Li Xiang, Gutierrez Davina V, Hanson M Gartz, Han Jing, Mark Melanie D, Chiel Hillel, Hegemann Peter, Landmesser Lynn T, Herlitze Stefan
Department of Neurosciences, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17816-21. doi: 10.1073/pnas.0509030102. Epub 2005 Nov 23.
Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated Ca2+ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.
快速无创控制神经元兴奋性的技术对于分析和理解神经元网络及动物行为至关重要。为开发这些工具，我们证明了两种光激活信号蛋白，即脊椎动物大鼠视紫红质4（RO4）和绿藻通道视紫红质2（ChR2），可用于控制神经元兴奋性并调节突触传递。脊椎动物视紫红质与Gi/o、百日咳毒素敏感途径偶联，以调节G蛋白门控内向整流钾通道和电压门控Ca2+通道。在培养的海马神经元中，光介导的RO4激活通过体树突膜超极化在毫秒内降低神经元放电，并且当在突触前位点激活时可调节突触传递和双脉冲易化。相比之下，ChR2的体树突激活使神经元充分去极化以诱导立即产生动作电位，该动作电位精确跟随ChR2激活直至20Hz的光刺激频率。为证明这些构建体可用于调节完整生物体中的网络行为，用任一构建体对胚胎鸡脊髓进行电穿孔，从而能够精确控制已知对运动回路形成很重要的自发爆发活动的发作频率。因此，光激活的脊椎动物RO4和绿藻ChR2能够在培养的神经元和完整的脊椎动物脊髓中以精确、可逆和无创的方式在毫秒至秒的时间内对神经元功能进行拮抗控制。
