Robust Optogenetic Inhibition with Red-light-sensitive Anion-conducting Channelrhodopsins

Reviewer #1 (Public Review):

Summary:
The authors of this manuscript characterize new anion conducting that is more red-shifted in its spectrum than prior variants called MsACR1. An additional mutant variant of MsACR1 that is renamed raACR has a 20 nm red-shifted spectral response with faster kinetics. Due to the spectral shift of these variants, the authors proposed that it is possible to inhibit the expression of MsACR1 and raACR with lights at 635 nm in vivo and in vitro. The authors were able to demonstrate some inhibition in vitro and in vivo with 635 nm light. Overall the new variants with unique properties should be able to suppress neuronal activities with red-shifted light stimulation.

Strengths:
The authors were able to identify a new class of anion conducting channelrhodopsin and have variants that respond strongly to lights with wavelength >550 nm. The authors were able to demonstrate this variant, MsACR1, can alter behavior in vivo with 635 nm light. The second major strength of the study is the development of a red-shifted mutant of MsACR1 that has faster kinetics and 20 nm red-shifted from a single mutation.

Weaknesses:
The red-shifted raACR appears to work much less efficiently than MsACR1 even with 635 nm light illumination both in vivo (Figure 4) and in vitro (Figure 3E) despite the 20 nm red-shift. This is inconsistent with the benefits and effects of red-shifting the spectrum in raACR. This usually would suggest raACR either has a lower conductance than MsACR1 or that the membrane/overall expression of raACR is much weaker than MsACR1. Neither of these is measured in the current manuscript.

There are limited comparisons to existing variants of ACRs under the same conditions in the manuscript overall. There should be more parallel comparison with gtACR1, ZipACR, and RubyACR in identical conditions in cultured cell lines, cultured neurons, and in vivo. This should be in terms of overall performance, efficiency, and expression in identical conditions. Without this information, it is unclear whether the effects at 635 nm are due to the expression level which can compensate for the spectral shift.

There should be more raw traces from the recordings of the different variants in response to short pulse stimulation and long pulse stimulation to different wavelengths. It is difficult to judge what the response would be like when these types of information are missing.

Despite being able to activate the channelrhodopsin with 635 nm light, the main utility of the variant should be transcranial stimulation which was not demonstrated here.

Figure 3B is not clearly annotated and is difficult to match the explanation in the figure legend to the figure. The action potential spikings of neurons expressing raACR in this panel are inhibited as strongly as MsACR1.

For many characterizations, the number of 'n's are quite low (3-7).

Reviewer #2 (Public Review):

Summary:
The authors identified a new chloride-conducting Channelrhodopsin (MsACR1) that can be activated at low light intensities and within the red part of the visible spectrum. Additional engineering of MsACR1 yielded a variant (raACR1) with increased current amplitudes, accelerated kinetics, and a 20nm red-shifted peak excitation wavelength. Stimulation of MsACR1 and raACR1 expressing neurons with 635nm in mice's primary motor cortices inhibited the animals' locomotion.

Strengths:
The in vitro characterization of the newly identified ACRs is very detailed and confirms the biophysical properties as described by the authors. Notably, the ACRs are very light sensitive and allow for efficient in vitro inhibition of neurons in the nano Watt/mm^2 range. These new ACRs give neuroscientists and cell biologists a new tool to control chloride flux over biological membranes with high temporal and spatial precision. The red-shifted excitation peaks of these ACRs could allow for multiplexed application with blue-light excited optogenetic tools such as cation-conducting channelrhodopsins or green-fluorescent calcium indicators such as GCaMP.

Weaknesses:
The in-vivo characterization of MsACR1 and raACR1 lacks critical control experiments and is, therefore, too preliminary. The experimental conditions differ fundamentally between in vitro and in vivo characterizations. For example, chloride gradients differ within neurons which can weaken inhibition or even cause excitation at synapses, as pointed out by the authors. Notably, the patch pipettes for the in vitro characterization contained low chloride concentrations that might not reflect possible conditions found in the in vivo preparations, i.e., increasing chloride gradients from dendrites to synapses.

Interestingly, the authors used soma-targeted (st) MsACR1 and raACR1 for some of their in vitro characterization yielding more efficient inhibition and reduction of co-incidental "on-set" spiking. Still, the authors do not seem to have utilized st-variants in vivo.

