This placebo-controlled rodent study (n=10) investigated the epigenetic factors driving neuroplasticity and contextual fear extinction in response to the serotonergic hallucinogen DOI (2mg/kg). Using a transgenetic knockout mouse strain that lacked 5-HT2A receptors, this study demonstrates that this pathway is essential for inducing long-lasting alterations in frontal cortex gene expression and chromatin organization that outlast the acute antidepressant action of this psychedelic and its presence in the organism.
Abstract
“Introduction: Clinical evidence suggests a potential therapeutic effect of classic psychedelics for the treatment of depression. The most outstanding and distinct characteristic is the rapid and sustained antidepressant action with one single exposure to the drug. However, the biological substrates and key mediators of psychedelics’ enduring action remain unknown.
Methods: Here, we aimed to elucidate the molecular mechanism responsible for the post-acute effects of the psychedelic phenethylamine DOI using mouse behavior models relevant to depression, anxiety and stressor-related disorders, as well as its implication in crucial plasticity phenotypes including frontal cortex dendritic spine structure and gene expression.
Results: Here, we show that a single administration of the psychedelic DOI produced 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 led to changes in chromatin organization particularly at enhancer regions of genes involved in synaptic assembly that stretched for days after the psychedelic exposure. DOI-induced alterations in neuronal epigenome overlapped with genetic loci associated with schizophrenia, depression and attention deficit hyperactivity disorder.
Discussion: Together, these data support the notion that epigenetic-driven changes in synaptic plasticity operate as the mechanistic substrate of psychedelic’s long-lasting antidepressant action but also warn on the limitations in individuals with underlying risk for psychosis.”
Authors: Mario de la Fuente Revenga, Bohan Zhu, Christopher A. Guevara, Lynette B. Naler, Justin M. Saunders, Zirui Zhou, Rudy Toneatti, Salvador Sierra, Jennifer T. Wolstenholme, Patrick M. Beardsley, George W. Huntley, Chang Lu & Javier González-Maeso
Summary
INTRODUCTION
Psychiatric conditions such as depression, anxiety, and stressor-related disorders affect millions of individuals worldwide. Current treatments take several weeks to months to show clinically relevant improvements, and are often accompanied by undesirable side effects.
Psychedelics, such as mescaline and DOI, are psychoactive compounds that profoundly affect various mental domains, particularly sensory perception and thought processes. However, recent pilot clinical trials suggest that psychedelics may represent a promising long-lasting treatment for patients with depression and other psychiatric conditions.
Preclinical assays in rodent models have evaluated the effects of psychedelics as rapid-acting antidepressant medications. The molecular mechanism responsible for the post-acute effects of psychedelics remain largely unexplored, but include dendritic spine structure, epigenetic alterations, and associated gene expression.
RESULTS
Psychedelics accelerate fear extinction via 5-HT2AR. DOI, a psychedelic yet relatively selective 5-HT2A/2CR agonist, reduces behavioral despair or passivity as a model of depression at least 24 h after its administration, whereas behaviors in models of anxiety, recognition memory, and sensorimotor gating remain unchanged.
5-HT2AR-deficient mice showed a modest yet statistically significant increase in fear during fear acquisition, whereas no differences were observed during the fear expression session. DOI had no effect on fear generalization in 5-HT2AR+/+ mice, but reduced freezing time during the generalization session in 5-HT2AR/ littermates. A different cohort of 5-HT2AR+/+ mice was administered DOI or vehicle 24 h before the fear-acquisition protocol. DOI reduced freezing time during the fear generalization and context fear extinction sessions.
Psychedelics enhance cortical dendritic density via 5-
The size and shape of dendritic spines are closely correlated with synaptogenesis and functional plasticity. Psychedelics can promote rapid structural plasticity in frontal cortex pyramidal neurons, although whether 5-HT2AR-dependent signaling is necessary for this particular phenotype remains a topic of intense debate. Here, we measured dendritic spine density in cortical pyramidal neurons in 5-HT2AR/ mice and controls. Our findings suggest a lower spine density in 5-HT2AR/ mice as compared to 5-HT2AR+/+ controls.
Based on the findings of dendritic spine structural plasticity, we next determined whether DOI also regulates persistent synaptic functional plasticity in frontal cortex pyramidal neurons. DOI enhanced synaptic plasticity in L2/3 frontal cortex pyramidal neurons.
Enhancers are highly dynamic epigenomic regulatory elements involved in plasticity and neurodevelopmental processes. They are also highly differentiating of various brain locations and functions. We examined gene ontology terms and enriched transcription factor binding motifs associated with differential enhancers that showed change from the control due to DOI exposure at one or more of the three time points. We divided all differential enhancers into 6 clusters with various patterns of variation after DOI administration.
To directly interrogate the functional relevance of these findings, we examined the GO terms and TF motifs associated with each of the clusters. We found that DOI administration leads to a decrease in synapse organization and assembly, as well as an increase in glutamate NMDA receptor activity.
DOI administration decreased enhancer intensity in clusters 4 – 6, indicating a compensatory mechanism. Cluster 3 shows enrichment in GO terms associated with rhythmic processes, and cluster 6 has synaptic plasticity-related genes heavily enriched.
Transcriptomic variations induced by DOI appear to be much more transient than epigenomic changes, and only a small number of genes exhibit a pattern of expression that correlates with enhancer dynamics.
We analyzed the overlap between H3K27ac peaks and SNPs associated with depression and eleven other traits. Three GWAS loci datasets had statistically significant overlap with both differential and static H3K27ac peaks, suggesting that epigenomic changes due to DOI exposure altered activity at these loci.
We used weighted correlation network analysis to detect co-expression modules with highly correlated genes and performed GO analysis on the genes included in these modules to associate these modules with sample traits or experimental groups.
The eigengene of module blue was overexpressed in both 24 and 48 h groups before decreasing to the control level on 7 days. This gene cluster is associated with axonogenesis, heart process, learning or memory, and histone modification. Previous studies suggested that Inpp4a may play an important role in the development of epilepsy, and that loss of function of Inpp4a increases severity of asthma, which may explain the mechanism underlying the effect of DOI preventing allergic asthma in a mouse model.
The expression of 5-HT2AR genes in module yellow shows opposite trend to that in module blue, and this module is also enriched with genes related to hypoxia-inducible factor 1 (HIF-1) signaling pathway.
DISCUSSION
Previous observations in rodent models of depression showed that psychedelics lead to long-lasting effects on synaptic plasticity and behavior models of depression. The present study provides direct evidence that psychedelics affect the 5-HT2AR and causes long-lasting effects on frontal cortex gene expression and chromatin organization.
The subjective effects of psychedelics may be necessary or complementary for their post-acute clinically relevant outcomes in patients with severe psychiatric disorders such as depression.
Psilocybin can induce subjective effects such as increased well-being and diminished ego functioning, which are difficult to control with placebo in the clinical setting. Rodent models of depression and stressor-related disorders provide an unparalleled platform for characterizing non-subjective effects of psychedelics, which can be segregated from uniquely human subjective processes such as attribution of mystical meaning to the psychedelic experience.
The epigenome and transcriptome of the frontal cortex are significantly changed following DOI administration. The epigenome changes persist for at least 7 days after DOI administration, and may constitute the molecular basis for the long-lasting effects.
We found significant overlap between peaks of H3K27ac in the mouse brain epigenome and 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.
Synaptic plasticity events have been linked to different adaptive and maladaptive traits, including increased dendritic spine density in the nucleus accumbens and schizophrenia-relevant features in rat models. The frontal cortex, ventral striatum, several thalamic nuclei, and the hypothalamus are all enriched in 5-HT2AR, and the post-acute effects of psychedelic DOI on the epigenomic landscape in the frontal cortex may contribute to drug addiction, psychotic symptoms, or hallucinogen persisting perception disorder. Previous work showed that 5-HT2AR agonism is not a sufficient condition for psychedelic action, and that a single dose of psychedelics induces frontal cortex 5-HT2AR-dependent expression of genes associated with cell morphogenesis, neuron projection, and synapse structure. A single dose of DOI leads to changes in frontal cortex synaptic plasticity and behavior models of fear extinction. It is plausible that activation of 5-HT2AR 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.
