对海马体突触处基于成像荧光蛋白的探针进行成像。
Imaging phluorin-based probes at hippocampal synapses.
作者信息
Royle Stephen J, Granseth Björn, Odermatt Benjamin, Derevier Aude, Lagnado Leon
机构信息
School of Biomedical Sciences, University of Liverpool, Liverpool, UK.
出版信息
Methods Mol Biol. 2008;457:293-303. doi: 10.1007/978-1-59745-261-8_22.
Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles.
准确测量突触小泡的胞吐作用和内吞作用对于理解突触传递的分子基础至关重要。将对pH敏感的绿色荧光蛋白（pHluorin）与各种突触小泡蛋白融合，使得实时研究突触小泡循环成为可能。在培养的海马神经元突触处，已经对两种这样的探针，即突触pHluorin和sypHy进行了成像。这些报告分子与分子干扰技术（如RNA干扰）相结合，使得对参与突触小泡循环的分子进行研究成为可能。在此，作者描述了海马神经元的培养和转染方法、在突触处对基于pHluorin的探针进行成像以及对pHluorin信号进行分析，直至单个突触小泡的分辨率。
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