Imaging phluorin-based probes at hippocampal synapses.
Imaging phluorin-based probes at hippocampal synapses.
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DOI：
10.1007/978-1-59745-261-8_22
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发表时间：
2008
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准确测量突触囊泡的胞吐和胞吞作用对于理解突触传递的分子基础至关重要。将pH敏感的GFP（pHluorin）与各种突触囊泡蛋白融合，使得能够实时研究突触囊泡再循环。两个这样的探针，synaptopHluorin和sypHy已在培养的海马神经元的突触成像。将这些报告基因与分子干扰技术（如RNAi）相结合，可以研究参与突触囊泡再循环的分子。在这里，我们描述的方法培养和转染海马神经元，成像的pHluorin为基础的探针在突触和分析的pHluorin信号下降到单个突触囊泡的分辨率。
Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive GFP (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 we 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.