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

摘要 本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 （广义上定义为神经元活动的减少）。即刻早期基因的转录 像c-Fos和Arc这样的IEG（IEG）已经被最广泛地用于将神经元活性转化为稳定的， 可跟踪的组织学标签，以允许结构和功能询问。现有活动目标 通过直接检测IEG或工程化的IEG启动子的方法被优化， 检测到神经活动的持续增加然而，它们通常对以下情况不太有效： 标记神经元活性的抑制。因此，为了更好地了解双向大脑， 因此，重要的是要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作为逆活动标记。 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 快速、双向、可跟踪。我们假设，如果我们可以反向识别PTM， 通过无偏筛选与神经元激活相关，这些变化可能发展为 IAM报告行为动物的神经抑制。我们建立了一个原始的光遗传- 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亚单位α 1或pPDH与神经元活性呈负相关。我们的中央 一个假设是检验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IAM与全脑清除相结合， 光片成像和多路标记，使细胞ID兼容工具能够进行无偏分析 全脑抑制我们组建了一个调查小组， 活动依赖性工具开发、电路映射、电生理学和蛋白质组学， 行为。这些新工具的开发和传播将为我们带来新的视角， 了解大脑的回路动力学。

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.