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Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus

Up-regulation of hspb11 transgene accompanies muscle hyperactivity.(A,B) Lateral views of the trunk region of a muscle-specific calcium sensor line Tg([-505/-310]unc45b:GCaMP5A) at 72 hpf. The fluorescent channel images for calcium (green) are merged with the bright field transmission images (gray) for non-contractile resting state (A) and excitatory state (B), exhibiting low and high fluorescent reporter levels, respectively. Anterior side is oriented to the left. Scale bar: 50 μm. (C–H) Profiles of calcium levels in the trunk skeletal muscle from individual embryos treated with either DMSO solvent control (Ctrl, C), dibromoethane (DBE, D), azinphosmethyl (APM, E), propoxur (PPX, F), galanthamine (GAL, G) or veratridine (VER, H) plotted as a function of time (temporal resolution: 2.5 minutes). The number of examined embryos is shown at the top-right of each panel. (I–J) Calcium profiles shown in the panels (C–H) were analyzed for the height of individual calcium spikes (I) and the temporal interval of two adjacent calcium spikes (J) and presented in whisker-box charts with individual data shown as gray dots. Black dots represent outliers. (I) One-way ANOVA showed significant differences of calcium spike height (F[5,315] = 4.1464, p = 0.00116) between solvent control (n = 55 spikes), dibromoethane (n = 16 spikes), azinphosmethyl (n = 89 spikes), propoxur (n = 45 spikes), galanthamine (n = 20 spikes), veratridine (n = 96 spikes). Significant differences over the solvent control were found with azinphosmethyl (TukeyHSD test from here onward, **p = 0.002698), propoxur (*p = 0.01845), galanthamine (**p = 0.009827) and veratridine (*p = 0.01391) treatments, while dibromoethane showed no difference over the control treatment (p = 0.9317). (J) Significant differences of calcium spike intervals were detected (one-way ANOVA, F[5,256] = 12.422, p = 8.405 × 10−11) between solvent control (n = 42 intervals), dibromoethane (n = 12 intervals), azinphosmethyl (n = 75 intervals), propoxur (n = 34 intervals), galanthamine (n = 16 intervals), veratridine (n = 83 intervals). Azinphosmethyl (TukeyHSD test from here onward, **p = 0.003285) and veratridine (***p = 1.0 × 10−7) treatments significantly shortened the interval of calcium spikes, while dibromoethane (p = 0.9744) galanthamine (p = 0.9895) and propoxur (p = 0.9999) were comparable with the control treatment.
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f5: Up-regulation of hspb11 transgene accompanies muscle hyperactivity.(A,B) Lateral views of the trunk region of a muscle-specific calcium sensor line Tg([-505/-310]unc45b:GCaMP5A) at 72 hpf. The fluorescent channel images for calcium (green) are merged with the bright field transmission images (gray) for non-contractile resting state (A) and excitatory state (B), exhibiting low and high fluorescent reporter levels, respectively. Anterior side is oriented to the left. Scale bar: 50 μm. (C–H) Profiles of calcium levels in the trunk skeletal muscle from individual embryos treated with either DMSO solvent control (Ctrl, C), dibromoethane (DBE, D), azinphosmethyl (APM, E), propoxur (PPX, F), galanthamine (GAL, G) or veratridine (VER, H) plotted as a function of time (temporal resolution: 2.5 minutes). The number of examined embryos is shown at the top-right of each panel. (I–J) Calcium profiles shown in the panels (C–H) were analyzed for the height of individual calcium spikes (I) and the temporal interval of two adjacent calcium spikes (J) and presented in whisker-box charts with individual data shown as gray dots. Black dots represent outliers. (I) One-way ANOVA showed significant differences of calcium spike height (F[5,315] = 4.1464, p = 0.00116) between solvent control (n = 55 spikes), dibromoethane (n = 16 spikes), azinphosmethyl (n = 89 spikes), propoxur (n = 45 spikes), galanthamine (n = 20 spikes), veratridine (n = 96 spikes). Significant differences over the solvent control were found with azinphosmethyl (TukeyHSD test from here onward, **p = 0.002698), propoxur (*p = 0.01845), galanthamine (**p = 0.009827) and veratridine (*p = 0.01391) treatments, while dibromoethane showed no difference over the control treatment (p = 0.9317). (J) Significant differences of calcium spike intervals were detected (one-way ANOVA, F[5,256] = 12.422, p = 8.405 × 10−11) between solvent control (n = 42 intervals), dibromoethane (n = 12 intervals), azinphosmethyl (n = 75 intervals), propoxur (n = 34 intervals), galanthamine (n = 16 intervals), veratridine (n = 83 intervals). Azinphosmethyl (TukeyHSD test from here onward, **p = 0.003285) and veratridine (***p = 1.0 × 10−7) treatments significantly shortened the interval of calcium spikes, while dibromoethane (p = 0.9744) galanthamine (p = 0.9895) and propoxur (p = 0.9999) were comparable with the control treatment.

