1. Author Response:

    Reviewer #1 (Public Review):

    The paper is a tour-de-force across multiple techniques and model systems from classical forward screening in C. elegans over ChIP to targeted CRISPR mutagenesis. The data is of a very high quality and supports most of the authors' claims strongly and convincingly. Finally, the manuscript is well written and, in spite of complex experiments and genetics, interesting and easy to comprehend.

    • CAMTA, as the name CaM-binding transcription activator implies, have been studied previously and across many different organisms including plants, mice and humans. It was thus presumed and in part shown that CAMTAs regulate transcription depending on CaM levels.
    • The authors confirm that the gene cmd-1 (encoding CaM) is directly regulated by Camt-1 by using a combination of cell-specific RNAseq and ChIP. This allows them to identify three binding sites upstream of the cmb-1 gene that bind to Camt-1.
    • Moreover, the authors show that overexpression of CaM in the nervous system fully rescues the observed behavioral phenotypes.
    • Importantly, the authors make another discovery. They show that CaM can directly repress its own transcription by binding to specific residues of Camt-1. Thereby, the authors argue, Camt-1 is used to precisely and bidirectionally regulate CaM levels dependent on the cell, animal's state etc.

    The reported data are interesting and, in particular, the aspect that CAMTAs likely act as activators AND repressors is a novel aspect previously not appreciated. In spite of all these strengths, a potential weakness is that it remains open whether this mechanism is primarily a house-keeping mechanism or is indeed, as the authors speculate, regulated by internal and external factors that might, through CAMTA, make cells more or less responsive to Ca2+-CaM signaling.

    We are grateful for and encouraged by our reviewer’s comments. We think that our discovery that CAMTAs regulate CaM expression is thought provoking.

    Reviewer #2 (Public Review):

    Vuong-Brender, Flynn, and de Bono report a detailed analysis of the function of a highly conserved calcium-calmodulin-dependent transcriptional regulator in the function of the C. elegans sensory nervous system. The C. elegans homolog of this factor - CAMT-1 - emerged from a genetic screen for mutants defective in a sensory-driven aggregation behavior. The authors find that multiple chemosensory modalities are disrupted by loss of CAMT-1, and this factor has distributed functions in the nervous system, including in interneurons that receive inputs from sensory neurons. A major finding of this study is that many of the effects of CAMT-1 mutation can be linked to a critical role for CAMT-1 in regulating expression of calmodulin itself. This finding is supported by multiple lines of experimentation, including a demonstration that the effects of losing CAMT-1 can be compensated by restoring expression of calmodulin. The authors further show that what is true for CAMT-1 and calmodulin in C. elegans also applies to Drosophila, indicating that CAMT-1 is a regulator of calmodulin expression whose function has been conserved throughout evolution. This manuscript has many strengths. Key hypotheses are tested using quantitative and technically independent experimental methods. The case that CAMT-1 is a regulator of calmodulin expression is built carefully and, for the most part, the logic of the argument is made clearly and supported by compelling data. Another strength of the manuscript is its candid exposition of data that do not fit neatly into the most simple and accessible model. It is refreshing to see authors who freely admit that they haven't neatly wrapped up every question in a field. The loose ends in this study do not impact the authors' main conclusions. However, some observations seem to consume more bandwidth than warranted, and the authors should consider reorganizing the manuscript so that the loose ends do not distract from the main thread of the narrative. The paper does have a few minor weaknesses that could be addressed. These are listed below.

    We thank our reviewer for their thoughtful review.

    Specific comments:

    1. The initial description of the isolation of camt-1 mutants seemed a bit disorganized. A description of the gene and gene product preceded descriptions of the mutants. Also, some mutants were mentioned in the text but not presented in the corresponding figure. The authors should consider minor changes to better communicate how the mutations were cloned.

    We have sought to do this.

