Morphological and swimming behavior changes of zebrafish larvaes after aconitine exposure. (A) Mortality of zebrafish larvaes exposed to aconitine at different concentrations (0.937–30 μM), n = 30. (B–F) Effects of exposure to aconitine on morphological parameters of zebrafish larvaes. Images were taken under a stereomicroscope (32× or 100×), n = 15. (D) Body length (μm). (E) Ocular distance (μm). (F) The surface area of eyes (μm²). (G–N) Effects of exposure to aconitine on neurobehavioral of zebrafish larvaes, n = 10. (G) Behavior trajectories. (H) Distance travelled (mm). (I) Movement speed (mm/s). (J) Time mobile (s). (K) Angular velocity (degree/s). (L) Meander (degree/mm). (M) CW rotation. (N) Frequency of visits to edge zone (%). The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups.

Cell damage of zebrafish larvaes and SH-SY5Y cells caused by aconitine. (A) The images of aconitine-treated larvaes by DCFH-DA (100×), n = 6. (B) The images of aconitine-exposed larvaes by TUNEL assay (100×), n = 3. (C) Cell viability of SH-SY5Y cells after 24 h or 48 h aconitine exposure, n = 8. (D) The images of aconitine-treated SH-SY5Y cells by DCFH-DA (200×), n = 3. (E) SH-SY5Y cell apoptosis aconitine-induced based on Annexin V/PI double staining by flow cytometry, n = 3. All photographs were taken under a stereoscopic fluorescence microscope or confocal microscope. The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups.

Alteration of dopamine and its metabolites levels in zebrafish larvaes and SH-SY5Y cells after aconitine exposure. (A) Dopamine, DOPAC and HVA levels and the turnover of dopamine after larvaes were treated with 1 μM aconitine for 0.5, 3, 6, and 24 h, respectively. (B) Dopamine, DOPAC and HVA contents and the turnover of dopamine after larvaes were incubated with aconitine at doses of 0.5, 1 and 2 μM for 24 h. (C) Intracellular dopamine, DOPAC and HVA levels and the turnover of dopamine at doses of 50, 100, and 200 μM for 24 h in SH-SY5Y cells. (D) Extracellular dopamine, DOPAC and HVA levels and the turnover of dopamine at doses of 50, 100, and 200 μM for 24 h in SH-SY5Y cells. The values are expressed as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups.

Effects of aconitine on TH, MAO, DAT and VMAT2 expressions in zebrafish larvaes and SH-SY5Y cells. (A) The mRNA expressions of th, mao, dat, vmat2 with aconitine exposure in zebrafish larvaes, n = 6. (B) TH and VMAT2 positive cells were investigated using immunostaining and Tg (vmat2:GFP) transgenic zebrafish larvaes, respectively, n = 5. (C) Effect of different exposure times of 1 μM aconitine on the protein expression of TH, n = 4. (D) The protein levels of TH, MAO-B, DAT and VMAT2 with aconitine exposure in zebrafish larvaes, n = 4. (E) The mRNA expressions of th, mao-b, dat, vmat2 with aconitine exposure in SH-SY5Y cells, n = 3. (F) The protein levels of TH, MAO-B, DAT, and VMAT2 with aconitine exposure in SH-SY5Y cells, n = 7. All photographs in which the green fluorescence represented the immune-reactive cells were taken under a confocal microscope (100×). The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups.

Regulation of DR-mediated AC/cAMP/PKA signalling pathway by aconitine in zebrafish larvaes and SH-SY5Y cells. (A) Expression of DR mRNA with aconitine exposure in zebrafish larvaes, n = 3. (B) D1R, D2R, AC, p-PKA, and PKA protein expressions with aconitine exposure in zebrafish larvaes, n = 7. (C) The cAMP contents were examined with aconitine exposure by ELISA assays in zebrafish larvaes, n = 3. (D) Changes of intracellular Ca²⁺ after treatment with aconitine in zebrafish larvaes, n = 5. (E) Expression of DR mRNA with aconitine exposure in SH-SY5Y cells, n = 5. (F) D1R, D2R, AC, p-PKA and PKA protein expressions with aconitine exposure in SH-SY5Y cells, n = 5. (G) The cAMP contents were examined with aconitine exposure by ELISA assays in SH-SY5Y cells, n = 3. All photographs were taken under a confocal microscope (100×). The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups.

FIGURE 6. Effects of SCH23390 and sumanirole on behavioral changes induced by aconitine in zebrafish larvaes. (A–E) SCH23390 inhibited aconitine-stimulated excitatory behaviors, n = 10. (F–J) Sumanirole suppressed aconitine-induced excitatory behaviors, n = 10. The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. aconitine-treated groups.

Effects of SCH23390, sumanirole and H-89 on intracellular Ca²⁺ changes induced by aconitine in zebrafish larvaes. (A) Effects of SCH23390 on intracellular Ca²⁺ changes induced by aconitine. (B) Sumanirole suppressed aconitine-induced the increase of intracellular Ca²⁺. (C) Pretreatment with SCH23390 and sumanirole inhibited the rise of intracellular Ca²⁺ aconitine-stimulated. (D) H-89 inhibited the increase of intracellular Ca²⁺ induced by aconitine. All photographs were taken under a confocal microscope (100×). The values are expressed as mean ± SD, n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. aconitine-treated groups.

FIGURE 8. Effects of SCH23390 and sumanirole on aconitine-induced the activation of AC/cAMP/PKA pathway in SH-SY5Y cells. (A–B) SCH23390 and sumanirole inhibited aconitine-induced cytotoxicity in SH-SY5Y cells, respectivevly, n = 9. (C–F) SCH23390 and sumanirole suppressed aconitine-mediated the activation of AC/cAMP/PKA pathway, n = 5. The values are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control groups. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. aconitine-treated groups.

Schematic diagram shows that aconitine induces neurological impairment via dopamine homeostasis disruption and dopamine receptors-mediated AC/cAMP/PKA pathway activation in zebrafish larvaes and SH-SY5Y cells.