Effect of Psilocybin and Ketamine on Brain Neurotransmitters, Glutamate Receptors, DNA and Rat Behavior

Mood and anxiety disorders remain leading causes of global disability, and current antidepressants often require weeks to take effect and fail in treatment-resistant cases. Recent clinical work has reported rapid antidepressant effects of ketamine and serotonergic psychedelics such as psilocybin, but the precise mechanisms that underlie these benefits and possible toxicities are incompletely understood. Earlier research implicates glutamate-mediated synaptic plasticity—via NMDA receptor modulation for ketamine and 5-HT2A receptor activation for classical psychedelics—as a shared pathway, yet direct comparative data on neurotransmitter dynamics, receptor adaptations and potential genotoxic effects are limited. Wojtas and colleagues set out to address these gaps by directly comparing a single administration of ketamine and two doses of psilocybin in rats. They measured extracellular dopamine (DA), serotonin (5-HT), glutamate and GABA in the frontal cortex and the reticular nucleus of the thalamus using in vivo microdialysis, assessed DNA damage in frontal cortex and hippocampus with the alkaline comet assay, quantified selected NMDA and AMPA receptor subunit proteins by Western blot, and evaluated behaviour in the open field, light–dark box and forced swim tests. Behavioural and molecular end points (except microdialysis) were assessed at least 24 h after dosing to distinguish persistent from acute effects, and MDMA was included as a comparator in the genotoxicity assays.

Adult male Wistar Han rats (280–350 g) were used across experiments; animals were acclimatised and housed under controlled temperature and humidity with a 12:12 light:dark cycle. Experimental procedures received local institutional approval and complied with relevant European regulations. The extracted text does not clearly report group sizes for all experiments; where n is important it appears to have been provided in figures or supplementary material not fully present in the extraction. Drugs were administered intraperitoneally (ip). Psilocybin was tested at 2 and 10 mg/kg, ketamine at 10 mg/kg, and MDMA at 10 mg/kg (the latter as a known inducer of oxidative DNA damage). Solutions were prepared fresh on the day of experiment. Microdialysis was performed in freely moving animals: probes were implanted into the frontal cortex (AP +2.7, L +0.8, V −6.5 mm from dura) and the reticular nucleus of the thalamus (AP −1.5, L +2.1, V −5.0 mm). After a 7-day recovery, artificial cerebrospinal fluid was perfused at 2 µL/min, five baseline samples were collected every 20 min following a 2 h washout, and dialysates were collected for 240 min after drug injection. Probe placement was validated histologically. Extracellular DA and 5-HT were quantified by HPLC with electrochemical detection; glutamate and GABA were measured by HPLC after OPA derivatisation. Limits of detection for the neurotransmitters are reported in the text. For receptor protein analysis, frontal cortex tissue was collected and processed for Western blotting; GluN2A, GluN2B, GluA1 and GluA2 were probed and normalised to GAPDH, with analyses performed 24 h after drug administration. The alkaline comet assay was used to assess oxidative DNA damage in frontal cortex and hippocampus seven days after dosing; damage was expressed as tail moment. Behavioural testing (open field, light–dark box, forced swim test) was performed 24 h post administration to reduce acute drug effects. Statistical analyses employed repeated-measures ANOVA for microdialysis data and one- or two-way ANOVA for other outcomes, with Tukey's post hoc tests and significance at p < 0.05; outliers were removed using Grubb's test.

