Abstract
Nonhallucinogenic psychoplastogens, such as tabernanthalog (TBG), are being developed as potentially safer, more scalable alternatives to psychedelics for promoting neuronal growth and treating various brain conditions. Currently, it is unclear whether 5-hydroxytryptamine 2A (5-HT2A) receptors and immediate early gene (IEG) activation have a role in the neuroplasticity-promoting effects of nonhallucinogenic psychoplastogens. Here, we use pharmacological and genetic tools in rodents to show that nonhallucinogenic psychoplastogens promote cortical neuroplasticity through the same biochemical pathway—involving 5-HT2A, TrkB, mTOR and AMPA receptor activation—as classic psychedelics and that TBG-induced cortical spinogenesis is required for the sustained antidepressant-like behavioral effect of TBG. In contrast to psychedelics, TBG does not induce an immediate glutamate burst or IEG activation. As these effects have been assumed to be necessary for psychedelic-induced neuroplasticity, our results shed light on the mechanisms by which certain psychoplastogens can promote cortical neuroplasticity in the absence of hallucinogenic effects.
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Data availability
Data are available in figshare at the following link: https://doi.org/10.6084/m9.figshare.26999302 (ref. 98). Data for snRNA-seq experiments are available in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), with accession no. GSE277053.
Code availability
Custom scripts from UNRAVEL (https://github.com/b-heifets/UNRAVEL) are available in Zenodo at https://zenodo.org/records/13377341 (ref. 99). Custom code for snRNA-seq analysis can be found in GitHub at https://github.com/NordNeurogenomicsLab. Custom MATLAB scripts for fiber photometry and two-photon imaging are available in Figshare at https://doi.org/10.6084/m9.figshare.26999302 (ref. 98).
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Acknowledgements
This work was supported by funds from the National Institutes of Health (NIH) (R01GM128997, R35GM148182 and R01DA056365 to D.E.O.; DP2MH136588 to C.K.K.; R35GM119831 to A.S.N.; R44MH119989 to D.G.W.; R35GM133421 to J.D.M.; R01MH130591 and P50DA042012 to B.D.H.), three NIH training grants (T32GM113770 to C. Ly, T32GM099608 to E.V.B. and T32MH112507 to L.P.C.), the Camille and Henry Dreyfus Foundation (D.E.O.), a Hellman Fellowship (D.E.O.), the Boone Family Foundation (D.E.O. and J.A.G.), the WoodNext Foundation (D.E.O.), a sponsored research agreement with Delix Therapeutics (D.E.O.), a Canadian Institutes of Health Research postdoctoral training award (202210MFE-491520-297096 to J.M.), a UC Davis Provost’s Undergraduate Fellowship (S.D.P.) and a UC Davis Human Genetics Focus Group grant (I.K.A., E.M.F. and H.H.). Delix Therapeutics funded the studies conducted at the various contract research organizations. This project used the Biological Analysis Core of the UC Davis MIND Institute Intellectual and Developmental Disabilities Research Center (U54 HD079125). The Olympus FV1000 confocal microscope used in this study was purchased using an NIH Shared Instrumentation Grant (1S10RR019266-01). We thank the MCB Light Microscopy Imaging Facility (a UC Davis Campus Core Research Facility) for the use of this microscope. Several of the drugs used in this study were provided by the National Institute on Drug Abuse Drug Supply Program. We thank H.T. Warren, L.E. Dunlap and A. Vernier for synthesizing TBG, 5-MeO and 6-F-DET. We also thank Y. Gallegos and E. Blaes for tissue clearing and light-sheet imaging support and J. Gonzalez-Maeso for providing 5-HT2AR KO animals.
