Fig. 4

(A) Glucose responses to spontaneous muscle twitches. We generated a zebrafish line that expresses iGlucoSnFR broadly (β-actin promoter) and the red calcium indicator jRGECO1a in muscle (α-actinin promoter). Non-anesthetized, non-paralyzed fish were imaged 5–6 days post-fertilization (dpf) for spontaneous responses to muscle twitches. Representative averaged traces from volumetric time-lapse confocal imaging of fed and never-fed fish are presented as indicated. Flin, fluorescence response divided by a linear baseline. (B) Imaging of insulin responses. 13 dpf zebrafish broadly expressing iGlucoSnFR (β-actin promoter) are shown. (B1) Schematic illustrating anatomy. Note injection pipette poised for injection into the posterior cardinal vein (PCV). (B2) Red fluorescence channel immediately post-injection with insulin and sulforhodamine-101 (SR101) as a marker dye. The entire vasculature became transiently fluorescent red after injection, demonstrating efficacy of systemic injection. (B3 and B4) iGlucoSnFR fluorescence before and after insulin injection. ROIs on brain (“B”) and muscle (“M”) as indicated are plotted in (C). Two channels: Ex 488 nm, Em 505–550; Ex 561, Em 565–650; objective EC Plan-Neofluar 10×/0.3 NA M27, imaging rate ∼1 s per image; 512 × 512 pixels. (C) Insulin-induced fluorescence changes. Red trace indicates injection of dye or dye plus insulin. Green trace indicates iGlucoSnFR fluorescence measured from muscle (ROI “M” in panel B). Blue trace indicates iGlucoSnFR fluorescence measured from brain (ROI “B” in panel B). Small, artifactual changes in fluorescence appear with introduction of the pipette (dye-only injection). Sustained fluorescence increases arise after insulin injection. (D) Imaging organism-scale responses of iGlucoSnFR to epinephrine addition. 5 dpf fish expressing both iGlucoSnFR (all cells, β-actin promoter) and jRGECO1a (muscle cells, α-actinin promoter). Top two images (before and after epinephrine, sagittal section) show false-color raw intensities from a volumetric time-lapse iGlucoSnFR movie average-projected in z. ROIs on muscle (“M”) and brain (“B”) are plotted in (E). Bottom image shows ΔF/F after addition of epinephrine, with muscle showing the greatest changes. 5 dpf; 15 planes at 15-μm steps; two channels: Ex 488 nm, Em 505–550; Ex 561, Em 565–650; objective EC Plan-Neofluar 10×/0.3 NA M27, imaging rate ∼10 s per volume; 512 × 256 pixels. (E) Epinephrine-induced fluorescence changes. Blue trace indicates iGlucoSnFR fluorescence measured from hindbrain (ROI “B” in panel D). Green trace indicates iGlucoSnFR fluorescence measured from muscle (ROI “M” in panel D). Red trace indicates jRGECO1a fluorescence measured from the same muscle ROI. F0 was defined as the averaged intensity value just before addition of epinephrine to the bath, as indicated. Note the delayed increase of neuronal activity subsequent to glucose increases. (F) Liver iGlucoSnFR responses to glucose and epinephrine. Ratio image (iGlucoSnFR/mRuby2) is shown of average of two confocal places (20 μm total) over eight time points (80 s) of a 7 dpf fish expressing mRuby-tagged iGlucoSnFR in liver (fabp10a promoter) before and after epinephrine addition. Plotted area in (G) is indicated by white box. 20 planes at 10-μm steps; two channels: Ex 488 nm, Em 505–550; Ex 561, Em 565–650; objective Plan-Apochromat 20×/0.8 NA M27, imaging rate ∼10 s per volume; 1,024 × 512 pixels. (G) Glucose and epinephrine-induced fluorescence ratio changes in liver. Glucose and epinephrine were added to bath at indicated times. ROIs adjacent in x, y, and z showed similar responses. (H) Imaging brain-scale epinephrine responses. Single coronal section confocal plane (after stack registration) is shown of a 5 dpf fish expressing both iGlucoSnFR (all cells, β-actin promoter) and jRGECO1a (neurons, elavl3 promoter) before and after epinephrine addition. ROIs on muscle (“M”) and cerebellum (“B”) are plotted in (I). 5 dpf; 40 planes at 3.3-μm steps; two channels: Ex 488 nm, Em 505–550; Ex 561, Em 565–650; objective Plan-Apochromat 20×/0.8 NA M27, imaging rate ∼25 s per volume; 512 × 256 pixels. (I) Epinephrine-induced fluorescence changes in the brain. Green trace indicates iGlucoSnFR fluorescence measured from muscle (ROI “M” in panel H). Blue trace indicates iGlucoSnFR fluorescence measured from cerebellum (ROI “B” in panel H). Red trace indicates jRGECO1a fluorescence from same cerebellum ROI. F0 was derived from the averaged intensity values just before addition of epinephrine to the bath.