率。在原来的出版策略，由威克沙姆等。4和贝尔等人。9许可证标记神经元的任何项目上最初感染TVA-表达细胞，在这里VSV被设计仅传输到电视A-细胞（ 图1B）。该病毒是第一个假RABV-G允许感染下游TVA表达神经元的神经元。这个第一群细胞感染后，病毒发布只能感染TVA-表达细胞。由于跨突触的病毒传播是有限的TVA-细胞的自定义类型的细胞连接的情况下，存在可以被探索和高分辨率。这些实验是在图2中所示的实验流程图。在这里，我们展示一个模型电路，在小鼠视网膜的方向选择性。我们考察爆无长突细胞（SACS）的连通性视网膜神经节细胞（RGC）。
Protocol
1。使病毒从cDNA恢复的VSV的cDNA使用牛痘-T7系统10 前一天进行实验，分割成60毫米菜含有DMEM + 10％FBS BsrT7细胞。每道菜的种子2E6细胞。 BsrT7细胞来源于BHK21，或幼仓鼠肾细胞。 暖用1mM镁和1mM氯化钙的PBS至37℃。 获取一个小等分vTF7-3，表达T7聚合酶的牛痘病毒，和解冻至室温。 vTF7-3是一种传染性的牛痘病毒表达T7聚合酶11，并应使用仅在生物安全级别2遏制。这种表…
Discussion
使用病毒来研究神经回路是一个相对较高的吞吐量，连接神经元的分析方法。但是，生成，VSV和RABV病毒颗粒是不平凡的。虽然提供了上面列出的用于从cDNA拯救病毒的协议，它仍然是一个低概率事件。水平的每一个的N，P和L的质粒中需要被精确地调整，需要做的工作，以确保病毒救援和许多试验和复制。将一个完整的VSV基因组RNA的核苷酸颗粒的形成是一个低概率事件，因此可能需要多次尝试。这?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们想感谢肖恩·惠兰与拯救重组VSV变种的协助，以及技术援助的Didem的戈兹贝达和Ryan Chrenek为。这项工作是由霍华德休斯医学研究所（CLC），NS068012-01（KTB）。
Materials
| Reagent | Company | Catalogue number | |
| Tissue Culture | |||
| Baby Hamster Kidney (BSRT7) cells | available upon request | ||
| vaccinia (vTF7-3) | available upon request | ||
| pN, pP, pl plasmids | available upon request | ||
| Calcium Chloride | Sigma | C1016 | |
| Magnesium Chloride | Sigma | M8266 | |
| HEK 293T cells | Open Biosystems | HCL4517 | |
| 60 mm TC-Treated Culture Dish | Corning | 430166 | |
| 75 cm2 Rectangular Canted Neck Cell Culture Flask with Vent Cap | Corning | 430641 | |
| Media : DMEM (Dulbecco’s Modified Eagle Medium) | Invitrogen | 12491-015 | |
| 1 M HEPES pH 7.4 | Gibo | 15630-080 | |
| FBS: Fetal Bovine Serum | Gibco | 10437-028 | |
| PKS | Invitrogen | 15140-163 | |
| Lipofectamine 2,000 Transfection Reagent | Invitrogen | 11668-019 | |
| Syringe: 5 ml Luer-Lock syringe | Sigma | Z248010-1PAK | |
| Syringe Filters | Nalgene | 190-2520 | |
| PEI: High Potency Linear PEI | Polysciences | 23966 | |
| Viral Centrifugation | |||
| Corning 150 ml Tube Top Vacuum Filter System, 0.45 μm Pore | Corning | 430314 | |
| Thinwall, Ultra-Clear, 38.5 ml, 25 x 89 mm ultracentrifuge tubes | Beckman-Coulter | 344058 | |
| Ultracentrifuge | Beckman-Coulter | optima XL-80K | |
| SW28 Ultracentrifuge rotor | Beckman-Coulter | 342207 | |
| Mouse Injection | |||
| Capillary micropipets | Drummond | 5-000-2005 | |
| Stereotax | Narishige | SR-5M | |
| Micromanipulator | Narishige | SM-15 | |
| Ump injector | World Precision Instruments | Sys-Micro4 | |
| Four channel microcontroller | World Precision Instruments | UMP3 | |
| M.TXB Bench Motor with C.EMX-1 Dial Control, 115 Volt | Foredom | M.TXB-EM | |
| H.10 Handpiece, Quick Change | Foredom | H.10 | |
| Step Drill, 0.5 mm | Foredom | A-58005P | |
| Microelectrode holder | World Precision Instruments | MEH2S | |
| Ketamine | Henry Schein | 995-2949 | |
| Xylazine | Henry Schein | 4015809TV | |
| Buprenorphine | Henry Schein | 1118217 | |
| 1 ml syringe | Becton-Dickinson | 309628 | |
| 30 gauge injection needle | Becton-Dickinson | 305106 | |
| Protective Ophthalmic Ointment | Doctors Foster and Smith | 9N-014748 | |
| Ethanol | Sigma | 493511 | |
| Iodine | Sigma | PVP1 | |
| Surgery and Dissection tools | |||
| Scissors | Fine Science Tools | 91402-12 | |
| Standard Forceps | Fine Science Tools | 11000-12 | |
| Fine Forceps | Fine Science Tools | 11255-20 | |
| Vannas spring scissors | Fine Science Tools | 15000-00 | |
| Scalpel handle | Fine Science Tools | 10003-12 | |
| Scalpel blades | Fine Science Tools | 10015-00 | |
| Sutures | Robbins Instruments | 20.SK640 | |
| Dissection and antibody staining | |||
| paraformaldehyde | Sigma | P6148 | |
| Phosphate Buffered Saline | Sigma | P4417 | |
| Triton X-100 | Sigma | T9284 | |
| Donkey Serum | Jackson Immunoresearch | 017-000-121 | |
| Antibodies | |||
| Antibodies | millipore | AB144P | |
| Anti-gfp | Abcam | ab13970 | |
| Donkey anti-chicken Dylight 488 | Jackson immunoresearch | 703-545-155 | |
| Donkey anti-chicken Alexa Fluor 647 | Jackson immunoresearch | 705-605-147 | |
| DAPI | Invitrogen | D1306 | |
| Tissue mounting | |||
| Superfrost plus microscope slides | Fisher | 12-550-100 | |
| Cover glass 22 x 22, 0 thickness | Electron Microscopy Sciences | 72198-10 | |
| Silicone elastomer | Rogers Corp | HT-6220 | |
| Clear nail polish | Electron Microscopy Sciences | 72180 | |
| Prolong Gold antifade reagent | Invitrogen | P36930 |
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Cite This Article
Beier, K., Cepko, C. Viral Tracing of Genetically Defined Neural Circuitry. J. Vis. Exp. (68), e4253, doi:10.3791/4253 (2012).