Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases

  1. Hanako Hagio
  2. Wataru Koyama
  3. Shiori Hosaka
  4. Aysenur Deniz Song
  5. Janchiv Narantsatsral
  6. Koji Matsuda
  7. Takashi Shimizu
  8. Shoko Hososhima
  9. Satoshi P Tsunoda
  10. Hideki Kandori
  11. Masahiko Hibi

  • Curated by eLife

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    This manuscript provides a valuable resource for scientists who wish to manipulate second messengers in zebrafish using optogenetics. The authors provide solid evidence, based on behaviour, monitoring of heart beat and imaging, that several of the opsins tested can have an effect in larval fish. Opsins that lack an effect are also described. As the second messengers affected by the tools are found in multiple cell types, the results should be of interest of scientists working in a variety of areas.

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Abstract

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited Gt CCR4 and Kn ChR, cation channelrhodopsins from algae, Be GC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria ( Oa PAC) or bacteria ( b PAC), to control cell functions in zebrafish. Optical activation of Gt CCR4 and Kn ChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of Be GC1 or PACs achieved it at long latencies. Activation of Gt CCR4 and Kn ChR in cardiomyocytes induced cardiac arrest, whereas activation of b PAC gradually induced bradycardia. Kn ChR activation led to an increase in intracellular Ca 2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

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  1. Version published to 10.7554/elife.83975 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This paper aims to test whether a series of light activated ion channels (GtCCRT4, KnChR) and enzymes that regulate second messengers (BeGC1, bPac, OaPac) can be used to manipulate cells in the zebrafish.

    Among the strengths of the paper are the use of several independent methods to test whether the tools are functional - e.g. electrophysiology of mammalian cells for GtCCR4, calcium and cAMP imaging in zebrafish cells in vivo, behaviour tests (tail movement) and monitoring of heart beat. Multiple transgenic lines were established, to select for lines with optimal expression levels. Experiments are carried out in two cell types - reticulospinal neurons in the hindbrain and cardiomyocytes.

    The authors have largely achieved their aim of determining whether the rhodopsins can be used in zebrafish. They demonstrate that the cation channel KnChR is particularly sensitive in triggering depolarization of the reticulospinal neurons, as indicated by tail movement. They show that the photoactivatable adenylyl cyclase bPAC and cation channels have an effect on heartbeat. Two other photoactivatable enzymes OaPAC and BeGC1 have no effect on heartbeat, although it is not evident whether this is due to lack of effect on cAMP and cGMP levels.

    The abstract sets out to investigate the role of second messengers, emphasizing the need for specificity. However, KnChR is not specific for Na+. As noted by Tashiro et al, the channel can also conduct H+, Ca2+ and Mg2+. The knowledge gap that is being addressed by the manuscript thus needs to be reframed. The concluding statement of the abstract, that the tools tested here can be used to investigate second messengers, is not accurate given the broad conductance of KnChR.

    We agree with the reviewer. We changed the title to “Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases” and revised the abstract and introduction, accordingly. The last sentence of the abstract was modified to “These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.”

    The tools described here have been tested previously in other species, either in cultured mammalian cells (GtCCR4, KnChR, OaPAC) or in vivo (bPAC and BeGC1). The current work thus does not introduce novel tools, but provides evidence that some of these tools can be used in zebrafish. Overall, the lines characterized here will be of use to scientists using zebrafish as the experimental system in a variety of areas.

    We appreciated the positive comments from the reviewer. It was worthwhile generating and analyzing so many transgenic zebrafish.