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Contributions
L.P.C. performed the in vitro dendritogenesis and spinogenesis assays with assistance from J.V.; L.P.C. and S.D.P. performed Golgi staining; E.V.B. performed the electrophysiology studies; S.B.J. performed the spine ablation study; I.K.A. performed the tail suspension test in 5-HT2AR KO animals with assistance from L.P.C.; H.D.H. and C.A.M. performed the pig behavior and receptor occupancy studies under the supervision of G.M.K.; L.P.C. performed the head-twitch response studies; I.K.A. performed the fiber photometry and two-photon imaging studies with assistance from R.S. and J.M.; I.K.A. dosed the animals and collected tissue for the tissue clearing experiments with assistance from C.K.K.; C.R., D.G.W., N.G. and S.P.G. contributed to the tissue clearing and light-sheet microscopy data; A.B.C. and D.R.R. analyzed the light-sheet microscopy data; E.M.F. performed the snRNA-seq experiments and the data analysis with assistance from I.K.A., H.H., S.A.L. and N.S.; C. Ly performed early pilot studies demonstrating that nonhallucinogenic analogs of psychedelics can promote plasticity through the activation of 5-HT2A receptors; M.C. and N.A.P. collected and analyzed the data from the 5-HT2 radioligand binding, aequorin, IP1, TrkB, MAO-A, NET/SERT radioligand binding, microdialysis, pharmacokinetic, unstressed FST, chronic IFNα, CSD and SmartCube assays performed by contract research organizations; J.J.H. performed BRET experiments under the supervision of J.D.M.; D.R., C.H. and E.O. performed the c-Fos immunohistochemistry in slices, and R.M. assisted with data analysis; I.K.A. performed pretreatment c-Fos immunohistochemistry studies with assistance from R.S.; G.Q., K.R., C. Liston, J.A.G., B.D.H., C.K.K., A.S.N. and D.E.O. supervised various aspects of this project and assisted with data analysis; D.E.O. conceived the project and wrote the manuscript with input from all authors.
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Competing interests
D.E.O. is a cofounder of Delix Therapeutics, Inc., serves as the chief innovation officer and head of the scientific advisory board, and has had sponsored research agreements with Delix Therapeutics. Delix Therapeutics has licensed TBG-related technology from the University of California, Davis. C. Liston is a member of the scientific advisory board of Delix Therapeutics. The sponsors of this research were not involved in the conceptualization, design, decision to publish or preparation of the manuscript. However, several employees of Delix Therapeutics are coauthors of this article as they collected and analyzed data and approved the final version of the manuscript. Within the last 3 years, G.M.K. has been an advisor for Sanos, Onsero, Pangea Botanica, Gilgamesh and Seaport. At the time of manuscript submission, H.D.H. was employed by H. Lundbeck A/S. B.D.H. is on the scientific advisory board of Osmind and Journey Clinical and is a paid advisor to Arcadia Medicine, Inc. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Pharmacology of TBG at 5-HT2 receptors.
(a–c) Radioligand binding (RLB), aequorin, and IP1 assays conducted using human 5-HT2A (a), 5-HT2B (b), and 5-HT2C (c) receptors, unless noted otherwise. The specific assay and agonist/antagonist mode is indicated in the title of each graph. For 5-HT2A and 5-HT2C RLB assays, [3H]-DOI was used while [125I]-DOI was used for 5-HT2B RLB assays. (d) BRET-based assays of Gq dissociation and β-arrestin2 recruitment demonstrate that TBG is a partial agonist of 5-HT2ARs. Data represent mean ± s.e.m. from two or more replicates. For additional details on statistics, see Table S4.
Extended Data Fig. 2 Photoablation of a random set of dendritic spines in the mPFC of mice does not produce antidepressant-like effects.
((a) Photoablation of VEH-induced dendritic spines in the PFC does not produce antidepressant-like effects in the TST. VEH = vehicle. Data are presented as mean ± s.e.m. ns = not significant, calculated using an unpaired two-tailed Student’s t-test. Without photoactivation, N = 9; with photoactivation, N = 10. For additional details on statistics, see Table S4. (b) Placement of AS-PaRac1 virus (green) and fiber optic implant into the mPFC for photoablation experiments. Left: low magnification (10x), scale bar = 500 μm. Right: high magnification (63x), scale bar = 50 μm.
Extended Data Fig. 3 Bona fide nonhallucinogenic psychoplastogens promote cortical plasticity in vitro through activation of 5-HT2A receptors.
