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

基本信息

  • 批准号:
    10708944
  • 负责人:
  • 金额:
    $ 11.66万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目摘要 为了编码感官体验并产生特定的行为,哺乳动物的大脑依赖于功能性的 分布在大脑多个区域的神经元网络之间的耦合。成像技术, 可以在全脑范围内观察分子事件,因此需要了解这些分子是如何发生的。 互联网作为一个整体发挥作用。荧光遗传编码钙指示剂(GECIs) 能够从明确定义的细胞类型中光学记录钙动力学(尖峰活动的可靠测量); 但是在小体积(~ 1 mm 3)上操作,限制了对局部神经回路中的神经信号的访问。更广泛 区域(~ 10 x 10 mm 2)可以用宽视场光学方法访问;但光散射将成像限制在亚 小鼠的皮质深度。另一方面,像功能性磁共振成像(fMRI)这样的技术可以在大脑中进行激活映射。 任意大的脑容量;但随后的信号只给出了基于神经活动的间接指示, 血流动力学参数的局部变化。组织学技术如c-fos免疫染色也可以 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 捕获动态演变的网络属性。这些共同的限制造成了一个关键的技术差距 用于大规模监测完整哺乳动物大脑内的神经活动。在这个项目中,我们将试点 为了解决上述挑战,我们将开发基因工具,用MRI对钙进行成像;我们将研究 他们忠实地监测培养的神经元中钙动力学的能力。拟议的传感器将基于 锰结合蛋白类称为钙卫蛋白,我们在2021年推出了第一个基于蛋白质的 MRI钙传感器。这些第一代传感器对超过5%的钙浓度作出响应 μM,远大于神经元在活动期间经历的典型动态范围(0.1-1 μM)。 从亲本传感器蛋白开始,并建立在一组强大的初步数据基础上,我们的目标是 为了优化钙传感的灵敏度,使其达到神经元范围(具体目标1),建立方法, 在哺乳动物神经元中遗传表达传感器,检查潜在的传感器相关毒性, 如果需要的话,采取补救措施,最后表明我们可以使用这些传感器来动态成像钙信号 在用激动剂和光遗传学刺激的神经元中(具体目标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点,包括细胞特异性靶向,长期表达, 获得广泛的神经科学方法,从轴突追踪和BBB交叉病毒, 转基因动物因此,这里的工作将为以前不可能的体内研究奠定基础, 功能耦合网络涉及从学习到执行控制和决策的一切, 以及这些连接在疾病状态下是如何改变的。

项目成果

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Arnab Mukherjee其他文献

Arnab Mukherjee的其他文献

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{{ truncateString('Arnab Mukherjee', 18)}}的其他基金

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
  • 财政年份:
    2022
  • 资助金额:
    $ 11.66万
  • 项目类别:
Engineering fluorescence and magnetic resonance reporter genes for imaging biological function in hypoxic cells and in vivo
工程化荧光和磁共振报告基因,用于缺氧细胞和体内生物功能成像
  • 批准号:
    10687183
  • 财政年份:
    2019
  • 资助金额:
    $ 11.66万
  • 项目类别:
Engineering fluorescence and magnetic resonance reporter genes for imaging biological function in hypoxic cells and in vivo
工程化荧光和磁共振报告基因,用于缺氧细胞和体内生物功能成像
  • 批准号:
    10266048
  • 财政年份:
    2019
  • 资助金额:
    $ 11.66万
  • 项目类别:
Engineering fluorescence and magnetic resonance reporter genes for imaging biological function in hypoxic cells and in vivo
工程化荧光和磁共振报告基因,用于缺氧细胞和体内生物功能成像
  • 批准号:
    10000120
  • 财政年份:
    2019
  • 资助金额:
    $ 11.66万
  • 项目类别:
Engineering fluorescence and magnetic resonance reporter genes for imaging biological function in hypoxic cells and in vivo
工程化荧光和磁共振报告基因,用于缺氧细胞和体内生物功能成像
  • 批准号:
    9797597
  • 财政年份:
    2019
  • 资助金额:
    $ 11.66万
  • 项目类别:

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