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

 描述(申请人提供)：破译哺乳动物大脑中的神经回路如何产生感知、认知和行为，是了解神经系统如何运作的核心。神经电路在广泛的空间和计算尺度上运行，从整合多个大脑区域信息的高级电路，到在专门的大脑结构内执行简单输入/输出转换的微电路。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极其困难的，因为大多数神经系统结构包含许多类型的神经元，这些神经元与轴突和树突密不可分地交织在一起。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。通过对带有外源包膜蛋白(如ENVA)的G缺失狂犬病载体进行伪分型，该载体选择性地转导基因工程设计的表达ENVA受体的靶神经元。如果这些细胞也表达狂犬病糖蛋白，载体就会逆行一个突触步骤，并直接传递突触前神经元。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，或者 神经科学工具(例如，钙指示剂或光门离子通道)，以监测或操纵电路活动。因此，G-缺失狂犬病载体系统允许对电路中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电路与行为联系起来的假设。这项技术使神经元电路的研究发生了革命性的变化，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，索尔克研究所目前生成狂犬病媒介的基因转移、靶向和治疗(GT3)核心被立即注射病毒试剂的请求淹没，需求超过了生产能力。这个R24应用程序建议扩展GT3核心的能力，以维护、传播和分发所有G-缺失狂犬病媒介变体和辅助媒介(目标1)。GT3核心还将在创新时将新开发的工具纳入技术平台(目标2)。建立这个中央狂犬病生产设施将降低试剂成本(通过规模经济)，并提高研究结果的重复性。实验室之间的成本分担机制将使小等分的分配成为可能，促进中试实验，并消除 新实验室采用的技术。新产生的试剂将立即分发给神经科学界，不受发表限制，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.