Carandini Matteo, Shimaoka Daisuke, Rossi L Federico, Sato Tatsuo K, Benucci Andrea, Knöpfel Thomas
UCL Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom,
UCL Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom, RIKEN Brain Science Institute, Wako City 351-0198, Japan, and
J Neurosci. 2015 Jan 7;35(1):53-63. doi: 10.1523/JNEUROSCI.0594-14.2015.
Genetically encoded voltage indicators (GEVIs) promise to reveal the membrane potential of genetically targeted neuronal populations through noninvasive, chronic imaging of large portions of cortical space. Here we test a promising GEVI in mouse cortex during wakefulness, a challenging condition due to large hemodynamic activity, and we introduce a straightforward projection method to separate a signal dominated by membrane voltage from a signal dominated by hemodynamic activity. We expressed VSFP-Butterfly 1.2 plasmid in layer 2/3 pyramidal cells of visual cortex through electroporation in utero. We then used wide-field imaging with two cameras to measure both fluorophores of the indicator in response to visual stimuli. By taking weighted sums and differences of the two measurements, we obtained clear separation of hemodynamic and voltage signals. The hemodynamic signal showed strong heartbeat oscillations, superimposed on slow dynamics similar to blood oxygen level-dependent (BOLD) or "intrinsic" signals. The voltage signal had fast dynamics similar to neural responses measured electrically, and showed an orderly retinotopic mapping. We compared this voltage signal with calcium signals imaged in transgenic mice that express a calcium indicator (GCaMP3) throughout cortex. The voltage signal from VSFP had similar signal-to-noise ratios as the calcium signal, it was more immune to vascular artifacts, and it integrated over larger regions of visual space, which was consistent with its reporting mostly subthreshold activity rather than the spiking activity revealed by calcium signals. These results demonstrate that GEVIs provide a powerful tool to study the dynamics of neural populations at mesoscopic spatial scales in the awake cortex.
基因编码电压指示剂（GEVIs）有望通过对大部分皮质空间进行无创、长期成像来揭示基因靶向神经元群体的膜电位。在此，我们在清醒小鼠的皮质中测试了一种很有前景的GEVI，清醒状态是一个具有挑战性的条件，因为存在大量的血液动力学活动，并且我们引入了一种直接的投影方法，以将由膜电压主导的信号与由血液动力学活动主导的信号分离。我们通过子宫内电穿孔在视觉皮质的第2/3层锥体细胞中表达了VSFP-Butterfly 1.2质粒。然后，我们使用两台相机进行宽场成像，以测量指示剂的两种荧光团对视觉刺激的响应。通过对这两种测量值进行加权求和与求差，我们清晰地分离了血液动力学信号和电压信号。血液动力学信号显示出强烈的心跳振荡，叠加在类似于血氧水平依赖（BOLD）或“内在”信号的缓慢动态变化之上。电压信号具有与电测量的神经反应相似的快速动态变化，并显示出有序的视网膜拓扑映射。我们将此电压信号与在整个皮质中表达钙指示剂（GCaMP3）的转基因小鼠中成像的钙信号进行了比较。来自VSFP的电压信号与钙信号具有相似的信噪比，它对血管伪影更具抗性，并且它在更大的视觉空间区域上进行整合，这与其主要报告阈下活动而非钙信号所揭示的动作电位发放活动一致。这些结果表明，GEVIs为研究清醒皮质中介观空间尺度上神经群体的动态变化提供了一个强大的工具。
