Journal: Gene Therapy

Article Title: Recent development of AAV-based gene therapies for inner ear disorders

doi: 10.1038/s41434-020-0155-7

Figure Lengend Snippet: The brief history of gene therapy in hearing diseases.

Article Snippet: TMC −/− mice [ ] , AAV2/1-Cba-Tmc , P0–2 Route: RWM , ~20–30 dB (110 dB of control) , IHCs and OHCs/restore the sensory transduction current of HCs, SCs, and SGs..

Techniques: Injection, Mouse Assay, Control, Membrane, In Utero, Transduction

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , Primary structures of red Ca 2+ sensors. Mutations are indicated in R-GECO1 numbering. b , Fluorescence intensities of red Ca 2+ sensors in Ca 2+ -free and Ca 2+ -saturated conditions in HEK293T cell lysate. n = 4 wells in each sensor. Tukey’s post hoc test following one-way ANOVA. Ca 2+ -free jRGECO1a vs Ca 2+ -free RCaMP3: P = 2.6 × 10 −5 ; Ca 2+ -free XCaMP-R vs Ca 2+ -free RCaMP3: P = 1.9 × 10 −7 . Ca 2+ -saturated jRGECO1a vs Ca 2+ -saturated RCaMP3: P = 1.3 × 10 −9 ; Ca 2+ -saturated XCaMP-R vs Ca 2+ -saturated RCaMP3: P = 1.4 × 10 −12 . c-e , Excitation and emission spectra of jRGECO1a, XCaMP-R, and RCaMP3 in Ca 2+ -free and Ca 2+ -saturated states. n = 4 wells in each condition. f , Comparison of excitation spectra of red Ca 2+ indicators. g-i , Ca 2+ titration curves (g), K d values (h), and Hill coefficients (i) of red Ca 2+ sensors. n = 4 wells in each sensor. Tukey’s post hoc test following one-way ANOVA. K d values (h) of jRGECO1a vs RCaMP3: P = 9.0 × 10 −1 ; XCaMP-R vs RCaMP3: P = 8.0 × 10 −6 . Hill coefficients (i) of jRGECO1a vs RCaMP3: P = 3.8 × 10 −2 ; XCaMP-R vs RCaMP3: P = 6.1 × 10 −8 . j , Normalized fluorescence of red Ca 2+ sensors in Ca 2+ -free and Ca 2+ -saturated states as a function of pH. Ca 2+ -saturated jRGECO1a: pKa = 6.3, Ca 2+ -free jRGECO1a: pKa = 9.0, Ca 2+ -saturated XCaMP-R: pKa = 5.3, Ca 2+ -free XCaMP-R: pKa = 8.6, Ca 2+ -saturated RCaMP3: pKa = 6.1, Ca 2+ -free RCaMP3: pKa = 9.4. n = 4 wells in each condition. k,l , One-photon (k) and two-photon (l) bleaching curves of jRGECO1a, XCaMP-R and RCaMP3 expressed in HEK293T cells. n = 63 neurons (jRGECO1a, one-photon), n = 91 neurons (XCaMP-R, one-photon), n = 76 neurons (RCaMP3, one-photon), n = 94 neurons (jRGECO1a, two-photon), n = 98 neurons (XCaMP-R, two-photon), n = 93 neurons (RCaMP3, two-photon). All shaded areas and error bars denote SEM.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Fluorescence, Comparison, Titration

