Optimization of a GCaMP calcium indicator for neural activity imaging.
Optimization of a GCaMP calcium indicator for neural activity imaging.
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DOI：
10.1523/jneurosci.2601-12.2012
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发表时间：
2012-10-03
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影响因子：
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通讯作者：
Looger LL
中科院分区：
文献类型：
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作者：
Akerboom J;Chen TW;Wardill TJ;Tian L;Marvin JS;Mutlu S;Calderón NC;Esposti F;Borghuis BG;Sun XR;Gordus A;Orger MB;Portugues R;Engert F;Macklin JJ;Filosa A;Aggarwal A;Kerr RA;Takagi R;Kracun S;Shigetomi E;Khakh BS;Baier H;Lagnado L;Wang SS;Bargmann CI;Kimmel BE;Jayaraman V;Svoboda K;Kim DS;Schreiter ER;Looger LL
遗传编码钙指标（GECIs）是系统神经科学的有力工具。最近在蛋白质工程方面的努力显着提高了GECIs的性能。最先进的单波长GECI，GCaMP3，已被部署在许多模式生物体，并可以可靠地检测三个或更多个动作电位（AP）在短脉冲在几个系统在体内。通过蛋白质结构测定、靶向诱变、高通量筛选和一系列体外测定，我们将GCaMP3的动态范围提高了数倍，从而创建了一个“GCaMP5”传感器家族。我们测试了GCaMP5在几个系统：培养的神经元和星形胶质细胞，小鼠视网膜，并在体内的小杆线虫化学感觉神经元，果蝇幼虫神经肌肉接头和成人触角叶，斑马鱼视网膜和顶盖，和小鼠视觉皮层。信噪比提高了至少2 - 3倍。在视觉皮层中，两种GCaMP5变体检测到的视觉刺激反应细胞是GCaMP3的两倍。通过将体内成像与电生理学相结合，我们表明GCaMP5荧光比其前身GCaMP3提供了更可靠的神经元活性测量。GCaMP5可以更灵敏地检测体内神经活动，并可广泛应用于细胞成像。
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.