Validation of mature cortical neuron properties and effect of psilocin on cell surface-located 5-HT2A receptor presentation.

(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. (B) Cortical neurons expressed the cell surface-located 5-HT2A receptor (ex5-HT2A-R) validated by immunofluorescence. (B – C) Representative ex5-HT2A-R staining showed decrease in TAU+ axonal receptor presentation 10 minutes after 10 μM psilocin trigger (10 min Psi) compared to the untreated condition (Ctrl), that may indicate receptor complex formation or internalization. (B) Scale bar: 50 μm (C), close up scale bar: 10 μm. (D) Representative ex5-HT2A-R staining. After 24 hrs, both the axonal and dendritic receptor density was significantly decreased, speaking for a subacute down-regulation of the receptor, scale bar: 50 μm (E) close up scale bar: 5 μm. (F) Decrease in TALT axonal receptor localization 10 minutes after 10 μM psilocin trigger compared to the control condition was significant. After 24 hrs, both the axonal and dendritic receptor density was significantly decreased, speaking for a down-regulation of the receptor. For Ctrl N = 180 neurites, for 10 min N = 180 neurites, for 24 hrs N = 135 – 140 neurites were calculated. Two control cell lines each with two biological batches for MAP2+ and TAU+ ex5-HT2A-R were included (except of 24hrs for Ctrl 3 with one biological batch). Kruskal-Wallis-Test for independent samples was calculated. Significance levels against the respective control and for multiple group comparisons. Bonferroni-correction, adjusted p <.05, mean ± SD. (G) Graphic illustration of data in (F), generated with BioRender.com.

Psilocin-induced increase in BDNF level was 5-HT2A receptor 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 A/ = 613 neuntes; Psi with N = 529 neuntes). (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-mediated process. Ketanserin monotreatment provoked a significant reduction in BDNF density compared to ketanserin co-treatment with psilocin. Three Ctrl cell lines were included in the analysis, each with one biological batch (Ctrl with N = 144 neurites; Psi with N = 147 neurites; Psi + Ket with N = 147 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 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 <.05.

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 hours a first wave of upregulation of selected GO genes based on DESeq2 normalized counts. Log2fold changes of each differentially expressed genes between two conditions are shown as dots. Zscores 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. Shaded area is indicating SD. (D) Chord plots showing significant genes appearing in at least 4 GO terms (of 6 selected GO terms) 1 day after psilocin administration and appearing in at least 3 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; CO, Cellular Compartment; KEGG, Kyoto Encyclopedia of Genes and Genomes. TPM, transcripts per kilobase million. Significance levels against the respective control ns: p-adj. >.05, *: p-adj.<=.05, **: p-adj.<=.01, ***: p-adj.<=0.001.

Psilocin induced neurite branching.

(A-C) Cells were transduced with AAV CamKIla 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 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 are *p <.05.

Psilocin-induced increase in synaptic strength and synaptogenesis.

(A) Total number of evoked action potentials (eAPs) significantly increased at day 7 after 24hrs permanent psilocin administration (Psi 24hrs; day 7) and was increased 7 days after a 10 minutes psilocin trigger (Psi 10min; day 7). AP amplitudes stayed constant. Representative traces for total number of APs. One Ctrl cell line with 2 biological batches was included in the analysis. Ctrl 3 with N = 28 cells, Psi 10min; day 7 with N = 29 cells, Psi 24hrs; day 7 with N = 34 cells. (B) Increase in sEPSCs amplitude and frequency 7 days after 10 minutes and 24hrs permanent psilocin administration. One Ctrl cell line with 2 biological batches was included in the analysis. Ctrl 3 with N = 19 cells, Psi 10min; day 7 with N = 20 cells, Psi 24hrs; 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 24hrs; day 7 with N = 14 cells. Increase in mEPSCs amplitude 10 days after 24hrs permanent psilocin administration, Ctrl 3 with N = 15 cells, Psi 24hrs; day 11 with N = 20 cells. (D) Significant increase in sEPSCs amplitude 6 days after 96hrs permanent 10 μM psilocin administration (Psi 96hrs; day 10). Ctrl 3 with N = 18 cells, Psi 96hrs; day 10 with N = 13 cells. Representative traces for control and psilocin condition. For all experiments Mann-Whitney-U-test for independent samples was calculated, Mean ± SEM. Significance levels against the respective control are *p <.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), closeup: 10 μm. Trend in synapsin (G) density and (H) intensity increase 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 Kruskal-Wallis-Test for independent samples was calculated. Bonferroni-correction, adjusted p <.05, mean ± SD. Significance levels against the respective control are *p <.05.

Primary antibodies used for immunocytochemistry (ICC).

Secondary antibodies used for ICC.

Fluorescent probes.

Primary antibodies used for Western blotting (WB).

IR-dye conjugated secondary antibodies used for WB. Secondary antibodies were diluted 1:15,000.

Primers for RT)-PCR.

Validation of iPSC and neural progenitor properties and ex5-HT2A-R 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 generated with BioRender.com. (B) iPSCs expressed pluripotent marker S0X2 and 0CT4. Neuronal progenitors expressed S0X2, PAX6 and NESTIN and were negative for F0XA2 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. (E-F) The decrease in ex5HT2A-R expression was prevented by inhibiting CME with dynasore co-treatment and monotreatment (“Psi + D” and “D”) showing in the close-up (E) axonal ex5-HT2A-R accumulation in aggregates, scale bar: 50 μm, close up-scale bar: 5 μm.

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 (A/= 75 neurites). (C) Representative image of a neuronal network 24 hrs after an “artificial” (10 min 10 μM) and a “physiological” (6 hrs 100nM) 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 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 TALT and dendritic MAP2+ BDNF density. Scale bar: 50 μm. (F) BDNF density was significantly increased for axonal TAU+ and dendritic MAP2+ 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 Kruskal-Wallis-Test for independent samples was calculated. Post hoc Wilcoxon rank sum test. Bonferron¡-correction, adjusted p < .05, mean ± SD. Significance levels against the respective control are *p <.05.

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 hours 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. Zscores 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. Shaded area is indicating SD. TPM, transcripts per kilobase million. Significance levels against the respective control ns: p-adj. >.05, *: p-adj.<= .05, **: p-adj.<=.0”\, ***: p-ad;.<=.001.

Psilocin-induced increase in synaptic strength.

(A) Increase in mEPSCs amplitude at day 7 after 24hrs permanent psilocin administration. Ctrl 2 with N = 16 cells, Psi 24hrs; day 7 with N = 14 cells. (B) Significant increase in sEPSCs amplitude 6 days after 96 hrs permanent 10 μM psilocin administration (Psi 96hrs; day 10). Ctrl 2 with N = 8 cells, Psi 96hrs; day 10 with N = 9 cells. For all experiments Mann-Whitney-U-test for independent samples was calculated, Mean ± SEM. Significance levels against the respective control are *p <.05.