Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
Round 1
Reviewer 1 Report
The authors summarize in a concise way the currently available optogenetic tools. Especially the tables are very useful for the reader. The review is well written.
Here just some minor comments:
Line 21: "protein engineering" has a very wide meaning; to build tools for neuroscience is just a tiny part of it. Maybe change to "engineering of optogenetic tools"?
Line 36: "keeping the complex cellular milieu ... undisturbed" Can we be so sure about this? Maybe we should be still careful about this because we simply do not know how much we disturb the system when we over-express a protein.
Line 58: Maybe cite original paper of Junichi Nakai (G-CaMP).
It might be also interesting to mention faster calcium indicators like https://www.nature.com/articles/ncomms3170
Line 82:
Line 145: Please explain the inferior photophysical properties leading to imaging artifacts.
Line 146: Maybe add https://www.nature.com/articles/nmeth.3185
Line 219-230: This chapter needs some work. There is a big difference between K+ and Na+ on one hand an Ca2+ on the other. Typically extracellular Ca2+ is 2mM and intracellular only 50nM - more than 4 orders of magnitude. K+ and Na+ however is all in the mM range - extracellular and intracellular - with only 1 or 2 orders of magnitude difference between inside and outside. The K+ and Na+ concentration during an AP barely changes ! See, for example, Johnston and Wu "Foundations of Cellular Neurophysiology". This makes the task of K+ and Na+ imaging so much more difficult. Please adapt the paragraph accordingly.
Line 244: Funny sentence .. please correct.
Line 247: remove "(ref. )" ... but keep the reference
Line 251: isn't it the biophysical characteristics instead of the biochemical?
Line 270: should read ", the so called electrochromic effect,"
Line 318: "wide-field"
Line 352: "under a cheap and simple"
Line 353: "was the first "
Line 510: "signal. Very"
Author Response
Reviewer#1
The authors summarize in a concise way the currently available optogenetic tools. Especially the tables are very useful for the reader. The review is well written.
We thank the reviewer for the kind comment. We hope readers from several disciplines will find our coverage interesting and invigorating.
Here just some minor comments:
Line 21: "protein engineering" has a very wide meaning; to build tools for neuroscience is just a tiny part of it. Maybe change to "engineering of optogenetic tools"?
To be more specific, we replaced “protein engineering” with “engineering optogenetic indicators”. Please see line 21 of the revised manuscript.
Line 36: "keeping the complex cellular milieu ... undisturbed" Can we be so sure about this? Maybe we should be still careful about this because we simply do not know how much we disturb the system when we over-express a protein.
The reviewer makes an excellent point that additional studies are needed to fully appreciate the effects of heterologous protein overexpression. We agree that it is incompletely understood how genetically encoded tools affect cellular physiology. Therefore, we have revised the text to “minimizing disturbance to the complex cellular milieu” (lines 36-37).
Line 58: Maybe cite original paper of Junichi Nakai (G-CaMP).
We cite the original G-CaMP paper along with other publication reporting first generation of calcium sensors. Please see lines 52-53 of the revised manuscript.
Line 82: It might be also interesting to mention faster calcium indicators like https://www.nature.com/articles/ncomms3170
Indeed, generation of the first GECIs with faster calcium binding kinetics was achieved by structure-guided mutagenesis of the GFP-CaM interface and the CaM-M13 peptide interactions reported in the indicated paper. A similar strategy was used later to develop the jGCaMP7 series of GECIs. It is important to acknowledge the earlier efforts on rational design of improved sensors, correspondingly we revised the text to cite the https://www.nature.com/articles/ncomms3170 paper. Please see line 82-84.
Line 145: Please explain the inferior photophysical properties leading to imaging artifacts.
We provided the most common example of the undesired photophysical properties inherent to red fluorescent proteins, which results in imaging artifact. Please see line 152.
Line 146: Maybe add https://www.nature.com/articles/nmeth.3185
Thank you for pointing this paper out, we have cited it accordingly. Please see line 153 of the revised manuscript.
Line 219-230: This chapter needs some work. There is a big difference between K+ and Na+ on one hand an Ca2+ on the other. Typically extracellular Ca2+ is 2mM and intracellular only 50nM - more than 4 orders of magnitude. K+ and Na+ however is all in the mM range - extracellular and intracellular - with only 1 or 2 orders of magnitude difference between inside and outside. The K+ and Na+ concentration during an AP barely changes ! See, for example, Johnston and Wu "Foundations of Cellular Neurophysiology". This makes the task of K+ and Na+ imaging so much more difficult. Please adapt the paragraph accordingly.
