Aconitine (purity>98%) was purchased from Chengdu Must Bio-Technology Co., Ltd (China) and prepared with dimethyl sulfoxide (DMSO) (Shanghai Solarbio Bioscience and Technology Co., Ltd, China) into the mother liquor with a concentration of 200 mM. Dopamine and its metabolites, namely 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) standards (more than 97% purity), were obtained from Sigma (United States). D1R antagonist SCH23390, D2R agonist sumanirole, and PKA antagonist H-89 were provided by MCE (United States). Anti-TH was acquired from Sigma-Aldrich (United States). Primary antibodies against MAO-B, VMAT2, and D1R were obtained from Abcam (England). Primary antibodies against D2R, DAT, and adenylate cyclase (AC) were purchased from Santa Cruz Biotechnology (United States). PKA, p-PKA, and β-actin rabbit monoclonal antibodies were obtained from Cell Signaling Tech (Danvers, MA, United States). Trizol for RNA extraction was acquired from Invitrogen (United States). All primers for PCR were provided by Biological Engineering Technology Company (Shanghai, China). First-strand cDNA Synthesis kit and SYBR Green Quantitative kits for real-time polymerase Chain Reaction (RT-PCR) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China).

Wild type AB zebrafish (Danio rerio) were purchased from the China Zebrafish Resource Center. Tg (vmat2:GFP) transgenic zebrafish were provided by S. Huang’s laboratory. Adult zebrafish were bred in accordance with the routine procedures of the zebrafish breeding center at Chengdu University of Traditional Chinese Medicine (Li et al., 2020). The females and males (1:2) were placed in a breeding tank overnight and were initially separated by partitions that were removed next morning after 30 min of illumination. After the removal of the dead eggs and faecal residual, the fertilized eggs were collected and transferred to a sterile culture dish. Zebrafish larvaes at six dpf were used for the following experiments. The animal experiments involved in this study were approved by the Animal Research Ethics Committee of Chengdu University of Traditional Chinese Medicine (SYXK 2016-04).

Aconitine was diluted with fish-raising water to different concentrations with less than 0.1% DMSO in each group. Larvaes at six dpf were placed in a 24-well plate with the density of 10 embryos per well, and were exposed to 0.937, 1.875, 3.75, 7.5, 15, or 30 μM of aconitine for 24 h, respectively. Three parallel samples were set for each concentration following the similar criteria with the vehicle controls. The fish with no visible movement (such as the flapping of gills, no response to touching the tail) was dead, and the dead larvaes in each group was collected to calculate the median lethal concentration (LC50) value of aconitine on zebrafish larvaes.

According to the LC50 of aconitine, the larvaes were treated with 0.5, 1, and 2 μM of aconitine in morphological observation and the following experiments. Potential teratogenicity of aconitine was estimated by monitoring the morphological defects of larvaes at seven dpf under a stereomicroscope. Several morphological indices, including body length (μm), ocular distance (μm), and surface area of the eyes (μm²), were evaluated using ImageJ.

TMR (red)-TUNEL cell apoptosis detection kit (Servicebio, China) was used to investigate whether aconitine induces the apoptosis of neuronal cells in zebrafish larvaes (Quan et al., 2019). Larvaes at six dpf were treated with aconitine following the methods in Section 2.4. The larvaes were fixed overnight at 4°C in 4% paraformaldehyde (PFA). After washing with PBS, the larvaes were orderly dehydrated by 95%, 90%, 80%, and 70% ethanol for 5 min each and then stored in 100% methanol overnight at -20°C. The larvaes were rehydrated and treated with proteinase K (20 μg/ml) at 37°C for 25 min, and then washed three times with PBS. The samples were added with 0.1% triton to permeabilize the tissues for 20 min at room temperature. The larvaes were then incubated at 37°C in a TUNEL reaction mixture containing TDT enzyme, dUTP, and buffer (1:5:50) for 2 h. After incubation, the nuclei were counterstained with DAPI at room temperature for 10 min. The larvaes were washed with PBS (three times, 5 min each) and covered with an anti-fade mounting medium. Apoptotic cells staining red and nuclei staining blue were observed under a fluorescence microscope.