Microdialysis in the frontal cortex showed dose- and drug-dependent changes across monoamines and amino acids. Psilocybin at 2 mg/kg increased extracellular DA to approximately 200% of baseline, while ketamine 10 mg/kg produced a larger DA elevation (up to about 500% of baseline). Repeated-measures ANOVA indicated significant treatment, time and interaction effects, and AUC analyses confirmed increased total DA release for psilocybin 2 mg/kg and ketamine (one-way ANOVA, F 3,39 = 192, p < 0.001). Both psilocybin doses (2 and 10 mg/kg) and ketamine increased frontal cortex 5-HT, with psilocybin producing rises to roughly 200–250% of baseline and ketamine up to about 350% (repeated-measures ANOVA significant; one-way ANOVA F 3,29 = 537, p < 0.0001). Glutamate responses were dose-dependent for psilocybin: 2 mg/kg slightly decreased extracellular glutamate to about 80% of baseline, whereas 10 mg/kg increased glutamate markedly (to ~300%); ketamine increased glutamate to roughly 150% (one-way ANOVA F 3,33 ≈ 327, p < 0.0001). GABA in the frontal cortex was elevated by psilocybin in a dose-dependent manner (≈120% for 2 mg/kg, ≈200% for 10 mg/kg) and by ketamine (≈180%); these changes reached statistical significance (one-way ANOVA F 3,35 = 276, p < 0.0001). In the reticular nucleus of the thalamus, neither psilocybin nor ketamine altered extracellular glutamate concentrations (no significant changes in AUC). By contrast, psilocybin dose-dependently increased GABA (up to ~170% with 10 mg/kg), while ketamine produced no significant GABA effect there (one-way ANOVA F 3,22 = 33, p < 0.0001 for GABA AUC differences among groups). Western blotting of frontal cortex tissue at 24 h showed that psilocybin 10 mg/kg increased the GluN2A (GluN2A/NR2A) protein level by about 40% versus control (one-way ANOVA F 3,44 = 9.3, p < 0.0001). Ketamine produced a non-significant ~30% decrease in GluN2A (p ≈ 0.16). GluN2B levels did not change significantly, and AMPA subunits GluA1 and GluA2 showed no differences between treatments. The comet assay showed oxidative DNA damage (tail moment) seven days after dosing. Psilocybin 10 mg/kg significantly increased DNA damage in both the frontal cortex and hippocampus, whereas psilocybin 2 mg/kg did not. Ketamine 10 mg/kg induced DNA damage in the hippocampus only. MDMA (10 mg/kg), used as a comparator, produced pronounced DNA damage in both regions. One-way ANOVA confirmed significant treatment effects in frontal cortex (F 4,26 = 39, p < 0.0001) and hippocampus (F 4,26 = 24, p < 0.0001). Behavioural testing at 24 h revealed limited effects. In the open field, ketamine reduced locomotor measures (time walking and crossings), whereas psilocybin had no significant effect on these parameters (one-way ANOVA F 3,35 values significant). In the light–dark box, time spent in the dark exceeded time in the light across groups, but there were no treatment effects on anxiety measures; however, psilocybin 2 mg/kg and ketamine reduced ambulatory distance in both light and dark zones (two-way ANOVA showed treatment effects on locomotion). In the forced swim test, psilocybin 2 mg/kg increased immobility and swimming time and reduced climbing time, while ketamine increased swimming time only; notably, none of the treatments produced an unambiguous reduction in immobility that would indicate an antidepressant-like effect at the 24 h timepoint (one-way ANOVA F 3,76 indicated significant treatment effects for immobility, climbing and swimming times).

Wojtas and colleagues interpret their findings as supportive of a shared role for glutamate release in the rapid actions of ketamine and serotonergic psychedelics, with mechanistic divergence at upstream targets. Their microdialysis data show that ketamine (10 mg/kg) and high-dose psilocybin (10 mg/kg) both elevate extracellular glutamate in the frontal cortex, consistent with ketamine's blockade of NMDA receptors on GABAergic interneurons and psychedelics' activation of cortical 5-HT2A receptors on pyramidal cells. Both drugs also produced robust increases in DA and 5-HT, though the authors note that pathways linking glutamate, serotonergic and dopaminergic systems differ between substances and may involve AMPA receptor-mediated effects on dorsal raphe or ventral tegmental circuits. An unexpected observation was the parallel increase in extracellular GABA alongside glutamate in the frontal cortex. The authors suggest several explanations: ketamine may block subsets of interneurons differentially (with GluN2D-containing interneurons being particularly sensitive), glutamate bursts may secondarily drive GABA release, or distinct neuronal populations and receptor subtypes might explain the co-elevation. In the reticular nucleus of the thalamus, psilocybin increased GABA but ketamine did not, a result the investigators deem surprising and warranting further study given the reticular nucleus's role in thalamocortical gating. At the receptor level, a single high dose of psilocybin increased GluN2A protein in frontal cortex at 24 h, whereas GluN2B and AMPA subunits were unchanged; ketamine produced non-significant decreases in GluN2A. The authors propose that increased GluN2A may relate to 5-HT2A-driven plasticity and dendritic spine changes attributed to psychedelics. Concerning genotoxicity, higher-dose psilocybin produced oxidative DNA damage in frontal cortex and hippocampus at 7 days, while ketamine affected the hippocampus; MDMA produced the strongest damage. The authors link these effects to excessive glutamate release leading to excitotoxicity and oxidative stress. Behaviourally, no clear anxiolytic or antidepressant-like effects were observed at 24 h in naive rats; ketamine reduced locomotion in the open field. The investigators note several caveats that may account for the lack of behavioural efficacy: testing at 24 h may be too early or too late for certain persistent effects, the forced swim test may not be suitable to detect psychedelic-related antidepressant-like actions, and using naive animals rather than stress or depression models could blunt observable benefits. They recommend further work in stress models and with varied timepoints to better characterise therapeutic versus adverse consequences. The authors acknowledge that single-dose studies have limits and call for additional research to clarify dose-response, timing and safety profiles.

Wojtas and colleagues conclude that a single administration of psilocybin and ketamine profoundly alters thalamo-cortical neurotransmission, albeit via distinct molecular targets: psilocybin primarily through 5-HT2A receptor activation and ketamine through blockade of NMDA receptors on interneurons. Both drugs facilitate glutamate release, which the authors propose drives increases in cortical DA and 5-HT and may underpin synaptic plasticity. However, higher doses produced indicators of oxidative DNA damage, suggesting potential genotoxic risks linked to glutamate-induced excitotoxicity. The neurochemical changes observed at 24 h did not translate into clear anxiolytic or antidepressant-like behavioural effects in naive rats, and the investigators recommend further studies in stress models and with different dosing/time windows to elucidate therapeutic mechanisms and safety.