A viral toolbox for conditional and transneuronal gene expression in zebrafish
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Evaluation Summary:
This manuscript puts forward a new toolkit of viruses for manipulation and visualization of zebrafish neural circuits. The authors overcome several challenges in the field and present a set of resources likely to be of high value to the zebrafish community.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.
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Author Response
Reviewer #2 (Public Review):
This manuscript is of interest for neuroscientists studying neural circuit mapping in late larval, juvenile, and adult zebrafish. The work adapts and refines methods for retrograde viral tracing in zebrafish, using conditional and transneuronal DNA cargoes, to gauge the structure, connectivity, and function of neurons. Overall, the methods described in the paper, combined with a suite of viral constructs that are made available, represent a practical advance for virus-based neural circuit mapping in zebrafish, although a few aspects of experimental design and data interpretation require strengthening.
This work provides methodological refinements and new constructs for retrograde neuronal tracing and functional testing of circuit elements in zebrafish. The authors of the manuscript put …
Author Response
Reviewer #2 (Public Review):
This manuscript is of interest for neuroscientists studying neural circuit mapping in late larval, juvenile, and adult zebrafish. The work adapts and refines methods for retrograde viral tracing in zebrafish, using conditional and transneuronal DNA cargoes, to gauge the structure, connectivity, and function of neurons. Overall, the methods described in the paper, combined with a suite of viral constructs that are made available, represent a practical advance for virus-based neural circuit mapping in zebrafish, although a few aspects of experimental design and data interpretation require strengthening.
This work provides methodological refinements and new constructs for retrograde neuronal tracing and functional testing of circuit elements in zebrafish. The authors of the manuscript put impressive efforts into developing methods that are compatible with currently available transgenic zebrafish lines. The authors developed the methods based on previously-described herpes simplex virus 1 (HSV1) and pseudotyped rabies virus (RV) with deleted G protein (RVΔG) as neuronal labeling tools. First, they explore and assess temperature's effect on viral infection efficiency. The results indicate that a temperature close to the viral host temperature is optimal. Second, they engineered HSV1 into the UAS system that either contained TVA or codon-optimized glycoprotein (zoSDG). In the lines that contained TVA, the authors delivered HSV1-UAS containing TVA to Gal4 zebrafish lines for specific cell type delivery. With Gal4/UAS, they expanded the tool to adapt the transgenic zebrafish system that is widely used. Because EnvA/TVA works as a system, they then inject EnvA- RVΔG to target neurons where TVA is prelocated for specific labeling. Because of the deleted glycoprotein in RV, the reproducibility of the virus was limited. Therefore, they showed another experiment that complemented the EnvA- RVΔG by co-injection of the HSV1 containing zoSDG (HSV1[UAS:zoSADG]) as a helper virus to assist RVΔG in the transneuronal spread. Using the resulting retrograde migration of RV, the authors visualized the firstorder upstream connections labeled by HSV1-TVA+ neurons. Appropriate for a methodological paper, the function of the viruses are well described and their properties are well documented. In some cases, however, supporting data are thin or anecdotal, and do not always sufficiently support the manuscript's claims and conclusions. Further data, more nuanced interpretations, and/or more circumspect discussion points are needed to address these concerns.
Strengths:
- HSV1 contains double-stranded DNA that can incorporate into the genome without using a complicated process to increase replication efficiency.
- Specific gene targeting with the EnvA-TVA system increases accuracy during gene delivery. The expanded toolkit enhances the targeting strategy to include a diversity of useful constructs for the structural and functional assessment of neural circuits.
- By making their toolbox compatible with the Gal4/UAS system, the authors leverage a large collection of Gal4 lines already available to the zebrafish community.
- The toolbox for virus-based circuit mapping is relatively immature in the zebrafish model. The methods and reagents introduced here complement the current anterograde tracing using VSV. They also fill a gap in viral tracing for circuit mapping in adult zebrafish, as the immune system in juveniles and adults tended to reduce the viral spread efficiency using other approaches.
