Ultrasonic Genetically Encoded Calcium Indicators for Whole-Brain Neuroimaging

用于全脑神经影像的超声波基因编码钙指示剂

• 批准号: 10166018
• 负责人: Mikhail Shapiro
• 金额: $ 214.21万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166018
• 关键词: 3-Dimensional; Acoustics; Address; Air; Amino Acids; Animal Model; Animals; BRAIN initiative; Bacteria; Binding; Biosensor; Blood flow; Brain; Brain imaging; Brain region; Calcium; Calcium Signaling; Calmodulin; Cell model; Development; Dimensions; Disease; Engineering; Enzymes; Fiber; Gases; Genes; Genetic; Goals; Human; Image; Imaging technology; Implant; In Vitro; Kinetics; Label; Light; Mammalian Cell; Maps; Measurement; Measures; Methods; Microscopy; Modality; Molecular; Molecular Genetics; Monitor; Mus; Nanostructures; Nature; Neurons; Neurosciences; Neurosciences Research; Optics; Peptides; Photometry; Physiologic pulse; Population; Preparation; Property; Protein Engineering; Proteins; Reporter; Research; Research Personnel; Resolution; Science; Serotyping; Signal Transduction; Specificity; Techniques; Technology; Ultrasonics; Ultrasonography; Vesicle; analog; awake; base; calcium indicator; experimental study; falls; hemodynamics; imaging modality; in vivo; in vivo imaging; mechanical properties; molecular imaging; nano; nervous system disorder; neural circuit; neural network; neuroimaging; neuropsychiatric disorder; neurotransmission; programs; promoter; relating to nervous system; response; sensor; sound; spatiotemporal; tool; two-dimensional; viral gene delivery

SUMMARY A major goal of the BRAIN initiative is to develop neural imaging technologies enabling whole-brain imaging of specific molecular signals such as neuronal calcium. Currently, fluorescent genetically encoded calcium indicators (GEGIs) combined with advanced microscopy techniques enable single-neuron Ca2+ imaging in volumes smaller than 1 mm3, typically at depths shallower than 1 mm. Alternatively, GECIs combined with implanted fiber photometry enable point-measurements of the aggregate activity of genetically defined neuronal populations in deep brain regions with dimensions on the order of 200 µm. While both of these optical approaches have proven their great value in enabling neuroscience discoveries, they fall short of providing simultaneous whole-brain access to neural signals. If a technology could be developed for whole-brain Ca2+ imaging it would have a transformative impact on neuroscience research. In this project, we will address this ambitious goal by developing ultrasonic genetically encoded calcium indicators (UGECIs) and showing that we can use them to image brain-wide calcium in mice. Ultrasound has unique advantages as a modality for neural imaging due to its ability to penetrate much deeper than light (several cm) while providing relatively high spatial (tens of µm) and temporal (ms) resolution. This potential has been demonstrated by hemodynamic functional ultrasound (fUS), which uses ultrafast Doppler imaging of blood flow to visualize neural activity with 100 µm and 100 ms resolution. fUS has shown that ultrasound imaging of neural activity is possible in species ranging from mice to humans and is compatible with awake, behaving, freely moving animals. However, hemodynamics provide only an indirect measure of neural activity. In contrast, the measurement of calcium with GECIs provides access to a molecular signal integral to neuronal excitation, and allows the probing of specific cellular populations. This project will combine the whole-brain coverage of ultrasound with the molecular and genetic specificity of GECIs by developing the ultrasound versions of these tools. Our proposal to develop UGECIs arises from a long-standing research program in our lab to develop the first genetically encoded reporters and sensors for ultrasound. These constructs are based on gas vesicles (GVs), a unique class of genetically encoded air-filled protein nanostructures derived from buoyant bacteria, which we discovered are capable of scattering sound waves and thereby producing ultrasound contrast. We recently showed that GVs can be engineered to incorporate molecular binding domains allowing their protein shells to change their mechanical properties and resulting ultrasound contrast in response to molecules such as calcium. Building on these advances, we will develop UGECIs, express them in the mouse brain and use them to image brain-wide calcium signals using new ultrasound techniques allowing rapid 2-dimensional and 3-dimensional molecular imaging. The resulting technology will provide neuroscience researchers with revolutionary capabilities for whole-brain molecular neural imaging.

概括 大脑倡议的主要目标是开发神经成像技术，以实现全脑成像 特定的分子信号，例如神经元钙。目前，荧光遗传编码的钙 指标（gegis）与先进的显微镜技术相结合，使单神经元Ca2+成像在 小于1 mm3的体积通常在较浅的深度下，大于1 mm。或者，Gecis与 植入的纤维光度法启用一般定义神经元的骨料活性的点测量 尺寸为200 µm的深脑区域的种群。而这两种光学方法 已经证明了它们在使神经科学发现中的巨大价值，他们没有同时提供 全脑访问神经信号。如果可以为全脑CA2+成像开发技术 对神经科学研究有变革性的影响。在这个项目中，我们将通过 开发超声遗传编码的钙指标（ugecis），表明我们可以使用它们 小鼠的图像大脑范围钙。超声波具有独特的优势，作为神经成像的一种方式 在提供相对高空间（数十万µm）的同时，可以穿透比光（几厘米）深得多的能力 临时（MS）分辨率。血液动力学功能超声（FUS）已证明了这一潜力， 它使用超快多普勒对血流的成像来可视化100 µM和100 ms分辨率的神经活动。 FUS表明，从小鼠到人类的物种，神经元活性的超声成像是可能的 与清醒，行为自由的动物兼容。但是，血液动力学仅提供间接 神经元活性的度量。相反，用GECI的钙测量可访问分子 信号是神经元兴奋的积分，并允许探测特定的细胞群体。这个项目将 将超声的全脑覆盖与GECI的分子和遗传特异性结合在一起 开发这些工具的超声版本。我们开发ugecis的建议是由长期存在的 我们实验室中的研究计划是为超声开发第一个遗传编码的记者和传感器。这些 结构基于燃气蔬菜（GVS），这是一类奇特的基因编码空气蛋白 源自浮力细菌的纳米结构，我们发现它们能够散射声波和 从而产生超声对比度。我们最近表明，可以设计GVS以合并 分子结合结构域，允许其蛋白质壳改变其机械性能并产生 超声反应与分子（例如钙）的对比。在这些进步的基础上，我们将发展 ugecis，在鼠标大脑中表达它们，并使用新的 超声技术允许快速的二维和三维分子成像。结果 技术将为全脑分子神经元提供神经科学研究人员提供革命性的能力 成像。