    1. In Fig. 2 npr-1 baselines vary a great deal between panels A, B, and C. It is not clear why npr-1 behavior is this variable, and the authors do not mention this obvious feature of their data. Data presented in Fig. 2 indicate that heat-shock-induced expression of camt-1 restores a defect in basal locomotion, but it is unclear whether it restores O2-sensitivity - the effect of oxygen on speed of transgenics seems the same +/- heatshock (compare black traces in panels 2B and 2C). We understand the concern of the reviewer. Since the design of these experiments was different from the rest (with only one shift in O2 concentration), we repeated them with 3 O2 changes, bringing them in line with the rest of the manuscript. The results are presented in the new Figure 2. We observed a more consistent baseline speed between different conditions, however some differences still exist (for example between panel 2A and 2B). One explanation is that for heatshock experiments we keep npr-1 animals at lower temperature (20 degree Celsius, panels 2B and 2C) to minimize basal activity of the heatshock promoter, whereas in the rescue experiment in Figure 2A, and in the rest of the manuscript, animals were kept at 22 oC. Figure 2B-C of our original submission used worms raised at 15 oC for the heatshock experiment, which may explain the greater discrepancy in npr-1 speed values. Heatshock also modifies slightly the response of the npr-1 control animals to O2.

    Regarding whether heat-shock-induced expression of camt-1 restores O2 responses, we found that the npr-1; camt-1; dbExhsp-16p::camt-1 heat-shocked strains aggregated much more than npr-1; camt-1 heat-shocked animals. However, the rescue is not complete. Thus expressing camt-1 using heatshock-induced expression restores some O2 sensitivity which correlates well with the partial rescue of the baseline in Figure 2C. We have noted this in the results.

    1. Unlike other datasets, the responses of wild-type AFDs to CO2 do not look particularly convincing (panel 3C). There is clearly an effect of camt-1 mutation on AFD calcium, but the AFD responses seem qualitatively different from the responses of BAGs to CO2 or URXs to O2. The authors might consider moving these data to a supplementary figure and tempering their description of wild-type AFDs as CO2-sensors.

    The data on AFD has been moved to Figure 3 – figure supplement 1. We should add that we agree that in the absence of an identified CO2 sensor expressed in AFD, we cannot be sure that AFD neurons are primary CO2 sensors. Although the AFD CO2-evoked responses are retained in mutants defective in synaptic transmission, they may very well still be indirectly evoked by other neurons.

    1. The authors candidly present data that do not conform to a simple model for how camt-1 affects behavior. Loss of camt-1 increases calcium in sensory neurons that activate the speed-controlling interneuron RMG. However, RMG calcium is reduced in camt-1 mutants. This inversion in the effect of camt-1 mutation might be caused by a homeostatic mechanism, as the authors propose. It might be possible to test this hypothesis by testing whether reducing excitatory input into RMGs elevates resting calcium in camt-1 mutants, for example via mutations that affect sensory transduction.

    In the interest of simplifying the manuscript, and given other comments, we have now removed the RMG Ca2+ imaging data. However, this is an interesting way of testing what is going.

    1. In Fig. 4H RMG data are presented as fractional ratio change - all other imaging data are presented as absolute ratios of YFP and CFP fluorescence. It is not clear why these data are treated differently. It is also no clear that these data are consistent with data shown in Fig. 3F. Which dataset represents the effect of camt-1 mutation on RMG calcium? More measurements might be warranted.

    As highlighted above we have removed the RMG imaging data from the paper. .

    1. Nice experiments show that regulation of calmodulin in Drosophila requires a CAMT-1 homolog. The bar graphs showing unity for values normalized to themselves are a bit odd - perhaps there's a more compact way to plot these data.

    We have sought to address this question in two ways. First, we have further buttressed our results by performing in situ immunofluorescence staining of dissected fly retinas with a calmodulin antibody. We see a significant decrease in calmodulin expression in fly CAMTA mutants compared to controls.

    Prompted by this comment, we also realized we omitted an explanation of how we normalized the data for the qPCR graphs in the figure legend. This was done using rRNA as a control. The Yamamoto lab had previously used the same control to normalize CAMTA expression in wild type and mutant flies. We add a note saying this.

    1. ChIPseq analysis of CAMT-1 is also quite nice. Is there a sequence motif for CAMT-1 binding that emerges from this study? If so, how does this motif compare to motifs from studies of CAMT-1 homologs in other species?