Weaknesses:
- One of the major concerns of using this method is temperature increase. In zebrafish, temperature increase has been used as a heat stressor and is known to accelerate and facilitate development at larvae stage also cause lethality. Because of this accelerated development, the neurons labeled with HSV1 under heated conditions might not be the consequence of efficient virus infection, but rather a byproduct of faster migration and differentiation of neurons and other cells. Although the authors stated that adult zebrafish could tolerate higher temperatures (see item 5, below), this is not the normal condition for mapping circuits function, and the virus, as indicated in the manuscript, is also used in larvae. Further justification will be required to convince the audience that the use of high temperatures is generally adaptable, including for mapping circuits involved in other circuits. This is especially a concern for the HPA, because of the challenges in distinguishing the stress is from HSV1-induced oxidative stress from heat-induced neural stress.
The reviewer raises the possibility that increased expression after injection of HSV1 at higher temperatures may reflect increased proliferation (accelerated development) rather than increased infection efficiency. This scenario implies that a substantial fraction of labeled neurons were infected as progenitors, which then divided and differentiated into neurons. This possibility, although formally possible, appears extremely unlikely for the following reasons.
The difference in the number of labeled neurons is very large. If this difference were due to a difference in the speed of development, there should be an enormous difference in (brain) size between fish kept at different temperatures. If present, this size difference should be easily observable, at least in larvae. However, we did not observe an obvious size difference.
Viral infection was studied primarily in adult fish where neurogenesis still occurs, but at low rates compared to development. Nevertheless, the difference in labeled neurons was very large.
Many labeled neurons showed elaborated morphologies and long-range projections. It appears very unlikely that such neurons and their projections can arise by differentiation from precursors within the given incubation time.
The quantification in Fig. 1C was performed specifically for neurons with long-range projections in adult fish. The virus was injected into the OB while the neuronal somata were located in the dorsal telencephalon. If these neurons arose from precursors at the injection site, it would have to be postulated that these precursors migrated to the dorsal telencephalon, differentiated into neurons, and developed projections back to the OB. It is extremely unlikely that this can occur within the time of incubation. Moreover, there is no biological evidence for the migration of neuronal precursors or differentiating neurons from the OB to the dorsal telencephalon.
To further confirm that a speed-up of development cannot account for the observed difference in labeling we performed another variation of the experiment shown in Fig. 1: adult fish injected with HSV1[LTCMV:DsRed] into the OB were first kept at 36 deg for 3 days and then kept at 26 deg for 3 days before analysis (“36→26”). In these fish, DsRed expression in dorsal telencephalic neurons was indistinguishable from fish that were kept at 36 deg for the full period of 6 days. Fish that underwent the opposite temperature shift (“26→36”), in contrast, did not express DsRed in dorsal telencephalic neurons (Fig. 1C), despite the fact that they spent the same amount of time at each temperature.
Hence, the time at increased temperature per se cannot account for the difference in expression, indicating that temperature affects the process of infection. The new results have now been integrated into Fig. 1.
We cannot rule out that the temperature change affects stress levels and the HPA axis. However, as discussed in more detail below, swimming behavior was almost unchanged and obvious signs of stress were not observed. Moreover, please note that the temperature change can be restricted to the time around the virus injection, while any effects on behavior or neural activity will typically be examined several days later. Hence, effects of transgene expression will usually be evaluated at the standard laboratory temperature, long after the temperature change and the injection procedure.
- HSV1 infects various cell types, not limited to neurons. The authors in the manuscript mentioned the high infection rate of cells. They did not categorize whether all infected cells were neurons or mixed neurons and glia. The authors briefly mention glia in the RNA sequencing data, but knowing the cell types and location is critical for circuit mapping. In Figure S2A-D, it seems that some of the cells around the midline could be radial glia. Cell migration from the midline is abundant, with radial-glia at the early stage guiding neurons from the ventricular zone to the mantle regions. How do authors ensure that the increased infection at higher temperatures does not include glia with the elevated immune response?
We do not claim that HSV1 infects only neurons. Indeed, HSV1 probably also infects glia, and the cells labeled in Fig. S2 are likely to include radial glia. However, this is not necessarily a disadvantage as additional specificity can be created by methods such as the Gal4 system. In fact, enhancing cell type specificity was a main motivation to combine HSV1 with the Gal4 system. A broad selectivity of the virus itself may then actually be considered an advantage because it allows for targeting of a broad spectrum of possible cell types. For example, HSV1 in combination with a transgenic line expressing Gal4 in glia (e.g., Tg[gfap:Gal4]) may be used to specifically interrogate glia cells if desired. We now discuss this issue of cell type specificity more specifically in the revised manuscript (ln 103-107; ln 339ff).
