A viral system for light-dependent trapping of activated neurons

一种光依赖性捕获激活神经元的病毒系统

• 批准号: 9056270
• 负责人: MICHAEL R DREW
• 金额: $ 22.15万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9056270
• 关键词: Achievement; Address; Animals; Anxiety; Automobile Driving; Award; BRAIN initiative; Behavior; Behavioral; Binding; Binding Sites; Biological Assay; Brain; Brain Mapping; Brain region; Calcium; Cell physiology; Cells; Chemicals; Chemistry; Clinical; Code; Complex; Cyclic AMP-Responsive DNA-Binding Protein; Deposition; Development; Disease; Dissection; Doxycycline; Elements; Erythromycin; Event; FOS gene; Forskolin; Fright; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Techniques; Genomics; Goals; Green Fluorescent Proteins; Hippocampus (Brain); Hunger; Immediate-Early Genes; Implant; In Vitro; Intervention; Label; Lasers; Learning; Ligands; Light; Lighting; Mediating; Memory; Mental Depression; Methods; Modeling; Molecular Genetics; Mus; Neurobiology; Neurons; Neurosciences; Pharmacogenetics; Phloretin; Physiologic pulse; Population; Property; Protein Binding; Proteins; Psyche structure; Regulation; Reporter; Reporting; Research Personnel; Risk; Rodent; Signal Transduction; System; Techniques; Testing; Tetracyclines; Time; Transgenic Animals; Vanillic Acid; Viral; Virus; Visible Radiation; addiction; advanced system; awake; base; cell assembly; cell type; cellular targeting; conditioned fear; conditioning; dentate gyrus; design; excitatory neuron; extracellular; fluorophore; gene induction; granule cell; high reward; high risk; in vivo; innovation; meetings; mental state; neurochemistry; neuronal circuitry; neurotransmission; novel; optical fiber; optogenetics; promoter; public health relevance; recombinase; relating to nervous system; response; sensor; tool

 DESCRIPTION (provided by applicant): A central goal in neuroscience is to identify cellular ensembles supporting mental and behavioral states, but these ensembles cannot be defined a priori. The dentate gyrus (DG), for example, contains more than 1M granule cells, which are essentially indistinguishable from each other, but less than 5% of these seemingly identical neurons are active during any one behavioral event, suggesting that the associated mental states are each mediated by a small subset of neurons. We propose to develop a novel method for identifying and gaining genetic access to such transient, behaviorally-relevant assemblies of neurons in awake animals. The key unique features of our approach are (1) its temporal precision is unprecedented because it is the first neuronal tagging technique that matches the timescale of naturalistic behavior; and (2) its ability to label multiple cell populations in the sme animal enables the comparison of state-specific cell ensembles. Our novel molecular-genetic technique first identifies activated neurons on the basis of elevated intracellular calcium and then tags them using light. Light application is especially attractive because it is temporally precise: just as other optogenetic methods have aided neuronal circuit analysis by approximating the timescale of cell activity, so too will a light-dependent labeling technique illuminate functional cell assemblies. The technique will be entirely virus-based, so it is usable across species without relying on transgenic animals. Under this award we will establish the technique by developing and testing two critical innovations: (1) a synthetic bidirectional promoter system, and (2) caging chemistry for multi-wavelength visible light regulation of promoter function. Ultimately this technique will be used to elucidate the neuronal substrates of diverse mental states, such as fear, hunger, depression, anxiety, and addiction, thereby advancing the exploration of critical brain networks. This high-risk, high-reward project comprises multiple innovative features. Elements of the nascent reporter system described here, such as promoter strength, mechanism for regulating gene expression, choice of activating ligand, caging chemistry, and in vivo ligand and light delivery represent starting point that will benefit from extensive optimization. Once existing reporter components have been sufficiently refined, we envision replacing fluorescent reporters with recombinases, so that actuators can be expressed in identified cells for testing neuronal function. Other features, including the development of novel caged ligands, as well as additional methods for brain-wide activity reporting will also be addressed following achievement of our Aims. Despite the inherent risks, we are confident that our proposed system represents a fundamental and much-needed departure from existing techniques. We believe that our approach will evolve from its present status as a promising endeavor into a widely-used tool with the support of the BRAIN Initiative.

 描述（由适用提供）：神经科学中的一个核心目标是识别支持精神和行为状态的细胞集合，但是这些集合不能先验定义。例如，齿状回（DG）包含超过1M的颗粒细胞，这些细胞本质上是无法区分的，但是在任何一种行为事件中，这些看似相同的神经元中的少于5％都活跃，这表明相关的精神状态每个人都由一小部分神经元介导。我们建议开发一种新的方法，以识别和获得遗传获取在清醒动物中神经元的这种短暂性，行为相关的组件。我们方法的主要独特特征是（1）它的临时精度是前所未有的，因为它是与自然主义行为的时间范围相匹配的第一个神经元标记技术； （2）它在中小型企业中标记多个细胞群体的能力可以比较状态特异性细胞集合。我们的新型分子遗传技术首先根据升高的细胞内钙来识别激活的神经元，然后使用光标记它们。光应用特别有吸引力，因为它暂时是精确的：就像其他光遗传学方法通过近似细胞活性的时间尺度来帮助神经元电路分析一样，光依赖性标记技术也会照亮功能性细胞组件。该技术将完全基于病毒，因此在不依赖转基因动物的情况下可以在各种物种上使用。在此奖项下，我们将通过开发和测试两个关键创新来建立该技术：（1）合成双向启动子系统，以及（2）用于多波长可见光调节启动子功能的笼子化学。最终，该技术将用于阐明多样性精神状态的神经元基质，例如恐惧，饥饿，抑郁，焦虑和成瘾，从而推进关键大脑网络的探索。这个高风险的高级项目包括多个创新功能。此处描述的新生记者系统的要素，例如启动子强度，确定基因表达的机制，活化配体的选择，笼子化学和体内配体和光递送代表的起点代表了从广泛优化中受益的起点。一旦现有的记者组件得到了足够的精制，我们就会设想用重组酶代替荧光报告，因此可以在确定的细胞中表达执行器以测试神经元功能。在实现我们的目标之后，还将解决其他特征，包括开发新型笼中配体以及大脑活动报告的其他方法。尽管有继承的风险，但我们相信我们的拟议系统代表了与现有技术的基本和急需的不同。我们认为，在大脑计划的支持下，我们的方法将从其当前的地位发展成为一种广泛使用的工具。