Anterograde Mono-synaptic Tracing - Brain Case ()

HSV anterograde mono-synaptic tracing system: a useful tool for studying output neural circuits

Herpes simplex virus (HSV) is a member of the alpha-herpesvirinae subfamily of the family Herpesviridae and can specifically infect the nervous system. HSV has a wide range of animal infection hosts and can be used to analyze the neural circuits of mice, rats, non-human primates and other animals. The HSV genome is a linear double-stranded DNA molecule, about 152kb, so it can carry larger fragments of foreign genes. The virus is currently divided into type 1 and type 2 based on differences in antigenicity. The neurotropic and transneuronal propagation properties of HSV-1 make it an ideal tool for tracing neural circuits. The McIntyre-B strain of HSV-1 can spread retrogradely across neurons, while the H129 strain can spread anterogradely across neurons.

H129, as an anterograde tracer across multiple levels of synapses, has been reported to be used in various animal models to study different neural circuits. Recombinant H129 viruses expressing fluorescent proteins can be used to label output neural networks. The expression of fluorescent signals greatly facilitates the use of H129 as a tracer. HSV type 1 strain H129 (HSV-1H129) can not only mark the connections between different central brain areas, but also mark the connections between the periphery and different central brain areas.

However, for a specific brain region or cell population, the results of labeling with tracers across multiple levels cannot distinguish direct one-level output neural circuits from multi-level output neural circuits. Therefore, anterograde mono-transsynaptic tracer systems are urgently needed for neuroscience research.

The principle of the HSV anterograde transsynaptic labeling system

HSV-ΔTK-hUbC-tdTomato is obtained by knocking out the thymidine kinase (TK) gene based on HSV-tdTomato (HSV1 strain H129 genome integrating a tdTomato gene). Due to the knockout of the TK gene, its ability to spread across synapses in vivo is limited. In the presence of a TK-expressing helper virus, HSV-ΔTK-hUbC-tdTomato can spread from the initial neurons to the postsynaptic neurons, thereby labeling the immediate downstream neurons directly innervated by a given brain area or specific type of neurons. To prevent retrograde labeling, the research team led by Professor Xu Fuqiang at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, developed a rigorous and practical monosynaptic anterograde tracing recombinant virus H129 system. They embedded the anti-Her2 scFv (single-chain antibody variable) region into the gDΔ6–38 region of the H129 envelope glycoprotein D (gD) to infect neurons that only express the Her2 receptor. This achieved specificity in targeting the initial site of H129 to Her2-expressing cells, thereby eliminating the reuptake ability of the original H129 strain at axon terminals.

Figure 1. Diagram illustrating anterograde mono-synaptic neuronal tracing

Application

Studying specific brain regions and neuronal types' anterograde monosynaptic output circuits (see case studies).

Case display

Example 1: Tracing neurons directly innervated by nRT-PV neurons

• Experimental animals : PV-cre mice
• Viral vector : H129-ΔTK-tdT and helper virus AAV-DIO-TK-GFP
• Experimental methods and results : Cre-dependent TK helper virus was first injected into the nRT brain area of ​​PV-cre mice, and H129-ΔTK-tdT was injected 3 weeks later. Then some mice were sacrificed on day 25 post-infection to identify the presynaptic cells involved in synaptic transmission. The remaining mice were sampled on day 32, and signals could be detected in multiple brain areas, including VP, Po, VM, etc.

Figure 2. Tracing neurons downstream directly innervated by nRT^PV neurons. (derived from Jiang Haifei’s doctoral thesis at Wuhan Institute of Virology)

Example 2: Using the Hs06 anterograde trans-monosynaptic labeling and tracing system to study the next-level neural circuits of SC

• Experimental animals : C57BL/6 Mice
• Viral vector : H129-dgD-hUbC-mCherry-P2A-scHer2::gd and helper virus rAAV-DIO-EGFP-T2A-Her2CT9, rAAV-DIO-cmgD, rAAV-hSyn-Cre
• Experimental methods and results : To confirm Hs06 virus system could realize anterograde trans-monosynaptic tracing without retrograde spreading, we performed tracing experiment initial form superior colliculus (SC) (Fig.3). It had been proved that SC only received projection form V1 but not projected back. Neurons in SC project to many regions including pretectal nucleus and LGN. In this experiment, we observed neurons labeled by Hs06 in SC projecting regions but not V1 (Fig.3C-G). These results suggested that the system could anterograde trans-monosynaptic spread exclusively with the assistance of Her2CT9 and gD.

Figure 3. Rigorous anterograde trans-monosynaptic tracing of genetic defined neurons with retargeted HSV1 H129, Peng Su Fuqiang Xu. doi: https://doi.org/10.1101/2020.12.01.407312

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