A new anterograde trans-synaptic tracer based on Sindbis virus

Abstract

Mapping neural circuits is critical for understanding the structure and function of the nervous system. Engineered viruses are a valuable tool for tracing neural circuits. However, current tracers do not fully meet the needs for this approach because of various drawbacks, such as toxicity and characteristics that are difficult to modify. Therefore, there is an urgent need to develop a new tracer with low toxicity and that allows for long-term studies. In this study, we constructed an engineered Sindbis virus (SINV) expressing enhanced green fluorescent protein (EGFP) reporter gene (SINV-EGFP) and found that it had no significant difference in biological characterization compared with the wild-type Sindbis virus in BHK-21 cells and neurons in vitro. We injected the virus into the visual circuit of mouse brain and found that the virus infected neurons in the local injected site and anterogradely spread in the neural circuits. Although the efficiency of transmission was limited, the findings demonstrate that SINV can be used as a new anterograde tracer to map neural circuits in mouse brain and that it spreads exclusively in the anterograde direction. Further, use of SINV in mouse brain research will provide longer time windows for circuit tracing than is possible with herpes simplex virus and vesicular stomatitis virus tracers.

Keywords: Sindbis virus; anterograde; lateral geniculate nucleus; mouse brains; neural circuit; neurons; retina; superior colliculus; synapse; tracer.

Figure 1

The biological characteristics of the SINV-EGFP in vitro. (A) Diagram of pSINV-EGFP (a) and pSINV-WT (b) genome structures. (B) The kinetics of virus production. Fluorescent images of BHK-21 cells after transfecting pSINV-EGFP and pSINV-WT at different time points. Fluorescence signals could be detected at 12 hpt and increased with time in the pSINV-EGFP group. No fluorescence was detected in the pSINV-WT group. Virus titers at different time points post-transfection were measured by plaque assay. (C) The single-step growth curves of both viruses. These viruses were collected and titered on BHK-21 cells at different time points post-infection. (D) Plaque sizes of both viruses. The sizes of the viruses were not significantly different. (E) Fluorescent images of cultured primary neurons and BHK-21 cells after infecting with SINV-EGFP. All cells were infected and expressed EGFP. C: Capsid; E3, E2, E1, 6K: structural protein; EGFP: enhanced green fluorescent protein; hpt: hours post-injection; MOI: multiplicity of infection; NSP1–4: nonstructural protein 1–4; UBC: ubiquitin C promoter.

Figure 2

Injection into the LGN of mice to characterize virus trans-synaptic properties. (A) Diagram of circuits between retinal ganglion cells, LGN, V1 and V2. Data are expressed as mean ± SD. (B) Positive cells of different brain areas at 96 hours post-injection. (C–E) SINV-EGFP spread transsynaptically in the anterograde direction from injected sites to primary output V1 and secondary output V2 by 96 hpi. A large number of neurons were labeled in the LGN (C), and positive cells were also detected in V1 (D) and V2 (E). The experiments were repeated three times. EGFP: Enhanced green fluorescent protein; hpi: hours post-injection; LGN: lateral geniculate nucleus; RGCs: retinal ganglion cells; V1: visual cortex area 1; V2: visual cortex area 2.

Figure 3

Injection into the retina of mice to characterize the virus. We injected 2 μL SINV-EGFP into the vitreous body of the eye of mice, and mice were sacrificed at 96 hpi. (A) Diagram of neural circuit between the RGCs of retina, the LGN and V1. (B) At 96 hpi of subretinal cells, we isolated and expanded retinal cells. RGC axons were labeled by SINV-EGFP. (C, D) Positive signals were detected in the LGN and V1, which indicates that the virus anterogradely spread in the neural circuit. The experiments were repeated three times. EGFP: Enhanced green fluorescent protein; hpi; hours post-injection; LGN: lateral geniculate nucleus; RGCs: retinal ganglion cells; V1: visual cortex area 1.

Figure 4

The time course of SINV-EGFP spread within the neural circuit. The superior colliculus (SC) was chosen as the injection site. (A) Diagram of the SC outputs in the neural circuits. (B) The virus was injected into the SC (150 nL, 3 × 10⁹ PFU/mL) in adult mice. Three adult mice per group were used for SC injection, and were sacrificed at 24, 48, 72, and 96 hpi. Positive cells were first observed at 24 hpi, and the number of labeled neurons in the injection sites increased with time. In the thalamic nucleus (LP), many positive cells were detected at 48 hpi, and in the LA, positive cells were detected at 96 hpi. (C) The positive cells within each brain area were quantified in every three whole-brain slices. Data are expressed as mean ± SD (n = 3 animals for each time point). (D) The lethality after intracranial injection with SINV-EGFP (150 nL, 3 × 10⁹ PFU/mL) and VSV-mNeonGreen (150 nL, 2.5 × 10⁸ PFU/mL). Survival rate of SINV and VSV infected mice. Two viruses were injected intracranially into the SC of adult mice. The percent survival was analysed by Log-rank test (n = 5 in each group). EGFP: Enhanced green fluorescent protein; hpi: hours post-injection; LA: lateral amygdala; LP: lateral posterior thalamic nucleus; SC: superior colliculus.