Cre-Dependent Anterograde Transsynaptic Labeling and Functional Imaging in Zebrafish Using VSV With Reduced Cytotoxicity.

Stanislav Kler,Sujatha Narayan,Y Albert Pan,Misha B Ahrens,Manxiu Ma

doi:10.3389/fnana.2021.758350

Abstract

The small size and translucency of larval zebrafish (Danio rerio) have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory (vglut2a positive) and inhibitory (gad1b positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5–8 days post fertilization (2–5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.

Highlights

Deciphering the connectivity patterns of neurons within the brain, both structural and functional, is necessary to fully understand how the brain functions
Expanding from mice, we found that vesicular stomatitis virus (VSV) can be used for polysynaptic anterograde or retrograde polysynaptic transsynaptic tracing in zebrafish (Mundell et al, 2015; Beier et al, 2016)
One notable difference between Tracer with Restricted Anterograde Spread (TRAS) and TRAS-M51R was in the retinal ganglion cells (RGCs) terminals in the optic tectum neuropil, where labeling is more robust in TRAS-M51R labeled animals

Summary

Introduction

Deciphering the connectivity patterns of neurons within the brain, both structural and functional, is necessary to fully understand how the brain functions. The use of viral vectors in zebrafish, an increasingly important model for neuroscience, has been much more limited, as many of the common viral vectors have either no infectivity (AAV) or reduced ability to spread (rabies) when applied to zebrafish (Zhu et al, 2009; Dohaku et al, 2019). To address this limitation, we explored the use of vesicular stomatitis virus (VSV), which has been successfully used as polysynaptic and monosynaptic transneuronal tracers in mice (Beier et al, 2013a,b). The unrestricted spread of replication-competent VSV makes it difficult to disambiguate between monosynaptic and polysynaptic connections

Methods

Results

Conclusion