3)One limitation with HSV1 is that it resides inside neurons for an unpredictable length of time before expression, which increases the latency for induction of TVA. This extended latency could reduce sample size or lead to missed temporal windows. This caveat should be discussed.
We agree that the delay between HSV1 injection and transgene (TVA) expression may, in principle, decrease the efficiency of Rabies infection and retrograde tracing. We therefore performed a set of experiments in which we injected the Rabies virus 2 or 4 days after the HSV1. However, we observed lower, rather than higher, rates of Rabies infection, possibly because the sites of the two injections were not precisely identical. Hence, the advantage of staggered injections, if any, appears to be offset by variability in the location of injections, at least in our hands. Moreover, previous applications in rodents also reported high efficiency of Rabies infection when the Rabies virus was applied at the same time as the TVA expression construct (Vélez-Fort et al. 2014; Wertz et al. 2015). We now show results in Figure 4 – figure supplement 1 and discuss these issues briefly in the revised manuscript (ln 271-274; ln 677ff).
4). In the manuscript, to achieve transneuronal labeling, the fish were exposed to three viruses across two injections. The approach also includes exposure to chronicle heat, selection of TVA+ neurons from the first round of injection, and long periods of incubation between steps in the protocol. This is both labor-intense and potentially challenging for the animals' health and survival. Because the rates of lethality and poor health are not quantified for times after the first injection, and because the efficiency of the labelling approach (assessed at the animal level) are not reported, it is difficult to judge whether the approach is efficient enough for experimental work, where a large n of animals will be necessary for multiple treatments. This is particularly the case for phenotyping where mutant lines may be predisposed to adverse effects from heat or other manipulations and interventions. The manuscript would ideally show the number of fish that 1) were injected, 2) were infected with the virus, 3) survived until the timepoint for data collection, and 4) yielded publishable data. The possible limitations for studying mutants, especially those susceptible to heat and infection, should be discussed.
We agree that more information on the success rate and survival rates is desired. Previously, we had not explicitly reported survival rates because these were very high, and we apologize for not mentioning this explicitly. In the revised manuscript, we have now addressed this issue more specifically.
Please note that all fish used in experiments are represented by individual data points in the figures (except for a very low number of fish that did not survive the injection); no fish were excluded from the analysis. This is now pointed out explicitly in Methods (“Statistical analysis”). Hence, the data in the figures show directly how many fish were infected with the virus (point 2 above; 100% of injected fish) and how many neurons were labled in each fish. In all fish, images were acquired and the number of labeled neurons was quantified, implying that all fish yielded “publishable data” (point 4 above).
The survival rate (points 1 and 3 above) was very close to 100% in adult fish, and very few fish were lost during the injection. This has now been quantified systematically for all experimental conditions. We directly compared the survival of fish that were not injected, injected with buffer, and injected with virus, either at standard laboratory temperature (typically 26 deg for adults, 28.5 deg for larvae) or at elevated temperature (36 or 35 deg, respectively). The results are shown in Figure 1 – figure supplement 1.
In adult fish, survival rates were 100% under all conditions after single injections of HSV1 viruses. In larvae, some mortality was observed under control conditions that was slightly enhanced at elevated temperatures. We speculate that this is an indirect effect because larvae were kept in petri dishes in stagnant medium and water quality degrades more rapidly at higher temperatures. In any case, survival rates one week after injection were still relatively high (~50%). Moreover, for the first 3 days, survival rates were >90%. This appears particularly relevant because two or three days of exposure to high temperature are sufficient to achieve efficient expression. Survival rates were still 80 – 90% after two injections of HSV1 or after injections of rabies virus. Hence, the temperature shift should be compatible with a broad spectrum of practical applications. No effect of the HSV1 itself was detected on survival rates.
- The current videos do not provide a rigorous demonstration that animals routinely tolerate elevated temperatures or infection (S Movies 1-3). Rates of survival for these cohorts and quantification of their swim behavior (such as distance travelled) with statistics would be more convincing. This criticism applies even more strongly to the single video of a sick fish (S Movie 4), which the authors use to support a claim of a targeted circuit manipulation using TeTx.
