INTRODUCTION: Psilocybin and ketamine are fast-acting antidepressants, but their precise neurochemical actions and safety profiles differ. Both enhance monoaminergic and aminoacidergic neurotransmission but via distinct mechanisms.

AIM(S): To determine how psilocybin versus ketamine affect neurotransmitter release, molecular outcomes and rat behavior.

METHOD(S): Male Wistar rats were injected with psilocybin or ketamine. In vivo microdialysis examined extracellular DA, 5-HT, glutamate and GABA in cortical and limbic structures. Western blot was used to assess the expression of selected receptors. Locomotion, anxiety/depression-like behaviors and psychotogenic potential were tested using open-field, light–dark box, prepulse inhibition, head-twitch and forced swim tests.

RESULTS: Both compounds affected extracellular concentrations of examined neurotransmitters. High-dose psilocybin caused DNA damage in frontal cortex and hippocampus; ketamine affected only hippocampus. Neither drug produced clear antidepressant or anxiolytic behaviors. Low doses of psilocybin also elevated monoamines and GABA but did not cause DNA damage. Low-dose psilocybin produced an anxiolytic response without causing psychotomimetic effects.

CONCLUSIONS: Ketamine and psilocybin both acutely boost cortical monoamine and GABA levels, but their regional effects and safety differ. Importantly, high doses of psilocybin carried genotoxic risk in cortex and hippocampus that ketamine did not, whereas low doses of psilocybin were anxiolytic without toxicity. These results suggest that ketamine and psilocybin engage overlapping but distinct neurotransmitter systems and receptors. Dose is critical: high doses of psilocybin require caution due to neurotoxicity, whereas low sub-psychedelic doses may offer anxiety relief safely.

FINANCIAL SUPPORT: Supported by the National Science Centre, Poland (grant no. 2020/37/B/NZ7/03753) and statutory funds of the Maj Institute of Pharmacology, Polish Academy of Sciences.