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

Animals

All experimental procedures were performed using protocols that were approved by the Institutional Animal Care and Use Committee at Kyoto University (Lif-K23008), University of Yamanashi (A1-20), RIKEN (W2022-2-012) and The University of Tokyo (P18-118). Wild-type mice (C57BL/6N, ICR) and A2A-Cre ((B6.FVB (Cg)-Tg (Adora2a-cre) KG139Gsat/Mmucd)) mice were group-housed and kept on a 12-h light–dark cycle with ad libitum food and water. The housing conditions were controlled at room temperature of approximately 22–24 °C and a relative humidity of 40–60%. Wild-type mice used in this study were purchased from Japan SLC. Experiments were performed using both male and female sex between 0 and 20 weeks of age.

Cell lines

HEK293T cells were obtained from the American Type Culture Collection (CRL-11268). Cells were cultured in DMEM (08458-16, Nacalai) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 50 units per ml penicillin and 50 μg ml−1 streptomycin (26252-94, Nacalai) at 37 °C and 5% CO2 in a humidified atmosphere. Escherichia coli (E. Coli) DH5α and DH10B, and Stbl3 cells were obtained from Toyobo (DNA-9303), Invitrogen (18297010) and Invitrogen (C737303), respectively. Bacteria were incubated in Lysogeny Broth (LB) medium supplemented with antibiotics at 37 °C.

Plasmids

To develop cAMP sensors, PKA-R1α (amino acids 108–186 and 190–381) was obtained from a mouse cDNA library, and cpGFP and RSET domain were subcloned from GCaMP6f (Addgene plasmid, 52924). The F2A sequence was from XCaMP-R24.The RPL10 domain was from GCaMP6m-RPL10a22. G-Flamp1 was synthesized (GENEWIZ). To develop red Ca2+ sensors, the cpRFP domain was synthesized (GENEWIZ), and RSET, RS20 and CaM domains were obtained from jRGECO1a26. For site-directed mutagenesis, plasmid libraries were made using the inverse PCR method with PrimeSTAR Max DNA polymerase (R045A, Clontech), In-Fusion HD Cloning Kit (639650, Clontech), and primers which included NNK codons, where K = G or T. For expression in E. coli, cAMP sensors were subcloned into a pBAD vector45. For optogenetic stimulation, ChRmine-mScarlet-Kv2.1 was synthesized (GENEWIZ). For expression in HEK293T cells, Ca2+ or cAMP sensors were subcloned into a plasmid encoding CAG promoter and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). For expression of GPCRs–P2A–mCherry in HEK293T cells, pTRE3G-HA signal-Flag–GPCRs–P2A–mCherry-reverse-PGK–TetOn3G was made of Tet-ON 3G inducible expression system (Clontech) and PRESTO-Tango GPCR Kit46.

In vitro fluorometry for cAMP sensor screening

The plasmids for bacterial expression of cAMP sensors were transformed into E. coli strain DH10B. E. coli cells were plated and cultured at 37 °C on an LB agar plate with ampicillin and 0.0004% arabinose. Each colony was used to inoculate 1.5 ml of LB liquid medium with ampicillin and 0.2% arabinose and grown at 37 °C overnight. After centrifugation, cells were resuspended in 150 µl suspension buffer (20 mM MOPS (pH 7.2), 100 mM KCl, 1 mM dithiothreitol (DTT), cOmplete EDTA-free protease inhibitor cocktail; 11836170001, Roche), sonicated at 4 °C, and centrifuged. The supernatant was collected. For fluorometry, the supernatant was diluted 20-fold with the suspension buffer. The diluted supernatant was applied to 96-well plates. cAMP (A2381, Tokyo Chemical Industry) was added to a final concentration of 300 µM for cAMP-saturated conditions. Fluorometric measurements were performed on a microplate reader (Spark, TECAN) at room temperature at 485 nm of excitation (bandwidth of 20 nm) and 535 nm of emission (bandwidth 20 nm).

In vitro cAMP fluorometry for HEK cell lysate

HEK293T cells were incubated on six-well plates. Next, 1 µg DNA was transfected using X-tremeGENE HP DNA Transfection Reagent (6366244001, Roche). Two days after the transfection, the cells were harvested into the 150 µl suspension buffer described above, sonicated at 4 °C, and centrifuged. The supernatant was collected. For cAMP-saturated conditions, the supernatant was diluted 20-fold with suspension buffer, and cAMP was added to a final concentration of 300 µM. Fluorometric measurements were performed at 485 nm or 450 nm of excitation (bandwidth 20 nm) and 545 nm of emission (bandwidth 20 nm). Excitation and emission spectra were taken at 555 nm and 460 nm, respectively. For measurement of cAMP affinity, suspension buffer with 0, 3, 10, 30, 100, 300, 1,000, 3,000, 10,000, 30,000, 100,000 and 300,000 nM cAMP was prepared. Then, the supernatant was diluted 40-fold with each suspension buffer. For measurement of cGMP affinity, suspension buffer with 0, 100, 300, 1,000, 3,000, 10,000, 30,000, 100,000, 300,000 and 1,000,000 nM cGMP (ab120805, Abcam) was prepared. Then, the supernatant was diluted 40-fold with each suspension buffer. For measurement of pKa, the supernatant was diluted 40-fold with pH buffer (20 mM citrate (for pH 5–6), 20 mM MOPS (for pH 6.5–10))25, and cAMP was added to a final concentration of 100 µM. pKa values were determined from the inflection point of a sigmoid fit to fluorescence versus pH.

