使用跨突触伙伴的哺乳动物 GFP 重组 (mGRASP) 来绘制小鼠大脑中的突触连接。
Using mammalian GFP reconstitution across synaptic partners (mGRASP) to map synaptic connectivity in the mouse brain.
机构信息
1] Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. [2].
1] Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. [2] Neuroscience program, University of Science and Technology, Daejeon, Korea. [3].
出版信息
Nat Protoc. 2014 Oct;9(10):2425-37. doi: 10.1038/nprot.2014.166. Epub 2014 Sep 18.
Many types of questions in neuroscience require the detection and mapping of synapses in the complex mammalian brain. A tool, mammalian GFP reconstitution across synaptic partners (mGRASP), offers a relatively easy, quick and economical approach to this technically challenging task. Here we describe in step-by-step detail the protocols for virus production, gene delivery, brain specimen preparation, fluorescence imaging and image analysis, calibrated substantially and specifically to make mGRASP-assisted circuit mapping (mGRASPing) practical in the mouse brain. The protocol includes troubleshooting suggestions and solutions to common problems. The mGRASP method is suitable for mapping mammalian synaptic connectivity at multiple scales: microscale for synapse-by-synapse or neuron-by-neuron analysis, and mesoscale for revealing local and long-range circuits. The entire protocol takes 5-6 weeks, including time for incubation and virus expression.
许多类型的神经科学问题都需要在复杂的哺乳动物大脑中检测和绘制突触。一种工具，哺乳动物 GFP 重组跨越突触伙伴（mGRASP），为这项技术上具有挑战性的任务提供了相对简单、快速和经济的方法。在这里，我们详细描述了逐步的病毒生产、基因传递、脑组织准备、荧光成像和图像分析方案，这些方案经过了实质性和专门的校准，使 mGRASP 辅助的回路映射（mGRASPing）在小鼠大脑中变得实用。该方案包括故障排除建议和常见问题的解决方案。mGRASP 方法适用于在多个尺度上绘制哺乳动物突触连接：用于突触对突触或神经元对神经元分析的微尺度，以及用于揭示局部和远程回路的中尺度。整个方案需要 5-6 周，包括孵育和病毒表达的时间。
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