1. Response to Reviewer #3 (Public Review):

    We have amended the methods section to state all AAVs were serotype 8; we thank the reviewer for bringing this to our attention.

    Regarding the INTERSECT ChR2 virus, we initially did not use an injection site marker for our INTERSECT ChR2 AAV injections. However, after failing to observe any YFP+ cells, we began including a second virus to confirm that our coordinates were correct. We are aware of the notion of competition between viral vectors (and have included this as a point of discussion within the revised manuscript) and used this as a last resort.

    We also agree that we should have included titers for each virus. We have included a table within the Materials and methods section which lists the titer information for each AAV.

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  2. Response to Reviewer #2 (Public Review):

    We agree with the reviewer; the utility of tTARGIT AAVs in other models is of primary importance. This thinking dictated both the timing of our submission and why we submitted the manuscript to eLife (due to their broad readership and open access policy). We are eager to distribute these tools to the field and let other neuroscientists assess their usefulness.

    Anecdotally, we begun to use tTARGIT AAVs in another neuronal population (using the same Slc17a6FlpO line in conjunction with a distinct Cre driver) and have observed similar levels of transgene expression.

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  3. Reviewer #3 (Public Review):

    The combination of Cre and Flp recombinase dependent system is powerful in manipulating specific intersectional neurons and has been successfully used in many systems. However, the system cannot express target genes sufficiently in some neurons, e.g., the LepRbVMH neurons. This paper solved this problem by developing a novel AAVs system, in which two AAVs were used, the "Driver" AAV permits Flp dependent expression of tTA, and the "Payload" AAV permits TRE-driven and Cre dependent expression of target gene. Because there two AAVs used, it is also expected to increase the capacity to incorporate more transgenes into the AAV system. The novel system to manipulate the intersectional neurons described in this work is an important addition to the current tools. It should be an excellent resource for the neuroscience community.

    This paper is nicely written and compared the previous intersectional approach of AAV-EF1α-Con/Fon-hChR2(H134R)-EYFP with their novel tTARGIT approach in labelling LepRbVMH neurons. The data convincingly demonstrated that the tTARGIT system can label many more cells. Small caveats include the author co-injected AAV-hSYN-Flex(Lox)-hM3Dq-mCherry as an injection site marker with AAV-EF1α-Con/Fon-hChR2(H134R)-EYFP, the serotypes of these AAVs were not reported. It is well known that different serotypes of AAVs infect different types of neurons with a different efficiency. Furthermore, the combination of the different AAV might affect each other's infection, leading to low expression of one type of AAV. The titres of AAVs also make a big difference to many AAVs, which were not reported in this paper. These information are important for other investigators if they would adopt the tTARGIT system in their own research.

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  4. Reviewer #2 (Public Review):

    The research community has been frustrated by difficulties in using AAVs to obtain robust experimental access to neurons co-expressing Cre and Flp recombinase (often called the intersectional approach). In many cases, the approach is sufficiently inefficient as to not be usable. This is in part due to difficulties in designing AAVs that will efficiently express protein-encoded tools in a Cre-ON/Flp-ON fashion, and in part due to the relative inefficiency of Flp recombinase. This present study presents a new intersectional approach for solving this problem. The approach involves co-injecting two AAVs into sites in the brain where Cre/Flp-co-expressing neurons reside - in this case, neurons in the ventromedial nucleus of the hypothalamus (VMH) which co-expresses VGLUT2 (Slc17a6)-Flp and Leptin receptor (Lepr)-Cre. One of the AAVs, in a Flp-dependent fashion, expresses the tTA transcriptional activator, while the other AAV, in a tTA and Cre-dependent fashion, expresses the protein-encoded tool. This new system produced robust expression in neurons co-expressing Flp and Cre in the VMH which previously could not be accomplished using existing intersectional AAVs. The authors also demonstrate a Flp-ON/Cre-OFF version of this approach. Finally, by using these tools the authors show, as was suspected based on prior work, that the Lepr/Vglut2-coexpressing VMH neurons increase brown fat thermogenesis and energy expenditure when stimulated. The results presented very strongly support the effectiveness of this new approach. The only weakness of this study is that, at this point in time, the universality of this approach for all Cre/Flp-co-expressing neurons is unknown. Its effectiveness was only evaluated in VMH neurons. While it is expected that this approach will work for most or all Cre/Flp-co-expressing neurons, there is anecdotal evidence of this or that AAV approach not working in this or that neuron.

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  5. Reviewer #1 (Public Review):

    This paper describes the development of a suite of viral vectors that allow expression (either on or off) of genes of interest depending on both Cre and Flp expression. They demonstrate that their system can solve the problem encountered with the other approach and use it for mapping axonal projections of the glutamatergic, LepR-expressing neurons and the consequences of chronic activation of these neurons on food intake and energy expenditure. The results are significant and clearly presented. The failure of the other system (INTRSECT) for their application is not clearly understood, but authors say that it may be due to low expression of Cre or Flp in these neurons; however, Supp Fig. 1 shows that it Lepr-Cre and Slc17a6-FLPo were sufficient to activate a transgenic reporter (Supp Fig. 1). The authors reveal that they probably could have used Nr5a1-Cre mice manipulate the activity of these VMH neurons. Nevertheless, it is worthwhile having multiple methods to attack a specific problem because of unforeseen complications with particular methods.

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  6. Evaluation Summary:

    The combination of Cre and Flp recombinase dependent system is powerful in manipulating specific intersectional neurons and has been successfully used in many systems. However, the system cannot express target genes sufficiently in some neurons, e.g., the LepRb VMH neurons. This paper solved this problem. It is therefore an important technical advance.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

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