Zebrafish larvaes at six dpf were transferred into 96-well plates (one fish/well) and treated with 100 μl of aconitine (0.5, 1, or 2 μM) per well for 24 h. After 0.5, 3, 6, 12, and 24 h, the swimming behavior of larvaes was monitored by the automated video tracking system (Noldus Co., Netherlands). After 5 min of habituation without light, the behavioral trajectories of larvaes for 10 min were captured and used the Ethovision XT12 software (Noldus Co., Netherlands) to track the swimming activity at a rate of 15 positions per second. Total distance travelled, movement speed, movement time, angular velocity, meander, clockwise (CW) rotation, frequency of visits to edge zone, and the behavioral trajectories were also obtained through the Ethovision XT12 software.

Human neuroblastoma (SH-SY5Y) cells were purchased from Procell Life Science and Technology (Wuhan, China) and routinely cultured in the mixed medium of MEM: F12 (1:1) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Sigma) at 37°C with 5% CO₂. The culture medium was renewed once every 2 days. Aconitine was diluted with less than 0.1% DMSO to 25–400 μM of concentration range for the following experiments.

Cell viability was performed using the CCK-8 assay kit (Biosharp Life Sciences, China) according to the protocol. The cells (5×10³ cells/well) were seeded in a 96-well plate. After treatment with different concentrations of aconitine (25, 50, 100, 150, 200, 300, and 400 μM) for 24 or 48 h, the cells were incubated with 10 μl of CCK-8 solution for 2 h in the dark at 37°C. The absorbance was determined at 450 nm under an automated microplate spectrophotometer (Spectra Max, MD, United States).

The Annexin V-FITC/PI double staining kit (Solarbio Life Sciences, China) was performed to quantify cell apoptosis according to the manufacturer’s instruction. The cells were incubated with aconitine at doses of 50, 100, and 200 μM for 24 h, and washed twice with cold PBS. The collected samples were centrifuged at 300 g for 5 min, and the supernatant was removed. The cells were resuspended with the binding buffer at a concentration of 1 × 10⁶ cells/mL. The cell suspensions were added with 5 μl of Annexin V-FITC, mixed gently, and incubated for 10 min at room temperature in the dark. All samples were in the dark incubated for 5 min at room temperature after adding 5 μl of PI solution. Annexin V-FITC binding and PI staining were evaluated by a flow cytometer (Ex = 488 nm, Em = 530 nm) (FACSVerse, BD Biosciences, United States). Total 10,000 events were analyzed for each independent experiment.

DCFH-DA staining was used to determine intracellular ROS levels in vivo and in vitro. The larvaes were placed in a 12-well plate with athe density of 10 fishes per well, and incubated with 0.5, 1, and 2 μM of aconitine for 24 h. The probe solution was diluted following the instructions. After staining with 25 μM of working solution (freshly prepared) at 28°C for 1 h in the dark, zebrafish larvaes were washed with fresh fish water three times and were taken photos under a stereoscopic fluorescence microscope (Leica, M165 FC, Singapore). In vitro, SH-SY5Y cells in each group were treated with aconitine at doses of 50, 100, and 200 μM for 24 h. The cells were incubated with 10 μM working solution prepared by serum-free medium for 1 h at 37°C. After the cells were gently washed with PBS two times, the fluorescence images were captured under a laser scanning confocal microscope (Leica TCS-SP8 SR, Germany). The intensity of fluorescence was analyzed with the ImageJ software.

