Brain-wide mapping of neuronal inhibition by novel inverse activity markers

摘要 该项目旨在开发第一个用于检测神经元抑制的反向活动标记物(IAM (广义定义为神经元活动减少)。即刻早期基因的转录 像c-Fos和Arc这样的IEGs是最广泛用于将神经元活动转化为稳定的， 可追踪的组织学标签，允许结构和功能审问。现有活动目标 通过直接检测IEG或工程IEG启动子的方法被优化为 检测神经活动的持续增加。然而，它们通常不太有效地 标记神经元活动的抑制。因此，为了更好地了解双向脑 活动，重要的是有一套新的标记物来标记神经元活动的减少 与传统的IEG相反，我们在这里建议将其称为逆活性标记。 我们的目标是开发基于蛋白质翻译后修饰(PTM)的IAM，这是已知的 做到快速、双向、可追踪。我们假设，如果我们能反向识别PTM 与通过无偏见筛选的神经元激活相关，这些变化可以发展为 Iam将报告行为动物的神经抑制。我们建立了一个原创的光遗传学- 蛋白质组学筛选平台，从中我们发现丙酮酸的磷酸化 脱氢酶E1亚基Alpha1或pPDH与神经元活性呈负相关。我们的中央 假设是检验pPDH是否可以作为第一个反映神经活动抑制的IAM 在体外和体内。该方法的开发目标是将IAMS与全脑清理相结合， LightSheet成像和多路复用标记，以实现与细胞ID兼容的工具，以进行无偏见的分析 全脑抑制。我们组建了一支具有公认专业知识的调查团队 依赖活动的工具开发、电路映射、电生理学和蛋白质组学以及 行为。这些新颖工具的开发和传播将带来新的视角 了解大脑的电路动力学。

Abstract This project aims to develop the first Inverse Activity Marker (IAM) for detecting neuronal inhibition (broadly defined as the decrease of neuronal activities). The transcription of immediate early genes (IEGs) like c-Fos and Arc has been the most widely used for translating neuronal activity into stable, trackable histological labels to allow structural and functional interrogations. Existing activity targeting methods, either through direct detection of IEGs or engineered IEG promoters, are optimized for detecting the sustained increase of neural activity. However, they are generally less effective for labeling the inhibition of neuronal activity. Therefore, to better understand the bi-directional brain activities, it is important to have a set of new markers to label the decrease of neuronal activity opposite to the conventional IEGs, which we propose here as the Inverse Activity Marker. We aim to develop IAMs based on protein post-translational modifications (PTMs), which are known to be rapid, bi-directional, and trackable. We hypothesize that if we can identify PTMs inversely correlated with neuronal activation through unbiased screens, these changes could be developed into IAMs to report neural inhibition in behaving animals. We established an original optogenetic- proteomics screening platform, from which we discovered that the phosphorylation of pyruvate dehydrogenase E1 subunit Alpha 1 or pPDH inversely correlated with neuronal activity. Our central hypothesis is to test whether pPDH can serve as the first IAM to reflect the inhibition of neural activity in vitro and in vivo. The method development goal is to integrate IAMs with whole-brain clearing, lightsheet imaging, and multiplexed labeling to enable a cell-ID compatible tool for unbiased profiling of brain-wide inhibition. We assembled a team of investigators with well-recognized expertise in activity-dependent tool development, circuit mapping, electrophysiology, and proteomics and behaviors. The development and dissemination of these novel tools will bring new perspectives to understanding the circuit dynamics of the brain.