Rabies Virus Pseudotyped with CVS-N2C Glycoprotein as a Powerful Tool for Retrograde Neuronal Network Tracing

Abstract

Background: Efficient viral vectors for mapping and manipulating long projection neuronal circuits are crucial in brain structural and functional studies. The glycoprotein gene-deleted SAD strain rabies virus pseudotyped with the N2C glycoprotein (SAD-RV(ΔG)-N2C(G)) shows high neuro-tropism in cell culture, but its in vivo retrograde infection efficiency and neuro-tropism have not been systematically characterized. Methods: SAD-RV(ΔG)-N2C(G) and two other broadly used retrograde tracers, SAD-RV(ΔG)-B19(G) and rAAV2-retro were respectively injected into the VTA or DG in C57BL/6 mice. The neuron numbers labeled across the whole brain regions were counted and analyzed by measuring the retrograde infection efficiencies and tropisms of these viral tools. The labeled neural types were analyzed using fluorescence immunohistochemistry or GAD67-GFP mice. Result: We found that SAD-RV (ΔG)-N2C (G) enhanced the infection efficiency of long-projecting neurons by ~ 10 times but with very similar neuro-tropism, compared with SAD-RV (ΔG)-B19(G). On the other hand, SAD-RV(ΔG)-N2C(G) showed comparable infection efficiency with rAAV2-retro, but had a more restricted diffusion range, and broader tropism to different types and regions of long-projecting neuronal populations. Conclusions: These results demonstrate that SAD-RV(ΔG)-N2C(G) can serve as an effective retrograde vector for studying neuronal circuits. Key words：Viral vector, N2C Glycoprotein, Neuronal circuits, Retrograde tracing Background In the central nervous system, distinct brain regions work corporately through particular circuit connections to process different and complex information [1-6]. Neuronal

AAV9-Retro Mediates Efficient Transduction with Axon Terminal Absorption and Blood-brain Barrier Transportation

“…The stereotactic coordinates for VTA were: AP: -3.20 mm; ML: ± 0.45 mm; DV: -4.30 mm from the bregma; For CPu: AP: +0.38 mm; ML: ±2.00 mm; DV: -3.50 mm from the bregma. 8-10 weeks old C57BL/6 mice (20-25 g) were used for aav virus injection, and standard injection process was referred to previously reported method [11]. For single VTA site injection, mice were divided into two groups (N=3 in each group), 300 nl of rAAV2-Retro-CaMKIIa-EGFP, and rAAV9-Retro-CaMKIIa-EGFP viruses were infused into the VTA of each group, respectively; For mixed viral injection into VTA, rAAV2-Retro-CaMKIIa-mCherry and rAAV9-Retro-CaMKIIa-EGFP viruses were mixed at the particles ratio of 1:1 and injected into VTA at 300 nl; For single CPu site injection, mice were divided into two groups (N=3 in each group), 300 nl of rAAV2-Retro-CaMKIIa-EGFP and rAAV9-Retro-CaMKIIa-EGFP viruses were infused into the CPu region of each group, respectively.…”

“…Viral vectors, especially which permit e cient gene transfer to central nervous system (CNS) from axonal terminals or across the blood-brain barrier, are useful for analyzing structure and function of speci c neuronal circuits around the injection site [1][2][3][4][5][6][7][8][9][10][11], and have become one of the most potential and promising therapy tools by delivery therapeutic genes to a distant target area [5,12]. Compared with traditional retrograde tracers, viral vectors can express genes in speci c neuron groups [9,13], and have been widely used to monitor and manipulate neuronal activities by expressing optogenetic [14,15], chemogenetic [16,17] and calcium-sensitive functional probes [18][19][20].…”

Neuronal Network Dissection with Neurotropic Virus Tracing

“…In the present issue of Neuroscience Bulletin, Zhu and collaborators [8] compared the efficiency of retrograde gene transduction and neurotropism in three widely-used retrograde virus tracers. They found that the SAD strain of rabies virus [SAD-RV(DG)-N2C(G)], packaged with the N2C glycoprotein from the CVS strain [9], has a retrograde efficiency comparable to rAAV2-retro, but has a broader tropism in different neural types and regions, especially in subcortical regions.…”

“…They found that the SAD strain of rabies virus [SAD-RV(DG)-N2C(G)], packaged with the N2C glycoprotein from the CVS strain [9], has a retrograde efficiency comparable to rAAV2-retro, but has a broader tropism in different neural types and regions, especially in subcortical regions. However, rAAV2-retro is more suitable for cortical neural circuit tracing [8]. On the other hand, HSV1 strain H129, widely used as an anterograde tracer, also efficiently infects upstream innervating neurons through axon terminal uptake and displays a clear retrograde labeling phenotype, indicating that there are two types of starter cell: locally infected neurons in the injection site and retrogradely infected neurons [10].…”

“…In neural circuit tracing, the qualitative and quantitative analyses of labeled images are also necessary for further analysis. Based on the results [8,10], different neurotropic viruses have different labeling characteristics in the infected cerebral regions. Results from a single tool might be incomplete and inadequate, thus needing verification with multiple techniques.…”