Lysergic acid diethylamide (LSD) is a well-known psychoactive substance that transiently alters mind and perception. During the last few years, renewed interest has been seen in the use of LSD in psychiatric research and practice (Liechti, 2017). Modern experimental studies in humans have reported LSD’s subjective, autonomic, and endocrine effects (Schmid et al., 2015; Carhart-Harris et al., 2016a; Dolder et al., 2016; Strajhar et al., 2016; Liechti et al., 2017), functional brain activation patterns (Carhart-Harris et al., 2016b; Lebedev et al., 2016; Tagliazucchi et al., 2016; Preller et al., 2017), and pharmacokinetics (Dolder et al., 2015b, 2017). However, many aspects of LSD’s pharmacological effects remain unclear, including the phenomenon of the rapid development of tolerance (i.e., tachyphylaxis) to its psychological and physiological effects with repeated administration (Passie et al., 2008). Early studies that employed repeated daily administration of 100 μg LSD in humans described partial tolerance with the second dose and complete tolerance with the third and subsequent doses (Cholden et al., 1955; Abramson et al., 1956; Belleville et al., 1956). The effects of LSD reappeared only after a substance-free interval of 4 days. Tolerance to LSD is believed to result from serotonin 5-HT_2A receptor downregulation (Nichols, 2016). LSD potently binds to the 5-HT_2A receptor (Wacker et al., 2017) where it acts as a partial agonist (Rickli et al., 2016). Pretreatment with the 5-HT_2A receptor antagonist ketanserin completely prevented all perceptual and mind-altering effects of LSD in humans (Kraehenmann et al., 2017; Preller et al., 2017), indicating that the hallucinogenic effects of LSD are primarily mediated through 5-HT_2A receptors. Similar to humans, tolerance to the behavioral effects of LSD is also observed in rats (Buchborn et al., 2015). Consistent with a role for 5-HT_2A receptors in the development of tolerance, daily LSD administration for 3 days decreased 5-HT_2A receptor binding in the rat frontal cortex (Buckholtz et al., 1985, 1990; Gresch et al., 2005). However, another study reported only a non-significant trend toward a reduction of frontocortical 5-HT_2A receptor binding in rats during the development of tolerance to LSD (Buchborn et al., 2015). Additionally, no effects of acute LSD on 5-HT_2A receptor mRNA expression were found in the rat prefrontal cortex, hippocampus, or midbrain (Nichols and Sanders-Bush, 2002), although the effects of repeated LSD administration were not studied. Instead, adaptations in glutamate receptors were observed (Buchborn et al., 2015). Indeed, a key mechanism of action of LSD is the activation of frontal cortex glutamate transmission secondary to 5-HT_2A receptor stimulation (Gonzalez-Maeso et al., 2008; Moreno et al., 2011; Buchborn et al., 2015). Thus, adaptive changes that underlie tolerance to LSD may be reflected by alterations in the expression of genes that are involved in glutamatergic signaling or genes that regulate 5-HT_2A receptor function or its downstream signaling pathways. Acute LSD increased the expression of immediate early genes in the rat prefrontal cortex, including EGR2 (i.e., a gene that is involved in cognition and neural plasticity) and several others genes (Nichols and Sanders-Bush, 2002; Nichols et al., 2003; Nichols and Sanders-Bush, 2004). Further animal studies showed that LSD also increased the expression of EGR2 and EGR1 in the mouse cortex via 5-HT_2A receptor stimulation (Gonzalez-Maeso et al., 2003, 2007). Finally, LSD was shown to produce a characteristic transcriptome signaling pattern in normal but not HTR2A^-/- mice (Gonzalez-Maeso et al., 2007).

Despite the renewed interest in the clinical use of LSD, no human studies on the effects of LSD on gene expression have been performed. Therefore, we investigated the effects of a single dose of LSD on the expression of the 5-HT_2A receptor gene (HTR2A) and EGR1-3 genes. The EGR1 and EGR2 genes were studied based on preclinical data (Gonzalez-Maeso et al., 2003, 2007), and the EGR3 gene was studied because it has been shown to regulate HTR2A expression (Maple et al., 2015).

Acute changes in gene expression cannot be determined in heathy human brain tissue, as biopsy or postmortem tissue is not available. However, mRNA level changes in blood for candidate genes including EGR and HTR2A can cautiously be used as peripheral markers of transcription alterations in the CNS in response to interventions or to characterize patient groups (Sullivan et al., 2006; Mohr and Liew, 2007; Desjardins et al., 2008; Belzeaux et al., 2010; Rollins et al., 2010; Rivera-Baltanas et al., 2014; Cattane et al., 2015). The EGR and HTR2A genes are expressed in peripheral blood cells (Stefulj et al., 2000; Fukuda et al., 2006; Inoue et al., 2011; Cattane et al., 2015). The peripheral mRNA expression of several genes including the HTR2A gene were shown to have relatively similar expression profiles as in brain tissues (Glatt et al., 2005; Fukuda et al., 2006; Sullivan et al., 2006; Desjardins et al., 2008; Rollins et al., 2010; Yubero-Lahoz et al., 2015; GTExPortal, 2017). In healthy humans, HTR2A gene expression levels in whole blood correlated with 5-HT metabolite levels in the cerebrospinal fluid (Luykx et al., 2016). Peripheral EGR1 expression was increased in patients with schizophrenia compared with healthy controls (Cattane et al., 2015).

Therefore, we expected altered HTR2A expression in response to LSD. Additionally, we hypothesized that LSD acutely increases EGR1 and EGR2 gene expression in humans similarly to rodents.

Three samples after 24 h had insufficient amounts of RNA to be included in the analysis (Table 2). The expression patterns of the HTR2A, EGR1, EGR2, and EGR3 genes were unchanged 1.5 and 24 h after the administration of LSD compared with placebo (Figure 1 and Table 2). Expression did also not change over time after LSD and placebo (HTR2A: F_2,24 = 0.02, P = 1.0 and F_2,26 = 1.24, P = 0.3; EGR1: F_2,24 = 1.14, P = 0.3 and F_2,26 = 1.9, P = 0.2; EGR2: F_2,24 = 1.20, P = 0.3 and F_2,26 = 2.67, P = 0.09; EGR3: F_2,24 = 1.17, P = 0.2 and F_2,24 = 0.08, P = 0.9, respectively). Sex did not moderate the effects of LSD or placebo on gene expression. Additionally, the findings were similar if the data was standardized to the mean age, body weight or plasma concentration of LSD.

The key finding of the present study was that acute LSD administration did not alter the expression of the HTR2A and EGR1-3 genes in humans using peripheral blood cells as peripheral biomarker possibly reflecting central gene expression. The lack of an acute effect of LSD on HTR2A gene expression in humans is consistent with a study in rats that reported no changes in HTR2A gene expression in different brain areas (Nichols and Sanders-Bush, 2002). However, 5-HT_2A receptor availability may also be altered independently of HTR2A gene expression (e.g., by receptor internalization or moderation of its activity). Several studies in rats (Buckholtz et al., 1985, 1990; Gresch et al., 2005; Buchborn et al., 2015) reported a decrease in 5-HT_2A receptor binding in the prefrontal cortex or consistent trend effects (Buchborn et al., 2015) after repeated LSD administration. Unknown, however, is whether lower binding also occurs after single-dose administration. The present findings of no changes in EGR1 and EGR2 gene expression in human blood samples after acute LSD administration contrast with preclinical findings. Specifically, LSD rapidly increased EGR1 and EGR2 expression in the cortex in rats (Nichols and Sanders-Bush, 2002, 2004; Nichols et al., 2003) and mice (Gonzalez-Maeso et al., 2003, 2007). We expected similar rapid increases in EGR1 and EGR2 expression in humans. Importantly, however, we evaluated gene expression in human blood samples, whereas the animal studies evaluated gene expression in brain tissue. Thus, it is possible that LSD alters gene expression in the brain and not in blood.

Tolerance to repeated LSD administration reportedly begins with the second daily dose of LSD, and complete tolerance develops within 3–4 days of repeated LSD administration in humans according to older studies (Cholden et al., 1955; Abramson et al., 1956; Belleville et al., 1956; Passie et al., 2008; Nichols, 2016) that need to be replicated. In the present study, we found no evidence of acute pharmacological tolerance within 12 h of acute LSD administration at a dose of 100 μg as documented in detail elsewhere (Dolder et al., 2017). Similarly, no acute tolerance was observed after single-dose administration of 200 μg LSD in humans within 24 h (Dolder et al., 2015b, 2017). Thus, after one dose of LSD, subjective effects of LSD were self-reported by the participants as long as LSD was present in plasma, and the subjective effects did not decline more rapidly than the plasma concentrations of LSD (Dolder et al., 2017). This is consistent with the view that LSD directly activates 5-HT_2A receptors to produce its mind-altering effects as long as it is present in the effect compartment (i.e., the brain) and assuming largely similar plasma and effect compartment kinetics. The finding of no acute tolerance in the participants in the present study (Dolder et al., 2017) also indicates that no relevant counterregulatory neuroadaptations occurred or were evident with the first 12–24 h after LSD administration. A recent study showed that LSD dissociates very slowly from the 5-HT_2A receptor, and the authors proposed that the high potency and long effect duration of LSD could be linked to a unique receptor interaction (Wacker et al., 2017). However, the LSD concentration-effect relationship (Dolder et al., 2017) shows that the presence of LSD in the body sufficiently accounted for the duration of its subjective effects. Doubling the LSD dose resulted in prolongation of the effect by approximately 3 h, consistent with its half-life of approximately 3 h (Dolder et al., 2017). In contrast to the pharmacokinetic–pharmacodynamic relationship of LSD (Dolder et al., 2017), other psychoactive substances, such as 3,4-methylenedioxymethamphetamine (MDMA), exhibit very marked acute pharmacological tolerance, with a rapid decline of subjective and physiological effects of MDMA within 4 h despite continuously high plasma levels and a relatively long half-life of 8 h (Hysek et al., 2010; Vizeli and Liechti, 2017).

The present study has several limitations. First, we assessed the effects of only an acute single dose of LSD on gene expression and tolerance. Further repeated dose administration studies need to be conducted. Second, we used only a moderate single dose of LSD, and the study sample was relatively small. Third, we assessed gene expression only at 1.5 and 24 h after LSD administration. Therefore, we may have missed effects that may have occurred between these time points. In fact, increases in EGR2 were observed only up to 5 h in rats (Nichols and Sanders-Bush, 2002; Gonzalez-Maeso et al., 2003; Nichols et al., 2003). Fourth, all changes in gene expression that are caused by LSD that have been reported to date have been observed animal brains, whereas our study focused solely on human blood cells as a peripheral biomarker of the central nervous system (Sullivan et al., 2006; Mohr and Liew, 2007; Desjardins et al., 2008; Belzeaux et al., 2010; Rollins et al., 2010; Rivera-Baltanas et al., 2014; Cattane et al., 2015).

In summary, an acute single dose of LSD in humans did not acutely alter the expression of the HTR2A and EGR1-3 genes in peripheral mononuclear blood cells and thus did not influence potential markers of neuroadaptation.

Each of the authors participated in this research by contributing to the conception and design of the study (PD and ML), study management (PD, FM, and SB) performance of laboratory experiments (EG) and statistical analysis and interpretation (PD, EG, and ML).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.