Fifty larvaes per sample were diluted in 30 μl of PBS, and then added with 6 μl of 0.1 mM perchloric acids after tissue homogenization, following 10 min of incubation on ice. The homogenates were centrifuged at 4°C with 14,000 g for 15 min 400 µl of supernatant was transferred to 1.5 ml of Eppendorf tubes, in which the same amount of 0.1 M perchloric acid was added on ice for 10 min, following centrifugation at 14,000 g (4°C) for 15 min. The supernatant was filtered through a 0.22 μm microporous membrane and stored in a brown sample bottle for HPLC analysis. In vitro, the cells seeded in a new culture dish were treated with different concentrations of aconitine (50, 100, and 200 μM) for 24 h. The cells were collected and centrifuged after wash with PBS twice, following resuspension with 30 μl of PBS. The cell samples were collected following the similar methods with the larvae samples. Next, dopamine, DOPAC, and HVA standards with the concentration of 100 μg/ml and the calibration mixture were prepared with ultra-pure water. The calibration concentrations ranged from 0.5 ng/ml to 500 ng/ml of dopamine and DOPAC, and from 2 ng/ml to 2000 ng/ml of HVA. The levels of dopamine and its metabolites were measured by HPLC (Enayah et al., 2018). Ultimate 3,000 system (ThermoFisher, United States) equipped with a diode array detector was used for HPLC analysis. Chromatographic separation was performed using a ChromCore C18 chromatographic column (4.6 mm × 250 mm, 5 μm, NanoChrom Technologies, China). The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid at the flow rate of 1 ml/min with the column temperature of 25°C. The results were expressed as a nanogram of analyte per milligram of protein in the homogenate. Total protein was measured by the BCA protein quantification kit. The ratio of DOPAC + HVA to dopamine contents was calculated to reflect dopamine turnover that is a specific index of dopamine metabolism (Thiffault et al., 2000).

Tg (vmat2:GFP) transgenic zebrafish embryos at one dpf were cultured in dishes with 0.03% 1-phenyl 2-thiourea (PTU) that inhibits the formation of melanin. The larvaes of seven dpf were anaesthetized (tricaine, 0.4%) and covered with 3% methylcellulose after different concentrations of aconitine treatment for 24 h. The zebrafish’s position was fixed and the brain of larvaes were photographed by LSCM (Olympus FV-1200, Japan).

Whole-mount immunofluorescence staining was used to observe the number of TH-positive cells in zebrafish larvaes. Wild type AB larvaes were treated with aconitine for 24 h, washed with cold PBS three times, and fixed with 2 ml of 4% formaldehyde overnight. After wash three times with TBST, the samples were punched with 1 ml of working fluid (1.25% NaOH: H₂O₂ = 4:1) for 20 min, added with 2.0 ml of methanol, and placed in a horizontal shaker for 15 min. The larvaes were blocked with 2% nonfat-dried milk (TBST +2% BSA) for 1 h, following incubation with the mouse-anti-TH antibody (1:500) at 4°C overnight. Alexa Fluor 488 labelled goat-anti-mouse antibody (1:200) was applied at room temperature for 1 h in the dark. The embryos were washed three times with TBST for total 30 min. The density of fluorescence was observed under LSCM.

Fluo-4 AM, a fluorescent dye (Servicebio, China), is usually applied to detect cytosolic Ca²⁺. After aconitine treatment, the larvaes were incubated with 40 μM of Fluo-4 AM containing 0.8% DMSO in egg water at room temperature for 30 min in the dark. The larvaes were then washed three times with fresh fish water, and the images were photographed under LSCM.

Zebrafish larvaes at six dpf were transferred to a 6-well plate (30 larvaes/well) and incubated with aconitine for 24 h. The larvaes were then washed twice with cold PBS and transferred to a new centrifuge tube. Each sample was added with 200 μl of RIPA containing PMSF and protease Inhibitor Cocktail (1:100) and homogenized for 2 min at -20°C with 70 Hz (Jingxin, China). The homogenates were centrifuged at 12,000 g, 4°C for 15 min, and the supernatant was collected. In vitro, SH-SY5Y cells were seeded in a culture dish as described previously. After aconitine administration, the cells were harvested and washed twice with cold PBS, lysed by RIPA on ice for 30 min, and centrifuged at 14,000 g, 4°C for 15 min. The supernatant was collected to 500 µl of centrifuge tube. After the protein was quantified with the BCA protein quantification method, the lysates were denatured with the loading buffer at 95°C for 5 min. An equal amount of protein (20–50 μg/well) was electrophoresed on 10% SDS-PAGE gel. The proteins were then electroblotted to PVDF membranes and blocked with 5% non-fat skim milk diluted in TBST (0.1% Tween/TBS) for 2 h. The membranes were then incubated with the primary antibody overnight, washed three times (5 min each time), and incubated again with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The bands on the membranes were visualized with the SuperLumia ECL HRP substrate kit (Abbkine, China). The acquired images were analyzed using Gel Pro3.0 (Media Cybernetics). Total protein level was normalized using the level of β-actin protein in each sample and presented as a percentage of the controls.

