Engineering a calcium reporter gene for magnetic resonance imaging of neural activity

设计用于神经活动磁共振成像的钙报告基因

• 批准号: 10575714
• 负责人: Arnab Mukherjee
• 金额: $ 30.79万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10575714
• 关键词: Address; Agonist; Animals; Area; Axon; Behavior; Binding Proteins; Biophysics; Blood flow; Brain; Brain imaging; Brain region; Buffers; Calcium; Calcium Signaling; Cell Line; Cells; Chemicals; Coupled; Coupling; Data; Decision Making; Development; Disease; Emotional; Engineering; Escherichia coli; Event; FOS gene; Fluorescence; Functional Magnetic Resonance Imaging; Future; Generations; Genetic; Genetic Engineering; Goals; Hippocampus (Brain); Histological Techniques; Image; Imaging Device; Imaging Techniques; Ionophores; Learning; Lens Fiber; Leukocyte L1 Antigen Complex; Libraries; Location; Magnetic Resonance; Magnetic Resonance Imaging; Mammalian Cell; Manganese; Measures; Mediating; Methods; Microscopy; Modality; Modeling; Molecular; Monitor; Mus; Mutagenesis; Neurobiology; Neurons; Neurosciences; Operative Surgical Procedures; Optical Methods; Optics; Patients; Pattern; Property; Protein Engineering; Proteins; Rattus; Reporter; Reporter Genes; Research; Resolution; Rodent; Sensory; Signal Transduction; Specificity; Stimulus; Techniques; Technology; Testing; Time; Toxic effect; Transgenic Animals; Variant; Viral Vector; Virus; Work; base; blood-brain barrier crossing; brain volume; calcium indicator; cell type; contrast imaging; executive function; experience; fluorescence imaging; hemodynamics; imaging approach; imaging modality; in vivo; light scattering; multi-photon; neural circuit; neuroregulation; neurosurgery; neurotoxicity; neurotransmission; optical fiber; optogenetics; primary outcome; protein expression; relating to nervous system; response; screening; sensor; tool

PROJECT SUMMARY To encode sensory experience and generate specific behaviors, the mammalian brain relies on functional coupling between networks of neurons distributed over multiple regions of the brain. Imaging techniques, which can observe molecular events at a brain-wide scale, are therefore needed to understand how these interconnected networks function as a whole. Fluorescent genetically encoded calcium indicators (GECIs) enable optical recording of calcium dynamics (a reliable measure of spiking activity) from well-defined cell types; but operate over small volumes (~ 1 mm3), limiting access to neural signals in localized neural circuits. Broader areas (~ 10 x 10 mm2) may be accessed with widefield optical methods; but light scattering limits imaging to sub- cortical depths in mice. On the other hand, techniques like functional MRI (fMRI) permit activation mapping in arbitrarily large brain volumes; but the ensuing signal gives only an indirect indication of neural activity based on localized changes in hemodynamic parameters. Histological techniques like c-fos immunostaining can also reveal whole-brain activation patterns; but only at a single time point per subject, thus failing to adequately capture dynamically evolving network properties. These collective limitations create a critical technological gap for large-scale monitoring of neural activity inside the intact mammalian brain. In this project, we will pilot steps to address the above challenge by developing genetic tools for imaging calcium with MRI; and we will examine their ability to faithfully monitor calcium dynamics in cultured neurons. The proposed sensors will be based on a manganese-binding protein class known as calprotectin, which we introduced in 2021 as the first protein-based MRI sensor for calcium. These first generation sensors are responsive to calcium concentrations exceeding 5 μM, which is much larger than the typical dynamic range (0.1-1 μM) experienced in neurons during activity. Starting with the parental sensor protein and building on a strong set of preliminary data, our goals here will be to optimize sensitivity for calcium sensing down to the neuronal range (Specific Aim 1), establish methods to genetically express the sensors in mammalian neurons, examine potential sensor-associated toxicity and take remedial steps if needed, and finally show that we can use these sensors to dynamically image calcium signals in neurons stimulated with agonists and optogenetics (Specific Aim 2). The primary outcome of this work will be the development of new neuroscience tools for directly observing calcium dynamics, while combining brain-wide access with all the advantages of genetic encoding, including cell-specific targeting, long-term expression, and access to a wide array of neuroscience methods ranging from axon-tracing and BBB-crossing viruses to transgenic animals. The work here will thus set the stage for previously impossible in vivo studies on identifying functionally coupled networks involved in everything from learning to executive control and decision making, as well as how these connections are modified in disease states.

项目总结 为了编码感觉体验和产生特定的行为，哺乳动物的大脑依赖于 分布在大脑多个区域的神经元网络之间的耦合。成像技术，它 可以在全脑范围内观察分子事件，因此需要了解这些 互连的网络作为一个整体发挥作用。荧光基因编码钙指示剂(GECI) 能够从明确定义的细胞类型中光学记录钙动态(一种可靠的尖峰活性测量)； 但工作在小体积(~1mm3)上，限制了对局部神经电路中神经信号的访问。范围更广 可以用广域光学方法访问区域(~10x10mm2)；但光散射将成像限制在亚 小鼠的皮质深度。另一方面，像功能磁共振成像(FMRI)这样的技术允许在 任意大的大脑体积；但随后的信号仅给出神经活动的间接指示，基于 血流动力学参数的局部改变。C-fos免疫染色等组织学技术也可以 揭示全脑激活模式；但仅在每个受试者的单个时间点，因此未能充分 捕获动态变化的网络属性。这些集体限制造成了严重的技术差距 用于大规模监测完好的哺乳动物大脑内的神经活动。在这个项目中，我们将试点步骤 通过开发用于核磁共振成像钙的基因工具来应对上述挑战；我们将研究 他们能够忠实地监测培养的神经元中的钙动态。拟议的传感器将基于 被称为钙保护素的锰结合蛋白类，我们于2021年推出，作为第一种基于蛋白质的 核磁共振钙质传感器。这些第一代传感器对钙浓度超过5 μM，这比神经元在活动过程中经历的典型动态范围(0.1-1μM)大得多。 从父母的感受器蛋白开始，以一组强大的初步数据为基础，我们的目标将是 为了优化钙离子感应的敏感度(具体目标1)，建立以下方法 在哺乳动物神经元中以遗传方式表达传感器，检测潜在的传感器相关毒性并服用 如果需要，可以采取补救措施，最后证明我们可以使用这些传感器动态成像钙信号 在用激动剂和光遗传学刺激的神经元中(特定目标2)。这项工作的主要成果将是 开发新的神经科学工具，直接观察钙动力学，同时结合全脑 利用遗传编码的所有优势，包括细胞特异性靶向、长期表达和 获取广泛的神经科学方法，从轴突追踪和血脑屏障交叉病毒到 转基因动物。因此，这项工作将为以前不可能进行的体内识别研究奠定基础。 功能耦合网络涉及从学习到执行控制和决策的所有方面，如 以及这些连接在疾病状态下是如何改变的。