Effects of serotonergic psychedelics on synaptogenesis and immediate early genes expression - comparison with ketamine, fluoxetine and lithium

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Effects of serotonergic psychedelics on synaptogenesis and immediate early genes expression – comparison with ketamine, fluoxetine and lithium View ORCID Profile Yana Vella , View ORCID Profile Kateřina Syrová , View ORCID Profile Aneta Petrušková , Isis Koutrouli , View ORCID Profile Viera Kútna , View ORCID Profile Jan Pala , View ORCID Profile Marek Nikolič , View ORCID Profile Klára Šíchová , View ORCID Profile Vladimír Mazoch , View ORCID Profile Radek Jurok , View ORCID Profile Martin Kuchař , View ORCID Profile Zdeňka Bendová , View ORCID Profile Tomáš Páleníček doi: https://doi.org/10.1101/2024.08.07.606965 Yana Vella 1 National Institute of Mental Health , Klecany, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yana Vella Kateřina Syrová 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kateřina Syrová Aneta Petrušková 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aneta Petrušková Isis Koutrouli 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Viera Kútna 1 National Institute of Mental Health , Klecany, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Viera Kútna Jan Pala 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jan Pala Marek Nikolič 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marek Nikolič Klára Šíchová 1 National Institute of Mental Health , Klecany, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Klára Šíchová Vladimír Mazoch 1 National Institute of Mental Health , Klecany, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vladimír Mazoch Radek Jurok 3 Forensic Laboratory of Biologically Active Compounds, Department of Chemistry of Natural Compounds, University of Chemistry and Technology , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Radek Jurok Martin Kuchař 1 National Institute of Mental Health , Klecany, Czechia 3 Forensic Laboratory of Biologically Active Compounds, Department of Chemistry of Natural Compounds, University of Chemistry and Technology , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martin Kuchař Zdeňka Bendová 1 National Institute of Mental Health , Klecany, Czechia 4 Department of Physiology, Faculty of Science, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zdeňka Bendová Tomáš Páleníček 1 National Institute of Mental Health , Klecany, Czechia 2 Third Faculty of Medicine, Charles University , Prague, Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tomáš Páleníček For correspondence: Tomas.Palenicek{at}nudz.cz Abstract Full Text Info/History Metrics Preview PDF Abstract Background Recent evidence suggests that psychedelics are able to induce rapid and long-lasting antidepressant effects. The generally acknowledged explanation for these traits is the phenomenon of neuroplasticity, although exact underlying molecular mechanisms remain unclear. Aims This study investigates selected neuroplastic effects of psilocin, lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) in direct comparison with ketamine, fluoxetine and lithium after acute (1 h) and/or prolonged (24 h) treatment in vitro . Methods Rat primary cortical cultures were treated with 10 µM psilocin, 1 µM lysergic acid diethylamide (LSD), 90 µM N, N-dimethyltryptamine (DMT), 1 µM ketamine, 10 µM fluoxetine and 5 mM lithium. Analysis of synaptic puncta was performed; puncta of presynaptic marker synapsin I/II, postsynaptic density protein 95 (PSD-95), and their co-localization (established synapse) were assessed 24 h after drug treatment. Next, expressions of immediate early genes (IEGs) encoding activity-regulated cytoskeleton-associated protein ( Arc ), early growth response 1 ( Egr1 ), and neuronal PAS (Per-ArntSim) domain protein 4 ( Npas4 ) were analysed 1 and 24 h after drug treatments. Results Psilocin increased synaptic puncta count and induced Arc expression. The effect to promote synaptogenesis was comparable to ketamine and lithium; ketamine additionally increased PSD-95 puncta count. LSD and DMT didn’t induce any significant effect. Interestingly, fluoxetine had no effect on synaptic puncta count, but upregulated Egr1 and Npas4 . Conclusions Psilocin demonstrated a significant neuroplastic effect comparable to that of ketamine and lithium, adding another piece of evidence to its profile as a promising therapeutic agent. Introduction After years of complete marginalization of classic psychedelics, research on their pharmacology is gaining robust support regarding their therapeutic potential on a plethora of neuropsychiatric conditions including depression, anxiety, post-traumatic stress disorder, addiction, anorexia, or chronic pain ( Cameron