retro-Tango enables versatile retrograde circuit tracing in Drosophila

  1. Altar Sorkaç
  2. Rareș A Moșneanu
  3. Anthony M Crown
  4. Doruk Savaş
  5. Angel M Okoro
  6. Ezgi Memiş
  7. Mustafa Talay
  8. Gilad Barnea

  • Curated by eLife

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    Sorkac et al. present a novel genetically encoded retrograde synaptic tracing method that has the potential for unbiased identification of presynaptically connected neurons. Retro-Tango is based on the previously developed anterograde method trans-Tango, promising high applicability and rendering the significance of this contribution important. The strength of the evidence is convincing.

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Abstract

Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed in Drosophila melanogaster , there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here, we describe retro -Tango, a method for transsynaptic, retrograde circuit tracing and manipulation in Drosophila . In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validate retro -Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of the Drosophila hemibrain. Our experiments establish retro -Tango as a key method for circuit tracing in neuroscience research.

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  1. Version published to 10.7554/elife.85041 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Sorkac et al. devised a genetically encoded retrograde synaptic tracing method they call retro-Tango based on their previously developed anterograde synaptic tracing method trans-Tango. The development of genetically encoded trans-synaptic tracers has long been a difficult stumbling block in the field, and the development of trans-Tango a few years back was a breakthrough that was immediately, widely, and successfully applied. The recent development of the retrograde tracer method BActrace was also exciting for the field, but requires lexA driver lines and required by its design the test of candidate presynaptic neurons instead of an unbiased test for connectivity.

    Retro-Tango now provides an unbiased retrograde tracer. They cleverly used the same reporter system as for trans-Tango by reversing the signaling modules to be placed in pre-synaptic neurons instead of post-synaptic neurons. Therefore, synaptic tracing leads to the labeling of pre-synaptic neurons under the regulation of the QUAS system. Using visual, olfactory as well sexually dimorphic circuits authors went about providing examples of specificity, efficiency, and usefulness of the retro-Tango method. The authors successfully demonstrated that many of the known pre-synaptic neurons can be successfully and specifically labelled using the retro-Tango method.

    Most importantly, because it is based on the most used, very well tested and widely adopted trans-Tango method, retro-Tango promises to not just be a clever development, but a really widely and well-used technique as well. This is an outstanding contribution.

    We would like to thank Dr. Hiesinger for his very kind words and for the overall appreciation of the contribution of the development of retro-Tango to the field. We are also grateful for the suggestions below aimed at improving the clarity of our manuscript. We individually address the points raised by Dr. Hiesinger below.

    Reviewer #2 (Public Review):

    Tools that enable labeling and genetic manipulations of synaptic partners are important to reveal the structure and function of neural circuits. In a previous study, Barnea and colleagues developed an anterograde tracing method in Drosophila, trans-TANGO, which targets a synthetic ligand to presynaptic terminals to activate a postsynaptic receptor and trigger nuclear translocation of a transcription factor. This allows the labeling and genetic manipulation of cells postsynaptic to the ligand-expressing starter cells. Here, the same group modified trans-TANGO by targeting the ligand to the dendrites of starter cells to genetically access pre-synaptic partners of the starter cells; they call this method retro-TANGO. The authors applied retro-TANGO to various neural circuits, including those involved in escape response, navigation, and sensory circuits for sex peptides and odorants. They also compared their retro-TANGO data with synaptic connectivity derived from connectivity obtained from serial electron microscopy (EM) reconstruction and concluded that retro-TANGO can allow trans-synaptic labeling of presynaptic neurons that make ~ 17 synapses or more with the starter cells.

    Overall, this study has generated and characterized a valuable retrograde transsynaptic tracing tool in Drosophila. It's simpler to use than the recently described BAcTrace (Cachero et al., 2020) and can also be adapted to other species. However, the manuscript can be substantially strengthened by providing more quantitative data and more evidence supporting retrograde specificity.

    We thank Dr. Luo for his kind words and his assessment of the value of retro-Tango as a new tool in the transsynaptic labeling toolkit in Drosophila. We followed the suggestions of Dr. Luo for providing more quantitative data and addressing the specificity and directionality of retro-Tango. We strongly believe that the implementation of his suggestions did enhance the quality of our manuscript.

    Reviewer #3 (Public Review):

    This is a valuable addition to the currently available arsenal of methods to study the Drosophila brain.

    There are many positives to the present manuscript as it is:

    (i) The introduction makes a clear and fair comparison with other available tracing methods.