We have now quantified swimming behavior using two approaches. First, we compared the mean swimming speed between the six experimental groups used to determine effects of temperature and HSV1 on survival rates. Swimming was quantified at 27 deg after keeping fish at either 27 deg or at 36 deg for seven days. No significant difference in swimming behavior was observed (Figure 1 – figure supplement 2).
In addition, we quantified swimming behavior of fish at room temperature (25 – 26 deg) or 36 deg. Fish were kept in groups of five and individual fish were tracked using a machine learning-based tracking software (DeepLabCut). This allowed us to quantify different behavioral components. We found that mean swimming speed was higher at 36 deg and fish stayed slightly higher in the water column. However, social distance and the visual appearance of swimming were not obviously different. Swimming speed was normal again when fish were returned to normal temperature after seven days at 36 deg. These data are now shown in Figure 1 – figure supplement 2A,B.
- FACS sorting and transcriptomics is a very complex and not wholly informative approach for judging stress at the cellular and organismal level. First, stress level is best assessed with high temporal resolution and best measured through blood or whole body (for larvae) cortisol measurements. Second, it is best to judge stress circuits in zebrafish in the diencephalon-mesencephalon, for the HPA. Cellular stress could best be measured with IHC for oxidative stress in infected cells and for apoptotic cells in the wake of infections. Taking measurements from OB neurons, with RNA sequencing that followed the elimination of dead cells during tissue disassociation and cell sorting, could have missed elements of the stress process. The sequencing result from only live cells in the OB may not provide the most reliable evidence.
We believe that there is a misunderstanding here. We did not analyze stress at the organismal level or activation of the HPA axis. In fact, we compared cells collected from the same individuals, which rules out any differences in organismal stress levels between samples. Organismal stress is not a topic of this study; addressing this is clearly beyond the scope of this study.
The transcriptomics experiments were specifically designed to examine cellular stress caused by Rabies infection. We agree that the transcriptomics approach has limitations but we feel that the data nevertheless contain valuable information. Together with other findings (morphology, calcium imaging), they support the conclusion that infection by the Rabies virus (in the absence of G) does not cause excessive cellular toxicity on the timescales of our experiments, consistent with results from other species. We agree that it is possible that the Rabies virus has more subtle effects on cellular stress levels (or immune responses) but a detailed analysis of such effects is beyond the scope of the present study. This is now discussed explicity (Results: ln 224-228; Discussion: ln 372-375). It is also possible that toxicity would occur on longer timescales. This may be expected based on findings in rodents but still leaves a broad time window for anatomical and functional experiments. This is now discussed explicity (ln 378-381).
- The down-regulation in stress markers needs further discussion. Under chronic stress of heat exposure, exacerbation of HPA axis function could reduce glucocorticoids.
Please note that control and infected cells were from the same animals. The temperature regime can therefore not explain the differences in gene expression. Please also note that animals were not exposed to elevated temperature for days prior to cell collection.
Please also note that the down-regulation of genes was broad, affecting not only stress-related genes. Indeed, stress-related genes were not downregulated more frequently than other genes. Gene groups that were down-regulated most frequently are associated with immune responses. We therefore conclude that the downregulation of genes does not specifically reflect a stress response, and we speculate that it may reflect a general immune-related response. However, this is very hypothetical, and further studies are needed to understand the processes behind the observed pattern of gene regulation.
This is now stated clearly in the revised text (ln 224-228; ln 372-375).
- Although it cannot be addressed for larvae, it is critical to report the sex ratio for your adults, since hormones affect stress and circuits formation.
Adult fish of both sexes in approximately a 50:50 ratio were used to ensure that there is no sexdependent bias in the data. However, the exact sex ratio in each experiment has not been recorded. This is now stated explicitly in Methods.
Reviewer #3 (Public Review):
Satou et al. report a viral toolbox by:
- Inventing a novel way through temperature-dependence of HSV1-mediated gene expression for adult and larval zebrafish;
- Employing Gal4/UAS system to achieve cell types specific expression in this model;
- Combining the modified rabies viruses and HSV1 for transneuronal tracing of neural circuits in zebrafish that is kept in a higher temperature environment.
This toolbox in the manuscript will be of great interest to the neuroscience field when they are using zebrafish as a model.
