Dual-color optical activation and suppression of neurons with high temporal precision

Reviewer #1 (Public Review):

Summary
In this manuscript, the authors generate an AAV-deliverable tool that generates action potentials in response to red light, but not blue light, when expressed in neurons. To do this, they screen some red light-excitatory/blue light-inhibitory opsin pairs to find ones that are spectrally and temporally matched. They first show that this works with Chrimson and GtACR2, however, they expand their search after finding that the tau-off (inactivation after light cessation) kinetics of these two opsins are not well-matched. They directly examine a small set of options based on a literature search and settle on a variant of red light-excitatory Chrimson and blue light-inhibitory ZipACR. To more closely match the kinetics of this pair, the authors create a structure homology model of the ZipACR retinal binding pocket and use this to guide the generation of a small mutagenesis panel, leading to a more optimized ZipACR mutant. They then show that a bicistronically expressed fusion arrangement of these opsins, plus some functional peptides, can drive action potentials up to 20hz with red light and does not do so with blue light, in hippocampal cells transduced by AAV. They also show function in vivo, in a mouse, using a physiological readout. They conclude that their new tool may be useful for complex experimental designs requiring multiple optical channels for write-in/read-out.

The major advantage claimed by the authors over existing tools is the temporal time-locking of their inhibitory opsin - this is driven by the contrast between the tau-off kinetics of their ZipACR variant compared to gtACR2, which is used by the leading competitor tool (BiPOLES).

Big thoughts
While the authors were carefully thoughtful about the potential influence of temporal kinetics on the efficiency of a tool such as this one, there were no experiments conducted that made use of the unique properties of this molecular strategy. To understand why they embarked on this engineering program, I was required to put on my neuroscientist hat and contemplate this question myself:

First, experimental designs where I require multiple optical channels of control. This appears to be aligned with the author's thoughts, as they state, correctly, that opsins utilizing retinal as a light-sensing chromophore are universally activated by blue light (the so-called 'blue shoulder'). Therefore, their tool may be useful for stimulating multiple populations using a blue excitatory opsin in neuron A and their tool for red excitation of neuron B - or, in the author's own words, "A potential solution to the problem of cross-talk...". Yet, there are no data presented that showcases their new tool for this purpose (e.g. Vierock, Johannes, et al. "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons." Nature Communications 12.1 (2021): 4527. Figure 4f-I; 6). The same set-up could be imagined for green GECI (or equivalent) imaging of cells in the same volume that their tool is being used in - for instance, interleaving red stimulation light and blue imaging light, (perhaps) without the typical concern of imaging light bleed-through activating the opsin itself.

Second, for high-frequency temporal control over both excitation and inhibition in the same neuron. The red light turns the cell on, and blue light turns the cell off (see, for instance, Zhang, Feng, et al. "Multimodal fast optical interrogation of neural circuitry." Nature 446.7136 (2007): 633-639. Figure 2; Vierock as above, Figure 4a,b). Again, here the authors are long on theory ("The new system...can drive time-locked high-frequency action potentials in response to red pulses") and short on data. While they do show that red light = excitation and blue light = inhibition, they neither show 1) all-optical on/off modulation of the same cell; nor 2) high-frequency inhibition or excitation (max stim rate of 20hz, which is the same as the BiPOLES paper used for their LC stimulation paradigm; Vierock, as above, Figure 7a-d).

Despite these major shortcomings, the further development and characterization of tandem opsins, such as this one, is of interest to the community. There is ongoing work by the BiPOLES team to create new iterations (e.g. Wahid, J., et al. "P-15 BiPOLES2 is a bidirectional optogenetic tool with a narrow activation spectrum and low red-light excitability." Clinical Neurophysiology 148 (2023): e16.). To make the case that the tool described in this manuscript is worth the effort that the authors are requesting the neuroscience community invest in trialing it in their own hands, they must revise the manuscript to show that their approach is both 1) different in some way when compared to BiPOLES (it is my understanding that they did not do this, as per the supplementary alignment of the BiPOLES sequence and the sequence of the BiPOLES-like construct that they did test) and 2) that the properties that the investigators specifically tailored their construct to have confer some sort of experimental advantage when compared to the existing standard.

There are a number of additional concerns and clarifications that will strengthen the manuscript that are communicated directly to the authors through this peer-review process.

Reviewer #2 (Public Review):

Summary:
One often wishes to combine activation of a neural population via red light with simultaneous modulation of a different population via blue light, or simultaneous imaging of a blue-excited fluorescent reporter. The problem is that all red-shifted opsins have an action spectrum with a long blue tail, leading to spurious opsin activation by blue light.

This valuable paper presents a clever solution to this problem, by pairing an engineered blue-shifted inhibitory chloride-conducting opsin with a red-shifted excitatory opsin. The combined effect is excitation by red light and shunting inhibition by blue light. The paper is very thorough, with convincing spectroscopic and patch clamp characterization of the tools, and tests in brain slices and in vivo. This tool is likely to be useful in the neuroscience community.

Strengths:
The methods are solid, including the complete characterization of each tool separately, as well as the combination in vivo. The array of testing gives a strong degree of confidence that this tool will work as expected.

Weaknesses:
There are two discussion points and one experimental point which would make the paper stronger.

1. In the Introduction or Discussion, the authors could better motivate the need for a red-shifted actuator that lacks blue crosstalk, by giving some specific examples of how the tool could be productively used, e.g. pairing with another blue-shifted excitatory opsin in a different population, or pairing with a GFP-based fluorescent indicator, e.g. GCaMP. The motivation for the current tool is not obvious to non-experts.