In vitro Ca2+ fluorometry for HEK cell lysate

Cell incubation, transfection and collection were performed as described above. For Ca2+-saturated or Ca2+-free conditions, the supernatant was diluted 20-fold with the Ca2+-EGTA buffer (30 mM MOPS (pH 7.2), 100 mM KCl, 10 mM EGTA, 10 mM CaCl2 and 1 mM DTT) or EGTA buffer (30 mM MOPS (pH 7.2), 100 mM KCl, 10 mM EGTA and 1 mM DTT), respectively. Fluorometric measurements were performed at 560 nm of excitation (bandwidth of 20 nm) and 610 nm of emission (bandwidth of 20 nm). Excitation and emission spectra were taken at the emission of 635 nm and at the excitation of 520 nm, respectively. For measurement of Ca2+ affinity, the supernatant was diluted 40-fold with a series of solutions with free Ca2+ concentration ranges from 0 nM to 3,900 nM (ref. 27). For measurement of pKa, the supernatant was diluted 40-fold with pH buffer (20 mM citrate (for pH 5–6), 20 mM MOPS (for pH 6.5–10)) containing 2 mM CaCl2 or 2 mM EGTA25. pKa values were determined from the inflection point of a sigmoid fit to fluorescence versus pH.

In vitro kinetics analysis

The plasmids for bacterial expression of cAMPinG1, which included His-tag sequence, were transformed into E. coli strain DH10B. E. coli cells were plated and cultured at 37 °C on an agar plate with ampicillin. A colony was used to inoculate 200 ml of LB liquid medium with ampicillin and 0.2% arabinose and grown at 18 °C for 44 h. After centrifugation, cells were resuspended in 10 ml suspension buffer (25 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT and cOmplete EDTA free (Sigma-Aldrich)), sonicated at 4 °C and centrifuged. The supernatant was collected.

Coverslips (no. 1, 15 mm diameter, Matsunami) were washed with 1% acetic acid solution for 6 h. Then, they were immersed in silanized solution containing 50% ethanol, 2% mercaptosilane and 1% acetic acid overnight, followed by drying at 143 °C for 1 h. They were immersed sequentially in reducing solution containing 50% ethanol, 2.5 mM ethylenediaminetetraacetic acid, 2 mM DTT and 100 mM phosphate buffer, pH = 7.0 for 1 h, 2.5 mg ml−1 maleimido-C3-NTA solution for 2 h, and 50 mM nickel sulfate solution for 1 h (ref. 9). The resultant nickel complex-coated coverslips were incubated in the supernatant including cAMPinG1 protein for 1 h at 4 °C. The cAMPinG1-binding coverslips were placed on the stage of an FVMPE-RS (Olympus) microscope equipped with a water-immersion ×25 objective lens (N.A.: 1.05, Olympus), a femtosecond laser (Insight DS+, Spectra-Physics) and GaAsP detector (Hamamatsu Photonics) with a 495–540-nm emission filter (Olympus). The laser was tuned at 920 nm. For measurement of binding kinetics (Extended Data Fig. 2g), cAMP was microperfused to the surface of the coverslips from 0 μM to 2 μM with a micropipette and a microinjector (BEX). Images (288 × 18 µm2, 128 × 8 pixels) were collected at 81.6 Hz. For measurement of dissociation kinetics (Extended Data Fig. 2h), cAMP was microperfused from 0.5 μM to 0 μM. Images (576 × 108 µm2, 256 × 48 pixels) were collected at 12.7 Hz.

cAMP imaging in HEK293T cells

For time-lapse imaging (Fig. 1h–j), HEK293T cells were incubated in 35-mm glass-bottom dishes. DNA encoding the sensors (1 µg) was transfected as described above. One day after the transfection, the culture medium was replaced with Tyrode’s solution (129 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM HEPES–NaOH, pH 7.4, 2 mM CaCl2, 2 mM MgCl2). Imaging for cAMPinG1 was performed using an LSM 880 confocal microscope (Carl Zeiss) with an air-immersion ×20 objective lens (N.A.: 0.80, Carl Zeiss). Then, 405-nm and 488-nm lasers were used for excitation in turns. Forskolin (16384-84, Nacalai) was added to a final concentration of 50 µM.

For single-timepoint imaging for GPCRs (Fig. 6), HEK293T cells were incubated in 96-well glass-bottom plates. Around 0.1 µg DNA encoding the sensors was transfected as described above. One day after the transfection, the culture medium was replaced with Tyrode’s solution. Imaging was performed using an LSM 880 confocal microscope with the ×20 objective lens. For tetracycline-dependent expression of GPCRs–P2A–mCherry, doxycycline was added to a final concentration of 100 ng ml−1 3 h before the imaging. Twenty minutes before the imaging, the culture medium was replaced with Tyrode’s solution with or without forskolin, dopamine (14212-71, Nacalai), clozapine (12059, Cayman), adrenocorticotropic hormone (AP3295, AdooQ,) or serotonin (18961-41, Nacalai). Lasers (405 nm and 488 nm) were used for ratiometric cAMP imaging, and 561-mn and 633-nm lasers were used for the visualization of GPCR-expressing cells.

