Subcellular targeting of light-responsive opsins for mapping and manipulating neu

描述(由申请人提供)：本提案的目标是开发将光激活蛋白，如通道视紫红质II(ChR2)和卤视紫红质(NpHR)，靶向于神经元中特定的亚细胞室。ChR2和NpHR都会对光产生电流：离子通道ChR2对蓝光做出反应时会传递去极化的阳离子电流，而NpHR则会对黄光做出反应而产生超极化的氯电流。表达这些蛋白质的神经元可以被光有效地兴奋或抑制。因此，ChR2和NpHR可以在特定的神经元群体中表达，以确定这些细胞的活动是否足以和必要地驱动一种功能，例如一种行为。通过结合膜片钳记录突触后细胞和刺激表达ChR2的突触前神经元，ChR2已被用于在脑片中映射突触电路。ChR2也可以用来刺激体内空间模式的神经元，例如确定运动图。在电路映射中使用光激活蛋白的一个主要限制是它们倾向于将非特异性定位到不同的神经元隔间。ChR2和NpHR似乎在轴突和树突中表达得同样好。在大多数神经组织中，树突和局部和远程轴突混合在一起。轴突中这些蛋白质的存在意味着光刺激可以产生非局部效应。因此，在不刺激邻近ChR2阳性轴突的情况下，几乎不可能从一个ChR2阳性细胞刺激树突，而邻近的ChR2阳性轴突可能来自遥远的和功能无关的神经元。同样，动作电位的传播可以被NpHR阳性轴突的光刺激所阻断。因此，通过将NpHR和ChR2靶向树突和体细胞，产生可以从轴突中排除的NpHR和ChR2的版本是非常有兴趣的。在其他应用中，将光激活蛋白特异性地靶向轴突是有利的。在这里，我们建议产生编码信号的多肽，这些信号将光激活蛋白靶向特定的亚细胞室，从而允许神经元被激活或被抑制，以便进行神经电路分析。公共卫生相关性：光激活蛋白有可能用于治疗抑郁症和癫痫等神经系统疾病。在这项资助中开发的技术将使光激活蛋白质的活动更加精确，并且不太可能产生不想要的影响。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop methods to target light-activated proteins, such as Channel Rhodopsin II (ChR2) and Halorhodopsin (NpHR), to specific subcellular compartments in neurons. Both ChR2 and NpHR generate electrical currents in response to light: ChR2, an ion channel, passes depolarizing cation current in response to blue light, whereas NpHR, a pump, generates hyperpolarizing Cl- currents in response to yellow light. Neurons expressing these proteins can be efficiently excited or inhibited with light. ChR2 and NpHR can thus be expressed in specific neuronal populations to determine if activity in these cells is sufficient and necessary to drive a function, for example a behavior. ChR2 has been used to map synaptic circuits in brain slices, by combining patch clamp recording of postsynaptic cells with stimulation of presynaptic neurons expressing ChR2. ChR2 can also be used to stimulate neurons in a spatial pattern in vivo, for example to determine motor maps. A major limitation to the use of light-activated proteins in circuit mapping is their tendency to localize nonspecifically to different neuronal compartments. ChR2 and NpHR appear to be expressed equally well in axons and dendrites. In most neural tissues, dendrites and local and long-range axons are intermingled. The presence of these proteins in axons means that photostimulation can have non-local effects. Thus, it is virtually impossible to stimulate dendrites from one ChR2-positive cell without also stimulating neighboring ChR2-positive axons that can arise from distant and functionally unrelated neurons. Similarly, action potential propagation can be blocked by photostimulation of NpHR-positive axons. It is therefore of great interest to generate versions of NpHR and ChR2 that can be excluded from axons, by targeting them to dendrites and somata. In other applications it is advantageous to specifically target light- activated proteins to axons. Here we propose to generate peptides encoding signals that target light-activated proteins to specific subcellular compartments allowing neurons to be activated or inhibited for neural circuit analysis. PUBLIC HEALTH RELEVANCE: Light activated proteins have the potential to be used for treatment of neurological diseases such as depression and epilepsy. The technology developed in this grant will make the actions of light activated proteins both more precise and less likely to produce unwanted effects.