Opto- and chemogenetic neural activity recording with diverse reporters

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控制。 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 穿过大脑最近的研究已经产生了许多用于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上和行为期间，使细胞神经回路的重建成为可能。 分辨率然而，记录整个脑体积的神经活动以重建神经回路是一个非常困难的方法。 在处理较大的大脑、不太专业的显微镜或刺激和行为时， 这不利于同时成像。在这些情况下，能够永久记录的策略 在限定的时间窗口期间观察神经活动以用于稍后的读出将是非常有益的。 在这个建议中，我们描述了一种永久记录神经活动的方法，使用Ca 2+依赖 基因编码底物的酶促活化。烟草蚀纹病毒（TEV） 蛋白酶，因为它在脊椎动物系统中没有内源性底物，它已被用于 活的神经元，它已被广泛重新设计，以记录不同的结合伙伴之间的相互作用， 其分裂片段在没有重组结合配偶体的情况下解离。我们提出了一个战略， 工程化Ca 2+依赖性TEV蛋白酶，其涉及酶的分裂形式，每一半连接 与Ca 2+结合伴侣钙调蛋白和M13结合。这些TEV片段被设计成使得在不存在TEV的情况下， 神经活动，它们将保持分离和不活动，但在神经活动和高Ca 2+的存在下， 钙调蛋白和M13的结合将允许TEV重建和酶活性。一旦激活， TEV蛋白酶可以切割多种遗传编码的底物，所述底物将TEV蛋白酶活性与TEV蛋白酶活性相关联。 所需基因的转录或荧光团的直接产生。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（例如，通过表达可光活化的 响应于一种刺激或行为，光转换mOrange，然后记录mOrange的光响应。 第二刺激）。最后，可以用模块化的方法控制分裂的TEV蛋白酶和底物的相互作用。 光遗传学工具，以提供精确的时间控制的时间段的记录。

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.