Most importantly, critical in vivo control experiments, such as negative controls like GFP or positive controls like NpHR, are missing. These controls would exclude potential behavioral effects due to experimental artifacts. Moreover, in vivo electrophysiology could have confirmed whether targeted neurons were inhibited under optogenetic stimulations.

Some of these concerns stem from the fact that the pulsed raACR stimulation at 635 nm at 10Hz (Fig. 3E) was far less efficient compared to MsACR1, yet the in vivo comparison yielded very similar results (Fig. 4D).

Also, the cortex is highly heterogeneous and comprises excitatory and inhibitory neurons. Using the synapsin promoter, the viral expression paradigm could target both types and cause differential effects, which has not been investigated further, for example, by immunohistochemistry. An alternative expression system, for example, under VGLUT1 control, could have mitigated some of these concerns.

Furthermore, the authors applied different light intensities, wavelengths, and stimulation frequencies during the in vitro characterization, causing varying spike inhibition efficiencies. The in vivo characterization is notably lacking this type of control. Thus, it is unclear why the 635nm, 2s at 20Hz every 5s stimulation protocol, which has no equivalent in the in vitro characterization, was chosen.

In summary, the in vivo experiments did not confirm whether the observed inhibition of mouse locomotion occurred due to the inhibition of neurons or experimental artifacts.

In addition, the author's main claim of more efficient neuronal inhibition would require them to threshold MsACR1 and raACR1 against alternative methods such as the red-shifted NpHR variant Jaws or other ACRs to give readers meaningful guidance when choosing an inhibitory tool.

The light sensitivity of MsACR1 and raACR1 are impressive and well characterized in vitro. However, the authors only reported the overall light output at the fiber tip for the in vivo experiments: 0.5 mW. Without context, it is difficult to evaluate this value. Calculating the light power density at certain distances from the light fiber or thresholding against alternative tools such as NpHR, Jaws, or other ACRs would allow for a more meaningful evaluation.

Author Response

Reviewer #1 (Public Review):

Summary:

The authors of this manuscript characterize new anion conducting that is more red-shifted in its spectrum than prior variants called MsACR1. An additional mutant variant of MsACR1 that is renamed raACR has a 20 nm red-shifted spectral response with faster kinetics. Due to the spectral shift of these variants, the authors proposed that it is possible to inhibit the expression of MsACR1 and raACR with lights at 635 nm in vivo and in vitro. The authors were able to demonstrate some inhibition in vitro and in vivo with 635 nm light. Overall the new variants with unique properties should be able to suppress neuronal activities with red-shifted light stimulation.

Strengths:

The authors were able to identify a new class of anion conducting channelrhodopsin and have variants that respond strongly to lights with wavelength >550 nm. The authors were able to demonstrate this variant, MsACR1, can alter behavior in vivo with 635 nm light. The second major strength of the study is the development of a red-shifted mutant of MsACR1 that has faster kinetics and 20 nm red-shifted from a single mutation.

Weaknesses:

The red-shifted raACR appears to work much less efficiently than MsACR1 even with 635 nm light illumination both in vivo (Figure 4) and in vitro (Figure 3E) despite the 20 nm red-shift. This is inconsistent with the benefits and effects of red-shifting the spectrum in raACR. This usually would suggest raACR either has a lower conductance than MsACR1 or that the membrane/overall expression of raACR is much weaker than MsACR1. Neither of these is measured in the current manuscript.

Thank you for addressing this crucial issue. We posit that the diminished efficiency of raACR in comparison to MsACR1 WT can be attributed to the tenfold acceleration of its photocycle. As noted by Reviewer 1, the anticipated advantages associated with a red-shifted opsin, particularly in in vivo preparations, are offset by its accelerated off-kinetics. Consequently, the shorter dwell time of the open state leads to a reduced number of conducted ions per photon. Nevertheless, the operational light sensitivity is not drastically altered compared to MsACR WT (Fig. 3C). We believe that the rapid kinetics offer interesting applications, such as the precise inhibition of single action potentials through holography.

There are limited comparisons to existing variants of ACRs under the same conditions in the manuscript overall. There should be more parallel comparison with gtACR1, ZipACR, and RubyACR in identical conditions in cultured cell lines, cultured neurons, and in vivo. This should be in terms of overall performance, efficiency, and expression in identical conditions. Without this information, it is unclear whether the effects at 635 nm are due to the expression level which can compensate for the spectral shift.

