Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor
Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor
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DOI：
10.1002/jnr.24433
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发表时间：
2019-08-01
影响因子：
4.2
通讯作者：
Yellen, Gary
中科院分区：
文献类型：
--
作者：
Diaz-Garcia, Carlos Manlio;Lahmann, Carolina;Yellen, Gary
葡萄糖是大脑能量的重要来源。最近，基因编码的荧光生物传感器的发展已经允许从单个神经元和星形胶质细胞的葡萄糖动力学的真实的时间可视化。这种方法的一个主要困难，即使是比率传感器，是缺乏一个实用的方法来转换这种测量到实际的浓度在离体脑组织或在体内。荧光寿命成像提供了克服这一点的策略。在先前的研究中，我们报告了用于监测神经元葡萄糖水平响应于刺激的变化的终身葡萄糖传感器iGlucoSnFR-TS（当时称为SweetieTS）。这种基因编码的传感器是通过将嗜热栖热菌葡萄糖结合蛋白与单体荧光蛋白T-蓝宝石的环状排列变体相结合而产生的。在此，我们提供了有关iGlucoSnFR-TS设计和表征以及pH和温度灵敏度的更多详细信息。为了准确估计葡萄糖浓度，传感器必须在与实验相同的温度下校准。我们发现，当细胞外葡萄糖浓度在2-10 mM范围内时，来自急性脑切片的海马神经元中的细胞内葡萄糖浓度类似于标称外部葡萄糖浓度的20%（类似于0.4-2 mM）。我们还测量了体内细胞溶质神经元葡萄糖浓度，发现清醒小鼠皮质神经元中的葡萄糖浓度范围为0.7-2.5 mM。
Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is similar to 20% of the nominal external glucose concentration (similar to 0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of similar to 0.7-2.5 mM in cortical neurons from awake mice.