Journal: Nature Communications

Article Title: Optogenetic stimulation of G s -signaling in the heart with high spatio-temporal precision

doi: 10.1038/s41467-019-09322-7

Figure Lengend Snippet: Generation of JellyOp expressing mouse ESCs and light-induced cAMP production in cardiomyocytes. a Plasmid for expression of JellyOp in fusion with the 1D4 rhodopsin epitope and with an internal ribosome entry site (IRES) for co-expression of the green fluorescence protein (GFP) under control of the chicken β-actin promoter (CAG). b Immunostaining of a transgenic ESC colony expressing GFP (green) and JellyOp (red: 1D4 rhodopsin epitope staining) (nuclear staining in blue, bars: 10 μm; insert: 5 µm). c GFP positive (green) cardiomyocytes indicated by α-actinin (yellow) staining (nuclear staining in blue, bar: 20 µm). d cAMP levels in JellyOp and GFP EBs after illumination (2.9 mW mm −2 , 5 min, MDL: 100 µM MDL-12,330A) or isoprenaline (Iso, 1 µM, 5 min) application ( n = 5–12, Welch ANOVA: p = 0.0014, Games–Howell post-test: * p < 0.05, ** p < 0.01). e Relationship between cAMP levels and light intensity in lactate-purified (see Methods) cardiomyocytes fitted with Hill equation ( n = 5–12). Error bars: S.E.M.

Article Snippet: After permeabilization with 0.2% Triton X-100 (Fluka) for 10 min and blocking with 5% donkey serum (Jackson ImmunoResearch) for 20 min, primary antibodies against the 1D4 rhodopsin-epitope (1:400, StressMarq) or α-actinin (1:400, Sigma-Aldrich) diluted in 0.5% donkey serum were applied for 2 h at room temperature.

Techniques: Expressing, Plasmid Preparation, Fluorescence, Immunostaining, Transgenic Assay, Staining, Purification

Journal: Nature Communications

Article Title: Optogenetic stimulation of G s -signaling in the heart with high spatio-temporal precision

doi: 10.1038/s41467-019-09322-7

Figure Lengend Snippet: JellyOp function in isolated mouse ventricular cardiomyocytes. a Overview of GFP fluorescence in an intact JellyOp mouse heart. b Section of the left ventricle with membrane-bound JellyOp-1D4 epitope signals (yellow) in GFP (green) positive cardiomyocytes (nuclear staining in blue, bar: 20 µm, insert: 5 µm). c Heart weight (HW) to femur length (FL) ratio in JellyOp (Jelly) and wild-type (WT) mice (>6 months of age, n = 5, two-sided unpaired Student's t -test: p = 0.8915). d , e Representative changes in peak L-type Ca 2+ current ( I Ca,L ) in a JellyOp ventricular cardiomyocyte upon illumination ( d , blue bar, 100 µW mm −2 , 10 s) and original traces taken from time points indicated ( e ). f , g Statistical analysis of maximal I Ca,L before (Contr., black) and after illumination ( f , blue, 100 µW mm −2 , 10 s, n = 8, two-sided paired Student's t -test: p = 0.0035) or isoprenaline application ( g , red, 1 µM, 75–100 s, n = 13, two-sided paired Student's t -test: p = 0.00003). h Original trace (upper panel) of cell length measurement from an electrically stimulated isolated JellyOp ventricular cardiomyocyte upon illumination (blue bar, 100 µW mm −2 , 10 s) with corresponding fractional shortening (FS, middle panel) and relaxation constant τ rel (lower panel). i , j Single traces of relative cell length in % of resting length ( i ) and normalized to maximal shortening ( j ) evoked by electrical stimulation (red line) before (1, black), 40 s (2, light blue) and 80 s (3, dark blue) after illumination (time points indicated in ( h )). k , l Statistical analysis of maximal fractional shortening ( k ) and relaxation speed ( τ rel , l ) before (black) and after (blue) illumination (100 µW mm −2 , 10 s, n = 10, two-sided paired Student's t -test, p = 0.0011 ( k ); Wilcoxon signed rank test, p = 0.004 ( l )). m Delay from illumination onset to maximal effect on fractional cell shortening (FS, dark blue) and relaxation speed ( τ rel , light blue) ( n = 8, Wilcoxon signed rank test, p = 0.04). Error bars: S.E.M.

Article Snippet: After permeabilization with 0.2% Triton X-100 (Fluka) for 10 min and blocking with 5% donkey serum (Jackson ImmunoResearch) for 20 min, primary antibodies against the 1D4 rhodopsin-epitope (1:400, StressMarq) or α-actinin (1:400, Sigma-Aldrich) diluted in 0.5% donkey serum were applied for 2 h at room temperature.

