在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合
Summary
目前的协议演示了一个简单的方法来跟踪腹腔四分位区域 （VTA） 谷氨酸投影到海马。VTA谷氨酸神经元的光刺激与CA1记录相结合，以演示VTA谷氨酸终端如何调节CA1假定金字塔发射率在体内。
Abstract
大脑中神经元亚群的光遗传调节使研究人员能够解剖体内和前体内的神经回路。这为确定神经元类型在神经回路中的作用及其在信息编码中相对于学习的意义提供了前提。同样，该方法也可用于测试清醒和麻醉动物中两个或两个以上相连的大脑区域的生理意义。目前的研究展示了VTA谷氨酸神经元如何调节麻醉小鼠CA1（海马）中假定金字塔神经元的发射速率。该协议采用腺相关病毒 （AAV） 依赖 VTA 谷氨酸神经元的标签，用于追踪海马层中的 VTA 早熟谷氨酸终端。AAV载体所蕴藏的光控制蛋白（通道罗多普辛;hChR2）和荧光蛋白（eYFP）的表达允许对VTA谷氨酸终端进行机前追踪，并可对VTA谷氨酸神经元细胞体（在VTA中）进行光刺激。高阻抗急性硅电极被放置在CA1中，以检测体内对VTA光刺激的多单元和单单元反应。这项研究的结果表明，在海马体（CA1，CA3和DG）中，早熟VTA谷氨酸终端的层依赖分布。此外，VTA谷氨酸神经元的光刺激增加了假定CA1金字塔单位在体内的发射和爆发率。
Introduction
在过去的十年中，开发了一系列遗传工具，以提高神经元类型调制的特异性，并绘制复杂的神经网络图谱1。值得注意的是，神经元细胞中具有内在感染和复制能力的神经毒性病毒已被部署用于表达或消融神经元亚类型的特定蛋白质。当含有荧光蛋白或基因编码突触活性指标时，转染AAV载体标签，并划出大脑区域2、3的神经网络。AAV 结构中的发起人选择指导具有一定特定性的神经元类型（促进者依赖表达）的载体表达。然而，通过Cre-lox重组，AAV结构被部署在神经元标记4，5，6，7的更大特异性。值得注意的是，在AAV载体中包装的光活化微生物蛋白和荧光蛋白可以用各种神经元亚型8来表达，非常适合成像、神经元型电路追踪和光调制9、10。
AAV 构建立体注射到大脑区域（或细胞核）中，驱动报告人蛋白质在索马、树突和轴突终端中的表达。AAV的神经表达含有记者基因（eYFP），有助于神经元细胞体的标签和分析跟踪投影到和从其他大脑区域11，12，13，14。AAV-eYFP结构携带光控制蛋白（例如hChR2），可以作为成像6，15和基于刺激的生理跟踪神经投影的工具，以目标大脑区域在体内16。根据 AAV 血清类型，神经元标记的方向可能是机前或逆行11，12。先前的研究已经证实，AAV5在神经元12中会异常旅行。因此，表达hChR2的细胞体的光刺激在大脑（目标）17的其他地方产生先天效应。
在这里，AAV（血清型5）与CaMKII®促进器被用来表达eYFP（记者）和hChR2（蛋白）在VTA谷氨酸神经元和轴向投影。这项研究的结果证明了VTA-谷氨酸早产终端在CA1、CA3和DG海马区域的层依赖分布。此外，与基线值相比，VTA 谷氨酸神经元的光刺激增加了 CA1 多单元和单单元活体中的发射速率。该协议使用经济实惠的工具和商用软件，可以提高从神经回路跟踪实验获得的数据质量。
Protocol
所有实验和动物处理程序都得到了路易斯安那州立大学兽医学院机构动物护理和使用委员会（IACUC）的批准。 1. 实验动物 使用5至6周大的小鼠。 在12小时交替光和暗循环的标准条件下，每笼有3至5只动物。食物和水应该提供。 2. 颅骨切除术和动物制备 注：本节描述了小鼠颅骨切除术的术前和术前手术程序。使用标准…
Representative Results
羚生追踪 AAV表达通过C57BL/6小鼠注射后21天（图2）的记者蛋白（eYFP）免疫荧光成像进行验证。在海马区成功标记了早产VTA谷氨酸投影，也通过在DG、CA3和CA1层的eYFP检测（图6a+d：电影2和3）。 VTA 谷氨酸预测海马调节 CA1 活动 VTA谷氨酸神经元的光刺激增?…
Discussion
在过去的十年中，AAV结构的设计有了显著的进步。因此，更多的神经元特异性促进剂已被纳入AAV血清型阵列，以改善转染特异性14。通过结合荧光蛋白、运输器、受体和离子通道的基因，AAV 的库现在用于成像、神经调节和突触活动检测。在商业上可用的AAV结构中，基因编码的荧光和离子通道（opsin）的组合允许神经解剖学和电生理学的神经回路14，18，19…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由哥伦比亚广播公司桥接赠款授予OOM资助。OOM、PAA 和 AS 设计了这项研究并进行了实验。AS 和 PAA 分析了结果。欧姆和PAA准备了手稿。我们感谢卡尔·迪塞罗斯博士（斯坦福大学）提供AAV供我们使用。
Materials
| 3% Hydrogen peroxide | Fisher chemical | H312 | |
| AAV-CaMKIIα-ChR2-eGYP | Addgene | Plasmid #26969 | |
| BNC cable | Amazon | ||
| BNC Splitter | Amazon | ||
| Ceramic Split Mating Sleeve for Ø1.25mm Ferrules. | Thorlabs | ADAL1-5 | |
| Drill | Dremel | LR 39098 | |
| Gelatin coated slides | Fisher scientific | OBSLD01CS | |
| Hamilton's syringe (Neuros) | WPI Inc. | 06H | |
| Head stage adapter | Neuronexus | Adpt-Q4-OM32 | |
| High impedance silicon probe | Neuronexus | Q1x1-tet-5mm-121-CQ4 | |
| INTAN 512ch Recording Controller | INTAN | RHD2000 | |
| Iodine solution | Dynarex | 1425 | |
| Isoflurane | Piramal | NDC 66794-017-25 | |
| Ketamine | Spectrum | K1068 | |
| LED Driver | Thorlabs | LEDD1B | |
| LED light source (470 nm)-blue light | Thorlabs | M470F3 | |
| Micromanipulator | Narishige | M0-203 | |
| Optic fiber | Thorlabs | CFMLC14L05 | |
| Pan head philips screw (M0.6 X 2mm) | Amazon | M0.6 X 2mm | |
| Pre-amplifier headstage (32 Channel) | INTAN | C3314 | |
| Stereotaxic frame | Kopf | 1530 | |
| TTL pulser | Prizmatix | 4031 | |
| Urethane | Sigma | U2500 | |
| Xylazine | Alfa Aesar | J61430 | |
| Software | Company | Version | |
| Graphpad Prism | |||
| Intan Recording Controller | |||
| Neuroexplorer | |||
| Plexon Offline Spike Sorter | |||
| ACSF Composition: | |||
| oxygenated ACSF with 95% Oxygen/5%CO2 constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2 and 25 Glucose). |
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Citar este artigo
Shrestha, A., Adeniyi, P. A., Ogundele, O. M. Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus. J. Vis. Exp. (163), e61282, doi:10.3791/61282 (2020).