Protein ticker-tapes for brain-wide neural recordings

项目摘要/摘要 用于全脑神经记录的蛋白质自动收报机磁带 行为来自广泛分布的神经元集合的相互作用活动；但所有现有的工具 为了测量大脑活动，只对这些动态中的一小部分进行了采样。在这里，我们提出了一种基于蛋白质的 记录神经活动两个关键指标全脑动态的方法：即刻早期基因(IEG) 表达和钙离子浓度。这项建议侧重于体外概念验证；后续工作将侧重于 在体内应用，如果有保证的话。 树轮和自动收报机的磁带。今天在树上拍摄的核心可以揭示几个世纪前的气候事件。 本提案采用这一思想来记录神经激活的历史。这个想法是慢慢形成的 生长的细胞内蛋白细丝，其位置相关的颜色反映了IEG活性或钙离子的历史 动力学。高分辨率的体外成像读到了这段历史。实现这一目标的步骤包括： 1)将蛋白质纤维设计成分子自动收报带。HaloTag受体(HT)将与 在细胞中形成线性生长结构的丝状形成蛋白(FFP)。按顺序添加不同的- 大脑中的有色HaloTag染料将在生长的细丝中产生彩色条纹，绘制出纤维生长的图谱 到挂钟时间了。蛋白质工程将用于优化成核、生长、标记和机械 这些纤维的特性。 2)记录蛋白质纤维上的IEG活性。一个HT-FFP基因将在一个构成成分下表达 启动子和IEG启动子下的EGFP-FFP。IEG激活将导致光纤中的绿色条纹，其 计时将参考不同颜色的HaloTag条纹来确定。启动子与蛋白质稳定性 将进行优化，以实现高分辨率和长时基IEG记录。 3)记录蛋白质纤维上的钙动态变化。羟色胺受体将被改造成含有TEV 蛋白酶识别基序(称为HT*)，当被切割时，它会使HT失活。融合蛋白，HT*-FFP，和 钙依赖的蛋白酶CaTEV将在神经元中共表达，两者都在构成启动子的作用下。 升高的钙离子将促使羟色胺的蛋白水解性断裂，导致暗带进入细丝(羟色胺*)。 灯丝内的标签将被周围的晶体保护，使其不被蛋白质降解)。 这项工作的结果将是对研究全脑神经的新方法的概念的体外证明 不需要电极或光学接触活体组织的记录。

PROJECT SUMMARY/ABSTRACT Protein ticker-tapes for brain-wide neural recordings Behavior emerges from the interacting activity of widely distributed ensembles of neurons; but all existing tools for measuring brain activity sample only a small subset of these dynamics. Here we propose a protein-based approach to record brain-wide dynamics of two key measures of neural activity: immediate early gene (IEG) expression and Ca2+ concentration. This proposal focuses on in vitro proof of concept; follow-up efforts will focus on in vivo application if warranted. Tree rings and ticker tapes. A core taken in a tree today can reveal climate events from centuries past. The present proposal adapts this idea to record the history of neural activation. The idea is to form slowly growing intracellular protein filaments whose position-dependent color reflects the history of IEG activity or Ca2+ dynamics. High-resolution imaging ex vivo reads this history. The steps to achieve this goal are: 1) Engineer protein fibers as molecular ticker-tapes. The HaloTag receptor (HT) will be fused to filament-forming proteins (FFPs) that form linearly growing structures in cells. Sequential addition of different- colored brain-permeant HaloTag dyes will create colored stripes in the growing filaments, mapping fiber growth to wall-clock time. Protein engineering will be used to optimize the nucleation, growth, labeling and mechanical properties of these fibers. 2) Recording IEG activity on protein fibers. A HT-FFP gene will be expressed under a constitutive promoter and eGFP-FFP under an IEG promoter. IEG activation will lead to green stripes in the fiber, whose timing will be determined by reference to the different-colored HaloTag stripes. Promoters and protein stability will be optimized to achieve high-resolution and long time-base IEG recordings. 3) Record Ca2+ dynamics on protein fibers. The HT receptor will be engineered to contain a TEV protease recognition motif (termed HT*) which inactivates the HT when cleaved. A fusion protein, HT*-FFP, and a Ca2+-dependent protease, CaTEV, will be co-expressed in neurons, both under constitutive promoters. Elevated Ca2+ will drive proteolytic cleavage of HT*, leading to incorporation of dark bands in the filament (HT* labels within the filament will be protected from proteolysis by the surrounding crystal). The outcome of this work will be an in vitro proof of concept of a new approach to brain-wide neural recording which does not require electrodes or optical access to the live tissue.