Total RNA of zebrafish larvaes and SH-SY5Y cells were extracted by using Trizol following the manufacturer’s protocols. The concentration and purity of RNA were measured by the NanoDrop 2000 spectrophotometer (Thermo, United States). RNA was reversely transcribed to single-strand cDNA by using the first-strand cDNA synthesis kit. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed on a CFX Connect fluorescence quantitative PCR instrument (BIO-RAD, United States) using the SYBR Green Quantitative kit. The reaction mixture (20 μl) contained 10 μl of 2×qPCR Mix, 0.6 μl of 10 μM each of the forward and reverse primers, 1 or 2 μl of template cDNA, and 7.8 or 6.8 μl of diethylpyrocarbonate (DEPC) H₂O. The primer sequences are shown in Supplementary Table S1. PCR amplification was performed under the following conditions: 95°C for 15 min, followed by 39 cycles of 95°C for 10 s and 60°C for 30 s. The melt curve assay was conducted with the increasing temperature from 60 to 95°C by 0.3°C every 5 s. The relative mRNA expression levels were standardized with the β-actin and represented by a ratio of the expression in aconitine-treated groups versus that in the controls.

All statistical analyses were performed in SPSS 22.0 statistical software (IBM Corp). The normality of data were assessed using the Kolmogorov Smirnov test. The Levene’s test was used to evaluate the homogeneity of variance. The differences between groups were analyzed using the one-way analysis of variance (ANOVA) following the least-significant difference (LSD) or Tamhane’s T2 post-hoc comparison. All data were expressed as mean ± standard deviation (SD) of at least three independent experiments. The difference was considered to be significant at the p < 0.05 level.

The results of acute toxicity test showed that zebrafish larvaes presented high mortality in an obvious concentration-dependent manner after treatment with aconitine (Figure 1A). Compared with the control group, the aconitine groups at doses of 0.937, 1.875, and 3.75 μM showed mortality percentages of 3.33%, 6.67%, and 20%, respectively. With the increasing doses of aconitine, the mortality rates gradually increased to 100%. The calculated LC50 of aconitine on larvaes for 24 h was 5.156 μM with 95% CI: 4.611–5.747 μM. The teratogenic effects of aconitine on the morphology of larvaes were investigated 24 h after administration. As a result, lighter pigmentation, body curvature, shorter body length and ocular distance, and increased surface area of the eyes in the aconitine groups were observed, compared with those in the control groups (Figures 1B–F). The locomotion and orientation of larvaes was examined at five different periods (0.5, 3, 6, 12, and 24 h) to reflect the behavior pattern of larvaes. Treatment with aconitine significantly increased the distance travelled and movement speed of larvaes after 3 h. The swimming distance and speed in 1 and 2 μM of aconitine groups reached the peak value at 12 and 6 h, respectively, following a decreasing trend. The above parameters in the 2 μM group significantly lowered, but the levels in the 1 μM group were still higher than those in the control group after 24 h (Figure 1H,I). The effect of aconitine treatment on time mobile was similar to the distance travelled and movement speed (Figure 1J). But the swimming time of the 2 μM group was fastest at 12 h and then dropped to the level below the control group. The angular velocity of the 2 μM group reached the maximum at 12 h, whereas this parameter continuously increased in the 1 μM group (Figure 1K). The meander of 1 and 2 μM groups reached the peak at 12 h and then decreased to the level of the control group, while the meander of 0.5 μM group increased in a time-dependent manner (Figure 1L). The CW rotation of animals exposed to 1 and 2 μM of aconitine for 12 h significantly increased compared with that of the control (Figure 1M). Moreover, the frequency of visits to the edge zone dose and time-dependently increased after treatment with aconitine for 6 h (Figure 1G,N). The larvaes in 1 and 2 μM of aconitine groups gradually showed unbalance swimming, upside down of head, tail violent tremor, intermittent in situ “circular” violent rotation, and other abnormal behaviors with the increasing exposure time, compared with those in the control groups. Given these, aconitine exposure resulted in dramatic alterations of the spontaneous movement and motor coordination in zebrafish larvaes.

