This mice study found that the anti-depressant effects from psilocybin (1mg/kg) are possibly independent of the psychedelic/hallucinogen effects by pre-treating the mice with ketanserin (which blocks the acute effects) and finding similar anti-depressant effects as in the psilocybin only group (measured through activity patterns and synaptic action).
Abstract
“Depression is a widespread and devastating mental illness and the search for rapid-acting antidepressants remains critical. There is now exciting evidence that the psychedelic compound psilocybin produces not only powerful alterations of consciousness, but also rapid and persistent antidepressant effects. How psilocybin exerts its therapeutic actions is not known, but it is widely presumed that these actions require altered consciousness, which is known to be dependent on serotonin 2A receptor (5-HT2AR) activation. This hypothesis has never been tested, however. We therefore asked whether psilocybin would exert antidepressant-like responses in mice and, if so, whether these responses required 5-HT2AR activation. Using chronically stressed male mice, we observed that a single injection of psilocybin reversed anhedonic responses assessed with the sucrose preference and female urine preference tests. The antianhedonic response to psilocybin was accompanied by a strengthening of excitatory synapses in the hippocampus—a characteristic of traditional and fast-acting antidepressants. Neither behavioral nor electrophysiological responses to psilocybin were prevented by pretreatment with the 5-HT2A/2C antagonist ketanserin, despite positive evidence of ketanserin’s efficacy. We conclude that psilocybin’s mechanism of antidepressant action can be studied in animal models and suggest that altered perception may not be required for its antidepressant effects. We further suggest that a 5-HT2AR–independent restoration of synaptic strength in cortico-mesolimbic reward circuits may contribute to its antidepressant action. The possibility of combining psychedelic compounds and a 5-HT2AR antagonist offers a potential means to increase their acceptance and clinical utility and should be studied in human depression.”
Authors: Natalie Hesselgrave, Timothy A. Troppoli, Andreas B. Wulff, Anthony B. Cole & Scott M. Thompson
Notes
This study was featured in a press release by the University of Maryland and also examined on Psychedelic Science Review, and a video by Asher Brandt.
Multiple articles in recent years have been investigating this line of thought, with Cameron and colleagues (2020) being the most prominent. If psychedelics (or analogs of them) can have antidepressant effects (for now in mice) without the need for many hours of therapy (during the acute effects), would that possibly make them cheaper and more accessible?
“If 5-HT2AR activation is not necessary, then the combination of psilocybin and a 5-HT2AR antagonist safe for human use, such as ketanserin, offers a potential means to eliminate, attenuate, or shorten the duration of psilocybin-induced alterations of perception while retaining its therapeutic benefits.”
One hypothesis that this paper puts forth, is that psilocybin may elicit its anti-depressive effects through other serotonin (5-HT) receptors than the 5-HT2A receptors. As ketanserin blocks the effect of psilocybin on this receptor, it is hypothesized that other receptors such as the 5HT1B or AMPA receptors could be responsible for the observed effects.
The Study
Mice (8 weeks old, C57BL/6J) were chronically exposed to stress. Normally they would be attracted to water with sucrose (think Gatorade) and female urine. Both these urges were suppressed after chronic stress exposure.
The study was broken into four different groups. The first group received nothing (but stress). The second group only received ketanserin. The third group received only psilocybin. And the fourth group received ketanserin and psilocybin.
The mice in the last two groups significantly improved their urges, whilst in the last group, there were no signs of activation of the 5-HT2A receptor (as indicated by the head-twitch response, usually also seen as a proxy for psychedelic/hallucinogenic effects).
“Significant improvements in both sucrose and female urine sniffing preferences were observed in mice having both few and many head twitches, consistent with our conclusion that the restoration of reward behavior by psilocybin was independent of 5-HT2AR activation.”
When investigating the brainwaves of the mice, the researchers “observed that ketanserin pretreatment greatly attenuated the decrease in LFP delta power in response to this high concentration of psilocybin, providing another strong positive control of its efficacy as a 5-HT2AR antagonist in the hippocampus under our experimental conditions.” The structural strengthening of synapses in the brain (also named functional connectivity in humans) is possibly responsible for the anti-depressant effects of psilocybin/psychedelics. The effect on AMPA/NMDA ratios were not influenced by the pretreatment with ketanserin.
Finally, the study looked at the forced swim test, but found no effects here (it does look to be a different group of mice and not using ketanserin here; see figure 4 in the appendix).
Limitations and Implications
To be clear, this study was done in mice and should be replicated in humans. The effects were only observed up to 48 hours later and the effects of a psychedelic experience may still be needed for enduring effects.
Still, this paper indicates that the acute psychedelic experience may not be necessary which could greatly increase access.
In the future, new compounds can be made that specifically target synaptic strengthening with fewer perceptual effects.