    Reviewer #2 (Public Review):

    Optogenetic proteins are important tools for circuit neuroscience. The authors characterize five proteins, GtCCR4, KnCHR2, BeGC1, bPAC, and OaPAC with respect to their ability to suppress normal cell excitability and compare the results to those for the more established GtACR1 and CrChR2[T159]. The study makes use of expression in the zebrafish heart and hindbrain, as well as in a cell line. Electrophysiology in the cell line demonstrates that GtCCR photo-activation induces similar currents as CrChR2 activation and shows less signs of desensitization. Using a transgenic vsx2:Gal4 zebrafish line, immunohistochemistry shows that the tools are expressed. When activated, they triggered the expected behavioral responses (swimming) at short latency (<4s). This was true even for the three tools that are guanylyl or adenylyl cyclases (BeGC1, bPAC, OaPAC) and thus affect cell excitability only indirectly. At the tested light intensity, the Klebsormidium nitens channelrhodopsin (KnChR) had the shortest latency (<0.5 s) and highest (100%) probabilities of inducing locomotion. When expressing the tools in the zebrafish heart, brief illumination (100 ms) induces brief (100 ms - 1500 ms) suppression of the heartbeat. Notably, also tools that evoke depolarization induce heartbeat suppression. Heartbeat movies and calcium imaging demonstrate that this is caused by prolonged cardiomyocyte contraction. The optogenetic guanylyl and adenylyl cyclases were not effective in perturbing zebrafish heartbeat (except for bPAC over longer time scales).

    Given the large number of optogenetic proteins available to date and the challenge of employing them in well-controlled neuroscience experiments, this study presents an important contribution for neuroscientists performing optogenetic research in animal models. Two light-gated cation channels, GtCCR4 and KnChR, are tested for the first time in vivo. The evidence supporting the claims regarding heartbeat and induced swimming behavior is solid. Since GtCCR4 is more Na+-selective than other channelrhodopsins, it should allow better control of experimental variables and is a valuable addition to the optogenetic tool box. The created transgenic zebrafish lines will be useful for the zebrafish neuroscience community.

    The expression in zebrafish was compared using immunohistochemical staining (of a single Gal4 driver line). From this experiment alone, it is difficult to judge the expression level, the in vivo visibility of the fluorescence under the microscope, and the proportion of target cells that do express the optogenetic gene of interest.

    The evidence for optogenetically induced alteration of swimming behavior is compelling. However, the associated neuronal responses and their dependence on different light intensity levels remain uncharacterized. Therefore, if anyone plans to use these tools to investigate a neural circuit in the future, the needed light levels and the specificity of the manipulation would still need to be determined.

    We stimulated neuronal ND7/23 cells, reticulospinal V2a neurons or cardiomyocytes expressing microbial optogenetic tools at various light intensities and examined their effects on neuronal activities and behaviors (tail movements and cardiac arrest). These data are shown in revised Figure 1, Figure 1-supplement 1, Figure 3, Figure 3-supplements 2, 3, Figure 5, and Figure 5-supplements 1, 2. We described the data on page 12, line-page 13, line 1 and page 14, lines 10-13 in the revised manuscript.

    For the optogenetic guanylyl and adenylyl cyclases, which clearly were able to alter behavioral responses, the signaling and circuit mechanisms that lead to neuronal depolarization remain unknown, but possible activation pathways are discussed.

    Reviewer #3 (Public Review):

    In this study, the authors set out to test several new optogenetic tools in zebrafish. They motivate the study by citing differences in ion selectivity of channelrhodopsins and the potential utility of photoactivatable anenylyl and guanylyl cyclases to control cell functions. Although the study provides some useful new information about the utility of these tools in zebrafish, the characterization is limited and there are serious caveats around interpretation of behavioral responses.

    The latency of behavioral responses is often extremely long and there is a lack of control data from opsin negative animals, raising serious doubts as to whether these responses are optogenetically mediated.