For single-timepoint imaging for sensor comparison (Extended Data Fig. 9a–c), HEK293T cells were incubated in 96-well glass-bottom plates, transfected and imaged as described above. Imaging was performed to calculate the relative change (ΔR/R) in fluorescence ratio (R) in the absence or presence of 10 μM forskolin. For cAMPinG1 and G-Flamp1, R is the ratio of green fluorescence (491–553 nm of emission) with 488 nm of excitation to that with 405 nm of excitation. For cAMPFIRE-L, R is the ratio of cyan fluorescence (464–499 nm of emission) with 458 nm of excitation to yellow fluorescence (526–597 nm of emission) with 514 nm of excitation.

Binding assay in HEK293T cells

HEK293T cells were incubated in 35-mm glass-bottom dishes. DNA encoding the sensors (1 µg) was transfected as described above. One day after the transfection, the culture medium was replaced with Tyrode’s solution as described above. Imaging was performed using an LSM 880 confocal microscope with an oil-immersion ×40 objective lens (N.A.: 1.30, Carl Zeiss). Images (53.1 × 53.1 µm2, 512 × 512 pixels) were collected using 488-nm and 561-nm lasers.

Fluorescence lifetime measurement

HEK293T cells in 96-well glass-bottom plates were prepared as described above. Twenty minutes before the imaging, the culture medium was replaced with Tyrode’s solution with or without 10 µM forskolin. Imaging was performed using a TCS SP8 FALCON microscope (Leica) at a pulse frequency of 80 MHz with an air-immersion ×20 objective lens (N.A.: 0.75, Leica). Excitation was 488 nm by white-light laser and emission was 500–550 nm. Images (233 × 233 µm2, 256 × 256 pixels) were collected. The lifetime was analyzed using the LAS X FLIM/FCS software (Leica).

Two-photon Ca2+ imaging in HEK293T cells

HEK293T cells were incubated in 35-mm glass-bottom dishes. A mixture of 0.8 µg DNA encoding the red Ca2+ sensors and 0.2 µg DNA of pCMV-mCerulean was transfected as described above. One day after the transfection, the culture medium was replaced with Tyrode’s solution as described above. Thirty seconds after bath application of ionomycin (Cayman Chemical, 11932) to a final concentration of 5 µM, two-photon imaging was performed with an FVMPE-RS (Olympus) equipped with a water-immersion ×25 objective lens (N.A.: 1.05, Olympus), a femtosecond laser (Insight DS+, Spectra-Physics) and two GaAsP detectors (Hamamatsu Photonics) with 495–540-nm and 575–645-nm emission filters (Olympus). Images (339 × 339 µm2, 1,024 × 1,024 pixels, single optical section) were collected. The laser was tuned to 880 nm for mCerulean and 1,040 nm at the front aperture of the objective for the red Ca2+ sensors.

Photostability analysis

HEK293T cells were prepared in 96-well glass-bottom plates as described above. For one-photon bleaching measurements (Extended Data Fig. 4k), imaging was performed using an LSM 880 confocal microscope with a ×20 objective lens immersed with Tyrode’s solution. A 561-nm laser was used for excitation. Images (170 × 170 µm2, 512 × 512 pixels) were collected at 0.6 Hz. For two-photon bleaching measurement (Extended Data Fig. 4l), imaging was performed using a FVMPE-RS microscope with the ×20 objective lens in Tyrode’s solution. A 1,040-nm laser was used for excitation. Images (255 × 255 µm2, 256 × 256 pixels) were collected at 0.82 Hz.

Stable cell lines generation

Lentiviral particles were produced by transfection of the packaging plasmids with polyethylenimine into HEK293T cells47. Lentivirus-infected HEK293T cells were dissociated and isolated into multi-well plates. Single clones with bright fluorescence were picked, grown and stored at −80 °C. To establish a triple stable cell line (Extended Data Fig. 9), cAMPinG1 single stable cell line was infected with lentivirus encoding TRE3G–DRD1–EF1a–TetOn3G–P2A–mCherry–NLSx3 and TRE3G–MC3R–EF1a–TetOn3G–P2A–iRFP670 (ref. 48).

Cell proliferation assay

The HEK293T cells were seeded on six-well plates with 2 ml DMEM and 10% FBS or 1.5% FBS and incubated at 37 °C. The 1.5% FBS group was a positive control of slow cell proliferation. Twenty hours after the beginning of the culture, cells in half of the wells were harvested, and the number of cells was counted using a counting chamber as a timepoint of zero. Sixty hours after the beginning of the culture, cells in the other half of the wells were harvested and counted as a timepoint of 48 h.

In utero electroporation

ICR pregnant mice (Japan SLC) were anesthetized with an anesthetic mixture (0.075 mg ml−1 medetomidine hydrochloride, 0.40 mg ml−1 midazolam and 0.50 mg ml−1 butorphanol tartrate) and administered at 100 μl per 10 g of body weight intraperitoneally. Then, 2.0 µl of purified plasmid (1.0 µg µl−1 final concentration in each sensor) was injected into the right lateral ventricle of embryos at embryonic day 15. pCAG-green cAMP sensors-WPRE (cAMPinG1-NE, cAMPinG1mut-NE, G-Flamp1) and pCAG-RCaMP3-WPRE were delivered to induce the expression of cAMP and Ca2+ indicators in L2/3 pyramidal neurons in the V1. After soaking the uterine horn with warm saline (37 °C), each embryo’s head was carefully held between tweezers with platinum 5-mm disk electrodes (CUY650P5, Nepagene). Subsequently, five electrical pulses (45 V, 50-ms duration at 1 Hz) were delivered by an electroporator (NEPA21, Nepagene)49,50. Electroporated mice were used for cAMP and calcium imaging 4–10 weeks after birth.

