Abstract
Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.
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Data availability
cAMPFIRE constructs and their corresponding sequences have been deposited to Addgene (nos. 182279–182286). cAMPFIRE DNA sequences have been deposited to GenBank (access nos. ON986214, ON986215, ON986216 and ON986217 for cAMPFIRE-L, cAMPFIRE-M, cAMPFIRE-H and cAMPFIRE-L-R279E, respectively). Source data are provided with this paper.
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Acknowledgements
We thank Ryohei Yasuda (Max Planck Florida) for 2pFLIM acquisition software; Jin Zhang (University of California, San Diego) for ICUE3 cDNA; Kees Jalink (Netherlands Cancer Institute) for Epac-SH187 cDNA; Laurinda Jaffe (University of Connecticut) and Viacheslav Nikolaev (University of Hamburg) for Epac1-camps and Epac2-camps300 cDNA; Lei Ma, Vivek Unni and Sydney Boutros (Oregon Health & Science University, OHSU) and Josh Melander (Stanford University) for training and advice on in vivo surgery; Kevin Wright (Vollum Institute) for immunoprecipitation reagents; Caitlynn De Preter for contributing to in vitro data collection; Makayla Freitas for assistance in spectrophotometer measurements; the OHSU Shared Resources Biophysics Core for use of the stopped-flow device; and Bart Jongbloets, Michael Muniak, Lei Ma, Yang Chen, and the entire Zhong lab and Mao lab at OHSU for helpful comments and discussions. Fly stocks from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used. This work was supported by NIH BRAIN Initiative awards R01NS104944 (H.Z. and T.M.) and RF1MH120119 (H.Z. and T.M.), National Institute of Neurological Disorders and Stroke (NINDS) R01 grants R01NS127013 (H.Z.), R01NS081071 (T.M.), and R01NS104299 (B.Y.), and National Institute of General Medical Sciences (NIGMS) R01 grant R01GM138862 (I.B.).
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H.Z. conceived the project. C.I.M., L.B.-E., T.M. and H.Z. designed the experiments. M.R.W., C.I.M. and H.Z. performed structural analyses of Epac. C.I.M. and L.B.-E. performed in vitro experiments with assistance from H.Z., except that C.C.C. performed electrophysiological recordings. C.I.M. and L.B.-E. carried out mouse in vivo imaging experiments. M.Q. generated critical reagents. J.C., A.B., E.W. and I.B. expressed and purified recombinant proteins. E.R.C. performed all experiments and data analysis associated with fly imaging, and E.R.C. and B.Y. wrote the corresponding legends and methods. C.I.M., L.B.-E., T.M. and H.Z. analyzed the data and wrote the manuscript, with edits and comments by all authors. T.M. and H.Z. secured the funding and supervised the project.
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Nature Methods thanks Martin Lohse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling editor: Nina Vogt, in collaboration with the Nature Methods team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Correcting the perinuclear localization of the Epac-SH187 sensor.
a, Crystal structure of the NPL sequence in Epac2A (top) and the alignment of NPL sequences between Epac1 with Epac2. The positions tested are highlighted in the structure (red) and at the alignment (asterisks). b, Representative images of the indicated mutants compared to Epac-SH187 in HEK cells. From left to right starting from the top, n (cells/coverslips) = 69/29, 50/5, 59/29, 18/3, 12/2, 12/2, 37/4. c, Response time course (left) and quantification of the response of indicated constructs to the indicated stimuli in HEK cells. From left to right, n = 20, 13, and 11. Dark lines and bars indicate mean. Shaded area indicates s.e.m. d, Representative images of the indicated mutant compared to Epac-SH187 in L2/3 pyramidal neurons. Single two-photon optical sections are shown. n (cells/mice) = 18/5 and 20/8 for Epac-SH187 and L777A/K778A, respectively.