Mentions: To correlate muscle physiology more directly with hspb11 up-regulation, we established a muscle specific Ca2+ biosensor line Tg([-505/-310]unc45b:GCaMP5A). We put the GCaMP5a cassette29 under the control of regulatory elements of the unc45b gene (Etard et al., unpublished), which confer skeletal muscle-specific reporter gene expression (Fig. 5A–B). We exposed embryos at 72 hpf with solvent control, with chemicals that induce hspb11 transgene expression (azinphosmethyl, propoxur, galanthamine and veratridine; Fig. 3A–C,F) and with dibromoethane, which does not induce hspb11 transgene expression (Fig. 3L), and measured Ca2+ fluctuations under a confocal microscope for 16 hours (Fig. 5C–H). To quantify the effects of chemicals on the dynamics of Ca2+ fluctuations, we compared the height of the Ca2+ peaks (Fig. 5I) as well as the time intervals between two adjacent Ca2+ peaks (Fig. 5J). In comparison with the control treatment, exposure to azinphosmethyl and galanthamine led to a significant increase in the height of calcium spikes (**p < 0.01, Fig. 5I). Similar effects were observed with propoxur and veratridine (*p < 0.05, Fig. 5I). In terms of the frequency of the calcium spikes, significantly reduced time intervals were observed with azinphosmethyl and veratridine in comparison to the control (**p < 0.01, Fig. 5J). Dibromoethane, which does not induce hspb11 transgene expression (Fig. 3L), showed no effects on the calcium dynamics compared to the control group (Fig. 5I–J). These increases of calcium spike intensity and frequency are hallmarks of muscle hyperactivity. Thus, we identified induction of muscle hyperactivity as a common feature of chemicals that up-regulate the hspb11 transgene.


Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

Up-regulation of hspb11 transgene accompanies muscle hyperactivity.(A,B) Lateral views of the trunk region of a muscle-specific calcium sensor line Tg([-505/-310]unc45b:GCaMP5A) at 72 hpf. The fluorescent channel images for calcium (green) are merged with the bright field transmission images (gray) for non-contractile resting state (A) and excitatory state (B), exhibiting low and high fluorescent reporter levels, respectively. Anterior side is oriented to the left. Scale bar: 50 μm. (C–H) Profiles of calcium levels in the trunk skeletal muscle from individual embryos treated with either DMSO solvent control (Ctrl, C), dibromoethane (DBE, D), azinphosmethyl (APM, E), propoxur (PPX, F), galanthamine (GAL, G) or veratridine (VER, H) plotted as a function of time (temporal resolution: 2.5 minutes). The number of examined embryos is shown at the top-right of each panel. (I–J) Calcium profiles shown in the panels (C–H) were analyzed for the height of individual calcium spikes (I) and the temporal interval of two adjacent calcium spikes (J) and presented in whisker-box charts with individual data shown as gray dots. Black dots represent outliers. (I) One-way ANOVA showed significant differences of calcium spike height (F[5,315] = 4.1464, p = 0.00116) between solvent control (n = 55 spikes), dibromoethane (n = 16 spikes), azinphosmethyl (n = 89 spikes), propoxur (n = 45 spikes), galanthamine (n = 20 spikes), veratridine (n = 96 spikes). Significant differences over the solvent control were found with azinphosmethyl (TukeyHSD test from here onward, **p = 0.002698), propoxur (*p = 0.01845), galanthamine (**p = 0.009827) and veratridine (*p = 0.01391) treatments, while dibromoethane showed no difference over the control treatment (p = 0.9317). (J) Significant differences of calcium spike intervals were detected (one-way ANOVA, F[5,256] = 12.422, p = 8.405 × 10−11) between solvent control (n = 42 intervals), dibromoethane (n = 12 intervals), azinphosmethyl (n = 75 intervals), propoxur (n = 34 intervals), galanthamine (n = 16 intervals), veratridine (n = 83 intervals). Azinphosmethyl (TukeyHSD test from here onward, **p = 0.003285) and veratridine (***p = 1.0 × 10−7) treatments significantly shortened the interval of calcium spikes, while dibromoethane (p = 0.9744) galanthamine (p = 0.9895) and propoxur (p = 0.9999) were comparable with the control treatment.
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Related In: Results  -  Collection