    We used the MEME algorithm, (motif-based sequence analysis tools (https://meme-suite.org/) to seek enriched sequence motifs in our ChIPSeq data. This identified a series of enriched motifs, although none coincided with the peaks at the CMD-1 promoter. However, we did observe sequences resembling the mouse CAMTA1 binding site at the centre of each of the three CAMT-1 binding peaks upstream of cmd-1. We now say this is the discussion.

    1. Figure 7 shows that CMD-1 inhibits cmd-1 expression via interaction with CAMT-1. These data are interesting, but it is not clear how this effect can be related to prior data showing that forced expression of CMD-1 can compensate for loss of CAMT-1. The authors behavioral and physiological studies suggest that in vivo CAMT-1 promotes CMD-1 expression. In Figure 7, they suggest that CAMT-1 inhibits expression of CMD-1, but there is no clear link to behavior or physiology for this repressor-function of CAMT-1. The manuscript might be more clear without these data, and the absence of these data would not affect the overall impact of the study.

    We agree that the feedback control of cmd-1 gene expression by CMD-1 interacting with CAMT-1 is a part of the story that has not been fully developed. Given the feedback from our reviewers and Editors to give these findings less prominence, but not remove them entirely, we moved the data into supplementary information. We have also altered the main text and the legend of Figure 7 to explicitly say that further experiments are needed to establish if this feedback is relevant under physiological conditions.

    Reviewer #3 (Public Review):

    Vuong-Brender et al present a thorough study investigating how CaM-binding transcription activators (CAMTAs) in C. elegans and Drosophila are required for numerous behaviors and proper neuronal function. The study is strong in how it uses a variety of approaches to study a major underlying mechanism for CAMTA. First, they use reporters, mutant analysis, and heat-shock rescue to show how cart-1 is expressed widely in neurons and functions in adults in several behaviors. They used transcriptional profiling to show that cart-1 is required to upregulate CaM in subsets of neurons in worm. They next use ChIP-seq to zero in on where worm CAMT-1 binds regulatory regions upstream of the CaM gene cmd-1 to promote its expression. They find that overexpression of CaM compensates for behavioral and neuronal response deficits in a cart-1 mutants. Lastly, they propose that when CaM highly expressed, it may down regulate its own expression by binding CART-1.

    We thank our reviewer for their critique of our work.

    1. Overall, I feel that the study is excellent and most conclusions are justified by evidence. However, I do not think the title is supported by the data. It currently is listed as: CAMTA TUNES NEURAL EXCITABILITY AND BEHAVIOR BY MODULATING CALMODULIN EXPRESSION. The authors show evidence that camt-1 is required for the normal function of neurons and behavior by promoting expression of CaM. Their only evidence that camt-1 downregulates CaM is a more artificial situation where CaM is overexpressed. I don't think they provide any evidence that camt-1 is used to "tune" behavior or neuron activity up and down in a wild-type strain. Tuning implies that the molecule modulates a physiological system bidirectionally in a natural situation. I suggest using a more accurate title that better fits the experimental evidence.

    We have changed the title to ‘Neuronal Calmodulin levels are controlled by CAMTA transcription factors’. We hope this more neutral title is appropriate to describe our findings.

    1. They show ample evidence that cart-1 appears to promote the expression of cmd-1 in most cases. This includes showing that overexpression of cmd-1 suppresses the behavioral and imaging phenotypes of cart-1. But they didn't perform the more straight forward epistasis test with the cart-1;cmd-1 double mutant in worm or fly , presumably because there is no viable loss-of-function allele in the coding area of the cmd-1 gene. It would help the readers understand why this simpler experiment was not performed if they explain this in the paper. A good place would be near line 220, where they generate hypomorphic promoter alleles using CRISPR. If they have tried to make their own loss-of-function alleles by mutating the coding area of cmd-1, but it resulted in presumed lethality, this might be mentioned here too.

    This is a good point, and one that we had overlooked. cmd-1 loss of function mutations do indeed confer lethality. We have added a sentence to say:

    ‘Straightforward comparison of camt-1 and cmd-1 loss of function phenotypes was not possible, since disrupting cmd-1 confers lethality (7, 8).’

