This rodent study (2022) assessed the effects of a single administration of ketamine and psilocybin on the extracellular levels of neurotransmitters in the rat frontal cortex and reticular nucleus of the thalamus. Ketamine and psilocybin increased the levels of dopamine, serotonin, glutamate and GABA extracellular levels in the frontal cortex, while psilocybin also increased GABA in the reticular nucleus. Interestingly, no antidepressant -anxiety effects were observed.
Abstract
“Clinical studies provide evidence that ketamine and psilocybin could be used as fast-acting antidepressants, though their mechanisms and toxicity are still not fully understood. To address this issue, we have examined the effect of a single administration of ketamine and psilocybin on the extracellular levels of neurotransmitters in the rat frontal cortex and reticular nucleus of the thalamus using microdialysis. The genotoxic effect and density of glutamate receptor proteins were measured with comet assay and Western blot, respectively. An open field test, light–dark box test and forced swim test were conducted to examine rat behaviour 24 h after drug administration. Ketamine (10 mg/kg) and psilocybin (2 and 10 mg/kg) increased dopamine, serotonin, glutamate and GABA extracellular levels in the frontal cortex, while psilocybin also increased GABA in the reticular nucleus of the thalamus. Oxidative DNA damage due to psilocybin was observed in the frontal cortex and from both drugs in the hippocampus. NR2A subunit levels were increased after psilocybin (10 mg/kg). Behavioural tests showed no antidepressant or anxiolytic effects, and only ketamine suppressed rat locomotor activity. The observed changes in neurotransmission might lead to genotoxicity and increased NR2A levels, while not markedly affecting animal behaviour.”
Authors: Adam Wojtas, Agnieska Bysiek, Agnieska Wawrzczak-Bargiela, Zuanna Szych, Iwona Majcher-Maslanka, Monika Herian, Marzena Mackowiak & Krystyna Golembiowska
Summary
- Introduction
Mood and anxiety disorders are among the leading causes of disability worldwide. Ketamine, a non-competitive NMDA receptor antagonist, can alleviate the symptoms of major depressive disorder only hours after administration, and is also effective in treatment-resistant patients.
Psychedelics can be divided into two main categories by structure: indoleamines and phenylalkylamines. The former bind to several subtypes of 5-HT receptors, while the latter bind mainly to the 5-HT2 receptor family.
Psychedelics, such as psilocybin, induce a rapid antidepressant effect and last for up to 6 months after a single administration. They are non-addictive and seem to exhibit fewer side effects than ketamine.
Ketamine and psilocybin have antidepressant properties, but their mutual properties are still not fully understood. Detailed comparison studies should be conducted. Psychedelic drugs can disrupt sensory input to the cortex and alter pyramidal cells’ signaling. We examined the effect of ketamine and psilocybin on dopamine, serotonin, glutamate and GABA in the rat frontal cortex and reticular nucleus of the thalamus using in vivo microdialysis and Western blot analysis. Psilocybin and ketamine affect neurotransmitters differently, and their effects may be related to genotoxicity, behavioral disturbances and adaptive changes in glutamate receptors.
Psilocybin and ketamine significantly increased extracellular levels of dopamine in the rat frontal cortex. The effects were dependent on dose, sampling period, and interaction between treatment groups and sampling period.
Psilocybin and ketamine increased extracellular 5-HT levels in the rat frontal cortex. The effects were significant for both doses and the interaction between treatment groups and sampling period.
The extracellular glutamate level was decreased by 2 mg/kg psilocybin and increased by 10 mg/kg psilocybin and ketamine. The total effects were significant decreased by 2 mg/kg psilocybin and significantly increased by 10 mg/kg psilocybin and ketamine.
The extracellular level of GABA was significantly increased by psilocybin and ketamine, and the effects were mediated by the treatment group, sampling period, and interaction between treatment groups and sampling period.