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , Top, primary structure of RCaMP3 with amino acids mutated relative to R-GECO1 (in R-GECO1 numbering). Bottom, tertiary structures of R-GECO1 (PDB 4I2Y ) with amino acids mutated in RCaMP3 (sphere). b , Δ F/F of red Ca 2+ sensor in HEK lysate. n = 4 wells in each sensor. Tukey’s post hoc test following one-way ANOVA. jRGECO1a versus RCaMP3: P = 5.8 × 10 −11 ; XCaMP-R versus RCaMP3: P = 4.0 × 10 −13 . c , Two-photon (1,040 nm) fluorescence in live HEK cells with ionomycin. n = 360 (jRGECO1a), n = 267 (XCaMP-R), n = 376 (RCaMP3) cells. Tukey’s post hoc test following one-way ANOVA. jRGECO1a versus RCaMP3: P = 6.9 × 10 −14 ; XCaMP-R versus RCaMP3: P = 6.9 × 10 −14 . d , Ca 2+ imaging with whole-cell patch-clamp recording in acute brain slices. e , Representative jRGECO1a and RCaMP3 responses to single action potentials (APs, vertical lines). Thin lines denote individual traces (jRGECO1a: 12 trials, RCaMP3: 10 trials), and thick lines denote average response. f – h , Δ F/F ( f ), rise time ( g ) and half-decay time ( h ) for single APs. n = 7 neurons (jRGECO1a), n = 6 neurons (RCaMP3). Unpaired two-tailed t -test. P = 4.7 × 10 −2 ( f ); P = 9.7 × 10 −1 ( g ); P = 4.6 × 10 −1 ( h ). i , Ca 2+ imaging under a cell-attached recording in vivo. j , Representative trace of simultaneous measurement of RCaMP3 fluorescence and APs in vivo. The number of APs for each event is indicated below the trace. The image shows a neuron expressing RCaMP3 with the recording pipette. Scale bar, 20 μm. k , Δ F/F of jRGECO1a (152 events from 12 cells) and RCaMP3 (228 events from 15 cells) for single APs. Thin lines represent individual traces, and thick lines represent the average traces. l , m , Half-rise time ( l ) and half-decay time ( m ) for single APs. n = 12 cells (jRGECO1a), n = 15 cells (RCaMP3). Unpaired two-tailed t -test. P = 8.8 × 10 −1 ( l ); P = 2.1 × 10 −1 ( m ). n , Δ F/F in response to APs (jRGECO1a, n = 152, 198, 124 and 44 events for 1, 2, 3 and 4 APs, respectively; RCaMP3, n = 228, 139, 159 and 17 events for 1, 2, 3 and 4 APs, respectively). Unpaired two-tailed t -test. P = 4.5 × 10 −58 (1AP); P = 1.8 × 10 −93 (2AP); P = 1.2 × 10 −60 (3AP); P = 3.8 × 10 −17 (4AP). o , Mesoscale Ca 2+ imaging using FASHIO-2PM. p , Left, a representative full field of view. Scale bar, 500 μm. Right, magnified images and Ca 2+ traces of 12 representative neurons. Scale bar, 10 μm. Error bars denote the s.e.m. Boxes indicate the 25th and 75th percentiles, solid lines indicate the median, dashed lines indicate the mean, and whiskers indicate the total range of data.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Fluorescence, Imaging, Patch Clamp, Two Tailed Test, In Vivo, Expressing, Transferring

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , Schematic of AAVs. AAVs encoding RCaMP3 and cAMPinG1-ST were co-injected into the L2/3 of the V1. b , Schematic of the experimental procedure. Sequential excitation at 940 nm and 1,040 nm was used for dual-color imaging of cAMPinG1-ST and RCaMP3. Three optical planes spaced 30 µm apart were imaged at 3.4 Hz per plane using a piezo objective scanner. c , Representative images of RCaMP3 and cAMPinG1-ST. Scale bar, 50 μm. d , Single-trial traces of RCaMP3 and cAMPinG1-ST. Cells are sorted according to Δ F/F of RCaMP3 during running. n = 461 cells in 1 mouse. e , Single-trial traces of RCaMP3 and cAMPinG1-ST of two representative cells. The box indicates the period of forced running. The cell number on the top corresponds to the number in d . f , Averaged fluorescence transients of RCaMP3 (magenta) and cAMPinG1-ST (green). n = 137 cells in 1 mouse (left), n = 324 cells in 1 mouse (right). g , Cumulative plot of mean cAMP Δ F/F of motion-related (green) and non-related (black) cells during forced running. n = 137 cells in 1 mouse (green), n = 324 cells in 1 mouse (black). Kolmogorov–Smirnov test. P = 1.1 × 10 −16 . h , Averaged Δ F/F of RCaMP3 (magenta) and cAMPinG1-ST (green) during forced running. n = 3 trials, n = 3 mice. Paired two-tailed t -test. P = 1.5 × 10 −2 . All shaded areas and error bars denote the s.e.m.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Injection, Imaging, Fluorescence, Two Tailed Test

