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

基本信息

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
项目类别：
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

项目摘要

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 BRAIN 计划的一个主要目标是开发神经成像技术，实现全脑成像特定的分子信号，例如神经元钙，目前，荧光基因编码钙。指示器 (GEGI) 与先进的显微镜技术相结合，可实现单神经元 Ca2+ 成像体积小于 1 mm3，通常深度小于 1 mm，或者与 GECI 结合使用。植入式光纤光度测定法可以对基因定义的神经元的聚合活动进行点测量这两种光学方法都在大脑深部区域中实现了尺寸约为 200 µm 的群体。已经证明了它们在实现神经科学发现方面的巨大价值，但它们无法同时提供如果可以开发出一种全脑 Ca2+ 成像技术，那么它将会实现全脑获取神经信号。对神经科学研究产生变革性影响在这个项目中，我们将通过以下方式实现这一雄心勃勃的目标。开发超声波基因编码钙指示剂 (UGECIs) 并表明我们可以使用它们超声作为神经成像方式具有独特的优势。能够比光穿透更深（几厘米），同时提供相对较高的空间（几十微米）和时间（毫秒）分辨率已通过血流动力学功能超声（fUS）得到证明，它使用超快多普勒血流成像以 100 µm 和 100 ms 分辨率可视化神经活动。 fUS 表明，对从小鼠到人类的各种物种的神经活动进行超声成像是可能的与清醒、行为正常、自由活动的动物相容，但是血流动力学仅提供间接的信息。相比之下，使用 GECI 测量钙可以获取分子信息。信号是神经兴奋的组成部分，并允许探测特定的细胞群。将超声的全脑覆盖与 GECI 的分子和遗传特异性结合起来我们开发 UGECI 的提议源于一个长期存在的问题。我们实验室的研究计划旨在开发第一个超声波基因编码发生器和传感器。结构基于气体囊泡 (GV)，这是一类独特的基因编码的充满空气的蛋白质来自浮力细菌的纳米结构，我们发现它们能够散射声波从而产生超声对比，我们最近表明 GV 可以被设计为合并。分子结合域允许其蛋白质壳改变其机械特性并产生在这些进展的基础上，我们将开发对钙等分子的超声对比。 UGECIs，在小鼠大脑中表达它们，并使用它们对全脑钙信号进行成像超声技术可实现快速 2 维和 3 维分子成像。技术将为神经科学研究人员提供全脑分子神经的革命性能力成像。