AAV production and injection

Recombinant AAVs were produced using HEK293T cells and purified by AVB Sepharose51. The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 1013 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 1013 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 1012 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. 3a–d); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 1013 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 1013 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. 3e–h); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 1013 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 1013 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. 3m–o); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 1013 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 1013 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. 3d–n); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 1013 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 1013 GC per ml) for two-photon mesoscale Ca2+ imaging (Fig. 3o,p); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 1013 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 1013 GC per ml) for inclusion counting (Extended Data Fig. 5a,b); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 1013 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 1013 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 1013 GC per ml) for two-photon imaging in the V1 (Figs. 4 and 5a–g). AAV2/1-eSyn-cAMPinG1-NE (7.0 × 1012 GC per ml) and AAV2/1-eSyn-G-Flamp1 (7.0 × 1012 GC per ml) for confocal imaging in fixed tissues (Extended Data Fig. 5f, g); AAV2/1-hSyn-iCre (2.0 × 1010 GC per ml), AAV2/1-CAG-DIO-cAMPinG1-NE (3.0 × 1013 GC per ml), AAV2/1-CAG-DIO-RCaMP3 (1.0 × 1013 GC per ml, for an infection marker) and AAV2/1-CAG-DIO-ChRmine-mScarlet-Kv2.1 (1.0 × 1012 GC per ml) for cAMP imaging and optogenetic stimulation (Fig. 5h–k); AAV2/1-eSyn-cAMPinG1-NE (7.0 × 1012 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 1013 GC per ml, for an infection marker) for fiber photometry in the V1 (Extended Data Fig. 6e–g); AAV2/1-gfaABC1D-cAMPinG1-NE (1.0 × 1013 GC per ml) and AAV2/1-gfaABC1D-RCaMP3 (1.0 × 1013 GC per ml) for astrocyte imaging (Extended Data Fig. 6h–j). AAV2/1-eSyn-G-Flamp1 (3.0 ×1013 GC per ml) for two-photon imaging in the V1 (note that the G-Flamp1 fluorescence diminishes when injecting a mixture of G-Flamp1 and RCaMP3 AAVs for coexpression; Extended Data Fig. 7c,d); AAV2/1-eSyn-cAMPinG1-NE (7.0 × 1012 GC per ml), AAV2/1-eSyn-cAMPinG1mut-NE (7.0 × 1012 GC per ml) or AAV2/1-eSyn-G-Flamp1 (7.0 × 1012 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 1013 GC per ml) for fiber photometry in the dorsal striatum (Extended Data Fig. 8).

Stereotaxic virus injection was performed to C57BL/6N male mice aged 4–6 weeks anesthetized by the anesthetic mixture described above except for two-photon mesoscale imaging. A2A-Cre transgenic mice were used for the slice experiments for local dopamine application (Extended Data Fig. 3e–h). A micropipette was inserted into the right V1 (A/P −3.85 mm, M/L +2.7 mm from bregma, D/V −0.30 mm from the pial surface), the barrel cortex (A/P −1.0 mm, M/L −3.0 mm from bregma, D/V −0.20 mm from the pial surface), the right dorsal striatum (A/P +0.5 mm, M/L +1.8 mm from bregma, D/V −1.5 mm from the pial surface), the nucleus accumbens (A/P +1.3 mm, M/L ±1.25 mm from bregma, D/V −4.5 mm) or the medial prefrontal cortex (A/P +1.8 mm, M/L +0.3 mm from bregma, D/V −2.4 mm). Then, the virus solution (volume: 500−1,000 nl) was injected. Carprofen (5 mg per kg of body weight, Zoetis) was administered intraperitoneally just after the injection experiment. Mice were subjected to imaging after 4–12 weeks of the injection.

cAMP imaging in acute brain slices

For cAMP imaging with forskolin/IBMX bath application (Extended Data Fig. 3a–d), AAV (AAVPHP.eB-CaMKII-Cre and AAV2/1-CAG-DIO-cAMPinG1, or AAV2/1-CAG-DIO-cAMPinG1mut) was injected into the V1 at a total volume of 500 nl (ref. 52). For cAMP imaging with local dopamine application (Extended Data Fig. 3e–h), AAV (AAV2/1-eSyn-cAMPinG1mut-NE, AAV-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS) was injected into the nucleus accumbens at a total volume of 1,000 nl. After 2 weeks of expression, mice were killed by rapid decapitation after anesthesia with isoflurane. The brains were immediately extracted and immersed in gassed (95% O2/5% CO2) and ice-cold solution containing: 220 mM sucrose, 3 mM KCl, 8 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3 and 25 mM glucose. Acute coronal brain slices (280 μm thick) of the visual cortex were cut in gassed ice-cold solution with a vibratome (VT1200, Leica). Brain slices were then transferred to an incubation chamber containing gassed artificial cerebrospinal fluid (ACSF) containing: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM CaCl2, 2 mM MgCl2 and 20 mM glucose at 34 °C for 30 min and subsequently maintained at room temperature before transferring them to the recording chamber and perfused with the ACSF solution described above, except using 2 mM CaCl2 and 1 mM MgCl2 at 30–32 °C. cAMP imaging was performed with an upright microscope (BX61WI, Olympus) equipped with an FV1000 laser-scanning system (FV1000, Olympus) and a water-immersion ×60 objective (N.A.: 1.0, Olympus), a femtosecond laser (MaiTai, Spectra-Physics) and a GaAsP detector (Hamamatsu Photonics) with a 500–550-nm emission filter (Semrock). The laser was tuned at 940 nm (2 mW at the front aperture of the objective). For cAMP imaging with forskolin/IBMX bath application (Extended Data Fig. 3a–d), images (105.6 × 105.6 µm2, 640 × 640 pixels) were taken every 30 s. During the imaging, forskolin (067-02191, Wako) and IBMX (3758, Tocris) were added to a final concentration of 25 µM and 50 µM, respectively. For cAMP imaging with local dopamine application (Extended Data Fig. 3e–h), images were taken at 2 Hz. During the imaging, 1 µM dopamine was microperfused with a micropipette.