Summary
Psilocybin, a psychedelic compound, produces powerful alterations of consciousness, but also rapid and persistent antidepressant effects in mice. These effects were not prevented by pretreatment with the 5-HT2A/2C antagonist ketanserin, suggesting that a 5-HT2AR – independent restoration of synaptic strength in cortico-mesolimbic reward circuits may contribute to its antidepressant action.
depression | hallucination | hallucinogen | psychedelic | serotonin
Psilocybin has been used by humans for millennia for spiritual and medicinal purposes. Clinical research has recently begun to provide evidence supporting its use as a therapeutics for numerous neuropsychiatric disorders, including obsessive-compulsive disorder, posttraumatic stress disorder, and treatment-resistant depression.
Psilocin, the active metabolite of psilocybin, is a potent agonist at almost all 5-HTRs, comparable to serotonin. It is possible that psilocybin relieves depressive symptoms through rapid activation of some other critical 5-HTRs.
Psychedelic drugs have been studied in mouse models of several behavioral disorders, but their effects have not been studied in well-validated animal models of schizophrenia.
Significance
Psychedelic compounds such as psilocybin may be useful in treating several psychiatric diseases, but they can also cause strong alterations in consciousness. We found that psilocybin has fast-acting antidepressant-like properties in mice.
S.M.T., T.A.T., A.B.W., A.B.C., and N.H. designed research, performed research, analyzed data, and wrote the paper. S.M.T. is listed as an inventor on a provisional patent on psychedelics combined with 5-HT2R antagonists to treat psychiatric disease.
Human depression results from a combination of genetic susceptibility and environmental factors, such as stress. Anhedonia is a core symptom of depression, and compounds that restore responses to rewarding stimuli in stressed animals are effective antidepressants.
We tested the hypothesis that psilocybin exerts an antidepressant-like response in chronically stressed mice and found that 5-HT2R activation is not necessary for this response.
Results
We exposed 8-wk old male C57BL/6J mice to a chronic multi-modal stress paradigm and tested their hedonic state with two well characterized appetitive choice tasks involving different senses. Psilocybin restored their preference for sucrose solution and female urine 24 to 48 h after injection.
We gave stress-susceptible mice an injection of ketanserin (2 mg/kg, i.p.) followed 1 h later by psilocybin (1 mg/kg, i.p.), and observed that ketanserin had no significant effect on sucrose and female urine preferences following stress, whereas psilocybin significantly increased head twitching in vehicle-pretreated mice.
Psilocybin induces head twitches, which require 5-HT2AR activation. However, psilocybin’s antianhedonic actions are independent of 5-HT2AR activation, as shown by the increased sucrose and female urine sniffing preferences in mice with both few and many head twitches.
Psilocybin decreases low-frequency oscillations in humans and rats via activation of 5-HT2ARs. In mice, ketanserin preinjection greatly attenuates this decrease in low-frequency oscillations in response to psilocybin, providing another strong positive control of ketanserin’s efficacy as a 5-HT2AR antagonist.
Psilocybin restored hedonic state by promoting synaptic connectivity in the prefrontal cortex in stress-naive mice via a 5-HT2AR – dependent mechanism. This was measured by AMPA/NMDA ratios at the archetypical stress-sensitive excitatory synapse formed by temporoammonic inputs to the distal dendrites of CA1 pyramidal cells.
Psilocybin administration promotes persistent synaptic strengthening in a depression-relevant brain region days after its elimination from the body, much like the persistent effects of psilocybin on human brain functional connectivity.
Pretreatment with ketanserin did not impair the ability of psilocybin to restore AMPA/NMDA ratios in the hippocampal slices of mice, and the behavioral response to psilocybin was independent of 5-HT2R activation.
Psilocybin did not affect the forced swim test in unstressed male or female C57BL/6J mice, but future studies are needed to determine if it has antidepressant-like effects in female mice.
Discussion
Our results provide evidence that psilocybin exerts rapid beneficial action in well-studied and well-validated models of chronic stress-induced deficits in depression-relevant hedonic behaviors. These effects recapitulate the rapid antidepressant actions of psilocybin in humans.
Psychedelic compounds alter consciousness through activation of 5-HT2ARs. However, pretreatment with ketanserin sufficiently attenuated the activation of 5-HT2ARs at the time we administered psilocybin, and our preclinical results suggest that 5-HT2ARs may not be required for an antidepressant response to psilocybin. If 5-HT2AR activation is not necessary, then ketanserin, a 5-HT2R antagonist, may be able to reduce the anhedonic effects of psilocybin while retaining its therapeutic benefits.
