RubyACRs Enable Red-Shifted Optogenetic Inhibition in Freely Behaving Drosophila

Reviewer #1 (Public review):

Summary:

This study by Bushey et al., focuses on two newly released red-shifted anion-Channelrhodopsins (A1ACR and HfACR, referred as Ruby-ACRs) in Drosophila. Here, the authors use a combination of electrophysiology, calcium imaging, and behavioral analyses to demonstrate the advantages of Ruby-ACRs over previous optogenetic silencers like the green-shifted GtACR1 and the blue-shifted GtACR2: higher photocurrent, faster kinetics, and operating at a light spectrum range that prevents unwanted behavioral effects in the fly. The availability of these new red-shifted silencers constitutes a great addition to the Drosophila genetic toolkit.

Strengths:

(1) The authors generate both UAS and LexAop RubyACR reagents and test them in a variety of preparations (electrophysiological recordings, calcium imaging, different behavioral paradigms) that cover the breadth of the fly research environment.

(2) The optical stimulation parameters are carefully measured and characterized. Especially impressive is that they managed to titrate over both wavelength and intensity across their various assays. This provides a comprehensive dataset to the community.

(3) Tools are made available to the community through the stock center.

Weaknesses:

(1) The authors could better describe their construct and choice of parameters for the chosen construct. I am specifically wondering about the following points:

a) Why use that particular backbone (not the most commonly used one across recent literature (pJFRC7 is more common).

b) Why do the CsChrimson and GTACR1 have a Kir sequence in it, and why did the authors not put this in the RubyACRs? I would also prefer if authors don't refer to GtACR1 as GTACR-Kir in text (e.g., in line 72); instead, they should either refer to it as GtACR1 or GtACR1-kir-mVenus (based on the full genotype mentioned in their table at the end). Same for CsChrimson-kir. From what I understand, this is just a Kir trafficking sequence and not the entire Kir sequence, which can confuse the readers.

c) Finally, I would also encourage authors to deposit plasmids on Addgene.

(2) Figure 2 is interesting, but it is a bit unfortunate that there is a YFP baseline in most of the samples here (except Chrimson88; this should also be mentioned). I wonder how the YFP baseline impacts this data. Could the high intensity stimulation (red light) lead to bleaching of YFP or tdTomato that reduces the baseline in the green channel? All this also makes me wonder if authors tried tagging the RubyACRs with other fluorophores or non-fluorescent tags and how that impacted their functioning. Non-YFP-tagged versions would be more useful for applications involving GCaMP imaging.

(3) Another point for Figure 2: Since RubyACRs seem to have such a broad activation range, I wonder how much the imaging light (920nm) impacts the baseline in these experiments. If there were plots without the red light stimulation and just varying imaging light intensity, that could be useful to the research community.

(4) Also, for Figures 2C - D, in the methods authors indicate that the stimulation light intensities were progressively increased. Could this lead to desensitization of opsin? Wouldn't randomized intensities be a better way to do this? Perhaps it should be mentioned as a caveat.

(5) In Figure 3E the bottom middle panel Vglut-Gal4,GtACR1 shows a major increase in walking at light onset. This seems very different than all other conditions, and I could not find any discussion of this. It would help if some explanation were provided for this.

Reviewer #2 (Public review):

Summary:

Bushey et al. investigate the feasibility of using RubyACRs, specifically A1ACR1 and HfACR1 (described previously in (Govorunova et al., 2020)) as red-shifted inhibitory opsins in Drosophila melanogaster. The study employs a wide range of techniques to demonstrate successful neuronal inhibition. Electrophysiology experiments established that HfACR1 was most effective at hyperpolarizing cells, compared to A1ACR1 and GtACR1; both RubyACRs also appeared to be more effective than GtACR1 when the latter was actuated by green light. The authors further demonstrate successful neuronal inhibition using calcium imaging. RubyACRs were also shown to be useful in in vivo behavioral setups, specifically in spontaneous locomotion, associative learning, and courtship paradigms. In the courtship assay, in particular, the authors test multiple wavelengths of light at various light intensities, thus providing a rigorous analysis of the RubyACRs' efficacy under different light conditions.