    1. I am most worried about the potential caveats with the calcium imaging experiments. As the authors note, it is challenging to infer absolute levels of calcium using the ratiometric sensor cameleon across different individuals and genotypes. However, the authors do not note that the YFP/CFP FRET signal from cameleon might be perturbed because it uses calmodulin to bind calcium. At the end of their study (line 244), they provide evidence that calmodulin may bind to CART-1 to suppress its own expression when calmodulin is highly expressed. This is worrisome because cameleon is probably expressed highly in some or most of these strains. The authors may want to re-examine neuronal activity for a subset of experiments with a method that is independent of a calmodulin-based sensor (if possible).

    We agree that this is a potential concern. As suggested by our referee, we therefore repeated some of our Ca2+ imaging experiments using a genetically-encoded Ca2+ indicator that does not contain CaM. We opted to use TN-XL, an indicator that uses troponin C as the Ca2+ binding moiety, and which has previously been used successfully in C. elegans. We imaged CO2-evoked Ca2+ responses in BAG sensory neurons, in wild type and in camt-1 mutant animals. The data obtained using TN-XL recapitulated what we observed using YC3.60 (BAG).

    1. The title of "Fig 3 - Figure supplement 1" is confusing because it suggests that they measured the levels of YC2.60 cameleon, when in fact they measured a separate GFP reporter, albeit using the same promoter. So they could clarify the figure title.

    The reviewer is right – our heading was confusing. We have changed it, and now say: ‘Expression from the gcy-37 promoter is reduced when CAMT-1 is overexpressed.’

    1. D. Bazopoulou, A. R. Chaudhury, A. Pantazis, N. Chronis, An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons. Sci Rep 7, 9403 (2017).
    2. M. S. Choi et al., Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.). J Biol Chem 280, 40820-40831 (2005).
    3. J. Han et al., The fly CAMTA transcription factor potentiates deactivation of rhodopsin, a G protein-coupled light receptor. Cell 127, 847-858 (2006).
    4. N. Bouche, A. Scharlat, W. Snedden, D. Bouchez, H. Fromm, A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277, 21851-21861 (2002).
    5. T. Yang, B. W. Poovaiah, A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. J Biol Chem 277, 45049-45058 (2002).
    6. E. Kodama-Namba et al., Cross-modulation of homeostatic responses to temperature, oxygen and carbon dioxide in C. elegans. PLoS Genet 9, e1004011 (2013).
    7. V. Au et al., CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3 (Bethesda) 9, 135-144 (2019).
    8. A. Karabinos et al., Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy. Eur J Cell Biol 82, 557-563 (2003).
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  2. Evaluation Summary:

    Calcium-calmodulin (CaM) signaling plays an essential role within and outside of the nervous system. Moreover, it is conserved from plants to humans. While a lot is known about the mechanisms of cellular calcium level fluctuations, how CaM levels are regulated is less clear. In this manuscript, Vuong-Brender and colleagues characterize a, likely, conserved role of the transcription factor CAMT-1 in the homeostatic regulation of CaM levels and show how it impacts animal behavior and nervous system function. The paper is a tour-de-force across multiple techniques and model systems. The data is of a very high quality and supports most of the authors' claims strongly and convincingly.

    (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. The reviewers remained anonymous to the authors_.)_

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  3. Reviewer #1 (Public Review):

    The paper is a tour-de-force across multiple techniques and model systems from classical forward screening in C. elegans over ChIP to targeted CRISPR mutagenesis. The data is of a very high quality and supports most of the authors' claims strongly and convincingly. Finally, the manuscript is well written and, in spite of complex experiments and genetics, interesting and easy to comprehend.

    - CAMTA, as the name CaM-binding transcription activator implies, have been studied previously and across many different organisms including plants, mice and humans. It was thus presumed and in part shown that CAMTAs regulate transcription depending on CaM levels.
    - The authors confirm that the gene cmd-1 (encoding CaM) is directly regulated by Camt-1 by using a combination of cell-specific RNAseq and ChIP. This allows them to identify three binding sites upstream of the cmb-1 gene that bind to Camt-1.
    - Moreover, the authors show that overexpression of CaM in the nervous system fully rescues the observed behavioral phenotypes.
    - Importantly, the authors make another discovery. They show that CaM can directly repress its own transcription by binding to specific residues of Camt-1. Thereby, the authors argue, Camt-1 is used to precisely and bidirectionally regulate CaM levels dependent on the cell, animal's state etc.