We apologize for the imprecise language regarding ion flux during an action potential. We have updated the text to reflect the reviewer’s correct comment on the difference in magnitude between changes in calcium compared to changes in potassium and sodium. We decided to remove the comparison to calcium to avoid confusion. We appreciate the reviewer’s excellent comment on why these biophysical properties make K+ and Na+ imaging inherently difficult, and have accordingly added a comment to reflect this (please see lines 238-243 of the revised text).
Line 244: Funny sentence .. please correct.
We apologize for the imprecision. We initially included this sentence to place voltage indicators in the context of other indicators we discussed. After deliberation, we decided to delete this sentence.
Line 247: remove "(ref. )" ... but keep the reference
We apologize for the error and have corrected the manuscript.
Line 251: isn't it the biophysical characteristics instead of the biochemical?
We replaced “biochemical” with “biophysical”.
Line 270: should read ", the so called electrochromic effect,"
We apologize for the error and have corrected the manuscript.
Line 318: "wide-field"
We apologize for the error and have corrected the manuscript.
Line 352: "under a cheap and simple"
We apologize for the error and have corrected the manuscript.
Line 353: "was the first "
We apologize for the error and have corrected the manuscript.
Line 510: "signal. Very"
We apologize for the error and have corrected the manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This concise review paper reviewed the progress of the optogenetic tools in the application of neuroscience. This paper is up to date, should be helpful for the field.
However, at least two key references are missed, 1) “Channelrhodopsin-2, a directly light-gated cation-selective membrane channel” , 2003 2) “eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications”, 2008, it would be great if the authors can redo the literature search to include all the key references.
some minor typos and grammatical errors need to be corrected.
Author Response
This concise review paper reviewed the progress of the optogenetic tools in the application of neuroscience. This paper is up to date, should be helpful for the field.
We thank the reviewer for the kind comment. We hope readers from several disciplines will find our coverage interesting and invigorating.
However, at least two key references are missed, 1) “Channelrhodopsin-2, a directly light-gated cation-selective membrane channel” , 2003 2) “eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications”, 2008, it would be great if the authors can redo the literature search to include all the key references.
While the focus of the review is on the very recent progress in optogenetics, we agree that both of these papers report key discoveries in the field. We have cited them in the revised version of the manuscript (see line 281). In addition, we have cited the key publications in the field of calcium imaging reporting first proof of principle GECIs (please see lines 52-53).
Some minor typos and grammatical errors need to be corrected.
We have further improved the text by fixing several typos as well as grammatical errors.
Author Response File: Author Response.docx
Reviewer 3 Report
The review by Piatkevich and colleagues focusses on recent advances in the development of genetically encoded fluorescence indicators in neuroscience. Clearly, this field is moving at a rapid pace, and it is of critical importance to the community to get an expert review on the suitability of the respective indicator for the individual research questions. The authors cover all relevant classes of indicators in great detail, with a particular emphasis on the biophysical properties of the sensors. The review is rather comprehensive and written with great insight into the biophysical mechanisms. The quality of the figures is high, and the tables, albeit complex, contain the key parameters of each indicator. Consequently, this review will be a great resource for neuroscientist. I fully support publication, but there are a few comments which will need to be addressed:
1. Title: The authors rightfully state in line 39, that this review is about genetically encoded fluorescence indicators of neuronal activity. Yet, in the title, the term “Optogenetic tools” is used. This is simply incorrect; optogenetics refers to tools allowing to manipulate neuronal activity, such as ChR2 or ArchT, not for indicators. It is absolutely critical to change the title, so that the readers are not being misled. This review is about indicators, not about actuators.
2. Lines 11, and 28: See comment 1, the authors refer to indicators, not actuators, so the term “optogenetics” is not correct
3. Line 31: “Additionally, genetically encoded probes generate limited toxicity,…” This is a very general statement, which is incorrect. The authors rightfully state later in the text (lines 61-63), that particularly GCaMP variants are discussed to exhibit quite substantial cytotoxicity, particularly in transgenic animals, so this sentence needs to be rephrased.
4. Lines 93 – 96. The authors discuss the stoichiometry of calcium binding. In my view, this is a very relevant aspect also in terms of the linearity of indicators: Synthetic indicators such as Oregon-Green-BAPTA contain only one binding site, so therefore display a linear relationship between the number of action potentials and increase of fluorescence, while GCaMPs contain 4, and NTnC two. I suggest that the authors include this aspect in their review.