    In other words, many of these responses may not result from optogenetic activation of V2a cells, but instead arise from indirect effects such as visual stimulation of the animal. Previous zebrafish studies have shown swimming responses in opsin-negative control animals at latencies above ~100 ms and used a 50 ms cut-off for optogenetically evoked swims. One can see evidence suggestive of this issue in the authors' data: latency data for GtCCR4 appears bimodal with a cluster of short latency swims and a second spread at latencies >2s; this could be a mix of fast optogenetic and slow artifactual responses. As the authors have already tested opsin negative control animals, they should examine the latency distribution of these responses. The long latency is even more striking in the case of BeGC1, pPAC and OaPAC where in all cases mean latency exceeds 2 seconds. No short latency responses are apparent and the delay is too long to be solely a result of second messenger action (e.g. activation of cyclic nucleotide gated ion channels). In any case, no explanation is provided.

    We understand the reviewer’s concern that the responses were too slow. However, the neurons responded after accumulation of cAMP or cGMP, which bind and activate CNG in the neurons. Similar delayed responses were observed when G protein-coupled bistable rhodpsins were activated in reticulospinal V2a neurons (please see the accompanying manuscript).

    We compared the latency of zebrafish larvae expressing each tool with those not expressing the tool. The data are shown in Figure 3, Figure 3-supplement 1, Figure 5, Figure 6, Figure 7, and Figure 7-supplement 1. Statistically, we considered responses within 8 s after the start of light stimulation as positive, and significant differences in responses were observed depending on the presence or absence of tool expression, suggesting that tail movements were induced by tool activation. In the absence of tool expression, spontaneous movements were occasionally observed, but they did not often occur within 8 s. We have described the data on page 15, line 20-page 16, line 4 in the revised manuscript.

    Although this study is motivated by the need to precisely control the flux of specific ions and modulate specific second messenger pathways, there is almost no characterisation of these processes in zebrafish cells. As such, the degree to which these tools are useful to "precisely control second messengers in vivo" is unclear and the lack of mechanistic data also leaves open questions about unexpected aspects of behavioral results (e.g. the long latency of presumed cyclic-nucleotide induced behavior, above).

    We believe that the description "controlling second messengers" was misleading. Since Reviewer #3 has taken issue with this aspect, we note that this paper does not provide a detailed analysis of second(ary) messengers. We have restructured the entire manuscript to focus on optogenetic regulation of zebrafish neurons and cardiomyocytes rather than on "control messenger regulation".

    Finally, there is little comparison with other commonly used optogenetic actuators. CrChR2[T159C] is used as the only control but more recent tools (e.g. CoChR, Chrmine, ChroME) are not considered. Thus, beyond showing that the new tools have behavioral effects in zebrafish, the usefulness of this report for researchers wanting to compare and select between tools is limited.

    We examined the activity of CoChR and ChrimsonR in neuronal ND7/23 cells. In addition, we generated transgenic zebrafish expressing CoChR or ChrimsonR, and examined their activity in V2a neurons and cardiomyocytes. We thereby compared the activity of GtACR4, KnChR, and CrChR2[T159C] with that of CoChR and ChrimsonR. The data are shown in Figure 1, Figure 1-supplement 1, Figure 2, Figure 3, Figure 3-supplement 3, and Figure 5-supplements 1, 2. We described the data for CoChR and ChrimsonR in the relevant part of the Result section (pages 8-14) and discussed a comparison on page 18, lines 2-16 in the revised manuscript.

    We found that KnChR was a more potent optogenetic tool than GtCCR4, CrChR2, and ChrimsonR in zebrafish reticulospinal V2a neurons. Optogenetic activity of KnChR was comparable to that of CoChR in both reticulospinal V2a neurons and cardiomyocytes (Figures 1, 3, 5). Truncation of KnChR prolonged the channel open lifetime by more than 10-fold (Tashiro et al. , 2021) (Figure 1). KnChR conducts various monovalent and bivalent cations, including H+, Na+, and Ca2+, while KnChR has a higher permeability to Na+ and Ca2+ and a higher permeability ratio of Ca2+ to Na+ than CrChR2 (Tashiro et al. , 2021). These properties may contribute to the high photo-inducible activity of KnChR. Activation of KnChR may induce influx of more cations with a longer channel open time than CrChR2 and ChrimsonR, leading to stronger cell depolarization. Optogenetic activity of KnChR was comparable to that of GtCCR4 in cultured cells, but higher than GtCCR4 in zebrafish reticulospinal V2a neurons and cardiomyocytes. While the exact reason is unclear, it is possible that the expression of functional KnChR protein may be high in zebrafish cells.