We compared MsACR1 and raACR with GtACR1 in ND cells in supplemental figure 4. We concur that further comparisons could be useful to emphasise both the strengths of MsACRs and applications where they may not be as suitable. We are currently in the process of outlining a separate article. We firmly believe that each ACR variant occupies a distinct application niche, which necessitates a more comprehensive electrophysiological comparison to provide valuable insights to the scientific community.

There should be more raw traces from the recordings of the different variants in response to short pulse stimulation and long pulse stimulation to different wavelengths. It is difficult to judge what the response would be like when these types of information are missing.

We appreciate Reviewer 1's feedback and have compiled a collection of raw photoresponses, encompassing various pulse widths and wavelengths, which can be found in the Supplementary materials (Supplementary Figures 4 and 5).

Despite being able to activate the channelrhodopsin with 635 nm light, the main utility of the variant should be transcranial stimulation which was not demonstrated here.

We concur with Reviewer 1's assessment that MsACR prime application is indeed transcranial stimulation. However, it's worth emphasising that the full advantages of transcranial optical stimulation become most apparent when animals are truly freely moving without any tethered patch cords. Our ongoing research in the laboratory is dedicated to the development of a wireless LED system that can be securely affixed to the animal's skull. We aim to demonstrate the potential of these novell optogenetic approaches in the field of behavioural neuroscience in the coming year.

Figure 3B is not clearly annotated and is difficult to match the explanation in the figure legend to the figure. The action potential spikings of neurons expressing raACR in this panel are inhibited as strongly as MsACR1.

We have enhanced the figure caption and annotations for clarity. The traces presented in Figure 3B are intended to demonstrate the overall effectiveness of each variant. However, it is in the population data analysis, as depicted in Figure 3E, where the meaningful insights are revealed.

For many characterizations, the number of 'n's are quite low (3-7).

We acknowledge Reviewer 1's suggestion regarding the in vivo data and agree with the importance of including more animals, as well as control animals. However, we are committed to adhering to the principles of the 3Rs (Replacement, Reduction, Refinement) in animal research, and given the robustness of our observed effects, we will add animals to reach the minimal number of animals per condition (n = 2) to minimise unnecessary animal usage while ensuring statistical power. We will continue to adhere to the established standards in the field, aiming for a range of 3 to 7 cells per condition, sourced from at least two independent preparations, to ensure the robustness and reliability of our in vitro data.

Reviewer #2 (Public Review):

Summary:

The authors identified a new chloride-conducting Channelrhodopsin (MsACR1) that can be activated at low light intensities and within the red part of the visible spectrum. Additional engineering of MsACR1 yielded a variant (raACR1) with increased current amplitudes, accelerated kinetics, and a 20nm red-shifted peak excitation wavelength. Stimulation of MsACR1 and raACR1 expressing neurons with 635nm in mice's primary motor cortices inhibited the animals' locomotion.

Strengths:

The in vitro characterization of the newly identified ACRs is very detailed and confirms the biophysical properties as described by the authors. Notably, the ACRs are very light sensitive and allow for efficient in vitro inhibition of neurons in the nano Watt/mm^2 range. These new ACRs give neuroscientists and cell biologists a new tool to control chloride flux over biological membranes with high temporal and spatial precision. The red-shifted excitation peaks of these ACRs could allow for multiplexed application with blue-light excited optogenetic tools such as cation-conducting channelrhodopsins or green-fluorescent calcium indicators such as GCaMP.

Weaknesses:

The in-vivo characterization of MsACR1 and raACR1 lacks critical control experiments and is, therefore, too preliminary. The experimental conditions differ fundamentally between in vitro and in vivo characterizations. For example, chloride gradients differ within neurons which can weaken inhibition or even cause excitation at synapses, as pointed out by the authors. Notably, the patch pipettes for the in vitro characterization contained low chloride concentrations that might not reflect possible conditions found in the in vivo preparations, i.e., increasing chloride gradients from dendrites to synapses.

We appreciate Reviewer 2’s feedback regarding missing control experiments. We will respond to these concerns in another section of our manuscript, as suggested. Regarding the chloride gradient, we understand the concerns of Reviewer 2, yet we chose these ionic conditions, particularly as they were used in the initial electrical characterization of GtACR1 in a neuronal context (Mahn et al., 2016). We will make sure to provide this context in our manuscript to justify our choice of ionic conditions.