5. Lines 102-103: “The different types of organization of…” Please rephrase
6. Lines 221-222: “Similar to calcium, intracellular sodium levels are on the order of the magnitude lower than extracellular.” This is incorrect: the concentration difference of extra- vs intracellular calcium ranges at 4-5 orders of magnitude (extracellular 1-2 mM, intracellular 1 * 10-4 mM), of sodium about 1 order of magnitude (145 mM vs 12 mM)
7. Line 227:”…dramatically decreasing cytoplasmic potassium levels”. While it is true, that upon each action potential, there is an efflux of potassium, the relative amount of ions is relatively small; otherwise a neuron could not perform high frequency AP firing, as the activity of the Na-K-ATPase is relatively slow. Only after excessive AP firing, these concentration change sare notable.
8. Chapter 4, Voltage sensors: I fully agree with the description of the advantages and disadvantages of voltage sensors: Yet, in my view, one important aspect is missing: Due to the short duration of the voltage changes upon an action potential, high frame rates in the kHz range are needed. This is currently achievable only in vitro. The calcium elevations upon an action potential are much longer-lived, so the sampling rate can be lower, and this makes calcium indicators the currently only option in in vivo application. I suggest including this aspect.
9. Line 361: “In the past few years the field of GECIs…” Maybe this is a typo? Should it not read “GEVI”?
10. Lines 386-387: Certainly, neurotransmitter release can initiate second messenger cascades postsynaptically, in case of metabotropic receptors, but, similarly important, neurotransmitters can evoke direct ion flux upon binding to ionotropic receptors, this aspect will need to be included as well.
11. Line 619. In my view, brain clearing and tissue expansion are post mortem techniques, so how can fluorescent indicators be of any use in these techniques?
Author Response
The review by Piatkevich and colleagues focusses on recent advances in the development of genetically encoded fluorescence indicators in neuroscience. Clearly, this field is moving at a rapid pace, and it is of critical importance to the community to get an expert review on the suitability of the respective indicator for the individual research questions. The authors cover all relevant classes of indicators in great detail, with a particular emphasis on the biophysical properties of the sensors. The review is rather comprehensive and written with great insight into the biophysical mechanisms. The quality of the figures is high, and the tables, albeit complex, contain the key parameters of each indicator. Consequently, this review will be a great resource for neuroscientist. I fully support publication, but there are a few comments which will need to be addressed:
We thank the reviewer for the kind comment. We agree the field is moving swiftly and we hope readers from several disciplines will find our coverage interesting and invigorating.
1. Title: The authors rightfully state in line 39, that this review is about genetically encoded fluorescence indicators of neuronal activity. Yet, in the title, the term “Optogenetic tools” is used. This is simply incorrect; optogenetics refers to tools allowing to manipulate neuronal activity, such as ChR2 or ArchT, not for indicators. It is absolutely critical to change the title, so that the readers are not being misled. This review is about indicators, not about actuators.
We agree with the Review that the title “Advances in Engineering and Application of Optogenetic Tools for Neuroscience” can be misleading for readers setting the wrong expectations that this review includes both optogenetic actuators and optogenetic indicators, while we focus on indicators and only briefly mentioning optogenetic actuators. Therefore, we change the title correspondingly replacing tools “tools” for “indicators”. Please see Line 3 of the revised manuscript, as well as the response to comment 2.
2. Lines 11, and 28: See comment 1, the authors refer to indicators, not actuators, so the term “optogenetics” is not correct.
Although the term “optogenetics” was coined over 15 years ago, there is no commonly accepted definition. In this review, we used term “optogenetics” and “optogenetic tools” in the broader sense as it was defined by the pioneers of light-sensitive genetically encoded molecules for the study of neurons and neuronal networks. For example, Deisseroth et al. 2006 gave the following definition: “Optogenetic technology combines genetic targeting of specific neurons or proteins with optical technology for imaging or control of the targets within intact, living neural circuits”. A similar definition was also accepted by other scientists in the field, such as Knopfel et al. 2010, “The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes”. We cite both publications in the relevant context (please see lines 28-29). Therefore, we think that using the term optogenetics in relation to genetically encoded fluorescent indicators is correct—and indeed is used by founders of the field. However, since the review focuses on indicators to monitor neuronal activity while only briefly mentioning actuators, we entirely agree is important to be precise in referring to the corresponding classes of the molecules to avoid any confusion. Therefore, throughout the text we used the term optogenetic indicators to refer to genetically encoded optical indicators of neuronal activity, please see lines 21,
3. Line 31: “Additionally, genetically encoded probes generate limited toxicity,…” This is a very general statement, which is incorrect. The authors rightfully state later in the text (lines 61-63), that particularly GCaMP variants are discussed to exhibit quite substantial cytotoxicity, particularly in transgenic animals, so this sentence needs to be rephrased.
