Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators.
Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators.
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DOI：
10.1371/journal.pone.0001796
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发表时间：
2008-03-19
期刊：
影响因子：
3.7
通讯作者：
Svoboda K
中科院分区：
文献类型：
--
作者：
Mao T;O'Connor DH;Scheuss V;Nakai J;Svoboda K
基因编码的钙指示剂(GECI)有望在选定的神经元群体和特定的细胞隔间中监测[Ca2+]。将Geci荧光与神经元活动联系起来需要定量的描述。我们已经在哺乳动物锥体神经元中表征了一种有前景的新的遗传编码的钙指示剂-GCaMP2。单个动作电位(17±10%ΔF/F[Mean±SD])可在部分(但不是全部)神经元中检测到荧光变化。高频动作电位序列产生了强健的反应(在83赫兹，40个动作电位序列的反应为302±50%)。来自宫内电穿孔小鼠的急性脑片的反应类似，表明长期表达不会干扰GCaMP2的功能。膜靶向的GCaMP2版本并不比非靶向的GCaMP2产生更大的信号。我们进一步将GCaMP2靶向树突棘，以监测突触NMDA受体激活引起的钙积聚。我们观察到强健的ΔF/F反应(范围：37%-264%)对单个脊椎去刺激物，这与通过体细胞贴片移液管测量的NMDA受体电流相关。GCaMP2的一个主要缺点是其基线荧光低。我们的结果表明，GCaMP2是对以前版本的GCaMP的改进，可能适合于检测体内高频动作电位和突触电流的爆发。
Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca2+] 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% ΔF/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 Ca2+ accumulations evoked by activation of synaptic NMDA receptors. We observed robust ΔF/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.
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影响因子：
4.8
作者：
Mainen, ZF;Maletic-Savatic, M;Svoboda, K
通讯作者：
Svoboda, K
影响因子：
64.8
作者：
Mainen, ZF;Malinow, R;Svoboda, K
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影响因子：
25
作者：
Oertner, TG;Sabatini, BL;Svoboda, K
通讯作者：
Svoboda, K
影响因子：
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作者：
Diez-Garcia, Javier;Akemann, Walther;Knopfel, Thomas
通讯作者：
Knopfel, Thomas
影响因子：
4.8
作者：
Heim, N;Griesbeck, O
通讯作者：
Griesbeck, O