Mao Tianyi, O'Connor Daniel H, Scheuss Volker, Nakai Junichi, Svoboda Karel
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
PLoS One. 2008 Mar 19;3(3):e1796. doi: 10.1371/journal.pone.0001796.
Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.
基因编码钙指示剂（GECIs）有望在特定神经元群体和特定细胞区室中监测[Ca(2+)]。将GECI荧光与神经元活动相关联需要进行定量表征。我们已经在哺乳动物锥体神经元中对一种有前景的新型基因编码钙指示剂——GCaMP2进行了表征。在一些但并非所有神经元中都能检测到对单个动作电位的荧光变化（17±10% ΔF/F [平均值±标准差]）。高频动作电位序列产生了强烈的反应（在83 Hz时，40个动作电位序列的反应为302±50%）。来自子宫内电穿孔小鼠的急性脑片的反应相似，这表明长期表达不会干扰GCaMP2的功能。膜靶向版本的GCaMP2产生的信号并不比非靶向版本的大。我们进一步将GCaMP2靶向树突棘，以监测由突触NMDA受体激活引起的Ca(2+)积累。我们观察到对单个棘突解笼刺激有强烈的ΔF/F反应（范围：37%-264%），这些反应与通过体细胞膜片钳测量的NMDA受体电流相关。GCaMP2的一个主要缺点是其基线荧光较低。我们的结果表明，GCaMP2比GCaMP的先前版本有所改进，可能适合在体内检测高频动作电位和突触电流的爆发。