(a) Maximum numbers of crossings (Nmax) of Sholl plots obtained from rat embryonic cortical neurons (DIV6) treated with compounds (1 μM). Data for hallucinogens and their nonhallucinogenic congeners are shown in red and blue, respectively. Box plots depict the median, interquartile range, and minimum/maximum. Compound-induced dendritogenesis is blocked by pre-treatment with ketanserin (KETSN) (10 μM). (b) Representative images of rat embryonic cortical neurons treated with psychoplastogens in the presence and absence of KETSN. (c) Maximum numbers of crossings (Nmax) of Sholl plots obtained from wild type (WT) and 5-HT2AR KO mouse embryonic cortical neurons (DIV6) treated with compounds (10 μM). (d) Maximum numbers of crossings (Nmax) of Sholl plots obtained from rat embryonic hippocampal neurons (DIV6) treated with compounds (10 μM). BDNF (50 ng/mL) was used as a positive control. (e–g) Maximum numbers of crossings (Nmax) of Sholl plots obtained from rat embryonic cortical neurons (DIV6) treated with hallucinogenic and nonhallucinogenic serotonergic psychoplastogens (1 μM) for 72 h in the presence or absence of various blocking agents (10 μM). (e) Inhibition of mTOR with rapamycin, (f) TrkB with ANA-12, or (g) AMPA receptors with DNQX prevents psychoplastogen-induced neuronal growth. Numbers within bar graphs indicate the number of neurons analyzed per treatment. (h) Spinogenesis induced by LSD and LIS in cortical cultures is blocked by co-treatment with KETSN. (i) Representative images of secondary branches of rat embryonic cortical neurons (DIV21) after compound treatment (1 μM) with or without KETSN (10 μM) pre-treatment. VEH = vehicle; LSD = lysergic acid diethylamide; LIS = lisuride; DMT = N,N-dimethyltryptamine; 6-F-DET = 6-fluoro-N,N-diethyltryptamine; 5-MeO = 5-methoxy-N,N-dimethyltryptamine; TBG = tabernanthalog, KETSN = ketanserin. Boxplots represent median, first quartile, third quartile, and min/max values. Bar graphs represent mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to VEH controls or VEH – blocker controls, calculated using a one-way ANOVA with Dunnett’s post hoc test (a, c, d, e, f, g, h). For additional details on statistics, see Table S4.
Extended Data Fig. 4 TBG exhibits limited efficacy at several targets implicated in the effects of antidepressants.
(a) Functional assays of TrkB activation demonstrate that the positive control (BDNF) is a full agonist, while TBG has no effect in either agonist or positive allosteric modulator (PAM) mode. (b) TBG is a weaker inhibitor of MAO-A than clorgyline by 3 orders of magnitude. (c) TBG binds to the NET with an affinity that is 4 orders of magnitude lower than desipramine. (d) TBG binds to the SERT with an affinity that is 2 orders of magnitude lower than fluoxetine. Data represent mean ± s.e.m. from two or more replicates. For additional details on statistics, see Table S4.
Extended Data Fig. 5 TBG produces minimal changes in neurotransmitter and neuromodulator levels in the PFC of rats.
Microdialysis studies from the PFC of rats were conducted following intraperitoneal administration of TBG. Concentrations of dopamine (DA), norepinephrine (NE), serotonin (5-HT), glutamate (Glu), and GABA in dialysate samples were determined by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) detection using stable labelled isotopes as internal standards. Data represent mean ± s.e.m. from 8–9 animals per treatment group (3 mg/kg = 9 male rats, 10 mg/kg = 9 male rats, 30 mg/kg = 8 male rats). For additional details on statistics, see Table S4. In the rat FST, both 3 and 10 mg/kg doses produced antidepressant-like effects (see ED Fig. 6c). Thus, the 30 mg/kg dose should be considered supratherapeutic.
Extended Data Fig. 6 TBG produces rapid and sustained antidepressant effects consistent with those of psychedelics.
(a) Pharmacokinetic studies demonstrate that TBG reaches μM concentrations in the rat brain. (b) Experimental design for an unstressed FST. (c–d) Quantification of FST behaviors in rats 24 h (c) and 7 d (d) after dosing. (e) Three weeks of chronic, intermittent interferon alpha (IFN-α) administration induces a depressive phenotype in the rat FST. A single dose of KET and TBG, but not FLX, was able to rescue the deficit 24 h after treatment. (f) Ten days of chronic social defeat (CSD) stress leads to social avoidance in mice. A single dose of KET, PSY, and TBG was able to rescue social preference deficits measured 24 h after treatment. (g) In the SmartCube® assay in mice, TBG produces a mixed behavioral signature. (h) Clustering analysis using de-correlated feature extracts revealed that the behavioral signature of TBG was more consistent with that of psilocybin/psilocin than selective serotonin reuptake inhibitors (SSRIs). VEH = vehicle; KET = ketamine; TBG = tabernanthalog, FLX = fluoxetine; PSY = psilocybin. Data are reported as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to VEH or VEH without IFN-α or stress controls, calculated using a one-way ANOVA with Dunnett’s post hoc test (c, d, e) or a two-way ANOVA with Dunnett’s post hoc test (f). For additional details on statistics, see Table S4.
Extended Data Fig. 7 Expression of c-Fos in mouse brain slices.
