A multicolor suite for deciphering population coding of calcium and cAMP in vivo

cAMP is a universal second messenger regulated by various upstream pathways including Ca2+ and G-protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, a sensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a sensitive red Ca2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons. We found Ca2+-related cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-related cAMP responses. Overall, our multicolor suite will facilitate analysis of the interaction between the Ca2+, GPCR and cAMP signaling at single-cell resolution both in vitro and in vivo.

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Reviewers' Comments:
Reviewer #1: Remarks to the Author: In this study, the authors developed a high-affinity, large fluorescence intensity change cAMP sensor and an improved red calcium sensor.They characterized and demonstrated the functionality of these new sensors in in vitro and in vivo applications, making some interesting biological observations in the process.They demonstrate the broad applicability of these tools for studying the brain and cell lines, and it is clear these tools could be applied to study other organs.Barring one potential concern (major comment #1) and one possible overinterpretation of the data (major comment #2), this study is a fantastic achievement.Overall, this is an exciting paper that could have an immediate impact on both the neuroscience field as well as a broader biology community especially if DNA and viral reagents are made easily accessible.
Major comments: 1) A possible concern for this sensor is the amount of pH sensitivity, which was not reported and can typically happen with excitation ratiometric sensors that switch between A-band and B-band excitation (see Yellen & Mongeon, Current Opinion in Chemical Biology, 2015).It's important to address this particularly as it is a Methods paper and will help future research users of this sensor that may not be aware of such issues.Can the authors provide a pH sensitivity curve similar to Figure 1f at the very least for cAMPinG1, though a comparison across all 4 sensors would be extremely valuable to the community?a.Including the cAMPingG1 mutant in the pH sensitivity assay is important as it would address how much one can use the mutant data as confirmation that pH was not underlying any observed in vivo cAMP dynamics.
2) Using ChRmine to directly stimulate neurons, the authors conclude that cell-intrinsic spiking can lead to calcium influx and cAMP production.However, as synaptic transmission is not blocked in these in vivo studies and given the long delay between stimulation and observed cAMP increase, it's possible that the increase is mediated by G protein-coupled receptor activation.For example, the stimulated neuron may drive local interneurons or glial cells via glutamate release and these stimulated cells then release a neuromodulator back onto the stimulated cell.a. Unless I missed something, there isn't sufficient evidence to conclude that calcium direction drives cAMP production in a cell-autonomous way.I suggest rewording the abstract, introduction, discussion and other places where the authors say "Ca2+-induced" or conclude that it must be calcium driving cAMP and instead say correlated with calcium.In the discussion it would be fair to speculate on this mechanism, but also speculate on polysynaptic evoked neuromodulator release.
3) It is not clear to me what the questions in Lines 50 -52 are exactly.What does "cAMP population pattern" exactly mean?I think this needs rewording especially as this is a key sentence of the introduction that addresses outstanding questions in the field that these new tools could answer.4) While other cAMP biosensors were recently published, the major accomplishment of cAMPinG1 is achieving the higher affinity for cAMP while also maintaining a large fluorescence change.However, the authors did not show how the higher affinity was necessary or important for biological studies, which would be necessary to demonstrate this tools advantage over existing methods like g-Flamp.It might be that the sensor is partially saturated and limited in its dynamics range by ceiling effects.For example, is the fact that the response magnitude is so much smaller in slices (Extended Figure 3) reflective of a ceiling effect of the sensor?Addressing this point directly would really clarify to the research community cases when it would be necessary to have this higher affinity.a. Could the authors add discussion in the manuscript about what is known about the actual concentrations of cAMP in different neuronal (spines, dendrites, cilia, soma, nucleus) and glial compartments.This will address how well tuned the dynamic range of cAMPingG1 for certain applications.b.Do the authors possibly have any direct comparison of g-Flamp and cAMPingG1 in similar in vivo studies as in Figure 2, 4, or 5? It would really highlight the advantage of using cAMPingG1.For example, it might even be that the suppression of cAMP seen in Figure 5 e,f is only possible with the higher affinity of cAMPinG1.

Minor comments:
1) It is becoming more apparent in the field the importance of quantitative biosensor measurements in vivo.Have the authors measured whether there is a change in lifetime of cAMPingG1?
2) Line 40 is overlooking several studies that have recorded cAMP dynamics in vivo.I agree that there is still much to discover, but there are several studies that have revealed in vivo cAMP dynamics including: 8) For Figure 4f, were non-motion responsive cells confirmed to be responsive at other points in the recording.Are the authors confident these neurons express sufficient rcamp3 and are truly nonresponive versus just not expressing the red calcium sensor well.9) Can the authors please add what the inter-stimulus interval was in Figure 5a.
