Neuroscience Center, University of Helsinki, FIN-00014, Helsinki, Finland.
BMC Neurosci. 2009 Nov 30;10:141. doi: 10.1186/1471-2202-10-141.
Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.
We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.
The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.
神经元兴奋性、突触效能以及细胞信号转导的普遍变化通常是由于关键分子插入质膜（PM）而产生的。许多用于监测 PM 插入的技术要么缺乏空间分辨率，要么缺乏时间分辨率。
我们通过补充重复的细胞外酸化方案改进了基于延时全内反射荧光（TIRF）显微镜和 pHluorin 标记的成像方法。我们通过展示在培养的海马神经元中短暂激活 NMDA 受体（“化学 LTP”）诱导包含 pHluorin 标记的 GluR-A（翻转）亚基的谷氨酸受体持续的 PM 插入，说明了该方法的适用性。
重复酸化技术提供了一种更准确的监测荧光标记分子的 PM 插入部分的方法，并提供了良好的时间和空间分辨率。
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