DCFH-DA staining was applied to determine intracellular ROS levels of zebrafish larvaes. Aconitine incubation at doses of 1 and 2 μM for 24 h significantly increased ROS production in the brain of zebrafish larvaes, compared with that in the control group (Figure 2A). TUNEL experiments were performed to observe whether aconitine induced cell apoptosis in the brain of zebrafish larvaes. The red fluorescence density of 2 μM aconitine greatly enhanced in the enlarged images compared to the control group, suggesting that aconitine could induce apoptosis (Figure 2B). In vitro, the cytotoxicity of aconitine on SH-SY5Y cells was assessed by the CCK-8 assay. Cell viability were significantly reduced in a dose and time-dependent manner after aconitine incubation (Figure 2C). After incubation with DCFH-DA probes, the green fluorescence in the 200 of μM aconitine exposure group also strengthened in cells (Figure 2D). Results from flow cytometry analysis demonstrated that aconitine dose-dependently induced apoptosis of SH-SY5Y cells (Figure 2E). Hence, aconitine could trigger ROS overproduction and cell apoptosis, leading to neurotoxicity in zebrafish larvaes and SH-SY5Y cells.

To explore the relationship between aconitine-induced behavioral changes and dopamine homeostasis, we evaluated the turnover of dopamine in vivo and in vitro. First of all, dopamine, DOPAC, and HVA contents in zebrafish were detected at different time points after exposure to 1 μM of aconitine. As a result, dopamine level initially decreased at 0.5 h of aconitine treatment and the dopamine turnover that reflects dopamine metabolic rate significantly increased in zebrafish larvaes. However, aconitine induced a significant increase of dopamine and DOPAC levels at 24 h compared to the control, whereas HVA level significantly reduced, indicating that dopamine turnover went down (Figure 3A). In addition, zebrafish larvaes at six dpf were treated with different concentrations of aconitine for 24 h to explore the dose-response relationship between aconitine and dopamine turnover. Dopamine levels significantly increased in aconitine treatment groups compared with that in the control group, while DOPAC level only increased in the 1 and 2 μM of aconitine groups and HVA level decreased significantly after aconitine treatment (Figure 3B). The results also revealed that aconitine decreased significantly dopamine turnover in a dose-dependent manner. In vitro, Figure 3C,D, the iterative experiments revealed the significant effects of aconitine on intracellular and extracellular levels of dopamine, DOPAC, and HVA. Both intracellular and extracellular dopamine contents dose-dependently increased after aconitine incubation. As for the metabolites of dopamine, intracellular DOPAC level also increased in a dose-dependent manner, whereas intracellular HVA level significantly decreased. Extracellular DOPAC and HVA alterations were exactly the opposite of intracellular changes. Nevertheless, both intracellular and extracellular dopamine turnover dose-dependently slowed down. Thus, aconitine could affect dopamine turnover to perturb dopamine homeostasis in vivo and in vitro.