Extended Data Fig. 2 Localization-correcting mutations reduce the tendency of the sensor to aggregate.
a & b, Representative images (a) and quantifications (b) of the number of aggregates per cell resulting from the indicated constructs. To ease the visualization of aggregates, saturated pixels (for display only) are showed in red. Single cpV: a sensor variant that is the same as Epac-SH187 with the exception that it only has a single cpVenus as the FRET acceptor. From left to right, n (cells) = 12, 58, 53, 48, and 41. Lines indicate mean. ***: P < 0.001 (from left to right, P = 3.3 × 10−10, 5.2 × 10−10, and 1.7 × 10−10) for all comparisons, two-tailed Welch test.
Extended Data Fig. 3 Candidate residues for tuning the affinity of cAMP binding.
a, Crystal structure analyses of the cAMP binding site of Epac2A suggests 10 positions that potentially affect the cAMP binding affinity. b, Alignment of a portion of the CNB domain of Epac1 with those of the CNG channel CNGA2 and CNGA3. Known positions at which mutations affect the affinity of the CNG channels to their ligand are shown (asterisks).
Extended Data Fig. 4 Responses of cAMPFIREs in absolute CFP/YFP ratios.
From left to right, n (cells) = 65, 53, 49, and 55. Black points indicate mean. The resting CFP/YFP ratios were (mean ± s.e.m): 0.164 ± 0.002 for Epac-SH187, and 0.222 ± 0.004 for cAMPFIRE-H; ***: P = 1.6 × 10−19, two-tailed Welch test.
Extended Data Fig. 5 pH dependence of cAMPFIRE responses.
Sensor signal (fluorescence ratio) in the absence (black; Apo) or presence (red) of 100 µM cAMP normalized to the dynamic range of the sensor at pH 7.3 under the same measurement conditions. Averaged data are presented as mean ± s.e.m. n = 3 measurements, each from a different protein aliquot, for each condition.
Extended Data Fig. 6 2pFLIM characterizations of cAMPFIREs in HEK cells and as immunoprecipitated proteins.
a, Representative images and lifetime decay curves of cAMPFIRE-M in HEK cells in the presence of the indicated stimuli. Representative of 10 cells from 4 coverslips. b & c, 2pFLIM response traces (b), and NE concentration-response curves in HEK cells fitted by the Hill equation (c), of Epac-SH187 and cAMPFIRE-L, M, and H in HEK cells. Hill coefficients = 0.32, 0.51, 0.79, and 0.84, respectively, for Epac-SH187 and cAMPFIRE-L, M, and H. From low to high concentrations, the n (cells) is: for cAMPFIRE-L, 8, 8, 8, 13, 8, and 8; for cAMPFIRE-M, 6, 8, 8, 10, 9, and 8; for cAMPFIRE-H, 8, 14, 7, 11, and 8; and for Epac-SH187, 9, 8, 11, 4, and 8. Dark lines and shaded areas in panel b, and points and error bars in panel c indicate mean and s.e.m., respectively. d, 2pFLIM response amplitudes of each sensor in HEK cells elicited by 25 µM forskolin and 50 µM IBMX. From left to right, n (cells) = 32, 45, 41, and 48. Bars indicate mean. ***: P < 0.001 for all comparisons, from left to right, P = 1.7 × 10−18, 1.1 × 10−28, and 8.2 × 10−39, dF = 35, 53, and 60, two-tailed Welch test. e, Representative lifetime decay curves of cAMPFIRE-L immunoprecipitated onto Protein A agarose beads in the presence of the indicated cAMP concentrations. Representative of 3 beads. f, 2pFLIM concentration-response curves of cAMPFIRE proteins immunoprecipitated onto Protein A agarose beads in response to cAMP. Points and error bars indicated mean and s.e.m., respectively. For cAMPFIRE-L, -M, and -H, respectively, n = 3, 4 and 4 beads; Hill coefficients = 0.65, 0.68, and 0.82.