Human studies with healthy volunteers and TRD patients reveal persistent increases in functional connectivity in the same circuits after psilocybin administration. The 5-HT1BRs underlying this synaptic response to psilocybin remain to be determined, but their definition could reveal a strategy for developing alternatives to psilocybin that are biased toward synaptic strengthening relative to perceptual alteration.
Materials and Methods
Male C57BL/6J mice were bred in-house and used in the stress-induced anhedonia experimental protocols of this study. Ketanserin’s activity was tested through in vivo electrophysiology and locomotion effects.
Chronic Multimodal Stress (CMMS) was used to induce an anhedonic-like phenotype in mice. The stress was initiated in the morning hours, between 9 and 10 AM, near the onset of the animals’ light cycle.
Hedonic behavior was assessed using the SPT and FUST prior to stress, after 10 to 14 d of CMMS, and 24 h after drug injection. The preference for 1% sucrose solution over tap water was calculated for each night.
Mice were individually transferred to empty, freshly made cages and allowed to habituate for 15 min. One hour later, two cotton swabs were added to the cage, one soaked in freshly collected urine from male mice and the other with urine from female mice in estrous.
Mice with a preference for sucrose of >65% at baseline and >70% following CMMS were considered stress susceptible. Mice with a female urine preference of >65% at baseline and >70% following CMMS were classified as resilient.
Mice exposed to CMMS and treated with vehicle, ketanserin, vehicle-psilocybin, and ketanserin-psilocybin were scored for head twitches. Individual head twitches were defined as events detected by at least two of the three scorers.
8-wk-old male mice were pretreated with ketanserin or saline and injected with psilocybin. Their locomotion was recorded in an open field arena.
A separate cohort of 9-wk-old male and female mice were used for the forced swim test. They were injected with vehicle or psilocybin and the first swim session occurred 24 h after injection.
In vitro electrophysiology was performed on 400 mm thick hippocampal slices prepared from euthanized mice. The slices were allowed to recover for 60 min at room temperature in ACSF before recording.
Extracellular recording was used to quantify AMPA/NMDA ratios because whole-cell recording is complicated by stress-induced changes in dendritic structure and electrotonic influence on recordings of distal TA-CA1 synapses. Field excitatory postsynaptic potentials were acquired using Clampex software, amplified, filtered, and digitized, and then DNQX was washed onto the slice for 15 min to block the AMPA component of the fEPSP and reveal the NMDA component.
AMPA/NMDA ratios of the TA-CA1 fEPSPs were calculated as described previously (24) and were used to measure synaptic strength across slices from different mice. AMPA/FV and NMDA/FV ratios were also calculated as an independent measure of synaptic strength.
Mice were anesthetized with isoflurane and placed in a stereotactic frame. Local field potentials were recorded with a Q4 silicon probe in the pyramidal layer of the hippocampal CA1 region and psilocybin was injected into the mice 30 min after LFP recordings were initiated.
Psilocybin and ketanserin were administered to rats 60 min prior to injection with either psilocybin or ketanserin. Psilocybin was given at 1 mg/kg and ketanserin at 2 mg/kg, consistent with previous rodent studies.
Statistical analysis was performed using Student’s t tests, one-, two-, and three-way ANOVAs, and Holm-Sidak multiple-comparison corrections. The results were not statistically different and were therefore pooled.
Data Availability. All study data are included in the article and/or SI Appendix.
The authors thank several people for their assistance with the experiments, including Jonathan Fischell, Adam Puche, Dongil Keum, Mary Kay Lobo, Hyungwoo Nam, Todd Gould, Sarah Clark, and Tara LeGates.
Psychedelic psychiatry’s brave new world, psilocybin with psychological support for treatment-resistant depression, a randomized clinical trial, and the abuse potential of medical psilocybin according to the 8 factors of the controlled substances act are all discussed. Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action, and affects facial recognition, goal-directed behavior, and mood state toward positive relative to negative emotions. The potency of hallucinogens in the mouse head-twitch response assay correlates with their behavioral and subjective effects in other species, and psychedelics, but not ketamine, produce persistent antidepressant-like effects in a rodent experimental system for the study of depression.
Ketamine and psilocybin induce behavioral effects in mice, including locomotor sensitization, activation of serotonin 2A receptors, and visual hallucinations. DOI, a hallucinogen, reduces low-frequency oscillations in rat prefrontal cortex and is reversible by antipsychotic drugs. Chronic stress induces a selective decrease in AMPA receptor-mediated synaptic excitation at hippocampal temporoammonic-CA1 synapses, and antidepressant-induced spine formation sustains rescue of prefrontal circuit dysfunction. Serotonin excites hippocampal CA1 GABAergic interneurons at the stratum radiatum-stratum lacunosum moleculare border, and psilocybin induces UP states in the brain slice of rat prefrontal cortex. Psilocybin lacks antidepressant-like effect in the Flinders Sensitive Line rat.