The strength is these novel methods will offer more experimental opportunities and will facilitate more exciting basic scientific discoveries. However, some concerns still exist as below:
- What's the mechanism of temperature-dependence expressions with these HSV1 and rabies virus in this study? At least the authors should discuss it. Have the authors done experiments like this: after getting enough gene expression from these viruses when maintaining these fishes in 35-37 degree, bring them back to normal temperature as they usually live to see what happen? Does this higher temperature help the fish brain cells get infected with more viral particles or just help increase the expression level? Or does just the higher temperature help produce more proteins?
The question raised by the reviewer is indeed interesting. We agree that it would be useful to know whether host-like temperature enhances the entry of the virus into the host cell (infection), viral replication/protein synthesis, or both. In the original manuscript, we reported results from a first experiment to address this question. In this experiment, we injected HSV1 at 26 deg and then increased temperature to 36 deg 3 days after infection (“2636”). This protocol yielded low expression. We have now also performed the reverse temperature shift (“3626”), as suggested by the reviewer. This protocol yielded high expression, comparable to the expression observed when fish were kept at 36 deg throughout (see Fig. 1). Together, these results suggest that temperature affected primarily the infection. However, additional, more advanced analyses are required to resolve to what extent temperature affects viral infection and viral replication/protein synthesis. This is now discussed explicitly (ln 332ff).
- The authors should address or discuss more whether the higher temperature affects these fishes' brain activity? The reason is if someone will use this method for a most important experiment like GCaMP7s calcium imaging, in order to get good expression with these viruses that authors described in the manuscript they should raise the temperature but they have no idea about whether these higher temperatures affect the behavior or brain activity in some special brain regions they are interested in.
Please note that, in most applications, the temperature is increased only transiently around the time of injection for two or three days. Thereafter, fish can be transferred back to normal laboratory temperature without compromising transgene expression. Any follow-up experiments, e.g. analyses of behavior or neuronal activity, can therefore be performed at standard laboratory temperature, after fish were kept at this temperature for a few days. The temperature change should therefore have only minor, indirect effects on the results of behavioral or physiological experiments. We apologize if this was not evident and discuss this now explicitly (ln 334ff).
In addition, we have analyzed the swimming behavior of zebrafish in more detail at elevated temperatures (36 deg for adults; 35 deg for larvae) and observed only minor differences in swimming behavior. These results are now reported in Figure 1 – figure supplement 2A. Moreover, we compared swimming behavior between control fish (kept at standard laboratory temperature) and fish that underwent a transient temperature change to 36 deg for 7 days before the test. No significant difference in swimming behavior was observed between groups (Figure 1 – figure supplement 2B). We therefore conclude that no obvious effects of temperature are observed at least at the behavioral level.
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Evaluation Summary:
This manuscript puts forward a new toolkit of viruses for manipulation and visualization of zebrafish neural circuits. The authors overcome several challenges in the field and present a set of resources likely to be of high value to the zebrafish community.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Satou and colleagues present a novel repertoire of viral tools for visualization, tracing, and manipulating neuronal circuits in zebrafish. Though viral tools have revolutionized mammalian neuroscience, they have not gained similar traction in fish due to technical difficulties with both infection and cell death. The various viral manipulations presented in this manuscript promise to overcome both of these challenges, and the authors have gone to great lengths to evaluate viral efficacy. The resources are therefore likely to be of tremendous value and interest to the zebrafish community.
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Reviewer #2 (Public Review):
This manuscript is of interest for neuroscientists studying neural circuit mapping in late larval, juvenile, and adult zebrafish. The work adapts and refines methods for retrograde viral tracing in zebrafish, using conditional and transneuronal DNA cargoes, to gauge the structure, connectivity, and function of neurons. Overall, the methods described in the paper, combined with a suite of viral constructs that are made available, represent a practical advance for virus-based neural circuit mapping in zebrafish, although a few aspects of experimental design and data interpretation require strengthening.
This work provides methodological refinements and new constructs for retrograde neuronal tracing and functional testing of circuit elements in zebrafish. The authors of the manuscript put impressive efforts …
Reviewer #2 (Public Review):
This manuscript is of interest for neuroscientists studying neural circuit mapping in late larval, juvenile, and adult zebrafish. The work adapts and refines methods for retrograde viral tracing in zebrafish, using conditional and transneuronal DNA cargoes, to gauge the structure, connectivity, and function of neurons. Overall, the methods described in the paper, combined with a suite of viral constructs that are made available, represent a practical advance for virus-based neural circuit mapping in zebrafish, although a few aspects of experimental design and data interpretation require strengthening.
