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: 10373012
• 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/10373012
• 关键词: Behavior; Binding; Brain; Calmodulin; Cell Nucleus; Cells; 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.

项目概要/摘要 大脑对我们的思想、感觉和行为的控制源于神经回路，这些神经回路执行逻辑 基于神经活动的时间模式和神经元作为电路的连接性的操作 穿过大脑。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，专门的显微镜系统可以对整个大脑体积进行成像 在神经活动的时间尺度和行为过程中，能够重建细胞的神经回路 解决。然而，记录整个大脑体积的神经活动以重建神经回路是一个 在处理更大的大脑、不太专业的显微镜或刺激和行为时面临重大挑战 不利于并发成像。在这些情况下，启用永久记录的策略 在定义的时间窗口内了解神经活动对于以后的读出将是非常有益的。 在本提案中，我们描述了一种使用 Ca2+ 依赖性永久记录神经活动的方法 基因编码底物的酶促激活。我们的重点是烟草蚀刻病毒 (TEV) 由于蛋白酶在脊椎动物系统中没有内源性底物，因此已被用于 活神经元，它已被广泛重新设计以记录不同结合伙伴之间的相互作用， 在没有重组结合伴侣的情况下，其分裂片段会解离。我们提出一项战略 设计一种 Ca2+ 依赖性 TEV 蛋白酶，该酶涉及酶的分裂版本，每一半都附着 Ca2+ 结合伙伴钙调蛋白和 M13。这些 TEV 片段的设计使得在没有 神经活动它们将保持独立且不活动，但在神经活动和高 Ca2+ 存在的情况下， 钙调蛋白和 M13 的结合将允许 TEV 重建和酶活性。一旦激活，分裂 TEV 蛋白酶可以裂解多种基因编码底物，这些底物将 TEV 蛋白酶活性与 所需基因的转录或直接产生荧光团。因此，使用此策略将使用户能够 控制由给定刺激或行为激活的神经元（例如，通过表达光遗传学工具来响应 TEV 活动）或可视化在不同时期内活跃的神经元（例如，通过表达可光激活的 mOrange 响应一种刺激或行为，对 mOrange 进行光电转换，然后记录 第二次刺激）。最后，分裂的 TEV 蛋白酶和底物相互作用可以通过模块化控制 光遗传学工具可对记录时间段提供精确的时间控制。