As mentioned above, aconitine disrupted dopamine homeostasis by decreasing the dopamine turnover both in vivo and in vitro. The dopamine metabolism-related function, including dopamine synthesis, degradation, transport, and reuptake, were examined to further explore the underlying mechanisms. As a rate-limiting enzyme of dopamine synthesis, TH protein expression was measured after treatment with 1 μM of aconitine for different times. As shown in Figure 4A,D, the protein and mRNA levels of TH remarkably increased in a concentration-dependent manner after aconitine treatment for 24 h. TH protein expression also increased in a time-dependent manner, and the gray value at 24 h increased to 2.9 times of that in untreated condition (Figure 4C). The mao mRNA expression significantly decreased in all treatment groups. However, the MAO-B protein expression in 1 and 2 μM of aconitine groups significantly increased, compared with that in the control group. The mRNA and protein expression levels of DAT significantly decreased in 1 and 2 μM of aconitine groups. The vmat2 mRNA expression in the 0.5 μM group had a sharp rise and then and decreased significantly in 1 and 2 μM groups, compared with that in the control group. The VMAT2 protein levels had a significant reduction in all treatment groups. Whole-mount immunofluorescence staining and Tg (vmat2:GFP) transgenic zebrafish were used to evaluate the expression of TH and VMAT2 in dopaminergic neurons of zebrafish larvaes, respectively (Figure 4B). Similar to the results of protein expression, the number of TH-positive neurons of the telencephalon greatly increased, while the fluorescence intensity of mesencephalon decreased in Tg (vmat2:GFP) transgenic zebrafish after different concentrations of aconitine exposure (Figure 4D). In SH-SY5Y cells (Figure 4E,F), the TH mRNA and protein expressions significantly increased in the aconitine treatment groups compared with that in the control group. In addition, the mRNA and protein levels of MAO-B significantly increased in a concentration-dependent manner, whereas both DAT and VMAT2 mRNA and protein expression levels significantly decreased after aconitine treatment.

It is well known that DRs can regulate the content of cAMP to mediate the downstream signal transduction (Beaulieu and Gainetdinov, 2011). Therefore, we further investigated the effects of aconitine on the DRs-mediated AC/cAMP/PKA signalling pathway. The mRNA expression of D₁-like dopamine receptors (drd1a, drd1b and drd5a) and D₂-like dopamine receptors (drd2a, drd2b, drd2l, drd3, drd4a, drd4b, and drd4-rs subtypes) was assessed to clarify the states of dopamine receptors after aconitine exposure in zebrafish (Yamamoto et al., 2015; Shontz et al., 2018). As depicted in Figure 5A, the drd1a mRNA levels remarkably rose only in 1 μM treatment group, compared with that in the control group, while the drd1b mRNA levels significantly increased in all aconitine-treated groups. The mRNA expression levels of drd2a and drd2b were significantly attenuated by aconitine in a concentration-dependent manner. The transcript levels of drd3 and drd4-rs significantly reduced only in the 2 μM group compared with that in the control group. By contrast, 0.5 and 1 μM of aconitine significantly increased the drd4a levels. Results in Figure 5B showed that the D1R protein levels significantly increased in 0.5 and 1 μM groups. Interestingly, aconitine at different concentrations has a bidirectional regulation effect on the D2R protein expression. Results indicated that the expression of D2R protein significantly increased at a low concentration (0.5 μM) and reduced at a high concentration (2 μM) in zebrafish larvaes. The AC protein levels were dose-dependently increased by aconitine treatment. Aconitine also significantly enhanced the phosphorylation levels of PKA in all aconitine groups. The content of cAMP in larvaes was detected by ELISA. As a result, the levels of cAMP significantly increased in 1 and 2 μM groups (Figure 5C). As shown in Figures 5D, 1 μM of aconitine significantly increased the Ca²⁺ fluorescence intensity in the brain of larvaes after 24 h of exposure.

