This rodent study investigated the biological substrates of the enduring effects of the psychedelic DOI on the frontal cortex. It finds that a single dose produced rapid structural changes in dendritic spines and sustained alterations in chromatin organisation related to synaptic plasticity, potentially explaining long-lasting antidepressant actions.
Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics' enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics' long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.
Depressive, anxiety and stress-related disorders are highly prevalent and current pharmacotherapies, including monoamine reuptake inhibitors, are limited by delayed onset of action and a substantial proportion of treatment resistance. Psychedelic compounds (phenethylamines such as DOI and mescaline, and tryptamines such as psilocybin and DMT) produce marked acute alterations in perception and cognition, and recent clinical pilot studies have reported rapid and sometimes long-lasting improvements in mood and anxiety measures following single administrations. Despite promising clinical signals, the molecular mechanisms that might underlie durable antidepressant-like effects of psychedelics—and whether these rely on the same receptor targets implicated in acute hallucinogenic effects—remain incompletely understood. This study set out to characterise post-acute molecular and synaptic plasticity changes in mouse frontal cortex following a single systemic dose of the phenethylamine psychedelic DOI, and to test the extent to which those changes depend on the serotonin 5-HT 2A receptor (5-HT 2A R). The investigators combined behavioural assays relevant to depression, anxiety and fear learning with dendritic spine imaging, electrophysiological measures of synaptic plasticity, cell-type-specific epigenomic profiling (H3K27ac ChIP-seq) and RNA-seq from neuronal nuclei, and computational analyses including enhancer clustering, motif and gene-network analysis. The goal was to link persistent chromatin and synaptic alterations to lasting behavioural effects observed after the drug has been cleared from brain tissue.
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Adult male mice (10–20 weeks) were used across experiments. Where relevant, the study employed wild-type strains (129S6/SvEv and C57BL/6) and 5-HT 2A R knockout (5-HT 2A R −/−) mice derived from heterozygote crossings; animals were randomly allocated to treatment groups and housed under standard conditions. DOI (2,5-dimethoxy-4-iodoamphetamine hydrochloride, 2 mg/kg, i.p.) or saline vehicle was administered, with behavioural testing typically conducted 24 h after injection; for molecular time courses frontal cortex tissue was collected at 24 h, 48 h and 7 days post-injection. The investigators state that no formal sample-size calculation was performed; experimenters performing imaging were blind to treatment. Behavioural assays included locomotor activity (open-field monitoring), light–dark box preference, novel-object recognition, prepulse inhibition of startle (PPI), forced-swim test (immobility scored in last 4 min of 6 min session), and contextual fear acquisition, generalisation and extinction using two distinct contexts. For fear paradigms DOI was given either 10 min after the expression stage or 24 h before acquisition depending on the cohort. Dendritic spine structure was assessed by injecting AAV8-CaMKIIa-eYFP into frontal cortex pyramidal neurons, waiting ≥3 weeks for expression, then imaging apical dendritic segments in fixed slices and classifying spines automatically into thin, stubby or mushroom using NeuronStudio and a validated rayburst algorithm. Functional synaptic plasticity was measured by whole-cell patch-clamp recordings of L2/3 pyramidal neurons with an L4→L2/3 pairing protocol to induce long-term potentiation (LTP). Recordings were performed 24 h after DOI or saline, with EPSC amplitudes normalised to baseline. For epigenomic and transcriptomic profiling, neuronal nuclei were isolated from frontal cortex, NeuN+ nuclei were sorted by FACS, and H3K27ac ChIP-seq (MOWChIP-seq) and Smart-seq2 RNA-seq were performed using low-input protocols; six animals per condition were reported for genomic assays. Bioinformatic analyses included peak calling and enhancer prediction from H3K27ac, K-means clustering of differential enhancers across time points (FDR < 0.05), motif and gene-ontology enrichment, differential gene-expression analysis with DESeq2 (FDR < 0.05), weighted gene co-expression network analysis (WGCNA), and overlap testing between H3K27ac peaks and human GWAS loci using liftOver to hg19 and Monte Carlo randomisation. Statistical tests reported in the manuscript included two-way ANOVA, repeated-measures ANOVA and Student's t tests as appropriate.