Interestingly, the authors used soma-targeted (st) MsACR1 and raACR1 for some of their in vitro characterization yielding more efficient inhibition and reduction of co-incidental "on-set" spiking. Still, the authors do not seem to have utilized st-variants in vivo.

At the time of submission, due to the long-term absence of our lab technician, we were not able to produce purified viruses. Therefore, we decided to move on with the submission. We now produced the virus externally, and will provide the experiments.

Most importantly, critical in vivo control experiments, such as negative controls like GFP or positive controls like NpHR, are missing. These controls would exclude potential behavioral effects due to experimental artifacts. Moreover, in vivo electrophysiology could have confirmed whether targeted neurons were inhibited under optogenetic stimulations.

We have several non-injected control animals that we used to calibrate this particular paradigm and never saw similar responses. However, we acknowledge the suggestion of Reviewer 2 and will include the GFP-injected control as recommended.

Some of these concerns stem from the fact that the pulsed raACR stimulation at 635 nm at 10Hz (Fig. 3E) was far less efficient compared to MsACR1, yet the in vivo comparison yielded very similar results (Fig. 4D).

As outlined previously, the accelerated photocycle of raACR results in a reduction in photocurrent amplitude, consequently diminishing the potency of inhibition per photon. In the context of in vitro stimulation, where single action potentials are recorded, this reduction in inhibition efficiency is resolved. However, in the realm of in vivo behavioural analysis, the observed effect is not contingent on single action potentials but rather stems from the disruption of the entire M1 motor network. In this context, despite the reduced efficiency of the fast-cycling raACR, it still manages to interrupt the M1 network, leading to similar behavioural outcomes.

Also, the cortex is highly heterogeneous and comprises excitatory and inhibitory neurons. Using the synapsin promoter, the viral expression paradigm could target both types and cause differential effects, which has not been investigated further, for example, by immunohistochemistry. An alternative expression system, for example, under VGLUT1 control, could have mitigated some of these concerns.

Indeed, we acknowledge the limitations of our current experimental approach. We are in the process of planning and conducting additional experiments involving cre-dependent expression of st-MSACR and st-raACR in PV-Cre mice.

Furthermore, the authors applied different light intensities, wavelengths, and stimulation frequencies during the in vitro characterization, causing varying spike inhibition efficiencies. The in vivo characterization is notably lacking this type of control. Thus, it is unclear why the 635nm, 2s at 20Hz every 5s stimulation protocol, which has no equivalent in the in vitro characterization, was chosen.

We appreciate the valuable comment from the reviewer. The objective of our in vitro characterization is to elucidate the general effects of specific stimulation parameters on the efficiency of neuronal inhibition. For instance, we aim to demonstrate that lower light intensities result in less efficient inhibition, or that pulse stimulation may lead to a less complete inhibition, albeit significantly reducing the energy input into the system.

In the in vivo characterization, we face constraints such as animal welfare considerations and limitations in available laser lines, which prevent us from exploring the entire parameter space as comprehensively as in the in vitro preparation. Additionally, it is important to note that membrane capacitance tends to be higher in vivo compared to dissociated hippocampal neurons. Consequently, we have opted for a doubled stimulation frequency from 10 Hz to 20 Hz and the stimulation pattern of 2 seconds ”on” and 5 seconds “off”. This approach allows the animals to spend less time in an arrested state while still demonstrating the effect of MsACR and variants.

In summary, the in vivo experiments did not confirm whether the observed inhibition of mouse locomotion occurred due to the inhibition of neurons or experimental artifacts.

In addition, the author's main claim of more efficient neuronal inhibition would require them to threshold MsACR1 and raACR1 against alternative methods such as the red-shifted NpHR variant Jaws or other ACRs to give readers meaningful guidance when choosing an inhibitory tool.

The light sensitivity of MsACR1 and raACR1 are impressive and well characterized in vitro. However, the authors only reported the overall light output at the fiber tip for the in vivo experiments: 0.5 mW. Without context, it is difficult to evaluate this value. Calculating the light power density at certain distances from the light fiber or thresholding against alternative tools such as NpHR, Jaws, or other ACRs would allow for a more meaningful evaluation.