Opto- and chemogenetic neural activity recording with diverse reporters

使用不同的记者记录光和化学遗传学神经活动

• 批准号: 10133168
• 负责人: Scott T. Laughlin
• 金额: $ 34.32万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10133168
• 关键词: Behavior; Binding; Brain; Calmodulin; Cell Nucleus; Cells; Cleaved cell; Color; Cytoplasm; Engineering; Enzymes; Exposure to; Feeling; Genes; Genetic; Genetic Transcription; Goals; Health; Human; Image; Kinetics; Light; Link; Membrane; Methods; Microscope; Microscopy; Nature; Neurons; Peptide Hydrolases; Pharmaceutical Preparations; Population; Proteins; Reporter; Reporting; Resolution; Specimen; Stimulus; Substrate Interaction; System; TEV protease; Techniques; Thinking; Time; Tobacco Dependence; Variant; base; brain volume; design; enzyme activity; experimental study; fluorophore; genetic approach; improved; mind control; neural circuit; neural patterning; operation; optogenetics; reconstitution; reconstruction; relating to nervous system; response; small molecule; stem; success; tobacco etch virus; tool

PROJECT SUMMARY/ABSTRACT The brain's control of our thoughts, feelings, and behaviors stems from neural circuits, which perform logical operations based on the temporal patterns of neural activity and the connectivity of the neurons as the circuit traverses the brain. Recent studies have produced many strategies for visualizing and controlling a circuit's neural activity. In some cases, specialized microscopy systems have enabled imaging of entire brain volumes on the timescale of neural activity and during behavior, enabling the reconstruction of neural circuitry at cellular resolution. However, recording neural activity from whole brain volumes to reconstruct neural circuitry is a significant challenge when working with larger brains, less specialized microscopes, or stimuli and behaviors that are not conducive to concurrent imaging. In these situations, strategies that enable permanent recording of neural activity during defined time windows for later readout would be highly beneficial. In this proposal, we describe an approach for permanent recording of neural activity using Ca2+-dependent enzymatic activation of genetically encoded substrates. We focus our efforts on the Tobacco Etch Virus (TEV) protease because it does not have endogenous substrates in vertebrate systems, it has been employed in living neurons, it has been extensively re-engineered to record interactions between diverse binding partners, and its split fragments dissociate in the absence of reconstituting binding partners. We propose a strategy for engineering a Ca2+-dependent TEV protease that involves a split version of the enzyme with each half attached to the Ca2+-binding partners calmodulin and M13. These TEV fragments are designed so that in the absence of neural activity they will remain separate and inactive, but in the presence of neural activity and high Ca2+, the association of calmodulin and M13 will permit TEV reconstitution and enzyme activity. Once active, the split TEV protease can cleave a variety of genetically encoded substrates that link TEV protease activity with transcription of a desired gene or direct creation of a fluorophore. Thus, using this strategy will enable users to control neurons activated by a given stimulus or behavior (e.g., by expressing optogenetic tools in response to TEV activity) or to visualize neurons active during distinct epochs (e.g., by expressing a photoactivatable mOrange in response to one stimulus or behavior, photoconverting the mOrange, and then recording the second stimulus). Finally, the split TEV protease and substrate interactions can be controlled with modular optogenetics tools to provide precise temporal control over the time period of recording.

项目摘要/摘要 大脑对我们的思想，感觉和行为的控制源于神经回路 基于神经活动的时间模式和神经元的连通性作为电路的操作 遍历大脑。最近的研究产生了许多可视化和控制电路的策略 神经活动。在某些情况下，专门的显微镜系统已启用整个大脑体积的成像 在神经活动的时间尺度和行为期间，可以在细胞处重建神经回路 解决。但是，记录从全脑体积到重建神经回路的神经活动是一个 使用较大的大脑，较少的专业显微镜或刺激和行为时，重大挑战 不利于并发成像。在这些情况下，可以永久记录的策略 在定义的时间窗口期间，以供以后读数的神经活动将是非常有益的。 在此提案中，我们描述了一种使用Ca2+依赖性的永久记录神经活动的方法 遗传编码底物的酶促激活。我们将精力集中在烟草蚀刻病毒（TEV）上 蛋白酶因为它在脊椎动物系统中没有内源性底物，所以它已用于 活着的神经元，已广泛重新设计以记录各种约束伙伴之间的相互作用， 在没有重建结合伙伴的情况下，其分裂碎片将分离。我们提出了一个策略 工程Ca2+依赖性的TEV蛋白酶，涉及酶的拆分版本 到CA2+结合伙伴钙调蛋白和M13。这些Tev碎片的设计是为了使 神经活动将保持分开和不活跃，但是在存在神经活动和高Ca2+的情况下， 钙调蛋白和M13的关联将允许TEV重建和酶活性。一旦活跃，拆分 TEV蛋白酶可以裂解各种遗传编码的底物，将TEV蛋白酶活性与 所需基因的转录或直接创建荧光团。因此，使用此策略将使用户能够 由给定刺激或行为激活的控制神经元（例如，通过表达光遗传学工具以响应 TEV活动）或在不同的时期内可视化神经元（例如，通过表达光活化 摩根响应一种刺激或行为，使摩根摩根散发，然后记录 第二个刺激）。最后，可以通过模块化控制分裂的TEV蛋白酶和底物相互作用 光遗传学工具可在记录时间段内提供精确的时间控制。