Neuronal activity is known to rearrange and affect the function of neuronal circuits in the brain54. Plasticity promoting capacity is associated with upregulation of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF)5, and downregulation of plasticity opposing signaling, epitomized by the Nogo signaling system55,56. In addition, plasticity is affected by modulatory neurotransmitters such as serotonin57. Psychedelic compounds such as psilocybin, lysergic acid diethylamide (LSD), mescaline, 4-iodo-2,5-dimethoxyphenylisopropylamine (DOI) and N, N-dimethyltryptamine (DMT) have individual pharmacological properties, but all involve signaling through serotonergic receptors58. LSD has been shown to increase levels of immediate early genes Arc and c-Fos fivefold and twofold, respectively, in prefrontal cortex, with c-Fos mRNA expression additionally showing twofold increases in hippocampus and midbrain59. DOI stimulation of serotonergic receptors affects BDNF mRNA expression in a region-specific manner, with higher doses producing a robust increase in parietal cortex and decrease in the dentate gyrus60. A number of psychedelic compounds, the most robust being LSD, were shown to promote neuritogenesis, spinogenesis, and synaptogenesis in cultured cortical neurons9. Taken together, these observations provide strong evidence that psychedelic compounds have plasticity evoking effects; however, little is known regarding the effects of psilocybin on synaptic activity. As interest in the psilocybin’s therapeutic value for neurologically related diseases increases20,61, it has become important to investigate its cell-type specific actions of psilocybin in addition to its plasticity enhancing properties at the synaptic level.
To deepen our understanding of the impact of psilocybin on synaptic and structural plasticity we used hippocampal and cortical neuronal cell cultures to obtain time curves of the expression of number and intensity of structural synaptic proteins in response to psilocybin. Our results show that psilocybin rapidly and differentially affects pre- and postsynaptic protein puncta in a time- and region dependent manner. In hippocampal neuronal cultures, psilocybin rapidly increased the number of pre- and post-synaptic (Piccolo and Homer1) structures, peaking between 1 and 3 h, with normalization back to baseline levels at 6 h post-treatment. The number and intensity of presynaptic protein, Synapsin-1, was assessed at shorter and longer time course and the highest number of puncta was found 72 h post psilocybin treatment, indicating an even more prolonged effect of the drug. The rapid peak of plasticity might be a productive window to place more focus on. Psilocybin is currently investigated as an adjuvant therapy to other modes of treatment, and by finding the optimal timing between psilocybin treatment, and for instance therapy, the effect might be even larger. Increased fluorescent intensity levels of synaptic puncta expression have previously been correlated to increased levels of functional synapses50 as well as post-synaptic machinery necessary for active synapses51. Intensity levels of Homer1, Piccolo, and Synapsin-1 puncta mirror the increased numbers of puncta we observe, with the exception of 72 h post-psilocybin treatment for Synapsin-1. At this timepoint, Synapsin-1 puncta reached the highest levels, though the intensity of these puncta was not increased. This may be a reflection of synapses becoming dormant, which is a reversible phenomenon52 (Crawford and Mennerick, 2012). Whether or not other synaptic proteins exhibit similar expression patterns at the 72 h timepoint will need to be further explored.
Interestingly, psilocybin treatment of cortical neuronal cell cultures did not change post-synaptic puncta counts (Homer1) but resulted in both decreased pre-synaptic puncta counts (Piccolo) and colocalization of pre- and post-synaptic sites. Intensity levels of puncta expression did not change over the course of the psilocybin treatment in cortical neuronal cultures. A recent study of effects of psilocybin in rats in vivo reports transcriptional up- and down-regulation of genes related to plasticity, specifically expression of Nr4a1, PSD95, Sgk1, Arc, IkBα, and Egr262. Our findings in the cortical cell culture match results by Jefsen et al. (2021) showing no changes in Synapsin-1 or Homer1 mRNA expression in the prefrontal cortex of rats; however, our results diverge from their rat hippocampal data as they did not find Synapsin-1 or Homer1 mRNA expression level changes. This is unlikely to be a species-specific difference but may be due to the timing of sacrifice after psilocybin treatment (90 minutes).
Overall, our findings support recent observations that psilocybin has a potent effect on synaptic density, as shown from prior studies showing increased expression levels of synaptic vesicle protein 2a (SV2A) in pigs following a single dose of psilocybin. The increases in the presynaptic SV2A protein were significantly increased 7 days post administration in both hippocampus and prefrontal cortex63. Shao et al., (2021) recently utilized a mouse model of learned helplessness (depression) and showed that a single dose of psilocybin ameliorated behavioral deficits64. They also found the single dose to significantly increase dendritic spine size and density in the medial frontal cortex a day after psilocybin treatment and persisting up to a month. While rapid effects of psilocybin have been less tested, our studies now provide a context for more short-term changes in synaptic architecture. In cortical neurons, we show that psilocybin has a rapid effect on the pre-synaptic side, resulting in downregulation of Piccolo within the first 3 hours, while having relatively little effect on the post synaptic side. This might appear paradoxical, but serotonin has been proposed to function as an expectation error signal65. We hypothesize that psilocybin might increase plasticity by removing potentially erroneous synapses before new synapses can be formed.