Behavioural findings: DOI (2 mg/kg, i.p.) produced a reduction in immobility time in the forced-swim test 24 h after administration (t18 = 3.80, p < 0.01; 10 mice per group), with a trend for reduced immobility detectable at 7 days in a smaller cohort (t8 = 2.96, p < 0.05 reported for a 5-per-group comparison). DOI did not alter locomotor activity in a novel environment, preference in the light–dark test, novel-object recognition, or prepulse inhibition measured 24 h after injection. In contextual fear paradigms, DOI accelerated context fear extinction in 5-HT 2A R +/+ mice: DOI-treated wild-type animals showed a faster progressive reduction in freezing during extinction compared with vehicle-treated controls (extinction F[1,96] = 5.51, p < 0.05). This facilitation of extinction was absent in 5-HT 2A R −/− mice, supporting receptor dependence. When DOI was administered 24 h prior to acquisition in a separate cohort of 5-HT 2A R +/+ mice, acquisition and expression were not affected but DOI reduced freezing during fear generalisation and extinction, indicating lingering effects on associative learning beyond the acute presence of the drug. Dendritic spine and electrophysiology results: Structural analysis of frontal cortex pyramidal neurons revealed that a single DOI administration increased the density of immature/transitional spines (thin and stubby categories) 24 h post-dose, while mature mushroom spine density was unchanged. Vehicle-injected 5-HT 2A R −/− mice exhibited lower thin and stubby spine density relative to vehicle wild-type controls, and the DOI-induced spine increases were not observed in knockout mice, indicating that spine remodelling required 5-HT 2A R expression. Functionally, DOI enhanced cortical synaptic potentiation: LTP induced by pairing L4 stimulation with brief postsynaptic depolarisation produced greater magnitude potentiation in DOI-treated mice than in vehicle-treated mice (two-way ANOVA F[1,10] = 9.84, p < 0.05; averaged post-induction magnitude t9 = 3.05, p < 0.05). Reported LTP recordings came from n = 5 DOI-treated neurons (from 5 mice) and n = 6 vehicle neurons (from 5 mice). Epigenomic and transcriptomic results: Cell-type-specific H3K27ac profiling in NeuN+ frontal cortex nuclei revealed widespread, time-dependent changes at enhancer regions after DOI. K-means clustering divided differential enhancers into six clusters: clusters 1–3 showed net increases after DOI, clusters 4–6 decreases. Clusters 1 and 2 peaked at 48 h then declined by 7 days; cluster 3 showed a slower increase peaking at 7 days. Notably, 32.7% of differential enhancers (clusters 3 and 6) remained altered at 7 days, indicating persistent epigenomic remodelling that outlasted the drug. Gene-ontology enrichment linked many clusters to synapse organisation, axonogenesis and cell junction assembly; motif analysis highlighted transcription factors implicated in neuronal growth and plasticity (including Mef2B family members), and NFIL3 was a top motif in the cluster with circadian-process enrichment. By contrast, transcriptomic changes were largely transient. Only 3.7% of differentially expressed genes (DEGs, FDR < 0.05) showed sustained variation at 7 days, and 574 genes (4.2% of total DEGs) displayed expression patterns that correlated with enhancer dynamics (Spearman r > 0.4). Weighted gene co-expression network analysis (WGCNA) identified ten modules; the blue module correlated positively with 48 h post-DOI and negatively with control, containing 1,933 genes with GO terms including axonogenesis and learning/memory and hub genes such as Inpp4a, Nfasc and Cntnap2. The yellow module, positively correlated with control, was enriched for synaptic vesicle recycling and HIF-1 pathway genes. GWAS overlap: The authors tested overlap between H3K27ac peaks and human GWAS loci. Of 12 GWAS datasets examined, three psychiatric sets—schizophrenia, depression and ADHD—showed statistically significant overlap (p < 0.05) with both differential and static peak groups. Two additional sets (white-matter structure and Crohn disease) showed significant overlap only with differential peaks, suggesting DOI-induced epigenomic changes intersect human loci previously associated with some psychiatric and non-psychiatric traits.