et al., 2023 ; Carhart-Harris & Goodwin, 2017 ). As a positive control, psychedelic studies often comprise ketamine, a dissociative anaesthetic which produces psychoactive effects in subanaesthetic doses and has a confirmed efficacy as a fast-acting antidepressant. While classic psychedelics and ketamine aim at distinct receptor systems (serotonergic and glutamatergic, respectively) they share significant features beyond being psychoactive; they rapidly increase dendritogenesis, spinogenesis and synaptogenesis ( De la Fuente Revenga et al., 2021 ; Li et al., 2010 ; Ly et al., 2021 ; Moliner et al., 2023 ; Raval et al., 2021 ), and induce a downstream network reconfiguration through complex neuroplastic mechanisms, hence the term “psychoplastogens” has recently been proposed ( Ly et al., 2018 ). A growing body of research postulates a correlation between many neuropsychiatric disorders and disrupted neuroplasticity, accompanied by an atrophy of neurons in the cortical and limbic brain regions ( Rădulescu et al., 2021 ; Soeiro-de-Souza et al., 2012 ). Vice versa, findings have demonstrated that antidepressant drugs, including selective reuptake inhibitors (SSRIs), exert their therapeutic effects by reactivating a plasticity window in the adult cortex, which resembles juvenile-like brain processes. In such cases, neuronal networks are being shaped by sensory information through experience, by regulating the expression of genes related to plasticity and resilience, among other mechanisms ( Puglisi-Allegra et al., 2021 ; Umemori et al., 2018 ). Although these medicaments have been the mainstay of treatment for depression, they possess several drawbacks; the antidepressant effects take 6-8 weeks of adequate doses to become evident, various side effects can appear, and many patients are unresponsive to this first line treatment. In line with the delayed onset of antidepressant actions, the neuroplastic changes take place only after chronic administration. Lithium is a drug primarily used as a mood stabilizer in bipolar disorder, but also as an augmentation strategy option in the treatment of depression. Recent evidence confirms its robust neuroplastic effects, such as neuroprotection, glioprotection, elevated adult neurogenesis and synaptic plasticity ( Hammonds & Shim, 2009 ). It has been shown to elevate postsynaptic density protein 95 (PSD-95) in rat cortical and hippocampal cultures ( Kim & Thayer, 2009 ; Tai et al., 2022 ). In spite of that, use of lithium has some limitations considering its possible adverse effects ( Antoniadou et al., 2018 ; Cameron et al., 2023 ). In contrast to common antidepressants, psychedelics and ketamine present a robust and rapid therapeutic efficacy even in treatment-resistant depression patients, most likely by the increase of structural and functional plasticity in the prefrontal cortex (PFC), hippocampus, and other brain regions involved in emotions ( Krystal et al., 2019 ; Vargas et al., 2023 ). Depending on the drug and dosage regimen, sustained antidepressant effects are observed. Immediate early genes (IEGS) are a family of genes that are rapidly and transiently transcribed after a wide variety of signals. As opposed to the “late response” genes, IEGs expression does not depend on any precedent protein synthesis. They encode many functionally distinct proteins, such as transcription and growth factors, cytoskeletal proteins or signalling molecules, and play an essential role in cellular responses that contribute to long-term neuronal plasticity. Arc , Egr1 and Npas4 are three IEGs linked to synaptic plasticity processes. Arc is a postsynaptic effector protein localised primarily in the dendrites of excitatory glutamatergic neurons of cortex and hippocampus. Arc alters spine morphology and synaptic strength via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalisation, and therefore has a key role in synaptic plasticity and long-term memory. Psychedelics have been found to induce its expression (Nichols & Sanders-Bush, 2002; Pei et al., 2000 ). Egr1 is a transcription factor which, following synaptic activation, binds to the Arc promoter and activates its transcription in a non-dependent manner. Egr1 is a transcription suppressor of PSD-95 and modulates N-methyl-D-aspartate receptor (NMDAR)-dependent AMPAR trafficking ( Qin et al., 2015 ). Egr1 is specifically involved in memory encoding and synaptic organization at multiple levels, resulting in synaptic stabilization. Increase in Egr1 gene expression in murine cortex has been found after LSD administration, as well as after fluoxetine in vitro ( González-Maeso et al., 2003 , 2007 ; Levy et al., 2019 ). Npas4 is a transcription factor selectively activated by neuronal activity, directly regulating brain-derived