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a-c , Representative two-photon images and averaged traces of cAMPinG1-ST (a), cAMPinG1mut-ST (b), and G-Flamp1 (c) in response to forced running. n = 1156 cells in 3 mice (cAMPinG1-ST), n = 905 cells in 4 mice (cAMPinG1mut-ST), n = 74 cells in 4 mice (G-Flamp1). Scale bar, 50 μm. d , Averaged Δ F/F of cAMPinG1-ST, cAMPinG1mut-ST, and G-Flamp1 in response to forced running. n = 3 trials in 3 mice (cAMPinG1-ST), n = 4 trials in 4 mice (cAMPinG1mut-ST), n = 4 trials in 4 mice (G-Flamp1). Unpaired two-tailed t -test. cAMPinG1-ST vs cAMPinG1mut-ST: P = 2.6 × 10 −2 , cAMPinG1-ST vs G-Flamp1: P = 4.6 × 10 −2 . e , Schematic of the experimental procedure. cAMPinG1-NE was expressed in neurons in the V1. Fiber photometry was performed during forced running. Thirty minutes after intraperitoneal administration of propranolol, a β-adrenoceptor blocker, fiber photometry was again performed during the forced running. f , Averaged traces of cAMPinG1-NE in response to forced running before and after intraperitoneal administration of mock solution (left) or propranolol (right). n = 6 trials in 6 mice (mock), n = 6 trials in 6 mice (propranolol). g , Difference of Δ F/F in response to forced running before and after the intraperitoneal administration. n = 6 trials in 6 mice (mock), n = 6 trials in 6 mice (propranolol). Paired two-tailed t -test. P = 1.4 × 10 −2 . h , Schematic of the experimental procedure. i , Representative images of cAMPinG1 and RCaMP3. Scale bar, 50 μm. j , Averaged fluorescence transients of cAMPinG1 and RCaMP3. n = 63 cells in 3 mice. All shaded areas and error bars denote SEM.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Two Tailed Test, Fluorescence

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , Schematic of the experimental procedure. Moving gratings of 8 directions were used to induce cell-specific Ca 2+ transients in L2/3 neurons of the V1. b , Δ F/F of RCaMP3 and cAMPinG1-ST of 2 representative cells. n = 6 times in each cell. c , Direction-selective visual responses of RCaMP3 and cAMPinG1-ST of the two representative cells in b . d , Direction-selective visual responses of RCaMP3 and cAMPinG1-ST. Top, neurons showing the direction selectivity index (DSI) < 0.4 in Ca 2+ response. n = 94 cells in 3 mice. Bottom, neurons showing the DSI ≥ 0.4 in Ca 2+ response. n = 101 cells in 3 mice. e , Single-trial traces of RCaMP3 and cAMPinG1-ST of 2 representative cells. The box indicates the period of visual stimuli. f , Averaged traces of RCaMP3 (magenta) and cAMPinG1-ST (green). n = 53 cells in 1 mouse (left), n = 408 cells in 1 mouse (right). g , Δ F/F of RCaMP3 and cAMPinG1-ST during and after the visual stimuli. n = 12 trials, n = 3 mice. Paired two-tailed t -test. Responded (during stim) versus non-responded (during stim): P = 1.5 × 10 −7 ; responded (during stim) versus responded (after stim): P = 2.1 × 10 −4 ; non-responded (during stim) versus non-responded (after stim): P = 5.1 × 10 −4 . h , Schematic of AAVs for sparse expression of cAMPinG1-NE and soma-targeted ChRmine. i , Representative fluorescence images of cAMPinG1-NE and soma-targeted ChRmine. Scale bar, 10 μm. j , Δ F/F of cAMPinG1-NE in response to 1,040 nm of photostimulation. n = 11 neurons in 3 mice (ChRmine (+), green), n = 11 neurons in 3 mice (ChRmine (−), black). k , Δ F/F of cAMPinG1-NE in response to 1,040 nm of photostimulation. n = 11 neurons in 3 mice (ChRmine (+), photostim (+)), n = 11 neurons in 3 mice (ChRmine (−), photostim (+)), n = 9 neurons in 3 mice (ChRmine (+), photostim (−)). Tukey’s post hoc test following one-way ANOVA. ChRmine (+), photostim (+) versus ChRmine (−), photostim (+): P = 1.4 × 10 −2 ; ChRmine (+), photostim (+) versus ChRmine (+), photostim (−): P = 2.0 × 10 −3 . All shaded areas and error bars denote the s.e.m.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Two Tailed Test, Expressing, Fluorescence

