DESI-MSI-Based Multi-Organ Distribution Mapping of Psilocin in Zebrafish

Psilocybin, a psychedelic drug with reported anxiolytic and antidepressant potential, is rapidly metabolized to its active metabolite psilocin. However, a lack of adequate toxicity studies and tissue distribution studies currently restricts its development and application. This study combined behavioral assays in zebrafish with desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to systematically evaluate the acute neurotoxicity of psilocybin and characterize the in vivo spatial distribution of its active metabolite, psilocin. The novel tank test was used to evaluate zebrafish following a 4 h exposure to psilocybin at three different doses (20, 40, and 80 μM; n = 6 per group). Statistical analysis of the data was performed using ANOVA. Behavioral analyses revealed that exposure to psilocybin induced pronounced neurobehavioral alterations, including hyperactivity and disrupted swimming patterns, as evidenced by significant increases in the number of zone transitions and shuttle frequency. We established a DESI-MSI-based method for quantitative mapping and visualization of psilocin in zebrafish tissues. Methodological validation indicated that a linear relationship between ion intensity, spotted amount (R2 = 0.9947), and reproducibility (RSD < 15%) is suitable for quantitative analysis of psilocin in zebrafish tissues. Spatial distribution maps showed that following continuous exposure for 4 h, psilocin was widely distributed across multiple tissues, such as the eye, brain, heart, liver, and kidney, with marked accumulation in the brain and the periportal regions of the liver. Relative psilocin signal intensity revealed a dose-dependent increase in tissue drug levels. The dose-dependent increase in both behavioral hyperactivity and brain psilocin levels points to a consistent relationship, in line with a central site of action. Collectively, these findings demonstrate that DESI-MSI provides a visual and efficient strategy for studying drug distribution in biological tissues from exposed animals. The neurobehavioral toxicity phenotypes and distinct tissue distribution patterns of psilocin uncovered in this study offer critical insights into the biological effects and potential risks of this psychoactive substance.