For cAMP imaging with norepinephrine bath application (Extended Data Fig. 3i–l), in utero electroporation was performed as described above. Four weeks after birth, mice were killed by rapid decapitation after anesthesia with the anesthetic mixture described above. The brains were immediately extracted and immersed in gassed (95% O2/5% CO2) and ice-cold solution containing: 222 mM sucrose, 3.6 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 1.5 mM NaH2PO4 and 27 mM NaHCO3. Acute coronal brain slices (300 μm thick) of the visual cortex were cut in gassed ice-cold solution with a vibratome (VT1200, Leica). Brain slices were then transferred to an incubation chamber containing gassed ACSF containing: 126 mM NaCl, 3 mM KCl, 1.1 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2 and 10 mM glucose at room temperature for 30 min. The brain slices were transferred to the recording chamber and perfused with the ACSF solution described above. cAMP imaging was performed with FVMPE-RS (Olympus) equipped with a water-immersion ×25 objective lens (N.A.: 1.05, Olympus), a femtosecond laser (Insight DS+, Spectra-Physics) and two GaAsP detectors (Hamamatsu Photonics) with 495–540-nm emission filters (Olympus). The laser was tuned at 940 nm. Images (339 × 339 µm2, 256 × 256 pixels) with 36 optical planes with planes spaced 2 µm apart in depth were collected every 31 s. During the imaging, norepinephrine (A0906, Tokyo Chemical Industry) was added to a final concentration of 0.5 µM.

Electrophysiology for characterization of cAMPinG1

For characterization of cAMPinG1 in acute brain slices (Extended Data Fig. 3m–o), AAV (AAV2/1-eSyn-cAMPinG1-NE, AAVPHP.eB-hSyn-EGFP) was injected into the medial prefrontal cortex of mice aged 8 weeks at a volume of 500 nl. After 2 weeks of expression, mice were decapitated, and acute brain slices were prepared as described above. Slices were perfused with oxygenated ACSF (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose and 200 μM Trolox). Whole-cell recordings were performed by 5–6 MΩ glass pipettes. Patch pipettes were filled with an internal solution (120 mM potassium gluconate, 20 mM KCl, 10 mM disodium phosphocreatine, 4 mM ATP (magnesium salt), 0.3 mM GTP (sodium salt), 10 mM HEPES (pH 7.25, 293 mOsm). Electrophysiological data were acquired using a patch-clamp amplifier (MultiClamp 700B, Molecular devices) and stored using a Digidata 1440A converter and pCLAMP software (Molecular Devices). To assess the excitability, the spike number was measured by injecting pulses of increased intensity in steps of 25 pA (from 0 to 250 pA, 500 ms duration). For miniature excitatory postsynaptic current measurement, tetrodotoxin (0.2 μM to a final concentration), APV (50 μM) and picrotoxin (25 μM) were added and membrane potential voltage clamped at −70 mV after correction of liquid-junction potential was used.

Tissue preparation

Tissue blocks were cut into 50-μm-thick slices with a cryostat (CM1950, Leica) and floated in PBS. For nuclear staining, brain slices were incubated with DAPI for 10 min at room temperature before being mounted on slides. Imaging was performed using an LSM 880 confocal microscope with a ×20 objective lens and 405-nm, 488-nm and 561-nm lasers. For inclusion counting of Ca2+ sensors, images (142 × 142 µm2, 512 × 512 pixels) were acquired across multiple optical planes, each spaced 2 µm apart in depth from L2/3 neurons in the V1.

Simultaneous Ca2+ imaging and whole-cell recordings in acute brain slices

AAV (AAV2/1-eSyn-jRGECO1a, AAV2/1-eSyn-RCaMP3) was injected into the barrel cortex (A/P −1.0 mm, M/L −3.0 mm from the bregma, D/V −0.2 mm from the pial surface) at 20 nl min−1 at a volume of 500 nl. After 4 weeks of expression, mice were killed by rapid decapitation after anesthesia with pentobarbital (100 mg per kg body weight). The brains were immediately extracted and immersed in gassed (95% O2/5% CO2) and ice-cold ACSF containing: 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2 and 10.1 mM glucose. Acute coronal brain slices (300 μm thick) of the barrel cortex were cut in gassed, ice-cold ACSF with a vibratome (VT1200S, Leica). Brain slices were then transferred to an incubation chamber containing gassed ACSF at 30 °C for 60 min and subsequently maintained at room temperature before transferring them to the recording chamber at 35 °C.