Techniques: Isolation, Fluorescence, Staining

Journal: Nature Communications

Article Title: Optogenetic stimulation of G s -signaling in the heart with high spatio-temporal precision

doi: 10.1038/s41467-019-09322-7

Figure Lengend Snippet: JellyOp function in mouse atria of intact hearts. a Section through the right atrium shows GFP (green) and membrane-bound JellyOp-1D4 epitope signals (yellow, nuclear staining in blue, bar: 20 µm, insert: 5 µm). b Analysis of cAMP levels in atrial tissue from JellyOp mice in the dark and after illumination (2.86 μW mm −2 , 5 min) or stimulation with isoprenaline (Iso, 1 µM, 5 min) ( n = 4–6, one-way ANOVA: p = 0.0074, Bonferroni post-test: * p < 0.05). c , d Representative heart rate traces of a JellyOp heart during maximal light stimulation of the dorsal right atrium ( c , blue bar, 2 mW mm −2 , 90 s) or during perfusion with isoprenaline ( d , red bar; 1 µM, 4 min) (gray: original trace, black: running average values). e – g Statistical analysis of the maximal relative heart rate increase ( e ), activation time ( f , stimulation start to 80% of maximum rate), and deactivation time ( g , stimulation end to 50% of maximum rate) after illumination (blue, 2 mW mm −2 , 90 s) and perfusion with isoprenaline (red, 1 µM, 4 min) ( n = 5, two-sided paired Student's t -test: p = 0.0248 ( e ), p = 0.0058 ( f ), p = 0.0017 ( g )). h Relative heart rate increase after illumination of the dorsal right atrium with light of wavelengths of 470 nm and 400–700 nm in 50 nm steps (10 µW mm −2 , 1 s, n = 5) fitted with the Govardovskii equation (red line, R 2 = 0.98). Error bars: S.E.M.

Article Snippet: After permeabilization with 0.2% Triton X-100 (Fluka) for 10 min and blocking with 5% donkey serum (Jackson ImmunoResearch) for 20 min, primary antibodies against the 1D4 rhodopsin-epitope (1:400, StressMarq) or α-actinin (1:400, Sigma-Aldrich) diluted in 0.5% donkey serum were applied for 2 h at room temperature.

Techniques: Staining, Activation Assay

Journal: Chemical Senses

Article Title: Opposing expression pattern of opsin 3 and opsin 5 in the developing and adult nasal epithelium

doi: 10.1093/chemse/bjaf051

Figure Lengend Snippet: Immunohistochemistry analyzes of Opn3, Opn5, and Tubb3 expression in the nasal epithelium at E13.5 and E17.5 using Opn3 - eGFP and Opn5 -tdTomato mice. a–d) At E13.5, Opn3 is expressed in the Tubb3 + sensory epithelium ( N = 3), while no Opn5 expression is detected in the entire nasal epithelium ( N = 3). e–h) At E17.5, Opn3 is expressed in the Tubb3 + sensory epithelium ( N = 5), and Opn5 is expressed in a few scattered cells in the respiratory epithelium ( N = 5). White boxes indicate magnified images shown in (i–p). In (a, d, e and h), arrows indicate the vomeronasal organ, and arrowheads indicate migratory olfactory neurons. i–l) At e 17.5, Opn3 is co-localized with Tubb3, which marks postmitotic immature neurons, while no expression of Opn5 is detected in the sensory epithelium (SE). m–p) At E17.5, scattered Opn5 expression, but no Opn3 expression, is detected in the respiratory epithelium (RE; indicated with asterisks). Scale bars: a–d) 100 µm; e–h) 200 µm; i–p) 25 µm.

Article Snippet: Genetically modified mouse strains used were as follows: (i) an Opn3 - eGFP reporter line ( Tg[Opn3-EGFP]JY3Gsat ; MMRRC/GENSAT stock number 030727-UCD, Mutant Mouse Resource & Research Centers, University of California, Davis, California); (ii) an Opn5 Cre/+ ; Ai14 reporter line ( ); (iii) an Opn3 lacZ/lacZ line that functionally acts like an Opn3 −/− line, mixed background strain C57Bl6/6N/FVB/N ( ); and (iv) an Opn5 −/− line, mixed background strain C57/129/CD1/FVB ( ).