2. Simultaneous excitation and inhibition are not the same as non-excitation. The authors mentioned shunting briefly. Another possible issue is changes in osmotic balance. Activation of a Na+ channel and a Cl- channel will lead to net import of NaCl into the cell, possibly changing osmotic pressure. Please discuss.

3. The authors showed that in ZipT-IvfChr, orange light drives excitation and blue light does not. But what about simultaneous blue and orange light? Can the blue light overwhelm the effect of the orange light? Since the stated goal is to open the blue part of the spectrum for other applications, one is now worried about "negative" crosstalk. Please discuss and, ideally, characterize this phenomenon.
   3.1) Does the use of the new tool require careful balancing of the expression levels of the ZipT and the IvfChr? Does it require careful balancing of blue and orange light intensities?
   3.2) Also, many opsins show complex and nonlinear responses to dual-wavelength illumination, so each component should be characterized individually under simultaneous blue + orange light.
   3.3) I was expecting to see photocurrents at different holding potentials as a function of illumination wavelength for the co-expressed construct (i.e. to see at what wavelength it switches from being excitatory to inhibitory); and also to see I-V curves of the photocurrent at blue and orange wavelengths for the co-expressed constructs (i.e. to see the reversal potential under blue excitation). Overall, the patch clamp and spectroscopic characterization of the individual constructs was stronger than that of the combined constructs.

Reviewer #3 (Public Review):

This study addresses the important topic of dual-color optogenetic control of neuronal activity, which is challenging due to significant optical crosstalk between channelrhodopsins of different absorption colors and ion selectivity. However, Mermet-Joret et al. demonstrate in flies that simple coexpression of a strong blue light-activated inhibitory opsin, such as the chloride-selective channelrhodopsin GtACR2, can suppress the blue light activity of a red-shifted excitatory opsin, such as Chrimson, and allow dual-color optogenetic control of the expressing neuron. The same concept was previously discussed by Vierock et al. and led to the generation of BiPOLES, which combines both channels in a single fusion protein. In the present manuscript, the authors introduce an alternative combination of channels with accelerated off-kinetics that are coexpressed by a bicistronic expression cassette. The goal is to better match the duration of illumination and optogenetic manipulation in order to reduce potential side effects induced by prolonged channel opening.

The major novelty of this work lies in the choice of the employed ion channels: the excitatory cation channel vf-Chrimson and the inhibitory anion channel ZipACR, alongside their subsequent modifications (Fig. 2 - 4). Both channels belong to the fastest known ChRs, but the choice of ZipACR raises questions. First, it has a peak absorption at 515 nm that is 40 nm further red-shifted than GtACR2 tested in Figure 1 and accordingly important optical cross-talk with the coexpressed Chrimson channel. Second, it was reported to have reduced chloride selectivity, first by Govorunova et al. in 2017 and later also by Kato et al. in 2018. Both of these aspects are also mentioned by the authors but were not resolved through molecular engineering. Instead site-directed mutagenesis primarily focused on membrane expression and photoreceptor kinetics of the employed channels. Nonetheless, improving the membrane targeting of the vf-Chrimson channel by exchange of the N-terminus finally provided sufficient red light activation at low light intensities to reliably activate expressing neurons and allowed in combination with the decelerated ZipACR mutants dual color optogenetic control with millisecond time resolution. At higher light intensities inactivation of Chrimson and the optical crosstalk of both channels seem to limit its performance.

The experimental results are well presented; but, certain questions persist:

1. The enhanced vf-Chrimson could potentially be a highlight of the manuscript, serving broader applications. Yet, gauging the overall improvements of ivf-Chrimson in comparison to other Chrimson variants remains intricate due to several reasons. First, photocurrents from ivf-Chrimson seem smaller than those from C-Chrimson (Supplemental Figure 3), and a direct comparison with standard vf-Chrimson is absent. Second, while membrane expression of ivf-Chrimson appears enhanced in provided bright-field recordings, the quantitative analysis would necessitate confocal microscopy and a membrane marker (Supplemental Figure 2). Finally, other N-terminal modified Chrimson variants, like CsChrimson by Klapoetke et al. in 2014 and C1Chrimson by Oda et al. in 2018, have been generated. Comparing ivf-Chrimson to vf-CsChrimson or vf-C1Chrimson would be important to evaluate the benefits of the applied N-terminal modification.

2. The action spectra of ZipACR suggest peak absorption of ZipACR WT and its mutant at 525 - 550 nm (Fig. 3). This is even further red-shifted than previously reported by Govorunova et al. Further action spectra recordings differ for all constructs between recordings initiated with blue or red light (Supplementary Fig. 5). This discrepancy is unexpected and should be discussed. Additionally, the representative photocurrents of Zip(151V) in Fig. 3D1 do not align with the corresponding action spectrum in Fig. 3D2 as they show maximal photocurrents for 400 nm excitation.

3. The authors introduce two different bicistronic expression cassettes-ZipT-IvfChR and ZipV-IvfChR-without providing clear guidelines on their conditions of use. Although the authors assert that ZipT is slower and further red-shifted than ZipV, the differences in the data for both ACR mutants are small and the benefits of the different final constructs should be explained.

4. The ZipT/V-IvfChRs are designed as bicistronic constructs; yet, disparities in membrane trafficking and protein degradation between the two channels could lead to divergences in blue and red light photoresponses. For future applicants, understanding the extent of expression ratio variations across cells using the presented expression cassettes could be of significance and should be discussed.