Strengths:

The work provides the Drosophila field with a promising new tool. Red-shifted opsins are particularly advantageous in behavioral assays as red light penetrates the cuticle better than green or blue light, and provides less visual stimulation to the fly. It is also ideal for imaging as it allows for simultaneous optogenetic stimulation and GCamp imaging. A particular strength of the paper is the direct demonstration of RubyACR's capacity to inhibit neurons via electrophysiology and calcium imaging. Furthermore, inhibition effects in the three behavioral assays are strong and convincing. Given the apparent efficacy of RubyACRs and the advantages of a red-sensitive anion channelrhodopsin, this tool has great potential.

Weaknesses:

This work convincingly demonstrates the efficacy and potential utility of RubyACRs in Drosophila for imaging and behavior. However, the lethality/toxicity of RubyACRs is a relevant concern that should be addressed in-depth rather than glossed over, as it may pose a major obstacle to use. Discussing this issue in the present study will also help guide potential users and will set the stage for potential future efforts to ameliorate RubyACRs as optogenetic inhibitors.

Major concerns:

(1) Table 1 demonstrates high lethality in the RubyACRs compared to GtACR1. For example, in the MI04979-VGlut driver, GtACR1 expression resulted in 32.9% lethality, while HfACR1 expression resulted in 98.7% lethality. This lethality presents an obstacle to the potential adoption of this tool, and should be discussed in detail, rather than in passing. The authors might like to present "% lethality" rather than "% survived", as the former is more relevant when discussing the relative yield and health of flies that can be used in experiments.

(2) In Figure 3D, driver>opsin flies have lower locomotion during the baseline (i.e., dark) phase, compared to opsin-only controls or GtACR1 flies. For some comparisons, flies are walking around 10-fold slower. For example, in the case of VGlut-GAL4>HfACR1, test flies are walking at <1 mm/s, while "Empty" test flies are walking at ~10 mm/s. This suggests that, for these drivers, neuronal and/or network function is affected. It opens the possibility that the lethality and locomotor defects could be due to cell-autonomous toxicity. We ask the authors to provide a description of this effect in the Results and to discuss it in the Discussion. Relatedly, VGlut-GAL4>GtACR1 flies in red light exhibit a locomotion increase, but this data is not mentioned in the text. The use of differing scales for the Y-axes in these panels can be confusing when the reader is expected to compare velocity across different panels. It would be best if the y-axes were set to a single range, e.g., 0 to 12 mm/s.

(3) Lethality in broad drivers could result from cell-autonomous toxicity or neuronal dysfunction resulting from RubyACR expression. Ideally, the authors would address or even investigate the possible mechanisms of toxicity of the RubyACRs. Do cells and/or synapses expressing RubyACRs have normal morphology and function? For example, the authors could compare cell survival between flies with RubyACR expression and flies with a fluorescent protein with no opsin. The authors may also want to present lethality data for other, less broad drivers (such as MB320C, which was used for the associative memory assay) in order to demonstrate whether this problem is confined to broad drivers such as VGlut-GAL4, or if this is a problem with narrow drivers as well. If new experiments are not possible, these issues should at least be mentioned in the Discussion.

Minor concerns

(1) The specific method used for quantifying lethality is mentioned briefly in Table 1 but is not detailed in the Methods. The authors derive lethality by comparing to a sibling control group with either the opsin or the driver alone, but the opsin alone or driver alone may cause some lethality by themselves. We suggest the use of a viability assay, e.g. (Rockwell et al., 2019), which would give potential users a clearer picture of which developmental stage is most affected by opsin expression, as well as allow opsin-only, driver-only and experimental groups to be assessed separately (lethality would then be reported as the % of embryos that reach each stage of development, and eventually enclosure).

