Aravamudhan Pavithra, Raghunathan Krishnan, Dermody Terence S
Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, USA.
Center for Microbial Pathogenesis, UPMC Children's Hospital of Pittsburgh, Pittsburgh, USA.
Bio Protoc. 2020 Nov 20;10(22):e3825. doi: 10.21769/BioProtoc.3825.
Neurotropic reoviruses repurpose host machinery to traffic over long distances in neuronal processes and access distal replication sites. Understanding mechanisms of neuronal transmission is facilitated by using simplified primary neuronal culture models. Advances in the design of compartmentalized microfluidic devices lend robustness to neuronal culture models by enabling compartmentalization and manipulation of distinct neuronal processes. Here, we describe a streamlined methodology to culture sensory neurons dissociated from dorsal root ganglia of embryonic rats in microfluidic devices. We further describe protocols to exogenously label reovirus and image, track, and analyze transport of single reovirus particles in living neurons. These techniques can be adapted to study directed axonal transport of other neurotropic viruses and neuronal factors involved in signaling and pathology.
嗜神经呼肠孤病毒利用宿主机制在神经元突起中进行长距离运输并到达远端复制位点。使用简化的原代神经元培养模型有助于理解神经元传播的机制。通过对不同神经元突起进行分隔和操控，分隔式微流控装置设计的进展增强了神经元培养模型的稳定性。在此，我们描述了一种简化的方法，用于在微流控装置中培养从胚胎大鼠背根神经节解离的感觉神经元。我们还进一步描述了对外源标记呼肠孤病毒以及对活神经元中单个呼肠孤病毒颗粒的运输进行成像、追踪和分析的实验方案。这些技术可用于研究其他嗜神经病毒的定向轴突运输以及参与信号传导和病理学的神经元因子。