Extended Data Fig. 7 Transfection and imaging of L2/3 neurons in the somatosensory cortex.
a, Example brain slice images (representative of 21 mice) from a mouse transfected with cAMPFIRE-L via in utero electroporation. The sensor-transfected neurons are shown in cyan. b, A post hoc coronal section from an experimental mouse (representative of 7 mice) showing the edge of cranial window (arrowheads), which was marked during perfusion and dissection of the brain. The window was implemented on comparable coordinates for all experimental mice.
Extended Data Fig. 8 Characterizations of cAMPFIRE responses in neurons.
a, Response traces of cAMPFIRE-M in CA1 neurons in cultured hippocampal slices to 0.1 µM norepinephrine in the absence or presence of 1 µM propranolol, followed by wash. n (cells) = 4 for both. Dark lines indicate mean and shaded areas indicate s.e.m. b, Response traces of cAMPFIRE-L and its R279E mutant in CA1 neurons in cultured hippocampal slices to 1 µM norepinephrine. cAMPFIRE-L data are the same as those in Fig. 5b. n (cells) = 5 for R279E mutant. Dark lines indicate mean and shaded areas indicate s.e.m. c, 2pFLIM responses in absolute values of cAMPFIRE-L and cAMPFIRE-M to forskolin and IBMX in different cell types, indicating that different cell types exhibit different baseline and dynamic ranges. From left to right, n (cells) = 21, 10, 12, 18, 16, and 10. Black points indicate mean.
Extended Data Fig. 9 Additional electrophysiological characterizations of cAMPFIREs in CA1 neurons.
a & b, Quantification of the elicited AMPAR and NMDAR currents (in pA) of CA1 neurons transfected with cAMPFIRE-L or cAMPFIRE-M compared to adjacent untransfected control neurons. For both, n = 10 neuronal pairs for cAMPFIRE-L and 16 for cAMPFIRE-M. Blue points and error bars indicate mean and s.e.m., respectively. c & d, example traces (c), and amplitudes (d) of mEPSCs (n = 9 for -L and 7 for -M) of CA1 neurons transfected with cAMPFIRE-L and cAMPFIRE-M compared to adjacent untransfected control neurons. e, Quantification of the paired pulsed ratio (n = 11 for -L and 15 for -M) of CA1 neurons transfected with cAMPFIRE-L and cAMPFIRE-M compared to adjacent untransfected control neurons. n.s.: not significant, paired two-tailed Student’s t-test. Colored points and error bars indicate mean and s.e.m., respectively. For panels d and e from left to right, P = 0.55, 0.87, 0.12, and 0.42, dF = 8, 6, 10 and 14.
Extended Data Fig. 10 cAMPFIREs detect cAMP responses in Drosophila larvae.
a, Schematic showing experimental setup. Larvae were dissected to expose the intact central nervous system (boxed region) and imaged with a water immersion objective. b & c, Average traces (b) and collective responses measured from the gray shaded area in panel b (c) of abdominal leucokinin neuronal somas expressing cAMPFIRE-M following application of forskolin (40 µM) or DMSO-only control. In panel b, Dark lines indicate mean and shaded areas indicate s.e.m. Arrowhead indicates time of bath application. In panel c, bars indicate mean. ***: P = 1.1 × 10−5, two-tailed Wilcoxon Rank-Sum test. n = 10 larvae per condition. d & e, Average traces (d) and collective responses measured from gray shaded area in panel d (e) of fly larval nociceptive axon terminals in response to bath application of AITC (allyl isothiocyanate; 2.5 mM) followed by forskolin (40 µM) and IBMX (100 µM). AITC is a stimulant for larval nociceptors. In panel d, dark lines indicate mean and shaded areas indicate s.e.m. In panel e, bars indicate mean. n.s.: not significant (p = 0.49), *: P = 0.049, **: P = 0.0048, two-tailed one-way ANOVA with Tukey’s multiple comparisons. n = 6 larvae per condition.
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Massengill, C.I., Bayless-Edwards, L., Ceballos, C.C. et al. Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo. Nat Methods 19, 1461–1471 (2022). https://doi.org/10.1038/s41592-022-01646-5
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DOI: https://doi.org/10.1038/s41592-022-01646-5