Whole-cell recordings were performed in the L2/3 pyramidal neurons of the barrel cortex with glass recording electrodes (5–8 MΩ) filled with the intracellular solution containing: 130 mM K-gluconate, 4 mM NaCl, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na-GTP, 7 mM dipotassium-phosphocreatine and pH adjusted to 7.0 with potassium hydroxide (296 mOsm). Electrophysiological data were acquired using a patch-clamp amplifier (MultiClamp 700B, Molecular devices) filtered at 10 kHz and sampled at 20 kHz. Single action potentials were evoked by injecting a series of current pulses (2 ms in duration) through the patch pipette. Each trial was repeated, and the mean value was presented.

Calcium imaging was performed using an upright microscope (BX51WI, Olympus) with a water-immersion ×40 objective lens (N.A.: 0.8, Olympus). To acquire RCaMP3 and jRGECO1a images with LED light (MCWHLP1, Thorlabs), a U-MWIG3 fluorescence mirror unit (Olympus) was used. Fluorescent images were captured by a sCMOS camera (Orca-Flash 4.0 v3, Hamamatsu Photonics) controlled by HC Image software (Hamamatsu Photonics). Images were acquired at 50 Hz with 1 × 1 binning.

Simultaneous calcium imaging and cell-attached recordings in vivo

AAV (AAV2/1-eSyn-jRGECO1a, AAV2/1-eSyn-RCaMP3) was injected into the barrel cortex (A/P −1.0 mm, M/L −3.0 mm from the bregma, D/V −0.2 mm from the pial surface) at 20 nl min−1 at a volume of 500 nl. After 4 weeks of expression, mice were head-fixed and anesthetized with isoflurane (~1.5–2.0%) throughout the experiment, and body temperature was kept at 37 °C with a heating pad. A craniotomy was made in the barrel cortex. The exposed brain was covered with 1.5% agarose in ACSF containing the following: 150 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 2 mM CaCl2 and 1 mM MgCl2, pH 7.3. A glass coverslip was then placed over the agarose to suppress the brain motion artifacts. A glass electrode (5–8 MΩ) was filled with ACSF containing Alexa 488 (200 µM). jRGECO1a or RCaMP3-expressing neurons were targeted using two-photon microscopy (Movable Objective Microscope, Sutter) with a tunable laser (InSight X3, Spectra-Physics) and a water-immersion ×16 objective lens (N.A.: 0.80, Nikon). Fluorescence signals were collected using a GaAsP photomultiplier tube (Hamamatsu Photonics) with a 590–660-nm emission filter. After establishing the cell-attached configuration (20–100 MΩ seal), simultaneous spike recording and calcium imaging were performed at the soma (sampling rate = 30 Hz, 512 × 512 pixels). Electrophysiological data were acquired using a patch-clamp amplifier (MultiClamp 700B; Molecular devices) in current-clamp mode, filtered at 10 kHz, and sampled at 20 kHz. The laser was tuned to 1,040 nm (40 mW at the front aperture of the objective).

Fiber photometry

A 400-µm-diameter mono fiber-optic cannula (Kyocera) was implanted. A custom-made metal head plate was attached to the skull with dental cement. Mice were subjected to imaging after more than 2 days of the surgery.

Dual-color fiber photometry for green cAMP sensors and RCaMP3 was performed using the GCaMP and Red Fluorophore Fiber Photometry System (Doric) with 405-nm, 470-nm and 560-nm LED, 500–540-nm and 580–680-nm emission filter, and 400-µm-diameter 0.57-N.A. Mono Fiber-optic Patch Cords (Doric). Photometry data were recorded at a sampling rate of 30 Hz by lock-in amplifier detection. Mice were head-fixed during the recordings.

For cAMP imaging in the V1, on the first day, just after the first recording for 30-s forced running, 25 mg per kg body weight propranolol (168-28071, Wako) or mock solution was intraperitoneally administered. Thirty minutes later, the second recording was performed. On the subsequent day, the same two recordings were performed for the same mice, but with the reversed treatments. Half of the mice received propranolol on the first day and the other half received mock solution on the first day.

Cranial window implantation

Mice were anesthetized by the anesthetic mixture described above. Before surgery, dexamethasone sodium phosphate (2 mg per kg body weight, Wako) and carprofen (5 mg per kg of body weight) were administered to prevent inflammation and pain. During surgery, mice were put on a heating pad, and body temperature was kept at 37 °C. A custom-made stainless-steel head plate was fixed to the skull using cyanoacrylate adhesive and dental cement (Sun Medical) above the right visual cortex. A craniotomy was drilled with a 2.5-mm diameter, and the brain was kept moist with saline. A cover glass (3-mm diameter, no. 0 thickness, Warner Instruments) was placed over the craniotomy site with surgical adhesive glue (Aron Alpha A, Sankyo)49,50. The mice were subjected to imaging more than 18 h after the surgery.

