Neurotropic viruses have greatly enhanced our understanding of connectivity in nervous systems, and thanks to powerful methods for engineering their genomes they promise to become increasingly important in the future. Viruses have already been engineered to target specific neuronal subtypes, to direct transport of cargo in either the anterograde or retrograde direction, to cross variable numbers of synapses, to produce Golgi-like fills of targeted neurons and to deliver cargo that can be used to both monitor and manipulate neural activity. However, significant deficits in the neuroanatomical toolbox remain presenting investigators with plenty of opportunities for methodological innovation. This special issue attempts to survey recent advances in exploiting the unique properties of neurotropic viruses to map connectivity and assess its functional significance in a variety of neural structures. One of the most critical features of any neuroanatomical tracer is the direction it travels: so-called retrograde tracers are taken up by axon terminals and transported back to the cell body, whereas anterograde tracers travel in the opposite direction. When interpreting the results of a neuroanatomical experiment, it is clearly essential to be able to distinguish between the two directions of transport, and a recent survey of non-viral tracers made clear that, while many “classical” tracers have a predilection for one direction or the other, almost none of them are exclusively transported in a single direction (see Table 1 of Nassi et al. 2015). In this special issue, Huadong Wang and colleagues raise a similar concern for a viral tracer, H129, previously thought to be purely an anterograde tracer. They use a replication-deficient mutant of H129 (called ‘H306′) to show that this Herpes simplex virus can indeed be transported retrogradely and therefore that studies using this tool must be interpreted with caution, especially when survival times are long. Taking a step back, Kevin Beier provides an overview of differently directional viruses and strategies for engineering them to either change or improve their directional specificity. Like Wang et al., he provides evidence that the directional specificity of some viral tracers may have been overstated. But, more importantly, he provides a careful analysis of how the biological mechanisms of the virus interact with those of the neuron to produce directional specificity and, in some cases, trans-synaptic spread. He makes it clear that a more thorough understanding of viral replication, trafficking, shedding and infection will be critical for engineering better viral tracers. Two other contributions to the special issue focus on protocols that will be invaluable to practicing neuroanatomists. The use of G-deleted rabies virus to map the inputs to genetically defined populations of neurons is simple in concept, but, as Lavin, Jin and Wickersham remind us, “the devil is in the details.” And they provide a detailed protocol to this end, complete with the description of an ideal injection apparatus, instructions on the preparation of reagents, and a step-by-step surgical protocol. In a similar spirit, Eriko Kuramoto describes a method for using the Sindbis virus to label and reconstruct single axons and its application to study thalamocortical projection neurons. Though laborious, the method provides unique insights into neuronal circuitry by virtue of the beautifully elaborate reconstructions of axonal arborizations that it permits. Finally, as a counter-point to the labor-intensive microscopy-based methods required by virtually all existing neuroanatomical approaches, Justus Kebschull, reviews a new method that allows mapping the outputs of thousands of individual neurons in a single experiment, so-called “MAPseq.” The method uses Sindbis virus to introduce nucleic acid “barcodes” along with the sequence of a presynaptic protein that drags the barcode to the infected neuron’s synaptic outputs. Projections from a given neuron at the injection site to a given brain structure are established by DNA sequencing, matching up barcodes at the two locations. While the five articles in this special issue are by no means a complete representation of virus-based neuroanatomical approaches, they constitute an interesting sample of this space. Importantly, they show the range of what is currently possible—from the anatomical detail of single neuronal projections to high-throughput, sequence-based methods—and they provide insights into approaches for generating the next generation of viral tracing tools.

中文翻译：

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化学神经解剖学杂志特刊“使用病毒追踪研究大脑连接的新方法”

