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

Reviewer #1 (Public Review):

EnvA-pseudotyped glycoprotein-deleted rabies virus has emerged as an essential tool for tracing monosynaptic inputs to genetically defined neuron populations in the mammalian brain. Recently, in addition to the SAD B19 rabies virus strain first described by Callaway and colleagues in 2007, the CVS N2c rabies virus strain has become popular due to its low toxicity and high trans-synaptic transfer efficiency. However, despite its widespread use in the mammalian brain, particularly in mice, the application of this cell-type-specific monosynaptic rabies tracing system in zebrafish has been limited by low labeling efficiency and high toxicity. In this manuscript, the authors aimed to develop an efficient retrograde monosynaptic rabies-mediated circuit mapping tool for larval zebrafish. Given the translucent nature of larval zebrafish, whole-brain neuronal activities can be monitored, perturbed, and recorded over time. Introducing a robust circuit mapping tool for larval zebrafish would enable researchers to simultaneously investigate the structure and function of neural circuits, which would be of significant interest to the neural circuit research community. Furthermore, the ability to track rabies-labeled cells over time in the transparent brain could enhance our understanding of the trans-synaptic retrograde tracing mechanism of the rabies virus.

To establish an efficient rabies virus tracing system in the larval zebrafish brain, the authors conducted meticulous side-by-side experiments to determine the optimal combination of trans-expressed rabies G proteins, TVA receptors, and recombinant rabies virus strains. Consistent with observations in the mouse brain, the CVS N2c strain trans-complemented with N2cG was found to be superior to the SAD B19 combination, offering lower toxicity and higher efficiency in labeling presynaptic neurons. Additionally, the authors tested various temperatures for the larvae post-virus injection and identified 36{degree sign}C as the optimal temperature for improved virus labeling. They then validated the system in the cerebellar circuits, noting evolutionary conservation in the cerebellar structure between zebrafish and mammals. The monosynaptic inputs to Purkinje cells from granule cells were neatly confirmed through ablation experiments.

However, there are a couple of issues that this study should address. Additionally, conducting some extra experiments could provide valuable information to the broader research field utilizing recombinant rabies viruses as retrograde tracers.

(1) It was observed that many radial glia were labeled, which casts doubt on the specificity of trans-synaptic spread between neurons. The issues of transneuronal labeling of glial cells should be addressed and discussed in more detail. In this manuscript, the authors used a transgenic zebrafish line carrying a neuron-specific Cre-dependent reporter and EnvA-CVS N2c(dG)-Cre virus to avoid the visualization of virally infected glial cells. However, this does not solve the real issue of glial cell labeling and the possibility of a non-synaptic spread mechanism.

In addition, wrong citations in Line 307 were made when referring to previous studies discovering the same issue of RVdG-based transneuronal labeling radial glial cells.

"The RVdG-based transneuronal labeling of radial glial cells was commonly observed in larval zebrafish29,30".

The cited work was conducted using vesicular stomatitis virus (VSV). A more thorough analysis and/or discussion on this topic should be included. Several key questions should be addressed:

Does the number of labeled glial cells increase over time?
Do they increase at the same rate over time as labeled neurons?
Are the labeled glial cells only present around the injection site?
Can the phenomenon of transneuronal labeling of radial glial cells be mitigated if the tracing is done in slightly older larvae?
What is the survival rate of the infected glial cells over time?
If an infected glial cell dies due to infection or gets ablated, does the rabies virus spread from the dead glial cells?
If TVA and rabies G are delivered to glial cells, followed by rabies virus injection, will it lead to the infection of other glial cells or neurons?

Answers to any of these questions could greatly benefit the broader research community.

(2) The optimal virus tracing effect has to be achieved by raising the injected larvae at 36C. Since the routine temperature of zebrafish culture is around 28C, a more thorough characterization of the effect on the health of zebrafish should be conducted.

(3) Given the ability of time-lapse imaging of the infected larval zebrafish brain, the system can be taken advantage of to tackle important issues of rabies virus tracing tools.
a) Toxicity.
The toxicity of rabies viruses is an important issue that limits their application and affects the interpretation of traced circuits. For example, if a significant proportion of starter cells die before analysis, the traced presynaptic networks cannot be reliably assigned to a "defined" population of starter cells. In this manuscript, the authors did an excellent job of characterizing the effects of different rabies strains, G proteins derived from various strains, and levels of G protein expression on starter cell survival. However, an additional parameter that should be tested is the dose of rabies virus injection. The current method section states that all rabies virus preparations were diluted to 2x10^8 infection units per ml, and 2-5 nl of virus suspension was injected near the target cells. It would be interesting to know the impact of the dose/volume of virus injection on retrograde tracing efficiency and toxicity. Would higher titers of the virus lead to more efficient labeling but stronger toxicities? What would be the optimal dose/volume to balance efficiency and toxicity? Addressing these questions would provide valuable insights and help optimize the use of rabies viruses for circuit tracing.

b) Primary starters and secondary starters:
Given that the trans-expression of TVA and G is widespread, there is the possibility of coexistence of starter cells from the initial infection (primary starters) and starter cells generated by rabies virus spreading from the primary starters to presynaptic neurons expressing G. This means that the labeled input cells could be a mixed population connected with either the primary or secondary starter cells.

