Psilocin fosters neuroplasticity in iPSC-derived human cortical neurons
- Department of Translational Brain Research, Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim, Germany
- Hector Institute for Translational Brain Research (HITBR gGmbH), Germany
- German Cancer Research Center (DKFZ), Germany
- Department of Neuroanatomy, Mannheim Centre for Translational Neuroscience (MCTN), Medical Faculty Mannheim, Heidelberg University, Germany
- Institute for Psychopharmacology, Central Institute of Mental Health (ZI), University of Heidelberg/Medical Faculty Mannheim, Germany
- Molecular Neuroimaging, Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim, Germany
- German Center for Mental Health (DZPG), partner site Mannheim-Heidelberg-Ulm, Germany
- Department of Neurophysiology, Mannheim Center for Translational Neuroscience (MCTN), University of Heidelberg/Medical Faculty Mannheim, Germany
Figures
Figure 1 with 1 supplement
Validation of mature cortical neuron properties.
(A) hiPSCs were differentiated in vitro into glutamatergic cortical neurons over a neuronal progenitor step since the cerebral cortex is a key region for psychedelic effects and psychiatric disorders. Mature 40-day-old cortical neurons were glutamatergic (vGLUT2 expression) and expressed cortical layer markers like TBR1, CTIP2 and the neuronal markers NeuN, MAP2, and TAU. cFOS expression is a sign for neuronal activity. GFAP expression indicated a low amount of astrocytes. Scale bar: 50 µm, for GFAP staining 100 µm.
Figure 1—figure supplement 1
Validation of iPSC and neural progenitor properties and HTR2A gene expression.
(A). Timeline: hiPSCs were differentiated in vitro into glutamatergic cortical neurons over a neuronal progenitor step. Maturation of cortical neurons took about 6 weeks. Scheme created in BioRender,. (B) iPSCs expressed pluripotent marker SOX2 and OCT4. Neuronal progenitors expressed SOX2, PAX6 and NESTIN and were negative for FOXA2 as a midbrain marker. Scale bar: 50 μm. (C) RT-PCR analysis revealed expression of neuronal subtype markers, neurotransmitter receptors, neuronal activity markers, neuronal markers and synapse-associated genes. (D) RT-PCR of the HTR2A gene (also expressed in commercialized samples of fetal and adult brain) confirmed expression of 5-HT2A-R. Scale bar: 50 μm.
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Figure 1—figure supplement 1—source data 1
RT-PCR analysis revealed expression of neuronal subtype markers, neurotransmitter receptors, neuronal activity markers, neuronal markers and synapse-associated genes in cortical neurons (uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
RT-PCR analysis revealed expression of neuronal subtype markers, neurotransmitter receptors, neuronal activity markers, neuronal markers and synapse-associated genes in cortical neurons (uncropped).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
RT-PCR of the HTR2A gene (expressed in commercialized samples of fetal and adult brain) confirmed expression of 5-HT2A-R in cortical neurons (uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig1-figsupp1-data3-v1.zip
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Figure 1—figure supplement 1—source data 4
RT-PCR of the HTR2A gene (also expressed in commercialized samples of fetal and adult brain) confirmed expression of 5-HT2A-R in cortical neurons (uncropped).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig1-figsupp1-data4-v1.zip
Figure 2 with 1 supplement
Psilocin-induced increase in BDNF level was 5-HT2A-R and PKC- and endocytosis-mediated and induced activation of m-BDNF/TrkB-associated downstream pathway.