Reviewer #2: Remarks to the Author: Yokoyama et al develops a green single-fluorophore sensor for cAMP, called cAMPinG1, and a red single-fluorophore sensor, called RCaMP3.cAMPinG1 uses a de novo design, whereas RCaMP3 is developed by integrating a number of known mutations from two R-GECO variants into jRGECO1a.The authors show that these sensors can be used in vivo in a multiplex manner for single-cell imaging and fiber photometry in the context of animal behavior.Notably, the authors discover direction selective cAMPinG1 signals in neurons of the mouse visual cortex that correlate with the direction selectivity of the corresponding neurons' calcium activity.
New cAMP and calcium sensors with enhanced properties are of great interests to the field.However, the enthusiasm is dampened for a number of reasons.The improvement over existing sensors seems to be moderate and is not well demonstrated in cells and neurons in direct comparison to existing sensors.Neither sensor is sufficiently characterized.Although the biological observation regarding the direction selectivity of cAMPinG1 signals is interesting, it needs critical controls and is not clear whether it is achievable (or not) using existing sensors.The authors should better acknowledge the recent progresses of in vivo imaging of existing cAMP and red calcium sensors, and demonstrate that the current sensor can achieve what previous sensors cannot do in a biological context.Major comments.1.Since the major goal of cAMPinG1 is for in vivo imaging, excitation radiometry is not really applicable.2p lasers change wavelengths slowly, and 2p excitation equivalent to UV single photon excitation is associated with high background and photodamage.In fact, ratiometric excitation is not used in the demonstrated in vivo imaging experiments.With single wavelength excitation, the improvement in dynamic range is minimal.2. The excitation of existing sensors should be at or near their respective optimal wavelength.E.g., G-Flamp1 is thought to exhibit the maximal dF/F0 at 450 nm. 3. Another improvement of cAMPinG1 is the sensitivity.However, whether the enhanced sensitivity is beneficial needs to be better demonstrated.The referenced EC50s of PKA was determined in vitro.The value in vivo can be different.Where determination of the basal cAMP concentration inside a cell has been attempted, the values are typically ~0.5-1 µM.A sensor that is too sensitive would result in elevated baseline in vivo.Indeed, the dynamic range of cAMPinG1 inside an intact cell appears to be much smaller than in lysate (Fig. 1j and ED Fig 3).Therefore, the sensor needs to be compared sideby-side with existing sensors under the same condition in intact cells and in intact neurons in neuronal slices using physiologically relevant stimulant.4. Another potential drawback of too high a sensitivity is the potential side effect of buffering.The authors need to determine whether or not cAMP-sensitive neuronal properties (synaptic transmission, excitability, etc) are altered by the expression of the sensor.5. cAMPinG1 needs to be better characterized, such as its kinetics and pH sensitivity.Notably, since buffering can alter cAMP concentration and kinetics inside a cell, kinetic experiments should include measurements in intact cells, both to a puff and to a step increase of a physiological stimulant.6.In vivo cAMP imaging has been achieved (Oe et al, 2020; Massengill et al, 2022; Wang et al, 2022).This should be clearly described in the introduction.Additionally, it is the authors' burden to demonstrate that cAMPinG1 performs better than previous sensors to a degree that crosses the high bar of Nature Methods.At this time, there is essentially no comparison in vivo.7. The authors utilize both cAMPinG1-NE and cAMPinG1-ST in vivo and in vitro.How are these variants compared to the parental sensor?This is important given that cAMPinG1 achieves only 5% of the dynamic range (ED Fig. 3) under very strong conditions compared to Fig. 1e.Also, are there example images showing the subcellular distribution of the ST-variant? 8.The authors state that they added mutations to jRGECO1a to blue-shift the excitation spectra, generate a larger dynamic range and less aggregation.The aggregation data should be shown and quantified.Overall, the improvement as shown in Fig. 3a-e, and ED Fig. 4 seems to be moderate for the bar of Nature Methods.9.As the field comes to realize, RS20 instead of M13 is used in RGECOs and GCaMPs.Also, since the improvement on calcium sensor is on RGECO, why naming the new sensor RCaMP3?RCaMPs already exist, and they are based on different calmodulin binding peptide and different fluorophore.Naming this new variant to RCaMP3 will cause a lot of confusion to the users.Please consider a name along the line of RGECO.10.Since many mutations are introduced to the fluorescent protein in the new calcium sensor, whether the photostability and pH sensitivity have changed should be tested.11.How the new calcium sensor responds to different number of action potentials should be presented and compared to the existing sensors.12. Fig. 3d-h: Any explanation