  3. eLife

    eLife assessment

    This manuscript provides a valuable resource for scientists who wish to manipulate second messengers in zebrafish using optogenetics. The authors provide solid evidence, based on behaviour, monitoring of heart beat and imaging, that several of the opsins tested can have an effect in larval fish. Opsins that lack an effect are also described. As the second messengers affected by the tools are found in multiple cell types, the results should be of interest of scientists working in a variety of areas.

  4. eLife

    Reviewer #1 (Public Review):

    This paper aims to test whether a series of light activated ion channels (GtCCRT4, KnChR) and enzymes that regulate second messengers (BeGC1, bPac, OaPac) can be used to manipulate cells in the zebrafish.

    Among the strengths of the paper are the use of several independent methods to test whether the tools are functional - e.g. electrophysiology of mammalian cells for GtCCR4, calcium and cAMP imaging in zebrafish cells in vivo, behaviour tests (tail movement) and monitoring of heart beat. Multiple transgenic lines were established, to select for lines with optimal expression levels. Experiments are carried out in two cell types - reticulospinal neurons in the hindbrain and cardiomyocytes.

    The authors have largely achieved their aim of determining whether the rhodopsins can be used in zebrafish. They demonstrate that the cation channel KnChR is particularly sensitive in triggering depolarization of the reticulospinal neurons, as indicated by tail movement. They show that the photoactivatable adenylyl cyclase bPAC and cation channels have an effect on heartbeat. Two other photoactivatable enzymes OaPAC and BeGC1 have no effect on heartbeat, although it is not evident whether this is due to lack of effect on cAMP and cGMP levels.

    The abstract sets out to investigate the role of second messengers, emphasizing the need for specificity. However, KnChR is not specific for Na+. As noted by Tashiro et al, the channel can also conduct H+, Ca2+ and Mg2+. The knowledge gap that is being addressed by the manuscript thus needs to be reframed. The concluding statement of the abstract, that the tools tested here can be used to investigate second messengers, is not accurate given the broad conductance of KnChr.

    The tools described here have been tested previously in other species, either in cultured mammalian cells (GtCCR4, KnChR, OaPAC) or in vivo (bPAC and BeGC1). The current work thus does not introduce novel tools, but provides evidence that some of these tools can be used in zebrafish. Overall, the lines characterized here will be of use to scientists using zebrafish as the experimental system in a variety of areas.

  5. eLife

    Reviewer #2 (Public Review):

    Optogenetic proteins are important tools for circuit neuroscience. The authors characterize five proteins, GtCCR4, KnCHR2, BeGC1, bPAC, and OaPAC with respect to their ability to suppress normal cell excitability and compare the results to those for the more established GtACR1 and CrChR2[T159]. The study makes use of expression in the zebrafish heart and hindbrain, as well as in a cell line. Electrophysiology in the cell line demonstrates that GtCCR photo-activation induces similar currents as CrChR2 activation and shows less signs of desensitization. Using a transgenic vsx2:Gal4 zebrafish line, immunohistochemistry shows that the tools are expressed. When activated, they triggered the expected behavioral responses (swimming) at short latency (<4s). This was true even for the three tools that are guanylyl or adenylyl cyclases (BeGC1, bPAC, OaPAC) and thus affect cell excitability only indirectly. At the tested light intensity, the Klebsormidium nitens channelrhodopsin (KnChR) had the shortest latency (<0.5 s) and highest (100%) probabilities of inducing locomotion. When expressing the tools in the zebrafish heart, brief illumination (100 ms) induces brief (100 ms - 1500 ms) suppression of the heartbeat. Notably, also tools that evoke depolarization induce heartbeat suppression. Heartbeat movies and calcium imaging demonstrate that this is caused by prolonged cardiomyocyte contraction. The optogenetic guanylyl and adenylyl cyclases were not effective in perturbing zebrafish heartbeat (except for bPAC over longer time scales).

