Resources for Studying Neural Circuit Structure and Function with G-Deleted Rabies Viruses

 描述（由申请人提供）：破译哺乳动物大脑内的神经回路如何产生感知、认知和行为对于理解神经系统如何发挥作用至关重要。神经回路在广泛的空间和计算尺度上运行，从跨多个大脑区域整合信息的高级回路到在专门的大脑结构内执行简单输入/输出转换的微回路。每一层次的分析对于制定对环境条件的反应都很重要。然而，研究特定的神经回路是非常困难的，因为大多数神经系统结构包含许多类型的神经元，这些神经元具有不可分割地交织在一起的轴突和树突。为了克服这一障碍，开发了糖蛋白（G）缺失的狂犬病载体系统，以识别特定神经元群体的直接突触输入。通过用外源包膜蛋白（如EnvA）对G缺失的狂犬病载体进行假分型，该载体选择性地转导经基因工程改造以表达EnvA受体的靶神经元。如果这些细胞也表达狂犬病糖蛋白，载体正好逆行一步突触，直接转导突触前神经元。狂犬病基因组可以被改变以编码任何感兴趣的基因，包括荧光蛋白以揭示突触前细胞的细胞结构，或 神经科学工具（例如，钙指示剂或光门控离子通道）以监测或操纵回路活动。因此，G缺失的狂犬病载体系统允许对回路内的特定细胞类型进行精细操作，使研究人员能够测试将这些回路与行为联系起来的假设。这项技术彻底改变了神经元回路的研究，对这些尖端试剂产生了很高的需求。然而，专注于了解神经回路的实验室通常没有资源或专业知识来生产高质量的狂犬病载体或相关的辅助载体，而这些载体是进行这些实验所必需的。正因为如此，索尔克研究所的基因转移，靶向和治疗（GT3）核心，目前产生狂犬病载体，是淹没与要求准备注射病毒试剂和需求超过生产能力。该R24申请旨在扩大GT3核心的能力，以维持、繁殖和分发所有G缺失狂犬病载体变体和辅助载体（目标1）。GT3 Core还将在技术平台中整合新开发的工具，因为它们是创新的（目标2）。建立狂犬病中心生产设施将降低试剂成本（通过规模经济），提高研究结果的重现性。实验室之间的成本分担机制将能够分配小的等分试样，促进试点实验，并消除最大的障碍， 新实验室的技术吸收。新生成的试剂将立即分发给神经科学界，没有出版限制，从而加快发现的步伐。这些努力将扩大这项技术的影响，并确保研究电路的神经科学家配备最现代的分析工具。

 DESCRIPTION (provided by applicant): Deciphering how neural circuits within the mammalian brain give rise to perception, cognition, and behavior is central to understanding how the nervous system functions. Neural circuits operate over a vast range of spatial and computational scales, from high-level circuits that integrate information across multiple brain regions, to microcircuits that perform simple input/output transformations within a specialized brain structure. Each level of analysis is important for formulating responses to environmental conditions. However, studying a specific neural circuit is extremely difficult, as most nervous system structures contain many types of neurons with inextricably intertwined axons and dendrites. To overcome this obstacle, the glycoprotein (G)-deleted rabies vector system was developed to identify direct synaptic inputs to a particular neuronal population. By pseudo typing the G-deleted rabies vector with a foreign envelope protein, such as EnvA, the vector selectively transduces target neurons genetically engineered to express the EnvA receptor. If these cells also express rabies glycoprotein the vector travels retrograde exactly one synaptic step and transduces direct presynaptic neurons. The rabies genome can be altered to encode any gene of interest, including fluorescent proteins to reveal the cytoarchitecture of presynaptic cells, or neuroscience tools (e.g., calcium indicators or light-gated ion channels) to monitor or manipulate circuit activity. Thus, the G-deleted rabies vector system allows the fine- scale manipulation of specific cell types within a circuit, allowing investigators to test hypotheses linking these circuts to behavior. This technology has revolutionized the study of neuronal circuits, creating high demand for these cutting-edge reagents. Laboratories that focus on understanding neural circuits, however, typically do not have the resources or expertise to produce high quality rabies vectors or associated helper vectors that are necessary to perform these experiments. Because of this, the Salk Institute's Gene Transfer, Targeting, and Therapeutics (GT3) Core, which currently generates the rabies vectors, is inundated with requests for ready-to- inject viral reagents and demand exceeds production capacity. This R24 application proposes to expand the GT3 Core's capacity for maintaining, propagating, and distributing all G-deleted rabies vector variants and helper vectors (Aim 1). The GT3 Core will also incorporate newly developed tools into the technology platform as they are innovated (Aim 2). Establishing this central rabies production facility will lower reagent costs (through economies of scale) and improve the reproducibility of study findings. Between-lab cost sharing mechanisms will enable the distribution of small aliquots, facilitating pilot experiments and removing the greatest barrier to technology uptake by new laboratories. Newly generated reagents will be immediately distributed to the neuroscience community without publication restrictions, thereby speeding the pace of discovery. These efforts will broaden the impact of this technology and ensure that neuroscientists studying circuits are equipped with the most modern analytic tools.