(A, B) Representative image of a neuronal network for pre-treatment condition (Ctrl) and 24 hrs after a 10 min 10 µM short psilocin trigger (Psi). Scale bar: 50 µm, (B) close-up: 2 µm. (C) BDNF density was significantly increased 24 hrs after a 10 min 10 µM psilocin trigger (Psi). Four Ctrl cell lines were included in the analysis (Ctrl with N=613 neurites; Psi with N=529 neurites). (D) Representative image 24 hrs after the simultaneous treatment with psilocin and ketanserin (Psi + Ket) and single treatment with ketanserin (Ket), scale bar: 50 µm. (E) Ketanserin co-treatment (Psi + Ket) and ketanserin monotreatment (Ket) significantly reduced BDNF density compared to the 24 hrs monotreatment psilocin condition, suggesting a 5-HT2A-R-mediated process. Ketanserin monotreatment provoked a significant reduction in BDNF density compared to ketanserin co-treatment with psilocin. Two Ctrl cell lines were included in the analysis, each with one biological batch (Ctrl with N=99 neurites; Psi with N=102 neurites; Psi + Ket with N=102 neurites; Ket with N=102 neurites). (F) Representative image 24 hrs after the simultaneous treatment of 10 µM psilocin with chelerythrine (Psi + Chel) or dynasore (Psi + D), scale bar: 50 µm. (G) Chelerythrine (Psi + Chel, selective PKC inhibitor) or dynasore (Psi + D, inhibition of clathrin-coated vesicle invagination) co-treatment significantly reduced BDNF density compared to the psilocin monotreatment condition (Psi). Two Ctrl cell lines were included in the analysis, each with one biological batch (Ctrl with N=90 neurites; Psi with N=90 neurites; Psi + Chel with N=90; Psi + D with N=90 neurites). (H) Phosphorylated AKT (pAKT) protein level was increased 72 hrs after psilocin exposure (Psi), reversed by ketanserin co-treatment (Psi + Ket), (I) both effects were not significant. Ctrl cell line 3 was included in the analysis (Ctrl with N=4 data points, Psi with N=4 data points, Psi + Ket with N=4 data points), each with two biological batches. For all analyses, the Kruskal–Wallis test for independent samples was calculated. Post hoc Wilcoxon rank sum test. Bonferroni correction, adjusted p<.05, mean ± SD. Significance levels against the respective control and for multiple group comparisons are *p<0.05.
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Figure 2—source data 1
Western Blot: phosphorylated AKT (pAKT) protein level and AKT protein level after psilocin exposure (Psi) and psilocin and ketanserin treatment (Psi + Ket) (72 hrs post, uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-data1-v1.zip
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Figure 2—source data 2
Western Blot: phosphorylated AKT (pAKT) protein level and AKT protein level after psilocin exposure (Psi) and psilocin and ketanserin treatment (Psi + Ket) (72 hrs post, uncropped).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-data2-v1.zip
Figure 2—figure supplement 1
Influence of different treatment conditions on BDNF level.
(A) Representative image of a neuronal network for pre-treatment condition (DMSO) and 24 hrs after a 10 min 10 nM, 100 nM, 1 μM or 10 μM trigger showed the strongest increase in BDNF density for the 10 μM treatment condition. Scale bar: 50 μm. (B) BDNF density was exclusively significantly increased 24 hrs after 10 min 10 μM Psilocin trigger (10 μM). Ctrl cell line 1 was included in the analysis, with one biological batch (Ctrl with N=76 neurites; 24 hrs after a 10 min 10 nM) (N=75 neurites), respectively 100 nM (N=75 neurites), respectively 1 μM (N=75 neurites), respectively 10 μM (N=75 neurites). (C) Representative image of a neuronal network 24 hrs after an ‘artificial’ (10 min 10 μM) and a ‘physiological’ (6 hrs 100 nM) treatment condition. Scale bar: 50 μm. (D) BDNF density was significantly increased 24 hrs after a 10 min 10 μM psilocin trigger (10 min 10 μM) and 24 hrs after a 6 hrs 100 nM psilocin stimulation (6 hrs 100 nM). BDNF density was also significantly increased for the ‘artificial’ compared to the ‘physiological’ stimulation. Two Ctrl cell lines were included in the analysis, each with scale bar: 50 μm. (F) BDNF density was significantly increased for axonal TAU + and dendritic MAP2+ one biological batch (Ctrl with N=99 neurites; 10 min 10 μM with N=102 neurites; 6 hrs 100 nM with N=102 neurites). (E) Representative image of a neuronal network of the Ctrl condition and 24 hrs after a 10 min 10 μM psilocin trigger showed an increase in axonal TAU+ and dendritic MAP2+BDNF density. BDNF density for the psilocin condition. One cell line (Ctrl 3) was included in the analysis, with two biological batches (Ctrl with N=90 neurites; Psi with N=90 neurites). (G) BDNF density was significantly increased 24 and still 48 hrs (24 hrs; 48 hrs) after a 10 min 10 μM psilocin trigger. Three Ctrl cell lines were included in the analysis (Ctrl with N=520 neurites; 24 hrs with N=478 neurites; 48 hrs with N=170 neurites). (H) Endogenous phosphorylated (activated) TrKB (pTrkB) receptor, pro-BDNF and m-BDNF protein level increased 24 hrs after psilocin (Psi) administration. (I) Representative WB: phosphorylated AKT (pAKT) protein level, indicating an activation of pro-survival AKT pathway, was increased 24 hrs after psilocin (Psi) exposure. For all analyses, the Kruskal–Wallis test for independent samples was calculated. Post hoc Wilcoxon rank sum test. Bonferroni correction, adjusted p<0.05, mean ± SD. Significance levels against the respective control are *P<.05.