(a) Immunofluorescence images of c-Fos protein expression (3 fields of view per treatment) from various brain regions demonstrates that PTZ, 5-MeO, and TBG induce distinct c-Fos expression patterns. Female mice were administered VEH or compound (50 mg/kg) via i.p. injection 90 min prior to tissue collection. (b) Low magnification coronal slices demonstrating that PTZ and 5-MeO increase c-Fos expression in the hippocampus and SSC, respectively, while TBG produces minimal effects relative to the VEH control. (c) Quantification of c-Fos expression in select brain regions. (d) A 10 min pretreatment with TBG (50 mg/kg) does not block the effects of 5-MeO (10 mg/kg) on c-Fos protein expression in the SSC. (e) Neither BOL nor AAZ increase c-Fos expression in the mPFC (left) or SSC (right). TBG = tabernanthalog; 5-MeO = 5-methoxy-N,N-dimethyltryptamine; VEH = vehicle; PTZ = pentylenetetrazole; BOL = BOL-148 (2-Br-LSD); AAZ = AAZ-A-154. Abbreviations for brain regions are shown in Table S1. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, calculated using a one-way ANOVA with Tukey’s (c, d) or Dunnett’s (e) post hoc tests. For additional details on statistics, see Table S4.
Extended Data Fig. 8 Effects of 5-MeO on c-Fos and Npas4 immunofluorescence shown in mouse coronal slices.
Valid clusters from each contrast in Fig. 5 are shown in coronal brain slices overlayed with a wire frame version of the atlas with ABA coloring. Each cluster was randomly colored and labeled with its ID. Clusters shown in Fig. 5c are indicated by lines connecting the ID to the cluster. Major columns correspond to the immunolabel and minor columns represent effect directions. FDR q value thresholds were 0.05 (Npas4) and 0.005 (c-Fos). Voxel coordinates are indicated and can be related to the center of gravity in Table S2. VEH = vehicle; 5-MeO = 5-methoxy-N,N-dimethyltryptamine.
Extended Data Fig. 9 Various snRNA-seq metrics show high quality data with strong consistency across pools and samples from the PFC of mice.
(a) UMAP with cells colored by pool and treatment shows consistent spatial distribution in transcriptomic space and within each cluster. (b) Expression of housekeeping gene Actb shows concordance across samples depicted by Seurat’s normalized data of the single cell transform (SCT) assay. (c) Each pool is represented in each cluster with relatively equal proportions in accordance with the total cell number from that sample. There is some variability of the total cell count from each sample but there is no obvious relation to treatment. Cell type labels are positioned in the blue Pool2_TBG area for visibility. (d) Quality control metrics of filtered data are distributed in line with expected ranges and show consistency across batch and sample.
Extended Data Fig. 10 Marker gene expression and public atlas integration informs snRNA-seq cell typing from the PFC of mice.
(a) Canonical marker genes establish cellular identity and distinguish between layer specific neuronal subtypes. (b) L4/5_IT_Glut_2 and L4/5_IT_Glut_4 cells are distinguished by their expression of certain genes and those with the greatest fold change in both directions are shown. (c) Brain Initiative Cell Census Network (BICCN) snRNA-seq data of cortical regions is shown in a UMAP with subtype labels shown. (d) The nuclei from our data (red) projected onto the dimensionality reduction of the BICCN data (black) map to cortical subsets of the reference which enables label transfer of cell type annotations. Our final cell type annotations are a combination of label transfer approach with manual curation based on published information on marker genes. (e) Feature plots show cluster specific patterning of select marker gene expression. The scales denote the normalized expression level. (f) Feature plots show overlapping distribution of the Camk2a driver with Htr2a-expressing cells. (g) Quantification of the co-expression of Camk2a in Htr2a-expressing cells indicates a high percentage of Htr2a-expressing neuronal cells also express Camk2a.
Supplementary information
Supplementary Table 1
c-Fos expression values from mouse brain slices. Raw immunofluorescence values and fold change (FC) for individual replicates of slice immunohistochemistry experiments evaluating c-Fos expression are shown. Full names for brain region abbreviations used in Extended Data Fig. 7 are shown in column B. Average FC relative to vehicle control for each treatment and brain region is shown in orange.
