Opto- and chemogenetic neural activity recording with diverse reporters

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，它执行逻辑 基于神经活动的时间模式和作为电路的神经元的连接性的操作 遍历大脑。最近的研究已经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 在神经活动和行为的时间尺度上，使得能够在细胞内重建神经电路 决议。然而，从整个大脑体积记录神经活动以重建神经回路是一种 在使用较大的大脑、较不专业的显微镜或刺激和行为时面临重大挑战 这不利于并行成像。在这些情况下，实现永久记录的策略 在规定的时间窗内监测神经活动，以便以后读出，将是非常有益的。 在这项建议中，我们描述了一种使用钙依赖的永久记录神经活动的方法 基因编码底物的酶激活。我们专注于烟草蚀刻病毒(TEV) 因为它在脊椎动物系统中没有内源底物，所以它被用于 对于活的神经元，它已经被广泛地重新设计，以记录不同结合伙伴之间的相互作用， 并且它的分裂片段在没有重组结合伙伴的情况下解离。我们提出了一个战略，以 设计一种依赖于钙离子的TEV蛋白酶，它涉及酶的一个分裂版本，每一半连接在一起 钙结合伙伴钙调蛋白和M13。这些TEV片段的设计是为了在没有 神经活动它们将保持分离和不活动，但在神经活动和高钙存在的情况下， 钙调蛋白和M13的结合将允许TEV重建和酶活性。一旦激活，拆分 TeV蛋白水解酶可以裂解多种基因编码的底物，将TEV蛋白水解酶活性与 转录所需的基因或直接产生荧光团。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，通过表达光遗传工具来响应 TeV活动)或可视化神经元在不同时期的活动(例如，通过表达可光激活的 对一种刺激或行为作出反应，将莫兰热进行光转换，然后记录 第二个刺激方案)。最后，分裂的TEV蛋白酶与底物的相互作用可以用模块化的方法来控制 光遗传学工具，提供对记录时间段的精确时间控制。

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.