Differ from zebrafish, the mammals have five main subtypes of dopamine receptors, which are encoded by the genes drd1, drd2, drd3, drd4, and drd5 (Beaulieu et al., 2015). The effects of aconitine on the expression of these genes were evaluated in SH-SY5Y cells. As shown in Figure 5E, the drd1 mRNA expression significantly increased in a concentration-dependent manner, whereas the drd4 expression significantly decreased, compared to the controls. The drd2 mRNA levels significantly increased at 50 μM and significantly decreased at 200 μM. The mRNA expression of drd3 increased significantly after different concentrations of aconitine treatment. The drd5 mRNA expression significantly decreased at 50 μM. In vitro, the effects of aconitine on the AC/cAMP/PKA signalling pathway was similar to that of zebrafish larvaes (Figure 5F,G). After aconitine exposure, the protein levels of D1R, AC, and cAMP content were significantly enhanced, while the ratio of p-PKA to PKA was significantly increased, indicating that D1R-mediated AC/cAMP/PKA signalling pathway was activated. Meanwhile, aconitine significantly reduced the protein expression of the D2R in 100 and 200 μM groups in cells, which differ from the vivo results. In general, in vivo and in vitro studies showed that aconitine activated the AC/cAMP/PKA signalling pathway by regulating the expression of D1R and D2R.

The effects of SCH23390 (a selective inhibitor of D1R) and sumanirole (a highly selective D2R complete agonist) on the swimming behaviors affected by aconitine were evaluated. Based on the results of the locomotor behavioral test, we investigated the concentration and time of SCH23390 and sumanirole incubation prior to aconitine treatment (Supplementary Figure S1A, B). The larvaes were treated with 1 μM of aconitine for 24 h after the presence or absence of 10 μM SCH23390 or 10 μM sumanirole for 2 h. As shown in Figure 6A–D, the increases of distance, movement speed, and mobility time in response to aconitine stimulation were significantly inhibited at 0.5, 3, and 6 h after SCH23390 pretreatment. SCH23390 significantly attenuated the increase of frequency of visits to the edge zone induced by aconitine at 0.5-24 h (Figure 6E). Furthermore, aconitine-induced increases in the distance and mobility time were also effectively inhibited at 3, 6, and 12 h after sumanirole administration, while the movement speed was significantly lowered by sumanirole at 6 and 12 h (Figures 6F–I). In contrast to SCH23390, pretreatment with sumanirole barely reversed the increases of frequency of visits to the edge zone by aconitine-induced (Figure 6J).

The effects of SCH23390, sumanirole, and H89 (a selective inhibitor of PKA) on intracellular Ca²⁺ induced by aconitine treatment were determined. As shown in Figure 7A, although the green fluorescence of the aconitine group with the presence of SCH23390 was slightly weakened compared with that of the aconitine group, the difference had no significance. Fortunately, pretreatment with sumanirole effectively inhibited the increase of intracellular Ca²⁺ induced by aconitine in the brain of larvaes (Figure 7B). Co-pretreatment with SCH23390 and sumanirole further enhanced the inhibition of Ca²⁺ elevation in response to aconitine stimulation (Figure 7C). As another target molecule of the AC/cAMP/PKA signalling pathway, the effects of PKA intervention with H-89 on intracellular Ca²⁺ in the brain of larvaes were examined. Similar to DRs modulators, the pretreatment concentration and time of H-89 were also optimized (Supplementary Figure S1C). The larvaes were treated with 10 μM of H89 for 3 h before aconitine administration. The result showed that H-89 effectively reversed aconitine-induced an increase of intracellular Ca²⁺ zebrafish larvaes (Figure 7D).

DRs modulators SCH23390 and sumanirole were separately used to further elucidate the roles of D1R and D2R in aconitine-activated AC/cAMP/PKA pathway. Similar to in vivo experiments, the pretreatment concentration and time of DRs modulators in cells were also investigated (Supplementary Figure S2). SH-SY5Y cells were pre-incubated with 10 μM of SCH23390 for 2 h or 10 μM of sumanirole for 0.5 h before aconitine treatment, respectively. Aconitine-induced cytotoxicity of SH-SY5Y cells could be effectively attenuated by 10 μM of SCH23390 or 10 μM of sumanirole (Figure 8A, B). In addition, both pretreatment with SCH23390 and sumanirole inhibited the activation of the AC/cAMP/PKA pathway caused by aconitine (Figures 8C–F). These results indicated that aconitine-induced neurotoxicity in SH-SY5Y cells might partially attribute to the activation of the AC/cAMP/PKA/pathway by up-regulating D1R or down-regulating D2R.