neurotrophic factor expression ( Sun & Lin, 2016 ). The exact pathways activated by psychedelics have been under intense investigation in recent years. Despite that, to the best of our knowledge, no study has directly compared these promising agents to regular psychiatric medication in terms of their neuroplastic effects. In this study, psilocin, DMT and LSD were examined and compared to two of the most prescribed psychiatric drugs, fluoxetine and lithium, as well as with ketamine, a generally acknowledged psychoplastogen, in the ability to promote synaptogenesis and to enhance the gene expression of selected IEGs Arc , Egr1 and Npas4 . Methods Primary cortical culture isolation and maintenance All experiments were conducted in accordance with guidelines of the Directive 2010/63/EU of the European Parliament and approved by the Animal Care and Use Committee of the Ministry of Health (reference number MZDR 17637/2020-4/OVZ) in line with the Animal Protection Code of the Czech Republic 246/1992. NIMH is authorized for the use of experimental animals (accreditation ref. no. MZE-51088/2021-13114). Primary cortical cultures were prepared as previously described by Jorratt et al. (2023) with minor modifications. Briefly, embryos were extracted from time-mated female Wistar rats on embryonic day 18. The cortices were dissociated under the stereomicroscope VisiScope® 250 (VWR, USA) and put in ice-cold Dulbecco’s phosphate-buffered saline (DPBS, Gibco, 14190144). Then, the cortices were washed thrice with ice-cold DPBS and mechanically dissociated with a 0.9 mm needle (B. Braun, 4657519), followed by a 0.45 mm needle (B. Braun, 4657683), each needle thrice. The dissociated supernatant was transferred into a seeding medium containing advanced Dulbecco’s modified Earle’s medium (DMEM, Gibco, 12491015) supplemented with 10% fetal bovine serum (FBS, Biowest, S1810-500), 1% Antibiotic-Antimycotic solution (Gibco, 15240062) and 1% GlutaMAX supplement (Gibco, 35050061), and filtered twice through a 100-µm cell strainer (VWR, 732–2759). The singularized cells were counted using a haemocytometer and seeded on poly-L-lysine (Merck, P1274-25MG) pre-coated well plates. 24-well plates with inserted 13-mm, #1.5 coverslips (VWR, 631-0150) with a seeding density of 50,000 cells/well were used for immunocytochemistry (ICC), 6-well plates with a density of 500,000 cells/well were used for real-time quantitative polymerase chain reaction (qPCR). The cells were cultured in a humidified incubator at 37°C and 5% CO 2 . On the next day, the seeding medium was replaced with neurobasal medium (Gibco, 21103049), enriched with 2% B27 supplement (Gibco, 17504044), 1% Antibiotic-antimycotic and 1% GlutaMAX. Half of the growth medium was changed twice a week. Drugs and chemicals On day in vitro (DIV) 17, the cultures were treated for 24 h for the ICC analysis of colocalization, and for 1 h and 24 h for the qPCR analysis of changes in IEGs expression. The cells were treated with 10 µM psilocin (THC Pharm, Germany), 1 µM LSD fumarate (Alfarma, Czechia), 90 µM DMT fumarate (Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology, Czech Republic), 10 µM fluoxetine hydrochloride (Molekula Group, Czechia), 5 mM lithium chloride (Merck, Germany) and 1 µM ketamine (Merck, Germany). All drugs were of ≥ 99% purity and were diluted in sterile distilled water. Sterile distilled water was used as negative control (Ctrl). Immunocytochemistry Cells were quickly washed with pre-warmed (37°C) phosphate buffer saline (PBS) and fixed with pre-warmed 4% paraformaldehyde in PBS for 4 min, then rinsed 3 x 5 min with PBS. Next, blocking and permeabilization were done in one step with 10% FBS, 0.1% Triton X-100 in PBS for 45 min at RT. The coverslips were subsequently incubated with primary antibodies chicken anti-MAP2 (1:10,000, Abcam, ab5392), mouse anti-PSD-95 (1:500, Abcam, ab192757), and guinea-pig anti-synapsin I/II (1:1000, Synaptic systems, 106004) at 4°C overnight. The following day, coverslips were washed 3 x 5 min with PBS and incubated with secondary antibodies AlexaFluor (AF) 647 conjugated donkey anti-chicken (703-605-155), AF488 donkey anti-guinea pig (706-545148) and AF594 donkey anti-mouse (715-545-151) for 1 h at room temperature in the dark. All secondary antibodies were purchased from Jackson ImmunoResearch and diluted 1:500 for working solutions. After the incubation, coverslips were washed 3 x 5 min with PBS in the dark and rinsed with distilled water, dried from excess liquid, mounted onto microscope slides using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36941), and allowed to cure overnight. All the antibodies were diluted in the staining solution consisting of 0.1% Triton X-100 in PBS. Image acquisition and analysis Images