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f5: Up-regulation of hspb11 transgene accompanies muscle hyperactivity.(A,B) Lateral views of the trunk region of a muscle-specific calcium sensor line Tg([-505/-310]unc45b:GCaMP5A) at 72 hpf. The fluorescent channel images for calcium (green) are merged with the bright field transmission images (gray) for non-contractile resting state (A) and excitatory state (B), exhibiting low and high fluorescent reporter levels, respectively. Anterior side is oriented to the left. Scale bar: 50 μm. (C–H) Profiles of calcium levels in the trunk skeletal muscle from individual embryos treated with either DMSO solvent control (Ctrl, C), dibromoethane (DBE, D), azinphosmethyl (APM, E), propoxur (PPX, F), galanthamine (GAL, G) or veratridine (VER, H) plotted as a function of time (temporal resolution: 2.5 minutes). The number of examined embryos is shown at the top-right of each panel. (I–J) Calcium profiles shown in the panels (C–H) were analyzed for the height of individual calcium spikes (I) and the temporal interval of two adjacent calcium spikes (J) and presented in whisker-box charts with individual data shown as gray dots. Black dots represent outliers. (I) One-way ANOVA showed significant differences of calcium spike height (F[5,315] = 4.1464, p = 0.00116) between solvent control (n = 55 spikes), dibromoethane (n = 16 spikes), azinphosmethyl (n = 89 spikes), propoxur (n = 45 spikes), galanthamine (n = 20 spikes), veratridine (n = 96 spikes). Significant differences over the solvent control were found with azinphosmethyl (TukeyHSD test from here onward, **p = 0.002698), propoxur (*p = 0.01845), galanthamine (**p = 0.009827) and veratridine (*p = 0.01391) treatments, while dibromoethane showed no difference over the control treatment (p = 0.9317). (J) Significant differences of calcium spike intervals were detected (one-way ANOVA, F[5,256] = 12.422, p = 8.405 × 10−11) between solvent control (n = 42 intervals), dibromoethane (n = 12 intervals), azinphosmethyl (n = 75 intervals), propoxur (n = 34 intervals), galanthamine (n = 16 intervals), veratridine (n = 83 intervals). Azinphosmethyl (TukeyHSD test from here onward, **p = 0.003285) and veratridine (***p = 1.0 × 10−7) treatments significantly shortened the interval of calcium spikes, while dibromoethane (p = 0.9744) galanthamine (p = 0.9895) and propoxur (p = 0.9999) were comparable with the control treatment.
Mentions: To correlate muscle physiology more directly with hspb11 up-regulation, we established a muscle specific Ca2+ biosensor line Tg([-505/-310]unc45b:GCaMP5A). We put the GCaMP5a cassette29 under the control of regulatory elements of the unc45b gene (Etard et al., unpublished), which confer skeletal muscle-specific reporter gene expression (Fig. 5A–B). We exposed embryos at 72 hpf with solvent control, with chemicals that induce hspb11 transgene expression (azinphosmethyl, propoxur, galanthamine and veratridine; Fig. 3A–C,F) and with dibromoethane, which does not induce hspb11 transgene expression (Fig. 3L), and measured Ca2+ fluctuations under a confocal microscope for 16 hours (Fig. 5C–H). To quantify the effects of chemicals on the dynamics of Ca2+ fluctuations, we compared the height of the Ca2+ peaks (Fig. 5I) as well as the time intervals between two adjacent Ca2+ peaks (Fig. 5J). In comparison with the control treatment, exposure to azinphosmethyl and galanthamine led to a significant increase in the height of calcium spikes (**p < 0.01, Fig. 5I). Similar effects were observed with propoxur and veratridine (*p < 0.05, Fig. 5I). In terms of the frequency of the calcium spikes, significantly reduced time intervals were observed with azinphosmethyl and veratridine in comparison to the control (**p < 0.01, Fig. 5J). Dibromoethane, which does not induce hspb11 transgene expression (Fig. 3L), showed no effects on the calcium dynamics compared to the control group (Fig. 5I–J). These increases of calcium spike intensity and frequency are hallmarks of muscle hyperactivity. Thus, we identified induction of muscle hyperactivity as a common feature of chemicals that up-regulate the hspb11 transgene.

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus