The Effects of Hallucinogens on Gene Expression

This book chapter/review (2017) discusses the current state of knowledge on the molecular genetic responses to psychedelics within the brain in order to contribute to our understanding of how even single doses of psychedelics can have longer-term effects on brain and behavior.

Abstract

“The classic serotonergic hallucinogens, or psychedelics, have the ability to profoundly alter perception and behavior. These can include visual distortions, hallucinations, detachment from reality, and mystical experiences. Some psychedelics, like LSD, are able to produce these effects with remarkably low doses of drug. Others, like psilocybin, have recently been demonstrated to have significant clinical efficacy in the treatment of depression, anxiety, and addiction that persist for at least several months after only a single therapeutic session. How does this occur? Much work has recently been published from imaging studies showing that psychedelics alter brain network connectivity. They facilitate a disintegration of the default mode network, producing a hyperconnectivity between brain regions that allow centers that do not normally communicate with each other to do so. The immediate and acute effects on both behaviors and network connectivity are likely mediated by effector pathways downstream of serotonin 5-HT2A receptor activation. These acute molecular processes also influence gene expression changes, which likely influence synaptic plasticity and facilitate more long-term changes in brain neurochemistry ultimately underlying the therapeutic efficacy of a single administration to achieve long-lasting effects. In this review, we summarize what is currently known about the molecular genetic responses to psychedelics within the brain and discuss how gene expression changes may contribute to altered cellular physiology and behaviors.”

Authors: David A. Martin & Charles D. Nichols

Summary

Psychedelics alter perception and behavior by altering brain network connectivity and gene expression. These changes in brain network connectivity and gene expression are likely to contribute to long-lasting effects of a single administration of psychedelics.

1 Introduction

LSD and DOI activate 5-HT2A receptors, which cause changes in synaptic transmission and action potential firing. These changes are followed by changes in mRNA expression and protein translation.

The transcription of a number of genes and proteins within activated neurons is likely important for the long-term clinical phenomena that are observed following psychedelic administration. This transcriptional program can be used to gain insight into the potential long-term clinical benefits and risks of these drugs.

Psychedelic drugs modify several signaling pathways, including the ania3 transcript, which encodes for a protein associated with post-synaptic metabotropic glutamate receptor signaling.

2 Immediate Early Genes

The most well-studied gene expression changes observed following psychedelics administration involve induction of a variety of immediate early genes (IEGs), whose transcription is begun within minutes following neuronal stimulation. These IEGs ultimately provide a mechanism by which long-term structural and connective changes can occur at the synapse.

Psychedelics induce IEGs in rats through the expression of c-Fos, a protein that appears in neurons labeled with neuron-specific enolase. The expression of c-Fos is dose-dependent and depends on the amount of DOI administered.

DOI induces c-Fos expression in the parietal cortex that increases with dose and reaches a plateau at 12 mg/kg. This induction follows 5-HT2A activation in several cortical and sub-cortical regions.

The expression of the transcription factors c-Fos, ngf1c, and tis1 were increased in the cortex, hippocampus, and cerebellum following DOI administration. These increases were blocked by the 5-HT2 receptor antagonist, ketanserin.

Psychedelics induce several types of IEGs, including brain-derived neurotrophic factor (BDNF), which is involved in neuron development, experience-dependent plasticity, and modification of dendritic morphology. BDNF expression is regulated by 5-HT2A receptors, which are also involved in potentiation of active synapses and ketamine-mediated synaptogenesis.

DOI was found to induce arc mRNA expression in rats in a dose-dependent fashion, and the expression of arc mRNA was prevented by the 5-HT2 receptor antagonist ketanserin.

Arc protein was shown to follow a pattern of DOI induction similar to the mRNA, and was also shown to be overlapping with c-Fos staining, suggesting the same cells were producing these two IEGs. Arc also reduces AMPA receptor signaling and contributes to synaptic elimination.

3 IEG and 5-HT2A Receptor Expression: Early Studies

Early studies on the effects of psychedelics on IEGs failed to demonstrate that c-Fos was expressed in cells that were labeled with antisera against the 5-HT2A receptor. However, more recent studies have indicated that c-Fos expression occurs almost exclusively in cells that are positive for 5-HT2A mRNA expression.

The pattern of 5-HT2A receptor staining produced by one particular antibody does not match the staining pattern of other antibodies whose specificity was recently verified in mice lacking the 5-HT2A receptor.

Lack of 5-HT2A immunoreactivity in cells displaying DOI-induced IEG transcription implied that the effect of cellular activation by hallucinogens was indirect. However, more recent evidence suggests that cortical – cortical interactions are critical for hallucinogenic activity.

4 Identification of Tissues that Transcriptionally Respond to Psychedelics: Early Studies

Psychedelics activate a variety of cell types, including inhibitory GABAergic neurons, pyramidal neurons, and 5-HT2A receptors, which are expressed in cortical interneurons. These activations may provide important information about the mechanisms through which psychedelics cause their effects.

The effects of DOI on interneurons are variable, with no GABAergic, c-Fos+ cells located in the barrel cortex and little c-Fos and parvalbumin overlap in the orbital cortex and dorsal medial PFC following DOI.

DOI and LSD produce a variety of genetic responses in the cortex, including c-Fos mRNA and protein induction. The c-Fos response to LSD was first demonstrated in 1999, and a lower, but behaviorally active, dose of LSD was able to induce c-Fos in the anterior cingulate cortex.

Studies demonstrate that LSD induces c-Fos expression in many of the same areas as DOI, with the exception of a less robust response following lower doses of LSD (0.16 mg/kg) in areas of the frontal and parietal cortex.

5 IEG and 5-HT2A Receptor Expression in the Post-Genomic Era

The first unbiased microarray screen on the effects of LSD within the brain identified five genes upregulated by drug administration. These genes were validated by RNase protection as differentially expressed in the PFC.

LSD increased the expression of many genes, but only the nor1 gene remained at maximum levels through the final 5 h time point. The expression of these genes was unaffected by selective antagonism of the 5-HT1A receptor with WAY-100,635 but was significantly attenuated by 5-HT2A receptor selective antagonist M100,907.

We performed a second microarray screen using a different Affymetrix gene chip version, and identified and validated three additional transcripts increased by LSD in the rat PFC: map kinase phosphatase 1, core/enhancer binding protein b, and a novel gene, induced by lysergic acid diethylamide 1.

Neither c-Fos nor arc changes were identified in these two microarray screens. Furthermore, only 1 in 4 – 5 genes could be confirmed as differentially expressed by RNAse protection.

LSD induces a variety of genes, including nor1, sgk, ania3, and c/ebp-b, which are involved in synaptic plasticity. However, the manner in which these genes contribute to the downstream transcriptional, structural, and functional sequelae of neuronal activation remains poorly understood.

Gonzalez-Maeso and colleagues studied the transcriptional effects of several psychotropic agents in a cell culture system using 5-HT2A-expressing HEK293 cells. They found that DOI induced the transcription of several genes, and that these genes followed dose- and time-dependent responses.

LSD and DOI upregulated three genes in mouse somatosensory cortex, and lisuride upregulated three genes. DOI produced no gene expression changes in mice lacking the 5-HT2A receptor.

These data suggest that functional selectivity is occurring at the 5-HT2A receptor, and that c-Fos expression is not always correlated with overt behaviors. The two genes, egr-1 and egr-2, are members of the zinc family of transcription factors whose expression is correlated with LTP and implicated in modulation of synaptic plasticity and memory formation.

A subsequent report found that hallucinogens and non-hallucinogens can produce dissociable effects on transcription and behavior. The overall transcriptional response was largely eliminated in mice lacking the 5-HT2A receptor.

Studies performed in primary neuronal cultures found that c-Fos and egr-2 expression were increased by LSD, but only c-Fos was increased by R-lisuride. egr-2 expression was dependent on the type of cell used.

LSD induces transcription of egr-2 and c-Fos in neuronal cultures without a requirement for neuronal depolarization, indicating that the transcriptional response mediated by 5-HT2A receptor activation is dependent on the ligand, the cell type, and the environment.

6 Psychedelics, Gene Expression, and Cellular Signaling

The 5-HT2A receptor activates several pathways, including the Gaq-activated PLC-b pathway, the Gai/o-activated Gbc pathway, and the Rho-activated PLC-b pathway. These pathways can recruit different sets of genes, depending on the ligand used to activate the receptor and the nature of the cell it is expressed in.

Further exploration of the gene expression differences between LSD and lisuride in neuronal cell culture has found that both Gaq activation of PLC-b and activation of Gai/o/Gbc/Src are necessary for psychedelic relevant gene expression patterns.

Several studies have attempted to modify the behavioral response to psychedelics by disrupting signaling pathways downstream of 5-HT2A receptor stimulation. These studies have shown that multiple pathways downstream of receptor activation are important for transcriptional and behavioral effects of psychedelics in vivo.

IEG expression has frequently been used to measure perturbations of psychedelic drug mediated signaling. AMPA and NMDA receptor activation is necessary for neurons to be transcriptionally activated by DOI, and this is consistent with a preponderance of evidence implicating glutamate release as critical for the electrophysiological and behavioral responses to hallucinogens.

The characterization of metabotropic glutamate receptor 2 (mGluR2) influences on 5-HT2A signaling has also relied partly on measurement of IEG expression. The mGluR2/3 agonist LY35470 and the mGluR2/3 antagonist LY341495 both potentiate the upregulation of BDNF by 5 mg/kg DOI. Activation of mGlurR2/3 with LY379268 attenuates DOI-induced c-Fos expression in the mPFC, but not in the frontoparietal or somatosensory cortex, indicating that mGluR2 and not mGluR3 receptors are involved in psychedelic-induced c-Fos expression changes. The mechanism for the functional interaction between 5-HT2A and mGluR2 signaling is not completely understood, but presynaptic 5-HT2A receptors have been identified on thalamic inputs in the cortex.

7 Identification and Characterization of the Cortical Cellular Population Responsive to Psychedelics

The exact population of cells in the cortex that initiate the signaling that leads to psychedelic transcriptional and behavioral effects remains unknown. However, 5-HT2A receptor signaling within the Emx1 lineage is necessary and sufficient for at least some of the effects of psychedelics.

We found that 5% of cortical neurons directly respond to psychedelics in vivo by increasing transcription of immediate early and other genes. These cells have a higher level of HTR2A mRNA expression, which may result in higher 5-HT2A receptor levels rendering them more sensitive to agonists for this receptor. We found that 5 to 10% of inhibitory GABA neurons are transcriptionally activated by psychedelics, and that non-neuronal cells like astrocytes are also transcriptionally activated for genes like c-Fos. The transcriptional responses differed between brain regions analyzed.

8 Chronic Effects of Psychedelics

LSD produces long-lasting changes in gene expression and behavior when given chronically, including hyperactivity, reduced sucrose preference, and changes of social behaviors. These changes are persistent at full strength long after the drug is discontinued.

We performed RNA sequencing on RNA isolated from the mPFC of rats four weeks after cessation of a 90-day treatment protocol with LSD or saline. We found several hundred altered genes that were significantly concentrated in pathways related to neurotransmission, synaptic plasticity, and metabolism.

Long-term LSD treatment produces persistent connectivity modifications that are likely mediated through general plasticity mechanisms. The abnormal behaviors produced and the genes that are affected may make rats a useful platform to study mechanisms underlying certain psychiatric diseases.

9 Effects of Psychedelics Outside of the CNS

There has been little study of psychedelics outside of the central nervous system. However, the 5-HT2A receptor is widely expressed.

Psychedelics have been found to have effects on peripheral tissues, including effects on gene expression. They can inhibit inflammation mediated by the proinflammatory agent tumor necrosis factor alpha (TNF-a) in both cell culture and in whole animal. Although the precise mechanism for inhibition of transcription of proinflammatory genes remains to be elucidated, we believe that stimulation of 5-HT2A receptors with psychedelics inhibits signaling from the TNF-a receptor and inhibits activation of NF-jB.

10 Conclusion

Through the early 1970s, several clinical studies were conducted using psychedelic compounds for the treatment of mental disorders and addictions. Recently, renewed interest in these drugs has led to research into their potential use for inflammatory disorders like asthma.

Psychedelics have been recognized for their ability to occasion mystical-type experiences. One or two treatments with psilocybin can produce long-lasting positive behavioral and/or physiological changes through long-term alterations in gene expression.