    Given the large number of optogenetic proteins available to date and the challenge of employing them in well-controlled neuroscience experiments, this study presents an important contribution for neuroscientists performing optogenetic research in animal models. Two light-gated cation channels, GtCCR4 and KnChR, are tested for the first time in vivo. The evidence supporting the claims regarding heartbeat and induced swimming behavior is solid. Since GtCCR4 is more Na+-selective than other channelrhodopsins, it should allow better control of experimental variables and is a valuable addition to the optogenetic tool box. The created transgenic zebrafish lines will be useful for the zebrafish neuroscience community.

    The expression in zebrafish was compared using immunohistochemical staining (of a single Gal4 driver line). From this experiment alone, it is difficult to judge the expression level, the in vivo visibility of the fluorescence under the microscope, and the proportion of target cells that do express the optogenetic gene of interest.

    The evidence for optogenetically induced alteration of swimming behavior is compelling. However, the associated neuronal responses and their dependence on different light intensity levels remain uncharacterized. Therefore, if anyone plans to use these tools to investigate a neural circuit in the future, the needed light levels and the specificity of the manipulation would still need to be determined.

    For the optogenetic guanylyl and adenylyl cyclases, which clearly were able to alter behavioral responses, the signaling and circuit mechanisms that lead to neuronal depolarization remain unknown, but possible activation pathways are discussed.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, the authors set out to test several new optogenetic tools in zebrafish. They motivate the study by citing differences in ion selectivity of channelrhodopsins and the potential utility of photoactivatable anenylyl and guanylyl cyclases to control cell functions. Although the study provides some useful new information about the utility of these tools in zebrafish, the characterization is limited and there are serious caveats around interpretation of behavioral responses.

    The latency of behavioral responses is often extremely long and there is a lack of control data from opsin negative animals, raising serious doubts as to whether these responses are optogenetically mediated.
    In other words, many of these responses may not result from optogenetic activation of V2a cells, but instead arise from indirect effects such as visual stimulation of the animal. Previous zebrafish studies have shown swimming responses in opsin-negative control animals at latencies above ~100 ms and used a 50 ms cut-off for optogenetically evoked swims. One can see evidence suggestive of this issue in the authors' data: latency data for GtCCR4 appears bimodal with a cluster of short latency swims and a second spread at latencies >2s; this could be a mix of fast optogenetic and slow artifactual responses. As the authors have already tested opsin negative control animals, they should examine the latency distribution of these responses. The long latency is even more striking in the case of BeGC1, pPAC and OaPAC where in all cases mean latency exceeds 2 seconds. No short latency responses are apparent and the delay is too long to be solely a result of second messenger action (e.g. activation of cyclic nucleotide gated ion channels). In any case, no explanation is provided.

    Although this study is motivated by the need to precisely control the flux of specific ions and modulate specific second messenger pathways, there is almost no characterisation of these processes in zebrafish cells. As such, the degree to which these tools are useful to "precisely control second messengers in vivo" is unclear and the lack of mechanistic data also leaves open questions about unexpected aspects of behavioral results (e.g. the long latency of presumed cyclic-nucleotide induced behavior, above).

    Finally, there is little comparison with other commonly used optogenetic actuators. CrChR2[T159C] is used as the only control but more recent tools (e.g. CoChR, Chrmine, ChroME) are not considered. Thus, beyond showing that the new tools have behavioral effects in zebrafish, the usefulness of this report for researchers wanting to compare and select between tools is limited.

  7. Version published to 10.1101/2022.10.25.513731v1 on bioRxiv