In vivo two-photon imaging

In vivo two-photon imaging was performed with an FVMPE-RS (Olympus) equipped with a water-immersion ×25 objective (N.A.: 1.05, Olympus), a femtosecond laser (Insight DS+, Spectra-Physics) and two GaAsP detectors (Hamamatsu Photonics) with 495–540-nm and 575–645-nm emission filters (Olympus). For somatic cAMP imaging of cAMPinG1, cAMPinG1mut and G-Flamp1 expressed by in utero electroporation, images (339 × 339 µm2, 512 × 512 pixels, single optical section) were collected at 15 Hz in the awake condition. The laser was tuned to 940 nm (48.6 mW at the front aperture of the objective). For cAMPinG1-NE spine imaging, images (28.8 × 38.4 µm2, 96 × 128 pixels, single optical section) were collected at 7.5 Hz in the condition anesthetized lightly by isoflurane (0.5% vol/vol). The laser power was 23.5 mW at the front aperture of the objective. For RCaMP3 and cAMPinG1-ST imaging, sequential excitation at 940 nm and 1,040 nm was used for dual-color imaging. Images (339 × 339 µm2, 512 × 512 pixels) with three optical planes with plane spaced 30 µm apart in depth were collected at 3.4 Hz per plane using a piezo objective scanner (Olympus). The laser power was set to 47.6 mW for 940 nm excitation, and to 113.7 mW for 1,040 nm excitation. The imaging with visual stimuli (Fig. 5a–g) was followed by the imaging during forced running (Fig. 4) on the same cell population. For cAMP and Ca2+ imaging in astrocytes, images (288 × 384 µm2, 192 × 256 pixels) were collected at 1.6 Hz in the awake condition. The laser power for 940 nm of excitation was set to 26.5 mW, and for 1,040 nm of excitation, it was set to 52.2 mW. For single-cell cAMP imaging with optical stimulation using soma-targeted ChRmine, images (86.4 × 115.2 µm2, 192 × 256 pixels) were collected at 3.2 Hz in the awake condition with 940 nm of excitation. Next, 1,040-nm two-photon excitation was used for 4-s, 16-Hz spiral scanning with 11-µm diameter. The imaging with a 940-nm laser was temporally stopped during the optical excitation. The laser power for 940-nm excitation was set to 4.1 mW, and for 1,040 nm of photostimulation, it was set to 39.8 mW.

Two-photon mesoscale Ca2+ imaging with FASHIO-2PM

AAV (AAV2/1-eSyn-RCaMP3) was injected into the neonatal somatosensory cortex53. After 8 weeks of AAV injection, a 4.5-mm-diameter craniotomy was performed over an area including the primary somatosensory area of the right hemisphere. A head plate was also fixed to the skull above the cerebellum.

Two-photon imaging was performed with FASHIO-2PM29 equipped with a femtosecond laser (Chameleon Discovery, Coherent). The laser was tuned to 1,040 nm. The field of view was 3.0 × 3.0 mm2 (2,048 × 2,048 pixels). The sampling rate was 7.5 Hz. Laser power of 270 mW and 360 mW at the front of the objective lens was used to observe L2/3 and L5 neurons of awake mice, respectively.

Physical stimulation

Airpuff stimuli (2 Hz, 0.1 s duration, 40 times) were generated using a microinjector (BEX). For the forced running task, mice were head-fixed, and a custom-made treadmill was turned on during recordings. Moving grating stimuli were generated using the PsychoPy function in Python34. The gratings were presented with an LCD monitor (19.5 inches, 1,600 × 900 pixels, Dell), placed 25 cm in front of the center of the left eye of the mouse. Each stimulus trial consisted of a 4-s blank period (uniform gray at mean luminance) followed by a 4-s drifting sinusoidal grating (0.04 cycles per degree, 2 Hz temporal frequency). Eight drifting directions (separated by 45°, in order from 0° to 315°) were used. The timing of each moving grating stimulus and the initiation of imaging were monitored with a data acquisition module (USB-6343, National Instruments) controlled by LabVIEW (2021).

Analysis of in vitro experiments

In vitro fluorometry analysis was performed using Python (https://www.python.org/) and Excel (Microsoft). For both Ca2+ and cAMP sensors, the Kd value and Hill coefficient were calculated by fitting according to the Hill equation. For both Ca2+ and cAMP sensors, the pKa values were calculated by the inflection point of a sigmoid fit to fluorescence versus pH. For in vitro kinetics analysis (Extended Data Fig. 2g,h), half-rise time and half-decay time were calculated by single exponential fitting.

Image analysis

Image analyses were performed with ImageJ (National Institutes of Health) and Python. For somatic cAMP imaging of cAMPinG1, cAMPinG1mut and G-Flamp1 expressed by in utero electroporation (Fig. 2a–f), somatic cAMP imaging expressed by AAV (Extended Data Fig. 6a–d and Extended Data Fig. 7) and simultaneous RCaMP3 and cAMPinG1-ST imaging (Figs. 4 and 5a–g and Extended Data Fig. 7e), motion correction was performed with Suite2p toolbox (https://github.com/MouseLand/suite2p/)54. For cAMPinG1-NE spine imaging (Fig. 2g–i), mesoscale Ca2+ imaging (Fig. 3o,p and Extended Data Fig. 5c,d), Ca2+ and cAMP imaging in astrocytes (Extended Data Fig. 6h–j) and cAMP imaging with optical stimulation using soma-targeted ChRmine (Fig. 5h–k), motion correction was performed with TurboReg55.

Region of interest (ROI) detection for in vivo Ca2+ imaging was performed with Suite2p. ROI detection for HEK cell live imaging and in vivo cAMP imaging were performed with ImageJ and Cellpose56. ROIs for cAMPinG1-NE spine imaging (Fig. 2g–i), Ca2+ and cAMP imaging in astrocytes (Extended Data Fig. 6h–j) and cAMPinG1-NE imaging with soma-targeted ChRmine (Fig. 5h–k) were drawn manually. The dynamic range was calculated as ΔF/F = (F – F0)/F0, where F0 was the average fluorescence intensity before stimulations after the subtraction of background fluorescence. No bleaching correction was performed in any analyses except Ca2+ imaging in acute brain slices (Fig. 3d–h). No fluorescence cross-talk correction was performed.

