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

光响应视蛋白的亚细胞靶向用于映射和操纵神经元

• 批准号: 8298613
• 负责人: DONALD B ARNOLD
• 金额: $ 38.89万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8298613
• 关键词: Action Potentials; Affinity; Area; Axon; Behavior; Binding; Brain; Cations; Cells; Cytoskeleton; Dendrites; Diffusion; Distant; Epilepsy; Goals; Grant; Halorhodopsins; Health; Integral Membrane Protein; Ion Channel; Light; Location; MYO5A gene; Maps; Mediating; Mental Depression; Messenger RNA; Methods; Motor; Myosin ATPase; Neurons; Opsin; Pattern; Peptides; Population; Protein Binding; Proteins; Pump; Rhodopsin; Signal Transduction; Site; Slice; Specificity; Technology; Time; Tissues; Work; improved; in vivo; interest; nervous system disorder; neural circuit; neuronal cell body; novel; patch clamp; postsynaptic; presynaptic; relating to nervous system; response

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.

描述（由申请人提供）：本提案的目标是开发将光激活蛋白（例如通道视紫红质 II (ChR2) 和盐视紫红质 (NpHR)）靶向神经元中特定亚细胞区室的方法。 ChR2 和 NpHR 都会响应光而产生电流：ChR2（离子通道）响应蓝光而传递去极化阳离子电流，而 NpHR（泵）则响应黄光而产生超极化 Cl- 电流。表达这些蛋白质的神经元可以被光有效地激发或抑制。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动对于驱动功能（例如行为）是否足够且必要。通过将突触后细胞的膜片钳记录与表达 ChR2 的突触前神经元的刺激相结合，ChR2 已被用于绘制脑切片中的突触回路图。 ChR2 还可用于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非特异性地定位到不同的神经元区室。 ChR2 和 NpHR 在轴突和树突中表达得同样好。在大多数神经组织中，树突以及局部和远程轴突混合在一起。这些蛋白质在轴突中的存在意味着光刺激可以产生非局部效应。因此，实际上不可能刺激一个 ChR2 阳性细胞的树突而不同时刺激邻近的 ChR2 阳性轴突，这些轴突可能来自远处且功能不相关的神经元。同样，动作电位传播可以通过 NpHR 阳性轴突的光刺激来阻断。因此，通过将 NpHR 和 ChR2 靶向树突和体细胞，生成可以从轴突中排除的 NpHR 和 ChR2 版本非常有意义。在其他应用中，将光激活蛋白特异性靶向轴突是有利的。在这里，我们建议生成肽编码信号，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。公共健康相关性：光激活蛋白有潜力用于治疗抑郁症和癫痫等神经系统疾病。这笔资助中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良影响。