De La and colleagues interpret their results as providing evidence that a single systemic dose of the phenethylamine psychedelic DOI produces post-acute increases in immature dendritic spines, enhances cortical LTP and accelerates contextual fear extinction in mice, and that these synaptic and behavioural outcomes require expression of the 5-HT 2A R. They further argue that DOI induces persistent reshaping of the frontal cortex epigenomic landscape—particularly at enhancer regions marked by H3K27ac—that endures for at least 7 days and is larger in magnitude and longer-lasting than transcriptomic changes measured in bulk neuronal nuclei. The authors position these data as complementary to earlier findings of rapid structural plasticity following psychedelic exposure, and suggest a mechanistic schema in which transient transcriptional responses to acute 5-HT 2A R activation may engage downstream chromatin and enhancer changes that sustain synaptic remodelling and lasting behavioural effects. Enrichment of enhancer clusters in synapse- and axonogenesis-related GO terms and the identification of related TF motifs (for example, Mef2B and NFIL3) are cited as molecular correlates tying chromatin remodelling to plasticity and potentially to circadian effects. Limitations acknowledged by the investigators include analysis of the entire neuronal population rather than specific neuronal subtypes, which could obscure cell-type-specific signatures, and uncertainty about extrapolation to other brain regions implicated in addiction or psychosis. They also note unresolved questions about whether subjective hallucinogenic experiences are necessary for clinical benefits in humans and whether non-hallucinogenic 5-HT 2A R agonists (or effects of tryptamine-class psychedelics with different signalling profiles) produce similar plasticity and epigenomic outcomes. Finally, the authors highlight the overlap between DOI-linked epigenomic marks and GWAS loci for schizophrenia as a reason for caution when considering psychedelic use in individuals at risk of psychosis. Overall, they conclude that 5-HT 2A R plays a central role in the persistent synaptic and chromatin changes observed after DOI and that these findings may help to understand the long-lasting effects reported in clinical studies, while emphasising the need for further work to delineate cell-type specificity, broader brain-region effects and safety considerations.
This study did not generate new unique reagents Data and code availability d ChIP-seq and RNA-seq datasets are deposited in the Gene Expression Omnibus (GEO) repository with the following accession number: GEO: GSE161626. d This paper does not report original code. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Previous observations in rodent models clearly showed that a single administration of psychedelics leads to long-lasting effects on synaptic plasticity and behavior models of depression. Based on both pharmacological (de la Fuente Revenga et al., 2019) and genetic (Gonza ´lez-Maeso et al., 2007) tools, it is clear that the effects of psychedelics on mouse behavior models of human hallucinogenic potential such as head-twitch behavior are mostly 5-HT 2A R dependent. However, it remains an open question whether this serotonin receptor is responsible for the manifestation of phenotypes associated with their therapeutic-related effects on preclinical models of neuropsychiatric conditions. In the present study, our data provide direct evidence that the post-acute effects of the phenethylamine psychedelic DOI on mouse frontal cortex dendritic spine structure and contextual fear extinction are 5-HT 2A R dependent. Our data also suggest that a single administration of DOI leads to long-lasting alterations in frontal cortex gene expression and chromatin organization that outlast the acute action of this psychedelic and its presence in the organism. One of the still-open questions in the field is whether the subjective effects of psychedelics are necessary or complementary for their post-acute clinically relevant outcomes in patients with severe psychiatric disorders such as depression. Thus, it could be speculated that the fast-acting and long-lasting antidepressant properties induced by psychedelics is a consequence of complex psychological processes, such as waking consciousness, derealization, Report and diminished ego functioning. It has also been reported that psilocybin can induce mystical and spiritually significant experiences to which participants attribute increases in well-being. These subjective components of psychedelic drug action appear to be difficult to control with placebo in the clinical setting due to the overwhelming nature of the drug effect. Accordingly, recent studies highlight