嗜神经病毒极大地增强了我们对神经系统连通性的理解，并且由于强大的基因组工程方法，它们有望在未来变得越来越重要。病毒已经被设计为针对特定的神经元亚型，以顺行或逆行方向直接运输货物，穿过可变数量的突触，产生高尔基体样填充的目标神经元，并提供可用于两者的货物监测和操纵神经活动。然而，神经解剖学工具箱中的重大缺陷仍然为研究人员提供了大量方法创新的机会。本期特刊试图调查在利用嗜神经病毒的独特特性来绘制连接性并评估其在各种神经结构中的功能意义方面的最新进展。任何神经解剖示踪剂最关键的特征之一是它的行进方向：所谓的逆行示踪剂被轴突末端吸收并运输回细胞体，而顺行示踪剂则以相反的方向行进。在解释神经解剖学实验的结果时，能够区分两个运输方向显然是必不可少的，最近对非病毒示踪剂的一项调查表明，虽然许多“经典”示踪剂偏爱一个方向或者，几乎没有一个是专门在一个方向上运输的（见 Nassi 等人的表 1。2015）。在本期特刊中，王华东及其同事对病毒示踪剂 H129 提出了类似的担忧，H129 以前被认为是纯粹的顺行示踪剂。他们使用 H129 的复制缺陷突变体（称为“H306”）来表明这种单纯疱疹病毒确实可以逆行传播，因此必须谨慎解释使用这种工具的研究，尤其是在存活时间很长的情况下。退后一步，Kevin Beier 概述了不同方向的病毒以及对其进行改造以改变或提高其方向特异性的策略。像 Wang 等人一样，他提供的证据表明某些病毒示踪剂的方向特异性可能被夸大了。但是，更重要的是，他仔细分析了病毒的生物学机制如何与神经元的生物学机制相互作用以产生定向特异性，在某些情况下还会产生跨突触传播。他明确表示，更透彻地了解病毒复制、贩运、脱落和感染对于设计更好的病毒示踪剂至关重要。对特刊的另外两个贡献集中在对实践神经解剖学家来说非常宝贵的协议上。使用 G 缺失的狂犬病病毒将输入映射到基因定义的神经元群在概念上很简单，但正如 Lavin、Jin 和 Wickersham 提醒我们的那样，“细节决定成败”。他们为此提供了详细的方案，包括理想注射装置的描述、试剂制备说明、和一步一步的手术方案。本着类似的精神，Eriko Kuramoto 描述了一种使用 Sindbis 病毒标记和重建单个轴突的方法及其在研究丘脑皮质投射神经元中的应用。虽然费力，但该方法通过其允许的轴突分支的精美精心重建，提供了对神经元回路的独特见解。最后，作为对几乎所有现有神经解剖学方法所需的劳动密集型基于显微镜的方法的对比，Justus Kebschull 回顾了一种新方法，该方法允许在单个实验中映射数千个单个神经元的输出，即所谓的“ MAPseq。该方法使用辛德毕斯病毒引入核酸“条形码”以及突触前蛋白质的序列，该蛋白质将条形码拖到受感染神经元的突触输出端。从注射部位的给定神经元到给定大脑结构的投影是通过 DNA 测序建立的，匹配两个位置的条形码。虽然本特刊中的五篇文章绝不是基于病毒的神经解剖学方法的完整代表，但它们构成了这一领域的一个有趣样本。重要的是，它们展示了当前可能的范围——从单个神经元投射的解剖细节到高通量、基于序列的方法——并且它们提供了对生成下一代病毒追踪工具的方法的见解。从注射部位的给定神经元到给定大脑结构的投影是通过 DNA 测序建立的，匹配两个位置的条形码。虽然本特刊中的五篇文章绝不是基于病毒的神经解剖学方法的完整代表，但它们构成了这一领域的一个有趣样本。重要的是，它们展示了当前可能的范围——从单个神经元投射的解剖细节到高通量、基于序列的方法——并且它们提供了对生成下一代病毒追踪工具的方法的见解。从注射部位的给定神经元到给定大脑结构的投影是通过 DNA 测序建立的，匹配两个位置的条形码。虽然本特刊中的五篇文章绝不是基于病毒的神经解剖学方法的完整代表，但它们构成了这一领域的一个有趣样本。重要的是，它们展示了当前可能的范围——从单个神经元投射的解剖细节到高通量、基于序列的方法——并且它们提供了对生成下一代病毒追踪工具的方法的见解。它们构成了这个空间的一个有趣的样本。重要的是，它们展示了当前可能的范围——从单个神经元投射的解剖细节到高通量、基于序列的方法——并且它们提供了对生成下一代病毒追踪工具的方法的见解。它们构成了这个空间的一个有趣的样本。重要的是，它们展示了当前可能的范围——从单个神经元投射的解剖细节到高通量、基于序列的方法——并且它们提供了对生成下一代病毒追踪工具的方法的见解。