In conclusion, our study highlights the role of 5-HT2AR in the action of psychedelics and reveals persisting chromatin remodeling events following DOI administration. These findings could facilitate the understanding of psychopharmacological interventions.
STAR+METHODS
We used a whole-cell patch-clamp recording method, a novel object recognition test, a light-dark boxes preference test, a force-swimming test, a prepulse inhibition of startle test, and a dendritic spine analysis method to analyze our data.
The lead contact for this project is Javier Gonza lez-Maeso. Additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Locomotor activity
Locomotor activity was monitored 24h after injection of DOI or vehicle on a computerized three-dimensional activity monitoring system. The open field was cleaned with 1% Roccal-D in between sessions.
Light-dark boxes preference
Animals were injected with DOI (2 mg/kg, i.p.) or vehicle and were placed in a commercial open field with light and dark areas connected through a small opening. Their preference for light or dark environment was assessed 18h after drug administration.
Novel object recognition
Mice were tested with novel and familiar objects in an opaque rectangular plastic container. They were allowed to get acclimate to the behavioral room for the duration of the whole session. The test consisted of three stages, a habituation to the field, a 5 min acquisition trial and a 5 min recognition trial. The exploratory preference for the novel object was calculated as percentage of total exploratory time spent exploring the novel object during the recognition stage.
Prepulse inhibition of startle
Mice were presented with startling stimuli and allowed to acclimate to the testing room for 5 min. They were then randomly subjected to 65 trials in five different categories (13 of each): stimulus-only, no stimulus, 73 dB prepulse and stimulus, 77 dB prepulse and stimulus, 85 dB prepulse and stimulus.
Contextual fear acquisition and extinction
We conducted fear conditioning tests on mice using two commercially supplied Near Infrared Video Freeze Systems, each operating four test chambers. The mice were acclimated to the behavioral testing room for at least 1h in the 2 days prior to experimental sessions. Two different contexts were employed: A standard polycarbonate squared cage with metallic walls and shock-delivering grid on the floor, and B a modified chamber with black triangular adaptor and cardamom scent on the bedding. Experimental sessions were performed on two consecutive days. Fear-acquisition was established in Context A and mice received 6 unconditioned stimuli consisting of 2 s scrambled 0.70 mA footshocks delivered through the grid floor separated by 90 s inter-stimulus intervals. Mice were held in Context B for 2 hours prior to the extinction test. They were observed for freezing behavior over time in periods of 5 min in the absence of any aversive stimuli.
Dendritic spine analysis
Dendritic spine analysis assays were performed as previously reported (Ibi et al., 2017), with minor modifications. Mice were anesthetized with isoflurane during the surgery and perfusion, and eYFP was delivered bilaterally with a Hamilton syringe. AAV-infected pyramidal neurons in L5 expressing eYFP were imaged using a confocal fluorescence microscope with a 3 63 lens and a zoom of 2.5. Only one dendrite per neuron on 4-5 neurons per animal per experimental group was analyzed. NeuronStudio was used to quantitatively analyze spine size and shape. Spines were classified as either stubby, thin or mushroom based on aspect ratio, head-to-neck ratio and head diameter.
Whole-cell patch-clamp recordings and LTP induction
Whole-cell patch clamp recordings were obtained from neurons in layers 2/3 of somatosensory cortex from 8-10-week-old male mice. The mice received a single injection of either saline or DOI 24 hours prior to recording. Mice were deeply anesthetized with isoflurane, decapitated, and their brains were placed in chilled sucrose-artificial cerebrospinal fluid (aCSF). The aCSF was continuously bubbled with carbogen and the brains were sectioned and perfused with aCSF containing the GABAA receptor antagonist gabazine. L2/3 neurons were stimulated with a tungsten electrode in L4 and paired with L2/3 neurons in the same column. EPSCs were recorded at 70 mV for 3-5 mins, followed by brief membrane depolarization to 0 mV for 10 mins, then the holding potential was returned to 70 mV for 35-40 mins.
Nuclei isolation and sorting via FACS
Nuclei isolation was conducted using a published protocol. The following ingredients were used: mouse frontal cortex tissue, nuclei extraction buffer, protease inhibitor cocktail, phenylmethylsulfonyl fluoride, dithiothreitol, and ribonuclease inhibitor. The tissue was homogenized in a tissue grinder, filtered with a 40 mm cell strainer, and centrifuged at 1000 RCF for 10 min. The nuclei pellet was resuspended in 2% normal goat serum in Dulbecco’s PBS for 20 min. To separate NeuN+ and NeuN- fractions, 2 ng/ml anti-NeuN antibody conjugated with Alexa 488 was added into the nuclei suspension and incubated at 4 C for 1 h. A mixture of 26,000 NeuN+ nuclei, 1.8 M sucrose, 10 M CaCl2, and 3 M Mg(Ac)2 was added to 800 mL of ice-cold PBS for ChIP-seq experiment. 10,000 NeuN+ nuclei were used to produce each ChIP-seq library and two technical replicates were generated for each brain sample.
5000 NeuN+ nuclei were used to produce each RNA-seq library, and 6 brain samples were processed under each treatment condition. RNA was extracted using RNeasy Mini Kit and RNase-Free DNase Set, and cDNA was prepared using SMART-seq2 protocol. 11.4 mL of reverse transcript mix, 0.5 mL of RNase inhibitor, 4 mL of Superscript II first-strand buffer, 1 mL of DTT, 4 mL of 5 M Betaine, 0.12 mL of 1 M MgCl2, 0.58 mL of nuclease-free water, and 20 mL of first-strand mixture were mixed together. Generated cDNA was amplified by PCR, and 600 pg were used to produce an RNA-seq library using Nextera XT DNA Library Preparation Kit.
Sequencing
ChIP-seq and RNA-seq libraries were quantified using high sensitivity DNA analysis kit and Illumina HiSeq 4000, and 15 million reads were generated for each library.
ChIP-seq reads alignment and normalization
Sequencing reads were trimmed, aligned to the mm10 genome with Bowtie2, peaks were called using MACS2, blacklisted regions were removed to improve data quality using SAMtools, and a normalized signal was calculated.
Differential enhancers
We predicted enhancer regions by identifying H3K27ac peaks that did not intersect with promoters, and then combined consensus enhancer sets for each experimental group using Diffbind. We then identified differential enhancers between experimental groups using the Benjamini-Hochberg method.
K-means clustering, GO term analysis, TF motif analysis
We performed K-means clustering on differential enhancers and DEGs across experimental groups, and then linked enhancers to their nearest genes using ChIPSeeker. The resulting enhancer target genes were then analyzed using the GO biological processes enrichment test and motif analysis.
GWAS loci association
Peak locations were converted from mm10 to hg19 through UCSC’s liftOver tool, and GWAS datasets were obtained from NHGRI-EBI’s GWAS Catalog. Only sets that had at least 500 SNPs were selected, and only SNPs present in the 1000 Genomes Project Phase 3 European population were kept.
RNA-seq data analysis
Sequencing reads were trimmed and mapped to the GRCm38 genome by hisat2. Datasets with poor quality were discarded and DEGs were determined by pairwise comparison using DESeq2.
Weighted gene co-expression network analysis
We normalized gene expression values using log2(FPKM), ranked genes based on their median absolute deviation, and then constructed a signed co-expression network using the WGCNA package in R. The top 100 intramodular hub genes were identified using the softConnedticity function in the package.
QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analysis was performed with GraphPad Prism software version 9. Animals were randomly allocated into the different experimental groups, and statistical significance was assessed by two-way ANOVA followed by Sidak’s multiple comparison test.
Study details
Participants
10