the role for positive expectancy in predicting positive outcomes following psychedelic microdosing in healthy volunteers and increases in suggestibility under the effect of these drugs. Obviously, these intricate mental experiences, which rely upon subjective reports, would be difficult, perhaps impossible, to model in rodents. However, the recursiveness of the neocortex structure across the mammalian clade and some evolutionarily preserved behaviors inherently linked to constructs relevant to mental health offer an unparalleled platform for characterizing non-subjective effects of psychedelics. Our data in this study, along with previous observations, suggest that favorable outcomes of psychedelics in processes related to cortical dendritic spine structure and behavior models relevant to depression and stressor-related disorders can be observed in rodents and therefore segregated from uniquely human subjective processes such as attribution of mystical meaning to the psychedelic experience. It is then feasible that quantifiable molecular events and subjective experience concur in the therapeutic benefit of psychedelics in human. Studies like the present utilizing animal models can therefore offer valuable insights on the biological substrate of the equation. We found that a single administration of the psychedelic DOI leads to a post-acute (24 h after) increase on the density of immature and transitional dendritic spines that included thin and stubby, whereas the density of mature mushroom spines was unaffected. This effect of DOI on frontal cortex dendritic spine structure was not observed in 5-HT 2A R À/À mice, but it is important to remark that vehicle-injected 5-HT 2A R À/À mice also showed reduced density of frontal cortex thin and stubby dendritic spine density in the frontal cortex as compared to vehicle-injected 5-HT 2A R +/+ controls. Although further investigation is necessary to unravel the mechanisms underlying this alteration in mice lacking the 5-HT 2A R, a potential explanation may be related to changes among developmental compensatory pathways also involved in the alterations in fear conditioning and extinction observed in 5-HT 2A R À/À mice. Additionally, behavioral alterations upon post-acute DOI administration such as reduction in immobility time in the forced-swim test as well as changes in frontal cortex structural plasticity were observed in naive mice. Although interesting, additional investigation will be necessary using behavior models that fall within the current version of the RDoC matrix and paradigms such as early life stress or chronic social defeat in rodents. Our findings on the epigenomic and transcriptomic dynamics following DOI administration are significant. There have been no integrative studies of temporal changes in frontal cortex epigenome and transcriptome after psychedelic administration previously. Genome-wide epigenetic changes have long been speculated to be the layer of regulation that integrates both ge-netic and environmental factors (Gra ¨ff and Tsai, 2013;. We show that the epigenetic landscape can be exogenously reshaped by a drug that elicits unequivocal psychedelic effects in humans. At the genomic level, genetic variations in non-coding regions may lead to propensity for depression, schizophrenia, and other psychiatric conditions via long-range chromatin regulation such as enhancer-promoter interaction. In this study, we report that, in contrast to the fairly rapid and transient changes in the transcriptome, a large fraction of epigenetic changes in enhancer regions persist for at least 7 days after DOI administration and potentially constitute the molecular basis for the long-lasting effects. It is worth noting that our findings were established by studying the entire neuronal cell population. Further examination of individual cell types (e.g., excitatory and inhibitory neurons) may yield additional information on the roles of specific neuron types. We also studied the points of convergence between genetic loci associated with a number of human disorders and the footprint of DOI in the mouse brain epigenome. The peaks of H3K27ac showed significant overlap with GWAS-discovered variants associated with schizophrenia, depression, and ADHD. Although psychedelics have been shown to have fast-acting and long-lasting therapeutic effect on depression, their effects on schizophrenia and ADHD remain unknown even when taking into account some overlap between these three psychiatric disorders. The acute effects of psychedelics resemble some of the positive symptoms (i.e., hallucinations and delusions) in patients with schizophrenia, and chronic LSD administration alters rat frontal cortex expression of a group of genes relevant to schizophrenia. Markers