Techniques: Immunohistochemistry, Expressing

Journal: Chemical Senses

Article Title: Opposing expression pattern of opsin 3 and opsin 5 in the developing and adult nasal epithelium

doi: 10.1093/chemse/bjaf051

Figure Lengend Snippet: Analyzes of Opn3 -eGFP/ Opn5 -tdTomato double reporter expression in comparison with Tubb3 and Foxj1 double immunohistochemistry at P14 ( N = 4). a–c) At P14, the Opn5 expression has expanded in the respiratory epithelium, with opposing Opn3 expression observed in the sensory epithelium. Most Opn5 + respiratory epithelial cells were observed in the distal part of the nasal cavity. A few intermingled Opn3 + and Opn5 + cells were detected where the sensory and respiratory domains meet (indicated by arrowheads). d) The sensory epithelium defined by Tubb3 expression and the respiratory domain by Foxj1 expression mimic the Opn3 and Opn5 expression domains, respectively. Scale bar: 200 µm.

Article Snippet: Genetically modified mouse strains used were as follows: (i) an Opn3 - eGFP reporter line ( Tg[Opn3-EGFP]JY3Gsat ; MMRRC/GENSAT stock number 030727-UCD, Mutant Mouse Resource & Research Centers, University of California, Davis, California); (ii) an Opn5 Cre/+ ; Ai14 reporter line ( ); (iii) an Opn3 lacZ/lacZ line that functionally acts like an Opn3 −/− line, mixed background strain C57Bl6/6N/FVB/N ( ); and (iv) an Opn5 −/− line, mixed background strain C57/129/CD1/FVB ( ).

Techniques: Expressing, Comparison, Immunohistochemistry

Journal: Chemical Senses

Article Title: Opposing expression pattern of opsin 3 and opsin 5 in the developing and adult nasal epithelium

doi: 10.1093/chemse/bjaf051

Figure Lengend Snippet: Immunohistochemistry analyzes of Opn3, Opn5, Foxj1, Ker8, Sox2, Tubb3, and OMP expression in the nasal epithelium at adulthood using Opn3 - eGFP ( N = 5) and Opn5 -tdTomato mice ( N = 4). a, b) In the adult nasal epithelium, an increased number of Opn5 + cells were detected in a scattered pattern within the Foxj1 + respiratory epithelium. White box in (a) indicates the magnified image shown in b. c–e) In the olfactory sensory epithelium, Opn3 and Ker8 are co-expressed in sustentacular cells. f) Opn3 is co-expressed with Sox2 in sustentacular cell nuclei (indicated by an arrow), but seemingly not in Sox2 + globose basal cells (indicated by arrowheads). g, h) Opn3 is not expressed in Tubb3 + postmitotic immature ORNs (g) or OMP + mature ORNs (h). Scale bars: a) 200 µm; b) 100 µm; c–h) 50 µm.

Article Snippet: Genetically modified mouse strains used were as follows: (i) an Opn3 - eGFP reporter line ( Tg[Opn3-EGFP]JY3Gsat ; MMRRC/GENSAT stock number 030727-UCD, Mutant Mouse Resource & Research Centers, University of California, Davis, California); (ii) an Opn5 Cre/+ ; Ai14 reporter line ( ); (iii) an Opn3 lacZ/lacZ line that functionally acts like an Opn3 −/− line, mixed background strain C57Bl6/6N/FVB/N ( ); and (iv) an Opn5 −/− line, mixed background strain C57/129/CD1/FVB ( ).

Techniques: Immunohistochemistry, Expressing

Journal: Chemical Senses

Article Title: Opposing expression pattern of opsin 3 and opsin 5 in the developing and adult nasal epithelium

doi: 10.1093/chemse/bjaf051

Figure Lengend Snippet: Buried food tests using Opn3 or Opn5 wild-type and deficient mice. Olfaction was evaluated by the buried food test, with a maximum of 10 min (i.e. 600 s) per trial to find a hidden cookie piece. a–c) The graphs show the time to find buried food for Opn3 +/+ ( N = 10) and Opn3 −/− mice ( N = 9). a) The bars represent the average time of three independent trials. b) The bars represent the three individual trials, T1–T3, analyzed as a function of trial order. c) The bars represent time to find buried food between T1 and T3 for Opn3 +/+ mice ( P = 0.0076), and Opn3 −/− mice ( P = 0.097). d–f) The graphs show the time to find buried food for Opn5 +/+ ( N = 8) and Opn5 −/− mice ( N = 11). (d) The bars represent the average time of three independent trials. e) The bars represent the three individual trials, T1–T3. f) The bars represent time to find buried food between T1 and T3 for Opn5 +/+ mice ( P = 0.72) and Opn5 −/− mice ( P = 0.21). Data on all graphs are presented as the mean ± standard deviation. Statistical significances were calculated using unpaired two-tailed Student's t -tests (a, c, d, f) or repeated measures two-way ANOVA followed by Sidak's multiple comparisons test (b, e).