why jRGECO1a's response is only about 10% of the literature?Also, Although the new calcium sensor gives larger response, its noise also seems to be higher (although to a less degree than the signal).Why? 13.Under 2p excitation for the cAMP sensor, how much contamination of the signal from the calcium sensor are there in the cAMP signal channel?One limitation of nearly all red fluorescent protein is that they contain a green component due to bifurcating maturation.This needs to be quantified for multiplex imaging.14.For in vivo experiments, both the cAMP and calcium sensor response should be compared with existing sensors to judgment whether the new sensors present a true improvement and what is the degree of the improvement.Relevant figures include Fig. 2, Fig. 3j, Fig. 4, and Fig. 5. 15.Pharmacological manipulation should be included for in vivo experiments to demonstrate the cAMP response and to dissect the potential pathways controlling its activity.16. cAMP binding mutant should be used in Fig. 4 and 5 to verify that the sensor response is specific.17.Fig. 6: Couldn't the experiment be done with existing ratiometric cAMP sensors?Additional comments 1.For measurements of cAMP affinity, the methods on page 21 (lines 361-366) suggest that the supernatant containing the sensors and cAMP was diluted to reduce the cAMP concentration.This would also reduce the sensor concentration, making comparison between cAMP concentrations inaccurate.Please either redo the experiment or rewrite the text so that it is clear that the sensor concentration was not held constant.2. Fig. 3j, 4 and 5, example images before and after stimulation with sufficient magnification should be presented.3. ED Fig. 2e is not referenced in text.Also, a quantification would strengthen the experiment.4. Fig. 5e: Within the same cell, is there correlation between calcium amplitude and cAMPinG1 signal? 5. Fig. 5j: For ChRmine elicited cAMP response, was it elicited by a single 4-s spiral stimulation?Please clarify in text.Also, how many action potentials does this corresponding to? 6. "G-Flamp1 limited the number of cells that could be imaged and quantified cAMP dynamics in the mouse cortex".This need to be justified for cited.7. The reviewer disagrees with this assertation: "which made fast imaging or combined use with other sensors, such as Ca2+ indicators, generally challenging due to the required optical settings".
Minor comments 1. Page 7 line 119: The corresponding figure (Extended Data Fig. 3) shows application of forskolin + IBMX, but text reads only forskolin.2. Page 9 line 139: "one of the upstream of cAMP signaling" should read "one of the upstream modulators of cAMP signaling".Similarly, page 11, Line 187 should read "multiple upstream pathways, including neuromodulators".Also page 12, line 210.3. Page 14 last paragraph: Fig. 6a,b shows dopamine application but this is not discussed in the text.4. Page 15 first paragraph.Please define TFX and ADRB2 in the text. 5. Fig. 1e and 3b: please specify the used cAMP or calcium concentration in text or legend.6. Page 31 line 550: Is 18 hours correct here?That seems very soon after surgery.It seems unlikely that inflammation would be reduced enough to accurately see cells.7. ED Fig. 2d: please include cAMPinG1 cAMP concentration response curve on this plot as a comparison of cAMP vs cGMP sensitivity 8. Supplementary Table 1 should read Massengill et al., instead of Crystian et al. 9. cAMPinG1 exhibits a cAMP binding hill coefficient of 1.Any explanations given that there are two binding sites?Reviewer #3: Remarks to the Author: Main findings: The authors developed an ultrasensitive genetically encoded green cAMP indicator, cAMPinG1, with a larger dynamic range and higher cAMP affinity than existing green cAMP indicators, allowing for in vivo imaging of cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds.They also introduced an improved red calcium indicator, RCaMP3, enabling simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons.Dual-color imaging revealed that cell-specific cAMP transients represented specific information downstream of Ca2+ signaling as well as GPCR-induced cAMP responses.The authors demonstrated the application of cAMPinG1 imaging in cultured cells for GPCR biology and suggest that their multicolor suite for Ca2+ and cAMP imaging will allow examination of how the information encoded in action potentials, Ca2+, and GPCR signaling is integrated and stored as cAMP transients at the singlecell level in both in vitro and in vivo settings.
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Nina Vogt, PhD Senior Editor Nature Methods a. Zhang et al., Nature, 2021 b.Massengill et al., Nature Methods, 2022 3) Line 99 = typo, AMPingG1-ST is missing the "c" 4) Line 146 -it would help to more clearly explain why blue shifting the rcamp was beneficial 5) Line 182 -please clarify what you mean by "some parts of cells".This was confusing to me. 6) Supplemental Table 1 -for cAMPfire-H reference it should be Massengill et al., not Crystian et al. 7) For Figure 2, please put individual dots for data points on top of the bar graphs.