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Figure 2—figure supplement 1—source data 1
Western Blot: phosphorylated TrKB (pTrkB), pro-BDNF and m-BDNF protein level after psilocin exposure (Psi) (24 hrs post, uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Western Blot: phosphorylated TrKB (pTrkB), pro-BDNF and m-BDNF protein level after psilocin exposure (Psi) (24 hrs post, uncropped).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Western Blot: phosphorylated AKT (pAKT) protein level and AKT protein level after psilocin exposure (Psi) (24 hrs post, uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-figsupp1-data3-v1.zip
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Figure 2—figure supplement 1—source data 4
Western Blot: phosphorylated AKT (pAKT) protein level and AKT protein level after psilocin exposure (Psi) (24 hrs post, uncropped, labeled).
- https://cdn.elifesciences.org/articles/104006/elife-104006-fig2-figsupp1-data4-v1.zip
Figure 3 with 1 supplement
Psilocin displays fast and enduring changes of the genetic landscape.
(A) Enrichment in differentially expressed genes associated with synapse formation, neuronal plasticity, and axonogenesis GO terms 1 day after psilocin administration, and the effect 3 days later. (B) Psilocin induced within 24 hrs a first wave of upregulation of selected GO genes based on DESeq2 normalized counts. Log2fold changes of each differentially expressed gene between two conditions are shown as dots. Z scores indicate if more genes in the respective GO term are upregulated or downregulated, also indicated by height and color of the bars. (C) TPM-normalized mean (red line) of psilocin-induced temporal expression pattern of genes belonging to the indicated GO. The shaded area indicates SD. (D) Chord plots showing significant genes appearing in at least four GO terms (of six selected GO terms) 1 day after psilocin administration and appearing in at least three GO terms 3 days after psilocin administration. (E). Heat map (z-scaled normalized counts) showing expression of immediate early genes and AMPA/NMDA receptor genes throughout psilocin administration. (F) Differentially expressed genes that are up-/downregulated upon psilocin treatment showed a reversed effect upon co-treatment with ketanserin. Mean TPM-normalized expression values are shown (red line), shaded area indicates S.D. BP, biological process; CC, cellular compartment; KEGG, Kyoto Encyclopedia of Genes and Genomes; TPM, transcripts per kilobase million. The significance was assessed using a Wald test. Significance levels against the respective control ns: p-adj.>0.05, *p-adj. ≤ 0.05, **p-adj. ≤ 0.01, ***p-adj. ≤ 0.001.
Figure 3—figure supplement 1
Psilocin displays fast and enduring changes on GO terms related to learning, memory, and cognition.
(A) Heat map (z-scaled normalized counts) showing expression of genes that are grouped in the selected GO term related to ‘cognition’. Genes that are also present in the GO term related to ‘learning and memory’ are stated as ‘present’, indicating great overlay of the genes of the two GO terms. (B, C) Psilocin induced within 24 hrs a first ‘shaking’ wave of selected GO genes based on DESeq2 normalized counts. After 3 days those GO genes normalized. (B) Log2fold changes of each gene between two conditions are shown as dots. Z scores indicate if more genes in the respective GO term are upregulated or downregulated, indicated by height and color of the bars. (C) TPM-normalized mean (red line) of psilocin-induced temporal expression pattern of genes belonging to the indicated GO. The shaded area indicates SD. TPM, transcripts per kilobase million. The significance was assessed using a Wald test. Significance levels against the respective control ns: p-adj.> 0.05, *p-adj. ≤ 0.05, **p-adj. ≤ 0.01, ***p-adj. ≤ 0.001.
Figure 4
Psilocin-induced neurite branching.
(A–C) Cells were transduced with AAV CamKIIa p-hCHR2(134a)-mCherry. (A) Representative image for pre-treatment condition (Ctrl) and 48 hrs after a 10 min short psilocin trigger (48 hrs) for mCherry staining showed an increase in neurite intersections for the latter. (B) Singular analysis: 48 hrs after a 10 min 10 µM psilocin trigger, the number of primary (25 µm distance from soma) neurite intersections significantly increased compared to the untreated control condition (Ctrl). The number of intersections at 50 µm distance from soma significantly increased 24 hrs and 48 hrs after a 10 min 10 µM psilocin trigger. (C) Significant changes for the calculated total neurite length and for the total number of neurite intersections significantly increased 24 hrs and 48 hrs after a 10 min 10 µM psilocin trigger. Two control cell lines with two biological batches (Ctrl with N=43–48 neurites; 24 hrs with N=47–48 neurites; 48 hrs with N=31–35 neurites) were included. (D) Sholl analyses summary for results shown in figure (B). For all analyses, the Kruskal–Wallis test for independent samples was calculated. Post hoc Wilcoxon rank sum test. Bonferroni correction, adjusted p<0.05, mean ± SD. Significance levels against the respective control are *p<0.05.
Figure 5 with 1 supplement
Psilocin-induced increase in synaptic strength and synaptogenesis.
(A) Total number of evoked action potentials (eAPs) significantly increased at day 7 after 24 hrs permanent psilocin administration (Psi 24 hrs; day 7) and was increased 7 days after a 10 min psilocin trigger (Psi 10 min; day 7). AP amplitudes stayed constant. Representative traces for the total number of APs. One Ctrl cell line with 2 biological batches was included in the analysis. Ctrl 3 with N=28 cells, Psi 10 min; day 7 with N=29 cells, Psi 24 hrs; day 7 with N=34 cells. (B) Increase in sEPSCs amplitude and frequency 7 days after 10 min and 24 hrs permanent psilocin administration. One Ctrl cell line with 2 biological batches was included in the analysis, Ctrl 3 with N=19 cells, Psi 10 min; day 7 with N=20 cells, Psi 24 hrs; day 7 with N=21 cells. (C) Increase in mEPSCs amplitude 6 days after permanent 10 µM psilocin administration, Ctrl 3 with N=16 cells, Psi 24 hrs; day 7 with N=14 cells. Increase in mEPSCs amplitude 10 days after 24 hrs permanent psilocin administration, Ctrl 3 with N=15 cells, Psi 24 hrs; day 11 with N=20 cells. (D) Significant increase in sEPSCs amplitude 6 days after 96 hrs permanent 10 µM psilocin administration (Psi 96 hrs; day 10). Ctrl 3 with N=18 cells, Psi 96 hrs; day 10 with N=13 cells. Representative traces for control and psilocin conditions. For all experiments, the Mann–Whitney U-test for independent samples was calculated, mean ± SEM. Significance levels against the respective control are *p<0.05. (E, F) Representative dendritic PSD-95 and synapsin staining for the untreated condition (Ctrl), 4 days after permanent 10 µM psilocin administration (4 days) and 10 days after 4 days permanent 10 µM psilocin administration (10 days). (E) Scale bar: 50 µm (F), close-up: 10 µm. Trend in synapsin (G) density and (H) intensity increased 4 days after permanent psilocin administration. (I) PSD-95 particle per neurite length and (J) intensity per area and (K) synapsin/PSD-95 co-localization were significantly increased 4 and 10 days after 4 days permanent 10 µM psilocin treatment compared to an untreated control condition. (G–K) Ctrl with N=180 neurites, 4 days with N=180 neurites, 10 days with N=180 neurites. Two control cell lines, each with two biological batches, were included. For all analyses, the Kruskal–Wallis test for independent samples was calculated. Bonferroni correction, adjusted p<0.05, mean ± SD. Significance levels against the respective control are *p<0.05.
Figure 5—figure supplement 1
Psilocin-induced increase in synaptic strength.