were acquired with confocal laser scanning microscope Leica SP8 X on inverted microscope scope Leica DMi8 (Leica Microsystems, Germany). Plan apochromat oil objective Leica 63x/1.40 CS2 was used. The confocal pinhole was set to Airy1, and bidirectional scanning with line averaging 4 times was applied for Z-stacks at 0.3 μm Z-step size. The acquisition was done in 8-bit, 1024×1024 pixels, and 2.6x digital zoom. For every experiment, samples were imaged under identical software settings. Automatic threshold selection was performed, and binary pictures were analysed using the Synapse Counter plugin ( Dzyubenko et al., 2016 ) for Fiji (version 1.52e, Schindelin et al., 2012 ), following a protocol from Li et al. (2016) . Changes in IEGs expression Cells were lysed in RNA Blue reagent (TopBio, R013) and phenol-chloroform-based extraction of the homogenate was performed according to the manufacturer’s instructions. Complementary DNA (cDNA) was generated using the same amount of each total RNA sample within one batch. High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) was used according to the manufacturer’s instructions. qPCR was performed with CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using Luna Universal qPCR Master Mix (New England Biolabs, M3003L) according to the manufacturer’s instructions. IEGs signal was normalized to housekeeping genes glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) and peptidylprolyl isomerase A ( Ppia ), which were selected as an optimal reference via NormFinder tool (version 0.953, Andersen et al., 2004 ). Comparative Ct (ΔΔCt) method was used to determine the changes in mRNA levels of Arc , Egr1 , and Npas4 ( Livak & Schmittgen, 2001 ). The primers were designed in the NCBI Primer-BLAST tool ( Ye et al., 2012 ) or selected based on literature ( Adams et al., 2017 ; Heroux et al., 2018 ) and the sequences were chosen as follows: Arc (5’- GCAGAAAGCGCTTGAACTTG-3’, 5’-AGCGGGACCTGTACCAGAC-3’), Egr1 (5’- CATGCAGATTCGACACTGGAAG-3’, 5’-GTATGCTTGCCCTGTTGAGTCC-3’), Npas4 (5’- GCCACTATGTCTTCAAGCTCT-3’, 5’-CTGCATCTACACTCGCAAGG-3’), Gapdh (5’- GGCATGGACTGTGGTCATGA-3’, 5’-CAACTCCCTCAAGATTGTCAGC-3’), Ppia (5’- TCCTTTCTCCCCAGTGCTCAG-3’, 5’-TCAACCCCACCGTGTTCTTC-3’). Data and statistical analysis Statistical analyses were performed in GraphPad Prism (version 8.0.1, GraphPad Software, Inc., USA). Outliers were removed using the ROUT method with a 1 % Q value. Data violating the assumption of normality (Shapiro-Wilk test) were normalised by square root transformation. Homogeneity of variance was tested using the Brown-Forsythe test. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test were performed for the comparison of drug treatment effect compared to NC. Results Psilocin induces synaptogenesis Synaptic puncta were identified by the co-localization of synapsin I/II and PSD-95 puncta. Analysis of changes in synapsin I/II density did not show any effect of the treatment ( Fig. 1A ). Significant changes were observed in PSD-95 puncta density after the treatment [F (6, 330) = 5.55, p < 0.001], post hoc analysis revealed prominent effect of ketamine (p < 0.001, Fig. 1B ). Analysis of co-localized (i. e. synaptic) puncta also displayed significant effect of the drug treatment [F (6, 333) = 8.60, p < 0.001], further post hoc revealed strong effect of psilocin, lithium, and ketamine (all p < 0.001, Fig. 1C ). Download figure Open in new tab Figure 1. The effect of selected psychoplastogens on synaptogenesis. Quantification of presynaptic density ( A , synapsin puncta), postsynaptic density ( B , PSD-95 puncta) and their co-localization ( C , established synapses) after 24 h drug treatment of primary cortical neurons. n = 38–79 randomly chosen neurons per treatment from at least three biological replicates. One-way ANOVA followed by Dunnett’s post hoc test was performed, data are presented as mean fold change ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. D Representative images of primary dendrites of cortical neurons (DIV18) after 24 h treatment. Orange areas in the synapsin + PSD-95 merged images indicate colocalization. Green, synapsin I/II; red, PSD-95; cyan, MAP2. Psilocin induces gene expression of Arc after acute treatment After one hour ( Fig. 2A ), Arc expression was significantly affected by the treatment [F (6, 24) = 4.73, p < 0.01], post-hoc analysis showed significant effect of psilocin (p < 0.01). Expression of Npas4 was also significantly changed after one hour [F (6, 26) = 2.55, p < 0.05], although post hoc test did not reveal any specific drug treatment effect. Download figure Open in new tab Figure 2. Effect of selected psychoplastogens on the expression of IEG mRNA. A + B Assessment of the changes in mRNA expression of IEG Arc , Egr1 and Npas4 