This work provides methodological refinements and new constructs for retrograde neuronal tracing and functional testing of circuit elements in zebrafish. The authors of the manuscript put impressive efforts into developing methods that are compatible with currently available transgenic zebrafish lines. The authors developed the methods based on previously-described herpes simplex virus 1 (HSV1) and pseudotyped rabies virus (RV) with deleted G protein (RVΔG) as neuronal labeling tools. First, they explore and assess temperature's effect on viral infection efficiency. The results indicate that a temperature close to the viral host temperature is optimal. Second, they engineered HSV1 into the UAS system that either contained TVA or codon-optimized glycoprotein (zoSDG). In the lines that contained TVA, the authors delivered HSV1-UAS containing TVA to Gal4 zebrafish lines for specific cell type delivery. With Gal4/UAS, they expanded the tool to adapt the transgenic zebrafish system that is widely used. Because EnvA/TVA works as a system, they then inject EnvA- RVΔG to target neurons where TVA is prelocated for specific labeling. Because of the deleted glycoprotein in RV, the reproducibility of the virus was limited. Therefore, they showed another experiment that complemented the EnvA- RVΔG by co-injection of the HSV1 containing zoSDG (HSV1[UAS:zoSADG]) as a helper virus to assist RVΔG in the transneuronal spread. Using the resulting retrograde migration of RV, the authors visualized the first-order upstream connections labeled by HSV1-TVA+ neurons. Appropriate for a methodological paper, the function of the viruses are well described and their properties are well documented. In some cases, however, supporting data are thin or anecdotal, and do not always sufficiently support the manuscript's claims and conclusions. Further data, more nuanced interpretations, and/or more circumspect discussion points are needed to address these concerns.
Strengths:
1. HSV1 contains double-stranded DNA that can incorporate into the genome without using a complicated process to increase replication efficiency.
2. Specific gene targeting with the EnvA-TVA system increases accuracy during gene delivery. The expanded toolkit enhances the targeting strategy to include a diversity of useful constructs for the structural and functional assessment of neural circuits.
3. By making their toolbox compatible with the Gal4/UAS system, the authors leverage a large collection of Gal4 lines already available to the zebrafish community.
4. The toolbox for virus-based circuit mapping is relatively immature in the zebrafish model. The methods and reagents introduced here complement the current anterograde tracing using VSV. They also fill a gap in viral tracing for circuit mapping in adult zebrafish, as the immune system in juveniles and adults tended to reduce the viral spread efficiency using other approaches.Weaknesses:
1. One of the major concerns of using this method is temperature increase. In zebrafish, temperature increase has been used as a heat stressor and is known to accelerate and facilitate development at larvae stage also cause lethality. Because of this accelerated development, the neurons labeled with HSV1 under heated conditions might not be the consequence of efficient virus infection, but rather a byproduct of faster migration and differentiation of neurons and other cells. Although the authors stated that adult zebrafish could tolerate higher temperatures (see item 5, below), this is not the normal condition for mapping circuits function, and the virus, as indicated in the manuscript, is also used in larvae. Further justification will be required to convince the audience that the use of high temperatures is generally adaptable, including for mapping circuits involved in other circuits. This is especially a concern for the HPA, because of the challenges in distinguishing the stress is from HSV1-induced oxidative stress from heat-induced neural stress.
2. HSV1 infects various cell types, not limited to neurons. The authors in the manuscript mentioned the high infection rate of cells. They did not categorize whether all infected cells were neurons or mixed neurons and glia. The authors briefly mention glia in the RNA sequencing data, but knowing the cell types and location is critical for circuit mapping. In Figure S2A-D, it seems that some of the cells around the midline could be radial glia. Cell migration from the midline is abundant, with radial-glia at the early stage guiding neurons from the ventricular zone to the mantle regions. How do authors ensure that the increased infection at higher temperatures does not include glia with the elevated immune response?
3. One limitation with HSV1 is that it resides inside neurons for an unpredictable length of time before expression, which increases the latency for induction of TVA. This extended latency could reduce sample size or lead to missed temporal windows. This caveat should be discussed.