For cAMP imaging using acute brain slices (Extended Data Fig. 3), background subtraction was performed before calculating ΔF/F. ROIs were drawn manually around somata (Extended Data Fig. 3a–h) or with Cellpose (Extended Data Fig. 3i–l) in the time-series-averaged image. ΔF/F was calculated as (F − F0)/F0, where F is the fluorescence intensity at any timepoint and F0 is the average fluorescence before the drug application.

For in vivo cAMPinG1-NE spine imaging (Fig. 2g–i), the period after stimulation was defined as a 15-s period starting 10 s after the end of airpuff stimulation.

For two-photon Ca2+ imaging in HEK293T cells (Fig. 3c), ROIs drawn based on mCerulean images with Cellpose were used for both Ca2+ sensor and mCerulean images. The red fluorescence intensity was divided by mCerulean fluorescence intensity in each cell for normalization.

For Ca2+ imaging using acute brain slices (Fig. 3d–h), background subtraction and bleach correction were performed before calculating ΔF/F. ROIs were manually selected around somata in the time-series-averaged image. ΔF/F was calculated as (F − F0)/F0, where F is the fluorescence intensity at any timepoint and F0 is the resting baseline fluorescence measured 200 ms before stimulation. The peak amplitude was defined as the maximum value of ΔF/F after the stimuli. The rise and decay curves were fit to a single exponential. The rise time was defined as the time from the beginning of the stimulus to the timepoint of the peak fluorescence amplitude. The half-decay time was defined as the time from the maximum value of ΔF/F to half of that value.

For simultaneous Ca2+ imaging and cell-attached recordings in vivo (Fig. 3i–n), ROIs were manually selected around somata in the time-series-averaged image. ΔF/F was calculated as (FF0)/F0, where F is the fluorescence intensity at any timepoint and F0 is resting baseline fluorescence measured 200 ms before the action potentials. Action potentials were detected by cell-attached recording of the signal. Spike events (1–4 action potentials) were identified, ensuring that no other action potentials occurred in the 1-s period before and after the first action potential. The rise and decay curves were fit to a single exponential. The half-rise time was defined as the time from the beginning of the stimulus to the timepoint of the half of the peak fluorescence amplitude. The half-decay time was defined as the time from the maximum value of ΔF/F to half of that value.

For in vivo Ca2+ and cAMP imaging (Figs. 4 and 5a–g and Extended Data Figs. 6a–d and 7), ROIs for RCaMP3 and cAMP indicators were drawn independently using Suite2p and Cellpose, respectively. For simultaneous RCaMP3 and cAMPinG1-ST imaging (Figs. 4 and 5a–g), the cells that had ROIs for both RCaMP3 and cAMPinG1-ST were selected for further analysis. Because the imaging using visual stimulus (Fig. 5a–g) was followed by the imaging using forced running (Fig. 4) on the same cell population, the same ROIs were used for both analyses. Motion-related neurons were defined as neurons that showed Ca2+ ΔF/F values of more than 0.3 during the running period (Fig. 4). Ca2+ responses to 4 s of visual stimulation were defined as averaged ΔF/F during the 4-s stimulation. cAMP responses to 4 s of visual stimulation were defined as averaged ΔF/F during 2-s periods that started 2 s after the end of the visual stimulus (Fig. 5a–d). Neurons that responded to repetitive 8-s visual stimuli were defined as neurons that showed Ca2+ ΔF/F values of more than 0.3 during the stimulation period (Fig. 5e–g). The period after repetitive visual stimuli was defined as a 40-s period starting after the end of the stimuli. The DSI was calculated for cells showing Ca2+ responses. The preferred direction (θpref) of each cell was defined as the stimulus that induced the largest Ca2+ ΔF/F. The DSI was defined as DSI = (Rpref – Rpref+π)/(Rpref + Rpref+π), where Rpref and Rpref+π are ΔF/F values at the preferred (θpref) and the opposite (θpref + π) directions, respectively. Imaging frames with notable motion artifacts were removed and supplied with the preceding frames.

For cAMPinG1-NE imaging with soma-targeted ChRmine (Fig. 5k), cAMP ΔF/F was defined as averaged ΔF/F during a 10-s period starting 20 s after the end of optical stimulation.

Analysis of fiber photometry

Fiber photometry analysis was performed using Python. The cAMPinG1 signal was calculated as follows: (470-nm signal)/(405-nm signal). The 560-nm signal was recognized as the RCaMP3 signal.

For imaging in the V1 (Extended Data Fig. 6e–g), ΔR/R was calculated as (R − R0)/R0, where F is mean fluorescence intensity in the last 10 s of the running period and F0 is the mean fluorescence intensity in 10 s before the start of running. ΔR/R (pre) and ΔR/R (post) were defined as ΔR/R in response to running before and after the intraperitoneal administration, respectively.

For imaging in the dorsal striatum (Extended Data Fig. 8), the period during stimulation was defined as a 10-s running period, and the period after stimulation was defined as 10 s after the end of the running period.

Statics and reproducibility

All experiments were conducted with at least two biological replicates, often more, involving independent transfection and mice. All statistical analyses of the acquired data were performed with Python. For each figure, a statistical test matching the structure of the experiment and the structure of the data was employed. All tests were two tailed. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (P > 0.05) for all statistical analyses presented in figures. No statistical tests were done to predetermine the sample size. Data acquirement and analysis were not performed blind to the conditions of the experiments. Experimental sample sizes are mentioned in the figure panel and legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.