Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses

Abstract

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

Brain-wide monosynaptic connectivity mapping with ROInet-seq

“…Future implementations of ROInet-seq can benefit from improved versions of modified rabies virus. The N2C-RV strain both reduced cytotoxicity and increased transsynaptic efficiency (Reardon et al 2016; Sumser et al 2022), and a recent double-deletion modification to the B19 strain led to 90% survival of infected cells over 12 weeks and enabled long-term recordings of presynaptic and starter cells (Jin et al 2024). Combined improvements in neuronal survival and comprehensive network labelling could make BC-ΔG-RV suited to study functional problems in neuroscience, where a sensitive, quantitative approach to connectivity is required.…”

Characterizing and targeting glioblastoma neuron-tumor networks with retrograde tracing

“…One limitation of the experimental platform is the neurotoxic potential associated with the use of rabies virus for retrograde tracing over time. [91][92][93] This issue underscores the importance of utilizing and further adapting less toxic rabies-based labeling strategies for glioblastoma, 93,94 to enable longer observation periods without adverse effects on neuronal health. Further, rabies virus-mediated retrograde tracing did not label all synaptic inputs in previous work, illustrating that the labeled connected TUM are possibly still an underestimation of the entire neuronal connectome of glioblastoma.…”