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靶向光激活蛋白的方法，如通道视紫红质II （ChR2）和Halorhodopsin (NpHR)，用于神经元中的特定亚细胞区室。ChR2和NpHR都对光产生电流响应：ChR2是一个离子通道，在蓝光下通过去极化阳离子电流响应，而NpHR是一个泵，在黄光下产生超极化Cl-电流响应。表达这些蛋白的神经元可以被光线有效地激发或抑制。因此，ChR2和NpHR可以在特定的神经元群体中表达，以确定这些细胞中的活性是否足以和必要地驱动某种功能，例如行为。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已被用于绘制脑切片中的突触回路。ChR2也可用于刺激体内空间模式的神经元，例如确定运动地图。光激活蛋白在电路制图中使用的一个主要限制是它们倾向于非特异性地定位于不同的神经元室。ChR2和NpHR似乎在轴突和树突中同样表达良好。在大多数神经组织中，树突和局部和远端轴突是混合在一起的。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，几乎不可能刺激来自一个chr2阳性细胞的树突，而不刺激邻近的chr2阳性轴突，这些轴突可能来自远端和功能无关的神经元。同样，动作电位的传播也可以通过光刺激nphrr阳性轴突来阻断。因此，通过将NpHR和ChR2定位于树突和体细胞，产生可以从轴突中排除的NpHR和ChR2版本是非常有趣的。在其他应用中，光活化蛋白特异性靶向轴突是有利的。在这里，我们建议产生编码信号的肽，将光激活蛋白靶向特定的亚细胞区室，从而激活或抑制神经元以进行神经回路分析。公共卫生相关性：光激活蛋白有可能用于治疗神经系统疾病，如抑郁症和癫痫。这项拨款开发的技术将使光激活蛋白的作用更加精确，并且不太可能产生不必要的影响。

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.