    The reported data are interesting and, in particular, the aspect that CAMTAs likely act as activators AND repressors is a novel aspect previously not appreciated. In spite of all these strengths, a potential weakness is that it remains open whether this mechanism is primarily a house-keeping mechanism or is indeed, as the authors speculate, regulated by internal and external factors that might, through CAMTA, make cells more or less responsive to Ca2+-CaM signaling.

    Read the original source
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  4. Reviewer #2 (Public Review):

    Vuong-Brender, Flynn, and de Bono report a detailed analysis of the function of a highly conserved calcium-calmodulin-dependent transcriptional regulator in the function of the C. elegans sensory nervous system. The C. elegans homolog of this factor - CAMT-1 - emerged from a genetic screen for mutants defective in a sensory-driven aggregation behavior. The authors find that multiple chemosensory modalities are disrupted by loss of CAMT-1, and this factor has distributed functions in the nervous system, including in interneurons that receive inputs from sensory neurons. A major finding of this study is that many of the effects of CAMT-1 mutation can be linked to a critical role for CAMT-1 in regulating expression of calmodulin itself. This finding is supported by multiple lines of experimentation, including a demonstration that the effects of losing CAMT-1 can be compensated by restoring expression of calmodulin. The authors further show that what is true for CAMT-1 and calmodulin in C. elegans also applies to Drosophila, indicating that CAMT-1 is a regulator of calmodulin expression whose function has been conserved throughout evolution. This manuscript has many strengths. Key hypotheses are tested using quantitative and technically independent experimental methods. The case that CAMT-1 is a regulator of calmodulin expression is built carefully and, for the most part, the logic of the argument is made clearly and supported by compelling data. Another strength of the manuscript is its candid exposition of data that do not fit neatly into the most simple and accessible model. It is refreshing to see authors who freely admit that they haven't neatly wrapped up every question in a field. The loose ends in this study do not impact the authors' main conclusions. However, some observations seem to consume more bandwidth than warranted, and the authors should consider reorganizing the manuscript so that the loose ends do not distract from the main thread of the narrative. The paper does have a few minor weaknesses that could be addressed. These are listed below.

    Specific comments:

    1. The initial description of the isolation of camt-1 mutants seemed a bit disorganized. A description of the gene and gene product preceded descriptions of the mutants. Also, some mutants were mentioned in the text but not presented in the corresponding figure. The authors should consider minor changes to better communicate how the mutations were cloned.

    2. In Fig. 2 npr-1 baselines vary a great deal between panels A, B, and C. It is not clear why npr-1 behavior is this variable, and the authors do not mention this obvious feature of their data. Data presented in Fig. 2 indicate that heat-shock-induced expression of camt-1 restores a defect in basal locomotion, but it is unclear whether it restores O2-sensitivity - the effect of oxygen on speed of transgenics seems the same +/- heatshock (compare black traces in panels 2B and 2C).

    3. Unlike other datasets, the responses of wild-type AFDs to CO2 do not look particularly convincing (panel 3C). There is clearly an effect of camt-1 mutation on AFD calcium, but the AFD responses seem qualitatively different from the responses of BAGs to CO2 or URXs to O2. The authors might consider moving these data to a supplementary figure and tempering their description of wild-type AFDs as CO2-sensors.

    4. The authors candidly present data that do not conform to a simple model for how camt-1 affects behavior. Loss of camt-1 increases calcium in sensory neurons that activate the speed-controlling interneuron RMG. However, RMG calcium is reduced in camt-1 mutants. This inversion in the effect of camt-1 mutation might be caused by a homeostatic mechanism, as the authors propose. It might be possible to test this hypothesis by testing whether reducing excitatory input into RMGs elevates resting calcium in camt-1 mutants, for example via mutations that affect sensory transduction.

    5. In Fig. 4H RMG data are presented as fractional ratio change - all other imaging data are presented as absolute ratios of YFP and CFP fluorescence. It is not clear why these data are treated differently. It is also no clear that these data are consistent with data shown in Fig. 3F. Which dataset represents the effect of camt-1 mutation on RMG calcium? More measurements might be warranted.

