Acoustically targeted molecular control of cell type specific neural circuits in non-human primates

总结 控制大脑大面积区域的特定神经回路是大脑的主要技术目标 倡议为了实现这一目标，技术应该理想地提供空间、时间和单元的组合， 类型特异性和非侵入性，以促进它们在动物模型和最终在人类中的翻译 患者在这里，我们提出了一种方法来调制神经回路非侵入性的空间，细胞类型和 时间特异性这种方法，我们命名为声学靶向化学遗传学，或ATAC， 使用瞬时聚焦超声（FUS）血脑屏障开放（BBBO）在特定时间点激活神经元， 在大脑中的位置与病毒编码的工程受体，随后响应系统性 施用生物惰性化合物以激活或抑制这些神经元的活性。这项技术允许 一种使一个或多个特定的大脑区域能够被选择性调节的简短的、非侵入性的过程 使用口服生物可利用的化合物。在初步实验中，我们已经在小鼠中实施了这一概念， 使用ATAC非侵入性靶向编码化学发生DREADD受体的AAV 9病毒载体， 海马神经元，并表明这使得药理学抑制记忆形成。 在这个概念验证的基础上，我们现在将扩展ATAC在非人类灵长类动物中的工作。这一目标尤其 鉴于现有技术的成功相对有限，包括光遗传学和常规技术， 化学遗传学，在大型动物的强大行为神经调节。将ATAC扩展到更大的动物需要 超越核心概念的几项创新，包括进化病毒载体，以实现更有效和更交叉的 用FUS-BBBO转染神经元，开发超声方法克服颅骨畸变， 能够在大型动物中精确靶向，建立确认ATAC非- 功能成像的侵入性，并优化选择和药理学管理， 用于大型动物行为研究的化学遗传配体。在这个项目中，我们将首先建立基本的 ATAC在NHP中的功能，并将其与非侵入性功能成像相结合，为 ATAC性能。然后，我们将使用一种开创性的技术，用于病毒载体的体内进化， AAV病毒被特别优化以有效地将化学发生受体递送至靶向具有化学发生受体的脑区域。 FUS-BBBO。与此同时，我们会发展非临床影像引导和像差校正方法， NHP中FUS-BBBO的精确靶向和验证。这将使学术团体有可能没有 使用昂贵的临床FUS系统在较大的生物体中进行ATAC。最后，作为激励性的例子， 应用程序，我们将证明优化的ATAC范式可用于抑制多种不同的大脑 区域，可逆地和可重复地调节他们识别面孔的能力，并将其应用于 感觉运动回路来改变功能连接。我们还将展示其稳定性，可靠性和无毒性。

SUMMARY Controlling specific neural circuits across large areas of the brain is a major technology goal of the BRAIN Initiative. To achieve this goal, technologies should ideally provide a combination of spatial, temporal and cell- type specificity and be noninvasive to facilitate their translation across animal models and, ultimately, human patients. Here, we propose an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, which we have named Acoustically Targeted Chemogenetics, or ATAC, uses transient focused ultrasound (FUS) blood brain barrier opening (BBBO) to transduce neurons at specific locations in the brain with virally-encoded engineered receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds. In preliminary experiments, we have implemented this concept in mice by using ATAC to noninvasively target AAV9 viral vectors encoding chemogenetic DREADD receptors to excitatory neurons in the hippocampus, and showing that this enables pharmacological inhibition of memory formation. Building on this proof of concept, we will now scale ATAC to work in non-human primates. This goal is particularly important given the relatively limited success of existing technologies, including optogenetics and conventional chemogenetics, in robust behavioral neuromodulation in larger animals. Scaling ATAC to larger animals requires several innovations beyond the core concept, including evolving viral vectors for more efficient and intersectional transfection of neurons with FUS-BBBO, developing ultrasound methods to overcome skull aberrations and enable precise targeting in large animals, establishing ways of confirming the functionality of ATAC non- invasively with functional imaging, and optimizing the selection and pharmacological administration of chemogenetic ligands for large-animal behavioral studies. In this project, we will first establish the basic capabilities of ATAC in NHPs and integrate them with non-invasive functional imaging, setting a baseline for ATAC performance. Then, we will use a pioneering technology for in vivo evolution of viral vectors to develop AAV viruses specifically optimized to efficiently deliver chemogenetic receptors to brain regions targeted with FUS-BBBO. In parallel, we will develop non-clinical image guidance and aberration correction methods to enable precise targeting and verification of FUS-BBBO in NHPs. This will make it possible for academic groups without access to expensive clinical FUS systems to perform ATAC in larger organisms. Finally, as motivating example applications, we will demonstrate that the optimized ATAC paradigm can be used to inhibit multiple distinct brain regions in macaques, reversibly and repeatably modulating their ability to recognize faces and also apply it in a sensorimotor circuit to alter functional connectivity. We will also show its stability, reliability and non-toxicity.