Psilocin is an unspecific serotonergic agonist and individual serotonergic receptors respond to different concentrations of psilocybin. High affinity is associated with the 5-HT7, 5-HT1D, and the 5-HT2A,B, and C receptors, while there is minimal interaction with the 5-HT3 receptor11. Mouse neurons express mRNA most for all of these receptors (minimally for 5HT2B)66, while microglia most highly express mRNA for the 5-HT2B and C receptors67. As the concentration of psilocybin and its active metabolite psilocin reasonably differs over time due to metabolism in our cell cultures, activation of various serotonergic receptors, inhibitory as well as excitatory68, are likely occurring over the timescales in the present study. Further studies are needed to understand which concentration of psilocybin optimally heightens plasticity. Access to an increased level of plasticity could potentially serve as a primary mechanism for the therapeutic value of psilocybin. Furthermore, by knowing when plasticity is maximal after psilocybin treatment may allow other therapeutic interventions, e.g., post stroke rehabilitation. to be timed to these windows.
In a recent study using pigs69, a single dose of psilocybin was shown to significantly upregulate many immunological gene pathways in the PFC, as determined by RNA sequencing and gene set enrichment analysis (GSEA). The most enriched pathways included GO Immune Response, GO Regulation of Immune Response, GO Immune Effector Process, and GO Innate Immune Response, among others related to response to interferon proteins and cytokines. Although these results point to a clear immunologic effect of a single psilocybin administration, the research group was unable to verify several candidate genes using RTqPCR. Though no inflammatory challenge was applied in this study, these results clearly show a relationship between the brain’s immune response and psilocybin. Another recent study demonstrated immunomodulatory effects (morphological and protein expression changes) in primary microglia treated with DMT or psilocin. This study provides further evidence of direct action of serotonergic psychedelics on microglia 70.
Our study expands understanding of the anti-inflammatory capacity of psychedelics affecting the serotonergic system29,30. We used an immortalized microglial (IMG) cell line, previously characterized to recapitulate key features of primary microglial activation43, challenged with LPS as a model of neuroinflammation. We demonstrate a significantly reduced release of the canonical inflammatory protein TNF-α by IMG cells pretreated with a 10− 5 M psilocybin and subjected to an LPS challenge. The pleiotropic cytokine IL-6, possessing both pro- and anti-inflammatory properties, usually accompanies TNF-α in inflammation states71. Hence, we also examined its presence in cell culture media following an LPS challenge. Although IL-6 presence trended down at a high dose of psilocybin, this cytokine was not significantly reduced.
While studies associated with psilocybin’s anti-inflammatory effects are limited due to its rapid metabolic breakdown, an inflammation suppressing pharmacophore of other psychedelics which activate the 5-HT2A receptor was recently described using a rat model of asthma29. This study showed significant reductions of expression of inflammation-related genes in the lung, including TNF-α and other cytokines. Interestingly, a recent study showed significant increases in mRNA expression of IkBα (nuclear factor kappa light-chain-enhancer polypeptide gene enhancer of B-cells inhibitor, alpha) and Dusp1 (dual-specificity-phosphate 1) in hippocampus and PFC of a rat after single administrations of different doses of psilocybin62. Increases in protein levels was confirmed in hippocampus but not in the PFC. IkBα is an endogenous inhibitor of the transcription factor NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells)72, which is inducible by LPS73 and central to maximal production of TNF-α74. Interestingly, IkBα has been shown to play an important function in synaptogenesis and might be a common target for synaptic and anti-inflammatory effects75. Dusp1 has been previously shown to have potent anti-inflammatory effects. Dusp1 knock-out mice show 10-fold increases compared to wild type animals of several cytokines in blood serum, including TNF-a76. Importantly, Dusp1 promotes the formation of an anti-inflammatory phenotype in microglia77, with other studies further demonstrating similar effects78,79. Psilocybin’s induction of IkBα and Dusp1 is a possible mechanism whereby the drug inhibits inflammation. Further studies are needed to confirm this.
Altogether, current evidence in the literature and our studies represents possibilities for new classes of drugs that act on serotonergic receptors with anti-inflammatory capacity. Allosteric targeting of these receptors to activate downstream anti-inflammatory pathways but avoid psychedelic effects may be a fruitful area of research in the future.