基于神经亲病毒的示踪剂已广泛应用于神经回路的映射和操纵。然而,它们的嗜神经和神经毒性特性仍有待充分表征。
通过神经电路追踪,我们系统地比较了不同的多跨突触和单突触逆行病毒示踪剂,包括伪狂犬病病毒(PRV),狂犬病病毒(RV),和新设计的逆转录腺相关病毒(rAAV2-retro)示踪剂。(单细胞)RNA测序分析用于寻找可能的神经嗜性差异的归因,并比较糖蛋白缺失的RV(RVG)和rAAV2-retro之间由病毒感染引起的细胞毒性。通过免疫组织化学评估病毒毒性诱导的小胶质细胞激活和神经元蛋白变化。
多跨突触逆行病毒示踪剂,PRV和RV,当它们用于从the淋巴结进行中枢神经回路追踪时,表现出不同的神经嗜性。单突触逆行示踪剂,包括RVG和rAAV2复古,显示出不同的亲神经特性,当它们被应用于追踪下丘脑外侧区和内侧视前核的输入时。rAAV2-复古在大脑皮层表现出偏好,而RVG更喜欢标记基底神经节和下丘脑。值得注意的是,我们检测到rAAV2-retro在第5层和第6层对RVG的特定皮质层的明显偏好,当它们被注射到背侧外侧膝状核以标记初级视觉皮层的皮质丘脑神经元时。RV抗性神经元中TVA受体基因的互补使EnvA假型RV感染成为可能,支持受体归因于病毒神经嗜性。此外,RVG和rAAV2-retro在注射部位和逆行标记部位均产生神经毒性影响,而RVG感染的变化更为深刻。最后,通过结合rAAV2-retro,我们展示了一种概念证明策略,用于更全面地对特定目标核进行高阶电路追踪,RV,和rAAV示踪剂。
不同的多跨突触和单突触逆行病毒示踪剂在某些脑区表现出不同的神经嗜性,甚至皮质层偏好。与rAAV2-retro相比,在RV-ΔG感染下观察到更多的神经毒性。通过结合rAAV2-复古,RV,和rAAV示踪剂,可以实现高阶电路跟踪。我们的发现为适当应用病毒示踪剂描绘景观和剖析神经网络功能提供了重要参考。
Neurotropic virus-based tracers have been extensively applied in mapping and manipulation of neural circuits. However, their neurotropic and neurotoxic properties remain to be fully characterized.
Through neural circuit tracing, we systematically compared the
neurotropism discrepancy among different multi-trans-synaptic and mono-synaptic retrograde viral tracers including pseudorabies virus (PRV), rabies virus (RV), and the newly engineered retro adeno-associated virus (rAAV2-retro) tracers. The (single-cell) RNA sequencing analysis was utilized for seeking possible attribution to
neurotropism discrepancy and comparing cell toxicity caused by viral infection between glycoprotein-deleted RV (RV-∆G) and rAAV2-retro. Viral toxicity induced microglia activation and neuronal protein change were evaluated by immunohistochemistry.
Multi-trans-synaptic retrograde viral tracers, PRV and RV, exhibit differential
neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes. Mono-synaptic retrograde tracers, including RV-∆G and rAAV2-retro, displayed discrepant neurotropic property, when they were applied to trace the inputs of lateral hypothalamic area and medial preoptic nucleus. rAAV2-retro demonstrated preference in cerebral cortex, whereas RV-∆G prefers to label basal ganglia and hypothalamus. Remarkably, we detected a distinct preference for specific cortical layer of rAAV2-retro in layer 5 and RV-∆G in layer 6 when they were injected into dorsal lateral geniculate nucleus to label corticothalamic neurons in primary visual cortex. Complementation of TVA receptor gene in RV-resistant neurons enabled EnvA-pseudotyped RV infection, supporting receptors attribution to viral
neurotropism. Furthermore, both RV-∆G and rAAV2-retro exerted neurotoxic influence at the injection sites and retrogradely labeled sites, while the changes were more profound for RV-∆G infection. Finally, we demonstrated a proof-of-concept strategy for more comprehensive high-order circuit tracing of a specific target nucleus by combining rAAV2-retro, RV, and rAAV tracers.
Different multi-trans-synaptic and mono-synaptic retrograde viral tracers exhibited discrepant
neurotropism within certain brain regions, even cortical layer preference. More neurotoxicity was observed under RV-∆G infection as compared with rAAV2-retro. By combining rAAV2-retro, RV, and rAAV tracers, high-order circuit tracing can be achieved. Our findings provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.