Psilocybin and ketamine had no effect on glutamate levels in the rat reticular thalamus nucleus.
GABA extracellular level was slightly increased by psilocybin at a low dose, but potently increased by psilocybin at a dose of 10 mg/kg. Ketamine did not affect GABA extracellular level.
Psilocybin administration increased the GluN2A protein level by 40% over the control, whereas ketamine administration decreased the GluN2A protein level by 30%, but this effect was not statistically significant. The protein levels of two AMPA receptor subunits were not changed by administration.
Psilocybin did not produce DNA damage in the frontal cortex and hippocampus at 2 mg/kg, but at 10 mg/kg it did. Ketamine produced DNA damage only in the hippocampus, while MDMA caused potent damage of DNA in the frontal cortex and hippocampus.
The time spent in the dark compartment was longer than in the light zone for all groups of animals, but no difference was observed between treatments. Psilocybin and ketamine significantly decreased exploration of the dark and light zones.
Psilocybin and ketamine significantly increased immobility, swimming and climbing time, but not climbing time.
- Discussion
Ketamine and psychedelics share rapid antidepressant properties, indicating the existence of convergence between their mechanisms of action. Both drugs strongly affect DA, 5-HT and GABA levels, though the exact mechanisms responsible for this phenomenon may differ between the substances.
Low doses of ketamine used for clinical and preclinical studies can block NMDA receptors on subsets of GABA interneurons and reduce interneuron firing prior to increasing pyramidal neuron firing. However, we observe an increase in the extracellular levels of GABA in parallel with an increase in the extracellular level of glutamate in the frontal cortex.
Ketamine increased the extracellular 5-HT level in the prefrontal cortex of rats and increased the DA extracellular level in the frontal cortex of rats. Ketamine might indirectly elevate 5-HT levels through the stimulation of AMPA receptors located in DRN. Ketamine restores the activity of a circuit formed by DA and glutamate projections in the medial prefrontal cortex and ventral tegmental area after treatment with stress.
Serotonergic psychedelics act primarily on serotonergic receptors, and psilocybin is rapidly converted in the liver into psilocin, which binds to many 5-HT receptor subtypes. 5-HT2A and 5-HT1A receptors were found in the same pyramidal neurons in the rat frontal cortex, and 5-HT2A agonism leads to increased membrane excitability and 5-HT1A agonism leads to decreased membrane excitability. These opposite receptor actions correspond to different pharmacological responses.
Psilocybin increased DA release in the midbrain to a certain extent, but only for a limited time, and was unrelated to glutamate and GABA levels. This is difficult to explain on the basis of glutamatergic or GABAergic projections into midbrain DA neurons.
In our study, psilocybin increased 5-HT extracellular level in the rat frontal cortex at both doses. This increase may be due to the selective activation of 5-HT2A receptors, which enhances the release of glutamate, which acts on pyramidal AMPA receptors, stimulating a descending projection to DRN, thus increasing 5-HT release.
We tested the effects of psilocybin and ketamine on the extracellular levels of amino acids in the reticular nucleus of the thalamus, but found that only psilocybin increased extracellular GABA levels, while ketamine did not affect either glutamatergic or GABAergic neurotransmission.
Ketamine acts by blocking NMDA receptors on interneurons, which increases glutamate release, which stimulates mTOR signaling through the activation of AMPA receptors. Ketamine-like effects are mediated by the GluN2B subunit located on GABAergic interneurons, but not by GluN2A subunits in glutamatergic neurons.
Ketamine decreased the expression of the NR2B subunit in the somatosensory cortex, while the expression of Nr2a and Nr2b genes increased four weeks after cessation of treatment with LSD in the rat prefrontal cortex.
Compounds such as ketamine and serotonin psychedelics that promote rapid plasticity may induce excitotoxicity resulting in oxidative stress and neuronal atrophy. In our study, psilocybin and ketamine caused oxidative stress but not DNA damage.