    6. Nice experiments show that regulation of calmodulin in Drosophila requires a CAMT-1 homolog. The bar graphs showing unity for values normalized to themselves are a bit odd - perhaps there's a more compact way to plot these data.

    7. ChIPseq analysis of CAMT-1 is also quite nice. Is there a sequence motif for CAMT-1 binding that emerges from this study? If so, how does this motif compare to motifs from studies of CAMT-1 homologs in other species?

    8. Figure 7 shows that CMD-1 inhibits cmd-1 expression via interaction with CAMT-1. These data are interesting, but it is not clear how this effect can be related to prior data showing that forced expression of CMD-1 can compensate for loss of CAMT-1. The authors behavioral and physiological studies suggest that in vivo CAMT-1 promotes CMD-1 expression. In Figure 7, they suggest that CAMT-1 inhibits expression of CMD-1, but there is no clear link to behavior or physiology for this repressor-function of CAMT-1. The manuscript might be more clear without these data, and the absence of these data would not affect the overall impact of the study.

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  5. Reviewer #3 (Public Review):

    Vuong-Brender et al present a thorough study investigating how CaM-binding transcription activators (CAMTAs) in C. elegans and Drosophila are required for numerous behaviors and proper neuronal function. The study is strong in how it uses a variety of approaches to study a major underlying mechanism for CAMTA. First, they use reporters, mutant analysis, and heat-shock rescue to show how cart-1 is expressed widely in neurons and functions in adults in several behaviors. They used transcriptional profiling to show that cart-1 is required to upregulate CaM in subsets of neurons in worm. They next use ChIP-seq to zero in on where worm CAMT-1 binds regulatory regions upstream of the CaM gene cmd-1 to promote its expression. They find that overexpression of CaM compensates for behavioral and neuronal response deficits in a cart-1 mutants. Lastly, they propose that when CaM highly expressed, it may down regulate its own expression by binding CART-1.

    1. Overall, I feel that the study is excellent and most conclusions are justified by evidence. However, I do not think the title is supported by the data. It currently is listed as: CAMTA TUNES NEURAL EXCITABILITY AND BEHAVIOR BY MODULATING CALMODULIN EXPRESSION. The authors show evidence that camt-1 is required for the normal function of neurons and behavior by promoting expression of CaM. Their only evidence that camt-1 downregulates CaM is a more artificial situation where CaM is overexpressed. I don't think they provide any evidence that camt-1 is used to "tune" behavior or neuron activity up and down in a wild-type strain. Tuning implies that the molecule modulates a physiological system bidirectionally in a natural situation. I suggest using a more accurate title that better fits the experimental evidence.

    2. They show ample evidence that cart-1 appears to promote the expression of cmd-1 in most cases. This includes showing that overexpression of cmd-1 suppresses the behavioral and imaging phenotypes of cart-1. But they didn't perform the more straight forward epistasis test with the cart-1;cmd-1 double mutant in worm or fly , presumably because there is no viable loss-of-function allele in the coding area of the cmd-1 gene. It would help the readers understand why this simpler experiment was not performed if they explain this in the paper. A good place would be near line 220, where they generate hypomorphic promoter alleles using CRISPR. If they have tried to make their own loss-of-function alleles by mutating the coding area of cmd-1, but it resulted in presumed lethality, this might be mentioned here too.

    3. I am most worried about the potential caveats with the calcium imaging experiments. As the authors note, it is challenging to infer absolute levels of calcium using the ratiometric sensor cameleon across different individuals and genotypes. However, the authors do not note that the YFP/CFP FRET signal from cameleon might be perturbed because it uses calmodulin to bind calcium. At the end of their study (line 244), they provide evidence that calmodulin may bind to CART-1 to suppress its own expression when calmodulin is highly expressed. This is worrisome because cameleon is probably expressed highly in some or most of these strains. The authors may want to re-examine neuronal activity for a subset of experiments with a method that is independent of a calmodulin-based sensor (if possible).

    4. The title of "Fig 3 - Figure supplement 1" is confusing because it suggests that they measured the levels of YC2.60 cameleon, when in fact they measured a separate GFP reporter, albeit using the same promoter. So they could clarify the figure title.

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