    (ii) The authors do a systematic analysis of the factors that influence the labeling by retro-tango (age, temperature, male versus female, etc...)

    (iii) The authors acknowledge that there are some limitations to retro-TANGo. For example, the fact that retro-T does not label all the expected neurons as indicated by the EM connectome. This is fine because no technique is perfect, and it is very laudable that the authors did a serious study of what one should expect from retro-tango (for example, a threshold determined by the number of synapses between the connected neurons).

    We would like to thank the reviewer for the kind words and the positive assessment of our manuscript. In addition, we would like to acknowledge the reviewer for the recommendations below, which we followed and we think made our manuscript stronger.

  3. eLife

    eLife assessment

    Sorkac et al. present a novel genetically encoded retrograde synaptic tracing method that has the potential for unbiased identification of presynaptically connected neurons. Retro-Tango is based on the previously developed anterograde method trans-Tango, promising high applicability and rendering the significance of this contribution important. The strength of the evidence is convincing.

  4. eLife

    Reviewer #1 (Public Review):

    Sorkac et al. devised a genetically encoded retrograde synaptic tracing method they call retro-Tango based on their previously developed anterograde synaptic tracing method trans-Tango. The development of genetically encoded trans-synaptic tracers has long been a difficult stumbling block in the field, and the development of trans-Tango a few years back was a breakthrough that was immediately, widely, and successfully applied. The recent development of the retrograde tracer method BActrace was also exciting for the field, but requires lexA driver lines and required by its design the test of candidate presynaptic neurons instead of an unbiased test for connectivity.

    Retro-Tango now provides an unbiased retrograde tracer. They cleverly used the same reporter system as for trans-Tango by reversing the signaling modules to be placed in pre-synaptic neurons instead of post-synaptic neurons. Therefore, synaptic tracing leads to the labeling of pre-synaptic neurons under the regulation of the QUAS system. Using visual, olfactory as well sexually dimorphic circuits authors went about providing examples of specificity, efficiency, and usefulness of the retro-Tango method. The authors successfully demonstrated that many of the known pre-synaptic neurons can be successfully and specifically labelled using the retro-Tango method.

    Most importantly, because it is based on the most used, very well tested and widely adopted trans-Tango method, retro-Tango promises to not just be a clever development, but a really widely and well-used technique as well. This is an outstanding contribution.

  5. eLife

    Reviewer #2 (Public Review):

    Tools that enable labeling and genetic manipulations of synaptic partners are important to reveal the structure and function of neural circuits. In a previous study, Barnea and colleagues developed an anterograde tracing method in Drosophila, trans-TANGO, which targets a synthetic ligand to presynaptic terminals to activate a postsynaptic receptor and trigger nuclear translocation of a transcription factor. This allows the labeling and genetic manipulation of cells postsynaptic to the ligand-expressing starter cells. Here, the same group modified trans-TANGO by targeting the ligand to the dendrites of starter cells to genetically access pre-synaptic partners of the starter cells; they call this method retro-TANGO. The authors applied retro-TANGO to various neural circuits, including those involved in escape response, navigation, and sensory circuits for sex peptides and odorants. They also compared their retro-TANGO data with synaptic connectivity derived from connectivity obtained from serial electron microscopy (EM) reconstruction and concluded that retro-TANGO can allow trans-synaptic labeling of presynaptic neurons that make ~ 17 synapses or more with the starter cells.

    Overall, this study has generated and characterized a valuable retrograde transsynaptic tracing tool in Drosophila. It's simpler to use than the recently described BAcTrace (Cachero et al., 2020) and can also be adapted to other species. However, the manuscript can be substantially strengthened by providing more quantitative data and more evidence supporting retrograde specificity.

  6. eLife

    Reviewer #3 (Public Review):

    This is a valuable addition to the currently available arsenal of methods to study the Drosophila brain.

    There are many positives to the present manuscript as it is:
    (i) The introduction makes a clear and fair comparison with other available tracing methods.
    (ii) The authors do a systematic analysis of the factors that influence the labeling by retro-tango (age, temperature, male versus female, etc...)
    (iii) The authors acknowledge that there are some limitations to retro-TANGo. For example, the fact that retro-T does not label all the expected neurons as indicated by the EM connectome. This is fine because no technique is perfect, and it is very laudable that the authors did a serious study of what one should expect from retro-tango (for example, a threshold determined by the number of synapses between the connected neurons).

  7. Version published to 10.1101/2022.11.24.517859v1 on bioRxiv