after 1 h ( A ) and 24 h ( B ) drug treatment of primary cortical neurons. Data were collected from at least four biological replicates. One-way ANOVA followed by Dunnett’s post hoc test was performed. Data are presented as mean fold change ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. After 24 hours ( Fig. 2B ), Egr1 levels were significantly affected by the treatment [F (6, 21) = 8.34, p < 0.001], post hoc test showed significant effect of fluoxetine (p < 0.001). Npas4 expression was also significantly altered by the treatment [F (6, 20) = 7.59, p < 0.001], post hoc test revealed prominent changes following fluoxetine treatment (p < 0.001). Discussion The main finding of our study is that psilocin significantly promotes synaptogenesis and expression of the IEG Arc , and moreover, the effects of psilocin were comparable to those of lithium and ketamine. Surprisingly, we did not observe such effects after LSD and DMT treatment. Concerning the positive control treatments, ketamine also increased PSD-95 density and fluoxetine did not affect synaptic density, but upregulated Egr1 and Npas4 expression. Our finding that psilocin increases neuronal plasticity is in line with other studies ( Du et al., 2023 ; Moliner et al., 2023 ; Raval et al., 2021 ; Vargas et al., 2023 ). In accordance with Ly et al. (2018) , we did not observe any effect on PSD-95 density after the treatment with DMT, but we discovered the increased PSD-95 and synapse densities associated with ketamine treatment, which was described also in other studies ( Li et al., 2010 ; Ly et al., 2021 ). Interestingly, ( Ly et al., 2018 , 2021 ) observed also the effect of LSD on presynaptic and synaptic puncta count, as opposed to our results. The fact that we did not find any significant effect of LSD and DMT on synaptogenesis was unexpected. The explanation could be that we did not add dimethyl sulfoxide (DMSO) into the drug solution for the elimination of any noxious effect on the primary cells. However, since DMSO is a permeation enhancer, its lack in the solution might cause the non-significant results for LSD and DMT in our study. In the study of changes in IEGs mRNA expression after psychedelic treatment, we found a significant upregulation of Arc after the acute treatment with psilocin, and of Egr1 and Npas4 following the prolonged treatment with fluoxetine. In contrast to our study, 90 min psilocybin treatment did not exhibit any significant changes in Arc in the PFC, but decreased expression in the hippocampi of rats ( Jefsen et al., 2021 ). Another animal study demonstrates upregulated Arc expression following 90 min LSD treatment in PFC, but not in hippocampus (Nichols & Sanders-Bush, 2002). Davoudian et al. (2023) describe comparable elevation of IEG cFos in many brain regions after psilocybin and ketamine administration, with selected brain regions manifesting drug-preferential differences. Concerning Egr1 , it has been shown that an acute dose of psychedelics increases its expression ( Desouza et al., 2021 ; Liu et al., 2023 ), while chronic microdosing does not ( Cameron et al., 2019 ). A study using noribogaine observed upregulated Egr1 and Npas4 expression in male mice with serotonin 2A receptor (5-HT2AR) knockout, Npas4 expression in 5-HT2AR knockout females, and Egr1 expression in wild type females; indicating sex and genotype differences in IEGs expression ( Villalba et al., 2024 ). In this study, Npas4 was clearly upregulated in a 5-HT2AR activation-independent manner. On top of that, noribogaine has a polypharmacological profile and does not aim directly at 5-HT2AR, as opposed to psychedelics. Another reason for the divergent results with aforementioned studies could dwell in our in vitro model, which comprises the mixture of cortical cells from both male and female embryos. This aspect may be necessary to take into account for the following studies focusing on IEGs, as it could be the cause of major variances. Overall, our study reveals that psilocin indeed acts as a neuroplastic agent by enhancing both synaptogenesis and Arc expression. The co-localization results also indicate similar mechanisms by which psilocin, ketamine and lithium promote their neuroplastic effect on the synapse. On the other hand, our findings also suggest that the effects of fluoxetine on neuroplasticity are produced through different mechanisms than those of serotonergic psychedelics or ketamine. Author contributions All authors made a significant contribution to the design, acquisition, analysis, or interpretation of data for this study. AP and TP designed the project, YV and KS carried out the experiments, YV, KS and IK wrote the manuscript. VK performed the dissections and supervised the ICC assay, KŠ, VM and MN carried out the statistical analyses, JP trained the experimenters in confocal microscopy and supervised the setup, MK and RJ synthesized and provided the group with ketamine, TP and ZB supervised the project and manuscript writing. All authors gave final approval for the current version of this work to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Conflict of interest TP declares having shares in „Psyon s.r.o.