linked to white matter structural changes, which have been associated with depression, were also significantly altered upon DOI administration. Other than outcomes in the treatment of depression, synaptic plasticity events in diverse parts of the brain have been linked to different adaptive and maladaptive traits. Previous studies have clearly demonstrated that psychoactive drugs of abuse such as cocaine increase dendritic spine density within key components of the brain's reward circuitry, such as the nucleus accumbens. It has also been reported that the density of immature thin dendritic spines is increased in the frontal cortex of a genetic rat model that exhibits schizophrenia-relevant features (Sa ´nchez-Gonza ´lez et al., 2021). Brain regions enriched in 5-HT 2A R include the frontal cortex, ventral striatum, several thalamic nuclei, and the hypothalamus. Considering our previous findings suggesting that the 5-HT 2A R in frontal cortex pyramidal neurons is necessary and sufficient for the effects of psychedelics on psychosis-related behavior (Gonza ´lez-Maeso et al., 2007), here we focused our efforts on the post-acute effects of the psychedelic DOI in this particular brain region. Although our data provide evidence of long-lasting effects of a single administration of psychedelics on epigenomic landscape within neuronal nuclei in the frontal cortex, additional efforts will be required to establish whether these changes in synaptic plasticity and chromatin rearrangement can be extrapolated to other brain regions relevant to substance abuse, and whether other cortical and subcortical dendritic structural and epigenetic plasticity events contribute to either clinically beneficial endpoints or, alternatively, unwanted side effects such as drug addiction, psychotic symptoms, or hallucinogen persisting perception disorder. Our previous work focused on the fundamental paradox that all known psychedelics are 5-HT 2A R agonists, but 5-HT 2A R agonism is not a sufficient condition for psychedelic action-as exemplified by structurally related drugs such as lisuride or ergotamine that do not exhibit psychedelic activity. We reported that a single dose of psychedelics, such DOI, LSD, or psilocin, induces frontal cortex 5-HT 2A R-dependent expression of genes associated with cell morphogenesis, neuron projection, and synapse structure, such as Egr1, Egr2, IkBa, and N10. Other genes, such as c-Fos, were induced by either psychedelic or non-psychedelic 5-HT 2A R agonists. Our data here support a scheme whereby a single dose of the phenethylamine DOI leads to changes in frontal cortex synaptic plasticity and behavior models of fear extinction. Considering the associative learning processes involved in contextual fear conditioning and extinction, and the effect of DOI on the faster development of the later, it is plausible that activation of 5-HT 2A R by this psychedelic accelerates context fear extinction likely in part through alterations in chromatin state at enhancer regions of genes predominantly involved in synapse organization and assembly such as Mef2B, NeuroG2, and Atoh1-a gene network that may be located downstream of the genes showing transient transcription upon acute psychedelic administration. Recent observations have also reported that a putative non-hallucinogenic psychedelic analog referred to as tabernanthalog promotes neural plasticity and reduces rodent behavior models relevant to depression. Further work will be needed to unravel the cellular signaling and neural target mechanism that may link psychedelic-induced synaptic plasticity and behavior, as well as the role, if any, of 5-HT 2A R in these plasticity-related events induced by non-hallucinogenic 5-HT 2A R agonist variants. Similarly, it will be interesting to expand this analysis in future studies to test the extent to which signaling mechanisms unrelated to 5-HT 2A R are responsible for the effects of tryptamine psychedelics such as psilocybin on brain plasticity and long-lasting antidepressantrelated behavior. In conclusion, our study highlights the fundamental role of 5-HT 2A R in the action of psychedelics and unveils persisting chromatin remodeling events following DOI administration linked to lasting synaptic plasticity and behavioral events. If generalizable to other psychedelics currently in clinical studies, these findings could also facilitate the understanding of psychopharmacological interventions whose mechanisms of action are not fully understood. Last, the overlap of the epigenomic markers of the action of the psychedelic DOI with loci associated with schizophrenia reiterate the need for caution in the use of psychedelics in individuals at risk for psychosis.
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