We conducted behavioral experiments 24 h post administration to limit high concentrations of circulating drugs and metabolites in the system. Ketamine negatively affected locomotor behavior in the open field test, while psilocybin had no effect. Few studies have been conducted to assess the effect of either psilocybin or ketamine on behavior 24 h after injection, and the available data is limited. Furthermore, the time of test performance is critical for its outcome.
- Materials and Methods 4.1. Animals
Adult male Wistar Han rats were used in all experiments. They were housed in environmentally controlled rooms with a 12:12 light:dark cycle and had free access to tap water and typical laboratory food.
4.2. Drugs and Reagents
Ketamine, psilocybin and MDMA were dissolved in sterile water and given intraperitoneally in the volume of 2 mL/kg. The dose of ketamine was 10 mg/kg and the dose of psilocybin was 2 and 10 mg/kg. Ketamine, xylazine hydrochlorides and sodium pentobarbital were used for anesthetizing the animals, and O-phthalaldehyde was used for derivatization of glutamate to an electroactive compound. MDMA was purchased from Toronto Research Chemicals Inc.
4.3. Brain Microdialysis
Ketamine and xylazine were injected intramuscularly to anesthetize the animals. Microdialysis probes were implanted into the frontal cortex and reticular nucleus of thalamus and artificial cerebrospinal fluid was delivered at a flow rate of 2 L/min.
Extracellular DA and 5-HT levels were measured using an Ultimate 3000 System (Dionex), electrochemical detector Coulochem III (model 5300; ESA, Chelmsford, MA, USA) and a Hypersil Gold C18 analytical column (Thermo Fisher Scientific), and the data were processed using Chromax 2005 software on a personal computer.
4.5. Alkaline Comet Assay
The alkaline comet assay was performed on frontal cortices and hippocampi of animals 7 days after drug injection. The DNA damage was measured using the tail moment and the data was analyzed using OpenComet software.
4.6. Western Blotting
The brains of the animals were removed from the skull and frozen in liquid nitrogen. The frontal cortex was dissected and homogenized in lysis buffer and the protein concentrations were determined using a QuantiPro BCA Assay kit. Protein extracts were separated on 7.5% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were stained with Ponceau S and incubated overnight at 4 C with the following primary antibodies: rabbit anti-GluN2A, rabbit anti-GluA2, rabbit anti-GAPDH and peroxidase-conjugated secondary antibodies.
Anti-rabbit IgG antibody was used to detect immune complexes, and blots were visualized using enhanced chemiluminescence and scanned using a luminescent image analyzer. The levels of analyzed proteins were normalized for GAPDH protein.
4.7. Open Field (OF) Test
The open field test was performed in a dimly lit room with a 75 W light bulb placed at a height of 75 cm. Rats’ behavior was recorded for 10 min.
4.8. Light–Dark Box (LDB) Test
The light/dark exploration test was performed in the TSE Fear Conditioning System (TSE System, Germany). Rats were individually tested in single 10 min trials, and the behavioral responses during the test session were recorded using Fear Conditioning software (TSE, Bad Homburg, Germany).
4.9. Forced Swim Test (FST) in Rats
Rats were placed in a cylinder filled with water for 15 minutes, dried with towels, placed in a warmer enclosure for 15 minutes, and then returned to their home cages. Twenty-four hours after the first exposure to forced swimming, the rats were retested for five minutes under identical conditions.
4.10. Statistical Analysis
Drug effects on DA, 5-HT, glutamate and GABA release in the brain regions were analyzed using repeated measures ANOVA on normalized responses followed by Tukey’s post hoc test.
- Conclusions
Psilocybin and ketamine affect thalamo-cortical neurotransmission by activating 5-HT2A receptors and blocking subsets of NMDA receptors on GABA interneurons, respectively. This results in an increase of dopamine and 5-HT levels, which in turn regulate sensory information provided to the cortex by psilocybin.