Comparative evaluation of MPTP and rotenone as inducing agents for Parkinson's disease in adult zebrafish: Behavioural and histopathological insights

Abstract

Parkinson's disease (PD), a prevalent neurodegenerative disorder, is marked by dopaminergic neuron loss and motor impairments. This study aimed to establish and compare PD models in adult zebrafish using two neurotoxins, MPTP and rotenone, evaluating their impact on behaviour and histopathology. Zebrafish were exposed to MPTP via intraperitoneal injection at two different doses or to rotenone in water for 21 days. Behavioural assessments, including Novel Tank Diving Test, bradykinesia, and C-bend response, revealed progressive motor and anxiety-like impairments, with rotenone exhibiting stronger locomotor effects. Histopathological analyses confirmed dose-dependent neurodegeneration in brain regions, with MPTP showing localized damage and rotenone causing widespread but milder effects. While both neurotoxins induced PD-like phenotypes, rotenone produced more pronounced locomotor deficits, whereas MPTP triggered anxiety-like symptoms. In conclusion, our study demonstrates that MPTP induces significant locomotor dysfunction along with anxiety-like symptoms, while rotenone strongly impacts locomotion with mild anxiety effects. Both neurotoxins exhibited maximum effects at their highest doses and over a similar time frame (Day 14 to Day 22). These findings highlight the distinct neurotoxic mechanisms of MPTP and rotenone and their relevance in modelling PD pathogenesis. The zebrafish model provides a robust platform for studying neurodegenerative diseases and testing therapeutic interventions. Further studies are required to explore the molecular mechanisms underlying their neurotoxic effects and to validate these models for long-term and translational research.

Keywords: Behavioural analysis; Histopathology; MPTP; Parkinson’s disease; Rotenone; Zebrafish.

Graphical abstract

Fig. 1

Experimental procedure timeline. MPTP injections were administered on Days 1 and 14. Rotenone exposure lasted for 21 days. Swimming behaviour tests were conducted on Days 0, 14, and 22.

Fig. 2

a) Total distance travelled in novel tank (m), b) Average speed (m/s), c) Number of crossings in novel tank. The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3

Locomotion trajectories of test zebrafish in novel tank after exposure to different treatments (n = 10). a) Control, b) MPTP-A, c) MPTP-B, d) Rotenone-A, and e) Rotenone-B.

Fig. 4

a) Time spent in top zone (s), b) Distance travelled in the top zone (m), c) Time spent in the bottom zone (s), d) Distance travelled in the bottom zone (m). The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5

Latency to first entry to the top zone (s). The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05.

Fig. 6

a) Total distance travelled in the bradykinesia tank, b) Average speed of zebrafish, c) Absolute turn angle (°), and d) Number of lines crossing between quadrants by zebrafish. The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7

Locomotion trajectories of test zebrafish in bradykinesia tank after receiving various administrations. a) Control, b) MPTP-A, c) MPTP-B, d) Rotenone-A, and e) Rotenone-B.

Fig. 8

Number of C-Bend in response to external stimuli after exposure to different treatments. The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 9

Body weight alteration in different experimental groups on day 0, day 14 and day 22. The data were expressed as mean ± SEM based on biological replicates (n = 10). Statistical significance was assessed using a two-way ANOVA followed by Tukey's post hoc test. Significance levels were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 10

Histopathological alteration caused by MPTP and rotenone in zebrafish brain of different experimental groups a) Control, b) MPTP-A, c) MPTP-B, d) Rotenone-A, and e) Rotenone-B. All images captured at 20x magnification. Scale bar = 10 µm.