4. In the manuscript, to achieve transneuronal labeling, the fish were exposed to three viruses across two injections. The approach also includes exposure to chronicle heat, selection of TVA+ neurons from the first round of injection, and long periods of incubation between steps in the protocol. This is both labor-intense and potentially challenging for the animals' health and survival. Because the rates of lethality and poor health are not quantified for times after the first injection, and because the efficiency of the labelling approach (assessed at the animal level) are not reported, it is difficult to judge whether the approach is efficient enough for experimental work, where a large n of animals will be necessary for multiple treatments. This is particularly the case for phenotyping where mutant lines may be predisposed to adverse effects from heat or other manipulations and interventions. The manuscript would ideally show the number of fish that 1) were injected, 2) were infected with the virus, 3) survived until the timepoint for data collection, and 4) yielded publishable data. The possible limitations for studying mutants, especially those susceptible to heat and infection, should be discussed.
5. The current videos do not provide a rigorous demonstration that animals routinely tolerate elevated temperatures or infection (S Movies 1-3). Rates of survival for these cohorts and quantification of their swim behavior (such as distance travelled) with statistics would be more convincing. This criticism applies even more strongly to the single video of a sick fish (S Movie 4), which the authors use to support a claim of a targeted circuit manipulation using TeTx.
6. FACS sorting and transcriptomics is a very complex and not wholly informative approach for judging stress at the cellular and organismal level. First, stress level is best assessed with high temporal resolution and best measured through blood or whole body (for larvae) cortisol measurements. Second, it is best to judge stress circuits in zebrafish in the diencephalon-mesencephalon, for the HPA. Cellular stress could best be measured with IHC for oxidative stress in infected cells and for apoptotic cells in the wake of infections. Taking measurements from OB neurons, with RNA sequencing that followed the elimination of dead cells during tissue disassociation and cell sorting, could have missed elements of the stress process. The sequencing result from only live cells in the OB may not provide the most reliable evidence.
7. The down-regulation in stress markers needs further discussion. Under chronic stress of heat exposure, exacerbation of HPA axis function could reduce glucocorticoids.
8. Although it cannot be addressed for larvae, it is critical to report the sex ratio for your adults, since hormones affect stress and circuits formation.
-
Reviewer #3 (Public Review):
Satou et al. report a viral toolbox by:
Inventing a novel way through temperature-dependence of HSV1-mediated gene expression for adult and larval zebrafish;
Employing Gal4/UAS system to achieve cell types specific expression in this model;
Combining the modified rabies viruses and HSV1 for transneuronal tracing of neural circuits in zebrafish that is kept in a higher temperature environment.
This toolbox in the manuscript will be of great interest to the neuroscience field when they are using zebrafish as a model.
The strength is these novel methods will offer more experimental opportunities and will facilitate more exciting basic scientific discoveries. However, some concerns still exist as below:
What's the mechanism of temperature-dependence expressions with these HSV1 and rabies virus in this study? At …
Reviewer #3 (Public Review):
Satou et al. report a viral toolbox by:
Inventing a novel way through temperature-dependence of HSV1-mediated gene expression for adult and larval zebrafish;
Employing Gal4/UAS system to achieve cell types specific expression in this model;
Combining the modified rabies viruses and HSV1 for transneuronal tracing of neural circuits in zebrafish that is kept in a higher temperature environment.
This toolbox in the manuscript will be of great interest to the neuroscience field when they are using zebrafish as a model.
The strength is these novel methods will offer more experimental opportunities and will facilitate more exciting basic scientific discoveries. However, some concerns still exist as below:
What's the mechanism of temperature-dependence expressions with these HSV1 and rabies virus in this study? At least the authors should discuss it. Have the authors done experiments like this: after getting enough gene expression from these viruses when maintaining these fishes in 35-37 degree, bring them back to normal temperature as they usually live to see what happen? Does this higher temperature help the fish brain cells get infected with more viral particles or just help increase the expression level? Or does just the higher temperature help produce more proteins?
The authors should address or discuss more whether the higher temperature affects these fishes' brain activity? The reason is if someone will use this method for a most important experiment like GCaMP7s calcium imaging, in order to get good expression with these viruses that authors described in the manuscript they should raise the temperature but they have no idea about whether these higher temperatures affect the behavior or brain activity in some special brain regions they are interested in.
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