Prior exposure to methylphenidate attenuates ethanol-induced conditioned place preference in zebrafish

Methylphenidate (MPH) is a psychostimulant widely prescribed for the treatment of attention-deficit/hyperactivity disorder (ADHD) and narcolepsy. Its primary mechanism of action involves inhibition of dopamine and noradrenaline reuptake. The non-medical use of methylphenidate by individuals without neurodevelopmental disorders has been increasing and becoming widespread in contexts of misuse and as a nootropic. This translational study aims to investigate the long-term effects of repeated MPH exposure on the susceptibility to alcohol-induced addiction. The conditioned place preference test (CPP) was used to evaluate preference for an environment previously associated with alcohol, as an indicator of increased sensitivity to its rewarding effects. Adult zebrafish (Danio rerio) were used as the experimental model. This study was conducted in full accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, ensuring transparency, reproducibility, and animal welfare (Ethics Committee approval protocol 005/2024). Animals were randomly assigned to four groups: (1) Control (system water, weekly exposure for 30 days; no alcohol challenge in the CPP test); (2) Control + alcohol (system water for 30 days followed by a 0.5% alcohol challenge in the CPP test); (3) MPH (2 mg/L, twice weekly exposure for 30 days; no alcohol challenge); and (4) MPH + alcohol (MPH 2 mg/L twice weekly exposure for 30 days followed by a 0.5% alcohol challenge in the CPP test). Statistical analysis was performed using parametric tests. Data were analyzed by two-way analysis of variance (two-way ANOVA) followed by Tukey's post hoc test. Differences were considered statistically significant at p < 0.05. Animals exposed only to 0.5% alcohol during CPP exhibited a significant increase in preference, suggesting addictive-like behavior. In contrast, animals previously exposed to MPH showed responses comparable to control levels. This effect may be related to the role of MPH in impulse control and modulation of dopaminergic signaling within reward pathways.