(2) For the calcium imaging analysis in Figure 2, the U-shaped curve observed for mean ΔF/F0 for A1ACR1 and HfACR1 may not be due to actual desensitization for the channels, as the authors suggest (lines 143-145), but may be due simply to a shifting baseline. The authors use the 5-s period preceding stimulation onset as F0, but in some cases (e.g., HfACR1 at 250 uW/mm2), calcium fluorescence rises above baseline and remains high post-stimulation (ΔF/F0 of +0.5, which we observe is the same magnitude as the ΔF/F0 of -0.5 observed during inhibition), thus affecting the ΔF/F0 for subsequent trials. The authors should discuss this incomplete recovery in the text, or (if available) use a static channel instead to provide a stable F0 for calculating ΔF/F0. Alternatively, if the authors wish to rigorously test the hypothesis that high light intensity indeed results in desensitization of these channels, they may consider using different flies for each light intensity or longer inter-stimulus intervals.

(3) For Figure 3C (Flybowl assay), the authors mention that "simply expressing the opsins decreased baseline locomotor activity compared to empty driver lines". However, the "Empty" controls in 3C appear to refer to opsin-only controls, not driver-only controls. The driver-only controls are not presented in the figure. The use of "empty" differs between the text and the figure, as the text refers to "empty" driver lines, while the figure uses "empty" to apparently refer to opsin-only controls. We recommend changing the terminology across all figures to be unambiguous, e.g., by using "opsin-only" or "driver-only" as opposed to the ambiguous "empty". In addition, the fact that opsin-only controls move less than driver-only controls may suggest some toxicity as a result of the opsin-only construct; this should be discussed further.

(4) Figures 4 and 5 lack the reporting of driver-only controls.

(5) Figures 3 and 4 lack positive controls; that is, the benchmarking of the efficacy of RubyACRs in their respective behavioral paradigms against a known inhibitor, e.g., GtACR1 with green light. To confirm that this GtACR1 transgene is functional, the authors could include GtACR1 with green light as a positive control for these two figures, as they have done for Figure 5-supplement 2 and 3.

(6) Several citations are missing. In their discussion, the authors highlight that shorter wavelengths of light are more attenuated by tissue (lines 278-281); this should be accompanied by the relevant citations (Inagaki et al., 2014). Similarly, the claim that behavioral experiments exhibit greater sensitivity to shorter wavelengths should be substantiated (lines 281-283).

References:

Govorunova EG, Sineshchekov OA, Li H, Wang Y, Brown LS, Spudich JL. 2020. RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption. Proc Natl Acad Sci U S A 117:22833-22840.

Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, Anderson DJ. 2014. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325-332.

Rockwell AL, Beaver I, Hongay CF. 2019. A direct and simple method to assess Drosophila melanogaster's viability from embryo to adult. J Vis Exp e59996.

Reviewer #3 (Public review):

Summary:

This study by Bushey et al. adapts and evaluates two newly developed red-shifted optogenetic inhibitors, A1ACR1 and HfACR1, collectively referred to as RubyACRs, for neuronal silencing in Drosophila melanogaster. Traditional optogenetic inhibitors such as GtACR1 and GtACR2 are activated by green (~515 nm) and blue (~470 nm) light, respectively, which poses several limitations in Drosophila. Specifically, shorter-wavelength light suffers from reduced tissue penetration and increased absorption, and is visible to flies, potentially confounding behavioral assays, particularly those involving visual processing. In contrast, RubyACRs are activated by red light (~610-660 nm), which penetrates the cuticle more effectively and thus can be more potent in manipulating fly behavior. In the current manuscript, the authors first demonstrate that both A1ACR1 and HfACR1 can be robustly expressed in fly neurons and are properly trafficked to the plasma membrane. Upon red-light stimulation, both opsins produce strong and sustained hyperpolarization in larval motor neurons, outperforming GtACR1 in both magnitude and temporal dynamics. Next, using two-photon calcium imaging in the visual system, the authors further demonstrate that activation of RubyACRs significantly reduces GCaMP6s signal, indicating that they can reliably inhibit neuronal activity. Importantly, unlike reported in some mammalian studies, RubyACRs do not appear to trigger paradoxical depolarization at axon terminals in the fly visual system, as no evidence of aberrant depolarization is observed in motion-detecting Mi1 neurons.