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , b , Representative two-photon images and traces of RCaMP3 and cAMPinG1-ST of the cells in Fig. , indicated by arrows. Scale bar, 10 μm. c , Averaged traces of cAMPinG1-ST (left), cAMPinG1mut-ST (middle), and G-Flamp1 (right) in response to visual stimuli. n = 1156 cells in 3 mice (cAMPinG1-ST), n = 905 cells in 4 mice (cAMPinG1mut-ST), n = 74 cells in 4 mice (G-Flamp1). Four trials in each cell. d , Averaged Δ F/F of cAMPinG1-ST, cAMPinG1mut-ST, and G-Flamp1 in response to visual stimuli. n = 12 trials in 3 mice (cAMPinG1-ST), n = 16 trials in 4 mice (cAMPinG1mut-ST), n = 16 trials in 4 mice (G-Flamp1). Unpaired two-tailed t -test after removing outliers with Smirnov-Grubbs’ test ( P = 0.05). cAMPinG1-ST vs cAMPinG1mut-ST: P = 1.9 × 10 −5 , cAMPinG1-ST vs G-Flamp1: P = 7.2 × 10 −4 . e , Averaged Δ F/F of RCaMP3 and cAMPinG1mut-ST in cells which showed Ca 2+ response to visual stimuli. n = 486 cells in 4 mice. All shaded areas and error bars denote SEM.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Two Tailed Test

Journal: Nature Methods

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

doi: 10.1038/s41592-024-02222-9

Figure Lengend Snippet: a , Schematic of the experimental procedure. AAVs encoding RCaMP3 and green cAMP sensors were injected into the dorsal striatum (dStr). Dual-color fiber photometry was performed in the dStr during a forced running task. For dual-color imaging, we employed different excitation wavelengths: 560 nm for RCaMP3 imaging and 405 nm and 470 nm for cAMPinG1 ratiometric imaging. b , Representative single-trial traces of cAMPinG1-NE (left), cAMPinG1mut-NE (middle), G-Flamp1 (right), and RCaMP3 signals. c , Averaged fluorescence traces of cAMPinG1-NE (left), cAMPinG1mut-NE (middle), G-Flamp1 (right), and RCaMP3 signals. n = 9 trials in 1 mouse (cAMPinG1-NE), n = 9 trials in 1 mouse (cAMPinG1mut-NE), n = 9 trials in 1 mouse (G-Flamp1). d , Averaged Δ F/F of cAMPinG1-NE, cAMPinG1mut-NE and G-Flamp1 during and after the stimulation. n = 36 trials in 4 mice (cAMPinG1-NE), n = 27 trials in 3 mice (cAMPinG1mut-NE), n = 36 trials in 4 mice (G-Flamp1). Unpaired two-tailed t -test. cAMPinG1-NE vs cAMPinG1mut-NE (during stim): P = 3.7 × 10 −4 ; cAMPinG1-NE vs G-Flamp1 (during stim): P = 3.6 × 10 −5 ; cAMPinG1-NE vs cAMPinG1mut-NE (after stim): P = 3.1 × 10 −5 ; cAMPinG1-NE vs G-Flamp1 (after stim): P = 4.2 × 10 −5 . All shaded areas and error bars denote SEM.

Article Snippet: The final titers were: AAV2/1-CAG-DIO-cAMPinG1-NE (5.0 × 10 13 genome copies (GC) per ml), AAV2/1-CAG-DIO-cAMPinG1mut-NE (2.0 × 10 13 GC per ml) and AAVPHP.eB-CaMKII-Cre (1.0 × 10 12 GC per ml) for forskolin/IBMX bath application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAV2/1-CaMKII (0.3 kb)-loxFAS-H2B-mCherry-loxFAS (1.0 × 10 13 GC per ml) for local dopamine application in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1mut-NE (1.5 × 10 13 GC per ml) and AAVPHP.eB-hSyn-EGFP (2.0 × 10 13 GC per ml) for electrophysiology in acute brain slices (Extended Data Fig. ); AAV2/1-eSyn-NES-jRGECO1a (3.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (2.0 × 10 13 GC per ml) for one-photon and two-photon calcium imaging in the barrel cortex (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon mesoscale Ca 2+ imaging (Fig. ); AAV2/1-eSyn-NES-jRGECO1a (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for inclusion counting (Extended Data Fig. ); AAV2/1-eSyn-cAMPinG1-ST (1.0 × 10 13 GC per ml), AAV2/1-eSyn-cAMPinG1mut-ST (1.0 × 10 13 GC per ml) and AAV2/1-eSyn-RCaMP3 (1.0 × 10 13 GC per ml) for two-photon imaging in the V1 (Figs. and ).

Techniques: Injection, Imaging, Fluorescence, Two Tailed Test

Journal: Cell reports

Article Title: A brain stem circuit integrating reflexive and anticipatory salivation

doi: 10.1016/j.celrep.2026.117067

Figure Lengend Snippet: (A) Schematic of the parasympathetic pathways connecting the brain stem salivatory center to the parotid and submandibular salivary glands. A 2% Fluorogold solution was injected into a ganglion on the submandibular salivary duct for retrograde tracing. (B) Mouse brain atlas showing the inferior salivatory nuclei (IS), highlighted in red. Adapted from Franklin and Paxinos. (C) Venn diagram showing the overlap (orange) between Fluorogold-labeled neurons (green) and ChAT-expressing neurons (red) in the IS region. Percentages indicate the proportion of each neuronal population. n = 3 animals, male. (D) Representative images of the IS region from a Fluorogold tracing experiment. Shown is Fluorogold labeling (green) in a ChAT-Cre;Flex-TdTomato (red) animal. An arrowhead marks the facial nerve. Scale bar, 100 μm. (E) Mouse brain atlas showing the superior salivatory nuclei (SUSs), highlighted in red. Adapted from Franklin and Paxinos. (F) Venn diagram showing the overlap (orange) between Fluorogold-labeled neurons (green) and ChAT-expressing neurons (red) in the IS region. Percentages indicate the proportion of each neuronal population. n = 3 animals, male. (G) Representative images of the SUS region from a Fluorogold tracing experiment. Shown is Fluorogold labeling (green) in a ChAT-Cre;Flex-TdTomato (red) animal. An arrowhead marks the facial nerve. Scale bar, 100 μm. (H) Schematic of the optogenetic stimulation of the IS. ChAT neurons were transduced with Cre-dependent AAV-Flex-ChR2 in a ChAT-Cre mouse, and an optic fiber was implanted above the injection site. Right: quantification of the saliva secreted during a 10-min collection period without (white) or with (blue) photostimulation. Mice n = 6 animals, male. Values are represented as mean ± SEM; two-tailed Student’s t test, ** p < 0.01.

Article Snippet: Flex-TdTomato , The Jackson Laboratory , 7909.

Techniques: Injection, Retrograde Tracing, Labeling, Expressing, Transduction, Two Tailed Test