测绘激光扫描Photostimulation抑制神经回路
Summary
本文介绍了激光扫描photostimulation在表达绿色荧光蛋白在有限的抑制性神经元的人口的转基因小鼠的全细胞记录相结合的方法。该技术可以广泛的测绘和定量分析当地具体的抑制皮层神经元的突触电路。
Abstract
抑制性神经元皮质功能的关键。他们皮层神经元的整个人口约占20％，还可以进一步细分到他们的免疫组化，形态和生理特性1-4为基础的多元化亚型。虽然以前的研究发现，个别类型的抑制性神经元的内在属性，对他们的局部电路连接知识还比较有限3,5,6。由于每一个人的神经元的功能是通过其兴奋性和抑制性突触内皮层电路输入形，我们已经使用激光扫描photostimulation者（LSP）地图的本地电路连接到特定的抑制细胞类型。 LSP的与传统的电刺激或谷氨酸粉扑刺激相比，具有独特的优势，允许广泛的测绘和定量分析的功能单独记录的神经元3,7-9当地投入。激光通过谷氨酸uncaging photostimulation选择性激活的神经元perisomatically，不激活的情况下通过轴突或远端树突，以确保子层的映射决议。非常适合皮质电路分析的灵敏度和效率，从在一个大区域的许多刺激站点的映射输入的LSP。
在这里，我们引进的技术，并结合本地抑制电路映射，全细胞膜片夹紧的LSP。有针对性的具体的抑制细胞类型的录音方便使用在有限的抑制神经元群表达绿色荧光蛋白（GFP）的转基因小鼠皮层3,10，使记录的细胞类型一致的抽样针对性的细胞类型，并明确识别。由于LSP的映射，我们概述系统仪器，描述实验过程和数据采集，和鼠标初级躯体感觉皮层的电路映射的例子。在我们的实验显示，笼谷氨酸在脑片的紫外激光光解空间限制的区域被激活;同时电压钳记录允许photostimulation诱发的突触反应的检测。无论是兴奋性或抑制性突触输入有针对性的神经元的生成地图，通过扫描激光束激发数百个潜在的突触前网站。因此，选择LSP可以经过反复实验撞击到特定类型的抑制性神经元的突触输入的详细地图的建设。两者合计，photostimulation为基础的技术提供神经学家确定的局部皮层电路功能组织一个强大的工具。
Protocol
Discussion
Photostimulation基础测绘技术已有效地应用于分析皮质电路。激光扫描photostimulation全细胞记录相结合，使高分辨率的映射到单个神经元突触前输入源层分布的，因为从突触后神经元的突触前神经元在许多不同的地点的集群photostimulation同步录音提供兴奋性的空间分布的定量措施或抑制性输入。使用在已知的抑制细胞类型表达绿色荧光蛋白的转基因小鼠资助这种技术大大促进了我们目前的工作，对阐明…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
陈德良Huynh，安德鲁杰里林圣安东尼奥，我们感谢他们的技术援助。这项工作是由国家卫生赠款研究院DA023700和DA023700 – 04S1至第XX
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| transgenic mouse lines | Jackson lab or other sources | Please refer to Xu and Callaway (2009) | |
| GFP goggles | BLS Ltd., Hungary | ||
| vibratome | Leica Systems | VT1200S | |
| MNI caged glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate) | Tocris Bioscience, Ellisville, MO | Cat No. 1490 | |
| biocytin | B4261 | ||
| electrode puller | Sutter Instrument, Novato, CA | P-97 | |
| glass tubes for making electrodes | BF150-86-10 | ||
| Multiclamp 700B amplifier | Molecular Devices, Sunnyvale, CA | Multiclamp 700B | |
| digital CCD camera | Q-imaging, Austin, TX | Retiga 2000 | |
| Research microscope | Olympus, Tokyo, Japan | BW51X | |
| UV laser unit | DPSS Lasers, Santa Clara, CA | model 3501 | |
| Other equipment for Laser scanning phostimulation | Please refer to Xu et al. (2010) |
Solutions:
- Sucrose-containing artificial cerebrospinal fluid (ACSF) for slice cutting (in mM: 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH2PO4, 4 MgCl2, 0.5 CaCl2, and 24 NaHCO3).
- Recording ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose)
- Electrode internal solution (in mM: 126 K-gluconate, 4 KCl, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na, and 10 phosphocreatine; pH 7.2, 300 mOsm).
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