Ultrasonic Genetically Encoded Calcium Indicators for Whole-Brain Neuroimaging

摘要 大脑计划的一个主要目标是开发神经成像技术，使之能够对 特定的分子信号，如神经元钙离子。目前，荧光基因编码的钙 指示器(GEGI)与先进的显微技术相结合，使单个神经元钙离子成像成为可能 体积小于1 mm3，通常在深度小于1 mm的地方。或者，GECI与 植入的纤维光度法能够点测量遗传定义的神经元的聚集活动 大小约为200微米的脑深部区域中的种群。而这两种光学方法 已经证明了它们在促进神经科学发现方面的巨大价值，但它们不能同时提供 全脑获取神经信号。如果可以开发一种用于全脑钙离子成像的技术，它将 对神经科学研究产生了革命性的影响。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(UGECI)，并表明我们可以使用它们来 为小鼠全脑钙离子成像。超声作为一种神经成像手段具有独特的优势，因为它 能够穿透比光更深的地方(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。血流动力学功能超声(FUS)已经证实了这种潜力， 它使用血流的超快多普勒成像，以100微米和100毫秒的分辨率可视化神经活动。 FUS已经表明，在从老鼠到人类的各种物种中，神经活动的超声波成像是可能的，并且 与清醒的、行为正常的、自由活动的动物兼容。然而，血流动力学只提供了一个间接的 神经活动的量度。相反，使用GECI测量钙提供了访问分子的途径 信号是神经元兴奋的组成部分，并允许探测特定的细胞群体。这个项目将 将超声波的全脑覆盖范围与GECI的分子和遗传特异性相结合 开发这些工具的超声波版本。我们发展UGECI的建议源于一个长期存在的 我们实验室的研究计划开发了第一个用于超声波的基因编码记者和传感器。这些 构建基于气泡(GV)，这是一种独特的基因编码的充气蛋白质 来自浮力细菌的纳米结构，我们发现这种结构能够散射声波和 从而产生超声对比度。我们最近展示了GV可以被设计成包括 分子结合域允许它们的蛋白质外壳改变其机械性能并导致 超声对比剂对钙等分子的反应。在这些进展的基础上，我们将发展 UGECI，在小鼠大脑中表达它们，并使用它们来成像全脑钙信号，使用新的 超声波技术可实现快速的二维和三维分子成像。由此产生的 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。

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.