Fig. 4.
Validation of the model prediction of nicotine effects and HCN2-induced recovery of membrane voltage prepatterns. Representative CC2-DMPE:DiBAC4(3) images of stage ~15 Xenopus embryos: a untreated controls, b nicotine-exposed (0.1 mg/mL – stage 10–35), c Hcn2-WT mRNA microinjected (0.75 ng/injection) in both blastomeres at 2-cell stage, and d nicotine-exposed (0.1 mg/mL – stage 10–35) and Hcn2-WT mRNA microinjected (0.75 ng/injection) in both blastomeres at 2-cell stage. Control embryos show the characteristic hyperpolarization (solid yellow arrow) as previously reported28. Nicotine-treated embryos show reduced signal (depolarized) within the neural tube (hollow yellow arrow). Hcn2-WT mRNA microinjection show enhanced signal (hyperpolarized) within the neural tube (magenta arrows), in presence or absence of nicotine exposure. e Quantification of CC2-DMPE:DiBAC4(3) images of stage ~15 Xenopus embryos along the red dotted line as indicated in the inset illustration, along with electrophysiology based membrane voltage approximations (as previously reported in refs.27,28). The fluorescence intensity/membrane voltage pattern within the neural tube (indicated by the black dotted line in the inset illustration and corresponding lack dotted line in the graph) is significantly reduced (depolarization) in nicotine-exposed embryos in comparison to controls. Hcn2-WT mRNA microinjection significantly enhances the fluorescence intensity/membrane voltage patterns within the neural tube in comparison to controls. Nicotine-exposed embryos that are microinjected with Hcn2-WT mRNA maintain a significantly enhanced fluorescence intensity/membrane voltage pattern within the neural tube in comparison to only nicotine-treated embryos. N = 10 embryos for each treatment group at each of the indicated spatial distance in pixels were collected from multiple animals across independent clutches. Data is plotted as mean ± S.E.M. Data at black line (400 Pixels) was analyzed using one way ANOVA, *p < 0.05, **p < 0.01. (f) Quantification of peak fluorescence intensity and electrophysiology based membrane voltage approximations (as previously reported in ref. 27,28) from voltage reporter dye images (CC2-DMPE:DiBAC4(3)) of ~stage 15 Xenopus embryos within the neural tube at the intersection of the red and black dotted lines in the inset illustration in (a). Nicotine exposure significantly reduced (depolarizes) the neural tube peak intensity/membrane voltage in comparison to controls. Hcn2-WT mRNA microinjection, both in presence or absence of nicotine, significantly enhances (hyperpolarizes) the neural tube peak intensity/membrane voltage in comparison to controls. N = 10 embryos for each treatment group at each of the indicated spatial distance in pixels were collected from multiple animals across independent clutches. Data is plotted as mean ± SD and was analyzed using one way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001