Article Snippet: Genetically modified mouse strains used were as follows: (i) an Opn3 - eGFP reporter line ( Tg[Opn3-EGFP]JY3Gsat ; MMRRC/GENSAT stock number 030727-UCD, Mutant Mouse Resource & Research Centers, University of California, Davis, California); (ii) an Opn5 Cre/+ ; Ai14 reporter line ( ); (iii) an Opn3 lacZ/lacZ line that functionally acts like an Opn3 −/− line, mixed background strain C57Bl6/6N/FVB/N ( ); and (iv) an Opn5 −/− line, mixed background strain C57/129/CD1/FVB ( ).

Techniques: Standard Deviation, Two Tailed Test

Journal: Chemical Senses

Article Title: Opposing expression pattern of opsin 3 and opsin 5 in the developing and adult nasal epithelium

doi: 10.1093/chemse/bjaf051

Figure Lengend Snippet: Immunohistochemistry analyzes of Tubb3, Foxj1, OMP, Sox2 and Ker8 in the nasal cavity of adult Opn3 +/+ , Opn3 −/− , Opn5 +/+ , and Opn5 −/− mice ( N = 3 for each mouse genotype). a–h) No apparent differences in the expression of Tubb3, Foxj1, OMP, Sox2, or Ker8 in the sensory epithelium of Opn3 −/− compared to Opn3 +/+ mice. e, f) Arrows indicate the Sox2 + sustentacular cells, and arrowheads indicate Sox2 + globose basal cells. i–l) No apparent difference in the expression of Tubb3, Foxj1, or Sox2 in the respiratory epithelium of Opn5 −/− compared to Opn5 +/+ mice. k, l) Arrows indicate the Sox2 + positive cells in the respiratory cell layer. Scale bars: a, b, i, j) 100 µm; c–h, k, l) 50 µm.

Article Snippet: Genetically modified mouse strains used were as follows: (i) an Opn3 - eGFP reporter line ( Tg[Opn3-EGFP]JY3Gsat ; MMRRC/GENSAT stock number 030727-UCD, Mutant Mouse Resource & Research Centers, University of California, Davis, California); (ii) an Opn5 Cre/+ ; Ai14 reporter line ( ); (iii) an Opn3 lacZ/lacZ line that functionally acts like an Opn3 −/− line, mixed background strain C57Bl6/6N/FVB/N ( ); and (iv) an Opn5 −/− line, mixed background strain C57/129/CD1/FVB ( ).

Techniques: Immunohistochemistry, Expressing

Journal: Journal of Ophthalmology

Article Title: Regulation of Reentrainment Function Is Dependent on a Certain Minimal Number of Intact Functional ipRGCs in rd Mice

doi: 10.1155/2017/6804853

Figure Lengend Snippet: The ipRGCs could be immunohistochemically labeled using antibodies against melanopsin. (a) The laser scanning confocal view of ipRGCs in flat-mounted retina, showing melanopsin immunoreactivity and cell morphology. Bar = 75 μ m. (b) The bar graph comparing the numbers of ipRGCs per visual field (200x magnification) among the rd mice at different ages. The differences in the number of ipRGCs were not statistically significant (one-way ANOVA with Tukey's multiple comparison test, P > 0.05).

Article Snippet: The free-floating retinas were incubated in a blocking solution (0.3% Triton X-100 and 5% bovine serum albumin in PBS) for 1 hour at room temperature and were then incubated with a primary melanopsin antibody (polyclonal rabbit anti-melanopsin; Affinity BioReagents, Golden, CO, USA) at 1:500 dilution in PBS/0.3% Triton X-100/5% bovine serum albumin for 72 hours at 4°C.

Techniques: Labeling, Comparison

Journal: Journal of Ophthalmology

Article Title: Regulation of Reentrainment Function Is Dependent on a Certain Minimal Number of Intact Functional ipRGCs in rd Mice

