Highly efficient and super-bright neurocircuit tracing using vector mixing-based virus cocktail

Abstract

Despite significant progresses in dissecting neural circuits with recombinant adenoassociated viruses (rAAVs), compatible and efficient gene expressions of multiple vectors within the same cell still remains challenging. Here, we developed MAP-ENVIVIDERS, a recombinase system-dependent (including Cre-lox/Flp-FRT recombinase systems) viral copackaging strategy to ameliorate mutual suppression and enhance compatibility among different rAAVs to improve neurocircuit tracing.For neuron labeling with wild-type and Cre-driver line mice, MAP-ENVIVIDERS was ~5-fold and over 10-fold more sensitive than the mixture of independently packaged rAAVs, respectively, enabling us to identify new type of neurons and capture massive morphological details of individual neurons (for example, a cholinergic neuron with 9,500+ axonal branches). For trans-monosynaptic retrograde tracing with rabies virus-mediated systems, MAP-ENVIVIDERS achieved 10-to 70fold enhancement of tracing efficiency in 40% of all input brain regions compared with the previous strategies. Therefore, MAP-ENVIVIDERS provides powerful tools for elucidating morphological detail and organization of neurocircuits.

Simultaneously cholinergic projection in Ascending and Descending Circuits from Midbrain

“…To obtain the fine morphology of individual cholinergic neurons, we employed the Cre-dependent virus for sparse labeling (Sun et al, 2020), fMOST system for whole-brain imaging (Gong et al, 2016), and GTree software for single-cell reconstruction (Zhou et al, 2020) ( Figure 1A, B ). Firstly, we performed 100 nl CSSP-YFP on the PPN or LDT of ChAT-Cre mice.…”

“…For sparse labeling, we employed the CSSP-YFP (5.2 × 10 12 vg/mL) packed by Brain Case (Brain Case Co., Ltd., Shenzhen, China). CSSP-YFP virus is produced by co-packaging the rAAV-EF1α-DIO-Flp plasmid and rAAV-FDIO-EYFP plasmid with the ratio of 1:20,000 in single rAAV production step (Sun et al, 2020).…”

Whole-Brain Mapping the Direct Inputs of Dorsal and Ventral CA1 Projection Neurons

“…The retrograde trans-monosynaptic system consisted of the genetically modified EnvA-pseudotyped RV (RV-EnvA-G-DsRed, 2.0 × 10 8 infectious particles per ml) and the Credependent co-packaged rAAV helper virus (AAV2/2, 5.27 × 10 12 genomic copies per ml). Production of rAAVs and genetically modified RV were prepared as previously described (Sun et al, 2020). The co-packaged helper rAAV particles (AAV2/2) were constructed by cotransfection of two packaging plasmids AAV-EF1α-DIO-EGFP-TVA (GT) and AAV-EF1α-DIO-RVG into HEK293T cells at a ratio of 1:2.…”