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

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.

An applicable and efficient retrograde monosynaptic circuit mapping tool for larval zebrafish

“…The RV d G-based transneuronal labeling of radial glial cells was commonly observed in larval zebrafish 29,30 (see Figure 1D,G and Figure 2—figure supplement 1H). The viral spread to glial cells may cause concerns about the synaptic specificity of RV d G spread between neurons.…”

“…TVA and G) in starter cells is critical for the application of RV d G[EnvA]-based circuit tracing. Previous studies on zebrafish expressed TVA and G through generating transgenic fish lines 26,27 or injecting helper viral vectors 27,29,30 . Both approaches allow for genetic access to a specific types or groups of neurons through the GAL4-UAS system.…”

“…Recently, the herpes simplex virus (HSV1) was used as a helper virus to deliver TVA and rabies G, leading to an increased efficiency of around one input per starter neuron in the brain of adult zebrafish 27 . For larval zebrafish, efforts have been focused on the vesicular stomatitis virus (VSV), another rhabdovirus similar to RV but with anterograde spread 28–30 . However, these reported VSV tools for larval zebrafish appeared relatively high cytotoxicity and have not been iterated to utilize the EnvA-TVA system to achieve initial infection specificity 28–30 .…”

“…For larval zebrafish, efforts have been focused on the vesicular stomatitis virus (VSV), another rhabdovirus similar to RV but with anterograde spread 28–30 . However, these reported VSV tools for larval zebrafish appeared relatively high cytotoxicity and have not been iterated to utilize the EnvA-TVA system to achieve initial infection specificity 28–30 . Therefore, developing applicable efficient viral tracing methods is still an important mission in the field, and will boost the application of larval zebrafish in systems neuroscience research.…”