CLARITY: fully-assembled biology

描述(申请人提供)：研究具有局部精度和全局范围的完整系统是生物学中一个基本未实现的目标。例如，在对大脑的研究中，确定大脑小块连接的努力虽然是开创性的，但受到一个事实的挑战，即结果图将几乎无法解释，因为追踪到的连接的脑部来源和目的地，以及相应细胞在重大行为事件期间的活动仍将是未知的。相反，记录群体活动的分子、电生理或成像方法与记录的细胞的全脑连接模式无关，造成了对功能的巨大理解差距。我们现在已经为应对这一挑战奠定了基础，通过将化学工程、计算光学和分子遗传学整合在一种名为Clarity的方法中。我们将在行为脊椎动物中枢神经系统中开发这种方法，挑战速度和复杂性，但随着我们为斑马鱼、啮齿动物和灵长类动物开发平台，这种方法将变得适用于整个生物学。在目标1中，我们引入了化学工程工具，将散射和不渗透的组织快速转化为完整但透明和大分子可渗透(抗体相容)的形式。在目标2中，我们开发了活动读出技术，旨在提取体积活动(即使是在自由活动的哺乳动物中)，然后可以将其与全球连接和分子表型联系起来。这项变革性的技术将允许从大型和完整的生物组织或器官中快速提取系统信息(动力学、历史、配线和分子表型)，而无需拆卸，精度可达毫秒级和细胞分辨率。在目标3中，我们直接对表现良好的小鼠和斑马鱼进行研究，快速获得与疾病相关的恐惧状态下每个细胞的全脑活动模式，以此作为奖励。仅此一项就将是生物学上的一项里程碑式的成就，此外，这些数据还将与同一大脑中那些细胞的全分子和全球连接信息联系在一起，所有这些信息都可以公开获取，以供挖掘/搜索。最后，在目标4中，我们设计和构建了斑马鱼、老鼠和灵长类的在线数据集，以广泛地服务于社区。计算基础设施将解决对收集到的海量数据(所有生物学中最大的数据集之一)的处理和公共访问，包括脊椎动物大脑中每个神经元的活动记录 在链接到分子和全球布线信息的特定体验期间。我们在技术推广和教育方面经验丰富，并将利用这些经验来实现这项新技术的全面变革使命。 公共卫生相关性：精神疾病是美国和世界范围内导致残疾的主要原因，但许多大型制药公司正在关闭精神病学项目；取消项目的原因之一是缺乏对与疾病相关的状态(如恐惧和奖励)的神经回路层面的了解，这损害了对治疗目标的识别。事实上，观察有症状的大脑的细微细节而不失去更大范围的电路透视可能对人类健康很重要。这里提出的一套名为Clarity的新技术将直接应对这一挑战，提供亟需的跨规模、同时详细的全球视角，可能会导致对精神疾病的基本洞察。

DESCRIPTION (provided by applicant): Studying intact systems with both local precision and global scope is a fundamental unmet goal in biology. For example, in the study of the brain, efforts to determine connectivity of small patches of brain, though pioneering, are challenged by the fact that resulting maps will be nearly impossible to interpret because both the brainwide sources and destinations of traced wiring connections, as well as the activity of corresponding cells during behaviorally significant events, will remain unknown. Conversely molecular, electrophysiological, or imaging methods to record population activity are agnostic with regard to brain-wide wiring patterns of recorded cells, creating an enormous gap in understanding of function. We have now laid foundations for addressing this challenge, by integrating chemical engineering, computational optics, and molecular genetics in an approach termed CLARITY. We will develop the approach in the behaving vertebrate CNS, challenging for speed and complexity, but CLARITY will become applicable across biology as we develop platforms for zebrafish, rodents, and primates. In Aim 1, we bring chemical engineering tools to bear, rapidly transforming scattering and impermeable tissues into intact but transparent and macromolecule-permeable (antibody-compatible) form. In Aim 2, we develop activity-readout technology designed to extract volumetric activity (even in freely-moving mammals) that can then be linked to the global wiring and molecular phenotypes. This transformative technology will allow rapid extraction of systems information (dynamics, history, wiring, and molecular phenotypes) from large and intact biological tissues or organs without disassembly, down to millisecond-scale and cellular resolution. In Aim 3, we directly apply CLARITY to behaving mice and zebrafish, rapidly obtaining brain-wide activity patterns of every cell involved in disease-relevant states of fear an reward. This alone will be a milestone achievement in biology, but furthermore these data will also be linked to full molecular and global wiring information of those cells in the same brains, al publicly accessible for mining/searching. Finally in Aim 4 we design and build online datasets for zebrafish, mouse, and primate to broadly serve the community. Computational infrastructure will address handling and public access to the massive amount of data collected (among the largest datasets in all of biology), including the records of activity in every neuron in vertebrate brains during specific experiences linked to molecular and global wiring information. We are experienced with technology outreach and education, and will leverage this experience to achieve the full transformative mission of this new technology. PUBLIC HEALTH RELEVANCE: Psychiatric disease represents the leading cause of disability both in the U.S. and worldwide, but many major pharmaceutical companies are closing down psychiatry programs; among the reasons cited for this withdrawal is the lack of neural circuit-level understanding of disease-related states such as fear and reward, which impairs identification of targets for treatment. Indeed, it is therefore likely important for human health o observe fine details of symptomatic brains without losing larger-scale circuit perspective. The efforts proposed here, in a new suite of technologies called CLARITY, will directly address this challenge, providing the much-needed across-scales, simultaneously detailed and global perspective, that may lead to fundamental insights into psychiatric disease.