We agree that additional studies are needed to more fully appreciate the effects of genetically encoded indicators on normal cellular physiology. To clarify our point that this technique is suitable for longitudinal imaging, we rephrased the sentence as follows: “Additionally, genetically encoded probes are the only technique available to observe precisely the same cells longitudinally, permitting long-term monitoring of specific cellular processes, up to months”, please see lines 31-33.
4. Lines 93 – 96. The authors discuss the stoichiometry of calcium binding. In my view, this is a very relevant aspect also in terms of the linearity of indicators: Synthetic indicators such as Oregon-Green-BAPTA contain only one binding site, so therefore display a linear relationship between the number of action potentials and increase of fluorescence, while GCaMPs contain 4, and NTnC two. I suggest that the authors include this aspect in their review.
We agree with the Reviewer that a linear relationship between the number of action potentials and an increase of fluorescence is an important characteristic for all kinds of calcium sensors. We therefore added a sentence describing linearity of fluorescence changes in response to increasing number of action potentials. Please see lines 99-101 of the revised manuscript.
5. Lines 102-103: “The different types of organization of…” Please rephrase.
We have rephrased the text to be more precise on the description of the presented figure. Please see lines 108-110 of the revised text.
6. Lines 221-222: “Similar to calcium, intracellular sodium levels are on the order of the magnitude lower than extracellular.” This is incorrect: the concentration difference of extra- vs intracellular calcium ranges at 4-5 orders of magnitude v e (extracellular 1-2 mM, intracellular 1 * 10-4 mM), of sodium about 1 order of magnitude (145 mM vs 12 mM)
We apologize for the misinterpretation. We have corrected the text to reflect the estimates of sodium concentration in mM. Correspondingly, we removed the comparison to calcium to avoid confusion and included the estimates of sodium concentrations for neurons at rest. The current text now reads: “In neurons at rest, cytoplasmic sodium levels are an order of magnitude lower than extracellular (~10 mM intracellular vs ~150 mM extracellular)”. Please see line 228-229 of the revised manuscript.
7. Line 227:”…dramatically decreasing cytoplasmic potassium levels”. While it is true, that upon each action potential, there is an efflux of potassium, the relative amount of ions is relatively small; otherwise a neuron could not perform high frequency AP firing, as the activity of the Na-K-ATPase is relatively slow. Only after excessive AP firing, these concentration change sare notable.
We agree with the Reviewer’s excellent comment and have revised the text correspondingly to avoid misconception on potassium levels in neurons during action potentials. Please see line 233-235 of the revised manuscript.
8. Chapter 4, Voltage sensors: I fully agree with the description of the advantages and disadvantages of voltage sensors: Yet, in my view, one important aspect is missing: Due to the short duration of the voltage changes upon an action potential, high frame rates in the kHz range are needed. This is currently achievable only in vitro. The calcium elevations upon an action potential are much longer-lived, so the sampling rate can be lower, and this makes calcium indicators the currently only option in in vivo application. I suggest including this aspect.
We agree with the Reviewer that in vivo voltage imaging is extremely challenging in comparison to calcium imaging and it is important to acknowledge this aspect. Therefore, we extended Chapter 4 to include discussion of current limitation for voltage imaging in vivo. Please see lines 257-268.
9. Line 361: “In the past few years the field of GECIs…” Maybe this is a typo? Should it not read “GEVI”?
Thank you for catching this, we have corrected the typo, please see line 383.
10. Lines 386-387: Certainly, neurotransmitter release can initiate second messenger cascades postsynaptically, in case of metabotropic receptors, but, similarly important, neurotransmitters can evoke direct ion flux upon binding to ionotropic receptors, this aspect will need to be included as well.
We apologize for omitting the important effects of ionotropic receptors. We have updated the sentence accordingly: “Here, neurotransmitters bind to receptors on the post-synaptic cell, initiating second messenger signaling cascades (via metabotropic receptors) or evoking ion flux (via ionotropic receptors)”. Please see line 409.
11. Line 619. In my view, brain clearing and tissue expansion are post mortem techniques, so how can fluorescent indicators be of any use in these techniques?
Indeed, brain clearing and tissue expansion are post mortem techniques. We think these techniques are complementary to fluorescence indicator technology and thus are used in combination as post-hoc imaging techniques to enable (1) anatomical connectivity investigation, (2) cell type identify classification using multiplexed in situ hybridization, and other sequencing modalities. This combination of in vivo imaging and post-hoc analysis ultimately informs the molecular and morphological identity of the cells imaged. We clarified our point in the revised text and cited relevant papers. Please see lines 642-643.
Author Response File: Author Response.docx