It would be immensely interesting if time-lapse imaging could be utilized to observe the appearance of such primary and secondary starter cells. Assuming there is a time difference between the initial appearance of these two populations, it may be possible to differentiate the input cells wired to these populations based on a similar temporal difference in their initial appearance. This approach could provide valuable insights into the dynamics of rabies virus spread and the connectivity of neural circuits.

Reviewer #2 (Public Review):

The study by Chen, Deng et al. aims to develop an efficient viral transneuronal tracing method that allows efficient retrograde tracing in the larval zebrafish. The authors utilize pseudotyped-rabies virus that can be targeted to specific cell types using the EnvA-TvA systems. Pseudotyped rabies virus has been used extensively in rodent models and, in recent years, has begun to be developed for use in adult zebrafish. However, compared to rodents, the efficiency of the spread in adult zebrafish is very low (~one upstream neuron labeled per starter cell). Additionally, there is limited evidence of retrograde tracing with pseudotyped rabies in the larval stage, which is the stage when most functional neural imaging studies are done in the field. In this study, the authors systematically optimized several parameters of rabies tracing, including different rabies virus strains, glycoprotein types, temperatures, expression construct designs, and elimination of glial labeling. The optimal configurations developed by the authors are up to 5-10 fold higher than more typically used configurations.

The results are solid and support the conclusions. However, the methods should be described in more detail to allow other zebrafish researchers to apply this method in their own work.

Additionally, some findings are presented anecdotally, i.e., without quantification or sufficient detail to allow close examinations. Lastly, there is concern that the reagents created by the authors will not be easily accessible to the zebrafish community.

(1) The titer used in each experiment was not stated. In the methods section, it is stated that aliquots are stored at 2x10e8. Is it diluted for injection? Are all of the experiments in the manuscripts with the same titer?

1. The age for injection is quite broad (3-5 dpf in Fig 1 and 4-6 dpf in Fig 2). Given that viral spread efficiency is usually more robust in younger animals, describing the exact injection age for each experiment is critical.

(3) More details should be provided for the paired electrical stimulation-calcium imaging study. How many GC cells were tested? How many had corresponding PC cell responses? What is the response latency? For example, images of stimulated and recorded GCs and PCs should be shown.

(4) It is unclear how connectivity between specific PC and GC is determined for single neuron connectivity. In other images (Figure 4C), there are usually multiple starter cells and many GCs. It was not shown that the image resolution can establish clear axon-dendritic contacts between cell pairs.

Reviewer #3 (Public Review):

Summary:

The authors establish reagents and define experimental parameters useful for defining neurons retrograde to a neuron of interest.

Strengths:

A clever approach, careful optimization, novel reagents, and convincing data together lead to convincing conclusions.

Weaknesses:

In the current version of the manuscript, the tracing results could be better centered with respect to past work, certain methods could be presented more clearly, and other approaches worth considering.

Appraisal/Discussion:

Trans-neuronal tracing in the larval zebrafish preparation has lagged behind rodent models, limiting "circuit-cracking" experiments. Previous work has demonstrated that pseudotyped rabies virus-mediated tracing could work, but published data suggested that there was considerable room for optimization. The authors take a major step forward here, identifying a number of key parameters to achieve success and establishing new transgenic reagents that incorporate modern intersectional approaches. As a proof of concept, the manuscript concludes with a rough characterization of inputs to cerebellar Purkinje cells. The work will be of considerable interest to neuroscientists who use the zebrafish model.

Author response:

We are grateful to the reviewers for their insightful comments on our manuscript and are encouraged by their overall favorable assessments. For the eLife Version of Record, we will make the following revisions to address reviewers’ comments and broaden the applicability of our technique in the zebrafish research community:

(1) We will elaborate on various facets with additional details:

a) Experimental conditions | We will specify the transgenic background, injected plasmids, larval stage, viral type, and viral titer clearly for each related experiment.

b) Experimental methods | We will depict in more details on how to inject the virus into a target area in larval zebrafish.

c) Data analysis | We will provide more detailed information on the paired electrical stimulation-calcium imaging study and on identifying connected Purkinje cells and granule cells during circuit reconstruction.

d) Discussion | We will elaborate on trans-synaptic specificity concerning glial cell labeling, toxicity related to viral dose and temperature, and the potential issue of secondary starters and multi-step circuit tracing.

(2) We will address the issue of glial cell labeling by adding more discussion and characterization, including potential mechanisms and implications, cell distribution, labeling progress, survival, and capability for viral transmission as starter cells.

(3) We will modify the text of the manuscript to clarify additional points raised by the reviewers.

(4) We will provide public repositories for accessing both the items and information on zebrafish lines, plasmids, viral vectors, and reconstructed data generated in this study.

In the end, we will submit full responses to the reviewer comments along with the revised version of the manuscript.