“ and in “Společnost pro podporu neurovědního výzkumu s.r.o.”. TP founded „PSYRES” – Psychedelic Research Foundation and he reports consulting fees from GH Research and CB21-Pharma outside the submitted work. TP is also involved in clinical trials of Compass Pathways with psilocybin, MAPS clinical trial with MDMA and GH-Research clinical trial with 5-MeO-DMT outside the submitted work. Data Availability All data supporting described findings can be obtained from the corresponding author upon reasonable request. Acknowledgements This work was supported by a grant from the Czech Science Foundation (project 20-25349S), Czech Health Research Council (project NU21-04-00307), Long-term conceptual development of research organization (RVO 00023752), Ministry of the interior of the Czech Republic (project VK01010212), Specific University Research, Czech Ministry of Education, Youth and Sports (project 260648/SVV/2024), ERDF-Project Brain dynamics, No. CZ.02.01.01/00/22_008/0004643, Charles University research program Cooperatio-Neurosciences, Grant Agency of Charles University in Prague, Czech Republic (project GAUK 430122) and private funds obtained via PSYRES - Psychedelic Research Foundation (psyresfoundation.eu). References ↵ Adams , K. W. , Kletsov , S. , Lamm , R. J. , Elman , J. S. , Mullenbrock , S. , & Cooper , G. M . ( 2017 ). Role for egr1 in the transcriptional program associated with neuronal differentiation of pc12 cells . PLoS ONE , 12 ( 1 ). OpenUrl CrossRef PubMed ↵ Andersen , C. L. , Jensen , J. L. , & Falck Ørntoft , T. ( 2004 ). Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets . In CANCER RESEARCH (Vol. 64 ). ↵ Antoniadou , I. , Kouskou , M. , Arsiwala , T. , Singh , N. , Vasudevan , S. R. , Fowler , T. , Cadirci , E. , Churchill , G. C. , & Sharp , T . ( 2018 ). Ebselen has lithium-like effects on central 5-HT 2A receptor function . British Journal of Pharmacology , 175 ( 13 ), 2599 – 2610 . OpenUrl ↵ Cameron , L. P. , Benetatos , J. , Lewis , V. , Bonniwell , E. M. , Jaster , A. M. , Moliner , R. , Castrén , E. , McCorvy , J. D. , Palner , M. , & Aguilar-Valles , A . ( 2023 ). Beyond the 5-HT2A Receptor: Classic and Nonclassic Targets in Psychedelic Drug Action . Journal of Neuroscience , 43 ( 45 ), 7472 – 7482 . OpenUrl Abstract / FREE Full Text ↵ Cameron , L. P. , Benson , C. J. , Defelice , B. C. , Fiehn , O. , & Olson , D. E . ( 2019 ). Chronic, Intermittent Microdoses of the Psychedelic N, N-Dimethyltryptamine (DMT) Produce Positive Effects on Mood and Anxiety in Rodents . ACS Chemical Neuroscience , 10 ( 7 ), 3261 – 3270 . OpenUrl ↵ Carhart-Harris , R. L. , & Goodwin , G. M . ( 2017 ). The Therapeutic Potential of Psychedelic Drugs: Past, Present, and Future . Neuropsychopharmacology , 42 ( 11 ), 2105 – 2113 . OpenUrl CrossRef PubMed ↵ Davoudian , P. A. , Shao , L. X. , & Kwan , A. C . ( 2023 ). Shared and Distinct Brain Regions Targeted for Immediate Early Gene Expression by Ketamine and Psilocybin . ACS Chemical Neuroscience , 14 ( 3 ), 468 – 480 . OpenUrl ↵ de la Fuente Revenga , M. , Zhu , B. , Guevara , C. A. , Naler , L. B. , Saunders , J. M. , Zhou , Z. , Toneatti , R. , Sierra , S. , Wolstenholme , J. T. , Beardsley , P. M. , Huntley , G. W. , Lu , C. , & González-Maeso , J. ( 2021 ). Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice . Cell Reports , 37 ( 3 ). OpenUrl ↵ Desouza , L. A. , Benekareddy , M. , Fanibunda , S. E. , Mohammad , F. , Janakiraman , B. , Ghai , U. , Gur , T. , Blendy , J. A. , & Vaidya , V. A . ( 2021 ). The Hallucinogenic Serotonin2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex . Frontiers in Molecular Neuroscience , 14 . ↵ Du , Y. , Li , Y. , Zhao , X. , Yao , Y. , Wang , B. , Zhang , L. , & Wang , G . ( 2023 ). Psilocybin facilitates fear extinction in mice by promoting hippocampal neuroplasticity . Chinese Medical Journal , 136 ( 24 ), 2983 – 2992 . OpenUrl ↵ Dzyubenko , E. , Rozenberg , A. , Hermann , D. M. , & Faissner , A . ( 2016 ). Colocalization of synapse marker proteins evaluated by STED-microscopy reveals patterns of neuronal synapse distribution in vitro . Journal of Neuroscience Methods , 273 , 149 – 159 . OpenUrl CrossRef PubMed ↵ González-Maeso , J. , Weisstaub , N. V. , Zhou , M. , Chan , P. , Ivic , L. , Ang , R. , Lira , A. , Bradley-Moore , M. , Ge , Y. , Zhou , Q. , Sealfon , S. C. , & Gingrich , J. A . ( 2007 ). Hallucinogens