(A) Increase in mEPSC amplitude at day 7 after 24 hrs permanent psilocin administration. Ctrl 2 with N=16 cells, Psi 24 hrs; day 7 with N=14 cells. (B) Significant increase in sEPSCs amplitude 6 days after 96 hrs permanent 10 μM psilocin administration (Psi 96 hrs; day 10). Ctrl 2 with N=8 cells, Psi 96 hrs; day 10 with N=9 cells. For all experiments, the Mann–Whitney U-test for independent samples was calculated, mean ± SEM. Significance levels against the respective control are *p<0.05.
Figure 6
Simplified schematic of signal transduction pathways proposed to mediate psychedelic-induced neuroplasticity in the cortex.
Serotonergic psychedelics activate the 5-HT2A-R, triggering G-protein–coupled PLC and PLA2 pathways, which, similar to m-BDNF-TrkB activation, increase intracellular Ca²+ and PKC activity. BDNF–TrkB signaling further activates mTOR and NMDAR-dependent plasticity via the PI3K/AKT pathway. Psychedelic-induced glutamate release promotes mTOR activation and BDNF release through AMPARs, enhancing glutamatergic signaling and IEG (e.g., c-FOS) expression. 5-HT2A-R also interacts with PSD-95, while PI3K, ERK1/2, and CaMK activate CREB, stimulating BDNF transcription. Image created in BioRender, adapted from de Vos et al., 2021 and used in Schmidt et al., 2024. 5-HT, serotonin; AKT, protein kinase B; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BDNF, brain-derived neurotrophic factor; CaMK, Ca²+/calmodulin-dependent kinase; CREB, cAMP response element–binding protein; ERK1/2, extracellular signal–regulated kinase 1/2; IEG, immediate early gene; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NMDAR, N-methyl-D-aspartate receptor; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PSD-95, postsynaptic density protein 95; TrkB, tropomyosin receptor kinase B.
Author response image 1
Immunostaining for 5-HT2A receptor across cell types and peptide-blocking control.
(a) HEK293 cells display a positive immunofluorescent signal despite not endogenously expressing 5-HT2AR, indicating nonspecific antibody reactivity. (b) HeLa cells also exhibit a positive signal despite lacking endogenous 5-HT2AR expression, further demonstrating nonspecific antibody binding in non-expressing cell types. (c) Neural progenitor cells show clear positive 5-HT2AR staining. (d) iPSC-derived neurons exhibit robust and well-defined 5-HT2AR staining. (e) Application of the Alomone 5-HT2AR blocking peptide (#BLP-SR033) markedly reduces neuronal signal intensity, supporting epitope-specific binding.
Author response image 2
Western blot analysis of 5-HT2A receptor abundance and peptide-blocking control.
(a-b) In line with the immunofluorescence a single band is detected in iPSCs, HEK cells, neural progenitors, iPSC-derived neurons and (b) HeLa cells. (a) Preincubation of the primary antibody with the corresponding blocking peptide abolishes this band across all samples, consistent with specific binding of the antibody to its intended epitope.
Author response image 3
Lack of detectable 5-HT2AR expression in HEK and HeLa cells.
(a) Analysis of a human-only HEK293T single-cell RNA-seq dataset (10x Genomics; here, accessed 2025-11-25) shows no meaningful HTR2A expression, whereas other genes such as GAPDH, TP53, MYC, and ACTB are robustly detected. Consistently, evaluation of a “Barnyard” dataset - an equal mixture of human HEK293T and mouse NIH3T3 cells (10x Genomics; here, accessed 2025-1125) reveals only ~4 of ~10,000 droplets with minimal HTR2A signal, confirming the absence of meaningful expression.(b) (b) qPCR analysis further demonstrates no detectable HTR2A transcripts in iPSCs or HeLa cells (Ct > 36), while neural progenitors and iPSC-derived cortical neurons show expression when normalized to housekeeping genes GAPDH and TBP.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/104006/elife-104006-mdarchecklist1-v1.docx
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Supplementary file 1
List of primers and antibodies used.
- https://cdn.elifesciences.org/articles/104006/elife-104006-supp1-v1.docx
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Psilocin fosters neuroplasticity in iPSC-derived human cortical neurons
eLife 14:RP104006.
https://doi.org/10.7554/eLife.104006.3