Supplementary Table 2
Data for clusters from brain-wide mapping of c-Fos and NPAS4 expression in mice after treatment with 5-MeO or TBG (related to Fig. 5). The ‘Legend’ tab defines brain regions according to Allen Brain Atlas conventions. Numbers (‘1’, ‘2’, ‘2/3’, ‘3’, ‘4’, ‘5’, ‘6’, ‘6a’, ‘6b’) = cortical layers (except ‘ANcr1’, ‘ANcr2’, ‘CA1’, ‘CA3’, ‘CUL4, 5’). Abbreviations not included: {‘4, 5’: ‘lobules IV–V’, ‘a’: ‘anterior’, ‘agl’: ‘agranular’, ‘al’: ‘anterolateral’, ‘am’: ‘anteromedial’, ‘c’: ‘capsular or central nucleus’, ‘cr1’: ‘crus 1’, ‘cr2’: ‘crus 2’, ‘d’: ‘dorsal nucleus or dorsal’, ‘e’: ‘external nucleus or external’, ‘i’: ‘internal’, ‘ig’: ‘motor related (intermediate gray layer)’, ‘iw’: ‘motor related (intermediate white layer)’, ‘l’: ‘lateral’, ‘m’: ‘medial’, ‘me’: ‘median’, ‘mo’: ‘molecular layer’, ‘p’: ‘posterior or primary or parvicellular’, ‘p-bfd’: ‘primary (barrel field)’, ‘p-ll’: ‘primary (lower limb)’, ‘p-m’: ‘primary (mouth)’, ‘p-n’: ‘primary (nose)’, ‘p-tr’: ‘primary (trunk)’, ‘p-ul’: ‘primary (upper limb)’, ‘pl’: ‘posterolateral’, ‘pm’: ‘posteromedial’, ‘po’: ‘polymorph layer or preoptic’, ‘r’: ‘rostral’, ‘s’: ‘supplemental or secondary’, ‘sg’: ‘granule cell layer’, ‘v’: ‘ventral’, ‘vl’: ‘ventrolateral’}. For comparison, ‘info’ tabs summarize valid cluster IDs and associated post hoc significance (Holm–Šídák tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001), volume (mm3), center of gravity (CoG) coordinates in atlas space, approximate region, the top four regions and the percentage of volume that they occupy. To determine majority brain regions, if the top four regions accounted for >80% of cluster volume, they were reported as is. Otherwise, subregions at the finest layer of granularity were collapsed into parent regions until >80% volume was accounted for. The ‘Volume’ column was conditionally formatted such that the fill indicates relative cluster volume (white = largest) using log-transformed volumes normalized to the maximum. ‘Raw’ data tabs show cell densities (cell count/cluster volume). Drug treatment, sex and hemisphere are also listed. ‘Stats’ tabs summarize post hoc ANOVA results for each cluster. df, degrees of freedom; sumsq, sum of squares; meansq, mean sum of squares. p.value_(f) shows f-statistical significance. Groups, n’s, P values and adjusted post hoc test P values are shown in addition to group means. A relaxed FDR q value was used for TBG (q < 0.1) to observe valid clusters where TBG altered c-Fos expression. TBG did not affect NPAS4 expression even at relaxed q values.
Supplementary Table 3
Differentially expressed IEGs in mouse PFC following snRNA-seq. Each table is a comparison of the specified drug versus vehicle. Ct1 indicates the cell type annotation. Wilcoxon testing was conducted within each cell type. The tag of _all_cells indicates no subsets beyond cell type were made, whereas _Htr2a_cells indicates that the testing was done only within cells with any detectable expression of Htr2a. The columns with p_val_adj_neuroest_targets_and_randoms indicate multiple testing corrections with the Benjamini–Hochberg method done across all cell types for the 22 selected IEGs and the matched background genes. Pct.1 and Pct.2 are the percentages of cells expressing the gene within the treatment and vehicle groups, respectively.
Supplementary Table 4
Statistical parameters. Various statistical parameters are shown corresponding to the data presented in the figures.
Supplementary Video 1
5-MeO, but not TBG, robustly modulates IEG expression across the mouse brain (related to Fig. 5). Rotating 3D images depict valid clusters representing differences in IEG expression and labeled cell density across the brain in response to compound treatment. Major labels indicate the treatment with the immunolabeled IEG in parentheses (q < 0.005 for c-Fos and q < 0.05 for NPAS4). The direction of change relative to the vehicle control is also indicated. Valid cluster maps were mirrored to show a bilateral representation.
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Aarrestad, I.K., Cameron, L.P., Fenton, E.M. et al. The psychoplastogen tabernanthalog induces neuroplasticity without proximate immediate early gene activation. Nat Neurosci 28, 1919–1931 (2025). https://doi.org/10.1038/s41593-025-02021-1
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DOI: https://doi.org/10.1038/s41593-025-02021-1