Recruit Specific Cortical 5-HT2A Receptor-Mediated Signaling Pathways to Affect Behavior . Neuron , 53 ( 3 ), 439 – 452 . OpenUrl CrossRef PubMed Web of Science ↵ González-Maeso , J. , Yuen , T. , Ebersole , B. J. , Wurmbach , E. , Lira , A. , Zhou , M. , Weisstaub , N. , Hen , R. , Gingrich , J. A. , & Sealfon , S. C . ( 2003 ). Cellular/Molecular Transcriptome Fingerprints Distinguish Hallucinogenic and Nonhallucinogenic 5-Hydroxytryptamine 2A Receptor Agonist Effects in Mouse Somatosensory Cortex . The Journal of Neuroscience , 23 ( 26 ), 8836 – 8843 . OpenUrl Abstract / FREE Full Text ↵ Hammonds , M. D. , & Shim , S. S . ( 2009 ). Effects of 4-week treatment with lithium and olanzapine on levels of brain-derived neurotrophic factor, B-Cell CLL/Lymphoma 2 and phosphorylated cyclic adenosine monophosphate response element-binding protein in the sub-regions of the hippocampus . Basic and Clinical Pharmacology and Toxicology , 105 ( 2 ), 113 – 119 . OpenUrl ↵ Heroux , N. A. , Osborne , B. F. , Miller , L. A. , Kawan , M. , Buban , K. N. , Rosen , J. B. , & Stanton , M. E . ( 2018 ). Differential expression of the immediate early genes c-Fos, Arc, Egr-1, and Npas4 during long-term memory formation in the context preexposure facilitation effect (CPFE) . Neurobiology of Learning and Memory , 147 (August 2017), 128 – 138 . OpenUrl CrossRef PubMed ↵ Jefsen , O. H. , Elfving , B. , Wegener , G. , & Müller , H. K . ( 2021 ). Transcriptional regulation in the rat prefrontal cortex and hippocampus after a single administration of psilocybin . Journal of Psychopharmacology , 35 ( 4 ), 483 – 493 . OpenUrl CrossRef PubMed ↵ Jorratt , P. , Ricny , J. , Leibold , C. , & Ovsepian , S. V . ( 2023 ). Endogenous Modulators of NMDA Receptor Control Dendritic Field Expansion of Cortical Neurons . Molecular Neurobiology , 60 ( 3 ), 1440 – 1452 . OpenUrl ↵ Kim , H. J. , & Thayer , S. A . ( 2009 ). Lithium increases synapse formation between hippocampal neurons by depleting phosphoinositides . Molecular Pharmacology , 75 ( 5 ), 1021 – 1030 . OpenUrl Abstract / FREE Full Text ↵ Krystal , J. H. , Abdallah , C. G. , Sanacora , G. , Charney , D. S. , & Duman , R. S . ( 2019 ). Ketamine: A Paradigm Shift for Depression Research and Treatment . In Neuron (Vol. 101 , Issue 5 , pp. 774 – 778 ). Cell Press . OpenUrl CrossRef PubMed ↵ Levy , M. J. F. , Boulle , F. , Emerit , M. B. , Poilbout , C. , Steinbusch , H. W. M. , Van den Hove , D. L. A. , Kenis , G. , & Lanfumey , L. ( 2019 ). 5-HTT independent effects of fluoxetine on neuroplasticity . Scientific Reports , 9 ( 1 ). OpenUrl ↵ Li , H. , Aksenova , M. , Bertrand , S. J. , Mactutus , C. F. , & Booze , R . ( 2016 ). Quantification of filamentous actin (F-actin) puncta in rat cortical neurons . Journal of Visualized Experiments , 2016 ( 108 ). OpenUrl ↵ Li , N. , Lee , B. , Liu , R. J. , Banasr , M. , Dwyer , J. M. , Iwata , M. , Li , X. Y. , Aghajanian , G. , & Duman , R. S . ( 2010 ). mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists . Science , 329 ( 5994 ), 959 – 964 . OpenUrl Abstract / FREE Full Text ↵ Liu , J. , Wang , Y. , Xia , K. , Wu , J. , Zheng , D. , Cai , A. , Yan , H. , & Su , R . ( 2023 ). Acute psilocybin increased cortical activities in rats . Frontiers in Neuroscience , 17 . ↵ Livak , K. J. , & Schmittgen , T. D . ( 2001 ). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method . Methods , 25 ( 4 ), 402 – 408 . OpenUrl CrossRef PubMed Web of Science ↵ Ly , C. , Greb , A. C. , Cameron , L. P. , Wong , J. M. , Barragan , E. V. , Wilson , P. C. , Burbach , K. F. , Soltanzadeh Zarandi , S. , Sood , A. , Paddy , M. R. , Duim , W. C. , Dennis , M. Y. , McAllister , A. K. , Ori-McKenney , K. M. , Gray , J. A. , & Olson , D. E . ( 2018 ). Psychedelics Promote Structural and Functional Neural Plasticity . Cell Reports , 23 ( 11 ), 3170 – 3182 . OpenUrl ↵ Ly , C. , Greb , A. C. , Vargas , M. V. , Duim , W. C. , Grodzki , A. C. G. , Lein , P. J. , & Olson , D. E . ( 2021 ). Transient Stimulation with Psychoplastogens Is Sufficient to Initiate Neuronal Growth . ACS Pharmacology and Translational Science , 4 ( 2 ), 452 – 460 . OpenUrl ↵ Moliner , R. , Girych , M. , Brunello , C. A. , Kovaleva , V. , Biojone , C. , Enkavi , G. , Antenucci , L. , Kot , E. F. , Goncharuk , S. A. , Kaurinkoski , K. , Kuutti , M. , Fred , S. M. , Elsilä , L. V. , Sakson , S. , Cannarozzo , C. , Diniz , C. R. A. F. , Seiffert , N. , Rubiolo , A. , Haapaniemi , H. , … Castrén , E . ( 2023 ). Psychedelics promote plasticity by directly binding to BDNF receptor TrkB . Nature Neuroscience , 26 ( 6 ), 1032 – 1041 . OpenUrl PubMed Moreno , J. L. , Holloway , T. , Albizu , L. , Sealfon , S. C. , & González-Maeso , J . ( 2011 ). Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists . Neuroscience Letters , 493 ( 3 ), 76 – 79 . OpenUrl CrossRef PubMed Nichols , C. D. , & Sanders-Bush , E. ( 2002a ). A Single Dose of Lysergic Acid Diethylamide Influences Gene Expression Patterns within the Mammalian Brain . In Neuropsychopharmacology (Vol. 26 , Issue 5 ). Nichols , C. D. , & Sanders-Bush , E. ( 2002b ). A Single Dose of Lysergic Acid Diethylamide Influences Gene Expression Patterns within the Mammalian Brain . In Neuropsychopharmacology (Vol. 26 , Issue 5 ). ↵ Pei , Q. , Lewis , L. , Sprakes , M. E. , Jones , E. J. , Grahame-Smith , D. G. , & Zetterström , T. S. C. ( 2000 ). Serotonergic regulation of mRNA expression of Arc, an immediate early gene selectively localized at neuronal dendrites . In Neuropharmacology (Vol. 39 ). ↵ Puglisi-Allegra , S. , Ruggieri , S. , & Fornai , F. ( 2021 ). Translational evidence for lithium-induced brain plasticity and neuroprotection in the treatment of neuropsychiatric disorders . In Translational Psychiatry (Vol. 11 , Issue 1 ). Springer Nature . ↵ Qin , X. , Jiang , Y. , Tse , Y. C. , Wang , Y. , Wong , T. P. , & Paudel , H. K . ( 2015 ). Early growth response 1 (Egr-1) regulates N-methyl-daspartate receptor (NMDAR)-dependent transcription of PSD-95 and α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) trafficking in hippocampal primary neurons . Journal of Biological Chemistry , 290 ( 49 ), 29603 – 29616 . OpenUrl Abstract / FREE Full Text ↵ Rădulescu , I. , Drăgoi , A. , Trifu , S. , & Cristea , M . ( 2021 ). Neuroplasticity and depression: Rewiring the brain’s networks through pharmacological therapy (Review) . Experimental and Therapeutic Medicine , 22 ( 4 ). ↵ Raval , N. R. , Johansen , A. , Donovan , L. L. , Ros , N. F. , Ozenne , B. , Hansen , H. D. , & Knudsen , G. M . ( 2021 ). A single dose of psilocybin increases synaptic density and decreases 5-HT2A receptor density in the pig brain . International Journal of Molecular Sciences , 22 ( 2 ), 1 – 14 . OpenUrl ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , Kaynig , V. , Longair , M. , Pietzsch , T. , Preibisch , S. , Rueden , C. , Saalfeld , S. , Schmid , B. , Tinevez , J. Y. , White , D. J. , Hartenstein , V. , Eliceiri , K. , Tomancak , P. , & Cardona , A . ( 2012 ). Fiji: An open-source platform for biological-image analysis . In Nature Methods (Vol. 9 , Issue 7 , pp. 676 – 682 ). OpenUrl CrossRef ↵ Soeiro-de-Souza , M. G. , Dias , V. V. , Figueira , M. L. , Forlenza , O. V. , Gattaz , W. F. , Zarate , C. A. , & Machado-Vieira , R . ( 2012 ). Translating neurotrophic and cellular plasticity: From pathophysiology to improved therapeutics for bipolar disorder . Acta Psychiatrica Scandinavica , 126 ( 5 ), 332 – 341 . OpenUrl CrossRef PubMed Web of Science ↵ Sun , X. , & Lin , Y . ( 2016 ). Npas4: Linking Neuronal Activity to Memory . In Trends in Neurosciences (Vol. 39 , Issue 4 , pp. 264 – 275 ). Elsevier Ltd . OpenUrl CrossRef PubMed ↵ Tai , S. H. , Huang , S. Y. , Chao , L. C. , Lin , Y. W. , Huang , C. C. , Wu , T. S. , Shan , Y. S. , Lee , A. H. , & Lee , E. J . ( 2022 ). Lithium upregulates growth-associated protein-43 (GAP-43) and postsynaptic density-95 (PSD-95) in cultured neurons exposed to oxygen-glucose deprivation and improves electrophysiological outcomes in rats subjected to transient focal cerebral ischemia following a long-term recovery period . Neurological Research , 44 ( 10 ), 870 – 878 . OpenUrl ↵ Umemori , J. , Winkel , F. , Didio , G. , Llach Pou , M. , & Castrén , E . ( 2018 ). iPlasticity: Induced juvenile-like plasticity in the adult brain as a mechanism of antidepressants . In Psychiatry and Clinical Neurosciences (Vol. 72 , Issue 9 , pp. 633 – 653 ). Blackwell Publishing . OpenUrl CrossRef PubMed ↵ Vargas , M. V , Dunlap , L. E. , Dong , C. , Carter , S. J. , Tombari , R. J. , Jami , S. A. , Cameron , L. P. , Patel , S. D. , Hennessey , J. J. , Saeger , H. N. , Mccorvy , J. D. , Gray , J. A. , Tian , L. , & Olson , D. E . ( 2023 ). Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors . Science , 379 , 700 – 706 . OpenUrl CrossRef PubMed ↵ Villalba , S. , González , B. , Junge , S. , Bernardi , A. , González , J. , Fagúndez , C. , Torterolo , P. , Carrera , I. , Urbano , F. J. , & Bisagno , V . ( 2024 ). 5-HT2A Receptor Knockout Mice Show Sex-Dependent Differences following Acute Noribogaine Administration . International Journal of Molecular Sciences , 25 ( 2 ), 687 . OpenUrl ↵ Ye , J. , Coulouris , G. , Zaretskaya , I. , Cutcutache , I. , Rozen , S. , & Madden , T. L . ( 2012 ). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction . BMC Bioinformatics , 13 ( 134 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted August 09, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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