In the second part of the manuscript, the authors characterize the effects of RubyACRs on fly behavior (walking, learning, and courtship song). Using the inhibition of genetically labelled neurons that regulate these behaviors, the authors demonstrate that stimulation of RubyACRs leads to potent suppression of locomotion, courtship song, or dopamine-dependent associative learning.

Strengths:

Altogether, the experiments conducted in this manuscript demonstrate that RubyACRs are powerful tools for optogenetic inhibition in Drosophila, with advantages in spectral compatibility, behavioral specificity, and potential applications in vivo two-photon calcium imaging.

Weaknesses:

The manuscript is strong, but it can be further improved with a few additional analyses and minor revisions. Especially, a more detailed evaluation of RubyACRs with two-photon excitation will help clarify to what extent these opsins can be simultaneously used together with green GECIs, such as GCaMPs.

Author response:

We thank the reviewers for their thoughtful and thorough consideration of the work. We appreciate the positive reception they give the work, and plan to address several of the comments with further experiments. To outline that work (and ensure that we are on the right track to addressing those concerns), we summarize the core concerns that prompt new experiments:

(1) Does the YFP tag on the ACRs interfere with simultaneous GCaMP imaging of RubyACR-expressing cells and could bleaching of the YFP complicate interpretation of the experiments here?

We will test whether 920 nm (2p) and 650 nm (1p) excitation cause YFP bleaching that interferes with interpretation of inhibitory calcium (i.e. GCaMP) signals. Because the YFP tag enhances opsin sensitivity, we prioritized these tagged RubyACRs for initial characterization. FLAG-tagged ACRs are in progress, but will take time to fully characterize. Considering that the RubyACR-EYFP versions work very well, and in many cases people will want the YFP tag, either for visualizing expression or to maximize sensitivity, we feel the current work is a valuable contribution on its own. Indeed several labs have already requested these lines.

(2) Are the ACRs activated by two-photon illumination?

We will examine GCaMP signals at increasing 2p intensities to determine whether imaging unintentionally activates RubyACRs, as well as whether 2p illumination could be used for intentional opsin activation.

(3) How toxic is the expression of these opsins?

We will update the quantification of toxicity in Table 1 to include all the drivers we used in this study. In fact the toxicity we observed was primarily with the vGlut driver, which was why that was the only information in the table. The other drivers we used did not appreciably reduce survival rate, but showing the one case where it did have a big effect left a strong and understandably inaccurate impression that toxicity was a big pitfall. We note that the widely used CSChrimson has similar % survival to the RubyACRs when expressed with these vGlut drivers.

We also plan to examine whether ACR expression leads to cell-autonomous perturbations. We will determine whether expression leads to some frequency of neuronal cell death, and we will evaluate whether any morphological effects occur.

We will also clarify in the Discussion that potential toxicity may be driver-specific (as it is here) and should be evaluated case-by-case by investigators using the tool.

(4) Use functional imaging to confirm inhibition of the neurons used only for behavioral experiments (pIP10 & PPL1-γ1pedc)

We will perform these imaging experiments. One caveat is that inhibition may not be readily detectable with GCaMP, as the resting calcium levels in pIP10 and PPL1-γ1pedc neurons may already be quite low. This differs from the non-spiking Mi1 neurons, where inhibition was clearly observed with GCaMP. For this reason, we consider the behavioral results stronger evidence of efficacy, but we agree that imaging could provide useful supporting evidence, recognizing that a negative result would be difficult to interpret.

(5) Confirm that the GtACR1 will inhibit locomotion in the flybowl when activated with green light, its spectral peak.

We will perform this benchmark experiment. Please note that our intention with this study was to find an effective red-light activated opto-inhibitor because these wavelengths are much less perturbing to behavior. In that respect, regardless of GtACR1’s performance with green light, the RubyACRs clearly provide important new tools for Drosophila behavioral neuroscience.