Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin.
Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin.
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DOI：
10.1371/journal.pone.0012893
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发表时间：
2010-09-23
期刊：
影响因子：
3.7
通讯作者：
Yawo H
中科院分区：
文献类型：
--
作者：
Wen L;Wang H;Tanimoto S;Egawa R;Matsuzaka Y;Mushiake H;Ishizuka T;Yawo H
神经网络的光遗传学操作使人们能够揭示中枢神经系统中如何出现高阶功能。衣藻的一种视紫红质，通道视紫红质-1 （ChR1）在光电流动力学方面优于通道视紫红质-2 （ChR2）。利用增强光电流的ChR1变异体，光遗传学有望提高时间分辨率。ChR1的光电流延迟是通过与ChR2的第6螺旋结构域交换而克服的，通过进一步的分子改造产生了通道视紫红绿色受体（ChRGR）。当在全细胞膜片钳下测量表达HEK293细胞的ChRGR光电流时，它优先被绿光激活，具有快速的动力学和最小的脱敏。由于其动力学优势，ChRGR的使用将使人们能够按照释放光的强度（光电流箝位）预测的时间过程向神经元注入电流。利用Sindbis假病毒载体在小鼠运动皮质神经元中表达ChRGR。当振荡LED光信号扫频时，在皮层切片第5层锥体细胞中产生与振荡光同步的5 - 10 Hz动作电位。通过监测局部场电位（LFPs）在体内驱动表达chrgr的神经元，并利用小波分析研究光诱发反应的时频能量分布。振荡光在5 ~ 10 Hz的优先频率范围内增强了LFP的同相和同相响应。在运动皮层的一个区域，有许多c- fos免疫反应神经元的ChRGR呈阴性，这一事实证明了活动的扩散。光电流箝位研究表明，少数神经元的去极化唤醒了运动皮质网络在某个临界点上的激活状态。
Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5–10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5–10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state.
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