doi: 10.1155/2017/6804853

Figure Lengend Snippet: Intravitreal injection of melanopsin-SAP led to partial ablation of ipRGCs in a dose-dependent manner. (a) Immunohistochemically labeled ipRGCs using antibodies against melanopsin and comparison of the number of ipRGCs on flat-mounted retina following intravitreal injection of immunotoxin melanopsin-SAP in rd mice. (A–C) The number of ipRGCs per visual field (200x magnification) on flat-mounted retinas in different dose groups; (D–F) the results of the control eyes (PBS injection group). As the dose of melanopsin-SAP increased, the number of melanopsin-positive cells in the experimental eye decreased, while that in the control eye remained the same. Bar = 250 μ m. (b) Analysis of the survival rate of ipRGCs after immunotoxin injection. The ipRGC survival rate was defined as (ipRGC number of experimental eye)/(ipRGC number of control eye) × 100%. The survival rate of ipRGCs was significantly reduced in the 200 ng/ μ l and 400 ng/ μ l groups when compared with the 100 ng/ μ l group (one-way ANOVA with Tukey's multiple comparison test, P < 0.01). But the difference between the 200 ng/ μ l group and the 400 ng/ μ l group was not statistically significant (one-way ANOVA with Tukey's multiple comparison test, P = 0.933). ∗ P < 0.01.

Article Snippet: The free-floating retinas were incubated in a blocking solution (0.3% Triton X-100 and 5% bovine serum albumin in PBS) for 1 hour at room temperature and were then incubated with a primary melanopsin antibody (polyclonal rabbit anti-melanopsin; Affinity BioReagents, Golden, CO, USA) at 1:500 dilution in PBS/0.3% Triton X-100/5% bovine serum albumin for 72 hours at 4°C.

Techniques: Injection, Labeling, Comparison, Control

Journal: Journal of Ophthalmology

Article Title: Regulation of Reentrainment Function Is Dependent on a Certain Minimal Number of Intact Functional ipRGCs in rd Mice

doi: 10.1155/2017/6804853

Figure Lengend Snippet: The morphological examination of retinal sections after injection of melanopsin-SAP immunotoxin in rd mice. (a) Photomicrographs of 8 μ m paraffin sections of retina were used for H&E staining. No significant morphological differences were observed between the highest dose group and the control group. Bar = 100 μ m. (IPL = inner plexiform layer; INL = inner nucleus layer; GCL = ganglion cell layer). (b) The thickness of the INL was measured using IPP software (pixel). The difference in the INL thickness between the highest dose group and the control group was not statistically significant (independent sample t -test, P > 0.05).

Article Snippet: The free-floating retinas were incubated in a blocking solution (0.3% Triton X-100 and 5% bovine serum albumin in PBS) for 1 hour at room temperature and were then incubated with a primary melanopsin antibody (polyclonal rabbit anti-melanopsin; Affinity BioReagents, Golden, CO, USA) at 1:500 dilution in PBS/0.3% Triton X-100/5% bovine serum albumin for 72 hours at 4°C.

Techniques: Injection, Staining, Control, Software

Journal: Journal of Ophthalmology

Article Title: Regulation of Reentrainment Function Is Dependent on a Certain Minimal Number of Intact Functional ipRGCs in rd Mice

doi: 10.1155/2017/6804853

Figure Lengend Snippet: Entrainment and wheel-running periods of rd mice after injection of melanopsin-SAP immunotoxin. The bar below the actograms indicates the light (white) and dark (black) periods, and the light and dark periods are indicated by white and gray backgrounds, respectively. When the light/dark cycle was delayed by 8 h, (a) the rd mice in the PBS control group were capable of reentraining to the light/dark cycle, and they took 4.67 ± 0.79 days to complete the synchronization with the shifted cycle. (b) The rd mice in the 100 ng/ μ l group and (c) the rd mice in the 200 ng/ μ l group required 7.90 ± 0.55 days and 11.00 ± 0.79 days to complete the synchronization with the new light/dark cycle, respectively, indicating slower reentrainment. (d) The wild-type mice took 3.67 ± 0.29 days to complete the synchronization with the shifted cycle, and the locomotor activity of the wild-type mice was more robust than that of the rd mice. ∗ represents the day delay in the time of lights on and lights off. (e) The differences in the number of days needed for reentrainment were not statistically significant between the rd mice in the PBS control group and the wild-type group (two-way ANOVA, followed by Fisher's LSD post hoc test, P > 0.05). The number of days required for reentrainment were significantly increased in the 100 ng/ μ l and 200 ng/ μ l groups when compared with the PBS control group (two-way ANOVA, followed by Fisher's LSD post hoc test, P < 0.01). ∗ P < 0.01.

Article Snippet: The free-floating retinas were incubated in a blocking solution (0.3% Triton X-100 and 5% bovine serum albumin in PBS) for 1 hour at room temperature and were then incubated with a primary melanopsin antibody (polyclonal rabbit anti-melanopsin; Affinity BioReagents, Golden, CO, USA) at 1:500 dilution in PBS/0.3% Triton X-100/5% bovine serum albumin for 72 hours at 4°C.

Techniques: Injection, Control, Activity Assay